Expression of apoptosis protein caspase 3 in developing mouse cerebellum

目的:观察C57/BL6小鼠小脑发生发育过程中的凋亡蛋白caspase 3表达变化,探讨小脑发生发育的可能机制.方法:应用免疫组织化学技术、免疫印迹对胚龄(E)12、14、16、18、20 d和生后(P)1、3、7、14、21、28 d的仔鼠及生后2、3、6、15个月的C57/BL6小鼠小脑的形态变化及caspase 3的分布进行系统观察和定量分析.结果:免疫组织化学结果显示,E12 d～P21 d,caspase 3在小鼠小脑中均有阳性表达,主要分布在神经元的细胞质和细胞核中.E12 d～P7d,caspase 3表达逐渐增多,P14 d～P21 d逐渐减少.免疫印迹分析表明,E18 d～3个月,caspase 3蛋白表达先逐渐增加后逐渐减少,P7d达高峰,3个月后维持在较低水平.结论:小鼠小脑的片层化结构主要形成于胚胎期,出生后片层化结构逐渐发育成熟,小脑得以迅速发育长大.此外,在小脑发育中存在着大量的细胞凋亡现象,其可能在小脑的塑形中发挥重要的作用.     

海洛因成瘾、戒断、脱毒对大鼠前额叶皮质GDNF和p75NTR表达的影响

目的:研究大鼠海洛因成瘾、戒断、脱毒治疗期间,前额叶皮质(PFC)脑区中胶质细胞源性神经营养因子(GDNF)和神经营养因子受体p75(p75NTR)的表达变化。方法:正常雄性SD大鼠32只,随机分为海洛因处理组(n=24)和对照组(n=8),海洛因处理组又分为海洛因成瘾组(HDG)、纳洛酮戒断组(HWG)、美沙酮脱毒治疗组(MDG)。各组大鼠取PFC脑区,采用免疫组织化学技术、Western Blot检测及图像分析方法研究GDNF和p75NTR阳性细胞的反应和蛋白表达情况。结果:(1)免疫组化结果显示:与对照组相比较,成瘾组中GDNF、p75NTR阳性细胞免疫反应明显减弱,平均光密度明显降低(P 0. 05),纳洛酮戒断之后GDNF、p75NTR阳性细胞免疫反应强度显著增强,平均光密度增强(P 0. 05)。(2) Western Blot结果显示:GDNF、p75NTR蛋白表达水平在海洛因成瘾大鼠PFC脑区显著降低(P 0. 05);经纳洛酮戒断和美沙酮脱毒治疗后GDNF、p75NTR蛋白表达水明显有所升高(P 0. 05)。结论:在海洛因成瘾、戒断、脱毒过程当中,GDNF和p75NTR的变化趋势大致相同,二者均有负性调节作用。     

孕期酒精暴露诱导神经鞘磷脂合成酶2基因敲除小鼠海马苔藓细胞凋亡

目的 探讨神经酰胺对孕期酒精暴露所诱导的小鼠神经细胞凋亡的影响.方法 建立神经鞘磷脂合成酶2基因敲除(SMS-/-2)小鼠和野生型(WT)小鼠的孕期酒精暴露模型,将出生后的不同基因型仔鼠(共360只)分为对照组和酒精组.用酶学法检测生后0 d(P0)仔鼠血清神经鞘磷脂(SM)含量,利用免疫荧光染色法观察对照组与模型组各年龄点仔鼠齿状回苔藓细胞凋亡数量的变化,免疫印迹法检测P7、P14仔鼠海马组织Caspase-8、Caspase-3激活蛋白的相对表达量.结果 酒精暴露后仔鼠血清SM水平降低,且具有剂量依赖性(F=41.08,P<0.05);SMS-/-2仔鼠血清SM水平低于同组WT仔鼠(F=53.34,P<0.01).酒精诱导WT和SMS-/-2仔鼠苔藓细胞凋亡(F=15.61,P<0.05),有剂量依赖性和长时程效应,与同年龄、相同处理条件的WT仔鼠相比,SMS-/-2仔鼠苔藓细胞凋亡数量较多(F=11.72,P<0.05).Western blotting检测结果 与免疫荧光结果 一致.结论 SMS2基因缺失使血清SM水平降低,可引起神经酰胺在体内蓄积;神经酰胺促进酒精暴露诱导神经细胞凋亡的过程;孕期酒精暴露主要通过死亡受体途径诱导神经细胞凋亡的发生.     

