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【Stroke】含氢气生理盐水的神经保护作用

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这个帖子发布于11年零30天前，其中的信息可能已发生改变或有所发展。

该文首次采用注射含氢水的方法治疗脑损伤，是对去年日本Nature Med文章的改进和提高。原来的研究是采用呼吸一定浓度的氢气,达到治疗脑缺血再灌注损伤的目的，但是，通过呼吸的方法不仅在给药过程中存在爆炸的危险，而且需要比较特殊的设备，操作比较复杂，在临床上难以推广，因此，寻找更加实用的给药方法也是需要探讨的问题。经过理论推算，如果将纯氢气在生理盐水中溶解，经过一定的处理，使其达到饱和溶解，可制造出氢气的生理盐水饱和溶液，这样就可通过注射方法给药。

本文实现了这一目的.
中文摘要:
含氢气生理盐水对大鼠新生儿脑缺血的神经保护作用

脑缺血缺氧是新生儿脑损伤的重要因素。本研究目的是利用大鼠新生儿脑缺血模型，观察含氢气生理盐水对脑缺血后脑损伤的长期和短期保护作用。采用7天生大鼠，左恻颈总动脉结扎， 8%低氧37 ◦C处理90分钟，制备动物模型。分别在模型制备后即刻和8小时后腹腔注射5 ml/kg饱和氢气生理盐水。24小时后断头处死动物进行TTC、尼氏和TUNEL染色。利用caspase-3活性、MDA和Iba-1免疫组化染色评价细胞死亡、炎症和氧化损伤程度。利用自发活动实验、水迷宫检测5周后神经功能情况。结果发现，氢气生理盐水对能有效降低caspase-3活性、MDA, Iba-1水平、梗死体积，提高长期神经行为功能。氢气生理盐水可能具有治疗新生儿脑缺血等各类脑缺血性疾病的价值。

本文首次证明通过注射含氢盐水可以治疗缺血损伤, 从日本首先发表呼吸2%氢治疗脑缺血的文章以后, 很多研究相继发表,证明呼吸氢可以治疗肝缺血、心肌缺血和小肠移植损伤，通过引用含氢水可以治疗人类糖尿病后氧化损伤，可以治疗应激动导致的脑损伤，可以治疗基因模型动物的脑损伤和动脉硬化。这些研究表明，给一定浓度的氢是一种非常好的治疗氧化损伤的手段。但是，通过注射的方法在临床上将是更理想的给药手段。我们经过反复研究，制备出含氢液体，并用这种注射液治疗脑损伤，效果理想。我们将在其他不同模型中继续证明这种注射液的作用，并深入研究其作用机制。我们相信，本文章的发表将会有力推动氢生物学效应的研究。希望有兴趣的战友加入我们的团队。
我们将来将希望在，缺血、炎症、创伤和各类慢性损伤模型中进行该课题研究，请继续关注我们的工作，我们相信，这些研究将给人类健康带来一个新的希望。氢的系列药物将属于高效、低毒、广谱的治疗药物。
如果将来确实能实现临床应用,本文将具有十分重要的意义.

Neuroprotective Effects of Hydrogen Saline in Neonatal Hypoxia-ischemia Rat Model

Abstract
Cerebral hypoxia–ischemia (HI) represents a major cause of brain damage in
the term newborn. This study aimed to examine the short and long-term
neuroprotective effect of hydrogen saline (H2 saline) using an established
neonatal HI rat pup model. Seven-day-old rat pups were subjected to left
common carotid artery ligation and then 90 min hypoxia (8% oxygen at 37 ◦C).
H2 saturated saline was administered by peritoneal injection (5 ml/kg)
immediately and again at 8 h after HI insult. At 24 h after HI, the pups were
decapitated and brain morphological injury was assessed by
2,3,5-triphenyltetrazolium chloride (TTC), Nissl, and TUNEL staining. Acute
cell death, inflammation and oxidative stress were evaluated at 24 h by
studying caspase-3 activity, MDA measurement as well as Iba-1
immunochemistry in the brain. At 5 weeks after HI, spontaneous activity test
and Morris water maze test were conducted. We observed that H2 saline
treatment reduced the caspase activity, MDA, Iba-1 levels, the infarct ratio, and
improved the long-term neurological and neurobehavioral functions. H2
saline has potentials in the clinical treatment of HI and other ischemia-related
cerebral diseases.

