Effect of short-term dopamine reduction on insulin sensitivity and post-prandial insulin and glucose responses in Standardbred horses.

The role of dopamine in the regulation of insulin secretion in horses is poorly understood and requires further investigation. Pituitary pars intermedia dysfunction (PPID) is associated with decreased activity of dopaminergic neurons which normally suppress peptide hormone secretion from the pituitary pars intermedia. A high proportion of horses with PPID also have insulin dysregulation (ID), characterised by post-prandial hyperinsulinaemia and/or tissue insulin resistance, which are risk factors for the development of laminitis. The aim of this study was to investigate the effects of alpha-methyl-para-tyrosine (AMPT), a tyrosine hydroxylase inhibitor that reduces dopamine production, on insulin sensitivity and the post-prandial insulin response to a glucose-containing meal. Six healthy Standardbred horses were enrolled in a placebo-controlled randomised crossover study, in which one dose of AMPT (40 mg/kg BW) or placebo was administered orally, prior to performing an in-feed oral glucose test (OGT) and a frequently sampled intravenous glucose tolerance test (FSIGTT). Dopamine reduction by AMPT was confirmed by an increase in plasma prolactin concentration and the lack of post-prandial increase in plasma dopamine concentration compared to placebo. Post-prandial insulin responses, both peak and AUCi, were increased after AMPT compared to placebo (P=0.048 and P=0.005, respectively), without affecting blood glucose concentrations. However, one dose of AMPT did not appear to affect tissue sensitivity as assessed by the FSIGTT. This study confirmed that dopamine plays a role in the regulation of insulin secretion in horses, as it does in other species, whereby the post-prandial release of dopamine into the circulation may inhibit pancreatic insulin secretion. Further studies are required to evaluate different dosing protocols for AMPT, and to further investigate the links between PPID, ID and laminitis risk in horses.

Physiological and metabolic effects of short-term dopamine reduction in healthy horses using a tyrosine hydroxylase inhibitor (alpha-methyl-para-tyrosine).

Alpha-methyl-para-tyrosine (AMPT) is a reversible inhibitor of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. This study aimed to determine whether AMPT could reduce dopamine concentrations in horses. Six healthy adult Standardbred geldings were administered AMPT (40 mg/kg BW, orally) or placebo in a randomised crossover study design. Clinical examination findings were recorded, and blood samples were collected for up to 6 h after administration of AMPT or placebo, for measurement of blood glucose, plasma ACTH and cortisol concentrations, and plasma metabolomic analysis. Plasma prolactin concentration was determined as a proxy index of central dopamine reduction. No adverse clinical effects were detected after oral administration of AMPT, with heart rate, mean arterial pressure and blood glucose concentration not differing between AMPT treatment or placebo. Plasma prolactin concentration peaked 1 h after AMPT administration before returning to baseline at 2 h (for five horses) or 6 h (for one horse). Metabolomic analysis demonstrated a reduction in plasma dopamine (0.72-fold change; P=0.016) 1 h after AMPT treatment. Plasma ACTH and cortisol concentrations were not different between AMPT and placebo over time. A few metabolites associated with ketogenesis were increased, and certain amino acids decreased, at 1 h compared with baseline, for both AMPT treatment and placebo. Therefore, AMPT was effective in reducing both central and circulating dopamine concentrations in healthy horses following a single oral dose. Further pharmacokinetic and pharmacodynamic studies are warranted to optimise the dose and duration of AMPT treatment to achieve longer-term dopamine reduction. Plasma metabolomic findings suggested an interruption to energy flux at the time of sample collection, which may be relevant to nutritional studies in horses and warrants further investigation.

Initial development of a rapid, portable, stall-side ELISA for the measurement of equine adrenocorticotropic hormone.

