睡眠剥夺通过影响大鼠下丘脑组蛋白乙酰化调控神经元Orexin A的表达

目的:探讨睡眠剥夺(sleep deprivation,SD)对大鼠下丘脑组蛋白乙酰化水平的影响,并观察乙酰化修饰是否影响神经元下丘脑促觉醒肽Orexin A的表达。方法:成年雄性大鼠24只,采用改良多平台睡眠剥夺法建立大鼠SD模型,随机分为对照组(n=6)和SD组(n=18);Western Blot法检测大鼠下丘脑组蛋白乙酰化水平的变化;免疫荧光法观察下丘脑Orexin A神经元。结果:与对照组相比,SD后1 d,3 d及6 d,下丘脑组蛋白H3亚基9位赖氨酸(H3K9)、H3K14位点的乙酰化水平明显下降(P0.05),但三个时间点之间无显著差异(P0.05)。SD后3 d,下丘脑的Orexin A+神经元数目和对照组相比明显减少(P0.05),而给予组蛋白去乙酰化酶抑制剂SAHA(25 mg/kg,i.p.)可部分恢复SD减少的Orexin A~+神经元数(P0.05)。结论:睡眠剥夺可能通过影响大鼠下丘脑组蛋白的乙酰化修饰水平减少神经元Orexin A的表达。     

REM期睡眠剥夺对大鼠不同脑区组蛋白H3K9和H3K4甲基化水平的影响

目的:研究REM期睡眠剥夺对成年大鼠不同脑区包括海马、腹内侧前额叶皮质、下丘脑和中缝核群的组蛋白H3K9和H3K4三甲基化水平的影响。方法:将成年SD大鼠随机分为正常对照组(con)、睡眠剥夺1 d组(SD1 d)、睡眠剥夺3 d组(SD3 d)、睡眠剥夺6 d组(SD6 d)。采用改良式多平台水环境法建立REM期睡眠剥夺模型,分别于睡眠剥夺后1、3、6 d断头取脑,用免疫荧光染色与Western Blot检测大鼠海马、腹内侧前额叶皮质、下丘脑和中缝核群H3K9和H3K4三甲基化水平的变化,最终结果进行统计学分析。结果:Western Blot结果显示:(1)海马、腹内侧前额叶皮质三个SD组H3K9三甲基化水平均低于control组(P0.05),H3K4三甲基化水平均高于control组(P0.05);(2)下丘脑三个SD组H3K9三甲基化水平均低于control组(P0.01),但SD1 d组H3K4三甲基化水平高于control组(P0.05),SD3 d、SD6 d组H3K4三甲基化水平低于control组(P0.05);(3)中缝核群三个SD组H3K9和H3K4三甲基化水平均高于control组(P0.05)。对海马与下丘脑免疫荧光验证H3K9三甲基化结果与Western Blot结果趋势一致,且说明细胞无减少。结论:睡眠剥夺可能与海马、腹内侧前额叶皮质、下丘脑和中缝核群的组蛋白H3K9和H3K4三甲基化水平密切相关。     

睡眠剥夺对大鼠海马miRNAs的影响

目的:探讨睡眠剥夺(sleep deprivation,SD)对成年大鼠海马miR132,miR138,miR127,miR128及let-7b的影响。方法:成年雄性Sprague-Dawley30只随机分为对照组(con),睡眠剥夺8 h组(sd8h),睡眠剥夺1 d组(sd1),睡眠剥夺3 d组(sd3),睡眠剥夺6 d组(sd6);采用改良多平台睡眠剥夺法(MMPM)建立大鼠SD模型,SYBRA greenⅠ荧光定量RT-PCR检测大鼠海马miRNAs的变化。结果:RT-PCR结果显示:(1)海马sd8 h组和sd1组miR132水平均低于control组(P0.05),但sd3组高于control组(P0.05);(2)海马sd8 h、sd3组miR138水平均低于control组(P0.01)但sd1组高于control组(P0.01);(3)海马sd8 h、sd1、sd3组miR127水平均低于control组(P0.05),但sd6组高于control组(P0.05);(4)海马sd1组miRl28水平低于control组(P0.05),sd8h组高于control组(P0.05);(5)海马sd8h、sdl、sd3组let-7b水平均高于control组(P0.05)。结论:睡眠剥夺可能与海马的miR132,miR138,miR127,miR128及let-7b的水平有关。     

