DOI: 10.31038/JCRM.2018113

Abstract

Background: The Child Perceptions Questionnaire (CPQ) is the most commonly used measure of Oral Health-Related Quality of Life (OHRQoL) and its validity and reliability have been tested among children/adolescents aged 11 to 14 years in many languages and populations. This innovative study was aimed to validate the CPQ among adolescents aged 15 to 18 in the population survey of orthodontic anomalies in Lithuania.

Methods: Representative samples of adolescents aged 11–14 years (N=307), 15–16 years (N=721) and 17–18 years (N=563) were selected from public schools of Lithuania. The CPQ including four domains, namely oral symptoms (OS), functional limitations (FL), emotional well-being (EWB), and social well-being (SWB), was used to measure OHRQoL. A self-reported malocclusion and orthodontic examinations were used to assess malocclusion.

Results: The distributions of individual items and sum scores of the CPQ and its domains did not differ significantly between 11–14, 15–16 and 17–18 age groups of adolescents. Across all age groups, Cronbach’s alpha for the total CPQ was approximately equal to 0.90 indicating good internal consistency reliability; the total CPQ and all domains significantly correlated with oral health, oral well-being and global life satisfaction. Discriminant validity analysis revealed that adolescents with severe malocclusion suffered a greater impact on their emotional and social well-being than those without malocclusion, however, this relationship was more engaging in group of adolescents aged 15–18 than in 11–14-year-olds. A moderate agreement between child and parental reports was found for OS and FL domains.

Conclusions: The Lithuanian version of the CPQ for measuring OHRQoL among adolescents aged 15 to 18 years seems to be as valid and reliable as for adolescents aged 11 to 14 years.

Keywords

Oral health-related quality of life, Child Perceptions Questionnaire, validity, reliability, orthodontics, adolescents, Lithuania

Introduction

Oral health-related quality of life (OHRQoL) is a holistic concept which determines the subjective functional and psycho-social impacts of oral disease on overall well-being.[1–3] Measuring OHRQoL provides essential information when making clinical decisions for individuals and helping public health actions and policies to uncover the needs of the society in prevention and treatment of oral disorders.[3–5] Hence, OHRQoL has been widely studied over the past two decades and many tools have been developed, but mostly for adults.[6, 7]

Recently, increasing attention has been also paid to OHRQoL in children and adolescents. The OHRQoL instruments designed to assess the impact of oral conditions on the daily lives of children and adolescents have been developed ranging from measurement of patient-reported oral functional and psychosocial problems to subjective well-being relating to oral health. Systematic reviews [8, 9] identified at least three validated instruments to measure OHRQoL in children and adolescents: Child Oral Impact of Daily Performances index,[10] Child Oral Health Impact Profile,[11] and Child Perceptions Questionnaire.[12]

The most commonly used OHRQoL questionnaire is the Child Perceptions Questionnaire (CPQ). It was developed by Jokovic et al. [12] as the CPQ_11–14 for children aged from 11 to 14 and was originally validated in children with caries, malocclusion and craniofacial anomalies.[12] In terms of cognitive development, age specific versions of this tool have been produced.[13] The CPQ does also have an analogous Parental CPQ which can be used as a proxy to Child CPQ.[14] The original item pool of the CPQ consists of 37 items, but the authors have also determined the psychometric properties of its shortened forms.[15] All variations of the CPQ evaluate the frequency of oral and orofacial impacts on children OHRQoL at symptomatic, functional, emotional and social levels whereas other questionnaires focus on severity of oral impacts. To date, the CPQ has been translated, validated and adapted to suit a number of languages and socio-cultural contexts demonstrating its applicability and perfect psychometric properties on numerous clinical and epidemiological occasions.[16–22]

OHRQoL research among children and adolescent in Lithuania is still nascent and no measures have been validated to date. Given the positive CPQ properties and its high applicability for both clinical assessments and large-scale population studies we have chosen this instrument for measure of OHRQoL in our research. It was also considered that the original long form (37 items) of this instrument is more sensitive to changes in oral conditions rather than its short forms,[15] hence, the original questionnaire was taken in focus. As it is well known that every time as the measurement scale is used in a new context or with a different population group, it is necessary to test its psychometric properties.[23] Therefore, our recent study, like most other similar studies, has been focused on adaptation and validation of the CPQ_11–14 in Lithuanian adolescents aged 11–14 years. A detailed examination of psychometric characteristics including factorial analysis of the Lithuanian version of CPQ with a modified item of the oral pain showed that the instrument is valid to be used in further studies for measuring OHRQoL among 11–14-year-old adolescents in Lithuania (Kavaliauskienė A, Šidlauskas A, Zaborskis A., 2018. Manuscript under review).

This study is a part of a large research project aimed to examine extend of orthodontic anomalies and OHRQoL among children and adolescents aged from 11 to 18 years in Lithuania. Hence, there was a problem to choose an appropriate instrument to measure OHRQoL among adolescents up to the ages of 18. We hypothesized that the association between severity of oral disorders and OHRQoL in older adolescent samples (e.g. in 15–16- or 17–18-year-olds) is possibly more evident than in sample of adolescent aged 11–14 years. Therefore, the CPQ instrument to measure OHRQoL among older adolescents could be as much valid as it was valid for adolescents aged 11–14 years. Only a few relevant studies were conducted among adolescents over the age of 14 but none in population older than 16 years of age [24–27].

As a consequence, the aim of the present study was to validate the CPQ among adolescents aged from 15 to 18 in the population survey of orthodontic anomalies in Lithuania.

Methods and Material

The study followed a cross-sectional design and was a part of a larger research project aimed to examine OHRQoL among children and adolescents in Lithuania. It was conformed to the principles outlined in the Declaration of Helsinki. Ethical approval for the study was granted by the Kaunas Regional Biomedical Research Ethics Committee (reference number BE-2–27) and was in line with local practice for school survey distribution. Written informed consent for child’s participation in the study was sought from both parents prior to his/her participation in the research.

Target population was adolescents aged 11–18 years. The sample being studied was made up of students from 26 randomly selected public schools using random cluster (school, class) sampling and included approximately 2000 students. School authorities were contacted by researchers and informed about all aspects of the study. Parents were then asked to provide permission for their child to participate in the study.

Data was collected using both questionnaires and dental examinations. The self-completed questionnaires for students were administrated in school classrooms before dental examination by the classroom teaching staff to ensure a familiar and consistent environment. Confidentiality and anonymity of respondents was ensured. A total of 1591 students (80% of initial sample and 94% of those who had parents’ permission) presented correctly completed questionnaires. Those parents who gave consents were also asked to complete a self-report questionnaire about child’s oral health and well-being. The number of correctly completed parents’ questionnaires was 1365 (67% of invited parents).

The orthodontic examination was a part of the dental examination. It was carried out in randomly selected 20 of 26 schools. Students’ examination was performed according to the methodology of oral status evaluation recommended by the WHO under standardized conditions in the school’s medical offices using portable equipment for dental examination [28]. The orthodontic examination of all students was undertaken by one orthodontist (A.K.) who was trained and tested in reliability of accessing orthodontic status (U.K. Cardiff University School of Dentistry, 2012) and her assistant.

In the end, 911 students participated both in the questionnaire and dental surveys, and 1365 parents provided their completed questionnaires to students who participated in the questionnaire survey. The size of studied sample was adequate to the minimum calculated as necessary (N=969). Figure 1 presents flow diagram of data collection and also illustrates the sample structure by age of adolescents.

Figure 1. Flow-diagram of the data collection process and distribution of participants in three age groups.

Before the main study, a pilot test was carried out with a sample (N=48) of students in one school. It confirmed the feasibility of the methodology with only minor modification of questionnaire wording and confirmed the organization of data collection procedures.

The originally created self-reported questionnaires for students and parents consisted of items assessing oral health and OHRQoL as well as demographic and social aspects of adolescent health.

