DOI: 10.31038/EDMJ.2020443

Abstract

Advanced age and medical co-morbidity are strong predictors of mortality in COVID-19 infection. Yet few studies (to date) have specifically addressed risk factors associated with COVID-19 mortality in a high-risk subgroup of older US adults having one or more chronic diseases. Our hypothesis is that medications having ‘off-target’ anti-inflammatory effects may play a role in modulating the immune response in COVID-19 infection. We analyzed baseline risk factors associated with respiratory failure or death in 55 older adult US military veterans hospitalized for COVID-19 infection during (March-June 2020) the peak of the pandemic in New Jersey. Fifty-three percent (29/55) of patients experienced respiratory failure and thirty-one percent (17/55) died. In adjusted logistic regression analysis, baseline neutrophil to lymphocyte ratio (NLR) (P=0.0035) and body mass index (P=0.03) were significant predictors of the risk for respiratory failure. Age (P=0.05) and non-use (vs. use) of psychotropic medications having serotonin 2A receptor antagonist properties (odds ratio 5.06; 95% confidence intervals 1.18-21.7; P= 0.029) was each a significant predictor of an increased risk of death. There was a significant interaction effect of age and non-use (vs.. use) of psychotropic serotonin 2A receptor antagonist medications on the odds ratio (OR) for death (P=0.011). In selected, ventilator-dependent COVID-19 pneumonia patients treated with psychotropic serotonin 2A receptor antagonist medications to control agitation and ICU delirium, there was an apparent positive association between medication use and significant rise in the absolute lymphocyte count and decrease in the neutrophil: lymphocyte ratio. Taken together, these data are the first to suggest that certain psychotropic medications used in the treatment of chronic psychiatric illness and/or for acute delirium are inversely associated with mortality in severe COVID-19 infection by unknown mechanism which may involve (in part) immunomodulatory effects.

Introduction

According to the Centers for Disease Control [1], medical co-morbidity was associated with a substantially increased risk of admissions to the ICU due to severe COVID-19 infection. Hypertension, diabetes and advanced age were among the risk factors associated with increased mortality due to severe COVID-19 infection in a recent large study from metropolitan New York [2]. Older adult U.S. military veterans carry a substantial burden of medical co-morbidities including type 2 diabetes mellitus and hypertension [3]. Yet few studies to date have examined risk factors predictive of severe COVID-19 infection in patients having one or more chronic illnesses. The aim of the present study was to evaluate risk factors associated with poor outcome in COVID-19 infection requiring hospitalization in older adult US military veterans. The SARS-Cov-2 virus mediates hyper-inflammation and dysregulated immunity leading to ‘cytokine storm’ [4]. Inflammation predisposes to hypercoagulability and platelet-derived serotonin (5-HT) promotes neutrophil infiltration at sites of injury, each process is recognized as a ‘bad actor’ in severe COVID-19 infection [5]. The 5-hydroxytryptamine 2A receptor is expressed on platelets, innate and adaptive immune cells [6,7] and it was reported to ‘drive’ chronic inflammation in animal models of autoimmune diseases [8,9]. A secondary aim of the present study was to test whether psychotropic medications belonging (in part) to the class of 5-hydroxytryptamine 2A receptor antagonists (in current use as anti-depressant or atypical antipsychotic medications) may modify the risk of death in severe COVID-19 infection.

Patients and Methods

The retrospective study was reviewed and approved by the local Veterans Affairs New Jersey Healthcare System Institutional Review Board. Patients were consecutively selected from among those inpatients admitted to an acute medical ward or intensive care unit (ICU) at the Veterans Affairs New Jersey Healthcare System (VANJHCS), East Orange campus between late March 2020 and early June 2020, i.e. during the peak of the pandemic in New Jersey. Nearly all patients tested COVID-positive (n=53) by polymerase chain reaction of an oropharyngeal or nasopharyngeal swab. Two patients had a clinical picture consistent with COVID-19 pneumonia, but a negative COVID-19 PCR test and were included in the analysis. Ninety-six percent of patients were men. Forty-one of 55 patients (75%) had respiratory symptoms on admission. Thirty-four percent of patients were 74 years or older, nearly all patients had at least one co-morbidity, including forty percent of patients who had a baseline history of cardiovascular disease (Table 1).

Table 1: Baseline clinical characteristics in the study patients.

Risk Factor	Mean (SD)
Age (years)	70.6 (11.5)
BMI (kg/m2)	29.0 (7.5)
Race AA/NHW/H (%)	47/35/11
Co-morbidities	(%)
Hypertension (%)	71
Diabetes (%)	58
Cardiac disease (%).	40
COPD/Asthma (%)	20
ESRD (%)	17
Psychiatric illness (%)	20
Medications/Treatments	(%)
Psychotropic having Serotonin 2A receptor Antagonist activity (%)	45
ACEi/ARB (%)	44
Glucocorticoids (%)	33
Insulin (%)	25
Remdesivir (%)	5
Convalescent plasma (%)	2

N=55 patients; BMI: Body Mass Index; ACEi: Angiotensin Converting Enzyme Inhibitor; ARB: Angiotensin Receptor Blocker Medication; AA: African-American, NHW: Non-Hispanic White, H: Hispanic; COPD: Chronic Obstructive Pulmonary Disease; ESRD: End Stage Renal Disease.

Endpoint(s)

Twenty-nine of fifty-five patients (52.7%) experienced respiratory failure and there were 17 deaths which generally occurred within 2-3 months of the acute COVID-19 hospitalization. Unless a patient was readmitted to the same institution (VANJHCS), or had subsequent entries in the VANJHCS medical record, he or she was presumed to have survived acute episode of COVID-19 infection following his or her successful discharge home or to a lower-intensity, rehabilitative-type care facility.

Respiratory failure is defined as requiring intubation and mechanical ventilation or high-flow, concentrated oxygen, e.g. 50% oxygen via nasal cannula at 15 liters/min or > 50% oxygen via a non-rebreather mask.

Diabetic microvascular complications – retinopathy – macular edema, or proliferative retinopathy, nephropathy – >300 mg/g creatinine albuminuria; painful neuropathy – evidenced by treatment with gabapentin and clinical diagnosis determined by trained neurology staff. Neutrophil/Lymphocyte ratio (NLR) – is the average on two consecutive days of the absolute neutrophil count/absolute lymphocyte count which occurred at the nadir of the absolute lymphocyte count.

Clinical and other laboratory data was extracted from retrospective chart reviews. Body weight index (BMI), glycosylated hemoglobin value was based on prior results closest to the date of inpatient admission. Baseline insulin use or the use of angiotensin converting enzyme inhibitor or angiotensin receptor blocker medications was determined from baseline outpatient medication lists.

Patients Treated with Antipsychotic or Antidepressant Medications

Treatment with an atypical, second-generation antipsychotic medication (SGA) or an antidepressant medication that has antagonist activity at the 5-hydroxytryptamine (serotonin)2A receptor was evaluated as a possible risk factor for the outcome of death vs. survival after acute COVID-19 infection. Both atypical antipsychotics and certain anti-depressants (tricyclic antidepressants, trazadone, mirtazapine) share high-affinity antagonism on the 5-hydroxytryptamine 2A receptor. Total twenty patients were treated with a SAG antipsychotic medication (risperidone, olanzapine, quetiapine, aripiprazole) and five were treated with one of the 5-HT2AR antagonist antidepressant medications (trazadone, mirtazapine) either for an underlying mental health disorder (schizophrenia, schizoaffective disorder, major depressive disorder) or to manage acute agitation/delirium.

Statistics

Logistic regression analysis was used to model possible baseline risks factors associated with the occurrence of respiratory failure or death. Age and body mass index were included as covariates in the model when testing for an effect of other risk variables. In univariate regression analysis, other risk variables [NLR, diabetes mellitus, baseline use of various medication classes, baseline cardiac disease] were tested for association with the endpoint, and were included in the model if it reached 0.05 of significance level. Two way-interactions were checked among significant risk variables. Logistic regression was conducted using SAS 9.4 (SAS Institute Inc, Cary, NC).

Results

The baseline clinical characteristics in the study patients are shown in Table 1. The mean age was 70.6 ± 11.5 years old. More than half of the patients had diabetes mellitus (nearly all type 2 DM) and one in four patients were treated with insulin. Mean glycosylated hemoglobin was 7.7 ± 2.0% among diabetic patients and one-third had at least one microvascular complication (not shown in Table 1).

