Monthly Archives: December 2019

JC FAMILY Project: Development and feasibility of a pilot trial of a 15-minute Zero-time exercise community-based intervention to reduce sedentary behaviour and enhance physical activity and family communication in older people

DOI: 10.31038/ASMHS.2019362

Abstract

Objectives: We developed and tested a very brief Zero-time exercise (ZTEx) community-based intervention to reduce sedentary behaviour and enhance physical activity and family communication in older people. ZTEx uses a foot-in-the-door approach to integrate simple strength- and stamina-enhancing physical activity into daily life at anytime, anywhere, and by anybody.

Methods: A 15-minute ZTEx intervention mini workshop with demonstrations by interventionists and practice by participants was conducted in each of the 18 districts in Hong Kong for a total of 556 public housing estate residents from 2015 to 2016. 141 participants (87% female, 73% aged ≥ 50 years) completed the evaluation. Primary outcome: intention to increase physical activity. Secondary outcomes: perceived knowledge, attitude (intention and self-efficacy) and practice regarding simple strength- and stamina-enhancing physical activity (i.e. ZTEx), days spent engaged in >= 10-minute moderate or vigorous physical activities and family communication (encouraging and engaging family members in ZTEx), and sitting time.

Results: Participants were enthusiastic and enjoyed the workshops. Perceived knowledge and attitude regarding sedentary behaviour, ZTEx, and family communication significantly increased immediately after the workshops (Cohen’s d = 0.20 to 0.30, all p < 0.05). At the 2-week follow-up, doing ZTEx and encouraging family members to do ZTEx significantly increased by 0.7 days and 0.4 days (Cohen’s d = 0.18 and 0.26, p < 0.05) respectively.

Conclusion: Our findings show early evidence that a brief ZTEx community-based intervention is an innovative, enjoyable and effective approach to improve perceived knowledge, attitude, practice, and family communication regarding simple strength- and stamina-enhancing physical activity in older people.

Keywords

Zero-time Exercise, Physical Activity, Sedentary Behaviour, Brief Community-Based Intervention

Introduction

We describe the development and feasibility of a pilot trial of a brief theory- and community-based intervention to reduce sedentary behaviour and enhance physical activity and family communication in older Chinese people in Hong Kong, the most westernized and urbanized city with rapid aging in China. Physical activity has been shown to reduce the risk of non-communicable diseases such as cardiovascular disease, stroke, and diabetes [1], improve mental health [2], and delay the onset of dementia [3]. Despite the well-known importance of physical activity for physical and mental health, physical inactivity is major public health problem globally and in Hong Kong. Physical inactivity is especially of concern given population ageing: physical activity tends to decline and sedentary behaviour tends to increase with advancing age, and the World Health Organization has stated that the proportion of the worlds’ population aged over 60 years is set to nearly double from 12% to 22% between 2015 and 2050 [4].

New, simple, and cost-effective approaches are needed to promote healthy ageing, particularly to reduce sedentary time and enhance physical activity. The present very brief intervention utilized multiple theory-based strategies: (i) cognitive dissonance, to arouse participants’ intrinsic motivation regarding exercise autonomy; (ii) the ‘foot-in-the-door’ approach, to promote participants’ exercise self-efficacy by starting with simple physical activity; (iii) gamification, by transforming the fitness assessments into fun games to promote exercise intention; and (iv) family involvement, by giving simple and specific instructions to participants to share what they have just learned with family members and praise them during the process to enhance family communication.

Zero-time exercise (ZTEx) uses a foot-in-the-door approach to kick-start the integration of simple strength- and stamina-enhancing physical activity, such as simple movements and stretching while sitting or standing, into daily life. ZTEx includes easy, enjoyable and effective (3Es) exercises that do not require extra time, money or equipment, and can be done anytime, anywhere and by anybody [5]. This approach is in line with the suggestions from physical activity guidelines for Americans that moving more and sitting less will benefit nearly everyone, and some physical activity is better than none [6]. Examples of ZTEx while sitting include raising the feet and legs off the ground, pedalling both legs, and stretching. Examples while standing include raising both heels and standing on one leg. More examples of ZTEx are shown in our YouTube videos (https://www.youtube.com/watch?v=ym3nGLGE4fg). Our pilot trials on ZTEx for lay health promoters (n = 28), social service and related workers (n = 56) and individuals with insomnia (n = 37) showed increased physical activity and perceived well-being [5, 7, 8]. The foot-in-the-door approach is a compliance tactic, which offers the easiest first step to start with, the idea being that small demands are easier to meet [9]. This approach has been applied in various fields such as the promotion of tobacco control and regular physical activity [10, 11].

Cognitive dissonance refers to the feeling of mental conflicts that occurs when an individual holds inconsistent attitudes, beliefs, and behaviours; this can lead to an alteration in attitudes, beliefs or behaviours to reduce the discomforts and restore psychological balance [12]. The desire to avoid the cognitive dissonance induced by discrepancies between one’s thoughts (harms of sedentary behaviour and advantages of physical activity) and current behaviour (low levels of physical activity) can help to arouse intrinsic motivation to increase physical activity. Dissonance interventions have been applied to improve health outcomes and suggested for health interventions for older people [13].

The Jockey Club FAMILY Project was initiated and funded by The Hong Kong Jockey Club Charities Trust. It aimed to promote family communication and family health, happiness and harmony (3Hs) in Hong Kong (website: http://www.family.org.hk/) [14]. In 2015, the School of Public Health, The University of Hong Kong was invited by the Hong Kong Department of Health and the Estate Management Advisory Committee of to add ZTEx content to a series of health talks aimed at residents living in public housing estates (low rental housing for low income groups) across the 18 districts in Hong Kong. The total duration of each session was 60 minutes, and the School of Public Health team was invited to utilize about 15 minutes in the middle to conduct a very brief ZTEx community-based intervention (i.e. a mini workshop). We hypothesized that this brief intervention would promote the knowledge, attitude (intention and self-efficacy), and practice of simple strength- and stamina-enhancing physical activity (i.e., ZTEx), family communication through encouraging and engaging family members in ZTEx, and personal and family well-being.

The primary outcome was the participants’ intention to increase simple strength- and stamina- enhancing physical activity (ZTEx) immediately after the workshop. The secondary outcomes were participants’ perceived knowledge and attitude regarding ZTEx, sedentary behaviour, and family communication (encouraging and engaging family members in ZTEx), immediately after the workshop. We also assessed participants’ sitting time, levels of simple strength- and stamina-enhancing physical activity, moderate and vigorous physical activity, and family communication regarding ZTEx, and personal and family well-being through a phone follow-up 2 weeks later. Feedback from participants on the quality of the intervention content and onsite observations on participants’ responses and intervention implementation were recorded.

Methods

Participants

The inclusion criteria included: (i) aged 18 years or older, (ii) can read Chinese and speak Cantonese, and (iii) can complete a short questionnaire. The exclusion criteria included having serious health conditions that might prevent them from physical activity. 556 participants from the 18 public housing estates attended the mini workshops as part of the health talks. All participants were invited to join the trial. The research protocol was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster with registration number UW15-743, and was registered at the National Institutes of Health (http://www.clinicaltrials.gov; identifier number: NCT02645071).

Intervention

The brief ZTEx intervention was a 15-minute face-to-face session (mini workshop) designed by academic health professionals (a public health physician and a nurse). The same intervention was conducted at the public housing estate health talks in each of the 18 districts in Hong Kong from 2015 to 2016. The intervention was grounded in cognitive dissonance theory. We first introduced the phenomenon of physical inactivity in Hong Kong and emphasised the harms of sedentary behaviour. Then, we asked simple questions related to the participants’ physical activity habits, aiming to induce dissonance between their beliefs and behaviour and arouse intrinsic motivation regarding exercise autonomy.

We then utilized a foot-in-the-door approach to kick-start participants’ practice of easy-to-do and simple strength- and stamina-enhancing physical activity (ZTEx) in daily life. We demonstrated examples of ZTEx, and invited the participants to follow the actions and practice immediately. We gave simple and clear instructions and examples for how to integrate ZTEx into daily life and encouraged them to choose and create their own ZTEx (varying type, frequency, intensity and time) to increase their exercise self-efficacy and autonomy. A meta-analysis of 41 studies indicated that providing choice enhanced intrinsic motivation, effort, task performance, and perceived competence [15]. The brief intervention utilized experiential learning, which is a powerful learning tool [16]. Throughout the intervention, the participants were actively engaged, practicing ZTEx (in the form of fun games, explained below) together.

We then incorporated the positive psychology themes ‘happiness’ [17] and ‘praise’ [18] into group activities during the intervention. Two interactive and fun games were used to promote doing ZTEx as a norm in the group; as positive reinforcement, participants’ efforts and improvements were praised. The first game (the ‘single-leg–stance game’) transformed an assessment test into a group competition game [19]. All participants were invited to stand on one leg and count the time in seconds that they could effectively balance on one leg, up to a maximum of 120 seconds. We used informal physical fitness benchmarking, with the interventionist and participants counting out loud (001, 002, 003, …, 120) at a steady pace to obtain an ongoing estimate of the time for which they were able to maintain balance while standing on one leg. After the ‘game’, we revealed the age- and gender-specific reference values for the single-leg-stance and encouraged participants to compare their results with the normative data [19]. We explained the clinical relevance, highlighted the importance of balance to reduce the risk of falling, and emphasized that one can quickly improve balance with a few days’ practice.

For the second game (the ‘grip strength game’), participants were invited to hold a spoon between the handles of a handgrip by squeezing the handles together. Participants counted the number of seconds that they could effectively hold the spoon, up to a maximum of 60 seconds. After the game, we introduced a simple and clear health message on the relationship between grip strength and cardiovascular disease: “Every 5 kilograms decrease in grip strength is associated with a 9% and 7% higher risk of stroke and heart attack (such as myocardial infarction), respectively [20]”.

Before the close of the session, each participant received a leaflet with pictorial instructions and examples of ZTEx and a handgrip to bring home, which would serve as visual reminders to practise grip strength exercises and other ZTEx regularly. We highlighted the interest and positive feelings of achievement to strengthen participants’ intrinsic motivation for doing physical activity. Lastly, we emphasised the importance of regular physical activity for healthy ageing and the relationship between healthy ageing and individual and family well-being. We recommended that the participants should take two actions: (i) introduce ZTEx to family members using the leaflet; and (ii) engage in the ‘single-leg-stance game’ and ‘grip strength game’ with family members with competition among family members. To play the games with family members, participants could follow the examples practiced in the workshop (by counting the time duration out loud). We highlighted that such games can provide a good opportunity for positive family communication and expressing care toward family members. The interventionists suggested that participants and their family members could record their baseline scores as reference, monitor their own progress, and set realistic goals and make plans regarding physical activity. Family well-being (health, happiness and harmony) was expected to be enhanced through the fun games and positive family communication.

Measures

Our research staff closely observed the responses and interaction among the participants and the interventionist. Structured questionnaires were used to measure the outcomes at baseline, immediately after the session, and at a 2-week phone follow-up.

Perceived knowledge and attitude regarding sedentary behaviour and physical activity

We asked the participants to indicate the extent of their agreement to four statements about their own knowledge and attitude regarding sedentary behaviour and physical activity. Simple strength- and stamina-enhancing physical activity (i.e. ZTEx) was introduced briefly with some examples before participants answered the questions. The statements were: (i) “I understand the general concept of ZTEx” (perceived knowledge); (ii) “I intend to do ZTEx regularly” (intention); (iii) “I need to reduce my sedentary behaviour” (intention); and (iv) “I am confident that I can do ZTEx regularly” (self-efficacy).

We also asked participants to indicate the extent of their agreement with five statements regarding family communication and engaging family members in ZTEx. Three statements addressed exercise intention: “I think there is a need for my family members to reduce their sedentary behaviour”; “I intend to encourage my family to do ZTEx regularly”; and “I intend to engage in ZTEx with my family regularly”. Two statements addressed exercise self-efficacy: “I am confident that I can encourage my family to engage in ZTEx regularly”; and “I am confident that I can engage in ZTEx with my family regularly”. Responses were made on a 6-point Likert scale, ranging from 1 (strongly disagree) to 6 (strongly agree). Higher scores indicated greater exercise intention and self-efficacy.

Practice regarding sedentary behaviour and physical activity

Questions from the short form of the International Physical Activity Questionnaire – Chinese version (IPAQ-C) were used to assess participants’ level of sedentary behaviour and physical activity by asking for their self-reported sitting time and the number of days on which they engaged in moderate and vigorous physical activity, respectively (21). The questions were: “On a typical weekday in the last 7 days, how many hours per day did you typically spend seated?”; “During the last 7 days, on how many days did you do at least 10 minutes of moderate physical activity?”; and “During the last 7 days, on how many days did you do at least 10 minutes of vigorous physical activity?” [21]. We assessed the number of days on which participants performed simple strength- and stamina-enhancing physical activity by asking three questions. The questions were: “During the last 7 days, on how many days did you do simple strength- and stamina-enhancing physical activity?”; “During the last 7 days, on how many days did you encourage your family to do simple strength- and stamina-enhancing physical activity?”; and “During the last 7 days, on how many days did you do simple strength- and stamina-enhancing physical activity with your family?”. The responses ranged from 0 to 7 days.

Perceived well-being

Perceived personal well-being was assessed by asking two questions: “Do you think that you are healthy?” and “Do you think that you are happy?” [7]. Perceived family well-being was assessed by asking three questions: “Do you think that your family is healthy?”; “Do you think that your family is happy?”; and “Do you think that your family is harmonious?”. Each item allowed a response on a scale from 0 (not at all healthy/happy/harmonious) to 10 (very healthy/happy/harmonious). A higher score indicated a more positive perception of family well-being [22].

Reactions to intervention content

Participants were asked to grade the quality and utility of the mini workshop (i.e. intervention) and its contents through two questions: “How much did you like the workshop?”; and “How feasible will it be to incorporate the exercises you have learned into your daily life?”. Responses were made on an 11-point Likert scale, ranging from 0 (very unsatisfied / totally not feasible) to 10 (very satisfied / very feasible) [23].

Feedback on intervention implementation by on-site observers

We asked on-site observers to indicate the extent of their agreement with four statements regarding the quality of intervention implementation: “The time arrangement is suitable for the intervention”; “The location is suitable for the intervention”; “The room size is suitable for the intervention; and “The facilities and manpower can meet the needs of the intervention”. Responses were made on a 5-point Likert scale, ranging from 1 (strongly disagree) to 5 (strongly agree). On-site observers also rated the level of participant participation on two aspects: participants’ punctuality, and participants’ involvement. Responses were made on a 5-point Likert scale, ranging from 1 (very low) to 5 (very high). A higher score indicated better performance.

Statistical Analysis

Analyses were conducted using SPSS version 24.0. The calculation of sample size was based on the assumption that the intervention on the change of intention to do simple strength and stamina enhancing physical activity (ZTEx) with small effect size (Cohen’s d = 0.3) immediately after the workshop. Ninety subjects in a group were required for a power of 80% and a maximum error of 5% by paired t-test. It was estimated that 100 subjects would be needed for this single-group trial, assuming a small attrition rate. The paired t-test and Wilcoxon signed-rank test were used to compare the continuous parametric and non-parametric data between two time-points, respectively. The McNemar test was used to examine the changes in categorical data between two time-points. Following convention, an effect size of 0.2 to < 0.5 was considered as small, 0.5 to < 0.8 as medium, and 0.8 or above as large (Cohen, 1988). Statistical significance was determined by p < 0.05. By intention-to-treat analysis, missing data for participants who were lost to follow-up or declined to complete the questionnaires were replaced with the corresponding baseline values. Complete-case analysis was conducted for the participants with completed assessments at baseline, immediately after the workshop, and at the 2-week follow-up, to determine whether the results were consistent with intention-to-treat analysis. Sensitivity analysis was performed using complete-case analysis, which included participants who completed the 2-week follow-up and excluded those with missing data.

Results

During 2015 to 2016, we conducted 18 mini workshops with the same content and procedures for 556 public housing estates residents. Most of the participants were enthusiastic, actively involved, enjoyed the session and showed great appreciation. The average duration of mini workshop was 15 ± 4 minutes. We could not include 299 participants in the trial because most participants were older people with poor eyesight and needed assistance in answering questionnaires. The workshops needed to start on time, but we did not have enough time and manpower to obtain consent from all participants and help them to complete the questionnaires, even though most were willing to join. 96 refused to give their phone number for the 2-week follow-up. 20 agreed to join, but did not complete the baseline questionnaire.

Before the start of the workshops, 141 participants (87% female, 73% aged ≥ 50 years) agreed to join the trial and completed the baseline questionnaires. Immediately after the workshops, 117 participants completed the immediately post-intervention questionnaire; 24 declined to answer or were unable to complete the questionnaire as they left the venue immediately. At the 2-week phone follow-up, 79 participants completed the 2-week phone follow-up questionnaire; we were unable to contact 25 participants after three phone call attempts per participant and 13 refused to answer. (Figure 1 and Table 1) show the flow and characteristics of the participants, respectively. There was no significant difference in the characteristics between two groups, except that the participants who completed the 2-week follow-up did more vigorous physical activity than those who did not complete the follow-up (p <0.05).

Table 1. Characteristics of all participants, those who completed and those who did not complete the 2-week follow–up.

ASMHS-19-Agnes_HongKong_t1

ZTEx = Zero-time exercise refers to simple strength- and stamina-enhancing physical activity

Independent T-test and Mann-Whitney test to compare the difference of the continuous parametric data and non-parametric data, respectively; Chi-square test to compare the difference of the categorical data between two groups; Difference between two time points:*p < 0.05

a 6-point Likert scale:1 (strongly disagree); 2 ( disagree); 3 (slightly disagree); 4 (slightly agree); 5 (agree); 6 (strongly agree).

b 11-point Likert scale: ranging from 0 (not at all healthy/happy/harmonious) to 10 (totally healthy/happy/harmonious).

ASMHS-19-Agnes_HongKong_F1

Figure 1. The flow.

Perceived knowledge and attitude regarding sedentary behaviour and physical activity

Table 2 shows significant increases in participants’ intention to reduce sedentary behaviour and perceived knowledge and attitude (intention and self-efficacy) regarding ZTEx immediately after the workshops, with small effect sizes (Cohen’s d: 0.20 to 0.27, all p < 0.05).

Table 2. Participants’ perceived knowledge and attitude regarding sedentary behaviour and physical activity at baseline and immediately after workshop: intention-to-treat analysis (n = 141).

ASMHS-19-Agnes_HongKong_t2

ZTEx = Zero-time exercise refers to simple strength- and stamina-enhancing physical activity

Paried T-test to compare the difference of the continuous parametric data between two groups; Difference between two time points: *p < 0.05, **p < 0.01

a 6-point Likert scale, 1 (strongly disagree); 2 ( disagree); 3 (slightly disagree); 4 (slightly agree); 5 (agree); 6 (strongly agree).

Effect Size (Cohen’s d): small = 0.20, medium = 0.50 and large = 0.80

Attitude regarding family communication through encouraging and engaging family members in physical activity

Immediately after the workshops, participants’ attitude (intention and self-efficacy) regarding family members’ sedentary behaviour, encouraging family members to do ZTEx, and engaging family members to do ZTEx with them were significantly increased with small effect sizes (Cohen’s d: 0.21 to 0.30, all p < 0.05) (Table 2).

Practice regarding sedentary behaviour and physical activity

Table 3 shows that at the 2-week follow-up, participants’ number of days spent doing simple strength- and stamina-enhancing physical activity (i.e. ZTEx) increased significantly by 0.7 days (Cohen’s d: 0.26, p < 0.01), and days spent encouraging family members to do ZTEx increased significantly by 0.4 days (Cohen’s d: 0.18, p < 0.01), both with small effect size. However, sitting time, moderate or vigorous physical activity, and days spent doing ZTEx with family members did not change significantly.

Table 3. Participants’ practice regarding sedentary behaviour, physical activity, family communication and well-being at baseline and the 2-week follow-up (n = 141).

ASMHS-19-Agnes_HongKong_t3

ZTEx = Zero-time exercise refers to simple strength- and stamina-enhancing physical activity

Paried T-test to compare the difference of the continuous parametric data between two groups; Difference between two time points: NS= not significant, *p < 0.05

a 11-point Likert scale: ranging from 0 (not at all healthy/happy/harmonious) to 10 (totally healthy/happy/harmonious ).

Effect Size (Cohen’s d): small = 0.20, medium = 0.50 and large = 0.80

(Figure 2) shows the proportion of participants doing simple strength- and stamina-enhancing physical activity and encouraging their family members to do simple strength- and stamina-enhancing physical activity. At the 2-week follow-up, there were significant increases in the proportion of participants doing ZTEx on 1 day or more, 4 days or more, and 7 days per week. The percentage increase (the ratio of the increased value to the baseline value multiplied by 100) ranged from 14% to 41% (all p < 0.05). The proportion of participants encouraging family members to do ZTEx on 1 day or more, 4 days or more, and 7 days per week increased significantly with the percentage increases ranging from 12% to 71% (all p < 0.05).

ASMHS-19-Agnes_HongKong_F2

Figure 2. Proportion of participants doing simple strength- and stamina-enhancing physical activity (i.e. ZTEx) and encouraging their family members to do ZTEx.

aIncreased percentage =Percentage of participation at 2 weeks minus percentage of participation at baseline.

bRelative increase = (Increased percentage  divided by  percentage of participation at baseline) ×100%.

c p value  of  McNemar’s test for assessing the difference between baseline and 2 weeks ZTEx= Zero-time exercise refer simple strength-stamina –enhancing physical activity

Perceived well-being

Table 3 shows no significant changes in perceived personal well-being (health and happiness) and family well-being (family health, happiness, harmony) at the 2-week follow-up. The complete-case analysis showed similar findings to the intention-to-treat analysis, but with greater effect sizes (Cohen’s d: 0.12 – 0.36, all p <0.05) immediately after the workshop and at the 2-week follow-up.

Reactions to intervention content

All participants rated the workshops highly. Immediately following the workshops, the participants rated the quality of intervention content as 8.9 ± 1.3 on a scale of 0 to 10. The level of the utility of the intervention (feasibility of incorporating the exercises into daily life) was rated 8.9 ± 1.4 on a scale of 0 to 10.

Feedback on intervention implementation by on-site observers

The scores for time and location arrangement, on a scale of 1 to 5, were 4.2 and 3.8, respectively. The scores for the suitability of the room size and facilities and manpower were 3.8 and 3.9, respectively. Most workshops were held in the morning, which facilitated the elderly to join. However, a few venues could only be accessed via long staircases or were too small for all participants to practice the demonstrated ZTEx together.

Participants were actively involved during the intervention mini workshop and excited about the games; the score for participant involvement was 4.2. However, the score for participant punctuality was 3.5. This might be related to the accessibility (long staircases) of the venues and weather conditions (rainy days).

Discussion

To our knowledge, the current paper is the first report of a very brief 15-minute community-based ZTEx intervention for reducing sedentary behaviour, enhancing perceived knowledge, intention, self-efficacy, and practice of simple strength- and stamina-enhancing physical activity (ZTEx), and promoting positive family communication showing small effect sizes. This brief ZTEx intervention is probably the shortest community-based intervention for reducing sedentary behaviour and enhancing physical activity with outcome and process evaluation as well as follow-up assessment in the community. Our trial successfully used a collaborative community-academic research partnership work model from the FAMILY Project to implement a simple intervention in the community. The collaborative work model allowed us to maximize existing community resources to promote physical activity and family well-being. This culminated in the present pilot trial that demonstrated the feasibility of a brief intervention using simple demonstrations, practice and games to deliver short and specific messages and encourage participants to share the messages with family members. The participants appreciated the intervention and enjoyed the simple games.

Most studies on reducing sedentary behaviour and increasing physical activity reported in the literature engaged participants in time- and resource-demanding intensive physical activity programs [23, 24]. In contrast, this trial used a brief session ‘mini workshop’ approach to deliver simple and specific messages and content. Such easy-to-do exercises can facilitate integration into and application in various community activities and settings. Our brief, theory-based and structured intervention also supports the suggestion from a recent systematic review that brief interventions may be as effective as more intensive interventions [25]. Our ZTEx intervention is particularly suitable for older people who are unable to meet physical activity guidelines due to limiting factors such as age and chronic diseases. This has clinical and public health significance since increasing physical activity can facilitate healthy ageing, helping minimize the burden on health and social care [26]. Our trial is in line with the idea that brief interventions delivered in primary care have the potential to reduce the public health burden of inactivity at relatively low cost [27].

We encouraged participants to engage in physical activity according to their abilities and incorporated fun game elements, with emphasis on enjoyment throughout the process, aiming to inspire the participants and promote the likelihood of establishing healthy physical activity habits [28]. This approach is supported by findings from a systematic review of 14 studies on the acceptability of physical activity interventions to the older adults: fun and enjoyment of social interaction and enjoyment coming from being physically active are important motivators of being physically active and maintaining physical activity beyond an intervention [29]. Dissonance could have also contributed to increased motivation [30].

Our trial had several limitations. First, we did not include objective measurements of sedentary behaviour and physical activity. Second, as validated questionnaires were not available in the literature, we developed our own outcome-based questionnaire to assess the changes in knowledge, attitude and practice regarding ZTEx, measuring perceptions rather than actual knowledge and skills. Such perceptions can be affected by individual self-perception and personality, and may be prone to under or over estimation. Third, the trial design did not include a control group and social desirability bias might have exaggerated the positive findings. However, no significant changes in sitting time or moderate or vigorous physical activity was reported, suggesting that the responses of participants were not primarily driven by social desirability. The consistent findings from the intention-to-treat and complete-case analyses indicated robust results. Third, the follow-up duration was short (2 weeks) and the completion rate was low (56%); we could consider modifying the study with a longer follow-up period and providing incentive…

Several suggestions can be derived from our findings and experiences. Additional supporting activities, such as periodic electronic prompts of text, pictorial and video messages could be sent to the participants; this might strengthen the participants’ intention, self-efficacy and practice. The reinforcement created by mobile messaging may increase the likelihood of exercising and may extend the effectiveness of the intervention [31, 32]. Further dissemination might be achieved by encouraging the participants to share information about ZTEx with their friends and neighbours, thereby extending the influence of the intervention within the community. Studies on a larger scale with longer period and a control group (such as randomised controlled trials) are needed to assess the effectiveness of the intervention and the sustainability of these changes.

Conclusion

Physical inactivity demands urgent attention to achieve cost-effective healthy ageing to alleviate this significant public health problem. Our findings show early evidence that a brief ZTEx community-based intervention is an innovative, enjoyable and effective approach to improve perceived knowledge, attitude, practice, and family communication regarding simple strength- and stamina-enhancing physical activity in older people. Further trials on this simple and low-cost intervention to deliver a simple-to-do specific message is the first step to promoting other behavioural change in community settings.

Informed consent: Informed consent was obtained from all individual participants included in the study.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. “All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.” The research protocol was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster with registration number UW15-743, and was registered at the National Institutes of Health (http://www.clinicaltrials.gov; identifier number: NCT02645071).

Funding

The FAMILY Project was funded by The Hong Kong Jockey Club Charities Trust.

Acknowledgement

We would like to thank the Hong Kong Jockey Club Charities Trust for the funding support, the staff from Hong Kong Department of Health and the Estate Management Advisory Committee for their coordination and implementation and the participants for joining the community programs.

