DNA fragmentation precedes aberrant expression of cell cycle-related protein in rat brain after MCA occlusion.

Recent experiments suggest that apoptotic mechanisms are involved in neuronal cell death after ischemic injury. Although the exact mechanism that triggers activation of apoptotic machinery remains uncertain, in vitro studies revealed that forced expression of cell cycle-related proteins induced apoptosis. Thus, aberrant expression of such proteins might be related to ischemic neuronal death. In the present experiment, we investigated expression of cell cycle-related proteins, i.e., cyclin B1, cyclin D1, cdk4, and PCNA, in rat brain after transient MCA occlusion, and compared the temporal profile of the results with that of TUNEL study, which detects double strand breaks in DNA. There were no immunoreactivities for cyclin B1, cyclin D1, and PCNA in the brain with and without ischemia. As for cdk4, however, it became present at 1 and 3 days of reperfusion after 2 h of ischemia. On the other hand, TUNEL positive cells appeared as early as 3 h of reperfusion, which peaked at 1 and 3 days. These results indicate that aberrant expression of cdk4, but not cyclin B1, cyclin D1 or PCNA, actually takes place in the brain after MCA occlusion, but this is not the causative mechanism of apoptotic cell death in the brain with ischemia.     

Apoptosis and protein expression after focal cerebral ischemia in rat

We used double staining histochemistry to investigate the relationship between apoptotic cell death and selective protein expression associated with DNA damage (p53, Bax, MDM2, Gadd45), DNA repair (PCNA) and cell cycle proteins (cyclin A, cyclin D, cdk2, cdk4) in rats (n=6; control rats, n=5) subjected to transient (2 h) middle cerebral artery occlusion (MCAo) and 46 h of reperfusion. Few apoptotic cells were detected in the non-ischemic hemisphere of control rats. In ischemic animals, scattered apoptotic cells were present in the ischemic core and clustered apoptotic cells were present and localized to the inner boundary zone of the ischemic core. Proteins were preferentially localized to the cellular cytoplasm of control rats and in the non-ischemic hemisphere of rats subjected to MCAo. However, after MCAo these proteins were expressed and were preferentially localized to nuclei within the ischemic lesion. DNA damage induced proteins (wt-p53 and p53-response proteins) were preferentially expressed within apoptotic cells after ischemia. DNA repair proteins and cell cycle proteins were preferentially expressed within morphologically intact cells and in reversibly damaged cells in the ischemic areas. The selective expression of proteins associated with DNA damage, DNA repair and cell cycle observed in morphologically intact cells, ischemic injured cells and apoptotic cells suggests a differential role for these proteins in cell survival and apoptosis after stroke.     

Cell cycle proteins preceded neuronal death after chronic cerebral hypoperfusion in rats

Abstract

Objectives: To explore the re-expression of cell cycle related proteins and delayed neuronal death after chronic cerebral hypoperfusion in rats and to investigate the relationship between aberrant expression of cell cycle proteins and apoptotic cell death.

Methods: Rat model of chronic cerebral hypoperfusion was established by permanent bilateral common carotid arteries occlusion (2VO) in the retired rats. The apoptotic cells were assessed by TUNEL method. The expression of cell cycle related proteins, i.e. CDK4 and cyclin B1, were detected by immunohistochemical staining and Western blotting. A cyclin-dependent kinases (CDKs) inhibitor, roscovitine, was intracerebroventricularly administered 1 day before 2VO insult. Spatial learning behavior was assessed by the Morris water maze 7, 14 and 21 days after the surgery.

Results: Aberrant expression of CDK4 and cyclin B1 became present 7 days after 2VO insult surgery and last for a long period. On the other hand, TUNEL positive cells appeared as early as 14 days after the surgery and peaked at day 21. Furthermore, roscovitine significantly improve behavioral deficit in the Morris water maze test 7 and 14 days after the surgery.

Conclusion: These findings indicated that aberrant expression of CDK4 and cyclin B1 takes place in the brain after chronic cerebral hypoperfusion in retired rat, and aberrant expression of cell cycle proteins preceded neuronal death in this model. Our data also suggest that the CDK inhibitor, roscovitine, has therapeutic potential for the treatment of dementia caused by chronic cerebral hypoperfusion.     

