脑外伤后脑线粒体功能和结构改变及神经节苷脂治疗作用

目的从牛脑组织中提取神经节苷脂ＧＭ１,研究其对脑外伤后脑细胞呼吸功能和结构保护作用。方法把ＳＤ大鼠分成正常组,生理盐水治疗的损伤对照组及ＧＭ１治疗的损伤治疗组。结果发现对照组伤后８小时和１６小时Ⅲ态呼吸耗氧速率（Ｒ３）,呼吸控制率（ＲＣＲ）,磷／氧比（Ｐ／Ｏ）及氧化磷酸化效率（ＯＰＲ）有明显降低,治疗组则明显增加,且与正常组相近。对照组皮层神经元细胞和线粒体超微结构有明显损害,治疗组损害明显减轻。结论其可能机制与ＧＭ１保护膜脂和膜酶活性,维持膜内外离子平衡,减轻水肿及减少自由基形成等有关     

大鼠脑缺血后脑线粒体功能的改变

为探讨脑缺血再灌注后脑线粒体功能的改变，利用大鼠全脑缺血及再灌注动物模型，测试各组大鼠脑线粒体的呼吸控制率、膜流动性和呼吸链的标志酶琥珀酸脱氢酶及细胞色素氧化酶的活性。结果显示，脑缺血２０分钟后，脑线粒体的呼吸控制率和线粒体膜的流动性开始降低，至再灌注１小时降为最低。随着再灌注时间的延长，两者都有恢复，但始终不及假手术组水平。琥珀酸脱氢酶和细胞色素氧化酶的活性在脑缺血及再灌注期降低的程度更重，持续的时间更长。结论：缺血性脑损伤对脑线粒体膜的破坏作用可能是线粒体呼吸受抑制的主要原因     

急性脑外伤大鼠脑神经节苷脂含量的研究

目的：研究急性脑外伤后大鼠脑神经节苷脂含量的变化。方法：选取Ｗｉｓｔａｒ大鼠３６只，落体撞击法造成左顶脑挫裂伤，于伤后４、１２和２４小时测定脂结合唾液酸含量和神经节苷脂组分的相对百分比值变化。结果：急性脑外伤后大鼠脑组织中脂结合唾液酸含量随时间呈明显下降趋势。神经节苷脂组分的变化以ＧＤ３的持续下降及ＧＭ１的增加最为显著，而且受损２４小时后除ＧＤ１ａ外其余几种神经节苷脂含量均较受损１２小时后有显著降低。结论：急性脑外伤后脑内有自我调节机制来增加ＧＭ１的合成，但是这种自我调节是有限的，因此，建议早期使用神经节苷脂治疗急性脑外伤。     

缺血性脑损伤对脑线粒体膜流动性及呼吸功能影响的研究

缺血性脑损伤对脑线粒体膜流动性及呼吸功能影响的研究李露斯，汪青松，蔡昌启线粒体是重要而敏感的细胞器．脑线粒体呼吸功能决定神经元的存活力．生物膜是动态结构，膜流动性为生物发挥生理功能提供必要的条件．脑缺血再＿灌流对线粒体膜流动性及线粒体呼吸功能的影响，...     

GM1对大鼠全脑缺血再灌注中海马线粒体钙和钙调素的影响

用神经节苷脂（ＧＭ１）（１００ｍｇ／ｋｇ）处理器血管闭塞蛤脑缺血再灌注模型，观察缺血海马组织线粒体钙（Ｍｉｔｏｃｈｏｎｄｒｉａ Ｃａｌｃｉｎｍ、ＭＣａ）钙调素（Ｃａｌｍｏｄｕｌｉｎ，ＣａＭ）的含量变化及评价ＧＭ１对其变化的影响。结果表明。ＧＭ１能遏止缺血再灌注期ＭＣａ、ＣａＭ含量明显增高的变化，明显减轻缺血再灌注期的钙跨膜内流，具有肯定的脑保护作用。     