References

  1. World Health Organization. PA for health (2018) More active people for a healthier world: draft global action plan on PA 2018- 2030. Vaccine 2018.
  2. Schuch FB, Vancampfort D, Richards J, Rosenbaum S, Ward PB, et al. (2016) Exercise as a treatment for depression: A meta-analysis adjusting for publication bias. J Psychiatr Res 77: 42–51. [crossref]
  3. Livingston G, Sommerlad A, Orgeta V, Costafreda SG, Huntley J, et al. (2017) Dementia prevention, intervention, and care. The Lancet 390: 2673–2734. [crossref]
  4. World Health Organization. Ageing and health 2018 [Available from: https://www.who.int/news-room/fact-sheets/detail/ageing-and-health accessed December 8 2019.
  5. Lai A, Stewart S, Wan A, Thomas C, Tse J, et al. (2019) Development and feasibility of a brief Zero-time Exercise intervention to reduce sedentary behavior and enhance physical activity: A pilot trial. Health and social care in the community 27: 233–245. [crossref]
  6. Piercy KL, Troiano RP, Ballard RM, Carlson SA, Fulton JE, et al. (2018) The Physical Activity Guidelines for Americans. JAMA 320: 2020–2028. [crossref]
  7. Lai AYK, Stewart SM, Wan ANT, Shen C, Ng CKK, et al. (2018) Training to implement a community program has positive effects on health promoters: JC FAMILY Project. Transl Behav Med 8: 838–850. [crossref]
  8. Yeung WF, Lai AY, Ho FY, Suen LK, Chung KF, et al. (2018) Effects of Zero-time Exercise on inactive adults with insomnia disorder: A pilot randomized controlled trial. Sleep Medicine 52: 118–127. [crossref]
  9. Freedman JL, Fraser SC (1966) Compliance without pressure: The foot-in-the-door technique. Journal of Personality and Social Psychology 4: 195–202.
  10. Chan SSC, Cheung YTD, Wong DCN, Jiang CQ, He Y, et al. (2017) Promoting smoking cessation in China: A foot-in-the-door approach to tobacco control advocacy. Glob Health Promot 26: 41–49.
  11. Gomes AR, Morais R, Carneiro L (2017) Predictors of exercise practice: from intention to exercise behavior. International Journal of Sports Science 7: 56–65.
  12. Festinger L (1957) A theory of cognitive dissonance. Stanford, CA: Stanford University Press.
  13. Cooper J, Feldman LA (2019) Does cognitive dissonance occur in older age? A study of induced compliance in health elderly population. Psychology and Aging 34: 709–713.
  14. Chan SSC, Viswanath K, Au DWH, Ma CM, Lam WW, et al. (2011) Hong Kong Chinese community leaders’ perspectives on family health, happiness and harmony: A qualitative study. Health Education Research 26: 664–674.
  15. Patall EA, Cooper H, Robinson JC (2008) The effects of choice on intrinsic motivation and related outcomes: a meta-analysis of research findings. Psychol Bull 134: 270–300. [crossref]
  16. Kolb DA (2015) Experiential Learning: Experience as the source of learning and development. New Jersey: Pearson Education Ltd.
  17. Seligman ME (2002) Authentic Happiness: Using the new positive psychology to realize your potential for lasting fulfillment: Simon and Schuster.
  18. Peterson C, Seligman M (2004) Character strengths and virtues: A handbook and classification. Washington, DC: American Psychological Association.
  19. Newton R (1989) Review of tests of standing balance abilities. Brain Inj 3: 335–343.
  20. Leong DP, Teo KK, Rangarajan S, Lopez-Jaramillo P, Avezum A Jr, et al. (2015) Prognostic value of grip strength: findings from the Prospective Urban Rural Epidemiology (PURE) study. Lancet 386: 266–273. [crossref]
  21. Macfarlane D, Chan A, Cerin E (2010) Examining the validity and reliability of the Chinese version of the International Physical Activity Questionnaire, long form (IPAQ-LC). Public Health Nutrition 14: 443–450.
  22. Soong C, Wang M, Mui M (2015) A “Community Fit” Community-Based Participatory Research Program for Family Health, Happiness, and Harmony: Design and Implementation. JMIR Res Protoc 28: 4.
  23. Costa EF, Guerra PH, Santos TId (2015) Systematic review of physical activity promotion by community health workers. Preventive Medicine 81: 114–121.
  24. Justine M, Azizan A, Hassan V (2013) Barriers to participation in physical activity and exercise among middle-age and elderly individuals. Singapore Med J 54: 582–586.
  25. Orrow G, Kinmonth AL, Sanderson S, Sutton S (2012) Effectiveness of physical activity promotion based in primary care: systematic review and meta-analysis of randomised controlled trials. BMJ 344: 1389. [crossref]
  26. Aittasalo M, Miilunpalo S, Suni J (2004) The effectiveness of physical activity counseling in a work-site setting. A randomized, controlled trial. Patient education and counseling 55: 193–202.
  27. Lamming L, Pears S, Mason D (2017) What do we know about brief interventions for physical activity that could be delivered in primary care consultations? A systematic review of reviews. Prev Med 99: 152–163.
  28. Kwasnickaa D, Dombrowskic SU, Whited M (2016) Theoretical explanations for maintenance of behaviour change: A systematic review of behaviour theories. Health Psychology Review 10: 277–296.
  29. Devereux-Fitzgerald A, Powell R, Dewhurst A (2016) The acceptability of physical activity interventions to older adults: A systematic review and meta-synthesis. Social Science & Medicine 158: 14–23.
  30. Orcullo DJC, Teo HS, Member I (2016) Understanding cognitive dissonance in smoking behaviour: A qualitative study. International Journal of Social Science and Humanity 6: 481–484.
  31. Cole-Lewis H, Kershaw T (2010) Text messaging as a tool for behavior change in disease prevention and management. Epidemiol Rev 32: 56–69.
  32. Kendzor DE, Shuval K, Gabriel KP (2016) Impact of a mobile phone intervention to reduce sedentary behavior in a community sample of adults: A quasi-experimental evaluation. Journal of medical Internet research 18: 19.

Supplementary Table 1. Participants’ perceived knowledge and attitude regarding sedentary behaviour and physical activity at baseline and immediately after workshop: complete-case analysis (n=117).

ASMHS-19-Agnes_HongKong_t4

ZTEx = Zero-time exercise refers to simple strength- and stamina-enhancing physical activity

Independent T-test and Mann-Whitney test to compare the difference of the continuous parametric data and non-parametric data, respectively; Chi-square test to compare the difference of the categorical data between two groups; Difference between two time points: *p < 0.05, **p < 0.01

a 6-point Likert scale:1 (strongly disagree); 2 ( disagree); 3 (slightly disagree); 4 (slightly agree); 5 (agree); 6 (strongly agree).

b 11-point Likert scale: ranging from 0 (not at all healthy/happy/ harmonious) to 10 (totally healthy/happy/harmonious).

Effect Size (Cohen’s d): small = 0.20, medium = 0.50 and large = 0.80

Supplementary Table 2. Participants’ practice regarding sedentary behaviour, physical activity, family communication and well-being at baseline and the 2-week follow-up: complete-case analysis (n=79).

ASMHS-19-Agnes_HongKong_t5

ZTEx = Zero-time exercise refers to simple strength- and stamina-enhancing physical activity

Paried T-test to compare the difference of the continuous parametric data between two groups; Difference between two time points: NS = not significant, *p < 0.05, **p < 0.01

a 11-point Likert scale: ranging from 0 (not at all healthy/happy/harmonious) to 10 (totally healthy/happy/harmonious).

Effect size (Cohen’s d): small = 0.20, medium = 0.50 and large = 0.80

Anti-Glomerular Basement Membrane Disease Following Nephrectomy

DOI: 10.31038/IJNUS.2019112

Abstract

A 51 year old female presented with rapidly progressive renal failure and diffuse alveolar hemorrhage following nephrectomy for retroperitoneal fibrosis.  Anti-glomerular basement membrane (anti-GBM) antibodies returned strongly positive confirming a diagnosis of anti-GBM disease.  She was treated with corticosteroids, plasma exchange and cyclophosphamide.  To our knowledge, this is the first adult case of anti-GBM disease following nephrectomy.

Keywords

Vasculitis, Anti-GBM disease, nephrectomy

Introduction

Anti-Glomerular Basement Membrane (anti-GBM) disease is characterized by rapidly progressive glomerular nephritis with or without pulmonary hemorrhage [1]. It is usually monophasic in nature and disease severity correlates with antibody titer [1]. Despite the known pathogenicity of anti-GBM antibodies, and the correlation of disease severity with their titers, the underlying pathogenesis of disease remains unclear.  Here we describe a patient who developed anti-GBM disease following nephrectomy.

Case Report

A 51 year old female presented for a second opinion to our facility.  She had a past medical history of seronegative rheumatoid arthritis which was diagnosed in early 2013 after she presented with joint pain following a personal stressor.   She was treated with methotrexate and adalimumab with resolution of her presenting complaints.  Three years later, during routine laboratory monitoring for her methotrexate, she was found to have an acute rise in her creatinine to 6.44 mg/dL above her baseline of around 1.0 mg/dL.  She reported some associated symptoms of malaise and abdominal pain. She was admitted to a local hospital for workup of her acute kidney injury.  At that time, urinalysis was unremarkable and serologies for Antineutophilic Cytoplasmic Antibodies (ANCA) were negative.  She did have elevated inflammatory markers (ESR 72 mm/hr and CRP 18.7).  CT of the abdomen and pelvis was performed and demonstrated severe, long-standing hydronephrosis of the right kidney and moderate hydronephrosis of the left kidney with associated hydroureters bilaterally.  There was an amorphous 5.5 × 2.8 cm soft tissue mass surrounding the aorta which was obstructing both ureters (Figure 1).   The findings were consistent with retroperitoneal fibrosis.  She underwent ultrasound guided bilateral percutaneous nephrostomy tube and stent placement with improvement of her creatinine to 3.0mg/dL.  Percutaneous biopsy performed at the time of the procedure confirmed the diagnosis of retroperitoneal fibrosis with negative staining for IgG4.

IJNUS-19-104_Nicole Droz_f1

Figure 1. Retroperitoneal mass encasing the aorta with associated hydro-ureters. Yellow arrow = retroperitoneal mass; white arrows = ureters; AO = aorta

She was referred to our center for second opinion.  During her evaluation, her right kidney was determined to be non-functioning.  Urology recommended right sided nephrectomy and left sided ureterolysis and stent placement.  Her post-operative course was uncomplicated and she was discharged on hospital day three.  Histopathologic examination of the right kidney revealed interstitial fibrosis with tubular atrophy and moderate arteriolosclerosis consistent with her history of chronic obstructive nephropathy. Two weeks following discharge, she was re-admitted for symptoms of cough, dyspnea and oliguria. She was afebrile and normotensive but was tachycardic.  She was tachypneic with a respiratory rate of 41 and oxygen saturation of 91% on 50% hi-flow nasal cannula. Her physical examination revealed bilateral coarse crackles throughout both lung fields. She had no rashes, sinus abnormalities or musculoskeletal abnormalities and the rest of her examination was unremarkable. Her laboratory evaluation revealed a rise in creatinine to 7.8mg/dL.  Her hemoglobin declined to 8.6 from her baseline of 10.6.  Inflammatory markers were again elevated.  Her urinalysis did show a large amount of blood as well as a moderate amount of leukocytes. Urine culture later grew E. faecalis.  She was treated for sepsis secondary to urinary tract infection with vancomycin and levofloxacin but failed to have improvement in her kidney function and quickly became anuric and required dialysis.

During this time, she became progressively more hypoxemic requiring supplemental oxygen by high flow nasal cannula. CT of the chest demonstrated bilateral ground glass opacities (Figure 2). Bronchoscopy with BAL was performed demonstrating progressively more bloody aliquots indicative of diffuse alveolar hemorrhage.  Workup for infectious etiologies was negative.   Further serologic workup was obtained to assess for systemic diseases.  ANCAs were again negative, butanti-GBMantibodies were strongly positive at a titer of >200RU/ml. The patient was diagnosed with anti-GBM disease based on her clinical presentation and positive anti-GBM antibodies.  A repeat renal biopsy was deferred due to her solitary kidney status.  She was treated with corticosteroids, cyclophosphamide and plasma exchange with improvement in her pulmonary manifestations but unfortunately never achieved renal recovery and is awaiting renal transplantation. In the setting of her previously unremarkable renal biopsy, we speculate that her urologic procedure may have led to the development of anti-GBM disease.

IJNUS-19-104_Nicole Droz_f2

Figure 2. CT of the chest demonstrating bilateral ground glass opacities

Discussion

Anti-GBM disease is characterized by rapidly progressive glomerular nephritis with or without pulmonary hemorrhage. The disease is mediated by pathogenic autoantibodies directed against the non-collagenous domain of the α3 chain of type IV collagen.  Antibodies are generally IgG, but IgA and IgM have also been reported [1]. Although it is well known that the antibodies are pathogenic, the underlying pathogenesis of disease has not been elucidated.  There is a strong HLA association with the disease with an over representation of HLA-DR15 and HLA-DR4 alleles suggesting a possible genetic component.  Further, HLA-DR7 and HLA-DR1 seem to be protective as they are underrepresented in disease [2]. Others have postulated that because of the cryptic nature of the antigenic target in anti-GBM disease, disruption of collagen hexamer is necessary to initiate disease [3].  Anti-GBM disease has been reported after environmental exposures such as tobacco use or hydrocarbons, bacterial or viral infections and from other glomerular diseases like ANCA associated vasculitis and membranous nephropathy [4- 6].  These exposures may alter the configuration of the collagen hexamer exposing the cryptic antigen leading to disease.

There have also been reports of onset of Anti-GBM disease following macroscopic renal damage. To investigate this mechanism further, authors Takeuchi et al hypothesized that mechanical renal damage may be necessary for exposure of the cryptic antigen to induce antibody production.  In their retrospective study, they evaluated patients who had anti-GBM antibodies done at the time of diagnosis of hydronephrosis.  They identified 11 patients for inclusion into their study.  3 of these patients had elevated anti-GBM antibody titers.  In 1 patient, anti-GBM antibody levels returned to normal after treatment of hydronephrosis [7].  This study, although small, lends support to the paradigm that disruption of the collagen hexamer and cryptic antigen exposure is necessary to induce disease. Surgical procedures, such as lithotripsy, have also been implicated in provoking the disease [8–10].  To our knowledge, this is the first case of an adult patient diagnosed with Anti-GBM disease following nephrectomy.  Authors Hagan et al previously reported the first pediatric patient to present with Anti-GBM disease following nephrectomy for xanthogranulomatous pyelonephritis [11].  Our case report lends credence to the hypothesis that cryptic antigen exposure is necessary for Anti-GBM disease development. Although rare, Anti-GBM disease should be suspected in patients who presents with rapidly progressive glomerulonephritis with or without pulmonary hemorrhage following urologic procedures.  Early recognition is critical for prompt treatment and improved patient outcomes.

References

  1. Salama A, Levy J, Lightsone L (2001) Goodpasture’s disease. Lancet 358: 917–920.
  2. Fisher M, Pusey CD, Vaughan RW, Rees AJ (1997) Susceptibility to anti-glomerular basement membrane disease is strongly associated with HLA-DRB1 genes.  Kidney Int 51: 222–229. [crossref]
  3. Pedchenko V, Bondar O, Fogo A, Vanacore R, Voziyan P et al. (2010) Molecular Architecture of the Goodpasture Autoantigen in Anti-GBM nephritis. N Engl J Med 363: 343–354. [crossref]
  4. Wilson C, Smith R (1972) Goodpasture’s syndrome associated with influena A2 virus infection. Ann Intern Med 76: 91–94. [crossref]
  5. Beirne G, Brennan J (1972) Glomerulonephritis associated with hydrocarbon solvents. Archives of Environmental Health: An International Journal 25: 365–369.
  6. Kitagawa W, Miura N, Yamada H, Nishikawa K, Futenma A et al. (2005) The increase of antiglomerular basement membrane antibody following pauci-immune-type crescentic glomerulonephritis. Clin Exp Nephrol 9: 69–73. [crossref]
  7. Takeuchi Y, Takeuchi E and Kamata K (2015) A possible Clue for the Production of Anti-Glomerular Basement Membrane Antibody Associated with Ureteral Obstruction and Hydronephrosis. Case Rep Nephrol Dial 5: 87–95. [crossref]
  8. Guerin V, Rabian C, Noel LH (1990) Anti-Glomerular basement membrane disease after lithotripsy. Lancet 335: 856–857.
  9. Xenocostas A, Jothy S, Collins B, Loertscher R, Levy M (1999) Anti-glomerular basement membrane glomerulonephritis after extracorporeal shock wave lithotripsy. Am J Kidney Dis 33: 128–132. [crossref]
  10. Umekawa T, Kohri K, Iguchi M, Yoshioka K, Kurita T (1993) Glomerular Basement membrane antibody and extracorporeal shock wave lithotripsy.  Lancet 341: 556. [crossref]
  11. Hagan E, Mallett T, Convery M, McKeever K (2015) Anti-GBM disease after nephrectomy for xanthogranulomatous pyelonephritis in a patient expressing HLA-DR15 major histocompatibility antigens: a case report. Clin Nephrol 3: 25–30. [crossref]

Lactobacillary Endocervicitis – A Novel Cause of Chronic Cervicitis

DOI: 10.31038/AWHC.2019266

 

We are describing a not yet documented cause for mucopurulent endocervicitis which is triggered by the reaction of neutrophil granulocytes against lactobacilli and is referred to as “lactobacillary endocervicitis” in this contribution. Lactobacilli are a normal component of the vaginal flora.

Samples from 16 women with chronic cervicitis were collected by swabbing the portion. Direct microscopy of the samples showed Gram-positive rods among polymorphonuclear cells (more than 10 per high-power field). Many of the polymorphonuclear cells seemed to try to phagocytize the rods (Figures 1 and 2). All samples tested negative by polymerase chain reaction (PCR) for Chlamydia trachomatis, Neisseria gonorrhoea, Ureaplasma urealyticum, Ureaplasma parvum, M.hominis and M.genitalium. Random samples were also tested for Trichomonas vaginalis and/or human papillomavirus by PCR and resulted negative.  None of the women showed signs of bacterial vaginosis. Neither clue cells (by microscopy) nor Gardnerella vaginalis (by culture) were detected in these cases. The 16 observed patients with lactobacillary endocervicitis ranged in age from 25 to 36 years old and 10 were pregnant. In all cases, the cervicitis persisted over 6 weeks and repeated testing for specific agents of infection was performed during that time.

AWHC-19-149-Thomas Ulrich Krech_switzerland_F1

Figure 1. Gram-stain of cervical pus with neutrophils and lactobacilli which are partly phagocytized (red arrow) or captured by the filamentous net (blue arrow) of the neutrophils (high-power field magnification 100 times).

AWHC-19-149-Thomas Ulrich Krech_switzerland_F2

Figure 2. Papanicolau stain of mucous cervical secretion showing columnar cells (yellow arrow) of the endocervix and many neutrophils, some of them with phagocytized lactobacilli (red arrow) (high-power field magnification 60 times).

Chronic cervicitis was first described by Gilbert Strachan in 1929 [1] as the most common lesion seen in the gynaecological practice. The author described gonococci as a possible cause of cervicitis. For other agents of infection, like chlamydia and mycoplasma/ureaplasma, detection techniques were unavailable at the time. Other possible causes for chronic cervicitis are Herpes simplex – virus and Trichomonas vaginalis. About 50% of all diagnosed mucopurulent cervicitis cases are non-specific [2].

Our findings described in this report add another cause for purulent endocervicitis. Lactobacillary endocervicitis is mainly seen in women aged between 25 and 35 years and can become chronic if not treated. Our treatment recommendation was 1g Amoxicillin taken daily orally for 5 to 7 days. In contrast to treatment studies of non-specific purulent endocervicitis [3], our approach proved successful in 15 of our 16 cases. Follow-up investigation of the unresolved case showed the same phagocytized Gram-positive rods in the putrid cervical secretion as observed before Amoxicillin treatment. The lactobacillus in this case was identified by MALDI-TOF as Lactobacillus gasseri and tested sensitive to Ampicillin by Etest (MIC 0.047 mg/L). In another case with successful therapy with Amoxicillin 2 g 1–1-1 for 3 days, the same lactobacilli species was identified in samples taken 1 day before and 69 days after as Lactobacillus johnsonii and Lactobacillus rhamnosus. Amoxicillin tested active against most lactobacilli [4]. Upon completion of antibiotic treatment, preparations containing lactobacilli were locally administered in all cases to re-establish the normal vaginal flora. Our excellent results with the antibiotic therapy described in this report, support our findings that lactobacilli in the wrong place, namely the cervix, can play a role in mucopurulent endocervicitis, which is also often observed in pregnancy.

The pathomechanism of the here described lactobacillary endocervicitis is unclear. A possible explanation might be that there is a constant defence of lactobacilli at the entrance to the endocervix by local defence mechanisms involving neutrophils. If the neutrophil defence is overwhelmed because of changes like pregnancy which impacts the local hormone balance and the opening of the cervix nut mouth , inflammation of the cervix with mucopurulent pus discharge and thus chronic cervicitis can result.  Further studies must be performed to shed light on the frequency and importance of lactobacillary infection as a cause of chronic endocervicitis and to establish an optimal treatment regimen.

Acknowledment

We thank Dr. Jäggi Franziska, 4800 Zofingen/Switzerland and her patient for the follow-up samples after treatment.

References

  1. Gilbert I. Strachan (1929) The Pathology of Chronic Cervicitis. The British Medical Journal 2: 659–661. [crossref]
  2. Taylor SN, Lensing S, Schwebke J, Lillis R, Mena LA, et al. (2013) Prevalence and treatment outcome of cervicitis of unknown etiology.  Sex Transm Dis 40: 379–385. [crossref]
  3. Lusk MJ, Garden FL, Cumming RG, Rawlinson WD, Naing ZW, et al. (2016) Cervicitis: a prospective observational study of empiric azithromycin treatment in women with cervicitis and non-specific cervicitis. Int J STD AIDS 28: 120–126. [crossref]
  4. Ocaña V, Silva C, Nader-Macías ME (2006) Antibiotic Susceptibility of Potentially Probiotic Vaginal Lactobacilli Virginia Ocana Clara Silva, and Maria Elena Nader-Macıa. Infect Dis Obstet Gynecol 2006: 18182. [crossref]

The effects of hesperidin or naringin dietary supplementation on yoghurt quality parameters in dairy ewes – A preliminary study

DOI: 10.31038/NDN.2019114

Abstract

Stakeholders that are involved in the animal production chain, such as primary producers,processors, distributors, and retailers continuously seek for alternative ways of improving health benefits and technological properties of dairy products. Hesperidin and naringin belong to flavonoids and are well-known for their multifaceted properties. The aim of this preliminary study was therefore to examine the effects of flavonoids supplementation into the diets of dairy ewes on the quality parameters and oxidative stability of yoghurt manufactured by their milk. Thirty-six Chios ewes were allocated to four groups; the control group (C) was fed concentrates without supplementation, while the other three experimental groups received the same diet further supplemented with hesperidin (6000mg/kg), naringin (6000mg/kg), or α-tocopheryl acetate (200mg/kg). As indicated, no effects on yoghurt quality parameters and oxidative stability were observed in individual samples manufactured from milk collected after 7, 21 and 28 days of flavonoids dietary supplementation. In conclusion, inclusion of flavonoids in ewes’ diet does not appear to affect yoghurt quality characteristics.

Keywords

hesperidin; naringin; yoghurt quality; oxidative stability 31

Introduction

Dairy fermented foods, such as yoghurt, have gained a positive perception and enjoyed high popularity among the consumers due to their beneficial effects on human health [1]. Among others, consumption of these products improves immunity and results in a slight reduction in stomach pH that minimizes the risk of pathogen transit and the impacts of low gastric juice secretion [2]. At the same time, several peptides derived by proteolysis could lower blood pressure in hypertensive patients [3]. Yoghurt consists of a casein network aggregated through isoelectric precipitation by lactic acid bacteria, such as Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus. Fermentation is a chemical process in which specific enzymes break down organic substances into smaller compounds resulting in more digestible, stable and flavored foods with enhanced nutritional value [4]. Enrichment of animal products with natural bioactive compounds seems to improve their quality characteristics and fortifies consumers against oxidation effects. Dietary flavonoids have received significant attention in recent years due to their antioxidant, anti-inflammatory, anti-mutagenic and anti-clotting properties that are associated with a declined risk of cardiovascular diseases and cancer development [5, 6]. In general, levels of polyphenols in yoghurt are low and its enrichment with plant-derived additives could improve its phenolic content contributing in disease prevention and correction of deficiencies with minimal side effects [1].

Several pre- and post- fermentation approaches for adding polyphenols to yoghurt have already been implemented with positive effects on the derived product. Addition of polyphenols originated from bitter orange (Citrus aurantium l.) flowers [7], berry [8], apple [9], strawberry [10], green tea [11], peppermint, dill and basil [12], pomegranate peel [13] or juice [14], or grape seed [15] significantly increased yoghurt antioxidant capacity without other significant effects on its quality. According to the literature [16], the pre-fermentation application could introduce some advantages such as the promotion of starter cultures’growth. Alternative approaches are continuously evaluated in animal production systems with the intention to improve the nutritional value and the organoleptic properties of the derived products. However, no data exist describing the effects of flavonoids inclusion into the diets of dairy ewes on the quality of the derived yoghurt. The aim of the present study was therefore to investigate the effects of hesperidin or naringin or a-tochopheryl acetate dietary supplementation on the quality characteristics (colour, pH, syneresis and texture) and oxidative stability of yoghurt manufactured by ewe milk.

Methods & Materials

Animal and diets

The experimental design is described in detail by Simitzis et al. [17]. In brief, 36 lactating Chios ewes were allocated into 4 experimental groups based on their milk yield and body weight. One of the groups served as a control (C) and was fed with a basal concentrate diet, whereas the other three groups were offered the same diet further supplemented with hesperidin (hesperidin, TSI Europe NV, Belgium) at 6000 mg/kg concentrated feed (H), or naringin (naringin hydrate 98%, Alfa Aesar GmbH & Co KG, Germany) at 6000 mg/kg concentrated feed (N), or α-tocopheryl acetate (DSM Nutritional Products Hellas, Greece) at 200 mg/kg concentrated feed (VE). Methods used in the present experiment were approved by the bioethical committee of the Agricultural University of Athens (Permit Number: 23/20032013) under the guidelines of “Council Directive 2010/63/EU on the protection of animals used for scientific purposes”.

Milk samples and yoghurt preparation

Animals were milked twice a day at 6 am and 6 pm by a milking machine. Individual milk samples were  collected the day  before  the  beginning  and  at  the 7th, 14th, 21st and 28th day of  the experiment and obtained after mixing the volume of milk collected during the morning and evening milking. Individual traditional Greek yoghurt samples were separately manufactured by milk collected from each ewe during the sampling days, apart from day 14 due to technical reasons. The main stages of yoghurt production were: collection and filtration of raw ovine milk, heating to 95°C for 15 min without homogenization, transfer to closed 250 ml cups, cooling to 45 – 50°C, inoculation and mixing with 2% of a commercial thermophilic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus (Chr. Hansen, Denmark), incubation at 45 °C for about 3 h and storage at 5 °C [18].

Yoghurt quality parameters

Yoghurt quality parameters were assessed after one day of refrigerated storage. Colour was measured (3 measurements per sample) using a Miniscan XE (HunterLab, Reston, USA) chromameter set on the L* (lightness), α* (redness), b* (yellowness) system (CIE 1976, Commission International de l’ Eclairage). pH was determined using a pHM210 standard pHmeter (MeterLab, Radiometer, Denmark). Rheological measurements of yoghurt were implemented with a Shimadzu Testing Instrument, model AGS-500 NG (Shimadzu Corporation, Kyoto, Japan) equipped with  5 kg load cell. A plunger with a diameter of 25 mm was attached to the moving crosshead, which moved both downwards and upwards at a speed of 120 mm/min, was inserted to a depth of 20 mm below the yoghurt surface. The firmness (N) was calculated from the resulting curve and defined as the height of the peak force during the compression cycle. Syneresis of yoghurt was measured by emptying the contents of the plastic container (200 g) into a stretched cheese cloth, cutting crosswise into four pieces, draining in a funnel for 24 h at 4 °C, collecting the amount of whey drained off in a conical bottle and weighing in gram to provide an index of syneresis.

Antioxidant capacity was assessed after 10 and 20 days of refrigerated storage at 4°C on the basis of the malondialdehyde (MDA) levels formed during storage. MDA concentration was determined by using a third-order derivative spectrophotometric method [19]. In brief, 2 g of each yoghurt sample (two sub-samples per ewe) was homogenized (Edmund Buehler 7400 Tuebingen/H04, Germany) in 8 ml of aqueous trichloroacetic acid (TCA) (50 g/l) and 5 ml of butylated hydroxytoluene (BHT) in hexane (8 g/l), and the mixture was centrifuged for 3 min at 3000 × g. The top hexane layer was discarded, and a 2.5 ml of aliquot from the bottom layer was mixed with 1.5 ml of aqueous 2-thiobarbituric acid (TBA) (8 g/l) and further incubated at 70°C for 30 min. Following incubation, the mixture was cooled under tap water and submitted to third-order derivative (3D) spectrophotometry (Hitachi U3010 Spectrophotometer) in the range of 500–550 nm. The concentration of MDA (ng/ml milk) was calculated on the basis of the height of the third-order derivative peak at 521.5 nm by referring to the slope and intercept data of the computed least-squares fit of standard calibration curve prepared using 1,1,3,3-tetraethoxypropane (TEP), the MDA precursor.

