R. Ann Sheldon

Strain-related brain injury in neonatal mice subjected to hypoxia-ischemia.

The development of transgenic mice has led to an increase in the use of mice as models for human disease. We hypothesized that the degree of brain damage sustained by animals in a neonatal mouse model of hypoxia-ischemia depends on the strain used. We compared three strains of mice commonly used to generate transgenic strains (C57Bl/6, 129Sv and CD1), as well as three hybrids of these strains (C57Bl/6x129Sv, CD1xC57Bl/6, and CD1x129Sv). At postnatal day 7 (P7), pups were subjected to a modified Vannucci procedure for hypoxia-ischemia as follows: permanent ligation of right common carotid artery under halothane anesthesia, 2-h recovery period, exposure to 8% oxygen at 37 degreesC for varying durations (30, 60 or 90 min). After 5 days, animals were perfused with 4% paraformaldehyde, brains were removed, postfixed and examined histologically with cresyl violet and Perls iron stain to assess the degree of damage. Damage was assessed blindly using a score ranging from 0 (none) to 3 (infarct) in eight regions (ant-, mid-, and post- cortex, CA1, CA2, CA3 and dentate gyrus of the hippocampus, and striatum). We found significant differences in susceptibility to brain damage and mortality depending on the strain used. While determining the maximal degree of injury with the least amount of mortality for each strain, it was found that some strains (CD1) are particularly susceptible to brain damage in this model, while others (129Sv) are resistant.

Neonatal Mice Lacking Neuronal Nitric Oxide Synthase Are Less Vulnerable to Hypoxic–Ischemic Injury

We hypothesized that elimination of neuronal nitric oxide synthase (nNOS) by targeted disruption of the nNOS gene would result in amelioration of damage seen after hypoxia-ischemia in the developing brain since nitric oxide (NO) has been implicated in glutamate-mediated neurotoxicity after ischemia. Both wildtype and nNOS-deficient pups were subjected to focal ischemia followed by 1.5 h of hypoxia at Postnatal Day 7. Seven days later, brains of surviving animals were analyzed for damage. The nNOS-deficient pups (n = 17) had less histopathologic evidence of injury in both the hippocampus (P = 0.008) and the cortex (P = 0.0008) than the wildtype (n = 30) mice. When injured, the nNOS-deficient mice had damage that was limited to the hippocampus. These results support a role for neuronally produced NO in injury after perinatal hypoxia-ischemia.

Brain injury after perinatal hypoxia-ischemia is exacerbated in copper/zinc superoxide dismutase transgenic mice

The role of superoxide radical formation in the pathogenesis of perinatal hypoxic-ischemic injury was examined using transgenic (Tg) mice expressing three times normal amounts of copper/zinc-superoxide dismutase (CuZn/SOD). Fourteen litters of postnatal d 7 strain 218/3 mice were subjected to right common carotid artery ligation followed by 90 min of hypoxia in an 8% oxygen/humidified chamber maintained at 37°C. Both Tg mice (n = 32) and their nontransgenic (nTg) littermates (n = 30) survived the injury equally. Evaluation of infarcted brain areas measured by video image analysis of three coronal brain sections through the anterior hippocampus from each animal revealed that the Tg animals suffered brain infarction more frequently than did nTg mice. Blinded histologic scoring of cerebral cortex and striatum 5 d after injury revealed that Tg mice were more likely to have higher histologic severity scores than their nTg littermates (p = 0.0463, Mann-Whitney U test). These findings suggest that brain injury in perinatal hypoxia-ischemia may be mediated in part by free radical formation from excessive hydrogen peroxide or nitric oxide production.

Hsp70 Overexpression Sequesters AIF and Reduces Neonatal Hypoxic/Ischemic Brain Injury:

Apoptosis is implicated in neonatal hypoxic/ischemic (H/I) brain injury among various forms of cell death. Here we investigate whether overexpression of heat shock protein (Hsp) 70, an antiapoptotic protein, protects the neonatal brain from H/I injury and the pathways involved in the protection. Postnatal day 7 (P7) transgenic mice overexpressing rat Hsp70 (Tg) and their wild-type littermates (Wt) underwent unilateral common carotid artery ligation followed by 30 mins exposure to 8% O2. Significant neuroprotection was observed in Tg versus Wt mice on both P12 and P21, correlating with a high level of constitutive but not inducible Hsp70 in the Tg. More prominent injury was observed in Wt and Tg mice on P21, suggesting its continuous evolution after P12. Western blot analysis showed that translocation of cytochrome c, but not the second mitochondria-derived activator of caspase (Smac)/DIABLO and apoptosis-inducing factor (AIF), from mitochondria into cytosol was significantly reduced in Tg 24 h after H/I compared with Wt mice. Coimmunoprecipitation detected more Hsp70 bound to AIF in Tg than Wt mice 24 h after H/I, inversely correlating with the amount of nuclear, but not cytosolic, AIF translocation. Our results suggest that interaction between Hsp70 and AIF might have reduced downstream events leading to cell death, including the reduction of nuclear AIF translocation in the neonatal brains of Hsp70 Tg mice after H/I.

