成年大鼠嗅球和鼻腔嗅粘膜成鞘细胞的分离、培养与鉴定

目的　在嗅球成鞘细胞 (OECs)移植到脊髓可有助于损伤神经纤维再生的基础上 ,分别对嗅球和嗅粘膜的OECs进行培养 ,探索自体嗅粘膜作为OECs供体的可能性。　方法　根据OECs、成纤维细胞和星形胶质细胞贴壁时间的不同 ,采用差时贴壁方法分离出OECs,培养 14div后进行NGFRp75和GDNF的免疫细胞化学染色。　结果　按形态学和免疫组织化学特性 ,培养的嗅球和嗅粘膜OECs可分为 3类 :双极细胞、三级细胞和扁圆细胞 ,其中以双极细胞最多。嗅粘膜的双极成鞘细胞的突起更加细长。　结论　差时贴壁细胞分离法是一种简单、经济、实用的成鞘细胞分离方法。鼻腔嗅粘膜OECs的形态学和免疫细胞化学特性与嗅球OECs基本相同 ,本实验为临床开展自体嗅粘膜OECs修复脊髓损伤的研究提供参考     

GDNF、GFAP和NGFRp75在成年大鼠和金黄地鼠嗅球成鞘细胞的免疫组织化学研究

目的　研究GDNF在成年大鼠和金黄地鼠嗅球成鞘细胞的表达 ,探索成鞘细胞在中枢神经再生中的作用。　方法　用免疫组织化学ABC法 ,显示GDNF在成年大鼠和金黄地鼠嗅球成鞘细胞的表达和分布 ,同时用NGFRp75和GFAP染色作为阳性对照。　结果　在成年大鼠和金黄地鼠嗅球的纤维层和小球层内均可见深棕色的GDNF免疫组织化学反应的成鞘细胞。在小球层与纤维层分界处和小球层与分子层分界处及嗅小球之间密集分布 ,在嗅小球之内较稀疏。同时在同一嗅球组织的另两组切片的相同部位 ,分别出现GFAP和NGFRp75免疫反应性细胞 ,间接说明GDNF免疫反应的结构是嗅球成鞘细胞。　结论　嗅球成鞘细胞含有胶质细胞源性神经营养因子     

预变性成年大鼠嗅神经后嗅球嗅鞘细胞的培养

目的:对预变性大鼠嗅神经后嗅球嗅鞘细胞的形态学及免疫组化进行研究,探讨获取自体激活嗅鞘细胞简单而实用的方法.方法:建立嗅神经切断模型,大鼠(12只)均在显微镜下暴露左侧嗅球,而预变性嗅神经组(6只)切断大鼠左侧嗅神经,3 d后取材采用差速贴壁纯化培养方法培养嗅鞘细胞,在不同时间进行NGFRp75 免疫组化染色鉴定及形态学观察.结果:嗅鞘细胞发生代偿性肥大,数量及纯度高,此方法成功培养出自体激活的嗅鞘细胞.结论:预变性嗅神经是获取成年大鼠自体激活嗅球嗅鞘细胞的简单而实用的方法.     

大鼠嗅鞘细胞体外培养及免疫组化研究

目的：建立体外纯化培养嗅鞘细胞（olfactory ensheathing cells，OECs）的稳定方法并观察培养嗅鞘细胞（OECs）形态学特征。方法：原代培养成年雄性SD大鼠嗅球的嗅神经层和颗粒层中提取的OECs，利用差速贴壁和阿糖胞苷（Ara-c）抑制法除去混杂的成纤维细胞和星形胶质细胞以获得纯化的OECs。用P75抗体免疫组织化学染色鉴定OECs，计算纯化度。结果：此方法获得的OECs纯度可达93％以上。OECs的外形主要以突起细长的双极和三极为主，或无突起呈“煎蛋样”，随时间延长细胞生长排列呈现一定方向性。结论：此方法稳定易复制，可获得高纯度的OECs。     

人胚嗅球嗅鞘细胞原代培养的实验研究

目的拟建立人胚胎嗅球嗅鞘细胞的培养方法并探讨这些细胞在活性、纯度方面是否具备临床应用水平。方法用含有促进细胞生长的谷氨酰胺、转铁蛋白、生物素、硒酸纳的无血清纯化液纯化嗅鞘细胞,胰蛋白酶消化收集细胞,制成单细胞悬液。采用台盼蓝染色法进行活性测定,P75NGFR和S-100双标免疫荧光染色鉴定,以Hoechst复染鉴定OECs的纯度。结果可见比较典型的嗅鞘细胞:双极或梭形、多突起形和扁圆形,主要以梭形和多突起形细胞为主。细胞活性大于95%,纯度大于90%。结论实验建立的人胚胎嗅球嗅鞘细胞原代培养的方法可行,细胞活性大于95%,纯度和均一性已达到临床应用水平。     