Statistical analysis

The experimental unit was the animal since it was the smallest unit upon which, either the treatment was applied or the measurements were made. Data were subjected to repeated measures analysis of variance using the MIXED procedure of SAS software [20], with dietary treatment as fixed effect and the sampling day as the repeated factor. Significant differences were tested at 0.05 significance level and results are presented as least square means ± s.e.m.

Results

No significant effects of hesperidin, naringin or vitamin E dietary supplementation on yoghurt quality parameters were observed. As shown in Table 1, quality characteristics of yoghurt  manufactured by ewe milk that was individually collected after 7, 21 and 28 days of flavonoids dietary inclusion were not significantly different among the experimental groups. Values for colour parameters (L, a*, b*), pH, firmness and syneresis were not influenced after 7, 21 or 28 days of flavonoids incorporation into dairy ewes’ diets. At the same time, no significant effects on MDA values were found in yoghurt manufactured with milk samples collected from ewes after 7, 21 and 28 days of flavonoids dietary supplementation and stored at 4°C for 10 and 20 days (Table 2).

Table 1. Effect of hesperidin and naringin on yoghurt characteristics after 0, 7, 21 and 28 days of dietary supplementation in dairy ewes  

Day

Parameter

Treatment1

S.E.M.

P-value

C

H

N

E

L

95.07

94.97

95.16

94.47

0.25

0.243

Colour2 a*

-2.76

-2.89

-2.99

-3.00

0.07

0.084

0

b*

11.46

11.33

11.84

11.58

0.35

0.790

pH

4.24

4.23

4.25

4.49

0.14

0.542

Firmness (N)

0.87

0.90

0.84

0.68

0.08

0.095

Syneresis (%)

1.96

2.73

1.33

3.50

0.65

0.109

L

94.93

94.91

95.22

95.05

0.32

0.888

Colour  a*

-2.81

-2.73

-2.84

-2.71

0.07

0.565

7

b*

10.75

10.76

10.52

10.73

0.44

0.977

pH

4.32

4.51

4.44

4.38

0.13

0.768

Firmness (N)

0.79

0.81

0.72

0.88

0.11

0.773

Syneresis (%)

2.12

2.47

1.58

3.44

0.66

0.260

L

94.73

94.53

95.02

94.88

0.37

0.811

Colour  a*

-2.70

-2.84

-2.77

-2.66

0.07

0.282

21

b*

10.32

11.00

10.84

10.80

0.57

0.854

pH

4.30

4.19

4.18

3.94

0.23

0.720

Firmness (N)

0.81

0.75

0.87

0.92

0.10

0.730

Syneresis (%)

2.22

3.43

2.99

3.41

0.93

0.768

L

94.60

94.53

94.69

94.78

0.34

0.953

Colour  a*

-2.81

-2.67

-2.84

-2.79

0.08

0.435

28

b*

10.28

11.51

11.01

11.47

0.11

0.529

pH

4.30

4.44

4.10

4.20

0.18

0.591

Firmness (N)

0.58

0.79

0.89

0.91

0.10

0.133

Syneresis (%)

2.49

3.59

3.84

2.44

0.87

0.220

1 The control group (C) was fed with a commercial basal diet, whereas the other groups consumed the same diet, with the only difference that concentrated feed was uniformly supplemented with hesperidin (H) (6000mg/kg feed) or naringin (N) (6000mg/kg feed) or vitamin E (VE) (200mg/kg feed). 2L*; lightness, α*; redness, b*; yellowness

Table 2. MDA values (ng/g) in yoghurt manufactured from milk samples collected the day before, 7, 21 and 28 days after hesperidin and naringin dietary supplementation.

Milk Sampling (days)

Refrigerated Storage (days)

Treatment1

S.E.M.

P-value

C

H

N

VE

0

10

2.96

2.76

2.65

2.88

0.23

0.907

20

3.35

3.24

3.28

3.16

0.23

7

10

2.19

2.05

1.83

1.82

0.27

0.484

20

3.25

2.63

2.83

3.23

0.27

21

10

2.32

2.43

2.87

2.90

0.23

0.902

20

2.90

2.75

3.36

3.18

0.23

28

10

3.87

4.23

3.80

3.50

0.36

0.468

20

4.21

4.35

4.99

4.19

0.36

1 The control group (C) was fed with a commercial basal diet, whereas the other groups consumed the same diet, with the only difference that concentrated feed was uniformly supplemented with hesperidin (H) (6000mg/kg feed) or naringin (N) (6000mg/kg feed) or vitamin E (VE) (200mg/kg feed).

Discussion

There is always a challenge of improving health benefits and technological properties of dairy products. According to the literature, yoghurts inoculated with phenolic extracts display higher antioxidant capacity compared to the controls, possibly through the scavenging of free radicals [21, 22]. However, as far as the authors are aware, no data exist on the influence of antioxidants’ dietary supplementation on yoghurt characteristics manufactured by ewe milk. The available literature is mainly focused on the effects of flavonoids dietary inclusion on milk characteristics of dairy cows. As indicated by the previous researchers, milk quality parameters in dairy cows were not negatively influenced by the inclusion of propolis [23, 24] or alfalfa [25] or grape seed and grape marc meal [26] or green tea and curcuma [27] flavonoids extracts in their diets. As already pointed out, no significant effects of hesperidin and naringin dietary supplementation on the quality parameters (colour, pH, firmness and syneresis) of the derived yoghurt were observed. This finding may be partially associated with the fact that yield, composition, coagulation properties and fatty acid profile of sheep milk are not influenced by the incorporation of hesperidin or naringin in the diets of dairy ewes [17]. On the other hand, an improvement of milk oxidative stability is observed both in dairy cows [23, 24] and dairy ewes [17] after the addition of flavonoids in their diets. In contrast, no significant effects of hesperidin or naringin dietary supplementation on yoghurt oxidative stability were observed in the present study. Fermentation and post-acidification may have negatively affected the antioxidant potential of the examined flavonoids, since they are chemical processes in which enzymes break down organic substances into smaller compounds with different function and value [4]. As indicated in previous studies, the interactions between added bioactive compounds, milk proteins, polysaccharides (such as pectin) and the starter cultures might vary on a case-by-case basis [8], leading to different effects on yoghurt texture parameters [28]. At the same time, it could be suggested that dietary flavonoids supplementation did not affect bacterial growth, since no differences in yoghurt properties and especially pH values were observed.

Conclusions

As indicated by the results of the present study, dietary supplementation of dairy ewes with flavonoids at the examined levels does not improve yoghurt quality characteristics and oxidative stability.

Funding information

This research was funded by Hellenic State and European Union, within the framework of the Project “Thalis – The effects of antioxidant’s dietary supplementation on animal product quality”, MIS 380231.

References

  1. Gahruie HH, Eskandari MH, Mesbahi G, Hanifpour MA (2015) Scientific and technical aspects of yogurt fortification: A review. Food Sci. Human Wellness 4: 1–8.
  2. O’Connell J, Fox P (2001) Significance and applications of phenolic compounds in the production and quality of milk and dairy products: a review. Int Dairy J 11: 103–120.
  3. FitzGerald RJ, Murray BA, Walsh DJ (2004) Hypotensive peptides from milk proteins. J Nutr 134: 980S-988S. [crossref]
  4. Yildiz F (2009) Development and manufacture of yogurt and other functional dairy products. (1st ed.). CRC press, Taylor and Francis Group: Boca Raton, Florida, USA.
  5. Mozaffarian D, Wu JH (2018) Flavonoids, dairy foods, and cardiovascular and metabolic health: a review of emerging biologic pathways. Circ Res 122: 369–384. [crossref]
  6. Rodríguez-García C, Sánchez-Quesada C, Gaforio J (2019) Dietary Flavonoids as Cancer Chemopreventive Agents: An Updated Review of Human Studies. Antioxidants 8: 137. [crossref]
  7. Hashemi SMB, Amininezhad R, Shirzadinezhad E, Farahani M (2016) The Antimicrobial and Antioxidant Effects of Citrus aurantium L. Flowers (Bahar Narang) Extract in Traditional Yoghurt Stew during Refrigerated Storage. J Food Saf 36: 153–161.
  8. Sun-Waterhouse D, Zhou J, Wadhwa SS (2013) Drinking yoghurts with berry polyphenols added before and after fermentation. Food Control 32: 450–460.
  9. Sun-Waterhouse D, Zhou J, Wadhwa SS (2012) Effects of adding apple polyphenols before and after fermentation on the properties of drinking yoghurt. Food Bioprocess Tech 5: 2674–2686.
  10. Oliveira A, Alexandre EM, Coelho M, Lopes C, Almeida DP et al. (2015) Incorporation of strawberries preparation in yoghurt: Impact on phytochemicals and milk proteins. Food Chem 171: 370–378. [crossref]
  11. Najgebauer-Lejko D, Sady M, Grega T, Walczycka M (2011) The impact of tea supplementation on microflora, pH and antioxidant capacity of yoghurt. Int Dairy J 21: 568- 574.
  12. Amirdivani S, Baba AS (2011) Changes in yogurt fermentation characteristics, and antioxidant potential and in vitro inhibition of angiotensin-1 converting enzyme upon the inclusion of peppermint, dill and basil. LWT-Food Sci Technol 44: 1458–1464.
  13. El-Said MM, Haggag HF, El-Din HMF, Gad AS, Azza M.Farahat (2014) Antioxidant activities and physical properties of stirred yoghurt fortified with pomegranate peel extracts. Ann Agric Sci 59: 207–212.
  14. Trigueros L, Wojdyło A, Sendra E (2014) Antioxidant activity and protein–polyphenol interactions in a pomegranate (Punica granatum L.) yogurt. J Agric Food Chem 62: 6417- 6425.
  15. Chouchouli V, Kalogeropoulos N, Konteles SJ, Karvela E, Dimitris Makris P et al. (2013) Fortification of yoghurts with grape (Vitis vinifera) seed extracts. LWT-Food Sci Technol 53: 522–529.
  16. Sun-Waterhouse D, Zhou J, Wadhwa SS (2011) Effects of adding apple polyphenols before and after fermentation on the properties of drinking yoghurt. Food Bioprocess Tech 5: 2674–2686.
  17. Simitzis P, Massouras Th, Goliomytis M, Charismiadou M,  Moschou K et al. (2019) The effects of hesperidin or naringin dietary supplementation on the milk properties of dairy ewes. J Sci Food Agric 99: 6515–6521. [crossref]
  18. Kaminarides S, Stamou P, Massouras T (2007) Comparison of the characteristics of set type yoghurt made from ovine milk of different fat content. Int J Food Sci Technol 42: 1019- 1028.
  19. Botsoglou NA, Fletouris DJ, Papageorgiou GE, Vassilopoulos VN, Antonios Mantis J et al. (1994) A rapid, sensitive, and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissues, food, and feedstuff samples. J Agric Food Chem 42: 1931–1937.
  20. Sas/Stat (2011) Statistical Analysis Systems, Version 9.3, SAS Institute Inc., Cary, NC.
  21. Karaaslan M, Ozden M, Vardin H, Turkoglu H (2011) Phenolic fortification of yogurt using grape and callus extracts. LWT-Food Sci Technol 44: 1065–1072.
  22. Alenisan MA, Alqattan HH, Tolbah LS, Shori AB (2017) Antioxidant properties of dairy products fortified with natural additives: A review. J Assoc Arab Univ Basic Appl Sci 24: 101–106.
  23. Aguiar SC, Cottica SM, Boeing JS, Samensari RB, Santos GT et al. (2014) Effect of feeding phenolic compounds from propolis extracts to dairy cows on milk production, milk fatty acid composition and the antioxidant capacity of milk. Anim Feed Sci Technol 193: 148–154.
  24. Santos NW, Yoshimura EH, Machado E, Matumoto-Pintro PT, Paula Montanher F et al. (2016) Antioxidant effects of a propolis extract and vitamin E in blood and milk of dairy cows fed diet containing flaxseed oil. Livestock Sci 191: 132–138.
  25. Zhan J, Liu M, Su X, Zhan K, Zhang C et al. (2017) Effects of alfalfa flavonoids on the production performance, immune system, and ruminal fermentation of dairy cows. Asian-Australas J Anim Sci 30: 1416–1424. [crossref]
  26. Gessner DK, Koch C, Romberg FJ, Winkler A, Dusel G et al. (2015) The effect of grape seed and grape marc meal extract on milk performance and the expression of genes of endoplasmic reticulum stress and inflammation in the liver of dairy cows in early lactation. J Dairy Sci 98: 8856–8868. [crossref]
  27. Winkler A, Gessner DK, Koch C, Romberg FJ, Dusel G et al. (2015) Effects of a plant product consisting of green tea and curcuma extract on milk production and the expression of hepatic genes involved in endoplasmic stress response and inflammation in dairy cows. Arch Anim Nutr 69: 425–441. [crossref]
  28. Do Espírito Santo AP, Perego P, Converti A, Oliveira MND (2012) Influence of milk type and addition of passion fruit peel powder on fermentation kinetics, texture profile and bacterial viability in probiotic yoghurts. LWT-Food Sci Technol 47: 393–399.

Proficiency Monitoring of Allergen-Specific IgE macELISA – 2019

DOI: 10.31038/IJVB.2019332

Abstract

This study was designed to evaluate the reproducibility of a macELISA for the detection of allergen-specific IgE in dogs and cats. Nine different individuals across seven separate affiliated laboratories evaluated 21 predefined sera samples in a single blinded fashion. For evaluations completed by multiple operators, the average inter-operator variance was calculated to be 3.7% (range = 1.5%-4.7%). The average intra-assay variance among reactive assay calibrators in all laboratories was 4.1% (range = 0.3–11.9%). The overall inter-assay inter-laboratory variance evident with reactive calibrators was consistent among laboratories and averaged 10.4% (range 4.4 – 13.0%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose response profiles observed in each of the laboratories were indistinguishable. Correlation of EAU observed for individual allergens between and among all laboratories was strong (r > 0.90, p < 0.001). Collectively, the results demonstrated that the macELISA for measuring allergen-specific IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory, but among different operators and between laboratories using the same macELISA.

Keywords

IgE, ELISA, Proficiency, Atopy, Allergy, Immunotherapy, Cross-reactive Carbohydrate

Introduction

Stallergenes Greer maintains a proficiency monitoring program for laboratories that routinely run a monoclonal antibody cocktail based enzyme-linked immunosorbent assay (macELISA) for evaluation of allergen-specific IgE in serum samples [1–4]. The foundation for this program is based in the desire for inter-laboratory standardization and quality control measures that ensure the uniformity, consistency, and reproducibility of results among laboratories that perform the assays. This program, now in its tenth year, is designed to periodically evaluate the proficiency of laboratories and ensures that individual operators yield consistent and reproducible results. The first proficiency evaluations documented that inter-laboratory standardization and quality control measures in the veterinary arena are on the immediate forefront and that uniformity, consistency, and reproducibility of results between laboratories is achievable [2]. Similarly, reproducibility of results among ten different laboratories was documented in the proficiency evaluations subsequently completed [2–4]. The results presented herein summarize the comparative results observed in the affiliate laboratories for the most recent proficiency evaluations that were completed in 2019.

Materials and Methods

All serum samples, buffers, coated wells, calibrator solutions, and other assay components were aliquants of the respective lots of materials manufactured at Stallergenes Greer’s production facilities (located in Lenoir, NC, USA) and supplied as complete kits to the participating laboratories along with the exact instructions for completing the evaluations.

Participating Laboratories

Seven independent Veterinary Reference Laboratories (VRLs) participated in the 2019 proficiency evaluation of macELISA. Participating laboratories included three separate IDEXX laboratories located in Memphis, Tennessee, Ludwigsburg, Germany, and Markham, Ontario Canada. Other affiliated European laboratories that participated in this evaluation included Agrolabo (Scarmagno, Italy), Laboratories LETI (Barcelona, Spain), and Ceva Biovac (Beaucouzé, France). Stallergenes Greer Laboratories (Lenoir, NC) served as the prototype for evaluation of macELISA; the 2019 evaluations included results reported by three separate and independent operators. Because the performance characteristics of macELISA in Stallergenes Greer’s VRL have been well documented for use over an extended period [1–4], all results observed in the other participating laboratories were compared directly with the results observed in Stallergenes Greer’s reference laboratory.

Serum Samples

Separate pollen and mite reactive sera pools as well as non-reactive sera pools were prepared from cat and dog serum samples that previously had been evaluated using the macELISA for detection of allergen-specific IgE in dogs and cats. The allergen-specific reactivity of each sera pool ranged from nonreactive to multiple pollen or mite reactivity’s. These sera pools and admixtures of the pools were used to construct a specific group of samples that exhibited varying reactivity to the allergens included in the evaluation panel. Eighteen samples were included in the blinded evaluation conducted by each laboratory. Two known pollen reactive control samples and one non-reactive control sample were also included; replicates of these identical samples were included as unknown blinded samples. Also included in the array of samples was a five tube three-fold serial dilution of a highly pollen-reactive pool, diluted into non-reactive sera, which served to document the dose response evident within the assay. All samples were stored at -20°C for the interim between testing.

Calibrators

Grass pollen reactive calibrator solutions of predetermined reactivity in the macELISA were prepared as three-fold serial dilutions of a sera pool highly reactive to most pollen allergens. Replicates of each were evaluated in each assay run and served as a standard response curve for normalizing results observed with the various samples. All results were expressed as ELISA Absorbance Units (EAU) which are background-corrected observed responses expressed as milli absorbance.

Buffers

The buffers used throughout have been previously described [1–4], and included: a) well coating buffer: 0.05 M sodium carbonate bicarbonate buffer, pH 9.6; b) wash buffer: phosphate buffered saline (PBS), pH 7.4, containing 0.05% Tween 20, and 0.05% sodium azide; c) reagent diluent buffer: PBS, pH 7.4, containing 1% fish gelatin, 0.05% Tween 20 and 0.05% sodium azide. Unique to this year’s evaluation was the inclusion of a serum diluent that contains an inhibitor of antibodies that are cross reactive to carbohydrate determinants (CCD). The inhibitor for the CCD (BROM-CCD) is a preparation containing the carbohydrate components present in bromelain, which was prepared in house and remains a proprietary product of Stallergenes Greer (Lenoir, NC, USA) [5]. The serum diluent consists of the reagent diluent with BROM-CCD added at a concentration of 2.5 mG/mL.

Allergen Panel

The allergen panel was a 24 allergen composite derived from the array of allergens that are included in the specific panels routinely evaluated in the various laboratories; the composite allergen panel consisted of 4 grasses, 6 weeds, 6 trees, 5 mites, and 3 fungi. The protocol for coating and storage of wells has been previously described [1–4].

Sample Evaluations – macELISA

The operational characteristics and procedures for the macELISAs have been previously described [1–4]. Following incubation of allergen coated wells with an appropriately diluted serum sample, allergen-specific IgE is detected using a secondary antibody mixture of biotinylated monoclonal anti-IgE antibodies, streptavidin alkaline phosphatase as the enzyme conjugate, and p-nitrophenylphosphate (pNPP) as substrate reagent. Specific IgE reactivity to the allergens is then estimated by determining the absorbance of each well measured at 405 nM using an automated plate reader. All results are expressed as ELISA Absorbance Units (EAU), which are background-corrected observed responses expressed as milli absorbance [1].

Statistics

A coefficient of variation was calculated as the ratio of standard deviation and means of the responses observed for the calibrator solutions within different runs in multiple laboratories. Pearson’s correlation statistic was used for inter-laboratory comparison among individual allergens.

Results

The assay variance (% CV) observed with the calibrator solutions in the different laboratories are presented in Table 1 and are representative of the assay reproducibility in the various laboratories. The average intra-assay % CV among positive calibrators (#1–4) was 4.1% (range = 0.3–11.9%); differences among laboratories or between assays and within assay runs were not detected. No substantial difference in results among various operators were revealed. The average inter-operator variance documented for Stallergenes Greer technicians was calculated to be 3.1% (range = 0.3%-5.0%). While the allergens and serum are the same as the previous 2 proficiency tests, the incorporation of CCD inhibitors precludes direct comparison to prior tests. The results of the current evaluation (Table 1) show that the inter-assay variance among positive calibrators for all laboratories included in this evaluation was calculated to be 10.4% (range = 5.5–13.0%). The intra-assay variability of the negative calibrator #5 was 3.7% (range 0.2 – 7.6%), while the background ODs had the highest intra-assay variance overall (average 5.5%; range 0.1–13.9%). A negative response is classified as anything with an EAU below 150. Any analysis of results below this threshold, especially when looking at %CV and relative differences, should be done so cautiously.

To evaluate the strength of association with the magnitude of EAU results observed for each allergen among the different laboratories a Pearson’s correlation coefficient was determined (Microsoft Excel 2016) for each laboratory pair. Because the macELISA is designed to yield comparable responses in dog and cat samples, comparison of results among affiliate laboratories included both cat and dog samples as a single population of sera samples. These results
(Table 2) demonstrate that very high inter-laboratory correlation (r > 0.90; p<0.001) is evident between the results observed in Stallergenes Greer’s laboratory and those observed in six affiliate laboratories for all mites and pollen allergens. The correlation (Pearson’s) of results observed with the fungal allergens within or between any of the testing laboratories was also substantial. However, the majority of results for the fungal allergens fell within the lower range of reactivity or within the negative range of the response curve (< 150 EAU). Consequently, the correlation of results among laboratories for the fungal allergens was somewhat less than the correlation evident with the mite and pollen allergens. The overall correlation of results observed in the various laboratories are summarized in Table 3; a very strong correlation was demonstrated between and among the results of the participating laboratories.

Table 1. Calculated variance of macELISA calibrator solutions observed with different laboratory runs by multiple operators during the 2019 Proficiency evaluation.

N

Calibrator % CV*

BG

Variance

#1

#2

#3

#4

#5

% CV

Inter-Laboratory

266

5.5

12.5

13.0

10.8

9.0

12.1

Inter-Assay (Stallergenes Greer)

124

1.5

4.1

4.7

4.4

3.5

4.2

Intra-Assay

Stallergenes Greer #1

 28

1.3

3.8

4.4

4.2

3.8

4.2

Stallergenes Greer #2

 28

1.5

4.1

5.0

4.1

2.9

5.2

Stallergenes Greer #3

 28

1.3

3.8

4.4

4.2

3.8

4.2

Stallergenes Greer #4

 28

3.5

2.1

0.8

0.3

0.2

0.1

IDEXX Memphis

 28

1.3

3.8

4.4

4.2

3.8

4.2

IDEXX Canada

 28

1.3

1.8

2.1

2.5

2.8

13.9

IDEXX Germany

 28

1.3

3.8

4.4

4.2

3.8

4.2

Agrolabo

 28

3.8

11.8

11.9

10.5

7.6

5.9

Biovac

 28

6.3

4.7

7.4

7.1

4.8

5.1

LETI

 28

2.0

5.5

4.5

5.5

3.4

7.8

* Calibrator #1 is prepared as a dilution of a sera pool which is highly reactive to grass pollen allergens; calibrator #5 is a dilution of a negative sera pool. Calibrators #2 – #4 are prepared as a serial 3-fold dilution of calibrator #1.

† Background responses observed with diluent in place of serum sample.

There is no compelling evidence that the level of allergen-specific IgE correlates with severity of clinical disease [6–9]. However, an evaluation that purports to measure allergen-specific IgE should have a reduction in signal that is directly proportional to the dilution factor of the test ligand [10]. For an evaluation of the dose response in this ELISA, a five tube three-fold serial dilution of a highly pollen-reactive dog sera pool was included as unknown independent samples. To be expected, the magnitude of responses observed in each laboratory was reduced in direct proportion to dilution (data not shown). Results from the final tube in the dilution scheme yielded results that were indistinguishable from negative responses, indicating a dilution extinction of detectable response.

Discussion

The results of the present study demonstrate that the variability between and among the affiliate laboratories and technicians are indistinguishable from the results evident within and between runs completed in the laboratory of Stallergenes Greer. The intra-assay variance observed with the positive calibrators evident among the various runs within each of the laboratories remains relatively low and indistinguishable among the various laboratories. Likewise, the inter-assay variance within each laboratory remained relatively constant and the results from all laboratories were demonstrably similar and the CV of the positive responses was relatively constant across the entire range of reactivity tested. Thus, we conclude that any and all laboratories and technicians are equally proficient in providing consistent results for all allergens tested and the results are well within the acceptable variance limits (± 20%) established for this assay [1].

Over the past ten years we have documented the reproducibility and robust character of the macELISA. In our most recent report [4], we document that comparable reproducibility of results can be achieved for a panel of identical sera samples when evaluated across multiple years. For the present study we document that inclusion of BROM-CCD inhibitor in our serum diluent [5] does not affect the intra-assay or inter-assay variance of the test. Incorporation of a CCD inhibitor has been shown to be critical for reduction in false positives that occur due to the binding of certain IgE to these carbohydrate groups that are common among pollen allergens, thus leading to potential increases in signal for the assay.

Table 2. Inter-laboratory correlation of macELISA results observed with individual allergens in Stallergenes Greer Laboratory and the results observed in the individual affiliate laboratories.

Inter-Laboratory Coefficient of Correlation

Allergens

Stallergenes Greer vs

IDEXX

IDEXX

IDEXX

Memphis

Ludwiasburg

Markham

Biovac

Agrolabo

LETI

Mites

Acaris siro

1.000

1.000

0.999

0.973

0.983

0.981

Dermatophagoides farinae

1.000

0.995

1.000

1.000

1.000

1.000

Dermatophagoides pteronyssinus

0.999

0.999

0.994

0.971

0.914

0.989

Lepidoglyphus destructor

1.000

1.000

0.993

0.986

0.933

0.993

Tyrophagus putrescentiae

1.000

1.000

0.998

0.981

0.991

0.982

Grasses

June Grass (Poa pratensis)

1.000

1.000

0.996

0.981

0.981

0.992

Meadow fescue (Festuca pratensis)

0.998

0.983

0.999

0.999

0.999

0.999

Orchard Grass (Dactylis glomerata)

1.000

1.000

0.995

0.994

0.967

0.990

Perennial Rye (Lolium perenne)

1.000

1.000

0.997

0.978

0.970

0.990

Trees

Birch (Betula pendula)

1.000

1.000

0.989

0.986

0.951

0.965

Cypress (Cupressus sempervirens)

0.999

0.999

0.976

0.973

0.983

0.969

Hazelnut (Corylus avellana)

1.000

1.000

0.997

0.960

0.896

0.949

Olive (Olea europaea)

0.999

0.995

0.998

0.999

0.999

0.999

Populus mix (P. nigra, P. tremula, P. alba)

1.000

1.000

0.998

0.975

0.932

0.981

Willow Black (Salix discolor)

1.000

1.000

0.999

0.977

0.921

0.978

Weeds

English Plantain (Plantago lanceolata)

0.999

0.999

0.998

0.980

0.935

0.977

Lambs Quarter (Chenopodium album)

0.989

0.985

0.998

0.999

0.999

0.999

Mugwort (Artemisia vulgaris)

1.000

1.000

0.996

0.990

0.940

0.989

Pellitory (Parietaria officinalis)

1.000

1.000

0.997

0.983

0.944

0.958

Ragweed (Ambrosia trifida, A. artemisiifolia)

0.999

0.999

0.994

0.998

0.988

0.993

Sheep Sorrel (Rumex acetosella)

1.000

1.000

0.994

0.986

0.964

0.989

Fungi

Alternaria alternata

0.995

0.995

0.987

0.977

0.971

0.942

Aspergillus fumigatis

0.999

0.997

0.999

0.998

0.998

0.998

Cladosporium herbarum

0.997

0.997

0.969

0.934

0.952

0.977

Overall

1.000

0.998

0.997

0.972

0.981

0.987

*Pearson Correlation Coefficient (r); Good Correlation (r > 0.8, p<0.001)

Table 3. Inter-laboratory correlation of macELISA results observed among individual affiliate laboratories.

