Renin-Angiotensin-Aldosterone System (RAAS) and Hypothalamic-Pituitary

Renin-Angiotensin-Aldosterone System (RAAS) and Hypothalamic-Pituitary-

Adrenal Axis (HPAA) in Critically Ill Foals

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Katarzyna Agnieszka Dembek

Graduate Program in Veterinary Clinical Sciences

The Ohio State University

2012

Master's Examination Committee:

Associate Professor Ramiro Toribio; Advisor

Professor Catherine Kohn

Assistant Professor Samuel Hurcombe

Copyrighted by

Katarzyna Agnieszka Dembek

2012

Abstract

Sepsis is a major cause of morbidity and mortality in neonatal foals. Dysfunction of the hypothalamic-pituitary-adrenal axis (HPAA), manifested as relative adrenal insufficiency

(RAI), has been associated with sepsis in newborn foals. Information on the renin- angiotensin-aldosterone system (RAAS) is minimal in healthy or sick foals. The HPAA and RAAS are interactive systems, and a relationship between RAAS activation and RAI is well documented in critically ill children, but limited information exists in septic foals.

We hypothesized that in critically ill septic newborn foals the RAAS and HPAA will be activated by systemic inflammation and hypoperfusion and the degree of activation will be associated with severity of sepsis and mortality.

For this project, 167 (study 1) and 182 (study 2) sick and healthy foals of less than

7 days of age were included. Blood samples were collected on admission from septic

(sepsis score >12), sick non-septic (SNS), and healthy foals. Blood concentrations of cortisol, aldosterone, angiotensin-II (ANG-II), corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), adrenocorticotropic hormone (ACTH) and plasma renin activity were determined by immunoassays.

Aldosterone, ANG-II, ACTH and cortisol concentrations were higher in septic compared to healthy foals (P<0.05). AVP was higher and CRH was lower in septic than healthy

iii foals. Septic foals had higher ACTH/aldosterone and ACTH/cortisol ratios than healthy foals (P<0.05). No differences in plasma renin activity were found.

Sepsis activates RAAS and HPAA in newborn foals. RAAS activation in critically ill foals is characterized by increased aldosterone and ANG-II concentrations. Low CRH concentrations in septic foals were an unexpected finding. We propose that AVP (not

CRH) is the main ACTH releasing-hormone in critically ill foals. Septic foals demonstrated adrenocortical exhaustion with high ACTH/cortisol and ACTH/aldosterone ratios, indicating that RAI is not restricted to the zona fasciculata, but also involves the zona glomerulosa.

Dedicated to my parents for their love, endless support and encouragement

Acknowledgments

I would like to acknowledge my adviser, Dr. Ramiro Toribio for his intellectual support and assistance in my research realization.

Special thanks go to Dr. Catherine Kohn, I am endlessly grateful for her mentorship in the clinical setting of my residency.

I am thankful for the support and advice from Dr. Samuel Hurcombe for his clinical mentorship and advice. I am also indebted to Steven Naber and Sasha Bai for their statistical expertise.

I would like to thank all of the clinicians and technical staff at Hagyard Equine Medical

Center and Rood and Riddle Equine Hospital in Lexington, Kentucky for their dedication and support of this project.

I also found myself lucky to have Kate Onasch, Brandy Marlow, Krista Hernon and

Eason Hildreth III to help me in the lab and with data retrieval.

Without the support from the aforementioned people, this project would have been very difficult to complete.

Vita since 07/09 Residency and Graduate Teaching and Research Assistant, Veterinary Clinical Sciences, The Ohio State University, Columbus, OH, USA 01/07 - 05/09 Associate, Al- Khalediah Equine Hospital , Saudi Arabia

01/06 - 12/07 Internship, Lisadell Equine Hospital, Ireland

06/05-12/05 Internship, Equine Clinic, Warsaw University of Life Sciences, Poland 09/99 - 05/05 School of Veterinary Medicine, Warsaw University of Life Sciences, Poland

Fields of Study

Major Field: Veterinary Clinical Sciences

vii

Table of Contents

Abstract ...... iii

Acknowledgments...... vi

Fields of Study ...... vii

Table of Contents ...... viii

List of Tables ...... ix

Chapter 1: Introduction and Literature Review ...... 1

2.1 Materials and Methods ...... 12

2.2 Results ...... 15

2.3 Discussion ...... 19

Chapter 3. Hypothalamic-pituitary-adrenal axis in hospitalized foals...... 32

3.1 Materials and Methods ...... 32

3.2. Results ...... 35

3.3 Discussion ...... 37

References ...... 46

viii

List of Tables Table 2.1 Blood hormone and electrolyte concentrations, and hormone ratios in neonatal foals ...... 22

Table 2.2 Blood hormone concentrations and hormone ratios in surviving and nonsurviving septic foals ...... 24

Table 2.3 Hormone univariate analysis for survival among neonatal foals ...... 25

Table 2.4 Correlations (ρ) between hormones, serum electrolytes, IgG, creatinine and

BUN concentrations, and sepsis score in septic foals……………………………… 29

Table 2.5 Correlations (ρ) between hormones, serum electrolytes, IgG, creatinine and

BUN concentrations, and sepsis score in SNS foals...... 30

Table 2.6 Correlations (ρ) between hormones, serum electrolytes, IgG, creatinine and

BUN concentrations, and sepsis score in hospitalized foals ...... 31

Table 3.1Blood hormone concentrations and hormone ratios in neonatal foals ...... 41

Table 3.2 Blood hormone concentrations and hormone ratios in surviving and nonsurviving septic foals (values expressed as median and range) ...... 42

Table 3.3 Hormone univariate analysis for survival among neonatal foals ...... 43

Table 3.4 Correlations (ρ) between hormones, IgG, sepsis score, total protein, and glucose concentrations in septic foals...... 44

Table 3.5 Correlations (ρ) between hormones, IgG, sepsis score, total protein, and glucose concentrations in SNS foals...... 44

Table 3.6 Correlations (ρ) between hormones, IgG, sepsis score, total protein, and glucose concentrations in septic foals...... 44

Chapter 1: Introduction and Literature Review

1.1 Sepsis in neonatal foals

Sepsis/septicemia, defined as the presence of bacteria or bacterial toxins in the bloodstream can lead to organ dysfunction and death in newborn foals.1-4 Sepsis is the main cause of foal mortality in the first week of life, resulting in major economical losses to the equine industry.1,1-9 Most of clinical signs of sepsis and septic shock are caused by a cascade of inflammatory mediators released in response to bacterial toxins. This cascade leads to the Systemic Inflammatory Response Syndrome (SIRS). When the initial protective response against sepsis is overwhelmed, the end result is Multiple Organ

Dysfunction (MODS).1 SIRS is not restricted to bacterial insults but it may also be caused by viruses, fungi, protozoa, extensive trauma, hypoxia, ischemic disease and certain drugs (mostly anti-neoplastic). Any type of infection (bacterial, fungal or viral) can result in sepsis/SIRS, however, bacterial infections are the most frequent cause of severe sepsis in neonatal foals.1-4,6-9 The most common routes of infections in newborn foals include: respiratory tract, gastrointestinal tract, placenta, umbilical structures, and the integumentA number of pathogens are involved in pathogenesis of septicemia in

neonatal foals.3,4,9 Escherichia coli, Actinobacillus equuli, Salmonella spp. and Klebsiella pneumoniae are the most commonly isolated gram negative organisms, while

Streptococcus spp., Staphylococcus aureus, and Clostridium spp. are the most common gram positive organisms isolated in septic foals.2,4,9

Failure of transfer of passive immunity (FTPI) is an important predisposing factor to the development of neonatal septicemia.1,10-12 The neonatal foal is immunocompetent, but it is immunologically naïve at birth. It is able to respond to antigens but has not yet been challenged. Because mares have epitheliochorial placentation, the newborn foal depends on passive transfer of colostral immunity for the majority of their immunological protection for at least the first few days to weeks of life. Immunoglobulins are selectively absorbed from the gut by specialized enterocytes which are replaced within 24-48 hours of birth. Maximum absorption occurs in the first 6-813 hours post-partum and maximum blood concentrations of IgG occur at about 18 hours post-partum. The half-life of IgG in healthy foals is approximately 12 to 25 days.1,16 When challenged by disease, IgG concentrations may decline rapidly. Closure of the gut is stimulated by factors in colostrum, so it is essential that foals receive an adequate quantity of good quality colostrum. Foals with adequate transfer of IgG have serum concentrations greater than

800 mg/dl; FTPI is defined as serum IgG concentration less than 400 mg/dl, and partial

FTPI as a serum IgG from 400 to 800 mg/dl.1,2,10,11

FTPI (< 400 mg/dl of IgG) occurs in 3 to 20% of foals.1,13 The incidence of septicemia and mortality rate is positively correlated with FTPI, therefore, it is important to diagnose and treat FTPI within the first 24 h of live. Septic foals may consume large amounts of IgG and require a supplemental source of immunoglobulins.