孕期DEHP暴露对新生大鼠海马神经元凋亡的影响

目的:探讨孕鼠DEHP暴露对新生小鼠海马神经元凋亡的影响。方法:SD雌性孕鼠24只,随机分为对照组(Control)、DEHP低剂量组(DEHP-L)、DEHP中剂量组(DEHP-M)和DEHP高剂量组(DEHP-H),通过灌胃方法给予孕鼠不同剂量DEHP进行染毒,通过测量新生大鼠的体长、尾长和体重,观察孕期DEHP暴露对新生大鼠发育的影响;通过尼氏(Nissl)染色观察仔鼠海马神经元形态和排列方式的变化,通过TUNEL检测新生大鼠海马部位细胞凋亡,通过Western Blot检测新生大鼠海马组织active caspase-3、Bcl-2和Bax蛋白的表达。结果:与对照组相比,DEHP暴露可导致新生大鼠发育迟缓,海马神经元数量稀少、结构排列紊乱; TUNEL染色检测显示DEHP可导致仔鼠海马细胞凋亡增多(P 0. 05); Western Blot检测结果显示DEHP暴露组新生大鼠海马组织active caspase-3和Bax表达增加,Bcl-2表达下降(P 0. 05),Bax/Bcl-2比值随着DEHP浓度增加而增加。结论:孕期DEHP暴露会影响子代大鼠发育迟缓、海马细胞凋亡增加。     

电针对海洛因成瘾大鼠PAG内细胞凋亡和caspase-3表达的影响

目的:研究电针对海洛因成瘾大鼠中脑导水管周围灰质(periaqueductal gray,PAG)的细胞凋亡及caspase-3表达的影响。方法:大鼠被随机分为四组:正常对照组、海洛因成瘾组(成瘾组)、海洛因戒断组(戒断组)和海洛因戒断针刺组(针刺组)。按给药剂量逐日递增的原则皮下注射海洛因建立成瘾模型;针刺组大鼠成瘾后用韩氏穴位神经刺激器(HNAS)针刺双侧"足三里"和"三阴交"穴位,30min,每日1次,连续5d;免疫组织化学染色法测caspase-3的表达;流式细胞仪测细胞凋亡情况。结果:(1)与正常组相比,成瘾组、戒断组和针刺组大鼠PAG内的细胞凋亡和caspase-3的表达均增多(P0.05);(2)针刺组与成瘾组和戒断组相比,针刺组PAG内的细胞凋亡和caspase-3的表达均减少(P0.05)。结论:(1)海洛因成瘾导致大鼠PAG内细胞凋亡和caspase-3的表达增多;(2)HANS针刺对海洛因成瘾引起的大鼠细胞凋亡有抑制作用,并下调大鼠PAG内caspase-3的表达。     

长期甲醛暴露对幼年小鼠学习记忆能力的影响

目的:研究长期甲醛暴露对幼年小鼠学习记忆能力的影响及分子机制。方法:将72只幼年C57BL/6小鼠分为对照组(control)、低浓度甲醛暴露组(FA-L)、中浓度甲醛暴露组(FA-M)和高浓度甲醛暴露组(FAH)。小鼠进行为期30 d不同浓度的甲醛暴露,称量小鼠体重变化,然后通过Morris水迷宫实验检测各组小鼠学习记忆能力,利用Nissl染色观察小鼠海马神经元结构变化,利用商品化试剂盒检测海马组织中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,利用Western Blot检测active caspase-3和Bcl-2等分子的表达变化。结果:与对照组小鼠相比,甲醛暴露小鼠体重增长缓慢,学习记忆能力下降,海马神经元数量减少,海马组织中SOD活性下降,MDA生成增加,active caspase-3水平增加,同时Bcl-2表达下降。结论:长期甲醛暴露可导致小鼠学习记忆能力下降,其作用机制与氧化应激反应增加并导致海马细胞凋亡有关。     

