Differential regulation of cyclooxygenase-2 in the rat hippocampus after cerebral ischemia and ischemic tolerance

We investigated the temporal changes and cellular localization of cyclooxygenase-2 (COX-2) in the rat hippocampus during the induction of acquired ischemic tolerance by sublethal ischemia, and compared these changes with those occurring following transient forebrain ischemia. Adult male Sprague Dawley rats were subjected to either 10 min of lethal global ischemia with or without 3 min of sublethal ischemic preconditioning, or 3 min of ischemia only. A short (3 min) cerebral ischemia as well as lethal ischemia with preconditioning substantially and significantly upregulated COX-2 expression in dentate granule cells, as confirmed by immunoblot analysis. This became evident by 4 h, peaked at 1-3 days, and returned to the basal level around 7 days. COX-2 expression was also increased in CA2 and CA3 neurons, although with weaker staining intensity, but in CA1 neurons very weak immunoreactivity was transiently observed. In the ischemic hippocampus, however, in agreement with previous reports, COX-2 expression was induced strongly in vulnerable CA1 and hilar neurons as well as in resistant CA3 and dentate granule cells. These data demonstrated that COX-2 expression is upregulated in neuronal subpopulations destined to survive, i.e., in CA3 and dentate granule cells after ischemia and ischemia-tolerance induction, as well as in ischemia-vulnerable neurons, i.e., in CA1 neurons after lethal ischemia, suggesting that hippocampal neuronal subpopulations have differential sensitivity to COX-2 upregulation.     

听源性惊厥易感大鼠惊厥和点燃后前脑结构内的c—fos表达

为探讨听源性惊厥点燃和前脑结构的关系，用免疫细胞化学方法结合体视学分析，研究Ｗｉｓｔａｒ种系的听源性惊厥易感大鼠惊厥和点燃后，前脑结构内ｃ－ｆｏｓ表达的差异。结果显示，１．正常Ｗｉｓｔａｒ大鼠接受一次强音刺激后，海马，齿状回，杏仁核，内嗅皮质，嗅周皮质和额－顶皮质内未见Ｆｏｘ阳性神经元；２．Ｐ７７ＰＭＣ大鼠一次惊厥后，除海马，齿状回外，上述被检各区内可见广泛的Ｆｏｓ阳性神经元，其分布具有区域差异．     

Ischemic preconditioning decreases intracellular zinc accumulation induced by oxygen-glucose deprivation in gerbil hippocampal CA1 neurons

In normal gerbils, intracellular zinc ions ([Zn2+]i) and calcium ions ([Ca2+]i) accumulate in hippocampal CA1 neurons after global ischemia. We examined whether ischemic preconditioning modifies these changes in gerbil hippocampal slices. In normal slices, large increases in [Zn2+]i and [Ca2+]i were observed in the stratum radiatum of the CA1 area after oxygen-glucose deprivation. In preconditioned slices, there were significantly decreased peak levels of [Zn2+]i and [Ca2+]i in CA1. However, there were no differences in the peak levels of these ions in CA3 and dentate gyrus. These results suggest that modified [Zn2+]i and [Ca2+]i accumulation after an ischemic insult might be important for the mechanisms of ischemic tolerance induced by preconditioning.     

Behavioral evaluation of ischemic damage to CA1 hippocampal neurons: effects of preconditioning

In Mongolian gerbils, global forebrain ischemia induces enhanced locomotor activity and the disruption of nest building immediately after the insult, followed by damage to hippocampal neurons developing 3 days later. Preconditioning by a brief episode of sublethal ischemia induces the protection of CA1 hippocampal neurons against a lethal ischemic insult. We examined how preconditioning with 2-min ischemia affects disturbances in the nest building behavior and locomotor activity induced by the injurious 3-min ischemia. Morphological examination confirmed that preconditioning significantly reduced neuronal damage in CA1 evoked by injurious ischemia. Behavioral studies demonstrated that preconditioning reduced the locomotor hyperactivity and latency in nest building after test ischemia, in comparison to sham or naive animals. The results indicate that the nest building test and measurement of locomotor activity may be used for an early in vivo prediction of the extent of ischemic brain damage and tolerance induced by ischemic preconditioning.     

