Pathway-Focused PCR Array Profiling of Enriched Populations of Laser Capture Microdissected Hippocampal Cells after Traumatic Brain Injury

Cognitive deficits in survivors of traumatic brain injury (TBI) are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays) to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive) or surviving (Fluoro-Jade- negative) pyramidal neurons obtained by laser capture microdissection (LCM). In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER) stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.创伤性脑损伤（traumatic brain injury, TBI）幸存者的认知功能缺损，与海马体等脑区内的不可逆神经退行性变密切相关。针对凋亡神经元与存活神经元开展比较基因表达分析，可为潜在治疗靶点的发掘提供重要科学见解。本研究采用两款通路特异性PCR芯片（RT2 Profiler细胞凋亡与神经营养因子及其受体PCR芯片），对通过激光捕获显微切割（laser capture microdissection, LCM）获取的凋亡（荧光金阳性，Fluoro-Jade-positive）或存活（荧光金阴性，Fluoro-Jade-negative）锥体神经元中，创伤性脑损伤诱导的基因表达情况进行鉴定与验证。在细胞凋亡PCR芯片检测中，与邻近的存活神经元相比，凋亡神经元的细胞死亡、炎症及内质网（endoplasmic reticulum, ER）应激相关基因的表达水平显著升高。同时，相较于存活神经元，凋亡神经元中具备多效性功能的促存活基因的表达亦显著上调，这表明即便遭受不可逆损伤的神经元，亦能够启动保护性应答反应。在神经营养因子及其受体PCR芯片检测中，该芯片涵盖的基因通常在两组海马神经元中均有表达，但仅少数基因在凋亡与存活神经元间存在显著表达差异。针对筛选出的差异表达蛋白开展的免疫组织化学分析，验证了基因表达检测结果。本研究首次针对创伤性脑损伤后大脑中已鉴定的凋亡与存活神经元群体，开展了通路聚焦型PCR芯片表达谱分析。将可精准获取特定细胞的激光显微切割技术与通路聚焦型PCR芯片分析相结合，是一种兼具实用性与低成本优势的微阵列（microarrays）替代方案，可为靶向改善创伤性脑损伤诱导的神经退行性变的神经保护信号研究提供新视角。