Immunocytochemical localization of three vesicular glutamate transporters in the cat retina

Vesicular transporters play an essential role in the packaging of glutamate for synaptic release and so are of particular importance in the retina, where glutamate serves as the neurotransmitter for photoreceptors, bipolar cells, and ganglion cells. In the present study, we have examined the distribution of the three known isoforms of vesicular glutamate transporter (VGLUT) in the cat retina. VGLUT1 was localized to all photoreceptor and bipolar cells, whereas VGLUT2 was found in ganglion cells. This basic pattern of complementary distribution for the two transporters among known populations of glutamatergic cells is similar to previous findings in the brain and spinal cord. However, the axon terminals of S-cone photoreceptors were found to express both VGLUT1 and VGLUT2 and some ganglion cells labeled for both VGLUT2 and VGLUT3. Such colocalizations suggest the existence of dual modes of regulation of vesicular glutamate transport in these neurons. Staining for VGLUT2 was also present in a small number of varicose processes, which were seen to ramify throughout the inner plexiform layer. These fibers may represent axon collaterals of ganglion cells. The most prominent site of VGLUT3 immunoreactivity was in a population of amacrine cells; the axon terminals of B-type horizontal cells were also labeled at their contacts with rod spherules. The presence of the VGLUT3 transporter at sites not otherwise implicated in glutamate release may indicate novel modes of glutamate signaling or additional roles for the transporter molecule.     

Distribution of monoamine-synthesizing neurons in the human medulla oblongata

We have employed immunohistochemical and morphometric procedures to study the distribution of monoamine-synthesizing neurons in the medulla oblongata of the adult human, utilizing antibodies to tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), and phenylalanine hydroxylase (PH8). In the human brain, the antigen with which PH8 reacts occurs within neurons that presumably synthesize serotonin (Haan et al., '87). Neurons containing these antigens were mapped and counted in successive coronal sections with the aid of a computer-assisted procedure. The results indicate that monoamine-synthesizing neurons are distributed in the human brain in patterns broadly similar to those described for other species. TH-immunoreactive cells extended caudorostrally for approximately 32 mm commencing at the spinomedullary junction and ending 8 mm caudal to the pontomedullary junction. In coronal sections these TH-immunoreactive neurons were seen in the lateral medulla dorsal to the inferior olive extending in a continuous band to the dorsomedial medulla. Above the obex the majority of these cells apparently synthesize adrenaline since many PNMT-immunoreactive cells were also found in this region. There were few or no PNMT-immunoreactive cells caudal to the obex, indicating that the TH-immunoreactive cells in this region synthesize either noradrenaline or dopamine. Approximately 65% of these TH-immunoreactive neurons contained melanin pigment, whereas few or no PNMT-immunoreactive cells contained melanin pigment. PH8-immunoreactive cells extended throughout the rostrocaudal extent of the medulla oblongata (approximately 40 mm). In coronal sections the majority were found in the medullary raphe nuclei. However, many cells throughout the rostrocaudal extent of the medulla were found laterally intermingled with catecholamine-synthesizing neurons. Occasional neurons in the lateral medulla appeared to contain both PH8- and TH-immunoreactivity.     

血管组织工程种子细胞在再生医学中的应用*☆◇

背景：小口径人工血管替代人体小动脉和静脉一直未获得满意的效果，因此研制出一种拥有较高远期通畅率的小口径人工血管成为了一个重要的研究课题。 目的：综述种子细胞在血管组织工程的研究进展。 方法：以 “Vascular tissue engineering, Seeding cells”为检索词，应用计算机检索Pubmed 数据库1960/2009有关文章。纳入有关血管组织工程种子细胞的文献。排除原始文献设计方法简单、结果可靠性差、非英文文献及结果重复的文献，保留35篇文献做进一步分析。 结果与结论：内皮细胞和平滑肌细胞是目前常用的种子细胞。内皮细胞和平滑肌细胞共同培养的体系，模拟体内环境，保持内皮细胞和平滑肌细胞具有正常的分泌功能和表型。骨髓间充质细胞可被有效的分离和扩增，在特定培养条件下可以诱导分化为多种血管细胞。在再生医学和生物组织工程方面有强大的潜力。     

