Study designs

3.2.1 Study I

One of the previous difficulties in trying to diagnose RAI in horses was that the ideal dose of ACTH required for ACTH stimulation testing was unknown. Therefore, we performed a study to determine the lowest dosage of ACTH

Figure 3a. Figure 3b.

Figure 3a. University owned brood mare and foal used in project III and later adopted as a

potential sport horse.

Figure 3b. Client owned horse in the equine intensive care unit receiving intravenous fluids after having 2 meters of small intestine removed surgically the previous evening.

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required to produce a significant increase in cortisol concentrations in normal adult horses (Stewart et al., 2011). In a randomized crossover design, 5 ACTH stimulation tests were performed on each of 8 horses (4 mares and 4 geldings), with a 3-day wash-out period between tests. Four dosages (0.02, 0.1, 0.25 and 0.5µg/kg) of cosyntropin and saline as a negative control were administered IV into a jugular vein. Blood samples were collected before (0 minutes [baseline]) and 30, 60, 90, 120, 180, and 240 minutes after injection of cosyntropin or saline solution for measurement of serum cortisol at each time point and plasma ACTH at baseline. Complete blood counts were performed with an automated analyser (Advia 120 Haematology System, Siemens, Tarrytown, NY.) and differential cell counts were performed by hand at 0, 60, 120, and 240 minutes after injection of cosyntropin or saline solution for routine CBCs.

3.2.2 Study II

Eleven apparently healthy foals (6 colts and 5 fillies) were studied. All foals were full term (> 330 days gestation), born without assistance and had an immunoglobulin concentration (> 800 mg/dL or < 8 g/L) by 24 hours of age. Foals entered the study at 2 days of age and were 12 days of age by study completion. No foal had received any medication prior to the study except diazepam and butorphanol for placement of a central venous catheter the day prior to study commencement (Figure 3c). The foals weighed from 32 to 65 kg; 52 ± 11 kg at the commencement of the study and were weighed daily during the study. Foals gained 4.5 to 29 kg13 ± 7 kg by the end of the study. In a randomized crossover design, 5 ACTH stimulation tests were performed on each foal, with a 2-day washout period between tests. For each test, saline (0.9% NaCl) solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 µg/kg cosyntropinwas administered IV into the contralateral jugular vein via a transiently placed catheter. Blood samples were collected from the central venous jugular catheter immediately before (0 minutes [baseline]) and 30, 60, 90, 120, 180, and 240 minutes after injection of cosyntropin or saline solution for measurement of cortisol concentration each time point and plasma ACTH at baseline. Samples were also collected at 10 and 20 minutes in 6 of 11 foals for cortisol measurement. Blood was also collected before and 60, 120, and 240 minutes after injection of cosyntropin or saline solution for performance of a routine CBC.

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3.2.3 Study III

To investigate insulin dysregulation in horses with SIRS a subset of horses used in study IV that presented to the emergency service of the Auburn University J.T. Vaughan Large Animal Teaching Hospital that fit additional criteria of being > 5 years of age and diagnosed with SIRS were included. Serum insulin and glucose concentrations were measured at admission from archived frozen serum samples. Insulin concentrations were measured in duplicate using an radioimmunoassay and blood glucose was measured using a glucohexokinase colorimetric assay as previously described (Reimers et al., 1982),_{(de Laat et al.,} 2016). Horses were grouped by outcome (survival, hyperinsulinemia and hyperglycaemia).

3.2.4 Study IV

To determine the cortisol and ACTH concentrations and ACTH/cortisol ratios in sick horses throughout hospitalisation and any associations with survival, an ischemic gastrointestinal lesion and SIRS. One hundred and fifty-one horses (> 1 year of age) presenting to the emergency service of two university teaching hospitals (Auburn University and Colorado State University) were recruited over two summers (mid-May until mid-August) and serum cortisol and plasma ACTH concentrations measured at admission and on morning of days 2, 4 and 6 if the patient was still hospitalized. Data recorded included signalment, physical examination findings and routine bloodwork at admission (haematological and biochemical data); diagnosis, outcome, duration of hospitalisation, diagnosis of SIRS, SIRS score (see below) and presence of an ischemic gastrointestinal lesion

Figure 3c. Placing a central venous catheter in a heavily sedated 1 day old foal for later

collection of blood samples Photo: A.J. Stewart.

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for horses that underwent surgery or necropsy. For statistical analysis horses were grouped by outcome (survival, SIRS score, presence of SIRS, SIRS score and an ischemic lesion).

3.2.5 Study V

To identify adrenal dysfunction in critically ill adult horses, a subset of 58 patients enrolled for project IV were requested to be involved in ACTH testing. Within the first 2 hours of admission, blood was collected for baseline endogenous serum cortisol and plasma ACTH concentrations. A low dose ACTH stimulation test was then performed with administration of 0.1 µg/kg IV of previously diluted cosyntropin with blood was collected at 30 minutes (T = 30) for post ACTH cortisol measurement, as previously described (Stewart et al., 2011). This sampling occurred at admission and on the mornings of days 2, 4 and 6 if the patient was still hospitalized, having not been discharged or euthanized. Data recorded included signalment, physical examination findings and routine bloodwork at admission (haematological and biochemical data); diagnosis, outcome, duration of hospitalisation, diagnosis of SIRS, SIRS score and presence of an ischemic gastrointestinal lesion for horses that underwent surgery or necropsy. Criteria adapted from definitions utilized in human intensive care and in a study involving sick neonatal foals was used to define an inadequate adrenal response to the degree of illness (Annane et al., 2018; Hart et al., 2009b; Annane et al., 2006). An inappropriately low basal cortisol concentration was defined by 2 methods: 1) a physiologic stressed cortisol concentration calculated as lowest cortisol concentration (mean - 1SD) obtained after administration of a physiologic dose (0.1 µg/kg) of cosyntropin to healthy adult horses which calculates as an endogenous cortisol <9.7 µg/dL and 2) a more conservative value of less than the lowest basal cortisol concentration (mean - 1SD) obtained from healthy horses, which calculates as < 4.51µg/dL (Hart et al., 2011; Stewart et al., 2011). An inappropriately low delta-cortisol 30 minutes after the administration of 0.1 µg/kg of cortrosyn was defined by two methods: 1) a value less than the mean delta-cortisol obtained from healthy horses which was < 5.65 µg/dL (definition previously utilized in a study of neonatal foals) and a more conservative value less than the mean delta-cortisol- 1SD obtained from healthy horses which was < 4.15 µg/dL (Hart et al., 2011; Stewart et al., 2011). A horse was defined as having RAI/CIRCI if it had an inappropriately low basal or delta-cortisol and it was considered to be critically ill based on clinical parameters. Horses were grouped by outcome (survival, SIRS score, presence of SIRS and an ischemic lesion).