The Lithuanian version of the of CPQ_11–14, cross-culturally adapted and validated for Lithuanian adolescents aged 11–14 years (Kavaliauskienė A, Šidlauskas A, Zaborskis A., 2018. Manuscript under review), was employed to evaluate the impact of oral conditions on the quality of life of adolescents of all ages. This questionnaire, as originally proposed by Jokovic et al.,[12] was a 37-item scale consisting of four health domains (subscales), namely oral symptoms (OS, 6 items), functional limitations (FL, 9 items), emotional well-being (EWB, 9 items), and social well-being (SWB, 13 items). The items of the OS and FL domains were also included into the parents’ questionnaire in order to test their agreement with their child’s report on his oral health troubles (the domains EWB and SWB were not included into the parents’ questionnaire as parents may not know so well the feelings of their children). The items are scored on a 5-point Likert scale ranging from 0 (“never”) to 4 (“every day or almost every day”). In the analysis, the scores for each item were added together to obtain a sum scores of each sub-scale as well as the total CPQ scale. Then, the sum scores were standardized to a percentage score scale of 0 – 100% by dividing the sum score by the maximum score and multiplying by 100. Note that higher sum/percentage scores refer to worse OHRQoL.

The students were also asked to rate their oral health and the extent to which it affected their well-being. For each of these dimensions five sub-items were worded in the following way: “How would you describe health status of the following oral parts: – teeth; – lips; – gum; – oral mucosa; – jaws or joints?” and “Over the last three months, how much your overall life was affected by the conditions of the following oral parts: – teeth; – lips; – gum; – oral mucosa; – jaws or joints?” The responses were scored in the following way: with regard to an oral health rating: 0=excellent, 1=very good, 2=good, 3=fair and 4=poor; with regard to well-being: 0=not at all, 1=very little, 2=somewhat, 3=a lot and 4=very much. The final score computed the maximal score on all the sub-items of each dimension.

The global life satisfaction, or well-being, of adolescents was rated using the measurement technique from the Health Behaviour in School-aged Children (HBSC) study [28]. Children were asked to take a look at the drawn ladder, with steps numbered from zero (“0”) at the bottom to ten (“10”) at the top, with the instruction to suppose the top of the ladder represented the best possible life, and the bottom of the ladder represented the worst possible life. Then they were asked to indicate the step of the ladder at which they would place their lives at present. Thus, the response was scored from 0 to 10.

In the questionnaires, the respondents were asked to rate their malocclusion experience by answering to the question, whether they had ever noticed that their teeth were irregularly grew/situated or they had malocclusion. The answer categories were: 1-yes, I noticed just myself; 2-yes, this was confirmed by dentist; 3- no, I don’t have such disorders. In analyses, the first two categories were combined, thus, two sub-groups of respondents , correspondingly ‘not healthy’ and ‘healthy’, were selected.

During the orthodontic examination, the Index of Orthodontic Treatment Need (IOTN) and the Index of Complexity, Outcome and Need (ICON) were recorded according to the methodology by Richmond (2008).[30] The IOTN measure categorizes the severity of malocclusion based on the relative effect of the various deviant occlusal traits on the longevity of the dentition. The five grades were outlined. Grade 1 recorded small deviations from normal and was categorized as ‘no need of orthodontic treatment’. The deviant occlusal anomalies become more severe in Grades 2, 3 and 4, while grade 5 represented the most severe malocclusion (e.g., impacted teeth, large overjet greater than 9 mm, defects of cleft lip and palate) and was categorized as ‘very great need of orthodontic treatment’. Grade 4 and 5 were regarded as clinical need for treatment. The another indicator of malocclusion, ICON, is based on five components, which are incorporated into calculation of the ICON value by a following regression equation: ICON = (Aesthetic assessment ×7) + (Upper arch crowding/Upper spacing × 5) + (Crossbite × 5) + (Incisor open bite/Incisor overbite ×4) + (Bucal segment antero-posterior ×3). An ICON value of > 43 corresponds to severe malocclusion with a fundamental treatment need [30].

The statistical analysis was performed using the Complex Samples module of the SPSS statistical package (version 21; IBM SPSS Inc., Chicago, IL, 2012) which adjust for the complex cluster-stratified sampling method (schools, classes).[31] All reported p values were from two-sided statistical tests and p values ≤ 0.05 were considered statistically significant.

Missing data of the CPQ items was replaced with the personal mean if a health domain had not more than half blank items, otherwise the record was excluded from analysis. The distributions of the sum score of the CPQ and its domains were examined and found not to be normally distributed. Therefore, median and Interquartile Range (IQR) were used to describe these distributions and to test the null hypothesis that there is no difference in the CPQ scores between the malocclusion and non-malocclusion groups. Due to the same reasons, binary associations between variables were evaluated with non-parametric Spearman correlation coefficient.

A set of test was used for examination of psychometric properties of the CPQ.[32–34] The Cronbach’s alpha was used as measure of internal consistency reliability of the total instrument and its domains. Their values ≥ 0.70 were considered acceptable.[33, 34] Furthermore, other tests of internal reliability (inter-item and item-total correlations) were also investigated. Construct validity of the instrument was tested using Spearman correlation coefficient to assess the association between the scores of total scale as well as its domains and the respondents’ rating of their oral health, oral health related well-being, and global life satisfaction. Discriminant validity was tested by comparing the medians of scores between groups defined by malocclusion traits.

Test-retest reliability test of the instrument was not employed. Instead of this, we assessed agreement between children’s and their parents’ answers to the same questions of the OS and FL domains. The association between child and parental sum scores was assessed by Spearman correlation coefficient, and agreement between two groups of raters was evaluated by the Intra-class Correlation Coefficient (ICC) using two way mixed consistency method and the quadratic weighted kappa.[34] The quadratic weighted kappa was used due to high range of sum scores.

Results

In this study, the sample consisted of 1591 adolescents recruited in the questionnaire survey, among which 911 adolescents were examined by an orthodontist. Participants were divided into three groups by age: 11–14 years (N=307), 15–16 years (N=721) and 17–18 years (N=563) (see Figure 1). In this paper, we looked into the validity of the CPQ scale among adolescents 15–16- and 17–18-year-old, so these groups were more numerous than the reference group of 11–14-year-olds. A total of 927 (58.3%) individuals in the sample were female. The respondents represented both the urban area (68.5%) and rural area (31.5%).

The response rate to the items of the CPQ varied by domains and age groups from 97.1% to 100% with the highest rate (2.9%) of blanks in responses to the items of the SWB domain among 11–14-year-old adolescents. All unanswered questions were restored according to the accepted rules of methods.

The impacts, that is the items scored from 1 (‘1 or 2 times’) to 4 (‘every day or almost every day’), were reported most frequently in the OS domain (“Pain in teeth, lips, jaws or mouth” – 68.8%; “Food stuck in or between teeth” – 62.0%; “Bleeding gums” – 53.6%; “Bad breath” – 45.2%) and in the EWB domain (“Worried that he/she is not as good looking as others” – 38.3%; “Worried that he/she is not as healthy as others” – 28.8%). Comparing three age groups, there were insignificant differences in prevalence of answers to separate items.

Descriptive statistics of the total CPQ and its domains are presented in Table 1. Sum scores were found to be highly skewed and not to be normally distributed in all the health domains with a very noticeable floor effect, especially in the SWB domain. Out of the theoretical range of 0–100% of relative scores, their mean (except OS domain) and median did not exceed 20%. The distributions of individual items and sum scores of the CPQ and its domains did not differ significantly between adolescents of different age groups. In all age groups, the female adolescents than the male tended to report higher scores of the CPQ (poorer OHRQoL). The significant gender difference was observed for the EWB domain (in all age groups) and for the FL domain (in 17–18-year-olds) (data not presented).

