Perinatal asphyxia is a significant contributor to neonatal brain injury. In the clinical environment there is variability in the severity of neural injury in neonates with similar clinical histories. Whilst the duration and intensity of hypoxia is well known to influence the severity of neurological outcome, the global physiological responses to hypoxia may also affect the subsequent variability in neurological outcome. The first step in this project was to identify which physiological response/s during a constant global hypoxic-ischemic insult influence the severity of neurological outcome in the newborn piglet and to assess the relative importance of these responses. Hypoxia/hypercapnia was induced in anaesthetized piglets by reducing the fraction of inspired oxygen to 0.1 (10%) and the ventilation rate from 30-10 breaths per minute for 45 minutes. Neurological outcome was determined by using functional markers including aEEG amplitude and cerebral impedance, and the structural marker microtubule associated protein-2 immunohistochemistry at 6 hours post hypoxia. The results from the initial study indicated that there was significant variability in neurological outcome following a constant hypoxia/hypercapnia insult. Serum cortisol concentrations were highly variable at the end of hypoxia (mean ± SD; 240.7 ± 90.9 nmol/L) and associated with cardiovascular function (time heart rate below baseline; r = 0.69) and neurological outcome (r = 0.70). Cardiovascular function (time heart rate was below baseline) and neurological outcome were strongly associated (r = 0.77). In this study we observed an oscillating pattern in cardiovascular function during hypoxia in some animals. In the regression analysis variability in cortisol concentrations and cardiovascular function explained 68% of the variability in the severity of neurological outcome. Additional physiological factors did not improve the strength of the association with neurological outcome. The variability in the physiological responses, particularly cardiovascular and endocrine responses to hypoxia may be more important determinants of neurological outcome then previously recognised. Results from the initial study opened up several questions about the relationship between cortisol and cardiovascular function during hypoxia and the relationship to the subsequent neurological outcome. Since variability in cortisol concentrations was associated with both cardiovascular function and neurological outcome the second step of this thesis was to investigate what factors contributed to the variability in serum cortisol concentrations during hypoxia. It is important to understand why some individuals produce more cortisol than others, and as a result are protected against brain injury. The aim was to determine if the variability in serum cortisol concentrations was the result of variability in ACTH concentrations during acute global hypoxia. The results from this study showed that early changes in serum cortisol concentration (15 minutes) were not correlated with ACTH (R2 = 0.26, P = 0.1), however, later changes (30 – 45 minutes) were (R2 0.45 - 0.68). This suggests that the primary factor controlling serum cortisol concentrations before hypoxia and during later hypoxia is ACTH concentrations. These data suggest that other factors may control cortisol secretion during early hypoxia; a key mechanism responsible for these changes may be the activity of the sympathetic nervous system and the maturity of the adrenal medullae. Since, higher cortisol concentrations were associated with better cardiovascular function and neurological outcome. As a result of this observation the aim of this study was to determine if cortisol concentrations during hypoxia are the causative factor responsible for improved cardiovascular function and better neurological outcome. The results from this study showed that manipulating serum cortisol concentrations into high (<500nmol/l) and low (>50nmol/l) levels during hypoxia did not affect cardiovascular function (P = 0.68) or neurological outcome (P = 46). Within each group (low, high and control hypoxia group) there was significant variability in cardiovascular function during hypoxia, which was associated with neurological outcome. (r = -0.63, p = 0.01). This study showed that serum cortisol concentrations during hypoxia are not the causative factor impacting on improved cardiovascular function and neurological outcome. It is possible that factors affecting both cardiovascular function and cortisol production such as activity of the sympathetic nervous system, may be a possible mechanism behind the variability in neurological outcome and cardiovascular function. Additionally, this study showed that cortisol concentrations at 3 hours post hypoxia were associated with neurological outcome (r = -0.67, p = 0.01). The animals with poorer outcome may also be those with greater multi-organ damage and thus reduced ability to clear cortisol from the systemic circulation. In light of this finding it may be interesting to assess cortisol concentration in the human neonate at 3 hours post hypoxia and determine the relationship to neurological outcome. In the final study of this thesis the function of the cardiovascular system during hypoxia was investigated in more detail because of its strong association with neurological outcome (results observed in Chapter 2 and 4. Few researchers have reported on oscillations in cardiovascular function, particularly type-3 waves, during hypoxia in the neonate. The aim of this study was to determine the characteristics and occurrence of type-3 waves in the neonatal piglet and their relationship to neurological outcome following acute global hypoxia. The result showed that the development of type-3 waves in cardiovascular function occurred in 56% of animals. An oscillating pattern was significantly associated with better neurological outcome (p = 0.01) and a lower duration of hypotension during hypoxia (p = 0.02), and occurred more frequently in females (p = 0.024). The development of type-3 waves during acute global hypoxia is a key mechanism in promoting natural tolerance; and may be the result of greater activity, maturity or sensitivity of the sympathetic nervous system in females compared to males. This may explain the improved neurological outcome following hypoxia in the female neonate seen in the clinical setting.

