加兰他敏对间歇性低氧引起的认知损害的预防作用

目的:探究加兰他敏对间歇性低氧引起的认知损伤是否有保护作用,从而说明其对睡眠呼吸暂停综合症引起的认知损害是否有预防作用。方法:建立间歇低氧大鼠模型,行水迷宫试验检测行为功能变化,免疫组化检测海马神经元及胶质细胞数目的变化。结果:加兰他敏与间歇低氧模型组相比,行水迷宫的平均逃避潜伏期缩短,游泳总距离减少;免疫组化的结果海马神经元的数目有所增加,胶质细胞的数目减少。结论:加兰他敏对间歇性低氧引起的认知损伤有明显的改善作用,可能与减少神经元的丢失及减少胶质细胞的再生有关。所以对于诊断了睡眠呼吸暂停综合征(ASA)的患者,如果同时合并其他痴呆的易感因素,可预防性应用加兰他敏。     

慢性间断性低氧大鼠认知功能和脑胆碱能神经元的进行性变化

目的：探讨慢性间断性低氧（CIH）大鼠认知功能的进行性变化及其与脑胆碱能神经元变化的关系。方法：成年雄性SD大鼠40只,随机均分为对照组、慢性间断性低氧1,3,5周组。应用Morris水迷宫检测认知功能的变化;利用HE染色在光镜下计数前额叶皮层和海马坏死神经元数;利用免疫组化方法检测前额叶皮层和海马胆碱乙酰转移酶（ChAT）阳性表达。结果：CIH各组大鼠学习记忆能力呈进行性下降趋势;与对照组比较,CIH5w组出现明显学习记忆功能障碍（P〈0.05）。CIH各组前额叶皮层和海马变性坏死神经元数增多,且随低氧时间延长,上述改变呈慢性进行性加重趋势。CIH各组前额叶皮层和海马ChAT阳性表达逐渐下降;与对照组比较,CIH3w组和CIH5w组前额叶皮层和海马ChAT阳性表达明显减少,差异具有显著性（P〈0.05）。结论：慢性间断性低氧大鼠认知功能进行性下降与前额叶皮层和海马神经元病理性损伤、ChAT表达进行性减少有关。     

Aβ25～35诱导大鼠记忆减退与星形胶质细胞及NOS阳性细胞变化

目的探讨Aβ25～35诱导模拟人类Alzheimer`s病(AD)的大鼠病理模型中星形胶质细胞变化与一氧化氮合酶神经元损伤引起的老年性记忆减退之间的关系.方法双侧海马内注射β-淀粉样多肽25～35片段(Aβ25～35)制作大鼠AD模型,注射一周后采用NOS组化染色、GFAP免疫组化染色及NOS组化和GFAP双重染色分析大鼠海马GFAP与NOS的表达.结果海马内注射Aβ25～35后出现海马星形胶质细胞增生、肥大、数目明显多于对照组(P<0.05),并出现一氧化氮阳性星形胶质细胞;海马一氧化氮神经元数量较对照组显著减少(P<0.05).结论 AD模型大鼠学习记忆功能低下与Aβ神经毒性导致NOS阳性神经元损伤、死亡直接相关,反应性星形胶质细胞参与Aβ导致NOS神经元细胞毒性损伤作用,间接导致学习记忆能力减退.     

Aβ325～35诱导大鼠记忆减退与星形胶质细胞及NOS阳性细胞变化

目的　探讨Aβ2 5～ 3 5诱导模拟人类Alzheimer‘s病 (AD)的大鼠病理模型中星形胶质细胞变化与一氧化氮合酶神经元损伤引起的老年性记忆减退之间的关系。方法　双侧海马内注射 β-淀粉样多肽 2 5～ 3 5片段 (Aβ2 5～ 3 5 )制作大鼠AD模型 ,注射一周后采用NOS组化染色、GFAP免疫组化染色及NOS组化和GFAP双重染色分析大鼠海马GFAP与NOS的表达。结果　海马内注射Aβ2 5～ 3 5后出现海马星形胶质细胞增生、肥大、数目明显多于对照组 (P <0 0 5 ) ,并出现一氧化氮阳性星形胶质细胞 ;海马一氧化氮神经元数量较对照组显著减少 (P <0 0 5 )。结论　AD模型大鼠学习记忆功能低下与Aβ神经毒性导致NOS阳性神经元损伤、死亡直接相关 ,反应性星形胶质细胞参与Aβ导致NOS神经元细胞毒性损伤作用 ,间接导致学习记忆能力减退     