Table 1. Summary statistics of the Child Perceptions Questionnaire and its domains, by age groups

CPQ: Child Perceptions Questionnaire, OS: Oral Symptoms, FL: Functional Limitations, EWB: Emotional Well-Being, SWB: Social Well-Being, CI: Confidence Interval, IQR: Range from 1^th to 3^rd quartile, p: test to compare medians across age groups.

Assessments of internal consistency reliability of the CPQ and individual domains are displayed in Table 2. Cronbach’s alpha for the total CPQ was approximately equal to 0.90 in all three age groups indicating good internal consistency reliability. Despite the adolescent age, the lowest values of Cronbach’s alpha were observed in the OS and FL domains being acceptable value of internal consistency reliability. For the domains EWB and SWB, the coefficient ranged from 0.82 to 0.88, indicating good internal consistency reliability in all three age groups. There was a large range of inter-item correlation and inter-total correlation in all domains, but no noticeable difference in these figures was seen comparing age groups of respondents.

Table 2. Internal consistency of the Child Perceptions Questionnaire and its domains, by age groups

CPQ: Child Perceptions Questionnaire, OS: Oral Symptoms, FL: Functional Limitations, EWB: Emotional Well-Being, SWB: Social Well-Being, IIR: Inter-Item Correlation, ITR: Item-Total Correlation.

Table 3 displays the correlation between the CPQ sum scores and overall ratings of oral health and well-being, as well as with global life satisfaction indicating construct validity of the instrument. Across all age groups, total CPQ and all domains were found to be significantly (p<0.01) and positively correlated with oral health and oral well-being. The correlations between the global life satisfaction and the domains were significant too (a negative correlation value indicates that higher life satisfaction is related to lower rating of oral problems).

Table 3. Spearman correlation of the Child Perceptions Questionnaire and its domains with rating of oral health, oral well-being and global life satisfaction, by age groups

CPQ: Child Perceptions Questionnaire, OS: Oral Symptoms, FL: Functional Limitations, EWB: Emotional Well-Being, SWB: Social Well-Being, ^** p < 0.01.

Discriminant validity of the instrument was tested assessing CPQ scores in regard to the orthodontic treatment need (Table 4). Malocclusion traits were recorded in the orthodontic examination and were self-reported in the questionnaire survey. According to the ICON>43 criterion, the need for orthodontic treatment was established in 31.6%, 28.0% and 26.1% (p>0.05) of adolescents aged 11–14, 15–16 and 17–18 years respectively, and, according to the IOTN>3 criterion, the need for orthodontic treatment was established in 29.2%, 33.0% and 36.6% (p=0.049) of adolescents by corresponding age groups. Subjectively orthodontic anomalies (but not necessarily to be treated) were reported by 55.6%, 56.8% and 57.7% (p>0.05) of adolescents in corresponding age groups. Across age groups, there was seen a variation in the gradient of overall CPQ and domain sum scores by malocclusion traits. Adolescents with severe malocclusion (ICON>43 or IOTN>3) suffered a greater impact on their emotional and social well-being than those without malocclusion, however, this relationship was more engaging in groups of adolescents aged 15–16 years and 17–18 years than in 11–14-year-olds. Adolescents who subjectively reported malocclusion in comparison with their ‘healthy’ counterparts indicated significantly greater scores for all domains in the 15–16 age group and for the OS, EWB and SWB domains in the 17–18 age group, while only for the single EWB domain in 11–14 age group.

Table 4. Discriminant validity of the Child Perceptions Questionnaire and its domains for clinically recorded and self-reported malocclusion, by age groups

CPQ: Child Perceptions Questionnaire, OS: Oral Symptoms, FL: Functional Limitations, EWB: Emotional Well-Being, SWB: Social Well-Being, IQR: Range from 1^th to 3^rd quartile, p: Test to compare medians across groups (significant values are in bold).

It was possible to compare records of 1365 parents with records of their children who independently each from other assessed items of the OS and FL domains of child OHRQoL (Table 5). Across all age groups of adolescents, positive significant correlations between parental and children assessments were observed for sum scores of both domains whereas these correlations were evaluated as a moderate level. The moderate values of kappa and ICC also confirmed agreement between child and parental reports. These results suggest on reliability of two subscales of the CPQ in respect of repeatability by two different raters.

Table 5. Agreement between child and parental reports about oral symptoms and functional limitations

OS: Oral Symptoms, FL: Functional Limitations, CI: Confidence Interval, ^** p<0.01.

Discussion

This innovative study was aimed to validate the CPQ among adolescents aged from 15 to 18 in the population survey of orthodontic anomalies in Lithuania. As a reference age group was chosen a group of adolescents aged 11–14 years. The main findings of our study showed that the CPQ instrument is valid to adolescents aged 15–18 years as well as it is valid for adolescents aged 11–14 years.

According to the literature review, most of the OHRQoL studies has focused on 11–14-year-old adolescents rather on older teens. This fact is not surprising because OHRQoL is often the key motive for seeking orthodontic treatment and can considered the measurement for orthodontic treatment need and outcome.[35–37] It also relates to the fact that children of this age group make up the majority of orthodontic patients. During this age period, the whole body, including the jaws, develops intensively. So the orthodontic anomalies that has arisen in this age period can be successfully corrected, even it is assumed that it is not possible to complete a full course of orthodontic treatment before the premolars and second permanent molars have erupted at dental age 12 or 13 years.[38] Therefore, it increasingly recognized that more and more teenagers and young adults are seeking correction of their malocclusion, if this could not be done in early adolescence. Thus, orthodontists should be aware that such patients might expect orthodontic treatment to provide not only improved oral functioning and health but also enhancement of aesthetics, self-esteem and social life.[39]

More recently, a number of tools to measure OHRQoL has been developed and used in assessing an association between severity of malocclusion and patients’ perception of their oral health status. The standard CPQ_11–14 was developed to measure the OHRQoL among adolescents between the ages of 11 and 14 years in Canada [12] and soon validated in many languages and cultures, including such as China,[40] India,[16] Korea,[21] Saudi Arabia,[22] and others. The questionnaire was also adapted to Lithuanian adolescents (Kavaliauskienė A, Šidlauskas A, Zaborskis A., 2018. Manuscript under review). After examination its psychometric properties, the Lithuanian version of CPQ_11–14 showed good internal consistency, discriminant validity and acceptable agreement between children and parental responses to the same items. However, there are few studies in which the well-known CPQ would be used to measure OHRQoL in adolescents over the age of 14 years.[24–27] So we felt the lack of an instrument suitable for measuring OHRQoL throughout all adolescence period as the investigation of orthodontic anomalies among adolescents of Lithuania was targeted to the population aged from 11 to 18 years.

Adolescence is marked as a transitional period of rapid developmental changes and often perceived as a time of changing trajectories and health across the life course.[29, 41] It is reasonable that adolescents of the older stage are very different from those of the younger age stage. Older teen like young adults are capable of abstract thinking, reasoning about the past events and relating them with good or bad consequences in health.[41] Based on this assumption, we hypothesised that the CPQ instrument to measure OHRQoL among 15–18-year-old adolescents could be as much valid as it was valid for adolescents aged 11–14 years. The hypothesis was confirmed by all tests traditionally employed in questionnaire validation procedures.

Initially, it was found that the distribution of CPQ sum scores and its ratio between males and females did not differ significantly across age groups of adolescents. This may suggest that the impact of malocclusion over all adolescence does not decrease as age increases. However, our study was limited to adolescents up to 18 years, while other studies among adolescents and young adults demonstrated a negative association between age and impact on quality of life due to malocclusion.[42] Exploring gender differences, regardless of age, girls were found to be more emotionally concerned with their teeth aesthetic or, alternatively, boys may be less self-conscious about their appearance. Similar findings were reported by Peres et al.[43] who found females adolescents having greater dissatisfaction with their dental appearance (Peres et al., 2008) but in the other studies the gender difference was not established significant.[44]

Next, a good internal consistency reliability of the total CPQ with Cronbach’s alpha equal to 0.90 was established in both 15–16 and 17–18 age groups and was as high as in the 11–14 age group presented in our study or reported by other authors.[12] Despite the adolescent age, the alpha coefficient for the EBW and SWB domains was also greater than 0.80. Similarly to the other CPQ validation studies,[16,17] the lowest values of Cronbach’s alpha were observed for the OS and FL domains. Many methodologists[33, 34] recommend a minimum alpha coefficient between 0.65 and 0.8 (or higher in many cases), thus the obtained values that varied from 0.66 to 0.73 could be considered acceptable for these domains in all age groups.

