Brahma Manoja Kumar, Dohare Preeti, Varma Saurabh, Rath Srikanta Kumar, Garg Puja, Biswal Prasanta Kumar, Chowdhury Piyali Dhar, Ray Madhur
Division of Pharmacology, Central Drug Research Institute, Lucknow, India.
J Neurosci Res. 2009 May 1;87(6):1400-11. doi: 10.1002/jnr.21960.
Global ischemia was induced in gerbil by bilateral occlusion of the common carotid arteries for 5 min. Sodium ionophore monensin or sodium channel blocker tetrodotoxin (TTX) was administered at doses of 10 micorg/kg, i.p., 30 min before ischemia induction; the dose was repeated after 22 hr. Subsequently, brain infarct occurred, determined at 24 hr after occlusion. Large, well-demarcated infarcts were observed in both hemispheres, an important observation because it critically influences the interpretation of the data. Because nitric oxide (NO) production is thought to be related to ischemic neuronal damage, we examined increases in Ca(2+) influx, which lead to the activation of nitric oxide synthase (NOS). Then we evaluated the contributions of neuronal NOS, endothelial NOS, and inducible NOS to NO production in brain cryosections. The cytosolic release of apoptogenic molecules like cytochrome c and p53 were confirmed after 24 hr of reflow. TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) labeling detected the apoptotic cells, which were confirmed in neuron-rich cell populations. After 24 hr, all the ischemic changes were amplified by monensin and significantly attenuated by TTX treatment. Additionally, the nesting behavior and histological outcomes were examined after 7 day of reflow. The neuronal damage in the hippocampal area and significant decrease in nesting scores were observed with monensin treatment and reduced by TTX pretreatment after day 7 of reflow. To our knowledge, this report is the first to highlight the involvement of the voltage-sensitive Na(+) channel in possibly regulating in part NO system and apoptosis in a cytochrome c-dependent manner in global ischemia in the gerbil, and thus warrants further investigation.
通过双侧颈总动脉闭塞5分钟在沙鼠中诱导全脑缺血。在缺血诱导前30分钟腹腔注射剂量为10微克/千克的钠离子载体莫能菌素或钠通道阻滞剂河豚毒素（TTX）；22小时后重复该剂量。随后，在闭塞后24小时确定脑梗死情况。在两个半球均观察到界限清楚的大面积梗死灶，这一重要观察结果对数据解读有至关重要的影响。因为一氧化氮（NO）的产生被认为与缺血性神经元损伤有关，所以我们检测了导致一氧化氮合酶（NOS）激活的Ca(2+)内流增加情况。然后我们评估了神经元型NOS、内皮型NOS和诱导型NOS对脑冰冻切片中NO产生的作用。再灌注24小时后证实了细胞色素c和p53等凋亡分子的胞质释放。TUNEL（末端脱氧核苷酸转移酶dUTP缺口末端标记）标记检测到了凋亡细胞，这些细胞在富含神经元的细胞群体中得到证实。24小时后，莫能菌素放大了所有缺血性变化，而TTX处理则显著减轻了这些变化。此外，在再灌注7天后检查筑巢行为和组织学结果。再灌注7天后，观察到莫能菌素处理导致海马区神经元损伤且筑巢评分显著降低，而TTX预处理可减轻这种损伤。据我们所知，本报告首次强调了电压敏感性Na(+)通道可能部分以细胞色素c依赖的方式调节沙鼠全脑缺血中的NO系统和细胞凋亡，因此值得进一步研究。
