脑缺血再灌注[^3H]——三磷酸肌醇放射活性及突触体游离C …

本文研究了大鼠脑缺血再灌流时［＾３Ｈ］－三磷酸肌醇［＾３Ｈ］－ＩＰ３）放射活性及突触体游离Ｃａ＾２＋（［Ｃａ＾２＋］ｉ）的变化，并用苯甲基磺酰氟化物（ＰＭＳＦ）治疗，观察其对［＾３Ｈ］－ＩＰ３放射活性及突触体［Ｃａ＾２＋］ｉ的影响。结果：脑缺血１ｍｉｎ［＾３Ｈ］－ＩＰ３放射活性非常显著地增高。缺血２０ｍｉｎ、缺血２０ｍｉｎ再灌注１ｈ、６ｈ、２ｄ［＾３Ｈ］－ＩＰ３放射活性非常显著地降低。缺血２０ｍ，     

全脑缺血后PAF和细胞内钙离子的变化及相关机制研究

目的 探讨血小板活化因子（ＰＡＦ）在缺血性脑损伤中的作用机制。方法 大鼠全脑缺血再灌注后,分别应用放免法和Ｆｕｒａ－２／ＡＭ荧光法测定海马组织中ＰＡＦ、突触体游离钙（「Ｃａ＾２＋」ｉ）浓度。结果 缺血２０ｍｉｎ后,ＰＡＦ含量显著高于对照组,再灌注２４０ｍｉｎ时已降至对照组水平,再灌注４８０ｍｉｎ后出现迟发性升高。对应「Ｃａ＾２＋」ｉ值随所观察的再灌注时间延长而增加。Ｔｅｔｒａｎｄｒｉｎｅ（钙拮抗剂）能明显降低再灌注４８０ｍｉｎ时ＰＡＦ和「Ｃａ＾２＋」ｉ水平。结论 全脑缺血再灌主后,ＰＡＦ的异常代谢与「Ｃａ＾２＋」ｉ水平密切关联,协同参与了神经细胞损伤的发生及发展。     

氟桂嗪对实验性脑缺血的保护作用

目的研究氟桂嗪（ＦＮＺ）对实验性脑缺血的保护作用。方法应用Ｆｕｒａ－２荧光比值法测兔大脑中动脉阻断后不同时期血小板胞浆游离［Ｃａ２＋］ｉ浓度变化，观察ＦＮＺ对其变化的影响及对缺血脑水肿的保护作用。结果血小板［Ｃａ２＋］ｉ随缺血时间延长而递增。ＦＮＺ能明显降低脑缺血后的血小板［Ｃａ２＋］ｉ及脑水含量（Ｐ＜０．０１）。结论ＦＮＺ对脑缺血有保护作用     

改良Fura—2双波长荧光法测定兔脑缺血脑片[Ca^2＋]i

采用新型Ｃａ２＋荧光指示剂Ｆｕｒａ－２建立双波长法测定兔ＭＣＡＯ局灶脑缺血脑片游离钙（［Ｃａ２＋］ｉ），结果显示脑缺血后神经细胞内［Ｃａ２＋］ｉ明显升高。     

脑缺血海马透析自由基活性研究

研究脑反复缺血再灌流羟自由基活性动态变化，探讨脑缺血损伤的病理生理机制。方法采用Ｐｕｌｓｉｎｅｌｌｉ和Ｂｒｉｅｆｌｅｙ４血管关闭方法，５ｍｉｎ×３次缺血，用微透析针植人右侧海马ＣＡ１区，用水杨酸盐捕获生成稳定的加合物２，３和２，５二氧苯甲酸（２，３和２，５ＤＨＢＡ）。用高压液相（ＨＰＬＣ）电化学检测。结果腹腔内注射水杨酸盐和微管透析针内注射水杨酸盐，２，３和２，５ＤＨＢＡ出现相似变化。缺血期２，３和２，５ＤＨＢＡ稍降低，灌流２０ｍｉｎ显著增高，灌流１ｈ升高至顶峰，持续３ｈ。证明脑反复缺血后再灌流羟自由基显著增高，并且不是一个暂时的现象。结论自由基在脑缺血损害中起重要作用。     

