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. Author manuscript; available in PMC: 2017 Jul 1.
Published in final edited form as: Neurosci Res. 2016 Feb 3;108:24–33. doi: 10.1016/j.neures.2016.01.008

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

Xingju Nie a, Danielle W Lowe b, Laura Grace Rollins c, Jessica Bentzley b, Jamie L Fraser d, Renee Martin e, Inderjit Singh b, Dorothea Jenkins b
PMCID: PMC4903952  NIHMSID: NIHMS762644  PMID: 26851769

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 50 mg/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: Neonatal hypoxia ischemia, N-acetylcysteine, sex, neuroprotection, redox regulation

Graphical Abstract

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Introduction1

Hypothermia treatment for hypoxic-ischemic (HI) neonates decreases the incidence of severe neurodevelopmental outcomes at 12-24 months of age to approximately 45-55%, without apparent differences in efficacy between sexes (Eicher et al., 2005, Shankaran et al., 2008, Azzopardi et al., 2009, Simbruner et al., 2010). With hypothermia now recognized as the clinical standard of care, the addition of other neuroprotective agents to hypothermia treatment are being investigated to improve outcomes further (Barks et al., 2010, Liu et al., 2012, Hobbs et al., 2008, Fan et al., 2013). N-acetylcysteine (NAC) is a promising antioxidant therapy that impacts many pathways of injury and has established neuroprotective effects in animal models of HI and neuroinflammation (Jatana et al., 2006, Paintlia et al., 2004, Liu et al., 2010). Immune and inflammatory responses to HI are modulated by cellular redox status, and increased apoptotic signaling pathways are present in cells with low anti-oxidant reserves (Lu, 2009, Cook et al., 2004, Circu and Aw, 2010, Wang and Kaufman, 2012, Ten and Starkov, 2012, Jager et al., 2012).

In our previous work, hypothermia plus NAC 50 mg/kg/day improved infarct volumes and negative geotaxis performance in neonatal animals subjected to right common carotid artery ligation and 2 hours of hypoxia, although sex effects were not evaluated (Jatana et al., 2006). For translation to therapeutic trials, these findings need to be replicated with robust statistical power, and optimal timing and dosing regimens determined. Furthermore, increasing evidence from in vitro and in vivo studies point to different mechanisms of injury, cell death, neuroinflammation and possibly repair between males and females in neonatal animals, even though both sexes are exposed to significant maternal estrogen (Zhang et al., 2010, Offner et al., 2009, Liesz et al., 2009, Nijboer et al., 2007, Zhu et al., 2006, Wen et al., 2006, Park et al., 2006, Weis et al., 2012). These different injury mechanisms warrant analytical consideration of sex-specific treatment effects. We performed 3 experiments with NAC plus hypothermia to determine sex differences in mechanisms of neuroprotection, to choose dose and timing of NAC administration, and to determine long-term outcomes in a severe HI model.

Materials and Methods

Animal

Postnatal day (PND) 7 Sprague-Dawley rats were used for all experiments (Harlan, Indianapolis, IN). Animals were housed in the animal care facility of the Medical University of South Carolina (MUSC) with a 12/12 hour light/dark cycle, and given standard chow and water ad libitum. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals adopted by the National Institutes of Health and approved by the MUSC Animal Care and Use Committee.

Reagents

2, 3, 5-triphenyl-tetrazolium chloride (TTC) and paraformaldehyde (Sigma Chemical Co., St. Louis, MO); Novaplus™ (Isoflurane, USP) (Abbot Laboratories, North Chicago, IL); VECTASHIELD Hard Set™ Mounting Medium with DAPI (H-1500, Vector Laboratories, Burlingame, CA); anti-Activated Caspase 3 (Abcam, Cambridge, MA); N-Acetylcysteine (Acetadote, Cumberland Pharmaceuticals, Nashville, TN.