大鼠局灶性脑缺血/再灌注损伤后Caspase-1的表达

目的研究Caspase-1在大鼠脑缺血／再灌注损伤中的作用。方法用Longa法制备大鼠大脑中动脉缺血（2h）／再灌注模型，HE染色观察梗死灶的形成，分别用TUNEL染色及免疫组化技术检测鼠脑缺血中心区及半暗带凋亡细胞与Caspase-1的表达。结果在缺血中心区Caspase-1及凋亡细胞主要见于缺血再灌注损伤早期；在缺血半暗带凋亡细胞与Caspase-1于缺血再灌注损伤早期表达不明显，于缺血再灌注24-48h则明显表达。结论细胞凋亡机制参与了缺血后迟发性神经元死亡，Caspase-1参与了其损伤过程。     

Oxidative damage and breakage of DNA in rat brain after transient MCA occlusion.

As thrombolytic therapy for treatment of ischemic stroke was propagated, much attention has been paid to reperfusion brain injury. Oxidative stress is one of the most important factors that exacerbate tissue damage by reperfusion. Thus, we investigated the extent of oxidative damage in rat brain after transient middle cerebral artery (MCA) occlusion by immunohistochemical analysis for 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is one of the best markers of oxidative damage. Furthermore, in order to investigate its role in neuronal cell death, we performed terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) study, and compared the results with that of 8-OHdG immunohistochemistry. There was no immunoreactive 8-OHdG in sham-operated brain, but it became present in neurons of MCA territory at 3 h of reperfusion after 90-min ischemia. At 48 h after reperfusion, cerebral tissue of MCA territory was severely destroyed, and many cells in that area revealed TUNEL positivity. Some neurons in MCA territory showed mild immunoreactivity for 8-OHdG at that time, but it was strongest in neurons in the outer area of MCA territory. Those cells did not show TUNEL positivity, suggesting that 8-OHdG production is not necessarily followed by early cell death. Here, it was demonstrated that oxidative DNA damage occurs in more extended area than that where cell death is recognized. Although this damage does not cause early cell death, this might result in more prolonged cell dysfunction and eventual neuronal loss. Anti-oxidant therapy might be required for treatment of stroke in the future.     

Temporal profile of cytochrome c and caspase-3 immunoreactivities and TUNEL staining after permanent middle cerebral artery occlusion in rats

Abstract

Although apoptotic pathways play important roles in ischemic neuronal injury, exact mechanism of apoptotic enzyme cascade has not been fully studied. Immunohistochemical stainings for cytochrome c and caspase-3, and histochemical staining for a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNED were examined in a rat model of permanent middle cerebral artery (MCA) occlusion. Cytochrome c was strongly induced in neurons of the ischemic penumbra from 3 h after MCA occlusion, and caspase-3 began to be induced in the same area from 3 h with a peak at 8 h. Neuronal cells in MCA area became TUNEL positive at delayed time, reaching a peak at 24 h. Thus, the peak of induction of cytochrome c preceded that of caspase-3, and these two peaks were also precedence of the peak of DNA-fragmentation. Western blot analysis showed cytosolic expression of cytochrome c from mitochondria. This study demonstrated 7. Rapid release of cytochrome c from mitochondria to the cytosol, mainly in neurons of the cortex at 3 h after ischemia. 2. Subsequent peaks of caspase-3 and TUNEL in this order. These temporal profiles suggest a serial cascadic activation of apoptotic pathways in neuronal death after permanent MCA occlusion of rats. [Neurol Res 2000; 22: 223-228]     

Loss of bag-1 immunoreactivity in rat brain after transient middle cerebral artery occlusion

Although bag-1 is a strong apoptosis repressor protein, its functions in normal or injured brains are not fully understood. In the present study, we investigated expression of bag-1 protein in rat brain after transient middle cerebral artery (MCA) occlusion, and compared the results with that of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (TUNEL). Immunohistochemical analysis revealed that neuronal, choroid plexus, and ependymal cells were positively stained in the sham control brain. After 90 min of transient MCA occlusion, immunoreactivity for bag-1 progressively decreased from 3 to 48 h in the nuclei of neurons. Western blot analysis revealed that immunoreactive bag-1 was markedly decreased in the nuclear fraction. In contrast, cytosolic and mitochondrial fractions showed no or only slight change after the ischemia. TUNEL positive cells appeared at 48 h after the reperfusion, which was preceded by loss of bag-1 immunoreactivity. The present results suggest that bag-1 plays some roles in normal neuronal function, and its loss may be involved in neuronal cell death after ischemia.     