脑缺血选择性海马CA1区神经元损害的实验研究

采用Ｐｕｌｓｉｎｅｌｉ－Ｂｒｉｅｒｌｅｙ４血管阻塞脑缺血模型观察了大鼠全脑缺血２０ｍｉｎ再灌流８ｈ，ｃ－ｆｏｓ基因表达及再灌流７ｄ海马ＣＡ１区迟发性神经元损害。在缺血再灌流早期（８ｈ）海马ＣＡ１区极少ｃ－ｆｏｓ表达，而齿状回、海马ＣＡ３区、杏仁核大量ｃ－ｆｏｓ表达。缺血再灌流晚期（７ｄ）镀银染色显示海马ＣＡ１区神经元及其突触终末带呈黑色溃变相，而齿状回、海马ＣＡ３区、杏仁核呈金黄色正常相。相邻切片ＨＥ染色示缺血组海马ＣＡ１区核完整的锥体细胞数（５±２．６个／２００μｍ）与对照组（４０±２．９个／μｍ）比较差异有显著意义（Ｐ＜０．０１）。脑缺血诱导的ｃ－ｆｏｓ基因表达对于缺血易损海马ＣＡ１区迟发性神经元坏死可能起直接的调控作用。     

脑缺血病人血小板肌球蛋白轻链激酶和Ca2+、Mg2+-ATP酶活性及其与[Ca2+]i关系的研究

目的探讨急性缺血性脑血管病血小板肌球蛋白轻链激酶（ＭＬＣＫ）和Ｃａ２＋、Ｍｇ２＋－ＡＴＰ酶活性的变化及与血小板胞浆游离钙浓度［Ｃａ２＋］ｉ的关系。方法用３２Ｐ同位素掺入法和比色法分别测定５８例脑缺血病人，及３５名健康对照者血小板ＭＬＣＫ和Ｃａ２＋、Ｍｇ２＋－ＡＴＰ酶活性。用荧光钙指示剂Ｆｕｒａ－２负载血小板扫描的方法，测定脑缺血病人血小板［Ｃａ２＋］ｉ浓度。结果ＴＩＡ组和脑梗死组ＭＬＣＫ活性与对照组相比均有明显增加（Ｐ＜０．０１），而Ｃａ２＋Ｍｇ２＋－ＡＴＰ酶活性均低于对照组（Ｐ＜０．０５，Ｐ＜０．０１）。ＴＩＡ组和脑梗死组血小板静息［Ｃａ２＋］ｉ；均高于对照组；血小板静息［Ｃａ２＋］ｉ与血小板ＭＬＣＫ活性呈显著正相关（Ｐ＜０．０１），而与血小板Ｃａ２＋、Ｍｇ２＋－ＡＴＰ酶活性呈负相关（Ｐ＜０．０５）。结论血小板ＭＬＣＫ和Ｃａ２＋、Ｍｇ２＋－ＡＴＰ酶活性与急性缺血性脑血管病的发生有密切关系，ＭＬＣＫ活性的变化可能是脑缺血病人血小板活化的分子基础。     

兔大脑中动脉阻断后脑片细胞内游离钙的观察

兔大脑中动脉阻断后脑片细胞内游离钙的观察☆孙青芳赵卫国张天锡田恒力△张学新※目前，细胞内游离Ｃａ２＋（［Ｃａ２＋］ｉ）浓度变化是脑缺血研究领域的热点。由于成年动物神经元很难分离，因而给在体脑缺血动物模型［Ｃａ２＋］ｉ的研究带来困难。有关在体脑...     

