A time course for the focal elevation of synthesis of basic fibroblast growth factor and one of its high-affinity receptors (flg) following a localized cortical brain injury.

Traumatic injury to the CNS initiates transient and unsuccessful regeneration of damaged neural pathways, accompanied by reactive gliosis, angiogenesis, and deposition of a dense fibrous glial/meningeal scar at the wound site. Basic fibroblast growth factor (basic FGF) is a CNS protein with potent effects on neurons, glia, fibroblasts, and vascular endothelial cells. Hybridization and immunocytochemical methods were used to examine temporal and spatial changes in distribution and levels of basic FGF protein and mRNA and also of its receptor mRNA (flg), following a defined wound to the cerebral cortex of adult rat brains. In the injured brain, a rapid, transient increase in basic FGF mRNA and protein is readily detectable within 7 d of surgery and thereafter declines in the tissues bordering the lesion. The increased expression is localized to multiple cell types including macrophages, neurons, astrocytes, and vascular endothelial cells. The changes in immunoreactive basic FGF parallel changes in the bioactivity of extracted heparin-binding proteins, which include basic FGF. Focal increases in flg mRNA appear 7 d after injury and subside by 14 d. The changes in local basic FGF synthesis, concentration, localization, and bioactivity suggest that this growth factor may contribute to the cascade of cellular events that occur in CNS wound repair.     

Oligodendroglial reaction following spinal cord injury in rat: Transient upregulation of MBP mRNA

The reaction of oligodendrocytes in response to traumatic injury of the CNS are poorly understood. In the present report we studied changes in the expression of a major constituent of CNS myelin, myelin basic protein (MBP), by immunohistochemistry and in situ hybridization from 6 h up to 2 weeks following partial transection of the spinal cord in adult rats. MBP immunohistochemistry showed degeneration of myelin at the lesion center and signs of myelin breakdown in necrotic foci in the dorsal and ventral funiculi proximal and distal to the lesion. In situ hybridization revealed that mRNA for MBP was downregulated at the local lesion site within the first day following injury, probably reflecting oligodendrocytes to undergo cell death. From 2 days on, however, MBP mRNA was conspicuously upregulated at the border of the lesion area. This “reactive” response of surviving oligodendrocytes, as indicated by increased levels of MBP mRNA, peaked around 8 days. At this time, oligodendrocytes displaying strong MBP in situ signal formed stripe-like structures which were oriented radially toward the lesion center and arranged in parallel to neurofilament-positive axons. At around 2 weeks post-injury, MBP mRNA at the border of the lesion area was again downregulated to levels comparable to uninjured controls. These results show that traumatic injury of the spinal cord induces a “reactive” response of surviving oligodendrocytes adjacent to lesion sites. This response might represent an important component of local repair mechanisms. GLIA 23:278–284, 1998. © 1998 Wiley-Liss, Inc.     

Expression of protein zero is increased in lesioned axon pathways in the central nervous system of adult zebrafish

The immunoglobulin superfamily molecule protein zero (P0) is important for myelin formation and may also play a role in adult axon regeneration, since it promotes neurite outgrowth in vitro. Moreover, it is expressed in the regenerating central nervous system (CNS) of fish, but not in the nonregenerating CNS of mammals. We identified a P0 homolog in zebrafish. Cell type-specific expression of P0 begins in the ventromedial hindbrain and the optic chiasm at 3-5 days of development. Later (at 4 weeks) expression has spread throughout the optic system and spinal cord. This is consistent with a role for P0 in CNS myelination during development. In the adult CNS, glial cells constitutively express P0 mRNA. After an optic nerve crush, expression is increased within 2 days in the entire optic pathway. Expression peaks at 1 to 2 months and remains elevated for at least 6 months postlesion. After enucleation, P0 mRNA expression is also upregulated but fails to reach the high levels observed in crush-lesioned animals at 4 weeks postlesion. Spinal cord transection leads to increased expression of P0 mRNA in the spinal cord caudal to the lesion site. The glial upregulation of P0 mRNA expression after a lesion of the adult zebrafish CNS suggests roles for P0 in promoting axon regeneration and remyelination after injury.     