The final confirmation of a diagnosis of septicemia is a positive blood culture. This test also provides antimicrobial treatment options, based on bacterial susceptibility. Results of the blood culture cannot be obtained for at least 72 hs1,14 which delays diagnoses and treatment. In order to obtain an earlier indication of sepsis, a sepsis scoring system was developed.17 The score was reported to have 93% sensitivity and 86% specificity14. A positive correlation was observed between the sepsis score and positive blood culture result, and both parameters were significantly associated with mortality.1,4,12,14

Septic foals are often hypovolemic and hypotensive due to decreased fluid intake, inflammation, vasoplegia, myocardial depression and ongoing fluid losses. Hypovolemia is common in critically ill horses and neonatal foals for a number of reasons including fluid loss into the gastrointestinal tract; endotoxin-induced capillary damage; fluid and protein leakage into the interstitial space; cytokine-mediated reduction in cardiac contractility and vasodilation.1 Mechanisms for sepsis-related vascular dysfunction include the production of nitric oxide (NO) induced by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) in response to infections and refractoriness to vasoconstriction. Excessive formation of NO, prostaglandin E2

15-19 (PGE2) and leukotrienes leads to vascular damage and hypotension.

The hypothalamic-pituitary-adrenal axis (HPAA) and the renin-angiotensin-aldosterone system (RAAS) are both activated in response to sepsis-related stress and hypotension.5,16,20 We, as well as others, have recently shown that HPAA activation is associated with severity of sepsis and mortality in septic foals.5,21-24 While presented as separated systems, in reality the HPAA and RAAS are highly interactive, sharing

hormonal factors such as ACTH, vasopressin (AVP), angiotensin-II (ANG-II), and aldosterone.

1.2 Renin-Angiotensin-Aldosterone System

The main function of the RAAS is to maintain tissue perfusion by increasing blood pressure and intravascular fluid volume. Hypotension and hyponatremia stimulate the juxtaglomerular apparatus in the kidneys to secrete renin. The juxtaglomerular apparatus is formed by cells in the macula densa (extraglomerular mesangial cells located between afferent and efferent arterioles of glomerulus), smooth muscle, and juxtaglomerular cells

(renin secreting cells) in the afferent arteriole. The juxtaglomerular apparatus contributes to the coordination between glomerular filtration and tubular reabsorption through the mechanism of tubuloglomerular feedback (TGF). The TGF mechanism refers to a series of events whereby changes in the Na+, Cl-, and K+ concentrations in the tubular fluid are sensed by the macula densa via the Na+-K+-2Cl- cotransporter in its luminal membrane. A decrease in Na+, K+ and Cl- concentrations in the distal tubules indicates a reduced glomerular filtration which increases prostaglandin production and renin release from juxtaglomerular apparatus. High Na+, K+ and Cl-concentrations in distal tubules increases local adenosine release, causing vasoconstriction of the afferent arteriole and decrease in renin release. Renin is a proteolytic enzyme that cleaves hepatic angiotensinogen to angiotensin I (ANG-I), which is primarily converted in the lungs by angiotensin- converting enzymes (ACE) into ANG-II.15,17,18,20,25 ANG-II (an eight amino acid peptide) is a potent vasoconstrictor, and also causes aldosterone secretion from the adrenal gland

cortex, AVP from the pituitary gland, and triggers thirst. Aldosterone secretion is also activated by hyperkalemia and ACTH.21,22,26

Steroidogenesis requires oxidative enzymes located in both mitochondria and smooth endoplasmic reticulum. The rate-limiting step in this process is the transport of free cholesterol from the cytoplasm into the mitochondria. In the mitochondria, cholesterol is converted to pregnenolone by an inner membrane enzyme CYP11A1 (cholesterol side- chain cleavage enzyme) that is primarily stimulated by ANG-II (aldosterone synthesis) and ACTH (cortisol synthesis).14,27,28 ACTH induces the hydroxylation of pregnenolone into 17-OH-pregnenolone, which is transported to the endoplasmic reticulum for conversion to 11-deoxycortisol. 11-deoxycortisol moves back to mitochondria where another hydroxylation takes place to produce the final product, cortisol. Aldosterone biosynthesis is completed in the mitochondria, where deoxycorticosterone undergoes

11β- and 18-hydroxylation, followed by 18-oxidation.

Aldosterone is the main adrenocortical mineralocorticoid and its function is to increase renal reabsorption of Na+ and water, and potassium elimination. In the distal nephron, aldosterone binds to mineralocorticoid receptors to stimulate the expression of genes involved in Na+, K+ , and water transport, including sodium and potassium channels,

Na+/K+-ATPase, and aquaporins.27,29 Reclamation of sodium (and water) by aldosterone, as well as efferent arteriolar vasoconstriction by ANG-II, aim to maintain volume and perfusion to vital organs and offset hypotension associated with septic, hypovolemic and neurogenic shock. 15,27

1.3. Hypothalamic-Pituitary-Adrenal Axis and Arginine Vasopressin Function.

Environmental and physiological stress induces the release of Corticotropin-Releasing

Hormone (CRH) from the paraventricular nucleus of the hypothalamus into the hypophyseal portal system. CRH stimulates the corticotrophs of the anterior pituitary gland to release Adrenocorticotropic Hormone (ACTH) into systemic circulation.

Arginine vasopressin (AVP) is a neural hormone, synthesized in the cell bodies of the magnocellular neurons in the supraoptic and paraventricular nuclei and transported down the axons of these neurons to the posterior pituitary lobe (neurohypophysis). There are three types of AVP receptors: V1, V2, and V3. The vasoconstrictor effects of AVP are mediated by V1 receptors in blood vessels. V2 receptors mediate the anti-diuretic effect by promoting the translocation of aquaporins (water channels) from cytoplasmic endosomes to the luminal cell membrane of the principal cells in the collecting

30,31 30,32,33 ducts. V3 receptors mediate ACTH secretion in the anterior pituitary. There is some evidence that AVP is an ACTH-releasing hormone in horses.26,34,35

ACTH stimulates the zona fasciculata of the adrenal cortex to secrete cortisol and to lesser extend the zona glomerulosa to release aldosterone. Cortisol production and the expression of the steroidogenic enzymes are directly stimulated by CRH in fetal adrenals.36,37 Depending on the physiological needs, cortisol secretion is under the control of a negative feedback mechanism at the level of the hypothalamus (CRH) and pituitary gland (ACTH).26,38-41 Cortisol is the main glucocorticoid and its function is to increase protein catabolism, hepatic glycogenesis, gluconeogenesis, and to modulate the inflammatory response. The effects of cortisol are initiated by binding to glucocorticoid

receptors, and the cortisol–receptor complex acts as a transcription factor to promote protein and enzyme synthesis.32,42,43

1.4. RAAS dysfunction in critical illness

Irreversible adrenal insufficiency or hypoadrenalism (Addison’s disease) results from failure of the adrenal gland to produce adrenocortical hormones (mineralocorticoid and glucocorticoid). The consequences of adrenocortical insufficiency are many, including decreased organ perfusion, electrolytes imbalance and increased mortality rate. Addison disease is well described in people and other species, but has not been documented in horses. Physiological partial aldosterone resistance (pseudohypoaldosteronism) associated with hyponatrema, hyperkalemia and high urinary sodium loss has been reported in newborn children.29 This resistance can be in part explained by the low renal expression of mineralocorticoid receptors in human neonates.44 To date, pseudohypoaldosteronism has not been reported in foals.