脂多糖LPS诱导的神经炎症对小鼠认知功能的影响及相关机制分析

为研究脂多糖(lipopolysaccharide,LPS)对小鼠认知功能的影响,本研究将18只C57BL/6小鼠随机分为两组,即正常对照组和LPS处理组,每组各9只小鼠。在腹腔注射LPS 24 h后行水迷宫实验检测小鼠行为学变化。此外,本研究采用Western blotting和免疫组化检测小鼠脑内小胶质细胞特异性标记物离子通道相关钙衔接蛋白(ionized calcium binding adapter,IBA1)的表达,TUNEL凋亡法检测小鼠脑内神经元凋亡情况。实验结果发现LPS处理后小鼠认知功能下降。Western blotting和免疫组化结果均提示LPS处理后小鼠脑中IBA1表达量增加,小胶质细胞激活;TUNEL提示LPS处理后小鼠脑内出现大量凋亡神经元,由此推断LPS可能通过激活小胶质细胞,扩大神经炎症反应,增加神经元凋亡导致小鼠认知功能下降。     

β-淀粉样肽诱导小胶质细胞培养上清致海马神经元损伤模型的建立

目的：建立β淀粉样肽（Aβ1-40）诱导激活小胶质细胞的上清致海马神经元损伤的细胞模型,并初步研究神经元损伤的机制。方法：用不同浓度的可溶性Aβ1-40诱导激活小胶质细胞,光镜下观察不同时间点的细胞形态,ELISA检测其分泌的肿瘤坏死因子仪;用激活后的小胶质细胞条件培养基刺激海马神经元,光镜下观察细胞形态,Western blot检测刺激后海马神经元内诱导型一氧化氮合酶（iNOS）和硝基酪氨酸（NT）的表达水平,ELISA检测海马神经元内胱冬蛋白酶-3（caspase-3）活性来评价神经元的损伤程度。结果：终浓度为10μmol／L的Aβ1-40与小胶质细胞孵育24h后,取上清液加到培养的海马神经元,孵育24-72h,海马神经元较对照组形态有明显变化;经Western blot检测,神经元内iNOS、NT表达明显增加;ELISA检测神经元内caspase-3活性明显增高。结论：小胶质细胞被Aβ1-40激活后,其释放物有明显的致神经元损伤效应,表明建立了神经元损伤模型。     

脑益康药物血清对谷氨酸诱导海马神经元损伤的保护作用

目的:探讨脑益康药物血清对谷氨酸(Glu)诱导的海马神经元损伤的保护作用。方法:大鼠海马神经元培养后,采用形态学观察、MTT法及DAPI染色法检测脑益康药物血清对Glu损伤细胞活力的影响,采用RT-PCR和免疫组化方法检测脑益康药物血清对Glu损伤细胞PTEN表达的影响。结果:脑益康药物血清可明显提高Glu损伤的海马神经元的细胞活力,减少PTEN的表达。结论:脑益康药物血清对Glu诱导的海马神经元损伤有保护作用,其机制可能与减少PTEN表达,抑制神经元凋亡有关。     

间歇性低氧对大鼠海巴长时程增强电位的影响

目的：研究间歇性低氧对大鼠海马神经元突触可塑性的影响。方法：大鼠受间歇性低氧处理后,用脑立体定位仪定位,观察海马时程增强电位（LTP）的变化。结果：间歇性低氧大鼠LTP幅值显著低于对照组。结论：间歇性低氯可影响LTP幅值,提示间歇性低氧可能使大鼠海马神经元的突触可塑性发生变化。     

CNTF对烧伤大鼠血清引起大鼠海马神经元细胞毒性的影响

应用整体和离体神经元培养,观察CNTF对烧伤大鼠海马神经元及烧伤血清引起海马神经元损伤的影响,结果表明,大鼠烧伤后海马组织神经元数目减少,NO含量升高;烧伤大鼠血清可引起培养的海马神经元细胞存活率下降,培养液中NO含量升高;CNTF能降低烧伤大鼠海马组织中NO的含量,保护海马神经元,并能提高培养的海马神经元的存活率,减少培养液中NO含量,其作用呈剂量依赖性;CNTF对神经元存活率的影响与NO含量呈显著负相关,提示CNTF对烧伤大鼠血清引起的海马神经元损伤有保护作用,其作用机制可能是通过抑制NO的神经毒性。     

