Cytokine dysregulation in aged horses and horses with pituitary pars intermedia dysfunction

BACKGROUND: Equine pituitary pars intermedia dysfunction (PPID) is the result of a loss of dopaminergic inhibition of the pars intermedia secondary to neurodegeneration of periventricular hypothalamic neurons. The pathologic events contributing to development of neurodegeneration or clinical signs in equids with PPID are unknown. Chronic inflammation may contribute to initiation or progression of PPID. HYPOTHESIS: Horses with PPID have a distinct systemic cytokine profile compared with that of normal adult or aged horses. The cytokine profile of healthy aged horses differs from that of adult horses. ANIMALS: Aged horses with PPID, healthy aged-matched controls, and adult controls (n = 14 per group). METHODS: Total leukocyte cytokine expression was determined by quantitative polymerase chain reaction (PCR), and tumor necrosis factor (TNF)-alpha plasma concentration was determined by enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cell (PBMC) TNF-alpha response after endotoxin (lipopolysaccharide [LPS]) treatment was assessed by ELISA. RESULTS: Aged healthy horses had increased expression of interleukin (IL)-6, IL-8, and interferon-gamma as well as PBMC TNF-alpha release after LPS stimulation compared with healthy adult horses. In contrast, aged horses with PPID had increased IL-8 expression, but expression of other cytokines was similar to that of healthy adult horses, not age-matched controls. CONCLUSIONS AND CLINICAL IMPORTANCE: Aged horses show evidence of a proinflammatory state that may contribute to development of age-associated diseases. Horses with PPID have increased expression of IL-8, which may influence the ability of horses with PPID to respond to bacterial pathogens. The general decrease in proinflammatory cytokine expression observed in horses with PPID may be the outcome of high plasma concentrations of anti-inflammatory hormones.     

Systemic and pituitary pars intermedia antioxidant capacity associated with pars intermedia oxidative stress and dysfunction in horses

OBJECTIVE: To determine whether a deficiency in systemic or local (pars intermedia) antioxidant capacity is associated with pituitary pars intermedia oxidative stress and pituitary pars intermedia dysfunction (PPID) in horses. SAMPLE POPULATION: Blood samples from 20 horses with PPID and 20 healthy client-owned horses, archived paraffin-embedded adrenal gland and substantia nigra tissues from 20 horses, and pituitary gland tissue from 16 horses. PROCEDURES: Total glutathione, superoxide dismutase, and glutathione peroxidase activities were determined in RBCs. Accumulation of a systemic marker of oxidative stress (3-nitrotyrosine) was assessed in plasma and formalin-fixed, paraffin-embedded adrenal gland and substantia nigra tissues. Local antioxidants (total and manganese superoxide dismutase, glutathione peroxidase, and total glutathione) were measured in pars intermedia tissues. RESULTS: No significant differences existed in systemic antioxidant enzyme activity or accumulation of 3-nitrotyrosine between horses with PPID and control horses. In pituitary gland tissues, glutathione peroxidase activity was increased in horses with oxidative stress, whereas total glutathione concentration and superoxide dismutase activity remained unchanged. There was an age-associated decrease in manganese superoxide dismutase activity in the pars intermedia. CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence of systemic accumulation of oxidative stress markers or deficiencies in antioxidant capacity in horses with PPID, suggesting that these are unlikely to be major predisposing factors in the development of PPID. Manganese superoxide dismutase activity in the pars intermedia decreased significantly with increasing age. Role of an age-associated decrease in antioxidant capacity for the pars intermedia in the development of PPID in horses warrants further investigation.     

Agreement in histologic assessments of the pituitary pars intermedia in aged horses

OBJECTIVE: To evaluate concordance among veterinary pathologists in the assessment of histologic findings in the pars intermedia of pituitary gland sections from aged horses with mild signs suggestive of pituitary pars intermedia dysfunction (PPID). Sample Population-10 pituitary glands from aged horses. PROCEDURE: 7 pathologists were provided with signalment, clinical signs, and a single H&E-stained pituitary gland section from 10 aged horses with mild signs suggestive of PPID. Pathologists described histologic findings for each section and stated whether findings were consistent with PPID. Agreement among pathologists and with antemortem diagnostic test results was calculated. RESULTS: Overall, only fair agreement was found among the pathologists as to which horses had histologic findings consistent with disease (mean +/- SE kappa value, 0.34 +/- 0.069). Interpretation of individual sections varied, with minimal agreement (4 or 5/7 pathologists) for 5 of 10 sections evaluated. Postmortem assessment was in agreement with an antemortem endocrine diagnostic test result 79% of the time. CONCLUSIONS AND CLINICAL RELEVANCE: Validation of antemortem diagnostic testing for PPID in horses often relies on the results of postmortem histologic evaluation. The lack of consensus in histologic interpretation of pituitary glands from aged horses with mild clinical signs in our study indicates that postmortem histologic evaluation of pituitary glands is an inappropriate standard in validation of antemortem diagnostic tests for detection of early PPID. Caution should be used when interpreting diagnostic test results in horses in which early PPID is suspected.     

