神经生长因子对兔局灶性脑缺血再灌注神经功能修复的影响和MR影像学评价

目的:研究神经生长因子(NGF)对兔局灶性脑缺血再灌注损伤神经功能修复的影响,并利用MR成像技术进行评价。方法:采用兔大脑中动脉阻断(MCAO)局灶性脑缺血再灌注模型,分别在缺血2h再灌注损伤后0、1、3和6h应用微量进样器将NGF立体定向导入梗死灶周,于再灌注72h,应用MR影像学、TTC染色和流式细胞术评价兔梗死体积、水肿体积、表观弥散系数(ADC)及神经功能缺损和凋亡状态。结果:缺血再灌注0h、1h、3h和6h灶周给予NGF,梗死体积分别比对照组下降50.1%、48.4%、37.6%和13.7%。同时再灌注3h内应用NGF水肿体积、神经功能缺损评分、灶周凋亡率及caspase-3含量明显下降,梗死中心区及灶周ADC比率(ADCR)升高;再灌注6h后给药,则无明显作用。相关分析显示各组灶周ADCR与灶周凋亡率具有明显负相关。结论:NGF对兔局灶性脑缺血再灌注损伤有保护作用,抑制caspase-3活性的表达和细胞凋亡是其保护机制之一,MR影像学检查可作为定量评价基因疗效的可靠指标。     

神经生长因子对兔局灶性脑缺血再灌注损伤的治疗时间窗及MR影像学评价

目的研究神经生长因子(NGF)对脑缺血再灌注损伤保护作用的有效时间窗,同时利用MR成像技术对其进行评价。方法采用兔大脑中动脉阻断(MCAO)局灶性脑缺血2 h再灌注72 h模型,分别于缺血再灌注0、1、36、h应用微量进样器将NGF立体定向导入梗死灶周,并于再灌注不同时间点应用MR影像学、TTC染色和流式细胞术评价家兔梗死体积、神经功能缺损和凋亡状态。结果缺血再灌注0、13、h组梗死灶周注射NGF后,经MRI检查所测梗死体积分别为(229.9±17.1)(、260.7±24.2)、(314.6±25.3)mm3,与对照组[(468.6±29.7)mm3]比较差异有统计学意义(均P<0.01);缺血再灌注6 h组梗死体积为(441.1±14.8)mm3,与对照组比较差异无统计学意义(P>0.05)。采用TTC染色所测梗死体积与MRI检查结果一致。脑缺血再灌注3 h内注射NGF,其神经功能缺损评分明显降低,凋亡率明显下降;再灌注6 h后注射NGF则无明显作用。结论NGF对兔局灶性脑缺血再灌注损伤的有效时间窗为再灌注损伤3 h内,MR影像学检查可作为定量评价基因疗效的可靠指标。     

Estrogen inhibits Fas-mediated apoptosis in experimental stroke

Estrogen is protective in experimental cerebral ischemia, yet the mechanism remains unclear. Fas-mediated apoptosis has been shown to be induced after cerebral ischemia and significantly contribute to ischemic brain damage. In this study, we tested if estrogen is protective against cerebral ischemia by suppressing Fas-mediated apoptosis. 17β-estradiol-treated and untreated ovariectomized (OVX) female mice were subjected to 2 h middle cerebral artery occlusion (MCAO). Expression of Fas and Fas-associated death domain (FADD) were measured at 3, 6 and 12 h of reperfusion by RT-PCR and Western blot, respectively. Post-ischemic activities of caspase-8 and -3 activities, the two downstream effectors of Fas-induced apoptosis, were also assayed at same time points by ELISA. Finally, Fas antibody-induced cell death in primary cortical neurons was assayed by fluorescence activated cell sorter (FACS) in the presence and absence of estradiol. Our data showed that estradiol-treated OVX female mice sustained smaller infarct compared to untreated OVX mice. Ischemia upregulated Fas and FADD expression, and increased caspase-8 and -3 activities in OVX female mouse cortex, which were significantly attenuated by estradiol. Estradiol also significantly inhibited Fas antibody-induced neuronal cell apoptosis. Our data suggests that inhibition of ischemia-induced Fas-mediated apoptosis is an important mechanism of neuroprotection by estrogen in cerebral ischemia.     

