The molecular cloning of glial fibrillary acidic protein in <Emphasis Type="Italic">Gekko japonicus</Emphasis> and its expression changes after spinal cord transection

The glial fibrillary acidic protein (GFAP) is an astrocyte-specific member of the class III intermediate filament proteins. It is generally used as a specific marker of astrocytes in the central nervous system (CNS). We isolated a GFAP cDNA from the brain and spinal cord cDNA library of Gekko japonicus, and prepared polyclonal antibodies against gecko GFAP to provide useful tools for further immunochemistry studies. Both the real-time quantitative PCR and western blot results revealed that the expression of GFAP in the spinal cord after transection increased, reaching its maximum level after 3 days, and then gradually decreased over the rest of the 2 weeks of the experiment. Immunohistochemical analyses demonstrated that the increase in GFAP-positive labeling was restricted to the white matter rather than the gray matter. In particular, a slight increase in the number of GFAP positive star-shaped astrocytes was detected in the ventral and lateral regions of the white matter. Our results indicate that reactive astrogliosis in the gecko spinal cord took place primarily in the white matter during a short time interval, suggesting that the specific astrogliosis evaluated by GFAP expression might be advantageous in spinal cord regeneration.     

Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 following acute spinal cord contusion in rats.

To investigate the possible role of vascular endothelial growth factor (VEGF) in the injured spinal cord, we analyzed the distribution and time course of the two tyrosine kinase receptors for VEGF, Flt-1 and Flk-1, in the rat spinal cord following contusion injury using a weight-drop impactor. The semi-quantitative RT-PCR analysis of Flt-1 and Flk-1 in the spinal cord showed slight upregulation of these receptors following spinal cord injury. Although mRNAs for Flt-1 and Flk-1 were constitutively expressed in neurons, vascular endothelial cells, and some astrocytes in laminectomy control rats, their upregulation was induced in association with microglia/macrophages and reactive astrocytes in the vicinity of the lesion within 1 day in rats with a contusion injury and persisted for at least 14 days. The spatiotemporal expression of Flt-1 in the contused spinal cord mirrored that of Flk-1 expression. In the early phase of spinal cord injury, upregulation of Flt-1 and Flk-1 mRNA occurred in microglia/macrophages that infiltrated the lesion. In addition, the expression of both receptors increased progressively in reactive astrocytes within the vicinity of the lesion, predominately in the white matter, and almost all reactive astrocytes coexpressed Flt-1 or Flk-1 and nestin. These results suggest that VEGF may be involved in the inflammatory response and the astroglial reaction to contusion injuries of the spinal cord via specific VEGF receptors.     

Modulation of NADPH-diaphorase and glial fibrillary acidic protein by progesterone in astrocytes from normal and injured rat spinal cord

Progesterone (P4) can be synthesized in both central and peripheral nervous system (PNS) and exerts trophic effects in the PNS. To study its potential effects in the spinal cord, we investigated P4 modulation (4 mg/kg/day for 3 days) of two proteins responding to injury: NADPH-diaphorase, an enzyme with nitric oxide synthase activity, and glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. The proteins were studied at three levels of the spinal cord from rats with total transection (TRX) at T10: above (T5 level), below (L1 level) and caudal to the lesion (L3 level). Equivalent regions were dissected in controls. The number and area of NADPH-diaphorase active or GFAP immunoreactive astrocytes/0.1 mm(2) in white matter (lateral funiculus) or gray matter (Lamina IX) was measured by computerized image analysis. In controls, P4 increased the number of GFAP-immunoreactive astrocytes in gray and white matter at all levels of the spinal cord, while astrocyte area also increased in white matter throughout and in gray matter at the T5 region. In control rats P4 did not change NADPH-diaphorase activity. In rats with TRX and not receiving hormone, a general up-regulation of the number and area of GFAP-positive astrocytes was found at all levels of the spinal cord. In rats with TRX, P4 did not change the already high GFAP-expression. In the TRX group, instead, P4 increased the number and area of NADPH-diaphorase active astrocytes in white and gray matter immediately above and below, but not caudal to the lesion. Thus, the response of the two proteins to P4 was conditioned by environmental factors, in that NADPH-diaphorase activity was hormonally modulated in astrocytes reacting to trauma, whereas up-regulation of GFAP by P4 was produced in resting astrocytes from non-injured animals.     