Hypoxia-ischemia animal model

PND 7 rat pups were randomized to experimental groups within litters. Liters were limited to 10 pups to ensure equal maternal access. We used the modified Levine model of HI injury with unilateral ligation of the right common carotid artery and 2 hour exposure to 8% oxygen atmosphere for all experiments as previously described (Jatana et al., 2006, Geddes et al., 2001). At the end of hypoxia, pups were exposed to systemic hypothermia (30±0.5°C) or normothermia (36.3±0.5°C) in temperature controlled chambers for 2 h (Jatana et al., 2006). Rectal temperatures were in the target range of 33.5-34.5°C for the hypothermic rats, and 36.5-37.5°C for the normothermic group, similar to that used in clinical trials of therapeutic hypothermia. Sham operated animals underwent anesthesia and a neck incision without ligation or hypoxia and received normothermia and intraperitoneal saline. All treatment groups were administered NAC or vehicle (saline) intraperitoneal 1 h after hypoxia (1 h after initiation of hypothermia). Animals were removed from respective temperature chambers briefly for the injection and replaced. NAC or saline injections were repeated daily until sacrifice: Sham, Vehicle (VEH) and hypothermia alone (HYPO) groups received saline; Hypothermic NAC rats received 50 or 150 mg/kg/day of NAC (HNAC 50, HNAC 150). Control groups were limited to sham surgery, untreated HI injury (VEH), and the clinical standard of care hypothermia after HI (HYPO), as prospective therapies would be used only in addition to hypothermia clinically. Daily weights were recorded from PND 7 until sacrifice.

Experimental design

Presented in FIG. 1. Experiment 1: For caspase 3 activation by paraffin-embedded immunohistochemistry, 31 rats were randomized to Sham(n=2), VEH(n=8), HYPO(n=8), HNAC 50(n=7), HNAC 150 (n=4 survived, n=2 expired) with sacrifice at 24h. For cytokine and inflammatory mediator expression by quantitative RT-PCR in flash frozen brain tissue, an additional 44 rats were randomized to the 5 groups (4-5 rats per sex per treatment) with sacrifice at 24h. Experiment 2: PND 7 rats (n=184) were randomized into 9 groups to evaluate for timing of NAC administration based on hours after onset of hypothermia: Sham (n=12), VEH (n=20) 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) for 48h infarct volumes. Infarct volumes were measured at 48 h, comparing volumes within NAC doses of 50 and 150 mg/kg/d, to select the most effective timing for each dose for further study, considering possible sex effects. Experiment 3: PND 7 rats were randomized to Sham, VEH, HYPO, HNAC 50 1h, HNAC 150 1h, HNAC 150 3h (n=33, 5-8 rats per treatment group), and saline or NAC were administered once daily for 6 weeks. Mortality, physical characteristics, neurological reflexes, and strength and coordination testing were measured until sacrifice at 6 weeks after HI.

Figure 1.

Figure 1

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Outline of experimental design.

Randomization, Power analyses and Outcome Measures

Each litter was randomized to include pups of each sex and treatment group. For Experiment 1, a minimum of n=4 per sex to observe a 50% difference by Wilcoxon signed rank test (80% power, a=0.05) was based on previous work looking at iNOS mRNA levels after HI (Park et al., 2006). For Experiment 2, effect sizes for differences in infarct volume stratified by sex were estimated from a report by Bona et. al., which reported differential sex effects of hypothermia and 50% reduction in gross morphology score in female PND 7 HI rats (Bona et al., 1998a). For median infarct volumes by sex with 50% effect size by Wilcoxon signed rank test (80% power, a=0.05), a required sample size of 18 rats per treatment group, (9 per sex). This number was verified by an independent investigator (F. Silverstein, personal communication) based on the variability of injury observed in this animal model. For Experiments 2 and 3 behavioral and sensorimotor function studies, a minimum of n=5, balanced by sex, was based on previous behavioral differences detected at 7 days post injury by our lab by Wilcoxon signed rank test (80% power, a=0.05) (Jatana et al., 2006).

