Sex differences in postischemic neuronal necrosis in gerbils

Twenty-four hour postischemic neuronal necrosis was compared in male vs. female Mongolian gerbils subjected to a 3-h period of severe incomplete hemispheric ischemia produced by unilateral carotid occlusion. The incidence of stroke-prone males was 42.9% versus 26.7% for the females. Among the stroke-prone animals, the males displayed significantly greater neuronal necrosis at 24 h after ischemia compared to the females in the cerebral cortex and CA1 region of the hippocampus. In the CA1 region of the stroke-prone males, only 2.0% of the normal neuronal population remained by 24 h compared to 36.8% in the stroke-prone females (p less than 0.02). In the cerebral cortex, the males had only 19.9% of normal versus 58.2% in the females (p less than 0.05). In a second series of mechanistic experiments, no differences in cortical blood flow (CBF) were disclosed between preselected male and female stroke-prone animals before, during, or for 2 h after ischemia. As with the CBF, the extent of cortical extracellular hypocalcia during ischemia did not differ significantly. However, the degree of postischemic recovery of cortical extracellular calcium was significantly better in the females from 30 min to 2 h after reperfusion. In the same experiments, hemispheric vitamin E levels were measured at the 2 h time point as an index of postischemic brain lipid peroxidation. No difference in baseline vitamin E levels was observed between male and female sham-operated gerbils. In the males subjected to 3 h of ischemia plus 2 h of reperfusion, the hemispheric vitamin E decreased by 43.5% compared to the sham-operated males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevention of postischemic hyperthermia prevents ischemic injury of CA1 neurons in gerbils

Halothane-anesthetized Mongolian gerbils were submitted to 5-min bilateral carotid artery occlusion. After ischemia, halothane anesthesia was continued for various periods of up to 85 min, and the degree of CA1 neuronal injury was estimated 7 days later by counting the number of surviving pyramidal cells. During ischemia and postischemic halothane anesthesia, rectal and cranial temperature was kept at control level (37.7 and 37.0 degrees C, respectively) using a feedback-controlled heating system. When anesthesia was discontinued after ischemia, transient hyperthermia occurred. In animals with 0- and 15-min postischemic halothane anesthesia, both cranial and rectal temperature rose by approximately 1.5 degrees C, and the number of surviving CA1 neurons amounted to less than 25% of control. After 45- or 85-min postischemic anesthesia, hyperthermia was significantly reduced and the number of surviving neurons increased to 65 and 89%, respectively. The protective effect of postischemic anesthesia was lost when anesthetized animals were submitted to the same hyperthermic profile as nonanesthetized ones, using a feedback-controlled heating system (16% surviving neurons in hyperthermia vs. 89% in normothermia, respectively). These observations demonstrate that postischemic anesthesia with 1% halothane protects against delayed neuronal death by preventing postischemic hyperthermia and not by its anesthetic effects.     

巴曲酶对沙土鼠海马CA1区神经元保护作用及其机制的研究

目的探讨巴曲酶对沙土鼠前脑缺血再灌注神经元保护作用的可能机制及时效关系。方法实验动物随机分为10组,分别为缺血再灌注后0h、1h、3h、6h、12h、24h、48h和72h组、假手术组及对照组,各个时间点分别给予腹腔注射巴曲酶8BU／kg,采用免疫组化SP法检测海马CA1区的Bcl-2、eNOS、VEGF及Akt蛋白的表达,光镜下计算沙土鼠海马CA1区的神经元阳性细胞数。并行电镜下神经元超微结构的观察。结果使用巴曲酶后,Bcl- 2、eNOS、VEGF及Akt蛋白表达明显增加,神经元阳性细胞数在巴曲酶各时间点与对照组比较差异有显著性(P< 0．01),而巴曲酶0h-24h时间点之间比较差异无统汁学意义(P>0．05);Bcl-2、eNOS及VEGF神经元阳性细胞数在0h-24h时间点与48h-72h时间点比较差异有统计学意义(P<0．05);超微结构显示0h-24h时间点较48h及 72h时间点抗细胞凋亡明显。结论巴曲酶脑保护作用的机制可能是通过上调Bcl-2、eNOS、VEGF及Akt蛋白的表达;72h内使用巴曲酶具有神经元保护作用,但在0h-24h时间点使用较48h及72h时间点保护作用更为明显。     

Delayed neuronal death in hippocampal CA1 pyramidal neurons after forebrain ischemia in hyperglycemic gerbils: amelioration by indomethacin

