Diabet Med. 2012 Aug;29(8):986-9. Hyperbaric oxygen therapy improves peripheral insulin sensitivity in humans. Wilkinson D, Chapman IM, Heilbronn LK. SourceHyperbaric Unit, Royal Adelaide Hospital, The University of Adelaide, Adelaide, SA, Australia. Abstract AIM: Hyperbaric oxygen therapy is known to reduce fasting blood glucose in individuals with Type 2 diabetes. However, the mechanisms of this effect are not clear. The aim of this study was to determine whether peripheral insulin sensitivity by hyperinsulinaemic euglycaemic clamp is increased in patients presenting for hyperbaric oxygen therapy. METHODS: Participants were non-obese individuals without Type 2 diabetes (n=5) or obese patients with Type 2 diabetes (n=5). Patients were given 100% oxygen at 2.0 absolute atmospheres for 2 h, six sessions per week for 5 weeks. RESULTS: Peripheral insulin sensitivity was increased in the whole cohort (P=0.04). Subsequent analysis revealed that this was significant at both treatment 3 (+37.3 ± 12.7%, P=0.02) and treatment 30 (+40.6 ± 12.6%, P=0.009). HbA(1c) was significantly reduced in subjects without diabetes only (P0.05). CONCLUSION: Insulin sensitivity increased within 3 days of hyperbaric oxygen treatment and this was maintained for 30 sessions. This increase in insulin sensitivity is equivalent to that observed following moderate weight loss. The mechanisms underlying the insulin-sensitizing effect of hyperbaric oxygen require further elucidation.
关于高压氧治疗疾病经常有一些传奇故事，例如曾有的人把高压氧作为提高高考成绩的手段，有的人认为高压氧有美容效果，也有人甚至把高压氧作为治疗百病的良药，但许多所谓的效果并没有明确的客观证据。 认为高压氧可治疗多种疾病的人认为，氧气是人体必须的物质，而由于氧气难以溶解，人体需要依赖血红蛋白才能满足组织对氧气运输数量的需要，普通的吸氧由于氧分压限制，很难能大幅度提高机体摄取氧气的能力，而高压氧可以大幅度提高溶解氧气的比例，极大地提高了纠正组织缺氧的能力，所以只要存在缺血缺氧，就可以使用高压氧。 由于氧气本身属于自由基，存在很强的毒性，当高压氧在压力超过部分组织，如肺组织耐受的程度，如果长时间持续呼吸高压氧，必然导致组织中毒，或者说氧化损伤。不过这种氧的毒性，需要一定的暴露时间和压力，如果限制暴露时间和压力，仍可以避免发生氧气中毒。因此为了既充分利用氧气纠正缺氧的好处，同时避免发生中。临床上的一贯作法是间歇性使用一定压力限制的高压氧。例如，每天呼吸 1 到 2 次，压力低于 2.5ATA ，持续时间 1-2 小时。具体使用情况或根据病情需要可能有所不同。 许多疾病都因为组织缺血缺氧，根据高压氧治疗疾病的基本原理，就是纠正缺氧，那么有多种治疗效果就值得期待。但不幸地是，临床上确定效果总是要按照客观证据，许多临床的研究表明，高压氧治疗并不是那么有效，甚至有时候存在不利的影响，这正是这一领域经常遇到的尴尬局面。 最近一些年，关于高压氧的效应研究，有人提出一种观点，高压氧治疗疾病可能不只是纠正缺氧那么简单，人体作为一种复杂的生物系统，对外来不良刺激有固有的适应能力，氧气本身的毒性也有可能成为这种不良刺激的一种类型，由于高压氧本身在临床上应用的安全性得到广泛认可，只是对有效性存在争议。那么如果从这个角度认识，对解释一些看上去比较意外的效果应该是一种好的策略。于是一种利用氧气有毒刺激机体产生对抗其他损伤因素的观点，就是高压氧预适应的研究遍出现了。并逐渐被临床上广泛接受和认可，作为一种解释效果的常见“机制”。 最近来自美国 Connecticut, 大学的一项研究表明，高压氧暴露具有预防紫外线导致的皮肤损伤，或者说具有美容效果。 研究采用每周（一、三、五） 3 次照射紫外线，每周 2 次（周一和三）或 4 次（周一到周四）高压氧治疗，连续进行 22 周，结果发现，没周 4 次的高压氧对紫外线引起的皮肤细胞增殖和凋亡有显著的抑制效果，对皮肤皱纹深度有明显的限制作用。当然这个研究不能作为建议用高压氧作为美容的常规手段，但对一些可能暴露在过度紫外线照射的人群，不失为一种理想的方法。显然用纠正缺氧来解释美容效果就比较牵强了。本研究作者认为，这种效果的根本原因是由于氧气的预适应效果。更令人称奇的是，作者观察到紫外线暴露可以引起肝脏组织的氧化应激，而高压氧也可以限制这种应激。为什么皮肤照射可以引起肝脏的反应，作者认为是氧化损伤导致一些系统反应信号导致的全身应激，也就是说，照紫外线可能会导致全身的应激反应。 预适应和皮肤损伤美国.pdf Hyperbaric oxygen preconditioning protects skin from UV-A damage AshleyM.Fuller 1 , CharlesGiardina 1 , LawrenceE.Hightower 1 , GeorgeA.Perdrizet 2 and CassandraA.Tierney 1 Abstract Hyperbaric oxygen therapy (HBOT) is used for a number of applications, including the treatment of diabetic foot ulcers and CO poisoning. However, we and others have shown that HBOT can mobilize cellular antioxidant defenses, suggesting that it may also be useful under circumstances in which tissue protection from oxidative damage is desired. To test the protective properties of hyperbaric oxygen (HBO) on a tissue level, we evaluated the ability of a preconditioning treatment regimen to protect cutaneous tissue from UV-A-induced oxidative damage. Three groups of hairless SKH1-E mice were exposed to UV-A 3days per week for 22weeks, with two of these groups receiving an HBO pretreatment either two or four times per week. UV-A exposure increased apoptosis and proliferation of the skin tissue, indicating elevated levels of epithelial damage and repair. Pretreatment with HBO significantly reduced UV-A-induced apoptosis and proliferation. A morphometric analysis of microscopic tissue folds also showed a significant increase in skin creasing following UV-A exposure, which was prevented by HBO pretreatment. Likewise, skin elasticity was found to be greatest in the group treated with HBO four times per week. The effects of HBO were also apparent systemically as reductions in caspase-3 activity and expression were observed in the liver. Our findings support a protective function of HBO pretreatment from a direct oxidative challenge of UV-A to skin tissue. Similar protection of other tissues may likewise be achievable