RAAS dysfunction during sepsis has received extensive attention in critically ill humans.15,15-18,20,25,45,46,46,47 An association between RAAS activation with multiple organ failure and survival has been documented in septic human patients.15,15-18,46-49 A dissociation of renin and aldosterone production has been reported in 20-30 % of critically ill patients.47 Hyperreninemic hypoaldosteronism results from impairment of zona glomerulosa and is characterized by normal production of ANG-II, hypercortisolemia and decreased concentrations of 18-hydroxycorticosterone

(aldosterone precursor).47

Critically ill children with meningococcal sepsis often have low aldosterone concentrations and elevated renin activity (96.7% of patients have aldosterone value below the range for healthy children).49 Explanations for hypoaldosteronemia during critical illness include: disruption of aldosterone synthesis from destruction of the zona glomerulosa, proinflammatory cytokine inhibition of aldosterone release, and damage to adrenal cortex from hemorrhage and ischemia.

Previous studies have shown that the aldosterone response to hypovolemia is different in neonatal foals compared to adult horses, likely due to differences in neonatal sodium and water regulation.20,50 Healthy human and equine neonates have higher aldosterone and renin concentrations than adults, which may reflect relative insensitivity of the distal tubules to aldosterone.

There is data on the RAAS in pregnant mares and adult horses under different exercise conditions,50-52 and some work has been published in healthy foals;20,53 however, information on the RAAS response to illness is lacking in both adult horses and newborn foals.

1.5. HPAA dysfunction and adrenal insufficiency in critical illness

Adrenal insufficiency (AI) can be classified based on the level of the HPAA that is affected. Primary AI: caused by damage to the adrenal gland cortex; secondary AI: caused by impaired ACTH secretion by the pituitary gland, and tertiary AI: caused by reduced CRH secretion by the hypothalamus. The most common type of AI in people is

iatrogenic AI: caused by chronic glucocorticoid administration leading to suppression of

HPA axis.

Relative adrenal insufficiency (RAI) or critical illness-related corticosteroid insufficiency (CIRCI), defined as an inappropriately low basal cortisol concentration or inadequate production of cortisol in relation to increased ACTH concentration during severe illness, is common in septic children and foals.5,21-24,54,55

Adrenal insufficiency can result from HPAA dysfunction at one or several levels and may be transient or permanent. Addison disease is the most common manifestation of permanent HPAA impairment. Genetic defects in adrenocortical enzymes leads to cortisol deficiency in people. Transient HPAA hypofunction is common in critically ill patients with a variety of conditions such as trauma, respiratory distress syndrome, surgeries and sepsis/septic shock. Severe sepsis is known to induce an abnormal HPAA response, resulting in profound hormonal changes in ACTH, cortisol and AVP concentrations in people and other species.56-60

ACTH and ACTH/cortisol ratios were lower whereas baseline cortisol was both higher and lower in septic than in non-septic humans.56,61 The mortality rate was higher in critically ill patients with hypercortisolemia. 58,59,62-67 Dissociation between the levels of

ACTH and cortisol during fetal, postnatal and adult life has been studied in humans and other species.5,19,26,38,41,68-71 In addition, clinical studies report the dissociation of ACTH and cortisol (RAI) in sepsis, inflammation and trauma. Cytokines, vasoactive factors and neuropeptides seem to play an important role in the modulation of the adrenal response to severe stress, and act independently of ACTH.5,22,24,61-63,67,72-75

Recently, high concentrations of ACTH and cortisol were reported in neonatal septic and non-septic hospitalized foals.5,21-24,26,39,40,54,76 Nonsurvival of septic foals was associated with both higher and lower cortisol concentrations and with a high ACTH/cortisol ratio, which suggest that some critically ill foals develop RAI.5,21,22,24,26,39,40,76

Elevated blood AVP concentrations is common in the acute stage of sepsis associated with systemic hypotension in humans.63,77,78 Similar findings were reported in septic foals, where AVP and AVP/ACTH ratio were significantly higher than in sick non-septic and healthy foals.5,26,39

While there is evidence that sepsis affects cortisol secretion in critically ill foals

(RAI),5,21-24,76,79 the aldosterone response to severe illness remains to be evaluated. We believe that adrenal insufficiency affects both the zona fasciculata and zona glomerulosa of the adrenal cortex. In addition, sex steroids produced by the zona reticularis of the adrenal cortex are likely to be affected in sepsis. Clinical studies demonstrated that dehydroepiandrosterone (DHEA) concentrations were low in septic shock, whereas the cortisol/DHEA ratio might be a prognostic indicator of adrenal insufficiency.65,80,81

The aim of this study was to investigate the RAAS and HPAA response in healthy and hospitalized (septic and sick non-septic) newborn foals, and to determine their association with clinical findings. We hypothesized that sepsis will lead to RAAS and HPAA activation that will be associated with severity of disease and mortality. We also proposed that a number of septic foals will have inappropriately low aldosterone concentrations

(based on ACTH and electrolyte concentrations), indicating that mineralocorticoid deficiency is part of the RAI that occurs in critically ill newborn foals.

The purpose of our second study (Chapter 3) was to determine the CRH, AVP, ACTH and cortisol concentrations in septic foals, sick foals with conditions other than sepsis, and healthy foals. We also sought to determine if there was any association among

ACTH regulating hormones (CRH and AVP) in neonatal hospitalized foals.

Chapter 2: Renin-Angiotensin-Aldosterone System Response and Ratios in Hospitalized

Newborn Foals

2.1 Materials and Methods

Animals

Foals ≤7 days old of any breed or sex admitted to 3 equine hospitals from one foaling season were included. Hospitalized foals were classified into 2 groups: septic and sick non-septic (SNS) foals. Foals in the septic group had a sepsis score of ≥12, a positive blood culture, or both.14,26 Foals in the SNS group were presented for illnesses other than sepsis (e.g. hypoxic ischemic encephalopathy, meconium impaction, failure of transfer of passive immunity or orthopedic conditions) requiring hospitalization. These foals had negative blood cultures and a sepsis score of ≤11. The control group consisted of 24-72- hour-old foals, classified as healthy based on physical examination, a normal CBC, serum biochemistry, serum immunoglobulin G (IgG) concentrations (>800 mg/dL) and a sepsis score of (0-4). Foals with a history of receiving glucocorticoids, intravenous crystalloid fluids, or plasma before admission were excluded from the study. Foals with uroabdomen were also excluded from the statistical analysis.

Survival was defined as discharged alive from the hospital. Foals that died or were euthanized due to a grave medical prognosis were defined as non-survivors. Foals euthanized for other reasons such as financial constraints were excluded from the study.

This study was approved by Veterinary Clinical Trials Office and adhered to the principles of humane treatment of animals in veterinary clinical investigations, as stated by the American College of Veterinary Internal Medicine and National Institute of Health guidelines.

Data Collection

Clinical history obtained upon presentation included expected foaling date, duration of pregnancy, parity of the mare, maternal illnesses, premature lactation, observed or assisted parturition, dystocia, passing and appearance of fetal membranes, and medications (mare and foal). Clinical data collected from the foal included signalment

(sex, age, breed), physical examination findings, CBC, biochemistry profile, fibrinogen concentrations, IgG concentrations, and blood culture results. Endocrine measurements included plasma renin activity (PRA), and ANG-II, ACTH, aldosterone, and cortisol concentrations. For consistency, the sepsis score was calculated by the first author for each foal, based on history, physical examination, and laboratory findings.

Sampling

Blood samples for hormone measurements from hospitalized foals were obtained on admission (within 1 hour of hospitalization) by sterile jugular venous catheterization.

Samples from healthy foals were obtained during routine examination of newborn foals.

Blood was collected in serum clot and aprotinin-EDTA tubes. Aprotinin was added to inhibit protease-mediated degradation of hormones (500 kU/mL of blood). Samples were centrifuged at 2,000 × g for 10 minutes at 4°C. Serum and plasma were aliquoted and stored at -80°C until analyzed. Blood samples for CBC, serum biochemistry, fibrinogen, and IgG concentrations were processed within 2 hours post collection.

Hormone Concentrations

Serum aldosterone and cortisol concentrations were determined using human-specific radioimmunoassays , validated for horses.2,4,27 Plasma ACTH concentrations were determined with an immunochemiluminometric assay validated for horses.28 Plasma

ANG-II concentrations were measured with a multi-species ANG-II radioimmunoassay , after peptide extraction using phenylsilylsilica solid-phase columns . Intra and inter-assay coefficients of variation for this assay, for equine samples were 8.3 and 10.7%, respectively. Parallelism for equine samples diluted 1:2-1:16 ranged from 84-103%.

Determination of PRA was based on the generation of angiotensin-I (ANG-I) at 4°C and

37°C for 1 h, measured with a radioimmunoassay and expressed as ng/ml/hr of generated

ANG-I. Intra and inter-assay coefficients of variation for this assay, for equine samples were 7.6 and 9.4%, respectively.

