Vitamin D improves functional outcomes in neonatal hypoxic ischemic male rats treated with N-acetylcysteine and hypothermia.

Hypothermia treatment neuroprotects approximately 50% of neonates who present with moderate to severe hypoxic ischemic encephalopathy (HIE). N-acetylcysteine (NAC), a potent antioxidant, is neuroprotective in combination with hypothermia in neonatal hypoxia-ischemia (HI) female rats, but less protective in males. Vitamin D is a neurosteroid, which may provide immunomodulation and improve outcomes for both sexes. We investigated the efficacy of this combination of drugs with hypothermia after severe HI, as well as potential mechanisms of vitamin D effects in the transition to chronic inflammation. DOL 7 rats were randomized to sham, or HI and hypothermia treated with either saline (HYPO), NAC (50 mg/kg/d, HNAC), or HNAC plus 1,25-dihydroxy-vitamin D3 (0.1 µg/kg/d, HNAC + VitD) daily for 2 weeks. A second set of animals were randomized and treated for 11 days to investigate vitamin D metabolism and inflammatory mediators. Rats treated with HNAC + VitD performed significantly better on tests of strength and use of affected limb, adaptive sensorimotor skills, motor sequence learning, and working memory than either HYPO or HNAC, particularly benefiting male rats. Significantly fewer rats in the HNAC + VitD group had severe hemispheric volume loss. HI injury decreased serum vitamin D at 11 days and induced the enzyme that deactivates vitamin D in the hippocampus, particularly in males. Persistent vitamin D dysregulation was seen in both hippocampi in males, which was not reversed by hypothermia. Vitamin D in combination with hypothermia and NAC supports functional recovery in both sexes of neonatal rats significantly better than hypothermia alone or hypothermia and NAC in this severe HI model.

Sex-specific effects of N-acetylcysteine in neonatal rats treated with hypothermia after severe hypoxia-ischemia.

Approximately half of moderate to severely hypoxic-ischemic (HI) newborns do not respond to hypothermia, the only proven neuroprotective treatment. N-acetylcysteine (NAC), an antioxidant and glutathione precursor, shows promise for neuroprotection in combination with hypothermia, mitigating post-HI neuroinflammation due to oxidative stress. As mechanisms of HI injury and cell death differ in males and females, sex differences must be considered in translational research of neuroprotection. We assessed the potential toxicity and efficacy of NAC in combination with hypothermia, in male and female neonatal rats after severe HI injury. NAC 50mg/kg/d administered 1h after initiation of hypothermia significantly decreased iNOS expression and caspase 3 activation in the injured hemisphere versus hypothermia alone. However, only females treated with hypothermia +NAC 50mg/kg showed improvement in short-term infarct volumes compared with saline treated animals. Hypothermia alone had no effect in this severe model. When NAC was continued for 6 weeks, significant improvement in long-term neuromotor outcomes over hypothermia treatment alone was observed, controlling for sex. Antioxidants may provide insufficient neuroprotection after HI for neonatal males in the short term, while long-term therapy may benefit both sexes.

Fetal and Neonatal Effects of N-Acetylcysteine When Used for Neuroprotection in Maternal Chorioamnionitis.

OBJECTIVE: To evaluate the clinical safety of antenatal and postnatal N-acetylcysteine (NAC) as a neuroprotective agent in maternal chorioamnionitis in a randomized, controlled, double-blinded trial. STUDY DESIGN: Twenty-two mothers >24 weeks gestation presenting within 4 hours of diagnosis of clinical chorioamnionitis were randomized with their 24 infants to NAC or saline treatment. Antenatal NAC (100 mg/kg/dose) or saline was given intravenously every 6 hours until delivery. Postnatally, NAC (12.5-25 mg/kg/dose, n = 12) or saline (n = 12) was given every 12 hours for 5 doses. Doppler studies of fetal umbilical and fetal and infant cerebral blood flow, cranial ultrasounds, echocardiograms, cerebral oxygenation, electroencephalograms, and serum cytokines were evaluated before and after treatment, and 12, 24, and 48 hours after birth. Magnetic resonance spectroscopy and diffusion imaging were performed at term age equivalent. Development was followed for cerebral palsy or autism to 4 years of age. RESULTS: Cardiovascular measures, cerebral blood flow velocity and vascular resistance, and cerebral oxygenation did not differ between treatment groups. Cerebrovascular coupling was disrupted in infants with chorioamnionitis treated with saline but preserved in infants treated with NAC, suggesting improved vascular regulation in the presence of neuroinflammation. Infants treated with NAC had higher serum anti-inflammatory interleukin-1 receptor antagonist and lower proinflammatory vascular endothelial growth factor over time vs controls. No adverse events related to NAC administration were noted. CONCLUSIONS: In this cohort of newborns exposed to chorioamnionitis, antenatal and postnatal NAC was safe, preserved cerebrovascular regulation, and increased an anti-inflammatory neuroprotective protein. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00724594.