Pituitary pars intermedia dysfunction (PPID) is a neurodegenerative disease of senior horses. Loss of dopaminergic inhibition of the melanotropes of the pars intermedia leads to increased concentrations of pro-opiomelanocortin (POMC)-derived peptides. Diagnosis is challenging due to pre-analytical variables, such as sample storage, handling, and time to analysis. Our objective was to develop an ELISA for ACTH measurement, which could ultimately form the basis for a stall-side equine ACTH test. We selected 2 ACTH-specific monoclonal antibodies, CBL57 and EPR20361-248, based on the recognition of separate epitopes, strong and rapid color change, and minimal background interference, including no cross-reactivity with themselves, each other, and the test reagents. CBL57 was chosen as the detection antibody (or secondary antibody). EPR20361-248, functionalized on superparamagnetic iron oxide beads, was chosen as the capture antibody (or primary antibody) to bind ACTH in plasma. The incorporation of magnetic beads marks the initial stage in establishing a platform that could potentially be utilized in the field, similar to other stall-side tests. The concentrations of antibodies, magnetic beads, and incubation durations were optimized. Our immunoassay detected unglycosylated rat recombinant ACTH. Further studies are ongoing to optimize and validate our assay using equine plasma and serum samples.

The detection of ACTH and insulin in equine plasma by solid-phase extraction and micro-flow LC/MSMS.

Previous liquid chromatography/mass spectrometry (LC/MS) methods for the detection of insulin and other similar peptide hormones in equine plasma relied on the use of antibody affinity extraction. As a result, these methods were not suitable for routine high-throughput analysis. A solid-phase extraction (SPE) method incorporating size exclusion as well as reversed-phase interactions allows the selective extraction of peptide hormones such as adrenocorticotropic hormone (ACTH), insulin and their synthetic analogues from equine plasma with approximately 80% extraction efficiencies. This extraction was combined with on-column derivatisation with acetic anhydride, followed by tryptic digestion and analysis by micro-LC/MSMS for high-sensitivity peptide hormone detection. The analysis of tryptic peptides provides greater sensitivity and more robust chromatography compared with the analysis of intact insulin and ACTH. For quantitative analysis, isotopically labelled internal standards of target peptides can be prepared in the laboratory through the use of deuterated acetic anhydride. The utility of the method was assessed for the analysis of ACTH and insulin in samples from horses suffering from pituitary pars intermedia dysfunction (PPID).

Strongyle egg shedding and egg reappearance periods in horses with pituitary pars intermedia dysfunction.

Pituitary pars intermedia dysfunction (PPID) is the most common endocrine disorder of older horses. Immune dysfunction in horses with PPID could increase susceptibility to infectious diseases, including strongyle infections; however, few data are available. The aim of this study was to determine if horses with PPID had increased strongyle faecal egg counts (FEC) compared with control horses, over a fourteen-week period in Victoria, Australia. Clinical signs and plasma adrenocorticotropic hormone (ACTH) concentrations were used to categorise horses into PPID (n=14) or control (n=31) groups. Faecal samples were collected for FEC determination prior to anthelmintic treatment, and fortnightly post-treatment for each horse. Generalised linear mixed modelling, using a gamma distribution, was used to compare differences between groups in the repeated measures study. The confounding variable of age was controlled for as a fixed effect. Following anthelmintic treatment, mean FEC was greater for the PPID group compared to the control group on day 56 (405 ± 756 eggs per gram [EPG] vs 40 ± 85 EPG, p=0.05) and day 70 (753 ±1598 EPG vs 82 ±141 EPG, p=0.04). There were no differences in mean FEC between groups on days 84 and 98. Cumulative FEC (day 14 to day 98) was significantly greater for the PPID horses than control horses (2118 ± 4016 EPG vs 798 ± 768 EPG, p<0.0001). Group egg reappearance period was shorter for PPID horses (day 56 post-anthelmintic treatment) compared to control horses (day 70) and 30% of the PPID horses reached a FEC threshold of >200 EPG on day 42, compared to 0% of control horses (p=0.02). These results suggest that the rate of a re-established patent infection between groups could be different due to a comprised immune response in PPID horses or differences in the host-parasite relationship regarding encysted stage larvae. However, despite differences between groups, some horses with PPID consistently had no detectable or low FEC (<200 EPG) during the study period. These findings highlight the importance of individual FEC monitoring to determine if anthelmintic treatment is required, in line with sustainable parasite management practices.