Immunohistochemistry (IHC) for Activated Caspase 3 and Infarct volume

Paraffin-embedded rat brain sections harvested 24 h after HI injury were prepared for immunohistochemistry, using standard protocols with a 1:100 dilution of anti-activated caspase-3 and 1:1000 dilution of fluorescent-conjugated secondary antibody, and mounted with aqueous mounting media containing DAPI. Three high power fields were counted in intact penumbral tissue (parietal cortex) for caspase-labeled and total cells. Infarct volume was quantified using 2-, 3-, 5-triphenyl-tetrazolium chloride (TTC, in saline) staining as previously described using Image J analysis software (NIH) (Jatana et al., 2006).

Quantitative Real Time reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Brains were rapidly dissected and snap frozen 24 h after HI injury. Total RNA was extracted (RNeasy Lipid Tissue Mini Kit, QIAGEN, MD) and reverse transcribed (iScript cDNA Synthesis Kit, Bio-Rad, CA) with cycles of 5 minutes at 25°C, 30 minutes at 42°C, and 5 minutes at 85°C. The primer sets for β-actin, COX2, CXCL1, CXCL2, ICAM-1, IL-1β, IL-4, IL-6, INF-γ, iNOS, MBP, MMP9, MMP13, nNOS, PPARα, and TNF-α are included in Supplemental Table 1. Real-time PCR was conducted using a Bio-Rad iCycler (Bio-Rad, Hercules, CA) with the following protocol: activation of iTaq DNA polymerase at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 30 s and 63°C for 1 min. Gene expression level in each sample were quantified against a standard curve, normalized to the sample β-actin units and calculated according to the following formula: relative target gene product units =sample target gene units/sample β-actin units.

Early Neurobehavioral Reflexes

Negative Geotaxis tests the righting reflex after being placed in a head down position on a board positioned at 45°. In Experiment 2, rat pups had 20 seconds to turn >90° towards the top of the board, or the maximum time was assigned. A more difficult version of the test was utilized in Experiment 3 with animals having to climb up the board with both forelimbs reaching the top edge of the board within a time limit of 30 seconds. Day of appearance was the first day in which the rat reached the top under 30 seconds.

Physical characteristics, reflexes, coordination and strength testing

In Experiment 3, Protocols for testing the long term development of strength, coordination and physical reflexes were followed as previously described (Lubics, Reglodi et al. 2005): Weights were recorded daily from PND 7-21 then twice weekly from PND 22-49. Neurological reflexes (eye twitch, ear twitch, righting reflex, limb placing, grasp, gait, cliff aversion, negative geotaxis) and physical characteristics (eye opening, ear unfolding, incisor eruption) were recorded daily from PND 8-21.

Rope suspension test

To measure forearm strength in the affected limb, rats were suspended by both forelimbs (two arm suspension) and only the affected forelimb (one arm suspension) on a 1/6″ diameter nylon rope above a foam pad. The duration of suspension was recorded. If the rat held on for entire 30 seconds, or pulled itself to the end of the rope and climbed onto the post, a time of 30 seconds was recorded. After testing two-arm suspension, rats had a 10 minute rest before the unaffected limb was immobilized with tape, and one arm suspension was assessed. The test was performed twice weekly from PND 14 to 49.

Grid walking

To analyze hindlimb placing deficits, rats were placed on a 32cm × 40cm grid with a mesh size of 4cm2, raised 12 inches above the benchtop, and observed for 1 minute. The total number of steps and the number of times a limb fell though the mesh opening (foot faults) were recorded.

Statistical analysis

For infarct volumes and gene expression, non-parametric Kruskal-Wallis tests were used to determine differences between groups with Mann-Whitney post-hoc testing. Changes in daily measures of physical characteristics, reflexes, negative geotaxis, righting reflex, gait, weight, rope suspension, and grid walking were analyzed over time using Univariate and General Linear Mixed Regression models for repeated measures with Tukey's post-hoc analysis. Negative geotaxis and gait were analyzed by absolute time and the first day the rat achieved a recorded time of <29 seconds (Day of Appearance). Righting reflex was analyzed by absolute time and the day on which the rat righted within 1 second (Day of Appearance). For rope suspension data, square root transformation preceded mixed model analysis with repeated measures. For Experiment 2, the decision was made a priori to compare the 3 control groups with the earliest NAC dosing, and then to examine timing of the two NAC does separately. For Experiment 3, comparisons were made within control (Sham, VEH and HYPO) and hypothermia treatment groups (HYPO, HNAC dosing groups), since hypothermia is the clinical standard of care. Significance was defined as p<0.05. All statistical calculations were done with SPSS version 20.0.

