<i>In vitro</i> gene knockdown with siRNA oligonucleotides.

<p>Knockdown of (A) neuronal nitric oxide synthase (nNOS) and (B) glutathione peroxidase-1 (GPx-1) with <i>Silencer</i> pre-designed siRNAs (Ambion) in H19-7 rat hippocampal progenitor cells. All three nNOS and GPx-1 siRNA oligos significantly reduced gene expression in cultured cells.</p

Inhibition of Rock and Lim kinase in Rho-Rock-Lim kinase pathway by AAV-nNOS siRNA suggests a mechanism for AAV nNOS-mediated neuroprotection.

<p>(T) = Traumatic brain injury, (N) = nNos (neuronal nitric oxide synthase), (G) = GPx-1 (Glutathione peroxidase-1). Within the pathways, genes that are labeled green are down-regulated and genes that are in red are considered up-regulated compared to uninjured control expression.</p

Neurobehavioral assessment after AAV siRNA treatment.

<p>Neurobehavioral assessment after AAV siRNA treatment.</p

Gene expression analysis of AAV treated rat brains.

<p>(A) Heat map from microarray analysis (rat Agilent whole genome arrays) of hippocampal gene expression two weeks after siRNA virus treatment. NNOS knockdown has strong effects on injury-associated gene expression. The effects of GPx-1 knockdown were more subtle. (B) AAV mediated gene silencing was very specific; only nNOS knockdown had effects on the canonical nitric oxide signaling pathway. (C) Confirmation of virus induced changes in genes affected by AAV siRNA treatments. All selected gene targets of nNOS knockdown were confirmed and four of six gene targets of GPx-1 knockdown showed expression trends that matched the microarray results.</p

Stereological assessment of neuronal injury.

<p>GPx-1 (2) and nNOS (3) were selected for <i>in vivo</i> assessment of neurodegeneration based on initial results of the qPCR and neurobehavioral tests. Only AAV nNOS (3) significantly reduced numbers of degenerating, Fluoro-Jade-positive (FJ+) cells in the TBI rat hippocampus.</p

Quantitative real-time PCR analysis of laser captured hippocampal neurons.

<p>(A) Laser capture microdissection of AAV siRNA treated hippocampal pyramidal neurons for subsequent quantitative real time PCR (qPCR) analysis. Nissl stained image showing the precise capture of pyramidal neurons for subsequent qPCR analysis of nNOS or GPx-1 expression. (B, C) QPCR analysis of nNOS and GPx-1 expression in laser captured neurons from brains of all control, TBI and virus-treated rats two weeks after siRNA virus treatment.</p

Comparison of AAV-mediated gene silencing on three canonical downstream cell signaling pathways essential for neuronal function.

<p>(A) Ephrin receptor signaling (B) CREB signaling (C) GABA receptor signaling.</p