Multiple modes of action of tacrolimus (FK506) for neuroprotective action on ischemic damage after transient focal cerebral ischemia in rats

While the immunosuppressant tacrolimus (FK506) is known to be neuroprotective following cerebral ischemia, the mechanisms underlying its neuroprotective properties are not fully understood. To determine the mode of action by which tacrolimus ameliorates neurodegeneration after transient focal ischemia, we therefore evaluated the effect of tacrolimus on DNA damage, release of cytochrome c, activation of microglia and infiltration of neutrophils following a 60-min occlusion of the middle cerebral artery (MCA) in rats. In this model, cortical brain damage gradually expanded until 24 h after reperfusion, whereas brain damage in the caudate putamen was fully developed within 5 h. Tacrolimus (1 mg/kg) administered immediately after MCA occlusion significantly reduced ischemic damage in the cerebral cortex, but not in the caudate putamen. Tacrolimus decreased both apoptotic and necrotic cell death at 24 h and reduced the number of cytochrome c immunoreactive cells at 8 h after reperfusion in the ischemic penumbra in the cerebral cortex. In contrast, tacrolimus did not show significant neuroprotection for necrotic cell death and reduction of cytochrome c immunoreactive cells in the caudate putamen. Tacrolimus also significantly decreased microglial activation at 8 h and inflammatory markers (cytokine-induced neutrophil chemoattractant and myeloperoxidase [MPO] activity) at 24 h after reperfusion in the ischemic cortex but not in the caudate putamen. These results collectively suggest that tacrolimus ameliorates the gradually expanded brain damage by inhibiting both apoptotic and necrotic cell death, as well as suppressing inflammatory reactions.     

大鼠局灶性脑缺血后Bcl-2、Bax的表达与细胞凋亡的关系

目的探讨缺血性脑损伤中bcl－2基因家族与细胞凋亡的关系。方法用线栓法制成Wistar大鼠大脑中动脉（MCA）阻塞-再通模型，应用免疫组化方法和TUNEL法，分别对大鼠局灶性缺血脑组织Bcl－2、Bax免疫反应阳性细胞和凋亡细胞的分布进行观察。结果大鼠MCA闭塞2h再通48h后，TUNEL阳性细胞主要分布在梗死灶周围的内侧尾壳核和额顶部皮层，与Bcl－2和BaX阳性细胞数在该区域的明显增加基本一致。同时，额顶部皮层Bcl－2／Bax阳性细胞数的比例较其他脑区及对照组明显降低。结论Bcl－2和Bax在缺血后表达的比例对缺血性脑损伤中细胞生存与凋亡可能起重要的调控作用。     

Neuroprotective effect of tacrolimus (FK506) on ischemic brain damage following permanent focal cerebral ischemia in the rat

We investigated the neuroprotective effect of tacrolimus (FK506) on the ischemic cell death with respect to cytochrome c translocation and DNA fragmentation, which are pivotal events in the necrotic and apoptotic signaling pathway, using permanent focal cerebral ischemia in rats. Immunohistochemically, cytochrome c was observed in the cytoplasm as early as 1 h after middle cerebral artery (MCA) occlusion in the infarcted hemisphere. Cytosolic release of cytochrome c after MCA occlusion was also confirmed by Western blot analysis and enzyme immunoassay. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) showed DNA fragmentation evolving in the ipsilateral cortex and the caudate putamen after 3 and 6 h, respectively, following MCA occlusion. Tacrolimus (1 mg/kg, i.v.), administered immediately after MCA occlusion, significantly attenuated the release of cytochrome c in the ischemic region, the number of TUNEL-positive cells in the ischemic penumbra zone, and the size of cortical ischemic lesions. This study demonstrated that tacrolimus ameliorated the accumulation of cytochrome c in the cytosol and the increase of TUNEL-positive cells induced by cerebral ischemia, indicating that the neuroprotective action of tacrolimus on ischemic brain injury caused by permanent focal cerebral ischemia could partially be attributed to the attenuation of the activation of the apoptotic execution machinery.     