Effects of progesterone in the spinal cord of a mouse model of multiple sclerosis

The spinal cord is a target of progesterone (PROG), as demonstrated by the expression of intracellular and membrane PROG receptors and by its myelinating and neuroprotective effects in trauma and neurodegeneration. Here we studied PROG effects in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis characterized by demyelination and immune cell infiltration in the spinal cord. Female C57BL/6 mice were immunized with a myelin oligodendrocyte glycoprotein peptide (MOG_40–54). One week before EAE induction, mice received single pellets of PROG weighing either 20 or 100 mg or remained free of steroid treatment. On average, mice developed clinical signs of EAE 9–10 days following MOG administration. The spinal cord white matter of EAE mice showed inflammatory cell infiltration and circumscribed demyelinating areas, demonstrated by reductions of luxol fast blue (LFB) staining, myelin basic protein (MBP) and proteolipid protein (PLP) immunoreactivity (IR) and PLP mRNA expression. In motoneurons, EAE reduced the expression of the alpha 3 subunit of Na,K-ATPase mRNA. In contrast, EAE mice receiving PROG showed less inflammatory cell infiltration, recovery of myelin proteins and normal grain density of neuronal Na,K-ATPase mRNA. Clinically, PROG produced a moderate delay of disease onset and reduced the clinical scores. Thus, PROG attenuated disease severity, and reduced the inflammatory response and the occurrence of demyelination in the spinal cord during the acute phase of EAE.     

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.     

Role of macrophages during Theiler's virus infection.

Theiler's virus, a murine picornavirus, causes a persistent infection of the central nervous system with chronic inflammation and primary demyelination. We examined the nature of infected cells at different times postinoculation (p.i.) with a combined immunocytochemistry-in situ hybridization assay. The virus was found in the gray matter of the brain, mostly in neurons, during the first week p.i. During the following weeks, the virus was present in the spinal cord, first in the gray and white matter, then exclusively in the white matter. Approximately 10% of infected cells were astrocytes at any time during the study. Infected oligodendrocytes were first noticed on day 14 p.i. and amounted to approximately 6% of infected cells. The number of infected macrophages increased with time and reached a plateau by day 21 p.i., when at least 45% of infected cells were macrophages. The role of blood-borne macrophages during infection was studied by depleting them with mannosylated liposomes containing dichloromethylene diphosphonate. The virus did not persist in the majority of the mice treated with liposomes. These mice showed only minimal mononuclear cell infiltration and no demyelination.     

Intrathecal Fas ligand infusion strengthens immunoprivilege of central nervous system and suppresses experimental autoimmune encephalomyelitis

Fas ligand (FasL) is an essential molecule strongly expressed in some immunoprivileged sites, but is expressed at very low levels in normal CNS. In this study, acute experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats with guinea pig myelin basic protein. Intrathecal infusion of recombinant FasL before EAE onset dose dependently suppressed acute EAE and alleviated pathological inflammation in lumbosacral spinal cord. This treatment greatly increased apoptosis in CNS inflammatory cells, but did not inhibit systemic immune response to myelin basic protein. Systemic administration of a similar dose of rFasL was ineffective. In vitro, encephalitogenic T cells were highly sensitive to rFasL-induced cell death, and activated macrophages were also susceptible. In addition, in vitro rFasL treatment potentiated the immunosuppressive property of rat cerebrospinal fluid. We conclude that intrathecal infusion of rFasL eliminated the initial wave of infiltrating T cells and macrophages, and therefore blocked the later recruitment of inflammatory cells into CNS. Although Fas receptor expression was observed on spinal cord neurons, astrocytes, and oligodendrocytes, no damage to these cells or to the myelin structure was detected after rFasL infusion.     

Immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to Ia-positive cells with dendritic morphology

The rat central nervous system (CNS) during experimental allergic encephalomyelitis (EAE) was analyzed immunohistochemically from the preclinical to recovery stage by using monoclonal antibodies specific for rat T lymphocyte subsets and Ia antigen. Through combination of the avidin-biotin technique and carefully selected fixative, cells with dendritic morphology (DC) and infiltrating mononuclear cells were clearly and intensely demonstrated in the CNS parenchyma during EAE. In normal and complete Freund's adjuvant (CFA)-injected controls, there were no inflammatory foci. Ia (OX3)-positive parenchymal cells were not detected, whereas W3/25 stained DC that were located mainly in the white matter and W3/13 stained axons. At the preclinical stage, 11 days after CNS/CFA sensitization, a few clusters of Ia+ DC were detected in some sections of the spinal cord. The number of Ia+ DC increased as clinical signs developed (P less than 0.001). In rats with a clinical score of 1 or 2, Ia+ DC were mainly located in the perivascular region and closely associated with infiltrating T lymphocytes. However, at moribund state (score 3), Ia+ DC were evenly distributed in gray and white matter on almost all sections of the spinal cord. In recovered rats, the numbers of inflammatory foci and Ia+ DC were less than those in clinical EAE rats (P less than 0.001). Rats without clinical signs throughout the course also contained a few clusters of Ia+ DC. Double immunofluorescent staining with OX3 and anti-glial fibrillary acidic protein (GFAP) antiserum demonstrated that Ia+ DC were negative for GFAP. Their morphology and distribution were similar to those of nucleoside diphosphatase-positive cells, suggesting that Ia+ DC are microglia. In contrast to DC, no astrocytes or endothelial cells express detectable levels of Ia antigen in control and clinical EAE rats. These findings suggest that brain cells other than Ia+ DC may not be involved in the local immune interaction. Ia+ DC may play a significant role in antigen presentation in the CNS with EAE.     

The role of dorsal root ganglia activation and brain-derived neurotrophic factor in multiple sclerosis

Multiple sclerosis (MS) is characterized by focal destruction of the white matter of the brain and spinal cord. The exact mechanisms underlying the pathophysiology of the disease are unknown. Many studies have shown that MS is predominantly an autoimmune disease with an inflammatory phase followed by a demyelinating phase. Recent studies alongside current treatment strategies, including glatiramer acetate, have revealed a potential role for brain-derived neurotrophic factor (BDNF) in MS. However, the exact role of BDNF is not fully understood. We used the experimental autoimmune encephalomyelitis (EAE) model of MS in adolescent female Lewis rats to identify the role of BDNF in disease progression. Dorsal root ganglia (DRG) and spinal cords were harvested for protein and gene expression analysis every 3 days post-disease induction (pdi) up to 15 days. We show significant increases in BDNF protein and gene expression in the DRG of EAE animals at 12 dpi, which correlates with peak neurological disability. BDNF protein expression in the spinal cord was significantly increased at 12 dpi, and maintained at 15 dpi. However, there was no significant change in mRNA levels. We show evidence for the anterograde transport of BDNF protein from the DRG to the dorsal horn of the spinal cord via the dorsal roots. Increased levels of BDNF within the DRG and spinal cord in EAE may facilitate myelin repair and neuroprotection in the CNS. The anterograde transport of DRG-derived BDNF to the spinal cord may have potential implications in facilitating central myelin repair and neuroprotection.     

Spread of a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes.

Mouse hepatitis virus strain JHM (MHV-JHM) causes a chronic encephalomyelitis in susceptible mice, with histological evidence of demyelination in the spinal cord. After intranasal inoculation, virus spreads retrogradely to several brain structures along neuroanatomic projections to the main olfactory bulb. In the absence of experimental intervention, mice become moribund before the spinal cord is infected. In this study, infusions of anti-MHV neutralizing monoclonal antibodies were administered to protect mice from the MHV-JHM-induced acute encephalitis and to allow survival until virus spread to the spinal cord. Under these conditions, virus was observed to enter specific layers (primarily laminae V to VII) in the gray matter of the upper spinal cord, consistent with transneuronal spread. While the brain structures which are the sources for virus spread to the spinal cord cannot be determined with certainty, the ventral reticular nucleus is likely to be important since it is consistently and extensively labeled in all mice and receives projections from subsequently infected areas of the spinal cord. After initial entry into the gray matter, virus rapidly spread to the white matter of the spinal cord. During the early stages of this process, extensive infection of astrocytes was noted, suggesting that cell-to-cell spread via these glial cells is an important part of this process. Reports from other laboratories using cultured cells strongly suggested that astrocytes serve as important regulators of oligodendrocyte function and, by extrapolation, have a major role in vivo in the processes of both demyelination and remyelination. Thus, our results not only outline the probable pathway used by MHV-JHM to infect the white matter of the spinal cord but also, with the assumption that infection of astrocytes leads to subsequent dysfunction, raise the possibility that infection of these cells contributes to the demyelinating process.     