Statistics

Data set was tested for normality by the Shapiro-Wilk statistic. ACTH and cortisol concentrations in healthy foals and aldosterone concentrations in all 3 groups of foals were normally distributed. The remainder of the data was not normally distributed.

Medians and ranges were calculated for continuous variables. Comparisons between groups of foals were carried out with the Kruskal-Wallis statistic and the Dunn’s post-test was used to compare each group individually. The Mann-Whitney-U test was used to compare survivors and non-survivors within each group. Significance was set at P < 0.05.

Correlations were determined with the Spearman rank order (ρ). Continuous variables were categorized by cutoff values based on distribution within a group, and analyzed using logistic regression for binomial distribution. Clinical data analyzed included sodium, potassium, chloride, L-lactate, IgG and hormone concentrations. Crude odds ratios and 95% confidence intervals were calculated and based on categories. The dependent variables were survival and non-survival. Data was analyzed with statistical software.

2.2 Results

Study Population

A total of 167 neonatal foals (133 hospitalized; 34 healthy foals) of ≤ 7 days of age were included. The median age of all hospitalized foals at admission was 10 hours. For healthy controls the median age was 24 hours.

Fifty six percent of hospitalized foals (74/133) were classified as septic and 44% (59/133) as SNS. The survival rate in septic foals was 55% (41/74) and in SNS was 88% (52/59).

Forty one percent (30/74) of septic foals had positive blood cultures. The median sepsis score for all hospitalized foals was 8 and 14 for septic foals.

Breeds representing hospitalized foals included Thoroughbred (n=92), Quarter Horse

(10), Standardbred (12), Appaloosa (6), Saddlebred (2), Warmblood (3), Friesian (1),

American Paint Horse (2), Arabian (2), Belgian (2) and Percheron (1). All healthy foals were Thoroughbreds (34).

Of the hospitalized foals, 54% (72/133) were colts and 46% (61/133) fillies.

Hormone concentrations

Results of PRA, ANG-II, aldosterone, cortisol and ACTH concentrations for all foals are presented in Table 1. Septic foals had significantly higher ANG-II, aldosterone, cortisol and ACTH concentrations compared to healthy foals (P<0.05). ANG-II and ACTH concentrations were higher in septic than SNS foals (P<0.05). Aldosterone concentrations were not different between SNS and healthy foals. SNS foals had higher cortisol concentrations than healthy foals (P<0.05). There were no statistical differences in PRA between foal groups.

Septic foals that died had lower cortisol concentrations than survivors (P<0.01); however, aldosterone concentrations were not different between surviving and non-surviving septic foals (Table 2).

In the hospitalized population, foals with aldosterone concentrations in the range of 25-

186.8 pg/mL were more likely to survive (OR=2.7) than foals with aldosterone concentrations >428.4 pg/mL (Table 3). Foals with ACTH concentrations in the range of

10-26.6 pg/mL and 26.7-152 pg/mL were 5.4 and 2.7 times more likely to survive than those with ACTH concentrations >153 pg/mL, respectively (Table 3).

Serum sodium and potassium concentrations were not different between foal groups, but septic foals had lower chloride concentrations than healthy foals (Table 1). Of interest, foals with potassium concentrations of 2.1-3.6 mEq/L and 3.7-4.5 mEq/L were more likely to survive (OR=8.8 and OR=2.4) than foals with potassium concentrations >4.6 mEq/L, respectively (Table 3). Foals with sepsis scores of 0-6 and 7-14 were 81 and 5.4 times more likely to survive than those with sepsis scores >15, respectively (Table 3).

Hormone ratios and correlations

For each foal group the hormone ratios were determined (Tables 1–2). We found no differences in PRA/ANG-II, ANG-II/aldosterone, and PRA/aldosterone ratios between groups. Septic foals had significantly higher ACTH/aldosterone and ACTH/cortisol ratios than healthy foals (P<0.05). The ACTH/cortisol ratio was also higher in septic than SNS foals (P<0.05), and higher in non-surviving than surviving septic foals (P<0.05) (Table

2).

In septic foals, aldosterone concentrations were positively correlated with ANG-II,

ACTH and cortisol. In hospitalized and SNS foals, aldosterone concentrations were positively correlated with ANG-II and cortisol (Tables 4-6). PRA was not associated with any of the hormones.

Association of hormones with electrolytes, creatinine, L-lactate, BUN and sepsis score in hospitalized (septic and SNS) foals

In septic foals, aldosterone was positively correlated with potassium, creatinine and BUN concentrations and negatively correlated with IgG and L-lactate concentrations (Table 4).

ACTH and cortisol concentrations were positively correlated with sodium concentration in septic foals. ANG-II and ACTH concentrations were negatively correlated with IgG concentrations in septic foals. ACTH was positively correlated with L-lactate concentrations. ANG-II concentrations were positively correlated with creatinine concentrations in septic foals (Table 4).

In SNS foals, aldosterone concentrations were positively correlated with potassium, BUN and sepsis score, and inversely correlated with chloride, sodium and IgG concentrations

(Table 5). ACTH was positively correlated with chloride concentration and the sepsis score. ACTH and cortisol concentrations were positively correlated with sodium and creatinine concentrations (Table 5). Cortisol was positively correlated with L-lactate concentration. ANG-II was inversely correlated with IgG concentration and positively correlated with BUN and L-lactate.

No correlations between PRA and electrolytes, L-lactate, sepsis score and IgG were found.

We found no association between hormones with electrolyte, L-lactate, creatinine and

BUN concentrations in healthy foals.

2.3 Discussion

In the study reported here, we documented that during critical illness in newborn foals, there is activation the RAAS and HPAA. Septic foals had higher ANG-II, aldosterone, cortisol and ACTH concentrations compared to SNS and healthy foals. We also showed that RAI, in addition to a high ACTH/cortisol ratio, was characterized by inappropriately low aldosterone concentrations (high ACTH/aldosterone ratio). This poor glucocorticoid and mineralocorticoid response is consistent with CIRCI, as documented in critically ill children.

Sepsis is a stressful event that activates the HPAA and RAAS to maintain organ function by modulating a multitude of homeostatic processes, including tissue perfusion, defense, inflammation, and tissue metabolism. Activation of the HPAA, characterized by increased ACTH and cortisol concentrations, is well documented in sick foals.2,4,8,9

RAI has been associated with mortality in septic human patients.29-31 Similarly, RAI seems to be common in septic and premature foals.2,4,8,10,31 The reported prevalence of

RAI is up to 50% in septic foals and 60% in septic human patients.31,32

In the study reported here, basal ACTH, aldosterone, cortisol, ANG-II concentrations and

ACTH/cortisol and ACTH/aldosterone ratios were increased in septic foals. Similar to recent studies,2,10 a high ACTH/cortisol ratio was associated with mortality in the foals of our study. Of interest, septic foals also had a high ACTH/aldosterone ratio indicating that

RAI is a complex process that affects multiple layers of the adrenal cortex. Abnormally low cortisol and aldosterone concentrations support CIRCI in septic foals.

From our current understanding on adrenal gland anatomy and physiology, there is no reason to believe that adrenal gland insufficiency is limited to the zona fasciculata since

the adrenocortical layers share circulation and innervation, and ACTH stimulates both the zona glomerulosa and the zona fasciculata.5,19 In critically ill humans, hypoperfusion of the adrenal cortex produces a transient dysfunction of the zona glomerulosa.5,19 Systemic inflammation, cytokines, poor perfusion, and hypotension may affect both glucocorticoid and mineralocorticoid production.5,19,32,33

Dissociation of aldosterone and renin (hyperreninemic hypoaldosteronism), characterized by an inappropriately low aldosterone/renin ratio has been associated with mortality in critically ill humans (adults and children).34,35 However, we found no differences in PRA and PRA/hormone ratios between foal groups. The lack of differences in PRA between groups could be explained with a poor renin response to sepsis and hypotension in neonatal foals, or could be analytical. Unlike other endocrine factors, it is recommended that PRA is determined in fresh samples (impractical for this type of study), as this is a dynamic test and factors such as sample collection, storage, temperature changes, and processing can alter enzymatic activity. Although not designed to assess plasma renin activity, perhaps the direct renin assay that measures enzyme concentrations would have been a better method to assess circulating renin in stored equine plasma samples.36,37

Aldosterone secretion is controlled by ANG-II, ACTH, sodium, and potassium concentrations. ANG-II and potassium are considered the main regulators of aldosterone secretion.12,33 However, ACTH is necessary for aldosterone production because it stimulates the synthesis of pregnenolone, the common precursor for all adrenal steroids.