焦辉医生博文 http://www.sciencenet.cn/blog/user_content.aspx?id=13926 所提高压氧治疗的适应症和时间：脑外伤是半个月，缺氧性脑病为20次以内，煤气中毒是7次以内，另外要掌握一下颅内压力，颅压正常可以做，高颅压或低颅压患者都不适合做。抽搐和气管切开不能做。不知依据来自何处？你是否有这方面的临床经验？我是北京天坛医院高压氧科主任，神经病学博士，神经病学研究生导师，主任医师。我专门从事高压氧神经病学的临床和科研工作，你上文所提的内容应该是指高压氧医学中的各种疾病的治疗剂量学问题，其内涵是很专业的，也极其复杂，也正是我们目前一直在潜心研究的内容，然结论远不像您文中表述的那么片面和轻率，甚至荒谬！ 从某种意义上来说，生命就是氧化还原反应。人类的大多数疾病究其发病原因、发生发展的病理过程，或因或果，都与缺氧密切相关。世界著名物理学家，有美国氢弹之父之称的爱德华?泰勒曾不无遗憾的说：It is not entirely impossible, that, perhaps sometime in the next decade, professors of medicine will have difficulty in explaining why the treatment with (hyperbaric) oxygen was not widely adopted much earlier.（很可能在十年以后某个时期，医学专家们将难以解释为什么没有在临床上更早一点的推广采用氧或高压氧来治疗疾病。） 我国有神外巨人之称的王忠诚院士在亲身做过20多次高压氧治疗后曾经动情的说：高压氧治疗的疗效真的很神奇，应当加强基础和临床研究！ 目前，美国高压氧神经病学专家曾多次报道高压氧治疗小儿颅脑损伤，其治疗次数高达100-200次以上，有关文献也证实，颅脑损伤患者通常高压氧治疗60次以上效果最为理想。我们北京天坛医院高压氧科主要从事众多神外病人颅脑损伤患者的复苏和恢复治疗，每日治疗患者50-60人次，所得剂量学认识也与你所引述大相径庭！另外，就说来我院救治的一氧化碳中毒迟发性脑病患者，只要一发病就来我院治疗的，几乎所有患者均经过我们合理用药和60次以上高压氧治疗病情达到临床痊愈，同时核磁结构及功能影响均显著好转（当然影像学改变与临床症状改善并不同步）；再说，您提到高压氧治疗颅脑损伤患者可引起脑萎缩、脑室增大...我想大概是您的臆断吧，不知是否有循证学依据？我认为，各种颅脑损伤，特别是全脑性缺血缺氧性损伤在各种治疗挽救可挽救脑组织基础上未能完全挽救的损伤脑组织都会逐渐出现坏死、软化，进而被吸收，经一段时间后在影像学上均会出现不同程度脑萎缩和病灶区域软化现象，是病情的自然发展结果，并不是高压氧治疗导致的。此外，作为一名神经科医生应该知道，导致脑室扩大的原因很多，例如常见的有因蛛网膜下腔出血后影响了脑脊液的循环导致的交通性脑积水，各种原因导致的梗阻性脑积水都会在脑损伤之后一定时期可能发生，造成影像学上的不同脑室的扩大现象。当然全脑损伤的一段时间后，因出现不同程度的脑萎缩，也会在影像学上表现为不同程度的脑室扩大现象。作为一名称职的神经科医生在患者损伤的初期就应该应该知道这些现象可能会发生，而且要在采用各种治疗手段积极挽救患者受损伤脑组织，使其最大程度的不至于形成坏死、软化或凋亡，造成将来脑萎缩，脑积水，甚至颅内高压的发生。一旦发现脑室扩大就要及时鉴别诊断清楚是否存在颅内高压，如果确定由脑脊液压力过高所致，应及时给予梗阻清除、脑室腹腔分流等颅脑手术，然后再继续采取内科手段，如高压氧、药物和康复治疗。 焦辉医生高压氧是急救治疗手段，不是越多越好 http://www.sciencenet.cn/blog/user_content.aspx?id=13926 一文所谈极不科学！我想这些专业问题应当具体情况具体分析，她应该抱有科学的态度才对！不该武断结论、客观上导致误导广大患者吧！
刚开始我们本来希望做对神经干细胞的影响,可是实验进展不太顺利, 就采用本学校生化的模型进行了这个研究. 主要从内皮细胞增生角度, 从HIF-VEGF进行分析.这个研究费力但效果不理想. 将来要从肝缺血方面进行深入研究更合适. Hyperbaric oxygen preconditioning promotes angiogenesis in rat liver after partial hepatectomy Ping Ren a , 1 , ZhiMing Kang b , 1 , GuoJun Gu b , Yun Liu b , WeiGang Xu b , HengYi Tao b , John H. Zhang c , XueJun Sun b , , and Hui Ji a , , a Department of Pharmacology, China Pharmaceutical University, Nanjing, 210009, PR China b Department of Diving Medicine, Faculty of Naval Medicine, Secondary Military Medical University, Shanghai, 200433, PR China c Department of Neurosurgery, Loma Linda University, Loma Linda, California, USA Received 25 October 2007; accepted 5 June 2008. Available online 24 June 2008. Abstract Hyperbaric oxygen preconditioning (HBO-PC) increases the level of HIF-1 (hypoxia inducible factor-1) and its target gene VEGF (vascular endothelial growth factor) which is involved in angiogenesis. Liver regeneration is an angiogenesis-dependent process. We hypothesized that HIF-1 and VEGF mediated the angiogenesis effect of HBO-PC on regenerating rat liver. Male Sprague Dawley rats received HBO-PC followed by 70% partial hepatectomy. Proliferation of hepatocytes and endothelial cells was evaluated by BrdU (bromodeoxyuridine) staining. Microvascular density was assessed by immunohistochemistry. mRNA expression of HIF-1 was assessed by quantitative RT-PCR and protein levels of HIF-1 and VEGF were assessed by western blot. HIF-1 DNA-binding activity was determined with an ELISA-based kit. HBO-PC increased the proliferation index of endothelial cells and microvascular density at 48h after partial hepatectomy. The protein level and DNA-binding activity of HIF-1 and the protein level of VEGF were increased by HBO-PC before and after partial hepatectomy. Partial hepatectomy alone also increased proliferation index and the expressions of HIF-1 and VEGF. Our results indicated that the angiogenesis effect of HBO-PC on liver after partial hepatectomy could be achieved by increased HIF-1 activity and VEGF expression. However, the angiogenic effect of HBO-PC is moderate and HBO-PC failed to produce additional effect on the enhancement of HIF-1 and VEGF induced by partial hepatectomy alone. Keywords: Angiogenesis; Hyperbaric oxygen preconditioning; Hypoxia inducible factor-1; Vascular endothelial growth factor Article Outline Introduction Materials and methods Animals and study protocols Liver regeneration HBO exposure Immunohistochemistry Western blot analysis HIF-1 DNA-binding assay RNA isolation and quantitative real-time PCR Data analysis Results Wet regeneration Proliferation index BrdU Microvascular density HIF-1 VEGF Discussion Acknowledgements References 全文
这个文章的投稿过程比较复杂,而且这个杂志收费比较厉害.但比较认真. 我们从NO途径来探讨预适应效果,从机制上有了一点深入. J Neurotrauma. 2009 Jan 18. Hyperbaric Oxygen Preconditioning Attenuates Early Apoptosis after Spinal Cord Ischemia in Rats. Wang L , Li W , Kang Z , Liu Y , Deng X , Tao H , Xu W , Li R , Sun X , Zhang JH . 1 Department of Anesthesiology, Changhai Hospital Affiliated to Second Military Medical University , Shanghai, P.R. China ., 2 Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University , Shanghai, P.R. China ., 3 Department of Physiology and Pharmacology, Loma Linda University School of Medicine , Loma Linda, California. Abstract This study tested the hypothesis that spinal cord ischemic tolerance induced by hyperbaric oxygen preconditioning (HBO-PC) is mediated by inhibition of early apoptosis. Male Sprague-Dawley rats were preconditioned with consecutive 4 cycles of 1-h HBO exposures (2.5 atmospheres absolute , 100% O(2)) at a 12-h interval. At 24 h after the last HBO pretreatment, rats underwent 9 min of spinal cord ischemia induced by occlusion of the descending thoracic aorta in combination with systemic hypotension (40 mmHg). Spinal cord ischemia produced marked neuronal death and neurological dysfunction in animals. HBO-PC enhanced activities of Mn-superoxide dismutase (Mn-SOD) and catalase, as well as the expression of Bcl-2 in the mitochondria in the normal spinal cord at 24 h after the last pretreatment (before spinal cord ischemia), and retained