No abstract

Demyelination contributes to the physiological and behavioral deficits after contusive spinal cord injury (SCI). Therefore, remyelination may be an important strategy to facilitate repair after SCI. We show here that rat embryonic day 14 spinal cord-derived glial-restricted precursor cells (GRPs), which differentiate into both oligodendrocytes and astrocytes, formed normal-appearing central myelin around axons of cultured DRG neurons and had enhanced proliferation and survival in the presence of neurotrophin 3 (NT3) and brain-derived neurotrophin factor (BDNF). We infected GRPs with retroviruses expressing the multineurotrophin D15A (with both BDNF and NT3 activities) and then transplanted them into the contused adult thoracic spinal cord at 9 d after injury. Expression of D15A in the injured spinal cord is five times higher in animals receiving D15A-GRP grafts than ones receiving enhanced green fluorescent protein (EGFP)-GRP or DMEM grafts. Six weeks after transplantation, the grafted GRPs differentiated into mature oligodendrocytes expressing both myelin basic protein (MBP) and adenomatus polyposis coli (APC).UltrastructuralanalysisshowedthatthegraftedGRPsformedmorphologicallynormal-appearingmyelinsheathsaroundtheaxonsinthe ventrolateral funiculus (VLF) of spinal cord. Expression of D15A significantly increased the percentage of APC ϩ oligodendrocytes of grafted GRPs (15-30%). Most importantly, 8 of 12 rats receiving grafts of D15A-GRPs recovered transcranial magnetic motor-evoked potential responses, indicating that conduction through the demyelinated VLF axons was restored. Such electrophysiological recovery was not observed in rats receiving grafts of EGFP-GRPs, D15A-NIH3T3 cells, or an injection of an adenovirus expressing D15A. Recovery of hindlimb locomotor function was also significantly enhanced only in the D15A-GRP-grafted animals at 4 and 5 weeks after transplantation. Therefore, combined treatment with neurotrophins and GRP grafts can facilitate functional recovery after traumatic SCI and may prove to be a useful therapeutic strategy to repair the injured spinal cord.

After spinal cord injury (SCI), the absence of an adequate blood supply to injured tissues has been hypothesized to contribute to the lack of regeneration. In this study, blood vessel changes were examined in 28 adult female Fischer 344 rats at 1, 3, 7, 14, 28, and 60 days after a 12.5 g x cm NYU impactor injury at the T9 vertebral level. Laminin, collagen IV, endothelial barrier antigen (SMI71), and rat endothelial cell antigen (RECA-1) immunoreactivities were used to quantify blood vessel per area densities and diameters in ventral gray matter (VGM), ventral white matter (VWM), and dorsal columns (DC) at levels ranging 15 mm rostral and caudal to the epicenter. This study demonstrates an angiogenic response, defined as SMI71/RECA-1-immunopositive endothelial cells that colocalize with a robust deposition of basal lamina and basal lamina streamers, 7 days after injury within epicenter VGM. This angiogenesis diminishes concurrent with cystic cavity formation. GAP43- and neurofilament- (68 kDa and 210 kDa) immunopositive fiber outgrowth was associated with these new blood vessels by day 14. Between 28 and 60 days after injury, increases in SMI71-immunopositive blood vessel densities were observed in the remaining VWM and DC with a corresponding increase in vessel diameters up to 15 mm rostral and caudal to the epicenter. This second angiogenesis within VWM and DC, unlike the acute response observed in VGM, did not correspond to any previously described changes in locomotor behaviors in this model. We propose that therapies targeting angiogenic processes be directed at the interval between 3 and 7 days after SCI.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to signal through a multicomponent receptor complex consisting of the Ret receptor tyrosine kinase and a member of the GFR␣ family of glycosylphosphatidylinositol-anchored receptors. In the current model of GDNF signaling, Ret delivers the intracellular signal but cannot bind ligand on its own, while GFR␣s bind ligand but are thought not to signal in the absence of Ret. We have compared signaling pathways activated by GDNF in two neuronal cell lines expressing different complements of GDNF receptors. In a motorneuron-derived cell line expressing Ret and GFR␣s, GDNF stimulated sustained activation of the Ras/ERK and phosphatidylinositol 3-kinase/Akt pathways, cAMP response element-binding protein phosphorylation, and increased c-fos expression. Unexpectedly, GDNF also promoted biochemical and biological responses in a line of conditionally immortalized neuronal precursors that express high levels of GFR␣s but not Ret. GDNF treatment did not activate the Ras/ERK pathway in these cells, but stimulated a GFR␣1-associated Src-like kinase activity in detergent-insoluble membrane compartments, rapid phosphorylation of cAMP response element-binding protein, up-regulation of c-fos mRNA, and cell survival. Together, these results offer new insights into the dynamics of GDNF signaling in neuronal cells, and indicate the existence of novel signaling mechanisms directly or indirectly mediated by GFR␣ receptors acting in a cell-autonomous manner independently of Ret.

