Assessment of the hypothalamic‐pituitary‐adrenocortical axis function using a vasopressin stimulation test in neonatal foals

Abstract Background Bacterial sepsis is the leading cause of death in foals and is associated with hypothalamic‐pituitary‐adrenocortical axis (HPAA) dysfunction. HPAA function can be evaluated by an arginine‐vasopressin (AVP) stimulation test. Hypotheses/Objectives Administration of AVP will stimulate a dose‐dependent rise in systemic adrenocorticotropin‐releasing hormone (ACTH) and cortisol in neonatal foals. There will be no response seen in corticotropin‐releasing hormone (CRH) and baseline AVP will be within reference interval. Animals Twelve neonatal foals, <72 hours old. Methods HPAA function was assessed in foals utilizing 3 doses of AVP (2.5, 5, and 7.5 IU), administered between 24 and 48 hours of age in this randomized cross‐over study. Cortisol, ACTH, CRH and AVP were measured at 0 (baseline), 15, 30, 60 and 90 minutes after AVP administration with immunoassays. The fold increase in cortisol and ACTH was calculated at 15 and 30 minutes compared to baseline. Results All doses of AVP resulted in a significant increase in cortisol concentration over time, and a dose‐dependent increase in ACTH concentration over time. ACTH and cortisol were significantly increased at 15 and 30 minutes, respectively after all 3 doses of AVP compared to baseline (P < .01). There was no change in endogenous CRH after stimulation with AVP. Conclusion and Clinical Importance Administration of AVP is safe and results in a significant rise in ACTH and cortisol in neonatal foals. A stimulation test with AVP (5 IU) can be considered for HPAA assessment in septic foals.

4][5][6] After input from the sympathetic nervous system, arginine-vasopressin (AVP) and corticotropin-releasing hormone (CRH) are released from the hypothalamus and elicit an increase in ACTH from the anterior pituitary. 7TH then acts on the adrenal cortex for cortisol synthesize and release. 8,96]10 RAI results in abnormal cortisol production, cellular activity, and metabolism.These processes can lead to hypoglycemia, electrolyte abnormalities and hypotension. 4,5,10evalence of RAI in septic foals is (40%-60%), with affected foals being more likely to develop multiple organ dysfunction syndrome (MODS) and less likely to survive. 2,3,11The gold standard diagnostic method in horses utilizes a dynamic ACTH stimulation and cortisol response test. 12,13However, this diagnostic test has remained controversial, and produces inconsistent results in horses. 3,6,10,11,14This gap in our current understanding of the HPAA in horses is highlighted by the lack of a more appropriate test to evaluate HPAA function effectively.
AVP has been implicated as a contributing factor driving fluctuations in both ACTH and cortisol in horses and may be more important for short-term control compared to CRH. 8,[15][16][17][18] We propose that AVP is an important stimulus of ACTH and cortisol production and release in neonatal foals and might be more effective in assessing HPAA function.AVP stimulation tests have been performed for HPAA assessment in cattle and demonstrated heightened peaks in ACTH and cortisol in hyperexcitable individuals. 191][22][23][24][25] No studies have been conducted investigating an AVP stimulation test and HPAA response in foals.
The purpose of this study was to investigate the HPAA response to AVP in neonatal foals during the first 72 hours of life in this crossover study and determine a safe and effective dose of AVP for HPAA assessment.Our hypotheses were that (1) administration of AVP would elicit a dose-dependent rise in both serum cortisol and plasma ACTH in neonatal foals, and that (2) there would be no clinically apparent adverse effects on physical examination parameters.Lastly (3), we predicted that there would be no effect on endogenous CRH and baseline AVP would be within reference interval.

