Studies on expression of FSH and its anti-apoptotic effects on ischemia injury in rat spinal cord

Studies indicated that many tissues could express FSH. New functions of FSH have been recognized beyond reproduction regulation. However, no report has been made about the expression and function of FSH in rat spinal cord. Double-labeled immunofluorescence stain and in situ hybridization were used to study the co-localization of FSH with its receptor and co-localization of FSH with GnRH receptor in rat spinal cord. Spinal cord ischemia injury models were built, TUNEL stain and Fas immunostaining were made to observe the anti-apoptotic effects of FSH to neurons induced by spinal cord ischemia injury. The results found that some neurons and glias of rat spinal cord showed both FSH immunoreactivity and FSH mRNA positive signals; not only FSH and its receptor but also FSH and GnRH receptor co-located in cells of both gray matter and white matter; treatment with certain concentration of FSH before ischemia–reperfusion injury, less TUNEL positive cells and Fas positive cells were found in motor neurons of ventral gray matter in FSH experiment group than that in control group. These suggested that rat spinal cord could express FSH, it is also a target organ of FSH; FSH might exert functions through its receptor by paracrine or autocrine effects; GnRH in spinal cord might regulate FSH positive neurons through GnRH receptor; FSH might inhibit ischemia induced neuron apoptosis by down-regulating Fas expression in spinal cord.     

Age-related changes of the GABA-B receptor in the lumbar spinal cord of male rats and penile erection

Dorsal horn neurons of lumbosacral spinal cord innervate penile vasculature and regulate penile erection. GABAergic system is involved in the regulation of male sexual behavior. Because aging is frequently accompanied by a progressive decline in erectile function, the aim of this work was to examine age-related changes of the GABA-B receptor in the lumbar spinal cord. Sprague-Dawley rats of 10 and 21 days old, 3, 9 and 20 months old were used. GABA-B receptors were evaluated by quantitative autoradiography using [3H]-Baclofen as ligand with or without GABA (10 microM) to determine the non-specific binding. Ten days after birth a homogeneous neuroanatomical distribution pattern was found in the gray matter, however at 20-day-old adult distribution emerged becoming heterogeneous with the highest binding values at layers II-III and X. In dorsal layers a significant decrease was observed in 9-month-old rats while layer X showed an earlier decrease (21-day-old). GABA-B receptor affinity showed significant age-dependent and regional increase. The GABA-B receptor decrease in aged rats seems not to be related to this receptor inhibitory function in penile erection. Moreover the changes found in GABA-B receptor binding anatomical distribution may indicate its role in the morphological development of the lumbar spinal cord rather than in the decline of the erectile function.     

Upregulation of Vascular Endothelial Growth Factor Receptor-3 in the Spinal Cord of Lewis Rats with Experimental Autoimmune Encephalomyelitis

We investigated the spatiotemporal expression of vascular endothelial growth factor receptor–3 (VEGFR-3) in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. VEGFR-3 mRNA and protein were constitutively expressed in gray matter neurons and in a few white matter astrocytes. Induction of VEGFR-3 occurred predominantly in perivascular infiltrated macrophages in the spinal cord white matter during the inductive phase of EAE. VEGFR-3 expression was also induced in activated microglial cells in the gray and white matter, mainly in the peak phase. In addition, reactive astrocytes in the white matter, but not in the gray matter, expressed VEGFR-3 as disease severity increased. These data suggest that VEGFR-3 is involved in the recruitment of monocytic macrophages and in glial reactions during EAE.     

The extracellular matrix molecule tenascin C modulates expression levels and territories of key patterning genes during spinal cord astrocyte specification

The generation of astrocytes during the development of the mammalian spinal cord is poorly understood. Here, we demonstrate for the first time that the extracellular matrix glycoprotein tenascin C regulates the expression of key patterning genes during late embryonic spinal cord development, leading to a timely maturation of gliogenic neural precursor cells. We first show that tenascin C is expressed by gliogenic neural precursor cells during late embryonic development. The loss of tenascin C leads to a sustained generation and delayed migration of Fgfr3-expressing immature astrocytes in vivo. Consistent with an increased generation of astroglial cells, we documented an increased number of GFAP-positive astrocytes at later stages. Mechanistically, we could demonstrate an upregulation and domain shift of the patterning genes Nkx6.1 and Nkx2.2 in vivo. In addition, sulfatase 1, a known downstream target of Nkx2.2 in the ventral spinal cord, was also upregulated. Sulfatase 1 regulates growth factor signalling by cleaving sulphate residues from heparan sulphate proteoglycans. Consistent with this function, we observed changes in both FGF2 and EGF responsiveness of spinal cord neural precursor cells. Taken together, our data implicate Tnc in the regulation of proliferation and lineage progression of astroglial progenitors in specific domains of the developing spinal cord.     