Lymphopenia is a characteristic laboratory feature in severe COVID-19 disease. The neutrophil to lymphocyte ratio (NLR) was reported to be a prognostic marker in COVID-19 infection: a threshold NLR value >5.0 vs. ≤5.0 demonstrated high sensitivity and specificity for differentiating between severe and mild COVID-19 infection [10]. In univariate regression analysis, the neutrophil: lymphocyte ratio (Odds ratio: 1.232; 95% confidence intervals 1.079-1.406; P=0.002) was a significant predictor of the risk for respiratory failure (Table 2A).

In multivariate logistic regression that adjusted for age and body mass index, baseline NLR (OR 1.2554; 95% CI: 1.0776-1.4624; P=0.0035) and BMI (OR 1.1429; 95% CI: 1.0131-1.12893; P=0.030) was each a significant predictor of the risk of respiratory failure in COVID-19 infection (Table 2B).

Table 2: Univariate (A) and multivariate (B) logistic regression of risk factors associated with Covid-19 respiratory failure.

Variable	Odds Ratio	95% CI	P-value
NLR	1.232	1.079-1.406	0.002
Age (years)	1.039	0.355-3.038	0.944
BMI (kg/m2)	1.081	0.991-1.179	0.231
Diabetes (yes/no)	1.031	0.983-1.081	0.212
Insulin (yes/no)	1.890	0.540-6.617	0.319
ACEi/ARB (yes/no)	1.108	0.381-3.224	0.851

N=55; NLR: Neutrophil to Lymphocyte Ratio; CI: Confidence Interval.

Variable	Odds Ratio	95% CI	P-value
NLR	1.255	1.078-1.462	0.0035
BMI (kg/m2)	1.143	1.013-1.129	0.030
Age (years)	1.052	0.993-1.134	0.079

N=55; NLR: Neutrophil to Lymphocyte Ratio; CI: Confidence Interval.

We next evaluated risk predictors of COVID-19 mortality. In univariate logistic regression analysis, the use of an antipsychotic or antidepressant medication having 5-HT2A receptor antagonist properties (OR 0.198; 95% CI:0.038-0.634; P=0.0094) was a significant predictor of a decreased risk of death (Table 3). Age (OR 1.076; 95% CI 1.015-1.141; P=0.0139) was a significant predictor of an increased risk of death (Table 3). Baseline cardiac disease (OR 3.095; 95% CI 0.948-10.109; P=0.0613) was a nearly significantly predictive of an increased risk of death (Table 3).

Table 3: Univariate logistic regression analysis of risk factors associated with Covid-19 death.

Variable	Odds Ratio	95% CI	P-value
Serotonin 2A R Blocker med* (yes/no).	0.156	0.038-0.634	0.0094
Age (years)	1.076	1.015-1.141	0.014
Cardiac disease (yes/no)	3.095	0.948-10.109	0.061
NLR	1.031	0.992-1.072	0.125
African-Am vs. other race or ethnicity	0.700	0.220-2.225	0.546
Diabetes (yes/no)	0.734	0.232-2.350	0.599
Glucocorticoids (yes/no)	2.489	0.753-8.223	0.135
Acei/ARB (yes/no)	0.606	0.186-1.975	0.406
Insulin (yes/no)	0.289	0.057-1.469	0.101
BMI (kg/m2)	0.994	0.921-1.072	0.876

BMI: Body Mass Index; dz: Disease; *Second generation atypical antipsychotics, tricyclic antidepressants, trazadone or mirtazapine; CI: Confidence Interval, N=55 patients.

In multi-variate logistic regression analysis, non-use of antipsychotic or antidepressant medications having 5-HT2AR antagonist properties (OR 5.058; 95% CI: 1.180-21.689; P=0.029) and age (OR 1.062; 95% CI: 1.000-1.128; P=0.050) was each a significant predictor of an increased risk of death in COVID-19 infection (Table 4A).

There was a significant interaction effect of age and 5-HT2A receptor-blocking classes of medication on the risk of death in COVID-19 infection (P=0.0112) (not shown in Table 4). Use of 5-HT2A receptor blocking medications was associated with low mortality rate (0/18) in younger patients (≤72 years old) and the overall mortality rate (3/8) increased among patients aged 73-95 years old treated with a 5-HT2A receptor blocking class of medication (not shown in Table 4).

Table 4: Multivariate logistic regression analysis of risk factors associated with Covid-19 death.

Variable	Odds Ratio	95% CI	P-value
Serotonin 2A R Blocker med* (no/yes)	5.058	1.180-21.689	0.029
Age (years)	1.062	1.000-1.128	0.050

N=55 patients; *Second generation atypical antipsychotics, tricyclic antidepressants, trazadone or mirtazapine; CI: Confidence Interval.

In representative patients having hypoxemic respiratory failure, treatment with psychotropic medications having 5-HT2A receptor blocking activity (to control agitation and/or delirium on the ventilator in the ICU) was associated with an abrupt increase in the absolute lymphocyte count and a decrease in the NLR.

Case 1

A 59-year old female with systemic lupus erythematosus developed hypoxemic respiratory failure. She manifested a high level of systemic inflammation, an initial C-reactive protein level > 320 mg/L, normal (1-10) and 2 weeks later it was still 189 mg/L. She was treated for 3 weeks with a nightly dose of an oral SGA to manage agitation/delirium on the ventilator in the ICU. Her absolute lymphocyte count increased from 0.9 to 3.7 K/cm2 in association with SGA treatment (solid line, Figure 1). She was discharged to a rehabilitation center in stable condition.

Figure 1: A 59-year old woman with systemic lupus erythematosus who developed hypoxemic respiratory failure. Solid line indicates nightly treatment with an oral second-generation anti-psychotic medication. The absolute lymphocyte count increased from 0.9 to 3.7 K/cmm in association with SGA treatment. The patient was discharged to a rehabilitation center in stable condition.

Case 2

A 55-year old man with T2DM experienced hypoxemic respiratory failure. The initiation of nightly atypical antipsychotic medication was associated with an abrupt rise in absolute lymphocyte count (solid bar, Figure 2A). The absolute lymphocyte count was significantly higher (Figure 2B) and the NLR was significantly lower (Figure 2C) during 7 days on SGA treatment compared to corresponding mean level 4 days before initiation of SGA treatment.

Figure 2: A 55-year old man with type 2 diabetes mellitus (T2DM) who developed hypoxemic respiratory failure. A) The initiation of treatment with nightly atypical, second-generation antipsychotic medication was associated with an abrupt rise in absolute lymphocyte count (solid bar). B) The absolute lymphocyte count was significantly higher and C) the NLR was significantly lower during 7 days on SGA treatment (Medication) compared to corresponding mean level on 4 consecutive days before initiation of SGA treatment (Medication).

Case 3

A 76-year old man with T2DM and polyneuropathy developed hypoxemic respiratory failure and was intubated. During the initial 15 days he was treated with a combination of D2 receptor blockers (Haldol) and benzodiazepines to control agitation/delirium in the ICU on the ventilator (dashed line, Figure 3A). Three days after successful extubation (arrow, Figure 3A) the drug regimen was changed and he received an 18-day course of an oral SGA medication nightly to control agitation/delirium (solid line, Figure 3A). He was later discharged (in stable condition) from the hospital. The mean absolute lymphocyte count was significantly higher during a 16-day period on daily treatment with an SGA compared to the mean level during a comparable, preceding 16-day period on intermittent D2 receptor antagonist and/or benzodiazepine medications (Figure 3B).

The mean neutrophil/lymphocyte ratio (NLR) was significantly lower (6.6 vs. 18.0; P < 0.001) during the period on treatment with an SGA (atypical antipsychotic medication) compared to the corresponding preceding period on D2R antagonist and/or benzodiazepine medications to control agitation and delirium (Figure 3C).

Figure 3: A 76-year old man with T2DM who developed hypoxemic respiratory failure and was intubated. A) During initial 15 days he was treated with a combination of D2 receptor blockers (Haldol) and benzodiazepines to control agitation/delirium in the ICU on the ventilator (dashed line). Following extubation (arrow) the drug regimen was changed to a nightly dose (for 18 consecutive days) of an oral SGA medication to control agitation/delirium (solid line). He was later discharged (in stable condition) from the hospital. B) The mean absolute lymphocyte count was significantly higher during a 16-day period on daily treatment with an SGA compared to the mean level during a comparable, preceding 16-day period on intermittent D2 receptor antagonist and/or benzodiazepine medications. C) The mean neutrophil/lymphocyte ratio (NLR) was significantly lower (6.6 vs. 18.0; P < 0.001) during the period on treatment with an SGA (atypical antipsychotic medication) compared to the corresponding preceding period on D2R antagonist and/or benzodiazepine medications to control agitation and delirium.

