Substance P fibres in the anterior segment of the rabbit eye

Some laboratories have obtained diverging results regarding the presence of substance P fibres in the cornea possibly because different antibodies were used. This has been further investigated by comparing results with several antibodies on identically treated sections from the anterior segment of rabbit eyes. In the uvea all antisera gave identical results showing substance P fibres in the iris and ciliary processes. In the cornea, on the other hand, polyclonal rabbit or guinea-pig antibodies gave high background fluorescence and no immunofluorescent fibres were detected. In contrast, the background staining was low with the monoclonal antibody so that substance P immunoreactive fibres could be demonstrated subepithelially, intraepithelially and in the corneal stroma.     

猪眼小梁组织眼前节灌流培养和组织块培养

目的了解猪眼小梁组织的灌流眼前节培养和组织块培养在青光眼研究中的应用。方法采用恒速眼前节灌流培养和组织块培养法培养猪眼小梁组织,用光学显微镜观察不同培养方法保存的小梁组织的状态。结果恒速(0.1mL/h)灌流猪眼前节,眼压可以稳定在正常眼压范围(10~12mmHg);在一定范围内,随着流速增加眼压能逐步升高。灌流培养的组织大体形态保留完好,组织层次清晰,细胞状态好。组织块原位培养的小梁组织,周边组织结构保留完好,细胞状态尚可,但是小梁网间隙不如灌流培养的小梁网间隙饱满。结论灌流眼前节培养法可作为短期高眼压模型,更接近小梁网的生理状态,小梁网的正常充盈是维持其功能和结构的必要条件。     

Novel mutations in the TIGR gene in early and late onset open angle glaucoma

A gene for juvenile onset, open angle glaucoma (JOAG) has been localized to chromosome 1q21-31 in several families. Mutations in the trabecular meshwork-induced glucocorticoid response protein (TIGR) gene, which maps to this region, recently have been found in families segregating both JOAG and a later onset form of primary open angle glaucoma (POAG). We have analysed the TIGR gene in two families; one Spanish family segregating autosomal dominant JOAG and an Irish family with a later onset form of autosomal dominant POAG. We have found a G-T transversion in the first base of codon 426 in all affected members of the Spanish family, which results in a valine to phenylalanine amino acid substitution. We have also found a G-A transition at the first base of codon 367 that segregates through all but one branch of the Irish family and results in a glycine to arginine amino acid substitution. Members of this family that carry the Gly367Arg change also share a common haplotype that is neither present in any of the unaffected members of the family, nor in the branch that does not segregate the mutation. Identification of further mutations in the TIGR gene increases its importance in the etiology of open angle glaucoma. Hum Mutat 11:244–251, 1998. © 1998 Wiley-Liss, Inc.     

Nitrergic and peptidergic innervation in the developing rat heart

The phenotypic expression and anatomic distribution of nitrergic and peptidergic innervation in the developing rat heart was localized by reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and immunohistochemistry using antibodies against neuronal isoform of nitric oxide synthase (nNOS), neuropeptide Y (NPY) and calcitonin-gene-related peptide (CGRP). NPY-immunoreactive nerve fibers showed the earliest expression by 16 days of gestation, with preferential innervation of the nodal and perinodal areas, followed by the innervation of the valves and ventricles by postnatal day 7. NPY immunoreactivity was also localized to a large proportion of the intrinsic cardiac ganglia from 16 days of gestation onwards with a progressive increase in the number of neuronal cell bodies per ganglia with age. CGRP-positive nerve fibers appeared by 19 days of gestation and were less dense during the gestational and early postnatal periods, and showed a quantitative increase in density by 7 days, followed by a decrease by 3 weeks postnatal. None of the intrinsic ganglia were stained positive for CGRP, indicating the extrinsic sensory origin of these stained fibers. Nitrergic innervation paralleled the sensory innervation, with the cardiac ganglia and nerve fibers showing a positive labeling from 19 days of gestation onwards. NADPH-d and nNOS were partially co-localized. Double-label immunohistochemistry showed that a considerable proportion of sensory CGRP-immunopositive fibers were also immunoreactive for NOS. The results of the present study show that neuropeptides and nitric oxide are expressed by the late gestational period and that autonomic efferent innervation precedes sensory and nitrergic innervation in the developing heart. Accepted: 4 January 2000     