Dermatitis, cellulitis, and osteomyelitis caused by Aspergillus nidulans in a horse with pituitary pars intermedia dysfunction.

Clinical and histologic examination of a 12-y-old client-owned Quarter Horse gelding with pituitary pars intermedia dysfunction revealed dermatitis, cellulitis, and osteomyelitis caused by Aspergillus nidulans, confirmed by a PCR assay. This novel presentation of a fungal disease in a horse was characterized by aggressive local invasion and failure to respond to all medical therapy attempted over a 1-y period. Treatments included systemic and topical antifungals, anti-inflammatories, and use of cellular matrices. Surgical excision was not attempted but should be strongly considered early in the disease process in similar cases if clean margins can be achieved. Postmortem findings were of locally aggressive disease with no dissemination.

Factors affecting measurement of basal adrenocorticotropic hormone in adult domestic equids: A scoping review.

Measurement of basal adrenocorticotropic hormone (ACTH) concentration is the most commonly used diagnostic test for pituitary pars intermedia dysfunction (PPID). Although several pre-analytical and analytical factors have been reported to affect basal ACTH concentrations in equids, the extent to which these have been evaluated in the context of PPID diagnosis is unclear. The objectives of this scoping review were to identify and systematically chart current evidence about pre-analytical and analytical factors affecting basal ACTH concentrations in adult domestic equids. Systematic searches of electronic databases and conference proceedings were undertaken in June 2022, repeated in October 2022 and updated in August 2023. English language publications published prior to these dates were included. Screening and data extraction were undertaken individually by the authors, using predefined criteria and a modified scoping review data extraction template. After removal of duplicates, 903 publications were identified, of which 235 abstracts were screened for eligibility and 134 publications met inclusion criteria. Time of year, exercise, breed/type and transportation were the factors most frequently associated with significant increases in ACTH concentration (n = 26, 16, 13 and 10 publications, respectively). Only 25 publications reported inclusion of PPID cases in the study population, therefore the relationship between many factors affecting basal ACTH concentration and diagnostic accuracy for PPID remains undefined. However, several factors were identified that could impact interpretation of basal ACTH results. Findings also highlight the need for detailed reporting of pre-analytical and analytical conditions in future research to facilitate translation of evidence to practice.

A transmandibular lateral transsphenoidal navigated surgical approach to access a pituitary macroadenoma in a warmblood mare.

A 16-year-old warmblood mare was referred with a progressive history of behavioral changes and left-sided blindness. Following neuroanatomical localization to the forebrain, magnetic resonance imaging of the head revealed a well-delineated, 4.5 cm in diameter, round pituitary mass causing marked compression of the midbrain and optic chiasm. Euthanasia was recommended but declined by the owners. Veterinary specialists and a human neurosurgeon collaboratively prepared for surgical case management. A novel navigated transmandibular lateral transsphenoidal approach was developed to access the region of the sella turcica and practiced on cadaver specimens. The horse was anesthetized and placed in sternal recumbency with the head above the heart line. Using a cone beam computed tomography (CBCT)-coupled navigation system, a navigated pin traversing the vertical ramus of the mandible and the lateral pterygoid muscle was placed in a direct trajectory to the predetermined osteotomy site of the basisphenoid bone. A safe corridor to the osteotomy site was established using sequential tubular dilators bypassing the guttural pouch, internal and external carotid arteries. Despite the use of microsurgical techniques, visualization of critical structures was limited by the long and narrow working channel. Whilst partial resection of the mass was achieved, iatrogenic trauma to the normal brain parenchyma was identified by intraoperative imaging. With consent of the owner the mare was euthanized under the same general anesthesia. Post-mortem magnetic resonance imaging and gross anatomical examination confirmed partial removal of a pituitary adenoma, but also iatrogenic damage to the surrounding brain parenchyma, including the thalamus.

Clinical implications of imprecise sampling time for 10- and 30-min thyrotropin-releasing hormone stimulation tests in horses.