Discussion

Biomarkers that predict an increased risk of severe COVID-19 infection can help guide therapy in selected patients. The present finding that the baseline neutrophil to lymphocyte ratio was a significant predictor of the risk for respiratory failure in older adult hospitalized patients suffering with COVID-19 pneumonia is consistent with other reports of the prognostic value of the NLR [10]. Yet, to our knowledge, this is the first report that treatment with certain psychotropic medications having 5-HT2A receptor blocking properties was associated with substantially lower mortality in severe COVID-19 infection. The apparent association between the use of these classes of medications (to control delirium and agitation in the ICU) and higher absolute lymphocyte count and lower NLR suggests a possible immunomodulatory role for 5-HT2AR antagonists. In a mouse model of autoimmune hepatitis, treatment with the 5-HT2AR blocking anti-depressant medication mirtazapine substantially lowered hepatic inflammation [8]. Other evidence derived from in vitro and animal studies [6,7,9] suggests that 5-hydroxytryptamine 2A receptor antagonism may mediate an anti-inflammatory effect in part by interfering with the elaboration of pro-inflammatory cytokines from innate immune cells, e.g. macrophages. Despite unexplained lymphopenia, T-cell activation is another characteristic immunologic feature in severe COVID-19 infection [4]. The 5-hydroxytryptamine 2A receptor is widely expressed on peripheral immune cells (B cells, T cells) and vascular cells and among the diverse reported effects of 5-HT and/or the 5-HT2A receptor are: T-cell activation [11] and increases in pro-inflammatory cytokines IL-6, interferon-gamma, and interleukin-2 [7,8].

Second-generation, atypical antipsychotic medications, tricyclic antidepressants and other antidepressant medications useful in treatment-resistant depression (trazadone, mirtazapine) all share high affinity for the 5-HT2A receptor. Several of the SGA medications also target the D2 dopamine receptor, the H1 histamine receptor, the alpha-1 adrenergic receptor, and the muscarinic acetylcholine receptor-making it difficult to ascribe a putative beneficial effect solely to actions at the 5-HT2A receptor. Yet (in our study) even administration of low doses of the antidepressant trazadone (relatively selective for 5-HT2AR antagonist activity) appeared to significantly upregulate lymphocyte count and downregulate the NLR within a time period (7-8 hours) consistent with the half-life of the medication.

Many of the patients in the present study having co-morbid psychiatric illness were treated (before and during COVID-19 infection) with an SGA or anti-depressant medication having serotonin 2A receptor antagonist properties. These patients tended to experience a less severe form of illness in which the baseline NLR was generally < 10. Mean baseline NLR did not differ significantly between patients who were treated or not treated with psychotropic medication having serotonin 2A receptor antagonist properties. In our multi-variate logistic regression model that included adjustment for baseline NLR, 5-HT2AR antagonist medication use (vs.. non-use) was still a significant predictor of a lower risk of mortality. Taken together, there did not appear to be a selection bias toward use of 5-HT2AR antagonist medications in patients with less severe form of COVID-19 infection.

The present retrospective study was not designed to test for a possible causal relationship between the use of 5-HT2AR antagonist-like medications and mortality in severe COVID-19 infection. There are caveats important in the interpretation of the present findings: first, patients having co-morbid schizophrenia or major depression were generally younger compared to the overall patient cohort and second, certain genetic and/or environmental factors (including unknown immune factors) previously associated with an increased risk of psychiatric disorders may have independently affected mortality risk in COVID-19 pneumonia.

The study has several limitations. It was small and the results may only apply to the experience of older adult men having a number of different co-morbidities (diabetes, advanced age, hypertension, cardiovascular disease) previously associated with a higher risk of death in COVID-19 infection. More study in a larger group of patients (including women and patients having fewer co-morbidities) is needed to determine the generalizability of the findings.

In summary, advanced age and the non-use of certain antipsychotic and anti-depressant medications having shared antagonist activity on the 5-HT2A receptor was each a significant predictor of an increased risk of death in a small cohort of hospitalized, older adults who experienced COVID-19 infection requiring hospitalization. A future randomized trial may help determine whether an apparent association between the use of these classes of ‘anti-inflammatory’ medications and improved immunological parameters may translate into lower COVID-19 mortality in a subset of severely-affected patients.

Acknowledgements

We thank the dedicated and caring nurses, physicians, hospital workers and administrators at the Veterans Affairs New Jersey Healthcare System for their tireless and compassionate efforts on the part of the veterans and other patients affected by the COVID-19 pandemic. The views expressed here are solely those of the authors and do not represent the official position of the U.S. Department of Veterans Affairs or the US Government.

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DOI: 10.31038/EDMJ.2020442

Abstract

Pituitary abscess (PA) caused by an infectious process is a rare cause of Sellar mass. The clinical features and radiological appearance of PA as an intra- or supra-sellar mass are similar to many other pituitary lesions, and so they are often misdiagnosed as pituitary tumor.

70% of cases occur in a previously healthy pituitary gland. These are classified as primary pituitary abscesses, persumbly secondary to either hematogenous spread or as an extension from an adjacent infective focus such as meningitis, sphenoid sinusitis, Cavernous sinus thrombophlebitis or contaminated cerebrospinal fluid (CSF) fistula.

The rest are secondary abscesses, and arise from pre-existing lesions, such as an adenoma, apoplexy in a tumor, a craniopharyngioma, or a complicated Rathke’s cleft cyst and lymphoma. The risk factors are for PA are immunosuppression, previous irradiation or surgical procedures to the pituitary gland [1].

In almost 50% of cases, the pathogenic microorganism causing the infection is not isolated. A history of recent meningitis sinusitis or head surgery can be the source [2].

Correct diagnosis before surgery is difficult and is usually confirmed intra- or post-operatively. The early surgical intervention allows appropriate antibiotic therapy and hormone replacement resulting in reduced mortality and morbidity. A long term follow-up is recommended because of the high risk of recurrence and of postoperative hormone deficiencies.

Keywords

Pituitary abscess, Papilledema, Panhypopituitarism, Rathke’s cleft cyst, Propionibacterium acnes

Introduction

A pituitary abscess (PA) represents 0.2%-0.6% of all pituitary lesions and can be life threatening. It can have a prolonged disease course. The first case was reported by Heslop in 1848, and so far, <300 cases have been reported worldwide [3]. It is an infectious process that presents as a mass in the Sella. Clinical features and the radiological appearance of the PA as an intra or suprasellar mass are similar to many other pituitary lesions, so it is often misdiagnosed as a cystic pituitary tumor, craniopharyngioma, and Rathke’s cyst. It can be life-threatening if not appropriately diagnosed or treated, and the outcome is difficult to predict. Fortunately, the majority of the cases have a chronic course. The disease has a higher prevalence in females between the age of 12 to 76 years. The average period it takes to diagnose from the onset of symptoms is around 8 years.

PA can occur as a primary disease or can be secondary to infections caused by either hematogenous spread or as an extension from an adjacent infected tissue such as meningitis, sphenoid sinusitis, Cavernous sinus thrombophlebitis or contaminated cerebrospinal fluid fistula 70% of cases occur in a previously healthy pituitary gland. These are classified as primary pituitary abscesses, and the rest are secondary abscesses that arise from pre-existing lesions, such as an adenoma, apoplexy in a tumor, a craniopharyngioma or a complicated Rathke’s cleft cyst and lymphoma [4].

In almost 50% of cases, the pathogenic microorganism causing the infection cannot be isolated. A history of recent meningitis sinusitis or head surgery can be the source [2].

Correct diagnosis before surgery is difficult and is usually confirmed intra- or post-operatively. The early surgical intervention allows appropriate antibiotic therapy and hormone replacement resulting in reduced mortality and morbidity. A long term follow-up is recommended because of the high risk of recurrence and postoperative hormone deficiencies.

We present 2 cases of pituitary abscess in young women. One presented with bilateral papilledema and the other with panhypopituitarism. Both had a sellar mass on an MRI scan, and the diagnosis was made intra-operatively. Microbiological culture in both cases was positive for Propionibacterium acnes (P.acnes). P.acnes is a gram-positive organism, a part of the normal skin microbe. This organism is most commonly isolated from wounds following craniotomies after Staphylococcus aureus and streptococcus epidermidis. Low-grade infections can manifest between 3-36 months.