Thus, adrenal resistance to ACTH has the potential to result in mineralocorticoid deficiency. Limited information exists on the ACTH/aldosterone ratio and aldosterone response to ACTH stimulation test in healthy and critically ill foals. DeClue and Martin

reported that in healthy cats, cortisol and aldosterone are released in response to ACTH stimulation.38 Interestingly, exogenous ACTH did not increase aldosterone secretion in healthy adult horses.39 To our knowledge, the ACTH/aldosterone ratio and aldosterone response to ACTH stimulation test has not been evaluated in foals. However, preliminary work from our group indicates that administration of synthetic ACTH (cosyntropin) to healthy foals causes a modest release of aldosterone.

Low cortisol and aldosterone concentrations during illness is consistent with CIRCI and represents the basis for hydrocortisone and fludrocortisone replacement therapy in children diagnosed with this condition.21,40,41 Furthermore, an inappropriate aldosterone response to sepsis may contribute to some of the electrolyte abnormalities (hyponatremia, hypochloremia, hyperkalemia) and mortality of critically ill foals.

In the present study, low serum chloride and high potassium, but not sodium concentrations were associated with increased aldosterone concentrations. It is possible that in critically ill foals renal tubular chloride concentrations are more important than sodium to activate the RAAS. Low chloride concentrations in the renal distal tubules induce renin release in other species.12 Due to the fact that we found no differences in

PRA, it is also possible that direct adrenal activation by potassium at this stage of life may be important in foals.

In the study reported here, L- lactate concentrations were elevated and associated with cortisol, aldosterone, ANG-II and ACTH concentrations. L-lactate is a good indicator of global tissue perfusion and has been shown to increase in sepsis, trauma and systemic inflammatory response in foals, horses and people.12,42-46

From our understanding of RAAS physiology, we assume that multiple factors, including hypotension, ANG-II, ACTH, chloride, and potassium concentrations, contributed to

RAAS activation in hospitalized foals.

This study provides evidence that the RAAS and HPAA are activated in sick foals. Our data also support that RAI in critically ill foals is characterized by inappropriately low aldosterone and cortisol concentrations. Functional studies evaluating the adrenal gland response to various stimuli (ACTH, ANG-II, potassium) will enhance our understanding of endocrine response to sepsis in foals.

Table 2. 1 Blood hormone and electrolyte concentrations, and hormone ratios in neonatal foals (values expressed as median and range)

Variables Septic (n=74) SNS (n=59) Healthy (n=34)

PRA (ng/mL/h) 6.82 (1-20) 6.52 (1-15) 5.89 (3-16)

ANG-II (pg/mL) 31.99 (3-863)*§ 13.42 (2-291) 28.55 (10-83)

ALD (pg/mL) 368 (25-1515)* 254.67 (38-1343) 202.55 (55-678)

Cortisol (µg/dL) 13.68 (1-74)* 10.7 (1-48)* 4.7 (1-14)

ACTH (pg/mL) 176 (10-1250)*§ 37 (11-647) 21.4 (10-40)

BUN (mg/dL) 22 (9-98)** 19 (4-69) 18 (12-31)

Creatinine (mg/dL) 3.1 (1-21)**§ 2.3 (1-22) 1.85 (1-3)

Continued 22

Table 2.1 Continued Variables Septic (n=74) SNS (n=59) Healthy (n=34)

Na+ (mEq/L) 138 (128-155) 137 (107-147) 137.5 (132-143)

K+ (mEq/L) 4.1 (2-6) 3.9 (2-7) 4.1 (3-5)

Cl- (mEq/L) 96 (76-107)** 97.9 (81-105)* 99 (92-105)

Lactate mmol/L 5.7 (1.3-18.9)**§ 4.4 (1.3-17.9) 2.1 (0.8-4.8)

ACTH/cortisol 11 (1-208.5)*§ 5.94 (1-30.9) 5.08 (1.23-22.35)

ACTH/ALD 0.4 (0.02-3.7)* 0.12 (0.01-2.45) 0.09 (0.02-0.42)

ANG-II/ALD 0.1 (0.01-3.8) 0.07 (0.01-1.2) 0.13 (0.03-0.84)

PRA/ANG-II 0.17 (0.01-0.9) 0.09 (0.04-0.93) 0.4(0.11-1.17)

ALD/K+ 187.39 (29.34-326.57)** 161.64 (35.7-319) 60 (19.22-188)

ALD/Cl- 8.05 (0.8-17.97)** 6.2 (1.1-16.1) 2.27 (0.85-6.98)

ALD/Na+ 5.43 (0.6-11.56)* 4.34 (0.8-10.92) 1.63 (0.63-4.97)

ANG-II/K+ 10 (2.39-156.8)*§ 3.78 (2.15-67.18) 3 (2.7-12.55)

ANG-II/Cl- 0.40 (0.10-10.39)*§ 0.12 (0.79-2.63) 0.12 (0.11-0.47)

ANG-II/Na+ 0.28 (0.06-6.68)*§ 0.08 (0.05-1.86) 0.09 (0.08-0.34)

PRA/K+ 1.8 (0.79-7.37) 0.77 (0.27-2.8) 1.48 (1.09-3.51)

PRA/Cl- 0.09 (0.03-0.23) 0.03 (0.01-0.11) 0.05 (0.46-0.14)

PRA/Na+ 0.06 (0.02-0.14) 0.03 (0.01-0.08) 0.04(0.03-0.11)

PRA, plasma renin activity; ALD, aldosterone; ACTH, adrenocorticotropic hormone; ANG-II, angiotensin-II; SNS, sick non-septic ,* P<0.05 compared to healthy foals; **P<0.01 compared to healthy foals; § P<0.05 compared to SNS foals

Table 2.2 Blood hormone concentrations and hormone ratios in surviving and nonsurviving septic foals (values expressed as median and range).

Hormones Surviving (n=41) Non-surviving (n=33)

PRA (ng/mL/h) 10 (8.17-19.69) 7.2 (4.3-8.8)

ANG-II (pg/mL) 123.31 (18.15-862.55) 23.8 (9.81-44.06)

ALD (pg/mL) 338.3 (125-1515) 385(144.3-1401)

Cortisol (µg/dL) 21.7 (6.9-36.7) 5.02 (1.97-6.63)*

ACTH (pg/mL) 97.8 (17.5-718) 147 (26.4-267.4)

ACTH/ALD 0.26 (0.02-0.59) 0.34 (0.08-1.95)

ACTH/cortisol 3. 87 (2.23-19.52) 28.58 (3.98-53.18)*

ANG II/ALD 0.18 (0.04-0.58) 0.04 (0.03-0.05)

PRA/ALD 0.02 (0.01-0.03) 0.02 (0.01-0.03)

PRA/ ANG II 0.16 (0.01-0.56) 0.18 (0.16-0.9)

PRA, plasma renin activity; ALD, aldosterone; ACTH, adrenocorticotropic hormone;

ANG-II, angiotensin-II

* P<0.05;

Table 2.3 Hormone univariate analysis for survival among neonatal foals

95% Confidence Variable Range Crude Odds ratio for survival Interval

25-186.8 2.7* 1.01-7.42

ALD (pg/mL) 186.9-428.3 1.5 0.61-3.84

428.4-1515 referent

1-5.34 0.68 0.24-1.9

Cortisol (ng/dL) 5.35-14.03 0.21 0.21-1.63

14.03-74 referent

10-26.6 5.4* 1.7-16.8

ACTH (pg/mL) 26.7-152 2.7* 1.03-7.13

153-1250 referent

2.1-12.5 0.5 0.18-1.57 ANG-II 12.6-38.4 1.12 0.31-3.19 (pg/mL) 38.5-863 referent

0.1-4.0 0.7 0.1-3.5 PRA 4.1-8.0 1.4 0.2-10.0 (ng/mL/h) 8.1-20 referent

Continued

Table 2.3 Continued

Variable Range Crude Odds ratio for survival 95% Confidence Interval 0.2-4.14 6.7* 1.9-23.14 ACTH/cortisol 4.15-11.0 5.2* 1.64-16.57 11.1-112.8 referent 0.01-0.16 3.06* 1.08-8.68 ACTH/ALD 0.17-0.65 1.87 0.5-6.01 0.66-2.62 referent 5.34-60.09 2.5 0.4-16.5 ALD/PRA 60.1-114.27 1.4 0.26-8.01 114.28-337.76 referent 8.1-31.94 2.8* 1.05-7.5 ALD/K+ 31.95-84.81 1.02 0.45-2.33 84.82-351.86 referent 0.25-1.94 3.02* 1.09-8.33 ALD/Cl- 1.95-4.6 1.3 0.76-4.9 4.7-19.17 referent 0.17-1.39 3.2* 1.32-9.48 ALD/Na+ 1.4-3.16 1.55 0.61-3.9 3.17-11.56 referent