Cellular and subcellular distribution of NMDA receptor subunit NR2B in the retina

Immunocytochemical studies showed the presence of staining for the N-methyl-D-aspartate (NMDA)-R2B glutamate receptor subunit at multiple sites in the cat retina. Reaction product in photoreceptor cells was localized at the inner/outer segment junction and in the axon terminals. Staining within the inner retina was limited to ganglion cells and their dendrites ramifying throughout the inner plexiform layer. These cells were seen to receive synaptic input from cone bipolar cells in both sublaminae. As with other glutamate receptor subunits, this immunoreactivity was typically confined to a single postsynaptic element at a cone bipolar dyad complex. Immunocytochemical localization of the NMDA-R1 subunit, considered to be an essential component of functional receptors, showed a widespread distribution across the retina including all the sites where NMDA-R2B staining was seen. Immunoprecipitation and Western blot analysis were used to confirm the presence of the NR2B receptor protein and its association with the NR1 subunit in both proximal and distal retinal layers. The findings suggest that NMDA-R2B subunits are positioned for multiple functions within the retina.     

Stem cell and precursor cell therapy

Strategies for cell replacement therapy have been guided by the success in the hematopoietic stem cell field. In this review, we discuss the basis of this success and examine whether this stem cell transplant model can be replicated in other systems where stem cell therapy is being evaluated. We conclude that identifying the most primitive stem cell and using it for transplant therapy may not be appropriate in all systems. We suggest alternative strategies such as progenitor cell replacement, inductive factors, bioengineering organs, in utero transplants, or any approach that takes advantage of the unique properties of the tissue and the stem cell type which, are more likely to provide effective functional replacement.     

GABA-like immunoreactivity in the macaque monkey retina: a light and electron microscopic study

The distribution of GABA-like immunoreactivity in the macaque monkey retina was studied by using postembedding techniques on semithin and ultrathin sections. At the light microscopic level, both inner and outer plexiform layers showed strong GABA-like immunoreactivity in the central retina. All the horizontal cells, some bipolar cells, 30-40% of amacrine cells, occasional interplexiform cells, and practically all displaced amacrine cells were labeled. In the peripheral retina (beyond 5 mm eccentricity), the outer plexiform layer and the horizontal cells were not labeled, but all other cell types showed the same labeling pattern as in the central retina. Synapses of the inner plexiform layer involving a pre- or postsynaptic GABA-labeled process were studied electron microscopically. Synapses involving a GABA-labeled presynaptic amacrine cell process made up 80% of the synapses observed. These GABA-labeled amacrine processes synapsed onto amacrine, bipolar, and ganglion cell processes as well as onto amacrine and ganglion cell bodies. Synapses involving a postsynaptic GABA-labeled process made up 20% of the synapses studied. The GABA-like immunoreactive processes were postsynaptic to bipolar cells at the dyads and to amacrine cells at conventional synapses.     

Distribution of neuropeptidelike immunoreactivities in the guinea pig olfactory bulb

The distribution of neuropeptidelike immunoreactivities in the adult guinea pig olfactory bulb was studied immunohistochemically with antisera raised against neurotensin (NT), substance P (SP), methionine-enkephalin-Arg6-Gly7-Leu8 (ENK), somatostatin (SOM), neuropeptide Y (NPY), and cholecystokinin-8 (CCK). In the main olfactory bulb, NT-like immunoreactive (NT-IR) neurons were found among periglomerular cells. In addition, a few periglomerular cells showed ENK-like immunoreactivity. Granule cells displaying SP- or ENK-like immunoreactivities and short axon cells with SOM- or NPY-like immunoreactivities were observed in the deeper half of the granule cell layer. SOM-IR short axon cells were also seen in the external plexiform layer. Dense NT- or NPY-IR fibers were distributed in superficial lamina of the granule cell layer, and sparse SP- or CCK-IR fibers were found in the glomerular layer. In the accessory olfactory bulb, some mitral, periglomerular, and granule cells showed NT-like immunoreactivity. SP- or ENK-IR granule cells were also observed. These results are discussed in relation to laminar organization of the olfactory bulb. The most characteristic features of peptide distribution in guinea pigs, as compared with that of rats in previous studies, were the relative abundance of NT-IR structures and the lack of SP- and CCK-IR juxtaglomerular and tufted cells.     

