As reported in other immature animal hypoxia-ischemia models, the evolution of the neonatal stroke injury is quite prolonged.18, 19
In this model, we observed progressive atrophy in the ipsilateral hemisphere over the three weeks following neonatal stroke at P12. Acutely, the majority of cells died in an environment where edema developed; later, over the following weeks increased apoptosis was visible in the injured hippocampus and neocortex. These results suggest that neonatal stroke has long lasting effects on neuronal viability and supports the existence of a prolonged potential therapeutic-window for alleviating the progression of cell-death after such an injury.
In order to better understand how ischemia affects neuronal activity, we monitored the temporal profile of c-fos expression. In this context, c-fos expression can serve as a marker of several processes, including neuronal hyper-activity following seizure, 20
excessive glutamate response following cerebral ischemia 21
A clear up-regulation in c-fos expression was observed 3 hours after stroke in all regions of the ipsilateral hippocampus. The CA3 and CA1 areas are the regions most susceptible to excitotoxic cell-death in the immature brain, 23
whereas the DG remains partially preserved. At one week after ligation (P19), ectopic foci of c-fos expression in CA1 were noted in and around areas of cell-death. At T14d (P26) and T21d (P33) after ligation, when hippocampal atrophy peaked, we observed c-fos expression restricted to scattered pyramidal cells of the ipsilateral hippocampus. This delayed and abnormal c-fos expression can be interpreted as resulting from hyper- or abnormal activity following neonatal ischemia that can contribute to the brain injury, as it has been proposed by others working with rat perinatal models of hypoxia ischemia.24-27
Several recent papers have reported that Notch-1 activation occurs in response to cerebral ischemia in very different cell types: in the germinal zones of the SVZ 9
and SGZ 10
where it has been shown to contribute to the maintenance of the progenitor pool 28
, and in the neurons of the cortex 8
where it is thought to contribute to neuronal damage.8
Neonatal mice with acute seizures following double unilateral carotid ligation showed a strong increase in Notch-2 receptor expression in the granule and pyramidal layers of the ipsilateral hippocampus, specifically in regions with ectopic c-fos expression, which subsequently became atrophic. Interestingly Hes5, a conical target of the Notch pathway, was also up-regulated in the hippocampus following ischemia (data not shown), indicating that up-regulation of Notch-2 led to pathway activation.
The widespread induction of Notch-2 in the GCL acutely after neonatal stroke injury, was distinct from Notch-1 which remained largely restricted to the SGZ.10
Interestingly, one week following injury, Notch-1 and Notch-2 still had very different cellular patterns of expression in the ipsilateral hippocampus; Notch-2 was aberrantly increased in injured hippocampal neurons, whereas Notch-1 was localized to reactive glia. This finding suggests a differential role for the two receptors in response to neonatal ischemia.
We have shown that OGD challenge in vitro
induced Notch-2 activation in primary hippocampal neurons, similarly to what has been seen with Notch-1 in cortical cultures, 8
and we have shown that most of the cells that had aberrant Notch-2 activation were also positive for the apoptotic marker cleaved-caspase-3. Utilizing a gain of function experiment, we demonstrated that over-expression of the transcriptionally active form of Notch-2 (NICD2) was neurotoxic under basal conditions to a comparable level as after OGD treatment alone. Furthermore, under OGD conditions the control transfected and NICD2 transfected cultures had similar levels of cell-death.
In conclusion this work demonstrates that neonatal ischemia induced by unilateral carotid ligation in P12 mice is a clinically relevant model that produces long lasting anatomical and molecular changes in the hippocampus and cortex. The prolonged and ectopic c-fos expression in regions of ongoing cell death is of particular interest for the possible identification of sites with prolonged abnormal neuronal activity and/or cell demise. Future research using this model may link these sites to the process of post-ischemic epileptogenesis, or alternatively to the focus of new regenerative strategies. In addition, Notch-2 appears to be rapidly and persistently induced in postmitotic neurons by ischemic injury. The work reported here suggests that this aberrant induction of Notch-2 may be neurotoxic. We anticipate that identifying the downstream effectors of Notch-2 after ischemic brain injury could lead to the development of better therapeutic agents, which might help contain the neuronal damage resulting from Notch-2 over-activation.