Kelsey A. Hart

Hypothalamic-pituitary-adrenal axis dysfunction in hospitalized neonatal foals.

BACKGROUND Transient hypothalamic-pituitary-adrenal (HPA) axis dysfunction occurs frequently in critically ill humans and impacts survival. The prevalence and impact of HPA axis dysfunction in critically ill neonatal foals are not well characterized. HYPOTHESES (1) HPA axis dysfunction occurs in hospitalized neonatal foals, and is characterized by inappropriately low basal serum cortisol concentration or inadequate cortisol response to exogenous adrenocorticotropic hormone (ACTH); (2) hospitalized foals with HPA axis dysfunction have more severe disease and are less likely to survive than hospitalized foals with normal HPA axis function. ANIMALS Seventy-two hospitalized foals and 23 healthy age-matched foals. METHODS Basal ACTH and cortisol concentrations were measured and a paired low-dose (10 microg)/high-dose (100 microg) cosyntropin stimulation test was performed at admission in hospitalized foals. HPA axis dysfunction was defined as (1) an inappropriately low basal cortisol concentration or (2) an inadequate increase in cortisol concentration (delta cortisol) after administration of cosyntropin, with cut-off values for appropriate basal and delta cortisol concentrations determined from results obtained in healthy age-matched foals. RESULTS Forty-six percent of hospitalized foals had an inappropriately low basal cortisol concentration and 52% had an inadequate delta cortisol concentration after administration of the 100 microg dose of cosyntropin. An inadequate delta cortisol response to the high (100 microg) dose of cosyntropin was significantly correlated with shock and multiple organ dysfunction syndrome in hospitalized foals, and with decreased survival in a subgroup of septic foals. CONCLUSIONS AND CLINICAL IMPORTANCE HPA axis dysfunction occurs frequently in hospitalized neonatal foals, and negatively impacts disease severity and survival.

Hypothalamic-Pituitary-Adrenal Axis Assessment in Healthy Term Neonatal Foals Utilizing a Paired Low Dose/High Dose ACTH Stimulation Test

BACKGROUND Hypothalamic-pituitary-adrenal (HPA) axis function is dynamic in the neonatal foal. The paired low dose/high dose cosyntropin (ACTH) stimulation test allows comprehensive HPA axis assessment, but has not been evaluated in neonatal foals. HYPOTHESIS Foal age will significantly affect cortisol responses to a paired 10 and 100 microg dose cosyntropin stimulation test in healthy neonatal foals. ANIMALS Twenty healthy neonatal foals. METHODS HPA axis function was assessed in 12 foals at birth and at 12-24, 36-48 hours, and 5-7 days of age. At each age, basal cortisol and ACTH concentrations were measured and cortisol responses to 10 and 100 microg cosyntropin were assessed with a paired ACTH stimulation test protocol. Eight additional 36-48-hour-old foals received saline instead of 10 microg cosyntropin in the same-paired ACTH stimulation test design. RESULTS At birth, foals had significantly higher basal cortisol and ACTH concentrations and higher basal ACTH : cortisol ratios compared with foals in all other age groups. A significant cortisol response to both the 10 and 100 microg doses of cosyntropin was observed in all foals. The magnitude of the cortisol response to both doses of cosyntropin was significantly different across age groups, with the most marked responses in younger foals. There was no effect of the paired ACTH stimulation test design itself on cortisol responses. CONCLUSIONS AND CLINICAL IMPORTANCE A paired 10 and 100 microg cosyntropin stimulation test can be used to evaluate HPA axis function in neonatal foals. Consideration of foal age is important in interpretation of HPA axis assessment.