The chondroitin sulphate proteoglycan brevican is upregulated by astrocytes after entorhinal cortex lesions in adult rats

The chondroitin sulphate proteoglycan brevican is one of the most abundant extracellular matrix molecules in the adult rat brain. It is primarily synthesized by astrocytes and is believed to influence astroglial motility during development and under certain pathological conditions. In order to study a potential role of brevican in the glial reaction after brain injury, its expression was analysed following entorhinal cortex lesion in rats (12 h, 1, 2, 4, 10, 14 and 28 days and 6 months post lesion). In situ hybridization and immunohistochemistry were employed to study brevican mRNA and protein, respectively, in the denervated outer molecular layer of the fascia dentata and at the lesion site. In both regions brevican mRNA was upregulated between 1 and 4 days post lesion. The combination of in situ hybridization with immunohistochemistry for glial fibrillary acidic protein demonstrated that many brevican mRNA-expressing cells are astrocytes. In the denervated zone of the fascia dentata, immunostaining for brevican was increased by 4 days, reached a maximum by 4 weeks and remained detectable up to 6 months post lesion. Electron microscopic immunocytochemistry showed that brevican is a component of the extracellular matrix compartment. At the lesion site a similar time course of brevican upregulation was observed. These data demonstrate that brevican is upregulated in areas of brain damage as well as in areas denervated by a lesion. They suggest a role of brevican in reactive gliosis and are compatible with the hypothesis that brevican is involved in the synaptic reorganization of denervated brain areas.     

Elevated expression of B-50 (GAP-43)-mRNA in a subpopulation of olfactory bulb mitral cells following axotomy

Neurons in the central nervous system regenerate poorly or not at all. In contrast neurons of the peripheral nervous system have the ability to regrow their nerve fibers over considerable distances. Previously it has been suggested that the absence of the reinduction of the expression of growth associated proteins such as B-50 (GAP43) may be an important factor in the differential response of CNS and PNS neurons to injury. We studied B-50(GAP43) mRNA expression following lesioning of a class of CNS neurons, the olfactory bulb mitral cells. Expression of B-50 mRNA in approximately 40% of the mitral cells was upregulated in response to transection of their axons in the lateral olfactory tract (LOT). Enhanced expression persisted for 10 days postlesion but had virtually declined to control levels by 4 weeks after the lesion. A large proportion of the mitral cells gradually degenerated subsequent to LOT transection. Thus a subpopulation of mitral cells maintains their ability to upregulate B-50, a protein characteristic of growing axons, but enhanced B-50 expression is not accompanied by regeneration of the severed LOT. © 1993 Wiley-Liss, Inc.     

CD14 expression by activated parenchymal microglia/macrophages and infiltrating monocytes following human traumatic brain injury

The immune response in the central nervous system (CNS) is under tight control of regulatory mechanisms, resulting in the establishment of immune privilege. CNS injury induces an acute inflammatory reaction, composed mainly of invading leukocytes and activated microglial cells/macrophages. The generation of this robust immune response requires binding of receptors such as CD14, a pattern recognition receptor of the immune system. CD14, a surface molecule of monocytic cells, is up-regulated after monocyte stimulation and is involved in cellular activation. To examine CD14 expression in human brain lesions we investigated sections of brains obtained at autopsy from 25 cases following closed traumatic brain injury (TBI) and 5 control brains by immunohistochemistry. Detection of CD14 in controls demonstrated constitutive expression by perivascular cells, but not in parenchymal microglial cells, equivalent to known expression pattern of ED2 in rats. Following TBI, numbers of CD14(+) cells in perivascular spaces and in the brain parenchyma increased in parallel within 1-2 days, both at the lesion and in adjacent perilesional areas. The number of CD14(+) cells in perivascular spaces and in the brain parenchyma reached maximum levels within 4-8 days and remained elevated until weeks after trauma. In contrast to activated parenchymal microglia/macrophages, resting parenchymal microglial cells lacked CD14. Thus, early CD14 expression constitutes an essential part of the acute inflammatory CNS response following trauma.     

Modulation of apolipoprotein D and apolipoprotein E expression in rat hippocampus after entorhinal cortex lesion.