Hyperglycemia worsens ischemic-induced neuronal damage. Many reports argue the delayed neuronal cell death (DND) after forebrain ischemia in gerbils is due to apoptosis. We examined the effects of hyperglycemia and indomethacin on DND after forebrain ischemia in gerbils. Complete occlusion of both common carotid arteries was performed for 3.5 min followed by declamping and reperfusion. Blood glucose levels were maintained at 25-30 mmol/1 for 24 h after reperfusion in the hyperglycemic groups. We examined morphological changes consistent with DND using Nissel-stained sections and DNA fragmentation using TUNEL staining, at 12, 24, 36, 48, 60, 72, 84, 96, 108, 120 h, and 7 days after reperfusion. DND was noted 96-120 h after ischemia in normoglycemic group. Hyperglycemia enhanced the development of DND at an earlier stage (48-84 h after ischemia). TUNEL positive neurons were detected 72-108 h after reperfusion in normoglycemic group, but very few TUNEL positive neurons were detected in hyperglycemic group at 36-48 h. Indomethacin reduced the number of TUNEL-positive cells in normoglycemia and completely inhibited the appearance of TUNEL-positive cells under hyperglycemia. The number of viable neurons at 7 days after ischemia was markedly higher in indomethacin-treated groups than vehicle-treated group. Our results indicate that hyperglycemia worsens DND after forebrain ischemia in gerbils but such process is not associated with DNA fragmentation. Our results also showed that indomethacin provides a neuroprotective effect in normo- and hyperglycemic conditions.     

Exercise preconditioning exhibits neuroprotective effects on hippocampal CA1 neuronal damage after cerebral ischemia

Recent evidence has suggested the neuroprotective effects of physical exercise on cerebral ischemic injury. However, the role of physical exercise in cerebral ischemia-induced hippocampal damage remains controversial. The aim of the present study was to evaluate the effects of pre-ischemia treadmill training on hippocampal CA1 neuronal damage after cerebral ischemia. Male adult rats were randomly divided into control, ischemia and exercise + ischemia groups. In the exercise + ischemia group, rats were subjected to running on a treadmill in a designated time schedule(5 days per week for 4 weeks). Then rats underwent cerebral ischemia induction th rough occlusion of common carotids followed by reperfusion. At 4 days after cerebral ischemia, rat learning and memory abilities were evaluated using passive avoidance memory test and rat hippocampal neuronal damage was detected using Nissl and TUNEL staining. Pre-ischemic exercise significantly reduced the number of TUNEL-positive cells and necrotic cell death in the hippocampal CA1 region as compared to the ischemia group. Moreover, pre-ischemic exercise significantly prevented ischemia-induced memory dysfunction. Pre-ischemic exercise mighct prevent memory deficits after cerebral ischemia through rescuing hippocampal CA1 neurons from ischemia-induced degeneration.     

三羟异黄酮对大鼠海马CA1区神经元自发放电的影响

目的研究三羟异黄酮（genistein，GST）对静息状态下的海马脑片神经元活动的影响。方法应用细胞外记录单位放电技术。结果（1）在48个CA1区神经元放电单位给予GST（10，50，100μmol／L）2min，有46个放电单位（95．83％）放电频率明显降低，且呈剂量依赖性；（2）在9个CA1区神经元放电单位上，GST（50μmol／L）的抑制效应可被G蛋白激活的内向整流型钾通道（Gprotein—coupled inwardly rectifying K^＋channels，GIRK）阻断剂（tetraethylammonium，TEA）1mmol／L完全阻断；（3）10个放电单位灌流一氧化氮合酶抑制剂（N^G-nitro—L—arginine methyl ester，L—NAME）50μmol／L，有9个单位（90．0％）放电明显增加，在此基础上灌流GST（50μmol／L）2min，放电被抑制；（4）预先用0．2mmol／L的L—glutamate（L—Glu）灌流海马脑片，11个放电单位放电频率明显增加，表现为癫痫样放电，在此基础上灌流GST（50μmol／L）2min，其癫痫样放电被抑制。结论GST可抑制海马神经元自发放电，并可抑制由L—NAME和L—glutamate诱发的神经元放电。提示GST对中枢神经元通过降低其活动而具有一定程度的保护作用，这种作用与钾电流有关，似与其激动GIRK促进K^＋外流引起细胞膜超极化以及NO产生增加有关。     

Ischemia-induced changes of platelet endothelial cell adhesion molecule-1 in the hippocampal CA1 region in gerbils