LPS诱导海马星形胶质细胞活化与活化的星形胶质细胞对海马神经元的影响

探讨脂多糖(Lipopolysaccharide,LPS)对长时间存活大鼠海马内星形胶质细胞的反应以及对神经元的影响。方法:本实验用10只健康成年雄性SD大鼠,海马CA3区注射LPS 10μ1．7和14d后,尼氏染色观察神经元的变化,免疫组织化学染色结合图像分析方法观察海马CA3区注射部位胶质纤维酸性蛋白(glial fibrillary acidic protein GFAP)、的表达变化。结果:脂多糖可促进海马星形胶质细胞的活化,但并不能引起海马区神经元的损伤。结论:星形胶质细胞在脑损伤后的脑内炎症反应起了一定的作用,但并不能引起神经元的损伤。     

Chronic intermittent hypoxia alters NMDA and AMPA-evoked currents in NTS neurons receiving carotid body chemoreceptor inputs

Chronic exposure to intermittent hypoxia (CIH) has been used in animals to mimic the arterial hypoxemia that accompanies sleep apnea. Humans with sleep apnea and animals exposed to CIH have elevated blood pressures and augmented sympathetic nervous system responses to acute exposures to hypoxia. To test the hypothesis that exposure to CIH alters neurons within the nucleus of the solitary tract (NTS) that integrate arterial chemoreceptor afferent inputs, we measured whole cell currents induced by activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors in enzymatically dispersed NTS neurons from normoxic (NORM) and CIH-exposed rats (alternating cycles of 3 min at 10% O2 followed by 3 min at 21% O2 between 8 AM and 4 PM for 7 days). To identify NTS neurons receiving carotid body afferent inputs the anterograde tracer 4- (4-(dihexadecylamino)styryl-N-methylpyridinum iodide (DiA) was placed onto the carotid body 1 wk before exposure to CIH. AMPA dose-response curves had similar EC50 but maximal responses increased in neurons isolated from DiA-labeled CIH (20.1 +/- 0.8 microM, n = 9) compared with NORM (6.0 +/- 0.3 microM, n = 8) rats. NMDA dose-response curves also had similar EC50 but maximal responses decreased in CIH (8.4 +/- 0.4 microM, n = 8) compared with NORM (19.4 +/- 0.6 microM, n = 9) rats. These results suggest reciprocal changes in the number and/or conductance characteristics of AMPA and NMDA receptors. Enhanced responses to AMPA receptor activation could contribute to enhanced chemoreflex responses observed in animals exposed to CIH and humans with sleep apnea.     

Invited review: Neural network plasticity in respiratory control.

Respiratory network plasticity is a modification in respiratory control that persists longer than the stimuli that evoke it or that changes the behavior produced by the network. Different durations and patterns of hypoxia can induce different types of respiratory memories. Lateral pontine neurons are required for decreases in respiratory frequency that follow brief hypoxia. Changes in synchrony and firing rates of ventrolateral and midline medullary neurons may contribute to the long-term facilitation of breathing after brief intermittent hypoxia. Long-term changes in central respiratory motor control may occur after spinal cord injury, and the brain stem network implicated in the production of the respiratory rhythm could be reconfigured to produce the cough motor pattern. Preliminary analysis suggests that elements of brain stem respiratory neural networks respond differently to hypoxia and hypercapnia and interact with areas involved in cardiovascular control. Plasticity or alterations in these networks may contribute to the chronic upregulation of sympathetic nerve activity and hypertension in sleep apnea syndrome and may also be involved in sudden infant death syndrome.     

Biology of peripheral blood cells in obstructive sleep apnea--the tip of the iceberg

Obstructive sleep apnea (OSA), a highly prevalent breathing disorder in sleep, characterized by intermittent and recurrent pauses in respiration, has emerged as an independent risk factor for cardiovascular morbidity and mortality. Accumulated evidence implicates Leukocyte-endothelial cell activation and adhesion as critical components that induce inflammation and injury to the vasculature resulting in the development of cardiovascular complications. Similar cellular interactions were described in conditions of ischemia/reperfusion, and various components of the metabolic syndrome as hypercholesterolemia and hypertension. The hallmark of sleep apnea--the multiple cycles of hypoxia/reoxygenation--promote oxidative stress and inflammation. These facilitate increased interactions of blood cells with endothelial cells, resulting in endothelial cell injury and dysfunction. Such events can promote atherosclerosis and the development of cardiovascular morbidities in OSA. However, inter-individual differences in response to intermittent hypoxia and activation of anti-inflammatory cytokine profiles in T lymphocytes can serve as protective mechanisms.     