Apolipoprotein (apo) D is a member of the lipocalin family of proteins. Although its physiological function is unknown, apoD is thought to transport one or more small hydrophobic ligands. A second apolipoprotein, apoE is known to play an important role in lipid transport, and apoE genetic polymorphism has been shown to be associated with susceptibility to Alzheimer's disease. Both apoD and apoE are expressed in the central nervous system (CNS) and both proteins accumulate at sites of peripheral nerve injury due to increased local synthesis. The two proteins may have overlapping or complementary functions within nervous tissue. In order to define the role of apoD within the CNS, we have studied the regional distribution of apoD and apoE mRNA and protein within the normal rat brain and the changes in apoD and apoE expression in the hippocampus of rats after entorhinal cortex lesion (EC lesion). Within the brains of normal rats, apoD expression in the hippocampus was as high as 180-fold that of the liver. ApoD mRNA levels in other areas of the rat brain ranged from 40 to 120 times the hepatic levels. The distribution of apoE gene expression within the brain was similar to that of apoD, but was much lower than hepatic apoE expression. When rats were subjected to EC lesion, the apoD message increased by 54% at 4 days post lesion (DPL) in the ipsilateral region of hippocampus while apoE mRNA levels (ipsilateral and contralateral) decreased by 43%. At 6 to 8 DPL apoD mRNA in the ipsilateral hippocampus remained elevated (42% above controls) whereas the apoE mRNA levels increased to about 15% above those of controls. At 14 and 31 DPL, both apoD and apoE expression was similar to controls. The increase in immunoreactive apoD in hippocampal extracts was more dramatic. At 1 DPL, immunoreactive apoD levels were already 16-fold higher than those in extracts of non-lesioned animals and, at 31 DPL, levels were still 8-fold higher than those of control animals. Finally, we have demonstrated that the levels of apoD in the brains of apoE-deficient mice are 50-fold those of wildtype control mice. ApoD clearly has an important function within the CNS in both normal and pathological situations.     

Differential expression of neuropsin and protease M/neurosin in oligodendrocytes after injury to the spinal cord

Neuropsin and protease M/neurosin are serine proteases expressed by neurons and glial cells, and serve a variety of functions in the central nervous system (CNS). The current study demonstrates changes in the expression of these proteases following hemisection of the mouse spinal cord. Within unlesioned spinal cord, neuropsin mRNA expression was occasionally observed in the gray but not white matter, while the level of protease M/neurosin mRNA was higher in the white matter. After injury to the spinal cord, neuropsin mRNA expression was induced in the white matter in the area immediately adjacent to the lesion, peaking at 4 days post-injury and disappearing by 14 days. Enhanced expression of protease M/neurosin mRNA was observed throughout the white and gray matter surrounding the lesion, peaking at 4 days and persisting for 14 days. Neuropsin mRNA was expressed predominantly by CNPase-positive oligodendrocytes. Furthermore, most of these cells were also associated with immunoreactivity for protease M/neurosin protein. Within unlesioned spinal cord, most protease M/neurosin mRNA-expressing cells were CNPase-positive oligodendrocytes, and a substantial fraction of these cells also showed immunoreactivity for NG2, a marker for oligodendrocyte progenitors. After injury, protease M/neurosin mRNA expression within NG2-positive cells was significantly decreased, while the constitutive expression in CNPase-positive oligodendrocytes appeared to be preserved. These findings suggest that each subpopulation of oligodendrocytes based on the expression of neuropsin and protease M/neurosin has different roles in the response of the spinal cord to injury as well as in normal homeostasis.     

Expression of insulin-like growth factor-1 (IGF-1) and IGF-binding protein 2 (IGF-BP2) in the hippocampus following cytotoxic lesion of the dentate gyrus