睡眠剥夺后大鼠海马生化及病理变化

目的 :观察睡眠剥夺后大鼠海马超微结构的病理变化及一氧化氮 (NO)含量、超氧化物歧化酶 (SOD)活力变化。方法 :以小平台水环境法建立大鼠睡眠剥夺模型 ,并设立大平台对照组 ,分离海马 ,电镜观察海马神经细胞超微结构变化、测定海马组织匀浆NO含量和SOD活力。结果 :睡眠剥夺组大鼠海马NO含量及SOD活力均高于大平台组和正常对照组 (P <0 0 5 )。病理形态学改变 ,光镜电镜下海马神经元减少 ,核仁碎裂、胞质内细胞器减少。结论 :睡眠剥夺可引起海马NO含量和SOD活力增高 ,并且存在病理形态学改变     

睡眠剥夺促进大鼠海马神经元凋亡及相关基因表达

探讨睡眠剥夺引起的神经元凋亡与相关基因表达的变化。采用TUNEL染色观察了快眼动睡眠剥夺大鼠海马神经元形态学变化,应用原位杂交、Western blot法检测了快眼动睡眠剥夺大鼠海马bcl-2,bax mRNA,MAPKs表达的变化。结果表明：快眼动睡眠剥夺大鼠海马CAl,CA3区神经元阳性凋亡细胞数明显增多,bcl-2,bax mRNA表达明显增强,ERK活性降低,JNK蛋白表达量较对照组明显增高。提示睡眠剥夺可引起大鼠海马神经元凋亡。与凋亡相关的bcl-2,bax mRNA基因表达及MAPKs活性的变化可能涉及神经元的凋亡机制。     

出生前尼古丁暴露增加新生大鼠下丘脑orexin A及延髓内orexin 1型受体表达

流行病学调查显示,出生前暴露于烟雾环境是新生儿猝死综合征发生的首位原因,尼古丁是香烟烟雾中最主要的影响胎儿神经系统发育的成分。为了观察出生前尼古丁暴露对新生大鼠下丘脑orexin A(OXA)及延髓内orexin 1型受体(OX1R)表达的影响,本实验将20只雌性成年大鼠随机均分为二组,怀孕后第5 d开始每天分别皮下注射尼古丁6 mg/kg(模型组)或等量的生理盐水(对照组),直至分娩。随机选取模型组和对照组所产的新生大鼠(1～3 d),采用免疫组织化学方法和图像分析技术,观察新生鼠下丘脑内OXA及延髓内OX1R阳性神经元的分布情况。结果显示:两组新生大鼠下丘脑内OXA免疫阳性细胞均有表达,且都主要存在于下丘脑背内侧区与穹窿周围,模型组的新生大鼠OXA免疫阳性细胞的相对光密度(ROD)值高于对照组(P<0.05)。延髓内OX1R免疫阳性细胞在两组内均有广泛分布,主要分布在腹外侧区和舌下神经核。在这两个区域,模型组新生鼠的OX1R免疫阳性细胞的ROD值均高于对照组(P<0.001)。以上结果表明,出生前尼古丁暴露的新生大鼠,下丘脑OXA及延髓内OX1R的表达均上调,提示出生前尼古丁暴露改变了新生大鼠脑内OXA系统递质的释放和突触传递,这意味着脑内orexin系统参与出生前尼古丁暴露导致的各种疾患。     

不同时段睡眠剥夺对大鼠脑内c-fos表达的影响

目的 :探索睡眠剥夺的脑机制。方法 :该研究采用小平台水环境法建立大鼠睡眠剥夺模型 ,用fos蛋白免疫组化的方法测量脑中fos蛋白的表达 ,分组为白天睡眠剥夺 12h组、夜晚睡眠剥夺 12小时组、大平台对照组和正常单独饲养组 ,每组 4只。结果 :睡眠剥夺使fos蛋白在皮层的广泛区域表达 ,脑干中同异相睡眠有关的区域有较高表达 ,同夜晚睡眠剥夺 12小时相比 ,白天睡眠剥夺 12小时在视交叉上核同生物节律有关的区域表达。结论 :睡眠节律改变可影响脑内fos蛋白表达     

MAPKs在睡眠剥夺大鼠海马神经元内表达变化的研究

目的:探讨睡眠剥夺大鼠丝裂素活化蛋白激酶(MAPKs)表达的变化及可能的意义。方法:采用TUNEL和HE法观察了睡眠剥夺大鼠海马神经元的形态学变化,采用Westernblot法、β-液闪计数法观察海马神经元ERK和JNK表达的变化。结果:快眼动睡眠剥夺组海马神经元阳性凋亡细胞数增多,ERK活性1764.00±941.56,显著低于对照组(P＜0.05),快眼动睡眠剥夺组JNK蛋白表达量为87.5%,显著高于对照组(P<0.05)。结论:睡眠剥夺可引起大鼠海马神经元MAPKs活性的变化,而这些变化可能涉及神经元的凋亡机制。     