细胞外信号调节激酶在NGF/VEGF介导的神经保护作用中的动态变化及其调控机制

目的探讨细胞外信号调节激酶1(ERK1)在局灶性脑缺血/再灌注不同时间、不同脑区的动态时空变化,以及其在NGF/VEGF介导的神经保护作用中的调控表达机制。方法采用兔大脑中动脉阻断(MCAO)局灶性脑缺血再灌注模型,所有动物随机分为假手术组(n=6)、缺血/再灌注组(n=60)、因子干预组(n=40)。应用免疫组化检测ERK1在脑缺血/再灌注损伤不同脑区的动态表达,同时,应用免疫组化、流式细胞术和电镜检测caspase-3表达、凋亡和超微结构的变化。结果免疫组化分析显示,再灌注损伤1hERK1首先在海马CA3和齿状回(DG)表达增加,6h后其它脑区也相继增加,随再灌注时间延长而加剧,1~3d达高峰。再灌注1hcaspase-3活性表达在各脑区迅速增加,3d达高峰。应用神经保护剂(NGF/VEGF)后各脑区ERK1表达呈明显抑制,caspase-3表达同时被抑制。结论ERK信号通路可能通过调节死亡受体途径介导神经保护作用,抑制ERK信号途径可能是减轻脑缺血损伤过程中神经细胞死亡的有效方法。     

Exendin-4对MCAO再灌注导致的脑缺血/再灌注损伤具有神经保护作用

目的 探讨GLP-1受体激动剂Exendin-4腹腔给药对大脑中动脉闭塞(MCAO)再灌注所致大鼠脑缺血/再灌注损伤的神经保护作用。方法 SD大鼠术前1 h腹腔注射Exendin-4,MCAO再灌注24 h后进行神经功能缺损评分,TTC染色计算脑梗死体积,免疫荧光观察神经元和小胶质细胞生存数量及检测凋亡通路相关蛋白的相对表达水平。结果 Exendin-4能够保护由于MCAO再灌注后所致的脑缺血再灌注损伤,减少了脑梗死体积,降低皮层凋亡蛋白的相对表达水平,抑制神经元凋亡。结论 Exendin-4可以对MCAO再灌注所致脑缺血/再灌注损伤具有神经保护作用,该作用是通过抑制凋亡蛋白的产生,从而抑制细胞凋亡。     

Inhibition of caspase-3 activation and apoptosis is involved in 3-nitropropionic acid-induced ischemic tolerance to transient focal cerebral ischemia in rats

Our aim was to investigate the involvement of caspase-3 activation and apoptotic cell death in mitochondrial toxin 3-nitropropionic acid (3-NPA)-induced ischemic tolerance to transient focal cerebral ischemia in rats. Rats were administrated either vehicle control or 3-NPA ip doses of 20 mg/kg. Three days later, rats were exposed to 2 h of middle cerebral artery occlusion, followed by 24 h of reperfusion. Infarct volumes were assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 h after reperfusion. We measured neural cell apoptosis in the cerebral ischemic penumbra by terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and flow cytometry (FCM). Cleavage of the fluorogenic substrate zDEVD-afc was used to assay caspase-3 activity. Compared with the vehicle-injected group, pretreatment with 3-NPA reduced the infarct volume by 22.3% and decreased the number of TUNEL-positive neural cells and apoptotic percentages by 47% (p < 0.05) and 43.9% (p < 0.01), respectively. In terms of caspase-3 activity in ischemic penumbral tissues, the 3-NPA-pretreated group showed 13.9% (p < 0.05) less caspase-3 activity than the control group. The development of 3-NPA-induced ischemic tolerance in brain may be related to decreases in caspase-3 activation, which leads to decreased neural cell apoptosis.     