其他相关文献：

日本的第一篇文章
Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals - ►h4o.co.jp [PDF]
I Ohsawa, M Ishikawa, K Takahashi, M Watanabe, K … - Nature Medicine, 2007 - nature.com
... 5a). Figure 5: Inhalation of hydrogen gas protects against ischemia-reperfusion
injury. Figure 5 : Inhalation of hydrogen gas protects against ischemia-reperfusion
injury. (a) Rats inhaled H 2 and 30% O 2 for 1 h under the anesthetics N 2 O ...
被引用次数：14 - 相关文章 - 网页搜索 - 图书馆搜索 - 所有 5 个版本

日本的文章
Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing …
K Fukuda, S Asoh, M Ishikawa, Y Yamamoto, I Ohsawa … - Biochemical and Biophysical Research Communications, 2007 - Elsevier
We have recently showed that molecular hydrogen has great potential for selectively reducing cytotoxic reactive oxygen
species, such as hydroxyl radicals, and that inhalation of hydrogen gas decreases cerebral infarction volume by ...
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The hydrogen highway to reperfusion therapy - ►h4o.co.jp [PDF]
KC Wood, MT Gladwin - Nature Medicine, 2007 - nature.com
During the ischemic phase of thromboembolic stroke, a blood clot travels to and lodges in the distal blood vessels in
the brain, blocking blood flow to the oxygen-starved tissue for a period of hours. This is followed by the ...
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[HTML] ►Advances in Emerging Nondrug Therapies for Acute Stroke 2007
AB Singhal, EH Lo - Stroke, 2008 - ahalibrary.com
From the Neuroprotection Research Laboratory, Program in Neuroscience, Harvard Medical School, and Department of
Neurology, Massachusetts General Hospital, Boston, Mass. ... Numerous clinical trials of thrombolytic and ...
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这个也是我们的文章：
Hydrogen therapy reduces apoptosis in neonatal hypoxia–ischemia rat model
J Cai, Z Kang, WW Liu, X Luo, S Qiang, JH Zhang, S … - Neuroscience Letters, 2008 - Elsevier
Hypoxia–ischemia (HI) brain injury is a major cause of neuronal cell death especially apoptosis in the perinatal
period. This study was designated to examine the effect of hydrogen therapy on apoptosis in an established neonatal ...
相关文章 - 网页搜索 - 图书馆搜索 - 所有 2 个版本

日本的文章
Consumption of Molecular Hydrogen Prevents the Stress-Induced Impairments in Hippocampus-Dependent …
K Nagata, N Nakashima-Kamimura, T Mikami, I Ohsawa … - Neuropsychopharmacology, 2008 - nature.com
We have reported that hydrogen (H 2 ) acts as an efficient antioxidant by gaseous rapid diffusion. When water saturated
with hydrogen (hydrogen water) was placed into the stomach of a rat, hydrogen was detected at several M level in ...
相关文章 - 网页搜索 - 所有 3 个版本

日本的文章
Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia–reperfusion …
K Hayashida, M Sano, I Ohsawa, K Shinmura, K … - Biochemical and Biophysical Research Communications, 2008 - Elsevier
Inhalation of hydrogen (H 2 ) gas has been demonstrated to limit the infarct volume of brain and liver by reducing
ischemia–reperfusion injury in rodents. When translated into clinical practice, this therapy must be most ...
被引用次数：1 - 相关文章 - 网页搜索 - 图书馆搜索 - 所有 2 个版本

美国的文章
Hydrogen Inhalation Ameliorates Oxidative Stress in Transplantation Induced Intestinal Graft Injury.
BM Buchholz, DJ Kaczorowski, R Sugimoto, R Yang, Y … - American Journal of Transplantation, 2008 - pt.wkhealth.com
ovid_logo. Search: Advanced Search. Ovid News. Learn about Ovid’s new research content and products. Ovid Events.
Check out where Ovid is across the globe at industry conferences and events worldwide. ...
网页搜索 - 图书馆搜索

