BACKGROUND： Blood supply to the hippocampus is not provided by the middle cerebral artery. However, previous studies have shown that delayed neuronal death in the hippocampus may occur following focal cerebral ischem...BACKGROUND： Blood supply to the hippocampus is not provided by the middle cerebral artery. However, previous studies have shown that delayed neuronal death in the hippocampus may occur following focal cerebral ischemia induced by middle cerebral artery occlusion. OBJECTIVE： To observe the relationship between reactive changes in hippocampal astrocytes and delayed neuronal death in the hippocampal CA1 region following middle cerebral artery occlusion. DESIGN, TIME AND SETTING： The immunohistochemical, randomized, controlled animal study was performed at the Laboratory of Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from July to November 2007. MATERIALS： Rabbit anti-glial fibrillary acidic protein （GFAP） （Neomarkers, USA）, goat anti-rabbit IgG （Sigma, USA） and ApoAlert apoptosis detection kit （Biosciences Clontech, USA） were used in this study. METHODS： A total of 42 healthy adult male Wistar rats, aged 3–5 months, were randomly divided into a sham operation group （n = 6） and a cerebral ischemia/reperfusion group （n = 36）. In the cerebral ischemia/reperfusion group, cerebral ischemia/reperfusion models were created by middle cerebral artery occlusion. In the sham operation group, the thread was only inserted into the initial region of the internal carotid artery, and middle cerebral artery occlusion was not induced. Rats in the cerebral ischemia/reperfusion group were assigned to a delayed neuronal death （＋） subgroup and a delayed neuronal death （–） subgroup, according to the occurrence of delayed neuronal death in the ischemic side of the hippocampal CA1 region following cerebral ischemia. MAIN OUTCOME MEASURES： Delayed neuronal death in the hippocampal CA1 region was measured by Nissl staining. GFAP expression and delayed neuronal death changes were measured in the rat hippocampal CA1 region at the ischemic hemisphere by double staining for GFAP and TUNEL. RESULTS： After 3 days of ischemia/reperfusion, astrocytes with abnormal morphology were detected in the rat hippocampal CA1 region in the delayed neuronal death （＋） subgroup. No significant difference in GFAP expression was found in the rat hippocampal CA1 region at the ischemic hemisphere in the sham operation group, delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup （P 〉 0.05）. After 7 days of ischemia/reperfusion, many GFAP-positive cells, which possessed a large cell body and an increased number of processes, were activated in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression in the hippocampal CA1 region was greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.01）. Moreover, GFAP expression was significantly greater in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.01）. After 30 days of ischemia/reperfusion, GFAP-positive cells were present in scar-like structures in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression was significantly greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.05）. GFAP expression was significantly lower in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.05）. The delayed neuronal death rates were 42% （5/12）, 33% （4/12） and 33% （4/12） at 3, 7 and 30 days, respectively, followingischemia/reperfusion. No significant differences were detected at various time points （χ2 = 0.341, P 〉 0.05）. CONCLUSION： The activation of astrocytes was poor in the hippocampal CA1 region during the early stages of ischemia, which is an important reason for delayed neuronal death. Glial scar formation aggravated delayed neuronal death during the advanced ischemic stage.展开更多
BACKGROUND： Proteasome dysfunction has been reported to induce abnormal protein aggregation and cell death. OBJECTIVE： To investigate the effect of proteasome changes on delayed neuronal death in CA1 and dentate gyr...BACKGROUND： Proteasome dysfunction has been reported to induce abnormal protein aggregation and cell death. OBJECTIVE： To investigate the effect of proteasome changes on delayed neuronal death in CA1 and dentate gyrus （DG） regions of the rat hippocampus following transient cerebral ischemia. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment. The study was performed at the Department of Biochemistry and Molecular Biology, Norman Bethune Medical College of Jilin University, from September 2006 to May 2008. MATERIALS： Rabbit anti-19S S10B polyclonal antibody was purchased from Bioreagents, USA; propidium iodide and fluorescently-labeled goat anti-rabbit IgG were purchased from Jackson Immunoresearch, USA; hematoxylin and eosin staining solution was purchased from Sigma, USA; LSM 510 confocal microscope was purchased from Zeiss, Germany. METHODS： A total of 40 healthy Wistar rats, male, 4 months old, were randomly divided into sham surgery group （n = 8） and model group （n = 32）. Ischemic models were established in the model group by transient clamping of the bilateral carotid arteries and decreased blood pressure. After 20 minutes of global ischemia, the clamp was removed to allow blood flow for 30 minutes, 4, 24 and 72 hours, respectively, with 8 rats at each time point. The bilateral carotid arteries were not ligated in the sham surgery group. MAIN OUTCOME MEASURES： Neuronal death in the CA1 