A novel neuroprotective strategy for ischemic stroke: transient mild acidosis treatment by CO2 inhalation at reperfusion

Acidosis is one of the key components in cerebral ischemic postconditioning that has emerged recently as an endogenous strategy for neuroprotection. We set out to test whether acidosis treatment at reperfusion can protect against cerebral ischemia/reperfusion injury. Adult male C57BL/6 J mice were subjected to 60-minute middle cerebral arterial occlusion followed by 24-hour reperfusion. Acidosis treatment by inhaling 10%, 20%, or 30% CO₂ for 5 or 10 minutes at 5, 50, or 100 minutes after reperfusion was applied. Our results showed that inhaling 20% CO₂ for 5 minutes at 5 minutes after reperfusion-induced optimal neuroprotection, as revealed by reduced infarct volume. Attenuating brain acidosis with NaHCO₃ significantly compromised the acidosis or ischemic postconditioning-induced neuroprotection. Consistently, both acidosis-treated primary cultured cortical neurons and acute corticostriatal slices were more resistant to oxygen–glucose deprivation/reperfusion insult. In addition, acidosis inhibited ischemia/reperfusion-induced apoptosis, caspase-3 expression, cytochrome c release to cytoplasm, and mitochondrial permeability transition pore (mPTP) opening. The neuroprotection of acidosis was inhibited by the mPTP opener atractyloside both in vivo and in vitro. Taken together, these findings indicate that transient mild acidosis treatment at reperfusion protects against cerebral ischemia/reperfusion injury. This neuroprotection is likely achieved, at least partly, by inhibiting mPTP opening and mitochondria-dependent apoptosis.     

Cannabinoid CB(2) receptor activation decreases cerebral infarction in a mouse focal ischemia/reperfusion model.

Cannabinoid CB(2) Receptor (CB(2)) activation has been shown to have immunomodulatory properties without psychotropic effects. The hypothesis of this study is that selective CB(2) agonist treatment can attenuate cerebral ischemia/reperfusion injury. Selective CB(2) agonists (O-3853, O-1966) were administered intravenously 1 h before transient middle cerebral artery occlusion (MCAO) or 10 mins after reperfusion in male mice. Leukocyte/endothelial interactions were evaluated before MCAO, 1 h after MCAO, and 24 h after MCAO via a closed cranial window. Cerebral infarct volume and motor function were determined 24 h after MCAO. Administration of the selective CB(2) agonists significantly decreased cerebral infarction (30%) and improved motor function (P<0.05) after 1 h MCAO followed by 23 h reperfusion in mice. Transient ischemia in untreated animals was associated with a significant increase in leukocyte rolling and adhesion on both venules and arterioles (P<0.05), whereas the enhanced rolling and adhesion were attenuated by both selective CB(2) agonists administered either at 1 h before or after MCAO (P<0.05). CB(2) activation is associated with a reduction in white blood cell rolling and adhesion along cerebral vascular endothelial cells, a reduction in infarct size, and improved motor function after transient focal ischemia.     

骨髓间充质干细胞对局灶脑缺血再灌注损伤的超早期干预

摘要：目的　探讨骨髓间充质干细胞(BMSCs) 对大鼠脑缺血再灌注损伤保护作用及机制。方法　雄性SD大鼠72只,以线栓法制成缺血再灌注模型,随机分为2组：溶剂对照组;BMSCs移植组。在缺血再灌注后1天、3天、6天分别行神经功能检测、TTC染色、免疫组化法检测Survivin、caspase-3表达,TUNEL法检测凋亡细胞表达。结果　与溶剂对照组比较,在梗死脑组织中移植骨髓间充质干细胞BMSCs后,可使大鼠神经功能有所恢复,在大鼠大脑中动脉局灶脑缺血90分钟再灌注3天及6天神经功能评分比较高,有统计学意义;TTC染色脑梗死体积百分比在缺血再灌注第3天、第6天较溶剂对照组分别减少2.13%和2.10%,有统计学意义。单纯BMSCs移植组比较对照组在缺血再灌注后1天、3天、6天缺血侧皮层Survivin表达增高、caspase-3表达降低,凋亡细胞减少,均有统计学意义。结论　脑缺血部位脑实质单纯 BMSCs 移植能够改善缺血后神经功能,减少脑缺血后梗死体积,可能通过增加脑缺血再灌注损伤部位Survivin蛋白的表达,降低凋亡相关蛋白Caspase-3表达,减少凋亡细胞数量发挥作用     

Normobaric hyperoxia delays perfusion/diffusion mismatch evolution, reduces infarct volume, and differentially affects neuronal cell death pathways after suture middle cerebral artery occlusion in rats.