The cholinergic innervation of normal and transplanted superior colliculus in the rat: an immunohistochemical study

The distribution of choline acetyltransferase was determined in normal and transplanted rat superior colliculus with choline acetyltransferase immunohistochemistry. This distribution was compared to the pattern of histochemically detected acetylcholinesterase activity. To determine cholinergic input to the superficial superior colliculus, double labelling experiments combining retrograde tracing methods and choline acetyltransferase immunohistochemistry were carried out. No choline acetyltransferase-containing neurons were observed in the rat superior colliculus. A dense network of choline acetyltransferase-immunoreactive fibres and terminals was seen in the intermediate layers of the normal superior colliculus. The distribution was patchy and very similar to the pattern of acetylcholinesterase activity. Occasional fibres and terminals were seen in the deep tectal laminae. The superficial layers contained a low number of choline acetyltransferase-stained fibres and terminals but a comparatively high level of acetylcholinesterase activity. Following a unilateral injection of a tracer into the superficial superior colliculus, retrogradely labelled choline acetyltransferase-immunoreactive neurons were found in the dorsal and ventral subnuclei of the ipsilateral parabigeminal nucleus. As in the normal superior colliculus, choline acetyltransferase-positive neurons were not found in tectal transplants. However, choline acetyltransferase-immunoreactive fibres and terminals were seen in grafts but only in those which had extensive connections with the host midbrain. The pattern of staining most closely resembled that seen in the intermediate layers of the normal superior colliculus. The similar arrangement of choline acetyltransferase and acetylcholinesterase activity in the intermediate layers of normal rat superior colliculus provides further evidence for cholinergic innervation to these layers, probably originating in the dorsal and pedunculopontine tegmental nuclei. The data from the double labelling experiments indicate that the choline acetyltransferase-immunoreactive terminals observed in the superficial layers represent the terminal field of an ipsilateral cholinergic projection from the parabigeminal nucleus. Tectal grafts receive cholinergic innervation from the host. The evidence suggests that much of this input derives from the cholinergic nuclei in the brainstem tegmentum which normally project to the intermediate tectal layers.     

Substance P-like immunoreactive nerves in the anterior segment of the rabbit, cat and monkey eye

The indirect immunofluorescence technique demonstrates a substance P-like immunoreactive innervation to the anterior segment of the rabbit, cat and monkey eye. In all three species there is a sparse, but definite, corneal innervation. For the rabbit, substance P-like immunoreactive nerves to the aqueous outflow apparatus are found chiefly in the pectinate ligament. In the cat, this innervation is somewhat more extensive, being seen in the septae of the ciliary cleft as well. The monkey has a more plentiful innervation to the outflow apparatus than either the cat or the rabbit. Substance P-like immunoreactive nerves are visible in the trabecular meshwork and at the inner and outer walls of Schlemm's canal. For all three animals, the iris contains immunoreactive nerve fibers to the sphincter muscle, to the large blood vessels and to the anterior stromal melanocytes. In the ciliary body, the ciliary processes receive a constant innervation; it is somewhat more dense in the rabbit. Some of the large ciliary body blood vessels also are innervated. Ciliary body melanocytes are innervated; it was not possible to determine whether or not immunoreactive fibers innervate the ciliary muscle cells as well. The present study extends prior knowledge of the innervation of the eye. Taken with the known physiologic effects of substance P, it indicates a series of potential roles for this peptide in the vegetative processes of the eye.     

成人眼小梁网内皮细胞具有干细胞/祖细胞的属性

背景：干细胞/祖细胞的研究为干细胞治疗疾病提供了可能,寻找正常人眼内小梁网干细胞/祖细胞,探究小梁网干细胞的属性能为原发性开角型青光眼的干细胞/祖细胞治疗提供新的思路。 目的：探索小梁网内皮干细胞/祖细胞的属性。 方法：选取成人眼球20例,其中10例眼球标本来源于因急性外伤大失血死亡后摘除的眼球,眶内容物剜除的眼球7例,3例来源于眼后段黑色素瘤患者手术摘除眼球。所有标本行苏木精-伊红染色以及免疫组化染色观察。 结果与结论：小梁网内皮细胞中阳性表达nestin,阴性表达凝血因子Ⅷ,CD31和CD34,而角膜内皮细胞中弱阳性表达nestin,Schlemm管内皮细胞未见nestin阳性表达,但凝血因子Ⅷ,CD31,CD34均呈强阳性表达。表明小梁网内皮细胞均具有干细胞/祖细胞的属性,小梁网nestin蛋白阳性的内皮细胞相似于表达nestin的血管内皮祖细胞。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

