Experimental Stroke and Neurotrophins: Regulation, function and gene transfer of neurotrophins in rat and mouse models of focal cerebral ischemia

Abstract: Stroke caused by focal cerebral ischemia is a major cause of death and neurological disability in humans. The forebrain region most commonly affected by ischemic incidents is that supplied by the middle cerebral artery. In this thesis, neuronal cell death has been investigated using immunohistochemistry for identification of specific neurons and unbiased counting methods after transient occlusion of the middle cerebral artery (MCAO). Our studies show that striatal projection neurons and parvalbumin-containing interneurons are vulnerable, whereas cholinergic and NOS-containing interneurons are highly resistant to MCAO. The high potency of the neurotrophin family of neurotrophic factors to inhibit neuronal death in immature cell systems and the widespread expression of these factors throughout the adult brain, suggest that the neurotrophins might be critical regulators of neuronal survival. Rapid and profound increase of BDNF gene expression has been shown in the frontal and cingulate cortices after MCAO. We show here that also the BDNF protein levels are elevated in the same cortical regions after the ischemic insult, but also further dynamic changes in subcortical regions strongly suggesting anterograde transport of BDNF in forebrain neurons. In our further studies, the ability of the neurotrophins to change the vulnerability of striatal neurons to ischemic damage was investigated in the MCAO model. First, the resistant cholinergic interneurons in striatum respond to MCAO with expression of the p75 neurotrophin receptor (p75NTR). However, the vulnerability of the cholinergic interneurons to MCAO was not increased in a mice strain mutant for the P75NTR, which strongly argue against a major role of this receptor for the prominent resistance of these interneurons to ischemic insults. In order to directly investigate the neuroprotective properties of the neurotrophins, the vulnerability of striatal neurons to focal cerebral ischemia was determined after increasing the levels of BDNF and NGF by gene transfer to the striatum. Increased survival of striatal projection neurons was found after intrastriatal grafting of NGF-secreting genetically modified neural stem cells and after direct transfer of the BDNF gene to striatal cells using an adenoassociated viral (AAV) vector. Furthermore, viral transfer of the BDNF or NGF gene to striatal cells increased survival of parvalbumin-containing interneurons after MCAO. Our data support a role for the neurotrophins as a protective factor against ischemic damage. To assess the functional significance of increased neuronal survival in striatum after focal cerebral ischemia, we utilized different behavioral tests for striatal motor function. We found a strong correlation between scores from these behavioral tests and the extent of striatal damage, and in particular skilled forelimb use in the staircase paradigm was the best predictor of damage. Viral delivery of the neurotrophins lead to improvements in ischemia-induced motor impairments, which appeared to be due to increase in the survival of striatal neurons treated with BDNF. In conclusion, this thesis shows that focal cerebral ischemia causes dynamic changes of the BDNF and p75NTR levels. Gene transfer-induced elevation of neurotrophin levels increases the survival of striatal neurons to ischemic damage, which indicates a neuroprotective role for these factors in the mature brain.