Inter-laboratory Coefficient of Correlation*

Laboratory

Stallergenes Greer

IDEXX Memphis

IDEXX Germany

IDEXX
Canada

Ceva
Biovac

Agrolabo

LETI

Stallergenes Greer

1

0.992

0.993

0.997

0.972

0.981

0.986

IDEXX Memphis

0.992

1

0.991

0.990

0.971

0.983

0.990

IDEXX Germany

0.993

0.991

1

0.994

0.960

0.979

0.988

IDEXX Canada

0.997

0.990

0.994

1

0.964

0.980

0.986

Biovac

0.972

0.971

0.960

0.964

1

0.979

0.972

Agrolabo

0.981

0.983

0.979

0.980

0.979

1

0.983

LETI

0.986

0.990

0.988

0.986

0.972

0.983

1

*Pearson Correlation Coefficient (r); Good Correlation (r > 0.8, p<0.001)

The positive response threshold for this assay has repeatedly been documented to be 150 EAU [1–4]. Simply stated, samples shown to have values less than 150 EAU should be considered non-reactive to a given allergen; samples with values in the 150–4000 EAU range can be considered to exhibit specific IgE reactivity that is proportional to serum concentration. We have previously documented that a three-fold increase in allergen-specific IgE content is required to affect an approximate two-fold increase in EAU. If we assume a relative concentration of 1 is required to effect an EAU signal of 150 then the relative concentration of allergen-specific IgE evident in the range of 150–300 EAU will be approximately 1–3, the relative concentration in the 301–600 EAU range will be 3–9, the 601–1200 EAU range will be 9–27, the 1200–2400 EAU range will be 27–81, while the relative concentration of IgE needed to effect a maximal signal will be greater than 150. This being the case, it is unlikely that a highly reactive serum sample will be detected as non-reactive at a 1:5 dilution. The variance evident in the low level range of responses dictates that true borderline positive samples might be identified as false negative responses and this tendency might compound the likelihood of false negative responses. However, a serum sample at a 1:5 dilution makes detection of false positive results seem rather remote. Further, EAU values in the range of 0 -150 cannot be differentiated and comparison of the reproducibility of results within this range is moot (i.e. beyond the scope of the assay), except they are defined as negative responses. Only when EAU values are within the range of defined reactivity (150 – 4000 EAU) can the magnitude of response be used to compare the reproducibility of an assay.

We have demonstrated a continued reliability and reproducibility of our macELISA with the open publication of our proficiency testing procedures and results. We encourage other groups to determine and document similar findings; however, we emphasize the importance of identifying results below the cutoff of 150 EAU merely as non-reactive and consequently negative responses. The reproducibility of the assay for these responses need to be defined only as negative and their numerical values become meaningless; comparison of EAU values are meaningful for reactive samples only. Because the magnitude of specific responses is dependent on the concentration of allergen-specific IgE that can span a wide range, a better means of comparison of repeat results for individual samples in an assay of this sort is to evaluate the correlation (perhaps Pearson statistic) of results that might exist.

The lack of a regulatory mandated quality assurance program for serum allergen-specific IgE testing in companion animals, that independently monitors performance of all laboratories and assay formats, prompts Stallergenes Greer to accept the responsibility for continued evaluation of laboratories that routinely use the company’s assays. Information presented herein documents the continued commitment of Stallergenes Greer and its affiliate laboratories to providing a stream of information relating these results to the veterinary community.

Authors Contributions

Kevin Enck and Kenneth Lee contributed to the conception and design of the study; contributed to the acquisition, analysis, and interpretation of data; and drafted the manuscript. Karen Blankenship and Brennan McKinney manufactured all components used throughout the evaluation and contributed to acquisition of the data. Gerhard Kern, Elizabeth Roth, Janice Greenwood, Santiago Cerrato, Laurent Drouet, and Cecilia Tambone contributed to acquisition of the data. All authors gave final approval and agree to be accountable for all aspects of the work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding

Funding for this study was provided by Stallergenes Greer.

References

  1. Lee KW, Blankenship KD, McCurry ZM, Esch RE, DeBoer DJ, et al. (2009) Performance characteristics of a monoclonal antibody cocktail-based ELISA for detection of allergen-specific IgE in dogs and comparison with a high affinity IgE receptor-based ELISA. Vet Dermatol 20: 157–164. [crossref]
  2. Lee KW, Blankenship KD, McCurry ZM (2012) Reproducibility of a Monoclonal Antibody Cocktail Based ELISA for Detection of Allergen Specific IgE in Dogs: Proficiency Monitoring of macELISA in Six US and European Laboratories. Vet Immunol Immunopathol 148: 267–275.
  3. Lee KW, Blankenship K, McKinney B, Kern G, Buch J, et al. (2015) Proficiency monitoring of monoclonal antibody cocktail–based enzyme-linked immunosorbent assay for detection of allergen-specific immunoglobulin E in dogs. Journal of Veterinary Diagnostic Investigation 27: 461–469.
  4. Lee K, Blankenship K, McKinney B, Kern G, Roth E, et al. (2018) Proficiency Monitoring of Allergen Specific IgE macELISA – 2018. Integr J Vet Biosci 2: 1–6.
  5. Lee KW, Blankenship KD, McKinney BH, Morris DO (2019) Detection and Inhibition of IgE for cross-reactive carbohydrate determinants evident in an enzyme linked immunosorbent assay for detection of allergen specific IgE in the serum of dogs and cats. Vet Dermatol: Submitted
  6. DeBoer DJ, Hillier A (2001) The ACVD task force on canine atopic dermatitis (XVI): laboratory evaluation of dogs with atopic dermatitis with serum-based “allergy” tests. Vet Immunol Immunopathol 81: 277–287. [crossref]
  7. Gorman NT, Halliwell REW (1989) Atopic Diseases. In: Halliwell REW, Gorman NT (ed). Veterinary Clinical Immunology, WB Saunders, Philadelphia. Pg No: 232–252.
  8. Griffin CE, DeBoer DJ (2001) The ACVD task force on canine atopic dermatitis (XIV): clinical manifestation of canine atopic dermatitis. Vet Immunol Immunopathology 81: 255–69.
  9. Griffin CE, Hillier A (2001) The ACVD task force on canine atopic dermatitis (XXIV): allergen-specific immunotherapy. Vet Immunol Immunopathol 81: 363–383. [crossref]
  10. Tijssen P (1993) Processing of data and reporting of results of enzyme immunoassays. In: Burdon, RH, van Knippenberg PH (eds.). Practice and Theory of Enzyme Immunoassays, Elsevier, Amsterdam. Pg No: 385–421.

A Perspective: Regulation of Ku70 Cytosolic Function

DOI: 10.31038/CST.2019453

Introduction

Ku70 was first discovered as an auto-antigen [1, 2]. The majority of Ku70 studies focused on its DNA binding activity in the non-homologous end joining (NHEJ) DNA repair mechanism in the nucleus [3, 4]. However, my laboratory has, for many years, been exploring the roles Ku70 in the cytosol, especially its role in regulating the proapoptotic activity of Bax [5, 6]. Here, I will discuss the roles of Ku70 in regulating Bax activity in the cytosol, integrating some of our key findings with others to illustrate a path forward in understanding the roles of cytosolic Ku70 in cells.

Model of Cytosolic Ku70 as a Survival Factor Against Bax-Mediated Cell Death

Because of its function as a DNA repair factor, the site of Ku70’s activities was always considered to be in the nucleus. The first study, however, to show that Ku70 could be a cytosolic protein was from a yeast two hybrid screen searching for Bax binding proteins [7]. Bax is a pro- apoptotic protein belonging to the Bcl-2 family of proteins [8]. Activation of Bax plays an important role in both intrinsic and extrinsic apoptotic pathways. Bax’s activity can be regulated by binding to other Bcl-2 proteins, such as Bcl-2 or Bcl-XL [9]. The yeast two hybrid study demonstrated that Ku70 binds to Bax and also suppresses Bax proapoptotic activity. This study was followed by a study showing that Ku70’s binding to Bax is regulated by acetylation of two lysine resides at K539 and K542 of Ku70 [10]. When Ku70 is acetylated at these two lysine residues, Bax is released, resulted in its entrance into the mitochondria, triggering apoptosis. We have also established that depleting Ku70 triggers cell death, but cell death can be prevented by simultaneously depleting Bax, suggesting that Ku70 may be required to suppress Bax activity in cells such that Bax is activated without the suppressive effect of Ku70 [11].

To investigate how Ku70 acetylation is regulated, we used neuroblastoma (NB) neuroblastic (N-type) cells as a model, and established that by altering the acetylation status of cells, we could modulate Ku70-Bax complex formation or dissociation, protecting these cells from dying or inducing these cells to die, respectively [12]. We have confirmed the original observation by Cohen et al. [10] that the cAMP-response element binding protein (CREB) binding protein (CBP), a transcriptional activator and an acetyltransferase, acetylates Ku70 [11]. Mutation of K539 and K542 of Ku70 to arginine blocked histone deacetylase inhibitors (HDACIs) induced cell death and also blocked Bax release following HDACI treatment. Our studies showed that over expression of CBP induced cell death in a Ku70 dependent manner. In contrast, CBP depletion caused reduction of Ku70 acetylation, increasing the resistance of HDACI-induced cell death. Our results also indicated that p300, a homolog of CBP in human cells that shares many identical functions in cells with CBP [13], did not affect cell death when over expressed, suggesting that CBP plays a unique role in acetylating Ku70 in cells.

To further understand how Ku70 acetylation is regulated, we sought to identify the deacetylase that deacetylates Ku70 in the cytosol. Our results showed that HDAC6 bound to Ku70 and Bax [14]. HDAC6 is a class IIb HDAC containing two catalytic domains [15, 16]. HDAC6 is mainly localized in the cytosol and has been associated with many cell functions including tubulin stabilization, cell motility, and regulation of binding between Hsp90 and its cochaperone [17]. In NB cells, HDAC6 formed a complex with Ku70 and Bax, and depleting HDAC6 had similar effects to treatment with tubacin, a HDAC6 specific inhibitor [18]. Furthermore, depleting HDAC6 also increased Ku70 acetylation, releasing Bax from Ku70, causing cell death. Based on our findings, we proposed a model in which Ku70 may serve as a survival factor in suppressing Bax-induced cell death. We reasoned that, throughout life, cells continuously receive stimuli that affect cell viability. Some of these stimuli may be strong enough to trigger a high level of Bax activation leading to instant cell death while some minor stimuli may only activate a few molecules of Bax. As a protective measure and to conserve energy, cells may avoid dying when receiving weak signals that only activate a low level of Bax. Thus, to cope with these small sporadic Bax activation signals, cells may find ways to block these signals. We believe that Ku70 may act as one of these survival factors, blocking low level of Bax activation and thereby preventing premature cell death. While this model is compatible with the current data, it raises two important questions: 1. What is the stoichiometry of the binding between Ku70 and Bax? 2. Does this model apply to all cell types?

The model suggests that Ku70 needs to bind to activated Bax when activated Bax’s level increases. But how much do cytosolic Ku70 and Bax bind to each other in cells at basal levels? One possibility is that Ku70 and Bax do not bind to each other at basal levels, and thus there is plenty of Ku70 available to bind Bax when Bax is activated. However, the study by Sawada et al. stated that “a large proportion of the Bax population is associated with Ku70 in normal cells.” [7] This statement is not consistent with published results reporting that Bax is inactive and monomeric in the cytosol [19]. Using gel filtration chromatograph and cross-linking techniques, we have shown that the majority of Bax is monomeric and the majority of Ku70 is in complex with other factors, including its DNA binding partner Ku80 [20]. There is only a small amount of Ku70 binding to a small amount of Bax at basal levels. Most important, however, is that there is no free Ku70 or monomeric Ku70 found in the cytosol. Where is the additional free Ku70 coming from when the rest of Ku70 in cells is in complex with other factors? Our model suggests that if Ku70 acts as a survival factor in rescuing cells from Bax-induced killing, Ku70 has to be released from complexes that contain Ku70 so that it is available to bind to activated Bax. If so, there has to be another level of regulation of Ku70 availability in the cytosol for Ku70 to act as a survival factor. Studies have shown that Ku70 binds to several factors in the cytosol [21–23]. For example, the FAAD-like interleukin-1-b-converting enzyme (FLICE)-inhibitory protein (FLIP) is an antiapoptotic protein that blocks caspase 8 activation by death receptors [24]. FLIP binds to Ku70 in an acetylation-dependent mechanism. However, unlike Bax binding to the carboxyl terminal of Ku70, FLIP binds to the Ku80 binding domain of Ku70. When Ku70 is acetylated at K539 and K542, the same two lysines when acetylated regulate the binding of Bax, causing FLIP to be released. It is then polyubiquitinated and degraded, allowing caspase 8 to be activated thereby inducing cell death via the extrinsic pathway [24]. It is not clear whether Ku70 binds to Bax and FLIP simultaneously, however. Thus, the weak incoming apoptotic signals have to achieve at least two things: 1. to activate Bax, and 2. to release Ku70 from its binding proteins so that Bax can be inactivated. This means that there is another level of regulation of Ku70 availability in the cytosol that releases Ku70 from Ku70-containing complexes. The relative affinities of Ku70 to various complexes are not yet known. Thus, it is difficult to predict which complex that Ku70 is released from and what the mechanism of regulation is.

While the notion that Ku70 is acting as a survival factor is intriguing, the question remains: does it apply to all cell types? Originally, we used the NB N-type SH-SY5Y cells to establish the model in which Ku70 is acting as a survival factor [11, 12]. Our results demonstrated that when Ku70 was depleted using Ku70 specific siRNA in SH-SY5Y cells, the cells would die in a Bax- dependent manner (rescued by simultaneously depleting Bax). However, previous studies by others have shown that in other cell types, such as HeLa and HEK293, depleting Ku70 using Ku70 specific siRNA did not induce cell death [7, 10]. Our results, together with the results in HeLa cells and HEK293 cells, suggest that there may be at least two cell types in terms of Ku70 regulating Bax function: one is Ku70-depletion sensitive, and one is Ku70-depletion less sensitive.

In a more extended study, we depleted Ku70 using Ku70-specific siRNA in a panel of N- type NB cells (including SH-SY5Y cells as a positive control), S-type NB cells (SHEP-1), HEK293T cells, and a couple of ovarian cancer cells [20]. We found that except for the N-type NB cells, depletion of Ku70 did not trigger cell death in these cells. More interesting, however, is the finding that the Ku70-depletion less sensitive cells were also less sensitive to the HDACI treatment. This was not due to the fact that Ku70 was not acetylated following treatment of these cells. In fact, Ku70 was also acetylated in these cells following HDACI treatment. However, even though Ku70 was acetylated, our results showed that Bax did not dissociate from Ku70, contrary to what we observed in SH-SY5Y cells: HDACI treatment induced Ku70 acetylation, separating Bax and inducing cell death. Our results can clearly distinguish between two types of cells in terms of their sensitivity to cell death after Ku70 depletion: one is sensitive and one is less sensitive.

In the Cohen et al. paper, they described that the two lysine residues (K539 and K542) on Ku70 that are important for regulating Bax binding are localized at the linker regions of Ku70, not within the Bax-binding domain at the carboxyl terminal of Ku70 (residues 578–609) [10]. Acetylation of these two lysine residues induces a conformational change of the Bax-binding domain of Ku70 resulting in Bax dissociation. Based on our current results in Ku70-depletion less sensitive cells, we suggest that some other factors in these cells must block the conformational change of Ku70 upon acetylation of these two lysine residues. Our gel filtration chromatograph data suggest that these factors must be small because the patterns of Ku70 and Bax gel filtration chromatograph are similar in HEK-293T cells and SH-SY5Y cells [20]. Another less likely possibility is that Ku70 depletion less sensitive cells may have higher levels of the anti-apoptotic Bcl-2 family of proteins that suppress activation of Bax and its association with Ku70 when Ku70 is acetylated. More work is needed to define these two cell types and how that knowledge can be used in targeted therapies in the treatment of cancer.

Conclusion

Our model that Ku70 acts as a survival factor for Bax-dependent cell death only in certain selected cell types is intriguing, and suggests that it could be beneficial to target Ku70-Bax complex as a therapeutic endpoint. It may be possible to manipulate the association or dissociation of Ku70 and Bax in these Ku70-depletion sensitive cells and preserve these specific cells from Bax-mediated cell death or to induce these cells to die, respectively, without affecting the Ku70-depletion less sensitive cells. A five-residue peptide corresponding to Ku70 (residues 596–600) has been demonstrated to bind Bax and block Bax-induced cell death [25]. Thus, this strategy provides a rationale for screening small molecules that mimic the Ku70-binding domain of Bax and block the interaction between Ku70 and Bax by competing with Bax for Ku70 binding, resulting in inducing cell death in these Ku70-depletion sensitive cells. Agents that block Bax binding to Kui70 resulting in cell death may be tested in clinical settings, either alone or in combination with radiotherapy or DNA damaging agents, to target cancer cells that are sensitive to Ku70 depletion, like that in N-type NB cells.

Acknowledgment

RPSK was partly supported by a grant from the National Institute of Health (R21 AG051820-02).

References

  1. Mimori T, Hardin JA (1986) Mechanism of interaction between Ku protein and DNA. J Biol Chem 261: 10375–10379.
  2. Rathmell WK, Chu, G (1994) Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks. Proc Natl Acad Sci U S A 91: 7623–7627.
  3. Dvir A, Peterson SR, Knuth MW, Lu H, Dynan WS (1992) Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II. Proc Natl Acad Sci USA 89:  11920–11924.
  4. Gottlieb TM, Jackson SP (1993) The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72: 131–142.
  5. Subramanian C, Opipari AW Jr, Castle VP, Kwok RP (2005) Histone deacetylase inhibition induces apoptosis in neuroblastoma. Cell Cycle 4: 1741–1743.
  6. Hada M, Kwok RP (2014) Regulation of ku70-bax complex in cells. J Cell Death 7: 11–13.
  7. Sawada M, Sun W, Hayes P, Leskov K, Boothman DA, et al. (2003) Ku70 suppresses the apoptotic translocation of Bax to mitochondria. Nat Cell Biol 5: 320–329.
  8. Cory S, Adams JM (2002) The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2: 647–656.
  9. Doerflinger M, Glab JA, Puthalakath H (2015) BH3-only proteins: a 20-year stock-take. FEBS J 282: 1006–1016.
  10. Cohen HY, Lavu S, Bitterman KJ, Hekking B, Imahiyerobo TA, et al. (2004) Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax- mediated apoptosis. Mol Cell 13: 627–638.
  11. Subramanian C, Jarzembowski JA, Opipari AW Jr, Castle VP, Kwok RP (2007) CREB-binding protein is a mediator of neuroblastoma cell death induced by the histone deacetylase inhibitor trichostatin A. Neoplasia 9: 495- 503.
  12. Subramanian C, Opipari AW Jr, Bian X, Castle VP, Kwok RP (2005) Ku70 acetylation mediates neuroblastoma cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci USA 102: 4842–4847.
  13. Goodman RH, Smolik S (2000) CBP/p300 in cell growth, transformation, and development. Genes Dev 14: 1553–1577.
  14. Subramanian C, Jarzembowski JA, Opipari AW Jr, Castle VP, Kwok RP (2011) HDAC6 deacetylates Ku70 and regulates Ku70-Bax binding in neuroblastoma. Neoplasia 13: 726–734.
  15. Grozinger CM, Hassig CA, Schreiber SL (1999) Three proteins define a class of human histone deacetylases related to yeast Hda1p. Proc Natl Acad Sci USA 96: 4868–4873.
  16. Verdel A, Khochbin S (1999) Identification of a new family of higher eukaryotic histone deacetylases. Coordinate expression of differentiation-dependent chromatin modifiers. J Biol Chem 274: 2440–2445.
  17. Lee YS, Lim KH, Guo X, Kawaguchi Y, Gao Y, et al. (2008) The cytoplasmic deacetylase HDAC6 is required for efficient oncogenic tumorigenesis. Cancer Res 68: 7561–7569.
  18. Cole PA (2008) Chemical probes for histone-modifying enzymes. Nat Chem Biol 4: 590–597.
  19. Vogel S, Raulf N, Bregenhorn S, Biniossek ML, Maurer U, et al. (2012) Cytosolic Bax: does it require binding proteins to keep its pro-apoptotic activity in check? J Biol Chem 287: 9112–9127.
  20. Hada M, Subramanian C, Andrews PC, Kwok RP (2016) Cytosolic Ku70 regulates Bax-mediated cell death. Tumour Biol 37: 13903–13914.
  21. Mazumder S, Plesca D, Kinter M, Almasan A (2007) Interaction of a cyclin E fragment with Ku70 regulates Bax-mediated apoptosis. Mol Cell Biol 27: 3511–3520.
  22. Renault TT, Manon S (2011) Bax: Addressed to kill. Biochimie 93: 1379–1391.
  23. Westphal D, Dewson G, Czabotar PE, Kluck RM (2011) Molecular biology of Bax and Bak activation and action. Biochim Biophys Acta 1813: 521–531.
  24. Kerr E, Holohan C, McLaughlin KM, Majkut J, Dolan S, et al. (2012) Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis. Cell Death Differ 19: 1317–1327.
  25. Gomez JA, Gama V, Yoshida T, Sun W, Hayes P, et al. (2007) Bax-inhibiting peptides derived from Ku70 and cell-penetrating pentapeptides. Biochem Soc Trans 35: 797–801.

Perceptual-Cognitive Training Can Improve Cognition in Older Adults with Subjective Cognitive Decline

DOI: 10.31038/ASMHS.2019361

Abstract

Introduction: Subjective cognitive decline (SCD) in older adults are an early risk indicator for Alzheimer’s disease or other forms of dementia, making older adults with SCD a target population for proactive interventions. The aim of this study was to determine if perceptual-cognitive training (PCT) can serve as a proactive intervention and enhance cognition in older adults with SCD.

Method: Forty-seven subjects aged 60–90 years of age were assigned to control and treatment groups using a randomised controlled trial. All the participants were asked to complete three neuropsychological assessments over a three-month period. The first assessment was prior to the PCT (T1). The second assessment (T2) was performed immediately after either seven weeks of PCT (treatment group), or after seven weeks of no training (control group). Four weeks after the completion of the PCT, a third assessment (T3) was performed to determine the veracity and persistence of any PCT benefits on cognitive performance.

Results: The results indicate a significant difference between groups at T1 and T2, wherein the treatment group has improved scores in memory tasks (e.g., CVLT-II: Immediate Free Recall; Short-Term Memory Recall, and Long-Term Memory Recall), working memory task (e.g., Digit Span Backward) and cognitive flexibility task (e.g., D-KEFS Verbal Fluency Category Switching and D-KEFS Verbal Fluency Letter Fluency). Within the treatment group the PCT scores of the last session were also significantly correlated with processing speed and cognitive flexibility. Furthermore, higher scores in memory performance were related to faster processing speeds.

Conclusion: These data suggest that PCT may serve as a proactive intervention to enhance memory, working memory and cognitive flexibility in older adults with SCD.

Keywords

Subjective cognitive decline, Perceptual-cognitive training, NeuroTracker, Memory, Processing speed, Cognitive flexibility, Working memory

Introduction

North America has a growing aging population that will introduce unique challenges for the health care system in the coming century [1]. In Canada, for example, 22.3% of the population is currently over 60 years old, and this is estimated to increase to 32.5% by 2050 [2]. While a life expectancy beyond 60 years of age has increased by about 25 years, only the first 18 years of this period are likely to be spent in good health, including good cognitive functioning [2, 3]. Generally, it is difficult to separate normal cognitive aging from pathological cognitive decline. For many people cognitive decline is associated with relatively minor and sporadic cognitive difficulties (e.g. processing speed, attention, working memory, cognitive flexibility, and episodic memory), considered normal within the spectrum of typical cognitive aging [4–6]. For some, cognitive changes are serious enough to be noticed by other people and confirmed by neuropsychological tests while these changes still do not interfere with daily life or independent function (i.e., Mild Cognitive Impairment). For others [7], cognitive decline is associated with severe cognitive deficits that impede the ability to live independently (i.e., Dementia).

Subjective cognitive decline (SCD) is a common complaint of the elderly population and may also be the earliest manifestation of Alzheimer or other forms of dementia [8]. Considerable evidence, from both behavioral and neurobiological sources, suggests that the basic cognitive domains most affected by age are executive function and memory [9, 10]. Although many older adults complain of increased memory lapses as they age not all kinds of memory are affected by normal ageing [10]. The most susceptible to brain damage and the most affected by normal aging is episodic memory [11,12]. For example, older adults tend to show more deficits on tests of free recall, to a somewhat lesser degree of difficulty in cued recall, and minimal difficulty in recognition memory. Furthermore, older adults often out-perform on attentional tasks that require flexible control, dividing or switching of attention among multiple inputs or tasks [13]. Indeed, older adults face greater difficulties in performing higher-level cognitive tasks that involve manipulation, reorganization, or integration of the contents of working memory. It seems likely that attentional resources [14], processing speed [6, 15] and the ability to inhibit irrelevant information [16] are all important functions for effective performance of these higher-level cognitive tasks.

There are many evidences that non pharmacological treatments, such as neurocognitive rehabilitation (e.g. brain stimulation techniques, computerized neurocognitive training tools), may be more effective than traditional cognitive stimulation in reducing or delaying cognitive decline in older adults [17–20]. A systematic review by Kueider and colleagues [18] assessed the efficacy of various computerized cognitive training tools, in comparison to traditional paper-and-pencil cognitive training approaches in older adults. The main benefits of the technological based training interventions were improvements in memory [21, 22], processing speed [23–27] and attention [28, 29]. Indeed, computerized cognitive training was found to be as effective as the traditional cognitive training but less labour-intensive alternative. Furthermore, computerized cognitive training had increased compliance in older adults, possibly because it is easy to access, can be used directly from home, is non-invasive, relatively inexpensive and does not require particular technological skills [18]. Therefore, introducing preventive treatments such as cognitive training programs, may have several significant benefits for an aging population [30].

Perceptual-Cognitive Training, also called Neurotracker, is a technology that was designed to enhance elite athlete performance by training their ability to track and focus on multiple moving objects in the three-dimensional visual field. This form of neurocognitive training engages visual scanning, sustained attention, divided attention, processing speed, working memory, inhibition ability, and cognitive flexibility [31–34]. Memory decline in older adults has been linked to deficits in executive processes (e.g. attention, inhibitory function, cognitive flexibility, working memory) due to their involvement in higher-level cognitive tasks [6, 9, 35]. PCT has been shown to improve different cognitive abilities in both healthy and pathological populations of young and old adults [34, 36, 37]. It was postulated that PCT may reduce or reverse the age-related cognitive decline and the aim of this study is to verify if PCT can enhance cognition in older adults with SCD.

Methods and Materials

Participants

A sample of 73 participants, between 60 and 90 years of age, was recruited using word of mouth referral and flyer distribution in the Capital Regional District (CRD) encompassing the southern tip of Vancouver Island. Print and web-based advertising were also used through the Institute on Aging and Lifelong Health at the University of Victoria. Participants were recruited from 30th of June 2017 to 13th of March 2018. The first follow-up was done on 18th of August 2017 and continued until 10th of May 2018. Socio-demographic information was collected from all participants at the baseline session (e.g., age, gender, level of education, and medical history) by completing an intake form approved by ethics committee of University of Victoria. All participants were screened for any medical, neurological, or psychiatric conditions known to affect cognitive performance in the first interview. The Mini Mental State Examination [38] was used as a screening tool (cut-off ≥ 26) to minimize the risk of including persons with preclinical dementia but as well to quantify the subjective cognitive complaints. Two tests, Activities of Daily Living [39] and Instrumental Activities of Daily Living [40], were administered to exclude subjects with possible dementia and to ensure that they were able to attend the testing and the training sessions at the University of Victoria. All participants were screened for SMCs using the Memory Complaint Questionnaire [41] and only the participants with a score of 25 or above were included in this study. All participants were screened for depression using the Geriatric Depression Scale [42] with cut-off ≥ 10, and for anxiety using the Geriatric Anxiety Inventory [43] with cut-off > 9. On self-report of a diagnosis, seven participants did not meet the inclusion criteria (i.e. one had ADHD, four subjects had Macular Degeneration; one had Anxiety Disorder, one had PTSD) and were not included in this research study. Following participant screening, only 66 subjects (female n = 48, 72.2%), aged 60 years and over (MeanAge = 73.32, SDAge = 7.58) satisfied the inclusion criteria and were enrolled in the study (e.g. over a three-month period). Eighteen subjects declined their participation to the study due to a long commitment time required. One female participant dropped out during the study due to a neurological event (e.g. a concussion outside the testing environment) and her data was removed from the analysis. The remaining 47 subjects (see Figure 1) were randomly assigned to either the treatment or control group and all subjects completed the follow-up. The method used to generate the allocation sequence was self-selection (i.e. we generated a random assignment based on the participant’s availability to commit to the study). The treatment group consisted of 25 participants between the ages of 61 and 89 years of age (female = 16; male = 9) whereas the control group consisted of 22 older adults, ages 60 to 90 years (female = 15; male = 7).