RESULTS

Experiment 1: HNAC treatment shows sex differences in inflammatory mediator expression

HNAC 1h decreased cell death at 24 h

Brain sections stained for activated caspase 3 displayed significantly fewer apoptotic cells in the penumbra of the ipsilateral cortex at 24 hours after HI injury in the HNAC animals compared with HYPO rats (n=4-8/group, FIG. 2A, B). Despite having two HNAC 150 1h animals die, there was no increased cell death observed in the remaining HNAC 150 animals. As females are reported to have greater caspase-mediated apoptosis than male rats after moderate HI (Mirza et al., 2015), we explored differences between females and males within treatment groups in our severe HI model. Males and females had similar caspase 3 activation in the penumbra within all groups (n=2-4 per sex, per group, data not shown), similar to findings by other investigators in severe HI (Askalan et al., 2015).

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

Figure 2

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(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<0.04). Sham(n=2), VEH(n=8), HYPO(n=8), HNAC 50(n=7), HNAC 150 (n=4 survived, n=2 expired), Kruskal-Wallis with Mann-Whitney post-hoc.

HNAC decreases iNOS expression in ipsilateral hemisphere in both sexes

Expression of inflammatory mediators in the ipsilateral hemisphere were analyzed at 24h after HI (n=8-10/group, FIG. 3A and Supplemental FIG. 1). HNAC 50 treatment resulted in significantly lower iNOS expression than HYPO alone in the combined sex analysis (p=0.009), while nNOS expression was significantly higher with HNAC 50 than HYPO alone (p=0.037).

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

Figure 3

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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<0.037) for both iNOS and nNOS. (B) iNOS expression: Male Veh animals had significantly higher iNOS expression than HNAC 50 males, and tended to have higher iNOS than females Veh animals (p=0.057). HNAC treatment tended to reduce iNOS vs. HYPO (p=0.063) in females. Hypothermia alone had no significant effect on iNOS expression in this severe model. (C) nNOS expression: Within the VEH group, nNOS expression was significantly higher in females than males. Within females, HNAC 50 1h nNOS expression was significantly higher than HYPO. (D) MBP expression: Within the VEH group, MBP expression was significantly higher in females than males. In HYPO females MBP expression was lower compared to VEH females. Within the HNAC 50 or 150 1h groups, males and females were not significantly different in expression of iNOS, nNOS, or MBP. n= 4-5 rats per sex per treatment, Kruskal-Wallis with Mann-Whitney post-hoc.

Our data show sex differences in nNOS and MBP expression. VEH HI females have higher nNOS and MPB expression than males and tend toward lower iNOS, as shown by other investigators (Park et al., 2006, Bonnin et al., 2012, McCullough et al., 2005). Previous reports also show nNOS is neuroprotective in female, but not male rats (McCullough et al., 2005), while male rats have greater neural and white matter injury after HI (Nijboer et al., 2007, Zhu et al., 2006, Bona et al., 1998a, Park et al., 2006, Weis et al., 2012) No gender differences in NOS or MBP expression were observed after hypothermia or HNAC treatment (FIG. 3B-C).