Inhalation of hydrogen gas attenuates left ventricular remodeling induced by intermittent hypoxia in mice

Sleep apnea syndrome increases the risk of cardiovascular morbidity and mortality. We previously reported that intermittent hypoxia increases superoxide production in a manner dependent on nicotinamide adenine dinucleotide phosphate and accelerates adverse left ventricular (LV) remodeling. Recent studies have suggested that hydrogen (H(2)) may have an antioxidant effect by reducing hydroxyl radicals. In this study, we investigated the effects of H(2) gas inhalation on lipid metabolism and LV remodeling induced by intermittent hypoxia in mice. Male C57BL/6J mice (n = 62) were exposed to intermittent hypoxia (repetitive cycle of 1-min periods of 5 and 21% oxygen for 8 h during daytime) for 7 days. H(2) gas (1.3 vol/100 vol) was given either at the time of reoxygenation, during hypoxic conditions, or throughout the experimental period. Mice kept under normoxic conditions served as controls (n = 13). Intermittent hypoxia significantly increased plasma levels of low- and very low-density cholesterol and the amount of 4-hydroxy-2-nonenal-modified protein adducts in the LV myocardium. It also upregulated mRNA expression of tissue necrosis factor-α, interleukin-6, and brain natriuretic peptide, increased production of superoxide, and induced cardiomyocyte hypertrophy, nuclear deformity, mitochondrial degeneration, and interstitial fibrosis. H(2) gas inhalation significantly suppressed these changes induced by intermittent hypoxia. In particular, H(2) gas inhaled at the timing of reoxygenation or throughout the experiment was effective in preventing dyslipidemia and suppressing superoxide production in the LV myocardium. These results suggest that inhalation of H(2) gas was effective for reducing oxidative stress and preventing LV remodeling induced by intermittent hypoxia relevant to sleep apnea.     

Invited review: Physiological consequences of intermittent hypoxia: systemic blood pressure.

One of the major manifestations of obstructive sleep apnea is profound and repeated hypoxia during sleep. Acute hypoxia leads to stimulation of the peripheral chemoreceptors, which in turn increases sympathetic outflow, acutely increasing blood pressure. The chronic effect of these repeated episodic or intermittent periods of hypoxia in humans is difficult to study because chronic cardiovascular changes may take many years to manifest. Rodents have been a tremendous source of information in short- and long-term studies of hypertension and other cardiovascular diseases. Recurrent short cycles of normoxia-hypoxia, when administered to rats for 35 days, allows examination of the chronic cardiovascular response to intermittent hypoxia patterned after the episodic desaturation seen in humans with sleep apnea. The result of this type of intermittent hypoxia in rats is a 10- to 14-mmHg increase in resting (unstimulated) mean blood pressure that lasts for several weeks after cessation of the daily cyclic hypoxia. Carotid body denervation, sympathetic nerve ablation, renal sympathectomy, adrenal medullectomy, and angiotensin II receptor blockade block the blood pressure increase. It appears that adrenergic and renin-angiotensin system overactivity contributes to the early chronic elevated blood pressure in rat intermittent hypoxia and perhaps to human hypertension associated with obstructive sleep apnea.     

The short-term and long-lasting changes in DNA and RNA synthesis in the cerebral cortex cells of animals subjected to hypoxia and nerve tissue transplantation]

The DNA and RNA synthesis in the cells of the brain cortex of intact rats and animals subjected to hypoxia, hypoxia with subsequent transplantation or by the local brain injury has been investigated. The DNA synthesis changes insignificantly in the case of hypoxia, it enhances slightly in the area of the injury and increases much more after transplantation. The RNA synthesis decreases considerably immediately after hypoxia and decreases much more 120 days later. Using the ultracentrifuge method it has been found that under the effect of hypoxia the number of nervous cells decreases, the number of glial cells does not change. The local injury in the nervous tissue enhances abruptly the synthesis in neurons and glial cells in the hypoxia-exposed animals, the embryonic nervous tissue transplantation normalizes the number of neurons in the specimens under study and the RNA synthesis in the neurons and glial cells.     