Normobaric hyperoxia (NBO) has been shown to extend the reperfusion window after focal cerebral ischemia. Employing diffusion (DWI)- and perfusion (PWI)-weighted magnetic resonance imaging (MRI), the effect of NBO (100% started at 30 mins after middle cerebral artery occlusion (MCAO)) on the spatiotemporal evolution of ischemia during and after permanent (pMCAO) and transient suture middle cerebral artery occlusion (tMCAO) was investigated (experiment 3). In two additional experiments, time window (experiment 1) and cell death pathways (experiment 2) were investigated in the pMCAO model. In experiment 1, NBO treatment reduced infarct volume at 24 h after pMCAO by 10% when administered for 3 h (P>0.05) and by 44% when administered for 6 h (P<0.05). In experiment 2, NBO acutely (390 mins, P<0.05) reduced in situ end labeling (ISEL) positivity in the ipsilesional penumbra but increased contralesional necrotic as well as caspase-3-mediated apoptotic cell death. In experiment 3, CBF characteristics and CBF-derived lesion volumes did not differ between treated and untreated animals, whereas the apparent diffusion coefficient (ADC)-derived lesion volume essentially stopped progressing during NBO treatment, resulting in a persistent PWI/DWI mismatch that could be salvaged by delayed (3 h) reperfusion. In conclusion, NBO (1) acutely preserved the perfusion/diffusion mismatch without altering CBF, (2) significantly extended the time window for reperfusion, (3) induced lasting neuroprotection in permanent ischemia, and (4) although capable of reducing cell death in hypoperfused tissue it also induced cell death in otherwise unaffected areas. Our data suggest that NBO may represent a promising strategy for acute stroke treatment.     

Lateral intracerebroventricular injection of Apelin-13 inhibits apoptosis after cerebral ischemia/reperfusion injury

Apelin-13 inhibits neuronal apoptosis caused by hydrogen peroxide, yet apoptosis following cerebral ischemia-reperfusion injury has rarely been studied. In this study, Apelin-13 (0.1 μg/g) was injected into the lateral ventricle of middle cerebral artery occlusion model rats. TTC, TUNEL, and immunohistochemical staining showed that compared with the cerebral ischemia/reperfusion group, infarct volume and apoptotic cell number at the ischemic penumbra region were decreased in the Apelin-13 treatment group. Additionally, Apelin-13 treatment increased Bcl-2 immunoreactivity and decreased caspase-3 immunoreactivity. Our findings suggest that Apelin-13 is neuroprotective against cerebral ischemia/reperfusion injury through inhibition of neuronal apoptosis.     

Anemonin Alleviates Nerve Injury After Cerebral Ischemia and Reperfusion (I/R) in Rats by Improving Antioxidant Activities and Inhibiting Apoptosis Pathway

In the present study, we aimed at evaluating the potential neuroprotective effect and the underlying mechanism of anemonin against cerebral ischemia and reperfusion (I/R) injury. Anemonin was administered to rats by the intraperitoneally (i.p.) route once daily for 7 days before middle cerebral artery occlusion (MCAO). Focal cerebral ischemia was induced by 90 min of MCAO followed by 24 h of reperfusion. After that, animals were sacrificed by decapitation, brain was removed, and various biochemical estimations, neurological status, and assessment of cerebral infarct size were carried out. MCAO followed by 24 h of reperfusion caused a significant increase in infarct size, neurological deficit score, malondialdehyde (MDA) content, reactive oxygen species (ROS) level, and DNA fragmentation, as well as a decrease in the activities of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GPx), and Na⁺, K⁺-ATPase in the brain. Furthermore, elevated Bax expression, increased caspase-3 cleavage, and decreased Bcl-2 expression were observed in nontreated rats in response to focal cerebral I/R injury. However, pretreatment with anemonin significantly reversed these levels of biochemical parameters, reduced cerebral infarct size, and improved the neurologic score in cerebral ischemic animals. Additionally, a wide distribution of anemonin in plasma and brain tissues and the brain-to-plasma partition coefficient (Ri) ratio of 0.7 at 90 min indicated that this compound could penetrate the blood-brain barrier (BBB). These results showed that pretreatment with anemonin provided a significant protection against cerebral I/R injury in rats by, at least in part, its antioxidant action and consequent inhibition of apoptosis.     

