大蒜素对全脑缺血再灌注大鼠脑保护机制的研究

目的：通过大蒜素预处理,观察全脑缺血再灌注大鼠海马区ICAM-1 的表达,从而探讨大蒜素的脑保护机制。方法：雄性 Wistar 大鼠30 只,随机分为5 组：假手术组、缺血再灌注组、缺血再灌注+ 大蒜素10、20、30 mg/kg 组。采用四血管闭塞法制备大 鼠全脑缺血再灌注模型,于再灌注24 h 取出海马,硫堇染色观察海马组织的形态学改变,免疫组织化学染色测定海马CA1 区 ICAM-1 免疫反应阳性细胞面积和积分光密度值。结果：通过给予大鼠全脑缺血8 min 再灌注24 h处理,海马CA1 区组织形态学 改变显著,神经元密度明显降低;ICAM-1的表达显著增加。静脉给予大蒜素可使缺血再灌注海马组织形态学改变明显改善,存活 神经元数目增加,ICAM-1 表达显著较少。结论：大蒜素可以通过减少ICAM-1 的表达抑制全脑缺血再灌注后的炎症损失从而发 挥脑保护作用。     

小鼠短暂前脑缺血海马中半胱天冬酶-３酶原表达的变化

通过测定脑缺血再灌注时海马中半胱天冬酶-3酶原(procaspase-3)的表达变化, 从细胞凋亡的角度探讨脑缺血再灌注损伤的分子生物学机制及procaspase-3的活化机制.将C57BL/6N小鼠随机分为假手术组(正常对照组)、缺血再灌注组(I/R组), 后者夹闭双侧颈总动脉20 min后再通血流, 建立前脑缺血再灌注模型, 分别于再灌注6 h、12 h、24 h和48 h取海马.采用蛋白免疫印迹(Western blotting)方法检测海马中procaspase-3的表达变化.结果显示, 12 h I/R及24hI/R组海马中总procaspase-3水平与假手术组相比有明显升高, 且差异有统计学意义(P<0.05),24 h I/R组海马中去磷酸化水平与假手术组相比有明显升高, 且差异有统计学意义(P<0.05),而各组procaspase-3磷酸化水平与假手术组相比差异无统计学意义.结果提示, 脑缺血再灌注损伤诱发procaspase-3表达增加,其中procaspase-3去磷酸化水平高明显, 提示脑缺血再灌注损伤可能诱发procaspase-3去磷酸化, 继而促进procaspase-3转化为活性形式.     

大鼠局灶性脑缺血再灌注损伤中iNOS在大脑皮层和海马的表达

目的研究局灶性脑缺血再灌注损伤中iNOS在不同脑区的表达.方法用改良的血管内栓线技术制造大鼠局灶性脑缺血与再灌注模型,应用免疫组织化学技术检测脑组织中的iNOS的表达.结果 (1)脑缺血再灌注损伤24h后,缺血组缺血侧大脑皮层、海马CA1区、CA3区神经元iNOS的表达显著增强,与正常对照组比较有显著性差异(P<0.05);(2)脑缺血再灌注损伤24h后,缺血组对照侧大脑皮层、海马CA1区、CA3区神经元iNOS的表达也明显增强,与正常对照组比较有显著性差异(P<0.05);(3) 与对照侧比较,脑缺血再灌注大鼠缺血侧皮质的iNOS表达显著增强(P<0.05),而海马CA1区、CA3区缺血侧的iNOS表达与对照侧相比无显著性差异(P>0.05).结论局灶性脑缺血再灌注损伤后,缺血侧皮层和海马iNOS表达显著升高,未缺血脑区(对照侧)iNOS反应性也较对照组者升高.     