Receptor binding and gene expression of several members of the IGF gene family were examined in the rat brain following lesion of the hippocampal dentate gyrus granular cells by intradentate colchicine injection. Dentate granular cell loss was accompanied by extensive reactive gliosis in the lesioned hippocampus and damaged overlying cortex, as verified by the increase in GFAP mRNA and BS-1 lectin binding. At 4 days post-lesion, ¹²⁵I-IGF-2 binding was dramatically increased within the lesioned dentate gyrus and damaged overlying cortex, and corresponded temporally and anatomically with increased IGF-BP2 gene expression following the lesion. Increased IGF-BP3 gene expression was only observed in the overlying cortex at 10 days post-lesion, and corresponded with an increase in ¹²⁵I-IGF-1 binding at the injured surface of the cortex. Type-2 IGF receptor mRNA expression was reduced to background levels in the lesioned dentate gyrus, suggesting that IGF-BP2 was a major component of the observed increase in ¹²⁵I-IGF-2 binding. In situ hybridization also revealed a prominent increase in IGF-1 mRNA expression by 4 days post-lesion, which was localized within the lesioned dentate gyrus and damaged cortical areas, and was shown to be expressed by microglia. While no IGF-2 mRNA expression was observed within the CNS, either prior to, or following the lesion, IGF-2 mRNA expression was observed in the choroid plexus, meningeal membranes, and in blood vessel endothelium, providing a potential source for the transport of IGF-2 into the CNS. In the injured CNS, increased IGF-BP2 expression may act to maintain or transport IGF-1 or IGF-2, as well as modulate the local autocrine and paracrine actions of the IGFs. Increased microglial IGF-1 expression following colchicine treatment correlates with the timing of a number of post-traumatic events within the CNS, suggesting that IGF-1 may have a role as a neuroprotectant for surviving neurons and signal for local neuronal sprouting, as well as a role in reactive astrogliosis. © 1996 Wiley-Liss, Inc.     

VEGF mRNA Induction Correlates With Changes in the Vascular Architecture Upon Spinal Cord Damage in the Rat

The multiple cellular and molecular processes induced by injury to the central nervous system (CNS) are still poorly understood. In the present study, we investigated the response of the vasculature and the expression of mRNA for the angiogenic vascular endothelial growth factor (VEGF) following X-irradiation of the spinal cord in the newborn and following traumatic spinal cord injury in the adult rat. Both lesion models induced changes in the density and the distribution pattern of blood vessels: while X-irradiation led to a permanent local increase in vascular density in the fibre tracts of the exposed segments, a transient local sprouting of vessels was induced upon traumatic spinal cord injury. In situ hybridization showed that an increase of VEGF mRNA anticipated and overlapped with the vascular responses in both lesion models. In addition to the temporal correlation of VEGF expression and vascular sprouting, there was a clear correlation in the spatial distribution patterns. Following X-irradiation, the expression of VEGF mRNA was restricted to the fibre tracts, precisely the areas where the changes in the vasculature were observed later on. Upon transection in the adult animal, VEGF was mainly detectable at the border of the lesion area, where the transient increase in vascular density could be observed. Interestingly, according to the type of lesion applied, astrocytes (X-irradiation) or inflammatory cells (presumably microglial cells or macrophages; traumatic lesion) are the cellular sources of VEGF mRNA. Our results strongly indicate that VEGF is crucially involved in mediating vascular changes following different types of injury in the CNS.     

Increased expression of the close homolog of the adhesion molecule L1 in different cell types over time after rat spinal cord contusion

The close homolog of the adhesion molecule L1 (CHL1) is important during CNS development, but a study with CHL1 knockout mice showed greater functional recovery after spinal cord injury (SCI) in its absence. We investigated CHL1 expression from 1 to 28 days after clinically relevant contusive SCI in Sprague-Dawley rats. Western blot analysis showed that CHL1 expression was significantly up-regulated at day 1 and further increased over 4 weeks after SCI. Immunohistochemistry of tissue sections showed that CHL1 in the intact spinal cord was expressed at low levels. By 1 day and through 4 weeks after SCI, CHL1 became highly expressed in NG2(+) cells. Hypertrophic GFAP(+) astrocytes also expressed CHL1 by 1 week after injury. The increase in CHL1 protein paralleled that of NG2 in the first week and GFAP between 1 and 4 weeks after injury. At 4 weeks, NG2(+) /CHL1(+) cells and GFAP(+) /CHL1(+) astrocytes were concentrated at the boundary between residual spinal cord tissue and the central lesion. NF200(+) spinal cord axons approached but did not penetrate this boundary. In contrast, CHL1(+) cells in the central lesion at 1 week and later colabeled with p75 and NG2 and were chronically associated with many NF200(+) axons, presumably axons that had sprouted in association with CHL1(+) Schwann cells infiltrating the cord after contusion. Thus, our study demonstrates up-regulation of CHL1 in multiple cell types and locations in a rat model of contusion injury and suggests that this molecule may be involved both in inhibition of axonal regeneration and in recovery processes after SCI.     