基于双光子共聚焦成像的大鼠小梁网三维重建及有限元分析

青光眼是一组威胁视神经视觉功能、与眼压升高密切相关的眼病。目前认为临床上大部分眼压的升高与房水流出阻力增加有关,特别是小梁网流出阻力的增加。因此,研究高眼压下影响房水外流阻力的重要区域小梁网的形态学信息尤为重要。通过前房灌注的方法制造大鼠急性高眼压动物模型,将6只SD大鼠分成两组(A组和B组),B组大鼠处死后于左眼球加压60 mmHg处理,其余眼球均为未加压对照组,利用双光子共聚焦成像系统采集正常眼压和高眼压下小梁网组织的断层序列图,通过图像处理方法,定量分析眼压对小梁网组织孔隙率变化的影响。通过三维重建获取正常眼压下的小梁网结构模型,并利用有限元方法,探讨眼压对于小梁网组织形态结构的影响。结合实验数据与模拟计算的结果,综合分析眼压的变化对于小梁网外流阻力的影响。高眼压组数据显示,部分小梁与周围组织融合,胶原纤维出现塌缩,越是靠近前房的小梁组织损伤越严重。有限元分析结果表明,孔隙越多的区域变形越大,而且压力越大,小梁变形程度越明显。眼内压升高可能会引起房水外流通道结构发生异常,主要表现为小梁网胶原纤维发生塌缩。高眼压与正常眼压情况相比,小梁网区域外流阻力增大的可能性较大。     

Spatial association of prolyl oligopeptidase, inositol 1,4,5-triphosphate type 1 receptor, substance P and its neurokinin-1 receptor in the rat brain: an immunohistochemical colocalization study

Prolyl oligopeptidase (POP) is a serine endopeptidase which hydrolyzes proline-containing peptides shorter than 30 amino acids. It has been suggested that POP is associated with cognitive functions, possibly via the cleavage of neuropeptides such as substance P (SP). Recently, several studies have also linked POP to the inositol 1,4,5-triphosphate (IP(3)) signaling. However, the neuroanatomical interactions between these substances are not known. We used double-labeled immunofluorescence to determine the POP colocalization with SP, SP receptor (neurokinin-1 receptor, NK-1R) and IP(3) type 1 receptor (IP(3)R1) in the rat brain. Furthermore, since striatal and cortical GABAergic neurons are involved in SP neurotransmission, we studied the coexpression of POP, SP and GABA by triple-labeled immunofluorescence. POP was moderately present in IP(3)R1-containing cells in cortex; the colocalization was particularly high in the thalamus, hippocampal CA1 field and cerebellar Purkinje cells. Colocalization of POP with SP and NK1-receptor was infrequent throughout the brain, though some POP and SP coexpression was observed in cerebellar Purkinje cells. We also found that POP partially colocalized with SP-containing GABAergic neurons in striatum and cortex. Our findings support the view that POP is at least spatially associated with the IP(3)-signaling in the thalamus, hippocampus and cerebellar Purkinje cells. This might point to a role for POP in the regulation of long-term potentiation and/or depression. Moreover, the low degree of colocalization of POP, SP and its NK-1R suggests that a transport system is needed either for POP or SP to make hydrolysis possible and that POP may act both intra- and extracellularly.     

Glycosaminoglycans of the trabecular meshwork of the eye in primary juvenile glaucoma

Significant changes in the qualitative and quantitative composition of glycosaminoglycans (decreased content of sulfated glycosaminoglycans and increased content of collagen-bound proteoglycanes) in the trabecular meshwork of the eye in primary juvenile glaucoma indicate fibrosis of the juxtacanalicular tissue, which was detected in pathomorphological examination of the operation material. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 3, pp. 354–357, March, 2007     

The monoaminergic innervation of the amygdala in the squirrel monkey: an immunohistochemical study