ASMHS-2019_Brian R. Christie_F1

Figure 1. Flow diagram summarizing patient recruitment and progress in the study. Seventy-three individuals were initially assessed to take part in the study, of these, 18 declined to participate and 7 were excluded for not meeting inclusion criteria. The remaining 48 subjects were randomly assigned to treatment and control groups. Both groups received identical assessments, however only the treatment group received perceptual-cognitive training. Only one individual from the treatment group did not complete the training and subsequent follow-up assessments.

Procedure

This clinical study, using a parallel design, was approved on 27th June 2017 by the University of Victoria Human Research Ethics Board. The authors confirm that all ongoing and related trials for this intervention are registered (NCT03763344). This study was not registered before the enrolment of participants since UVic Research Ethics Board did not consider this study as a clinical trial but as a research study on sub-clinical population (e.g. Subjective cognitive decline).

All participants provided their informed written consent prior to participating in this study. Participants from both the treatment and control groups received a total of three neuropsychological assessments over a three-month period (see Figure 1). All the data were collected in the Concussion Laboratory of the Division of Medical Sciences, at the University of Victoria. All the tests were administered by a Doctoral Student in Clinical Neuropsychology. Considering that an essential methodological component of the training studies [44] is the use of standardized neuropsychological tests, validated and reliable measures were used. The primary outcome measure was California Verbal Learning Test, Second Edition (i.e., standard and alternate forms) [45–47]. The secondary outcome measures were Digit Span, D-KEFS Trail Making Test, D-KEFS Verbal Fluency Test (both standard and alternate forms) [45–47], and Stroop Test. Each assessment was 50–60 minutes in duration and was administered by a Doctoral Student in Clinical Neuropsychology. The first assessment was administered at baseline (T1). Then, each subject of the treatment group underwent seven weeks of perceptual cognitive training, while the control group completed seven weeks without formal training. The intervention consisted of 14 sessions of PCT each lasting 25–30 min, twice per week for seven weeks. After the seven-week time period, a second neuropsychological assessment was performed on both groups (T2). After eleven weeks, a follow-up assessment was conducted to verify whether the benefits of cognitive training endure over time (T3). We offered the PCT to both groups but at different time points (e.g. the control group engaged in the training after the follow-up assessment).

Neuropsychological Tests

Episodic memory

California Verbal Learning Test Second Edition (CVLT-II; D. C. Delis, Kramer, Kaplan, & Ober, 2000) [48] CVLT-II is a multiple-trial list-learning task that measures individuals’ episodic memory and auditory learning ability. CVLT is considered a sensitive tool in identifying subtle episodic memory difficulties. This test assesses recall and recognition of two-word lists over immediate and delayed memory trials. Standard and alternate forms of these lists exist, each with different lists of words to avoid practice effects. Each form contains two lists: list A and list B. List A is composed of 16 words divided in four different semantic categories (e.g., furniture, vegetables, methods of transportation, and animals); whereas words from the same semantic category are never presented consecutively. There are five trials using List A, and each trial requires the participant to immediately recall as many words from the list as possible. List B is a 16-word interference list, which includes different categories. List B is presented once, following the five trials of immediate recall of List A. Immediately after presentation of List B, short-delay free recall of List A is administered. Between the short-delay recall and long-delay recall, there is a 20-minute delay, which is filled with non-verbal testing (e.g., D-KEFS TMT; Stroop Test). After the non-verbal testing, long-delay free recall of List A and a recognition task (yes/no format) are administered. This list included words from both List A and List B, as well as other distractor words, where the examinee is required to identify only the words belonging to List A.

Executive Function

Delis–Kaplan Executive Function System Trail Making Test (D-KEFS TMT) (Delis, Kaplan, & Kramer, 2001) [49] is a pencil and paper task, used to evaluate aspects of cognition including processing speed, motor speed and cognitive flexibility. It involves a series of five conditions: visual scanning, number sequencing, letter sequencing, number-letter switching, and motor speed. In the visual scanning condition, examinees must cross out all the threes that appear on the response sheet mixed with other numbers. In the number sequencing condition, examinees draw a line connecting the numbers 1–16 in counting order while avoiding distractor letters that appear on the same page. The letter sequencing condition requires examinees to connect the letters A through P, with distractor numbers presented on the page. In the number-letter switching condition, examinees switch back and forth between connecting numbers in counting order and letters in alphabetical order (i.e., 1, A, 2, B, etc., to 16, P). This condition requires the ability to switch mentally between numerical and alphabetical sequences and provides an assessment of the participant’s cognitive flexibility. Finally, the examinee completes a motor speed condition in which he/she has to trace over a dotted line connecting circles on the page as quickly as possible. This final section assesses their graphomotor speed.

Delis–Kaplan Executive Function System Verbal Fluency Test (D-KEFS VFT) (Delis, Kaplan, & Kramer, 2001) [49] is a short test of verbal functioning that measures processing speed and cognitive flexibility. There are three conditions: Letter Fluency, Category Fluency, and Category Switching. In all three conditions, the examinees are given 60 seconds to generate as many words following a semantic cue (e.g., specific category), a phonemic cue (e.g. starting with a certain letter) or alternating between two categories a task, which requires a certain amount of mental flexibility.

Digit Span Test is a measure of working memory consisting of 16 trials; eight in Digit Span Forward, and eight in Digit Span Backward. In both conditions, the examiner reads out a series of numbers, ranging from 2–9 digits in sequence. In the forward condition, the participant is asked to repeat the numbers verbatim as stated by the examiner at the end of each trial. In the backward condition, the participant is asked to repeat the numbers in the reverse order stated by the examiner.

Stroop test is used to measure selective attention, psychomotor speed and cognitive flexibility [50]. In this study, the Stroop test was delivered using the Encephal App [51], which adheres to the same principles as the classic Stroop version [52]. Subjects are required to identify the ink colour of discordant-colour words (red, blue, or green). The task consists of two parts: the Stroop effect turned off (i.e. the examinees name the colour of the ink of a set of number signs) and the Stroop effect turned on (i.e. series of colour words “Red”, “Blue”, “Green” are presented in an incongruent coloured ink). In this task, the examinee must inhibit the automatic tendency of reading in order to name correctly the colour of the ink. The placement of the words and number signs are randomized and change position on the screen with each new stimulus. The order of the responses on the bottom of the screen that examinees need to respond to are randomized and shifts in order with each new stimulus. The examinees are not instructed that the order of the response options shift with each new screen, requiring more focus and mental flexibility to the changing stimuli.

Perceptual-Cognitive Training

NeuroTracker is a computerized perceptual-cognitive training system developed by Jocelyn Faubert of University of Montreal [33, 53, 54]. This training is based on a computerized 3D Multiple Object Tracking (3D-MOT) model that follows two principles: isolation and overloading. Isolation training uses limited and consistent cognitive load, while overloading challenges the subject by training them at levels beyond their current ability in order to increase cognitive functioning. Previous studies have indicated that the training effect is reduced if isolation and overloading are not applied to the task [55, 56].

Each PCT session consists of three series of 20 trials in which the subject wears 3D glasses and tracks four spheres among four identical distractors that move in a 3D volumetric cube on the screen. In the first phase, all eight spheres are stationary on the screen, then the four targets briefly change to red and after two seconds revert to yellow. The four target spheres must be tracked as they moved in a linear trajectory for eight seconds. After this, the spheres stop moving and the subject is asked to identify the four targets.

The sessions are based on a staircase procedure [57], in which an algorithm shifts the speed of the target spheres in regard to the participants’ performance (i.e., overloading principle). If all targets were correctly identified, the speed of the movement of the spheres increases by 0.05log, whereas with each incorrect response the speed decreases by 0.05log.

Data Analysis

IBM SPSS Statistics v22.0 and R Software were used for the statistical analyses. Descriptive statistics were computed and full statistical diagnostics carried out to check for adequate distributions, out-of-range values, missing values and outlier checks well as overall standard deviations and standard errors values. Such diagnostics were iteratively conducted on the data collected upon completion of the three assessments: prior the intervention (T1), after the seven weeks of training (T2), and four weeks post-intervention (T3). In particular, box plots for each group and dependent measures were used to identify critical outliers pre-, post-training, and after a month of follow-up. It was decided to constrain outliers values with more than 3 standard deviations above or below the mean. The Trimming method [58, 59] was used to replace the outliers found by the second-highest value from the respective cognitive task group (e.g. CVLT-II, D-KEFS VFT) or by the second-lowest value from the tasks measured in seconds (e.g. D-KEFS TMT, Stroop Test). Data of a subject that dropped out in the middle of the intervention for a concussion reason was removed. Following up the statistical diagnostics and data screening, a first series of independent t-tests were performed on the data at T1 to verify that both groups were equal at baseline in terms of age, education, global cognitive efficiency (MMSE), memory complaints, and leisure activities prior to the intervention. Next, a factorial between-within subject differences (i.e. treatment and control differences across time T1, T2 and T3) were examined by a Doubly Factorial MANOVA. Finally, univariate Within-Subjects Contrasts further examined cognitive abilities that displayed a linear trend in the treatment group (p < .05).

Research expectations were 1) to support a construction of a balanced design with no multivariate or univariate F test differences at baseline (T1) between the two groups); 2) to detect significant multivariate and univariate effects at T1 and T2 between groups (expectation is that treatment group would perform better); 3) to identify some linear trends for the experimental group across T2 and T3. Notably, testing for significant multivariate results at T2 and T3 (if any) might also provide some indication on the potential future use of a linear composite of such DVs to study differences across patients instead of relying on single univariate measures. A one way repeated measures (RM) ANOVA (Time: Session 1 to Session 14) to analyse the PCT performance for the treatment group. Additionally, a series of stepwise linear regressions were used to verify if PCT training scores predicted cognitive performance for the treatment group. Where appropriate, the assumption of sphericity was tested and where violations occurred a Greenhouse-Geisser correction was applied.

Results

Descriptive statistics

The analyses were performed at the group level on all 47 subjects that concluded the study. The data of the participant that dropped-out was removed from the analysis. An independent t-test was performed between the control and the treatment groups and showed no differences (all p > .05) for age, global cognitive efficiency, and memory complaints prior to the intervention (see Table 1).

Table 1. Demographic information for control (n = 22) and treatment (n = 25) groups.

Control group (n = 22)

Treatment group (n = 25)

U test / t-test

Variables

M (SD)

95% CI

M (SD)

95% CI

t

p

Age

72.14 (6.23)

69.37

74.9

74.36 (8.73)

70.75

77.96

1.01

.137

Education

15.73 (2.81)

14.47

16.97

16.40 (4.03)

14.73

18.06

.65

.516

MMSE

29.27 (.70)

28.96

29.58

29.24 (1.30)

28.7

29.77

-.10

.914

Mdn

Mdn

U test

p

MAC-Q

26 (7)

27 (8)

212

.170

MMSE: Mini Mental State Examination; MAC-Q: Memory Complaint Questionnaire

Similarly, no differences (all p > .05) were found between groups at baseline for the major components that could contribute to their cognitive reserve (education and leisure activities). Further, the Multivariate difference analysis at baseline (T1) shows no differences (all p > .05) between groups in terms of cognitive functioning. Overall such results would well represent an experimental condition of favorable balanced design (Table 2).

Table 2. Multivariate difference at baseline (T1) between groups.

Cognitive variables

Control group

Treatment group

Pairwise comparisons

M (SD)

M (SD)

p

F value

CVLT-II List A IFR

52.50 (2.42)

55.28 (2.26)

.410

.703

CVLT-II List A SDFR

10.54 (.79)

11.92 (.74)

.210

1.617

CVLT-II List A LDFR

11.32 (.63)

11.72 (.59)

.644

.216

CVLT-II List A LDR

15.50 (.17)

15.13 (.16)

.131

2.269

CVLT-II List A LDR FPE

2.22 (.46)

1.25 (.43)

.134

2.332

DIGIT SPAN F.

6.50 (.23)

6.80 (.21)

.343

.920

DIGIT SPAN B.

5.23 (.28)

5.70 (.26)

.244

1.396

TOTAL DIGIT SPAN

11.68 (.44)

12.48 (.40)

.190

1.794

D-KEFS TMT: VS

24.36 (1.40)

25.96 (1.26)

.390

.764

D-KEFS TMT: NS

46.07 (3.58)

38.80 (3.36)

.145

2.197

D-KEFS TMT: LS

42.73 (3.75)

37.92 (3.53)

.560

.871

D-KEFS TMT: NLS

94.90 (10.82)

98.24 (10.15)

.823

.050

D-KEFS TMT: MS

27.18 (2.32)

31.03 (2.18)

.232

1.467

D-KEFS VFT: LF

42.64 (2.12)

44.64 (1.99)

.495

.473

D-KEFS VFT: CF

37.64 (1.83)

38.90 (1.71)

.625

.242

D-KEFS VFT: CS

11.72 (.74)

11.36 (.70)

.720

.132

STROOP TEST OFF

83.35 (3.43)

85.75 (3.21)

.612

.261

STROOP TEST ON

100.12 (4.30)

103.63 (4.03)

.560

.354

*CVLT-II List A IFR – Immediate Free Recall Trials 1–5; CVLT-II List A SDFR – Short-Delay Free Recall; CVLT-II List A LDFR – Long-Delay Free Recall; CVLT-II List A LDR – Long-Delay Yes/No Recognition; CVLT-II List A LDR FPE – Long-Delay Recognition False Positive Errors; DIGIT SPAN F. – Digit Span Forward; DIGIT SPAN B. – Digit Span Backward; D-KEFS TMT:VS – Visual Scanning; D-KEFS TMT: NS-Number Sequencing; D-KEFS TMT: LS – Letter Sequencing; D-KEFS TMT: NLS – Number-Letter Switching; D-KEFS TMT: MS – Motor Speed; D-KEFS VFT: LF – Letter Fluency; D-KEFS VFT: CF – Category Fluency; D-KEFS VFT: CS – Category Switching.

Factorial Multivariate Analysis

A Factorial Doubly MANOVA was conducted (i.e. 2×3 groups: control, experimental; time: T1, T2 and T3) to examine the transferability of PCT benefits on cognitive performance. Using Wilk’s lambda, there was a significant multivariate effect of interaction between groups and time for the cognitive variables considered in this study Λ =.401, F = (38, 144) = 2.20, p= .000, ɳp2 = 1. To further explore this significant MANOVA interaction a set of separate follow-up univariate ANOVAs (simple main effects analysis) on the cognitive variables revealed significant treatment effects between groups on CVLT-II Immediate Free Recall Trials 1–5; CVLT-II Short-Delay Free Recall; CVLT-II Long-Delay Free Recall; CVLT-II Recognition; D-KEFS VFT Letter Fluency, D-KEFS VFT Category Switching, D-KEFS TMT Visual Scan, D-KEFS TMT (Table 3). Notably, due to the exploratory nature of such analysis all such individual F-value tests have to be further investigated to confirm the various target variable contributions to the MANOVA model findings so far.

Table 3. Univariate test between groups in time.

Sum of Squares

df

Mean Square

F

p

Partial Eta Squared

Observed Power

CVLT-II List A/B IFR

290.16

2

145.080

3.247

.043*

.067

.605

CVLT-II List A/B SDFR

36.008

2

18.004

5.016

.009**

.100

.803

CVLT-II List A/B LDFR

44.905

2

22.452

6.433

.002**

.125

.895

CVLT-II List A/B LDR

4.715

2

2.357

4.474

.014*

.090

.753

CVLT-II List A/B LDR FPE

7.668

2

3.844

.829

.440

.018

1.658

DIGIT SPAN F.

.172

2

.086

.165

.848

.004

.075

DIGIT SPAN B.

3.190

2

1.595

1.716

.186

.037

.352

TOTAL DIGIT SPAN

3.341

2

1.671

1.256

.290

.027

.267

D-KEFS VFT: LF

245.452

2

122.726

3.752

.027*

.077

.672

D-KEFS VFT: CF

18.428

2

9.124

.397

.673

.009

.112

D-KEFS VFT: CS

48.512

2

24.256

3.551

.033*

.073

.647

D-KEFS TMT:VS

103.179

2

51.590

3.753

.027*

.077

.672

D-KEFS TMT:NS

532.787

2

266.394

2.210

.116

.047

.440

D-KEFS TMT:LS

203.779

101.890

1.056

.352

.023

.230

D-KEFS TMT: NLS

2.953.761

2

1.476.880

2.457

.091

.052

.483

D-KEFS TMT: MS

260.530

2

130.265

2.740

.070

.057

.529

STROOP TEST OFF

242.613

2

121.306

1.016

.366

.022

.222

STROOP TEST ON

202206

2

101103

.658

.520

.014

.157

*indicates significance at the 0.05 level **indicates significance at the 0.01 level

CVLT-II List A IFR – Immediate Free Recall Trials 1–5; CVLT-II List A SDFR – Short-Delay Free Recall; CVLT-II List A LDFR – Long-Delay Free Recall; CVLT-II List A LDR – Long-Delay Yes/No Recognition; CVLT-II List A LDR FPE – Long-Delay Recognition False Positive Errors; DIGIT SPAN F. – Digit Span Forward; DIGIT SPAN B. – Digit Span Backward; D-KEFS TMT:VS – Visual Scanning; D-KEFS TMT: NS-Number Sequencing; D-KEFS TMT: LS – Letter Sequencing; D-KEFS TMT: NLS – Number-Letter Switching; D-KEFS TMT: MS – Motor Speed; D-KEFS VFT: LF – Letter Fluency; D-KEFS VFT: CF – Category Fluency; D-KEFS VFT: CS – Category Switching.

Treatment-Control Groups differences

To dissect further the univariate F tests main effects analyses discussed above, a series ofsimple contrasts comparisons across the treatment and control groups were carried out separately at T2 and T3 respectively. At T2 a evaluations significant difference was observed in the scores of CVLT-II long delay recognition memory task between control (M=15.15; SE=.15) and treatment (M=15.79; SE=.14) groups F(25)=7.190, p=.010 at T2. The observed power of this significant difference represents a large-sized effect (Table 4). A significant difference was also noticed in verbal cognitive flexibility performance, such as D-KEFS verbal fluency category switching task, between the control (M=10.83; SE=.66) and treatment (M=12.64; SE=.62) groups F(25)=4.065, p=.050 at T2. The observed power of this significant difference represents a medium-sized effect (Table 4). A significant difference was observed in sustained attention task, such as STROOP TEST OFF, between the control (M=78.75; SE=3.20) and treatment (M=87.53; SE=3.01) groups F(25)=4.065, p=.050 at T2. The observed power of this significant difference represents a medium-sized effect (Table 4). Furthermore, it seems to be a trend of higher performance for the treatment group compared to the control group in retrieving words in a memory task such as CVLT-II Immediate Free Recall (e.g. CVLT-II List A/B IFR). Although this difference represents a medium-sized effect, it does not reach statistical significance (p < .05).

Table 4. Pairwise comparisons between groups T2.

Cognitive variables

Control group

Treatment group

Pairwise comparison

M (SE)

M (SE)

p

F

Partial Eta Squared

Observed Power

CVLT-II List A/B IFR

52.73 (2.15)

58.04 (2.01)

.078

3.254

.67

.423

CVLT-II List A/B SDFR

10.50 (.67)

11.84 (.63)

.154

2.097

.045

.294

CVLT-II List A/B LDFR

10.97 (.71)

12.36 (.67)

.158

2.062

.044

.290

CVLT-II List A/B LDR

15.15 (.15)

15.79 (.14)

.010*

7.190

.138

.747

CVLT-II List A/B LDR FPE

3.73 (.92)

1.70 (.87)

.113

2.616

.113

.353

DIGIT SPAN F.

6.59 (.23)

6.72 (.22)

.690

.161

.004

.068

DIGIT SPAN B.

5.09 (.31)

5.16 (.29)

.870

.027

.001

.053

TOTAL DIGIT SPAN

11.64 (.45)

11.90 (.42)

.693

.158

.004

.068

D-KEFS VFT: LF

41.00 (2.31)

44.80 (2.16)

.236

1.445

.031

.218

D-KEFS VFT: CF

40.46 (1.82)

39.92 (1.71)

.831

.046

.001

.055

D-KEFS VFT: CS

10.83 (.66)

12.64 (.62)

.050*

4.065

.083

.505

D-KEFS TMT: VS

23.49 (1.22)

23.20 (1.15)

.865

.029

.001

.053

D-KEFS TMT: NS

34.23 (2.35)

36.40 (2.21)

.503

.455

.010

.101

D-KEFS TMT: LS

37.99 (3.63)

37.23 (3.41)

.880

.023

.001

.053

D-KEFS TMT: NLS

93.64 (7.92)

86.77 (7.43)

.530

.400

.009

.095

D-KEFS TMT: MS

27.31 (1.85)

25.17 (1.74)

.403

.713

.016

.131

STROOP TEST OFF

78.75 (3.20)

87.53 (3.01)

.050*

4.002

.082

.499

STROOP TEST ON

96.08 (4.50)

104.7 (4.22)

.169

1.952

.042

.277

*indicates significance at the 0.05 level

VLT-II List A IFR – Immediate Free Recall Trials 1–5; CVLT-II List A SDFR – Short-Delay Free Recall; CVLT-II List A LDFR – Long-Delay Free Recall; CVLT-II List A LDR – Long-Delay Yes/No Recognition; CVLT-II List A LDR FPE – Long-Delay Recognition False Positive Errors; DIGIT SPAN F. – Digit Span Forward; DIGIT SPAN B. – Digit Span Backward; D-KEFS TMT:VS – Visual Scanning; D-KEFS TMT: NS-Number Sequencing; D-KEFS TMT: LS – Letter Sequencing; D-KEFS TMT: NLS – Number-Letter Switching; D-KEFS TMT: MS – Motor Speed; D-KEFS VFT: LF – Letter Fluency; D-KEFS VFT: CF – Category Fluency; D-KEFS VFT: CS – Category Switching.

At T3 significant differences between groups were observed in the scores of CVLT-II immediate free recall memory task F(25)=8.545, p=.005, CVLT-II short delay free recall F(25)=15.690, p=.000, and CVLT-II long delay free recall task F(25)=13.007, p=.001. The number of words recalled by the treatment group is higher compared to controls and the observed power of these significant differences represents a large- sized effect (Table 5). A significant difference between groups at T3 was also noticed in the scores of working memory task (i.e. Digit Span Backward) F(25)=5.700, p = .112. The number of digits repeated by the participants of the treatment group is higher compared to controls and the observed power of this significant difference represents a large-sized effect (Table 5). Similarly, a significant difference between groups at T3 was also noticed in a verbal task that requires a certain amount of cognitive flexibility (i.e. D-KEFS verbal fluency category switching task) F(25)=7.032, p=.011. In this task the participants of the treatment group generate a higher number of words compared to controls and the observed power of this significant difference represents a large- sized effect (Table 5).

Table 5. Pairwise comparisons between groups T3.

Cognitive variables

Control group

Treatment group

Pairwise comparison

M (SE)

M (SE)

p

F

Partial Eta Squared

Observed Power

CVLT-II List A/B IFR

51.94 (2.43)

61.68 (2.27)

.005**

8.545

.160

.816

CVLT-II List A/B SDFR

9.46 (.65)

12.96 (.61)

.000**

15.690

.259

.972

CVLT-II List A/B LDFR

10.18 (.63)

13.32 (.60)

.001**

13.007

.224

.942

CVLT-II List A/B LDR

15.20 (.22)

15.29 (.20)

.566

.334

.007

.087

CVLT-II List A/B LDR FPE

2.42 (.47)

1.24 (.44)

.075

3.325

.069

.430

DIGIT SPAN F.

6.64 (.22)

6.84 (.21)

.505

.451

.010

.101

DIGIT SPAN B.

5.27 (.25)

6.08 (.23)

.021*

5.700

.112

.647

TOTAL DIGIT SPAN

11.96 (.41)

12.92 (.39)

.093

2.943

.061

.389

D-KEFS VFT: LF

40.91 (2.48)

49.20 (2.32)

.019*

5.952

.117

.665

D-KEFS VFT: CF

39.18 (1.60)

39.40 (1.50)

.921

.010

.000

.051

D-KEFS VFT: CS

10.41 (.70)

12.76 (.61)

.011*

7.032

.135

.737

D-KEFS TMT: VS

25.23 (1.24)

22.64 (1.17)

.135

2.311

.049

.319

D-KEFS TMT: NS

35.96 (2.64)

32.32 (2.48)

.321

1.009

.022

.166

D-KEFS TMT: LS

39.51 (2.70)

33.01 (2.53)

.086

3.081

.064

.404

D-KEFS TMT: NLS

100.22 (7.85)

81.12 (7.37)

.083

3.150

.065

.412

D-KEFS TMT: MS

26.36 (1.70)

24.70 (1.60)

.475

.520

.011

.109

STROOP TEST OFF

78.75 (3.20)

83.43 (2.83)

.246

1.382

.030

.210

STROOP TEST ON

93.70 (4.17)

97.19 (3.92)

.541

.380

.008

.093

*indicates significance at the 0.05 level **indicates significance at the 0.01 level

VLT-II List A IFR – Immediate Free Recall Trials 1–5; CVLT-II List A SDFR – Short-Delay Free Recall; CVLT-II List A LDFR – Long-Delay Free Recall; CVLT-II List A LDR – Long-Delay Yes/No Recognition; CVLT-II List A LDR FPE – Long-Delay Recognition False Positive Errors; DIGIT SPAN F. – Digit Span Forward; DIGIT SPAN B. – Digit Span Backward; D-KEFS TMT:VS – Visual Scanning; D-KEFS TMT: NS-Number Sequencing; D-KEFS TMT: LS – Letter Sequencing; D-KEFS TMT: NLS – Number-Letter Switching; D-KEFS TMT: MS – Motor Speed; D-KEFS VFT: LF – Letter Fluency; D-KEFS VFT: CF – Category Fluency; D-KEFS VFT: CS – Category Switching.

Furthermore, it seems to be a trend of higher performance for the treatment group compared to the control group in tasks such as long-delay memory recognition (e.g. CVLT-II List A/B LDR FPE), working memory (e.g. Total Digit Span), visual cognitive flexibility (e.g. D-KEFS TMT: LS) and visual processing speed (e.g. D-KEFS TMT: NLS), but did not reach statistical significance (p < .05).

Descriptive Trend analysis across groups

For exploratory purposes the descriptive linear trends over the 3 time periods (T1, T2 and T3) are reported in (Figures 2, 3 and 4). The figures 2 and 3 show the upwards increase in the estimated marginal means for “CVLT Long Delay Memory Recall” (i.e. episodic memory) and “D-KEFS VF Category Switching” (i.e. cognitive flexibility) between the treatment group versus the control group. The latter one instead depicts the downward and expected linear trend of “D-KEFS TMT Number-Letter Switching” (i.e. cognitive flexibility). Such descriptive trends (Table 6) mirror various results in the dissected MANOVA pairwise comparisons across the groups and time windows. Clearly more research is needed to further understand potential clinical impact of such potential trends. Nevertheless, such trends are encouraging and require further research in the near future. Such trends, if present could be highly relevant to verify the magnitude of improvement across different time periods and adequate clinical design tailored to such processes.

Table 6. Linear trend analysis results of the cognitive performance in the treatment group.

Cognitive variables

Treatment group (n = 25)

T1 M (SD)

T2 M (SD)

T3 M (SD)

F

p

ɳp2

Power

CVLT-II List A/B IFR

55.28 (2.26)

58.04 (2.01)

61.68 (2.27)

15.23 (1, 24)

.001**

.388

.963

CVLT-II List A/B SDFR

11.92 (.74)

11.84 (.63)

12.96 (.61)

3.84 (1, 24)

.062

.138

.469

CVLT-II List A/B LDFR

11.72 (.59)

12.36 (.67)

13.32 (.60)

17.45 (1, 24)

.000**

.421

.980

CVLT-II List A/B LDR

15.13 (.16)

15.79 (.14)

15.29 (.20)

.775 (1, 24)

.388

.031

.135

CVLT-II List A/B LDR FPE

1.25 (.43)

1.70 (.87)

1.24 (.44)

.002 (1, 24)

.962

.000

.050

DIGIT SPAN F.

6.80 (.21)

6.72 (.22)

6.84 (.21)

.033 (1, 24)

.857

.001

.054

DIGIT SPAN B.