and DG regions was observed by hematoxylin-eosin staining. Proteasome expression in CA1 and DG region neurons was detected by immunohistochemistry. RESULTS： Hematoxylin-eosin staining showed neuronal death in the CA1 region alone at 72 hours of reperfusion following ischemia. In comparison to the sham surgery group, a significant decrease in proteasome expression was observed, by immunohistochemistry, in the CA1 and DG regions in the model group, following 30 minutes, 4, 24, and 72 hours of reperfusion （P 〈 0.01）. After 72 hours of reperfusion following ischemia, proteasome expression had almost completely disappeared in the CA1 region. In contrast, neurons of the DG region showed minimized proteasome expression at 24 hours, with a slight increase at 72 hours （P 〈 0.01）. CONCLUSION： The alteration of proteasome following ischemia/reperfusion in the neurons of hippocampal CA1 and DG regions reduces the ability of cells to degrade abnormal protein, which may be an important factor resulting in delayed neuronal death following transient cerebral ischemia.展开更多
The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not bee...The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions （CA1-3） between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group, p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.展开更多
BACKGROUND： Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase （nNOS） to induce increased nitric oxide levels, resulting in brain neuronal i...BACKGROUND： Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase （nNOS） to induce increased nitric oxide levels, resulting in brain neuronal injury. OBJECTIVE： To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING： Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006. MATERIALS： A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China. METHODS： The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups： sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup. MAIN OUTCOME MEASURES： nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride （TTC） and immunohistochemistry. RESULTS： In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet （P 〈 0.01）. At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet （P 〈 0.05）. When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet. CONCLUSION： nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time.展开更多
Stroke is a debilitating disease that affects millions each year. While in many cases cerebral ischemic injury can be limited by effective resuscitation or thrombolytic treatment, the injured neurons wither in a proce...Stroke is a debilitating disease that affects millions each year. While in many cases cerebral ischemic injury can be limited by effective resuscitation or thrombolytic treatment, the injured neurons wither in a process known as delayed neuronal death (DND). Mounting evidence indicates that DND is not simply necrosis played out in slow motion but apoptosis is triggered. Of particular interest are two groups of signal proteins that participate in apoptosis cyclin dependent kinases (CDKs) and p53 among a myriad of signaling events after an ischemic insult. Recent investigations have shown that CDKs, a family of enzymes initially known for their role in cell cycle regulation, are activated in injured neurons in DND. As for p53, new reports suggest that its up regulation may represent a failed attempt to rescue injured neurons, although its up regulation was previously considered an indication of apoptosis. These observations thus rekindle an old quest to identify new neuroprotective targets to minimize the stroke damage. In this review, the author will examine the evidence that indicates the participation of CDKs and p53 in DND and then introduce pre clinical data to explore CDK inhibition as a potential neuroprotective target. Finally, using CDK inhibition as an example, this paper will discuss the pertinent criteria for a viable neuroprotective strategy for ischemic injury.展开更多
Transient ischemia in the whole brain leads to neuronal loss/death in vulnerable brain regions. The striatum, neocortex and hippocampus selectively loose specific neurons after transient ischemia. Just 5 minutes of tr...Transient ischemia in the whole brain leads to neuronal loss/death in vulnerable brain regions. The striatum, neocortex and hippocampus selectively loose specific neurons after transient ischemia. Just 5 minutes of transient ischemia can cause pyramidal neuronal death in the hippocampal cornu ammonis (CA) 1 field at 4 days after transient ischemia. In this study, we investigated the effects of 5-minute (mild), 15-minute (severe), and 20-minute (lethal) transient ischemia by bilateral common carotid artery occlusion (BCCAO) on behavioral change and neuronal death and gliosis (astrocytosis and microgliosis) in gerbil hippocampal subregions (CA1-3 region and dentate gyrus). We performed spontaneous motor activity test to evaluate gerbil locomotor activity, cresyl violet staining to detect cellular distribution, neuronal nuclei immunohistochemistry to detect neuronal distribution, and Fluoro-Jade B histofluorescence to evaluate neuronal death. We also conducted immunohistochemical staining for glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 (Ibal) to evaluate astrocytosis and microgliosis, respectively. Animals subjected to 20-minute BCCAO died in at least 2 days. BCCAO for 15 minutes led to pyramidal cell death in hippocampal CA1-3 region 2 days later and granule cell death in hippocampal de匚tate gyrus 5 days later. Similar results were not found in animals subjected to 5-minute BCCAO. Gliosis was much more rapidly and severely progressed in animals subjected to 15-minute BCCAO than in those subjected to 5- minute BCCAO. Our results indicate that neuronal loss in the hippocampal formation following transient ischemia is significantly different according to regions and severity of transient ischemia. The experimental protocol was approved by Institutional Animal Care and Use Committee (AICUC) of Kangwon National University (approval No. KW-180124-1) on May 22, 2018.展开更多