突触素在大鼠嗅球的分布

用免疫组织化学方法观察突触素在大鼠嗅球各层的分布。结果显示：小球层染色最深，其次为分子层，再其次是颗粒层，僧帽细胞胞体周围未见有明显阳性颗粒，纤维层和髓层不着色。     

经改良的取材和差速贴壁法培养纯化嗅黏膜嗅鞘细胞

目的:采用经改良的取材和差速贴壁法培养纯化嗅黏膜嗅鞘细胞(OECs),以探求更加简单、高效的嗅黏膜OECs取材和培养纯化方法.方法:成年雄性SD大鼠6只,采用经改良的方法剥取嗅黏膜,然后用改良差速贴壁法培养纯化嗅黏膜OECs.倒置相差显微镜下观察细胞生长情况以及形态并照相,培养7 d、14 d细胞行NGFRp75免疫细胞化学染色鉴定,并根据免疫细胞化学染色结果计算细胞纯度.结果:体外培养的嗅黏膜OECs形态主要有扁圆形或油煎蛋形、梭形或双极、多突起形.嗅黏膜OECs NGFRp75免疫细胞化学染色阳性.培养7 d嗅黏膜OECs纯度为90%,培养14 d嗅黏膜OECs纯度为85%.嗅黏膜OECs最长可以存活35 d.结论:经改良的取材和差速贴壁法培养纯化嗅黏膜OECs具有简单、高效的优点,培养的嗅黏膜OECs的纯度完全可以达到细胞移植的要求.     

突触素在大鼠嗅球的分布

用免疫组织化学方法观察突触素在大鼠嗅球各层的分布。结果显示:小球层染色最深,其次为分子层、再其次是颗粒层。僧帽细胞胞体周围未见有明显阳性颗粒,纤维层和髓层不着色。     

大鼠嗅球内成鞘细胞的形态特征和分布

嗅成鞘细胞(OECs)是嗅神经束外包裹的髓鞘细胞，是介于雪旺氏细胞和少突胶质细胞之间的一种独特的胶质细胞。近来许多学者的研究表明嗅成鞘细胞具有可以促进神经元轴突生长的功能。目的：利用Nissl、Luxol、Mallory三种经典染色法，观察成年大鼠嗅球内OECs的形态特征和分布，通过记数进行统计分析，     

中间丝蛋白vimentin在非洲爪蛙嗅球发育过程中的表达及意义

中间丝蛋白vimentin在嗅球发育中作用还没有被阐明,本研究目的是探讨vimentin在嗅球发育中时空模式及其意义。选择23～63期幼年和成年非洲爪蛙,做包含嗅神经和嗅球的连续冰冻切片,切片厚20μm,然后进行vimentin和胶质原纤维酸性蛋白(GFAP)免疫荧光染色,荧光显微镜观察。结果显示,vimentin在发育早期开始表达,一直维持整个变形期及成年。在49期以前,脑室前脚附近的放射性胶质细胞可见呈直的平行状的免疫反应。在变形前期和变形期,嗅球的每一层均可见到vimentin免疫反应,在嗅小球及僧帽细胞层vimentin阳性的放射胶质突起形态上呈丛状。在成年脑室附近的颗粒细胞层vimentin染色强度占优势,这反映了其在不同发育期的功能。结果提示,vimentin阳性的放射性胶质细胞,在早期及变形期可能为嗅神经元轴突的进入和嗅小球的形成提供适应的环境。而在成年,这种激活的胶质细胞可能与嗅球保持很强的再生能力有关。     

Ontogenesis of the axonal circuitry associated with the olfactory system of the rat embryo.

The prenatal development of axonal connections in the rat olfactory system was studied using DiI. On day 16 (E16), the olfactory and vomeronasal nerves extended from the olfactory epithelia to the olfactory bulb (OB), the terminal nerve to the telencephalic septum, while axons of mitral and tufted cells reached the anterior olfactory nucleus (AO). Axons from the AO were also seen in the anterior commissure. On day E16(8) (at 16 days, 8 h), axons were anterogradely followed from the dorsal OB through the lateral olfactory tract (lo) to the bed nucleus of the accessory olfactory tract. At E18(0), crystals implanted in the olfactory epithelium labeled the mitral cell layer and the lo.     