人骨髓间充质干细胞向多巴胺神经元分化的体外研究

目的探讨人骨髓间充质干细胞(hMSC)向神经元和多巴胺神经元分化的潜能。方法分离和纯化hMSCs;在体外以WHI-P131预处理和碱性成纤维细胞生长因子预诱导后,全反式维甲酸和胶质细胞源性神经营养因子联合诱导hMSCs向神经元和多巴胺神经元分化。光镜下观察其分化过程中hMSCs的形态变化,免疫组化检测诱导前后细胞是否表达神经元和多巴胺能神经元标志蛋白。结果诱导后的hMSCs能分化成为具有典型神经元形态的细胞,并明显表达抗人神经巢蛋白(nestin)[(54.2±3.7)%]和神经元特异性烯醇化酶(NSE)[(77.0±5.7)%],低表达胶质纤维酸性蛋白(GFAP)[(8.8±2.4)%];对照组细胞这些表达均为阴性;而且相当部分hMSCs表达酪氨酸羟化酶(TH)[(36.5±15.8)%]和多巴胺转运体(DAT)[(26.0±14.2)%]。结论在适当条件下,hMSCs可分化成为神经元样细胞和多巴胺神经元样细胞。     

大鼠骨髓间充质干细胞体外诱导为肝细胞样细胞***

背景：目前体外诱导骨髓间充质干细胞分化为肝细胞样细胞的研究主要集中在不同诱导因子的诱导作用,而微环境的诱导作用研究较少。 目的：观察肝细胞生长因子、胎肝细胞对骨髓间充质干细胞体外诱导分化为肝细胞样细胞的影响。 方法：采用密度梯度离心法分离大鼠骨髓间充质干细胞,反复贴壁法纯化扩增骨髓间充质干细胞;采用Ⅰ型胶原酶消化法分离孕3周大鼠胚胎肝脏细胞,差速贴壁法纯化肝脏细胞;阴性对照组骨髓间充质干细胞只加入含体积分数为10%胎牛血清的L-DMEM培养基。诱导组在阴性对照组基础上加入一定浓度肝细胞生长因子或者与胎肝细胞共培养进行诱导分化。 结果与结论：诱导组白蛋白、甲胎蛋白水平均高于非诱导组(P < 0.01),诱导组糖原染色阳性,免疫细胞化学染色CK-18阳性,而非诱导组糖原染色、CK-18均阴性。结果显示肝细胞生长因子和胎肝细胞均可在体外诱导大鼠骨髓间充质干细胞分化为具有肝细胞样细胞表型和功能的类肝细胞。     

周围神经组织工程中种子细胞的最新研究进展

典型的组织工程化人工神经主要包括种子细胞、支架材料以及有助于细胞生长、分化的细胞外基质，其中数量足够、不引起免疫排斥且有再生活力的种子细胞是其前提和基础。目前常用的种子细胞有雪旺氏细胞（SCs）、嗅球成鞘细胞（OECs）、骨髓基质细胞（BMSCs）和神经干细胞（NSCs）等。SCs一直是周围神经损伤修复研究领域的热点，OECs、BMSCs和NSCs修复周同神经损伤也在起步中。本从组织工程方面就上述四种细胞的最新研究进展进行综述，同时就存在的问题进行讨论。[第一段]     

Analysis of T regulator cell surface markers and functional properties in multiple sclerosis

The relative proportions as well as cell surface and functional properties of T suppressor (T8⁺) and T helper (T4⁺) cells in peripheral blood mononuclear cells (MNCs) of MS patients were analyzed. The proportion of T8 cells compared to normal controls was suggestively lower in patients during relapses and significantly lower in those with progressive disease. The density of T8⁺ antigen on cells of MS patients with active disease as measured by median fluorescence intensity (MFI) was also decreased compared to controls and stable MS patients. Using OKT8-mAb modulated MNCs as a model, we found that reduction of T8 antigen density results in substantial discrepancies between FACS and microscope methods for enumeration of T8⁺ cells. Levels of pokeweed mitogen-induced IgG secretion by MNCs of MS patients did not correlate with proportion of T8⁺ cells within the MNCs, but rather with the functional activity of the T8⁺ cells of given individuals, as tested in an in vitro suppressor assay using constant numbers of T8⁺ cells.     

