姜黄素预防高原缺氧大鼠认知功能障碍的机制研究

目的探讨姜黄素（Curcumin）预防高原缺氧大鼠认知功能障碍的机制。方法将24只成年雄性sD大鼠随机分为健康对照组、模型组（model组）、姜黄素[按体重60mg／（kg·d）]防治组（curcumin组）。造模后,给予姜黄素防治,采用Morris水迷宫实验方法检测各组大鼠学习记忆功能变化,并运用Westernblot技术检测海马Bcl-2、Bax及活化的caspase-3等凋亡相关蛋白表达水平的变化。结果①模型组大鼠寻找平台的潜伏期明显长于对照组,具有统计学意义（P〈0．05）,姜黄素组寻找平台的潜伏期相对于模型组显著缩短（P〈0．05）。撤离平台后,模型组大鼠在平台所在象限的停留时间明显短于对照组（P〈0．05）,姜黄素干预后大鼠在平台所在象限的停留时间较模型组显著延长（P〈0．05）;②姜黄素可显著减轻慢性缺氧导致的Bax、活化的easpase．3表达水平升高级Bcl-2表达水平下降。结论姜黄素可显著改善高原缺氧大鼠认知功能障碍,其机制可能和减轻海马神经元的凋亡有关。     

低压低氧预适应对大鼠急性重复缺氧耐受的影响

目的研究低压低氧预适应(HHP)对SD大鼠缺氧耐受的影响。方法雄性SD大鼠随机分为对照组及HHP组,分别检测对照组以及HHP后1、3、5和7d时大鼠急性重复缺氧耐受时间以及致死性缺氧死亡时间,并进行比较分析。结果急性重复缺氧耐受实验检测发现,HHP后大鼠出现喘式呼吸的时间较对照组有所延长,其中HHP后第3天,HHP处理组大鼠出现喘式呼吸时间明显延长(P<0.05);致死性缺氧时间HHP处理组与对照组比较无显著差异(P>0.05)。结论 HHP在一定程度上能够改善大鼠耐受缺氧的能力,尤其是在HHP后第3天最为明显。     

Acute ultrastructural response of hypoxic hypoxia with relative ischemia in the isolated brain

Summary The acute cortical response to surgical brain isolation and subsequent extracorporal normoxic or 30 min hypoxic (PaO₂=20 mm Hg) perfusions (hypoxic hypoxia with relative ischemia) was evaluated. Cerebral blood flow, arterial pH and CO₂ were maintained constant during both perfusions; only the arterial oxygen content was changed. The isolated brain model used in this and previous investigations produces no qualitative ultrastructural changes in the neocortex following brain isolation and normoxic perfusion. However, the acute cortical structural response to 30 min of hypoxic hypoxia with relative ischemia demonstrated a number of important observations. Hypoxic hypoxia produced ultrastructural responses common to cerebral ischemia such as nuclear chromatin clumping, nucleolar condensation and cytoskeletal breakdown. Although neuronal abnormalities seen after 30 min of hypoxic hypoxia were similar to those acute neuronal changes observed following complete cerebral ischemia without recirculation, they differed three ways: (a) mitochondrial swelling and microvacuolation were observed in many cortical pyramidal neurons. (b) Glycogen particles within astroglial processes were observed even after a 30-min period of hypoxic hypoxia. (c) Perivascular astroglial swelling was minimal despite considerable perineuronal swelling. In contrast, incomplete cerebral ischemia produces mitochondrial changes similar to those in hypoxic hypoxia but also causes the depletion of tissue glycogen and perivascular glial swelling. Thus, hypoxic hypoxia with relative ischemia produces a unique acute ultrastructural response compared to either complete or incomplete cerebral ischemia.Supported by an NIH grant NS05961     

Ultrastructural investigation of the CA1 region of the hippocampus after transient cerebral ischemia in gerbils