Perinatal asphyxia is a significant contributor to neonatal brain injury. In the clinical environment there is variability in the severity of neural injury in neonates with similar clinical histories. Whilst the duration and intensity of hypoxia is well known to influence the severity of neurological outcome, the global physiological responses to hypoxia may also affect the subsequent variability in neurological outcome. The first step in this project was to identify which physiological response/s during a constant global hypoxic-ischemic insult influence the severity of neurological outcome in the newborn piglet and to assess the relative importance of these responses. Hypoxia/hypercapnia was induced in anaesthetized piglets by reducing the fraction of inspired oxygen to 0.1 (10%) and the ventilation rate from 30-10 breaths per minute for 45 minutes. Neurological outcome was determined by using functional markers including aEEG amplitude and cerebral impedance, and the structural marker microtubule associated protein-2 immunohistochemistry at 6 hours post hypoxia. The results from the initial study indicated that there was significant variability in neurological outcome following a constant hypoxia/hypercapnia insult. Serum cortisol concentrations were highly variable at the end of hypoxia (mean ± SD; 240.7 ± 90.9 nmol/L) and associated with cardiovascular function (time heart rate below baseline; r = 0.69) and neurological outcome (r = 0.70). Cardiovascular function (time heart rate was below baseline) and neurological outcome were strongly associated (r = 0.77). In this study we observed an oscillating pattern in cardiovascular function during hypoxia in some animals. In the regression analysis variability in cortisol concentrations and cardiovascular function explained 68% of the variability in the severity of neurological outcome. Additional physiological factors did not improve the strength of the association with neurological outcome. The variability in the physiological responses, particularly cardiovascular and endocrine responses to hypoxia may be more important determinants of neurological outcome then previously recognised. Results from the initial study opened up several questions about the relationship between cortisol and cardiovascular function during hypoxia and the relationship to the subsequent neurological outcome. Since variability in cortisol concentrations was associated with both cardiovascular function and neurological outcome the second step of this thesis was to investigate what factors contributed to the variability in serum cortisol concentrations during hypoxia. It is important to understand why some individuals produce more cortisol than others, and as a result are protected against brain injury. The aim was to determine if the variability in serum cortisol concentrations was the result of variability in ACTH concentrations during acute global hypoxia. The results from this study showed that early changes in serum cortisol concentration (15 minutes) were not correlated with ACTH (R2 = 0.26, P = 0.1), however, later changes (30 – 45 minutes) were (R2 0.45 - 0.68). This suggests that the primary factor controlling serum cortisol concentrations before hypoxia and during later hypoxia is ACTH concentrations. These data suggest that other factors may control cortisol secretion during early hypoxia; a key mechanism responsible for these changes may be the activity of the sympathetic nervous system and the maturity of the adrenal medullae. Since, higher cortisol concentrations were associated with better cardiovascular function and neurological outcome. As a result of this observation the aim of this study was to determine if cortisol concentrations during hypoxia are the causative factor responsible for improved cardiovascular function and better neurological outcome. The results from this study showed that manipulating serum cortisol concentrations into high (<500nmol/l) and low (>50nmol/l) levels during hypoxia did not affect cardiovascular function (P = 0.68) or neurological outcome (P = 46). Within each group (low, high and control hypoxia group) there was significant variability in cardiovascular function during hypoxia, which was associated with neurological outcome. (r = -0.63, p = 0.01). This study showed that serum cortisol concentrations during hypoxia are not the causative factor impacting on improved cardiovascular function and neurological outcome. It is possible that factors affecting both cardiovascular function and cortisol production such as activity of the sympathetic nervous system, may be a possible mechanism behind the variability in neurological outcome and cardiovascular function. Additionally, this study showed that cortisol concentrations at 3 hours post hypoxia were associated with neurological outcome (r = -0.67, p = 0.01). The animals with poorer outcome may also be those with greater multi-organ damage and thus reduced ability to clear cortisol from the systemic circulation. In light of this finding it may be interesting to assess cortisol concentration in the human neonate at 3 hours post hypoxia and determine the relationship to neurological outcome. In the final study of this thesis the function of the cardiovascular system during hypoxia was investigated in more detail because of its strong association with neurological outcome (results observed in Chapter 2 and 4. Few researchers have reported on oscillations in cardiovascular function, particularly type-3 waves, during hypoxia in the neonate. The aim of this study was to determine the characteristics and occurrence of type-3 waves in the neonatal piglet and their relationship to neurological outcome following acute global hypoxia. The result showed that the development of type-3 waves in cardiovascular function occurred in 56% of animals. An oscillating pattern was significantly associated with better neurological outcome (p = 0.01) and a lower duration of hypotension during hypoxia (p = 0.02), and occurred more frequently in females (p = 0.024). The development of type-3 waves during acute global hypoxia is a key mechanism in promoting natural tolerance; and may be the result of greater activity, maturity or sensitivity of the sympathetic nervous system in females compared to males. This may explain the improved neurological outcome following hypoxia in the female neonate seen in the clinical setting.