Sex differences in inflammatory mediator expression were present within hypothermia treated animals but not within HNAC animals

Analyses of inflammatory mediators demonstrated expression differences based on sex in endothelial activation (ICAM, MMP9) and neutrophil chemotactic (CXCL1/CXCL2, IL-6) factors (p<0.05). Male HYPO rats showed significantly lower expression of MMP9, ICAM, and CXCL1, compared with female HYPO rats (FIG. 4A-C). Male HYPO rats also showed a trend toward lower expression of chemotactic proteins CXCL2/ GROβ (monocytes) and IL-6 (neutrophils) than HYPO females (FIG. 4D-E). However, when NAC was added to hypothermia, levels of ICAM and MMP9 (FIG. 4A-B) and CXCL1/ GROα expression (FIG. 4C) decreased in females to near male levels. Taken together, these findings suggest that hypothermia regulates transcription of these mediators of endothelial activation and immune response differentially in males and females. However, the addition of NAC modulates markers of endothelial-immune activation observed in hypothermia treated female rats. MMP13, COX2, IFN-g, TNF-a, and IL-4 were not different among treatment groups (Supplemental FIG. 1).

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

Figure 4

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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.

Experiment 2: Infarct volume and negative geotaxis improve with NAC 50 treatment when started during hypothermia in females

NAC 50 1h decreased infarct volumes in female neonatal rats when given during hypothermia

The combined sex analysis of infarct volumes at 48 hours did not show definitive neuroprotective effect of NAC 50 mg/kg/d administered 1 h after the start of hypothermia (n=20-28, p= 0.079; FIG. 5A). In females, there were significant differences in infarct volumes between treatments (p=0.0198, FIG. 5B). HNAC 50 provided significant neuroprotection, and HYPO showed a strong trend towards neuro-protection, compared to VEH female animals in this severe model. However, median infarct volumes in female animals given HNAC 150 were significantly increased compared to HNAC 50 or HYPO females, and equal to VEH. Male rats exhibited no improvement with either NAC dose over hypothermia alone in 48h infarct volumes (FIG. 5C). For neurobehavioral reflex testing, there were no overall treatment differences in negative geotaxis at 48h after HI injury in the combined gender analysis. However, among female rats, both HNAC 1h treated groups performed significantly better than HYPO alone in the time to rotate >90° (p<0.036) , but were not significantly different than VEH (Supplemental FIG. 2). Taken together, results from Experiments 1 and 2 show that low dose NAC in combination with hypothermia improve mediators of inflammation after severe HI in the first 24 hours after HI injury. In addition, the data show smaller infarct volumes at 48 hours in female HI rats exposed to hypothermia and NAC compared to vehicle animals, while hypothermia alone provided no neuroprotection.

Figure 5. Infarct volumes at 48h.

Figure 5

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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.

Delay of high dose NAC administration improves infarct volumes

To determine the effects of delaying NAC treatment after hypothermia, we compared 1, 3, and 5 h NAC dosing (50 and 150mg/kg) after the initiation of hypothermia treatment. Among HNAC 50 groups, longer delays in administration did not improve infarct volumes at 48h (FIG. 6A). Using higher dose NAC, infarct volume increased with NAC administration after hypothermia (FIG. 6B), consistent with toxicity of this dose when given during hypothermia. There were no sex differences within the 3 and 5h groups for either NAC dose.

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

Figure 6

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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.

Experiment 3: Long-term sensorimotor function improves with HNAC 50 mg/kg/day for 6 weeks

Long-term treatment with NAC 50 mg/kg was well tolerated

Based on the results of Experiment 2, HNAC 50 1h, HNAC 150 1h, and HNAC 150 3h q24h for 6 weeks (n=5-8) doses were selected to evaluate long-term behavioral outcomes and toxicity. Weight was measured twice weekly, as failure to gain weight is a measure of toxicity. HNAC 50 rats had consistent weight gain throughout the study, similar to sham. However, HNAC 150 1h rats demonstrated decreased rate of weight gain, starting 24d after injury (data not shown). Three pups in the HNAC 150 1h and 1 pup in the HNAC 150 3h treatment groups lost weight and exhibited decreased activity prior to demise during weeks 5-6, and were excluded from those analyses. There were no differences in development of physical characteristics among hypothermia groups.