日本的文章
Hydrogen-rich pure water prevents superoxide formation in brain slices of vitamin C-depleted SMP30/ …
Y Sato, S Kajiyama, A Amano, Y Kondo, T Sasaki, S … - Biochemical and Biophysical Research Communications, 2008 - Elsevier
Hydrogen is an established anti-oxidant that prevents acute oxidative stress. To clarify the mechanism of hydrogen’s
effect in the brain, we administered hydrogen-rich pure water (H 2 ) to senescence marker protein-30 (SMP30)/ ...
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日本的文章
Inhibitory Effect of Electrolyzed Reduced Water on Tumor Angiogenesis
J Ye, Y Li, T Hamasaki, N Nakamichi, T Komatsu, T … - Biological & Pharmaceutical Bulletin, 2008 - J-STAGE
VEGF gene expression is initiated by extracellular signals in- cluding growth factors, mitogens, phorbol ester,
cytokines and extracellular stresses. The first three of these exogenous signals activate the Ras-Raf-MEK-ERK ...
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容易被遗漏的一篇早期法国文献
Anti-inflammatory properties of molecular hydrogen: investigation on parasite-induced liver …
B Gharib, S Hanna, OMS Abdallahi, H Lepidi, B … - Comptes Rendus de l'Academie des Sciences Series III …, 2001 - Elsevier
... All rights reserved. Anti-inflammatory properties of molecular hydrogen: investigation
on parasite-induced liver inflammation. Première mise en évidence des propriétés
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sunxjk 编辑于 2008-11-13 15:45

• 丁香热议——谈恋爱遇见住院总：短信不回，电话不接，还以为ta出轨了！

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本人已认领该文编译，48小时后若未提交译文，请其他战友自由认领。

2008-11-11 08:24

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• 丁香热议——谈恋爱遇见住院总：短信不回，电话不接，还以为ta出轨了！

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1. Introduction
Cerebral hypoxia–ischemia (HI) represents a major cause of brain damage in the term newborn. Although the mechanisms involved in HI were not completely understood, neuronal cell death either necrosis or apoptosis may play a critical role (Martin et al., 2000; Northington et al., 2001). Specially, apoptosis represents a treatable target which may occur in penumbra areas for days after the initial insult (Pulera et al., 1998). However, there is no specific treatment which is available to HI patients (Perlman, 2006).

Inflammation and oxidative stress are the two major causes of apoptosis identified after ischemic brain injury including neonatal HI (Kriz, 2006). Microglia was involved in the inflammatory process induced by the HI (Stoll et al., 1998). Allograft inflammatory factor-1 (AIF-1) in microglia is an index for the activation of microglia (Postler etal., 2000). Oxidative stress after HI damages to DNA, membrane and proteins and contribute to apoptotic changes. The malondialdehyde (MDA) is the product of lipid membrane oxidation and a marker of the oxidative damage.

Hydrogen gas was found to be protective in the brain, heart and liver after ischemia-reperfusion damage (Ohsawa et al., 2007; Fukuda et al., 2007; Hayashida et al., 2008). Hydrogen gas neutralizes free radicals and reduces oxidative stress. However, application of hydrogen gas presents a clinical issue for safety and convenience. In this study, we produced and tested long term neuroprotective effect of intraperitoneal application of saturated hydrogen saline (H2 saline) in an established neonatal HI model.

2. Results
2.1 Nissl staining
We tested three different doses of hydrogen saline in the treatments to identify the proper dose in the Nissl staining. Figure 1 shows representative samples of Nissl staining from the cerebral cortex and hippocampus of pups 24 h after HI insult. Extensive neuronal changes in the cortex and CA1 sector of the hippocampus were noticed with features of considerable dark, pyknotic neurons in HI group (B1-4). More Nissl stained cells (D1-4) were observed in H2W group than in HI group. We used 5 ml/Kg injection for the following studies because it was more effective than the other two dosages.

2.2 TTC staining
Figure 2 shows representative photographs of TTC-stained sections from 8-day-old rats in each group, 24 h after the initial HI insult. The infarct ratio in HI group (10.8%) is markedly higher than that in H2W group (0.99%) which is not significant from normal controls.

2.3 Content of MDA
The content of MDA in each group was detected at 24 h after HI. The content of MDA in HI group (9.00±0.92) significantly increased compared to the control rats (3.52±1.50). However, H2 saline administration dramatically suppressed the production of MDA (4.5±1.96) in rats after HI when compared that in the HI group.