Evaluation of suspected pituitary pars intermedia dysfunction in horses with laminitis

OBJECTIVE: To determine prevalence and clinical features of pituitary pars intermedia dysfunction (PPID) in horses with laminitis. DESIGN: Case series. ANIMALS: 40 horses with laminitis. PROCEDURES: Horses with laminitis that survived an initial episode of pain and were not receiving medications known to alter the hypothalamic-pituitary-adrenal axis were tested for PPID by evaluation of endogenous plasma ACTH concentration. Signalment, suspected cause, month of onset and duration of laminitis, Obel grade of lameness, pedal bone rotation, physical examination findings, results of endocrine function tests, treatment, outcome, and postmortem examination findings were recorded. RESULTS: Prevalence of PPID as defined by a single high plasma ACTH concentration was 70%. Median age of horses suspected of having PPID (n = 28) was 15.5 years, and median age of horses without PPID (12) was 14.5 years. Laminitis occurred most frequently in horses with and without suspected PPID during September and May, respectively. Chronic laminitis was significantly more common in horses suspected of having PPID. In horses suspected of having PPID, the most common physical examination findings included abnormal body fat distribution, bulging supraorbital fossae, and hirsutism. Five horses suspected of having PPID had no clinical abnormalities other than laminitis. Seventeen horses suspected of having PPID that were treated with pergolide survived, and 3 horses that were not treated survived. CONCLUSIONS AND CLINICAL RELEVANCE: Evidence of PPID is common among horses with laminitis in a primary-care ambulatory setting. Horses with laminitis may have PPID without other clinical signs commonly associated with the disease.     

Suspensory ligament degeneration associated with pituitary pars intermedia dysfunction in horses

In older horses, pituitary pars intermedia dysfunction (PPID) and suspensory ligament (SL) degeneration are common. The aim of the present study was to identify histopathological changes in the SL in horses with PPID. SLs of four horses with clinical signs of PPID (17–26 years of age) were compared with SLs from four old horses (18–31 years of age) and three young horses (4–9 years of age). In horses with PPID, there was reduced longitudinal arrangement of collagen fibres in SLs, along with inclusions of cartilage, extracellular matrix and haemorrhage, as well as significant proteoglycan accumulations between SL fibres. These changes are similar to the degeneration of connective tissues in Peruvian Paso horses with SL degeneration and in humans with Cushing's disease or after long term high dose corticosteroid treatments. These findings indicate an association between degeneration of the SL and PPID.     

Evaluation of the combined dexamethasone suppression/ thyrotropin-releasing hormone stimulation test for detection of pars intermedia pituitary adenomas in horses

BACKGROUND: A combined dexamethasone (DEX) suppression/thyrotropin-releasing hormone (TRH) test (DEX/TRH test) has been developed to evaluate horses for presence of a pars intermedia pituitary adenoma (PIPA), but to the authors' knowledge, the accuracy of this test has not been previously determined. HYPOTHESIS: The sensitivity and specificity of the DEX/TRH test can be determined by comparing test results with histopathologic examination findings. ANIMALS: Age of 42 horses of various breeds ranged from 2 to 33 years. METHODS: Plasma cortisol concentration was measured before and 24 hours after IV administration of 40 microg of DEX/kg of body weight, and before and 30 minutes after IV administration of 1 mg of TRH that had been given 3 hours after the injection of DEX. Results of the DEX/TRH test were considered positive if either the plasma cortisol concentration exceeded 10 ng/mL 24 hours after DEX administration, or if the change in plasma cortisol concentration 30 minutes after injection of TRH was > or = 66% above the 3-hour baseline. Diagnosis of PIPA was determined by histologic examination of the pituitary gland. RESULTS: PIPA was detected in 17 of 42 (40%) horses. The DEX/TRH test had sensitivity, specificity, positive predictive value, and negative (NPV) predictive value of 88, 76, 71, and 90%, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: The combined DEX/TRH test was more sensitive than either of its component tests and had a high NPV, but was not as specific as the TRH component alone (92%). The DEX/TRH test should be used to screen older horses for PIPA.     