胞外无钙时电刺激诱发海马脑片释放去甲肾上腺素

大鼠海马脑片用［３Ｈ］去甲肾上腺素（［３Ｈ］ＮＡ）孵育后，用无钙含２ｍｍｏｌ／ＬＥＧＴＡ任氏液灌流，用电刺激或高Ｋ＋诱发［３Ｈ］ＮＡ释放。电刺激诱发的［３Ｈ］ＮＡ释放被ＴＴＸ有力地抑制，被ＢＡＰＴＡ—ＡＭ显著地减弱，但ＴＴＸ和ＢＡＰＴＡ—ＡＭ对高Ｋ＋诱发的［３Ｈ］ＮＡ释放无作用。电刺激和高Ｋ＋诱发的［３Ｈ］ＮＡ释放均被蛋白激酶Ｃ（ＰＫＣ）的激活剂佛波醇脂加强，并被α２－自身受体激动剂ｃｌｏｎｉｄｉｎｅ所削弱。结果提示：在胞外无钙时，电刺激诱发海马脑片释放［３Ｈ］ＮＡ是来自囊泡的胞吐；Ｎａ＋进入神经末梢和内Ｃａ２＋释放参与这一诱发释放过程，但不参与高Ｋ＋诱发［３Ｈ］ＮＡ释放机制。     

化学致痫剂马桑内酯导致大鼠海马神经细胞内Ca^2＋升高

于培养的新生太鼠海马神经细胞,应用ｆｕｒａ－２荧光检测技术,观察了致痫剂马桑内酯对单个海马神经细胞内钙离子浓度（［Ｃａ＾２＋］ｉ）的影响。培养液内马桑内酯浓度达１０＾－８ｍｏｌ／Ｌ时可引起［Ｃａ＾２＋］ｉ增加,［Ｃａ＾２＋］ｉ随给药浓度的提高而升高。马桑内酯浓度达５×１０＾－６ｍｏｌ／Ｌ时,［Ｃａ＾２＋］ｉ维持于稳定的高水平上。当胞外无Ｃａ＾２＋时,马桑内酯仍可引起［Ｃａ＾２＋］ｉ的升高;Ｌ－型钙     

Alterations of 45Ca accumulation and [3H]inositol 1,4,5-trisphosphate binding using autoradiography in the exo-focal postischemic brain areas of the rat.

We studied the alterations of calcium accumulation and intracellular signal transduction using autoradiography of the second messenger system in order to clarify the mechanisms of the delayed neuronal changes in the remote areas of rat brain after transient focal ischemia. Chronological changes of 45Ca accumulation and [3H]inositol 1,4,5-trisphosphate (IP3) binding sites were determined after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by different periods of recirculation. After the ischemic insult, 45Ca accumulation extended to the lateral segment of the caudate putamen and to the cerebral cortex, both supplied by the occluded MCA. One day after the ischemia, [3H]IP3 binding sites decreased significantly compared with the control values in these ischemic areas. Moreover, 3 days after the ischemia, 45Ca accumulation was first detected in the ipsilateral thalamus and the substantia nigra, which lay outside the ischemic areas. In the substantia nigra, a significant decrease of [3H]IP3 binding sites and concurrent 45Ca accumulation were observed. In the thalamus, however, there was not alteration until 1 week after the ischemia, and then [3H]IP3 binding sites increased significantly 2 weeks (P less than 0.05) and 4 weeks (P less than 0.01) after the ischemia. Based on the present study, we speculate that different mechanisms associated with signal transduction systems may be responsible for exo-focal postischemic delayed neuronal changes in the thalamus and the substantia nigra. The increase of [3H]IP3 binding sites of the thalamus in the chronic stage may be new evidence of plasticity related to neurotransmission.     

Ontogeny of influence of clonidine on high potassium-induced release of noradrenaline and specific [3H]clonidine binding in the rat brain cortex

A high (20 mM) K+-induced, Ca2+-dependent release of L-[3H]-noradrenaline (NA) from cerebral cortex slices was detected in rats on day 18 of gestation. Depolarization-evoked release (% of total radioactivity) increased with age between day 18 of gestation and day 70 of postnatal life. Clonidine (0.1 microM) significantly (p less than 0.05) reduced a high K+-induced L-[3H]NA release on days 7 and 70 but not on day 1. Both KD and Bmax of specific [3H]clonidine binding to cerebral cortex membranes rapidly increased between days 1 and 7, followed by a gradual increase to the adult level. Presynaptic alpha 2-adrenoceptors which regulate NA release seems to become sensitive to alpha 2-agonists and to reach functional maturity between days 1 and 7 after birth in the cerebral cortex of rats.     