The correlation coefficients in the construct validity analysis were significant in all age groups. So construct validity of the questionnaire in survey of older adolescents was in any case as high as that found among the youngest adolescents. Compared with other studies,[17, 20] which considered the CPQ valid for the population being assessed, the correlations between the respondents’ global rating of oral health and well-being and the CPQ sum scores outlined in our study were higher in many cases. The construct validity of the questionnaire for all age groups was also confirmed by the significant relationship between CPQ of sum scores and the adolescent’s global life satisfaction that is an essential dimension of young people well-being.[45] The relationship indicated that adolescents, regardless of their age, were more likely to report lower global life satisfaction when they felt any oral health-related complaints.

A discriminant validity of the CPQ was examined comparing the distribution of the CPQ scores between groups of adolescents with regard to their subjectively perceived and objectively measured orthodontic status. We found that malocclusion experience has a negative impact on the adolescents’ perceptions but its strength (difference in the CPQ distribution) differed by the method of definition of severity of malocclusion and the age of adolescents. Adolescents who reported malocclusion complaints themselves (were ‘not healthy’ in respect to orthodontic status) were more likely to provide greater perceptions of oral health-related problems than adolescents with clinically defined need for orthodontic treatment. This finding shows that a malocclusion can be perceived differently by the affected person, and a person’s degree of awareness of their malocclusion might not be related to its severity.[42] The findings of the study also suggest that young adolescents when evaluating their malocclusion by orthodontist mainly suffer emotional problems, as their OHRQoL might not be related so much with severity of malocclusion. Previous studies examining the impact of malocclusion on children (young adolescents) oral health-related perceptions have been also equivocal. Systematic reviews of literature on this issue reported studies that claimed evidence for a clear inverse association of malocclusion with OHRQoL.[46–48] At the same time, they reported studies with no clear relationship indicating that the strength of the association differed depending on the age of studied sample and cultural environment. In part, our findings confirmed this suggestion indicating that in older adolescents clinically defined need for orthodontic treatment may have a significant effect on perceived OHRQoL in more domains. Therefore, in respect of discriminant validity, the CPQ had no disadvantages both in the younger and older adolescents groups.

Finally, test-retest reliability of the CPQ instrument was not assessed due to organizational and logical reasons. With regard to organizational reasons, a retest appeared problematic as organizing another dental examination session at all of the schools participating in our study would have a complex endeavour. With the respect to logical reasons, a retest of the same students was replaced with an alternative analysis that included comparison of children’s and their parents’ answers to the same questions of the OS and FL sub-scales. Such comparison was not performed for the EWB and SWB sub-scales, because some parents may have limited knowledge about their children’s OHRQoL, particularly the impact on social and emotional well-being.[49]. As in other similar studies in this field,[49–51] findings of the present study confirmed an agreement between child and parental reports suggesting on reliability of the PCQ in respect of its repeatability by two different raters.

As an advantage of this study may be the fact that data were collected in cross-sectional population survey of representative adolescents’sample but not within sample of patients attending dental treatment as in several studies.[12, 24,52] The adolescents completed their questionnaires at school anonymously without any influence of their parents’ and dentist’s opinion, thus, adolescents could express their own feelings towards their QoL. That was an important condition comparing children’s and their parent responses, as well as their perceptions and orthodontic measures. This is the first study on OHRQoL among adolescents ever to be carried in Lithuania.

In terms of the limitations of our study, we conducted oral examination with respect to orthodontic disorders without assessing of dental cariousness and periodontal conditions that would have a considerable impact on OHRQoL in adolescence.[53–55] The sample was not also homogenous with respect to previously conducted orthodontic treatment. So, the possible confounding effects of these conditions on the participants’ OHRQoL were not considered in the analysis. Another limitation of the research is that we worked on the “long form” (37 items) of the original CPQ_11–14 together with other scales, including such as eating behaviour and self esteem. Our experience from the HBSC study [29] showed that an increase of number of items in the questionnaire may affect respondent’s accuracy providing inaccurate answers, which may, consequently, reduce reliability of the tested scale. Finally, test-retest reliability of the CPQ instrument was replaced with an alternative analysis that included comparison of children’s and their parents’ responses to the same questions of the OS and FL domains. This approach is not free from limitations, especially in relation to its accuracy because children and parents may not share the same views about illness and health.[56]

Conclusions

The Lithuanian version of the CPQ showed good internal consistency and construct and discriminant validity in all age groups of adolescents, consequently, it seems to be a valid instrument for measuring OHRQoL among adolescents aged 15 to 18 years as well as among adolescent aged 11 to 14 years.

Acknowledgements: The authors would like to thank all reviewers for their thoughtful comments in the manuscript. A gratitude is also expressed to the schoolchildren and their parents for their participation in this study as well as to the teachers for their help during fieldwork.

Competing interests: The authors declare that they have no competing interests.

Funding: This study was funded by the Lithuanian University of Health Sciences.

Abbreviations

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DOI: 10.31038/JCRM.2018112

Abstract

Background: The present study focuses on the evaluation of the protective effect on retinal neovascularization exerted by melanocortin receptor 1–5 (MCR 1–5) agonists in a mouse model of proliferative retinopathy induced by intravitreal injection of vascular endothelial growth factor 165 (VEGF165).

Methods: Intravitreal injections of the MCR1 agonist BMS-470539 and of the MCR5 agonist PG-901 were performed 7 days before the VEGF165 injection and 1 day after VEGF165 injection. Fluorescein angiography (FAG), immunohistochemical and biochemical analysis were performed at different time points during the follow up.

Results: FAG showed a marked vascular tortuosity accompanied by retinal neovascularization in mice receiving VEGF165 intravitreal injection and the absence of neovascularization in mice treated with MCR 1–5 agonists, confirmed by CD34 antigen immunostaining. The biochemical analysis of retinal VEGFR2 gene and protein expression showed that MCR 1–5 agonists significantly reduced VEGFR2 levels, increasing in parallel retinal mir-150 expression.

Conclusions: According to these evidences, we propose that intravitreal injections of melanocortin receptor agonists up-regulate mir-150 expression, and this consequently reduces VEGFR2 expression levels with the endpoint being the blockade of VEGF165 induced retinopathy.

Keywords

intravitreal injection; neovascularization; melanocortin receptors; retina; VEGF;

Introduction

Some of the major eye diseases that cause blindness worldwide are related to intraocular neovascularization. Among these conditions we must consider age-related macular degeneration (AMD), retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vascular occlusions (RVO), which are all linked to ocular ischemia that causes progressively elevated intraocular levels of vascular endothelial growth factor (VEGF) with secondary retinal, subretinal or intravitreal neovascularization leading to progressive visual impairment [1–4].

VEGF is the main player in neovascularization during proliferative retinopathy, it plays a key role as a potent inducer and modulator of proliferation, permeability and survival of endothelial cells. Four VEGF proteins are known VEGFA, -B, -C, -D that act through interaction with the VEGFR1, -2, and -3 receptor subtypes [5,6]. Among these, VEGFR2 is the main receptor that modulates endothelial cell proliferation, migration and in vivo angiogenesis [5,6]. Instead, VEGF-C and -D are involved in the modulation of cell adhesion and cell migration and in extracellular matrix degradation; VEGF-B and PIGF are involved in lymphangiogenesis [7]; VEGF-A promotes the process of neovascularization and increased vascular permeability. In particular, VEGF164(165) isoform is primarily implicated in the pathogenesis of major eye diseases that recognize ocular ischemia as a prime mover [8–11].