Erythropoietin Improves Functional and Histological Outcome in Neonatal Stroke

Neonatal stroke is a condition that leads to disability in later life, and as yet there is no effective treatment. Recently, erythropoietin (EPO) has been shown to be cytoprotective following brain injury and may promote neurogenesis. However, the effect of EPO on functional outcome and on morphologic changes in neonatal subventricular zone (SVZ) following experimental neonatal stroke has not been described. We used a transient focal model of neonatal stroke in P10 rat. Injury was documented by diffusion weighted MRI during occlusion. Immediately upon reperfusion, either EPO (5U/gm) or vehicle was administered intraperitoneally and animals were allowed to grow for 2 wk. Sensorimotor function was assessed using the cylinder rearing test and then brains were processed for volumetric analysis of the SVZ. Stroke induced SVZ expansion proportional to hemispheric volume loss. EPO treatment markedly preserved hemispheric volume and decreased the expansion of SVZ unilaterally. Furthermore, EPO treatment significantly improved the asymmetry of forelimb use following neonatal stroke. This functional improvement directly correlated with the amount of preserved hemispheric volume. These results suggest EPO may be a candidate in the treatment of neonatal stroke.

Regulation of hypoxia-inducible factor 1α and induction of vascular endothelial growth factor in a rat neonatal stroke model

Stroke is a devastating condition occurring in at least 1 in 4000 live births in the neonatal period. Since hypoxia-inducible factor (HIF)-1alpha can modulate ischemic injury via induction of target genes that may protect cells against ischemia, and is induced after preconditioning by hypoxia in the neonatal rat brain hypoxia-ischemia model, we evaluated whether HIF-1alpha is induced after focal ischemia-reperfusion, a model for neonatal stroke. We developed an ischemia-reperfusion model in postnatal day 10 (P10) rats by transiently occluding the middle cerebral artery (MCA) for 1.5 h. The MCA territory was reperfused for 0, 4, 8, or 24 h and the expression of HIF-1alpha and its target gene, vascular endothelial growth factor (VEGF), were delineated. HIF-1alpha protein and VEGF protein peaked at 8 h, and declined subsequently at 24 h in injured cortex following 1.5 h of MCA occlusion. Double-immunolabeling indicated that both HIF-1alpha and VEGF are expressed together in neurons with a similar time course of expression. The presence of HIF-1alpha and VEGF after moderate ischemia-reperfusion injury suggests potential avenues to exploit for neuroprotection.

Mesenchymal Stem Cell Transplantation Attenuates Brain Injury After Neonatal Stroke

Background and Purpose— Brain injury caused by stroke is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. Mesenchymal stem cells (MSC) have been shown to improve outcome after neonatal hypoxic-ischemic brain injury mainly by secretion of growth factors stimulating repair processes. We investigated whether MSC treatment improves recovery after neonatal stroke and whether MSC overexpressing brain-derived neurotrophic factor (MSC-BDNF) further enhances recovery. Methods— We performed 1.5-hour transient middle cerebral artery occlusion in 10-day-old rats. Three days after reperfusion, pups with evidence of injury by diffusion-weighted MRI were treated intranasally with MSC, MSC-BDNF, or vehicle. To determine the effect of MSC treatment, brain damage, sensorimotor function, and cerebral cell proliferation were analyzed. Results— Intranasal delivery of MSC- and MSC-BDNF significantly reduced infarct size and gray matter loss in comparison with vehicle-treated rats without any significant difference between MSC- and MSC-BDNF–treatment. Treatment with MSC-BDNF significantly reduced white matter loss with no significant difference between MSC- and MSC-BDNF–treatment. Motor deficits were also improved by MSC treatment when compared with vehicle-treated rats. MSC-BDNF–treatment resulted in an additional significant improvement of motor deficits 14 days after middle cerebral artery occlusion, but there was no significant difference between MSC or MSC-BDNF 28 days after middle cerebral artery occlusion. Furthermore, treatment with either MSC or MSC-BDNF induced long-lasting cell proliferation in the ischemic hemisphere. Conclusions— Intranasal administration of MSC after neonatal stroke is a promising therapy for treatment of neonatal stroke. In this experimental paradigm, MSC- and BNDF-hypersecreting MSC are equally effective in reducing ischemic brain damage.

