Neonatal stroke in mice causes long-term changes in neuronal Notch-2 expression that may contribute to prolonged injury.

BACKGROUND AND PURPOSE: Notch receptors (1-4) are membrane proteins that, on ligand stilumation, release their cytoplasmic domains to serve as transcription factors. Notch-2 promotes proliferation both during development and cancer, but its role in response to ischemic injury is less well understood. The purpose of this study was to understand whether Notch-2 is induced after neonatal stroke and to investigate its functional relevance. METHODS: P12 CD1 mice were subjected to permanent unilateral (right-sided) double ligation of the common carotid artery. RESULTS: Neonatal ischemia induces a progressive brain injury with prolonged apoptosis and Notch-2 up-regulation. Notch-2 expression was induced shortly after injury in hippocampal areas with elevated c-fos activation and increased cell death. Long-term induction of Notch-2 also occurred in CA1 and CA3 in and around areas of cell death, and had a distinct pattern of expression as compared to Notch-1. In vitro oxygen glucose deprivation treatment showed a similar increase in Notch-2 in apoptotic cells. In vitro gain of function experiments, using an active form of Notch-2, show that Notch-2 induction is neurotoxic to a comparable extent as oxygen glucose deprivation treatment. CONCLUSIONS: These results suggest that Notch-2 up-regulation after neonatal ischemia is detrimental to neuronal survival.

Chronic brain injury and behavioral impairments in a mouse model of term neonatal strokes.

Stroke in term neonates remains a significant cause of long-term neurological morbidity. This study was designed to assess the relationships between ischemic stroke induced by permanent unilateral carotid ligation in P12 CD1 mice and the structural and functional outcomes in the young mice as a consequence. After P12 ischemic strokes, mice were behaviorally tested using accelerated rotorod, spontaneous alternation on a T-maze, open-field, and cylinder tests between P33 and P39. Brain injury was scored by histology at P40 with cresyl violet-stained coronal sections and computerized quantification of the ischemic injury. The ligation-injured mice were not different from controls on cylinder testing for asymmetric use of their forelimb, or on rotorod measures. In the spontaneous alternation task, however, injured mice demonstrated significantly lower rates of alternation indicating a deficit in working memory. Open-field testing repeated on two consecutive days revealed that the ligated mice were less active than the controls and that they failed to habituate to the open field environment between sessions indicating a learning deficit. Overall, our results demonstrate that ischemia induced by our neonatal stroke model produces behavioral deficits that are consistent with the brain injury.

Gabapentin neuroprotection and seizure suppression in immature mouse brain ischemia.

Stroke is a major cause of neurologic morbidity in neonates and children. Because neonatal and pediatric stroke frequently present with seizures, the question of which anticonvulsant best blocks acute ischemic seizures and reduces injury is clinically relevant. The purpose of this study was to determine the extent to which gabapentin is neuroprotective and suppresses acute seizures in this model of ischemic injury in the immature brain. Postnatal day 12 CD1 mice underwent right common carotid artery ligation and immediately after ligation received a 0, 50, 100, 150, or 200 mg/kg dose of gabapentin intraperitoneally. Acute seizure activity was behaviorally scored and hemispheric brain atrophy measured. In vehicle-treated mice, severity of acute seizures correlated with hemispheric brain atrophy 4 wks later. Gabapentin significantly decreased acute seizures at 200 mg/kg and reduced brain atrophy at doses of 150 and 200 mg/kg but not at lower doses. These results suggest that gabapentin effectively reduces acute seizures and injury after ischemia in the immature brain. When analyzed by animal sex, the data suggest that gabapentin may more effectively reduce acute seizures and injury in male pups vs. female pups.

Neural stem cells reduce brain injury after unilateral carotid ligation.

Neonatal stroke presents with seizures and results in neurologic morbidity, including epilepsy, hemiparesis, and cognitive deficits. Stem cell-based therapy offers a possible therapeutic strategy for neonatal stroke. We developed an immature mouse model of stroke with acute seizures and ischemic brain injury. Postnatal day 12 CD1 mice received right-sided carotid ligation. Two or 7 days after ligation, mice received an intrastriatal injection of B5 embryonic stem cell-derived neural stem cells. Four weeks after ligation, hemispheric brain atrophy was measured. Pups receiving stem cells 2 days after ligation had less severe hemispheric brain atrophy compared with either noninjected or vehicle-injected ligated controls. Transplanted cells survived, but 3 out of 10 pups injected with stem cells developed local tumors. No difference in hemispheric brain atrophy was seen in mice injected with stem cells 7 days after ligation. Neural stem cells have the potential to ameliorate ischemic injury in the immature brain, although tumor development is a serious concern.