Ischemic intensity influences the distribution of delayed infarction and apoptotic cell death following transient focal cerebral ischemia in rats

The aim of this study was to investigate whether the apoptotic process contributes to the delayed infarction that follows a middle cerebral artery (MCA) occlusion of 20 min (mild ischemia group) and to compare this with the delayed component of infarct following 2 h of MCA occlusion (severe ischemia group). Adult male Sprague-Dawley rats underwent left MCA occlusion for either 20 min or 2 h and were reperfused for 12, 24 and 72 h. On 2,3,5-triphenyltetrazolium chloride-stained coronal sections, delayed infarction was observed to develop in the whole MCA territory after mild ischemia, and also in the frontoparietal cortex after severe ischemia. At 24 h after 20 min of MCA occlusion, characteristic apoptotic features, including chromatin condensation and apoptotic bodies were frequently observed by electron microscopy. In both ischemic groups, Hoechst 33342 staining showed typically condensed and fragmented nuclei in the area showing delayed infarction, where TdT-dUTP nick end labeling (TUNEL)-positive cells were also significantly increased. Caspase-3 activity was also found to be elevated 24 and 72 h after reperfusion and this peaked at 24 h in both groups. These findings suggest that ischemic severity may influence the distribution of delayed infarction, and that apoptosis is the underlying pathophysiologic mechanism.     

Increase in p53 protein expression following cortical infarction in the spontaneously hypertensive rat

Using stroke-prone spontaneously hypertensive (SH-SP) rats with permanent occlusion of the middle cerebral artery (MCA), we investigated the expression of wild type p53 (wt-p53) protein and the occurrence of DNA fragmentation in cerebral neurons after ischemia. Three days following MCA occlusion, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL staining) revealed a distinct pattern of nuclear staining in many neurons around the ischemic core. On the lesioned side of the cerebral cortex one day after MCA occlusion, wt-p53 immunoreactivity was observed specifically in the cortical neurons, in the same regions as the TUNEL staining. Mutant type p53 (mt-p53) immunoreactivity was not observed at any time following MCA occlusion. These findings suggest that wt-p53 dependent cell death of cortical neurons occurred in the ischemic periphery following cerebral ischemia and that this pathway for the induction of cell death may play an important role in the exaggeration of cerebral ischemic injury.     

大鼠局灶性脑缺血再灌注后Fas表达与细胞凋亡关系的研究

目的 :本实验通过观察缺血再灌注后大鼠脑组织中 Fas的表达及细胞凋亡的情况 ;初步探讨脑缺血后 Fas表达与细胞凋亡的关系 ,以及其发生的机理。方法 :采用 TUNEL和免疫组化方法观察细胞凋亡及 Fas蛋白在脑缺血再灌注后随时间延长动态变化情况 ,并利用图像分析测定二者的免疫强度。结果 :脑缺血再灌注后神经细胞过度地表达 Fas,以缺血半暗带明显 ,且在缺血再灌注后 6小时达高峰 ,于 2 4小时开始下降。而细胞的凋亡于缺血再灌注后 6小时开始增加 ,于 2 4小时达高峰 ,于 36小时略有下降 ,凋亡细胞分布也以缺血半暗带明显。结论 :脑缺血再灌注可引起Fas的过度表达和神经细胞凋亡增加 ,而缺血后 Fas的过度表达是诱导细胞凋亡的重要的因素之一     

Augmentation of endoplasmic reticulum stress in cerebral ischemia/reperfusion injury associated with comorbid type 2 diabetes

Abstract

<title/>

Objective: Diabetes is one of the major risk factors for ischemic stroke and is reported to aggravate the ischemic brain damage in different experimental models as well as clinical situations. However, the mechanisms underlying the exacerbated ischemia/reperfusion (I/R) brain injury associated with comorbid diabetes are still not clear. This study investigated the role of endoplasmic reticulum (ER) stress in pathophysiology of aggravated I/R brain injury associated with diabetes.

Methods: Focal cerebral ischemia was induced by middle cerebral artery occlusion for 2 hours followed by 22 hours of reperfusion in high-fat diet-fed and low-dose streptozotocin-treated type 2 diabetic rats. Immunohistochemistry and western blotting analysis were performed to detect the changes in expression of various ER stress and apoptotic markers such as 78 kDa glucose-regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153), and caspase-12. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed to detect the extent of DNA fragmentation and cell death.