Serum Free Cortisol Fraction in Healthy and Septic Neonatal Foals

BACKGROUND Relative cortisol insufficiency occurs in septic foals and impacts survival. Serum free (biologically available) cortisol concentration might be a better indicator of physiologic cortisol status than serum total cortisol concentration in foals. HYPOTHESES In septic foals, (1) low free cortisol concentration correlates with disease severity and survival and (2) predicts disease severity and outcome better than total cortisol concentration. ANIMALS Fifty-one septic foals; 11 healthy foals; 6 healthy horses. METHODS In this prospective clinical study, foals meeting criteria for sepsis at admission were enrolled. University-owned animals served as healthy controls. Basal and cosyntropin-stimulated total cortisol concentration and percent free cortisol (% free cortisol) were determined by chemiluminescent immunoassay and ultrafiltration/ligand-binding methods, respectively. Group data were compared by ANOVA, Mann-Whitney U-tests, and receiver operator characteristic curves. Significance was set at P < .05. RESULTS Basal % free cortisol was highest in healthy foals at birth (58 ± 8% mean ± SD), and was higher (P ≤ .004) in healthy foals of all ages (33 ± 6 to 58 ± 8%) than in adult horses (7 ± 3%). Cosyntropin-stimulated total and free cortisol concentrations were lower (P ≤ .03) in foals with shock (total = 6.2 ± 8.1 μg/dL; free = 3.5 ± 4.8 μg/dL versus total = 10.8 ± 6.0 μg/dL; free = 6.9 ± 3.3 μg/dL in foals without shock) and in nonsurvivors (total = 3.8 ± 6.9 μg/dL; free = 1.9 ± 3.9 μg/dL versus total = 9.1 ± 7.7 μg/dL; free = 5.5 ± 4.4 μg/dL in survivors). Free cortisol was no better than total cortisol at predicting disease severity or outcome in septic foals. CONCLUSIONS AND CLINICAL IMPORTANCE Serum free cortisol is impacted by age and illness in the horse. There is no advantage to measuring free over total cortisol in septic foals.

Adrenocortical insufficiency in horses and foals.

The adrenal cortices produce various steroid hormones that play vital roles in several physiologic processes. Although permanent adrenocortical insufficiency is rare in all species, emerging evidence in both human and equine medicine suggests that transient reversible adrenocortical dysfunction resulting in cortisol insufficiency frequently develops during critical illness. This syndrome is termed relative adrenal insufficiency (RAI) or critical illness-related corticosteroid insufficiency (CIRCI) and can contribute substantially to morbidity and mortality associated with the primary disease. This review discusses the mechanisms, diagnosis, and clinical consequences of adrenocortical insufficiency, with particular focus on the current understanding of RAI/CIRCI in horses and foals.

Effects of Low-Dose Hydrocortisone Therapy on Immune Function in Neonatal Horses

Low-dose hydrocortisone (LDHC) therapy modulates inflammatory responses in adults and improves outcomes in some septic adults and neonates, but its immunologic effects have not been evaluated in neonates. The objective of this study was to evaluate effects of LDHC therapy on ex vivo immune function in neonatal horses (foals). We hypothesized that LDHC treatment would dampen proinflammatory responses without impairing neutrophil function. Hydrocortisone (1.3 mg/kg/d i.v.) was administered to foals in a tapering 3.5 d course. Peripheral blood leukocytes were collected from foals before, during, and after hydrocortisone treatment. A separate group of age-matched untreated foals served as controls. Endotoxin-induced peripheral blood mononuclear cell gene expression of inflammatory cytokines was measured by real-time quantitative RT-PCR. Neutrophils were incubated with labeled, killed Staphylococcus aureus or Escherichia coli for assessment of phagocytosis, and with phorbol myristate acetate, zymosan, or endotoxin for measurement of reactive oxygen species (ROS) production. Neutrophil phagocytosis and ROS production were similar in both groups. Foals receiving hydrocortisone had significantly decreased endotoxin-induced expression of TNF-α, IL-6, IL-8, and IL-1β. These data suggest that this LDHC treatment regimen ameliorates endotoxin-induced proinflammatory cytokine expression in neonatal foals without impairing innate immune responses needed to combat bacterial infection.