Continued

Table 2.3 Continued

Variable Range Crude Odds ratio for survival 95% Confidence Interval 0.67-3.1 0.45 0.14-1.04 ANG/K+ 3.2-10.38 0.69 0.24-2.27 10.39-191.82 referent 0.20-0.13 0.56 0.19-1.69 ANG/Cl- 0.14-0.42 0.32 0.32-3.33 0.43-10.4 referent 0.01-0.09 0.55 0.19-1.69 ANG/Na+ 0.1-0.32 1.20 0.36-4.04 0.33-6.68 referent 0.04-1.04 0.55 0.10-2.83 PRA/K+ 1.05-2.14 0.75 0.14-4.03 2.15-7.37 referent 0.0-0.04 0.75 0.4-4.03 PRA/Cl- 0.05-0.09 0.65 0.13-3.75 0.1-0.24 referent 0.0-0.03 0.75 0.14-4.03 PRA/Na+ 0.04-0.06 0.69 0.13-3.75 0.07-0.14 referent

Continued

Table 2.3 Continued

Variable Range Crude Odds ratio for survival 95% Confidence Interval 107-135 0.68 0.28-1.66 Na + (mEq/L) 136-139 0.95 0.37-2.44 139-155 referent 76-95 0.9 0.4-2.23 Cl - (mEq/L) 96-99 1.01 0.4-2.49 100-107 referent 2.1-3.6 8.8* 2.5-30.9 K + (mEq/L) 3.6-4.5 2.4* 1.01-6.27 4.6-7 referent 0.8-2.8 42.22** 5.24-339.7

2.9-4.9 4.76* 1.68-13.477 Lactate mmol/L 5.0-18.9 referent 1-1.9 3.16* 1.27-7.87 Creatinine 2-3.3 2.15 0.89-5.1 (mg/dL) 3.4-22 referent 0-6 81* 9.8-679.0 Sepsis score 7-14 5.4* 2.2-13.3 15-21 referent

PRA, plasma renin activity; ALD, aldosterone; ACTH, adrenocorticotropic hormone; ANG-II, angiotensin II; BUN, blood urea nitrogen, * P<0.05

Table 2.4 Correlations (ρ) between hormones, serum electrolytes, IgG, creatinine and BUN concentrations, and sepsis score in septic foals.

ANG-II ALD Cortisol ACTH Na+ K+ Cl- L-lactate IgG Sepsis score Creatinine BUN Variable

-0.09 0.19 0.29 -0.01 0.17 -0.16 -0.10 -0.29 -0.28 -0.27 0.08 -0.19 PRA 0.35* 0.16 0.28 -0.28 -0.13 -0.15 0.22 -0.43* 0.10 0.35* 0.09 ANG-II 0.43** 0.32* 0.07 0.41** -0.10 -0.33* -0.41* 0.15 0.33* 0.30* ALD 0.28* 0.49** -0.09 0.27 -0.17 -0.03 -0.08 0.01 0.15 Cortisol

0.29* 0.27 0.26 0.26* -0.39* 0.20 0.12 0.11 ACTH

PRA, plasma renin activity; ALD, aldosterone; ACTH, adrenocorticotropic hormone; ANG-II, angiotensin-II; BUN, blood urea

nitrogen *P<0.05; **P<0.01

Table 2.5 Correlations (ρ) between hormones, serum electrolytes, IgG, creatinine and BUN concentrations, and sepsis score in

SNS foals.

ANG- L- Sepsis Variable ALD Cortisol ACTH Na+ K+ Cl- IgG Creatinine BUN II lactate score

PRA 0.14 0.30 0.38 0.26 0.12 -0.05 -0.30 -0.18 -0.23 -0.11 0.39 0.38

ANG-II 0.40* -0.01 -0.13 -0.23 -0.15 -0.28 0.44* -0.43* 0.04 0.29 0.37*

ALD 0.46** 0.01 -0.23* 0.41** -0.36** 0.06 -0.29* 0.30* 0.27 0.55**

Cortisol 0.67** 0.43** -0.04 0.19 0.54* -0.03 -0.05 0.43* 0.20

ACTH 0.30* 0.20 0.33** 0.26 -0.46** 0.29* 0.51** 0.16

PRA, plasma renin activity; ALD, aldosterone; ACTH, adrenocorticotropic hormone; ANG-II, angiotensin-II; SNS, sick non-

septic; BUN, blood urea nitrogen* P<0.05; ** P<0.01

Table 2.6 Correlations (ρ) between hormones, serum electrolytes, IgG, creatinine and BUN concentrations, and sepsis score in hospitalized foals

Variable ANG-II ALD Cortisol ACTH Na+ K+ Cl- L-lactate IgG Sepsis score Creatinine BUN

PRA 0.42 0.25 0.32 0.06 0.25 -0.05 -0.08 -0.02 -0.27 -0.05 0.20 0.07

ANG-II 0.41** 0.14 0.26 -0.15 0.02 -0.17 0.05 -0.44** 0.04 0.34** 0.28**

ALD 0.43** 0.21* -0.10 0.44** -0.25* 0.20 -0.43** 0.16 0.34** 0.44**

Cortisol 0.39** 0.17 0.18 0.19 0.13 -0.15 -0.07 0.15 0.18

ACTH 0.24* 0.12 0.15 0.22 -0.43** 0.39** 0.30** 0.19

1 PRA, plasma renin activity; ALD, aldosterone; ACTH, adrenocorticotropic hormone; ANG-II, angiotensin-II; BUN, blood urea

nitrogen

* P<0.05; ** P<0.0

Chapter 3. Hypothalamic-Pituitary-Adrenal Axis in Hospitalized Foals

3.1 Materials and Methods

Animals

Foals ≤7 days old of any breed or sex admitted to The Ohio State University Galbreath

Equine Center, Hagyard Equine Medicine Institute and Rood & Riddle Equine Hospital from one foaling season were included. Hospitalized foals were classified into 2 groups: septic and sick non-septic (SNS) foals. Foals in the septic group had a sepsis score of

≥12, a positive blood culture, or both.14,48 Foals in the SNS group were presented for illnesses other than sepsis (e.g. hypoxic ischemic encephalopathy, meconium impaction, failure of transfer of passive immunity or orthopedic conditions) requiring hospitalization. These foals had negative blood cultures and a sepsis score of ≤11. The control group consisted of 24-72-hour-old foals, classified as healthy based on physical examination, a normal CBC, serum biochemistry, serum immunoglobulin G (IgG) concentrations (>800 mg/dL) and a sepsis score of (0-4). Foals with a history of receiving corticosteroids, intravenous crystalloid fluids, or plasma before admission were excluded from the study. Survival was defined as discharged alive from the hospital. Foals that died or were euthanized due to a grave medical prognosis were defined as non-survivors.

Foals euthanized for other reasons such as financial constraints were excluded from the study.

This study was approved by the OSU Veterinary Clinical Trials Office, the Institutional

Animal Care and Use Committee, and adhered to the principles of humane treatment of animals in veterinary clinical investigations, as stated by the American College of

Veterinary Internal Medicine and National Institute of Health guidelines.

Data Collection

(sex, age, breed), physical examination findings, CBC, biochemistry profile, fibrinogen concentrations, IgG concentrations, and blood culture results. Endocrine measurements included corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), adrenocorticotropin (ACTH), and cortisol concentrations. For consistency, the sepsis score was calculated by the graduate student for each foal, based on history, physical examination, and laboratory findings.

Sampling

Blood samples for hormone measurements from hospitalized foals were obtained on admission (within 1 hour of hospitalization) by sterile jugular venous catheterization.