The introduction in clinical practice of various anti-VEGF drugs injected intravitreally (bevacizumab, ranibizumab, aflibercept) [12] for the management of the main eye diseases has positively changed the evolution and prognosis of diseases such as AMD,ROP, DR and RVO. In fact, an open research of new strategies and tools has been undertaken nowadays.

Previous studies by Rossi et al.,(2016) indicated that one of these strategies could be that of the modulation of the activity of the retinal endogenous melanocortin pathway and receptors. Indeed, he showed that an agonism at melanocortin receptor 1,5 (MCR1–5) attenuates retinal damage in a mouse model of streptozotocin (STZ) induced diabetic retinopathy (DR) by modulating the pattern of cytockine and chemochine expression [13]. Subsequently, Maisto el al. evidenced that this MCR1–5 agonism restores antioxidant enzyme levels in high glucose primary retinal cells and protects the photoreceptors from the glucose induced damage [14]. These receptors, therefore, appeared to be good candidates for targeted therapy of diabetic retinopathy and possibly its proliferative form as well. Noteworthy, these melanocortin receptors have already been accredited to control a wide range of biological activities including inflammation, oxidative stress, energy homeostasis, sexual function, pain and immune response [13].

Based on this experience, the present study aimed to evaluate the protective effect exerted by MCR 1–5 agonists on retinal vascularization in a mouse model of retinopathy induced by intravitreal injection of VEGF165.

Materials and Methods

BMS-470539 and PG-901 were used as MCR1 and MCR5 agonist respectively [15] and were supplied by Professor Grieco (Pharmacy Department, University of Naples Federico II).

Animals

All the experimental procedures were performed according to Animal Care Ethical Committee of the University of Campania, in accordance with ARVO Statement for the use of Animals in Ophthalmic and Vision Research.

C57BL/6 mice (Harlan, Italy) aged 7 to 10 weeks were housed in standard cages with 24-hours light-dark cycle, humidity and temperature automatically controlled. C57BL/6 mice were divided into 2 settings: the first setting was composed of 2 groups (n = 12 animals per group), treated respectively with sham intravitreous injections of 5μl balanced saline (0.1 ml) and 5μl intravitreal injection of VEGF165 (10μg, Sigma-Aldrich, Italy) [10,11] on day 0. The two groups were monitored by FAG in order to ascertain the time course of the vascular modifications. The second setting was composed of the following 2 groups (n = 8 animals per group): (1) mice treated with 5μl intravitreal injection of the MC1 receptor agonist BMS-470539 (33 μmol) [16] 7 days before the injection of VEGF165 and 1 day after VEGF165 injection; (2) mice treated with 5μl intravitreal injection of the MC5 receptor agonist PG-901 (7.32 nM) [17] 7 days before the VEGF165 injection and 1 day after VEGF165 injection, following pioneering in house experience.

Intravitreal injections

Mice were anesthetized by pentobarbital (45 mg/kg in saline i.p.). Tropicamide (5%) was instilled into the right eye of each animal, in order to induce papillary dilation, and tetracaine (1%) was topically applied for local anaesthesia. Physiological saline, VEGF165 or MC receptor agonists (5 μL volume) were injected intravitreally into the right eye using sterile syringes fitted with a 30-gauge needle (Microfine; Becton Dickinson AG, Meylan, France), as previously described [10,11,18]. Briefly, a transconjunctival 30-gauge, 1.5-mm needle was introduced behind the limbus in the superior temporal quadrant. When the needle was visualized at the vitreous, the various substances were injected in the various experimental groups. After the needle was withdrawn, the scleral entrance site was compressed with a cotton tip for 1 min and a topical antibiotic ointment was applied.

The following MC receptor ligands were used at the indicated doses as selected from the reported publications: BMS-470539, 33 μmol; PG-901, 7.32 nM [16,17].

Fluorescein Angiography (FAG)

For FAG assessment the animals were restrained without anesthesia by grasping firmly at the base of the tail and grasping the scruff of the neck. FAG was performed by using a Topcon TRC-50DX apparatus (Topcon, Japan) following intraperitoneal injection of 10% fluorescein sterile solution (1 mL/kg body weight, AK-Fluor; Akorn Inc., USA). Fundus photographs were captured in order to display the retinal vasculature and to evaluate the early typical alterations of microangiopathy.

Follow-up

In the first setting, FAG was performed after papillary dilatation on all the animals receiving the intravitreal injection of balanced saline or VEGF165 in order to detect the appearance of early vascular disorders. At 7 the day time point, when FAG evidenced neovascularization in VEGF165 mice, 4 animals out of the 12 for each group were randomly chosen, sacrificed and assayed for VEGFR2 and other biochemical parameters. The remaining 8 animals were monitored with FAG at the 14 day time point, when neovascularization was still evident. Again, 4 mice out of the 8 were randomly chosen, sacrificed and assayed for same biochemical parameters as at 7 day time point. The remaining 4 animals were monitored by FAG at 28 days, when no signs of neovascularization were evidenced.

In the second setting, mice receiving intravitreal injection of BMS-470539 and PG-901 seven days prior and one day after VEGF165 were monitored by FAG at 7 and 14 day time points. At 7 days, 4 animals out of the 8 for each group were randomly chosen, sacrificed and assayed for VEGFR2 and other biochemical parameters. The remaining 4 animals were sacrificed and assayed for same biochemical parameters at 14 day time points.

For the biochemical analysis, the eyes were displaced forward by placing curved forceps around the posterior part and cut in two halves. On one half of each eye the cornea was cut using a sharp blade or scalpel, and the retina was squeezed through the cut together with residual pigment epithelium and lens by applying gentle pressure with the forceps. Dissected retina was placed in cooled PBS, freed from non retinal tissue using the forceps, and immediately frozen in liquid nitrogen and stored at −80°C for subsequent biochemical analysis. The other half of each eye was fixed by immersion in 10% neutral buffered formalin and paraffin-embedded for immunohistochemistry.

Immunohistochemistry

CD34 antibody was used as marker of neo-angiogenesis (sc-74499 Santa Cruz Biotec, USA). The ocular tissues were incubated with the primary antibody, washed in PBS, and incubated with secondary antibody. The ocular samples were analyzed by an expert pathologist (variability 6%); every sample was visualized at 200x magnification. The number of CD34 positive particles per area was analyzed in 20 microscopic fields and expressed as percentage of positive stained area/total area.

Total RNA isolation; mir-150 and VEGFR2 mRNA expression levels

The extraction of total RNA, including small RNAs, was performed according the MiRNeasy Minikit protocol (Qiagen, Italy). Syn-cel-miR-39 miScript miRNA Mimic 5 nM (Qiagen, Italy) was added to each sample before the extraction as internal control. The total RNA concentration and integrity were determined by Nanodrop ND-1000 UV spectrophotometer (Nano-Drop® Technologies, Thermo Scientific, USA).

The qRT-PCR analysis for mir-150 was performed on Mastercycler personal (eppendorf, Germany) using miScript II Reverse Transcription Kit (Qiagen, Italy) and on CFX96 Real Time

System cycler (BioRad, Italy) using miScript SYBR Green Kit (Qiagen, Italy) and miScript Primer Assays specific for Syn-cel-miR-39 and mir-150 (Qiagen, Italy).