神经节苷脂GM1对小鼠脑缺血复灌后记忆障碍的实验治疗作用

目的探讨脑缺血复灌后连续应用ＧＭ1对改善记忆障碍的疗效。方法采用小鼠夹闭双侧颈总动脉的脑短暂缺血复灌模型．缺血１５ｍｉｎ后复灌开始即连续给予ＧＭ１,７ｄ后进行开场行为、抑制性回避反应及Ｙ-水迷宫试验．检测动物行为及认知功能．然后脑片观察海马ＣＡ１区锥体细胞密度。结果ＧＭ１组（１０ｍｇ／Ｋｇ·ｄ．共７ｄ）;抑制性回避反应经首次训练后２４ｄ再次检测,错误次数明显减少（从２.１±１．１４到０．７±０．４６）、Ｙ－水迷宫试验正确次数明显增加（从０．７±０．９到１．５±１．０）,而对照组两次检测结果均无显著差异。脑片观察ＧＭ１明显抑制脑缺血复灌后迟发性神经元死亡．ＧＡ１区锥体细胞数为７８±１５／ｍｍ。显著高于对照组（５３±６．１／ｍｍ）。结论复灌开始后连续应用ＧＭ１治疗对脑缺血造成的记忆障碍有明显改善作用,这可能与其减少迟发性神经元死亡有关。     

左归丸对大鼠慢性脑缺血线粒体呼吸链酶复合物活性的影响

目的探讨左归丸对慢性脑缺血线粒体呼吸链酶复合物活性的影响及其机制。方法将实验大鼠随机分为对照组、假手术组、慢性脑缺血组和左归丸治疗组,各8例。后两组大鼠采用双侧颈总动脉结扎法建立慢性脑缺血大鼠模型。左归丸治疗组于术后3周开始每天予以左归丸液灌胃,剂量为4.0 g/kg/d。术后6周,测试各组大鼠脑皮质线粒体活性氧(ROS)水平及线粒体呼吸链酶复合物活性测定。结果与对照组及假手术组比较,慢性脑缺血组大鼠的脑的线粒体ROS水平明显增高(P0.01),呼吸链酶复合物Ⅰ-Ⅳ活性均有不同程度的明显降低(P0.01);接受左归丸治疗组的大鼠的脑的线粒体ROS水平明显降低(P0.05),呼吸链酶复合物Ⅳ(COX)活性得到明显改善(P0.05)。结论左归丸可能通过修复线粒体COX的活性水平,改善线粒体功能,减少ROS的生成,从而起到对慢性缺血的保护作用。     

中枢神经系统感染患者血浆与脑脊液中内皮素变化的研究

目的探讨中枢神经系统感染（ＣＮＳＩ）时血浆与脑脊液（ＣＳＦ）中内皮素（ＥＴ）的变化。方法采用放射免疫法测定６６例ＣＮＳＩ患者血浆和ＣＳＦ中的ＥＴ－１水平。结果ＣＮＳＩ患者ＣＳＦ中的水平较对照组高，对照组与病毒脑、结脑组相比有显著性差异（Ｐ分别＜０．００１、０．０５），与化脑组相比无显著性差异（Ｐ＞０．０５），血浆中的水平与ＣＮＳＩ关系不明显（Ｐ＞０．０５）；ＣＮＳＩ伴重度脑功能障碍者ＣＳＦ和血浆中ＥＴ－１水平明显高于伴轻中度脑功能障碍者（Ｐ＜０．００１）或无脑功能障碍者（Ｐ＜０．００１），轻中度脑功能障碍又明显高于无脑功能障碍者（Ｐ＜０．００１）。结论ＣＳＦ和血浆中ＥＴ－１水平与ＣＮＳＩ的病种无关，而与脑损伤的程度有关，其含量增高可作脑实质损伤的重要指标，ＥＴ－１可能是脑实质损伤的重要因素     

