神经节苷脂联合参附注射液对新生大鼠缺氧缺血性脑损伤的保护作用

目的探讨神经节苷脂(GM1)联合参附注射液对新生大鼠缺氧缺血性脑损伤(HIBD)的神经保护作用。方法将新生7d SD乳鼠90只随机分为假手术组、缺血缺氧组(HIBD组)、缺血缺氧+GM1干预组(GM1组)、缺血缺氧+参附干预组(参附组)及缺血缺氧+GM1及参附干预组(合用组)。在给药后不同时间点,采用免疫组化法半定量测定脑组织中Bcl-2及Bax含量。结果假手术组海马CA1区可见少量Bcl-2及Bax表达;HIBD后该区Bcl-2及Bax表达均增加,且Bax表达较明显;分别应用GM1、参附注射液治疗后该区Bcl-2表达进一步增加,而Bax蛋白水平下降;联合应用两种药物治疗后该区Bcl-2表达增加、Bax表达下降的趋势更为明显。结论新生大鼠脑缺氧缺血后腹腔注射神经节苷脂或参附注射液均可不同程度减轻大鼠脑损伤,两种药物联合应用对其脑损伤的改善作用更为明显。     

脉络宁对新生大鼠缺氧缺血性脑损伤后nNOS和c-Fos表达的影响

目的：检测新生大鼠缺血缺氧后神经元型一氧化氮合酶（nNOS）和c-Fos免疫活性表达改变及脉络宁对其的影响。方法：结扎7d龄大鼠右侧颈总动脉1h，然后暴露在8％氧和92％氮的混合气体中2h，建立缺氧缺血脑损伤模型。用免疫组织化学方法检测并比较缺血缺氧后和脉络宁处理后c-Fos和nNOS的免疫活性。结果：缺血缺氧6h后c-Fos表达达高峰；与缺氧缺血组相比，脉络宁处理组c-Fos阳性神经元数量增加，表达c-Fos的时间延长；与假手术组相比，缺氧缺血组nNOS表达水平增高，脉络宁处理组则下降。结论：脉络宁可抑制nNOS表达，增强c-Fos表达，可能对缺血缺氧性脑损伤有保护作用。     

脉络宁对新生大鼠缺氧缺血性脑损伤后nNOS和c-Fos表达的影响

目的:检测新生大鼠缺血缺氧后神经元型一氧化氮合酶(nNOS)和c-Fos免疫活性表达改变及脉络宁对其的影响。方法:结扎7d龄大鼠右侧颈总动脉1h,然后暴露在8%氧和92%氮的混合气体中2h,建立缺氧缺血脑损伤模型。用免疫组织化学方法检测并比较缺血缺氧后和脉络宁处理后c-Fos和nNOS的免疫活性。结果:缺血缺氧6h后c-Fos表达达高峰;与缺氧缺血组相比,脉络宁处理组c-Fos阳性神经元数量增加,表达c-Fos的时间延长;与假手术组相比,缺氧缺血组nNOS表达水平增高,脉络宁处理组则下降。结论:脉络宁可抑制nNOS表达,增强c-Fos表达,可能对缺血缺氧性脑损伤有保护作用。     

神经生长因子对新生大鼠缺血缺氧性脑损伤的保护作用研究

目的探讨神经生长因子(never growth factor,NGF)对新生大鼠缺氧缺血性脑损伤(hypoxic-ische-mic encephalopathy,HIE)的保护作用。方法将7d龄的Wistar大鼠,随机分成NGF治疗组、HIE组和假手术组,在制成HIE模型前半小时腹腔注射NGF或0.9%氯化钠溶液各1次,缺血缺氧2h后再腹腔注射等量的NGF或0.9%氯化钠溶液,放回母鼠处喂养44h后处死取出大脑并固定,应用免疫组织化学-SP法研究p53蛋白和bcl-2在新生大鼠及缺血缺氧后脑中表达及与凋亡的关系。结果 p53蛋白在假手术组中基本无表达,HIE组p53蛋白表达明显高于假手术对照组,NGF干预后p53蛋白下降显著,而bcl-2正好相反,HIE组较假手术组表达降低,NGF干预组bcl-2表达显著增加。结论 NGF能减轻缺血缺氧性脑损伤。     