We observed chronological changes of platelet endothelial cell adhesion molecule-1 (PECAM-1), final mediator of neutrophil transendothelial migration, immunoreactivity, and protein level in the gerbil hippocampus proper after 5 min of transient ischemia. One day after ischemic insult, PECAM-1 immunoreactivity and protein level increased slightly in the hippocampus proper. Thereafter, PECAM-1 immunoreactivity and protein level increased significantly in the hippocampus proper by 4 days after ischemic insult. Especially, PECAM-1 in the hippocampal CA1 region was higher than that in the CA2/3 region. Five days after ischemic insult, PECAM-1 immunoreactivity decreased compared to the 4 days post-ischemic group. However, the RNA levels of PECAM-1 in the hippocampus proper were significantly decreased in the 4 days post-ischemic groups compared to that in the sham-operated group. This result suggests that the increase of PECAM-1 and decrease of PECAM-1 RNA in the CA1 region 4 days after ischemia may be associated with transmigration of neurotrophil.     

Adenosine treatment delays postischemic hippocampal CA1 loss after cardiac arrest and resuscitation in rats

Resuscitation from cardiac arrest results in reperfusion injury that leads to increased postresuscitation mortality and delayed neuronal death. One of the many consequences of resuscitation from cardiac arrest is a derangement of energy metabolism and the loss of adenylates, impairing the tissue's ability to regain proper energy balance. In this study, we investigated the effects of adenosine (ADO) on the recovery of the brain from 12 min of ischemia using a rat model of cardiac arrest and resuscitation. Compared to the untreated group, treatment with adenosine (7.2 mg/kg) initiated immediately after resuscitation increased the proportion of rats surviving to 4 days and significantly delayed hippocampal CA1 neuronal loss. Brain blood flow was increased significantly in the adenosine-treated rats 1 h after cardiac arrest and resuscitation. Adenosine-treated rats exhibited less edema in cortex, brainstem and hippocampus during the first 48 h of recovery. Adenosine treatment significantly lowered brain temperature during recovery, and a part of the neuroprotective effects of adenosine treatment could be ascribed to adenosine-induced hypothermia. With this dose, adenosine may have a delayed transient effect on the restoration of the adenylate pool (AXP = ATP + ADP + AMP) 24 h after cardiac arrest and resuscitation. Our findings suggested that improved postischemic brain blood flow and ADO-induced hypothermia, rather than adenylate supplementation, may be the two major contributors to the neuroprotective effects of adenosine following cardiac arrest and resuscitation. Although adenosine did not prevent eventual CA1 neuronal loss in the long term, it did delay neuronal loss and promoted long-term survival. Thus, adenosine or specific agonists of adenosine receptors should be evaluated as adjuncts to broaden the window of opportunity in the treatment of the reperfusion injury following cardiac arrest and resuscitation.     

Expression of neurotrophin receptors by proliferating glia in postischemic hippocampal CA1 sector of adult monkeys

Transient global cerebral ischemia elicits a nearly total neuronal cell death in the hippocampal CA1 sector, accompanied by a marked microglial and astroglial proliferation. The molecular mechanisms regulating the postischemic glial reaction in the primate brain remain obscure. Here we present in situ evidence that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for the TrkA ligand nerve growth factor (NGF) and the tyrosine kinase TrkB, a receptor for brain-derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus.     

Role of the hippocampal CA2 region following postischemic hypothermia in gerbil

To investigate the changes in the principal subunit of N-methyl-D-aspartate (NMDA) receptor 1 (NR1) following the transient ischemia and postischemic hypothermia, in situ hybridization was used in the gerbil hippocampus. One of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, Glutamate receptor 2 (GluR2) was also investigated to compare with NR1. Even at 1 day, NR1 and GluR2 mRNAs in the CA1 region were reduced following ischemia. Although postischemic hypothermia prevented almost all the neuronal cell death by ischemia and inhibited the reduction of NR1 and GluR2 mRNAs in the CA1 region after 7 days, the downregulation of NR1 mRNA in the CA2 region was observed even at 1 day. This change was specific for NR1 and not for GluR2. These results suggest that the changes in NR1 and GluR2 receptors at the mRNA level would occur in spite of postischemic hypothermia. The phenomenon in the CA2 region may play an important role to rescue neuronal cell death by ischemia.     