神经节苷脂GM1对大鼠脑外伤后脑细胞呼吸功能和结构保护作用

把SD大鼠分成正常组、生理盐水治疗的损伤对照组及GM_1治疗的损伤治疗组。结果发现对照组伤后8h和16h。脑线粒体呼吸功能明显降低,治疗组则明显好转。对照组皮层神经元细胞和线粒体超微结构有明显损害,治疗组损害明显减轻。其可能机制与GM_1保护膜脂和膜酶活性,维持膜内外离子平衡,减轻水肿及减少自由基形成等有关。     

大鼠缺血性脑线粒体损伤机制的研究

目的　研究缺血性脑线粒体功能损伤机制。　　方法　采用改良Zieve法测定脑PLA2 活力、HPLC测定线粒体磷脂含量、荧光分光光度计测脑线粒体膜流动性。　　结果　脑缺血 2 0min及再灌流早期PLA2 活性显著增强 ,随着再灌流时间延长PLA2 活性明显下降 ;缺血 2 0min再灌流 1h组线粒体卵磷脂、脑磷脂、心磷脂含量与膜流动性降低。　　结论　线粒体磷脂含量与膜流动性密切相关 ,PLA2 参与缺血性脑线粒体功能损伤。     

Polydatin prevents the induction of secondary brain injury after traumatic brain injury by protecting neuronal mitochondria

Polydatin is thought to protect mitochondria in different cell types in various diseases. Mitochondrial dysfunction is a major contributing factor in secondary brain injury resulting from traumatic brain injury. To investigate the protective effect of polydatin after traumatic brain injury, a rat brain injury model of lateral fluid percussion was established to mimic traumatic brain injury insults. Rat models were intraperitoneally injected with polydatin(30 mg/kg) or the SIRT1 activator SRT1720(20 mg/kg, as a positive control to polydatin). At 6 hours post-traumatic brain injury insults, western blot assay was used to detect the expression of SIRT1, endoplasmic reticulum stress related proteins and p38 phosphorylation in cerebral cortex on the injured side. Flow cytometry was used to analyze neuronal mitochondrial superoxide, mitochondrial membrane potential and mitochondrial permeability transition pore opened. Ultrastructural damage in neuronal mitochondria was measured by transmission electron microscopy. Our results showed that after treatment with polydatin, release of reactive oxygen species in neuronal mitochondria was markedly reduced; swelling of mitochondria was alleviated; mitochondrial membrane potential was maintained; mitochondrial permeability transition pore opened. Also endoplasmic reticulum stress related proteins were inhibited,including the activation of p-PERK, spliced XBP-1 and cleaved ATF6. SIRT1 expression and activity were increased; p38 phosphorylation and cleaved caspase-9/3 activation were inhibited. Neurological scores of treated rats were increased and the mortality was reduced compared with the rats only subjected to traumatic brain injury. These results indicated that polydatin protectrd rats from the consequences of traumatic brain injury and exerted a protective effect on neuronal mitochondria. The mechanisms may be linked to increased SIRT1 expression and activity, which inhibits the p38 phosphorylation-mediated mitochondrial apoptotic pathway. This study was approved by the Animal Care and Use Committee of the Southern Medical University, China(approval number: L2016113) on January 1, 2016.     

Insulin protects against amyloid beta-peptide toxicity in brain mitochondria of diabetic rats