Samples from healthy foals were obtained during routine examination of newborn foals via jugular venipuncture. Blood was collected in serum clot and aprotinin-EDTA tubes and was kept refrigerated on ice. Aprotinin was added to inhibit protease-mediated degradation of hormones (500 kU/mL of blood). Samples were centrifuged at 2,000 × g for 10 minutes at 4°C. Serum and plasma were aliquoted and stored at -80°C until analyzed. Blood samples for CBC, serum biochemistry, fibrinogen, and IgG concentrations were processed immediately.

Hormone Concentrations

Plasma CRH and serum cortisol concentrations were determined using radioimmunoassays,i,ii validated for horses.5,21,34,35,51 Plasma ACTH concentrations were determined with an immunochemiluminometric assayiii validated for horses.82 Plasma

AVP concentrations were measured with an AVP radioimmunoassayiv (validated for horses5), after peptide extraction using phenylsilylsilica solid-phase columns.v

Statistics

Data set was tested for normality by the Shapiro-Wilk statistic. ACTH and cortisol concentrations in healthy foals were normally distributed. The remainder of the data was not normally distributed. Medians and ranges were calculated for continuous variables.

Comparisons between groups of foals were carried out with the Kruskal-Wallis statistic and the Dunn’s post-test was used to compare each group individually. The Mann-

Whitney-U test was used to compare survivors and non-survivors within each group.

Significance was set at P < 0.05. Correlations between continuous variables were determined with the Spearman rank order (ρ). Continuous variables were categorized by cutoff values based on distribution within a group, and analyzed using logistic regression for binomial distribution. Clinical data analyzed included total protein, glucose, IgG and hormone concentrations. Crude odds ratios and 95% confidence intervals were calculated and based on categories. The dependent variables were survival and non-survival. Data was analyzed with statistical software.vi, vii

3.2. Results

Study Population

A total of 182 neonatal foals (164 hospitalized; 18 healthy foals) of ≤ 7 days of age were included. The median age of all hospitalized foals at admission was 14 hours. For healthy controls the median age was 24 hours.

Thirty seven percent of hospitalized foals (61/164) were classified as septic and 63%

(103/164) as SNS. The survival rate in septic foals was 60% (36/61) and in SNS was 85%

(88/103). Thirty eight percent (23/61) of septic foals had positive blood cultures. The median sepsis score for all hospitalized foals was 9.

Breeds representing hospitalized foals included Thoroughbred (n=101), Quarter Horse

(15), Standardbred (12), Appaloosa (6), Saddlebred (4), Warmblood (7), Morgans (5),

American Paint Horse (5), Arabian (4), Belgian (3) and Percheron (2). All healthy foals were Thoroughbreds (18).

Of the hospitalized foals, 48% (78/164) were colts and 52% (86/164) fillies.

Hormone concentrations

Results of CRH, AVP, ACTH and cortisol concentrations for all foals are presented in

Table 1. Septic foals had significantly lower CRH concentrations compared to healthy and SNS foals (P<0.01). ACTH and AVP concentrations were higher in septic than healthy and SNS foals (P<0.01). SNS foals had higher ACTH concentrations than healthy foals (P<0.01). Cortisol concentrations were higher in septic and SNS than healthy foals.

CRH and AVP concentrations were not different between SNS and healthy foals. CRH,

ACTH, cortisol and AVP concentrations were not different between surviving and nonsurviving septic foals (Table 2).

Serum glucose and IgG concentrations were lower in septic than in SNS and healthy foals

(Table 1). Plasma total protein was lower in septic than healthy foals (Table 1).

Septic foals that died had lower IgG concentrations than surviving foals (Table 2).

In the hospitalized population, foals with AVP concentrations in the range of 0.416-1.58 pg/mL were more likely to survive (OR=14.3) than foals with AVP concentrations >3.35 pg/mL (Table 3). Foals with ACTH concentrations in the range of 10-24 pg/mL were 4.5 times more likely to survive than those with ACTH concentrations >171 pg/mL (Table

3). In addition, foals with cortisol concentrations in the range of 1.2-3.27 µg/dL were more likely to survive (OR=3.17) than foals with cortisol concentrations >8.06 µg/dL

(Table 3).

Hormone ratios and correlations

For each foal group the hormone ratios were determined (Tables 1–2). The CRH/ACTH and CRH/cortisol ratios were significantly lower in septic foals than SNS and healthy foals. The CRH/ACTH ratio was also lower in SNS than healthy foals. Further, septic foals had significantly higher ACTH/cortisol ratios than healthy foals (P<0.05). The

AVP/cortisol ratios were higher in septic than SNS and healthy foals (P<0.05). Septic foals that died had higher AVP/ACTH ratios than surviving foals (Table 2).

In septic foals, ACTH was positively correlated with AVP and inversely correlated with

IgG (Table 4).

In SNS foals, CRH concentrations were negatively correlated with cortisol and the sepsis score. ACTH concentrations were positively correlated with cortisol and AVP in SNS and hospitalized foals (Table 5 and 6).

In SNS and hospitalized foals, cortisol concentrations were positively correlated with

AVP and sepsis score (Table 5 and 6). Sepsis score was positively correlated with ACTH, cortisol and AVP concentrations in hospitalized foals (Table 6).

In hospitalized foals, CRH concentrations were inversely correlated with ACTH, cortisol and sepsis score (Table 6).

3.3 Discussion

In the current study we documented that HPAA activation during sepsis is characterized by decreased CRH and increased AVP, ACTH, and cortisol concentrations. Hospitalized

foals with lower concentrations of ACTH, cortisol and AVP were more likely to survive.

We also demonstrated primary and relative adrenal insufficiency in critically ill foals.

CRH is the major stress hormone synthesized by parvocellular neurons of the hypothalamic paraventricular nucleus. AVP is a 9-amino acid peptide produced by the magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus.

HPAA activation in response to sepsis associated stress is characterized by release of

CRH, ACTH, AVP and cortisol in neonatal foals and humans.21,22,24,54,61-64,67,76 In septic foals of our study, ACTH, cortisol and AVP concentrations were increased. An unexpected finding was that CRH concentrations were low in the same foals. Elevation in

AVP concentrations in septic foals are likely to be the result of sepsis associated hypovolemia, hypotension and hypothalamic stimulation by proinflammatory mediators and endotoxins.5,26 In addition, AVP is the main pituitary ACTH secretagogue in horses.26

We do not have a precise pathological explanation for decreased CRH concentrations in the septic foals of our study; however, we propose that in critically ill foals AVP is more important in ACTH release than CRH. These results differ from some results of HPAA function studies in other species, where CRH, ACTH and cortisol were proportionally increased in response to stress.83-85

Activation of the HPAA by endotoxin (LPS) has been studied in septic humans and animals models.84,86-88 Endotoxin increases the expression and release of cytokines such as IL-1, IL-6, TNF-α from macrophages and monocytes. These cytokines work together

to stimulate the HPAA. LPS significantly increases the concentrations of CRH, ACTH and cortisol and its effect has been demonstrated to be dose and time-depended in septic humans and other species.61,84,86 Endotoxins also down-regulate the mRNA coding for

AVP and CRH in anterior pituitary gland in rodents.86 Further, it is suggested that CRH plays a more important role in the early phase of the ACTH response whereas the non-

CRH-dependent mechanism such as AVP become more dominant at a later stage. In a study on repeated LPS injection in rats, the main findings included: a decrease in basal

CRH; an increase in AVP and pituitary POMC. Those results suggest a central adaptation of the HPAA during chronic exposure to LPS.86,89

In addition to cytokines, other hormones and neurotransmitters such as histamine, serotonine, catecholamines, bradykinin, endothelin-1 and prostaglandins have been suggested to contribute to HPAA activation during sepsis. Recent studies in rodents have shown that cyclo-oxygenase, nitric oxide and leptin can modulate the response of HPAA to inflammatory stimuli.89 Similar studies are lacking in critically ill equine neonates.

Critical illness-related corticosteroid insufficiency (CIRCI) characterized by an inadequate production of cortisol in relation to increased demand during sepsis is common in critically ill human and other species neonates.5,21,56,64,90 Severe illness may result in decreased CRH, ACTH and cortisol synthesis through damage to the hypothalamus, pituitary gland or adrenal gland. A variety of tests have been advocated, including basal cortisol concentration, ACTH-stimulation test, the cortisol- to- endogenous ACTH ratio, CRH stimulation test, the metyrapone (cortisol synthesis inhibitor) test and insulin tolerance test.21,75,90,91 Effects of CRH administration on

cortisol concentrations have been studied in healthy adult horses,92 dogs with hyperadrenocorticism and CIRCI,90,93,94 and in septic human adults and neonates,56,83,95-97 however, limited information exists on central (secondary and tertiary) adrenal insufficiency in critically ill foals. In the current study, we have demonstrated the adrenal insufficiency at the level of adrenal gland, which is characterized with increased

ACTH/cortisol and AVP/cortisol rations in septic foals. Interestingly, the AVP/ACTH ratio was not different among foals groups, which suggest an appropriate response of the pituitary gland to stress in septic foals.