Indomethacin ameliorates ischemic neuronal damage in the gerbil hippocampal CA1 sector

The purpose of our experiment was to examine whether the cyclooxygenase inhibitor indomethacin ameliorates neuronal injury in the gerbil hippocampal CA1 sector following 5 minutes of forebrain ischemia. Thirty minutes before bilateral carotid artery occlusion, Mongolian gerbils were injected intraperitoneally with 1 (n = 10), 2 (n = 10), 5 (n = 12), or 10 (n = 7) mg/kg of indomethacin. Seven days after occlusion, the gerbils were perfusion-fixed and neuronal density in the hippocampal CA1 sector was assessed. The mean +/- SEM neuronal density in nine unoperated normal gerbils was 307 +/- 9/mm, in 10 untreated ischemic gerbils 55 +/- 21/mm, and in seven vehicle-treated ischemic gerbils 15 +/- 9/mm. The mean +/- SEM neuronal density in ischemic gerbils treated with 1, 2, 5, or 10 mg/kg indomethacin was 132 +/- 28/mm, 154 +/- 29/mm, 176 +/- 30/mm, and 136 +/- 39/mm, respectively. Indomethacin at any dose significantly ameliorated ischemic neuronal damage in the gerbil hippocampal CA1 sector.     

Adinazolam affects biogenic amine release in hippocampal CA1 neuronal circuitry.

The new triazolobenzodiazepine, adinazolam, which has dual anxiolytic and antidepressant activities, was studied for its effects on hippocampal CA1 norepinephrine and serotonin release in chloral hydrate-anesthetized rats, with in vivo voltammetry. Norepinephrine signals were further characterized in vivo by the detection of a significantly increased norepinephrine signal (mean = 25.8%) (p less than 0.003) after intraperitoneal administration of the alpha 2 adrenoreceptor antagonist, yohimbine, and by the detection of a significantly decreased norepinephrine signal (mean = 20.1%) (p less than 0.037) after intraperitoneal administration of the alpha 2 adrenoreceptor agonist, clonidine. Time course studies showed that the anxiolytic-antidepressant drug adinazolam (10 mg/kg IP) significantly decreased hippocampal norepinephrine release (mean = 26.2%) (p less than 0.007). The norepinephrine signal was further significantly decreased by adinazolam (mean = 16.4%) (p less than 0.009) after an additional 2 mg/kg IP injection. Serotonin release, which was detected with norepinephrine in sequence, was also significantly decreased by adinazolam (10 mg/kg IP) (mean = 22.4%) (p less than 0.002). The supplemental dose of adinazolam (2 mg/kg IP), however, did not significantly alter serotonin release any further (p less than 0.307). The findings show that the mechanism of action of adinazolam occurs simultaneously on presynaptic release mechanisms for norepinephrine and for serotonin in CA1 region of hippocampus. These findings implicate that noradrenergic and serotonergic release mechanisms may be responsible in part for the dual anxiolytic-antidepressant efficacy of adinazolam.     

Drug effects on calcium homeostasis in mouse CA1 hippocampal neurons

Voltage-dependent Ca2+ channels (VDCC) are important in control of neuronal excitability, synaptic transmission, and many other cellular process. Even the slightest alteration in Ca2+ currents can have a considerable impact on the neuronal function. However, it is still unknown whether Ca2+ currents are affected by neurotoxic drugs such as lead, cobalt, zinc, cadmium, thallium, lanthanum, and aluminum. We have characterized the effects of neurotoxic drugs on Ca2+ homeostasis in CA1 hippocampal C57BL mice. Fura 2-AM fluorescence photometry was used to measure intracellular Ca2+ concentration ([Ca2+]i) in the presence and absence of neurotoxic drugs (10 microM) in response to KCl application. The peak [Ca2+]i due to KCl application was reduced in the presence of lead (60%), cobalt (35%), zinc (62%), cadmium (71%), thallium (27%), and lanthanum (66%). By contrast, in the presence of aluminum the peak [Ca2+]i was either increased (46%) or it was not affected. These results indicate that neurotoxic drugs could block the entry of calcium into CA1 neurons via VDCC.     

Sustained calpain activation associated with lysosomal rupture executes necrosis of the postischemic CA1 neurons in primates