2.4 TUNEL staining
Photos in Figure 3 were the representative graphs with different magnifications in TUNEL staining in samples collected at 24 h after HI. At higher magnification, the nuclei of cells were clearly stained in both hippocampus and cortex. The results indicated that TUNEL-positive cells were markedly increased in cortex and hippocampus after HI insult (B1-4). While the administration of H2 saline dramatically reduced the number of TUNEL-positive cells (C1-4). A few TUNEL-positive cells were identified in samples from normal control pups (A1-4).

2.5 Caspase-3 activity
The activity of caspase-3 was measured at 24 h after HI insult as shown in Figure 5. The activity was 1.18±0.23 in cortex and 0.93±0.22 in hippocampus in HI group. H2 saline significantly suppressed the activity of caspase-3 in the cortex (0.12±0.09) and hippocampus (0.09±0.10) which were consistent with the results of TUNEL staining.

2.6 Immunohistochemical analysis
Iba1-positive cells had small nuclei, scant cytoplasm, and thin, branched processes in Figure 4. This kind of cell was found throughout the parenchyma of the white and grey matter. The distinctive morphology and widespread distribution of these cells were highly consistent with the classical descriptions of ramified microglia. H2 saline treatment dramatically decreased the number of Iba1-positive cells (B1,B2). Samples were collected at 24 h after HI.

2.7 Body weight
There was no disparity in body weight between the three groups in the first 15 days after HI insult (Figure 5). In the later 20 days, a significant difference was found between the HI and HI+H2W group, while there was no statistic difference between control and H2 saline treatment group.

2.8 Function test
The Postural Reflex Test shows an HI insult affects the sensorimotor function of the rats. Score 0 represent normal sensorimotor function, whereas Score 1 and Score 2 represent a deficiency in sensorimotor function. All of the control animals scored a Score 0. 22.22% of the HI animals scored a Score 0, 77.78% a Score 1 and 66.67% a Score 2. 63.64% of the HI+H2W scored a Score 0, 36.36% a Score 1 and 9.09% a Score 2. A Chi-squared test showed that there was a significant difference in the distribution between the groups.

2.9 The locomotor activity
In order to display the time dependency of a behavior, spontaneous activity was tested. Fig 6 shows the trace of spontaneous behavior and the statistic. The observation indicated all rats experienced HI insult moved less as time in the chamber increased. However, the rats in HI group were more active and the move time for HI+H2W group, not different from normal controls, was more than that in HI group. The average total distance in HI+H2W group was less than that in HI group.

2.10 Morris Water Maze
The escape latency was measured at 24 h after HI insult. The escape latency was 12±1.23 sec in control group and 27±3.21 sec in HI group. Administration of H2 saline shortened the time (15±1.89 sec) required for rats to reach the platform compared with HI group during the water maze testing. But the time in rats of H2 saline group was not different from that in normal rats. H2 saline improved spatial recognition and learning that had declined by HI.

3. Discussion
We investigated the neuroprotective effect of peritoneal administration of saturated H2 saline in neonatal HI rats. The short term results indicated H2 saline treatment significantly reduced the infarct ratio, increased the number of survival neurons, reduced the number of apoptotic cells, suppressed caspase-3 activity, prevented activation of microglia, and decreased the level of oxidative stress (MDA). These short term effects were translated into long-term neurological functional improvements at 5 weeks after HI insult. These observations indicated that H2 saline may be a potential therapeutic option for neonatal brain disorders.