This study compared the status of brain mitochondria isolated from 12-week streptozotocin (STZ)-diabetic rats versus STZ-diabetic animals treated with insulin during a period of 4 weeks. Brain mitochondria isolated from 12-week citrate (vehicle)-treated rats were used as control. For that purpose, several mitochondrial parameters were evaluated: respiratory indexes (respiratory control ratio (RCR) and ADP/O ratio), transmembrane potential (DeltaPsim), repolarization lag phase, repolarization level, ATP, glutathione and coenzyme Q (CoQ) contents, production of H2O2, ATPase activity, and the capacity of mitochondria to accumulate Ca2+. Furthermore, the effect of Abeta1-40 was also analyzed. We observed that STZ-induced diabetes promoted a significant decrease in mitochondrial CoQ9, ATPase activity, and a lower capacity of mitochondria to accumulate Ca2+ when compared with control and insulin-treated diabetic rats. The presence of 4 microM Abeta1-40 induced a significant decrease in RCR in the three groups of rats. However, this peptide induced a significant increase in the repolarization lag phase and a significant decrease in the repolarization level in control and diabetic animals without insulin treatment. Furthermore, this peptide exacerbated significantly the production of H2O2 in STZ-diabetic rats, this effect being avoided by insulin treatment. Our data show that although diabetes induces some alterations in brain mitochondrial activity, those alterations do not interfere significantly with mitochondria functional efficiency. Similarly, insulin does not affect basal mitochondria function. However, in the presence of amyloid beta-peptide, insulin seems to prevent the decline in mitochondrial oxidative phosphorylation efficiency and avoids an increase in oxidative stress, improving or preserving the function of neurons under adverse conditions, such as Alzheimer's disease.     

Mitochondrial response to calcium in the developing brain

Developmental differences in mitochondrial content and metabolic enzyme activities have been defined, but less is understood about the responses of brain mitochondria to stressful stimuli during development. Cerebral mitochondrial response to high Ca(2+) loads after brain injury is a critical determinant of neuronal outcome. Brain mitochondria isolated from 16-18-day-old rats had lower maximal, respiration-dependent Ca(2+) uptake capacity than brain mitochondria isolated from adult rats in the presence of ATP at both a pH of 7.0 and 6.5. However, in the absence of ATP, immature brain mitochondria exhibited greater Ca(2+) uptake capacity at pH 7.0 and 6.5, indicating a greater resistance of immature brain mitochondria to Ca(2+)-induced dysfunction under conditions relevant to those that exist during acute ischemic and traumatic brain injury. Acidosis reduced the maximal Ca(2+) uptake capacity in both immature and adult brain mitochondria. Cytochrome c was released from both immature and adult brain mitochondria in response to Ca(2+) exposure, but was not affected by cyclosporin A, an inhibitor of the mitochondrial membrane permeability transition. Developmental changes in mitochondrial response to Ca(2+) loads may have important implications in the pathobiology of brain injury to the developing brain.     

Neuroprotective effects of tempol, a catalytic scavenger of peroxynitrite-derived free radicals, in a mouse traumatic brain injury model.

We examined the ability of tempol, a catalytic scavenger of peroxynitrite (PN)-derived free radicals, to reduce cortical oxidative damage, mitochondrial dysfunction, calpain-mediated cytoskeletal (alpha-spectrin) degradation, and neurodegeneration, and to improve behavioral recovery after a severe (depth 1.0 mm), unilateral controlled cortical impact traumatic brain injury (CCI-TBI) in male CF-1 mice. Administration of a single 300 mg/kg intraperitoneal dose of tempol 15 mins after TBI produced a complete suppression of PN-mediated oxidative damage (3-nitrotyrosine, 3NT) in injured cortical tissue at 1 h after injury. Identical tempol dosing maintained respiratory function and attenuated 3NT in isolated cortical mitochondria at 12 h after injury, the peak of mitochondrial dysfunction. Multiple dosing with tempol (300 mg/kg intraperitoneally at 15 mins, 3, 6, 9, and 12 h) also suppressed alpha-spectrin degradation by 45% at its 24 h post-injury peak. The same dosing regimen improved 48 h motor function and produced a significant, but limited (17.4%, P<0.05), decrease in hemispheric neurodegeneration at 7 days. These results are consistent with a mechanistic link between PN-mediated oxidative damage to brain mitochondria, calpain-mediated proteolytic damage, and neurodegeneration. However, the modest neuroprotective effect of tempol suggests that multitarget combination strategies may be needed to interfere with posttraumatic secondary injury to a degree worthy of clinical translation.     