Electroacupuncture preconditioning at acupoint Baihui （GV20） can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynami...Electroacupuncture preconditioning at acupoint Baihui （GV20） can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 （Drp1） can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 （depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day）. Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.展开更多
In this study, we tried to verify the neuroprotective effect of Chrysanthemum indicum Linne（CIL） extract, which has been used as a botanical drug in East Asia, against ischemic damage and to explore the underlying m...In this study, we tried to verify the neuroprotective effect of Chrysanthemum indicum Linne（CIL） extract, which has been used as a botanical drug in East Asia, against ischemic damage and to explore the underlying mechanism involving the anti-inflammatory approach. A gerbil was given CIL extract for 7 consecutive days followed by bilateral carotid artery occlusion to make a cerebral ischemia/reperfusion model. Then, we found that CIL extracts protected pyramidal neurons in the hippocampal CA1 region（CA1） from ischemic damage using neuronal nucleus immunohistochemistry and Fluoro-Jade B histofluorescence. Accordingly, interleukin-13 immunoreactivities in the CA1 pyramidal neurons of CIL-pretreated animals were maintained or increased after cerebral ischemia/reperfusion. These findings indicate that the pre-treatment of CIL can attenuate neuronal damage/death in the brain after cerebral ischemia/reperfusion via an anti-inflammatory approach.展开更多
Calbindin D-28K （CB）, a Ca2＋-binding protein, maintains Ca2＋ homeostasis and protects neurons against various insults. Hyperthermia can exacerbate brain damage produced by ischemic insults. However, little is repo...Calbindin D-28K （CB）, a Ca2＋-binding protein, maintains Ca2＋ homeostasis and protects neurons against various insults. Hyperthermia can exacerbate brain damage produced by ischemic insults. However, little is reported about the role of CB in the brain under hyperthermic condition during ischemic insults. We inves- tigated the effects of transient global cerebral ischemia on CB immunoreactivity as well as neuronal damage in the hippocampal formation under hyperthermic condition using immunohistochemistry for neuronal nuclei （NeuN） and CB, and Fluoro-Jade B histofluorescence staining in gerbils. Hyperthermia （39.5 ＋ 0.2~C） was induced for 30 minutes before and during transient ischemia. Hyperthermic ischemia resulted in neu- ronal damage/death in the pyramidal layer of CA1-3 area and in the polymorphic layer of the dentate gyrus at 1, 2, 5 days after ischemia. In addition, hyperthermic ischemia significantly decreaced CB immunoreac- tivity in damaged or dying neurons at 1, 2, 5 days after ischemia. In brief, hyperthermic condition produced more extensive and severer neuronal damage/death, and reduced CB immunoreactivity in the hippocampus following transient global cerebral ischemia. Present findings indicate that the degree of reduced CB immu- noreactivity might be related with various neuronal damage/death overtime and corresponding areas after ischemic insults.展开更多
To study the effect of anisodamine on neuronal death and hydroxyl radical (OH·) production during forebrain ischemia reperfusion in gerbils Methods The tested gerbils were divided into 3 groups, including sham...To study the effect of anisodamine on neuronal death and hydroxyl radical (OH·) production during forebrain ischemia reperfusion in gerbils Methods The tested gerbils were divided into 3 groups, including sham operated, control and anisodamine groups. In each group, there were 8 animals for biochemical examination and 6 animals for histologic study. Forebrain ischemia was induced by occlusion the bilateral common carotid arteries for 10 min in gerbils. 2,3 and 2,5 DHBA outputs were determined by high performance hiquid chromatography coupled with electrochemical detection. Behavioral change was tested by open field test and neuronal death was assessed by histological examination.Results The exploratory activities of gerbils in the control group were significantly higher than those in the anisodamine group on all test days The amount of viable looking neurons in the medial, middle and lateral CA1 sectors in anisodamine group were 41%±12%, 50%±21% and 67%±15% of the sham operated gerbils, respectively, being significantly higher than those in the control group (3%±2%, 4%±3% and 7%±4% of sham, P <0 01) The 2,3 DHBA outputs in the control group increased by 5 fold of the sham operated gerbils after reperfusion for 60 min, but the 2,3 DHBA outputs in the anisodamine group were only 2 4 fold of sham operated gerbils, being significantly lower than that in the control group ( P <0 01) The 2,5 DHBA outputs in the control group were significantly higher than those in the sham operated group ( P <0 05) Conclusion Anisodamine has inhibitory effects on neuronal death and OH·production during cerebral ischemia reperfusion in gerbils展开更多