| Animals
Twelve neonatal foals (6 healthy and 6 admitted to the hospital), 24-48 hours of age were included in the study, with a median gestation length of 330 days (range, 322-335 days).A statistical power analysis was performed for sample size estimation, based on data from a pilot study comparing cortisol concentration before and after administration of AVP 5 IU in healthy foals.With an alpha = 0.05 and power = 0.80, the projected sample size needed with this effect size (1.16) is approximately N = 9 per group.Thus, our sample size of N = 12 per group was adequate for the main objective of this study.
Foals were recruited for the study from December 2020 to June 2021, and were determined to be not critically ill based on physical exam parameters, sepsis scores, CBC (Advia 120 Hematology Analyzer, Siemens Healthineers, Malver, Pennsylvania), and serum biochemistry analysis (Cobas C 501, Roche Diagnostics Corporation, Indianapolis, Indiana) values within acceptable reference intervals. 26e study sample consisted of 6 hospitalized foals managed for failure of transfer of passive immunity, flexural limb deformities, and birth via Cesarean section, and the remaining 6 were housed on their farm of birth.All foals were determined to have an intact

| Study design
A randomized cross-over repeated measures design was used to compare systemic CRH, ACTH and cortisol response to 3 different doses of AVP (2.5, 5, and 7.5 IU) in neonatal foals.8][29][30][31][32] Injectable vasopressin (Vasostrict) was supplied as a sterile aqueous injection in glass vials (20 IU/mL), (Vasostrict, Par Pharmaceutical Companies, Inc, Spring Valley, New York).Baseline sampling was performed at 24 hours of age, and foals subsequently received 3 doses of AVP intravenously (2.5, 5, and 7.5 IU) every 12 hours.The 2.5 IU dose of vasopressin was diluted in 20 mL of sterile saline, and both 5 and 7.5 IU doses were diluted in a 250 mL bag of sterile saline for administration.Additional blood samples were collected at 15, 30, 60, and 90 minutes after each AVP infusion.The median dosage of AVP administered to each foal based on body weight was 0.06 IU/kg (range, 0.04-0.11)for the 2.5 IU dose, 12 IU/kg (range, 0.08-0.18)for the 5 IU dose, and 0.18 IU/kg (range, 0.12-0.27)for the 7.5 IU/kg.The order of stimulation tests was randomized within the individual foals, and baseline AVP before each stimulation test was compared.

| Sample collection
Jugular catheters were placed in all foals.AVP was administered over a period of 5 minutes for the 2.5 IU dose, and 10 minutes for the 5 and 7.5 IU doses.Serum and plasma were collected into serum clot and chilled EDTA-aprotinin tubes (500 k U/mL of blood), respectively, centrifuged at 2000g at 4 C for 15 minutes, aliquoted, and stored at À80 C until analysis.Inc, Burlingame, California).Both cortisol and CRH RIA measurements have been previously validated in horses. 33,34Plasma AVP concentrations were measured using a multispecies enzyme immunoassay with a detection limit of 2.11 ng/mL and a working range between 4.09 and 1000 pg/mL (Arg-Vasopressin Enzyme Immunoassay Kit (AVP) [Multispecies], Arbor Assays, Ann Arbor, Michigan).

| Measurement of blood hormone concentrations
Plasma samples were extracted with the provided extraction solution following the manufacturer's instructions, before running the kit.The AVP intra-assay and inter-assay CVs were 8.9% and 8.6%, respectively.Plasma ACTH was measured using an automated chemiluminescent assay (CLIA), previously validated for equids (ACTH Immulite kit, Siemens Medical Solutions USA, Tarrytown, New York). 35pearman's rank correlation test was used to determine the effect of body weight on fold increase in cortisol and ACTH concentration 15 and 30 minutes after AVP stimulation.The cortisol and ACTH fold increase at 15 and 30 minutes after AVP stimulation were compared with the Friedman's test to determine the effect of order on dosing AVP.The cortisol and ACTH fold increase at 15 and 30 minutes were compared between foals presented to the hospital and healthy foals with 3-way ANOVA (fold increase, dose of AVP, and group of foals) and no difference was noted between 2 groups of foals.A comparison was made between these 2 groups to further confirm them as not critically ill, and to demonstrate an appropriately functioning HPAA.Relationships between categorical variables were analyzed using contingency tables and Fisher's exact test.Statistical analysis was performed using Prism and IBM SPSS Statistical Software (SPSS and GraphPad Software, Inc [version 7.0b], La Jolla, California).