Cyclosporin A attenuates acute mitochondrial dysfunction following traumatic brain injury

Experimental traumatic brain injury (TBI) results in a rapid and significant necrosis of cortical tissue at the site of injury. In the ensuring hours and days, secondary injury exacerbates the primary damage, resulting in significant neurological dysfunction. Recent reports from our lab and others have demonstrated that the immunosuppressant cyclosporin A (CsA) is neuroprotective following TBI. The opening of the mitochondrial permeability transition pore (MPTP) is inhibited by CsA, thereby maintaining the mitochondrial membrane potential and calcium homeostasis in isolated mitochondrial. In the present study we utilized a unilateral controlled cortical impact model of TBI to assess mitochondrial dysfunction in both isolated mitochondria and synaptosomes to elucidate the neuroprotective role of CsA. The results demonstrate that administration of CsA 15 min postinjury significantly attenuates mitochondrial dysfunction as measured using several biochemical assays of mitochondria integrity and energetics. Following TBI, mitochondria isolated from the injured cortex of animals treated with CsA demonstrate a significant increase in mitochondria membrane potential and are resistant to the induction of mitochondrial permeability transition compared to vehicle-treated animals. Similarly, synaptosomes isolated from CsA-treated animals demonstrate a significant increase in mitochondria membrane potential, accompanied by lower levels of intramitochondrial Ca2+ and reactive oxygen species production than seen in vehicle-treated animals. These results suggest that the neuroprotective properties of CsA are mediated through modulation of the MPTP and maintenance of mitochondria homeostasis. Amelioration of cortical damage with CsA indicates that pharmacological therapies can be devised which will significantly alter neurological outcome after injury.     

Inhibition of endoplasmic reticulum stress alleviates secondary injury after traumatic brain injury

Apoptosis after traumatic brain injury has been shown to be a major factor influencing prognosis and outcome. Endoplasmic reticulum stress may be involved in mitochondrial mediated neuronal apoptosis. Therefore, endoplasmic reticulum stress has become an important mechanism of secondary injury after traumatic brain injury. In this study, a rat model of traumatic brain injury was established by lateral fluid percussion injury. Fluorescence assays were used to measure reactive oxygen species content in the cerebral cortex. Western blot assays were used to determine expression of endoplasmic reticulum stress-related proteins. Hematoxylin-eosin staining was used to detect pathological changes in the cerebral cortex. Transmission electron microscopy was used to measure ultrastructural changes in the endoplasmic reticulum and mitochondria. Our results showed activation of the endoplasmic reticulum stress-related unfolded protein response. Meanwhile, both the endoplasmic reticulum stress response and mitochondrial apoptotic pathway were activated at different stages post-traumatic brain injury. Furthermore, pretreatment with the endoplasmic reticulum stress inhibitor, salubrinal(1 mg/kg), by intraperitoneal injection 30 minutes before injury significantly inhibited the endoplasmic reticulum stress response and reduced apoptosis. Moreover, salubrinal promoted recovery of mitochondrial function and inhibited activation of the mitochondrial apoptotic pathway post-traumatic brain injury. These results suggest that endoplasmic reticulum stress might be a key factor for secondary brain injury post-traumatic brain injury.     

The potential role of mitochondria in pediatric traumatic brain injury

Mitochondria play a central role in cerebral energy metabolism, intracellular calcium homeostasis and reactive oxygen species generation and detoxification. Following traumatic brain injury (TBI), the degree of mitochondrial injury or dysfunction can be an important determinant of cell survival or death. Literature would suggest that brain mitochondria from the developing brain are very different from those from mature animals. Therefore, aspects of developmental differences in the mitochondrial response to TBI can make the immature brain more vulnerable to traumatic injury. This review will focus on four main areas of secondary injury after pediatric TBI, including excitotoxicity, oxidative stress, alterations in energy metabolism and cell death pathways. Specifically, we will describe what is known about developmental differences in mitochondrial function in these areas, in both the normal, physiologic state and the pathologic state after pediatric TBI. The ability to identify and target aspects of mitochondrial dysfunction could lead to novel neuroprotective therapies for infants and children after severe TBI.     