Objective: To investigate the relationship between p53, p21 proteins and delayed neuronal death (DND) after reperfusion following forebrain ischemia in rats. Methods With four-vessel occlusion model of rats, the expre...Objective: To investigate the relationship between p53, p21 proteins and delayed neuronal death (DND) after reperfusion following forebrain ischemia in rats. Methods With four-vessel occlusion model of rats, the expression of p53, p21 proteins in brain tissue using labeled streptavindin-biotin immunohistochemical (LAAB) suming were observed. Re sults: The expression of p53, p21 proteins in brain was upregulated after reperfusion following 15 min forebrain ischemia and their distribution was similar. p53 and p21 proteins in brian sections was detected earlier in the white matter of hippocampal formation, thalamus, hypothalamus (6 h following reperfusion) than in the neuronal nuclei in cerebral cortex and CA1 region (24h), and the maximal induction was observed at 72 h following reperfusion. CA1 region suffered the most serious injury, where the positive expression of p53 and off proteins was most. Conclusion: Reperfusion following forebrain ischemia could upregulate the expression of p53 and p21 proteins in the brain region, suggesting that p53 and p21 proteins participate in and possibly promote the apoptosis of ’DND.展开更多
基金the National Outstanding Youth Foundation Program, No. 30725019 the Natural Science Foundation of Hubei Province, No. 2007ABA174
文摘BACKGROUND： Blood supply to the hippocampus is not provided by the middle cerebral artery. However, previous studies have shown that delayed neuronal death in the hippocampus may occur following focal cerebral ischemia induced by middle cerebral artery occlusion. OBJECTIVE： To observe the relationship between reactive changes in hippocampal astrocytes and delayed neuronal death in the hippocampal CA1 region following middle cerebral artery occlusion. DESIGN, TIME AND SETTING： The immunohistochemical, randomized, controlled animal study was performed at the Laboratory of Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from July to November 2007. MATERIALS： Rabbit anti-glial fibrillary acidic protein （GFAP） （Neomarkers, USA）, goat anti-rabbit IgG （Sigma, USA） and ApoAlert apoptosis detection kit （Biosciences Clontech, USA） were used in this study. METHODS： A total of 42 healthy adult male Wistar rats, aged 3–5 months, were randomly divided into a sham operation group （n = 6） and a cerebral ischemia/reperfusion group （n = 36）. In the cerebral ischemia/reperfusion group, cerebral ischemia/reperfusion models were created by middle cerebral artery occlusion. In the sham operation group, the thread was only inserted into the initial region of the internal carotid artery, and middle cerebral artery occlusion was not induced. Rats in the cerebral ischemia/reperfusion group were assigned to a delayed neuronal death （＋） subgroup and a delayed neuronal death （–） subgroup, according to the occurrence of delayed neuronal death in the ischemic side of the hippocampal CA1 region following cerebral ischemia. MAIN OUTCOME MEASURES： Delayed neuronal death in the hippocampal CA1 region was measured by Nissl staining. GFAP expression and delayed neuronal death changes were measured in the rat hippocampal CA1 region at the ischemic hemisphere by double staining for GFAP and TUNEL. RESULTS： After 3 days of ischemia/reperfusion, astrocytes with abnormal morphology were detected in the rat hippocampal CA1 region in the delayed neuronal death （＋） subgroup. No significant difference in GFAP expression was found in the rat hippocampal CA1 region at the ischemic hemisphere in the sham operation group, delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup （P 〉 0.05）. After 7 days of ischemia/reperfusion, many GFAP-positive cells, which possessed a large cell body and an increased number of processes, were activated in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression in the hippocampal CA1 region was greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.01）. Moreover, GFAP expression was significantly greater in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.01）. After 30 days of ischemia/reperfusion, GFAP-positive cells were present in scar-like structures in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression was significantly greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.05）. GFAP expression was significantly lower in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.05）. The delayed neuronal death rates were 42% （5/12）, 33% （4/12） and 33% （4/12） at 3, 7 and 30 days, respectively, followingischemia/reperfusion. No significant differences were detected at various time points （χ2 = 0.341, P 〉 0.05）. CONCLUSION： The activation of astrocytes was poor in the hippocampal CA1 region during the early stages of ischemia, which is an important reason for delayed neuronal death. Glial scar formation aggravated delayed neuronal death during the advanced ischemic stage.
