兔脑缺血后脑微血管和突触膜Na^＋K^＋—ATP酶活性变化

建立兔MCAO局灶脑块血实验模型。通过不连续梯度超迷离心分离脑微血管(CMV)和突触膜(SPM),用生化法分别测其Na~ ,K~ -ATP酶活性。结果发现CMV Na~ ,K~ -ATP酶活性在MCAO后先升后降,而SPM Na~ ,K~ -ATP酶活性别随时相递降,且两者与脑水含量变化均有密切关系。提示CMV和SPM Na~ ,K~ -ATP酶活性变化参与了脑缺血后早期脑水肿的发生发展。     

急性脑梗塞病人的红细胞膜ATP酶活性变化和自由基损伤

测定33例急性脑梗塞病人的红细胞膜Na~ K~-ATP酶和Ca~(2 )Mg~(2 )-ATP酶活性及血浆MDA含量,红细胞SOD活性,与29例正常老年人作比较,发现急性脑梗塞病人的红细胞膜Na~ K~ -ATP酶和Ca~(2 )Mg~(2 )-ATP酶活性均降低,而血浆MDA含量升高,红细胞SOD活性下降。说明急性脑梗塞病人存在红细胞功能受损和自由基损害。这些变化的研究,对于进一步探讨脑梗塞的病理生理及其防治可能具有重要意义。     

脑缺血后脑组织和血浆中内皮素的变化

建立兔大脑中动脉阻断(MCAO)脑缺血模型。将48只兔随机分为四组:C(正常对照和假手术对照组)和脑缺血 I_2、I_4、I_(24)组,每组12只,测脑 H_2O、Na~+、K~+、Ca~(2+)、内皮素(ET)和血浆 ET。结果示脑缺血侧脑皮质和血浆中 ET 含量显著高于对照组(P<0.001),且随时相递增(P<0.01),ET与脑 H_2O 含量之间呈显著正相关(P<0.05),提示 ET 可能参与脑缺血、脑水肿的发生发展。     

局灶脑缺血脑水肿时强啡肽的含量变化

应用兔大脑中动脉阻断(MCAO)局灶脑缺血模型,将实验动物随机分成4组:对照组C、脑缺血后2h、4h、24h(I_2、I_4、I_(21))组。放射免疫法测定脑组织和血浆中强啡肽(Dynorphin,Dyn)含量的时相变化,同时测脑组织H_2O、Na~+含量。结果发现MCAO后脑组织和血浆Dyn含量显增,且随时相递增,并与脑组织H_2O、Na~+变化呈正相关,提示Dyn可能参与脑缺血、脑水肿的发生发展。     

神经节苷脂（GM1）对局灶脑缺血的保护作用

在建立兔大脑中动脉阻断(MCAO)局灶脑缺血模型基础上,观察了神经节苷脂(GM_i)对脑缺血后1h,2h,4h脑片细胞内Ca~(2 )([Ca~(2 )]_i),Na~ ,K~ -ATPase,Ca~(2 ),Mg~(2 )-ATPase活性及脑水肿的影响。结果显示GM_1能降低[Ca~(2 )]_i含量,恢复两种ATPase活性及减轻脑水肿。这为应用GM_i治疗临床脑缺血脑水肿提供了实验依据。     

脑缺血后脑细胞线粒体LPO、SOD的变化

我们利用兔MCAO模型,分别测定脑缺血后4h(I_4)、24h(I_(24))脑组织的H_2o、Na~ 、Ca~(2 )、LPO,SOD以及线粒体的LPO,SOD,经与对照组(包括假手术和正常对照)对比,结果发现脑缺血后脑组织H_2o、Na~ 、C_a~(2 )LPO及线粒体LPO均明显增加,而SOD活性变化则相反地降低。结果提示脑缺血后脑细胞线粒体的LPO含量增加,而SOD活性则下降,说明脑缺血后线粒体产生的自由基参与了脑缺血脑损伤机制。     