Evaluation of pituitary gland anatomy and histopathologic findings in clinically normal horses and horses and ponies with pituitary pars intermedia adenoma

OBJECTIVE: To determine size and weight of the pituitary gland and associations between pituitary gland size and weight and sex and age in horses without clinical signs associated with pituitary pars intermedia adenoma (PPIA) and horses and ponies with PPIA. ANIMALS: Pituitary glands from 100 horses without clinical signs of PPIA and 19 horses and 17 ponies with PPIA. PROCEDURES: Pituitary glands were weighed, measured, and examined histologically by use of H&E stain. Masson trichrome and periodic acid-Schiff staining were used, when appropriate. Histologic lesions in the pars intermedia, pars distalis, or both were classified as no significant lesions, single or multiple cysts, focal or multifocal hyperplasia, single or multiple microadenomas, and adenoma. Relative pituitary weight (RPW) was calculated as pituitary weight (grams) divided by body weight (grams). RESULTS: There was an age-related increase in the presence of pituitary lesions in the pars distalis and pars intermedia in geldings, mares overall, and non-pregnant mares. Mean (+/-SD) RPW in horses with PPIA was not significantly different from ponies with PPIA (15+/-5.9 x 10(-6) and 16+/-72 x 10(-6), respectively). Maximum pituitary weight in a horse with PPIA was 13.9 g (RPW, 2.9 X 10(-5)). Plasma glucose concentration was positively correlated with RPW in ponies with PPIA. CONCLUSIONS AND CLINICAL RELEVANCE: Pituitary lesions may be a factor in horses with insulin resistance and laminitis before development of clinical signs of PPIA. Ovarian steroids may be involved in the pathogenesis of lesions in the pars intermedia.     

Comparison of hair follicle histology between horses with pituitary pars intermedia dysfunction and excessive hair growth and normal aged horses

Background –  Pituitary pars intermedia dysfunction (PPID) in older equids is commonly recognized by a long hair coat that fails to shed. Objective –  The aim of this study was to compare hair follicle stages in PPID‐affected horses with excessively long hair coats with the stages of normal aged horses (controls) and to compare hair follicle stages in PPID‐affected horses after 6 months of treatment with pergolide mesylate with those of control horses. Animals –  Eight PPID‐affected horses and four normal, age‐matched, control horses. Methods –  Skin biopsies were collected from the neck and rump of PPID‐affected and control horses. A diagnosis of PPID was established based on hair coat changes and supportive overnight dexamethasone suppression test results. Skin biopsies were repeated after 6 months of treatment with pergolide. The number of hair follicles in anagen (A) or telogen (T) was counted for each skin biopsy using transverse sections. Results –  Pretreatment biopsies had a greater percentage of A follicles (neck 96%, rump 95%) and a lower percentage of T follicles (neck 4%, rump 5%) in PPID‐affected horses than in control horses (A, neck 15%, rump 25%; and T, neck 85%, rump 75%). After treatment with pergolide, all PPID‐affected horses had improved shedding, and the percentages of A follicles (neck 69%, rump 70%) and T follicles (neck 31%, rump 30%) were not different from untreated control horses (A, neck 68%, rump 82%; and T, neck 32%, rump 18%). Conclusions –  These findings document that excessive hair growth (hypertrichosis) in PPID‐affected horses is due to persistence of hair follicles in A. Furthermore, treatment with pergolide improved shedding and reduced the percentage of A follicles in PPID‐affected horses.     

Preliminary study on the effects of pergolide on left ventricular function in the horses with pituitary pars intermedia dysfunction

BackgroundPituitary pars intermedia dysfunction (PPID), a neurodegenerative disease leading to reduced dopamine production, is a common disease in aged horses. The treatment is based on administration of the dopamine agonist pergolide. This drug has been related to valvular fibrosis in humans, but the cardiovascular effect of this drug has not yet been investigated in horses.ObjectivesTo determine whether pergolide induces valvular disease in horses or affects the cardiac function.MethodsStandard, tissue Doppler (TDE) and two-dimensional speckle tracking (STE) echocardiography were performed in horses with diagnosed PPID based on adrenocorticotropic hormone dosage. Measurements taken in horses treated with pergolide were compared with those from untreated horses with nonparametric t-tests. Furthermore, measurements from follow-up examinations performed at least three months after the initial exam were compared with a Wilcoxon signed rank test for repeated measurements in each group.ResultsTwenty-three horses were included. None of the 12 horses under treatment developed valvular regurgitation. Furthermore, no differences in the measurements of the left ventricular systolic or diastolic function could be seen between the group of horses with treatment and those without treatment. Measurements taken in the follow-up exam did not differ compared to those taken in the initial exam in both groups.ConclusionsNo changes of the left ventricular function assessed by TDE and STE could be shown in a small population of horses with confirmed PPID. Treatment with pergolide did not affect the ventricular function nor induce valvular disease.     