HNAC 50 improves long-term neuromotor tests, controlling for sex

Consistent with infarct volume results in this moderate to severe model, hypothermia treatment did not improve performance over VEH rats, with the exception of the grid-walking test (FIG. 7A,D,G,J). These neurobehavioral outcomes define the time course of long-term functional deficits in this severe HI model, and these results are comparable to published data (Lubics et al., 2005, Bona et al., 1998a, Fan et al., 2013). On strength testing by rope suspension over the 35 days of testing, HNAC 50 1h group displayed the best performance in both two-arm (FIG. 7B) and one-arm tests (FIG. 7E). In the two-arm suspension test, HNAC 50 1h and 150 3h rats tended to hold on longer than hypothermia alone early after injury (FIG. 7B, C). However, in the more difficult one arm test, significant improvement in HNAC 50 1 h rats performance compared with HYPO rats was detectable as early as day 17 (p=0.014) and persisted into 6th week after HI injury (FIG. 7F).

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

Figure 7

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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<0.00001). Among Hypothermia treatment groups, HNAC 50 and HNAC 150 3h tended to perform better over other groups between days 24-31 using two arms (B & C) although all groups improved over time. In one arm suspension testing (D, E, F) using only affected arm, duration of suspension for HNAC 50 1h rats were significantly better than HYPO as early as Day 17 (p=0.014), persisting to last week of testing (E). HNAC 50 1h rats had the longest times for one-arm rope suspension on day 49 (F) with mean suspension times of 90% of Sham time. In the negative geotaxis testing (G, H, I), VEH and HYPO rats took significantly more time to turn around and climb to the top of the board than Sham (G, p= 0.017 and 0.003, respectively). In NAC treatment groups (H), HNAC 50 consistently performed better than HYPO (p=0.018) over time and had earlier successful completion of negative geotaxis (I), with HNAC 50 1h rats completing the task 3 days earlier than HYPO rats. In grid walking (J, K, L), testing hindimb placing accuracy (foot faults to total steps) from days 14 – 49, HYPO rats were significantly different from Sham and VEH groups over time (p<0.0001), though there was no significant improvement with NAC treatment over hypothermia alone (K). In the sum of total grid walking steps, measuring locomotor activity from days 14 – 38 (H), HNAC 50 1h rats took more steps than HYPO (p=0.043) and HNAC 150 1 h (p=0.027, HNAC 150 1h: 572 steps, HNAC 50 1h: 617 steps). Error Bars indicate ± 1 SD. 5-8 rats per treatment group, mixed regression statistical models.

For negative geotaxis (FIG. 7H, I), HNAC 50 1h rats first completed the task on average 3 days earlier than HYPO rats (FIG. 7I) and consistently performed the best of all hypothermia groups throughout testing (FIG.7H). In an ambulatory test of locomotion and accuracy of hind limb placing (grid walking), HNAC 50 1h rats took significantly more total steps from days 14 to 38 compared to HYPO and HNAC 150 1 h rats (FIG. 7L). HNAC 50 1h rats had the highest number of total steps, and HNAC 150 1h the lowest. There were no additional improvements over hypothermia in grid walking foot-faults with any NAC dose (FIG. 7K).