基金Supported by:the Postdoctoral Foundation of China,No.20080440422International Cooperation G rant,No.20070721+1 种基金Outstanding Youth Grant of the Science and Technology Department of Jilin Province, No.20080139a grant from the Science and Technology Department of Changchun City,No.2007128
文摘BACKGROUND： Proteasome dysfunction has been reported to induce abnormal protein aggregation and cell death. OBJECTIVE： To investigate the effect of proteasome changes on delayed neuronal death in CA1 and dentate gyrus （DG） regions of the rat hippocampus following transient cerebral ischemia. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment. The study was performed at the Department of Biochemistry and Molecular Biology, Norman Bethune Medical College of Jilin University, from September 2006 to May 2008. MATERIALS： Rabbit anti-19S S10B polyclonal antibody was purchased from Bioreagents, USA; propidium iodide and fluorescently-labeled goat anti-rabbit IgG were purchased from Jackson Immunoresearch, USA; hematoxylin and eosin staining solution was purchased from Sigma, USA; LSM 510 confocal microscope was purchased from Zeiss, Germany. METHODS： A total of 40 healthy Wistar rats, male, 4 months old, were randomly divided into sham surgery group （n = 8） and model group （n = 32）. Ischemic models were established in the model group by transient clamping of the bilateral carotid arteries and decreased blood pressure. After 20 minutes of global ischemia, the clamp was removed to allow blood flow for 30 minutes, 4, 24 and 72 hours, respectively, with 8 rats at each time point. The bilateral carotid arteries were not ligated in the sham surgery group. MAIN OUTCOME MEASURES： Neuronal death in the CA1 and DG regions was observed by hematoxylin-eosin staining. Proteasome expression in CA1 and DG region neurons was detected by immunohistochemistry. RESULTS： Hematoxylin-eosin staining showed neuronal death in the CA1 region alone at 72 hours of reperfusion following ischemia. In comparison to the sham surgery group, a significant decrease in proteasome expression was observed, by immunohistochemistry, in the CA1 and DG regions in the model group, following 30 minutes, 4, 24, and 72 hours of reperfusion （P 〈 0.01）. After 72 hours of reperfusion following ischemia, proteasome expression had almost completely disappeared in the CA1 region. In contrast, neurons of the DG region showed minimized proteasome expression at 24 hours, with a slight increase at 72 hours （P 〈 0.01）. CONCLUSION： The alteration of proteasome following ischemia/reperfusion in the neurons of hippocampal CA1 and DG regions reduces the ability of cells to degrade abnormal protein, which may be an important factor resulting in delayed neuronal death following transient cerebral ischemia.
基金supported by 2013 Research Grant from Kangwon National University(120131480)Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education(NRF-2014R1A6A3A01056005)
文摘The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions （CA1-3） between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group, p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.
文摘BACKGROUND： Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase （nNOS） to induce increased nitric oxide levels, resulting in brain neuronal injury. OBJECTIVE： To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING： Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006. MATERIALS： A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China. METHODS： The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups： sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup. MAIN OUTCOME MEASURES： nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride （TTC） and immunohistochemistry. RESULTS： In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet （P 〈 0.01）. At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet （P 〈 0.05）. When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet. CONCLUSION： nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time.