Summary Ultrastructural damage leading to delayed neuronal death was investigated in the mid-CA1 region of the hippocampus from the stratum (str.) moleculare to oriens after transient bilateral forebrain ischemia in Mongolian gerbils. After ischemia for 5 min without recirculation, mild swelling of the peripheral part of the apical and basal dendrites was already apparent in the str. moleculare and str. oriens. Mitochondria in the dendrites were also swollen in the same area. During recirculation for 12 h to 3 days, swelling of the dendritic cytoplasm persisted with formation of microvacuoles, but swelling of mitochondria receded. Microvacuolation and loss of microtubules were also observed in the proximal part of the dendrites during this period, and swelling and disruption of internal cristae were observed in mitochondria after recirculation for 3 days. The dendrites became severely degenerated after recirculation for 4 days. In the pyramidal cell bodies, no abnormality was observed at the end of ischemia for 5 min, but disaggregation of polyribosomes and swelling of the endoplasmic reticulum were observed 12 h after recirculation. Proliferation of the endoplasmic reticulum in parallel arrays occurred after recirculation for 1 day and persisted. Severe degeneration of the pyramidal cell bodies was obvious after recirculation for 4 days. The findings observed in the present investigation suggested that the neuronal structure most vulnerable to ischemia was the peripheral part of the dendrites and postischemic neuronal damage occurred early in this part of the dendrites.Supported by the grant NS-06663 from the National Institutes of Health, U.S. Public Health Service     

Ultrastructural analysis of vascular features in cerebral cavernous malformations

Objective

Investigation of the structure of vascular malformations highlights the pathogenic mechanisms underlying their clinical behavior. One of the vascular malformations is called cerebral cavernous malformation (CCM). However, the ultrastructural features of the vascular malformations are not defined in detail.

Methods

We aimed to investigate the ultrastructural features of CCMs using transmission (TEM), scanning (SEM) electron microscopy, and also immunohistochemistry methods with antibodies against CCM proteins such as CCM2 and CCM3. CCM tissues (n = 6) microsurgically excised from patients for conventional indications.

Results

CCM2 and CCM3 were strongly detected in the vascular endothelium. However, there was a very weak immunostaining in stroma. SEM observations revealed that there were ruptures and damages in the luminal endothelium, possibly due to the damage of intercellular junctions. TEM observations also showed a few ruptures and detachments between the endothelium and basal lamina as observed with partially damages and disconnections. The architecture of pericytes showed protrusions and shrinkages. Our results suggest that the thin vessel walls of CCMs were lacking of subendothelial support and intact basal lamina underlying the endothelial cells.

Conclusion

This study is so far the first study attempting to show human CCM lesions with SEM. We believe that an understanding of the ultrastructural features of these lesions by light and electron microscopy techniques would help to understand the pathology of these diseases.     

Hypobaric hypoxia modifies constitutive nitric oxide synthase activity and protein nitration in the rat cerebellum

Ischemic hypoxia provokes alterations in the production system of nitric oxide in the cerebellum. We hypothesize that the nitric oxide system may undergo modifications due to hypobaric hypoxia and that may play a role in high altitude pathophysiology. Therefore, changes in the nitric oxide system of the cerebellum of rats submitted to acute hypobaric hypoxia were investigated. Adult rats were exposed for 7 h to a simulated altitude of 8235 m (27000 ft.) and then killed after 0 h or 1, 3, 5 and 10 days of reoxygenation. Nitric oxide synthase calcium-dependent and -independent activity, immunoblotting and immunohistochemistry of neuronal, endothelial, and inducible nitric oxide synthase, and nitrotyrosine were evaluated. Immunoreactivity for neuronal nitric oxide synthase slightly increased in the baskets of the Purkinje cell layer and in the granule cells, after 0 h of reoxygenation, although no changes in neuronal nitric oxide synthase immunoblotting densitometry were detected. Calcium-dependent activity significantly rose after 0 h of reoxygenation, reaching control levels in the following points, and being coincident with a peak of eNOS expression. Nitrotyrosine formation showed significant increments after 0 h and 1 day of reoxygenation. Nitrotyrosine immunoreactivity showed an intracellular location change in the neurons of the cerebellar nuclei and in addition, an appearance of nitration in the soma of the Purkinje cells was detected. No changes in inducible nitric oxide synthase activity, immunoblotting or immunohistochemistry were detected. We conclude that at least part of the nitric oxide system is involved in cerebellum responses to hypobaric hypoxia.     