Retinal anatomy and visual performance in a diurnal cone-rich laboratory rodent, the Nile grass rat (Arvicanthis niloticus)

Unlike laboratory rats and mice, muridae of the Arvicanthis family (A. ansorgei and A. niloticus) are adapted to functioning best in daylight. To date, they have been used as experimental models mainly in studies of circadian rhythms. However, recent work aimed at optimizing photoreceptor-directed gene delivery vectors (Khani et al. [2007] Invest Ophthalmol Vis Sci 48:3954-3961) suggests their potential usefulness for studying retinal pathologies and therapies. In the present study we analyzed the retinal anatomy and visual performance of the Nile grass rat (A. niloticus) using immunohistofluorescence and the optokinetic response (OKR). We found that approximately 35-40% of photoreceptors are cones; that many neural features of the inner retina are similar to those in other diurnal mammals; and that spatial acuity, measured by the OKR, is more than two times that of the usual laboratory rodents. These observations are consistent with the known diurnal habits of this animal, and further support its pertinence as a complementary model for studies of structure, function, and pathology in cone-rich mammalian retinae.     

Characterization of CGRP protein expression in “satellite‐like” cells and dendritic arbours of the mouse olfactory bulb

The main olfactory bulb (OB) is made up of several concentric layers, forming circuitries often involving dendro‐dendritic synapses. Important interactions between OB neurons occur in the external plexiform layer (EPL), where dendrites of tufted and Van Gehuchten cells form synapses with dendrites of deeper lying mitral, tufted, and granule cells. OB neurons display a variety of neurotransmitters. Here, the focus is on calcitonin gene‐related peptide (CGRP), a 37‐amino acid neuropeptide transmitter that is widely distributed in the central and peripheral nervous system. In the OB, CGRP‐immunoreactive (ir) cell bodies were mostly observed in the mitral cell layer (MCL) of normal mice, and their number increased following colchicine treatment. Sparsely distributed CGRP‐ir cell bodies were also found in the EPL and granular cell layer. Double‐immunofluorescence experiments revealed a lack of co‐localization between CGRP‐like immunoreactivity (LI) and corticotropin‐releasing factor‐ or galanin‐LI, two markers for mitral cells, and no CGRP‐LI was found in cholecystokinin‐, parvalbumin‐, or vasoactive intestinal polypeptide‐ir tufted/Van Gehuchten cells. CGRP‐ir cell bodies were not found to co‐localize glutamic acid decarboxylase 67 (GAD67)‐green fluorescence protein, γ‐aminobutyric acid (GABA)‐, or calretinin‐LI, although the possibility remains that CGRP‐ir cells may contain low levels of GABA and/or GAD67 not detected by our methodology. Dendrites of CGRP‐ir cells extensively ramified deep in the EPL and double‐immunofluorescence revealed them to be adjacent with, often apparently contacting, dendrites of granule, mitral, tufted, and Van Gehuchten cells. We propose that these CGRP‐ir cell bodies in the mouse OB are “satellite‐like” cells within and, occasionally, close to the MCL. J. Comp. Neurol. 518:770–784, 2010. © 2009 Wiley‐Liss, Inc.     

Dorsal raphe nucleus projecting retinal ganglion cells: Why Y cells?

Retinal ganglion Y (alpha) cells are found in retinas ranging from frogs to mice to primates. The highly conserved nature of the large, fast conducting retinal Y cell is a testament to its fundamental task, although precisely what this task is remained ill-defined. The recent discovery that Y-alpha retinal ganglion cells send axon collaterals to the serotonergic dorsal raphe nucleus (DRN) in addition to the lateral geniculate nucleus (LGN), medial interlaminar nucleus (MIN), pretectum and the superior colliculus (SC) has offered new insights into the important survival tasks performed by these cells with highly branched axons. We propose that in addition to its role in visual perception, the Y-alpha retinal ganglion cell provides concurrent signals via axon collaterals to the DRN, the major source of serotonergic afferents to the forebrain, to dramatically inhibit 5-HT activity during orientation or alerting/escape responses, which dis-facilitates ongoing tonic motor activity while dis-inhibiting sensory information processing throughout the visual system. The new data provide a fresh view of these evolutionarily old retinal ganglion cells.     