文摘Stroke is a debilitating disease that affects millions each year. While in many cases cerebral ischemic injury can be limited by effective resuscitation or thrombolytic treatment, the injured neurons wither in a process known as delayed neuronal death (DND). Mounting evidence indicates that DND is not simply necrosis played out in slow motion but apoptosis is triggered. Of particular interest are two groups of signal proteins that participate in apoptosis cyclin dependent kinases (CDKs) and p53 among a myriad of signaling events after an ischemic insult. Recent investigations have shown that CDKs, a family of enzymes initially known for their role in cell cycle regulation, are activated in injured neurons in DND. As for p53, new reports suggest that its up regulation may represent a failed attempt to rescue injured neurons, although its up regulation was previously considered an indication of apoptosis. These observations thus rekindle an old quest to identify new neuroprotective targets to minimize the stroke damage. In this review, the author will examine the evidence that indicates the participation of CDKs and p53 in DND and then introduce pre clinical data to explore CDK inhibition as a potential neuroprotective target. Finally, using CDK inhibition as an example, this paper will discuss the pertinent criteria for a viable neuroprotective strategy for ischemic injury.
基金supported by Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education(NRF-2016R1D1A1B01011790 to JHC)+3 种基金Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Science,ICT&Future Planning(NRF-2017R1A2B4009079 to MHW)Cooperative Research Program for Agriculture Science and Technology Development(Project No.PJ01329401to MHW) Rural Development Administration,Republic of Korea
文摘Transient ischemia in the whole brain leads to neuronal loss/death in vulnerable brain regions. The striatum, neocortex and hippocampus selectively loose specific neurons after transient ischemia. Just 5 minutes of transient ischemia can cause pyramidal neuronal death in the hippocampal cornu ammonis (CA) 1 field at 4 days after transient ischemia. In this study, we investigated the effects of 5-minute (mild), 15-minute (severe), and 20-minute (lethal) transient ischemia by bilateral common carotid artery occlusion (BCCAO) on behavioral change and neuronal death and gliosis (astrocytosis and microgliosis) in gerbil hippocampal subregions (CA1-3 region and dentate gyrus). We performed spontaneous motor activity test to evaluate gerbil locomotor activity, cresyl violet staining to detect cellular distribution, neuronal nuclei immunohistochemistry to detect neuronal distribution, and Fluoro-Jade B histofluorescence to evaluate neuronal death. We also conducted immunohistochemical staining for glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 (Ibal) to evaluate astrocytosis and microgliosis, respectively. Animals subjected to 20-minute BCCAO died in at least 2 days. BCCAO for 15 minutes led to pyramidal cell death in hippocampal CA1-3 region 2 days later and granule cell death in hippocampal de匚tate gyrus 5 days later. Similar results were not found in animals subjected to 5-minute BCCAO. Gliosis was much more rapidly and severely progressed in animals subjected to 15-minute BCCAO than in those subjected to 5- minute BCCAO. Our results indicate that neuronal loss in the hippocampal formation following transient ischemia is significantly different according to regions and severity of transient ischemia. The experimental protocol was approved by Institutional Animal Care and Use Committee (AICUC) of Kangwon National University (approval No. KW-180124-1) on May 22, 2018.
基金supported by the Natural Science Foundation of Shandong Province of China,No.ZR2015HM023a grant from the Science and Technology Plan Project of Shinan District of Qingdao City of China,No.2016-3-029-YY
文摘Electroacupuncture preconditioning at acupoint Baihui （GV20） can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 （Drp1） can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 （depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day）. Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.
基金supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science,ICT and Future Planning(NRF-2013R1A2A2A01068190)Hallym University Specialization Fund(HRF-S-13)
文摘In this study, we tried to verify the neuroprotective effect of Chrysanthemum indicum Linne（CIL） extract, which has been used as a botanical drug in East Asia, against ischemic damage and to explore the underlying mechanism involving the anti-inflammatory approach. A gerbil was given CIL extract for 7 consecutive days followed by bilateral carotid artery occlusion to make a cerebral ischemia/reperfusion model. Then, we found that CIL extracts protected pyramidal neurons in the hippocampal CA1 region（CA1） from ischemic damage using neuronal nucleus immunohistochemistry and Fluoro-Jade B histofluorescence. Accordingly, interleukin-13 immunoreactivities in the CA1 pyramidal neurons of CIL-pretreated animals were maintained or increased after cerebral ischemia/reperfusion. These findings indicate that the pre-treatment of CIL can attenuate neuronal damage/death in the brain after cerebral ischemia/reperfusion via an anti-inflammatory approach.