higher levels throughout the early reperfusion in the ischemic spinal cord. In parallel, superoxide and hydrogen peroxide levels in mitochondria were decreased, cytochrome c release into the cytosol was reduced at 1 h after reperfusion, and activation of caspase-3 and -9 was subsequently attenuated. HBO-PC improved neurobehavioral scores and reduced neuronal apoptosis in the anterior, intermediate, and dorsal gray matter of lumbar segment at 24 h after spinal cord ischemia. HBO-PC increased nitric oxide (NO) production. L-nitroarginine-methyl-ester (L-NAME; 10 mg/kg), a nonselective NO synthase (NOS) inhibitor, applied before each HBO-PC protocol abolished these beneficial effects of HBO-PC. We conclude that HBO-PC reduced spinal cord ischemia-reperfusion injury by increasing Mn-SOD, catalase, and Bcl-2, and by suppressing mitochondrial apoptosis pathway. NO may be involved in this neuroprotection. PMID: 19196076 全文下载
Hyperbaric oxygen preconditioning reduces ischemiareperfusion injury by inhibition of apoptosis via mitochondrial pathway in rat brain J.-S. Li a , 1 , W. Zhang a , 1 , Z.-M. Kang b , S.-J. Ding a , , , W.-W. Liu b , J.H. Zhang c , Y.-T. Guan a and X.-J. Sun b , , a Department of Neurology, Changhai Hospital, 168 Changhai Road, Shanghai 200433, PR China b Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433, PR China c Department of Neurosurgery, Loma Linda University, Loma Linda, CA, USA Accepted 5 January 2009. Available online 13 January 2009. Abstract This study examined the hypothesis that apoptotic inhibition via mitochondrial pathway was involved in hyperbaric oxygen preconditioning (HBO-PC)induced neuroprotection on ischemiareperfusion injury in rat brain. Male SpragueDawley rats (250 280 g, n =144) were divided into control, middle cerebral artery occlusion (MCAO) for 90 min, and HBO-PC plus MCAO groups. HBO-PC was conducted four times by giving 100% oxygen at 2.5 atm absolute (ATA), for 1 h at 12 h intervals for 2 days. At 24 h after the last HBO-PC, MCAO was performed and at 24 h after MCAO, neurological function, brain water content, infarct volume, and cell death were evaluated. Enzymatic activity of capase-3 and 9, and expression of cytochrome c , Bcl-2 and Bax proteins were performed in the samples from hippocampus, ischemic penumbra and core of the brain cortex, respectively. HBO-PC reduced brain edema, decreased infarction volume, and improved neurological recovery. HBO-PC reduced cytoplasm cytochrome c levels, decreased caspase enzyme activity, upregulated the ratio of Bcl-2 and Bax expression, and abated the apoptosis of ischemic tissue. HBO-PC protects brain tissues from ischemiareperfusion injury by suppressing mitochondrial apoptotic pathways. Key words: hyperbaric oxygenation; prevention; middle cerebral artery occlusion; apoptosis; ischemic penumbra Abbreviations: CCA, common carotid artery; ECA, external carotid artery; HBO, hyperbaric oxygen; HBO-PC, hyperbaric oxygen preconditioning; ICA, internal carotid artery; MCAO, middle cerebral artery occlusion; PBS, phosphate-buffered saline; TTC, 5-triphenyltetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling Article Outline Experimental procedures Experimental groups HBO-PC MCAO procedures Neurobehavioral functional scoring TTC staining Brain water content In situ labeling of DNA fragmentation Ischemic core and penumbra dissections Caspase activity assay Western blot Statistical analysis Results Neurological score TTC staining Brain water content TUNEL staining Activities of caspase-3 and 9 Western blot of cytochrome c, Bcl-2 and Bax Discussion Conclusion Acknowledgements Supplementary data References Hypoxic or ischemic preconditioning may have clinical potentials for neuroprotection for neurosurgery patients who undergo temporary clipping of major intracranial vessels during aneurismal or cerebral bypass surgery procedures. The neuroprotective effect of preconditioning has been established in animal models ( , and ) and observed in clinical cases after multiple transient ischemic attacks ( Sitzer et al., 2004 ). However, the safety concerns and practical feasibility have limited the application of preconditioning in practice. Hyperbaric oxygen (HBO) has been used for multiple neurological diseases ( , , and ) and proved a safe treatment modality in all age and gender groups, including neonates ( Calvert et al., 2004 ) and pregnant mothers ( Xiao et al., 2006 ). Hyperbaric oxygen preconditioning (HBO-PC) has been reported to increase ischemic tolerance against neuronal injury in animals ( , , and ) Recently, we have observed that a single dose of HBO-PC reduced hypoxicischemic brain injury in neonatal rats ( Freiberger et al., 2006 ), However, the dosage, the timing of application, and the mechanisms of HBO-PC remain to be determined ( and ). Therefore, we examined the neuroprotective effect and mechanisms of a short term HBO-PC in an established middle cerebral artery occlusion (MCAO) rat model. Experimental procedures Experimental groups The Experimentation Ethics Committee of the Second Military Medical University in Shanghai, China approved the animal protocols