红景天苷对缺氧缺血性脑损伤大鼠海马神经元自噬的影响

目的:探讨红景天苷(Sal)对缺氧-缺血(H/I)大鼠海马神经元自噬的影响。方法:36只成年雄性SD大鼠分为假手术组(Sham)、H/I组(H/I)、H/I联合Sal治疗组(H/I+Sal),利用双侧颈总动脉结扎及低氧处理制备H/I模型,通过腹腔注射给予大鼠Sal治疗3 d,Morris水迷宫实验检测大鼠学习记忆能力,TUNEL染色检测海马神经元凋亡,Western Blot检测微管相关蛋白1轻链3(LC3)的表达。结果:与sham组相比,H/I大鼠学习记忆能力受损明显(P 0. 05),海马细胞凋亡增加明显(P 0. 05),LC3-II/LC3-I比值增加(P 0. 05);经过Sal治疗后,与H/I大鼠相比,Sal治疗组大鼠学习记忆功能得到明显改善(P 0. 05),凋亡细胞减少(P 0. 05),LC3-II/LC3-I比值降低(P 0. 05)。结论:Sal能够通过抑制H/I模型大鼠海马神经元自噬减轻凋亡,发挥神经保护作用。     

红景天苷对中波紫外线诱导HaCaT细胞凋亡的影响

目的:探讨红景天苷对中波紫外线(UVB)诱导HaCaT细胞凋亡的影响及可能的分子机制。方法:将HaCaT细胞分为正常对照组、红景天苷处理组、UVB组和UVB+红景天苷组,先用UVB照射细胞,再用红景天苷处理HaCaT细胞24h,Real-time PCR和免疫印迹分别检测caspase 3、caspase 12、前细胞凋亡因子(CHOP)的mRNA及蛋白表达;MTT检测细胞增殖,流式细胞术检测细胞凋亡。结果:UVB处理组caspase 3、caspase 12、CHOP的mRNA和蛋白表达明显增加,红景天苷处理后明显降低。UVB+红景天苷组较UVB组细胞增殖显著增加,凋亡率减少23.22%。结论:红景天苷可通过抑制caspase 12、CHOP的表达,减少UVB引起的HaCaT细胞凋亡。     

注射海洛因、麻黄素对仔鼠大脑颞叶皮层结构及学习记忆能力的影响

目的 探讨海洛因和麻黄素对仔鼠大脑颞叶皮层结构及学习记忆能力的影响. 方法 108只仔鼠采用递增剂量腹腔连续注射海洛因和麻黄素,通过食饵迷宫观察仔鼠行为学变化,在光学显微镜和透射电镜下观察大脑颞叶皮层细胞结构的变化;用免疫组织化学方法观察Bax蛋白和角质细胞生长因子(KGF)在大脑颞叶皮层的表达;采用比色法检测大脑颞叶皮层胆碱乙酰基转移酶(ChAT)活性的变化. 结果 除麻黄素组5d时与对照组差异不显著(P>0.05)外,海洛因和麻黄素组各期仔鼠食饵迷宫试验的错误次数及总测试时间均高于对照组,差异显著或极显著(P<0.05或P<0.01);海洛因组仔鼠食饵迷宫的上述两项指标均高于麻黄素组,差异显著或极显著(P<0.05或P<0.01).注射海洛因、麻黄素5d、10 d、15 d、20d,仔鼠大脑颞叶皮层锥体神经元树突、轴突萎缩,胞体缩小,神经元核膜失去正常圆形轮廓,胞内细胞器结构不正常或空泡化;毛细血管周围基质溶解破坏,管腔狭窄;突触小泡减少;大脑颞叶皮层凋亡细胞、坏死细胞及Bax蛋白和KGF阳性表达细胞的数量均高于对照组,ChAT的活性低于对照组,差异显著或极显著(P<0.05或P<0.01);海洛因组仔鼠大脑颞叶皮层凋亡细胞、坏死细胞、Bax蛋白和KGF阳性表达细胞的数量及ChAT的活性与麻黄素组相比,差异显著或极显著(P<0.05或P<0.01).随着海洛因和麻黄素剂量的递增,仔鼠食饵迷宫试验的错误次数,大脑颞叶皮层凋亡细胞、坏死细胞及Bax蛋白和KGF阳性表达细胞的数量呈增多趋势. 结论海洛因和麻黄素显著影响仔鼠的学习记忆能力,其可能机制与大脑颞叶皮层结构受损及ChAT活性下降有一定的相关性.     