5.70 (.26)

5.16 (.29)

6.08 (.23)

2.087 (1. 24)

.161

.080

.284

TOTAL DIGIT SPAN

12.48 (.40)

11.90 (.42)

12.92 (.39)

1.160 (1, 24)

.292

.046

.179

D-KEFS VFT: LF

25.96 (1.26)

44.80 (2.16)

49.20 (2.32)

7.03 (1, 24)

.014*

.227

.721

D-KEFS VFT: CF

38.80 (3.36)

39.92 (1.71)

39.40 (1.50)

.306 (1, 24)

.585

.013

.083

D-KEFS VFT: CS

37.92 (3.53)

12.64 (.62)

12.76 (.61)

3.56 (1, 24)

.071

.129

.441

D-KEFS TMT: VS

98.24 (10.15)

23.20 (1.15)

22.64 (1.17)

8.90 (1, 24)

.006*

.271

.817

D-KEFS TMT: NS

31.03 (2.18)

36.40 (2.21)

32.32 (2.48)

3.45 (1, 24)

.075

.126

.431

D-KEFS TMT: LS

44.64 (1.99)

37.23 (3.41)

33.01 (2.53)

3.96 (1,24)

.058

.142

481

D-KEFS TMT: NLS

38.90 (1.71)

86.77 (7.43)

81.12 (7.37)

4.88 (1,24)

.037*

.129

.564

D-KEFS TMT: MS

11.36 (.70)

25.17 (1.74)

24.70 (1.60)

7.66 (1,24)

.011*

.242

.757

STROOP TEST OFF

85.75 (3.21)

87.53 (3.01)

83.43 (2.83)

.416 (1,24)

.525

.017

.095

STROOP TEST ON

103.63 (4.03)

104.7 (4.22)

97.19 (3.92)

2.65 (1,24)

.116

.100

.347

*indicates significance at the 0.05 level **indicates significance at the 0.01 level

VLT-II List A IFR – Immediate Free Recall Trials 1–5; CVLT-II List A SDFR – Short-Delay Free Recall; CVLT-II List A LDFR – Long-Delay Free Recall; CVLT-II List A LDR – Long-Delay Yes/No Recognition; CVLT-II List A LDR FPE – Long-Delay Recognition False Positive Errors; DIGIT SPAN F. – Digit Span Forward; DIGIT SPAN B. – Digit Span Backward; D-KEFS TMT:VS – Visual Scanning; D-KEFS TMT: NS-Number Sequencing; D-KEFS TMT: LS – Letter Sequencing; D-KEFS TMT: NLS – Number-Letter Switching; D-KEFS TMT: MS – Motor Speed; D-KEFS VFT: LF – Letter Fluency; D-KEFS VFT: CF – Category Fluency; D-KEFS VFT: CS – Category Switching.

ASMHS-2019_Brian R. Christie_F2

Figure 2. Linear trend analysis. Long-delay memory recall measured with CVLT-II List A/B Long-Delay.

ASMHS-2019_Brian R. Christie_F3

Figure 3. Linear trend analysis. Verbal cognitive flexibility measured with D-KEFS Verbal Fluency Test: Category Switching.

ASMHS-2019_Brian R. Christie_F4

Figure 4. Linear trend analysis. Visual cognitive flexibility measured with D-KEFS Trail Making Test: Number-Letter Switching.

Perceptual-cognitive training (PCT) performance analyses

A visual inspection of the PCT data suggested that the treatment group showed improvements in performance across sessions (Figure 3). To affirm this, for example, the PCT thresholds showed an apparent logarithmic trend, characteristic of a good learning curve (R2 = .92) [37]. Further, a one-way (Time: Session 1 to Session 14) RM ANOVA was used to statistically analyse PCT performance. This analysis revealed a significant change in performance F(1, 13) = 49.95, p = .000 from Session 1 to Session 14, corroborating the significant presence of a trend across the sessions (Figure 5).

ASMHS-2019_Brian R. Christie_F5

Figure 5. Average speed threshold scores with PCT from the treatment group participants (n= 25). Speed thresholds are plotted for subjects in the treatment group. Subjects received two training sessions a week over a 7 week period, for a total of 14 sessions. Note how subjects show a marked improvement in performance after session 2 that persists for the duration of the training period.

Relationship between PCT performance and enhancement in cognitive functioning in the treatment group

Finally, a series of stepwise regression were used to verify if PCT scores predicted cognitive performance for the treatment group. Results showed that PCT scores predicted increasing performance in Digit Span Backward task F(1, 23) = 17.429, p = .000b, with an R2 of .442. Further, results revealed a negative relationship between the performance in the last PCT session performance and in the D-KEFS TMT Visual Scanning (r = – .366; p = .036) and D-KEFS TMT Number Sequencing (r = – .364; p = .037). Similarly, a positive relationship was found between performance in the last PCT session and D-KEFS Letter Fluency (r = .387; p = .028) and CVLT-II Long Delay Recall (r = .391; p = .027) (Table 7).

Table 7. Bivariate correlation between the cognitive tasks in the control group.

Cognitive variables

1

2

3

4

5

6

7

8

9

10

11

12

13

1

1

2

r = .570**

1

3

r = .329

r = .526

1

4

r = -.620**

r = -.578**

r = -.308

1

5

r = -.438*

r = -.372

r = -.120

r = -.120

1

6

r = -.153

r = .005

r = .279

r = .284

r = .621**

1

7

r = -.393

r = -.208

r = -.102

r = .166

r = .557**

r = .400

1

8

r = -.399

r = -.358

r = -.251

r = .403

r = .294

r = .454*

r = .551*

1

9

r = -.177

r = -180

r = -.145

r = .195

r = .379

r = .217

r = .296

r = .366

1

10

r = .308

r = -.011

r = .050

r = -.389

r = -.363

r = -.271

r = -.332

r = -.244

r = -.022

1

11

r = .229

r = .203

r = .151

r = -.202

r = -.384

r = -.263

r = .033

r = -.216

r = -.339

r = .332

1

12

r = .234

r = -.228

r = -.148

r = .157

r = .051

r = .209

r = -.091

r = .115

r = .114

r = .033

r = .046

1

13

r = -.034

r = -.207

r = .082

r = .051

r = .128

r = .070

r = .080

r = .418

r = .457*

r = -.382

r = -.359

r = .418

1

*indicates significance at the 0.05 level **indicates significance at the 0.01 level

1. CVLT-II List A Immediate Free Recall Trials 1–5; 2. CVLT-II List A Long-Delay Free Recall; 3. CVLT-II List A Long-Delay Yes/No Recognition; 4. CVLT-II List A Long-Delay Yes/No Recognition False-Positives; 5. D-KEFS TMT: Visual Scanning; 6. D-KEFS TMT: Number Sequencing; 7. D-KEFS TMT: Letter Sequencing; 8. D-KEFS TMT: Number-Letter Switching; 9. D-KEFS TMT: Motor Speed; 10. D-KEFS VFT: Letter Fluency; 11. D-KEFS VFT: Category Fluency; 12. D-KEFS VFT: Category Switching; 13. Encephalapp Stroop Test: Stroop On

Discussion

The purpose of this study was to examine whether older adults with SCD would benefit from Perceptual-Cognitive Training. The results indicate a significant difference between treatment and control groups in tasks of episodic memory, working memory, cognitive flexibility and processing speed. After the 14 sessions of brain stimulation with PCT (T2) the treatment group performed better compared to controls in a task of episodic memory, such as retrieving the previous encoded abstract wordlist after a long delay (CVLT-II List A/B LDFR), and in a task of cognitive flexibility, such as generating words by alternating between two categories (D-KEFS VF CS). Furthermore, a trend of higher performance was noticed in the treatment group in another task of episodic memory, immediate free recall CVLT-II List A/B IFR).

One month follow-up after the Perceptual-Cognitive Training (T3), the benefits observed for the participants of the treatment group in retrieving words after a long delay were maintained and were significantly higher compared to controls. Furthermore, a significant major effect between groups was observed in others episodic memory tasks such as immediate free recall, (CVLT-II List A/B IFR) and short delay recall (CVLT-II List A/B SDFR). A significant major effect after a month follow-up was observed in treatment participants in a verbal cognitive flexibility task (D-KEFS VF CS) and a trend of higher performance was noticed in a visual cognitive flexibility task (D-KEFS TMT: NLS). Furthermore, the treatment group performed significantly better in a working memory task, such as repeating digits backward (Digit Span Backward) and showed a trend of better scores in Total Digit Span. Similarly, the treatment group performed better compared to controls in tasks of processing speed (D-KEFS TMT: LS; D-KEFS VF LF). Moreover, a trend of higher performance was noticed in the treatment group compared to controls in the accuracy and the number of words recognized from a bigger list after a long delay (CVLT-II List A/B LDR FPE). Specifically, the participants of the control group reported a greater number of false-positive errors after seven and twelve weeks of follow-up.

Previous studies have demonstrated that computerized cognitive training programs serve as powerful tools to enhance cognition in healthy older adults [18, 22, 23, 30]. The current study expands on these findings by showing additional benefits of computer training on cognition in older adults with subjective cognitive decline. Similar benefits in memory, processing speed, working memory and cognitive flexibility were found in previous studies on PCT intervention [33, 37, 60 ] in different populations (e.g. healthy young adults and students with neurodevelopmental conditions, healthy adults and adults with concussions, healthy older adults and older adults with subjective memory complaints). For example, a case study on an 80-year-old man with memory complaints, that underwent 32 sessions of training with PCT, showed improvements in working memory, episodic memory, processing speed, as well as reduction in cognitive complaints with positive impact on quality of life. Other work from our laboratory on healthy older adults indicated improvements in cognitive flexibility after just 7 sessions (i.e. 21 trials) of PCT [34]. Parsons et al., [33] found that students who performed 10 sessions of PCT improved in performance as investigated with standardized cognitive assessments of working memory and attention on visual information. Tullo et al., [37] observed that performing 15 sessions of PCT was associated with increased attentional abilities in students with neurodevelopmental conditions (e.g. Autism Spectrum Disorder, Attention-Deficit/Hyperactivity Disorder, Intellectual Disability, Specific Learning Disorder. Similarly, etc.). Vartanian and colleagues [60] trained military personal with the PCT and observed improved performances on working memory task compared to no improvements from participants who underwent PCT training.

Considerable evidence [6, 61, 62,], from both behavioral and neurobiological sources, suggests that age-related memory declines might be linked to deficits in executive functioning (EF), including inhibitory functions, working memory [63,64], and cognitive flexibility [4, 64, 65,]. Memory tasks involve the organization of new information, selective attention for the information that has to be encoded, the suppression of unnecessary information, and at times the maintenance and shifting of cognitive sets, so this is not surprising in many ways. Furthermore, in order to encode and retrieve new information cognitive efficiency relies on processing speed and working memory. Evidence suggests that slow processing speed or working memory difficulties [9, 66] in older adults impact on the accuracy of encoding new information and on the retrieval of it later on [9, 66]. This pattern of deficits in executive tasks associated with episodic memory decline is consistent with the view that underlying cognitive functions depend on multiple-interacting neural networks, including the medial temporal memory complex and prefrontal cortical executive system [67, 68]. Therefore, any memory enhancement obtained after PCT may be in part due to improvements in processing speed, working memory (i.e. brief sustained attention), and cognitive flexibility. The treatment group became significantly faster in processing new information, such as word production or connecting letters with distractor numbers presented on the page, faster in tasks that require certain mental flexibility, and better in encoding and retrieving an abstract wordlist after a short and long delay. The enhancement in these cognitive tasks was also associated with a significant correlation between improved processing speed and the performance in memory tasks. In contrast, we observed that the control group was slower in processing speed and retrieved fewer words compared to the treatment group. Further, no significant relationship between processing speed and memory task performance was observed in the control group. These findings are interesting and require further replication, possibly with the inclusion of a second control group of healthy older adults.

Consistent with some imaging studies, episodic memory functioning is the most robust neuropsychological predictor of dementia [69–71]. One recent study found that performance for immediate versus delayed episodic memory recall varies according to the temporal stage of disease progression [30, 72]. Contrary to the common view that delayed memory recall is the most sensitive measure of early dementia, Bilgel et al. found that immediate verbal recall measures in the CVLT were the first to decline in preclinical dementia, followed by delayed verbal recall on the same test closer to a diagnosis of mild cognitive impairment. Although research on PCT does not typically result in generalization of learning to daily living tasks in older adults [33, 37, 54, 73], an interesting result observed in our study is the transfer effect between PCT and episodic memory tasks. For example, the older adults with SCD from the treatment group showed a significant enhancement in episodic memory tasks such as learning abstract word lists and retrieving words after a short and a long delay period (e.g. 30 min). Although the benefits on memory tasks have no overlap with the trained cognitive functions of PCT and may thus be considered a far transfer [74, 75], this transfer was characterized by a medium-large effect size and a power above .80. This reflects the effectiveness of PCT, though little is known about the transfer effect between the PCT and memory performance in older adult with SMCs. That being said, PCT intervention may play a significant role in dementia prevention or cognitive decline but further research is needed to ensure reliability and validity. The concept of adult neurogenesis provides an interesting potential mechanism for the cognitive benefits observed in the treatment group, particularly since benefits were still observed in the follow-up testing a month later. Here the hypothesis would be that the PCT provides enough cognitive enrichment to enhance adult neurogenesis. This is similar to the effect observed in animals that exercise or are in enriched environments [76], which rely on increases in neurotrophin levels [77, 78]. Indeed, learning behaviours that involve the hippocampus have been shown to impact adult neurogenesis in animal models [79].

An increasing number of studies have examined how environmental and/or behavioural factors can modulate neurogenesis and subsequently effect hippocampal-dependent learning and memory in humans [80]. Indeed, exercise has even been shown to be beneficial for individuals with subjective memory complaints, enhancing medial temporal lobe thickness [81]. The time course for the increase in performance observed one month after testing corresponds well with the time course for new neurons generated in response to the PCT training to be incorporated into, and enhance, existing networks [82]. In addition, an increased activation of the neural structures and circuits was observed during PCT training in a recent fMRI investigation [34]. These neural areas are involved in executive function tasks. Thus it would be interesting for future studies to determine if PCT has the capacity to promote neuroplasticity, providing a mechanism through which it can enhance learning and memory processes [83].

A very positive aspect of the PCT intervention was the ability of older adults to be able to engage in this computerized training task, even if their performance was slower than in younger adult groups [84, 85]. The learning curve in our study indicates that PCT can be a good cognitive training tool for older populations. Moreover, as PCT involves an individualized dynamic and homeostatic adjustment of the training speed, the subjects found they could easily work with the program irrespective of their initial performance. Because each trial was based upon the participant’s performance in the prior trial, the software provided a continuous challenge that helped maintain a high level of engagement and motivation. Hence, participants can remain highly motivated to engage regularly in the training regimen. Therefore, the results should be replicated by further research on clinical older population to ensure reliability.

Limitations

The use of an inactive control group does not exclude the possibility that this empirical finding reflects a placebo effect [86], although, a greater significant difference in cognitive performance was observed between groups not only after PCT intervention but also at the second follow-up (T3), where both groups were on rest for 4 weeks (i.e. no intervention was administered). Therefore, the results should be replicated by further research to ensure reliability. A limitation of this study was the non-administration of the memory complaint rating scale (MAC-Q) after the PCT intervention (i.e. MAC-Q was only used to assess the inclusion/exclusion criteria of this study). In addition, research would benefit from using a quality of life questionnaire test to assess the transfer of these cognitive benefits on daily activities.

Conclusions

The current study demonstrated improved performance in older adults with SCD on measures of episodic memory, processing speed, working memory, and cognitive flexibility. The prolonged enhancement result observed over a month may hold promise for cognitive rehabilitation/neurogenesis, but it needs to be replicated to further support its validity, in both healthy samples and those with neurocognitive disorders or types of dementia. Further research is essential to examine structural neuroplasticity and transfer effects from the PCT to daily tasks. Taken together, the results of this study suggest that the PCT may be an effective tool for cognitive enhancement in preclinical and clinical populations of older adults.

Acknowledgement

We would like to thank all participants for their significant commitment to this study. Thank you also to the Christie Lab graduate and undergraduate students who assisted with data collection. A special thank you is reserved for Dr. Scott Hofer and the Institute on Aging and Lifelong Health. SM was supported by the Fondazione Banca del Monte di Lombardia for travel to Canada to conduct this research. The protocol can be accessed on Clinical.trials.gov with the following registry number: NCT03763344.

References

  1. WHO (2016) Ageing.
  2. Suzman R, Beard J (2011) Global Health and Aging. NIH Publication No 117737, 1: 273–277. https://doi.org/11-7737
  3. De Grey AD, Ames BN, Andersen JK, Bartke A, Campisi J et al. (2002) Time to talk SENS: critiquing the immutability of human aging. Ann N Y Acad Sci 959: 452–462.
  4. Kehagia AA, Murray GK, Robbins TW (2010) Learning and cognitive flexibility: Frontostriatal function and monoaminergic modulation. Current Opinion in Neurobiology 20: 199–204.
  5. Salthouse TA (2013) Effects of age and ability on components of cognitive change. Intelligence 41: 501–511.
  6. Salthouse TA, Atkinson TM, Berish DE (2003) Executive functioning as a potential mediator of age-related cognitive decline in normal adults. Journal of Experimental Psychology. General 132: 566–594.
  7. Dixon RA (2009) An epidemiological approach to cognitive health in aging. In Memory, Aging and the Brain: A Festschrift in Honour of Lars-Göran Nilsson. https://doi.org/10.4324/9780203866665
  8. Rabin LA, Smart CM, Amariglio RE (2017) Subjective Cognitive Decline in Preclinical Alzheimer’s disease. Annual Review of Clinical Psychology 13: 369–396.
  9. Goldberg E (2009) The new executive brain: Frontal lobes in a complex world. The New Executive Brain: Frontal Lobes in a Complex World. Journal of Economic Psychology 32: 688–690.
  10. Reuter-Lorenz PA, Park DC (2010) Human Neuroscience and the Aging Mind: A New Look at Old Problems. The Journals of Gerontology Series B: Psychological Sciences and Social Sciences 65B: 405–415.
  11. Craik FIM (2016) A functional account of age differences in memory. In Memory, Attention, and Aging: Selected Works of Fergus I. M. Craik, Routledge, New York Pg No: 340.
  12. Tulving E, Stevenson RJ, Boakes RA (2002) Episodic memory: from mind to brain\rA mnemonic theory of odor perception. Annu Rev Psychol
  13. McDowd JM, Shaw RJ (2000) Attention and aging: A functional perspective. In F. I. M. Craik & T. A. Salthouse (Eds.), The handbook of aging and cognition (p. 221–292). Lawrence Erlbaum Associates Publishers.
  14. Craik FIM, Byrd M (1982) Aging and Cognitive Deficits. In Aging and Cognitive Processes 191–211.
  15. Salthouse TA (1996) The Processing-Speed Theory of Adult Age Differences in Cognition. Psychological Review 103: 403–428.
  16. Hasher L, Zacks RT (1988) Working memory, comprehension, and aging: A review and a new view BT – The psychology of learning and motivation. The Psychology of Learning and Motivation 22: 193–225.
  17. Bherer L (2015) Cognitive plasticity in older adults: Effects of cognitive training and physical exercise. Annals of the New York Academy of Sciences 1337: 1–6.
  18. Kueider AM, Parisi JM, Gross AL, Rebok GW (2012) Computerized cognitive training with older adults: A systematic review. PLoS ONE 7.
  19. Straubmeier M, Behrndt EM, Seidl H, Özbe D, Luttenberger K et al. (2017) Non-Pharmacological Treatment in People With Cognitive Impairment. Deutsches Aerzteblatt Online 114: 815–821.
  20. Teixeira CVL, Gobbi LTB, Corazza DI, Stella F, Costa JLR et al. (2012) Non-pharmacological interventions on cognitive functions in older people with mild cognitive impairment (MCI). Archives of Gerontology and Geriatrics 54: 175–180.
  21. Garner J (2009) Conceptualizing the relations between executive functions and self-regulated learning. Journal of Psychology 143: 405–426.
  22. Hampstead BM, Sathian K, Phillips PA, Amaraneni A, Delaune WR et al. (2012) Mnemonic strategy training improves memory for object location associations in both healthy elderly and patients with amnestic mild cognitive impairment: A randomized, single-blind study. Neuropsychology 26: 385–399.
  23. Anguera JA, Boccanfuso J, Rintoul JL, Al-Hashimi O, Faraji F et al.  (2013) Video game training enhances cognitive control in older adults. Nature 501: 97–101.
  24. Berry AS, Zanto TP, Clapp WC, Hardy JL, Delahunt PB et al. (2010) The influence of perceptual training on working memory in older adults. PLoS ONE 5: 1–8.
  25. Bherer L, Kramer AF, Peterson MS, Colcombe S, Erickson K et al. (2006) Testing the limits of cognitive plasticity in older adults: Application to attentional control. ActaPsychologica 123: 261–278.
  26. Edwards JD, Wadley VG, Vance DE, Wood K, Roenker DL (2013) The impact of speed of processing training on cognitive and everyday performance. Aging & Mental Health 9: 37–41.
  27. Mozolic JL, Long AB, Morgan AR, Rawley-Payne M, Laurienti PJ (2011) A cognitive training intervention improves modality-specific attention in a randomized controlled trial of healthy older adults. Neurobiology of Aging 32: 655–668.
  28. Ball K, Berch DB, Helmers KF, Jobe JB, Leveck MD et al. (2002) Effects of cognitive training interventions with older adults: A randomized controlled trial. Journal of the American Medical Association. 288: 2271–2281.
  29. Kramer AF, Larish JL, Weber TA, Bardell L (1999) Training for executive control: Task coordination strategies and aging. Attention and Performance.
  30. Mortamais M, Ash JA, Harrison J, Kaye J, Kramer J et al. (2017) Detecting cognitive changes in preclinical Alzheimer’s disease: A review of its feasibility. Alzheimer’s and Dementia 13: 468–492.
  31. Assed MM, de Carvalho MKHV, Rocca CC de A, Serafim A de P (2016) Memory training and benefits for quality of life in the elderly: A case report. Dementia & Neuropsychologia 10: 152–155.
  32. Cavanagh P, Alvarez GA (2005) Tracking multiple targets with multifocal attention. TRENDS in Cognitive Sciences 9: 349–354.
  33. Parsons B, Magill T, Boucher A, Zhang M, Zogbo K et al. (2016) Enhancing Cognitive Function Using Perceptual-Cognitive Training. Clinical EEG and Neuroscience 47: 37–47.
  34. Spaner CR, Musteata S, Kenny RA, Gawryluk JR, Hofer S et al. (2019) 3-Dimensional Multiple Object Tracking Training Can Enhance Selective Attention , Psychomotor Speed , and Cognitive Flexibility in Healthy Older Adults. Ageing Sci Ment Health Stud 3: 1–12.
  35. Bottari C, Dassa CM, Rainville C, Dutil ÉL (2009) The criterion-related validity of the IADL Profile with measures of executive functions, indices of trauma severity and sociodemographic characteristics. Brain Injury 23: 322–335.
  36. Corbin-Berrigan LA, Kowalski K, Faubert J, Christie B, Gagnon I (2018) Three-dimensional multiple object tracking in the pediatric population: the NeuroTracker and its promising role in the management of mild traumatic brain injury. NeuroReport 29: 559–563.
  37. Tullo D, Guy J, Faubert J, Bertone A (2018) Training with a three-dimensional multiple object-tracking (3D-MOT) paradigm improves attention in students with a neurodevelopmental condition: a randomized controlled trial. Developmental Science 21.
  38. Folstein MF, Folstein SE, McHugh PR (1975) “Mini-mental state”. A practical method for grading the cognitive state of patients for the clinician. Journal of Psychiatric Research 12: 189–198.
  39. Mahoney FI, Barthel DW (1965) Functional Evaluation: the Barthel Index. Maryland State Medical Journal 14: 61–65.
  40. Lawton MP, Brody EM (1969) Assessment of older people: self-maintaining and instrumental activities of daily living. The Gerontologist 9: 179–186.
  41. Crook III TH, Feher EP, Larrabee GJ (1992) Assessment of memory complaint in age-associated memory impairment: the MAC-Q. International Psychogeriatrics 4: 165–176.
  42. Yesavage Ja, Brink TL, Rose TL, Lum O, Huang V et al. (1983) Development and validation of a geriatric depression screening scale: a preliminary report. Journal of Psychiatric Research 17: 37–49.
  43. Pachana NA, Byrne GJ, Siddle H, Koloski N, Harley E et al. (2007) Development and validation of the Geriatric Anxiety Inventory. International Psychogeriatrics 19: 103–114.
  44. Zokaei N, Mackellar C, Čepukaitytė G, Patai EZ, Nobre AC (2017) Cognitive training in the elderly: Bottlenecks and new avenues. Journal of Cognitive Neuroscience 29: 1473–1482.
  45. Cooper DB, Epker M, Lacritz L, Weine M, Rosenberg RN et al. (2001) Effects of practice on category fluency in Alzheimer’s disease. The Clinical Neuropsychologist 15: 125–128.
  46. Cooper DB, Lacritz LH, Weiner MF, Rosenberg RN, Cullum CM (2004) Category fluency in mild cognitive impairment: Reduced effect of practice in test-retest conditions. Alzheimer Disease and Associated Disorders 18: 120–122.
  47. Woods SP, Delis DC, Scott JC, Kramer JH, Holdnack JA (2006) The California Verbal Learning Test – second edition: Test-retest reliability, practice effects, and reliable change indices for the standard and alternate forms. Archives of Clinical Neuropsychology 21: 413–420.
  48. Delis DC, Kramer JH, Kaplan E, Ober BA (2000) California Verbal Learning Test.  2nd edn. Adult version. Manual. Test.
  49. Delis, D, Kaplan E, Kramer J (2001) Delis-Kaplan executive function system (D-KEFS). Canadian Journal of School Psychology 20: 117–128.
  50. Allampati S, Duarte-Rojo A, Thacker LR, Patidar KR, White MB et al. (2016) Diagnosis of Minimal Hepatic Encephalopathy Using Stroop EncephalApp: A Multicenter US-Based, Norm-Based Study. American Journal of Gastroenterology 111: 78–86.
  51. Bajaj JS, Heuman DM, Sterling RK, Sanyal AJ, Siddiqui M et al. (2015) Validation of EncephalApp, Smartphone-Based Stroop Test, for the Diagnosis of Covert Hepatic Encephalopathy. Clinical Gastroenterology and Hepatology 13: 1828–1835.
  52. Stroop JR (1935) Stroop color word test. J. Exp. Physiol
  53. Faubert J (2002) Visual perception and aging. Can J Exp Psychol 56: 164–176.
  54. Faubert J, Sidebottom L (2013) NeuroTracker System: Its role for perceptual-cognitive training of athletes and its potential impact on injury reductions and concussion management in sports. Journal of Clinical Sports
  55. Faubert J, Sidebottom L (2012) Perceptual-Cognitive Training of Athletes. Journal of Clinical Sport Psychology 6: 85–102.
  56. Van Merriënboer JJG, Sweller J (2010) Cognitive load theory in health professional education: design principles and strategies. Medical Education 44:  85–93.
  57. Hairston WD, Maldjian JA (2009) An adaptive staircase procedure for the E-Prime programming environment. Computer Methods and Programs in Biomedicine 93: 104–108.
  58. Field A (2013) Moderation, mediation and more regression. Discovering Statistic Using SPSS 4th (edn).
  59. Yantis S (2002) Stevens’ handbook of experimental psychology. Stephens’ Handbook of Experimental Psychology. https://doi.org/10.1026//1618-3169.50.2.155.
  60. Vartanian O, Coady L, Blackler K (2016) 3D multiple object tracking boosts working memory span: Implications for cognitive training in military populations. Military Psychology 28: 353–360.
  61. Glisky EL, Polster MR, Routhieaux BC (1995) Double Dissociation Between Item and Source Memory. Neuropsychology 9: 229–235.
  62. Hedden T, Gabrieli JDE (2004) Insights into the ageing mind: A view from cognitive neuroscience. Nature Reviews Neuroscience 5: 87–96.
  63. Johnson MK, Reeder Ja, Raye CL, Mitchell KJ (2002) Second thoughts versus second looks: an age-related deficit in reflectively refreshing just-activated information. Psychological Science : A Journal of the American Psychological Society / APS 13: 64–67.
  64. Mitchell KJ, Johnson MK, Raye CL, Greene EJ (2004) Prefrontal cortex activity associated with source monitoring in a working memory task. Journal of Cognitive Neuroscience 16: 921–934.
  65. Spiro RJ, Collins BP, Thota JJ, Feltovich PJ (2003) Cognitive Flexibility Theory: Hypermedia for Complex Learning, Adaptive Knowledge Application, and Experience Acceleration. Educational Technology 43: 5–10.
  66. Salthouse TA (1994) Aging associations: influence of speed on adult age differences in associative learning. Journal of Experimental Psychology Learning Memory and Cognition 20: 1486–1503.
  67. Abutalebi J, Green DW (2016) Neuroimaging of language control in bilinguals: Neural adaptation and reserve. Bilingualism 19: 689–698.
  68. Craik FIM, Bialystok E (2006) Cognition through the lifespan: Mechanisms of change. Trends in Cognitive Sciences 10: 131–138.
  69. Beck IR, Gagneux-Zurbriggen A, Berres M, Taylor KI, Monsch AU (2012) Comparison of verbal episodic memory measures: Consortium to establish a registry for alzheimer’s disease-neuropsychological assessment battery (CERAD-NAB) versus California verbal learning test (CVLT). Archives of Clinical Neuropsychology 27: 510–519.
  70. Hodges JR, Graham KS (2001) Episodic memory: insights from semantic dementia. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 356: 1423–1434.
  71. Peter J, Scheef L, Abdulkadir A, Boecker H, Heneka M et al. (2014) Gray matter atrophy pattern in elderly with subjective memory impairment. Alzheimer’s and Dementia 10: 99–108.
  72. Bilgel M, An Y, Lang A, Prince J, Ferrucci L et al. (2014) Trajectories of Alzheimer disease-related cognitive measures in a longitudinal sample. Alzheimer’s and Dementia 10: 735–742.
  73. Legault I, Allard R, Faubert J (2013) Healthy older observers show equivalent perceptual-cognitive training benefits to young adults for multiple object tracking. Frontiers in Psychology 4: 323.
  74. Klahr D, Chen Z (2011) Finding One’s place in transfer space. Child Development Perspectives 5: 196–204.
  75. Zelinski EM (2009) Far transfer in cognitive training of older adults. Restorative Neurology and Neuroscience 27: 455–471.
  76. Patten AR, Yau SY, Fontaine CJ, Meconi A, Wortman RC et al. (2015) The Benefits of Exercise on Structural and Functional Plasticity in the Rodent Hippocampus of Different Disease Models. Brain Plasticity: 1: 97–127.
  77. Boehme F, Gil-Mohapel J, Cox A, Patten A, Giles E et al. (2011) Voluntary exercise induces adult hippocampal neurogenesis and BDNF expression in a rodent model of fetal alcohol spectrum disorders. European Journal of Neuroscience 33: 1799–1811.
  78. Farmer J, Zhao X, van Praag H, Wodtke K, Gage FH et al. (2004) Effects of voluntary exercise on synaptic plasticity and gene expression in the dentate gyrus of adult male Sprague-Dawley rats in vivo. Neuroscience 124: 71–79.
  79. Epp JR, Chow C, Galea LAM (2013) Hippocampus-dependent learning influences hippocampal neurogenesis. Frontiers in Neuroscience 7: 57.
  80. Bruel-Jungerman E, Rampon C, Laroche S (2007) Adult hippocampal neurogenesis, synaptic plasticity and memory: facts and hypotheses. Reviews in the Neurosciences 18: 93–114.
  81. Siddarth P, Rahi B, Emerson ND, Burggren AC, Miller KJ et al. (2018) Physical activity and hippocampal sub-region structure in older adults with memory complaints. Journal of Alzheimer’s Disease 61: 1089–1096.
  82. Van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD et al. (2002) Functional neurogenesis in the adult hippocampus. Nature 415: 1030–1034.
  83. Sörman DE, Rönnlund M, Sundström A, Adolfsson R, Nilsson LG (2015) Social relationships and risk of dementia: A population-based study. International Psychogeriatrics 27: 1391–1399.
  84. Rush B, Barch D, Braver T (2006) Accounting for cognitive aging: Context processing, inhibition or processing speed? Aging, Neuropsychology, and Cognition 13: 588–610.
  85. Trick LM, Perl T, Sethi N (2005) Age-related differences in multiple-object tracking. Journals of Gerontology – Series B Psychological Sciences and Social Sciences 60: 102–105.
  86. Boot WR, Simons DJ, Stothart C, Stutts C (2013) The Pervasive Problem With Placebos in Psychology: Why Active Control Groups Are Not Sufficient to Rule Out Placebo Effects. Perspectives on Psychological Science 8: 445–454.