DISCUSSION

Oxidative stress is a major determinant of injury in HI and leads to accumulation of reactive oxygen species and dysregulated inflammatory cascades (Naik and Dixit, 2011). At the same time, the neonatal brain handles oxidative stress poorly, with antioxidant activities that are only half of adult levels at term gestational age (Aspberg and Tottmar, 1992, Sheldon et al., 2004). Glutathione, as the major intracellular antioxidant, is an in vivo CNS marker of the severity of oxidative stress (Satoh and Yoshioka, 2006). Glutathione is depleted due to excessive acute or chronic ROS and is closely associated with neuronal death. (Han et al., 1999, Filibian et al., 2012, Ceccon et al., 2000) N-acetylcysteine serves as a source of cysteine, a glutathione precursor, and scavenges oxygen free radicals with its thiol-reducing group, acting directly and indirectly as a potent antioxidant (Oda et al., 1999, Cuzzocrea et al., 1999, Diniz et al., 2006, Van Antwerpen et al., 2005, Lee et al., 2008a). Importantly, NAC crosses the blood brain barrier, where it reduces cerebral oxidative stress, preserves peroxisomes and restores myelination (Farr et al., 2003, Neuwelt et al., 2001, Paintlia et al., 2004, Lee et al., 2008b). NAC has been shown to improve both cellular and mitochondrial glutathione redox status and mitochondrial membrane potential, which leads to improved cell survival (Okouchi et al., 2009, Wang et al., 2007). These properties make NAC a strong candidate to ameliorate some of the cascades of inflammation and stimulate endogenous repair mechanisms after HI (Paintlia et al., 2004). In our neonatal HI model, neuromotor tests were improved with long-term, low dose NAC. Hypothermia alone did not demonstrate neuroprotective effects, similar to other reports using moderate to severe HI models (Bona et al., 1998b, Burnsed et al., 2015, Traudt et al., 2013, Sabir et al., 2012, Nedelcu et al., 2000). Our data add to that of other investigations that have found NAC to be neuroprotective in HI injury and in several adult and neonatal animal models of neuroinflammation (Lee et al., 2008b, Cuzzocrea et al., 2000, Wang et al., 2007, Lante et al., 2008). Thus, NAC in combination with hypothermia may provide functional neuroprotection after more severe HI injuries, in which hypothermia alone is not sufficient to improve outcomes.

Interestingly, in our short-term studies, low-dose NAC added to hypothermia preserved infarct volumes in female, but not in male, neonatal rats 48h after severe HI compared with vehicle controls. Sex differences in outcomes and injury pathways are increasingly recognized as important considerations in neuroprotection. We found sex differences in expression of factors that are secreted by activated endothelial cells and microglia (Okouchi et al., 2009, Lijia et al., 2012), and impact neutrophil recruitment (CXCL1/ GROα, CXCL2/GROβ) and invasion (ICAM), and tissue integrity (MMP9). These factors work cooperatively to increase leukocyte-endothelial interaction and infiltration early after HI (Lee and Lo, 2004, Gidday et al., 2005). Hypothermia down-regulated expression of these mediators in our male neonatal rats, as has been reported in other experiments using male animals (Wang et al., 2002, Sarcia et al., 2003, Bednarek et al., 2012, Liu et al., 2008, Lee et al., 2005). However, expression of iCAM, MMP9, and CXCL1 were significantly higher in our female hypothermic rats, and only with the addition of NAC were these mediators decreased to same levels as hypothermic males. Down regulation of MMP9 in particular, is important in females, since it triggers caspase-mediated apoptosis (the predominant pathway in female neurons), and worsens infarct volumes by cleaving extracellular matrix components and disrupting homeostatic integrin signaling (Du et al., 2004, Lee and Lo, 2004). Female neonatal rats have shown increased cytochrome c-mediated caspase activation and greater mitochondrial loss in the cortex and hippocampus at 2-18 hours after HI compared with males (Weis et al., 2012, Nijboer et al., 2007).

We also found overall differences in nNOS and iNOS expression, with reduced iNOS and restored nNOS expression to near Sham levels with the addition of NAC compared to hypothermia treatment alone. Indiscriminant iNOS activation under conditions of redox stress may result in uncoupling of nitric oxide (NO) production and electron transfer, leading to peroxynitrite (ONOO−) formation, oxidation of cysteine residues, and nitration of purines in DNA and tyrosine residues in proteins (Du et al., 2004, McCullough et al., 2005, Hagberg et al., 2004, Chen et al., 2003). In primary neuronal cultures exposed to ONOO− for 24 h, XY neurons showed greater reduction in intracellular glutathione and increased cell death compared with XX neurons (Du et al., 2004). Males may, therefore, be more susceptible to uncoupling of NO production and peroxynitrite oxidative stress than females. Under improving intracellular redox status, constitutive nNOS and eNOS produce low levels of endogenous, bioavailable NO, which may also utilized in post-translational modification of cell signaling proteins through a S-NO bond (nitrosylation) (Keynes and Garthwaite, 2004, Keynes et al., 2004, Martinez-Ruiz and Lamas, 2004, Matsumoto et al., 2003). A key apoptotic mediator, procaspase 3, is maintained in an inactive state by nitrosylation, while removal of the NO group leads to caspase 3 activation (Lai et al., 2011). HNAC treatment decreased iNOS expression and caspase 3 activation, while improving nNOS expression in both sexes of neonatal rats in our model, indicating that combined treatment may benefit some mechanisms of injury in both sexes.