Endoplasmic reticulum in the penumbra following middle cerebral artery occlusion in the rabbit

Caspase-12 has been localized to endoplasmic reticulum (ER) and showed to involve ER stress-induced apoptosis. In the present work we investigated the temporospatial alterations of caspase-12 immunoreactivity in the penumbra following cerebral ischemia/reperfusion in rabbit. Transient cerebral ischemia was produced by intraluminal occlusion of the middle cerebral artery for 2 h followed by 1 h, 6 h, 1 day, 3 days, 7 days and 14 days of reperfusion. Caspase-12 immunohistochemistry was first increased in the penumbra 1 h after reperfusion, with a peak at day 1 to day 3, and then gradually decreased to basal level at day 14. The number of TUNEL-positive cells and ultrastructural observation of brain sections in the penumbra showed a similar change at the same time points. ER mediated by caspase-12 participated in apoptosis induced by cerebral ischemia/reperfusion injury, which may provide a new area for therapeutic intervention to ameliorate outcomes following cerebral ischemia.     

Delayed treatment with MLN519 reduces infarction and associated neurologic deficit caused by focal ischemic brain injury in rats via antiinflammatory mechanisms involving nuclear factor-kappaB activation, gliosis, and leukocyte infiltration.

Secondary brain injury due to ischemia includes the infiltration of leukocytes into the brain parenchyma mediated by activation of nuclear factor-kappaB (NF-kappaB), which is activated by proteasome degradation. Neuroprotection with the proteasome inhibitor MLN519 has previously been reported to decrease ischemic brain injury in rats. The authors used higher doses of MLN519 to evaluate the neuroprotection therapeutic window after 24 hours of brain injury in rats as correlated to proteasome levels, activated NF-kappaB immunoreactivity, and leukocyte infiltration. Male Sprague-Dawley rats were subjected to 2-hour middle cerebral artery occlusion (MCAO) and recovery. MLN519 or vehicle was administered after injury with a single injection given in delayed increments of 2 hours (i.e., 4, 6, or 8 hours after MCAO). Treatment with MLN519 up to 6 hours after MCAO (4 hours after reperfusion) effectively reduced neuronal and astrocytic degeneration, decreased cortical infarct volume, and increased neurologic recovery. These effects were related to >80% reductions in blood proteasome levels, reduced neutrophil infiltration, and a decrease in activated NF-kappaB immunoreactivity. This improved neuroprotection profile and antiinflammatory effect of MLN519 provides an exciting avenue for potential treatment of focal ischemic brain injury in humans.     

Neuroprotection induced in vitro by ischemic preconditioning and postconditioning: modulation of apoptosis and PI3K-Akt pathways

Preconditioning and postconditioning are mild ischemic exposures before or after severe injurious ischemia, respectively, that elicit endogenous neuroprotective responses. Molecular mechanisms of neuroprotection through preconditioning and postconditioning are not completely understood. Here we optimized the in vitro oxygen and glucose deprivation (OGD) models of preconditioning and postconditioning in primary cortical neuron cultures that allow the studies of the corresponding molecular mechanisms of neuroprotection. We found that the cortical cells preconditioned with a single 45-min OGD treatment administered 24 h prior to injurious 2 h OGD were robustly protected after both 3 h and 16 h of reperfusion. For the postconditioning treatment, we found that three cycles of 15 min OGD followed by 15 min reperfusion, applied immediately after injurious 2 h OGD and prior to complete reperfusion, resulted in effective neuroprotection at both 3 h and 16 h of reperfusion. Using real-time RT–PCR arrays focused on genes of the apoptosis and PI3K–Akt pathways, we found that injurious OGD mainly induced apoptosis-related and repressed PI3K–Akt pathway-related genes after either 3 h or 16 h of reperfusion. Preconditioning treatment resulted in the activation of both pro-survival and anti-apoptotic pathways after 3 h of reperfusion and mainly anti-apoptotic pathway after 16 h of reperfusion. In contrast, the activation of PI3K–Akt pathway mainly contributed to the neuroprotective effect by the postconditioning treatment after 3 h of reperfusion, but differential gene expression likely contributed minimally, if at all, to the neuroprotection observed after 16 h of reperfusion. Among the novel markers of neuroprotection, Nol3 gene upregulation was observed after 3 h of reperfusion following either preconditioning or postconditioning treatments and after 16 h of reperfusion following preconditioning treatment.     