Synthetic adrenocorticotropic hormone stimulation tests in healthy neonatal foals

BACKGROUND Cosyntropin (adrenocorticotropic hormone [ACTH]) stimulation tests are used to evaluate adrenal function. Low-dose ACTH stimulation tests are the most accurate method for diagnosing relative adrenal insufficiency in critically ill humans but have not been evaluated in foals. HYPOTHESIS Peak serum cortisol concentrations in healthy foals will not be significantly different after intravenous administration of 1, 10, 100, and 250 microg of cosyntropin. ANIMALS 14 healthy neonatal foals, 3-4 days of age. METHODS A randomized cross-over model was used in which cosyntropin (1, 10, 100, or 250 microg) was administered intravenously on days 3 and 4 of life. Blood samples were collected before and 30, 60, 90, 120, and 150 minutes after administration of cosyntropin for determination of serum cortisol concentration. RESULTS Serum cortisol concentrations did not significantly increase after administration of 1 microg of cosyntropin. Cortisol concentration peaked 30 minutes after administration of 10 microg of cosyntropin and 90 minutes after 100 and 250 microg of cosyntropin. There was no relationship between cosyntropin dose and serum cortisol concentration at 30 minutes. Compared with the 10-microg dose, 100 and 250 microg of cosyntropin induced significantly greater cortisol concentrations at 90 minutes, at which point the 10-microg cosyntropin-dose cortisol values were indistinguishable from baseline. There was no significant difference in the area under the cortisol concentration curve between the 100- and 250-microg doses. No effect of day of testing or foal weight on peak cortisol concentration was detected. CONCLUSIONS AND CLINICAL IMPORTANCE The results of this study suggest that 10- and 100-microg doses of cosyntropin would be appropriate for evaluating adrenal function in neonatal foals.

The use of cortisol for the objective assessment of stress in animals: Pros and cons

Accurate determination of pain and stress in animals in clinical, experimental, and production settings is critical for the design and implementation of appropriate medical therapy, experimental protocols and handling procedures. Unfortunately, as animals cannot clearly verbally express stress responses, it is often difficult to quantify these parameters objectively in animal species (and even in humans in some settings). Methodologies frequently used to try to quantify pain and stress in animals include direct behavioral observation and use of behavior scoring systems, automated behavior recognition programs, and quantification of physiological responses to pain and stress such as changes in heart rate, blood pressure, and circulating ‘stress’ hormones (e.g. cortisol, b-endorphin, or catecholamines) (Ayala et al., 2012; Livingston, 2010; Schmidt et al., 2010). Unfortunately, all of these methods have some potential for error or misinterpretation, resulting in overor under-quantification of an animal’s pain and stress in some circumstances. Because there potential subjective interpretation of animal behavior is eliminated as a possible error source, quantification of physiological neuroendocrine responses to a stimulus may offer a more objective measurement of associated pain and stress than behavioral observation in most species. The hypothalamic–pituitary–adrenal (HPA) axis is a key component of the neuroendocrine response to environmental, emotional, and physiological stress across animal species. The HPA axis is activated when signals from the peripheral and central nervous systems are integrated in the hypothalamus, resulting in the release of corticotropin releasing hormone (CRH) followed by induction of release of the peptide hormone adrenocorticotrophic hormone (ACTH) from the anterior

Effect of Age, Season, Body Condition, and Endocrine Status on Serum Free Cortisol Fraction and Insulin Concentration in Horses

Background Increased free cortisol fraction is associated with insulin dysregulation (ID) in people with Metabolic Syndrome and Cushings Disease. Free cortisol has not been investigated in equine endocrine disorders. Hypotheses (1) In healthy horses, sex, age, body condition score (BCS), and season impact free cortisol; (2) free cortisol is increased in horses with Pituitary Pars Intermedia Dysfunction (PPID) or Equine Metabolic Syndrome (EMS). Animals Fifty‐seven healthy horses; 40 horses and ponies with PPID (n = 20) or EMS (n = 20). Methods Prospective study. Serum collected seasonally from healthy animals and archived serum from PPID and EMS animals was analyzed for insulin, total and free cortisol concentrations, and free cortisol fraction (FCF). Linear mixed models were used to determine effects of age, sex, season, and BCS on hormones in controls. Hormone measurements were compared between disease groups and age‐ and season‐matched controls with t‐tests. EMS and hyperinsulinemic PPID animals were combined in an ID (hyperinsulinemia) group. Results Free cortisol concentrations were increased in overweight/obese controls (0.3 ± 0.1 μg/dL) compared to lean controls (0.2 ± 0.1 μg/dL; P = .017). Mean FCF was significantly higher in animals with PPID (8.8 ± 5.8 μg/dL, P = .005) or ID (8.8 ± 10.2 μg/dL, P = .039) than controls (5.0 ± 0.9 μg/dL), but total cortisol concentrations were similar (P ≥ .350) (PPID: 4.2 ± 4.3 μg/dL; ID: 5.0 ± 4.5 μg/dL; controls: 4.6 ± 1.7 and 5.1 ± 2.1 μg/dL). Conclusions and Clinical Importance Increased FCF is associated with obesity in healthy horses and with ID (hyperinsulinemia) in horses and ponies with endocrine disease. Decreased plasma cortisol‐binding capacity could be a component of these endocrine disorders in horses.