Carotid body function and ventilatory responses in intermittent hypoxia. Evidence for anomalous brainstem integration of arterial chemoreceptor input

Obstructive sleep apnea is a frequent medical condition consisting in repetitive sleep-related episodes of upper airways obstruction and concurrent events of arterial blood hypoxia. There is a frequent association of cardiovascular diseases and other pathologies to this condition conforming the obstructive sleep apnea syndrome (OSAS). Laboratory models of OSAS consist in animals exposed to repetitive episodes of intermittent hypoxia (IH) which also develop cardiovascular pathologies, mostly hypertension. The overall OSAS pathophysiology appears to be linked to the repetitive hypoxia, which would cause a sensitization of carotid body (CB) chemoreflex and chemoreflex-driven hyperreactivity of the sympathetic nervous system. However, this proposal is uncertain because hyperventilation, reflecting the CB sensitization, and increased plasma CA levels, reflecting sympathetic hyperreactivity, are not constant findings in patients with OSAS and IH animals. Aiming to solve these uncertainties we have studied the entire CB chemoreflex arch in a rat model of IH, including activity of chemoreceptor cells and CB generated afferent activity to brainstem. The efferent activity was measured as ventilation in normoxia, hypoxia, and hypercapnia. Norepinephrine turnover in renal artery sympathetic endings was also assessed. Findings indicate a sensitization of the CB function to hypoxia evidenced by exaggerated chemoreceptor cell and CB afferent activity. Yet, IH rats exhibited marked hypoventilation in all studied conditions and increased turnover of norepinephrine in sympathetic endings. We conclude that IH produces a bias in the integration of the input arising from the CB with a diminished drive of ventilation and an exaggerated activation of brainstem sympathetic neurons.     

Obstructive sleep apnea and chronic intermittent hypoxia: a review

Hypoxia is an important topic both physiologically and clinically. Traditionally, physiology research has been focusing on the effect of acute and chronic sustained hypoxia and human adaptive response to high altitude. In the past 20 years, genetic studies by many have expanded our understanding of hypoxia to the molecular level. However, in contrast to our extensive knowledge about acute and chronic sustained hypoxia, we know relatively little about the effect of chronic intermittent hypoxia (CIH). In recent years, CIH has attracted more research attention because of the increasing prevalence of obesity and obstructive sleep apnea (OSA) in the western countries. Clinically, CIH is commonly seen in patients with sleep-disordered breathing including OSA, Cheyne-Stokes respiration and nocturnal hypoventilation. It was estimated that for OSA of at least mild severity prevalence estimates range from 3 to 28% in the general population. OSA is characterized by recurrent upper airway collapse during sleep leading to intermittent nocturnal hypoxia and sleep fragmentation. OSA is associated with significant mortality and morbidity including neurocognitive dysfunction, hypertension, many cardiovascular disorders and metabolic disorders such as diabetes and metabolic syndrome. The intermittent hypoxia in OSA closely mimics what is seen in the ischemia-reperfusion injury. Experimentally, there is no universally accepted definition for CIH. Laboratory protocols vary greatly in duration of hypoxia exposure, numbers of hypoxia episodes per day and the total number of days of exposure. Despite the lack of a uniform definition, recent data suggest that CIH may lead to multiple long-term pathophysiologic consequences similar to what we see in patients with OSA. Recent evidences also demonstrate that there are remarkable differences in the response of the physiologic systems to sustained hypoxia and intermittent hypoxia. This review is aimed to briefly discuss the clinical significance of sleep-disordered breathing and our current understanding of CIH.     

Nitric oxide deficit in chronic intermittent hypoxia impairs large conductance calcium-activated potassium channel activity in rat hippocampal neurons

Sleep apnea associated with chronic intermittent hypoxia (IH) impairs hippocampal functions but the pathogenic mechanisms involving dysfunction of nitric oxide (NO) and ionic channels remain unclear. We examined the hypothesis that hippocampal NO deficit impairs the activity of large conductance calcium-activated potassium (BK) channels in rats with chronic IH, mimicking conditions in patients with sleep apnea. A patch-clamp study was performed on hippocampal CA1 neurons acutely dissociated from IH and control rats. The levels of endogenous NO and intracellular calcium in the CA1 region of the hippocampal slices were measured respectively by electrochemical microsensors and spectrofluorometry. We found that the open probability of BK channels remarkably decreased in the CA1 pyramidal neurons in a time-dependent manner with the IH treatment, without changes in the unitary conductance and reversal potential. NO donors, SNP or DETA/NO, significantly restored the activity of BK channels in the IH neurons, which was prevented by blockade of S-nitrosylation with NEM or MTSES but not by inhibition of the cGMP pathway with ODQ or 8-bromo-cGMP. Endogenous NO levels were substantially lowered in the IH hippocampus during resting and hypoxia. Also, the level of protein expression of neuronal NO synthase was markedly lessened in the IH neurons with decreased intracellular calcium response to hypoxia. Collectively, the results suggest that the IH-induced NO deficit mediated by a down-regulation of the expression of neuronal NO synthase plays a causative role in the impaired activity of BK channels, which could account for the hippocampal injury in patients with sleep apnea.