Temporal Distribution of p300/CBP Immunoreactivity in the Adult Rat Spinal Dorsal Horn Following Chronic Constriction Injury (CCI)

p300 and its homolog cyclic AMP response element binding protein (CBP) are coactivators that were identified to participate in many biological processes including neural development and cognition. Their roles within the rodent spinal cord have not been reported systematically; in this study, their spatiotemporal distribution in the spinal cord of adult rat following chronic constriction injury (CCI) was studied. p300 and CBP expressed predominantly in nuclei in the gray matter of rat spinal cord. Rats undergoing CCI surgery showed increased p300/CBP immunoreactivity (IR) compared with normal control and sham-operated rats. The number of IR cells reached the peak at day 14 following CCI compared with those on day 3, 7, and 21, accompanied with significant behavioral changes of neuropathic pain. Cell-type determination by immunofluorescence at day 14 following CCI revealed that p300 and CBP expressed in neurons, but not in astrocytes or microglial cells. These results suggest that p300 and CBP are probably involved in the maintenance of neuropathic pain on spinal cord level. Furthermore, p300 and CBP may serve as a sensor only in neurons but not in astrocytes or microglia cells in the adult rat spinal cord.     

移植的5—羟色胺能神经元跨软脊膜迁入大鼠脊髓的研究

本文对移植的5-HT神经元从蛛网膜下腔跨软脊膜迁移进入脊髓作了初步研究。将含有5-HT细胞的胚胎中缝核组织小块或神经细胞悬浮液作为移植物,以5-HT免疫组织化学方法跟踪移植细胞,结果如下:(1)在低胸水平横切脊髓,10d后,横断脊髓内的5-HT纤维消失。(2)横切脊髓(方法同上)后,立即将中缝核组织小块移植在胸腰段脊髓的蛛网膜下腔,一月后.在横断脊髓内出现5-HT阳性神经元和纤维。5-HT纤维能在灰白质内延伸。(3)脊髓横断后,若以中缝核的细胞悬浮液代替组织小块,作上述移植,则在移植区附近的灰质内出现大量的5-HT阳性神经元。这些神经元在灰质内的分布范围与神经细胞悬浮液在蛛网膜下腔的移植范围相一致。迁入神经元能在灰质内重新形成5-HT阳性纤维网。(4)经上述移植后,灰质内出现的5-HT阳性纤维随远离细胞体而变得稀疏。白质内的5-HT阳性纤维远比灰质内稀少。本实验结果表明:移植在脊髓蛛网膜下腔的脑干5-HT细胞能跨软脊膜迁移进入脊髓。     

Excitotoxic cell death induces delayed proliferation of endogenous neuroprogenitor cells in organotypic slice cultures of the rat spinal cord

The aim of the present report was to investigate whether, in the mammalian spinal cord, cell death induced by transient excitotoxic stress could trigger activation and proliferation of endogenous neuroprogenitor cells as a potential source of a lesion repair process and the underlying time course. Because it is difficult to address these issues in vivo, we used a validated model of spinal injury based on rat organotypic slice cultures that retain the fundamental tissue cytoarchitecture and replicate the main characteristics of experimental damage to the whole spinal cord. Excitotoxicity evoked by 1 h kainate application produced delayed neuronal death (40%) peaking after 1 day without further losses or destruction of white matter cells for up to 2 weeks. After 10 days, cultures released a significantly larger concentration of endogenous glutamate, suggesting functional network plasticity. Indeed, after 1 week the total number of cells had returned to untreated control level, indicating substantial cell proliferation. Activation of progenitor cells started early as they spread outside the central area, and persisted for 2 weeks. Although expression of the neuronal progenitor phenotype was observed at day 3, peaked at 1 week and tapered off at 2 weeks, very few cells matured to neurons. Astroglia precursors started proliferating later and matured at 2 weeks. These data show insult-related proliferation of endogenous spinal neuroprogenitors over a relatively brief time course, and delineate a narrow temporal window for future experimental attempts to drive neuronal maturation and for identifying the factors regulating this process.     

The Expression of Nerve Growth Factor Receptor on Schwann Cells and the Effect of These Cells on Regeneration of Axons in Traumatically Injured Human Spinal Cord

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Increased expression of nitric oxide synthase interacting protein (NOSIP) following traumatic spinal cord injury in rats

Previous studies indicated that nitric oxide (NO) is involved in secondary damage of spinal cord injury (SCI), which worsens the primary physical injury to the central nervous systems. Recently, nitric oxide synthase interacting protein (NOSIP) has been identified to interact with neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase by inhibiting the NO production. However, its expression and function after a central nervous system injury remains unclear. In this study, we examined the expression and cellular localization of NOSIP in the spinal cord of an adult rat. Western blot analysis indicated that NOSIP protein levels increased at day1 post-injury and peaked at day 14. Double immunofluorescence staining showed that NOSIP was primarily expressed in neurons and glial cells in the intact spinal cord. Interestingly, this study also showed that the expression of NOSIP significantly increased in astrocytes after injury. Furthermore, injury-induced expression of NOSIP was co-expressed with proliferating cell nuclear antigen (PCNA) positive astrocytes after injury. We also showed the NOSIP was co-localized with nNOS in gray matter and white matter after SCI. All these data taken together suggested that NOSIP may play an important roles in astrogliogenesis after a spinal cord injury.     