of this study. All methods and animal procedures were met or exceeded all federal guidelines for the humane use of animals in research. All efforts were made to minimize the number of animals used and their suffering. A total of 144 male SpragueDawley rats weighing 250 280 g (Slaccas, Shanghai, China) were used. They were housed at a temperature of 2224 C and 12 h light/dark cycle controlled environment with free access to food and water prior to and following surgery. Rats were randomly assigned to one of the following three groups: control group ( n =24), MCAO group ( n =60), and HBO-PC plus MCAO group ( n =60). All animals were sacrificed at 24 h after MCAO. HBO-PC HBO-PC was administered by using 100% oxygen at 2.5 atmosphere absolute for 1 h at 12 h intervals four times in 2 days. The last HBO-PC was performed at 24 h before MCAO. Compression was performed at 1 kg/cm 2 /min and decompression was performed at 0.2 kg/cm 2 /min. None of the animals had seizures during or after HBO-PC. Chamber temperature was maintained between 22 and 25 C. Accumulation of CO 2 was prevented by using a small container with calcium carbonate crystals. To minimize the effects of diurnal variation, all exposures were started at 8:00 am . MCAO rats were placed in the same rodent chamber for 1 h at 12 h interval for 2 days in room air. MCAO procedures MCAO was produced by the filament model initially reported by Zea-Longa et al. (1989) with some modifications. Briefly, the rats were anesthetized with an i.p. injection of 2% pentobarbital sodium (40 mg/kg) and were allowed to breathe spontaneously. A supplemental anesthetic dose was added if necessary. Via a midline neck incision, the submandibular glands were separated to allow access to the right carotid artery. The common carotid artery (CCA), the external carotid artery (ECA), and the internal carotid artery (ICA) were isolated from connective tissues. The ECA and the proximal end of the CCA stump were cut and a suture loop was put around the distal end of the CCA. A microvascular clip was temporarily put on the ICA. A 0.24 mm diameter carbon fishing-line with a pretreated rounded tip was introduced via the CCA stump into the ICA. After removal of the clip, the filament was advanced further in the ICA until a resistance was felt at approximately 18.00.5 mm from the carotid bifurcation. The filament was fastened by tightening the loop around the distal CCA stump and the neck incision was closed. During surgery, a heating blanket was used to maintain the rectal temperature at 37.00.5 C. After 90 min MCAO, blood flow was restored by the withdrawal of the intraluminal suture and then the awaking rats were returned to their cages. Neurobehavioral functional scoring The neurological scoring systems proposed by Dean et al. (2003) and Ohlsson et al. (1995) were adopted with modifications as shown in Table 1 . All neurological evaluations were done blinded by a researcher without knowledge of the animal groups. The neurological testing was composed by thee sets: Zea-Longa score, beam-walking test, and prehensile traction test. The higher score represents severe deficits. Table 1. Neurological deficit score for rats Zea-longa score 0 The rat has no neurological defect 1 The rat is unable to extend affected forward limb 2 The rat circles while walking 3 The rat tumbles to its side because of hemiplegia while walking 4 The rat is unable to walk and unconsciousness is present 5 The rat is dead Beam-walking test 0 The rat crosses the beam with no foot slips 1 The rat crosses the beam with a few foot slips 2 The rat traverses the beam with more than 50% foot slips 3 The rat can traverse the beam, but the affected hind limb does not aid in forward locomotion 4 The rat falls down while walking 5 The rat is unable to traverse the beam but remains sitting across the beam 6 The rat falls down from the beam Prehensile traction test 0 The rat hangs on 5 seconds and brings rear limb up to rope 1 The rat hangs on 5 seconds, no third limb up to rope 2 The rat hangs on 3 to 4 seconds 3 The rat hangs on 0 to 2 seconds Full-size table Minimum score: 0, namely healthy rats; maximum score: 14, namely death. View Within Article TTC staining Infarct volume was determined by staining with 2,3,5-triphenyltetrazolium chloride (TTC). Briefly, the brains were quickly removed and placed at 20 C for 15 min, and then cut into five 2 mm coronal slices starting at 1 mm from the frontal pole. After incubation in 1% TTC in 0.2 mol/L phosphate-buffered saline (PBS) at 37 C for 30 min, the slices were fixed in 4% paraformaldehyde in 0.1 mol/L PBS. After 24 h, the sections were digitally photographed and infarction volumes were analyzed using image analysis system (Image J software ( http://www.quickvol.com ), a public domain image analysis program developed at the National Institutes of Health). The percentage of infarction (infarct ratio) was calculated by dividing the infarct volume by the total volume of the slices. Brain water content The brains were harvested and quickly separated to the left and right hemispheres, cerebellum and the brain stem. Brain samples were weighed