Effects of anisodamine on altered [Ca~(2+)]i and cerebral cortex ultrastructure following acute cerebral ischemia/reperfusion injury in rabbits

BACKGROUND： Calcium ion （Ca^2＋） overload plays an important role in cerebral ischemia/reperfusion injury. Anisodamine, a type of alkaloid, can protect the myocardium from ischemia and reperfusion injury by inhibiting intracellular calcium [Ca^2＋]i overload. OBJECTIVE： To investigate effects of anisodamine on [Ca^2＋]i concentration and cortex ultrastructure following acute cerebral ischemia/reperfusion in rabbits. DESIGN, TIME AND SETTING： Randomized and controlled trial was performed at the Department of Emergency, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from September to December 2006. MATERIALS： Forty healthy rabbits were used to establish models of acute cerebral ischemia/reperfusion. Anisodamine was provided by Lianyungang Dongfeng Pharmaceutical Factory; Fura-2 was purchased from Nanjing Jiancheng Bioengineering Institute; dual-wave length fluorescent spectrophotometry system and DM-300 software were provided by Bio-Rad, USA; OPTON-EM10C transmission electron microscope was product of Siemens, Germany. METHODS： Forty rabbits were randomly divided into the following groups： sham operation, ischemia, ischemia/reperfusion, and anisodamine, with ten rabbits in each group. Models of complete cerebral ischemia injury were established. In addition, blood was collected from the femoral artery of rats in the ischemia/reperfusion and anisodamine groups to induce hypotension and establish repeffusion injury models. The bilateral common carotid artery clamp was removed from the anisodamine group 20 minutes after ischemia, and anisodamine （10 mg/kg body mass） was injected via the femoral vein. Rabbits in the sham operation group underwent only venous cannulation. MAIN OUTCOME MEASURES： [Ca^2＋]i concentration was determined using a dual-wave length fluorescent spectrophotometry system, and cortical ultrastructure was observed following uranyl-lead citrate staining. RESULTS： The levels of [Ca^2＋]i in the ischemia and ischemia/reperfusion gro     

Arachidonic acid metabolism in gerbil cerebra: effects of ischemia and pentobarbital

Pentobarbital pretreatment may be used to predict biochemical events involved in ischemic brain damage following bilateral carotid artery ligations in the gerbil, since it reduces the subsequent edema and mortality. The effects of this anesthetic on the ischemia-induced modifications of cerebral arachidonic acid metabolism were investigated, in order to correlate observed alterations with tissue damage. Cerebral lipids were radiolabeled in vivo with [3H]arachidonic acid prior to 10 min of cerebral ischemia and 0-120 min of perfusion. Ischemia stimulated a 97.3% increase in unesterified [3H]arachidonate, which was due to the loss of label from choline, inositol, and ethanolamine glycerophospholipids. Tissue reperfusion stimulated further reductions in [3H]choline and [3H]inositol glycerophospholipids, while ethanolamine glycerophospholipid and triglyceride labeling increased. Inositol glycerophospholipid, but not choline glycerophospholipid, labeling returned to control level by 60 min of reperfusion. Pentobarbital pretreatment reduced the accumulation of [3H]arachidonate by 56.2% during ischemia. It increased the recovery of [3H]ethanolamine glycerophospholipids during the ischemic period and [3H]choline glycerophospholipids during the first 5 min of reperfusion. These effects accounted for the reduction of unesterified [3H]arachidonate observed during ischemia and reperfusion.     

Discrepancy between cell injury and benzodiazepine receptor binding after transient middle cerebral artery occlusion in rats