Humanin通过保护线粒体功能对缺氧诱导的神经元损伤发挥保护作用

BACKGROUND： Humanin is a 24-amino acid peptide isolated from the brain of an Alzheimer’s disease patient. Several studies have indicated that Humanin can protect cells against cytotoxicity induced by various insults.
OBJECTIVE： To investigate the protective role of Humanin on hypoxia-induced neuronal death, and to determine the most appropriate therapeutic concentration of Humanin.
DESIGN, TIME AND SETTING： Neuropathophysiological, randomized, controlled experiment, conducted at the Department of Physiology and Neurobiology, Shanxi Medical University, between March 2007 and October 2007.
MATERIALS： Newborn Wistar rats, 5,5＇,6,6＇ tetrachloro-1,1＇,3,3＇-tetraethyl- benzimidazolylcarbo- cyanine iodide （JC-1, USA）, calcein-acetoxymethylester （calcein-AM, USA）, and Humanin （Shanghai, China） were used in this study. METHODS： Primary cortical neurons were cultured with dulbecco＇s modified eagle＇s medium containing 15% fetal bovine serum. Cultures were divided into three groups： control, hypoxia, and hypoxia ＋ Humanin. Various concentrations of Humanin （1, 10, and 20 μmol/L） were added to the cultures 16 hours prior to hypoxia induction. For hypoxic conditions, cells were maintained at 37 ℃ within an incubator chamber filled with 95% N2 and 5% CO2 for 24 hours. Cells in the control group were cultured in normal oxygen.
MAIN OUTCOME MEASURES： Cell viability was determined through the use of the vital dye calcein-AM, and the number of live cells was determined. Mitochondrial membrane potential （△Ψm） was assessed using the fluorescent probe JC-1. Mitochondrial permeability transition pore （mPTP） opening was determined with calcein-AM in the presence of cobalt chloride.
RESULTS： （1） Cell viability： Hypoxia for 24 hours induced death in a large number of neurons. Pre- treatment with 10 μmol/L and 20 μmol/L Humanin, 16 hours prior to hypoxia, protected cells against hypoxia. However, 1 μmol/L Humanin provided little protection. （2） △Ψm： △Ψm was re-duced after 24-hour hypoxia, as assessed by JC-1 and a confocal microscope. Pretreatment with 20 μmol/L Humanin preserved the loss of △Ψm. （3） mPTP： Hypoxia induced the opening of mPTP. Pretreatment with 20 μmol/L Humanin repressed the opening of mPTP, as most of the calcein fluorescence remained in the mitochondria.
CONCLUSION： Humanin （20 μmol/L） protects neuronal cells from hypoxia-induced insults by in- hibiting the opening of mPTP and preserving △Ψm.     

Ultrastructural study of medullomyoblastoma

Summary A case of cerebellar medullomyoblastoma in a young boy was investigated by electron microscopy. The neuroectodermal component shows the characteristics of a desmoplastic medulloblastoma. The mesodermal component consists of more or less differentiated cross-striated muscle cells. Undifferentiated muscle cells are very similar to proliferated endothelial cells of blood vessels within the muscular component, so that an origin of this component from pluripotential endothelial cells of the vessel wall is suggested. This tumor is considered a malignant teratoid because of the derivation from two blastodermic layers and because of the midline localization in children suggesting a malformative origin.     

Ultrastructural aspects of myogenesis found in neoplasms

Summary The ultrastructure of myogenic cells occurring in neoplasms was investigated and aspects of differentiation (myofilament interactions and organization, and sarcotubular development) were characterized. The stages of muscle differentiation present were extremely similar to those reported during human fetal development prior to innervation. Exceptions, however, being: (1) the presence of fewer multinucleated cells; (2) the general lack of cell elongation and its apparent effect on myofibril orientation; and (3) evidence of a higher number of myofilaments in mononucleated cells. The findings were compared to those reported in normal human fetal development, human myogenic cells in vitro and the literature on mammalian and avian muscle development and discussed with regard to the influence of tension and innervation. The significance of degenerating myogenic cells found in these neoplasms is also discussed.     