The key neuroprotective effect of hydrogen is neutralizing free radicals, especially the hydroxyl radical and peroxynitrite anion (Ohsawa et al., 2007). The hydroxyl radical, •OH, is the neutral form of the hydroxide ion and the normal product in the cell metabolism. •OH has a high reactivity, making it a very dangerous radical with a very short in vivo half-life of approximately 10−9 s (Valko et al., 2007). In addition, cells produce both the superoxide anion (O2-•) and nitric oxide (NO) during the oxidative burst triggered during inflammatory processes (Dedon and Tannenbaum, 2004). Under these conditions, NO and O2-• may react together to produce significant amounts of oxidative active molecule, peroxynitrite anion (ONOO−), which is a potent oxidizing agent that can cause DNA fragmentation and lipid oxidation (Valko et al., 2007). ONOO− also reacts with carbon dioxide (CO2) present in biological fluids to form reactive intermediates that can oxidize thiols and nitrate phenolic compounds, such as tyrosine (Valko et al., 2007). After neonatal hypoxic and ischemic brain injury, reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as the •OH, O2-, hydrogen dioxide (H2O2), NO, ONOO–, appear to play a critical role in cell death. Among the ROS, •OH and ONOO– are much more reactive and react indiscriminately with nucleic acids, lipids and proteins. The brain has potent defenses including dietary free-radical scavengers (ascorbate, α-tocopherol), the endogenous tripeptide glutathione, and enzymatic antioxidants against ROS. However, there isn’t known detoxification system for •OH and ONOO–. Recently, Ohsawa et al (2007) found that molecular hydrogen can selectively reduce •OH and ONOO– in cell-free systems and exert a therapeutic antioxidant activity, in a rat middle cerebral artery occlusion model. Therefore, the ability of hydrogen to reduce or eliminate •OH and ONOO– may be responsible for the neuroprotective effect observed in this study.

Recent studies reported that hydrogen gas serves as an antioxidant to provide protective effects to ischemic insult in the brain and liver by selectively scavenging •OH and ONOO– (Ohsawa et al., 2007; Fukuda et al., 2007). A recent article also indicated that inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia–reperfusion injury (Hayashida et al., 2008) and intragastric administration of H2 saline prevented the stress-induced decline in learning and memory caused by chronic physical restraint (Nagata et al., 2008). In our previous study, we found inhalation of 2% hydrogen also provided the protective effects on the same HI neonatal model (Cai et al., 2008). Although gas containing 2% hydrogen is safe for clinical practice, this strategy was not convenient because of the requirement of chamber or face mask. Therefore, we produced H2 saline which can be injected quickly without safety risks.

We selectively studied two major mechanisms of HI injury, inflammation and oxidative stress. Inflammatory response can be mediated by activated microglia, the resident immune cells of the CNS, which normally respond to neuronal damage including neonatal HI and remove the damaged cells by phagocytosis (Stoll et al., 1998). Iba1 is a 147-amino-acid calcium-binding protein widely used as a marker for microglia (Ito et al., 2001). The Iba1 gene and protein are identical to AIF-1, a protein involved in various aspects of inflammation (Deininger et al., 2002). AIF-1 has been reported to play a role in microglia activation in neuro-inflammatory disorders and ischemic brain injury (Postler et al., 2000). Expression of AIF-1 was upregulated in response to apoptotic neuronal cell death and degeneration of injured central motoneurons (Tanaka et al., 1998), and sensory neurons (Ito et al., 1998). In the present study, H2 saline reduced brain injury possibly by suppression of inflammatory response induced by HI. In addition, H2 saline may reduce neuronal apoptosis to decrease the expression of AIF-1. We have observed similar results using hydrogen gas in another study (Cai et al., 2008). Furthermore, oxidative stress may lead to inflammation or may be enhanced by inflammatory actions. H2 saline reduced oxidative stress in the neonatal brain after HI may contribute to the reduction of inflammation and apoptosis.

Even though the different pharmacokinetics between hydrogen gas and hydrogen saline are not clear and deserve further investigation, the advantages of hydrogen saline over hydrogen gas application are the safety issues, the easiness of application, and the possible higher concentrations of hydrogen in saline than could be used in gas. It is established that hydrogen at a concentration above 4% is inflammable and dangerous, and application of hydrogen gas requires either a sealed chamber or mask. In a previous study that hydrogen water was administered into the stomach of rats (Nagata et al., 2008). Even though this gastric administration is safe, but hydrogen in water tends to evaporate and loss hydrogen in the stomach or intestine, and it is difficult to control concentration and absorption. Therefore, we used peritoneal injection that we can quickly withdraw and immediately inject hydrogen saline into animals to avoid losing hydrogen into environment. This hydrogen saline preparation is superior to hydrogen gas or hydrogen water, and higher and more accurate concentrations of hydrogen can be applied.