连续及部分睡眠剥夺96小时后大鼠脑干中c-Fos蛋白的表达

目的：探索连续及部分不同睡眠剥夺条件下的脑c-Fos蛋白的表达。方法：采用小平台水环境法建立大鼠睡眠剥夺模型，用Fos蛋白免疫组化的方法分别测量连续睡眠剥夺96小时组、部分睡眠剥夺96小时组、大平台对照组和正常单独饲养组脑干中Fos蛋白的表达，每组4只。结果：同连续睡眠剥夺组相比，部分睡眠剥夺组表达范围更广，在脑干网状结构及中缝的被盖背侧核、脑桥被盖网状核及脑桥吻侧网状核、脑桥尾侧网状核、丘脑的中线核群、板内核群和网状核均有所表达，但阳性程度低。结论：部分睡眠剥夺对脑的影响要小于连续睡眠剥夺。     

快速眼动睡眠剥夺对中枢5-羟色胺缺失小鼠orexin阳性神经元活性的影响

为探讨中枢5羟色胺(5-HT)的缺失对正常睡眠和快速眼动睡眠剥夺(REM sleep deprivation)情况下orexin阳性神经元活动的影响,本研究利用中枢5-HT神经元缺失的条件性基因敲除小鼠(Pet1-Cre/Lmx1b flox/flox CKO小鼠),采用小平台水环境法建立小鼠快速眼动睡眠剥夺模型,免疫组化方法观察野生型小鼠和中枢5-HT神经元缺失小鼠在正常睡眠状态及8 h快速眼动睡眠剥夺后下丘脑内orexin阳性神经元的数量,免疫组化双标法观察orexin/c-fos双标神经元占orexin阳性神经元的比例。结果显示:CKO小鼠睡眠剥夺前后orexin阳性神经元的数量未见明显差别,与野生型小鼠相比亦未见统计学差别;在正常睡眠状态下(对照组),CKO小鼠orexin/c-fos双标神经元的数量与野生型小鼠相当,但睡眠剥夺后明显低于野生型小鼠睡眠剥夺组。本研究结果提示,作为维持觉醒的重要神经递质5-HT的缺失可能降低了中枢神经系统的觉醒水平,致使睡眠剥夺不能提高促发和维持觉醒的orexin阳性神经元的活性。     

Expression of Orexin A and Its Receptor 1 in the Vestibular Glands of the Cattle Genital Tract

The hypothalamic peptide orexin A (oxA) binds specifically the G‐protein–coupled orexin receptor 1 (ox1R). It is involved in many physiological functions including the regulation of food intake, sleep–wake cycle, arterial blood pressure, heart rate, and sexual behavior. The localization of oxA in adrenal glands, stomach, bowel, pancreas, and testis has recently been assessed. Here, we provide the first evidence for the expression of oxA and ox1R in the vestibular glands of mammalian genital tract. Anat Rec, 2009. copy; 2008 Wiley‐Liss, Inc.     

Adenosine and the homeostatic control of sleep: effects of A1 receptor blockade in the perifornical lateral hypothalamus on sleep-wakefulness

The orexinergic neurons of the lateral hypothalamus (LH) are critical for wakefulness [McCarley RW (2007) Neurobiology of REM and NREM sleep. Sleep Med 8:302-330]. Recent evidence suggests that adenosine (AD), a homeostatic sleep factor, may act via A1 receptor (A1R) to control orexinergic activity and regulate sleep-wakefulness [Thakkar MM, Winston S, McCarley RW (2002) Orexin neurons of the hypothalamus express adenosine A1 receptors. Brain Res 944:190-194; Liu ZW, Gao XB (2006) Adenosine inhibits activity of hypocretin/orexin neurons via A1 receptor in the lateral hypothalamus: a possible sleep-promoting effect. J Neurophysiol]. To evaluate the role of AD in the orexinergic LH and its influences on sleep-wakefulness, we designed two experiments in freely behaving rats: First, we bilaterally microinjected 1,3-dipropyl-8-phenylxanthine (DPX) (1.5 pmol and 15 pmol), a selective A1R antagonist into the LH during the light cycle and examined its effect on spontaneous sleep-wakefulness. Second, we performed 6 h of sleep deprivation. Thirty minutes before the animals were allowed to enter recovery sleep, 15 pmol of DPX was bilaterally microinjected into the LH and its effects on recovery sleep were monitored. Microinjection of DPX into the orexinergic LH produced a significant increase in wakefulness with a concomitant reduction in sleep, both during spontaneous bouts of sleep-wakefulness and during recovery sleep. Local administration of DPX into the LH produced a significant increase in the latency to non-REM sleep during recovery sleep. However, total slow wave (delta) activity during non-REM sleep phase of recovery sleep remained unaffected after DPX treatment. This is the first study that implicates endogenous adenosine to have a functional role in controlling orexinergic tone and influencing the homeostatic regulation of sleep-wakefulness.     