Prevalence,risk factors and clinical signs predictive for equine pituitary pars intermedia dysfunction in aged horses

Reasons for performing study: Equine pituitary pars intermedia dysfunction (PPID) is an ageing‐related neurodegenerative disorder. The prevalence and risk factors for PPID using seasonally adjusted basal adrenocorticotropic hormone (ACTH) concentrations in aged horses have not been previously reported. Objectives: To determine the prevalence, risk factors and clinical signs predictive for PPID in a population of horses aged ≥15 years in Queensland, Australia. Methods: Owner‐reported data was obtained using a postal questionnaire distributed to an equestrian group. A subgroup of surveyed owners were visited and a veterinary physical examination performed on all horses aged ≥15 years. Blood samples were analysed for basal plasma alpha melanocyte‐stimulating hormone (α‐MSH) and ACTH concentrations, routine haematology and selected biochemistry. Aged horses with elevations above seasonally adjusted cut‐off values for basal plasma ACTH were considered positive for PPID. Positive horses were compared with their aged counterparts to determine risk factors and clinical signs associated with PPID. Results: Pituitary pars intermedia dysfunction was prevalent in aged horses (21.2%) despite owners infrequently reporting it as a known or diagnosed disease or disorder. Numerous clinical or historical signs were associated with an increased risk of PPID in the univariable model, but only age (odds ratio (OR) 1.18; 95% confidence interval (CI) 1.11–1.25, P<0.001) and owner‐reported history of hirsutism (OR 7.80; 95% CI 3.67–16.57, P<0.001) remained in the final multivariable model. There were no routine haematological or biochemical variables supportive of a diagnosis of PPID. Conclusions and potential relevance: Pituitary pars intermedia dysfunction occurs commonly in aged horses despite under‐recognition by owners. The increased risk of PPID with age supports that this is an ageing associated condition. Aged horses with clinical or historical signs consistent with PPID, especially owner‐reported hirsutism (delayed shedding and/or long hair coat), should be tested and appropriate treatment instituted.     

Adrenocorticotropin concentration following administration of thyrotropin-releasing hormone in healthy horses and those with pituitary pars intermedia dysfunction and pituitary gland hyperplasia

OBJECTIVE: To compare the effect of thyrotropin-releasing hormone (TRH) administration on endogenous ACTH concentrations in healthy horses and those with pituitary pars inter-media hyperplasia and compare the test with the dexamethasone suppression test (DST). DESIGN: Prospective case series. ANIMALS: 15 horses with clinical signs of pituitary pars intermedia dysfunction (PPID), 4 horses with equivocal signs of PPID, and 29 horses without signs of PPID. PROCEDURES: ACTH concentrations prior to and after administration of TRH were measured 61 times in 48 horses. Results of the DST (cortisol response) were compared with those of the TRH test in 29 horses. Thirty-three horses (24 with no clinical signs of PPID, 5 with clinical signs of PPID, and 4 with equivocal clinical signs of PPID) were euthanized and necropsied and their pituitary glands evaluated. RESULTS: ACTH concentrations increased in all horses, but magnitude and duration of increase were significantly higher in horses with PPID. Endogenous ACTH concentrations were influenced by season. The ACTH baseline concentrations and response to TRH were not correlated with results of the DST. Results of DST were abnormal only in clinically abnormal horses or those with pars intermedia hyperplasia, but were within reference range in 17 of 26 tests in these horses. CONCLUSIONS AND CLINICAL RELEVANCE: The ACTH response to TRH is a useful test for diagnosis of pituitary gland hyperplasia, particularly in horses in which baseline ACTH concentrations are within reference range. The DST was specific but not sensitive and was inconsistent for individuals, and results often did not agree with the TRH test response.     

Pathophysiology and clinical features of pituitary pars intermedia dysfunction

Our understanding of the development and progression of equine pituitary pars intermedia (PI) dysfunction has expanded over the last decade, although much remains to be explained. Degeneration of the hypothalamic periventricular dopaminergic neurons results in disinhibition of the endocrine cells of the PI, the melanotropes. As a result, the PI enlarges, compressing the adjacent lobes, the pars distalis and pars nervosa. The disinhibited melanotropes overproduce pro‐opiomelanocortin peptides, including α‐melanocyte stimulating hormone, β‐endorphin, corticotrophin‐like intermediate lobe peptide and adrenocorticotropin. The excess in PI hormones, perhaps in concert with a deficiency in other pituitary hormones, results in clinical signs of pituitary PI dysfunction. Evidence suggests both oxidative stress and accumulation of misfolded neuronal proteins contribute to damage to the periventricular neurons, although it is not clear if these pathologies actually initiate the disease or are downstream events.