Effects of AAV-mediated knockdown of nNOS and GPx-1 gene expression in rat hippocampus after traumatic brain injury.

Virally mediated RNA interference (RNAi) to knock down injury-induced genes could improve functional outcome after traumatic brain injury (TBI); however, little is known about the consequences of gene knockdown on downstream cell signaling pathways and how RNAi influences neurodegeneration and behavior. Here, we assessed the effects of adeno-associated virus (AAV) siRNA vectors that target two genes with opposing roles in TBI pathogenesis: the allegedly detrimental neuronal nitric oxide synthase (nNOS) and the potentially protective glutathione peroxidase 1 (GPx-1). In rat hippocampal progenitor cells, three siRNAs that target different regions of each gene (nNOS, GPx-1) effectively knocked down gene expression. However, in vivo, in our rat model of fluid percussion brain injury, the consequences of AAV-siRNA were variable. One nNOS siRNA vector significantly reduced the number of degenerating hippocampal neurons and showed a tendency to improve working memory. GPx-1 siRNA treatment did not alter TBI-induced neurodegeneration or working memory deficits. Nevertheless, microarray analysis of laser captured, virus-infected neurons showed that knockdown of nNOS or GPx-1 was specific and had broad effects on downstream genes. Since nNOS knockdown only modestly ameliorated TBI-induced working memory deficits, despite widespread genomic changes, manipulating expression levels of single genes may not be sufficient to alter functional outcome after TBI.

Effects of trauma, hemorrhage and resuscitation in aged rats.

Traumatic brain injury (TBI) is a leading cause of death in the elderly and the incidence of mortality and morbidity increases with age. This study tested the hypothesis that, after TBI followed by hemorrhagic hypotension (HH) and resuscitation, cerebral blood flow (CBF) would decrease more in aged compared with young rats. Young adult (4-6 months) and aged (20-24 months) male Sprague-Dawley rats were anesthetized with isoflurane, prepared for parasagittal fluid percussion injury (FPI) and randomly assigned to receive either moderate FPI (2.0 atm) only, moderate FPI+severe HH (40 mm Hg for 45 min) followed by return of shed blood, or sham FPI. Intracranial pressure (ICP), CBF, and mean arterial pressure (MAP) were measured and, after twenty-four hours survival, the rats were euthanized and their brains were sectioned and stained with Fluoro-Jade (FJ), a dye that stains injured neurons. After moderate FPI, severe HH and reinfusion of shed blood, MAP and CBF were significantly reduced in the aged group, compared to the young group. Both FPI and FPI+HH groups significantly increased the numbers of FJ-positive neurons in hippocampal cell layers CA1, CA2 and CA3 (p<0.05 vs Sham) in young and aged rats. Despite differences in post-resuscitation MAP and CBF, there were no differences in the numbers of FJ-positive neurons in aged compared to young rats after FPI, HH and blood resuscitation. Although cerebral hypoperfusion in the aged rats was not associated with increased hippocampal cell injury, the trauma-induced reductions in CBF and post-resuscitation blood pressure may have resulted in damage to brain regions that were not examined or neurological or behavioral impairments that were not assessed in this study. Therefore, the maintenance of normal blood pressure and cerebral perfusion would be advisable in the treatment of elderly patients after TBI.

Influence of stochastic gene expression on the cell survival rheostat after traumatic brain injury.

Experimental evidence suggests that random, spontaneous (stochastic) fluctuations in gene expression have important biological consequences, including determination of cell fate and phenotypic variation within isogenic populations. We propose that fluctuations in gene expression represent a valuable tool to explore therapeutic strategies for patients who have suffered traumatic brain injury (TBI), for which there is no effective drug therapy. We have studied the effects of TBI on the hippocampus because TBI survivors commonly suffer cognitive problems that are associated with hippocampal damage. In our previous studies we separated dying and surviving hippocampal neurons by laser capture microdissection and observed unexplainable variations in post-TBI gene expression, even though dying and surviving neurons were adjacent and morphologically identical. We hypothesized that, in hippocampal neurons that subsequently are subjected to TBI, randomly increased pre-TBI expression of genes that are associated with neuroprotection predisposes neurons to survival; conversely, randomly decreased expression of these genes predisposes neurons to death. Thus, to identify genes that are associated with endogenous neuroprotection, we performed a comparative, high-resolution transcriptome analysis of dying and surviving hippocampal neurons in rats subjected to TBI. We found that surviving hippocampal neurons express a distinct molecular signature--increased expression of networks of genes that are associated with regeneration, cellular reprogramming, development, and synaptic plasticity. In dying neurons we found decreased expression of genes in those networks. Based on these data, we propose a hypothetical model in which hippocampal neuronal survival is determined by a rheostat that adds injury-induced genomic signals to expression of pro-survival genes, which pre-TBI varies randomly and spontaneously from neuron to neuron. We suggest that pharmacotherapeutic strategies that co-activate multiple survival signals and enhance self-repair mechanisms have the potential to shift the cell survival rheostat to favor survival and therefore improve functional outcome after TBI.