局部脑缺血再灌注损伤小鼠脑组织TMEM166的表达及其与脑细胞凋亡的关系

目的:观察跨膜蛋白166(Transmembrane protein 166,TMEM166)基因在小鼠局部脑缺血再灌注损伤(MCAO)后的表达改变及其对脑细胞凋亡的影响。方法:C57/BL6J雄性小鼠50只,体重22-28 g,采用线栓法制作MCAO模型,缺血后动物随机分为再灌注6、12、24、48 h组。采用免疫组化染色的方法观察缺血侧大脑TMEM166、Caspase 3的阳性细胞数目,TUNEL标记法检测细胞凋亡情况,Western blot检测不同再灌注时间点TMEM166、Caspase 3蛋白水平的表达。结果:缺血侧TMEM166的表达随着再灌注时间的延长而增加,24 h达到高峰,48 h后逐渐下降;再灌注6 h后Caspase 3观察到有表达,同样随着再灌注时间而升高,24 h达到高峰。缺血侧细胞凋亡数量变化趋势与TMEM166基本一致,对侧大脑半球上述指标无明显变化。结论:小鼠局部脑缺血再灌注损伤时伴有TMEM166的表达升高,可能通过激活Caspase 3引起脑细胞凋亡。     

MK801对大鼠脑缺血再灌注后海马CA1区c-fos基因表达及神经元凋亡的影响

目的:研究MK801对大鼠脑缺血再灌注后海马CA1区c-fos基因表达及神经元凋亡的影响。方法:将大鼠随机分为正常对照组、脑缺血再灌注模型组和脑缺血用药后再灌注组,用药组在再灌注前经腹腔注射MK801(0.5mg/kg);分别于再灌注后2、6、24和48 h,采用免疫组化法和Western印迹观察海马CA1区c-fos基因的表达情况。结果:c-fos基因的表达在脑缺血再灌注后2 h即开始增强,至再灌注后6 h达到高峰,24 h表达降低,至48 h又至高峰,但较再灌注6 h后较低。用药组海马CA1区c-fos基因的表达均较模型组的相应时间段降低,具有显著性差异(P<0.01)。结论:海马CA1区c-fos基因在脑缺血再灌注后表达升高,MK801可降低脑缺血再灌注后c-fos基因的表达,对缺血再灌注后的脑组织具有保护作用。     

肢体缺血-再灌注大鼠脑组织iNOS基因表达的半定量检测

目的检测大鼠肢体缺血-再灌注后脑组织iNOS基因表达的变化.方法SD大鼠,随机分为肢体缺血-再灌注(I-R)组、肢体单纯缺血(I)组及正常对照(N)组,通过夹闭腹主动脉末端4?h,或/和开放2～24?h,复制I、I-R组动物模型,半定量RT-PCR方法检测脑组织iNOSmRNA表达的变化,免疫组化染色法观测脑组织内iNOS及过氧亚硝基阴离子(ONOO-)的硝基化产物硝基酪氨酸(NT)的生成与分布.结果N组脑组织iNOSmRNA未检出,I组及I-R组脑组织iNOSmRNA均有表达,再灌注2?h,iNOSmRNA表达量显著升高(P<0.01,vsN组),再灌注6?h达到高峰,随后下降,24?h仍有少量表达;I及I-R组脑组织均有iNOS阳性细胞,I-R组见于大脑皮层各区、海马、尾状核,I组仅见于皮层后肢区及尾状核.I-R组脑组织可见弥散分布的NT阳性神经元,I组偶见NT阳性神经元.结论大鼠肢体缺血-再灌后脑组织iNOS基因表达显著增强,且有时相变化特征.     

异丙酚对全脑缺血/再灌注大鼠海马iNOS表达的影响

目的观察异丙酚对全脑缺血/再灌注大鼠海马神经元诱导型一氧化氮合酶(iNOS)表达的影响,探讨异丙酚对迟发性脑神经元损伤保护作用机制。方法采用Pulsinelli-Brierley四血管阻断法制备全脑缺血模型。全脑缺血20min再灌注24h后断头取脑,采用Western blot方法检测大鼠海马iNOS的蛋白表达。结果与缺血/再灌注组相比较,异丙酚处理组大鼠海马iNOS蛋白表达明显降低,存活的神经元数目明显增加,统计结果差异均有显著性(P<0.05或0.01)。结论异丙酚通过抑制iNOS蛋白表达对大鼠脑迟发性神经元损伤起保护作用。     