VEGFR2 gene expression was measured by RT-PCR amplification on Mastercycler personal (eppendorf, Germany). cDNA synthesis was obtained using SuperScript III Reverse Transcriptase Kit (Invitrogen, USA) starting from 200 ng of total RNA. Aliquots of 2 μl cDNA were transferred into a 25 μl PCR reaction mixture containing dNTPs, MgCl2, reaction buffer, specific primers and GoTaq Flexi DNA polymerase (Promega, USA). Sequences for the mouse VEGFR2 mRNA from GeneBank (DNASTAR INC., USA) were used to design specific primer pairs for RT-PCR (Microtech, Italy). The housekeeping gene chosen was hypoxanthine-guanine phosphoribosyl transferase (HPRT). PCR products were resolved into 2.0% agarose gel. A semiquantitative analysis of VEGFR2 mRNA levels was carried out by the Gel Doc EZ UV System (Bio-Rad, USA).

Relative quantification of gene expression was normalized to Syn-cel-miR-39 for mir-150 and to HPRT for VEGFR2, using the 2^−ΔΔCt method.

Enzyme-Linked Immunosorbent Assay (ELISA)

Mouse VEGFR2/Flk-1 Quantikine ELISA Kit (R&D System, UK) was used in the homogenate of dissected retina in order to assess the ocular protein VEGFR2 expression levels, according to the manufacturer’s protocol.

Statistical Analysis

All values are expressed as mean ± standard error of the mean (SEM) of = 4 mice. Statistical analyses were assessed either by Student’s -test (when only two groups were compared) or one-way analyses of variance (ANOVA), followed by Dunnett’s post hoc test (more than two experimental groups). < 0.05 was considered statistically significant.

Results

Melanocortin Receptor1–5 activation prevents the development of retinal neovascularization

No significant vascular alterations were observed in the control group at any of the various time points (Figure 1a and Figure 2a). All the mice treated only with intravitreal injection of VEGF165 developed vascular tortuosity accompanied by retinal neovascularization at 7 days that persisted up to 14 days post-treatment, while it disappeared at the day 28 time point (Figure 1b and Figure 2b).

Figure 1. (a) Representative FAG images of a control eye. There are no vascular alterations at the various time points. (b) Representative FAG images of mice treated with intravitreal injection of VEGF165: a marked vascular tortuosity accompanied by retinal neovascularization is present at 7 days and persists until 14 days, while it disappears at the 28 day time point. Arrows indicate the formation of neovessels.

Figure 2. (a) Representative FAG images of a control eye (as Figure 1a). (b) Representative FAG images of mice treated with intravitreal injection of VEGF165 (as Figure 1b). (c) Representative FAG images of mice treated with intravitreal injection of BMS-470539 (33 μmol) 7 days before and 1 day after the injection of VEGF165: the mice did not develop retinal neovascularization during the follow-up but only an irregular vessel caliber appeared at 7 days and persisted until the end of the follow-up at 14 days. (d) Representative FAG images of mice treated with intravitreal injection of PG-901 (7.32 nM) 7 days before and 1 day after the injection of VEGF165: the mice did not develop retinal neovascularization but only an irregular vascular course and caliber was observed, accompanied by an increase in vascular permeability, resulting in dye leakage evident at 7 days and persisting until the end of follow-up at 14 days. Arrows indicate the formation of neovessels; arrowheads indicate vascular leakage.

Intravitreal injections of MC1 receptor agonist BMS-470539 (33 μmol) or MC5 receptor agonist PG-901 (7.32 nM) protect retinal vasculature, as demonstrated by FAG. In particular, mice treated with BMS-470539 (33 μmol) 7 days before and 1 day after the injection of VEGF165 did not develop retinal neovascularization during the follow-up, only an irregular vessel caliber appeared at 7 days and persisted until the end of the follow-up at 14 days (Figure 2c). The mice treated with PG-901 (7.32 nM) 7 days before and 1 day after VEGF165 injection did not develop retinal neovascularization during the follow-up, only an irregularity of the vascular course and caliber was observed. However, this was accompanied by an increase in the vascular permeability resulting in dye leakage which was evident at the 7 day time point and persisted until the end of follow-up at 14 days (Figure 2d).

Melanocortin MC1,5 receptor agonists reduce the retinal expression of the hematopoietic endothelial progenitor cell antigen CD34.

In order to confirm the development of retinal neovascularization at 7 and 14 days after VEGF165 intravitreal injection, immunohistochemistry for CD34 was performed in 4 mice per group randomly chosen. At 7 and 14 days, the analysis showed a significant (P<0.01) increase of CD34 immunostaining in mice receiving intravitreal VEGF165 compared to the mice receiving saline solution (Figure 3–4). In contrast, a marked decrease in the percentages of CD34 positive stained area/total stained area was observed at both time points in retinal tissue of mice receiving intravitreal injections of the MC1 agonist BMS-470539 (40.0 ± 5.15% at 7 days; 31.8 ± 4.3% at 14 days) or the MC5 agonist PG-901 (46.7 ± 4.7% at 7 days; 28.9 ± 4.7%) as calculated against the values quantified in VEGF165 mice (Figure 3 and Figure 4).

Figure 3.(a) Representative immunohistochemistry for CD34 in the retina of mice treated with vehicle, VEGF165(10 μg/5 μl), BMS-470539 (33 μmol) + VEGF165 and PG-901 (7.32 nM) + VEGF165, 7 days after VEGF165 intravitreal injection. BMS-470539 and PG-901 reduce CD34 immunostaining compared to rats receiving only VEGF165. Scale bar, 50 m. 200x magnification. (b) Graph showing the percentage of the total positive stained area for CD34 per total area analyzed at 200x magnification. Values are mean ± SEM of = 4 observations for each group. ** P < 0.01 vs Control; °° P < 0.01 vs VEGF165.

Figure 4. (a) Representative immunohistochemistry for CD34 in the retina of mice treated with vehicle, VEGF165(10 μg/5 μl), BMS-470539 (33 μmol) + VEGF165 and PG-901 (7.32 nM) + VEGF165, 14 days after VEGF165 intravitreal injection. BMS-470539 and PG-901 reduce CD34 immunostaining compared to mice receiving only VEGF165. Scale bar, 50 m. 200x magnification. (b) Graph showing the percentage of the total positive stained area for CD34 per total area analyzed at 200x magnification. Values are mean ± SEM of = 4 observations for each group. ** P < 0.01 vs Control; ° P < 0.05 vs VEGF165.

Melanocortin MC1,5 receptor activation decreases retinal VEGFR2 gene expression

qRT-PCR analysis of VEGFR2 gene expression in the retina showed that in mice receiving a single intravitreal injection of BMS-470539 and PG-901 7 days prior and first day after VEGF165, BMS-470539 and PG-901 significantly down-regulate VEGFR2 gene expression levels both at 7 and 14 day (P < 0.01) time points compared with the group receiving saline only (Figure 5a).

Figure 5. (a) RT-PCR analysis for VEGFR2 gene expression in retinal tissue 7 and 14 days after VEGF165 intravitreal injection. Treatment with BMS-470539 (33 μmol) and PG-901 (7.32 nM) significantly decreases VEGFR2 mRNA expression levels at 7 and 14 days, compared to mice receiving saline only. Results are expressed as mean ± SEM of = 4 observations for each group. ** P < 0.01 vs Control. A.U. = arbitrary units. (b) qRT-PCR analyzing mir-150 expression in retinal tissue 7 and 14 days after VEGF165 intravitreal injection. At 7 and 14 days, BMS-470539 (33 μmol) and PG-901 (7.32 nM) significantly increase mir-150 expression levels compared to mice receiving saline only. Values are mean ± SEM of = 4 observations for each group. ** P < 0.01 vs Control. A.U. = arbitrary units.

Melanocortin MC1,5 receptor activation increases mir-150 expression levels

Intravitreal injection of the MC1 agonist BMS-470539 and the MC5 agonist PG-901 7 days prior and first day after VEGF165, significantly up-regulate retinal mir-150 expression levels at 7 and 14 days (P < 0.01) time points compared with the group receiving saline only (Figure 5b). The single injection of VEGF165 into the vitreous does not affect qRT-PCR analysis of the retina for mir-150 expression levels (Figure 5b).