Case 1

A 14-year old South Asian girl presented with a one-month history of worsening frontal headaches that occurred daily, associated with vomiting, nausea, lethargy, photophobia, and sleep disturbance. Aside from well-controlled asthma, she has been previously healthy. There was no recent travel history or infectious contacts. On examination, she appeared alert and active. She had bilateral papilledema, suggesting raised intracranial pressure (ICP). She was apyrexial and systemically well. Her cerebral magnetic resonance imaging (MRI) scan revealed a soft tissue mass in the pituitary fossa extending up towards the optic chiasm, with mild edema in the optic nerve and tracts. The scan also showed an enlarged pituitary gland and thickened stalk. The findings suggest an inflammatory process like hypophysitis, particularly Langerhans cell histiocytosis (LCH) because of her age. There were no other features of LCH. She had a normal liver US and skeletal survey. She had no symptoms of Diabetes insipidus. Her pituitary hormones were normal, including the stimulated cortisol. Her Prolactin was elevated. Her serum sodium and osmolality were normal. Her ESR was slightly raised, but autoantibodies, serum tumor markers, ACE, and the Quantiferon tuberculosis test were negative. Her IgG4 subclass was normal (Table 1). The formal ophthalmology review did not show evidence of bilateral papilledema. Her symptoms improved with oral analgesics, and steroid treatment was not initiated.

Table 1: Results at initial presentation.

                    Short Synacthen test

Time	T=0	T-30	T=60
Cortisol (nmol/L)	186	452	594

                   Baseline tests

Test	Result	Normal range
IGF-1(nmol/L)	47.9	18.3 to 63.5
TSH (mU/L)	1.62	0.51-4.3
T4 (pmol/L)	13.3	10.8-19
LH (IU/L)	4.3	Follicular phase 2-13 Mid cycle 14-6 Luteal phase 1-11
FSH (IU/L)	1.8	Follicular phase 4-13 Mid cycle 5-22 Luteal phase 2-8 Postmenopause>25
ACTH (ng/L)	<3	0-50
Prolactin (mU/L)	806	102-496
Serum Na mmol/L	144	133-146
Serum Osmolality mOsmo/Kg	293	282-300
Random Urine Osmolality mOsmo/Kg	475	100-1400
LDH (u/L)	188	120 to 300
HCG (IU/L)	<1	0-1
alpha Fetoprotein (kU/L)	1	0-10
C-Reactive protein (mg/L)	1.5	0-5
ESR (mm/h)	28	1-12
Complement C3 (g/l)	1.1	0.75-1.65
Complement C4 (g/l)	0.29	0.14-0.54
Antinuclear antibodies	Negative
Angiotensin convert enzyme (U/L)	42	16-85
IgG4 (g/L)	0.04	0-1.3

 

A repeat MRI scan 3 months later discussed in a multidisciplinary meeting was reported to suggest Rathke’s cleft cyst abscess/ Pituitary abscess (Figures 1 and 2). She underwent a trans-sphenoidal endoscopic pituitary biopsy for diagnosis. The appearances suggested a Rathke’s left cyst and a pituitary abscess. Immunostaining for ACTH, FSH, LH, growth hormone, TSH and Prolactin, chromogranin, synaptophysin, and collagen IV was consistent with anterior pituitary tissue. Microbiological culture on prolonged incubation was positive for Propionibacterium with no acid-fast bacilli growth. TB culture was also negative. She received a 6-week course of antibiotics, including 2 weeks of intravenous ceftriaxone and oral metronidazole followed by 4 weeks of oral co-amoxiclav. Her headaches and vomiting deteriorated after biopsy with a peak CRP of 218 mg/L, which resolved following medical treatment. Imaging with MRI and baseline pituitary function blood tests has since been repeated following the 6 weeks to assess the management’s effectiveness, which showed normal results. The patient reported the resolution of headaches and able to resume full-time schooling.

Figure 1: MRI at presentation.

Figure 2: MRI 3 months after transphenoidal surgery.

Case 2

29 years old Caucasian fine arts student presented to the emergency department with fever, headaches, profuse sweating, tiredness, and blurring of vision. Her symptoms, particularly headaches, had worsened over the last 12 months. She had noticed polydipsia and polyuria. She also had amenorrhoea for twelve months. She was treated at her local hospital twice in the preceding 3 years with symptoms of headaches, fever, weight loss, and vomiting. She had a lumbar puncture 3 times to rule out a possibility of central nervous system infection. On both occasions, she was discharged home after empirical treatment with antibiotics for suspected meningitis. There was no other past medical history. There was no recent travel history or infectious contacts. She was not on any regular medications.

The initial pituitary MRI and contrast-enhanced MRI scan revealed the absence of the posterior pituitary bright spot and a thickened pituitary stalk with a deviation of infundibulum to the right. There was a homogenous hyperintense area within the pituitary gland with no discernable pituitary tissue. This area was hypointense on T2 (Figures 3-5). The differential diagnosis was apoplexy, hypophysitis or a proteinaceous cystic lesion replacing or compressing the pituitary gland. The optic nerves and the chiasm appeared normal. Her investigations confirmed her to have hypopituitarism with Diabetes insipidus (Table 2). Her lumbar puncture showed no CSF abnormality. Her tumor markers and Quantiferon for tuberculosis were negative. The case was discussed in multidisciplinary meeting (MDT) and with empirical diagnosis of hypophysitis, she was started on prednisolone with the replacement of deficient hormones, including Desmopressin. She showed no improvement in her clinical symptoms. A 3 month interval scan showed an increase in the size of the pituitary gland with further thickening of the stalk and optic chiasm displaced superiorly. After the second discussion in MDT, she had a pituitary biopsy. During surgery, soft yellow-white pus-like material was drained after dural incision. The microscopy showed necrotic material with a little amount of compressed anterior pituitary gland, chronic inflammation, and no evidence of adenoma or granuloma or giant cells was found. No acid-fast bacilli or organisms were seen on gram staining, and the culture for TB was negative. There was scanty growth of Propionibacterium acneformis. Her interval scan 3 months later showed complete resolution of the non-enhancing T1 hypertense pituitary tissue with a further decrease in the size of the pituitary gland. She remains on full hormones replacement. She had an insulin tolerance test that confirmed her growth hormone deficiency, and she is now on growth hormone replacement. She remains on hydrocortisone, Thyroxine, female hormone replacement, and Desmopressin.

Figure 3: MRI at presentation.

Figure 4: MRI 3 months later.

Figure 5: MRI post-surgery.

Table 2: Results at initial presentation.

                   Short Synacthen test

Time	T=0	T-30
Cortisol (nmol/L)	148	169

                   Baseline tests

Test	Result	Normal range
IGF-1(nmol/L)	12.7	11.9-40.7
TSH (mU/L)	1.35	0.27-4.20
T4 (pmol/L)	5.3	10.8-25.5
LH (IU/L)	3.1	Follicular phase 2-13 Mid cycle 14-96 Luteal phase 1-11
FSH (IU/L)	5.1	Follicular phase 4-13 Mid cycle 5-22 Luteal phase 2-8 Postmenopause>25
Oestradiol (pmol/L)	<92	92-1462
Prolactin (mU/L)	577	102-496
Serum Na mmol/L	142	133-146
Serum Osmolality mOsmo/Kg	301	275-295
Random Urine Osmolality mOsmo/Kg	154	100-1400
CSF-b HCG (IU/L)	<2	<2
CSF-alpha fetoprotein (µg/L)	<1	<1
C-reactive protein (mg/L)	1.4	0-5
ESR (mm/h)	3	1-12
Antinuclear antibodies	Negative
IgG4 (g/L)	<0.01	0-1.3

Discussion

A pituitary abscess is an infectious process characterized by the accumulation of purulent material in the sella turcica. It is rare, and can be a life-threatening condition unless promptly diagnosed and treated. We report 2 cases of secondary pituitary abscess in young women. The first case was due to abscess in the Rahtke’s cleft cyst (RCC), and the second was Pituitary gland abscess with a history of otitis media and repeated lumbar punctures for presumed meningitis.