姜黄素对自发性高血压大鼠脑缺血/再灌注后海马神经元损伤及RANTES表达的影响

目的：探讨姜黄素对自发性高血压大鼠（SHR）脑缺血/再灌注后认知功能及海马神经元损伤和调解活化正常T细胞表达和分泌的趋化因子（RANTES）表达的影响。方法：雄性Wistar-Kyoto大鼠（WKY）和SHR,随机分为5组：假手术组（W-Sham、S-Sham）、缺血/再灌注组（W-I/R、S-I/R）和姜黄素组（S-Cur）,各组按再灌注时间分为3h、12 h、1 d、3 d、7 d 5个亚组（n=6）。采用四血管阻断法制备全脑缺血/再灌注模型,HE染色观察海马CA1区神经细胞形态,Nissl染色计数海马CA1区平均锥体细胞密度,ELISA法检测海马RANTES表达,于再灌注后7 d观察行为学。结果：与假手术组大鼠比较,缺血/再灌注组大鼠学习和记忆能力下降,海马CA1区神经元损伤加重,海马RANTES蛋白表达上调（P〈0.05）;与W-I/R大鼠比较,S-I/R大鼠学习和记忆能力下降,海马CA1区神经元损伤加重,海马RANTES蛋白表达上调（P〈0.05）;姜黄素组大鼠学习和记忆能力明显改善,海马CA1区神经元损伤减轻,海马RANTES蛋白表达下调（P〈0.05）。结论：缺血/再灌注更易导致SHR海马神经元损伤。姜黄素减轻SHR脑缺血/再灌注海马神经元损伤,其机制可能与抑制RANTES蛋白的表达有关。     

姜黄素对自发性高血压大鼠海马缺血/再灌注损伤神经元凋亡核通路的影响

目的:探讨自发性高血压大鼠(SHR)脑缺血/再灌注损伤海马神经元凋亡c-Jun氨基末端激酶(JNK)核通路的变化特点,以及姜黄素对其保护作用可能机制。方法:雄性Wistar-Kyoto大鼠(WKY)和SHR,随机分为5组:WKY假手术组(W-Sham组)、缺血/再灌注组(W-I/R组)和SHR假手术组(S-Sham组)、缺血/再灌注组(S-I/R组)、姜黄素100mg/kg预处理组(S-Cur组),上述5个实验组按再灌注时间又分为再灌注2h、6h、1d、3d、7d5个亚组(n=6)。采用四动脉结扎法制备脑缺血/再灌注模型,以TUNEL法检测海马CA1区的细胞凋亡,免疫组化法分析海马CA1区c-jun、c-fos的动态变化。结果:S-Sham组大鼠海马CA1区TUNEL细胞数量和c-jun、c-fos表达高于W-Sham组(P0.05),S-I/R组TUNEL细胞数量和c-jun、c-fos表达高于S-Sham组及W-I/R组(P0.05);S-Cur组TUNEL细胞数量和c-jun、c-fos表达较S-I/R组明显降低(P0.05)。结论:缺血/再灌注更易导致SHR海马神经元凋亡。姜黄素可抑制SHR脑缺血/再灌注损伤海马神经元凋亡,其作用机制可能与抑制c-jun、c-fos蛋白的表达有关。     

缺血/再灌注小鼠海马组织cAMP和腺苷环化酶mRNA水平的变化

目的:观测缺血/再灌注小鼠海马组织环磷酸腺苷(cAMP)和腺苷环化酶(AC)mRNA水平,探讨缺血/再灌注发病的分子生物学机制.方法:通过双侧颈总动脉线结、连续3次缺血-再灌注,制作缺血/再灌注动物模型,并设立假手术组;术后29 d、30 d分别测试学习和记忆成绩;应用放射免疫法检测小鼠海马组织cAMP水平,应用原位杂交技术检测ACmRNA水平.结果:与假手术组比较,模型组学习和记忆成绩均降低(P＜0.05),且海马组织cAMP水平也降低(P＜0.05),海马CA1区AC mRNA阳性神经元面密度明显降低(P＜0.05).结论:海马组织cAMP和AC mRNA水平降低可能参与了缺血/再灌注后学习和记忆障碍的分子生物学发病机制.     