BACKGROUND: The thyrotropin-releasing hormone (TRH) stimulation test is used to diagnose pituitary pars intermedia dysfunction (PPID) using 10- or 30-min protocols. Imprecise sampling time for the 10-min protocol can lead to misdiagnoses. OBJECTIVES: To determine the effect of imprecise sampling time for the 30-min protocol of the TRH stimulation test. STUDY DESIGN: In vivo experiment. METHODS: Plasma immunoreactive adrenocorticotropin (ACTH) concentrations were measured 9, 10, 11, 29, 30 and 31 min after intravenous administration of 1 mg of TRH in 15 control and 12 PPID horses. Differences in ACTH concentrations between sampling times, variability in ACTH concentrations between protocols, and diagnostic classification of PPID were assessed using Friedman's test, Bland-Altman plots, and Fisher's exact test, respectively, with 95% confidence intervals reported and significance set at p < 0.05. RESULTS: Imprecise sampling time resulted in variable ACTH concentrations, but significant differences in absolute ACTH concentrations were not detected for imprecise sampling within each protocol or between protocols. Imprecise sampling changed PPID diagnostic classification for 3/27 (11 [4-28] %) horses for both protocols. Using the 30-min protocol as a reference, 1/12 (8 [1-35] %) horses returned a negative test result and 5/12 (42 [19-68] %) horses returned equivocal test results that would be considered positive in practice due to the presence of supportive clinical signs. MAIN LIMITATIONS: Limited sample size and inter-horse variability reduced the ability to detect small but potentially relevant differences. CONCLUSIONS: Overall, the impact of imprecise sampling was not significantly different between the 10- and 30-min TRH stimulation test protocols. However, diagnostic classification for PPID would have varied between the 10- and 30-min protocols in this population, if clinical signs had been ignored. Precise timing during TRH stimulation tests and contextual interpretation of ACTH concentrations remain fundamental for the diagnosis of PPID.

Pituitary pars intermedia dysfunction with pituitary gland melanotroph adenoma in a Grevy's zebra (Equus grevyi).

A 22-year and 9-month-old female Grevy's zebra (Equus grevyi) showed signs of polyuria, polydipsia, glucosuria, and muscle atrophy. Blood tests revealed hyperglycemia, hypertriglyceridemia, electrolyte imbalance, high levels of adrenocorticotropic hormone (ACTH) and cortisol, and low levels of hormones secreted by the pituitary pars distalis. Pathological examinations revealed a pituitary gland tumor and bilateral adrenal cortical hyperplasia. Pituitary tumor cells showed immunoreactivity for α-melanocyte-stimulating hormone and ACTH. The deposition of amyloid ß was observed in the parenchyma and vascular walls of the cerebrum. The zebra showed clinical signs of pituitary pars intermedia dysfunction and was histopathologically diagnosed with pituitary gland melanotroph adenoma. This case report provides insight into neoplastic and endocrine diseases associated with the aging of a zebra.

Prevalence and risk factors for laminitis within the Norwegian pony breed Nordlandshest/Lyngshest.

BACKGROUND: Laminitis is a systemic condition resulting in debilitating pain and structural changes within the feet, and hence has major welfare implications. Causes include endocrine and systemic inflammatory conditions. Ponies are frequently affected, and observations in the field suggest that occurrence of laminitis is also common in Norwegian breeds. The aim of this study was to estimate the prevalence and risk factors for laminitis within the Norwegian pony breed Nordlandshest/Lyngshest. RESULTS: The study was a cross-sectional study based on questionnaires sent to members of the Norwegian Nordlandshest/Lyngshest breed association. Questionnaires were received for 504 animals, of which 464 records were eligible and included in analyses. The population comprised 71 stallions, 156 geldings, and 237 mares, with an age between 1 and 40 years (median and interquartile ranges: 12 (6-18) years). The estimated 3-year period prevalence of laminitis was 8.4% (95% confidence interval (CI95): 6.0-11.3%), whereas lifetime prevalence was 12.5% (CI95: 9.6-15.9%). Mares had a significantly higher period- and lifetime prevalence of laminitis than male horses, and horses 10 years and older had a significantly higher prevalence than younger horses. The lifetime prevalence of laminitis was 3.2% in horses 9 years and younger, whereas from 17.3-20.5% in older horses. Multivariable logistic regression analysis identified age, sex, and regional adiposity as significantly (P < 0.05) associated with the 3-year period outcome of laminitis: Horses older than 9 years had a three-fold increase in the likelihood of having laminitis compared to the younger horses (Odds Ratio (OR)10-14 years = 3.37 (CI95 = 1.19-9.50), OR15-19 years = 3.06 (CI95 = 1.04-9.05), and OR>20 years = 2.70 (CI95 = 0.90-8.02). Mares were more than twice as likely (OR = 2.44 (CI95 = 1.17-5.12) to have laminitis compared to male horses, and horses with regional adiposity had increased odds (OR = 2.35 (CI95 = 1.15-4.82) of laminitis compared to horses without regional adiposity. CONCLUSIONS: Laminitis appears to be a considerable welfare issue in the Norwegian pony breed Nordlandshest/Lyngshest. The identified risk factors age, sex, and regional adiposity highlight the need for improved owner education and awareness of strategies to reduce laminitis risk.