亚低温干预下新生大鼠缺氧缺血性脑损伤COX 2基因表达变化及意义

目的：探讨亚低温干预下新生大鼠缺氧缺血性脑损伤（HIBD）时环氧化酶2（COX 2）基因表达变化及意义。方法120只7 d龄 SD大鼠,采用 Rice法建立新生大鼠缺氧缺血脑损伤模型,随机分为假手术组、常温组、亚低温组,每组又根据处死时间的不同分为1 h、6 h、12 h、24 h、48 h五个亚组,用实时荧光定量聚合酶链反应检测新生大鼠COX 2mRNA表达。结果假手术组COX 2mRNA表达甚微,且各组之间无明显变化;常温组 COX 2mRNA表达6 h开始上调且逐渐增加,24 h含量达高峰,24 h-48 h维持在高峰（P〈0.05）;亚低温组各组 COX 2mRNA表达与常温组各时间点比较均降低（P〈0.05）。结论 COX 2在新生大鼠 HIBD形成中发挥一定的作用,亚低温干预能降低 HIBD后不同时间脑组织 COX 2的表达活性,提示在恰当的时间窗内进行亚低温干预治疗对脑保护是有效的。     

大鼠缺氧缺血后海马神经元凋亡及凋亡抑制蛋白XIAP表达的变化

目的 探讨新生大鼠缺氧缺血性脑损伤(HIBD)后海马CA1区神经元凋亡及X连锁凋亡抑制蛋白(XIAP)表达的变化规律.方法选择48例新生Wistar大鼠为HIBD组,并选择12只新生大鼠分为假手术组(n=6)和正常对照组(n=6).HIBD制模后3h、6h、12h、24 h、48 h、72 h、7 d、14 d处死取材,每次处死6只,采用流式细胞技术检测3组大鼠海马神经元的凋亡率,并应用免疫组织化学染色方法检测各组海马CA1区XIAP的表达变化情况.结果 与假手术组相比,HIBD组缺氧缺血后3h海马神经元凋亡率即开始升高,但差异无统计学意义(P＞0.05);于缺氧缺血后12h凋亡率显著升高(P＜0.05),48 h时达高峰,72 h时开始下降,7d时明显低于高峰水平(P＜0.05),而与假手术组无显著差异,14 d时基本恢复至假手术组水平.正常对照组和假手术组海马CA1区有极少量XIAP阳性表达细胞.HIBD组于缺氧缺血3h时XIAP阳性表达显著升高(P＜0.05),之后逐渐升高,在48 h时达到高峰,72 h时开始逐渐下降,7d时已明显低于高峰水平(P＜0.05),但仍明显高于正常对照组和假手术组水平,在14 d时略高于正常对照组和假手术组,但差异无统计学意义(P＞0.05).XIAP蛋白表达与HIBD后各时间点阳性凋亡细胞出现呈负相关(r=-0.564,P＜0.05).结论 XIAP蛋白的过度表达在脑缺氧缺血后细胞凋亡的调控过程中起着重要作用,对HIBD损伤的脑神经细胞具有保护作用.     

电针对CUMS大鼠前额皮层星形胶质细胞GS、EAAT1、EAAT2的影响

目的观察电针对CUMS大鼠前额皮层星形胶质细胞谷氨酰胺合成酶(GS)、EAAT1、EAAT2的影响。方法将56只雄性SD大鼠随机分为正常组、空白组、针刺组和药物组,每组14只。除正常组常规饲养外,空白组、针刺组和药物组大鼠予慢性不可预见性温和刺激建立抑郁模型。造模成功后,空白组单笼饲养、只给予相同程度的捉抓;针刺组单侧交替取太冲、合谷穴予连续波、2 Hz电针刺激,1次/d、30 min/次,连续治疗35 d;药物组予利鲁唑4 mg/kg灌胃,1次/d,连续给药35 d。用旷场实验和糖水偏好实验评价大鼠行为学,用Western印迹技术从蛋白水平观察前额皮层星形胶质细胞高亲和性兴奋性氨基酸转运体EAAT1、EAAT2蛋白以及GS的变化,应用RT-PCR技术从mRNA水平检测EAAT1 mRNA、EAAT2mRNA、GS mRNA的变化。结果行为学药物组和电针组均能改善因CUMS造模导致的旷场实验的水平爬格、直立站立次数降低,治疗至第14天时可达正常水平(P0.05);糖水偏好率也于治疗第7天开始得到纠正(P0.05);电针组和药物组对行为学的改善程度相当(P0.05)。行为学的改善与前额皮层GS、EAAT1、EAAT2和EAAT1 mRNA、EAAT2 mRNA、GS mRNA升高相对应。结论电针上调前额皮层星形胶质细胞高亲和性兴奋性氨基酸转运体EAAT1、EAAT2和GS表达,提高谷氨酸摄取功能,从而改善抑郁症状,可能是针刺抗抑郁的机制之一。     