Peroxynitrite-mediated oxidative damage to brain mitochondria: Protective effects of peroxynitrite scavengers

Peroxynitrite-mediated oxidative damage has been implicated in brain mitochondrial respiratory dysfunction after traumatic brain injury (TBI), which precedes the onset of neuronal loss. The aim of this study was to investigate the detrimental effects of the peroxynitrite donor SIN-1 (3-morpholinosydnonimine) on isolated brain mitochondria and to screen penicillamine, a stoichiometric (1:1) peroxynitrite-scavenging agent, and tempol, a catalytic scavenger of peroxynitrite-derived radicals, as antioxidant mitochondrial protectants. Exposure of the isolated mitochondria to SIN-1 caused a significant dose-dependent decrease in the respiratory control ratio and was accompanied by a significant increase in state II respiration, followed by significant decreases (P < 0.05) in states III and V. These functional alterations occurred together with significant increases in mitochondrial protein carbonyl (PC), lipid peroxidation-related 4-hydroxynonenal (4-HNE), and 3-nitrotyrosine (3-NT) content. Penicillamine hydrochloride (10 microM) partially but significantly (P < 0.05) protected against SIN-1-induced decreases in states III and V. However, a 2.5 microM concentration of tempol was able to significantly antagonize a 4-fold molar excess (10 microM) concentration of SIN-1 as effectively as were higher tempol concentrations, consistent with the likelihood that tempol works by a catalytic mechanism. The protection of mitochondrial respiration by penicillamine and tempol occurred in parallel with attenuation of PC, 4-HNE, and 3-NT. These results indicate that SIN-1 causes mitochondrial oxidative damage and complex I dysfunction and that antioxidant compounds that target either peroxynitrite or its radicals may be effective mitochondrial protectants in the treatment of neural injury.     

Fasting is neuroprotective following traumatic brain injury

To determine the neuroprotective effect of fasting after traumatic brain injury (TBI) and to elucidate the potential underlying mechanisms, we used a controlled cortical impact (CCI) injury model to induce either a moderate or a severe injury to adult male Sprague Dawley rats. Tissue-sparing assessments were used to determine the level of neuroprotection of fasting, hypoglycemia (insulin 10 U), or ketone (1.66 mmoles/kg/day or 0.83 mmoles/kg/day; D-beta-hydroxtbutyrate) administration. Mitochondrial isolation and respiratory studies were utilized to determine the functionality of mitochondria at the site of injury. We also investigated biomarkers of oxidative stress, such as lipid/protein oxidation, reactive oxygen species (ROS) production, and intramitochondrial calcium load, as a secondary measure of mitochondrial homeostasis. We found that fasting animals for 24 hr, but not 48 hr, after a moderate (1.5 mm), but not severe (2.0 mm), CCI resulted in a significant increase in tissue sparing. This 24-hr fast also decreased biomarkers of oxidative stress and calcium loading and increased mitochondrial oxidative phosphorylation in mitochondria isolated from the site of injury. Insulin administration, designed to mimic the hypoglycemic effect seen during fasting did not result in significant tissue sparing after moderate CCI injury and in fact induced increased mortality at some injection time points. However, the administration of ketones resulted in increased tissue sparing after moderate injury. Fasting for 24 hr confers neuroprotection, maintains cognitive function, and improves mitochondrial function after moderate (1.5 mm) TBI. The underlying mechanism appears to involve ketosis rather than hypoglycemia.