吸收外来学生，功课共同开展研究 开展内皮素诱导大鼠脑缺血模型后1年，从山东来了两个学生，加入我们的小组。我感觉他们的基础比较差，就安排他们进行比较简单的实验，这个时候我们已经比较清楚高压氧预适应的效果非常好。主要问题是要用比较好模型和研究手段，那么我们就选择新生儿脑缺血模型，这个模型的特点是重复性非常好。另外美国有一个人也发表了文章开展这方面的研究。我们很快建立了模型，（该学生的 进步非常快，让我改变了过去的误解，基础差与是否优秀没有关系）。而且很快验证了 美国人的结果，结果很让我吃惊：效果太好了。于是我们选择细胞碉亡为指标，通过进行组织化学和酶活性测定等方法。确定了效果，并提出高压氧预适应效应与细胞碉亡的关系密切（这个想法很没有新意，但这个方向我们是比较早作的）。 文章投出后1个月就发表了，结果有一些数据没有用上，因为学生着急回去，没有继续延续，有一些遗憾。实际上这个方面有很多工作都可以继续下去。到目前，也只有美国和我们两篇文章。需要做的工作很多。我非常喜欢这个模型。后来我们用这个模型开展了呼吸氢和注射氢水的工作，都是开创性的，这个模型给我们许多惊喜。当然最近我们又做了一些这方面的工作。只是进度比较慢。 Brain Res. 2008 Feb 27;1196:151-6. Epub 2007 Dec 28. Links Mechanism of hyperbaric oxygen preconditioning in neonatal hypoxia-ischemia rat model. Li Z , Liu W , Kang Z , Lv S , Han C , Yun L , Sun X , Zhang JH . Department of Pathology, Weifang Medical College, Shandong, 261042, PR China. Hypoxic ischemic (HI) injury in neonates damages brain tissues. We examined the mechanism of hyperbaric oxygen preconditioning (HBO-PC) in neonatal HI rat model. Seven-day-old rat pups were subjected to left common carotid artery ligation and hypoxia (8% oxygen at 37 degrees C) for 90 min. HBO (100% O(2), 2.5 atmospheres absolute for 2.5 h) were administered by placing pups in a chamber 24 h before HI insult. Brain injury was assessed by the survival rate, 2,3,5-triphenyltetrazolium chloride (TTC), Nissl, TUNEL straining and caspase-3,caspase-9 activities after HI. In HBO preconditioned animals, survival rate was increased, infarct ratio was decreased, and the positive stained TUNEL cells were reduced, accompanied by the suppression of caspase-3 and -9 activities. These results indicate that a single HBO-PC appears to provide brain protection against HI insult via inhibition of neuronal apoptosis pathways. 新生儿脑病
意外收获，发现高氧可诱导机体对氧的敏感性增加 本来计划研究反复高压氧暴露对低氧耐受的机制，从NO途径进行探讨，但发现过去国外曾有人发现高压氧暴露能引起动物对氧的敏感性增强，就是更容易发生中毒，氧中毒本来就是我一直关注的课题，那么就顺便看一下，结果确实是这样的，而且没有人采用我们的观测手段，于是就从NOS和NO角度对氧敏感性进行了分析。文章很快被接受，十分意外。后来总结经验是，只要你的分析合理，实验结果可靠。意义有的时候不一定完全符合自己的判断。 这个实验比较简单，没有脑电测定和血流测定。 从这个文章我想到，采用一氧化碳和硫化氢研究氧中毒更可行。过去我们的研究生已经研究过一氧化碳，可惜没有在国外发表。文章可惜了。 Brain Res. 2008 Mar 27;1201:128-34. Epub 2008 Feb 5. Links Repetitive hyperbaric oxygen exposures enhance sensitivity to convulsion by upregulation of eNOS and nNOS. Liu W , Li J , Sun X , Liu K , Zhang JH , Xu W , Tao H . Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University, and Department of Neurology, Changhai Hospital, Shanghai, 200433, PR China. BACKGROUND: Repetitive hyperbaric oxygen (HBO) exposures as preconditioning methods produce ischemic tolerance, but may increase the risk of convulsions in patients. The purpose of this study was to investigate the mechanisms in increased sensitivity to convulsions and the role of nitric oxide (NO) and its synthases after repetitive HBO exposures. METHODS: Mice were randomly assigned into three groups: HBO group, hyperbaric air (HBA) group and normobaric air (NBA) group. Mice in HBO or HBA group were exposed to hyperbaric oxygen or hyperbaric air respectively for 60 min twice daily for 3 consecutive days (2.5 atmosphere absolute ). 