短暂前脑缺血小鼠海马脑红蛋白表达的动态变化

研究了小鼠短暂前脑缺血再灌注后不同时相点脑红蛋白表达的动态变化及其意义。用夹闭双侧颈总动脉的方法建立C57BL/6小鼠缺血再灌注动物模型;采用RT-PCR及Western blotting方法检测各组小鼠海马组织中脑红蛋白在转录和翻译水平表达的动态变化。结果显示,脑红蛋白在mRNA水平表达的动态变化为:与假手术对照组(100±0.00)比较,再灌注后6h(132.59±28.26,P<0.05)开始升高;24h(157.36±13.85,P<0.001)达高峰;48h(146.55±23.17,P<0.01)开始下降;72h(118.42±34.23,P>0.05)基本恢复至正常水平。脑红蛋白在蛋白水平表达的动态变化为:与假手术对照组(100±0.00)比较,再灌注后6h(111.46±23.54,P>0.05)轻微升高,24h(141.25±32.12,P<0.01)达高峰,48h(138.02±19.68,P<0.05)开始下降,72h(119.29±35.18,P>0.05)基本恢复至正常水平。结果提示,脑缺血再灌注各时相点的脑红蛋白mRNA及蛋白表达水平均增加,可能是机体的应激反应,但持续时间较短(48h以内)。     

Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

Comparative study on high fat diet-induced 4-hydroxy-2E-nonenal adducts in the hippocampal CA1 region of C57BL/6N and C3H/HeN mice

In the present study, we investigated the influences of a high fat diet (HD) fed for 12 weeks, on lipid peroxidation and antioxidant enzyme using 4-hydroxy-2E-nonenal (HNE)-modified proteins (HNE-mp) and Cu,Zn-superoxide dismutase (SOD1) in the hippocampal CA1 region (CA1) in C57BL/6N and C3H/HeN mice. Body weights and body weight gains were significantly higher in HD fed C57BL/6N mice than in low fat diet (LD) fed C57BL/6N and LD or HD fed C3H/HeN mice. In the HD fed C57BL/6N and C3H/HeN mice, HNE-mp immunoreactivity and protein levels were much higher than in the LD fed C57BL/6N or C3H/HeN mice. In particular, HNE-mp immunoreactivity and protein levels in HD fed C57BL/6N mice was higher than that in the HD fed C3H/HeN mice. SOD1 immunoreaction was detected in the non-pyramidal cells of C57BL/6N mice, while in the C3H/HeN mice SOD1 immunoreaction was observed in CA1 pyramidal cells. The SOD1 immunoreactivity in the LD fed C57BL/6N and C3H/HeN mice was slightly, but not significantly decreased compared to that in the HD fed C57BL/6N and C3H/HeN mice, respectively. In addition, ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactive microglia in the HD fed C57BL/6N showed hypertrophy of cytoplasm, which is the characteristics of activated microglia. These results suggest that HD fed C57BL/6N mice are more susceptible to lipid peroxidation in the CA1 than in LD fed C57BL/6N and LD or HD fed C3H/HeN mice without any differences of SOD1 expression. In Koo Hwang and Il Yong Kim have contributed equally to this article.     

Substrains matter in phenotyping of C57BL/6 mice

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.     

Increased microglial activation and astrogliosis after intranasal administration of kainic acid in C57BL/6 mice

Glutamate excitotoxicity plays a key role in inducing neuronal cell death in many neurological diseases. In mice, intranasal administration of kainic acid (KA), an analogue of the excitotoxin glutamate, results in hippocampal cell death and provides a well-characterized model for studies of human neurodegenerative diseases. In this study, we describe neurodegeneration and gliosis following intranasal administration of KA in C57BL/6 mice. By using Nissl's staining, neurodegeneration was found in area CA3 of hippocampus, and neuronal apoptosis was demonstrated by enhanced FAS(CD95/APO-1) expression detected by immunohistochemistry and Western blotting. Astrogliosis was exhibited by increased glial fibrillary acidic protein (GFAP) expression in the hippocampus and cortex. We also studied the profile of molecular expression on microglia in C57BL/6 mice. One and 3 days after KA administration, CD45, F4/80, CD86, MHCII, iNOS but not CD40 expression was enhanced or induced on microglia. In summary, KA administration results in an early microglial activation and a prolonged astrogliosis in C57BL/6 mice.     