Investigation of breed differences in plasma adrenocorticotropic hormone concentrations among healthy horses and ponies.

Plasma adrenocorticotropic hormone (ACTH) concentration is commonly measured to diagnose pituitary pars intermedia dysfunction (PPID). Several intrinsic and extrinsic factors affect ACTH concentrations, including breed. The objective of this study was to prospectively compare plasma ACTH concentrations among different breeds of mature horses and ponies. Three breed groups comprised Thoroughbred horses (n = 127), Shetland ponies (n = 131) and ponies of non-Shetland breeds (n = 141). Enrolled animals did not show any signs of illness, lameness or clinical signs consistent with PPID. Blood samples were collected 6 months apart, around the autumn equinox and spring equinox, and plasma concentrations of ACTH were measured by chemiluminescent immunoassay. Pairwise breed comparisons within each season were performed on log transformed data using the Tukey test. Estimated mean differences in ACTH concentrations were expressed as fold difference with 95 % confidence intervals (CI). Reference intervals for each breed group per season were calculated using non-parametric methods. In autumn, higher ACTH concentrations were found among non-Shetland pony breeds compared with Thoroughbreds (1.55 fold higher; 95 % CI, 1.35-1.77; P < 0.001), and in Shetland ponies compared with Thoroughbreds (2.67 fold higher; 95 % CI, 2.33-3.08; P < 0.001) and non-Shetland pony breeds (1.73 fold higher; 95 % CI, 1.51-1.98; P < 0.001). In spring, no differences were identified among breed groups (all P > 0.05). Reference intervals were similar among breed groups in spring, but upper limits for ACTH concentrations were markedly different between Thoroughbred horses and pony breeds in autumn. These findings emphasise that breed should be accounted for when determining and interpreting reference intervals for ACTH concentrations among healthy horses and ponies in autumn.

A silent corticotroph adenoma: making the case for a pars intermedia origin. Illustrative case.

BACKGROUND: Silent corticotroph adenomas (SCAs) are the only pituitary adenomas thought to originate from the pars intermedia. This case report presents the rare finding of a multimicrocystic corticotroph macroadenoma displacing the anterior and posterior lobes of the pituitary gland on magnetic resonance imaging (MRI). This finding supports the hypothesis that silent corticotroph adenomas may originate from the pars intermedia and should be considered in the differential for tumors arising from this location. OBSERVATIONS: A 55-year-old man presented with an episode of confusion and blurred vision. MRI demonstrated separation of the anterior and posterior glands by a solid-cystic lesion located within the pars intermedia that superiorly displaced the optic chiasm. Endocrinologic evaluation was unremarkable. The differential diagnosis included pituitary adenoma, Rathke cleft cyst, and craniopharyngioma. The tumor was confirmed to be an SCA on pathology and was completely removed through the endoscopic endonasal transsphenoidal approach. LESSONS: The case highlights the importance of preoperative screening for subclinical hypercortisolism for tumors arising from this location. Knowledge of a patient's preoperative functional status is critical and dictates their postoperative biochemical assessment to determine remission. The case also illustrates surgical strategies for resecting pars intermedia lesions without injuring the gland.