Activation of the unfolded protein response (UPR) is involved in the pathogenesis of numerous CNS myelin abnormalities; yet, its direct role in traumatic spinal cord injury (SCI)-induced demyelination is not known. The UPR is an evolutionarily conserved cell defense mechanism initiated to restore endoplasmic reticulum homeostasis in response to various cellular stresses including infection, trauma, and oxidative damage. However, if uncompensated, the UPR triggers apoptotic cell death. We demonstrate that the three signaling branches of UPR including the PERK, ATF6, and IRE1α are rapidly initiated in a mouse model of contusive SCI specifically at the injury epicenter. Immunohistochemical analyses of the various UPR markers revealed that in neurons, the UPR appeared at 6 and 24-h post-SCI. In contrast, in oligodendrocytes and astroglia, UPR persisted at least for up to 3 days post-SCI. The UPR-associated proapoptotic transcriptional regulator CHOP was among the UPR markers upregulated in neurons and oligodendrocytes, but not in astrocytes, of traumatized mouse spinal cords. To directly analyze its role in SCI, WT and CHOP null mice received a moderate T9 contusive injury. Deletion of CHOP led to an overall attenuation of the UPR after contusive SCI. Furthermore, analyses of hindlimb locomotion demonstrated a significant functional recovery that correlated with an increase in white-matter sparing, transcript levels of myelin basic protein, and Claudin 11 and decreased oligodendrocyte apoptosis in CHOP null mice in contrast to WT animals. Thus, our study provides evidence that the UPR contributes to oligodendrocyte loss after traumatic SCI.

After traumatic spinal cord injury (SCI), disruption and plasticity of the microvasculature within injured spinal tissue contribute to the pathological cascades associated with the evolution of both primary and secondary injury. Conversely, preserved vascular function most likely results in tissue sparing and subsequent functional recovery. It has been difficult to identify subclasses of damaged or regenerating blood vessels at the cellular level. Here, adult mice received a single intravenous injection of the Griffonia simplicifolia isolectin B4 (IB4) at 1-28 days following a moderate thoracic (T9) contusion. Vascular binding of IB4 was maximally observed 7 days following injury, a time associated with multiple pathologic aspects of the intrinsic adaptive angiogenesis, with numbers of IB4 vascular profiles decreasing by 21 days postinjury. Quantitative assessment of IB4 binding shows that it occurs within the evolving lesion epicenter, with affected vessels expressing a temporally specific dysfunctional tight junctional phenotype as assessed by occludin, claudin-5, and ZO-1 immunoreactivities. Taken together, these results demonstrate that intravascular lectin delivery following SCI is a useful approach not only for observing the functional status of neovascular formation but also for definitively identifying specific subpopulations of reactive spinal microvascular elements.