脑梗死急性期血小板超微结构的变化及意义

目的：探讨脑梗死急性期血小板超微结构的变化及其意义,方法：采用透射电镜对20例脑梗死急性期患者的血小板进行观察,结果：发病后24小时内的血小板超微结构改变明显,表现为伪足增多,聚集融合成片状;α颗粒明显减少;线粒体也显著减少,残留者肿胀,空泡化;血小板膜多处破裂;血小板病变的严重程度与脑梗死病灶的大小相一致。发病后2周血小板超微结构明显恢复,但3例面积脑梗 者未见恢复。结论脑梗死急性期血小板超微结构发生明显改变,尤其是α颗粒明显减少,可作为判断病情轻重及预后的一个客观指标。     

新生大鼠缺氧缺血脑损伤后bcl-x mRNA的表达

目的　探讨新生大鼠脑缺氧缺血后死亡相关基因 bcl- x m RNA表达的变化及其与脑缺氧缺血所致细胞凋亡的关系。方法　通过建立新生大鼠缺氧缺血性脑病动物模型 ,应用快速竞争性 RT- PCR技术对缺氧缺血后不同时间点的缺血侧大脑组织中 bcl- x m RNA的表达进行半定量分析 ,并在相同缺血基础上观察缺氧 1.5小时、2 .5小时和 3.5小时对 bcl- x m RNA表达的影响。结果　缺氧缺血后 ,新生大鼠脑 bcl- xs(bcl-x短型 ) m RNA的表达自缺氧结束后即刻即有明显增强 ,2 4小时达高峰 ,此后逐渐下降 ,7天时回复至正常基线水平。随着缺氧时间的延长即缺氧程度的加重 ,bcl- xs m RNA的表达有增强趋势 ,缺氧 1.5小时组、2 .5小时组及 3.5小时组之间 bcl- xs m RNA表达水平的差异均具显著性意义 (P<0 .0 1)。bcl- xs m RNA的表达高峰与缺氧缺血后脑细胞凋亡的高峰时相相吻合。缺氧缺血对 bcl- xl(bcl- x长型 ) m RNA的表达无影响。结论缺氧缺血可诱导新生大鼠脑 bcl- xs m RNA表达增强。在一定范围内 ,其表达强度与缺氧程度成正相关。 bcl-xs过表达在缺氧缺血后脑细胞凋亡的调控过程中起着一定的作用     

Ultrastructural relationships between noradrenergic nerve fibers and non-neuronal elements in the rat cerebral cortex

Pharmacological and biochemical data suggest that noradrenaline (NA)-containing fibers not only regulate the activity of cortical neurons but also influence the functional state of non-neuronal elements. In the present study, immunocytochemistry with an antiserum against NA, followed by silver-gold intensification of the immunoreaction end-product, was employed to examine the ultrastructural relationships between the NA fiber system and the intraparenchymal blood vessels, oligodendrocytes, and astrocytes in the rat visual cortex. Electron microscopy revealed a large number of fine varicose NA fibers to be in intimate contact with cortical capillaries. Examination of single thin sections showed that NA boutons were usually separated from the capillary wall by a fine astroglial sleeve. However, serial section analysis revealed that the continuity of the astrocytic end feet was interrupted at sites, resulting in direct apposition of the perivascular NA fibers to the capillary basal lamina. Noradrenergic fibers were found to contact both types of macroglial cells. Single or clustered oligodendrocytes in intimate contact with NA fibers were observed throughout the cortical depth. Individual contacts could be followed in more than six successive thin sections, and oligodendrocyte plasma membrane frequently exhibited a light thickening at the sites of the NA fiber apposition. NA fiber-astroglial relationships were largely encountered in supragranular layers. In these layers, astrocytic cell bodies were characteristically outlined by fine varicose NA fibers. However, no plasma membrane differentiations were observed at the sites of intimate NA fiber apposition. The present ultrastructural findings provide the anatomical substrate for the control exerted by the NA fiber system over cortical microvasculature and macroglia. © 1996 Wiley-Liss, Inc.     