on a precise electronic balance and placed in an oven at 100 C for 48 h. After 48 h, the samples were weighed again and the water content was calculated according to the following formula: 100 % . In situ labeling of DNA fragmentation The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay was performed on paraffin-embedded sections according to the manufacturer's instructions (Roche Molecular Biochemicals, Inc., Mannheim, Germany). The sections were dewaxed and rehydrated according to standard protocols, pretreated with proteinase K (20 g/mL in 0.01 mol/L PBS) for 15 min at room temperature. The slides were rinsed three times with PBS before they were incubated in TUNEL reaction mixture for 1 h at 37 C. We dried the area around the sample and added converter-AP to samples for 1 h at 37 C. After rinsing with PBS (5 min, three times), sections were stained in the dark with Nitroblue Tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP). For negative controls, the sections were incubated without terminal deoxynucleotidyl transferase. There were two distinct patterns of TUNEL staining. Some cells were densely labeled and showed clear apoptotic characteristics. Other cells were weakly labeled and considered to be necrotic cells. Only the densely labeled cells were counted as TUNEL positive cells. The ischemic core and penumbra of the cerebral cortex and CA1 of hippocampus were photographed (Leica, Germany) in each section. The data were represented as the number of cells per mm 2 . Ischemic core and penumbra dissections Ischemic core and penumbra were dissected according to well-established protocols in rodent models of unilateral proximal MCAO ( and ). Briefly, each hemisphere was cut longitudinally, from dorsal to ventral at 1.5 mm from the midline to exclude medial brain structures that were supplied primarily by the anterior cerebral artery. A transverse diagonal incision at approximately the 2 o'clock position separated the core from the penumbra. Caspase activity assay The activity of caspase-3 and 9 was measured with caspase-3 and caspase-9/CPP32 fluorometric Assay Kit (BioVision, Inc., USA). Briefly, brain samples from the ischemic core and penumbra or hippocampus were homogenized on the ice in ice-cold cell lysis buffer contained in the kit and centrifuged at 12,000 rpm for 15 min at 4 C, and then the supernatants were assayed for protein concentration by Enhanced BCA Protein Assay Kit. The treated samples were liquated and stored at 80 C until use. Equal amounts of the protein samples were incubated in a 96-well plate with 50 l of 2 Reaction buffer. Reactions were initiated by adding 5 l of the 1 mM DEVD-AFC substrate. After incubation in the dark at 37 C for 1 h, the plate was read in a fluorometer (Flexstation, Molecular Devices, USA) equipped with a 400-nm excitation filter and 505-nm emission filter. No tissue samples were added for negative controls. The results were expressed as a relative number (fluorescence intensity of sample/fluorescence intensity of negative controls). Western blot The sample proteins were extracted as described above. Equal amounts of the protein samples were loaded per lane and electrophoresed in 12% dodecylsulfatepolyacrylamide gel at 120 V (Mini-Protean III Electrophoresis System, Bio-Rad, USA) for 1 h. Proteins from gels were transferred at 110 mA for 100 min (for Bcl-2 and Bax) or at 70 V for 40 min (for cytochrome c ) onto a nitrocellulose filter membrane. Membranes were incubated overnight at 4 C with rabbit anti-Bcl-2 polyclonal antibody (1:1000 dilution, Chemicon International, Inc., USA), rabbit anti-Bax polyclonal antibody (1:1000 dilution, Stressgen Bioreagents, Corp., USA), cytochrome c antibody (1:1000 dilution, Cell Signaling Technology, Inc., USA), goat polyclonal -actin antibody (1:1000 dilution, Santa Cruz, Inc., USA), respectively, and then with horseradish peroxidaseconjugated secondary antibodies diluted at 1:1000 for 1 h at room temperature. The positive bands were revealed using enhanced chemiluminescence detection reagents (Pierce, USA) and autoradiography film. Protein bands were quantified by densitometry (Smartscape, Furi Co. Ltd., Shanghai, China). Statistical analysis Data are expressed as meanSD. Statistical analyses were made using one-way analysis of variance (ANOVA) with Tukey's post hoc inter-group comparisons. A value of P 0.05 was considered to denote statistical significance. Results Neurological score A significant increase in neurological score (8.622.29) was found in rats at 24 h after MCAO ( Fig. 1 ). HBO-PC alleviated the neurological injury to 4.872.29 ( P 0.01 vs. MCAO group). Animals in the control group showed no neurobehavioral functional deficit. Full-size image (17K) High-quality image (62K) Fig. 1.Beneficial effect of HBO-PC on neurobehavioral recovery after MCAO. Neurological scores were significantly lower (better) in HBO-PC animals as compared with MCAO group (** P 0.01, Student's t -test). Control n =24, MCAO n =47, HBO-PC n =55. View Within Article TTC staining Fig. 2 A shows representative photographs derived from postmortem TTC staining sections at 24 h. The