神经节苷脂GM_1对脑缺血脑保护作用的实验探讨

用神经节苷脂GM_1治疗实验性局灶性脑缺血大鼠,结果显示脑Na~ —K~ —ATP酶活性明显恢复,脑水肿明显减轻,光镜与电镜观察神经节苷脂GM_1治疗组鼠脑神经毡无明显水肿,病灶区大多数神经元及胶质细胞未见不可逆性损害,病理学改变明显较脑缺血生理盐水对照组轻,提示神经节苷脂GM_1对局灶性脑缺血性损害有一定的脑保护作用。     

硫酸镁对大鼠急性脑缺血再灌注损伤时ATP酶的影响

目的　探讨镁剂在动物实验性急性脑缺血再灌注 (cerebral ischemia-reperfusion,CIR)过程中对 ATP酶的影响。方法　选用 Wistar大鼠 ,按改良的 Pulsinelli法建立了大鼠颈总动脉 CIR损伤模型 ;CIR损伤 1 5 min后 ,断髓处死鼠 ,取额叶脑组织测定 ATP酶含量。结果　急性 CIR早期 ,脑中 Na -K -ATP酶活性降低极显著(P <0 .0 1 ) ,Mg2 -ATP酶和 Ca2 -ATP酶活性降低显著 (P <0 .0 5 ) ;预先应用 Mg SO4 能稳定 ATP酶的活性(Mg2 -ATP酶 ,P <0 .0 1 ;Ca2 -ATP酶、Na -K -ATP酶 ,P <0 .0 5 )。结论　 Mg SO4 对大鼠 CIR损伤的脑保护作用与防止脑内多种 ATP酶活性降低有关。     

大鼠脑缺血再灌注后线粒体ATP酶活性、形态学变化及丹参多酚酸的保护作用

目的观察注射用丹参多酚酸对大鼠脑缺血再灌注损伤后线粒体ATP酶活性、及形态学的影响。方法将54只雄性SD大鼠随机分成3组(n=18):假手术组(Sham组)、缺血再灌注组(IR组)、丹参多酚酸组(Salvianolate组)。HE染色法观察大脑神经元的组织病理学变化;TTC染色检测脑梗死体积;分光光度计发法线粒体丙二醛含量和线粒体Na~+/K~+ATP酶、Ca~(2+)ATP酶、Mg~(2+)ATP酶活性。结果假手术组大鼠脑组织红染,未见梗死灶;丹参多酚酸组神经细胞变性坏死程度、脑梗死面积较缺血再灌注组明显减轻;与缺血再灌注组相比,丹参多酚酸组线粒体丙二醛含量下降(P0.05)和线粒体Na~+/K~+ATP酶、Ca~(2+)ATP酶、Mg~(2+)ATP酶活性升高(P0.05)。结论注射用丹参多酚酸对缺血再灌注损伤有保护作用,其机制与提高线粒体ATP酶活性有关。     

灯盏花素对大鼠颅脑损伤脑组织线粒体ATP酶活性的影响

目的 探讨灯盏花素对创伤性颅脑损伤后脑组织线粒体ATP酶活性水平的影响。方法 建立自由落体脑挫裂伤模型,伤后立即腹腔注射灯盏花素注射液,采用生化检测的方法分别测定伤后4h、24h和48h及各自的对照组大鼠脑组织线粒体ATP酶活性水平。结果 颅脑损伤后大鼠脑组织线粒体Na~ -K~ ATP酶,Ca~(2 )-ATP酶及Mg~(2 )-ATP酶活性均明显下降(P<0.05),灯盏花素治疗后24h和48h脑组织线粒体ATP酶活性均高于各自时间点对照组(P<0.05)。结论 灯盏花素可以通过影响线粒体功能而减轻创伤性颅脑损伤后的继发性脑损害,从而改善预后。     

Effects of the hyperbaric oxygen treatment on the Na+,K+ -ATPase and superoxide dismutase activities in the optic nerves of global cerebral ischemia-exposed rats