吗啡依赖小鼠海马神经元c-fos的表达

目的:探讨吗啡依赖小鼠海马不同亚区神经元c-fos表达的差异。方法:以剂量递增法皮下注射吗啡建立吗啡依赖小鼠模型,腹腔注射纳洛酮诱发戒断症状。根据小鼠戒断反应中出现的跳跃次数、体重下降等指标评定戒断反应强度。采用免疫组织化学法显示吗啡依赖小鼠和正常对照组小鼠海马神经元c-fos的表达。结果:吗啡依赖组海马CA1区和齿状回Fos阳性神经元数目明显增加(P<0.05),CA3区无明显改变(P>0.05)。纳洛酮催促戒断组海马CA1和CA3区Fos阳性神经元数目明显增加(P<0.05),齿状回Fos阳性神经元数目增加更为明显(P<0.01)。吗啡依赖组与纳洛酮催促戒断组CA3区阳性神经元数目有显著性差异(P<0.05)。结论:海马神经元c-fos的表达增强可能与吗啡依赖对神经元的损伤有关。     

Interruption of supramammillohippocampal afferents prevents the genesis and spread of limbic seizures in the hippocampus via a disinhibition mechanism

In this study we describe the preventive effect of interruption of the supramammillohippocampal afferents on the Fos expression in the forebrain and epileptic discharges in the hippocampal electroencephalogram in rat model of kainic acid-induced limbic seizure. Little was known about the contribution of different degrees of neural activity of hippocampal principal cells to the genesis and spread of limbic seizures in the forebrain structures. Following kainic acid injection to the amygdala with or without concurrent injection of muscimol to the supramammillary nucleus, behavioral changes and electroencephalograms were observed in freely moving rats. The animals were processed for Fos immunocytochemical analysis at several time points. The latest expression of Fos at 2h was seen in hippocampal CA1-CA3, ventrolateral thalamic nuclei and mediodorsal caudate putamen, while the early Fos expression at 0.5h was seen in the piriform, entorhinal and other cortices, the thalamic midline nuclei and hypothalamic nuclei. Muscimol injection to the supramammillary nucleus prevented Fos expression in the CA1-CA3 region and reduced that in the forebrain regions with the latest Fos expression, but did not affect Fos expression in other forebrain regions with early Fos expression. This treatment also eliminated epileptic discharges and attenuated all waves in hippocampus.These findings indicate that an acute interruption of the facilitatory hypothalamic afferents by intrasupramammillary injection of muscimol may cause the inactivation of the disinhibition mechanism for hippocampal throughput at the dentate gyrus, resulting in the blockade of the genesis and spread of limbic seizures in the hippocampus.     

Environmental enrichment enhances recovery of function but exacerbates ischemic cell death.

Prior exposure to brief 'conditioning' episodes of ischemia protects hippocampal CA1 neurons against a subsequent more severe ischemic insult. However, protected cells exhibit abnormal function and as survival times are extended this ischemic tolerance dissipates and cells begin to die. In this study, we sought to determine whether environmental enrichment could alter the above pattern of delayed cell death and functional impairment in a gerbil model of ischemic tolerance. Gerbils received either ischemic preconditioning, 5 min of ischemia without preconditioning or sham surgery. Three days after ischemia, gerbils were placed in either an enriched environment or standard laboratory housing. Open field habituation was assessed 3, 7, 10, 30 and 60 days after ischemia. Subsequently, animals were trained in two versions (win-shift and win-stay) of a T-maze task. Following behavioral testing, extracellular CA1 field potential amplitudes and CA1 cell counts were determined. Initial open field activity was significantly higher in all experimental groups compared to sham animals (P<0.001). By 60 days, enriched ischemic preconditioned and enriched ischemic gerbils were not different than shams whereas non-enriched, ischemic preconditioned and ischemic gerbils continued to have higher activity scores (P<0.05). Preconditioned and enriched ischemic animals learned the win-shift T-maze problem as quickly as shams while non-enriched ischemic gerbils were severely impaired compared with all other groups (P<0.001). Only the sham and enriched preconditioned groups readily acquired the win-stay paradigm. CA1 field potential amplitudes were lower (P<0.05) in ischemic than sham gerbils irrespective of treatment. Surprisingly, CA1 cell counts were significantly lower (P<0.01) in enriched versus non-enriched ischemic preconditioned animals.These data demonstrate that early, intensive intervention after ischemia can improve functional outcome but that this is accompanied by increased brain damage. Careful consideration needs to be given to the timing of rehabilitation after stroke and related types of brain injury.     