The clinical presentation of PA is nonspecific, such as headaches, pituitary hypofunction, and visual disturbances, whereas the infection can be discreet and inconstant [5,6]. Symptoms can be acute, subacute, or chronic, explaining the late diagnosis; in some cases. Visual disturbance, including hemianopia, can be present in 50% of cases. Headache without a particular pattern is a regular feature (70-90%) and can be debilitating. Anterior pituitary hypofunction due to destruction and necrosis of the gland is the commonest presentation resulting in fatigue and amenorrhoea (54-85%). In one series, 28 out of the 33 patients had anterior pituitary hypofunction. Pituitary hormone deficiencies persist in the majority of patients following treatment Up to 70% of patients with PA can have central Diabetes insipidus. In contrast, fever with signs of meningeal irritation is reported in 25% of cases [5].

MRI is the imaging of choice for the pituitary lesions. PA can present as a suprasellar mass (65%) or as an intrasellar mass (35%). A typical PA appears as a single cystic or partially cystic mass that is hypointense on T1-weighted image and hyperintense in T2-weighted image. It can show a rim of enhancement after contrast gadolinium. The posterior pituitary bright spot is mostly absent in majority of the cases (Wang et al.). The lesion’s signal depends on protein, water, lipid content, and whether there is hemorrhage. Imaging can also show the invasion of an adjacent anatomical structure, peripheral meningeal enhancement, thickening of the pituitary stalk, and paranasal sinus enhancement [6].

Diffusion-weighted magnetic resonance imaging (DWI) is widely used to differentiate cerebral abscess from other necrotic masses. Brain abscesses typically show high intensity on DWI with decreased apparent diffusion coefficient (ADC) value in their central region. The high intensity on DWI is useful but not specific to PA because pituitary apoplexy can also exhibit high intensity on DWI [7]. The accuracy of DWI in PA remains controversial. In the Wang et al. case series, PA was misdiagnosed in one-third of the case [6]. The radiological differential diagnosis includes, Rathke’s cleft cyst, cystic pituitary adenoma, arachnoid, and dermoid cysts, metastases, glioblastoma multiforme, chronic hematoma, and multiple sclerosis [8]. Rathke’s cleft cyst mainly can mimic a pituitary abscess [9]. RCC is the second most common incidentaloma after adenomas and accounts for 20% of incidental pituitary lesions at autopsy. The incidence of RCCs in children was reported to be much lower than in adults. However, the prevalence is now believed to be much higher, especially among those with the endocrine-related disorder [10]. Gunes et al. reported the radiological appearance of RCC on MRI in 13.5% of the children who underwent MRI for the investigation of endocrine-related disorders. Patients with RCC are usually asymptomatic, but symptomatic RCC is more common in females in both adult and pediatric populations [11]. RCC can cause significant morbidity such as headache, visual disturbances, chemical meningitis, endocrine dysfunction (hypothyroidism, menstrual abnormalities, diabetes insipidus, adrenal dysfunction, and very rarely apoplexy). Short stature, growth deceleration, delayed puberty are also reported in children and adolescents.

The diagnosis of PA in most cases can only be confirmed after surgical exploration, due to overlapping of clinical signs, symptoms, imaging, and laboratory findings with other sellar lesions. Signs of inflammation are present in less than a third of the patients. The PA should be included in the differential diagnosis of patients with headaches or signs of pituitary dysfunction and patients with pituitary mass who develop signs of meningeal inflammation.

The main treatment for PA in patients with mass effect is Transsphenoidal excision (TSS) with decompression of sella and antibiotic therapy. This can result in the resolution of visual abnormalities. Treatment is effective for typical symptoms such as fever, headache, and visual changes. Patients with shorter duration of symptoms and those with primary abscess have better improvement in their pituitary dysfunction. Majority of the patients remain with pituitary dysfunction even after the treatment.

Antibiotic therapy should to started promptly even in the patients who are waiting for microbiology and histological confirmation for about 4–6 weeks [1,12]. Empirical treatment with ceftriaxone is indicated until the results are available. Hormone replacement is commenced depending on the hormone deficits including stress dose glucocorticoid therapy. Hypocortisolemia should be recognized among patients presenting with sellar masses, as early diagnosis and treatment improve survival and endocrinological outcome. Patients who suffer from the pituitary abscess may eventually have a good quality of life if they are diagnosed and treated early. A craniotomy is reserved for larger lesions with the suprasellar extension or where transsphenoidal surgery is ineffective [13]. In a series published with 66 patients, 81.8% of patients recovered completely, 12.1% of patients had at least one operation for recurrence, and only one patient had died [14].

There are widespread pathogenic microorganisms in abscesses. These include Gram-positive bacteria, Gram-negative bacteria, anaerobes, and fungi [8,11]. Streptococcus and Staphylococcus are the most predominant Grampositive bacteria, whereas Escherichia coli, Mycobacterium, and Neisseria have also been reported [3,10,11]. Aspergillus fumigatus is mostly isolated in cases of secondary PA. Immunosuppressed patients mostly have Candida and Histoplasma. Cultures are positive only in 50% of cases; therefore, broad-spectrum antibiotics are given as empirical treatment. The pathogen identification is important for the therapeutic management [15].

Both patients had culture-positive for Propionibacterium acnes (P. acnes). This organism is seated deeply in the pilosebaceous glands, mainly in the scalp and face. It is a slow-growing, pleomorphic, non-spore-forming gram-positive anaerobic bacillus that is a universal component of the normal skin microbiota. It is usually considered a contaminant of blood cultures but occasionally can cause serious infections, including postoperative central nervous system (CNS) infections. P. acnes are the most commonly isolated organism after Staphylococcus aureus and Staphylococcus epidermidis following craniotomies. In the presence of heavy infiltrates, the Gram stain is not reliable. Gram stain is only positive in about 10.5% of clinically significant infections with moderate growth. P. acnes behave in a less aggressive manner than other postsurgical organisms and only accounts for a small fraction of CNS infections [16]. P. acnes abscesses typically follow craniotomy, shunts, access to reservoirs, trauma, and foreign bodies. Granulomatous responses have been documented in the CNS following P. acnes infections.

P. acnes grow slowly in the laboratory. This can cause in a delay in diagnosis, missed diagnosis, or delay in treatment if specimens are not cultured for an extended period. Cultures may not grow for as long as 14 days, so samples should be held beyond the usual 5 to 7 days. Gram stain may not be a reliable technique for the rapid diagnosis of P. acnes infections. When there is evidence of an abundant inflammatory response in the Gram-stained smear, a more careful evaluation of cultures must be performed. Polymerase chain reaction for the 16S rRNA or mass spectrometry can be a useful tool for rapid identification and typing of P. acnes following recovery in culture. Propionibacterium is susceptible to antibiotics used for the treatment of anaerobic infections, including penicillin, erythromycin, lincomycin, and clindamycin, but not metronidazole, which is notably ineffective against P. acnes [17].

Patients with PA should be followed up with serial MRI of the pituitary, hormonal profile and visual fields at 3, 6, and 12 months after surgery. The recurrence rate is variable and depends on the nature of the abscess (primary or secondary. The majority of relapses are associated with either an immunological defect or previous pituitary surgery [12,18].

Conclusion

We presented 2 cases of unusual sellar mass from an abscess in an adolescent and a young adult due to P. acnes, both responded well to treatment.

The pituitary abscess should be included as the differential diagnosis of patients with a sellar or a suprasellar mass, headaches, pituitary dysfunction, and meningeal inflammation.

The diagnosis is difficult before surgery because of overlapping clinical signs, radiological and laboratory findings with other sellar lesions.

Broad-spectrum antibiotics should be started empirically even before the culture results are available.

Culture is positive only in 50% of cases, and in case of unusual bacteria like P. acnes, an extended culture is required for the confirmation of the diagnosis.

Pituitary dysfunction should be recognized and appropriately treated particularly glucocorticoid replacement.

Transsphenoidal surgery is the treatment of choice and this is followed by pronged 4-6 weeks of broad-spectrum antibiotic therapy.

Early and efficient surgical and medical management results in lower mortality and higher recovery of pituitary hormone function.

Patients should be followed up with MRI imaging, assessment of the hormone replacement if required, and visual field assessment because of a chance of recurrence.

References

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DOI: 10.31038/EDMJ.2020435

Abstract

Aims: Circulating neurotoxic autoantibodies to the 5-hydroxytryptamine 2A receptor were increased in older adult type 2 diabetes in association with certain neurodegenerative complications. The male Zucker diabetic fatty (ZDF) rat is a model system for studies of obese, type 2 diabetes mellitus. The aim of the current study was to test for (and compare) circulating neurotoxic autoantibodies to the 5-hydroxytryptamine 2A receptor in the Zucker diabetic fatty rat and age-matched lean Zucker rat strains.