The effects of hyperbaric oxygen (HBO) treatment on the Na+,K+ -ATPase and superoxide dismutase (SOD) activities were examined in the optic nerves of the rats exposed to global cerebral ischemia. Animals were exposed to global cerebral ischemia of 20-min duration and were either sacrificed or exposed to the first HBO treatment immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na+,K+ -ATPase activities measurement) or 2, 24, 48 or 168 h after ischemia (for SOD activities measurement). HBO procedure was repeated for 7 consecutive days. It was found that global cerebral ischemia induced a statistically significant decrease in the Na+,K+ -ATPase activity of the optic nerves, starting from 0.5 to 168 h of reperfusion. Maximal enzymatic inhibition was registered 24 h after the ischemic damage. The decline in the Na+,K+ -ATPase activity was prevented in the animals exposed to HBO treatment within the first 6 h of reperfusion. The results of the presented experiments demonstrated also a statistically significant increase in the SOD activity after 24, 48 and 168 h of reperfusion in the optic nerves of non-HBO-treated ischemic animals as well as in the ischemic animals treated with HBO. Our results indicate that global cerebral ischemia induced a significant alterations in the Na+,K+ -ATPase and SOD activities in the optic nerves during different periods of reperfusion. HBO treatment, started within the first 6 h of reperfusion, prevented ischemia-induced changes in the Na+,K+ -ATPase activity, while the level of the SOD activity in the ischemic animals was not changed after HBO administration.     

Neurochemical correlates of selective neuronal loss following cerebral ischemia: role of decreased Na, K-ATPase activity

In order to investigate the role of Na+,K(+)-ATPase in the development of neuronal necrosis following cerebral ischemia, ischemia was induced in gerbils by occluding the common carotid artery unilaterally for 10 min. A time-course analysis revealed that significant reductions of the Na+,K(+)-ATPase activity in the cerebral cortex and hippocampus were manifested at 15 min, 30 min, and 1 h, and returned to the control level one day following recirculation. No apparent alterations of the Mg(2+)-ATPase activity, on the other hand, were obtained throughout the experimental period. Furthermore, Scatchard analyses of [3H]ouabain binding to the cerebral cortex membranes disclosed that the Bmax values invariably decreased without any change of Kd values following ischemia. It has also been shown that treatment of the animals with an agent known to mitigate ischemic neuronal necrosis, i.e. BY-1949, significantly reversed such derangements. These results suggest that the recovery of decreased Na+,K(+)-ATPase activity shortly after ischemia exerts a protective effect against ischemic brain damage.     

缺血后处理对糖尿病大鼠局灶性脑缺血再灌注损伤线粒体的影响

目的观察缺血后处理(I-Post)对糖尿病大鼠局灶性脑缺血再灌注损伤线粒体超微结构和功能的影响,探讨I-Post诱导的脑保护的可能机制。方法采用链脲佐菌(STZ)腹腔注射建立糖尿病大鼠模型,在此基础上通过线栓法建立大鼠大脑中动脉阻塞/再灌注模型。SD糖尿病大鼠随机分为4组(n=10),空白对照组、假手术组、缺血再灌注组(I/R组)、缺血后处理组(I-Post组)。于缺血90min再灌注6h后电镜下观察线粒体超微结构、测定缺血侧脑组织线粒体中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、Na+/K+-ATPase和Ca2+-ATPase活性。结果缺血后处理能明显减轻I/R引起的线粒体超微结构的损伤,提高线粒体SOD、GSH-Px、Na+/K+-ATPase和Ca2+-ATPase的活性(P<0.05或0.0),降低MDA的含量(P<0.05)。结论线粒体可能在I-Post诱导的脑保护中起关键性作用,I-Post诱导的脑保护机制可能与SOD、Na+/K+-ATP酶、Ca2+-ATP酶和GSH-Px活性增加有关。     

Intrastriatal methylmalonic acid administration induces convulsions and TBARS production,and alters Na+,K+-ATPase activity in the rat striatum and cerebral cortex