Intravitreal injection of melanocortin MC1,5 receptor agonists reduces retinal VEGFR2 protein levels

To confirm gene expression data, retinal VEGFR2 protein levels were measured by ELISA assay, and fitting with RT-PCR results, VEGRF2 protein levels significantly decreased after BMS-470539 and PG-901 (P < 0.01, Figure 6) at 7 and 14 time points.

Figure 6. Retinal VEGFR2 protein expression levels (ng/ml) analyzed by Elisa assay 7 and 14 days after VEGF165 intravitreal injection. BMS-470539 (33 μmol) and PG-901 (7.32 nM) significantly decrease VEGFR2 protein expression levels at 7 and 14 days, compared to mice receiving saline only. Results are expressed as mean ± SEM of = 4 observations for each group. * P < 0.01 vs Control.

Discussion

This paper describes for the first time the protective role of the MC1–5 receptor agonists in a mouse model of retinopathy. The paper shows that a single intravitreal injection of the MC1 receptor agonist BMS-470539 or MC5 receptor agonist PG-901 7 days before and 1 day after the injection of VEGF165 into the vitreous prevents the development of retinal neovascularization as viewed by FAG.

Based on the results obtained in the present study, melanocortins and their receptors seem to be good candidates for treating VEGF dependent neovascularization of the retina having shown pivotal control of the development and progression of new vessels into the retina in the mouse model of retinopathy adopted here, although the single concentration of agonist used is a limitation of any cross-molecule comparison.

Melanocortins are endogenous peptides that possess a wide range of biological activities, including promotion of the resolution phase of inflammation effects in experimental models of inflammation-based diseases [19–27]. Their receptors are ubiquitously expressed into the eye and called MC1, MC3, MC4 and MC5. Rossi et al., in an experimental paper published in 2016 [13] showed that among the four melanocortin receptor subtypes expressed in the retina the MC1 and MC5 are the two involved in a positive control of diabetic retinopathy. Indeed, the activation of these receptors by specific agonists induces a series of downstream events such as reduction of inflammatory cytokines IL-1α, IL-1β, IL-6, chemokines MIP-1α, MIP-2α, MIP-3α and M1 macrophage that lead to reduced retinal damage. To this data, we add here a reduced retinal derangement from MC1,5 agonism exerted through reduced proliferation of new vessels in this tissue.

From the molecular point of view we noted that these structural changes were paralleled by biochemical alterations such as reduction in the expression of the VEGFR2 receptor within the retina both at 7 and 14 day time points. The down-regulation of VEGFR2 was caused by selective stimulation of MC1–5 receptors. Indeed, at the same time points it was not present in the control group treated with VEGF165 only. Thus, neovascularization induced by VEGF165 within the retina is hampered by selective activation of endogenous melanocortin MC1,5 receptors and reduced VEGFR2 receptor expression.

How this happens would require further investigation, but a novelty is provided here by the strict correlation between functional improvement of retinal vessel form, VEGFR2 expression and retinal expression of the miRNA mir-150. This mir-150, reported to attenuate retinal vascular overgrowth in diabetic mice through a down-regulation of VEGFR2 gene expression [27], is for the first time related to melanocortin MC1,5 receptors stimulation by the present study. MC1,5 receptors stimulation increases the expression of the retinal mir-150, consequently down-regulating VEGFR2 gene expression and protein levels. This finally improves the early vascular disorders induced by VEGF165 alone. Mirror of this mechanism, CD34 labeling within the retina was reduced. Particularly, CD34 labeling was increased by VEGF165 both at 7 and 14 day time points, confirming the early vascular disorders evidenced by FAG, while it was significantly reduced after MC1,5 agonists.

Noteworthy, CD34 is a transmembrane phosphoglycoprotein well known marker of hematopoietic endothelial progenitor cells (EPCs) proliferation [28], and shown to promote the formation of pathological, invasive vessels during neovascularization in a mouse model of oxygen-induced retinopathy [29]. Interestingly, the differentiation of these cells from the hematopoietic stem cells (HSCs) is associated with the up-regulation of vascular endothelial growth factor receptor 2 (VEGFR2), after which the HSCs are considered endothelial progenitor cells (EPCs), effectively [30–32].

Conclusions

In conclusion, there is positive interplay exerted through increase of mir-150 between retinal melanocortin MC1,5 receptors and VEGFR2 receptor in the model of VEGF165 induced retinopathy in order to prevent the local neoangiogenesis and retinopathy. From the translational point of view, it is tempting to speculate that the enhancement and reinforcement of the action of the endogenous melanocortins pathway can be a tool to prevent the development of retinal neovascularization, probably by amplifying its anti-inflammatory, anti-oxidant and anti-proliferative role. The potential systemic toxicity of the agents used in this study has not been investigated and would require deepening in order to reinforce their efficacy.

Acknowledgments: The authors are grateful to Professor Paolo Grieco (Pharmacy Department, University of Naples Federico II) for his support and acknowledge the precious funding of MIUR PRIN 2015 project.

Conflicts of Interest: The authors declare no conflict of interest.

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DOI: 10.31038/JCRM.2018111

Introduction

Diabetes affects more than 30 million people in the United States with type 2 diabetes accounting for 90–95% of cases (www.diabetes.org). Annual medical expense and disease-related societal burden from diabetes cost more than $245 billion. Most of the diabetic -related disabilities are from chronic diabetic complications in the cardiovascular, renal, retinal, and nervous systems. Among these, diabetic polyneuropathy occurs in approximately 60% of all diabetic patients [1, 2]. Diabetic polyneuropathy causes significant public health burden, serving as the leading cause of diabetes-related hospital admissions and non-traumatic amputations [1, 3, 4].

Patients with diabetic polyneuropathy frequently suffer from painful symptoms, termed as painful diabetic neuropathy (PDN) [2]. Clinically, PDN typically presents with length-dependent spontaneous pain with a combination of burning, tingling, electric-like, or achy sensations. It begins in the feet and extends proximally over time with bilateral and symmetric stocking distribution. Similar distal to proximal pattern of painful symptoms could develop at a later stage in the upper extremities. Patients with PDN also experience induced-pain, such as allodynia and hyperalgesia. Allodynia occurs when regularly innoxious stimuli, such as light touch, become painful, whereas hyperalgesia is increased nocuous sensitivity to painful stimuli, like pin prick. Despite the high morbidity of PDN [5, 6], the underlying molecular mechanisms of PDN are poorly understood [7]. Without targeting the key pathology that leads to the development of PDN, currently accepted medical approaches are only partially successful and are often ineffective [5, 8]. Inadequate control of PDN has significantly reduced quality of life for patients with diabetes [5, 6, 8]. In addition to suffering from painful symptoms, patients with PDN frequently develop insomnia, depression and anxiety, decreased mobility, psychomotor impairment and loss of work [5, 6, 8]. Clearly, more mechanism-specific therapies are urgently needed to effectively manage this common and important health problem.

Current treatment guidelines

Over the last three decades, basic science and clinical studies have generated significant amount of evidenced-based data to establish treatment guidelines for PDN. The 2006 and 2010 guidelines from the European Federation of Neurological Societies task Force (EFNS) [9, 10] and the 2011 guidelines from the American Academy of Neurology (AAN), the American Association of Neuromuscular and Electrodiagnostic Medicine, and American Academy of Physical Medicine and Rehabilitation [11] are the most thorough and up-to-date guidelines on this topic. Several class drugs including α2δ calcium channel antagonists (gabapentin, and pregabalin), anti-convulsants, tricyclic anti-depressants (TCAs), serotonin-norepinephrine reuptake inhibitors (SNRIs), opioids, and various other treatment modalities are discussed and recommended according to the quality of their supporting data. Each published clinical trial is classified according to its level of evidence, following guidelines such as the “AAN classification of recommendations” (www.AAN.com). Although there could be variations among these guidelines, trials deemed as class I are considered to have the highest quality of evidence with lowest risk of bias to support the application of the study drugs. The quality of evidence is decreased in high leveled classes; with class IV evidence has the highest bias potential and lowest supporting evidence for clinical use.