Altered circulating leukocytes and their chemokines in a clinical trial of therapeutic hypothermia for neonatal hypoxic ischemic encephalopathy*.

OBJECTIVES: To determine systemic hypothermia's effect on circulating immune cells and their corresponding chemokines after hypoxic ischemic encephalopathy in neonates. DESIGN: In our randomized, controlled, multicenter trial of systemic hypothermia in neonatal hypoxic ischemic encephalopathy, we measured total and leukocyte subset and serum chemokine levels over time in both hypothermia and normothermia groups, as primary outcomes for safety. SETTING: Neonatal ICUs participating in a Neurological Disorders and Stroke sponsored clinical trial of therapeutic hypothermia. PATIENTS: Sixty-five neonates with moderate to severe hypoxic ischemic encephalopathy within 6 hours after birth. INTERVENTIONS: Patients were randomized to normothermia of 37°C or systemic hypothermia of 33°C for 48 hours. MEASUREMENTS AND MAIN RESULTS: Complete and differential leukocyte counts and serum chemokines were measured every 12 hours for 72 hours. The hypothermia group had significantly lower median circulating total WBC and leukocyte subclasses than the normothermia group before rewarming, with a nadir at 36 hours. Only the absolute neutrophil count rebounded after rewarming in the hypothermia group. Chemokines, monocyte chemotactic protein-1 and interleukin-8, which mediate leukocyte chemotaxis as well as bone marrow suppression, were negatively correlated with their target leukocytes in the hypothermia group, suggesting active chemokine and leukocyte modulation by hypothermia. Relative leukopenia at 60-72 hours correlated with an adverse outcome in the hypothermia group. CONCLUSIONS: Our data are consistent with chemokine-associated systemic immunosuppression with hypothermia treatment. In hypothermic neonates, persistence of lower leukocyte counts after rewarming is observed in infants with more severe CNS injury.

Serum cytokines in a clinical trial of hypothermia for neonatal hypoxic-ischemic encephalopathy.

Inflammatory cytokines may mediate hypoxic-ischemic (HI) injury and offer insights into the severity of injury and the timing of recovery. In our randomized, multicenter trial of hypothermia, we analyzed the temporal relationship of serum cytokine levels in neonates with hypoxic-ischemic encephalopathy (HIE) with neurodevelopmental outcome at 12 months. Serum cytokines were measured every 12 hours for 4 days in 28 hypothermic (H) and 22 normothermic (N) neonates with HIE. Monocyte chemotactic protein-1 (MCP-1) and interleukins (IL)-6, IL-8, and IL-10 were significantly higher in the H group. Elevated IL-6 and MCP-1 within 9 hours after birth and low macrophage inflammatory protein 1a (MIP-1a) at 60 to 70 hours of age were associated with death or severely abnormal neurodevelopment at 12 months of age. However, IL-6, IL-8, and MCP-1 showed a biphasic pattern in the H group, with early and delayed peaks. In H neonates with better outcomes, uniform down modulation of IL-6, IL-8, and IL-10 from their peak levels at 24 hours to their nadir at 36 hours was observed. Modulation of serum cytokines after HI injury may be another mechanism of improved outcomes in neonates treated with induced hypothermia.