Selective neuronal death after transient forebrain ischemia in the Mongolian gerbil: a silver impregnation study

An important feature of ischemic brain damage is the exceptional vulnerability of specific neuronal populations and the relative resistance of others. Silver impregnation was used to delineate the extent and time-course of neuronal degeneration produced by 5 min of complete forebrain ischemia in the Mongolian gerbil. Lesions were confined to four brain regions: (1) hippocampal areas CA1, CA2-CA3a and CA4; (2) the dorsomedial portion of the lateral septal nucleus; (3) the dorsolateral portion of the striatum; and (4) the somatosensory neocortex. The ischemic lesion evolved with time in all four regions, but at different rates. Somatic argyrophilia developed rapidly in the striatum and hippocampal area CA4 (maximal in 24 h or less), at intermediate rates in the somatosensory neocortex, hippocampal areas CA1a and CA2-CA3a and the lateral septal nucleus (maximal in 2 days), and slowly in hippocampal area CA1b (maximal in 3 days). These results emphasize that the extent and rate of neuronal degeneration can vary even within a presumably homogeneous neuronal population, as evidenced by the different results in areas CA1a and CA1b. Similar results were obtained from analysis of brain sections stained with Cresyl Violet, hematoxylin-eosin or hematoxylin-eosin/Luxol Fast Blue. Terminal-like silver granules were observed in the projection fields of degenerated neurons. They also appeared, however, in the perforant path terminal zone of the hippocampal dentate molecular layer 1-2 days after transient ischemia and in stratum oriens and stratum radiatum of area CA1b prior to somatic degeneration. These granular deposits could not be clearly related to the degeneration of neuronal somata. Novel findings of this study include the degeneration of some dentate basket cells and lateral septal neurons and the appearance of terminal-like argyrophilia in the hippocampal formation without any obvious relation to somatic degeneration. Some of our results lend support to the hypothesis that ischemic neuronal cell death constitutes an excitotoxic process. Other results, however, suggest that the selective vulnerability of neurons to transient ischemia must involve factors beyond excitotoxicity.     

Effects of global cerebral ischemia and preconditioning on heat shock protein 27 immunocontent and phosphorylation in rat hippocampus.

Global cerebral ischemia, with or without preconditioning, leads to an increase in heat shock protein 27 (HSP27) immunocontent and alterations in HSP27 phosphorylation in CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (1, 4, 7, 14, 21 and 30 days) using 2 min, 10 min or 2+10 min of ischemia. The results showed an increase in HSP27 immunocontent of about 300% after 10 min of ischemia in CA1 and dentate gyrus. CA1, a hippocampal vulnerable area, showed an increase in HSP27 phosphorylation, parallel with immunocontent. In dentate gyrus, a resistant area, the increase in HSP phosphorylation was lower than immunocontent. After preconditioned ischemia (2+10 min), when CA1 neurons are protected to a lethal, 10 min insult, we observed an increase in HSP immunocontent and a decrease in phosphorylation in both regions of the hippocampus, suggesting that, when there is no neuronal death, HSP27 in a vulnerable area responds similarly to the resistant area.When dephosphorylated, HSP27 acts as a chaperone, protecting other proteins from denaturation. As it is markedly expressed in astrocytes, we suggest that HSP27 could be protecting hippocampal astrocytes, which could then be helping neurons to resist to the insult, maintaining tissue normal homeostasis.     