Methods: Plasma from lean and Zucker diabetic fatty rat (obtained at different developmental stages) was subjected to protein G affinity chromatography. The resulting immunoglobulin G fraction was tested for neurotoxicity (acute neurite retraction, accelerated neuron loss) in N2A mouse neuroblastoma cells and for binding to a linear synthetic peptide corresponding to the second extracellular loop of the 5-hydroxytryptamine 2A receptor.

Results: The male Zucker diabetic fatty rat (fa/fa) and two Zucker lean strains (+/?) and (fa/+) harbored autoantibodies to the 5-hydroxytryptamine 2A receptor which appeared spontaneously around 7-8.5 weeks of age. The circulating autoantibodies persisted until at least 25 weeks of age in the Zucker diabetic fatty rat and in the Zucker heterozygote (fa/+), but were no longer detectable in 25-week-old lean (+/?) Zucker rats. Autoantibody-induced acute neurite retraction and accelerated loss in mouse neuroblastoma N2A cells was dose-dependently prevented by selective antagonists of the 5-hydroxytryptamine 2A receptor. It was also substantially prevented by co-incubation with antagonists of RhoA/Rho kinase-mediated signaling (Y27632) or Gq11/phospholipase C/inositol triphosphate receptor-coupled signaling.

Conclusions: These data suggest that neurotoxic 5-hydroxytryptamine 2A receptor-targeting autoantibodies increase in the aging male Zucker diabetic fatty rat and in male Zucker lean rats harboring a heterozygous mutation, but not in age-matched, older Zucker lean rats lacking a known leptin receptor mutation. The Zucker genetic strain may be useful in studies of the role of humoral and/or innate immunity in late neurodegeneration.

Introduction

Type 2 diabetes mellitus is associated with an increased risk of late neurodegeneration [1,2] via mechanisms which may involve (in part) increased peripheral and central inflammation. Increased pro-inflammatory cytokines and innate immunity have been associated with early Parkinson’s disease [3] and dementia [4] through complex mechanisms. Leptin is a hormone released by fat cells that is important in energy metabolism. Dysfunction of the leptin system results in classic signs of type 2 diabetes mellitus, such as obesity, insulin resistance and high circulating insulin. Leptin is also a member of the type 1 cytokine family that includes interleukin-6 [5,6]. The leptin receptor is normally expressed on hematopoietic cells where it stimulates helper- T cell, and effector T cell functions and inhibits regulatory T cell function [7].

The male Zucker Diabetic Fatty rat (fa/fa) (ZDF) is a well-recognized animal model of obese type 2 diabetes mellitus and hypertension [8,9] in which hyperphagia causes morbid obesity leading to high level of pro-inflammatory adipocytokines [10]. In the ZDF rat, pro-inflammatory cytokines cause enhanced innate immunity [11], however, owing to its complete leptin receptor deficiency, helper T-cell function is reduced [12]. The lean heterozygous Zucker rat (fa/+) has haplo-sufficiency at the leptin receptor and a two-fold increased plasma leptin concentration [13] compared to the wild-type (+/+) lean Zucker rat. Since hyperleptinemia promoted helper-T cell-mediated autoantibody formation and worsened clinical outcome(s) in a mouse model of systemic lupus erythematosus [14], the lean heterozygote Zucker rat might be expected to exhibit higher autoantibody expression than in the ZDF (fa/fa) rat strain.

In prior studies in older adult (human) obese type 2 diabetes, patients harboring increased circulating autoantibodies to the 5-hydroxytryptamine 2A receptor (5-HT2AR) had an increased prevalence of co-morbid neurodegenerative complications including Parkinson’s disease and dementia [15]. Human plasma autoantibodies targeting the 5-HT2A receptor promoted long-lasting (agonistic) activation of Gq11/phospholipase C/inositol triphosphate receptor/Ca2+ signaling causing accelerated endothelial cell and neuronal cell death [16]. Since the 5-hydroxytryptamine 2A receptor is normally expressed on arterial smooth muscle cells [17] and on neurons in specific brain regions involved in the regulation of mood, thinking, perception, and sleep [18], altered signaling at the 5-HT2A receptor could have diverse peripheral and central effects.

The aim of the present study was to test for spontaneously-occurring autoantibodies to the 5-HT2A receptor in the circulation in male ZDF rat (fa/fa) and age-matched male lean Zucker rats either lacking a known leptin receptor mutation (+/?) or harboring a heterozygous leptin receptor mutation (fa/+). The developmental expression of autoantibody was compared in each genetic strain, and the mechanism of neurotoxicity downstream of 5-HT2AR binding was evaluated in mouse neuroblastoma N2A cells.

Materials and Methods

Animals

All procedures were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Veterans Affairs Medical Center (East Orange, New Jersey). Male ZDF (N=12) and lean (+/?) Zucker rats (N=12) were obtained from Charles River Laboratories (Kingston, NY) at approximately 6 weeks of age. All rats were single housed upon arrival, with modest enrichment (a PVC tube). Rats were provided ad libitum access to food and water and maintained in a 12 h light/dark cycle with lights on at 0630. All procedures occurred during the light phase of the cycle.

Blood Drawing

EDTA plasma from 10-week ZDF rat and 10-week lean, heterozygous (fa/+) Zucker rats was obtained from Charles River Laboratories. For all other time points, blood was collected from the retro-orbital plexus of ZDF, and lean (+/?) Zucker rats using a retro-orbital blood procedure. The blood was centrifuged at 1400 g x 10 minutes at 4 degrees C. The resulting plasma was stored at -4^oC for up to 2-3 weeks prior to protein G affinity chromatography.

Tail-Nick

Capillary blood glucose was determined from a drop of blood obtained after tail nick at sequential time intervals. A glucose dehydrogenase method (Accucheck Aviva, Roche Diagnostics, Inc) was used for the determination of blood glucose.

Protein G Affinity Chromatography

Protein-G affinity chromatography was carried out as previously reported for protein-A affinity chromatography in human plasma [16]. The pH of the 10 mm Tris binding buffer (6.5) and the 0.1 M citrate elution buffer (2.7) was adjusted to optimize binding and elution of rat immunoglobulin G. Protein-G eluate fractions were stored at 0-4 degrees C.

Mouse N2A Neuroblastoma Cells

Cells were cultured in DMEM with 10% fetal calf serum as previously reported [16].

Neurite Retraction and N2A Cell Survival Assays

Assays were performed as previously described [16].

Enzyme-Linked Immunoassay (ELISA)

The ELISA procedure was carried out as previously reported using an 18-meric linear synthetic peptide Q..N18 corresponding to the second extracellular loop of the human 5-HT2AR as the solid phase antigen [15].

Chemicals

All chemicals were obtained from Sigma Co., Inc. (St. Louis, MO).

Protein determination-Protein levels were determined using a bichinchonic assay method (BioRad, Inc.).

Statistics

Statistical analysis was performed using an unpaired Student’s t-test with an α cutoff level for significance of < 0.05.

Results

Occurrence of Circulating 5-HT2A Receptor Autoantibody in the ZDF Rat: Relation to Obesity and Diabetes

Mean body mass in ZDF rats significantly exceeded that in lean (+/?) Zucker rat at week 7, week 8.5, and week 12 (Figure 1A). There was no longer a significant weight difference between the strains at week 25 (Figure 1A). Diabetes (hyperglycemia) emerged between week 8-10 in the ZDF rat, but at week 7 the ZDF rat was still normoglycemic (Figure 1B). In contrast, lean (+/?) Zucker rats were normoglycemic at all ages tested (Figure 1B), not tested at week 7.

Figure 1: Age-dependent change in body mass(A) and capillary blood glucose concentration (B) in male Zucker fatty (ZDF) vs lean (+/?) (ZDL) rats. A) Body weight increased significantly in the male ZDF rat (starting at week 7) compared to control lean Zucker rat B) capillary blood glucose was determined by tail nick using glucose dehydrogenase method as described in Materials and Methods. Glucose was normal at week 7 in the ZDF rat, but increased significantly compared to ZDL rat starting at week 8.