We investigated postischemic alterations in benzodiazepine receptor, D1 dopamine receptor, and muscarinic acetylcholine receptor binding after transient middle cerebral artery (MCA) occlusion in rats using [3H]-flumazenil, [3H]-SCH23390, and [3H]-N-methyl-4-piperidyl benzilate ([3H]-NMPB), respectively, as radioligand. These ligand bindings were determined at 3 and 24 h and at 3 and 7 days after ischemia/reperfusion of MCA by using autoradiographic methods. Ischemic cell injury was clearly detected from 3 h after ischemia/reperfusion and progressively increased from 3-24 h after ischemia/reperfusion of MCA. The area of cell injury reached maximum at 24 h after ischemia/reperfusion of MCA. [3H]-SCH23390 binding was reduced to 47% of the contralateral side at 3 days after ischemia/reperfusion of MCA. After 7 days, [3H]-SCH23390 binding was further reduced by 20% in the striatum. [3H]-NMPB binding was slightly decreased in both the striatum and cerebral cortex at 3 days after ischemia/reperfusion of MCA, and [3H]-NMPB binding in the striatum and cerebral cortex were reduced to 42 and 62% of the contralateral side at 7 days after ischemia/reperfusion of MCA. [3H]-NMPB was also decreased at 24 h. In contrast, [3H]-flumazenil binding was not decreased in the striatum and cerebral cortex within 7 days after ischemia/reperfusion of MCA. These results suggest that [3H]-SCH23390 and [3H]-NMPB binding do not correlate with cell injury by ischemia/reperfusion, although vulnerability to ischemia/reperfusion was observed with these receptors. In addition, central benzodiazepine receptor imaging might be essentially stable to neuronal cell injury induced by transient focal cerebral ischemia in rats, in contrast to the results of PET studies.     

Synthesis of 4′-[3H]-Phospho-pantothenic acid and studies of its metabolism in structures of the brain

[³H]-Phospho-pantothenic acid ([³H]-PhPa) was synthesized by the method of heat activation of tritium with a specific radioactivity of 9.8 GBq/mmol. Distribution, transport, and biotransformation of [³H]-PhPA were studied after its intracerebroventricular administration (unilateral and bilateral) to male rats of the Wistar line at a dose of 0.6 μCi. Unchanged [³H]-PhPA was detected in the cerebro-spinal fluid 10–20 min after the administration. [³H]-PhPA was absorbed by brain structures with predominantly ipsilateral simultaneous transformation into metabolites in the following sequence: cortex of cerebral hemispheres > hippocampus > brain stem > frontal cortex > and cerebellum. Brain structures held up to 39–42% of the total radionuclide (10–20 min). The content of the radionuclide had decreased to 17% 6 h later. This decrease was not accompanied by a significant efflux of the radioactivity into the blood circulation. The maximum level of [³H]-PhPA was observed in the hippocampus and cortex of the cerebral hemispheres (37–41% of the total radioactivity). Biotransformation of the radionuclide proceeded via dephosphorylation and biosynthesis of phosphopantetheine (12–20%) and CoA (1.5–5%). The possibility of the direct transport of [³H]-PhPA through neuronal membranes and its deposition in the CNS as precursors of biosynthesis of CoA (pantothenate, PhPA, and phosphopantetheine) was proposed.     

Ion homeostasis in rat brain in vivo: intra- and extracellular [Ca2+] and [H+] in the hippocampus during recovery from short-term, transient ischemia.

Changes in intra- and extracellular [Ca2+] and [H+], together with alterations in tissue PO2 and local blood flow, were measured in areas CA1 and CA3 of the hippocampus during recovery (up to 8 h) after an 8-min period of low-flow ischemia. Restoration of blood supply was followed by an immediate rise in flow and tissue PO2 above normal, with large fluctuations in both persisting for up to 4 h. In area CA1, [Ca2+]i decreased rapidly from an ischemic mean value of 30 microM to a control mean level of 73.1 nM in 20-30 min, whereas normalization of [Ca2+]e took approximately 1 h. Recovery of [Ca2+]i was accelerated by preischemic administration of a calcium antagonist, nifedipine, and a free radical scavenger, N-tert-butyl-alpha-phenylnitrone (PBN), but not by MK-801, a blocker of N-methyl-D-aspartate receptors. There was a secondary rise in [Ca2+]i in many cells beginning approximately 2 h after reperfusion. This was attenuated somewhat by PBN but not clearly influenced by either nifedipine or MK-801. Changes of [Ca2+]i in area CA3 were much smaller and slightly slower than in area CA1 and were not affected by the drugs mentioned above. In both areas CA1 and CA3, pHe and pHi fell during ischemia to an average value of 6.2, from which there was a rapid initial recovery in the first 5-10 min when blood flow was restored. Thereafter tissue pH rose slowly and did not reach control levels for approximately 1 h, and in some microareas not at all. It is concluded that (a) effective mechanisms for restoring normal [Ca2+]i remain intact after 8 min of low-flow ischemia; (b) in neurons of area CA1, some insidious change in the homeostasis of calcium triggers a secondary rise in its free cytosolic concentration, which may be causally related to activation of irreversible cell damage; and (c) the changes in [Ca2+]i and [Ca2+]e during and following 8 min of ischemia can be adequately accounted for by movements of a fixed pool of Ca between intra- and extracellular compartments, and possible mechanisms are discussed.     