Location and distribution of Fos protein expression in rat hippocampus following acute moderate aerobic exercise

Hippocampal neurons are activated during endurance exercise; however, little attention has been given to the location and spatial distribution of these neurons. We have, therefore, used Fos protein expression to identify the location and distribution of hippocampal neurons that become activated during acute moderate aerobic exercise. Adult rats were assigned into trained running (TR), trained nonrunning (TNR), untrained nonrunning (UNR), and cage-bound (CB) groups. Rats in the TR and TNR groups were trained to run, for three 20-min running periods separated by 3 min rest, on a treadmill. Rats in the UNR group spent identical time on a nonactivated treadmill, while rats in the CB group remained in their home cages throughout the training and experimentation. After training to criterion performance for both TR and TNR groups, both groups were rested for 1 day. Rats in the TR were then run on the treadmill to criterion level, while those in TNR and UNR groups spent equivalent time on the nonactivated treadmill. Animals in all groups were then killed and their brains removed, sectioned, and processed for Fos protein immunocytochemistry. Fos-like immunoreactive (FLI) neurons were counted in the dentate and CA1-3 fields of the hippocampus. The total numbers of hippocampal FLI neurons, as well as FLI neurons in each hippocampal region, were compared among groups. The total numbers of FLI neurons in the hippocampus, as well as in individual regions, were significantly greater in the TR group compared with the other three groups. Similarly, significant differences were found between the TNR group when compared with UNR and CB groups. Conversely, a significant difference existed between UNR and CB only in the CA1 field, which may account for the significant difference in the total number of hippocampal FLI neurons between these two groups. These results show that Fos induction occurs in the hippocampus during moderate physical exercise. Furthermore, the importance of the incorporation of adequate controls to account for possible differences in expression of immediate early gene expression due to trained performing, trained nonperforming, and untrained groups is discussed. The results indicate that adequate control for nonexercise stimuli is necessary for studies of the effect of exercise on the brain when expression of immediate early genes such as c-fos is used as an outcome measure.     

Food restriction induces long-lasting recovery of spatial memory deficits following global ischemia in delayed matching and non-matching-to-sample radial arm maze tasks

Food restriction has been shown to be beneficial for a number of brain processes. In the current study, we characterized the impact of food restriction on hippocampal damage 70 days following ischemia. We assessed memory and cognitive flexibility of ad libitum fed (AL) and food-restricted (FR) animals using complex delayed non-matching- and matching-to-sample tasks in the radial arm maze. Our findings demonstrate that food restriction led to significant improvement of ischemia-induced memory impairments. FR ischemic animals rapidly reached comparable performance as both AL and FR sham animals in delayed-non-matching (win-shift) and matching (win-stay) radial arm maze tasks. They also made considerably fewer microchoices in the retention trials than AL ischemic animals. In contrast, AL ischemic rats showed persistent spatial memory impairments in the same paradigms. Assessment of basal and stress-induced corticosterone (CORT) secretion revealed no significant differences in baseline levels in AL and FR rats prior to or following global ischemia. However, FR animals showed a more pronounced attenuation of CORT secretion 45 min following restraint. Both FR and AL ischemic rats had comparable cell loss within CA1 and CA3 subfields of Ammon's horn (CA1 and CA3) at 70 days following reperfusion, although a trend toward increased CA3 cell survival was observed in FR ischemic rats. The functional sparing in the FR ischemic animals in the face of equivalent hippocampal cell loss suggests that food restriction somehow enhanced the efficacy of remaining hippocampal or extrahippocampal neurons following ischemia. In the current study, this phenomenon was not associated with diet- and or ischemia-related alterations of vesicular glutamate transporter 1 expression in various hippocampal regions although lower vesicular GABA transporter immunostaining was present in the CA1 stratum oriens and the CA3 stratum radiatum in FR sham and ischemic rats.     

Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons

Although large increases in neuronal intracellular calcium concentrations ([Ca(2+)](i)) are lethal, moderate increases in [Ca(2+)](i) of 50-200 nM may induce immediate or long-term tolerance of ischemia or other stresses. In neurons in rat hippocampal slice cultures, we determined the relationship between [Ca(2+)](i), cell death, and Ca(2+)-dependent neuroprotective signals before and after a 45 min period of oxygen and glucose deprivation (OGD). Thirty minutes before OGD, [Ca(2+)](i) was increased in CA1 neurons by 40-200 nM with 1 nM-1 microM of a Ca(2+)-selective ionophore (calcimycin or ionomycin-"Ca(2+) preconditioning"). Ca(2+) preconditioning greatly reduced cell death in CA1, CA3 and dentate during the following 7 days, even though [Ca(2+)](i) was similar (approximately 2 microM) in preconditioned and control neurons 1 h after the OGD. When pre-OGD [Ca(2+)](i) was lowered to 25 nM (10 nM ionophore in Ca(2+)-free medium) or increased to 8 microM (10 microM ionophore), more than 90% of neurons died. Increased levels of the anti-apoptotic protein protein kinase B (Akt) and the MAP kinase ERK (p42/44) were present in preconditioned slices after OGD. Reducing Ca(2+) influx, inhibiting calmodulin, and preventing Akt or MAP kinase p42/44 upregulation prevented Ca(2+) preconditioning, supporting a specific role for Ca(2+) in the neuroprotective process. Further, in continuously oxygenated cultured hippocampal/cortical neurons, preconditioning for 30 min with 10 nM ionomycin reduced cell death following a 4 microM increase in [Ca(2+)](i) elicited by 1 microM ionomycin. Thus, a zone of moderately increased [Ca(2+)](i) before a potentially lethal insult promotes cell survival, uncoupling subsequent large increases in [Ca(2+)](i) from initiating cell death processes.     

海马区星形胶质细胞的活化状态与缺血耐受性关系的研究

目的：观察海马区星形胶质细胞的活化与缺血耐受性的关系。方法：钳夹沙土鼠的双侧颈总动脉制造脑缺血模型,尼氏染色及免疫荧光法染色,观察海马锥体细胞迟发性神经元坏死及抗胶质纤维酸性蛋白（ＧＦＡＰ）染色的改变。结果：脑缺血组,ＣＡ１区锥体细胞几乎全产中丧失,只见很量少的细胞残存,零星散在分布;预缺血组,ＣＡ１区锥体细胞少部分丧失,大部分幸存。对照组有少量微弱的ＧＦＡＰ染色阳性细胞;脑缺血组有些ＧＦＡＰ染色     

Remote organ ischemic preconditioning protect brain from ischemic damage following asphyxial cardiac arrest

Ischemic preconditioning (IPC) is a phenomenon whereby an organ's adaptive transient resistance to a lethal ischemic insult occurs by preconditioning this organ with a sub-lethal/mild ischemic insult of short duration. Besides IPC, recent studies reported that a short sub-lethal ischemia and reperfusion in various organs can induce ischemic tolerance in another organ as well. This phenomenon is known as remote ischemic preconditioning (RPC). In the present study we tested the hypothesis that tolerance for ischemia can be induced in brain by RPC and IPC in a rat model of asphyxial cardiac arrest (ACA). RPC was induced by tightening the upper two-thirds of both hind limbs using a tourniquet for 15 or 30 min and IPC was induced by tightening bilateral carotid artery ligatures for 2 min. Eight minutes of ACA was induced 48 h after RPC or IPC. After 7 day of resuscitation, brains were extracted and examined for histopathological changes. In CA1 hippocampus, the number of normal neurons was 63% lower in cardiac-arrested rats as compared to the control group. The number of normal neurons in the 15 min RPC, 30 min RPC, and IPC groups was higher than the ACA group by 54, 70, and 67%, respectively. This study demonstrates that RPC and IPC are able to provide neuroprotection in a rat model of ACA. Besides direct application of RPC or IPC paradigms, the exploration of the mechanisms of observed neuroprotection by RPC and IPC may also lead to a possible therapy for CA patients.     