Detection of plasma immunoreactive 5-HT2AR autoantibody had a similar trajectory to diabetes in ZDF rats. At 7 weeks of age, ZDF rat had no detectable plasma immunoreactive 5-HT2AR autoantibody (Figure 1C). At 10-weeks of age, plasma from ZDF rat contained a two-fold, higher binding to 5-HT2AR (0.14 AU, N=2) compared to background levels (0.07 AU; Figure 2a). Plasma from lean ((+/?) Zucker rats harbored two-fold or slightly higher level of autoantibody binding to 5-HT2AR between 8.5-15 weeks of age which declined to undetectable levels at 25 weeks of age (Figure 2B).

Figure 2: Developmental expression of autoantibody binding to 5-HT2AR synthetic peptide in three Zucker rat strains. A 2 ug/mL concentration of the protein G eluate was tested for binding to the linear synthetic 18-meric 5-HT2A receptor peptide as described in Materials and Methods. Results are “mean +/- SE” in A) ZDF rat: 7-week (N=2); 8.5-week (N=3); 10-week (N=2); 25-week (N=3) B) lean (+/?) Zucker rat: 7-week (N=3); 8.5 week (N=3); 15-week (N=10); 25-week (N=6). C) lean heterozygote (fa/+) Zucker rat: 7-week (N=2); 10-week (N=5); 25-week (N=2).

Spontaneous IgG autoantibody to the 5-HT2A receptor was already present at 7 weeks of age in the heterozygous lean (fa/+) Zucker rat (Figure 2C) and levels at 10-weeks old and 25-weeks old were substantially higher than in age-matched ZDF rat (Figure2C). Autoantibody to the 5-HT2A receptor persisted into older age, i.e. 25 weeks old, in male ZDF and lean heterozygous Zucker rats, but disappeared at 25 weeks of age in the lean (+/?) Zucker rat lacking a known leptin receptor mutation. In a recent report [19], diabetes, obesity and hypertension were associated with substantially increased hazard rates for the occurrence of dementia, Parkinson’s disease and severe depression following traumatic brain injury in older adult veterans.

The present data suggest that male ZDF and heterozygous lean rat strains may be useful in modeling the effects of heightened innate and/or adaptive immunity, respectively, on the occurrence of neurodegeneration.

Zucker Diabetic Fatty Rat Plasma 5-HT2AR Autoantibody Inhibits N2A Cell Survival

We next tested for neurotoxicity in the autoantibody (from 10- week-old ZDF rat plasma) that displayed increased binding to the 5-HT2A receptor peptide. Autoantibody in plasma from the 10-week old ZDF rat caused dose-dependent inhibition of survival in N2A mouse neuroblastoma cells (Figure 3). A two and one-half microgram per milliliter concentration of the IgG caused approximately 40% neuroblastoma cell loss (after 24 hours incubation) compared to control, untreated cells (P< 0.01, Figure 3).

Figure 3: Dose-dependent neurotoxicity in the protein G eluate fraction from Zucker diabetes fatty rat plasma. The protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells (at the indicated concentrations) for 24 hours. % N2a cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of quadruplicate determinations; * P < 0.01 compared to cell survival in untreated cells.

Zucker Diabetic Fatty Rat Plasma 5-HT2AR Autoantibody Causes Acute N2A Neurite Retraction

A 120 nanomolar concentration (1.8 g/mL IgG) of the protein G- eluate fraction of plasma from 10-week-old male ZDF fatty rat caused significant 37% acute neurite length-shortening (after 15 minutes of incubation) of N2A mouse neuroblastoma cells (Figure 4A). Co-incubation with a 200 nanomolar concentration of M100907, a highly selective, potent antagonist of the 5-HT2AR, afforded 86% significant (P < 0.01) protection against autoantibody-induced neurite retraction (Figure 4A).

Figure 4: Neurite outgrowth inhibition (A) and accelerated N2A neuroblastoma cell loss (B) in autoantibody from ZDF rat plasma: neutralization by M100907, a highly selective 5-HT2AR antagonist. A) A 120 nM concentration of the protein-G eluate fraction of 10-week-old male ZDF rat plasma was incubated with N2A mouse neuroblastoma cells in the presence or absence of a 200 nM concentration of the selective 5-HT2AR antagonist M100907. % Basal neurite length was determined after 15 minutes as described in Materials and Methods B) A 160 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells in the presence or absence of the indicated concentration of M100907 for 24 hours at 37 deg C. Percent N2A cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations.

Pharmacologic Profile of Zucker Diabetic Fatty Rat Plasma 5-HT2AR Autoantibody Toxicity

A ~170 nanomolar concentration (2.5 µg/mL) of the protein G-eluate fraction of plasma from 10-week-old male ZDF rat was incubated with N2A mouse neuroblastoma cells in the presence or absence of a (500 or 1000) nanomolar concentration of M100907 for 24 hours at 37 deg C. M100907 caused significant (dose-dependent) protection against accelerated N2A cell loss, after 24 hours incubation with the ZDF rat IgG (Figure 4B).

Spiperone is a potent, but less selective 5-HT2AR antagonist. Spiperone at 500-1000 nanomolar concentrations afforded significant dose-dependent protection against ZDF rat IgG-induced accelerated N2A cell loss (Figure 5). Ketanserin is a less potent 5-HT2AR antagonist compared to M100907 or spiperone. Substantially higher concentration (2.5 µM) of ketanserin was required to significantly protect against ZDF rat IgG-induced N2A cell loss (Figure 6).

Figure 5: Potent, less selective 5-HT2AR antagonist (spiperone) dose-dependently prevented accelerated neuron loss induced by ZDF rat plasma autoantibody. A 160 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells in the presence or absence of the indicated concentration of spiperone for 24 hours at 37 deg C. Percent N2a cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations.

Figure 6: Higher concentration of ketanserin, a less potent, but selective 5-HT2AR antagonist dose-dependently prevented accelerated neuron loss induced by ZDF rat plasma autoantibody A-B) 120 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells in the presence or absence of the indicated concentration of ketanserin for 24 hours at 37 deg C. Percent N2a cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations.

The receptor antagonist profile and concentration associated with significant protection against ZDF IgG autoantibody-induced neuroblastoma cell loss is consistent with relative affinity constants on the 5-HT2A receptor (M100907 < spiperone<< ketanserin).

A one micromolar concentrations of SB-20471, a selective antagonist to the 5-HT2B receptor did not significantly prevent ZDF autoantibody-induced neurite retraction (Table 1). A ten micromolar concentration of either bosentan (an endothelin-1 receptor) or losartan (an angiotensin II type 1 receptor) antagonist did not significantly protect against ZDF IgG-induced acute N2A neurite retraction (Table 1). An 850 nanomolar concentration of prazosin (alpha 1 adrenergic R antagonist) did not afford significant protection against ZDF IgG autoantibody-induced acute neurite retraction (Table 1). Taken together, these data suggest that neurotoxicity in the 10-week old male ZDF rat IgG appears selective for the 5-HT2A receptor.

Table 1: Receptor specificity of prevention of ZDF rat IgG autoantibody-induced acute N2A neurite retraction.

Antagonist	Conc	GPCR	% of ZDF IgG-induced Neurite Retraction
M100907	200 nM	5HT2A	13% + 15%
SB-20471	1 µM	5HT2B	89% + 20%
Bosentan	10 µM	ET-1	95% + 20%
Losartan	10 µM	AT-1	81% + 5%
Prazosin	850 nM	A1-A	89% + 11%

A 160 nanomolar concentration of 10-week-old male Zucker fatty rat IgG autoantibodies was incubated in the presence or absence of the indicated GPCRs. Acute N2A neurite retraction was determined after 15 minutes as described in Materials and Methods. Results are mean +/- SD.

Involvement of Gq11/Phospholipase C/Inositol Triphosphate and Rho Kinase Signaling Pathways

Co-incubation of N2A cells with ZDF plasma IgG autoantibodies together with individual selective antagonists of Gq11 (YM-254890), phospholipase C (U73122), inositol triphosphate receptor (2-APB) signaling or Rho A/Rho kinase (Y27632) mediated signaling completely or nearly completely protected against acute neurite retraction (Table 2) consistent with involvement of Gq11/phospholipase C/inositol triphosphate and Rho kinase signaling pathways in mediating IgG neurotoxicity downstream of 5-HT2A receptor binding.

Table 2: Effect of signaling pathway inhibitors on ZDF rat IgG autoantibody-induced acute N2A neurite retraction.