infarct ratio was 0.210.04 in the MCAO group and 0.140.03 in the HBO-PC group ( P 0.05, Fig. 2 B). Full-size image (43K) High-quality image (368K) Fig. 2.Effect of HBO-PC on TTC staining at 24 h after MCAO. The infarction volume in the HBO group is significantly decreased as compared with MCAO group (* P 0.05, Student's t -test). Control n =4, MCAO n =7, HBO-PC n =9. View Within Article Brain water content The right hemisphere (infarct side) mean brain water contents were 0.770.01, 0.860.02, and 0.780.01, in the control, MCAO, and HBO-PC groups, respectively ( Fig. 3 ). The water content of the MCAO group was significantly higher than the control ( P 0.01) and HBO-PC groups ( P 0.01). No difference in brain water content was found between the control and HBO-PC groups ( P 0.05). Full-size image (36K) High-quality image (195K) Fig. 3.Effect of HBO-PC on brain water content. HBO-PC reduced significantly brain water content in the right hemisphere (** P 0.01 vs. MCAO group). Control n =4, MCAO n =9, HBO-PC n =10. View Within Article TUNEL staining There were more apoptotic neurons in the core ( Fig.4 aB, C) of HBO-PC rats than MCAO rats (80.4315.47 vs. 59.2314.35, P 0.05). HBO-PC reduced the number of TUNEL positive cells in the penumbra (84.5718.75 vs. 145.8217.02, P 0.01, Fig. 4 aD, E) and CA1 sector (67.5818.30 vs. 163.6820.35, P 0.01, Fig. 4 bB, C) when compared with MCAO group. Full-size image (69K) High-quality image (640K) Fig. 4.(a) TUNEL staining of the core (B, C) and penumbra (D, E) of the cortex. (b) TUNEL staining of CA1 sector (AC). Only the densely stained cells were counted as TUNEL positive cells. There were no TUNEL positive cells in the cortex and CA1 sector in control animals (a A, b A). TUNEL positive cells were observed in the ipsilateral cortex and hippocampus after MCAO. The chromatin congregated on the cell border and some cells formed apoptotic bodies which have different shapes and concentrated cytoplasm with characteristic triangles. The penumbra ( ## P 0.01 vs. MCAO group) and CA1 sector (** P 0.01 vs. MCAO group) of the HBO-PC group had fewer apoptotic cells; however, the core had more than the MCAO group (* P 0.05). n =4 In the control group, n =8 in the MCAO group, n =10 in the HBO-PC group. View Within Article Activities of caspase-3 and 9 At 24 h after MCAO the relative activities of caspase-3 and 9 in the ischemic core were similar for MCAO and HBO-PC, respectively ( Fig. 5 A, C, P 0.05). HBO-PC reduced caspase-3 and 9 activities in the penumbra ( Fig. 5 A, C, P 0.05) and in the hippocampus ( Fig. 5 B, D, P 0.05). Full-size image (73K) High-quality image (341K) Fig. 5.The enzymatic activity of caspase-9 (A, B) and caspase-3 (C, D). The enzymatic activity of caspase-3 or caspase-9 was reduced in the ischemic core (* P 0.05, HBO-PC group vs. MCAO group; ** P 0.01 HBO-PC group vs. control group, MCAO group vs. control group) than in the penumbra ( # P 0.05 HBO-PC group vs. MCAO group; ## P 0.01 HBO-PC group vs. control group, MCAO group vs. control group). The caspase activity is more pronounced in the hippocampus in MCAO group than the HBO-PC group (* P 0.05, ** P 0.01). n =4 In the control group, n =7 in the MCAO group, n =9 in the HBO-PC group. View Within Article Western blot of cytochrome c, Bcl-2 and Bax Western blot analysis of brain samples at 24 h after MCAO showed similar levels of cytoplasmic cytochrome c in the ischemic core ( Fig. 6 A) in MCAO and HBO-PC groups ( P 0.05). HBO-PC reduced cytochrome c in the penumbra and in the hippocampus ( Fig. 6 A) ( P 0.05). HBO-PC enhanced Bcl-2 levels in the ischemic core, penumbra and hippocampus ( Fig. 6 B). The level of Bax remains constant in all groups after MCAO ( Fig. 6 C). Full-size image (140K) High-quality image (636K) Fig. 6.Western blot analysis of cytochrome c (A), Bcl-2 (B) and Bax (C) protein. (A) Pretreatment with HBO decreased the expression of cytoplasmic Cytochrome c in the penumbra (** P 0.01 vs. MCAO group; ## P 0.01 vs. control group) and hippocampus ( # P 0.05 vs. MCAO group), but not the ischemic core (* P 0.05 vs. MCAO group). (B) Bcl-2 increased in the ischemic penumbra and hippocampus in the HBO-PC group ( # P 0.05, vs. MCAO group; ## P 0.01 vs. control group). but not the ischemic core (* P 0.05 vs. MCAO group). (C) Bax expression was enhanced after MCAO compared with control rats but no difference was found between MCAO and HBO-PC groups (* P 0.05, # P 0.05 vs. MCAO group). n =4 In the control group, n =9 in the MCAO group, n =9 in the HBO-PC group. View Within Article Discussion HBO-PC was neuroprotective in previous studies but a 10 days protocol was used to give HBO every other day for five treatments ( Wada et al., 2001 ) which is difficult to apply in clinical practice. In this study we observed that a condensed application of HBO-PC in 2 days produced a marked brain protection in an established focal cerebral ischemia model. Four applications of HBO-PC in 2 days suppressed brain edema, decreased cerebral infarction and improved neurological function at 24 h after MCAO. These outcomes that cytochrome c release was suppressed, caspase-3 and -9 activity