Because of the paucity of primate experimental models, the precise molecular mechanism of ischemic neuronal death remains unknown in humans. This study focused on nonhuman primates to determine which cascade necrosis or apoptosis is predominantly involved in the development of delayed (day 5) neuronal death in the hippocampal CA1 sector undergoing 20 min ischemia. We investigated expression, activation, and/or translocation of micro-calpain, lysosome-associated membrane protein-1 (LAMP-1), caspase-3, and caspase-activated DNase (CAD), as well as morphology of the postischemic CA1 neurons and DNA electrophoresis pattern. Immunoblotting showed sustained (immediately after ischemia until day 5) and maximal (day 3) activation of micro-calpain. The immunoreactivity of activated micro-calpain became remarkable as coarse granules at lysosomes on day 2, while it translocated throughout the perikarya on day 3. The immunoreactivity of LAMP-1 also showed a dynamic and concomitant translocation that was maximal on days 2-3, indicating calpain-mediated disruption of the lysosomal membrane after ischemia. In contrast, immunoblotting demonstrated essentially no increase in the activated caspase-3 at any time points after ischemia, despite upregulation of pro-caspase-3. Although expression of CAD was slightly upregulated on day 1 or 2, or both, it was much less compared with lymph node or intestine tissues. Furthermore, light and electron microscopy showed eosinophilic coagulation necrosis and membrane disruption without apoptotic body formation, while DNA electrophoresis did not show a ladder pattern, but rather a smear pattern. Sustained calpain activation and the resultant lysosomal rupture, rather than CAD-mediated apoptosis, may cause ischemic neuronal necrosis in primates.     

挤压伤至股骨干骨折对大鼠海马CA1区形态学变化

目的观察挤压伤(CS)至股骨干骨折后缺血/再灌注(I/R)引起的SD大鼠海马CA1区形态学的变化特征。方法建立大鼠CS模型,SD大鼠双侧后肢挤压6 h,再灌注0、6、12、24 h,4%多聚甲醛灌注,用刀片把脑组织从正中矢状位分为左右两半,自上丘至视交叉节段取脑组织,一半用于高尔基染色,一半制备成石蜡切片,分别用于HE染色和Nissl染色。结果挤压伤至股骨干骨折I/R后开始出现大鼠海马神经元疏松紊乱、肿胀变性,形态结构受损,凋亡数目增加,神经元大量丢失,海马CA1区树突棘密度减少,挤压再灌注12 h最为显著,24 h上述形态有所改善,但仍低于正常水平。结论 I/R引起SD大鼠海马CA1区神经元细胞受损,受损程度在解压后12 h达到高峰。     

Neuroprotective effects of GluR6 antisense oligodeoxynucleotides on transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 region

To investigate whether the kainate (KA) receptors subunit GluR6 is involved in the neuronal cell death induced by cerebral ischemia followed by reperfusion, the antisense oligodeoxynucleotides (ODNs) of GluR6 were used to suppress the expression of GluR6 by intracerebroventricular infusion once per day for 3 days before ischemia. Transient brain ischemia was induced by four-vessel occlusion in Sprague-Dawley rats. The effects of GluR6 antisense ODNs on the phosphorylation of MLK3 and JNK and the interactions of MLK3 and PSD-95 with GluR6 were examined by immunoprecipitation and immunoblotting. Our results show that GluR6 antisense ODNs can knock down the expression of GluR6 and suppress the assembly of the GluR6.PSD-95.MLK3 signaling module and, therefore, inhibit JNK activation and phosphoralation of c-jun. On the other hand, the GluR6 antisense ODNs also show a protective role against neuronal cell death induced by cerebral ischemia/reperfusion. Administration of GluR6 antisense ODNs once per day for 3 days before cerebral ischemia significantly decreased neuronal degeneration. In conclusion, our results demonstrate that kainate receptor subunit GluR6 plays an important role in neuronal death induced by cerebral ischemia followed by reperfusion.     

Comparison of midazolam effects on inhibitory postsynaptic currents in hippocampal CA3 with those in CA1

We compared the effects of midazolam, one of the popular benzodiazepines, on inhibitory postsynaptic currents in CA3 pyramidal cells with those in CA1 pyramidal cells in rat hippocampal slices. With all of the midazolam concentrations tested, the conductance of the evoked inhibitory postsynaptic current of the CA3 and CA1 pyramidal cells was significantly larger than the control, and increased in a dose-dependent manner. The normalized conductance of the inhibitory postsynaptic currents of the CA3 and CA1 pyramidal cells was not significantly different. However, we found that midazolam significantly prolonged the decay time of inhibitory postsynaptic currents of CA3 pyramidal cells. The results suggest that the differential effect of midazolam on the inhibitory postsynaptic currents could be due to the different decay kinetics, which depend on the gamma-aminobutyric acid type A receptor subunit compositions of the CA3 and CA1 pyramidal cells.     

Nicotine exerts differential effects on different CA1 hippocampal cell types

The effects of (−) nicotine hydrogen tartrate (NHT) were examined on several cell types in the CA1 region of rat hippocampus. The results indicate that nicotine may have a preferential net inhibitory effect on basket cells and an excitatory effect on oriens/alveus interneurons. The resultant effects of nicotine on pyramidal cells may thus be a product of complex local circuit interactions.