Altered neurotrophin expression profiles in the nucleus of the solitary tract of spontaneously hypertensive rats. Rattus norvegicus

Our previous findings suggest that the nucleus of the solitary tract (NTS), a pivotal region for regulating the set-point of arterial pressure, exhibits abnormal inflammation in pre-hypertensive and spontaneously hypertensive rats (SHRs) together with elevated anti-apoptotic and low apoptotic factor levels compared with that of normotensive Wistar–Kyoto (WKY) rats. Whether this chronic condition affects neuronal growth and plasticity in the NTS remains unknown. To unveil the characteristics of the neurodevelopmental environment in the NTS of hypertensive rats, we investigated the gene expression profile of neurotrophins and their receptors in SHRs compared to that of normotensive rat WKY. Overall design: The NTS was dissected from the brain of 6 SHRs and 6 WKY rats and the total RNA was extracted. In both groups of rats (SHRs & WKY rats, n = 6 each), a total of 2 ug mRNA extracts from each NTS were pooled together, treated with RNase-free DNAse I (Invitrogen Life technologies) to remove any genomic contamination, and further purified using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription was subsequently performed on 1 ug total RNA using SuperArray’s RT2 First Strand Kit (SABiosciences); the resulting cDNA was submitted for real-time quantitative PCR reactions on RT2 ProfilerTM PCR array plates using Superarray RT2 SYBR Green qPCR Master Mix (SAbiosciences) and iCycler iQ thermal cycler (Bio-rad), following the manufacturer’s instructions. The experiment was performed in duplicate in each group.

我们此前的研究结果显示，孤束核（nucleus of the solitary tract, NTS）作为调控动脉血压调定点的关键脑区，在高血压前期大鼠与自发性高血压大鼠（spontaneously hypertensive rats, SHRs）中存在异常炎症反应，且与正常血压Wistar-Kyoto（WKY）大鼠相比，其抗凋亡因子水平升高、促凋亡因子水平降低。目前尚不清楚这种慢性炎症状态是否会影响孤束核内的神经元生长与可塑性。为阐明高血压大鼠孤束核内神经发育微环境的特征，本研究对比了自发性高血压大鼠与正常血压Wistar-Kyoto大鼠的神经营养因子及其受体的基因表达谱。

总体实验设计：
从6只自发性高血压大鼠与6只正常血压Wistar-Kyoto大鼠的脑组织中分离孤束核并提取总RNA。两组大鼠（自发性高血压组与正常血压组，每组n=6）均将每只大鼠孤束核提取的2μg总mRNA混合，经无核糖核酸酶脱氧核糖核酸酶I（RNase-free DNase I，Invitrogen Life Technologies）处理以去除基因组DNA污染，随后按照试剂盒说明书使用RNeasy迷你试剂盒（RNeasy mini kit，Qiagen）进行进一步纯化。随后使用SuperArray RT2第一链合成试剂盒（SuperArray’s RT2 First Strand Kit，SABiosciences）以1μg总RNA进行逆转录反应；所得cDNA按照说明书使用SuperArray RT2 SYBR Green实时定量PCR预混液（Superarray RT2 SYBR Green qPCR Master Mix，SABiosciences）与iCycler iQ实时荧光定量PCR仪（iCycler iQ thermal cycler，Bio-rad），在RT2 Profiler™ PCR芯片板上完成实时定量聚合酶链反应（real-time quantitative PCR, qPCR）。每组实验均重复进行两次。