Epidemiological, Clinical Characteristics and Treatment of the Pelvic Inflammatory Disease in a University Hospital

DOI: 10.31038/AWHC.2019265

Abstract

Introduction: Pelvic inflammatory disease (PID) is a syndrome characterized by an infection that usually ascends from the vagina and cervix into the upper genital tract. Chlamydia trachomatis is the predominant causal agent of this type of infection. In Mexico, the prevalence of C. trachomatis infection is still unknown although some reports suggest a frequency of 4 to 10% in the open population, 16% in patients with a history of sexually transmitted diseases and 21% in patients with a PID diagnosis. The objective of the present study was to analyze the epidemiological characteristics of the cases with Pelvic Inflammatory Disease diagnosis attended by the Gynecology and Obstetrics service of the University Hospital “Dr. José Eleuterio González”.

Materials and methods: A descriptive, retrospective and cross-sectional study was carried out; including patients aged 18 to 35 years with a diagnosis of Pelvic Inflammatory Disease attended by the Gynecology and Obstetrics Department of the University Hospital “Dr. José Eleuterio González” from January 1, 2014 to October 1, 2018. The variables evaluated were demographic characteristics, gyneco-obstetric history, risk factors, clinical presentation and the treatment they received.

Results: 221 patients with a diagnosis of Pelvic Inflammatory Disease were included. The average age was 33 ± 7 years. 73 patients (33%) until the time of interrogation had only had one sexual partner, 93 patients (42%) had had 2 to 3 sexual partners and 131 patients (59.28%) mentioned that the IUD was their contraceptive of choice. Regarding the clinical symptoms presented by the patients at the time of the interrogation, 214 patients (96.83%) mentioned presenting pain and 202 patients (91.4%) presented vaginal discharge. 139 patients (63%) received a double antibiotic scheme, 57 patients (26%) received a triple antibiotic scheme. Of the patients who received double treatment scheme, 38 patients (17.19%) were treated with cephalothin and metronidazole. Of the patients who received triple antibiotic scheme, 28 patients (12.67%) were treated with cephalothin; gentamicin and metronidazole. 84 patients underwent a surgical procedure.

Conclusions: With these results, we can conclude the epidemiological characteristics of the patients who are diagnosed with pelvic inflammatory disease. Knowing these characteristics, we can carry out programs to detect risk factors and prevent the incidence and recurrence of PID, in addition to find the treatment scheme that has worked best according to the characteristics of our population.

Keywords

Pelvic Inflammatory Disease; Epidemiology; Treatment; Clinical

Introduction

Pelvic inflammatory disease (PID) is a syndrome characterized by an infection that usually ascends from the vagina and cervix into the upper genital tract. This entity may appear at any time during the woman’s reproductive life, being the adolescence the stage with the highest risk of presentation [1]. It is estimated that 857,000 cases of PID cases are diagnosed annually in the United States, corresponding to an annual incidence rate of 17.2 per thousand women between 15 and 44 years. Over 100,000 women become infertile every year because of this entity and a large proportion of ectopic pregnancies are associated with previous events of PID [2]. Infertility and ectopic pregnancy are caused by tubal damage. The greater the severity and the number of episodes, the greatejr the probability that they appear. Chlamydia trachomatis is the predominant causal agent of this type of infection. Chronic pelvic pain in these patients is secondary to abdominal adhesions [3]. In Mexico, the prevalence of C. trachomatis infection is still unknown although some reports suggest a frequency of 4 to 10% in the open population, 16% in patients with a history of sexually transmitted diseases and 21% in patients with a PID diagnosis using laboratory tests such as cell culture, direct immunofluorescence and molecular techniques [4–7]. The objective of the present study was to analyze the epidemiological characteristics of the cases with Pelvic Inflammatory Disease diagnosis attended by the Gynecology and Obstetrics service of the University Hospital “Dr. José Eleuterio González”.

Materials and Methods

Study Design and Ethical Aspects

A descriptive, retrospective and cross-sectional study was carried out. The Ethics and Research Committee of the Faculty of Medicine and University Hospital of the U.A.N.L. granted previous authorization.

Study population

The information will be recorded in an Excel program database, the variables that will be evaluated are: demographic characteristics: age, educational level and marital status. Gyneco-obstetric history: gravity, parity, age of first sexual intercourse, contraceptive methods, sexually transmitted diseases. Risk factors such as sexually transmitted diseases, previous episodes of IPD, relation to menstruation and clinical presentation such as pain in hypogastrium, abnormal vaginal leucorrhea, fever, nausea and vomiting, pain during intercourse, cervical hemorrhage, pain to the mobilization of the cervix and the treatment they received.

Statistical Analysis

A univariate analysis will be carried out applying the measures of central tendency (mean and median), and measures of dispersion (standard deviation or range), as appropriate for parametric and non-parametric variables, in addition to proportions for qualitative variables. A bivariate analysis will also be done for qualitative variables, as appropriate. An X2 will be made for qualitative variables, and a Student’s T for quantitative variables, with a p <0.05 for statistical significance; in addition, OR and 95% CI will be determined when appropriate. All this supported by pre-designed Excel sheets. For the Software the statistical program EPI-Info 7 is used, which is a free-use program that does not require a license for its management.

Sample size calculation

A sample size calculation was made with a formula for estimating a proportion in an infinite population based on a prevalence of 28% of patients with PID who are evaluated in our setting; for a bilateral confidence of 95% and an accuracy of 10%, we require at least 143 study subjects.

Results

221 patients with a diagnosis of Pelvic Inflammatory Disease were included. The average age was 33 ± 7 years. For the analysis, the data was divided into the risk factors presented by the patients, the clinic presentation at the time of the interrogation and the treatment that was performed: pharmacological or surgical. Among the risk factors, we asked about comorbidities, finding that 160 patients (66%) did not present any comorbidity at the time of interrogation, 28 patients (10.5%) had type 2 Diabetes Mellitus and 12 patients (2.7%) presented Arterial Hypertension in their different stages. In addition, 21 patients (8.60%) presented various comorbidities that because they were presented only in one patient, were not significant with the disease under study (Figure 1).

AWHC-19-148-RODOLFO MORALES AVALOS_ Mexico_F1

Figure 1. Comorbidities. T2DM, Type 2 Diabetes Mellitus; HBP, High Blood Pressure.

Regarding the non-pathological history such as smoking, it was found that 44 patients (19.91%) were active smokers and 177 patients (80.09%) denied having smoked. For the gyneco-obstetric history, the number of sexual partners was interrogated. 73 patients (33%) until the time of the interrogation had only had one sexual partner, 93 patients (42%) had had 2 to 3 sexual partners, 25 patients (11%) had had 4 to 5 sexual partners, 8 patients (3.62%) had had 5 to 10 sexual partners and 5 patients (11.31%) had had more than 10 sexual partners. (Figure 2) It was asked whether, as a contraceptive method they used an intrauterine device (IUD), 131 patients (59.28%) mentioned that the IUD was their contraceptive of choice and 88 patients (39.82%) mentioned not using the IUD as a contraceptive (Figure 3).

AWHC-19-148-RODOLFO MORALES AVALOS_ Mexico_F2

Figure 2. Number of sexual partners

AWHC-19-148-RODOLFO MORALES AVALOS_ Mexico_F3

Figure 3. Use of Intrauterine Device (IUD)

The clinical presentation among the patients at the time of the diagnosis was 214 patients (96.83%) presented pain and 202 patients (91.4%) presented vaginal discharge. 139 patients (63%) received a double antibiotic scheme and 57 patients (26%) received a triple antibiotic scheme. Of the patients who received a double treatment scheme, 38 patients (17.19%) were treated with cephalothin and metronidazole, 33 patients (14.9%) with cephalothin and gentamicin, 31 patients (14.03%) with doxycycline and metronidazole, 19 patients (8.6%) with clindamycin and gentamicin, 8 patients (3.62%) with clindamycin and metronidazole, 6 patients (2.71%) with gentamicin and metronidazole, 4 patients (1.81%) with levofloxacin and metronidazole (Figure 4).

AWHC-19-148-RODOLFO MORALES AVALOS_ Mexico_F4

Figure 4. Double antibiotic Scheme

Of the patients who received a triple antibiotic scheme, 28 patients (12.67%) were treated with cephalothin; gentamicin and metronidazole, 8 patients (3.62%) with ceftriaxone, metronidazole and levofloxacin, 7 patients (3.17%) with ampicillin, gentamicin and metronidazole, 5 patients (2.26%) with ceftriaxone, clindamycin and gentamicin, 5 patients (2.26%) with ceftriaxone, gentamicin and metronidazole and 4 patients (1.81%) with cephalothin, doxycycline and metronidazole (Figure 5).

AWHC-19-148-RODOLFO MORALES AVALOS_ Mexico_F5

Figure 5. Triple antibiotic Scheme

84 patients underwent a surgical procedure. 20 patients (9.05%) underwent a left salpingoophorectomy, 12 patients (5.43%) had an abdominal hysterectomy plus bilateral salpingoophorectomy, 11 patients (4.98%) had an abdominal hysterectomy, 9 patients (4.07%) had an abdominal washout. In addition, 8 patients (3.62%) underwent an abdominal hysterectomy plus right salpingoophorectomy, 8 patients (3.63%) had a left salpingectomy, 7 patients (3.17%) a right salpingoophorectomy, 6 patients (2.71%) underwent a right salpingectomy and 3 patients (1.36%) underwent abdominal hysterectomy plus left salpingoophorectomy (Figure 6).

AWHC-19-148-RODOLFO MORALES AVALOS_ Mexico_F6

Figure 6. Surgical Procedure. SO, Salpingoophorectomy; AH, Abdominal Hysterectomy.

Discussion

The risk factors for pelvic inflammatory disease are well known and include multiple sexual partners, women under 25, and sexually transmitted diseases [5, 6]. In our study, it was decided to analyze the characteristics of the patients who come to the clinic and they are diagnosed with PID. We found that the average age of patients is 26 to 33 years, age range that differs from other studies, where adolescents or women under 20 years are reported [7]. It has been described that one of the biggest risk factors for PID is the number of sexual partners [8–10]. The results in our study were not very different from what is reported in the literature, because we found that 93 patients had had 2 to 3 sexual partners. Tabacco L. et al. conducted a study on sexual behavior and the relationship between the number of sexual partners and the risk of developing PID. They found that most patients mention only having one sexual partner, causing condom use to decrease and thereby increasing the risk of a sexually transmitted disease [11]. This could explain the case for the 73 patients with only one sexual partner and a diagnosis for PID.

The intrauterine device is one of the most commonly used contraceptives in the population and is one of the risk factors for PID. Data from 12 randomized IUD studies, including more than 20,000 women and more than 5,000 women between 15 and 24 years showed a general PID rate of 1.6 cases per 1,000 women-years of use. After adjustment for confounding variables, the risk of PID rose only during the 20 days immediately after IUD insertion. Although the risk in the first 20 days after insertion was six times the baseline risk, the absolute risk of PID was small and cases of PID were unusual. After the insertion period, the risk of PID for all women returned to the initial risk during the next 8 years of follow-up study. The risk of PID in IUD users was inversely related to age, a finding probably related to a higher prevalence of STIs in younger women rather than the presence of an IUD [12].

In our study, 131 patients mentioned using the IUD as their contraceptive method of choice. However, the question remains if the insertion of the IUD was in a range of 4 months prior to the presentation of the symptoms of PID to be able to relate whether the IUD was the factor risk that in most cases caused the onset of symptoms. The polymicrobial etiology of PID is clearly accepted, so it must be treated with antibiotics that offer coverage against a broad spectrum of pathogens, the CDC has developed guidelines for treatment focused on the most common pathogens, Chlamydia trachomatis and Neisseria gonorrhoeae [13]. We conclude that in our hospital different treatment schemes are used, the most frequent was a double scheme of medications with cephalothin and metronidazole, prescribed scheme in 38 patients that differs from what the literature recommend. The CDC recommends a clindamycin and gentamicin scheme, which in our study was only prescribed in 19 patients, both groups with favorable clinical results. Regarding the triple scheme, the CDC recommends a combination of ceftriaxone, doxycycline and metronidazole or cefoxitin, doxycycline and metronidazole; The triple scheme prescribed more frequently in our study differs from what the CDC recommends because in 28 patients it was decided that cephalothin, gentamicin and metronidazole would be prescribed, a scheme with which favorable results were obtained.

Conclusion

With these results, we were able to know the epidemiological characteristics of the patients diagnosed with pelvic inflammatory disease. Knowing these characteristics, we can carry out programs to detect risk factors and prevent the incidence and recurrence of PID, in addition to find the treatment scheme that has worked best according to the characteristics of our population.

References

  1. Hernández Durán D, Diaz Mitjans O (2010) Enfermedad inflamatoria pélvica. Revista Cubana de Obstetricia y Ginecología 36: 613–631.
  2. Risser JM, Risser WL, Risser AL (2008) Epidemiology of infections in women. Infectious disease clinics of North America 22: 581–599.
  3. Bender N, Herrmann B, Andersen B, Hocking JS, van Bergen J, et al. (2011) Chlamydia infection, pelvic inflammatory disease, ectopic pregnancy and infertility: cross-national study. Sexually transmitted infections 87: 601–608.
  4. Villacreses S, Stefania S (2013) Prevalencia de las infecciones de transmisión sexual en mujeres de edad fértil, diagnosticadas por medio de estudios citológicos. Estudio realizado en Hospital Enrique C. Sotomayor de septiembre 2012 a febrero del 2013.
  5. Peláez Mendoza J (2012) Enfermedad inflamatoria pélvica y adolescencia. Revista Cubana de Obstetricia y Ginecología 38: 64–79.
  6. Barrantes S (2015) Enfermedad Pélvica Inflamatoria. Ginecologia y Obstetricia Revista Médica de Costa Rica y Centro America LXXII 614) 2015: 105–109.
  7. Valdés S, Essien J, Saavedra D, Bardales J (2006) Enfermedad inflamatoria pelviana en adolescentes. Clínica e Investigación en Ginecología y Obstetricia 33: 177–179.
  8. Bohm MK, Newman L, Satterwhite CL, Tao G, Weinstock HS (2010) Pelvic inflammatory disease among privately insured women, United States, 2001–2005. Sexually transmitted diseases 37: 131–136.
  9. Kreisel K (2017) Prevalence of pelvic inflammatory disease in sexually experienced women of reproductive age – United States, 2013–2014. Mobitdity and Mortality Weekly Report 66: 80–83.
  10. Trent M, Chung SE, Burke M, Walker A, Ellen JM (2010) Results of a randomized controlled trial of a brief behavioral intervention for pelvic inflammatory disease in adolescents. Journal of pediatric and adolescent gynecology 23: 96–101.
  11. Tabacco (2018) Relationship Status and Sexual Behaviors in Post- Pelvic Inflamatory Disease (PID) Affected Urban Young Women: A Sub-Study of Randomized Controlled Trial. In Arch Nurs Health Care. 4: 088.  
  12. Farley TMM, Rowe PJ, Meirik O, Rosenberg MJ, Chen JH (1992) Intrauterine devices and pelvic inflammatory disease: an international perspective. The Lancet 339: 785–788.
  13. Workowski KA, Berman SM (2007) Centers for Disease Control and Prevention sexually transmitted diseases treatment guidelines. Clinical Infectious Diseases 1: 73–76.

Gum Arabic and Tricalcium Phosphate as Encapsulating Agents during kiwifruit Drying

DOI: 10.31038/NDN.2019113

Abstract

The current interest in the healthfulness of fruits may be linked to its antioxidant capacity, related to the presence of bioactive compounds. Freeze-drying opens up new alternatives for fruit processing, mainly due to the scarce heat damage to the product. Nevertheless, the high cost of this process may recommend some pre-drying treatment. In this study, the effect that using pre-microwaves or hot air partial drying prior to freeze-drying has on the vitamin A, C and E content and antioxidant capacity of kiwifruit puree, with or without the presence of gum Arabic and tricalcium phosphate, was studied. Powders obtained from kiwifruit purees with solutes added showed a significant (p<0.05) higher content of vitamins and greater antioxidant capacity. Gum Arabic and tricalcium phosphate protect against loss caused by any studied drying treatments. Nevertheless,  microwaves and especially hot air partial drying treatments prior to freeze-drying caused significant vitamin and antioxidant capacity loss.

Keywords

microwaves; hot air; freeze-drying; antioxidant capacity; vitamins A, E and C

Introduction

Every day, our body is subjected to attack by many reactive oxygen species (ROS), formed not only during the physiological metabolism of energetic nutrients but also under infection, smoking, and pollution threaten [1, 2]. When the normal antioxidant defences are overwhelmed, ROS bring about a condition of oxidative stress [3], which means that ROS attack and oxidize portions of lipids, nucleic acids, and proteins, with the consequent impairment of physiological functions and the appearance of pathologies [1]. The contribution of antioxidant nutrients becomes of vital importance to rescuing the body from oxidative stress conditions [4]. Fresh vegetables are relevant reservoirs of vitamins and phenol compounds [5]. If introduced into the body at every meal, those nutrients provide consistent protection to physiological macromolecules by increasing the antioxidant capacity of the plasma [6, 7]. The kiwifruit is the edible berry of a cultivar group of the woody vines of several Actinidia species. The most common commercially available, green-fleshed kiwifruit is the ‘Hayward’ cultivar, which belongs to the Actinidia deliciosa species [8]. Kiwifruit are often promoted for their high vitamin C content, which probably contributes to the health benefits that include antioxidant, antiatherogenic and anticarcinogenic activities, as well as immunomodulation [9]. However, kiwifruit also contain other vitamins and minerals that may contribute to possible health benefits, including folate, potassium and magnesium, dietary fibre and phytochemicals [10]. The effects of kiwifruit consumption on hypertension and dyslipidemia have been tested in human trials. [11] found that the consumption of green kiwifruit resulted in a small but significant reduction in diastolic and systolic blood pressure in male smokers, the effect being strongest in those with hypertension. On the other hand, in subjects that were hypercholesterolemic, kiwifruit consumption did have a favourable effect on plasma high-density lipoprotein cholesterol (HDL-C) concentrations and the total cholesterol (TC): HDL-C ratio [12]. These results support the earlier findings that kiwifruit consumption increased HDL-C and decreased the TC: HDL-C ratio and also decreased the plasma triglyceride concentrations [13, 14].

In addition to the “mainstream” health targets commonly used to define the benefits from functional food, a small number of studies demonstrate more novel bioactive effects of kiwifruit, such as “natural” sleep aid or wound healingThe consumption of “Hayward” kiwifruit in the evening has been shown to improve sleep onset, duration, and efficiency in adults with self-reported sleep disturbances [15]. In addition, the application of kiwifruit fresh slices to burns has been shown to improve the healing process [16].

However, many people are not able to consume fresh fruit every day, due to their work conditions, distance from the markets of fresh products, or having very little time for shopping and cooking [17]. Therefore, they buy processed fruit and consume them every day. Powdered fruit products may be an alternative means of increasing fruit consumption in response to the increased demand for ready-to-eat foodstuffs. The benefits of handling, packaging and transporting fruit powder, the great stability of the product and the ease of final consumption could contribute to this end. Freeze-drying (FD) emerged as a drying method which generates high quality products with very low moisture content, good sensory and nutritional properties and a good capacity for rehydration. However, long processing times and high operation costs are necessary to obtain freeze-dried products with an adequate level of quality (18).  Different studies have been carried out into how to reduce processing costs, for example abolishing the cost of vacuum generation, combining technologies prior to or during FD operation or enhancing heat transfer [19–21]. On the other hand, freeze-dried fruit products are highly hygroscopic and are prone to suffering changes in their physical properties brought about by the environment and time. Therefore, adding biopolymers and other solutes has been shown to be necessary in order to increase the product stability by acting as a barrier to water adsorption [22, 23]. The aim of this study was to evaluate the impact of both the use of microwave (MW) or hot-air drying (HA) as pre-drying treatments prior to freeze-drying and the presence of gum Arabic (GA) and tricalcium phosphate (TCP) on both the vitamins and antioxidant capacity of kiwifruit powders.

Materials and Methods

Sample preparation and treatments

Kiwifruit (Actinidadeliciosa var. Hayward) was purchased in a local market in Valencia (Spain). The mean values (with standard deviation in brackets) of pH, ºBrix and water content of the kiwifruit used were 3.32 (0.09), 13.6 (0.7) and 84.0 (0.8) g/100g, respectively. The fruit pieces were peeled, washed with distilled water and triturated in a Thermomix (TM 21, Vorwerk, Spain). The obtained puree was divided into two parts and GA (Sigma CAS: 9000-01-5, Spain) along with TCP (Sigma CAS: 7758-87-04, Spain) were added to one of them. The quantities employed were: 1 kg GA/kg soluble solids of the liquid phase of the product [24] and 0.02 kg TCP/kg soluble solids of the liquid phase of the product [25, 26]. As a result, two different samples were obtained: kiwifruit and kiwifruit with solutes. A part of each sample was freeze-dried. To this end, a layer (5 mm thickness) of each sample was placed in a standardised aluminium plate (15 cm diameter and 5 cm height). Consecutively, samples were stored in a CVF 525/86 cryo freezer (-86ºC) (Ing. Climas, Spain) for the 24 h before being freeze-dried in a Lioalfa-6 Lyophyliser (Telstar, Spain) at 0.026 mbar and -56.6 ºCat condenser for 24 h. The obtained cakes were crushed (Thermomix TM 21, Vorwerk, Spain) to obtain kiwifruit powders, with and without solutes, which were named KS and K, respectively.

The rest of the samples of kiwifruit and kiwifruit with solutes was submitted to two different pre-drying methods, prior to FD, in order to reduce the initial water content: MW and HAD. According to preliminary experiments (data not shown), the final water content for pre-treatments was set at 65 g water/100 g sample. This moisture level led to a sample of good appearance with noticeable water loss, without the need for an excessively long pre-treatment time. To ensure this water content in the samples, their weight was recorded during the process. It allowed for the calculation of the water content at different drying times, provided the initial water content is known. Puree samples of kiwifruit and kiwifruit with solutes (300 g) were placed in standard recipients (25 cm diameter and 3 cm depth) made of Teflon (Mecaplast, Spain) or aluminium for MW and HAD, respectively. A household microwave (3038GC, Norm, China), set at a microwave power of 2 W/g and an oven (5141 AFW2, Moulinex, China), set at an air temperature of 40 ºC and an air velocity of 1.5 m/s, were used to carry out the drying pre-treatments. Microwave power and air temperature were selected by taking into account the minimal loss of antioxidant capacity obtained in preliminary experiments (data not shown). The treated samples were immediately cooled in ice water until the puree reached 30 °C. All four samples were freeze-dried as described above to obtain the corresponding powders, thus becoming freeze-dried kiwifruit puree with and without solutes added, pre-dried by MW or HAD, named as follows: KMW, KHAD, KSMW and KSHAD, respectively.

Analytical determinations

All the analyses described below were carried out in triplicate on fresh and freeze-dried kiwifruit samples. Despite the obtained results being referred to 100 g of each sample, the losses of vitamins, total phenolic compounds and antioxidant capacity suffered by the samples were calculated and also referred to 100 g of the kiwifruit’s own solutes (KS) in order to compare them(equations 1 and 2).

NDN-19-103_N. Martínez-Navarrete_Eq1

NDN-19-103_N. Martínez-Navarrete_Eq2

where: xwp is the water content of the powder (w/w), xGS/TS is the mass fraction of the grapefruit’s own solutes (GS) to total sample solutes, mGA, mCMC and mL are the mass of gum Arabic, carboxymethyl cellulose and liquidized grapefruit, respectively, in the sample and xwL is the water content of the liquidized grapefruit (w/w).

The mass fraction of water was obtained by vacuum drying the samples in a vacuum oven (Vaciotem, J.P. Selecta, Spain) at 60 ± 1 ºC under a pressure of < 100 mm Hg until constant weight. To determine the total solute content (TS) of the freeze-dried samples, the methodology described by [27] was followed. In turn, 1.5 g of each powder were rehydrated, in triplicate, and the TS of the bulk suspension were determined by oven drying (Vaciotem, J.P. Selecta, Spain) at 60 ºC until constant weight.