The monoaminergic innervation of the amygdala of the squirrel monkey (Saimiri sciureus) was studied by using immunohistochemical methods with primary antisera raised against serotonin, and the catecholamine synthesizing enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase. Serotonin was widely distributed within the amygdala including profuse terminal labeling in central, basolateral and cortical nuclear groups. The accessory basal and medial nuclei were the only two areas receiving relatively poor serotoninergic innervation. Tyrosine hydroxylase was more discretely distributed, with very dense to moderate terminal labeling in central, basal and lateral nuclei, but only scant labeling within accessory basal and corticomedial nuclei, except at the cortical transitional area where dense terminal labeling was noted. Dopamine-beta-hydroxylase immunoreactivity was moderate in central and corticomedial nuclei, but comparatively light in other nuclear groups. Phenylethanolamine-N-methyltransferase was only sparsely distributed in the amygdala. The findings of the present study reveal that the monoaminergic innervation of the primate amygdala is similar to that reported in rodents, although some conspicuous exceptions do exist. Whereas the noradrenergic and serotoninergic neuronal systems ramify profusely within the amygdala, the dopaminergic system appears to be more discretely and topographically organized.     

The cholinergic innervation of the rat substantia nigra: a light and electron microscopic immunohistochemical study

Summary Putative cholinergic axons and synaptic endings were demonstrated in the substantia nigra (SN) of the rat by light and electron microscopy on the basis of the localization of choline acetyltransferase (ChAT) immunoreactivity. The distribution of ChAT immunoreactivity in the SN as demonstrated by light microscopy revealed a modest network of ChAT-immunoreactive beaded axons in the SNc, in comparison to a relatively sparse distribution in the SNr. These axonal profiles were most dense in the middle of the rostral-caudal extent of the SNc and appeared to be concentrated in the middle third of the medial-lateral extent. By electron microscopy, unmyelinated, small diameter (0.25 m) ChAT-immuno-reactive axons were observed interspersed among numerous other non-immunoreactive axons in the SNc. ChAT-immunoreactive synaptic endings were observed in juxtaposition to small caliber (0.5 m) non-immunoreactive dendrites, and contained numerous spheroidal synaptic vesicles and occasional mitochondria. Synaptic contact zones were characterized by an accumulation of synaptic vesicles along the presynaptic membrane, and a prominent postsynaptic densification producing an asymmetrical pre-/postsynaptic membrane profile typical of excitatory synapses. These findings provide direct evidence for a cholinergic innervation of the SN, and suggest that this input may have an excitatory effect on neuronal elements in the SNc.     

青光眼相关生物力学研究进展

原发性青光眼已成为全球第二致盲眼病.其发病原因很多,病程发展的主要力学原因是各种因素引起的眼压升高,致使视觉细胞或视神经受到持续压迫,最终导致视野受损致盲.本文主要就闭角型青光眼相关的生物力学研究现状做一简要综述.     

Calcitonin gene-related peptidergic projection from the parabrachial area to the forebrain and diencephalon in the rat: an immunohistochemical analysis

We investigated ascending fiber projections of calcitonin gene-related peptide from the parabrachial area to the forebrain and diencephalon in the rat using immunocytochemistry. Destruction of the lateral portion of the dorsal parabrachial area resulted in a marked ipsilateral decrease in the fibers containing calcitonin gene-related peptide in the ventromedial hypothalamic nucleus, indicating that cells containing calcitonin gene-related peptide in the lateral portion of the dorsal parabrachial area projected to the ipsilateral ventromedial hypothalamic nucleus. Destruction of the ventral portion of the parabrachial area resulted in a marked decrease of fibers containing calcitonin gene-related peptide in the bed nucleus of the stria terminalis, the central amygdaloid nucleus and the lateral hypothalamus just medial to the crus cerebri (the far-lateral hypothalamus), and a less marked decrease in the ventromedial thalamic nucleus. This means that there are projections from cells containing calcitonin gene-related peptide in the ventral portion of the parabrachial area to the first three regions just mentioned, and to some extent to the last.     