AT4 receptor activation increases intracellular calcium influx and induces a non-N-methyl-D-aspartate dependent form of long-term potentiation

Although neonatal hypoxia can lead to brain damage, mild hypoxic episodes may be beneficial, as illustrated by tolerance induction by preconditioning, a process that might involve neurogenesis. To examine if brief hypoxia in newborn rats could stimulate the generation of neurons, pups were exposed for 5 min to 100% N₂. Cell density and apoptosis were monitored in various brain regions and cell proliferation was studied by the incorporation of bromodeoxyuridine. Hypoxia did not result in detectable cell death but promoted cell proliferation in the ensuing three weeks in the subventricular zone and hippocampal dentate gyrus, with increased cell density in hippocampus CA1 pyramidal cells and granular layer of the dentate gyrus. Newly generated cells expressed neuronal markers (NeuroD or neuronal nuclear antigen) and were able to migrate from germinative zones to specific sites, in particular from the subventricular zone to the CA1 layer along the posterior periventricle. Neurogenesis was associated with an early activation of the extracellular regulated kinase 1/2 pathway, and pre-hypoxic administration of U0126, an inhibitor of mitogen-activated protein kinase kinase, impaired hypoxia effect on cell proliferation. Neurobehavioral capacities of hypoxic rats paralleled those of controls, but early exposure to hypoxia was associated with significantly improved memory retrieval scores at 40 days. In conclusion, brief neonatal hypoxia may trigger delayed generation of potentially functional neurons without concomitant cell death. This may constitute an interesting model for studying cell key events involved in the induction of neurogenesis.     

低氧预处理对缺氧-复氧后体外培养大鼠海马神经元Fos、Jun表达和神经元凋亡的影响

目的　观察低氧预处理对体外培养大鼠海马神经元缺氧 复氧后Fos、Jun表达和神经元凋亡的变化。方法　取培养 12d的两组 (对照组和低氧预处理组 )神经元 ,同时置于缺氧环境 (0 90l LN2 、0 10l LCO2 )中培养 4h后取出 ,置含 0 10l LCO2 和空气的培养箱内复氧培养 2 4和 72h。于不同时间取出 ,观察神经元存活数 ,并分别用抗Fos和Jun抗血清进行免疫组织化学染色 ,观察Fos、Jun表达阳性和阴性神经元数目 ,计数Fos和Jun表达神经元所占百分率。并用原位末端标记 (TUNEL)法和流式细胞术分别检测缺氧 复氧对体外培养海马神经元凋亡的影响。　结果　缺氧 复氧后大鼠海马培养神经元中Fos、Jun表达阳性神经元百分率和凋亡神经元百分率均较缺氧前显著增加。经低氧预处理的海马神经元缺氧 复氧后Fos、Jun表达神经元和凋亡神经元百分率均较对照组明显减少。神经元损伤程度减轻 ,神经元存活数明显高于对照组。　结论　低氧预处理可使培养中海马神经元对缺氧产生耐受 ,减少缺氧 复氧后神经元Fos和Jun表达 ,减少缺氧 复氧后神经元凋亡。     

Antisense in vivo knockdown of synaptotagmin I and synapsin I by HVJ-liposome mediated gene transfer modulates ischemic injury of hippocampus in opposing ways

Neurotransmitter release during and after ischemic event is thought to be involved in excitotoxicity as a pathogenesis for the ischemic brain damage, which is mediated by excessive activation of glutamate receptors and attendant calcium overload. To ascertain the role of transmitter release from nerve terminals in promoting the ischemic neurodegeneration, we delivered antisense oligodeoxynucleotides (ODNs) to synaptotagmin I or synapsin I into the rat brain by using HVJ-liposome gene transfer technique. The antisense ODNs were injected into the lateralventricle in rats 4 days prior to transient forebrain ischemia of 20 min. With a single antisense treatment, long-lasting downregulation of the transmitter release relating protein levels at overall synaptic terminals was achieved. The antisense in vivo knockdown of synaptotagmin I prevented almost completely the ischemic damage of hippocampal CA1 neurons, while the in vivo knockdown of synapsin I markedly promoted the ischemic damage of CA1 pyramidal neurons and extended the injury to relatively resistant CA2/CA3 region. The modulation of ischemic hippocampal damage by the in vivo knockdown of synaptotagmin I or synapsin I suggests that transmitter release from terminals plays an important role in the evolution of ischemic brain damage and therefore the transmitter release strategy by the use of antisense ODNs-HVJ-liposome complex is reliable for neuroprotective therapies.