Vitamins A, C and E

The vitamins were determined by HPLC (Jasco equipment, Italy). The procedure employed to determine vitamin C was the reduction of dehydroascorbic acid to ascorbic acid, using DL-dithiothreitol as reductant reagent according to Igual et al., 2016. The HPLC method and instrumentation were: Ultrabase-C18, 5 μm (4.6×250 mm) column (AnálisisVínicos, Spain); mobile phase 0.1 % oxalic acid, volume injection 20 μL, flow rate 1mL/min, detection at 243 nm and at 25 ºC. AA standard solution (Panreac, Spain) was prepared.

Vitamins A and E were extracted twice in the hexane phase and the collected extract was dried under a stream of liquid nitrogen [28]. The dried extract was solubilized in 0.2 mL methanol. The HPLC method and instrumentation were: Ultrabase-C18, 5 μm (4.6 × 250 mm) column (Spain); mobile phase methanol/acetonitrile/chloroform (47:42:11, v/v/v), volume injection 20 μL, flow rate 1 mL/min, detection at 326 and 296 for vitamins A and E, respectively at 25 ºC. Standard curves of each reference compound (Fluka-Biochemika, USA) were used for quantification purposes.

Antioxidant capacity

Antioxidant capacity (AOC) was assessed using the free radical scavenging activity of the samples evaluated with the stable radical 2, 2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH). Briefly, the samples were diluted in methanol, homogenized and centrifuged (Selecta Medifriger-BL, Spain) at 400 x g for 10 min at 4 °C. 0.1 mL of supernatant was added to 3.9 mL of DPPH (Sigma-Aldrich, Germany) diluted in methanol (0.030 g/L). At 30 s intervals, a UV-visible spectrophotometer was used to measure the absorbance at 25 ºC and515 nm until the reaction reached the steady state. The percentage of DPPH was calculated following Eq. (3).

NDN-19-103_N. Martínez-Navarrete_Eq3

where Acontrol is the absorbance of the control (initial time) and Asample the absorbance of the sample at the steady state.

The final results were expressed as mmol trolox equivalents (TE) per 100 g sample using a trolox calibration curve in the range of 6.25–150 mM (Sigma-Aldrich, Germany).

Statistical analysis

An analysis of variance (ANOVA) was carried out to evaluate the differences among samples. When the p value was lower than 0.05, significant differences between samples were assumed. Furthermore, an analysis of the correlation between AOC and all the studied vitamins, with a 95 % significance level, was carried out. All statistical analyses were performed using Stat graphics Centurion XVI.II for Windows.

Results and discussion

Kiwifruit characterization

The vitamin A content (0.029 (0.002) mg/100 g) was lower than that of the rest of the quantified bioactive compounds. The vitamin E content was 2.10 (0.13) mg/100 g, similar to the values of this vitamin shown by [29] (2.5 mg /100g,). In general, citric fruit are considered a good source of vitamin C. However, the amount of vitamin C in kiwifruit (105 (5) mg/100 g) is about three times more than that present in 100 g of grapefruit [30] or twice more than orange [31]. The obtained results coincide with the values shown for kiwifruit var. Hayward by other authors [32–35]. One of the predominant mechanisms of the protective action of bioactive compounds is their antioxidant activity and the capacity to scavenge free radicals [36]. Kiwifruit showed 15.63 (0.15) mmol TE/100g of AOC.

The effect of solute addition and pre-drying on bioactive compound variation of freeze-dried kiwifruit powder

The losses of vitamins and AOC suffered by the samples as a consequence of each drying step have been plotted in Fig. 1 – 4. The loss of each component (ΔMi), referred to the fresh kiwifruit content, were calculated according to equations 4 and 5.

NDN-19-103_N. Martínez-Navarrete_Eq4

NDN-19-103_N. Martínez-Navarrete_Eq5

where: Mi: mass of compound i in the sample referred to the kiwifruit’s own solutes and superscripts: FK: fresh kiwifruit, P: pre-treated (mixed with the solutes and/or pre-dried by MW or HAD), FD: freeze-dried.

NDN-19-103_N. Martínez-Navarrete_F1

Figure 1. Mean value and standard deviation of Vitamin C losses suffered by the samples as a consequence of each process studied.

Letters indicate homogeneous groups established by the ANOVA (p < 0.05) for total process variation.

NDN-19-103_N. Martínez-Navarrete_F2

Figure 2. Mean value and standard deviation of Vitamin A losses suffered by the samples as a consequence of each process studied

Letters indicate homogeneous groups established by the ANOVA (p < 0.05) for total process variation.

NDN-19-103_N. Martínez-Navarrete_F3

Figure 3. Measn value and standard deviation of Vitamin E losses suffered by the samples as a consequence of each process studied

Letters indicate homogeneous groups established by the ANOVA (p < 0.05) for total process variation

NDN-19-103_N. Martínez-Navarrete_F4

Figure 4. Mean value and standard deviation of antioxidant capacity losses suffered by the samples as a consequence of each process studied.

Letters indicate homogeneous groups established by the ANOVA (p < 0.05) for total process variation.

As regards the vitamins studied (Figures 1 – 3), FD was observed to bring about the greatest vitamin loss in sample K. When comparing samples K and KS, a loss of all three of the vitamins studied was observed due to the mixing process when solutes were added to the kiwifruit puree. Nevertheless, solutes reduced the losses caused by FD. In general, the application of MW or HAD pre-treatment led to an increase in vitamin losses, especially when no solutes were present. In this way, GA+TCP can be confirmed to exert a protective effect on the vitamins studied throughout FD.As regards pre-drying treatments, the loss of vitamins A or C due to MW or HAD pre-treatments was of the same order, while the loss of vitamin E was greater when HAD was used. In fact both the KMW and KHAD samples even lost the entire amount of vitamin A present in the fresh kiwi. In these KMW and KHAD samples, the additional loss of the remaining vitamin C brought about by the freeze-drying process was almost negligible, while the loss of vitamin E was also promoted in sample KMW. When comparing these samples with KSMW and KSHAD, the loss of vitamins was reduced in the samples with solutes added, so that for vitamins A and C the loss was reduced in the range of 30 and 20 %, respectively and for vitamin E from 15 % to 40%, depending on whether the HAD or the MW pre-treatment was applied, respectively. As regards AOC (Fig. 4), a loss of about 5 % was observed caused by FD (sample K) or simply by sample formulation (sample KS). A 50–60 % loss of AOC was observed in pre-treated KMW and KHAD samples, due just to FD or to HAD and FD, respectively. This loss was reduced to 20–25 % when solutes were present (KSMW and KSHAD samples). Added solutes help the retention of nutritive properties of kiwifruit during pre-treatments and FD. Maltodextrins and gums are added during the production of food powders in order to act as encapsulating or wall materials, contributing to keeping the desired functional properties in the finished product, such as stability against oxidation, ease of handling, improved solubility, controlled release and extended shelf-life [37]. GA is the gum most often used as a flavour encapsulating material, mainly due to its solubility, low viscosity, emulsification characteristics and its good retention of volatile compounds [38–40].

Table 1 shows the mean value of vitamins and AOC contained in 15 g of the different obtained kiwifruit powders. About 15 g of kiwifruit powder is the equivalent to a fresh kiwi-fruit (≈85 g). The highest vitamin intake is provided by sample KS, despite the highest AOA being supplied by sample K. An intake of 15 g of K or KS powders or ≈ 23 g of any of the other powders is enough to meet the daily amount of vitamin C recommended by the World Health Organization (≈40 mg). KMW, KHAD and KSHAD samples also showed the lowest AOC. In order to explain the relationship among the different vitamins quantified in this study themselves and also between them and the AOC of the samples, correlation statistical analyses were performed using all the obtained data (Table 2). A significant relationship (p>0.05) between the AOC and any of the studied vitamins was observed, as was a closer relationship among the vitamins.

Table 1. Mean values (with standard deviation) of vitamins (mg/15 g) and antioxidant capacity (AOC) (mmolTE/15 g) of obtained powders: K (kiwifruit powders without solutes), KS (kiwifruit powders with solutes), KMW (kiwifruit powder without solutes added pre-dried by microwaves), KHAD (kiwifruit powder without solutes added pre-dried by hot air), KSMW (kiwifruit powder with solutes added pre-dried by microwaves) and KSHAD (kiwifruit powder with solutes added pre-dried by hot air).

Sample

Vitamin A

Vitamin C

Vitamin E

AOC

K

0,004 (0,0005)bc

43 (5)a

0.2990 (0,0012)d

12.24 (0,13)a

KS

0,006 (0,0003)a

40.5 (1.7)a

0.813 (0,012)a

7.55 (0,07)b

KMW

0d

27.5 (0.8)b

0.184 (0,015)e

6.86 (0,03)bc

KHAD

0d

27.7 (0.9)b

0.473 (0,012)c

5.5 (0.4)d

KSMW

0,004 (0,0004)b

26.8 (0.7)b

0.543 (0,004)b

6.05 (0,06)cd

KSHAD

0,003 (0,0002)c

25.3 (0.6)b

0.470 (0,003)c

5.5 (60.9)d

The same letter in superscript within columns indicates homogeneous group established by ANOVA (p<0.05).

Table 2. Pearson correlation coefficients among all studied components

Vitamin C

Vitamin A

Vitamin E

AOC

0,5787*

0,4728*

0,6143*

Vitamin C

0,7926*

0,8077*

Vitamin A

0,846*

* Correlation is significant at the 0.05 level.

Conclusions

An encapsulating effect of GA+TCP was observed both for the mixing with solutes and/or the pre-drying treatment and for the freeze-drying process. When solutes are present, a pre-drying treatment with MW may be recommended instead of HAD for the purposes of obtaining a freeze-dried product, as vitamin E and AOC are better preserved. Nevertheless, despite the fact that the pre-treatment will shorten the subsequent freeze-drying time and so reduce the energy costs, if the maximum preservation of the vitamin content and AOC is desired, the use of microwave or hot air drying pre-treatment should be avoided.

Acknowledgments

The authors thank the Ministerio de Economía y Competitividad for the financial support given through the Project AGL 2017-89251-R (AEI/FEDER-UE).

References

  1. Dean RT, Fu S, Stocker R, Davies MJ (1997) Biochemistry and pathology of radical- mediated protein oxidation. Biochem J 324: 1–18. [crossref]
  2. Finkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the biology of aging. Nature 408: 239–247. [crossref]
  3. Halliwell B, Cross CE (1994) Oxygen-derived species: Their relation to human disease and environmental stress. Environ Health Perspect 102: 5–12. [crossref]
  4. Sun J, Chu YF, Wu X, Liu RH (2002) Antioxidant and Antiproliferative Activities of Common Fruits. J Agric Food Chem 50: 7449–7454. [crossref]
  5. Rickman, J.C., Barret,D.M. & Bruhn, C.M (2007) Review: Nutritional comparison of fresh and frozen fruits.  Journal of Science and Food Agriculture 87: 930–944.
  6. Ninfali P, Mea G, Giorgini S, Rocchi M, Bacchiocca M (2005) Antioxidant capacity of vegetables, spices and dressings relevant to nutrition. British J Nutr 93: 257–266. [crossref]
  7. Prior RL, Gu L, Wu X, Jacob RA, Sotoudeh G et al. (2007) Plasma antioxidant capacity changes following a meal as a measure of the ability of a food to alter in vivo antioxidant status. J American Coll Nutr 26: 170–81. [crossref]
  8. Fiorentino A, D’Abrosca B, Pacifico S, Mastellone C, Scognamiglio M et al. (2009) Identification and assessment of antioxidant capacity of phytochemicals from kiwi fruits. J Agric Food Chem 57: 4148–4155. [crossref]
  9. Vissers MC, Carr AC, Pullar JM, Bozonet SM (2013) The bioavailability of vitamin C from kiwifruit. Adv Food Nutr Res 68: 25–147. [crossref]
  10. Drummond L (2013) The Composition and Nutritional Value of Kiwifruit. Adv Food Nutr Res 68: 33–57. [crossref]
  11. Karlsen A, Svendsen M,  Seljeflot I, Laake P, Duttaroy AK et al. (2013) Kiwifruit decreases blood pressure and whole-blood platelet aggregation in male smokers. J Human Hypertension 27: 126–130. [crossref]
  12. Gammon C, Kruger R, Minihane A, Conlon C, Von Hurst P et al. (2013) Kiwifruit consumption favourably affects plasma lipids in a randomised controlled trial in hypercholesterolaemic men. British J Nutr 109: 2208–2218. [crossref]
  13. Brevik A, Gaivão I, Medin T, Jørgenesen A, Piasek A et al. (2011) Supplementation of a western diet with golden kiwifruits (Actinidia chinensis var.’Hort 16A’:) effects on biomarkers of oxidation damage and antioxidant protection. Nutr J 10: 54. [crossref]
  14. Duttaroy AK, Jøorgensen A (2004) Effects of kiwifruits consumption in human volunteers on platelet aggregation and plasma lipids in vitro. Platelets 15: 287–292. [crossref]
  15. Lin HH, Tsai PS, Fang SC, Liu JF (2011) Effect of kiwifruit consumption on sleep quality in adults with sleep problems. Asia Pacific J Clinical Nutr 20: 169–174. [crossref]
  16. Kooshiar H, Abbaspour H, Motamed Al Shariati SM, Rakhshandeh H, Khajavi Rad A et al. (2012) Topical effectiveness of kiwifruit versus fibrinolysin ointment on removal of necrotic tissue of full-thickness burns in male rats. Dermatol Therapy 25: 621–625. [crossref]
  17. Bertuccioli A, Ninfali P (2014) The Mediterranean Diet in the era of globalization: The need to support knowledge of healthy dietary factors in the new socio-economical framework. Mediterranean J NutrMetab 7(1): 75–86.
  18. Menlik T, Özdemir MB, Kirmaci V (2010) Determination of freeze-drying behaviours of apples by artificial neural network. Expert Sys Appl 37: 7669–7677.
  19. Zhang M, Tang J, Mujumdarc AS, Wang, S (2006) Trends in microwave related drying of fruits and vegetables. Trends Food Sci Technol 17: 524–534.
  20. Pardo JM, Leiva DA (2010) Effects of different pre-treatments on energy consumption during freeze drying of pineapple pieces. Interciencia 35: 934–938.
  21. Huang L, Zhang, Mujumdar AS, Lim R (2011) Comparison of four drying methods for re-structured mixed potato with apple chips. J Food Eng 103: 279–284.
  22. Cozic C, Picton L, Garda M, Marlhoux F, Le Cerf D (2009) Analysis of arabic gum: study of degradation and water desorption processes. Food Hydrocoll 23: 1930–1934.
  23. Telis VN, Martínez-Navarrete N (2012) Biopolymers used as drying aids in spray drying and freeze drying of fruit juices and pulps. In V. Telis (Ed.). Biopolymer Engineering in Food Processing (pp. 279–326).London: CRC Press. Taylor & Francis Group.
  24. Mosquera LH, Moraga G, Martínez-Navarrete N (2012) Critical water activity and critical water content of freeze-dried strawberry powder as affected by maltodextrin and arabic gum. Food Res Inter 47: 201–206.
  25. Cubero N, Monferrer A, Villalta J (2002) Aditivos alimentarios. Madrid: A. Madrid Vicente.
  26. Jaya, S., & Das, H. (2009). Glass transition and sticky point temperatures and stability/mobility diagram of fruit powders. Food Bioprocess Technology 2: 89–95.
  27. Benlloch-Tinoco M, Varela P, Salvador A, Martínez-Navarrete N (2012) Effects of microwave heating on sensory characteristics of kiwifruit puree. Food Bioprocess Technol 5: 3021–3031.
  28. Igual M, García-Martínez E, Camacho MM, Martínez-Navarrete N (2016) Stability of micronutrients and phytochemicals of grapefruit jam as affected by the obtention process. Food Sci Technol Inter 22: 203–212. [crossref]
  29. Rush EC, Patel M, Plank LD, Ferguson LR (2002) Kiwifruit promotes laxation in the elderly. Asia Pacific J Clin Nutr 11: 164–168. [crossref]
  30. Igual M, García-Martínez E, Camacho MM, Martínez-Navarrete N (2010) Effect of thermal treatment and storage on the stability of organic acids and the functional value of grapefruit juice. Food Chem 118: 291–299.
  31. Sánchez-Moreno C, Plaza L, De Ancos B, Cano MP (2003) Quantitative bioactive compounds assessment and their relative contribution to the antioxidant capacity of commercial orange juices. J SciFood Agric 83: 430–439.
  32. De Ancos B, Cano MP, Hernández A, Monreal M (1999) Effects of microwave heating on pigment composition and color of fruit purees. J Sci Food Agric 79: 663–670.
  33. Schwartz M, Nuñez H, Muñoz AM (1999) Effect of concentration temperature of kiwifruit pulp on colour, chlorophyll and ascorbic acid. Archivos Latino Nutr 49: 44–48. [crossref]
  34. Antunes MDC, Dandlen S, Cavaco AM, Miguel G (2010) Effects of postharvest application of 1-mcp and postcutting dip treatment on the quality and nutritional properties of fresh-cut kiwifruit. J Agric Food Chem 58: 6173–6181. [crossref]
  35. Zolfaghari M, Sahari A, Barzegar M, Samadloiy H (2010) Physicochemical and enzymatic properties of five kiwifruit cultivars during cold storage. Food Bioprocess Technol 3: 239–246.
  36. Yahia  EM, Maldonado E, Svendsen M (2010) The contribution of fruit and vegetable consumption to human health. In: Yahia EM (Ed). Fruit and vegetable phytochemicals.(pp. 3–51), Wiley-Blackwell, USA.
  37. Jafari SM, Mahdavi-Khazaei K, Hemmati-Kakhki A (2016) Microencapsulation of saffron petal anthocyanins with cress seed gum compared with Arabic gum through freeze drying. Carbohydrate Polymers 140: 20–25.
  38. Madene A, Jacquot M, Scher J, Desobry S (2006) Flavour encapsulation and controlled release – a review. Inter J Food Sci Technol 41: 1–21.
  39. Righetto AM, Netto FM (2006) Vitamin C stability in encapsulated green West Indian cherry juice and in encapsulated synthetic ascorbic acid. J Sci Food Agric 86: 1202–1208.
  40. Desobry SA, Netto FM, Labuza TP (1997) Comparison of spray-drying, drum-drying and freeze-drying for β-carotene encapsulation and preservation. J Food Sci 62: 1158–1162.

New insights on the tectonic structure of the Southern Central Andes – Western Argentina – from seismic tomography

DOI: 10.31038/GEMS.2019113

Introduction

The geodynamic evolution of central Chile-Argentina is mainly controlled by the subduction of the oceanic Nazca plate beneath the continental South American lithosphere (Figure 1a). In particular, between 29°S and 34°S, the Nazca plate exhibits a flat-slab subduction (Figure 1b) around 100 km of depth, that extends eastward for hundreds of km before returning to a downward descent (Cahill and Isacks 1992). This condition causes an interruption in the Andean volcanic arc and an intense crustal deformation that continues toward the craton, beyond the foreland thrust belt. The outcome is a very active crustal seismicity and complicated structural framework. Despite the recent advances achieved through geological, seismological and geodetic studies (e.g. Siame et al. 2006; Anderson et al. 2007 and references therein), several aspects related to the geometry, type and contributions of each fault and its seismogenic rule, as well as of the slab geometry, have still not been satisfactorily explored. In order to study the seismotectonic characteristics of this flat-slab region, we analyzed data recorded by a local network, performing a simultaneous inversion of both the velocity structure and the hypocentre parameters. The availability of accurate hypocentre locations and finely tuned velocity structure allows us to improve the earthquake source characterization and the knowledge on the ongoing seismotectonics of the region (Figure 1).

Gems-19-103_Luciano Scarfì_F1

Figure 1. (a) Map of the global South American tectonics and (b) Nazca plate iso-depth contours after Ramos et al., 2002 (dashed lines). Red triangles represent active volcanic edifice; red rectangle marks the studied area. (c) Schematic tectonic cross-section showing the eastward migration of the main volcanic centres during the shallowing of the oceanic slab (after Ramos and Folguera 2009).

Tectonic Settings

The studied region, between 29° and 34° S, corresponds to a complex surface setting made up of the Principal Cordillera and Frontal Cordillera, which form the main part of the Andes, and, eastward, of the Precordillera, which is a thin-skinned thrust and fold belt, and Sierras Pampeanas, considered as crystalline basement uplifts (Figure 1b). The basement blocks of these geological provinces uplifted during the shallowing of the slab since 25 Ma, while the flat-subduction produced a progressive eastward migration of the orogenic front and volcanic arc. The gap in the volcanic arc, spatially correlated with the sub-horizontal subduction zone, has led some authors to infer that the flat subduction changes the thermal structure of the margin, displacing the hot asthenospheric wedge away from the trench and producing a progressive eastward migration of the orogenic front and the volcanic arc (see e.g. Gutscher, 2002). Earthquakes are distributed in two separate depth ranges: i) events with hypocentral depths of about 100 km are related to the Wadati-Benioff zone of the subducting slab; ii) earthquakes with hypocentral depth ranging down to about 40 km correspond to the crustal seismicity (Cahill & Isacks 1992).

Data

We analyzed seismicity recorded by the local network operated by 23 short period, vertical component, seismic stations, from the ‘‘Red Sismologica Zonal Nuevo Cuyo’’ belonging to ‘‘Red Nacional de Estaciones Sismologicas’’ of the INPRES (Instituto Nacional de Prevención Sísmica, Argentina). The initial data-set consists of more than 450 earthquakes with a magnitude between 3.4 and 4.8, occurring in the period 1995–1999. Because large uncertainties in the hypocentre locations can introduce instabilities in the inversion process, before including the earthquakes in the joint inversion, we filtered our database to match minimum requirements with respect to location quality criteria (i.e. GAP<240°, a minimum number of 8 P-phase recordings and travel time residuals < 0.5 s). The resulting data-set consists of about 290 earthquakes with 2965 P- and 2513 S-wave readings (Figure 2).

Gems-19-103_Luciano Scarfì_F2

Figure 2. Digital elevation model of the studied area (top) and a 3-D sketch of P-wave ray paths traced in the a-priori velocity model. Earthquakes and seismic stations are indicated by red circles and green triangles, respectively.

Calculation of the 3D velocity model and event locations

Seismic velocity modelling was carried out by the program LOTOS (Koulakov, 2009) that automatically sets up the inversion mesh according to the distribution of the seismic rays. Specifically, given a regular grid step, which should be smaller than the presumed size of the anomalies, the software is able to not install any node in the case of absence of rays, whereas in areas of higher ray number, it can increase the grid density up to the maximum value (in our case, 10 and 5 km for the horizontal and vertical direction, respectively). The inversion solution is further controlled by smoothing parameters which reduce the difference in the final values of neighbouring nodes. The ability to build such a dense (quasi continuous) mesh is fundamental for a “local earthquake tomography” in order to reduce the bias of the resulting models due to the grid configuration (i.e. too large spacing between nodes to suitably image earth heterogeneities). Fine-tuning of the inversion was then obtained by discarding events with travel-time residuals of more than 1.5 s for P- and 2.5 s for S-rays. Before performing the tomography, it has been necessary to define a set of parameters and in particular a preliminary guess for the 1D seismic velocities, which was derived from the model optimized for the same area by Scarfì et al. (2012). The accuracy of the final 3D model was assessed by numerical tests (see e.g. Scarfì et al., 2018), whose results clearly indicate that the VP structure is adequately recovered in the central zone of the studied region down to a depth of 50 km. Finally, earthquake locations were further enhanced by the tomoDDPS algorithm (Zhang et al., 2009) and the obtained 3D velocity model. This code has the advantage of using a combination of both absolute and differential arrival time readings. In case of clustered events, additional information is used to improve the relative locations: i.e., since the uncertainty of the velocity model along the station-hypocenter path is the same for all the earthquakes, the travel-time differences found at a common station can be attributed to the spatial offset between the events. This procedure produced better clustering and further reduced the residuals (RMS) of about 33%, with an average of 0.25 sec.

Results

Figure 3 shows the final hypocentre locations of all 264 well-locatable events and highlights that seismicity is mainly located in two clearly separate depth ranges: the first between 0 and 40 km and the second between 90 and 130 km. Representative cross-sections of the 3D VP model are shown in Figure 4. The obtained velocity images highlight vertical and lateral crustal heterogeneities which can be associated with the main tectonic features of the region. In particular, the sharp velocity contrasts along with the relocated events are consistent with previously recognized or supposed active faults in the area of Sierra Pie de Palo. We can hypothesize that at Sierra Pie de Palo seismicity mainly originates from an active west-dipping fault, striking in NNE-SSW direction and extending from its eastern boundary to about 30 km of depth, where, probably, it intercepts another sub-horizontal structure (see also Scarfì et al., 2012 and references therein). Other events are aligned along the Villicum-Pedernal thrust in the eastern Precordillera (Figures 3, 4).

Gems-19-103_Luciano Scarfì_F3

Figure 3. Final locations of the selected earthquakes in map view, N-S and W-E cross sections.

Gems-19-103_Luciano Scarfì_F4

Figure 4. Vertical sections through the P-wave velocity model. The trace of the section is reported in the sketch map. Contour lines are at 0.2 km/s intervals. Relocated earthquakes, within +/-15 km from the sections, are plotted as red circles.

Concerning the intermediate-depth seismicity of the Wadati-Benioff zone, we note that our relocated events are fairly well clustered between the Precordillera and Sierra Pie the Palo (Figure 5). The hypocentres highlight two sub-zones characterized by slightly different focal depths, i.e., the shallower events (90–100 km deep) are located in the eastern zone, the deeper events (depth of 100–120 km) lie in the western zone. This distribution suggests the formation of a bent in the slab geometry (Figure 3, 5).

Gems-19-103_Luciano Scarfì_F5

Figure 5. Final location of the whole dataset of the Wadati-Benioff zone events obtained by the calculated 3D velocity model, in map view, N-S and W-E cross sections.

Conclusion

Applying the techniques of the joint inversion of velocity and hypocentral parameters, we obtained precise and reliable hypocentre locations and information about the velocity structure within the area monitored by the “Red Sismologica Zonal Nuevo Cuyo” network. The results depict important structural features in the Sierra Pie de Palo area and also provide new constraints on the geometry of the subducted slab, within the limits of the study area. In conclusion, our specific analyses contribute to the understanding of the tectonic picture of the Southern-Central Andes region; however, further in depth studies and more data are needed to gain a better interpretation of the tectonic processes acting in the area.

Acknowledgement

We are especially grateful to G. Badi, from the Universidad Nacional de La Plata, and to M. Araujo, from the Instituto Nacional de Prevención Sísmica, Argentina, who provided the data.

References

  1. Anderson M, Alvarado P, Zandt G, Beck S (2007) Geometry and brittle deformation of the subducting Nazca Plate, Central Chile and Argentina. Geophysical Journal International 171: 419–434.
  2. Cahill T, Isacks BL (1992) Seismicity and shape of the subducted Nazca plate. Journal of Geophysical Research  97: 17503–17529.
  3. Gutscher MC (2002)  Andean subduction styles and their effect on thermal structure and interplate coupling.  J. South Am. Earth Sci. 15: 3–10.
  4. Koulakov I (2009) LOTOS code for local earthquake tomographic inversion: Benchmarks for testing tomographic algorithms. Bull. Seismol. Soc. Am. 99: 194–214.
  5. Ramos VA, Cristallini EO, Pérez DJ (2002) The Pampean flat-slab of the central Andes. Journal of South American Earth Sciences 15: 59–78.
  6. Ramos VA, Folguera A (2009) Andean flat-slab subduction through time. In: Murphy JB, keppie JD, Hynes AJ (eds). Ancient Orogens and Modern Analogues, Geological Society, London, Special Publications 327: 31–54.
  7. Siame LL, Bellier O, Sébrier M (2006) Active tectonics in the Argentina Precordillera and Western Sierra Pampeanas.  Revista de la Associacion Geologica Argentina  61: 604–619.
  8. Scarfì L, Raffaele R, Badi G, Ibanez JM, Imposa S et al. (2012) Seismotectonic features from accurate hypocentre locations in southern central Andes (western Argentina). Tectonophysics 518–521: 44–54.
  9. Scarfì L, Barberi G, Barreca G, Cannavò F, Koulakov I, et al. (2018) Slab narrowing in the Central Mediterranean: the Calabro-Ionian subduction zone as imaged by high resolution seismic tomography. Scientific Reports 8.
  10. Zhang H, Thurber C, Bedrosian P  (2009)  Joint inversion for Vp, Vs, and Vp/Vs at SAFOD, Parkfield, California. Geochem Geophys Geosyst 10.