Sympathetic and sensory innervation of the rat shoulder joint: a WGA-HRP tracing and CGRP immunohistochemical study

The innervation of the shoulder joint of the rat was investigated. Nerve origin was assessed by injection of a neuronal tracer (WGA-HRP) into the shoulder joint cavity and calcitonin gene-related peptide (CGRP), which is known to be present in some sensory neurons, was detected immunohistochemically with an anti-CGRP antibody. In the ipsilateral sympathetic and dorsal root ganglia, 133–312 and 12–55 nerve cell bodies were respectively labeled by injection of the tracer. In the sympathetic ganglia, 83% of all labeled cells were found in the stellate ganglion and 17% in the superior cervical ganglion. In the dorsal root ganglia, 75% of the labeled cells were found in C4 and the neighboring ganglia (C4–C5), while the rest were observed in C6–8 and T3. This suggested that the origin of sensory innervation for the shoulder joint was mainly in the mid-cervical cord. CGRP-immunoreactive fibers were found in the synovial capsule of the shoulder joint. These fibers were fine and resembled type 4 axons as classified by Brodai, i.e., nerve related to pain sensation. These findings indicate that sensory nerves from the mid-cervical cord and sympathetic nerves from the cervical ganglion are distributed to the shoulder joint. It is possible that these nerves are related to symptoms such as pain in patients with frozen shoulder or other diseases.     

The serotonin innervation of the cerebral cortex in the rat—an immunohistochemical analysis

The serotonergic innervation of the cerebral cortex in the rat has been studied by immunohistochemistry employing an antibody directed against the neurotransmitter, serotonin. The dorsal raphe, median raphe and B9 cell groups contain intensely labelled neuronal perikarya. Bundles of large diameter axons suggestive of fibers of passage are observed in successive sections as they ascend through the midbrain tegmentum, medial forebrain bundle, diagonal band and supracallosal stria en route to the cortex. In addition, a lateral pathway to the cerebral cortex traversing the ansa peduncularis is visualized. All regions of the cerebral cortex appear to be innervated by serotonergic axons which have a distinctive morphology: they are fine (0.1–0.5 μm), varicose, and extremely convoluted. Serotonergic axons of passage are thicker and comparatively straight. Throughout the lateral neocortex, as well as in the anterior cingulate cortex, serotonergic axons form a densely arborizing plexus through all cortical layers. Contrary to earlier reports, based on histofluorescence, describing a sparse innervation of the cortex with most of the fibers found in the molecular layer, the present study reveals that the innervation is relatively uniform across all cortical layers. In most of the cortex the density of serotonin-containing axons exceeds that of noradrenergic fibers. A distinctive and different pattern of serotonin innervation is found in the posterior cingulate cortex (cytoarchitectonic field RSg): the serotonergic axons are restricted largely to lamina I and III. A restricted laminar pattern also characterizes the innervation of the hippocampus; dense axonal plexuses occur in the outer rim of the dentate hilus and in the stratum lacunosum-moleculare. The serotonergic afferents to the cortex appear to have at least two different modes of distribution, a relatively uniform pattern in the anterior cingulate and the lateral neocortex and a restricted, laminar pattern in the posterior cingulate and the hippocampus.The density and extent of the serotonin innervation is such that the raphe neurons may contact every cell in the cortex. The widespread arborization of serotonin axons contrasts with the spatially restricted termination of thalamic afferents. The distribution of serotonin-containing fibers also differs substantially from the terminal patterns of noradrenergic and dopaminergic fibers. The differences in axonal morphology and distribution amongst the monoamine afferents reflect differences in their contributions to cortical circuitry. The present findings indicate that the serotonin-containing neurons may exert a profound and global, but not necessarily uniform, influence upon cortical function.     

大鼠小梁细胞的体外培养及弹性模量的测量

目的小梁网是由小梁薄片和其上的小梁细胞构成的网状结构,它对眼压和房水流出具有重要的调节作用,同时小梁细胞的力学特性和生物学特性与房水流出阻力密切相关。因此本研究主要探讨体外培养的大鼠小梁细胞的生物学特性并应用原子力显微镜测量其弹性模量,为今后建立高眼压动物模型并探究原发性开角型青光眼的发病机制提供理论依据。方法取3只SD大鼠双眼小梁网组织,应用消化法对小梁细胞进行体外混合培养。倒置相差显微镜和免疫组化SABC染色的方法确定小梁细胞并观察其生物学特性。应用原子力显微镜压痕方法测量细胞的弹性模量。结果大鼠小梁细胞10 d左右达到融合,细胞形态多样,免疫组化检测结果显示层粘连蛋白、纤维连接蛋白和神经元特异性烯醇化酶染色阳性。小梁细胞弹性模量为1. 02 kPa±0. 66 k Pa。结论消化法成功培养出大鼠小梁细胞,并利用原子力显微镜测得小梁细胞的弹性模量,为之后研究青光眼小梁细胞的特性奠定基础。