24 h after the last exposure, mice were exposed to HBO (100% O2, 6 ATA). The latency of convulsions was recorded. In addition, the levels of NO, NADPH-diaphorase, mRNA and protein expressions of NOS isoforms in hypothalamus and hippocampus were determined. RESULTS: Latency to seizures was significantly shortened in mice after six HBO pre-exposures. The level of NO in hypothalamus in HBO group was increased. The number of NADPH-d positive cells and the levels of protein and mRNA of eNOS and nNOS in hypothalamus and hippocampus were increased. CONCLUSION: After repeated HBO exposures, elevated NO may enhance the sensitivity to convulsions and this may lead to seizures during the subsequent oxygen exposures. Prevention of seizures is needed when HBO is used as preconditioning method. PMID: 18342297 全文
遇到障碍，回到脑缺血预防 第一篇文章完成后，我们投稿发生了问题，审稿专家认为我们的实验不够人道，我们是将多只动物放在低氧舱，观察致死时间，确实有点残忍，但这样的结果比较准确，实验组与对照组放在同样的条件下。这些结果我们反复多次进行研究。绝对可靠，效果十分好。遇到这样的问题，我们就暂时把文章放下，没有继续投稿，实际上这个决定是错误的，主要是当时的信心不足。 为了解决这个令人尴尬的问题，我们又回到经典的模型上，正好我在加拿大学习时掌握一种制备脑缺血的模型，这个模型的好处是稳定，几乎有100%成功，当然稳定性并不很好。这对我们的实验已经比较好。 进行预实验的研究，模型的成功率非常重要，因为失败的模型会影响实验结果的可靠性。不象治疗，我可以在模型结束后再分组。预实验的不能这样。 我把这个模型教会学生，就去做了，因为我们当时不知道采用什么样的暴露方案最有效，只能参考日本人的一个文章，采用5次10天的暴露方法，这个方案十分麻烦。给我们造成了不少困难。现在想想是当时不够大胆。先重复别人的也可以理解。因为我们已经发现了HIF这个分子很重要，我们就以这个分子为中心进行探讨。我们用这个模型确定了高压氧能诱导HIF，而且其活性是增加的。我们采用了DNA结合活性和PCR方法证明了这个观点。文章被美国应用生理学杂志接受，是目前我们发表的最好的文章。我一直很骄傲。 1: J Appl Physiol. 2008 Apr;104(4):1185-91. Epub 2008 Jan 3. Links Mechanism of ischemic tolerance induced by hyperbaric oxygen preconditioning involves upregulation of hypoxia-inducible factor-1alpha and erythropoietin in rats. Gu GJ , Li YP , Peng ZY , Xu JJ , Kang ZM , Xu WG , Tao HY , Ostrowski RP , Zhang JH , Sun XJ . Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433, People's Republic of China. We studied the effect of hyperbaric oxygen (HBO) preconditioning on the molecular mechanisms of neuroprotection in a rat focal cerebral ischemic model. Seventy-two male Sprague-Dawley rats were pretreated with HBO (100% O(2), 2 atmospheres absolute, 1 h once every other day for 5 sessions) or with room air. In experiment 1, HBO-preconditioned rats and matched room air controls were subjected to focal cerebral ischemia or sham surgery. Postinjury motor parameters and infarction volumes of HBO-preconditioned rats were compared with those of controls. In experiment 2, HBO-preconditioned rats and matched room air controls were killed at different time points. Brain levels of hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream target gene erythropoietin (EPO) analyzed by Western blotting and RT-PCR as well as HIF-1alpha DNA-binding and transcriptional activities were determined in the ipsilateral hemisphere. HBO induced a marked increase in the protein expressions of HIF-1alpha and EPO and the activity of HIF-1alpha, as well as the expression of EPO mRNA. HBO preconditioning dramatically improved the neurobehavioral outcome at all time points (3.0 +/- 2.1 vs. 5.6 +/- 1.5 at 4 h, 5.0 +/- 1.8 vs. 8.8 +/- 1.4 at 8 h, 6.4 +/- 1.8 vs. 9.7 +/- 1.3 at 24 h; P 0.01, respectively) and reduced infarction volumes (20.7 +/- 4.5 vs. 12.5 +/- 3.6%, 2,3,5-Triphenyltetrazolium chloride staining) after cerebral ischemia. This observation indicates that the neuroprotection induced by HBO preconditioning may be mediated by an upregulation of HIF-1alpha and its target gene EPO. PMID: 18174394 全文
标签: 脑缺血