基金supported by the Biomedical Technology Development Program of the NRF funded by the Korean Government,MSIP(NRF-2015M3A9B6066835)by the Bio-Synergy Research Project(NRF-2015M3A9C4076322)of the Ministry of Science,ICT and Future Planning through the National Research Foundation
文摘Calbindin D-28K （CB）, a Ca2＋-binding protein, maintains Ca2＋ homeostasis and protects neurons against various insults. Hyperthermia can exacerbate brain damage produced by ischemic insults. However, little is reported about the role of CB in the brain under hyperthermic condition during ischemic insults. We inves- tigated the effects of transient global cerebral ischemia on CB immunoreactivity as well as neuronal damage in the hippocampal formation under hyperthermic condition using immunohistochemistry for neuronal nuclei （NeuN） and CB, and Fluoro-Jade B histofluorescence staining in gerbils. Hyperthermia （39.5 ＋ 0.2~C） was induced for 30 minutes before and during transient ischemia. Hyperthermic ischemia resulted in neu- ronal damage/death in the pyramidal layer of CA1-3 area and in the polymorphic layer of the dentate gyrus at 1, 2, 5 days after ischemia. In addition, hyperthermic ischemia significantly decreaced CB immunoreac- tivity in damaged or dying neurons at 1, 2, 5 days after ischemia. In brief, hyperthermic condition produced more extensive and severer neuronal damage/death, and reduced CB immunoreactivity in the hippocampus following transient global cerebral ischemia. Present findings indicate that the degree of reduced CB immu- noreactivity might be related with various neuronal damage/death overtime and corresponding areas after ischemic insults.
基金ThisstudywassupportedbygrantsfromtheJiangsuProvincialHealthDepartment (No Z9717) JiangsuProvinceCommitteeofScienceandTechn
文摘To study the effect of anisodamine on neuronal death and hydroxyl radical (OH·) production during forebrain ischemia reperfusion in gerbils Methods The tested gerbils were divided into 3 groups, including sham operated, control and anisodamine groups. In each group, there were 8 animals for biochemical examination and 6 animals for histologic study. Forebrain ischemia was induced by occlusion the bilateral common carotid arteries for 10 min in gerbils. 2,3 and 2,5 DHBA outputs were determined by high performance hiquid chromatography coupled with electrochemical detection. Behavioral change was tested by open field test and neuronal death was assessed by histological examination.Results The exploratory activities of gerbils in the control group were significantly higher than those in the anisodamine group on all test days The amount of viable looking neurons in the medial, middle and lateral CA1 sectors in anisodamine group were 41%±12%, 50%±21% and 67%±15% of the sham operated gerbils, respectively, being significantly higher than those in the control group (3%±2%, 4%±3% and 7%±4% of sham, P <0 01) The 2,3 DHBA outputs in the control group increased by 5 fold of the sham operated gerbils after reperfusion for 60 min, but the 2,3 DHBA outputs in the anisodamine group were only 2 4 fold of sham operated gerbils, being significantly lower than that in the control group ( P <0 01) The 2,5 DHBA outputs in the control group were significantly higher than those in the sham operated group ( P <0 05) Conclusion Anisodamine has inhibitory effects on neuronal death and OH·production during cerebral ischemia reperfusion in gerbils
基金Supported by National Natural Science Foundation of China, No.3957263 and Natural Science Foundation of Guangdong province,No.
文摘Objective: To investigate the relationship between p53, p21 proteins and delayed neuronal death (DND) after reperfusion following forebrain ischemia in rats. Methods With four-vessel occlusion model of rats, the expression of p53, p21 proteins in brain tissue using labeled streptavindin-biotin immunohistochemical (LAAB) suming were observed. Re sults: The expression of p53, p21 proteins in brain was upregulated after reperfusion following 15 min forebrain ischemia and their distribution was similar. p53 and p21 proteins in brian sections was detected earlier in the white matter of hippocampal formation, thalamus, hypothalamus (6 h following reperfusion) than in the neuronal nuclei in cerebral cortex and CA1 region (24h), and the maximal induction was observed at 72 h following reperfusion. CA1 region suffered the most serious injury, where the positive expression of p53 and off proteins was most. Conclusion: Reperfusion following forebrain ischemia could upregulate the expression of p53 and p21 proteins in the brain region, suggesting that p53 and p21 proteins participate in and possibly promote the apoptosis of ’DND.