Delayed neuroprotection induced by sevoflurane via opening mitochondrial ATP-sensitive potassium channels and p38 MAPK phosphorylation

This study aimed to investigate the role of p38 MAPK phosphorylation and opening of the mitoK_ATP channels in the sevoflurane-induced delayed neuroprotection in the rat brain. Adult male Sprague-Dawley rats (250–300 g) were randomly assigned into four groups: ischemia/reperfusion (Control), sevoflurane (Sevo), 5-hydroxydecanoate (5-HD) + sevoflurane (5-HD + Sevo) and 5-HD groups and were subjected to right middle cerebral artery occlusion (MCAO) for 2 h. Sevoflurane preconditioning was induced 24 h before MCAO in sevoflurane and 5-HD + sevoflurane groups by exposing the animals to 2.4% sevoflurane in oxygen for 60 min. In control and 5-HD groups: animals were exposed to oxygen for 60 min at 24 h before MCAO. A selective mitoK_ATP channel antagonist, 5-hydroxydecanoate (5-HD, 40 mg/kg, i.p.), was administered 30 min before sevoflurane/oxygen exposure in the 5-HD + sevoflurane and 5-HD groups, respectively. Neurological deficits scores and the protein expression of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were evaluated at 24 and 72 h after reperfusion. Cerebral infarct size was evaluated at 72 h after reperfusion by 2,3,5-triphenyltetrazolium chloride staining. Sevoflurane preconditioning produced marked improvement neurological functions and a reduction in brain infarct volumes than animals with brain ischemia only. Sevoflurane treatment also caused increased phosphorylation of p38 MAPK at 24 and 72 h after reperfusion. These beneficial effects were attenuated by 5-HD. Blockade of cerebral protection with 5-HD concomitant with decrease in p38 phosphorylation suggests that mitoK_ATP channels opening and p38 phosphorylation participate signal transduction cascade of sevoflurane preconditioning and p38 MAPK activation may be a downstream of opening mitoK_ATP channels.     

Comparison of the anti-apoptotic effects of 15-and 35-minute suspended moxibustion after focal cerebral ischemia/reperfusion injury

Heat-sensitive suspended moxibustion has a neuroprotective effect against focal cerebral ischemia/reperfusion injury, but the underlying mechanisms remain unclear. The duration of heat-sensitive suspended moxibustion(usually from 30 minutes to 1 hour) is longer than traditional suspended moxibustion(usually 15 minutes). However, the effects of 15-and 35-minute suspended moxibustion in rats with cerebral ischemia/reperfusion injury are poorly understood. In this study, we performed 15-or 35-minute suspended moxibustion at acupoint Dazhui(GV14) in an adult rat model of focal cerebral ischemia/reperfusion injury. Infarct volume was evaluated with the 2,3,5-triphenyltetrazolium chloride assay. Histopathological changes and neuronal apoptosis at the injury site were assessed by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase d UTP nick end labeling assay. Caspase-9 and caspase-3 expression at the injury site was detected using immunofluorescent staining. Bax and Bcl-2 expression at the injury site was assessed using western blot assay. In the 35-minute moxibustion group, infarct volume was decreased, neuronal apoptosis was reduced, caspase-9, caspase-3 and Bax expression was lower, and Bcl-2 expression was increased, compared with the 15-minute moxibustion group. Our findings show that 35-minute moxibustion has a greater anti-apoptotic effect than 15-minute moxibustion after focal cerebral ischemia/reperfusion injury.     