| Study sample
The study sample consisted of 12 neonatal foals (5 females, 7 males), The second foal had growth of Stenotrophomonas rhizophila from jugular vein site, and no growth from an alternative location.Possible explanations for positive blood culture in conjunction with the low sepsis scores and no evidence of sepsis based on physical exam variables and blood work include sterile site contamination during blood collection, or a transient bacteremia that occurs in healthy neonatal foals. 36

| Endogenous AVP concentration before AVP stimulation tests
Baseline endogenous AVP concentration was 9.5 (5.8-13.2),10.9 (7.9-13.4),and 8.8 (7.11-14.16)pg/mL before stimulation with 2.5, 5, and 7.5 IU of AVP, respectively.There were no differences detected in basal AVP concentration before stimulation test with each dose of AVP (P > .05).There was a significant effect of both time and dose of AVP on ACTH concentration after administration of 2.5, 5, and 7.5 IU of AVP.
Plasma ACTH concentration increased 15 and 30 minutes after administration of all 3 doses of AVP compared to baseline (P < .01).For the 7.5 IU dose of AVP, plasma ACTH concentration was higher at 15 and 30 minute time points compared to 2.5 IU of AVP (P < .05).
Plasma ACTH concentration remained higher at 60 minutes compared to time 0 in response to 7.5 IU of AVP (P < .05).
3.5 | Change in systemic ACTH and cortisol concentrations after administration of 2.5, 5, and 7.5 IU of AVP in neonatal foals Change in systemic ACTH and cortisol concentration 15 and 30 minutes after AVP stimulation is presented in Figure 3A,B, respectively.ACTH response 15 minutes after AVP stimulation with 5 and 7.5 IU of AVP was higher compared to 2.5 IU dose of AVP (P < .05)and (P < .01),respectively.For the 7.5 IU of AVP, the fold increase in ACTH concentration 0-15 minutes was higher compared to 5 IU dose of AVP (P < .01).Fold increase in ACTH 0-15 minutes in response to 5 and 7.5 IU of AVP was higher than the fold increase in ACTH 0-30 minutes for the same doses of AVP (P < .01).There were no differences detected in the fold increase in cortisol concentration from baseline to 15 and 30 minutes after stimulation with all 3 doses of AVP (P > .05).Fold increase in cortisol concentration 0-30 minutes after administration of 7.5 IU of AVP was higher compared to the fold increase in cortisol concentration 0-15 minutes after the same dose of AVP (P < .01).The order of AVP dosing had no effect on the fold increases in cortisol and ACTH at 15 and 30 minutes after stimulation (P > .05).There was no statistical difference in cortisol and ACTH response to AVP among healthy foals and foals admitted to the hospital (P > .05).Finally, there was no effect of body weight on fold increase of cortisol and ACTH at 15 and 30 minutes after stimulation with all 3 doses of AVP (P > .05).

| Peak concentration and time to peak for cortisol and ACTH in neonatal foals
Peak and time to peak for cortisol and ACTH concentrations in neonatal foals are presented in Table 1.The time to peak for cortisol concentration was higher for 7.5 IU of AVP compared to 2.5 IU (P < .05).However, the cortisol peak concentrations were not different between 3 doses of AVP.
Peak ACTH concentration was higher for 7.5 IU of AVP compared to 2.5 IU and occurred at 15 minutes after the stimulation (P < .01).