Increased levels of tyrosine hydroxylase and glutamic acid decarboxylase in locus coeruleus neurons after rapid eye movement sleep deprivation in rats

Functional magnetic resonance imaging (fMRI) was used to determine the acute blood oxygen level dependent effect (BOLD) of neuroleptic drugs in healthy male subjects. Using a robust simultaneous visuo-acoustic stimulation paradigm fMRI measurements were obtained prior to as well as 1 h and 24 h after intravenous infusion of 5 mg haloperidol to six healthy young men. After the administration, subjects showed significantly reduced BOLD contrast in the middle occipital gyrus while BOLD contrast was increased in the lingual gyrus. This pattern normalised within 24 h. Our results emphasise the necessity to control for interactions through acute medication and confirm fMRI as a non-invasive method for studying cerebral psychopharmacological effects.     

食欲素A对人肝癌细胞Hep G2增殖和凋亡的影响及机制

目的研究食欲素A(orexin A)对人肝癌细胞Hep G2增殖和凋亡的影响,及其可能作用机制。方法以不同浓度的orexin A干预Hep G2细胞24h,MTT法检测细胞的增殖能力,筛选最适的orexin A作用浓度。使用食欲素受体1选择性拮抗剂SB408124(10μM)预处理细胞1 h,然后用最适浓度的orexin A干预细胞24h,实验设置为:正常对照组、溶剂对照组、orexin A组、SB408124+orexin A组。MTT法检测细胞的增殖情况,流式细胞术检测细胞周期和细胞凋亡率,Hoechst染色观察细胞的形态变化,Western blot检测Bax、Bcl-2、Cytochrome c和Cleaved-caspase-3蛋白的表达。结果 Orexin A以剂量依赖性方式明显抑制Hep G2细胞的生长,最适宜浓度为0.5μM,细胞G0/G1期百分比(72.50%±1.32%)明显高于S期(19.6%9±1.15%)。Orexin A促进Hep G2细胞凋亡,细胞凋亡率显著升高(0.01)。Orexin A显著上调Bax和Cleaved-caspase-3表达水平,下调Bcl-2和线粒体Cytochrome c表达水平。SB408124的干预能够减小orexin A对Hep G2细胞的影响。结论 Orexin A通过食欲素受体1抑制Hep G2细胞增殖并诱导其凋亡。     

Nuclear organization of orexinergic neurons in the hypothalamus of a lar gibbon and a chimpanzee

Employing orexin-A immunohistochemical staining we describe the nuclear parcellation of orexinergic neurons in the hypothalami of a lar gibbon and a chimpanzee. The clustering of orexinergic neurons within the hypothalamus and the terminal networks follow the patterns generally observed in other mammals, including laboratory rodents, strepsirrhine primates and humans. The orexinergic neurons were found within three distinct clusters in the ape hypothalamus, which include the main cluster, zona incerta cluster and optic tract cluster. In addition, the orexinergic neurons of the optic tract cluster appear to extend to a more rostral and medial location than observed in other species, being observed in the tuberal region in the anterior ventromedial aspect of the hypothalamus. While orexinergic terminal networks were observed throughout the brain, high density terminal networks were observed within the hypothalamus, medial and intralaminar nuclei of the dorsal thalamus, and within the serotonergic and noradrenergic regions of the midbrain and pons, which is typical for mammals. The expanded distribution of orexinergic neurons into the tuberal region of the ape hypothalamus, is a feature that needs to be investigated in other primate species, but appears to correlate with orexin gene expression in the same region of the human hypothalamus, but these neurons are not revealed with immunohistochemical staining in humans. Thus, it appears that apes have a broader distribution of orexinergic neurons compared to other primate species, but that the neurons within this extension of the optic tract cluster in humans, while expressing the orexin gene, do not produce the neuropeptide.