reduced, and apoptotic cell decreased were observed at cellular level and might be responsible for neuroprotection. These results are consistent with previous HBO-PC in other animal models that required 5 to 10 days' preconditioning ( and ). We chose an MCAO rat model for this study because similar major cerebral arteries may be temporarily clipped during aneurismal surgery or cerebral bypass. In addition, an MCAO rat model provides a clear ischemic core and penumbra ( Kaufmann et al., 1999 ) and the neuronal injury in the ischemic penumbra is reversible and may be salvaged by proper interventions. Temporary clipping of major cerebral arteries during the abovementioned neurosurgery procedures might result in a penumbra type of neuronal injury. Indeed, we have observed neuroprotective effect in the penumbra by HBO-PC in this study. Therefore, this 2-day HBO-PC protocol may have clinical potentials for patients undergoing major aneurismal surgery, spinal cord surgery or even for cardiac bypass or transplantation procedures. One of the major cell death patterns in the ischemic penumbra is apoptosis especially mediated by mitochondrial pathways ( Graham et al., 2001 ). In this study, we have observed a higher number of TUNEL-positive cells in the ischemic core in the HBO-PC group than in the MCAO group. This might indicate that more cells survived from necrosis in the ischemic core region after HBO-PC. However, it is the penumbra where HBO-PC exerted major neuroprotection. The penumbral cortex was mostly spared from infarction in the HBO-PC group. Since the blood supply of the hippocampus comes from many arteries, especially the anterior choroidal artery and posterior cerebral artery, the hippocampus represents a penumbra type of injury in this MCAO rat model. Similar neuroprotection by HBO-PC was observed in the hippocampus in TUNEL studies. Furthermore, not only the numbers of TUNEL positive cells decreased in the penumbra in HBO-PC group, but also the molecular events related to mitochondrial apoptotic pathways that were altered by HBO-PC. During the process of apoptosis, the release of cytochrome c from the mitochondria into the cytosol has been proposed as a trigger for the development of neuronal apoptosis ( Green et al., 1998 ). The cytochrome c release can activate caspase-3 by activating apoptotic protease activating factor 1 and complexes of procaspase-9. Caspase-3 then causes degradation of cytoskeleton, DNA fragmentation, and eventually cell death. Our observation made in this study indicates that HBO-PC reduced cytochrome c release, and markedly inhibited caspase activity in the penumbra and hippocampus. Two upstream apoptosis factors Bcl-2 and Bax in the mitochondrial pathways were examined in this study. Upregulation of Bcl-2 or downregulation of Bax ( Adams et al., 2007 ) has been found to attenuate apoptotic cell death previously. We have observed in this study that HBO-PC enhanced the Bcl-2 but did not change Bax expression in the penumbra and hippocampus, increasing Bcl-2/Bax ratio. 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这个工作是与长海医院合作, 我们已经找到比较合理的暴露方案: 1小时/12小时连续4次. 我们这个方案是采用脊髓缺血模型找到的, 于是我们就采用经典的脑缺血模型(插线法),重复是否效果理想. 我们从各种抗氧化酶是否受高压氧诱导来解释这个效应,这个思路是来自四军大脊髓缺血模型的结果. 文章很快完成并发表在Brain res. 从线粒体途径的研究发表在神经科学上. 文章比较好一些. Hyperbaric oxygen preconditioning induces tolerance against brain ischemiareperfusion injury by upregulation of antioxidant enzymes in rats Jiasi Li a , Wenwu Liu b , Suju Ding a , , , Weigang Xu b , Yangtai Guan a , John H. Zhang c and Xuejun Sun b , , a Department of Neurology, Changhai Hospital,174 Changhai Road, Shanghai 200433, PR China b Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai, 200433, PR China c Department of Neurosurgery, Loma Linda University, Loma Linda, California, USA Accepted 4 March 2008. Available online 12 March 2008. Abstract The present study examined the hypothesis that cerebral ischemic tolerance induced by hyperbaric oxygen preconditioning (HBO-PC) is associated with an increase of antioxidant enzyme activity. Male SpragueDawley rats (250280g, n =74) were divided into sham, middle cerebral artery occlusion (MCAO) for 90min, and MCAO plus HBO-PC groups. HBO-PC was conducted four times by given 100% oxygen at 2.5atmosphere absolute (ATA), for 1h at every 12h interval for 2days. At 24h after the last HBO-PC, MCAO was performed and at 24h after MCAO, neurological function and Nissl Staining were performed to evaluate the effect of HBO-PC. Malondialdehyde (MDA) content, activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) sampled from the hippocampus, ischemic penumbra or core of cortex were measured. HBO-PC decreased mortality rate, improved neurological recovery, lessened neuronal injury, reduced the level of MDA and increased the antioxidant activity of CAT and SOD. These observations demonstrated that an upregulation of the antioxidant enzyme activity by HBO preconditioning plays an important role in the generation of tolerance against brain ischemiareperfusion injury. Keywords: Hyperbaric oxygen; Prevention; Antioxidant enzyme; Ischemic tolerance; Reactive oxygen species Article Outline 1. Introduction 2. Results 2.1. Physiologic variables 2.2. Mortality rate and neurologic outcome 2.3. Nissl staining 2.4. MDA content 2.5. Antioxidant enzyme activities 3. Discussion 4. Experimental procedures 4.1. Animals and groups 4.2. HBO preconditioning 4.3. MCAO procedures 4.4. Neurologic functional scoring 4.5. Nissl staining 4.6. Ischemic core and penumbra dissections 4.7. Measurement of antioxidant enzyme activity and malondialdehyde content 4.8. Statistical analyses Acknowledgements Glossary References brain res