| DISCUSSION
This study investigated AVP stimulation tests in foals using a safe and effective dose for assessment of the HPAA.The results show that all 3 doses of AVP produced a significant rise in both ACTH and cortisol at 15 and 30 minutes, respectively, demonstrating AVP is a potent regulator of both hormones in foals. 8,16,37,38This is contrary to AVP stimulation in humans, where no significant change is seen in either hormone. 39Peak in ACTH was more pronounced for the 7.5 IU dose compared to the 2.5 IU dose, suggesting a dose-dependent effect between AVP and ACTH.1][42] The cortisol fold increase was significant at all 3 doses; however, the highest dose produced an even more pronounced fold increase, close to a 7-fold increase from baseline.
ACTH stimulation tests are still considered to be the gold standard for RAI or CIRCI detection in critically ill foals and people. 3,6,10,11wever, exogenous ACTH stimulation tests have resulted in a variable cortisol response in both healthy and septic foals, and critically ill humans, implicating it as a poor diagnostic tool in both species. 1,4,5,11,41,43In the current study, exogenous AVP administration not only resulted in a rise in endogenous ACTH, but also produced a significant increase in cortisol.The cortisol fold increase in this study is significantly higher after exogenous AVP administration compared to the cortisol fold increase after exogenous ACTH administration in previous studies in foals. 11,41Direct comparison between the AVP stimulation and previously investigated doses of ACTH is challenging as these are 2 different hormones with different mechanisms of action.A significant difference has been demonstrated between 1 and T A B L E 1 Cortisol and ACTH peak and time to peak after AVP stimulation (2.5, 5, and 7.5 IU) in 12 neonatal foals <72 hours old (median and interquartile ranges).10 μg of ACTH in cortisol response at 30 minutes, but no difference between 100 and 250 μg of ACTH in overall cortisol response until 120 minutes was reached. 446][47][48] This additional and direct pathway is a possible explanation for the heightened cortisol peak seen with AVP stimulation compared to ACTH stimulation.Future studies are needed to directly compare AVP and ACTH stimulation tests in a cross-over design, in the same study sample of foals.
Baseline ACTH and cortisol concentrations are within the reference interval in this study sample and are consistent with a healthy and functionally intact HPAA. 3,6,34,42,441 Again, there is a poor cortisol response after ACTH stimulation in healthy, unstressed foals. 3,11,41,44This is juxtaposed to our study that demonstrates a reliable and pronounced rise in cortisol with even the low dose (2.5 IU) of AVP.Our study shows that AVP is a potent HPAA stimulant on multiple levels, resulting in ACTH and cortisol release in foals.
Baseline cortisol, AVP, ACTH, and CRH concentrations were within the reference interval for all foals, further supporting their status as healthy. 6,34,42,44After AVP administration, no response was detected in CRH, indicating that AVP has a negligible effect on the hypothalamic release of CRH in neonatal foals.Additionally, a drop in CRH was not demonstrated throughout the rise of ACTH and cortisol, which would be expected because of the negative feedback loop of both hormones. 7However, the majority of CRH is released into the hypothalamic-hypophyseal portal vessels, as opposed to systemic circulation, which is a possible explanation for this finding. 6CRH is considered to be the main stimulus driving ACTH release in healthy humans, however, close correlation between these hormones in horses only occurs during times of critical illness such as with profound hypoglycemia. 17In healthy horses, AVP and ACTH release is tightly correlated with up to 92% of ACTH peaks occurring subsequent to an AVP peak even in the face of a stable pituitary venous CRH. 17,18In addition to an unchanged CRH seen in our study, no effect on baseline AVP was detected with previous AVP administration.Stimulation tests were performed both in the morning and the evening with no effect on the time of day seen in the cortisol responses, which is expected in foals of this age.Foals inherently lack a circadian rhythm until they are 20-40 days of age, at which point they develop a diurnal rhythm. 49,50e objective of this study was to investigate if an AVP stimulation test is an appropriate diagnostic tool to stimulate the HPAA and identify critically ill foals with RAI.We demonstrated that AVP reliably produces a significant HPAA stimulation in neonatal foals, with resulting rise in both cortisol and ACTH, as compared to previously published studies in adult horses.No clinically important adverse effects were observed with the 3 doses of AVP (2.5, 5, and 7.5 IU).A slight drop from baseline heart rate and pale mucous membranes were detected with all 3 doses; however, these changes were transient and persisted no longer than 15 minutes. 45,51There was no significant effect of dose on duration or severity of decline in heart rate. 5mitations of this study include the small sample size (n = 12).
The cross-over design minimized this limitation to improve statistical power.A second limitation was that 6 of the foals were subjected to stimulation while in hospital.However, there is much evidence from clinical examination, clinicopathologic data, sepsis scores and baseline hormone levels that support their healthy status.Statistical comparison showed there was no difference in cortisol and ACTH response between healthy foals and foals admitted to the hospital.However, the main goal of this study was to investigate HPAA response to AVP in healthy foals, and data regarding critically ill and septic hospitalized foals are warranted for comparison of hormone response levels between groups.A third limitation considered is the positive blood culture results identified in 2 foals, which may be interpreted as evidence of sepsis.Alternative explanations for this include site collection contamination or transient bacteremia that has been documented in otherwise healthy foals. 36Another limitation is that the 5 and 7.5 IU doses were diluted in 250 mL of saline and administered over 10 minutes, compared to a smaller dilution and more rapid infusion of the 2.5 IU dose (5 minutes).Initiation of blood collection began 15 minutes after the start of the infusion and potentially could have resulted in variation between peak hormone concentrations between the doses.Finally, foals were not monitored after the 90-minute study period for any possible delayed adverse effects.However, rapid Radioimmunoassays (RIA) were used to measure serum concentration of cortisol and plasma concentration of CRH (Cortisol Coated Tube radioimmunoassay kit, MP Biomedicals, LLC, Solon, Ohio; Corticotropin Releasing Hormone (CRH) [Human, Rat, Mouse, Canine, Feline] RIA Kit Assay Protocol, Phoenix Pharmaceuticals, Data were tested for normality by a Kolmogorov-Smirnov test and were noted to be not normally distributed.Data were presented as median and interquartile ranges.The Freidman's test was used to compare cortisol, ACTH, and CRH concentrations at baseline and 15, 30, 60, and 90 minutes after each dose of AVP to assess for the effect of time or dose of AVP on hormone concentration.Dunn's post hoc comparisons were made when relevant.A fold increase was defined as the ratio of an increased concentration at 15 and 30 minutes to the baseline concentration.Peak ACTH and cortisol concentration was defined as the single highest hormone concentration achieved in each individual foal.Time of peak cortisol and ACTH concentration was the time point at which peak hormone concentration was achieved in each foal for each dose of AVP.Differences in peak concentration and time of peak for cortisol and ACTH were analyzed with Kruskal-Wallis test with Dunn's post hoc test for multiple comparisons.Differences in fold increase in cortisol and ACTH concentration were analyzed with the Friedman's test with a Dunn's post hoc test for multiple comparisons.There was no effect of time or dose of AVP on endogenous CRH concentration in foals.Consequently, the fold increase as well as peak CRH and time of peak CRH were not calculated.