猫腰髓白质年龄相关的形态学变化

以青年成年猫(1-3龄,2-2．5 kg)和老年猫(12龄,3-3．5kg)L6段脊髓白质为研究对象,用 神经丝蛋白(NF)免疫染色显示神经纤维,用改良的Holzer结晶紫染色显示所有胶质细胞并用成年动物Golgi 法显示其形态,用胶质纤维酸性蛋白(GFAP)免疫染色显示星形胶质细胞。光镜下对青年猫与老年猫腰髓白质 中神经纤维和胶质细胞进行形态学观察和定量研究。与青年猫相比,老年猫腰髓白质中的神经纤维密度显著下 降(P相似文献   

Spatiotemporal Pattern of RNA-Binding Motif Protein 3 Expression After Spinal Cord Injury in Rats

RNA-binding motif protein 3 (RBM3) belongs to a very small group of cold inducible proteins with anti-apoptotic and proliferative functions. To elucidate the expression and possible function of RBM3 in central nervous system (CNS) lesion and repair, we performed a spinal cord injury (SCI) model in adult rats. Western blot analysis revealed that RBM3 level significantly increased at 1 day after damage, and then declined during the following days. Immunohistochemistry further confirmed that RBM3 immunoactivity was expressed at low levels in gray and white matters in normal condition and increased at 1 day after SCI. Besides, double immunofluorescence staining showed RBM3 was primarily expressed in the neurons and a few of astrocytes in the normal group. While after injury, the expression of RBM3 increased both in neurons and astrocytes at 1 day. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA) and active caspase-3 in injured spinal cords by western blot. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many RBM3-expressing cells and rare caspase-3 was observed in RBM3-expressing cells at 1 day after injury. Our data suggested that RBM3 might play important roles in CNS pathophysiology after SCI.     

LPA receptor expression in the central nervous system in health and following injury

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA_1–3) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA₁ was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA₂ showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA₃ showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA_1–3 are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.     

Prolonged Gray Matter Disease without Demyelination Caused by Theiler's Murine Encephalomyelitis Virus with a Mutation in VP2 Puff B

Theiler's murine encephalomyelitis virus (TMEV) is divided into two subgroups based on neurovirulence. During the acute phase, DA virus infects cells in the gray matter of the central nervous system (CNS). Throughout the chronic phase, DA virus infects glial cells in the white matter, causing demyelinating disease. Although GDVII virus also infects neurons in the gray matter, infected mice developed a severe polioencephalomyelitis, and no virus is detected in the white matter or other areas in the CNS in rare survivors. Several sequence differences between the two viruses are located in VP2 puff B and VP1 loop II, which are located near each other, close to the proposed receptor binding site. We constructed a DA virus mutant, DApBL2M, which has the VP1 loop II of GDVII virus and a mutation at position 171 in VP2 puff B. While DApBL2M virus replicated less efficiently than DA virus during the acute phase, DApBL2M-induced acute polioencephalitis was comparable to that in DA virus infection. Interestingly, during the chronic phase, DApBL2M caused prolonged gray matter disease in the brain without white matter involvement in the spinal cord. This is opposite what is observed during wild-type DA virus infection. Our study is the first to demonstrate that conformational differences via interaction of VP2 puff B and VP1 loop II between GDVII and DA viruses can play an important role in making the transition of infection from the gray matter in the brain to the spinal cord white matter during TMEV infection.     