诱导性多潜能干细胞的应用前景

背景：通过基因转染的方式将已分化的体细胞重编程为诱导性多潜能干细胞,是近年来干细胞领域一项令人瞩目的新技术。由于诱导性多潜能干细胞摆脱了材料来源和伦理学的限制,因此其出现为特异的细胞治疗,特别是再生医学带来新的曙光。 目的：从诱导性多潜能干细胞的制备流程、产生的限制条件与机制、患者诱导性多潜能干细胞的获得及应用前景等方面做一评述。 方法：由第一作者检索PubMed数据库(www.ncbi.nlm.nih.gov/PubMed)2006-01/2010-03有关诱导性多潜能干细胞制备、产生机制的文章,检索词“induced pluripotent stem cells”,限定语言种类为English;同时手工检索部分文章。排除重复性研究,最终纳入34篇符合标准的文献。 结果与结论：诱导性多潜能干细胞系的建立主要包括以下几个步骤：①重组因子的选择。②目的细胞的选择。③重组因子的导入。④重组因子在目的细胞内的表达。⑤诱导性多潜能干细胞的产生。⑥重组细胞的鉴定。DNA甲基化、组蛋白的修饰作用和甲基化以及p53基因的表达在体细胞重编程为多潜能干细胞的过程中具有重要作用。虽然诱导性多潜能干细胞技术的研究仍然处于初级阶段,但是毫无疑问其具有广阔的应用前景。特别是患者特异性和疾病特异性诱导性多潜能干细胞的获得对于更好地理解疾病的发病机制及药物安全性评价提供了巨大的细胞种子资源。     

Diversity of immune cell types in multiple sclerosis and its animal model: Pathological and therapeutic implications

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system with an autoimmune attack on the components of the myelin sheath and axons. The etiology of the disease remains largely unknown, but it is commonly acknowledged that the development of MS probably results from the interaction of environmental factors in conjunction with a genetic predisposition. Current therapeutic approaches can only ameliorate the clinical symptoms or reduce the frequency of relapse in MS. Most drugs used in this disease broadly suppress the functions of immune effector cells, which can result in serious side effects. Thus, new therapeutic methods resulting in greater efficacy and lower toxicity are needed. Toward this end, cell‐based therapies are of increasing interest in the treatment of MS. Several immunoregulatory cell types, including regulatory T cells, regulatory B cells, M2 macrophages, tolerogenic dendritic cells, and stem cells, have been developed as novel therapeutic tools for the treatment of MS. In this Review, we summarize studies on the application of these cell populations for the treatment of MS and its animal model, experimental autoimmune encephalomyelitis, and call for further research on applications and mechanisms by which these cells act in the treatment of MS. © 2017 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.     

Synaptic input to the on-off directionally selective ganglion cell in the rabbit retina

A physiologically identified on-off directionally selective (DS) ganglion cell with its preferred-null axis defined was stained with horseradish peroxidase (HRP) and prepared for electron microscopy. A continuous series of thin sections were used to examine the cell's synaptology. Although the DS cell dendrite received the majority of its synaptic input from a heterogeneous population of amacrine cell processes, a frequently observed synaptic profile consisted of a DS cell dendrite receiving synapses from a cluster of several amacrine cell processes. These clusters of processes were assumed to be from a fascicle of amacrine cells, most of which probably belonged to several different cholinergic starburst amacrine cells. The most frequently observed presynaptic profile within the clusters consisted of a synaptic couplet in which two processes synapsed with each other before one of them finally synapsed with the DS ganglion cell dendrite; occasionally, a chain of three serial synapses was seen. In addition, a specific microcircuit that has the potential to exert lateral feedforward inhibition was also observed. This microcircuit consisted of two cone bipolar cell terminal dyad synapses where one dyad contained an amacrine cell process making a reciprocal synapse and a DS ganglion cell dendrite receiving direct excitation; the other dyad synapse, found lateral to the first dyad, contained two amacrine cell processes that both made reciprocal synapses, but one fed forward to make a putative inhibitory synapse with the DS cell dendrite.