F
I G U R E 1 Serum cortisol concentrations before (time 0) and 15, 30, 60 and 90 minutes after intravenous administration of 2.5, 5, and 7.5 IU of AVP in neonatal foals, <72 hours old (median and interquartile ranges).**P < .01compared to time 0. F I G U R E 2 Plasma ACTH concentrations before (time 0) and 15, 30, 60 and 90 minutes after intravenous administration of 2.5, 5, and 7.5 IU of AVP in neonatal foals, <72 hours old (median and interquartile ranges).**P < .01compared to time 0; a*P < .057.5 compared to 2.5 IU dose of AVP within the same time point; b*P < .05compared to time 0 for 7.5 IU of AVP.
HPAA, and no signs of systemic illness.IgG concentrations were measured using an immunoturbidimetric assay (Rapid DVM Test II, Value Diagnostics, MAI Animal Health, Melksham, Wiltshire, UK) on EDTA-blood.Three foals were from North Carolina State University teaching herd, 3 of the foals were from Iowa State University teaching herd and the remainder of the foals (n = 6) were hospitalized at 8,11,15,42AVP stimulation with the 5 IU dose is ideal for assessment of both pituitary and adrenal responses, as demonstrated by this study.5 IU of AVP produced a significantly higher ACTH peak at 15 minutes compared to the 2.5 IU dose; whereas the 7.5 IU dose did not produce any clinically significant difference in either ACTH (8 vs 10-fold increase) or cortisol, compared to the 5 IU dose.Dynamic evaluation of both cortisol and ACTH, additionally can be used to differentiate between primary and secondary adrenal insufficiency for more targeted therapy.Future investigation into HPAA response to exogenous AVP in critically ill foals compared to healthy foals, and those that have a dysfunctional HPAA is warranted to establish cut-off values for cortisol and ACTH response to diagnose RAI.