Ultrastructural concomitants of anoxic injury and early post-anoxic recovery in rat optic nerve

To study the effects of anoxia on CNS white matter, we examined the ultrastructure of axons and glial cells in a white matter tract, the rat optic nerve, that was subjected to a standardized anoxic insult in vitro. Previous electrophysiological studies showed that in this model, action potential conduction is rapidly abolished by anoxia, and conduction is restored after reoxygenation in about 30% of axons following a 60-min anoxic period. The present study examined the ultrastructural correlates of anoxic injury and early post-anoxic recovery in this model. Optic nerves examined immediately following 60 min of anoxia displayed numerous large, apparently empty zones located within myelin sheaths adjacent to the axon. The myelin remained compact and retained its periodicity. In some regions, the extracellular space was enlarged. There was mitochondrial swelling with loss of normal cristae. There was also loss of microtubules and, to a smaller degree, of neurofilaments in large-diameter axons. Some nodes of Ranvier in anoxic optic nerves displayed detachment of terminal oligodendroglial loops or retraction of the myelin from the node; the presence of tongue-like processes, extending from nearby cells under the detached myelin loops, suggested a possible role of cell-mediated damage to the paranodal myelin. Bundles of dense astrocyte processes were present, and there was vesicular degeneration of perinodal astrocyte processes. In optic nerves that had been permitted to recover for 60 min in oxygenated Ringers following the anoxic period, empty zones were only rarely observed within myelin sheaths and, when present, were smaller than in optic nerves immediately following 60 min of anoxia. The axoplasm of large fibers continued to show loss of microtubules and neurofilaments, as well as mitochondrial swelling. Myelin appeared normal, and only rare paranodal oligodendroglial processes remained unattached from the axon membrane. These results provide support for the idea that, during anoxia, myelinated axons are damaged with significant injury to cytoskeletal elements, probably due to an influx of calcium. The ultrastructural results, together with our earlier observations on the physiological correlates of anoxia and re-oxygenation, suggest that the development of intramyelinic spaces or damage to paranodes lead to conduction block in the anoxic optic nerve. These results also suggest that repair of these structural abnormalities may provide a morphological basis for the early recovery of conduction that occurs after re-oxygenation.     

Ultrastructural and cytochemical characteristics of cultured rat Schwann cells

Summary Schwann cell cultures were established from sciatic nerve of 3 day-old rats. Described are the ultrastructural, histochemical and ultracytochemical properties of amyelic cultured rat Schwann cells. Ultrastructural characteristics of the cultured Schwann cells are compared to the Schwann cells of 3 day-old and adult rat sciatic nerve. These findings serve as a basis for comparison when studying experimentally induced alterations in the cultured Schwann cells as well as changes due to myelination in vitro.     

Ultrastructural pathology in emetine-induced myopathy

Summary Progressive myopathy developed in two women who consumed ipecac syrup containing emetine hydrochloride to induce vomiting as part of their anorexia nervosa. Muscle biopsy specimens were characterized by severe disruption of the sarcomeres. The ultrastructural spectrum extended from Z-band streaming to the formation of cytoplasmic bodies and also comprised abnormalities of the sarcotubular system, thus suggesting that muscle weakness may be related to both sarcomeric and sarcotubular lesions in this self-inflicted myopathy. It is tempting to suggest that muscle weakness may be correlated with or based on the pathology in sarcomeres and the sarcotubular system. As the myopathy is clinically reversible upon discontinuation of ipecac consumption the morphological findings should also be potentially reversible. Experimentally induced emetine myopathy may, thus, serve as a useful model to study morphological dynamics of sarcomeric lesions, which may be observed separately or simultaneously in a variety of spontaneously occurring human neuromuscular disorders.This study was presented at the 1986 Annual Meeting of the Deutsche Gesellschaft für Neuropathologie und Neuroanatomie, Mainz, Federal Republic of Germany     

SDF-1 activity on microvascular endothelial cells: consequences on angiogenesis in in vitro and in vivo models

The chemokine stromal cell-derived factor-1 (SDF-1) has been shown to be involved in cell migration. As the receptor CXCR-4 is expressed on endothelial cells and upregulated by angiogenic factors, we were prompted to study the effect of SDF-1 on angiogenesis in endothelial cells from microvasculature. This study demonstrates that SDF-1 induces an angiogenic effect in vitro, primarily in a tridimensional fibrin gel. The increase in capillary tube formation was evident after a 10-day incubation with SDF-1. This was associated with a mild increase in VEGF production by microvascular endothelial cells (ELISA and rt-PCR) and a potent chemotactic effect. SDF-1 also induced an in vivo angiogenic activity as shown in the model of the rabbit corneal pocket. However, the angiogenesis was located in an area rich in inflammatory cells. The results of our study suggest that these data underline the potential role of SDF-1 in angiogenesis as the microvascular endothelial cells were greatly involved in this process.     