Expression of Vascular Endothelial Growth Factor-C (VEGF-C) and Its Receptor (VEGFR-3) in the Glial Reaction Elicited by Human Mesenchymal Stem Cell Engraftment in the Normal Rat Brain

To determine whether vascular endothelial growth factor-C (VEGF-C) and its receptor (VEGFR-3) are involved in the glial reaction elicited by transplanted mesenchymal stem cells (MSCs), we examined the cellular localization of VEGF-C and VEGFR-3 proteins in the striatum of adult normal rats that received bone marrow-derived human MSCs. The MSC grafts were infiltrated with activated microglia/macrophages and astrocytes over a 2-week period post-transplantation, which appeared to parallel the loss of transplanted MSCs. VEGF-C/VEGFR-3 was expressed in activated microglia/macrophages recruited to the graft site, where the induction of VEGF-C protein was rather late compared with that of its receptor. VEGF-C protein was absent or very weak on day 3, whereas VEGFR-3 immunoreactivity was evident within the first three days. Furthermore, within three days, VEGF-C could be detected in the brain macrophages localized immediately adjacent to the needle track. At the same time, almost all the brain macrophages in both regions expressed VEGFR-3. Reactive astrocytes at the graft site expressed VEGFR-3, but not VEGF-C. These data demonstrated the characteristic time- and cell-dependent expression patterns for VEGF-C and VEGFR-3 within the engrafted brain tissue, suggesting that they may contribute to neuroinflammation in MSC transplantation, possibly through the recruitment and/or activation of microglia/macrophages and astrogliosis.     

Characterisation of transverse slice culture preparations of postnatal rat spinal cord: preservation of defined neuronal populations

Spinal cord injury induces degenerative and regenerative processes and complex interactions of neurons with non-neuronal cells. In order to develop an in vitro tool for the investigation of such processes, we prepared and characterised spinal cord slice cultures (SCSC) from Wistar rats (p0–12). SCSC were sustained in vitro up to 12 days and characterised by immunohistochemistry. Calbindin⁺ neurons, distributed across the entire gray matter, were visible also after longer culture periods. NeuN⁺ neurons were best preserved in the dorsal horn whereas large NeuN⁺ and choline acetyltransferase⁺ motoneurons in the ventral horn vanished after 3 days in vitro. Nestin immunoreactivity was found in animals of all age groups, either in cells interspersed in the ependymal lining around the central canal or in cells resembling protoplasmic astrocytes. Glial fibrillary acidic protein⁺ astrocytes, initially restricted to the white matter, invaded the gray matter of SCSC early during the culture period. Microglial cells, stained by Griffonia simplicifolia isolectin B₄, were rapidly activated in the dorsal tract and in the gray matter but declined in number with time. SCSC derived from p0 or p3 animals showed a better preservation of the cytoarchitecture than cultures derived from older animals. In summary, SCSC undergo degenerative changes, but they contain defined neuronal populations, the cytoarchitecture is partially preserved and the glial reaction is limited.     

Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shiverer) adult mice with comparisons among fixation methods

The peroxidase-antiperoxidase technique was used for immunocytochemical localization of carbonic anhydrase in the mouse spinal cord to detect whether this antigen was normally present in myelinated fibers, in oligodendrocytes in both white and gray matter, and in astrocytes, and to determine where the carbonic anhydrase might be localized in the spinal cords of dysmyelinating mutant (shiverer) mice. The most favorable methods for treating tissue were: 1) immersion in formalin-ethanol-acetic acid followed by paraffin embedding, or 2) light fixation with paraformaldehyde and preparation of vibratome sections. Carnoy's solution, followed by paraffin embedding, extracted myelin from the tissue, while aqueous aldehydes, when used before paraffin embedding, reduced staining everywhere except at sites of compact myelin. The latter conclusion was based, in part, on the almost complete loss of this antigen from the shiverer cord, where compact myelin is known to be virtually absent but where membrane-bound carbonic anhydrase was demonstrated enzymatically. When the optimal methods were used with normal mouse cords, carbonic anhydrase was found throughout the white matter columns and in the oligodendrocytes in gray and white matter. The staining of the white matter was attributed to myelinated fibers because of the similarity in distribution to both a histological myelin stain and the immunocytochemical staining for myelin basic protein. In the mutant mice the oligodendrocyte cell bodies and processes, which were stained in all areas of the spinal cord, were particularly numerous at the periphery of the sections. In contrast to the oligodendrocytes, the fibrous astrocytes appeared to lack carbonic anhydrase, or to have lower than detectable levels, since the astrocyte marker, glial fibrillary acidic protein, had a very different distribution from that of carbonic anhydrase. Even finer localization was obtained in vibratome sections, where the antibody against carbonic anhydrase permitted visualization of the processes connecting oligodendrocytes to myelinated fibers in the normal adult 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.