丰富环境对大鼠局灶性脑梗死后微血管新生的影响

目的 研究丰富环境对局灶性脑梗死后大鼠微血管新生的影响.方法 采用开颅电凝法制作SD大鼠右侧大脑中动脉缺血(MCAO)模型,术后24 h随机分为丰富环境组(EE组)和标准环境组(SE组).另设假手术组(Sham组).免疫组织化学法检测大鼠颅脑血管内皮生长因子(VEGF)、Ⅷ因子表达,测定微血管密度.结果 EE组和SE组大鼠大脑中动脉栓塞后,缺血区神经元变性、坏死,术后1 dVEGF在缺血周边区开始表达,3 d达高峰,EE组大鼠经干预后后期VEGF表达和微血管数目高于SE组大鼠.结论 丰富环境可促进脑梗死大鼠VEGF、Ⅷ因子表达,促进微血管新生,有利于脑损伤修复.     

HIF-1α及其抑制剂对新生大鼠缺氧缺血性脑损伤时神经细胞凋亡的影响

目的 探讨缺氧诱导因子-1α(HIF-1α) 及其抑制剂2-甲氧基雌二醇(2ME2)对新生大鼠缺氧缺血性脑损伤时神经细胞凋亡的影响.方法 将120只新生7 d龄Wistar大鼠随机分为假手术组(Sham组,n=8)、缺氧缺血模型组(HIBD组,n=56)、2ME2干预组(2ME2组,n=56),后两组采用Rice法建立模型后,根据断头取脑的时间不同又分为3 h、6 h、12 h、24 h、48 h、72 h和7 d7个亚组.应用HE染色观察脑组织的病理变化,免疫组化技术检测HIF-1α、Bax、Bcl-2的表达,TUNEL法计数脑细胞凋亡.结果 HE染色显示2ME2干预后脑组织的损伤减轻;免疫组化显示:HIF-1α在HIBD组于模型制备后3 h升高,12 h达高峰,之后下降,2ME2组表达趋势和HIBD组相同,表达水平显著下降.与HIBD组比较,2ME2组各时间点的Bcl-2表达显著升高,Bax表达显著降低,TUNEL标记的阳性细胞数明显减少.结论 在新生大鼠缺氧缺血急性期使用2ME2抑制HIF-1α的表达,引起Bax/ Bcl-2表达量比值降低,凋亡细胞数目减少,改善脑损伤.     

缺血缺氧性脑损伤对新生鼠脑内神经干细胞的影响

目的观察新生鼠缺血缺氧性损伤后皮层及海马神经干细胞的变化。方法采用新生7d龄SD大鼠制作新生鼠缺血缺氧性脑病模型（HIE），行免疫组化染色，巢蛋白（Nestin）标记神经干细胞。结果正常SD大鼠新生早期的皮层及海马存在一定数量的Nestin阳性细胞，随年龄增加而减少；缺血缺氧后．新生SD大鼠皮层损伤区及其周围、对侧镜区和双侧海马都出现Nestin阳性细胞增多，并以病灶边缘曼加突出，在皮层伤后3d即出现高峰，而海马的高峰期在伤后7d，然后都逐渐减少，至伤后28d均与正常组无差别。结论缺血缺氧能促使新生SD大鼠皮层及海马的神经干细胞增多。     

Development of brain damage after neonatal hypoxia-ischemia: Excitatory amino acids and cysteine