证明高压氧能诱导心脏新血管生长 05年的时候，国际上开展这个方面的实验室我们都比较熟悉：美国、日本、德国、英国和中国的四军大。从文章档次和数量上看,四军大的工作最出色.英国在心脏保护方面最系统.日本的工作最早,后来的工作都比较缓慢.而我们现在的进展最快.现在已经在数量上领先所有的实验室.但质量与四军大还有很大差距.需要继续深入研究. 这些实验室的特点是方向比较集中，但重点都不在高压氧这个方向上，都是在各自的领域涉及到高压氧。而我们可以不受这些限制。我们了解到英国已经有临床研究：在心脏手术前进行高压氧暴露，可有效预防手术造成的心肌缺血损伤，这个研究非常重要，说明这个手段确实能在临床上使用。这也是唯一的临床证据。 给这个领域的研究带来很好的期望。我们也希望能做一些动物实验证明对心脏的作用。正好我们了解到本校有个老师在心肌缺血方面研究基础很好，于是我派一个学生学习，结果很快，我们证证明明这个模型效果特别好。这个阶段，我们做了一些干细胞方面探讨，主要是我们发现高压氧能诱导新生血管生成。结果我们只用BRDU方面不能确定细胞的类型，审稿专家给我们提出这个问题。后来美国的张教授建议我们把这个方面的内容去掉，结果杂志同意发表了。这个文章的启发是，任何实验，要么别做，要做就全面，别留下太多漏洞。否则会浪费时间。当然这个想法十分功利。不提倡 Exp Biol Med (Maywood). 2008 Nov;233(11):1448-53. Epub 2008 Aug 14. Links Hyperbaric oxygen preconditioning alleviates myocardial ischemic injury in rats. Han C , Lin L , Zhang W , Zhang L , Lv S , Sun Q , Tao H , Zhang JH , Sun X . Department of Diving Medicine, Second Military Medical University, Shanghai 200433, People's Republic of China. It has been shown that after ischemia-reperfusion, application of hyperbaric oxygen (HBO) reduces cardiac injury. In this study we tested the hypothesis that HBO preconditioning reduces injury to the ischemic myocardium. One hundred and eight adult male Sprague-Dawley rats (250-280 g) were randomly divided into four groups: normoxia + sham surgery (CS), normoxia + permanent occlusion of the left anterior descending (LAD) coronary artery (CMI), HBO preconditioning + sham surgery (HS), and HBO preconditioning + permanent LAD occlusion (HMI). Rats receiving HBO preconditioning were intermittently exposed to 100% O(2) at 2.5 atmosphere absolute (ATA) for 60 min, twice daily for 2 days followed by 12 hrs of recovery in room air prior to the myocardial ischemic insult induced by LAD ligation. Rats in the normoxia group were time-matched with the HBO group and maintained under normoxic conditions prior to LAD occlusion. At 3 and 7 days after LAD occlusion, heart function parameters were measured by inserting a catheter into the left ventricle, infarct size was calculated using the method of TTC staining, myocardial capillary density was determined by immunohistochemical staining with a monoclonal anti-CD(31)/PECAM-1 antibody, and VEGF protein level was determined by Western blot analysis. At 3 days after LAD ligation, the infarct size of the HMI group was significantly smaller than that of the CMI group (26 +/- 2.5% vs. 38 +/- 3%, P 0.05). The heart function parameters including left ventricular systolic pressure (LVSP), +dP/dt(max) and -dP/dt(max) were significantly improved in the HMI group compared to the CMI group at 3 and 7 days after LAD occlusion. Capillary density and VEGF protein levels were significantly increased in the ischemic myocardium pre-exposed to HBO. We conclude that HBO preconditioning alleviates myocardial ischemia in rat model. 全文
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
以毒攻毒，提出高压氧预防低氧的设想 最近几年，发表的论文主要有两类，氧和氢。这些论文都有一点值得回忆的内容，从今天开始整理这些回忆，作为一段科研经历的总结。主要分：论文摘要、研究背景、优点与缺点等几个问题。尽量把全文放上，供大家批评。 这个文章是在04年开始进行的工作，是这个氧系列的第一个工作。 我们最基本的思路是，高压氧有毒性，有毒性的物质都能诱导机体产生抗毒能力，就是说高氧应该具有预适应效应。这个思路实际上我在1998年就已经想到了，可惜一直因为条件限制没有开展。04年获得系里的一个小基金支持，就开始了这个工作。实际上这个时候国内外已经有了一些这个思路的文章。 但没有关于低氧的文章，我们就实验反复进行高压氧处理是否能预防低氧（高原）损伤，结果非常令人吃惊，效果十分理想，沿着这个思路，我们寻找原因。HIF是低氧耐受最重要的蛋白，我们采用组化和蛋白测定方法证明，HIF参与这个过程，这应该是我们首先发现高氧能诱导 HIF的实验。可惜在06年有人发表了在肝脏也有这个效果。实际上我们的实验应该在他们之前。不过他们认为 HIF没有活性，这一点我们超过了他们。 这个工作最主要的缺点是，没有采用PCR的方法证明HIF的活性。设计不够严谨。 Brain Res. 2008 May 30;1212:71-8. Epub 2008 Mar 27. Links Up-regulated HIF-1alpha is involved in the hypoxic tolerance induced by hyperbaric oxygen preconditioning. Peng Z , Ren P , Kang Z , Du J , Lian Q , Liu Y , Zhang JH , Sun X . Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai, 200433, PR China. Hyperbaric oxygen preconditioning (HBO-PC) has been shown to be effective in preventing hypoxic injuries in many animal models. The aim of the present study was to examine the hypoxic tolerance induced by HBO-PC and to explore the role of hypoxia-inducible factor-1alpha (HIF-1alpha) in a global hypoxia model. Male mice received HBO-PC before hypoxia exposure and swimming. HBO-PC significantly prolonged the survival time and the tolerance time of swimming under normobaric hypoxia. HBO-PC increased the protein content of HIF-1alpha and erythropoietin (EPO) in the cerebral cortex and hippocampus and prevented the changes of blood brain barrier (BBB) permeability and brain edema caused by hypoxia exposure. The results suggested that HBO-PC induced hypoxic tolerance in mice via up-regulation of HIF-1alpha and its downstream genes. 全文下载： 1111 对视神经损伤的保护作用 baric oxygen preconditioning promotes survival of retinal ganglion cells in a ra.pdf
标签: 高压氧
