Visualization and quantification of NAD(H) in brain sections by a novel histo-enzymatic nitrotetrazolium blue staining technique.
Visualization and quantification of NAD(H) in brain sections by a novel histo-enzymatic nitrotetrazolium blue staining technique.
复制标题
DOI：
10.1016/j.brainres.2009.12.042
复制
发表时间：
2010-02-26
期刊：
影响因子：
2.9
通讯作者：
Kristian, Tibor
中科院分区：
文献类型：
--
作者：
Balan, Irina S.;Fiskum, Gary;Kristian, Tibor
关键词：
开发了一种用于可视化和定量脑组织中内源性 NAD(H) 的组织酶技术，该技术基于耦合酶循环反应，可还原硝基四唑蓝氯化物产生甲臜。使用的条件是烟酰胺腺嘌呤二核苷酸 (NAD(H)) 的内源水平是甲臜产生的速率限制因素。通过添加烟酰胺单核苷酸（NAD+糖水解酶的抑制剂），可以最大程度地减少解冻组织孵育过程中发生的 NAD+ 自发降解。使用在断头后立即和 30 分钟后从大鼠身上获得的大脑冷冻切片来确定单独缺血对大脑 NAD(H) 水平和神经解剖学分布的影响。缺血性损伤导致海马 CA1 锥体神经元层、顶叶皮层和纹状体中甲臜生成率下降 50% 以上，但海马 CA3 和齿状回 (DG) 亚区域则没有。通过利用脑样本高氯酸提取物中存在的 NAD(H) 的分光荧光法测量，证实了缺血引起的 NAD(H) 水平变化。这种新的组织酶技术适用于可视化和量化大脑中相对 NAD(H) 水平。该测定可用于识别可能导致神经退行性变的区域选择性 NAD(H) 分解代谢。
A histo-enzymatic technique for visualizing and quantifying endogenous NAD(H) in brain tissue was developed, based on coupled enzymatic cycling reactions that reduce nitrotetrazolium blue chloride to produce formazan. Conditions were used where the endogenous level of nicotinamide adenine dinucleotides (NAD(H)) was the rate limiting factor for formazan production. Spontaneous degradation of NAD+ that occurs during incubation of thawed tissue was minimized by the addition of nicotinamide mononucleotide, an inhibitor of NAD+ glycohydrolases. Cryostat sections of brains obtained from rats immediately after decapitation and at 30 min later were used to determine the effects of ischemia alone on brain NAD(H) levels and neuroanatomic distribution. The ischemic insult resulted in a greater than 50% decline in the rate of formazan generation in the CA1 pyramidal neuronal layer of the hippocampus and in the parietal cortex and striatum, but not in the CA3 and dentate gyrus (DG) subregions of the hippocampus. The ischemia-induced changes in NAD(H) levels were confirmed by utilizing spectrofluorimetric measurements of NAD(H) present in perchloric acid extracts of brain samples. This new histo-enzymatic technique is suitable for visualizing and quantifying relative NAD(H) levels in the brain. This assay could prove useful in identifying region-selective NAD(H) catabolism that may contribute to neurodegeneration.
登录
查看更多内容
DOI：
10.1016/j.vascn.2006.02.010
发表时间：
2006-11-01
影响因子：
1.9
作者：
Khalil, Philipe N.;Siebeck, Matthias;Fritz, Hans
通讯作者：
Fritz, Hans
影响因子：
12.7
作者：
LISZCZAK, TM;HEDLEYWHYTE, ET;ZERVAS, NT
通讯作者：
ZERVAS, NT
影响因子：
9.5
作者：
KLEIN, HH;SCHAPER, J;SCHAPER, W
通讯作者：
SCHAPER, W
影响因子：
4.7
作者：
SNELL, CR;SNELL, PH;RICHARDS, CD
通讯作者：
RICHARDS, CD
影响因子：
8.3
作者：
BEDERSON, JB;PITTS, LH;BARTKOWSKI, HM
通讯作者：
BARTKOWSKI, HM