PURPOSE: Methylmalonic acid (MMA) inhibits succinate dehydrogenase (SDH) and beta-hydroxybutyrate dehydrogenase activity in vitro. Acute intrastriatal administration of MMA induces convulsions through glutamatergic mechanisms probably involving primary adenosine triphosphate (ATP) depletion and free radical generation. In this study we investigated whether the intrastriatal administration of MMA causes lipoperoxidation and alteration in Na+, K+-ATPase activity ex vivo and characterized the electrographic changes elicited by the intrastriatal administration of this organic acid. METHODS: MMA-induced lipoperoxidation, alterations in Na+, K+-ATPase activity and electrographic changes were measured by measuring total thiobarbituric acid-reacting substances and inorganic phosphate release by spectrophotometry, and by depth electrode recording, respectively. RESULTS: We demonstrated that intrastriatal MMA (6 mmol) injection causes convulsive behavior and electrographically recorded convulsions that last approximately 2 h. Concomitant with the increase of thiobarbituric acid-reacting substances (TBARS) content, we observed a significant inhibition of Na+,K+-ATPase activity in the striatum, and activation of Na+,K+-ATPase activity in the ipsilateral cerebral cortex. Intrastriatal MMA injection increased the content of TBARS in the striatum measured 30 min (32.4 +/- 12.0%, compared with the noninjected contralateral striatum) and 3 h (39.7 +/- 5.1%, compared with the noninjected contralateral striatum) after MMA injection. TBARS content of the ipsilateral cerebral cortex increased after MMA injection at 30 min (42.1 +/- 6.0%) and 3 h (40.4 +/- 20.2%), and Na+,K+-ATPase activity in the ipsilateral cerebral cortex increased during ictal activity (113.8 +/- 18%) and returned to basal levels as electrographic convulsions vanished in the cortex. Interestingly, intrastriatal MMA administration induced a persistent decrease in Na+,K+-ATPase activity only in the injected striatum (44.9 +/- 8.1% at 30 min and 68.7 +/- 9.4 at 3 h). CONCLUSIONS: These data suggest that MMA induces lipoperoxidation associated with Na+,K+-ATPase inhibition or activation, depending on the cerebral structure analyzed. It is suggested that Na+,K+-ATPase inhibition may play a primary role in generating MMA-induced convulsions.     

Cerebral Na+,K+-ATPase activity during exposure to and recovery from acute ischemia

This study documents the Na+,K+-ATPase activity as well as selected parameters of oxidative metabolism and electrophysiological function in rat brain exposed to ischemia produced by electrocautery of the vertebral arteries and reversible occlusion of the carotid arteries. During a 0.5-h ischemic exposure in which the electroencephalograph (EEG) was abolished and energy metabolism severly compromised the Na+,K+-ATPase showed a capability for enhanced activity (120-140% of control). On recirculation, the Na+,K+-ATPase activity showed a phasic pattern, which was characterized by normal values at 0.25-2 h, increased values (115-125% of control) at 3-24 h, and, finally, normal values at 72 h of recirculation, respectively. The maintenance of Na+,K+-ATPase integrity was correlated with a gradual return of EEG activity and virtually complete restitution of the cerebral energy state during the 72 h of recirculation. Measurements of thiobarbituric acid reactive material and water soluble antioxidant during ischemia and recirculation gave no evidence of the presence of significant free radical lipid peroxidation in this model. It is concluded that Na+,K+-ATPase and its associated membrane lipids are not irreversibly damaged by ischemia in which the tissue lactacidosis is limited to less than 20 mumol g-1.     

Influence of lactate accumulation on Na,K-ATPase activity of ischemic and post-ischemic brain

In this study the cerebral Na+, K+-ATPase activity as well as selected parameters of oxidative metabolism and electrophysiological function were assessed in normoglycemic and hyperglycemic rats which were exposed to ischemia produced by electrocautery of the vertebral arteries and reversible occlusion of the carotid arteries. In hyperglycemic animals 0.5 h of ischemia was associated with massive accumulation of lactate (34 mumol X g-1) and enhanced Na+, K+-ATPase activity (116% control), whereas normoglycemic animals showed more moderate lactate accumulation (17 mumol X g-1) and normal Na+, K+-ATPase activity (102% control). In normoglycemic animals release of the carotid clamps and recirculation for 0.5-1.5 h was associated with a normalization of the lactate levels and a decrease in Na+, K+-ATPase activity (68-72% control). Restituted hyperglycemic animals showed metabolic changes which seemed related to the blood pressure, with hypotensive hyperglycemic animals showing continuing massive lactacidosis (30-35 mumol X g-1) and enhanced Na+, K+-ATPase activity (108-110% control), whereas normotensive hyperglycemic animals showed progressive decreases in lactate level (14-20 mumol X g-1) and normal or mildly suppressed Na+, K+-ATPase activity (88-97% control). These patterns of change suggest that the reperfusion of the post-ischemic hyperglycemic-hyperlactacidotic brain was inadequate or non-homogeneous.     