Daily endogenous cortisol production and hydrocortisone pharmacokinetics in adult horses and neonatal foals.

OBJECTIVE To compare daily endogenous cortisol production rate and the pharmacokinetics of an i.v. bolus of hydrocortisone between neonatal foals and adult horses. ANIMALS 10 healthy full-term 2- to 4-day-old foals and 7 healthy adult horses. PROCEDURES Blood samples were collected from each horse every 15 to 20 minutes for 24 hours for determination of 24-hour mean cortisol concentration. Afterward, dexamethasone (0.08 mg/kg) was administered i.v. to suppress endogenous cortisol production. Twelve hours afterward, hydrocortisone sodium succinate (1.0 mg/kg) was administered as a rapid i.v. bolus and serial blood samples were collected to determine hydrocortisone pharmacokinetics. Cortisol concentrations, daily cortisol production rate, and hydrocortisone pharmacokinetics were determined, and results were compared between adult horses and foals. RESULTS The mean ± SD 24-hour cortisol concentration was significantly lower in foals (20 ± 4 ng/mL) than in horses (26 ± 6 ng/mL), but the daily cortisol production rate was significantly greater in foals (6,710 ± 320 ng/kg/d) than in horses (2,140 ± 400 ng/kg/d). For hydrocortisone, foals had a significantly greater volume of distribution at steady state (1.92 ± 1.11 L/kg) and total body clearance (1.39 ± 0.108 L/kg/h) and significantly lower peak plasma concentration (1,051 ± 343 ng/mL) than did horses (0.58 ± 0.15 L/kg, 0.349 ± 0.065 L/kg/h, and 8,934 ± 3,843 ng/mL, respectively). CONCLUSIONS AND CLINICAL RELEVANCE Important differences were detected in cortisol production and metabolism between neonatal foals and adult horses consistent with lower plasma protein binding of cortisol in foals. This decrease may contribute to cortisol insufficiency during prolonged critical illness in neonatal foals.

Detection of endogenous cortisol in equine tears and blood at rest and after simulated stress.

OBJECTIVE To determine whether cortisol is present in equine tears at rest and during simulated stress and compare tear cortisol to serum free and total cortisol. MATERIALS AND METHODS Fourteen healthy adult horses were included. Paired tear total cortisol and serum total and free cortisol concentrations were measured with ELISA, chemiluminescent immunoassay, and ultrafiltration methodology, respectively, in 10 horses at rest once daily for five consecutive days. In an additional four horses, paired tear and serum samples were collected for cortisol measurement before and after adrenocorticotropic hormone (ACTH) stimulation (cosyntropin, 1 μg/kg IV). RESULTS Cortisol was detectable in equine tears at rest. Following ACTH stimulation, tear cortisol increased significantly from baseline at 60-120 min (P ≤ 0.001). Serum total and free cortisol also increased significantly at 30-180 min after ACTH stimulation (P ≤ 0.001). Both serum and tear cortisol returned to baseline concentrations by 360 min. Changes in tear cortisol were similarly associated with changes in serum total and free cortisol, although high tear cortisol concentrations suggest a portion of tear cortisol may be protein-bound. DISCUSSION Cortisol is present in equine tears and increases in concert with serum cortisol following ACTH stimulation. Further study is needed to determine whether endogenous cortisol in tears contributes to ocular pathology.