Level A treatments are strongly recommended with class I evidence or consistent findings from multiple studies of class II, III, or IV. They are recommended in clinical practice unless a clear and compelling rationale for an alternative approach is present. Level B treatments are with levels II, III, or IV evidence and findings are generally consistent. Generally, clinicians should follow this recommendation but should remain alert to new information and sensitive to patient preferences [12]. Level C, D, and U treatments do not have sufficient evidence to support their clinical practice.

The use of gabapentin, pregabalin, TCAs (such as amitriptyline), SNRIs (venlafaxine and duloxetine) are supported by EFNS with level A recommendation. In addition, controlled-release oxycodone is recommended by EFNS as effective with level A recommendation based on two class I studies. Tramadol alone or with acetaminophen were listed by the EFNS as level A effective treatments based on two class I studies. Level B recommendations from EFNS include Dextromethorphan (an agonist of N-methyl-D-aspartate receptor, 400 mg/d), Topical capsaicin 0.075% ointment that activates the transient receptor potential cation channel subfamily V member (TRPV) 1, isosorbide dinitrate spray (a vasodilator), type A botulinum toxin (BTX-A, blocks acetylcholine release) and levodopa (a dopamine precursor) [9, 10].

The guideline from AAN supports the use of pregabalin with level A recommendation. Gabapentin, sodium valproate (an anti-convulsant), venlafaxine, duloxetine, amitriptyline, dextromethorphan, morphine sulfate, tramadol, oxycodone, capsaicin 0.075% ointment, isosorbide dinitrate spray, electric stimulation and percutaneous nerve stimulation are presented as level B recommendations. Other anti-convulsants such as oxcarbazepine and lamotrigine; clonidine (an a2 adrenergic  agonist), pentoxifylline (a xanthine derivative), magnetic field treatment, low-intensity laser therapy, and Reiki therapy are not recommended [11].

Emerging treatments

One of the most promising new gene therapies for PDN is a DNA-based therapy using a plasmid DNA that contains the human hepatocyte growth factor (HGF) gene (VM202). VM202 enhances local expression of HGF to promote microvasculature growth and regenerate peripheral nerves to improve symptoms of PDN. A phase 3 study showed that PDN patients receiving 8 mg of VM202 injection per leg improved in all efficacy measures with 48.4 % of the patients experienced at least a 50% reduction in mean pain score in the treated group compared with 17.6 % in the placebo group after 3 months [13]. However, this analgesic effect was not statistically significant at 6 and 9 months. The study also demonstrated significant improvement in the brief pain inventory and the questionnaire portion of the Michigan Neuropathy Screening Instrument. Interestingly, the researchers noted that the largest reductions in pain were found among patients not on pregabalin or gabapentin. In addition, there were no significant adverse events attributable to VM202 and this treatment was deemed safe and well tolerated [13].

A network meta-analysis accumulated 25 randomized controlled trials for studying the effects of capsaicin 179 mg cutaneous patch (capsaicin 8% patch) on PDN. It was concluded that capsaicin 8% patch was significantly more effective than placebo with ≥30% pain reduction in PDN patients. In addition, capsaicin patch was statistically more efficacious when compared with pregabalin and gabapentin. It had similar efficacy while being compared with duloxetine [14].

Nerve growth factor (NGF) has been established as an essential factor for the development of nociceptive nerves. It also mediates the development of mechanical allodynia in animal model of type 2 diabetes [15]. Clinical trials using NGF neutralizing antibodies, including tanezumab and fulnatumab, have been reported with positive results for treating PDN. In the study that examined the effects of tanezumab in PDN, test subjects received subcutaneous tanezumab 20 mg or placebo on Day 1 and Week 8. Mean PDN pain reduction from baseline to Week 8 was greater with tanezumab vs placebo. However, differences in Patient’s Global Assessment of DPN were not significant [16].

Fulranumab, a fully human monoclonal anti-NGF antibody was also tested for PDN. In a phase II, double-blind, placebo-controlled trial, patients with moderate to severe PDN were randomized to treatments with fulranumab (1, 3, or 10 mg) or placebo administered subcutaneously every 4 weeks. Because of early study termination (clinical hold by the US Food and Drug Administration), only 77 of the planned 200 patients were enrolled. The primary endpoint, the mean reduction of average daily pain at week 12 compared with baseline, showed a positive dose-response relationship. The pair-wise comparison between the 10-mg group and placebo was significant. An exploratory responder analysis revealed that a greater proportion of patients in the 10-mg group reported ≥30% reduction in the average pain intensity compared with placebo at week 12. During the combined efficacy and safety extension phases, the top 3 treatment-emergent adverse events in the combined fulranumab group were arthralgia (11%), peripheral edema (11%), and diarrhea (9%). No cases of joint replacement or death were reported [17]. Despite early study termination, fulranumab treatment resulted in dose-dependent efficacy and was generally well tolerated.

ARA 290 is a nonhematopoietic peptide designed from the structure of erythropoietin. In this trial, ARA 290 (4 mg) or placebo were self-administered subcutaneously daily for 28 days and the subjects followed for an additional months without further treatment. During the 56-day observation period, subjects with ARA 290 treatments had improvement in hemoglobin A1c (Hb A1c) and lipid profiles. Neuropathic pain from PDN improved significantly in the ARA 290 group. In addition, subjects with >1 standard deviation reduction in mean corneal nerve fiber density (CNFD) showed a significant improvement in CNFD compared with no change in the placebo group [18].

Botulinum toxins (BoNTs) are used for treating multiple painful conditions. However, BoNTs are not yet approved for treating PDN in the United States. Multiple small-scaled clinical trials have provided evidence to support the use of type A BoNT (BTX-A) injections for PDN. A meta-analysis selected and analyzed the data from a class I [19] and class II [20] studies to examine the efficacy of BTX-A on PDN [21]. Combining the two qualifying studies, there were a total of 58 patients receiving a sum of 76 treatments for PDN randomly allocated to placebo or BTX-A treatments. The injected areas were identical in each trial with a fixed protocol using a 3 × 4 grid that was equally spaced to demarcate the injection sites on the dorsum of each foot. The class 2 study used OnabotulinumA while the class 1 study used AbobotulinumtoxinA. It was concluded that there was an improvement of 1.96 visual analogue scale points following treatment with BTX-A [21]. The results were concluded as clinically significant improvement of “minimum change in pain.” No serious adverse effects were reported in both trials. The meta-analysis evaluated the significance, low overall risk of bias, and almost no statistical heterogeneity support a correlation between Botox and improvement of pain scores for treating PDN [21]. However, further large scale controlled trials are needed to further establish the clinical efficacy and safety for this potential new indication for BTX-A.

Future study strategies

As reviewed in the current article, promising evidence support that several emerging treatments could be available for treating PDN in the near future. Other novel strategies are also under extensive study for developing new PDN treatments.

Animal studies have provided evidence that neurogenic inflammation in skin could be an important pathomechanisms for the development of PDN [22]. In a mouse model of type 2 diabetes, skin inflammatory cells (such as macrophages and Langerhans cells) could be activated by NGF signaling to target intraepidermal nerve fibers and be responsible for the development of pain behaviors. New evidence suggests that cytokine dysregulation could contribute to these skin inflammatory phenomena and suggest using immuno-modulatory therapies could be a novel treatment strategy for PDN [23].

Sodium channel NaV 1.7, NaV 1.8, and NaV 1.9 (encoded by SCN9A, SCN10A, and SCN11A respectively) are preferentially expressed in peripheral sensory neurons for nociception. Sodium channel Nav1.7 antagonists, including Xenon 402, CNV1014802, and PF-05089771, are being tested as new therapies for PDN [24]. Taken together, accumulating data from evidence-based studies shine light to the promising future of PDN management.

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