The effect of hypoglycemia on the function of the HPAA has been studied in horses and other species.34,84,98,99 Alexander et al reported that AVP is the primary signal for ACTH releases in horses, however the magnitude of ACTH release induced by hypoglycemia is determinate by CRH release.98 Likewise, our findings demonstrate changes in CRH and

AVP concentrations associated with hypoglycemia in some septic foals.

In this study, we demonstrated that relative adrenal insufficiency is attributed to adrenal gland exhaustion (primary insufficiency) as most critically ill foals had an adequate pituitary response. Dynamic studies are needed to determine the HPAA activation in particularly CRH response to stress in hospitalized foals.

Table 3. 1Blood hormones concentrations and hormone ratios in neonatal foals (values expressed as median and range)

Foal classification Septic (n=61) SNS (n=103) Healthy (n=18) Variables CRH (pg/mL) 35.5 (7.5-80.69)**§ 90.63 (36.8-231.65) 96.99 (74.55- 196.1) ACTH (pg/mL) 295.5(39.3-712.0)**§ 29.5 (13.9-647.2)** 26.5 (15-29.1) Cortisol (µg/dL) 11.68 (3.05-40.10)** 3.77 (1.21-20.9)** 1.68 (1.25-2.71) AVP (pg/ml) 4.86 (0.91-73.67)**§ 1.91 (0.88-23.37) 1.34 (0.416-1.87) CRH/ACTH 35.5 (7.56-80.69)**§ 90.63 (36.8- 98.9 (74.55-196.1) 231.65)* ACTH/cortisol 23.59 (0.98-208.55)* 10.7 (3.84-30.92) 13.2 (10.7-22.35) AVP/ACTH 0.062 (0.005-0.16) 0.048 (0.032-0.15) 0.054 (0.027- 0.064) AVP/CRH 0.3 (0.012-6.91)**§ 0.017 (0.011-2.04) 0.019 (0.011-0.2) CRH/AVP 3.01 (0.012- 58.5 (0.49-178.92) 84.9 (40.92- 79.44)**§ 268.85) CRH/cortisol 5.55 (0.48-44.6)**§ 23.9 (2.7-219.8) 59.09 (23.1-85.5) AVP/cortisol 1.27 (0.06-17.52)*§ 0.64 (0.09-2.73) 0.66 (0.14-1.42) Glucose (mg/dL) 108 (16-251)*§ 133 (25-261) 146 (101-168) IgG (mg/dL) 460 (0-1523)**§ 767 (0-2423) 1200 (493-1200) Total protein 4.5 (2.8-6.6)* 5 (3.3-6.7) 5.3 (4.2-6.1) (g/dL) ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; AVP, arginine vasopressin, * P<0.05 compared to healthy foals, **P<0.01 compared to healthy foals, § P<0.05 compared to SNS foals

Table 3.2 Blood hormone concentrations and hormone ratios in surviving and nonsurviving septic foals (values expressed as median and range)

Hormones Surviving (n=36) Non-surviving (n=25)

CRH (pg/mL) 24.21 (7.5-40.8) 30.1 (19.2-52.15)

ACTH (pg/mL) 334 (152-516.0) 178 (39.3-543.0)

Cortisol (µg/dL) 8.3 (3.05-13.67) 9.7 (4.08-40.1)

AVP (pg/mL) 27 (0.91-53.55) 2.97 (1.59-73.67)

CRH/ACTH 0.064 (0.049-0.079) 0.169 (0.045-1.08)

CRH/cortisol 6.9 (0.55-13.3) 3.26 (0.48-12.77)

ACTH/cortisol 89.9 (11.11-168.8) 18.3 (0.98-112.8)

AVP/ACTH 0.054 (0.005-0.103)* 0.04 (0.006-0.162)

Glucose (mg/dL) 153 (146-160) 95 (18-181)

IgG (g/dL) 650 (100-1200)* 335 (117.5-782)

Total Protein (g/dL) 4.4 (3.8-5.0) 4.4 (3.8-6.0)

ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; AVP, arginine vasopressin; * P<0.05

Table 3.3 Hormone univariate analysis for survival among neonatal foals

Crude Odds ratio for 95% Confidence Variable Range survival Interval 7.56-53.11 0.065 0.013-0.319 CRH (pg/mL) 53.12-93.43 0.48 0.082-2.849 93.44-492.92 referent 1.2-3.27 3.17* 1.035-9.75 Cortisol (ng/dL) 3.28-8.05 1.08 0.43-2.69 8.06-46.99 referent 10-24 4.5* 1.26-16.6 ACTH (pg/mL) 25-170 2.7 0.845-8.66 171-1250 referent 0.416-1.58 14.3* 1.64-128.44 AVP (pg/mL) 1.6-3.34 2.35 (0.635-8.72) 3.35-172.36 referent 0.67-10.72 2.2 0.54-7.48 ACTH/cortisol 10.73-22.35 3.5 0.804-15.71 22.36-284.53 Referent 0.005-0.047 2.6 0.57-11.9 AVP/ACTH 0.048-0.11 13.12* 1.36-126.3 0.11-6.07 referent 0.48-8.95 0.087 0.01-0.76 CRH/cortisol 8.96-32.58 0.174 0.019-1.616 32.59-219.83 referent 0-475 0.106 0.022-0.496 IgG (mg/dL) 476-1049 0.109 0.023-0.510 1050-2423 referent 16-107 0.448 0.17-1.13 Glucose (mg/dL) 108-146 0.946 0.34-2.57 147-292 referent 2.8-4.5 0.67 0.28-1.59 Total protein (g/dL) 4.6-5.3 0.82 0.32-2.11 5.4-6.7 referent

ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; AVP, arginine vasopressin; * P<0.05

Table 3.4 Correlations (ρ) between hormones, IgG, sepsis score, total protein, and glucose concentrations in septic foals.

Sepsis Total glucose Variable ACTH Cortisol AVP IgG score Protein CRH -0.19 -0.17 -0.15 0.1 -0.21 -0.05 0.18 ACTH -0.19 0.54* -0.38* 0.18 -0.24 -0.29

Cortisol -0.12 -0.12 0.03 0.14 0.06

AVP -0.19 0.14 -0.33 -0.15

ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; AVP,

arginine vasopressin, *P<0.05; **P<0.01

Table 3.5 Correlations (ρ) between hormones, IgG, sepsis score, total protein, and

glucose concentrations in SNS foals.

Sepsis Total Glucose Variable ACTH Cortisol AVP IgG score Protein CRH 0.11 -0.4* 0.03 -0.21 -0.47** 0.15 0.11 -0.08 0.12 ACTH 0.64** 0.65** 0.07 0.07 Cortisol 0.54** 0.16 0.42** -0.06 -0.06

AVP 0.04 0.19 -0.33 -0.13

ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; AVP,

arginine vasopressin, *P<0.05; **P<0.01

Table 3.6 Correlations (ρ) between hormones, IgG, sepsis score, total protein, and glucose concentrations in septic foals.

Variable ACTH Cortisol AVP IgG Sepsis Total Glucose score Protein CRH -0.23* -0.37** -0.09 0.11 -0.68** 0.17 0.24 -0.22 -0.22 ACTH 0.43** 0.72** -0.2 0.41** Cortisol 0.32* -0.03 0.3* -0.03 -0.02

AVP -0.14 0.29* -0.41** -0.23

ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; AVP, arginine vasopressin, *P<0.05; **P<0.01

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iCoat-A-Count Cortisol Radioimmunoassay, Siemens Healthcare Diagnostics, Los Angeles, CA ii CRH Radioimmunoassay, Phoenix Pharmaceuticals, CA iiiImmulite ACTH assay, Siemens Healthcare ivVasopressin Radioimmunoassay, ALPCO Diagnostics, Salem, NH vPhenylsilylsilica Extraction Columns, ALPCO Diagnostics, Salem, NH viPrism, version 4.0a, GraphPad Software Inc, San Diego, CA viiSAS version 9.1, SAS Institute Inc, Cary, NC