The aim of this study was to investigate the possible role of excitatory amino acids (EAAs) and cysteine in the development of brain damage after hypoxia-ischemia (HI) in neonates. In a rat model of neonatal HI, changes in extracellular (ec) amino acids in cerebral cortex were measured with microdialysis and correlated with the extent of brain damage at the site of probe placement. Extracellular concentrations of glutamate, aspartate and cysteine increased during HI and remained elevated during reperfusion. During HI the pattern of EAA changes was the same in the infarcted, undamaged and border zone regions. During reperfusion, however, the ec concentrations of glutamate, aspartate and cysteine were higher in infarcted and border zone areas compared to undamaged tissue. HI also produced a slight increase of tissue concentration of cysteine and decrease of tissue concentration of glutamate in parietal cortex of the HI hemisphere. The effect of cysteine on brain damage induced by HI and glutamate was also investigated. A subtoxic dose of cysteine potentiated glutamate toxicity in the arcuate nucleus and enhanced brain infarction after HI in neonatal rats. The results show that in neonatal HI the extracellular levels of EAAs during HI are not directly related to brain injury but the EAA levels during reflow predict the extent of infarction. Cysteine increases HI-induced brain injury and potentiates glutamate toxicity in neonatal rats. Speculatively, elevated level of cysteine during reperfusion may participate in the excitotoxic cascade leading to brain injury.     

Effects of progesterone on the neonatal brain following hypoxia-ischemia

Progesterone displays a strong potential for the treatment of neonatal hypoxic-ischemic encephalopathy since it has been shown to be beneficial in the treatment of the central nervous system injuries in adult animals. Here, we evaluated the effects of the administration of progesterone (10 mg/kg) in seven-days-old male Wistar rats submitted to neonatal hypoxia-ischemia (HI). Progesterone was administered immediately before ischemia and/or 6 and 24 h after the onset of hypoxia. The body weight of the animals, the volume of brain lesion and the expression of p-Akt and procaspase-3 in the hippocampus were evaluated. All animals submitted to HI showed a reduction in the body weight. However, this reduction was more remarkable in those animals which received progesterone before surgery. Administration of progesterone was unable to reduce the volume of brain damage caused by HI. Moreover, no significant differences were observed in the expression of p-Akt and procaspase-3 in animals submitted to HI and treated with either progesterone or vehicle. In summary, progesterone did not show a neuroprotective effect on the volume of brain lesion in neonatal rats submitted to hypoxia-ischemia. Furthermore, progesterone was unable to modulate p-Akt and procaspase-3 signaling pathways, which may explain the absence of neuroprotection. On the other hand, it seems that administration of progesterone before ischemia exerts some systemic effect, leading to a remarkable reduction in the body weight.     

Melatonin protects from the long-term consequences of a neonatal hypoxic-ischemic brain injury in rats

Abstract:  Among the main factors responsible for perinatal brain injury, inflammation, hypoxia-ischemia and formation of free radicals (FR) appear to play key roles. Melatonin, an endogenously produced indoleamine formed in higher amounts in adults than in neonates, is a potent FR scavenger as well as an indirect antioxidant. Herein, we examined whether melatonin provides significant protection against brain damage and its long-term consequences in a neonatal model of hypoxia-ischemia (HI). Seven day-old rats were subjected to permanent legation of the right common carotid artery followed to 2.5 hrs hypoxia 3 hrs later (HI). The neuroprotective effect of melatonin was evaluated 7 days after HI, or when rats reached adulthood, using behavioral and histological analyses. A beneficial effect was observed with 5 mg/kg melatonin administered before HI. The same dose repeated three times reduced further injury. A significant protective effect was found when 15 mg/kg melatonin was given 30 min before HI or when the same dose was given after HI and administration repeated after 24 and 48 hrs. The latter schedule of administration was used to assess the long-term protective effects. Melatonin did not affect growth rate and behavior at adulthood, but significantly improved the behavioral asymmetry and learning deficits induced by HI. Consistently, brain injury was significantly attenuated in the melatonin-treated ischemic group. The present study demonstrates that melatonin administration before or after HI in immature rats has an excellent and long-lasting benefit on ischemic outcomes suggesting that the drug could represent a potentially safe approach to perinatal brain damage in humans.     

Melatonin reduces endoplasmic reticulum stress and preserves sirtuin 1 expression in neuronal cells of newborn rats after hypoxia–ischemia

Conditions that interfere with the endoplasmic reticulum (ER) functions cause accumulation of unfolded proteins in the ER lumen, referred to as ER stress, and activate a homeostatic signaling network known as unfolded protein response (UPR). We have previously shown that in neonatal rats subjected to hypoxia–ischemia (HI), melatonin administration significantly reduces brain damage. This study assessed whether attenuation of ER stress is involved in the neuroprotective effect of melatonin after neonatal HI. We found that the UPR was strongly activated after HI. Melatonin significantly reduced the neuron splicing of XBP‐1 mRNA, the increased phosphorylation of eIF2α, and elevated expression of chaperone proteins GRP78 and Hsp70 observed after HI in the brain. CHOP, which plays a convergent role in the UPR, was reduced as well. Melatonin also completely prevented the depletion of SIRT‐1 induced by HI, and this effect was observed in the same neurons that over‐express CHOP. These results demonstrate that melatonin reduces ER stress induced by neonatal HI and preserves SIRT‐1 expression, suggesting that SIRT‐1, due to its action in the modulation of a wide variety of signaling pathways involved in neuroprotection, may play a key role in the reduction of ER stress and neuroprotection observed after melatonin.     