Judicious dosing of NAC may be an important factor in our observed functional neuroprotection with longer-term treatment. Low dose NAC has been shown to attenuate H2O2 production, inhibit NF-κβ, and decrease ICAM mRNA and protein expression in oxidatively-stressed endothelial cells (Mukherjee et al., 2007). While low dose NAC is beneficial to our hypothermic female HI rats, higher doses of NAC resulted in significantly worse infarct volumes in our study. High dose NAC is known to increase both reduced and oxidized glutathione, causing reductive stress and unfolded protein response in the endoplasmic reticulum (Zhang et al., 2012). The toxicity-related dose effects of NAC also depend on the temperature. At normothermia, 500 mg/kg of NAC was required to cause detrimental effects in adult HI rats (Khan et al., 2004). However, with hypothermia in our study, even the usual clinical anti-oxidant dose of NAC in acetaminophen overdose (150 mg/kg) in females showed worsening infarct volumes and increased death. Therefore, hypothermia seems to lower the threshold for both NAC activity and toxicity, possibly through decreased clearance and prolonged NAC half-life.

Improving upon hypothermia's neuroprotection is challenging, as it acutely regulates the very cascades affected by other neuroprotective agents (Fan et al., 2013, Fang et al., 2013). At the cellular level, normalization of cytoplasmic, mitochondrial and endoplasmic reticular oxidative balance is a crucial first step for restoration of homeostasis and growth factor signaling, and is an important therapeutic target in neurologic diseases (Victor et al., 2003, Jager et al., 2012, Ten and Starkov, 2012, Lu, 2009, Perrone et al., 2012). Our data in a severe HI PND7 model show short-term neuroprotection only in females treated with NAC plus hypothermia, and no neuroprotection with hypothermia alone compared with saline treated animals. Long-term treatment with NAC was required for functional improvement in male and female rats after severe HI, implying NAC has important effects on repair, neurogenesis, and synaptogenesis, which may be independent of sex. These studies provide a basis for further investigations of neuroprotection with NAC, but highlight the importance of sex considerations in neurotherapeutics.

Supplementary Material

NIHMS762644-supplement.docx (109.4KB, docx)

HIGHLIGHTS.

  • Low dose NAC with hypothermia decreases infarct volumes in female neonatal HI rats

  • NAC decreases ICAM, MMP-9, CXCL-1 expression in female rats after severe HI

  • Long-term NAC treatment may be necessary for functional improvement in both sexes

ACKNOWLEDGMENTS

This work was supported by NIH grants NS 054428, NS 022576 and NS 037766.

Footnotes

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1

Abbreviations

CXCL-1/GROα- Chemokine C-X-C ligand 1/Growth-regulated oncogene alpha

CXCL-2/GROβ- Chemokine C-X-C ligand 2 /macrophage inflammatory protein 2-alpha/ Growth-regulated protein beta

HI- Hypoxia ischemia

HNAC- hypothermia with N-Acetylcysteine treatment

HYPO- hypothermia treatment

ICAM- Intercellular adhesion molecule

IL-Interleukin

iNOS, nNOS- inducible or neuronal nitric oxide synthase

MBP - Myelin basic protein

MMP – Matrix metalloproteinase

NAC- N-Acetylcysteine

NO- nitric oxide

PND - Postnatal day

TTC - 2, 3, 5-Triphenyl-tetrazolium chloride

VEH- vehicle, saline treatment

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