Fishes in the superorder Ostariophysi, including fathead minnows (Pimephales promelas), possess specialized epidermal club cells that contain an alarm substance. Damage to these cells, as would occur during a predator attack, causes the release of the alarm substance and can indicate the presence of actively foraging predators to nearby conspecifics. For nearly 70 years, research involving epidermal club cells has focused on the alarm substance and the role it plays in predator/prey interactions. However, recent studies have indicated that there may be a connection between epidermal club cells and the fish immune system. Fish increase investment in epidermal club cells upon exposure to skin penetrating pathogens and parasites. In this study I tested for differences in epidermal club cell investment by fathead minnows exposed to the immunosuppressive effects of the glucocorticoid hormone cortisol. In experiment 1, fathead minnows were exposed to either a single intraperitoneal injection of corn oil or no injection at all. The purpose of this experiment was to determine whether corn oil, the vehicle for cortisol injections in later experiments, had an effect on epidermal club cell density. The treatments had no effect on epidermal club cell size, cell area, or epidermal thickness. In experiment 2, skin extract was prepared from the skin of corn oil injected and non injected fathead minnows as in experiment 1 to determine whether corn oil had an effect on the epidermal club cell alarm substance concentration. The treatments showed no significant differences in observed anti-predator behaviour, including change in shelter use, dashing and freezing. In experiment 3, fathead minnows were exposed to either a single intraperitoneal injection of cortisol or corn oil. The purpose of this experiment was to determine whether cortisol, a known immunosuppressant, had an effect on epidermal club cell investment. Fathead minnows exposed to a single cortisol injection had significantly reduced respiratory burst activity of kidney phagocytes indicating that there was suppression of the innate immune system. Furthermore, cortisol treated fathead minnows showed significantly lower numbers of epidermal club cells. The treatments had no effect on individual epidermal club cell area, epidermal thickness and serum cortisol levels after 12 days. The results from this experiment suggest that pharmacological cortisol injections in fathead minnows have a suppressive effect on the fish innate immune system. Furthermore, the findings that cortisol induced immunosuppression also influences epidermal club cell investment provides support for the hypothesis that epidermal club cells may function as part of the fish immune system.

Fishes in the superorder Ostariophysi, including fathead minnows (Pimephales promelas), possess specialized epidermal club cells that contain an alarm substance. Damage to these cells, as would occur during a predator attack, causes the release of the alarm substance and can indicate the presence of actively foraging predators to nearby conspecifics. For nearly 70 years, research involving epidermal club cells has focused on the alarm substance and the role it plays in predator/prey interactions. However, recent studies have indicated that there may be a connection between epidermal club cells and the fish immune system. Fish increase investment in epidermal club cells upon exposure to skin penetrating pathogens and parasites. In this study I tested for differences in epidermal club cell investment by fathead minnows exposed to the immunosuppressive effects of the glucocorticoid hormone cortisol. In experiment 1, fathead minnows were exposed to either a single intraperitoneal injection of corn oil or no injection at all. The purpose of this experiment was to determine whether corn oil, the vehicle for cortisol injections in later experiments, had an effect on epidermal club cell density. The treatments had no effect on epidermal club cell size, cell area, or epidermal thickness. In experiment 2, skin extract was prepared from the skin of corn oil injected and non injected fathead minnows as in experiment 1 to determine whether corn oil had an effect on the epidermal club cell alarm substance concentration. The treatments showed no significant differences in observed anti-predator behaviour, including change in shelter use, dashing and freezing. In experiment 3, fathead minnows were exposed to either a single intraperitoneal injection of cortisol or corn oil. The purpose of this experiment was to determine whether cortisol, a known immunosuppressant, had an effect on epidermal club cell investment. Fathead minnows exposed to a single cortisol injection had significantly reduced respiratory burst activity of kidney phagocytes indicating that there was suppression of the innate immune system. Furthermore, cortisol treated fathead minnows showed significantly lower numbers of epidermal club cells. The treatments had no effect on individual epidermal club cell area, epidermal thickness and serum cortisol levels after 12 days. The results from this experiment suggest that pharmacological cortisol injections in fathead minnows have a suppressive effect on the fish innate immune system. Furthermore, the findings that cortisol induced immunosuppression also influences epidermal club cell investment provides support for the hypothesis that epidermal club cells may function as part of the fish immune system.