The fine structure of the Golgi tendon organ

Summary The Golgi tendon organ (GTO) has a capsule composed of cells confluent with the perineural epithelial sheath surrounding the Ib afferent nerve. Fluids of the capsule lumen are isolated from extra-capsular fluids by the capsule wall; its tight-fitting collars form seals through which collagen bundles enter and leave the proximal and distal ends of the fusiform capsule. The capsule lumen between the sealed capsule openings, is divided into longitudinal compartments by delicate processes of septal cells. Collagen bundles spiral down through the longitudinal axis of the GTO. As they descend through the lumen, the bundles are distinctly separated from each other by fluid-filled spaces. These collagen bundles divide, twist, and regroup over short longtiudinal distances and unmyelinated axons interwine among them. Compartments which are densely filled with collagen are not innervated. Axon profiles appear in four forms: (I) myelinated branches of Ib axon, (II) unmyelinated axons surrounded by Schwann cell processes and basal lamina, (III) axons covered only by basal lamina, and (IV) axons with bare surfaces. We postulate that increased tensile forces on the collagen bundles caused by muscle contraction tighten the braided collagen bundles thereby squeezing and distorting the axon terminals. The proposed mechanical events are consistent with known discharge characteristics of Ib axons.     

Innervation of pulmonary alveoli of the mouse lung: An electron microscopic study

The nerve supply to the alveolar ducts and alveoli of mouse lungs was studied with the electron microscope. In the alveolar ducts, nerve bundles consisting of as many as nine unmyelinated axons surrounded by connective tissue fibers were usually located in the interstitium surrounding the openings of the alveoli. In the alveolar walls, nerve bundles consisting of as many as five unmyelinated axons were located in the interstitium between pneumocytes and the capillaries. The unmyelinated axons contained neurotubules and some mitochondria, and were partially or completely surrounded by Schwann cells. Two distinct types of enlarged nerve endings were identified. The first type contained many small mitochondria and was either associated with the type I pneumocyte or located alone in the interstitium. The second type was packed with numerous large dense-core vesicles and was in close contact with the type II pneumocyte. It is suggested that the first type of ending is sensory and the second type is motor in function.     

A Golgi study on the olfactory bulb in the lamprey, Lampetra japonica

The intrinsic organization of the olfactory bulb in the lamprey was studied using the rapid Golgi method. Although not as discrete as in many vertebrates, a laminar organization was recognized. From the periphery inward, the following layers were discernible: the layer of the olfactory fibers, the olfactory glomeruli with the mitral cells, the granule cells, and the ependymal cells. Just beneath the surface of the olfactory bulb, the olfactory fibers extended over the entire bulb forming a dense fiber plexus terminating in the olfactory glomeruli which were arranged in one to two layers internally to the layer of the olfactory fibers. The mitral cells formed no discrete layer and were located mainly around the olfactory glomeruli. The mitral cells in the lamprey were lacking in secondary dendrites, but had two or more primary dendrites which terminated in the olfactory glomeruli. The axons of the mitral cells proceeded inwardly and accumulated diffusely in the granule cell layer which occupied a wide area internally to the layer of the olfactory glomeruli with the mitral cells. The granule cell layer was composed of densely packed small spindle or fusiform axonless cells, the processes of which extended superficially to be distributed in the olfactory glomeruli. At the deepest region of the bulb was a layer of the ependymal cells lining the surface of the olfactory ventricle. The external and internal plexiform layers were not evident. Thus, while the major constituents of the olfactory bulb of the vertebrate could be identified in that of the lamprey, the general laminar organization seemed indiscrete.     

Ultrastructure of the pineal body of the common vampire bat,Desmodus rotundus

The type AB pineal body of the common vampire bat, Desmodus rotundus was recessed and lobulated, was extensively vascularized and intimately related to great veins, and was unassociated with the epithalamic region. The habenular and the posterior commmissures coursed anteriorly and were unassociated with the pineal. The saccular suprapineal recess of the third ventricle extended dorsally juxtaposed to the pineal body. These anatomical features are likely to make pinealectomies in the vampire more difficult to manage. The pineal parenchyma consisted of light pinealocytes surrounded by canaliculi of various sizes, often transmitting unmyelinated nerve fibers and glial processes. Desmosomes were common. The pinealocyte nuclei were large and highly infolded; characteristic cytoplasmic constituents included abundant dilated Golgi complexes associated with clear vesicles, numerous polyribosomes, few single cisternae of ribosome-studded rough endoplasmic reticulum, mitochondria, and occasional multivesicular bodies and lysosomes. Almost all pinealocytes exhibited centrioles and some, in addition, displayed basal bodies but rarely ciliary shafts. A conspicuous feature of the pinealocyte cytoplasm was the presence of branched bundles of intermediate filaments, especially in the perinuclear zone. Siderotic macrophages, lipofuscin-pigment-containing phagocytic cells, mast cells, myelin bodies, and both fenestrated and continuous capillaries were present. The perivascular compartment was densely packed with unmyelinated nerve bundles containing small to large fibers exhibiting axoaxonic densities. Other constituents of the perivascular compartment were club-shaped pinealocyte processes filled with clear vesicles, microtubules, an occasional mitochondrion, glial processes, and collagen fibers. “Synapselike” contacts were observed between the axons and pinealocyte processes. Abundant pinocytotic vesicles in the capillary endothelium indicated active pinocytosis. Myelinated nerve fibers were lacking. The pineal ultrastructure of Desmodus is in part unlike that reported for other mammals, including bats.     