Inhibition of the activity of excitatory amino acid transporter 4 expressed in Xenopus oocytes after chronic exposure to ethanol

Background: The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3K) in these effects. Methods: EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol‐containing solution for 24 to 96 hours. Membrane currents induced by l ‐aspartate were recorded using 2‐electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (μC). Results: Ethanol dose‐ and time‐dependently reduced EAAT4 activity. EAAT4 activity after a 96‐hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 ± 0.2 μC for control vs. 1.6 ± 0.2 μC for ethanol, n = 18, p < 0.05) of EAAT4 for l ‐aspartate. Preincubation of ethanol‐treated (50 mM for 96 hours) oocytes with phorbol‐12‐myrisate‐13‐acetate (100 nM for 10 minutes) abolished the ethanol‐induced decrease in EAAT4 activity. While staurosporine (2 μM for 1 hour) or chelerythrine (100 μM for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 μM for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups. Conclusions: The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri‐Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion.     

EPO对新生大鼠缺氧缺血性脑损伤时脑组织Caspase-9和Caspase-3的影响

目的 观察新生大鼠缺氧缺血性脑损伤(HIBD)时脑组织Caspase-9和Caspase-3的表达变化及促红细胞细胞生成素(EPO)对其表达的影响,从而探讨EPO发挥脑保护作用的可能机制.方法 将新生7 d SD大鼠120只随机分成3组,假手术组、HIBD组、rhEPO治疗组,每组根据不同时间点又分为5个亚组:6 h组、12 h组、24 h组、48 h组、72 h组,每组8只,用免疫组化的方法观察各组脑组织Caspase-9和Caspase-3的表达.结果 Caspase-9的表达在缺氧缺血6 h即增强,12 h时逐渐升高,24 h～72 h维持在高峰水平(P<0.01);Caspase-3的表达也在缺氧缺血6 h即增强,12 h时逐渐升高,24 h～48 h达高峰,72 h稍有降低(P<0.01);rhEPO治疗组各时间点Caspase-9和Caspase-3的表达水平较HIBD组均明显降低(P<0.01).结论 Caspase-9和Caspase-3参与了新生大鼠脑组织HIBD的发生发展过程,EPO可能通过降低新生大鼠缺氧缺血性脑损伤时脑组织Caspase-9和Caspase-3的表达发挥其脑保护作用.     

Possible expression of functional glutamate transporters in the rat testis

Neither expression nor functionality is clear in peripheral tissues with the molecular machineries required for excitatory neurotransmitter signaling by L-glutamate (Glu) in the central nervous system, while a recent study has shown that several Glu receptors are functionally expressed in the rat testis. This fact prompted us to explore the possible functional expression in the rat testis of the Glu transporters usually responsible for the regulation of extracellular Glu concentrations in the brain. RT-PCR revealed the expression, in the rat testis, of mRNA for five different subtypes of Glu transporters, in addition to that for particular subtypes of ionotropic and metabotropic Glu receptors. Glutamate transporter-1 (GLT-1) was different in the brain from that in the testis in terms of molecular sizes on Northern and Western blot analyses. In situ hybridization as well as immunohistochemical analysis showed localized expression of glutamate aspartate transporter at interstitial spaces and GLT-1 at elongated spermatids in the rat testis respectively. The expression of mRNA was localized for excitatory amino acid transporter-5 at the basal compartment of the seminiferous tubule in the rat testis. [(3)H]Glu was accumulated in testicular crude mitochondrial fractions in a temperature- and sodium-dependent saturable manner with pharmacological profiles similar to those shown in brain crude mitochondrial fractions. These results suggested that particular subtypes of central Glu transporters for the regulation of extracellular Glu concentrations in the rat testis could be constitutively and functionally expressed.