The neuroprotective effect of deferoxamine in the hypoxic-ischemic immature mouse brain

The iron chelator deferoxamine is efficacious in ameliorating hypoxic-ischemic brain injury in some models, perhaps by decreasing oxidative stress. Transgenic copper/zinc superoxide dismutase-1 (SOD1) overexpression in neonatal mice increases brain injury after hypoxia-ischemia compared to non-transgenic wildtype littermates because of increased oxidative stress. A neonatal mouse model of hypoxia-ischemia was used to examine histopathological damage, iron histochemistry and free iron concentration in the brains of SOD1 transgenic and non-transgenic littermates. Deferoxamine significantly decreased injury in non-transgenics compared to controls with a trend toward neuroprotection in the transgenics. There was no difference in free iron concentrations in the brains of SOD1 overexpressors or non-transgenics. Deferoxamine may protect the neonatal brain by a number of anti-oxidant mechanisms including iron chelation, enhancement of stress gene expression, or induction of other factors responsible for neuroprotection.

Manipulation of antioxidant pathways in neonatal murine brain.

To assess the role of brain antioxidant capacity in the pathogenesis of neonatal hypoxic-ischemic brain injury, we measured the activity of glutathione peroxidase (GPX) in both human-superoxide dismutase-1 (hSOD1) and human-GPX1 overexpressing transgenic (Tg) mice after neonatal hypoxia-ischemia (HI). We have previously shown that mice that overexpress the hSOD1 gene are more injured than their wild-type (WT) littermates after HI, and that H2O2 accumulates in HI hSOD1-Tg hippocampus. We hypothesized that lower GPX activity is responsible for the accumulation of H2O2. Therefore, increasing the activity of this enzyme through gene manipulation should be protective. We show that brains of hGPX1-Tg mice, in contrast to those of hSOD-Tg, have less injury after HI than WT littermates: hGPX1-Tg, median injury score = 8 (range, 0–24) versus WT, median injury score = 17 (range, 2–24), p < 0.01. GPX activity in hSOD1-Tg mice, 2 h and 24 h after HI, showed a delayed and bilateral decline in the cortex 24 h after HI (36.0 ± 1.2 U/mg in naive hSOD1-Tg versus 29.1 ± 1.7 U/mg in HI cortex and 29.2 ± 2.0 for hypoxic cortex, p < 0.006). On the other hand, GPX activity in hGPX1-Tg after HI showed a significant increase by 24 h in the cortex ipsilateral to the injury (48.5 ± 5.2 U/mg, compared with 37.2 ± 1.5 U/mg in naive hGPX1-Tg cortex, p < 0.008). These findings support the hypothesis that the immature brain has limited GPX activity and is more susceptible to oxidative damage and may explain the paradoxical effect seen in ischemic neonatal brain when SOD1 is overexpressed.

Acute hypoxia-ischemia results in hydrogen peroxide accumulation in neonatal but not adult mouse brain.

The neonatal brain responds differently to hypoxic-ischemic injury and may be more vulnerable than the mature brain due to a greater susceptibility to oxidative stress. As a measure of oxidative stress, the immature brain should accumulate more hydrogen peroxide (H2O2) than the mature brain after a similar hypoxic-ischemic insult. To test this hypothesis, H2O2 accumulation was measured in postnatal day 7 (P7, neonatal) and P42 (adult) CD1 mouse brain regionally after inducing HI by carotid ligation followed by systemic hypoxia. H2O2 accumulation was quantified at 2, 12, 24, and 120 h after HI using the aminotriazole (AT)-mediated inhibition of catalase spectrophotometric method. Histologic injury was determined by an established scoring system, and infarction volume was determined. P7 and P42 animals were subjected to different durations of hypoxia to create a similar degree of brain injury. Despite similar injury, significantly less H2O2 accumulated in P42 mouse cortex compared with P7 at 2, 12, and 24 h after HI. In addition, less H2O2 accumulated in P42 mouse hippocampus compared with P7 hippocampus at 2 h. Since immature neurons are more vulnerable to the toxic effects of H2O2 than mature neurons, this increased accumulation in the immature brain may explain why the neonatal brain may be more devastated, even after a milder degree of acute hypoxic-ischemic injury.