Periodic hypoxia, intermittent pain, and caffeine in male and female neonatal rats: Corticosterone, insulin resistance, and hepatic gene expression

Preterm infants experience multiple stressors including periodic neonatal hypoxia, maternal/caregiver separation, and acute pain from clinical procedures. Although neonatal hypoxia or interventional pain are associated with sexually dimorphic effects that may last into adulthood, the interaction of these common preterm stressors and with caffeine pretreatment remain unknown. We hypothesize that an interaction of acute neonatal hypoxia, isolation, and pain modeling the experience of the preterm infant will augment the acute stress response, and that caffeine routinely given to preterm infants will alter this response. Male and female rat pups were isolated and exposed to six cycles of periodic hypoxia (10% O2) or normoxia (room air control) and/or intermittent pain by administering needle pricks (or touch control) to the paw on postnatal (PD) days 1-4. An additional set of rat pups was pretreated with caffeine citrate (80 mg/kg IP) and studied on PD1. Plasma corticosterone, fasting glucose, and insulin were measured to calculate HOMA-IR (index of insulin resistance). Glucocorticoid-, insulin-, and caffeine-sensitive gene mRNAs were analyzed in the PD1 liver and hypothalamus to evaluate downstream markers of glucocorticoid action. Acute pain with periodic hypoxia led to a large increase in plasma corticosterone, which was attenuated by pretreatment with caffeine. Pain with periodic hypoxia led to a 10-fold increase in hepatic Per1 mRNA expression in males, which was attenuated with caffeine. The augmentation of corticosterone and HOMA-IR at PD1 after periodic hypoxia with pain suggests early intervention to attenuate the stress response may mitigate the programming effects of neonatal stress

Effect of a melanocortin type 2 Receptor (MC2R) antagonist on the corticosterone response to hypoxia and ACTH stimulation in the neonatal rat

The adrenal stress response in the neonatal rat shifts from ACTH-independent to ACTH-dependent between postnatal days 2 (PD2) and 8 (PD8). This may be due to an increase in an endogenous, bioactive, non-immunoreactive ligand to the MC2R. GPS1574 is a newly described MC2R antagonist that we have shown to be effective in vitro. Further experimentation with GPS1574 would allow better insight into this seemingly ACTH-independent steroidogenic response in neonates. We evaluated the acute corticosterone response to hypoxia or ACTH injection following pretreatment with GPS1574 (32 mg/kg) or Vehicle for GPS1574 in PD2, PD8, and PD15 rat pups. Pretreatment with GPS1574 decreased baseline corticosterone in PD2 pups, but increased baseline corticosterone in PD8 and PD15 pups. GPS1574 did not attenuate the corticosterone response to hypoxia in PD2 pups, and augmented the corticosterone response in PD8 and PD15 pups. GPS1574 augmented the corticosterone response to ACTH in PD2 and PD15 pups, but had no significant impact on the response in PD8 pups. Baseline adrenal Mrap and Star mRNA increased from PD2 to PD15, whereas Mrap2 mRNA expression was low and did not change with age. The data suggests that GPS1574 is not a pure MC2R antagonist, but rather acts as a bias agonist/antagonist. Its ability to attenuate or augment the adrenal response may depend on the ambient plasma ACTH concentration and/or developmental changes in early transduction steroidogenic pathway genes

Insulin sensitivity, leptin, adiponectin, resistin, and testosterone in adult male and female rats after maternal-neonatal separation and environmental stress

Care of premature infants often requires parental and caregiver separation, particularly during hypoxic and hypothermic episodes. We have established a neonatal rat model of human prematurity involving maternal-neonatal separation and hypoxia with spontaneous hypothermia prevented by external heat. Adults previously exposed to these neonatal stressors show a sex difference in the insulin and glucose response to arginine stimulation suggesting a state of insulin resistance. The current study used this cohort of adult rats to evaluate insulin resistance [homeostatic model assessment of insulin resistance (HOMA-IR)], plasma adipokines (reflecting insulin resistance states), and testosterone. The major findings were that daily maternal-neonatal separation led to an increase in body weight and HOMA-IR in adult male and female rats and increased plasma leptin in adult male rats only; neither prior neonatal hypoxia (without or with body temperature control) nor neonatal hypothermia altered subsequent adult HOMA-IR or plasma adiponectin. Adult male-female differences in plasma leptin were lost with prior exposure to neonatal hypoxia or hypothermia; male-female differences in resistin were lost in the adults that were exposed to hypoxia and spontaneous hypothermia as neonates. Exposure of neonates to daily hypoxia without spontaneous hypothermia led to a decrease in plasma testosterone in adult male rats. We conclude that neonatal stressors result in subsequent adult sex-dependent increases in insulin resistance and adipokines and that our rat model of prematurity with hypoxia without hypothermia alters adult testosterone dynamics

Programming of the adult HPA axis after neonatal separation and environmental stress in male and female rats

