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 caffeine pretreatment remains 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 homeostatic model assessment for insulin resistance (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.

Serum soluble urokinase plasminogen activator receptor in adolescents: interaction of chronic pain and obesity.

INTRODUCTION: Obesity in adolescents is increasing in frequency and is associated with short-term and long-term negative consequences that include the exacerbation of co-occurring chronic pain. OBJECTIVE: To determine whether the interaction between chronic pain and obesity would be reflected in changes in serum soluble urokinase plasminogen activator receptor (suPAR) concentrations, a novel marker of systemic inflammation associated with obesity, insulin resistance, and cardiovascular disease. METHODS: We measured serum suPAR levels in 146 adolescent males and females with no pain or obesity (healthy controls; n = 40), chronic pain with healthy weight (n = 37), obesity alone (n = 41), and the combination of chronic pain and obesity (n = 28). RESULTS: Serum suPAR (median [interquartile range]) was not increased by chronic pain alone (2.2 [1.8-2.4] ng/mL) or obesity alone (2.2 [2.0-2.4] ng/mL) but was increased significantly with the combination of chronic pain and obesity (2.4 [2.1-2.7] ng/mL; P < 0.019). This finding confirms the proposition that pain and obesity are inflammatory states that display a classic augmenting interaction. CONCLUSION: We propose that measurement of serum suPAR can be added to the armamentarium of serum biomarkers useful in the evaluation of mechanisms of inflammation in adolescent obesity and chronic pain.

Bedtime Salivary Cortisol and Cortisone by LC-MS/MS in Healthy Adult Subjects: Evaluation of Sampling Time.

The measurement of late-night salivary cortisol is a mainstay in the diagnosis of Cushing syndrome. Furthermore, the measurement of salivary cortisol is useful in assessing the cortisol awakening response. Because the salivary glands express 11-ß-hydroxysteroid dehydrogenase, the measurement of salivary cortisone may improve the performance of salivary corticosteroid measurements. We measured salivary cortisol by enzyme immunoassay (EIA) and salivary cortisol and cortisone by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in only 50 µL of saliva sampled from 54 healthy subjects (aged 20 to 64 years). We allowed patients to sample at their normal bedtime (2025 to 2400 hours) to answer a common question as to whether sampling at the normal bedtime is equivalent to the standard required sampling at 2300 to 2400 hours. We found that the salivary cortisol and cortisone results by LC-MS/MS correlated well with salivary cortisol measured with the US Food and Drug Administration-cleared EIA. Furthermore, the upper limit of normal of salivary cortisol by EIA for bedtime samples was lower than the previously published upper limit of normal with sampling required at 2300 to 2400 hours. There were no significant effects of age or sex on any of the salivary steroid measurements. We conclude that (i) salivary cortisol and cortisone can be reliably measured by LC-MS/MS in small volumes of saliva and (ii) that patients can be evaluated using saliva sampled at their normal bedtime, rather than being required to stay awake until 2300 to 2400 hours.

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.

Overexpression of cyclooxygenase-2 in rat oral cancers and prevention of oral carcinogenesis in rats by selective and nonselective COX inhibitors.

Oral squamous cell carcinomas induced in rats by 4-nitroquinoline-1-oxide (NQO) show substantial overexpression of cyclooxygenase-2 (COX-2) when compared with adjacent phenotypically normal oral tissues. By contrast, neither 5-lipoxygenase (LOX) nor 12-LOX is overexpressed in rat oral cancers. Two chemoprevention studies were done to test the resulting hypothesis that COX-2 is a useful target for oral cancer chemoprevention in the rat. In both studies, male F344 rats received drinking water exposure to NQO (20 ppm) for 10 weeks, followed by administration of chemopreventive agents from week 10 until study termination at week 26. In the first study, groups of rats were fed basal diet (control), or basal diet supplemented with the selective COX-2 inhibitor celecoxib (500 or 1,500 mg/kg diet), the nonselective COX inhibitor piroxicam (50 or 150 mg/kg diet), or the 5-LOX inhibitor zileuton (2,000 mg/kg diet). In the second study, rats were fed basal diet (control) or basal diet supplemented with nitric oxide-naproxen (180 or 90 mg/kg diet), a nonselective COX inhibitor that shows reduced gastrointestinal toxicity. When compared with dietary controls, celecoxib decreased oral cancer incidence, cancer invasion score, and cancer-related mortality. Piroxicam decreased cancer-related mortality and cancer invasion score, whereas nitric oxide-naproxen decreased oral cancer incidence and cancer invasion score. By contrast, zileuton showed no chemopreventive activity by any parameter assessed. These data show that both selective and nonselective inhibitors of COX-2 can prevent NQO-induced oral carcinogenesis in rats. The chemopreventive activity of COX inhibitors may be linked to overexpression of their enzymatic target in incipient oral neoplasms.