Cytodifferentiation of Wulzen's cone of mature male sheep hypophysis cerebri with special emphasis on its parenchymal correlations with the adjacent pars distalis, pars intermedia and pars nervosa.

Background: Hypophysis cerebri is considered the master endocrine gland as it plays a critical role in influencing and controlling the vitality of other endocrine organs via several hormones secretion. Aim: The present study was performed to clarify the localization of Wulzen's cone (WC) within sheep hypophysis and cytodifferentiation of the glandular cells filling cone parenchyma with particular emphasis on the cone correlations with adjacent pars distalis (pd), pars intermedia (pi), and pars nervosa (pn). Methods: Pituitaries were collected and processed histologically, then subjected to different combinations of special stains; Br-AB- OFG., PFA-AB-PAS-OG., PAS-Orange G., Orange G- Acid Fuchsin- Light Green, Bielschowsky technique, Masson's trichrome & Gomori's reticulin. Results: A sagittal section through the pituitaries revealed a well-developed cone of glandular cells protruding from the pi like a tongue plate towards the hypophyseal cleft in the neighborhood of the pd and behind the pn. Resembling the pd, various glandular cells were distinguished in the cone; chromophobes and chromophils of acidophils & basophils. The cone is mainly formed from acidophils intermingled with the chromophobes. Meanwhile, basophils were primarily localized at the most anterior & posterior parts of the cone. In front of the cone, pd were localized, resembling a wing-shaped and filled with several categorized glandular cells; chromophobes and chromophils. Upper to the cone, pi were localized and composed mainly of weakly basophilic cuboidal or polygonal cells arranged in parallel cords or follicles. Behind the cone, pn was localized as a ventral outpouching of the brain floor-like water drop. Unlike the cone, it was devoid of any glandular secretory cells or nerve cells but consisted mainly of unmyelinated nerve fibers, herring bodies, and pituicytes. Conclusion: WC is present and well-developed in sheep adenohypophysis. Various glandular cells were distinguished, filling the cone, chromophobes, and chromophils of acidophils & basophils that were typically similar to the glandular cells of pd but with different distributions.

Periodontal structures in horses with pituitary pars intermedia dysfunction: A histological evaluation.

Introduction: Pituitary pars intermedia dysfunction (PPID) and dental disorders are of major concern in horses older than 15 years. Although PPID in geriatric horses and dental disorders in all age groups are well described, a connection between this endocrine disease and pathological changes in equine dental structures has not yet been investigated. In humans, periodontitis is considered to be a complication of systemic diseases like diabetes mellitus type 2, obesity and various conditions leading to an impaired immune response. In PPID, cross links to insulin and immune dysregulations are proven. The aim of this study was to compare histological findings of the gingiva and the sub gingival periodontal ligament of PPID affected horses with control horses. Methods: In a case-control morphometric descriptive study, 145 dental locations of 10 PPID affected horses (27.3 ± 2.06 years) were compared with 147 dental locations of 10 controls (21.4 ± 4.12 years). Histological parameters were leukocyte infiltration, keratinization of gingival epithelium, blood vessel supply of the periodontium and structure of cementum. Results: The distribution and localization of gingival leukocyte infiltrations (LI) in PPID affected horses was more often multifocal to coalescing (p = 0.002) and reached into deeper parts of the periodontium, sometimes down to the sub gingival periodontal ligament (PDL). Aged animals of both groups showed higher prevalence (PPID: OR 1.66; controls: OR 1.15) for severe leukocyte infiltration in the PDL. PPID was not significantly associated with increased LI. The cementum bordering the soft tissue in interdental locations showed four times more irregularities in PPID affected horses than in controls which predisposes for interdental food impaction and periodontal diseases. Discussion: In summary, multifocal to coalescing leukocytes and irregular cementum are seen more often in PPID than in controls - however our findings mainly reflect an association of older age with periodontal disease.