Proliferation and migration of glial precursor cells in the developing rat spinal cord

The mechanisms that control the production and differentiation of glial cells during development are difficult to unravel because of displacement of precursor cells from their sites of origin to their permanent location. The two main neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. Considerable evidence supports the view that oligodendrocytes in the spinal cord are derived from a region of the ventral ventricular zone (VZ). Some astrocytes, at least, may arise from radial glia. In this study a 5-Bromo-2′-deoxyuridine (BrdU) incorporation assay was used to identify proliferating cells and examine the location of proliferating glial precursor cells in the embryonic spinal cord at different times post BrdU incorporation. In this way the migration of proliferating cells into spinal cord white matter could be followed. At E14, most of the proliferating cells in the periventricular region were located dorsally and these cells were probably proliferating neuronal precursors. At E16 and E18, the majority of the proliferating cells in the periventricular region were located ventrally. In the white matter the number of proliferating cells increased as the animals increased in age and much of this proliferation occurred locally. BrdU labelling showed that glial precursor cells migrate from their ventral and dorsal VZ birth sites to peripheral regions of the cord. Furthermore although the majority of proliferating cells in the spinal cord at E16 and E18 were located in the ventral periventricular region, some proliferating cells remained in the dorsal VZ region of the cord.     

Expression of ecotropic murine leukemia virus in the brains of C58/M, DBA2/J, and in utero-infected CE/J mice.

In C58 and AKR mice, endogenous N-tropic, ecotropic murine leukemia virus (MuLV) proviruses become activated in rare cells during embryogenesis. Resultant replication-competent progeny viruses then actively infect a large number of cells throughout the fetus, including cells in the developing central nervous system. By in situ hybridization analyses, we have assessed the presence of ecotropic MuLV RNA in the brains of C58 mice as a function of age. Only a few ecotropic MuLV-positive cells were observed in weanling mice, but the number of positive cells in the brain increased progressively with increasing age of the mice. Throughout the lives of the mice, the ecotropic MuLV RNA-positive cells were primarily located in well-defined white-matter tracts of the brain (commissura anterior, corpus callosum, fimbria hippocampi, optical tract, and striatum) and of the spinal cord. Cells of the subventricular zone also expressed ecotropic MuLV RNA, and in older mice a small number of positive cells were present in the grey matter. Infection of endogenous ecotropic MuLV provirus-less CE/J mice in utero with ecotropic MuLV clone AKR-623 resulted in the extensive infection of brain cells. The regional distribution of ecotropic MuLV RNA-containing cells was the same as observed in the brains of C58 mice, in which cells became infected by endogenously activated virus, but the number of positive cells was higher.     

Fluoro-Jade B evidence of induced ischemic tolerance in the rat spinal cord ischemia: physiological, neurological and histopathological consequences

Fluoro-Jade B, a marker of degenerating neurons, was used to label histopathological changes in the rat spinal cord after transient ischemia and ischemic preconditioning (IPC). To characterize postischemic neurodegenerations and consequent neurological changes, a particular attention was paid to the standardization of ischemic conditions in animals of both groups. 1. The control ischemic rats were submitted to a reversible occlusion of descending aorta by insertion and subsequent inflation of a 2F Fogarty catheter for 12 min. 2. In the IPC rats, an episode of short 3 min occlusion and 30 min reperfusion preceded the 12 min ischemia. Postischemic motor function testing (ambulation and stepping) was provided repeatedly for evaluation of neurological status 2 h and 24 h after surgery and at the end of postischemic survival, i.e. after 48 h. Fluoro-Jade B staining was used to demonstrate degenerated neurons. In the control rats, neurological consequences of histopathological changes in lumbosacral spinal cord, manifested as paraplegia, were present after 12 min ischemia. Thus, numbers of degenerated Fluoro-Jade B positive cells were visible in gray matter of the most injured L(4)-S(2) spinal cord segments. Slight motor function impairment, consequential from significant decreasing in Fluoro-Jade B-positivity in the L(4)-S(2) spinal cord segments of the IPC rats, was considered the pathomorpfological evidence that IPC induces spinal cord tolerance to ischemia. Our results are consistent with the previously published silver impregnation method for histopathological demonstration of ischemic degeneration.     

Early neurogenesis during caudal spinal cord regeneration in adult Gekko japonicus

Gekko japonicus undergoes dramatic changes in the caudal spinal cord after tail amputation. The amputation induces cell proliferation in the caudal ependymal tube. We performed hematoxylin and eosin staining at different time points in the regeneration process to investigate the morphological characterization of the regenerated appendages. The central canal extended to the blastema post-amputation and the cartilage and muscle tissue appeared 3 weeks after injury. We performed the bromodeoxyuridine (BrdU) incorporation assay to detect proliferating cells during the regeneration process. BrdU positive cells were detected in the peri-central canal. Furthermore, nestin and neuron-specific enolase (NSE) immunocytochemistry were applied to detect neural stem/progenitor cells and neurons. Two weeks after injury, nestin-positive cells undergoing proliferation were located outside of the ependymal tube, and NSE positive cells appeared after 3 weeks of amputation. These data suggest that neurogenesis is an early event during caudal spinal cord regeneration in gecko.