Alteration of voltage-dependent calcium channels in canine brain during global ischemia and reperfusion.

Elevated intracellular calcium (iCa2+) plays an important role in the pathophysiology of ischemic brain damage. The mechanisms by which iCa2+ increases are uncertain. Recent evidence implicates the voltage-dependent calcium channel (VDCC) as a likely site for the alteration in Ca2+ homeostasis during ischemia. The purpose of this study was to determine whether VDCCs are altered by global ischemia and reperfusion in a canine cardiac arrest, resuscitation model. We employed the radioligand, [3H]PN200-110, to quantitate the equilibrium binding characteristics of the VDCCs in the cerebral cortex. Twenty-five adult beagles were separated into four experimental groups: (a) nonischemic controls, (b) those undergoing 10-min ventricular fibrillation and apnea, (c) those undergoing 10-min ventricular fibrillation and apnea followed by spontaneous circulation and controlled respiration for 2 and (d) 24 h. Brain cortex samples were taken prior to killing of the animal, frozen immediately in liquid nitrogen, and crude synaptosomal membranes isolated by differential centrifugation/filtration. After 10 min of ischemia the maximal binding (Bmax) of [3H]PN200-110 increased to greater than 250% of control values (control Bmax 11.16 +/- 0.98; ischemic 28.35 +/- 2.78 fmol/mg protein; p less than 0.05). Bmax returned to near control values after 2 h of reperfusion but remained significantly greater than the control at 24 h. Although the affinity constant (Kd) (control = 0.12 +/- 0.03 nM) appeared to increase with ischemia and normalize with reperfusion, the changes were not statistically significant. We conclude that the binding of [3H]PN200-110 to L-type VDCCs is increased after 10 min of global ischemia/anoxia produced by ventricular fibrillation and apnea in the dog.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative brain autoradiography of [9,10-3H]palmitic acid incorporation into brain lipids

The distribution of radioactivity within brain metabolic compartments was examined following the intravenous injection of [9,10-3H]palmitate into awake rats. Brain radioactivity reached a maximum value by 15 min after [9,10-3H]palmitate injection and remained unchanged for at least 4 hr. Regional differences in radioactivity could be determined with high resolution by quantitative autoradiography, at the level of cell layers within the hippocampus and cerebral cortex, and between striosomes of the caudate nucleus. Regional brain radioactivities were converted to normalized regional radioactivities (k) by dividing them by the integrated plasma fatty acid radioactivity (integrated over the time course of the experiment). These values reflected incorporation mainly into brain phospholipids; radioactivity due to nonlipid components was minimal. Indeed, about 85% of brain radioactivity was within lipids between 5 min and 4 hr postinjection, the remainder being equally divided between protein-associated pellet and aqueous-soluble metabolites. The major lipids labeled were phospholipids, particularly phosphatidylcholine, which contained about 75% of phospholipid radioactivity. The results show that [9,10-3H]palmitate can be used to examine incorporation of plasma palmitate into individual brain regions via quantitative autoradiography. Furthermore, the tracer is a rather selective marker for phosphatidylcholine and can be used to examine turnover and synthesis of this phospholipid. [9,10-3H]palmitate has advantages over [U-14C]palmitate for autoradiographic studies of incorporation; following the 14C-tracer, significant label even at 4 hr after injection is in nonlipid compartments (glutamate and aspartate), and the long path length of 14C limits resolution at the cell layer level.