局部亚低温对脑缺血再灌注治疗时间的影响

目的观察有效再灌注时间窗内实施局部亚低温对不同时间点血管再通(再灌注)的影响,并探讨其机制。方法将150只雄性大鼠随机分为大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)24h组10只;常温再灌注组50只;MCAO 2h进行亚低温再灌注组50只;MCAO 3h进行亚低温再灌注组40只。动物均于再灌注24h后处死,但MCAO 24h组大脑中动脉闭塞24h后直接处死。采用TTC染色观察梗死体积,用干-湿质量法测定脑含水量,用原位末端标记(TUNEL)观察神经细胞凋亡的变化,采用免疫组化法检测大鼠模型的Bcl-2、Bax及水通道蛋白4(aquaporin4,AQP4)的表达。结果 MCAO 24h组的梗死体积、脑含水量、TUNEL阳性细胞数、及Bcl-2、Bax、AQP4的表达相比较,常温组2～3h有统计学差异(P<0.01),4～6h没有统计学差异(P>0.05);MCAO 2h亚低温再灌注组2～5h有统计学差异(P<0.01),6h没有统计学差异(P>0.05);MCAO 3h亚低温再灌注组3～4h有统计学差异(P<0.01),5～6h没有统计学差异(P>0.05)。亚低温再灌注组各时相点的梗死体积、脑含水量、TUNEL阳性细胞数、Bax、AQP4的表达均显著低于相应常温组,Bcl-2的表达显著高于常温组(P<0.01,P<0.05)。结论实施局部亚低温可延长再灌注治疗时间窗,而且亚低温开始时间越早,其延长时间窗的效果就越显著。这为解决在时间窗内就诊的患者因检查等错过再灌注治疗这一临床难题的解决提供了重要的实验室依据。其机制可能与抑制半暗带细胞的凋亡和改善微循环有关。     

VEGF治疗脑缺血再灌注损伤的分子机制研究

目的 通过检测血管内皮生长因子（VEGF）治疗兔脑缺血再灌注损伤的有关分子表达情况,探讨其分子机制.方法 采用兔大脑中动脉阻塞（MCAO）2h再灌注72h模型,在再灌注即刻,应用微量进样器将VEGF立体定向导入梗死灶周,于再灌注72h断头取脑,应用免疫组化方法检测缺血半暗带区caspase-3和细胞外信号调节激酶1（estracellular signal-regulated kinase,ERK1）的表达情况.结果 VEGF治疗后缺血半暗带区caspase-3和ERK1表达明显减低.结论 VEGF可能通过抑制caspase-3和ERK1表达发挥治疗作用.     

Expression of Bcl-2 and Bax after hippocampal ischemia in DHA + EPA treated rats

To determine the impact of ω3 fatty acids on post-ischemic expression of pro- and anti-apoptotic proteins in hippocampus, male rats were received 10 or 100 mg/kg [Docosahexaenoic acid (DHA) + Ecosapentaenoic acid (EPA); gavage; 21 days before ischemia to 2–10 days after ischemia]. Global cerebral ischemia reperfusion (IR) was performed using the four-vessel occlusion model; ischemia 8 min and reperfusion 6, 48 h and 10 days. IR increased Bcl-2 and Bax expression after 48 h (p < 0.05 and p < 0.01 vs. sham) and 10 days (only Bax; p < 0.05), without significant difference with DHA + EPA groups after 6 h. But after 48 h expression of Bcl-2 increased (p < 0.05 vs. IR) and Bax decreased (p < 0.05). At day 10 after ischemia expression of Bax in DHA + EPA acid groups was less than IR (p < 0.05) and in 100 mg/kg DHA + EPA group Bcl-2 expression was more than IR (p < 0.05). These data suggested that long-term gavage with DHA + EPA increase hippocampal neurons survival for days after ischemia, revealed by increased Bcl-2 and decreased Bax expressions.