Short Communication: Identification of equine corticotropin-like intermediate lobe peptide (CLIP) binding to an adrenocortipcotrophic hormone (ACTH) assay capture antibody.

A chemiluminescent immunoassay is commonly employed to measure adrenocorticotrophic hormone (ACTH) concentrations to assist pituitary pars intermedia dysfunction diagnosis. In a previous study, seasonally-dependent assay cross-reactivity to endogenous equine corticotropin-like intermediate lobe peptide (CLIP, ACTH 18-39) was suspected. The present study aimed to demonstrate binding of endogenous equine CLIP to the capture antibody of the ACTH chemiluminescent immunoassay. Liquid chromatography - mass spectrometry (LCMS) methods were optimised to identify selected ions from synthetic human ACTH, α-melanocyte stimulating hormone (α-MSH, ACTH 1-17) and CLIP. Synthetic ACTH and CLIP bound to the capture antibody of the chemiluminescent ACTH assay, but α-MSH did not. Equine endogenous CLIP was detected by LCMS in pony plasma taken in the autumn and could be eluted from the capture antibody of the ACTH chemiluminescent immunoassay. Further research is required to enable quantification of CLIP. Equine CLIP may alter measured ACTH concentrations in vivo.

Effect of sirolimus on insulin dynamics in horses.

BACKGROUND: Sirolimus, a mechanistic target of rapamycin inhibitor, suppresses insulin production in other species and has therapeutic potential for hyperinsulinemia in horses. HYPOTHESIS/OBJECTIVE: Determine the pharmacokinetics (PKs) of sirolimus and evaluate its effect on insulin dynamics in healthy and insulin dysregulation (ID) horses. ANIMALS: Eight Standardbred geldings. METHODS: A PK study was performed followed by a placebo-controlled, randomized, crossover study. Blood sirolimus concentrations were measured by liquid chromatography-mass-spectrometry. PK indices were estimated by fitting a 2-compartment model using nonlinear least squares regression. An oral glucose test (OGT) was conducted before and 4, 24, 72, and 144 hours after administration of sirolimus or placebo. Effects of time, treatment and animal on blood glucose and insulin concentrations were analyzed using mixed-effects linear regression. Sirolimus was then administered to 4 horses with dexamethasone-induced ID and an OGT was performed at baseline, after ID induction and after 7 days of treatment. RESULTS: Median (range) maximum sirolimus concentration was 277.0 (247.5-316.06) ng/mL at 5 (5-10) min and half-life was 3552 (3248-4767) min. Mean (range) oral bioavailability was 9.5 (6.8-12.4)%. Sirolimus had a significant effect on insulin concentration 24 hours after a single dose: median (interquartile range) insulin at 60 min (5.0 [3.7-7.0] µIU/mL) was 37 (-5 to 54)% less than placebo (8.7 [5.8-13.7] µIU/mL, P = .03); and at 120 min (10.2 [8.4-12.2] µIU/mL) was 28 (-15 to 53)% less than placebo (14.9 [8.4-24.8] µIU/mL, P = .02). There was minimal effect on glucose concentration. Insulin responses decreased toward baseline in ID horses after 7 days of treatment. CONCLUSION AND CLINICAL IMPORTANCE: Sirolimus decreased the insulinemic response to glucose and warrants further investigation.

Palatability, glycemic, and insulinemic responses to various carbohydrate formulations: Alternatives for the diagnosis of insulin dysregulation in horses?