The presence of nerve growth factor (NGF) mRNA and protein in the rat central nervous system is documented. Blot-hybridization analysis showed an abundance of NGF mRNA in the hippocampus, cerebral cortex, and olfactory bulb. Enzyme immunoassay confirmed significant levels of a NGF-like protein in the hippocampus and cerebral cortex. Bioassay of a NGF-like immunoaffinity-purified protein from these regions was physiologically indistinguishable from NGF. Immunohistochemistry revealed a widespread distribution of NGF-like reactivity in the adult brain, preferentially in fiber tracts. NGF mRNA accumulation began at birth, with adult levels reached 3 weeks postnatally. Enzyme immunoassay detected the presence of a NGF-like protein in the embryonic rat brain. Postnatally, the level of NGF-like protein reached a maximum at 3 weeks. Additionally, a distinct fetal form of NGF may exist.,B nerve growth factor (NGF) is essential for the development and maintenance of sensory and sympathetic neurons in the peripheral nervous system (PNS) (1, 2). However, both the presence and potential physiological function of NGF in the central nervous system (CNS) has remained elusive (3). The characteristic responses of peripheral adrenergic neurons following NGF or anti-NGF antibody injection have not been observed after similar application into the CNS (4-11).In the PNS, NGF is taken up by adrenergic terminals and also retrogradely transported to the cell body (2). In the CNS, injected NGF was specifically retrogradely transported from the hippocampus to the medial septum and the diagonal band of Broca (9) and also from the neocortex to the nucleus basalis (12), presumably in cholinergic neurons. NGF induced choline acetyltransferase in neonatal rat cortex, hippocampus, septum (10), and striatum (11), but not in adult hippocampus (13).Thus, NGF may have a physiological function in the CNS, and affect cholinergic neurons. Initial indication of endogenous NGF in the CNS was obtained by immunohistochemistry in the fetal rat (14, 15). NGF cDNA clones have been isolated from male mouse submaxillary glands (16,17), and Shelton and Reichardt (18) recently detected NGF mRNA in the rat brain. Subsequently, NGF mRNA and an NGF-like protein have been described in some regions of the adult rat brain (19).In the present study, we demonstrate the developmental time course as well as the adult regional distribution of both NGF mRNA and protein in the adult rat brain. Finally, using a bioassay, we show that the adult brain contains nerve growth-stimulating activity typical of purified NGF. Our results demonstrate that NGF is expressed in the adult rat CNS with regional specificity and suggests the existence of a fetal form of NGF. MATERIALS AND METHODSPreparation of RNA and Blot-Hybridization Analysis. Total RNA from the brains of Sprague-Dawley rats (Alab, Stockholm, Sweden) was prepared (20), and poly(A)+ RNA, isolated by oligo(dT) chromatography (21), was separated on 1% agarose gels containing 0.7% formaldehyde and transferred to nitrocel...

The infusion of BDNF and NT-3 into Schwann cell (SC) grafts promotes regeneration of brainstem neurones into the grafts placed in adult rat spinal cord transected at T8 (Xu et al., 1995b). Here, we compared normal SCs with SCs genetically modified to secrete human BDNF, grafted as trails 5 mm long in the cord distal to a transection site and also deposited in the transection site, for their ability to stimulate supraspinal axonal regeneration beyond the injury. SCs were infected with the replication-deficient retroviral vector pL(hBDNF)RNL encoding the human preproBDNF cDNA. The amounts of BDNF secreted (as detected by ELISA) were 23 and 5 ng/24 h per 106 cells for infected and normal SCs, respectively. Biological activity of the secreted BDNF was confirmed by retinal ganglion cell bioassay. The adult rat spinal cord was transected at T8. The use of Hoechst prelabelled SCs demonstrated that trails were maintained for a month. In controls, no SCs were grafted. One month after grafting, axons were present in SC trails. More 5-HT-positive and some DbetaH-positive fibres were observed in the infected vs. normal SC trails. When Fast Blue was injected 5 mm below the transection site (at the end of the trail), as many as 135 retrogradely labelled neurones could be found in the brainstem, mostly in the reticular and raphe nuclei (normal SCs, up to 22, mostly in vestibular nuclei). Numerous neurones were labelled in the ventral hypothalamus (normal SCs, 0). Also, following Fast Blue injection, a mean of 138 labelled cells was present in dorsal root ganglia (normal SCs, 46) and spinal cord (39 vs. 32) rostral to the transection. No labelled spinal neurones rostral to the transection were seen when SCs were not transplanted. Thus, the transplantation of SCs secreting increased amounts of BDNF improved the regenerative response across a transection site in the thoracic cord. Moreover, the enhanced regeneration observed with infected SCs may be specific as the largest response was from neurones known to express trkB.