碱性成纤维细胞生长因子对小鼠创伤性脑损伤后神经元凋亡及星形胶质细胞活化的影响

目的 观察碱性成纤维细胞生长因子(bFGF)对小鼠脑损伤后伤侧皮质和海马神经元凋亡及星形胶质细胞活化的影响. 方法 36只小鼠随机分为对照组、生理盐水组、bFGF组,每组各12只.采用自由落体打击装置建立脑损伤模型,分别于建模后3d和7d取脑(每时相点6只),应用免疫荧光双标和免疫印迹法检测大脑皮质、海马神经细胞凋亡因子caspase-3的变化,以及大脑皮质中胶质纤维酸性蛋白(GFAP)的表达. 结果 bFGF组皮质和海马中caspase-3的表达比生理盐水组和对照组减少(P<0.05);bFGF组皮质中GFAP表达比生理盐水组和对照组增加(P<0.05);生理盐水组与对照组的caspase-3及GFAP表达差异无统计学意义(P>0.05). 结论 bFGF能降低小鼠脑损伤后大脑皮质和海马caspase-3表达并增高大脑皮质GFAP表达,从而促进大脑皮质星形胶质细胞活化和抑制神经细胞凋亡.     

Cerebellar neuronal apoptosis in heroin-addicted rats and its molecular mechanism

Background: The overall objective of this study was to investigate neuronal apoptosis and expression of apoptosis related proteins (c-jun, cytc and Bax) in the cerebellum of rates with heroin addiction. Material/Methods: 40 adult male Sprague-Dawley rats which weighing 200-220 g were randomly divided into 5 groups (n = 8 per group): control group, 10-day heroin-addicted group, 20-day heroin-addicted group, 30-day heroin-addicted group and 40-day heroin-addicted group. Rats in the control group were treated with normal saline. Rats in the addiction groups (20 d, 30 d, 40 d) were all given subcutaneous injection with heroin for 15 days to induce heroin addiction. After injected with heroin for 15 days, rats were treated with naloxone at a dose of 5 mg/kg to induce abstinence for 30 mins to examine the addiction of rats. They were then continued to be treated with heroin for another 10 days, 20 days, 30 days, and 40 days respectively to establish heroin-addicted models. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was employed to identify apoptotic cells [6]. Immunohistochemistry and Western blot assay were also used in the study to examine the protein expressions of c-jun, cytc and Bax in the cerebellum. Results: Compared with the control group, the proportion of apoptotic neurons increased significantly in the heroin addiction groups (10 d, 20 d, 30 d, 40 d) (P < 0.05), also accompanied by markedly increased expressions of c-jun, cytc and Bax (P < 0.05) depending on doses of heroin in the cerebellum. Thus, the significant differences were observed in heroin addiction groups (10 d, 20 d,30 d, 40 d) and control group (P < 0.05). Conclusion: Long-term use of heroin may induce neuronal apoptosis in the cerebellum by raising the expressions of pro-apoptotic c-jun, cytc and Bax, which might be one of mechanisms underlying the heroin-induced cerebellum neuronal damage.     