Ultrastructural changes in skeletal muscle in ovine muscular dystrophy

Summary The initial ultrastructural changes in skeletal myofibers in ovine muscular dystrophy (MD) consisted of focal degeneration of myofibrils and the formation of Z-disc abnormalities, including nemaline rods, in adjacent sarcomeres. Peripheral and central sarcoplasmic masses, which occurred initially in large diameter fibers, contained a mixture of normal organelles and abnormal tubular and fibrillar formations. Vesiculate sarcolemmal nuclei with prominent nucleoli accumulated in central and subsarcolemmal locations in small clusters and short rows. Deformed individual nuclei were sometimes present within nuclear rows. Loss of the myofibrillar mass, increased density of small spherical nuclei, collections of fibrillar and tubular arrays, excessive folding of the sarcolemma and greatly reduced fiber diameter were seen in the end stage of the dystrophic process. Resting satellite cells were present at all stages of lesion development. The morphological progression of the lesions suggested an inherited inability to effectively replace lost myofibrils with ultimate exhaustion of the capacity for repair followed by pathological fiber atrophy.Supported in part by the Muscular Dystrophy Research Association of Western Australia     

Ultrastructural studies on the neuromuscular junctions of Becker's muscular dystrophy

Summary Ultrastructural studies on muscle biopsies from three patients with Becker's muscular dystrophy showed that the i.m. nerves presented loss or disarrangement of the neurofilaments and an increased number of glycogen granules and/or myelin figures not infrequently in the myelinated and unmyelinated nerve fibers. The neuromuscular junctions showed markedly widened sole-plate areas, and several terminal axons frequently abutted and formed neuromuscular junctions on the same fiber. The secondary synaptic clefts were markedly decreased in number and short in length in type I fibers but not in type II fibers. Most terminal axons showed no degenerative changes. Therefore, the participation of a neural factor might be suggested as the cause of Becker's muscular dystrophy, although it does not mean denervation in the conventional sense of an axonal degeneration.Supported by grant no. 83-02-31 from NCNMMD of the Ministry of Health a nd Welfare, Japan     

Architectural alterations in rat cerebral microvessels after hypobaric hypoxia

We performed 3-dimensional studies of vascular casts of the microvasculature of the cerebral cortex of rats that were exposed to three weeks of hypobaric hypoxia and of control rats. Scanning electron microscopy of the casts gave the qualitative impression of increased vascularity of the cerebral cortex, particularly the deeper layers, in hypoxic rats. Quantitative analysis of capillary segment lengths revealed a significant shift in the frequency distribution to longer lengths (from 77 ± 8 to 90 ± 14 μm) in the deep, but not in the superficial, layers of the cerebral cortex of hypoxic rats. These findings agree with previous results reporting increased capillary density in the brain after exposure to prolonged hypobaric hypoxia and suggest that capillary segment elongation plays a role in the increased capillary density in the deeper layers of the cerebral cortex.     

Elevated plasma S100B levels in high altitude hypobaric hypoxia do not correlate with acute mountain sickness

Abstract

Objectives:

Ascent to high altitude may result in a hypobaric hypoxic brain injury. The development of acute mountain sickness (AMS) is considered a multifactorial process with hypoxia-induced blood–brain barrier (BBB) dysfunction and resultant vasogenic oedema cited as one potential mechanism. Peripheral S100B is considered a biomarker of BBB dysfunction. This study aims to investigate the S100B release profile secondary to hypoxic brain injury and comment on BBB disturbance and AMS.

Methods:

A prospective field study of 12 subjects who ascended Mt Fuji (3700 m) was undertaken.

Results:

The mean baseline plasma S100B level was 0·11 μg/l (95% CI 0·09–0·12), which increased to 0·22 μg/l (95% CI 0·17–0·27) at the average of three high altitude levels (2590, 3700, and 2590 m on descent) (P < 0·001). The mean level for the seven subjects who experienced AMS rose from 0·10 to 0·19 μg/l compared to 0·12 to 0·25 μg/l for the five subjects who did not develop AMS (P = 0·33).

Conclusion:

Ascending to 3700 m resulted in elevated plasma S100B levels but this was not associated with AMS.