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

Abstract

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.

Keywords: N-acetylcysteine; Neonatal hypoxia ischemia; Neuroprotection; Redox regulation; Sex.

Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Figures

Figure 1

Outline of experimental design.

Figure 2. Activated caspase 3+ cells in ipsilateral cortex harvested 24h after HI injury

(A) Representative cortical sections showing caspase 3+ (green) and DAPI (blue) labeled cells. (B) Median and IQR of the percent of total cells that were activated caspase 3+, of three HPFs in ipsilateral cortex at 24 hour after HI. There are significantly fewer caspase 3+ cells in the cortex of HNAC 50 1h and HNAC 150 1h rats compared with HYPO animals (*p

Figure 3. PCR analysis of inflammatory mediators at 24h, normalized to β-actin

Median and 95% CI by treatment (A) and by sex (B-D). Irrespective of sex (A), HNAC 50 1h was significantly different than HYPO (* p

Figure 4. PCR analysis of inflammatory mediators at 24h by sex, normalized to β-actin

Box plot within sex, male treatment groups are represented in gray, females in white. Within the HYPO group, ICAM1 (A), MMP 9 (B), and CXCL1 (C) levels were significantly higher in females compared to males, with strong trends for CXCL2 (D) and IL6 (E) (p=0.057). With addition of NAC 50 1h to hypothermia, the increased expression of ICAM, MMP-9, CXCL1, CXCL2 in female HYPO rats was abolished, and expression in HNAC females was similar to that in males for these inflammatory mediators. n= 4-5 rats per sex per treatment, Kruskal-Wallis with Mann-Whitney post-hoc.

Figure 5. Infarct volumes at 48h

Median values are marked by bar. For the overall infarct volume (A), there was a trend towards a treatment effect (p = 0.0792), with HNAC 50 1h having the lowest median infarct volumes. In the female rats (B), treatment had a significant effect on infarct volumes (p = 0.0198): HNAC 50 mg/kg/d provided significant neuroprotection and HYPO had marginal neuro-protection vs. VEH (p= 0.059). HNAC 150 mg/kg/d exhibited increased median infarct volume compared to HYPO or HNAC 50, similar to VEH. No treatment effect was observed in males (C, p =0.5947). Sham (n=12), VEH (n=20) HYPO (n=28), HNAC 50 1h (n=20), HNAC 150 (n=21), Kruskal-Wallis with Mann-Whitney post-hoc.

Figure 6. Infarct volumes at 48h with delayed initiation of therapy

Median values (irrespective of sex) are marked by bar, HYPO group included for reference. With the HNAC 50 dose (A), there was no significant difference with delayed dose administration. However in the HNAC 150 dose (B), the infarct size significantly improved with delayed administration of the dose to either 3h or 5h compared to the 1h administration. HYPO (n=28), HNAC 50 1h (n=20), HNAC 50 3h (n=21), HNAC 50 5h (n=20), HNAC 150 (n=21), HNAC 150 3h (n=21), and HNAC 150 5h (n=21), Kruskal-Wallis with Mann-Whitney post-hoc.

Figure 7. Functional outcomes at 7 weeks in controls (left panels) and in NAC treatment groups (center and right panels)

For control groups, HI injury significantly decreased the time of rope suspension using two arms (A) and one arm (D) compared to Sham over time (p