BACKGROUND: Oral glycemic challenge (GC) tests are recommended for diagnosis of insulin dysregulation (ID). Various protocols are used, but all have limitations in terms of palatability, ease of use, variable composition, geographic availability, or some combination of these. HYPOTHESIS/OBJECTIVE: To evaluate newly developed formulations with defined carbohydrate composition for use as oral GCs. ANIMALS: Thirty-four horses and ponies in various metabolic states. METHODS: Our objectives were carried out in 2 separate cross-over experiments. First, the palatability and acceptance of various GCs (2 syrups, 1 granulate) offered for free intake were compared to glucose mixed in a chaff-based diet. Subsequently, syrups were administered by syringe and compared to an oral glucose test using naso-gastric tubing (tube OGT) to investigate the glycemic and insulinemic responses. Second, these variables were compared in the best performing GC-formulations (granulate further optimized to pelleted formulation and 1 syrup) and a tube OGT. All GCs were administered with equivalent amounts of 0.5 g glycemic carbohydrates per kg body weight. RESULTS: Only the GC pellets were consumed completely by all horses (consumption time 5 ± 2 min). When administered by syringe, the GC syrup also was well accepted. The insulin concentrations at 120 min correlated significantly between tube OGT and GC pellets (r = .717; P < .001) or GC syrup (r = .913; P < .001). The new GC syrup and GC pellets discriminate between healthy and ID horses. CONCLUSIONS AND CLINICAL SIGNIFICANCE: The GC pellets (DysChEq)™ and GC syrup can be used as palatable and well-accepted oral GC tests for assessment of ID in horses.

T2 hypointense signal discovered incidentally at the posterior edge of the adenohypophysis on MRI: its prevalence and morphology and their relationship to age.

PURPOSE: T2 hypointense signal at the posterior edge of the adenohypophysis (T2HSPA) on magnetic resonance imaging (MRI) is incidentally encountered. We aimed to investigate the prevalence and morphology of T2HSPA and their relationship to age. METHODS: A total of 212 cases between 3 and 88 years old were examined. Sagittal T2-weighted image (T2WI) was evaluated for the presence of T2HSPA, which classified by its morphology into two types (belt-like or nodal). The Wilcoxon rank sum test and chi-square test were used to evaluate the differences between the groups. The T2HSPA was extracted by ImageJ software and measured as a cross-sectional area (CSA) quantitatively by threshold setting. We examined the relationship between CSA of T2HSPA and age, and Spearman's correlation coefficients were used for statistical analysis. RESULTS: Of the 212 cases, 80 (37.7%) were identified with T2HSPA. The groups with T2HSPA were significantly younger than the groups without it (p = .01). Groups with belt-like T2HSPA were significantly younger than the groups with nodal T2HSPA (p = .01). There was a weak negative correlation between CSA of T2HSPA and age (p = .02). CONCLUSION: T2HSPAs were incidentally detected in 37.7% of all cases, tended to be more common in younger cases, and their morphology was related to age. They seem to have little clinical significance as they tend to decrease in size with age.

Short-Term Effects of Temperature and Thyrotropin-Releasing Hormone Stimulation on Adrenocorticotropin Stability in Horses.

Pituitary pars intermedia dysfunction (PPID) is diagnosed by increased basal or post thyrotropin-releasing hormone (TRH) stimulation ACTH concentrations. ACTH is known to be unstable; however, the effect of different temperatures and TRH stimulation on equine ACTH stability is poorly described. In total, 15 horses, including 8 PPID positive (ACTH > 35 pg/mL at baseline or >65 pg/mL 30 min after TRH stimulation), were divided into 2 groups: 9, including 5 PPID positive, with basal ACTH concentrations and 6, including 3 PPID positive, with post TRH stimulation ACTH concentrations. Whole blood was stored for 1 h at 4, 20, 30, 40, or 70 °C. After centrifugation, immunoreactive ACTH concentrations were determined using a chemiluminescent assay. Linear mixed effect models were used to detect the effects of temperature, PPID status, and TRH stimulation on the immunoreactive ACTH concentration. Temperature had a significant effect (p = 0.003) on immunoreactive ACTH concentrations, and this effect was greater in PPID-negative horses (p = 0.01), with the changes in immunoreactive ACTH concentrations being slightly unpredictably higher or lower than samples stored at 4 °C. Even at 20 °C, mean immunoreactive ACTH concentrations minimally changed by 5% in PPID horses and 12% in non-PPID horses after 1 h. No significant effect of TRH stimulation was identified. Although ACTH concentrations should ideally be determined from samples kept at 4 °C, samples inadvertently left at temperatures of up to 40 °C can provide valid results if analyzed within 1 h; however, this increases the risks of altered ACTH concentrations, occasionally influencing the diagnosis of PPID.