Maternal separation, hypoxia, and hypothermia are common stressors in the premature neonate. Using our rat model of human prematurity, we evaluated sexual dimorphisms in the long term effects of these neonatal stressors on behavior of the hypothalamic-pituitary-adrenal (HPA) axis in adult rats. Neonatal rats were exposed daily on postnatal days 2-6 to maternal separation with normoxia, with hypoxia allowing spontaneous hypothermia, with hypothermia per se, and with hypoxia while maintaining isothermia with external heat. The major findings were that (a) prior maternal-neonatal separation during the first week of postnatal life significantly attenuated the plasma ACTH and corticosterone response to restraint stress in adult male but not female rats, (b) prior neonatal hypothermia augmented the plasma ACTH and corticosterone response to restrain stress in adult male, but not female rats, and (c) changes in hypothalamic, pituitary, and adrenal mRNA expression did not account for most of these HPA axis effects. Most of the programming effects on adult HPA axis was attributed to prior maternal-neonatal separation alone (with normoxia) because the addition of hypoxia with spontaneous hypothermia, hypothermia per se, and hypoxia while preventing hypothermia during maternal-neonatal separation had minimal effects on the HPA axis. These results may inform strategies to prevent sexually dimorphic sequelae of neonatal stress including those due to medical interventions

Intermittent neonatal hypoxia elicits the upregulation of inflammatory-related genes in adult male rats through long-lasting programming effects

The long-term effects of neonatal intermittent hypoxia (IH), an accepted model of apnea-induced hypoxia, are unclear. We have previously shown lasting programming effects on the HPA axis in adult rats exposed to neonatal IH. We hypothesized that neonatal rat exposure to IH will subsequently result in a heightened inflammatory state in the adult. Rat pups were exposed to normoxia (control) or six cycles of 5% IH or 10% IH over one hour daily from postnatal day 2-6. Plasma samples from blood obtained at 114 days of age were analyzed by assessing the capacity to induce transcription in a healthy peripheral blood mononuclear cell (PBMC) population and read using a high-density microarray. The analysis of plasma from adult rats previously exposed to neonatal 5% IH versus 10% IH resulted in 2579 significantly regulated genes including increased expression of Cxcl1, Cxcl2, Ccl3, Il1a, and Il1b. We conclude that neonatal exposure to intermittent hypoxia elicits a long-lasting programming effect in the adult resulting in an upregulation of inflammatory-related genes

Glucocorticoid receptor antagonist alters corticosterone and receptor sensitive mRNAs in the hypoxic neonatal rat

Hypoxia, a common stressor with preterm birth, increases morbidity and mortality associated with prematurity. Glucocorticoids (GC) are administered to the preterm infant to improve oxygenation; prolonged use of GCs remains controversial. We evaluated a selective glucocorticoid receptor (GR) antagonist (CORT113176) in our neonatal rat model of human prematurity to assess how fasting and hypoxia-induced increases in neonatal corticosterone affects endogenous hormones and endocrine pancreas function. Neonatal rat pups at postnatal day (PD) 2, PD8, and PD15 were pretreated with CORT113176 and, after 60 min of separation and fasting, exposed to hypoxia (8% O2) or control (normoxia) for 30 or 60 minutes while fasting was continued. Plasma corticosterone, ACTH, glucose, and insulin were measured and fasting HOMA-IR (index of insulin resistance) calculated. Glucocorticoid and insulin receptor sensitive gene mRNAs were analyzed in liver, muscle, and adipose to evaluate target tissue biomarkers. CORT113176 pretreatment augmented baseline and hypoxia-induced increases in corticosterone and attenuated hypoxia-induced increases in insulin resistance at PD2. Normoxic and hypoxic stress increased the hepatic GR sensitive gene mRNAs, Gilz and Per1; this was eliminated by pretreatment with CORT113176. CORT113176 pretreatment decreased baseline insulin receptor sensitive gene mRNAs Akt2, Irs1, Pik3r1, and Srebp1c at PD2. We show that CORT113176 variably augments the stress-induced increases in corticosterone concentrations (attenuation of negative feedback) and that GR is critical for hepatic responses to stress in the hypoxic neonate. We also propose that measurement of Gilz and Per1 mRNA expression may be useful to evaluate the effectiveness of GR antagonism

Sex differences in adult rat insulin and glucose responses to arginine: programming effects of neonatal separation, hypoxia, and hypothermia

Acute neonatal hypoxia, a common stressor, causes a spontaneous decrease in body temperature which may be protective. There is consensus that hypothermia should be prevented during acute hypoxia in the human neonate; however, this may be an additional stress with negative consequences. We hypothesize that maintaining body temperature during hypoxia in the first week of postnatal life alters the subsequent insulin, glucose, and glucagon secretion in adult rats. Rat pups were separated from their dam daily from postnatal days (PD) 2-6 for the following 90 min experimental treatments: (1) normoxic separation (control), (2) hypoxia (8% O2) allowing spontaneous hypothermia, (3) normoxic hypothermia with external cold, and (4) exposure to 8% O2 while maintaining body temperature using external heat. An additional normoxic non-separated control group was performed to determine if separation per se changed the adult phenotype. Plasma insulin, glucose, and glucagon responses to arginine stimulation were evaluated from PD105 to PD133. Maternal separation (compared to non-separated neonates) had more pronounced effects on the adult response to arginine compared to the hypoxic, hypothermic, and hypoxic-isothermic neonatal treatments. Adult males exposed to neonatal maternal separation had augmented insulin and glucose responses to arginine compared to unseparated controls. Additionally, neonatal treatment had a significant effect on body weight gain; adults exposed to neonatal maternal separation were significantly heavier. Female adults had significantly smaller insulin and glucose responses to arginine regardless of neonatal treatment. Neonatal maternal separation during the first week of life significantly altered adult beta-cell function in a sexually dimorphic manner