A feature of this study is the evaluation of long term neurological and neurobehavioral functions. We have found that H2 saline does not only prevent or reduce early pathological changes such as infarction or biochemical changes such as inflammation and oxidative stress, but also produce long lasting functional improvement. This observation provides strong support for future clinical trials of H2 saline in neonatal HI or other brain injuries. Taken together, peritoneal administration of H2 saline reduces brain injury after neonatal HI possibly by attenuating the inflammation and oxidative stress, leading to reduction of apoptosis and improvement of long term neurological and neurobehavioral functions.

4. Experimental procedures
4.1 Experimental groups
7-day-old Sprague-Dawley rat pups were randomly assigned to the following three groups: 1) control group (no carotid ligation or hypoxia) (n=40), 2) HI group (carotid ligation and hypoxia) (n=80), 3) HI+H2W group (carotid ligation, hypoxia and H2 saturated saline treatment) (n=80). Pups in each group were obtained from different litters to obtain parity within the groups. The Animal and Ethics Review Committee at the Second Military Medical University evaluated and approved the protocol used in this study.

4.2 H2 saline therapy paradigms
Purified H2 was dissolved into normal saline for 2 h under 0.6 MPa. H2 saturated saline was administered by peritoneal injection (5 ml/kg) immediately and again at 8 h after HI insult.

4.3 Hypoxia–ischemia model
The model used in this study was based on the Rice–Vannucci model (Vannucci and Vannucci, 1997). Pups were housed with the dam under a 12:12 h light–dark cycle, with food and water available ad libitum throughout the studies. These neonatal rats were anesthetized by inhalation with Diethyl Ether. The rats were kept at a temperature of 37 °C as the left common carotid artery was exposed and ligated with 5-0 surgical sutures. After operation, the pups were returned to the holding container. Anesthesia and surgery time averaged 5 min per pup. Surgery was completed for an entire litter, and the pups were allowed to recover within their dams for 1 h (for rehydration via nursing). Then they were placed in a jar perfused with a humidified gas mixture (8% oxygen balanced nitrogen) for 90 min. Both the jar and mixture were kept at 37 °C to maintain a constant thermal environment. All surviving pups were returned to their dams after hypoxia exposure.

4.4 Measurement of infarct ratio
At 24 h after administration of H2 saline, the pups were decapitated and the left brain hemispheric volumes were measured. Briefly, the brains were quickly removed after decapitation and placed in cold saline for 5 min, cut at 2-mm intervals from the frontal pole into 5 coronal sections. After incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC) for 8 min at 37 °C, the brain slices were fixed in 4% formaldehyde for 24 h. The volumes of each of the sections were summed by an image analysis system (ImageJ, 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.

4.5 Nissl staining
For Nissl staining, the 4-μm sections were hydrated in 1% toluidine blue at 50 °C for 20 min. After rinsing with double distilled water, they were dehydrated and mounted with permount. The cortex and the CA1 area of hippocampus from each animal were captured and Imaging-Pro-Plus (LEIKA DMLB) was used to perform quantitative analysis of cell numbers.

4.6 TUNEL staining
TUNEL staining was performed on paraffin-embedded sections by using the in situ cell death detection kit (Roche). According to standard protocols, the sections were dewaxed and rehydrated by heating the slides at 60 °C. Then these sections were incubated in a 20 μg/ml proteinase K working solution 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. Dried area around sample and added Converter-AP on samples for 1 h at 37 °C. After rinsing with PBS (5 min, 3 times), sections were colourated in dark with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP).

4.7 Cell counting
Six visual fields (0.6 mm2) of the cerebral cortex and CA1 were photographed in each section. The number of staining cells in each field was counted at higher magnification (×40). The data were represented as the number of cells per high-power field.

4.8 Caspase-3 activity assay
Brain samples from the cortex and hippocampus were taken from the impaired hemispheres of neonatal rats 24 h after H2 saline administration. The activity of caspase-3 was measured with caspase-3/CPP32 Fluorometric Assay Kit (BIOVISION Research Products 980 Linda Vista Avenue, Mountain View, CA 94043 USA). Briefly, brain samples were homogenized in ice-cold cell lysis buffer and kept at 4 °C for 1 h. Brain homogenate was centrifuged (Eppendorf, 5810R) at 12,000 g for 15 min at 4 °C. The supernatant was collected and stored at −80 °C until use. Protein content was measured by using the Enhanced BCA Protein Assay Kit. 20-200 μg cell lysates were incubated in a 96-well plate with 2×Reaction Buffer (50 μl). The reaction was started by adding 1 mM DEVD-APC substrate (5 μl). After incubation in the dark at 37 °C, the plate was read in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter.