Unexpected Rescue of Alpha-synuclein and Multimerin1 Deletion in C57BL/6JOlaHsd Mice by Beta-adducin Knockout

Uniform genetic background of inbred mouse strains is essential in experiments with genetically modified mice. In order to assess Add2 (beta-adducin) function, its null mutation was produced in embryonic stem cells derived from 129Sv mouse and the subsequently obtained mouse mutants were backcrossed for 6 generations with C57BL/6JOlaHsd strain. Comparison of brain proteins between mutated and control animals by two-dimensional gels linked to mass spectroscopy analysis showed expression of Snca (alpha-synuclein) in the mutated animals, but unexpectedly not in the control C57BL/6JOlaHsd mice. Comparison between C57BL/6JOlaHsd and C57BL/6NCrl mice confirmed the presence of a deletion encompassing Snca and in addition Mmrn1 (multimerin1) loci in C57BL/6JOlaHsd strain. The segregation of mutated Add2 together with an adjacent part of the chromosome 6 derived from 129Sv mice, rescued the loss of these two genes in knockout mice on C57BL/6JOlaHsd background. The fact that Add2 knockout was compared with the C57BL/6JOlaHsd mouse strain, which is actually a double knockout of Snca and Mmrn1 emphasizes a need for information provided by commercial suppliers and of exact denominations of substrains used in research.     

Age-related loss of neuronal nicotinic receptor expression in the aging mouse hippocampus corresponds with cyclooxygenase-2 and PPAR gamma expression and is altered by long-term NS398 administration

Age-related changes in the mammalian dorsal hippocampus are associated with diminished expression of neuronal nicotinic acetylcholine receptors (nAChR), which is particularly severe in pathologies such as those associated with dementias, including Alzheimer's disease. Because the mouse is a useful model for age-related decline in nAChR expression in the basal forebrain and limbic system, we used immunohistochemistry to examine the influence of long-term (12-month) oral administration of nicotine and/or the cyclooxygenase-2 (COX-2) preferring non-steroidal anti-inflammatory drug (NSAID) NS398 on nAChR alpha4, alpha5, alpha7, and beta4 expression in the C57BL/6 mouse. Inhibitory neurons of the dorsal hippocampus that express nAChRs also constitutively express COX-2 and the peroxisome proliferator-antagonist receptor subtype gamma-2 (PPAR gamma2) which is also a target of NS398. Administration of NS398 correlated with retention of nAChR alpha4 and to a lesser extent nAChR beta4, but not nAChR alpha5 or alpha7, but nicotine exhibited no similar effect. Nicotine and NS398 co-administration abolished the NS398-related effect on nAChR alpha4 retention. These results provide evidence that the interaction during aging between oral administration of nicotine and NSAIDs are not straightforward and could even be antagonistic when combined.     

A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

Background

The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.

Results

We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.

Conclusions

Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.     

Differential expression of VGLUT3 in laboratory mouse strains: Impact on drug‐induced hyperlocomotion and anxiety‐related behaviors

The atypical vesicular glutamate transporter VGLUT3 is present in subpopulations of GABAergic interneurons in the cortex and the hippocampus, in subgroups of serotoninergic neurons in raphe nuclei, and in cholinergic interneurons in the striatum. C56BL/6N mice that no longer express VGLUT3 (VGLUT3^?/?) display anxiety‐associated phenotype, increased spontaneous and cocaine‐induced locomotor activity and decreased haloperidol‐induced catalepsy. Inbred mouse strains differ markedly in their sensitivity to anxiety and behavioral responses elicited by drugs. The purpose of this study was to investigate strain differences in VGLUT3 expression levels and its potential correlates with anxiety and reward‐guided behaviors. Five inbred mouse lines were chosen according to their contrasted anxiety and drugs sensitivity: C57BL/6N, C3H/HeN, DBA/2J, 129/Sv, and BALB/c. VGLUT3 protein expression was measured in different brain areas involved in reward or mood regulation (such as the striatum, the hippocampus, and raphe nuclei) and genetic variations in Slc17a8, the gene encoding for VGLUT3, have been explored. These five inbred mouse strains express very different levels of VGLUT3, which cannot be attributed to the genetic variation of the Slc17a8 locus. Furthermore, mice behavior in the open field, elevated plus maze, spontaneous‐ and cocaine‐induced locomotor was highly heterogeneous and only partially correlated to VGLUT3 levels. These data highlight the fact that one single gene polymorphism could not account for VGLUT3 expression variations, and that region specific VGLUT3 expression level variations might play a key role in the modulation of discrete behaviors.