Results: The diabetic rats subjected to I/R manifested significantly larger brain infarct volume and severe deterioration in neurological deficits than their normal, non-diabetic counterparts. There was a marked upregulation of GRP78 observed in brains of diabetic rats after 22 hours of reperfusion. Furthermore, augmentation of CHOP/GADD153 expression and activation of caspase-12 (ER stress-induced apoptotic factors) were observed in parallel with enhanced TUNEL-positive cells or DNA fragmentation in diabetic rats compared to normal rats following cerebral I/R.

Discussion: Taken together, the current experimental findings demonstrate that diabetes exacerbates brain I/R injury which may be mediated through enhanced ER stress and cell death involving CHOP/GADD153 and caspase-12 activation.     

Induction of PML immunoreactivity in rat brain neurons after transient middle cerebral artery occlusion

Abstract

Promyelocytic leukemia (PML) protein is involved in apoptotic death of cultured neuronal cells, but its role in ischemic brain damage remains uncertain. In this study, we investigated change of immunoreactivity for PML protein in rat brain after transient middle cerebral artery occlusion, and compared the results with that of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL). Western blotting analysis revealed that PML immunoreactivity was only scant in the sham-control brain, but it increased at 1 h and 1 day after reperfusion, and decreased in density thereafter. Immunohistochemical analysis revealed that nuclei of neurons were most densely stained. TUNEL positive cells appeared at 1 day and peaked at 3 days of reperfusion, indicating that PML protein induction preceded DNA fragmentation in neurons. The present results suggest that PML protein may be one of the key molecules in ischemic neuronal cell death. [Neurol Res 2001; 23: 772-776]     

Temporal profile of cytochrome c and caspase-3 immunoreactivities and TUNEL staining after permanent middle cerebral artery occlusion in rats

Although apoptotic pathways play important roles in ischemic neuronal injury, exact mechanism of apoptotic enzyme cascade has not been fully studied. Immunohistochemical stainings for cytochrome c and caspase-3, and histochemical staining for a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNEL) were examined in a rat model of permanent middle cerebral artery (MCA) occlusion. Cytochrome c was strongly induced in neurons of the ischemic penumbra from 3 h after MCA occlusion, and caspase-3 began to be induced in the same area from 3 h with a peak at 8 h. Neuronal cells in MCA area became TUNEL positive at delayed time, reaching a peak at 24 h. Thus, the peak of induction of cytochrome c preceded that of caspase-3, and these two peaks were also precedence of the peak of DNA-fragmentation. Western blot analysis showed cytosolic expression of cytochrome c from mitochondria. This study demonstrated 1. Rapid release of cytochrome c from mitochondria to the cytosol, mainly in neurons of the cortex at 3 h after ischemia. 2. Subsequent peaks of caspase-3 and TUNEL in this order. These temporal profiles suggest a serial cascadic activation of apoptotic pathways in neuronal death after permanent MCA occlusion of rats.     

丁苯酞对大鼠脑缺血再灌注损伤后的Fas／FasL蛋白表达的影响

目的研究大鼠脑缺血再灌注损伤后Fas和FasI。蛋白的表达和丁笨酞保护作用的机制。方法线栓法制备大鼠大脑中动脉缺血再灌注模型,SD大鼠随机分为正常对照组、假手术组、缺血再灌注组和丁苯酞组;应用尼氏染色显示存活神经元,末端转移酶介导的缺口末端标记（TUNEI。）方法检测程序化死亡细胞;免疫组织化学法观察脑缺血再灌注后6h、1d、3d、7d四个时间点脑组织中Fas和FasI．蛋白的表达变化。结果丁苯酞组皮层存活神经元数量多于缺血再灌注组,TUNEI．阳性细胞数减少,丁苯酞组在再灌注后各时间点的Fas和FasL阳性细胞数较缺血再灌注组少,差异均有统计学意义。结论丁苯酞可能通过抑制Fas／FasL蛋白的表达,从而减少神经元凋亡,减轻缺血再灌注后脑组织的损伤。     

Inhibition of apoptosis by hyperbaric oxygen in a rat focal cerebral ischemic model.