Transient upregulation of NCAM mRNA in astrocytes in response to entorhinal cortex lesions and ischemia

Axonal sprouting and synaptic reorganization play an important role in the adaptation of the CNS to injury. However, the molecular mechanisms underlying this neuronal plasticity are poorly understood. In the present study we used in situ hybridization to examine the expression of NCAM mRNA in normal hippocampus, and in response to entorhinal cortex (EC) lesions and transient global ischemia. Both neurons and astrocytes were labeled by digoxygenin-tagged cRNA probes which recognize all three major NCAM isoforms of the adult CNS. In contrast, NCAM180-specific probes labeled only neurons in the hippocampus. After unilateral EC lesion, a transient and anatomically restricted upregulation of NCAM120/140 mRNA in reactive astrocytes in the denervated molecular layer of the dentate gyrus was observed. This increase was only present 2–4 days after the lesion whereas the GFAP mRNA increase was present up to 30 days postlesion. Following global ischemia a similar, transient increase of NCAM120/140 mRNA labeling of reactive astrocytes was observed; this increase was anatomically restricted to CA1, where neuronal loss occurred. Results suggest that the transient upregulation of NCAM120/140 mRNA in reactive astrocytes shortly after injury might be an important molecular mechanism in the cascade of events underlying neuronal plasticity in the adult CNS.     

Expression of insulin‐like growth factors in remyelination following ethidium bromide‐induced demyelination in the mouse spinal cord

Insulin‐like growth factors, IGF‐I and IGF‐II, play important roles in development and myelination in the CNS, but little is known about the response of IGF after demyelination. The present study investigated the expression of IGF and their cognitive receptors in the process of remyelination following ethidium bromide (EBr)‐induced demyelination in the adult mouse spinal cord. The present results, in a quantitative real‐time PCR, showed significant increases in the levels of the mRNA for both IGF‐I and IGF‐II during both the demyelination and remyelination stages. The levels of IGF‐I receptor (IGF‐IR) mRNA increased from 10 days to 4 weeks after the EBr injection. The levels of IGF‐II receptor (IGF‐IIR) mRNA decreased for 6 days and then increased 10 days after the EBr injection. In situ hybridization studies showed the cells expressing IGF‐I mRNA to be mainly macrophage‐like cells, while those expressing IGF‐II mRNA were predominantly Schwann cell‐like cells invading the demyelinating lesion. The immunoreactivity for the IGF‐IR and IGF‐IIR increased in various kinds of cells within and around the demyelinating lesions from 6 days to 4 weeks after the EBr injection. These results suggest that locally produced IGF could partly be involved in some mechanisms underlying remyelination processes following the EBr‐induced demyelination in the mouse spinal cord.     

Injury-induced Regulation of Ciliary Neurotrophic Factor mRNA in the Adult Rat Brain

Ciliary neurotrophic factor (CNTF) is a pleiotropic molecule that acts as a neurotrophic factor for a wide range of embryonic neurons as well as a differentiation factor for sympathetic neuroblasts and O2A progenitor cells in culture. CNTF messenger RNA (mRNA) is present at very low levels in the normal adult rat central nervous system (CNS), but is dramatically up-regulated after an aspiration lesion of dorsal hippocampus and overlying cortex, in the area coincident with glial scar. The increased level of CNTF mRNA in lesioned hippocampus is maximal by 3 days and is sustained for up to 20 days, the longest time point examined. In contrast, mRNA levels for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were slightly decreased during the same period. In situ hybridization experiments revealed that cells expressing CNTF mRNA were concentrated at the margin of the wound, and also present within the gelfoam which filled the lesion cavity. This distribution of CNTF-expressing cells corresponded very closely to that of cells expressing high levels of glial fibrillary acidic protein mRNA at the wound site. Paralleling the observed increase in CNTF mRNA, increased levels of CNTF-like neurotrophic activity were apparent in soluble extracts of the lesioned tissues. This neurotrophic activity for ciliary ganglion neurons was completely blocked by the addition of neutralizing antiserum against CNTF. Basic fibroblast growth factor, which has been shown by others to increase after a similar lesion paradigm (Frautschy et al., Brain Res. , 553 , 291–299, 1991), does not contribute appreciably to this trophic activity. We conclude that CNTF is markedly increased as a function of injury to the CNS and that its expression is most likely restricted to reactive astrocytes in the glial scar.     