The functional organization of the fish olfactory system

Recent developments in the functional anatomy and physiology of the fish olfactory system reveal three parallel pathways from the sensory epithelium, via the olfactory bulb to the telencephalon. There are three morphological types of sensory neurones spread in a seemingly overlapping arrangement in the olfactory epithelium. The axons of each type of sensory neurones converge to a specific region of the olfactory bulb and connect to separate sets of relay neurones. The axons of these relay neurones leave in three bundles to the telencephalon. Each bundle conveys specific information that elicits sets of characteristic behaviour in response to odours involved in essential life processes in the fish. One pathway is tuned to social cues, another to sex pheromones, and the third to food odours.     

Differentiation of olfactory receptor cells in organ culture

Presumptive olfactory mucosa was excised from the heads of rat fetuses in the eleventh and twelfth days of gestation and explanted in organ cultures. At the time of explantation, the presumptive olfactory cells were recognizable by their long narrow apical processes and basally located nuclei. However at this stage they were in an early phase of differentiation as indicated by the large numbers of free ribosomes and virtual absence of microtubules and cilia in the apical cytoplasm. After three to eight days in culture, there was a progressive increase in the total number of cells in the epithelium. Differentiation in olfactory receptor cells was detectable by the appearance in the apical processes of axially oriented microtubules and centrioles or basal bodies. Some of which generated cilia. At their basal ends, the cytoplasm narrowed into axons and bundles of these axons, arranged in the unique manner of olfactory nerve axons, entered the connective tissue. Olfactory receptor cells, as defined by morphological criteria, differentiated under suitable organ culture conditions in the absence of any tissue from the central nervous system.     

The glial cells in the nerve fiber layer of the rat olfactory bulb

In mammals the olfactory receptor neurons are the only ones that are known to undergo continuous cell renewal in the adult animal. This means that the axon of each newly formed neuron must grow into the olfactory bulb to find its appropriate target cell. It is presumed that astrocytes ensheath the olfactory axons as they course through the nerve fiber layer of the bulb even though the cells in question differ ultrastructurally from typical astrocytes. The purpose of the present study was to examine the glial cells in the nerve fiber layer of the rat olfactory bulb in an effort to resolve this apparent discrepancy. Two morphologically distinct types of glial cell were found in the nerve fiber layer. One type, which resembled the typical astrocytes that are present in other areas of the cental nervous system, contained bundles of filaments in an electron-lucent cytoplasm. These cells also formed endfeet on blood vessels and formed part of the external glial limiting membrane. They did not, however, ensheath the olfactory axons. The cytoplasm of the other type of glial cell was denser than that of typical astrocytes and contained fewer filaments, which were seldom grouped into bundles. These cells also formed part of the glial limiting membrane at the surface of the bulb and were the only ones that ensheathed the olfactory axons. It is concluded that the cell ensheathing the olfactory axons in the nerve fiber layer of the rat olfactory bulb is a morphological variant of the typical astrocyte. One role of the former cell may be to support or encourage the growth of olfactory axons within the central nervous system.     

Evidence for a Notch1‐mediated transition during olfactory ensheathing cell development

Olfactory ensheathing cells (OECs) are a unique glial population found in both the peripheral and central nervous system: they ensheath bundles of unmyelinated olfactory axons from their peripheral origin in the olfactory epithelium to their central synaptic targets in the glomerular layer of the olfactory bulb. Like all other peripheral glia (Schwann cells, satellite glia, enteric glia), OECs are derived from the embryonic neural crest. However, in contrast to Schwann cells, whose development has been extensively characterised, relatively little is known about their normal development in vivo. In the Schwann cell lineage, the transition from multipotent Schwann cell precursor to immature Schwann cell is promoted by canonical Notch signalling. Here, in situ hybridisation and immunohistochemistry data from chicken, mouse and human embryos are presented that suggest a canonical Notch‐mediated transition also occurs during OEC development.