4.9 Detection of MDA
Lipid peroxidation levels were measured with the thiobarbituric acid (TBA) reaction. This method was used to obtain a spectrophotometric measurement of the color produced during the reaction to thiobarbituric acid (TBA) with MDA at 535 nm. For this purpose, 2.5 ml of 100 g/l trichloroacetic acid solution was added to 0.5 ml homogenate in centrifuge tube and placed in a boiling water bath for 15 min. The mixture was cooled and centrifuged at 1000× g for 10 min. Next, 2 ml of the supernatant was added to 1 ml of 6.7 g/l TBA solution in a test tube, and placed in a boiling water bath for 15 min. The solution was then cooled and its absorbance was measured with a spectrophotometer (UV-WFZ75, Shanghai, China). TBARS levels were expressed as nmol/mg protein in the brain.

4.10 Immunohistochemical procedures
Rats were perfused through the left ventricle of the heart with PBS and then with 4% paraformaldehyde in PBS. The fixed brains were immersed in 20% sucrose in PBS overnight, quickly frozen with dry ice powder, sliced into 12–14 μm sections with a cryostat, and stored at −80°C. For the Iba1 staining, sections were washed in PBS, incubated in 0.3% H2O2 in methanol for 30 min to inhibit endogenous peroxidase activity, washed in PBS, and blocked in PBS containing 1.5% normal goat serum and 1% BSA for 2 h at room temperature. The sections were then incubated with a rabbit anti-Iba1 polyclonal antibody (Abcam, UK) overnight at 4°C, washed in PBS, and incubated with a HRP-conjugated goat anti-rabbit IgG antibody for 2 h at room temperature. They were then incubated with 50 mM Tris-HCl (pH 7.2) containing 0.05% diaminobenzidine tetrahydrochloride (DAB) and 0.01% H2O2. For control staining, normal rabbit IgG was used as the primary antibody.

4.11 Function test
The postural reflex test (Bona et al., 1997) was used to evaluate functional recovery in the pups 5 weeks after injury. The examiner was blinded to the experimental protocols. The pups were held by the tail 50 cm above the table. Normal rats extend both forelimbs toward the table (Score 0). Pups with brain damage flex the forelimb contralateral to the damaged hemisphere (Score 1). Thereafter, the pups were put onto the table, and a lateral pressure was applied behind the shoulder of the pup until the forelimbs slid. This was repeated several times, and a reduced resistance to lateral force toward the contralateral side was considered abnormal (Score 2).

4.12 Spontaneous activity test
Locomotor activity testing was performed once per animal in 42×42 cm chambers equipped with a 16×16 grid of infrared LED-photodetector pairs in the x and y planes and an additional elevated set in the x plane to record rearing movements as described previously by Reed et al (Reed et al., 2002). Total distance, rest time and move time were recorded for a total of 3 min for each animal.

4.13 Spatial Learning
Morris water maze testing was performed in a tank of 122 cm diameter with the water temperature maintained at 21 °C. The water was tinted with white tempera paint to obscure the platform. A 10 cm×10 cm platform was hidden 1 cm below the surface of water. Entry points were varied. Each trial lasted until either the rats had found the fixed platform or for a maximum of 3 min. All rats were allowed to rest on the platform for 20 s and each rat was allowed 4 trials per day for 4 days. 2 days after training, the test was performed again and the examiner determined the time of swimming until the rats reached the platform. The time spent in each quadrant was recorded and retention of the spatial training was assessed.

4.14 Data analysis
All quantitative data are expressed as mean±SD. The significance of differences between means was verified by ANOVA followed by Tukey test. For analyzing the results of cell counting, a non-parametric Kruskal–Wallis ANOVA was used followed by Dunn's test. Chi-square analysis was used for the test based on the scoring system, spontaneous activity and water maze tests. Significance is defined as a probability of 0.05 or less.

2008-11-11 08:51

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• 丁香早知道 - 12.9 | 手术中医生突发心梗，竟让同事先给患者做手术！

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本人已认领该文编译，48小时后若未提交译文，请其他战友自由认领。

2008-11-11 13:20

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