Antagonist	Conc	Signaling  Intermediate	% of ZDF IgG-induced Neurite Retraction
U73122	10 µM	PLC	14% + 13%
YM-254890	1 µM	Gq11	14% + 5%
2-APB	50 µM	IP3R	0% + 0%
Y27632	10 µM	Rho kinase	0% + 0%

A 160 nanomolar concentration of 10-week-old male Zucker fatty rat IgG autoantibodies was incubated in the presence or absence of the indicated GPCRs. Acute N2A neurite retraction was determined after 15 minutes as described in Materials and Methods. Results are mean +/- SD as described in Materials and Methods.

Level and Titer of Autoantibody to 5-HT2A Receptor in Male Zucker Heterozygote (fa/+)

An identical (1.5 µg/mL concentration) of the IgG from Zucker heterozygote rat plasma (N=5) displayed two-fold significantly higher binding (to the 5-HT2A receptor peptide) compared to IgG in plasma from five ZDF rat plasmas (N=2, 10-week-old and N=3, 25-week-old rats) (Figure 7A). The titer of autoantibody to the 5-HT2A receptor peptide was more than two-fold higher in the 10-week old lean Zucker heterozygous rat compared to age-matched, 10-week old ZDF rat (Figure 7B).

Figure 7: A) 2 ug/mL concentration of the protein G eluate of ZDF rat and lean Zucker heterozygote plasma was tested for binding to linear, synthetic 18-meric 5-HT2A receptor peptide.
B) Titer of autoantibody binding to 5-HT2A receptor peptide was tested at the indicated concentrations in 10-week lean Zucker heterozygote (N=5) or 10-week ZDF (N=2) protein G eluate. Results are “mean +/- SE”. * P< 0.01

Bioactivity in the Antibody Fraction of Male Zucker Heterozygous (fa/+) Plasma

Dose-dilution curves of the protein-G eluate fraction from three, 10-week-old lean heterozygous Zucker heterozygous plasmas demonstrated potent inhibition of N2A cell survival at all IgG concentrations tested in two of three rats (Figure 8A). In one of the three rats, lean heterozygous Zucker rat #3, a component of significant N2A survival-promoting activity were observed at high IgG dilution (Figure 8A). ‘Functional selectivity’ is a unique property of the 5-hydroxytryptamine 2A receptor [20]-it refers to the ability of different 5-HT2AR ligands to preferentially activate alternative signaling pathways, e.g. PLC/IP3/Ca2+ signaling or a beta-arrestin 2/PI3-kinase survival pathway. Since the selective 5-HT2AR antagonist M100907 significantly protected against lean heterozygous Zucker rat IgG autoantibody-induced accelerated N2A cell loss (Figure 8B) the neurotoxicity is likely due to Gq11/PLC/IP3/Ca2+-mediated signaling occurring downstream of 5-HT2AR binding. More study is needed, however, to determine whether N2A survival-promoting activity in a subset of lean heterozygous Zucker rat IgG autoantibodies may result from selective activation of beta arrestin-2/PI3-kinase-mediated signaling. In a prior report [21], highest level of 5-HT2A receptor-binding occurring in the autoantibodies from a patient with lupus. The lupus IgG autoantibodies were not only neurotoxic, but (at certain concentrations) promoted N2A neuroblastoma cell survival dependent on PI3 kinase-mediated signaling [21].

Figure 8: A) Dose-dilution curves of three Zucker lean heterozygous protein G eluates on survival in N2A mouse neuroblastoma cells; B) Co-incubation of a representative lean heterozygous protein G eluate fraction with 1 µM concentration of the selective 5-HT2AR antagonist M100907. * P< 0.05.

Apparent MW of the Active Component in ZDF Protein G Eluates

The active component in the ZDF protein G eluate (which caused accelerated N2A cell loss) had an apparent MW > 10 kD (Figure 9A) consistent with an antibody or antibody fragment. There was no significant N2A cell survival-inhibitory activity present in the flow-through fraction following membrane dialysis of the ZDF protein G eluate on a 10 kilodalton Molecular Weight (MW) cutoff membrane (Figure 9A). The freshly-isolated, lean heterozygous Zucker rat (fa/+) protein G eluate fraction (SM, starting material) caused significantly increased N2A cell survival. Following dialysis on a 10 kD MW cutoff membrane, more than 90% of the N2A survival stimulatory activity was recovered in the fraction having an apparent MW > 10 kD, i.e. retained on the membrane (Figure 9B). The low MW (< 10 kD) flow-through fractions following membrane dialysis of the ZDF or lean heterozygous Zucker rat protein G eluate did not cause significant inhibition or stimulation of N2A survival (Figure 9A and 9B).

Figure 9: Apparent MW of peak neuroblastoma survival-inhibitory (A) and – stimulatory (B) activity in the plasma protein G eluate from ZDF or heterozygous lean ZDL rat. Apparent MW of peak neuroblastoma survival-inhibitory (A) or -stimulatory (B) activity in the protein G eluate from a representative ZDF (A) and lean heterozygote # 3 Zucker rat (B) plasma. A 160 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker rat plasma was dialyzed on a 10kD MW cutoff membrane. The resulting retained and flow-through fractions or starting material (SM) were incubated with N2a mouse neuroblastoma cells for 24 hours as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations. * P < 0.05 compared to control N2A cell without added test fractions.

Discussion

The male ZDF rat is a well-studied model of human obese type 2 diabetes mellitus. The animals undergo progressive gain in fat mass, with obesity arising around 4-weeks of age [9]. Hyperglycemia commences around week 8- 9, and chronic marked elevation in blood glucose concentrations (400-500 mg/dL) is associated with early hyperinsulinemia followed by later relative insulin deficiency [10]. Hypertension and severe hypertriglyceridemia manifest around week 9 in the ZDF rat [9,10]. In adult, long-standing human obese type 2 diabetes mellitus from the Veterans Affairs Diabetes Trial, the baseline presence of anti-endothelial cell autoantibodies was significantly associated with lack of baseline insulin therapy (i.e. relative insulin deficiency) and lack of baseline triglyceride-lowering medication [22]. Endothelial cell heparanase expression is upregulated in insulin deficiency [23] and by high level of pro-inflammatory cytokines [24]. Heparanase cleaves heparan sulfate side chains abundant on neurons and vascular cells, and heparan sulfate proteoglycan is a known target of humoral autoimmunity [25]. In older adult diabetes patients evidence suggested that autoantibodies targeting the 5-HT2AR were in many cases cross-reactive with heparan sulfate proteoglycan [26]. Taken together it is possible that certain metabolic derangements in the ZDF rat strain, (insulin deficiency, and high level of pro-inflammatory cytokines) may contribute in part to the long persistence of circulating 5-HT2A receptor autoantibodies at older ages.

Although the heterozygous (fa/+) Zucker rat is lean and non-diabetic, it demonstrated substantially higher titer of 5-HT2A receptor autoantibodies compared to age-matched ZDF rat. One possibility is that enhanced helper T-cell immunity in the lean heterozygote Zucker rat drives autoantibody formation which includes a subset (of autoantibodies) targeting the 5-HT2A receptor. Neurotoxicity in the lean heterozygote Zucker IgG autoantibodies (i.e. acute neurite retraction and accelerated N2A cell loss) was as high or higher than in ZDF IgG autoantibodies consistent with a prior report of a significant correlation between antibody binding level and level of acute neurite retraction in N2A cells [15]. Higher level of 5-HT2AR targeting autoantibodies in the lean heterozygote Zucker rat supports a role for upregulated adaptive humoral immunity (in part) due to a heterozygous leptin receptor mutation causing hyperleptinemia. More study is needed to determine whether high titer of autoantibodies in the lean heterozygous Zucker rat may predispose to immune complex formation that could bias signaling pathway activation downstream of 5-HT2A receptor binding.

In summary, 5-HT2A receptor autoantibodies increased in the circulation in male hypertensive ZDF rat between 8.5-10-weeks of age and persisted up until 25-weeks old or longer. The ZDF rat 5-HT2A receptor-targeting autoantibodies caused acute neurite retraction and accelerated mouse neuroblastoma cell loss by a mechanism involving activation of Gq11/PLC/IP3R pathway and RhoA/Rho kinase coupled signaling. The ZDF and lean heterozygous Zucker rat strains may be suitable animal models for investigations of the roles of obesity-associated inflammation and dysregulated humoral immunity, respectively, in the etiology of certain forms of late-onset neurodegeneration.

Funding

This project was supported by grants from the New Jersey Commission on Brain Injury Research (CBIR19PIL007) to MBZ and the Biomedical Laboratory Research and Development Service of the VA Office of Research and Development (I1BX004561) to KCHP. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs or the United States government.

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