Tenascin-C expression and axonal sprouting following injury to the spinal dorsal columns in the adult rat

We have examined the expression and distribution of the extracellular matrix molecule tenascin-C in and around lesions of the thoracic dorsal columns in adult rats 3 days to 8 weeks after injury, using in situ hybridization, immunofluorescence, electron microscopy and immunoelectron microscopy. Numerous tenascin-C mRNA+ cells were present in and around the lesion at 3 days; fewer were present at 14 days and almost none 30 days after injury. Most tenascin-C mRNA+ cells in the spinal cord around the lesion were GFAP+, but most of those within the lesion were not, suggesting that tenascin-C is produced in the injured spinal cord by a subpopulation of astrocytes and by other cells that invade the lesion; these cells may include meningeal cells, macrophages, and Schwann cells. From 3 to 30 days after injury, heavy tenascin-C immunoreactivity was present at the lesion site (especially transections), and there was lighter immunoreactivity around the lesion and in the degenerating dorsal column. The heaviest immunoreactivity was associated with collagen fibrils in areas of expanded extracellular space and with basal laminae (covering Schwann cells and some astrocytes) but tenascin-C was also found close to the surfaces of some OX-42+ macrophages/microglia, leptomeningeal cells, and capillaries. Neurofilament (NF)+ axons grew into the highly tenascin-C-immunoreactive lesion sites, indicating that tenascin-C does not prevent axonal growth into these areas. However, such axons were not coated with tenascin-C except where directly exposed to the extracellular space. J. Neurosci. Res. 49:433–450, 1997. © 1997 Wiley-Liss, Inc.     

Lesion-induced changes in the expression of ciliary neurotrophic factor and its receptor in rat optic nerve

There is evidence that ciliary neurotrophic factor (CNTF) is involved in reactive changes following lesions of the nervous system. To investigate, whether differences in the regulation of CNTF and CNTF receptor α (CNTFRα) contribute to the differences in PNS and CNS responses to injury, we have studied their expression on the mRNA and protein level in the rat optic nerve following a crush lesion to compare them with the situation in peripheral nerve. Seven days after the lesion, CNTF mRNA and protein levels were markedly decreased at the lesion site, concommitant with the disappearance of GFAP- and CNTF-immunopositive astrocytes. CNTF levels in proximal and distal parts were less affected. This was in contrast to the situation in the PNS, where CNTF was downregulated at and distal to the lesion site. Different from other CNS regions, optic nerve astrocytes expressed CNTFRα mRNA under normal conditions. Following lesion, CNTFRα was reduced substantially only in distal and proximal parts of the optic nerve but continued to be expressed at high levels at the lesion site, suggesting that GFAP-negative, CNTF-responsive cells are present there. Our results suggest that differences in lesion-induced changes in the optic and sciatic nerve reflect differences in the response to injury of astrocytes and Schwann cells. In the light of the known actions of CNTF in inducing astrogliosis, the expression pattern observed in the optic nerve indicates that CNTF and CNTFRα are involved in glial scar formation in the lesion area. GLIA 23:239–248, 1998. © 1998 Wiley-Liss, Inc.     

Semaphorin and neuropilin expression in motoneurons after intraspinal motoneuron axotomy

We have examined mRNA and protein distribution for the axon guidance molecules semaphorin3A, 3F, 4F and semaphorin receptors neuropilin-1 and 2, 1-21 days after intramedullary axotomy of rat lumbar spinal cord motoneurons. We show that semaphorin3A mRNA and protein are up-regulated in the scar and in motoneurons from 3 days and upto 3 weeks after injury. Neuropilin-1 mRNA showed no changed expression in axotomized motoneurons. Semaphorin3F mRNA expression was found in ventral roots after ventral funiculus lesion (VFL) and neuropilin-2 mRNA was found in affected motoneurons from 1 day after injury throughout the examined period. Semaphorin4F mRNA was first found in motoneurons 3 weeks after lesion. These results suggest semaphorin/neuropilin involvement in the injury response of intramedullary axotomized motoneurons.