In vivo and in vitro effects of homocysteine on Na,K-ATPase activity in parietal, prefrontal and cingulate cortex of young rats

In the present study we determined the effect of chronic administration of homocysteine on Na+,K+-ATPase activity in synaptic membranes from parietal, prefrontal and cingulate cortex of young rats. We also studied the in vitro effect of homocysteine on this enzyme activity and on some oxidative stress parameters, namely thiobarbituric acid-reactive substances (TBA-RS) and total radical-trapping antioxidant potential (TRAP) in the same cerebral structures. For the in vivo studies, we induced elevated levels of homocysteine in blood (500 microM), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3-0.6 micromol/g of body weight) twice a day at 8 h intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Rats were killed 12 h after the last injection. Chronic administration of homocysteine significantly decreased (50%) Na+,K+-ATPase activity in parietal, increased (36%) in prefrontal and did not alter in cingulate cortex of young rats. In vitro homocysteine decreased Na+,K+-ATPase activity and TRAP and increased TBA-RS in all cerebral structures studied. It is proposed that the alteration of Na+,K+-ATPase and induction of oxidative stress by homocysteine in cerebral cortex may be one of the mechanisms related to the neuronal dysfunction observed in human homocystinuria.     

Intrastriatal hypoxanthine administration affects Na,K-ATPase, acetylcholinesterase and catalase activities in striatum, hippocampus and cerebral cortex of rats

The aim of this study was to investigate the effects of a single intrastriatal injection of hypoxanthine, the major metabolite accumulating in Lesch-Nyhan disease, on Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities in striatum, cerebral cortex and hippocampus of rats at different post-infusion periods. Adult Wistar rats were divided in two groups: (1) vehicle-injected group (control) and (2) hypoxanthine-injected group. For Na(+),K(+)-ATPase activity determination, the animals were sacrificed 3h, 24h and 7 days after drug infusion. For the evaluation of acetylcholinesterase and catalase activities, the animals were sacrificed 30min, 3h, 24h and 7 days after hypoxanthine infusion. Results show regional and time dependent effects of hypoxanthine on Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities. The in vitro effect of hypoxanthine on the same enzymes in striatum was also investigated. Results showed that hypoxanthine inhibited Na(+),K(+)-ATPase, but not the activities of acetylcholinesterase and catalase in rat striatum. We suggest that these modification on cerebral biochemical parameters (Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities) induced by intrastriatal administration of hypoxanthine in all cerebral structures studied, striatum, hippocampus and cerebral cortex, could be involved in the pathophysiology of Lesch-Nyhan disease.     

Methylmalonate administration decreases Na+,K+-ATPase activity in cerebral cortex of rats

Buffered methylmalonate (MMA) was injected s.c. into rats twice a day at 8 h intervals from 5 to 25 days of age (chronic treatment), or into 10-day-old rats three times a day at 1 h intervals (acute treatment). Control rats received saline in the same volumes. Na+,K+-ATPase and Mg2+-ATPase activities were determined in the synaptic plasma membranes from cerebral cortex of rats. Na+,K+-ATPase activity was reduced by 30-40% in MMA-treated rats, whereas Mg2+-ATPase activity was not. In contrast, MMA at final concentrations ranging from 0.1 to 2.0 mM had no in vitro effect on these enzyme activities. However, when brain homogenates were incubated with 2 mM MMA before membrane preparation, Na+,K+-ATPase activity was decreased by 44%. Furthermore, this reduction was totally prevented by the simultaneous addition of glutathione and MMA, suggesting that oxidation of thiol groups or other oxidative damage to the enzyme could be responsible for this effect.