The activation of caspase-3 and DNA fragmentation in B cells phagocytosed by macrophages

Apoptotic signaling of mammalian cells involves two pathways: the death receptor and mitochondrial pathways. In this in vivo study, we investigated apoptotic signaling of B cells in mouse germinal centers (GCs) of gut-associated lymphoid tissues (GALTs) using transmission electron microscopy (TEM), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL), immunofluorescence of members of caspase family and cFLIP(L), and caspase activity assay. It was very difficult to ultrastructurally differentiate B cells undergoing apoptosis from B cells differentiating into memory cells or plasma cells among B cells constituting GCs. Isolated B cells in GCs showed no active form of caspase-3 or TUNEL immunoreactivity, but expressed cFLIP(L). Contrary to isolated B cells, apoptotic B cells phagocytosed by macrophages exhibited immunoreactivity of the active form of caspase-3 and TUNEL, but lacked the cFLIP(L) expression. The caspase activity assay in GALTs clearly showed intense activity of caspase-3, caspase-9, and caspace-8 that was high in order. Therefore, the death receptor pathway accompanying the increased activity of caspase-3 and caspase-8 may be blocked by the expression of cFLIP(L) in B cells of GALTs. Moreover, both the activation of caspase-3 and DNA fragmentation first occur only when B cells are phagocytosed by macrophages.     

组蛋白乙酰化酶p300和CREB结合蛋白在小鼠胚胎心发育中的时序表达

目的:探讨p300和CREB结合蛋白(CBP)在小鼠胚胎心发育过程中的时序表达规律.方法:选取胎龄7.5～18 d、出生1 d、3月小鼠正常心,采用免疫组织化学法观察p300和CBP在小鼠胎心发育中的表达分布及变化规律.结果:p300在胚胎心发育各个时期及出生1 d和3月成年鼠的心各部分均有很强表达.CBP在E7.5不表达;在E8.5～E9.5的心管中较强表达;E10.5～E16.5及E18和生后1 d心肌较强表达,房室瓣和小梁网较弱表达,室间隔肌部弱表达而膜部几乎不表达;3月成年鼠心肌和小梁较弱表达,室间隔极弱表达.结论:p300对发育早期的生心细胞诱导特化和中期的室间隔形成作用较CBP更大.     

Pre-administration of luteoline attenuates neonatal sevoflurane-induced neurotoxicity in mice

Sevoflurane is a widely used inhaled anesthetic, which triggers neuroapoptosis and oxidative damage in the developing central nervous system and cognitive dysfunction later in life. However, no effective therapeutic strategy for sevoflurane-induced deleterious effects is well developed. The purpose of the present study was to explore whether luteoline could attenuate neonatal sevoflurane exposure-triggered neurotoxicity. In this study, six-day-old C57BL/6 mice were pretreated with luteoline (30, 60 mg/kg) intraperitoneally for 30 min before exposed to 3% sevoflurane 6 h consecutively. We first examined the effects of luteoline on hippocampal neuron apoptosis, inflammation and oxidative stress 18 h post anesthesia. The spatial learning and memory performance was measured using Morris water maze test from postnatal day 31 to 38. The results showed that luteoline ameliorated neuronal apoptosis as evidenced by decrease of apoptotic cells, downregulation of the cleavage levels of caspase-3 and PRAP, and inactivation of caspase-3. Moreover, luteoline significantly decreased protein expressions of inflammatory cytokines (IL-1β, IL-18 and TNF-α), inhibited NF-кB/NLRP3 pathway (NF-кB, NLRP3, ASC and caspase-1) and suppressed NF-кB activity. Our analyses indicated that luteoline had a significant effect on decreasing the contents of ROS and MDA, elevating the activity of SOD, and ultimately improving spatial learning and memory deficits of mice. In summary, our findings confirm that the attenuation of luteoline on sevoflurane-induced spatial learning and memory impairment later is associated with inhibition of hippocampal neuron apoptosis, inflammation and oxidative stress early. Luteoline might be a potential therapeutic for sevoflurane anesthesia-induced neurobehavioral dysfunction.     