The hypothesis was tested that hyperbaric oxygen therapy (HBO) reduced brain infarction by preventing apoptotic death in ischemic cortex in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and subsequently were exposed to HBO (2.5 atmospheres absolute) for 2 h, at 6 h after reperfusion. Rats were killed and brain samples were collected at 24, 48, 72 h, and 7 days after reperfusion. Neurologic deficits, infarction area, and apoptotic changes were evaluated by clinical scores, 2,3,7-triphenyltetrazolium chloride staining, caspase-3 expression, DNA fragmentation assay, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL)-hematoxylin and eosin (H&E) costaining. In MCAO/R without HBO treatment animals, DNA fragmentation was observed in injured cortex at 24, 48, and 72 h but not in samples at 7 days after reperfusion. Double labeling of brain slides with NeuN and caspase-3 demonstrated neurons in the injured cortex labeled with caspase-3. TUNEL+H&E costaining revealed morphologic apoptotic changes at 24, 48, and 72 h after reperfusion. Hyperbaric oxygen therapy abolished DNA fragmentation and reduced the number of TUNEL-positive cells. Hyperbaric oxygen therapy reduced infarct area and improved neurologic scores at 7 days after reperfusion. One of the molecular mechanisms of HBO-induced brain protection is to prevent apoptosis, and this effect of HBO might preserve more brain tissues and promote neurologic functional recovery.     

Selective neuronal survival and upregulation of PCNA in the rat inner retina following transient ischemia

In this study we investigated the extent and time course of neuronal cell death and the regulation of the proliferating cell nuclear antigen (PCNA) in the different retinal cell layers following ischemia-reperfusion injury. Retinal ischemia was induced by controlled elevation of the intraocular pressure for a duration of 60 min. Changes in thickness and cell numbers in the retinal cell layers were analyzed at various time points (1 h to 4 weeks) after reperfusion. In parallel, apoptotic cell death was determined by the TUNEL method and the expression of PCNA analyzed by immunocytochemistry. In addition, we tested whether PCNA is expressed in neurons by double immunocytochemistry. The reduction in thickness was found to be less pronounced in the inner nuclear layer (INL). Correspondingly, cell numbers decreased by only 33% in the inner retina, but by more than 80% in the outer nuclear layer (ONL). Alterations in glial cell numbers did not contribute significantly to postischemic changes in the INL and ONL as assessed by using immunocytochemical markers for microglial and Müller cells. The time course of cell death determined by the TUNEL technique also differed markedly in the retinal layers being rapid and transient in the inner retina but delayed and prolonged in the ONL. PCNA immunoreactivity was undetectable in the normal retina, but was specifically induced in neurons of the inner retina within 1 h after reperfusion and was sustained for at least 4 weeks. We conclude that in contrast to photoreceptors in the ONL, a significant proportion of inner retinal neurons is resistant to ischemic insult induced by transiently increased intraocular pressure and that PCNA may possibly play a role in the selective postischemic survival of these cells.     

褪黑素对SD大鼠脑缺血再灌注损伤后c-fos表达的影响

目的探讨褪黑素在大鼠脑缺血再灌注损伤中的神经保护作用及可能机制。方法选取45只雄性SD大鼠,分为假手术组(5只)、脑缺血再灌注组(20只)、褪黑素干预组(20只);脑缺血再灌注组和褪黑素干预组根据时间点第6小时、第1天、第3天、第7天分为4个组,每组5只。采用Longa线栓法建立大鼠左侧大脑中动脉栓塞(MCAO)模型,采用HE染色检测脑组织的病理改变,TUNEL染色检测神经细胞的凋亡,免疫组织化学(免疫组化)法及蛋白质印迹法(Western Blotting)观察大鼠脑组织内c-fos表达情况。结果在脑缺血再灌注组的各时间点的HE染色显示,胶质细胞呈现程度不一的增生,神经元出现坏死;褪黑素干预能减轻脑缺血再灌注后胶质细胞增生及神经元的坏死。在TUNEL染色凋亡检测中,脑缺血再灌注组各时间点的神经细胞凋亡升高;褪黑素干预组各时间点的细胞凋亡数低于脑缺血再灌注组(P <0.05)。在免疫组化及蛋白质印迹检测中,脑缺血再灌注组c-fos表达增加,在第1天时达到高峰,之后表达逐步降低;在褪黑素干预组,c-fos表达趋势与缺血再灌注组一致,但表达水平比缺血再灌注组相应时间点低,差异有统计学意义(P <0.05)。结论褪黑素能够减轻脑缺血再灌注后神经元的损伤,降低c-fos的表达,表明褪黑素可能通过调控c-fos的表达在脑缺血再灌注中发挥神经保护作用。