Early life protein restriction alters dopamine circuitry

Adverse prenatal environment, such as intrauterine growth retardation (IUGR), increases the risk for negative neurobehavioral outcomes. IUGR, affecting approximately 10% of all US infants, is a known risk factor for attention deficit hyperactivity disorder (ADHD), schizophrenia spectrum disorders and addiction. Mouse dams were fed a protein deficient (8.5% protein) or isocaloric control (18% protein) diet through pregnancy and lactation (a well validated rodent model of IUGR). Dopamine-related gene expression, dopamine content and behavior were examined in adult offspring. IUGR offspring have six to eightfold over-expression of dopamine (DA)-related genes (tyrosine hydroxylase (TH) and dopamine transporter) in brain regions related to reward processing (ventral tegmental area (VTA), nucleus accumbens, prefrontal cortex (PFC)) and homeostatic control (hypothalamus), as well as increased number of TH-ir neurons in the VTA and increased dopamine in the PFC. Cyclin-dependent kinase inhibitor 1C (Cdkn1c) is critical for dopaminergic neuron development. Methylation of the promoter region of Cdkn1c was decreased by half and there was a resultant two to sevenfold increase in Cdkn1c mRNA expression across brain regions. IUGR animals demonstrated alterations in dopamine-dependent behaviors, including altered reward-processing, hyperactivity and exaggerated locomotor response to cocaine. These data describe significant dopamine-related molecular and behavioral abnormalities in a mouse model of IUGR. This animal model, with both face validity (behavior) and construct validity (link to IUGR and dopamine dysfunction) may prove useful in identifying underlying mechanisms linking IUGR and adverse neurobehavioral outcomes such as ADHD.     

Naloxone-precipitated withdrawal enhances ERK phosphorylation in prefrontal association cortex and accumbens nucleus of morphine-dependent mice

Mitogen-activated protein kinases (MAPK) can be activated by opioids such as morphine via opioid receptor, and their activations have been observed in synaptic plasticity, learning, memory and addiction. Long-term exposure to morphine may induce physical dependence, manifested as somatic withdrawal symptoms such as diarrhea, body weight loss, jumping and headshaking, when drug is deprived. Though morphine dependence and withdrawal have been extensively studied, their molecular mechanisms have not been fully elucidated. In the present study, the physical dependence on morphine was developed in mice by an intermittent, escalating procedure of morphine injections, and was measured by the body weight loss and the behavioral signs (jumping and headshaking). We found that the mice with chronic morphine administration experienced dramatic body weight loss, compared with the saline-treated controls. Naloxone-precipitated withdrawal led to more body weight loss, compared with spontaneous withdrawal. Naloxone-precipitated withdrawal mice showed significantly aggravated morphine-withdrawal symptoms (including jumping and heading shaking), compared with spontaneous withdrawal mice. MAPK pathway activities in the frontal association cortex (FrA), accumbens nucleus (Acb) and caudate putamen (CPu) were examined to probe into molecular mechanism for morphine dependence and withdrawal. Compared with saline-treated mice, morphine-dependent mice and spontaneous withdrawal mice, naloxone-precipitated withdrawal mice showed a significantly increased ERK phosphorylation in FrA and Acb, but not in CPu. However, the activities of other protein kinases in the MAPK pathway, including p38 and JNK, showed no changes in FrA, Acb and CPu of the mice during the chronic morphine dependence and withdrawal phases. These results suggest that the ERK phosphorylation in FrA and Acb may be associated with naloxone-precipitated withdrawal syndrome.     

Early inhibitory learning in the rat. I. Learning and memory development

The present status of knowledge on early inhibitory learning and memory is presented. The inhibitory learning (passive avoidance) can be mastered by rat pups several hours after parturition, provided specific characteristics of the newborn organism and its neurobehavioral capabilities are respected. The curve of numbers of trials to criterion displays, as already reported earlier, a generally decreasing trend which is, however, interrupted by a temporary inversion in both strains studied, but at different time, taking place in Long Evans between postnatal day 1 and 2, which is shifted in Wistar pups one day later. Present data show significant learning differences between strains at postnatal days 1-4; from day 5 onward no significant differences are evidenced up to postnatal day 11. A reliable 24-hour memory shown by positive values of the retention index (RI) is revealed from postnatal day 4, having in Long Evans higher RI values. Moreover, high positive RIs of 24-hour memory are found in animals trained on the day just before the inversion of the learning development. It is conjectured that the indirect development of the early inhibitory learning is due to the developmental reorganization within CNS, and the necessity to find out its underlying mechanisms is emphasized.