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This article reviews all historical literature in which rodent-derived myelinating cells have been engrafted into the contused adult rodent spinal cord. From 2500 initial PubMed citations identified, human cells grafts, bone mesenchymal stem cells, olfactory ensheathing cells, non-myelinating cell grafts, and rodent grafts into hemisection or transection models were excluded, resulting in the 67 studies encompassed in this review. Forty five of those involved central nervous system (CNS)-derived cells, including neural stem progenitor cells (NSPCs), neural restricted precursor cells (NRPs) or oligodendrocyte precursor cells (OPCs), and 22 studies involved Schwann cells (SC). Of the NSPC/NPC/OPC grafts, there was no consistency with respect to the types of cells grafted and/or the additional growth factors or cells co-grafted. Enhanced functional recovery was reported in 31/45 studies, but only 20 of those had appropriate controls making conclusive interpretation of the remaining studies impossible. Of those 20, 19 were properly powered and utilized appropriate statistical analyses. Ten of those 19 studies reported the presence of graft-derived myelin, 3 reported evidence of endogenous remyelination or myelin sparing, and 2 reported both. For the SC grafts, 16/21 reported functional improvement, with 11 having appropriate cellular controls and 9/11 using proper statistical analyses. Of those 9, increased myelin was reported in 6 studies. The lack of consistency and replication among these preclinical studies are discussed with respect to the progression of myelinating cell transplantation therapies into the clinic.

Within the cervical and lumbar spinal enlargements, central pattern generating (CPG) circuitry produces the rhythmic output necessary for limb coordination during locomotion. Long propriospinal neurons that inter-connect these CPGs are thought to secure hindlimb-forelimb coordination, ensuring that diagonal limb pairs move synchronously while the ipsilateral limb pairs move out-of-phase during stepping. Here, we show that silencing long ascending propriospinal neurons (LAPNs) that interconnect the lumbar and cervical CPGs disrupts left-right limb coupling at each girdle. These perturbations to interlimb coordination occurred independent of the locomotor rhythm, did not affect intralimb coordination, and did not disrupt the speed-dependent (or any other) principal features of locomotion. Strikingly, the functional consequences of silencing LAPNs are highly context-dependent; the phenotype was not expressed during swimming, treadmill stepping, exploratory locomotion, or walking on an uncoated, slick surface. Together, these data show that LAPNs provide temporal information important for interlimb coordination in a flexible, context-driven manner.

CD36 is a pleiotropic receptor involved in several pathophysiological conditions, including cerebral ischemia, neurovascular dysfunction and atherosclerosis, and recent reports implicate its involvement in the endoplasmic reticulum stress response (ERSR). We hypothesized that CD36 signaling contributes to the inflammation and microvascular dysfunction following spinal cord injury. Following contusive injury, CD36(-/-) mice demonstrated improved hindlimb functional recovery and greater white matter sparing than CD36(+/+) mice. CD36(-/-) mice exhibited a reduced macrophage, but not neutrophil, infiltration into the injury epicenter. Fewer infiltrating macrophages were either apoptotic or positive for the ERSR marker, phospho-ATF4. CD36(-/-) mice also exhibited significant improvements in injury heterodomain vascularity and function. These microvessels accumulated less of the oxidized lipid product 4-hydroxy-trans-2-nonenal (4HNE) and exhibited a reduced ERSR, as detected by vascular phospho-ATF4, CHOP and CHAC-1 expression. In cultured primary endothelial cells, deletion of CD36 diminished 4HNE-induced phospho-ATF4 and CHOP expression. A reduction in phospho-eIF2α and subsequent increase in KDEL-positive, ER-localized proteins suggest that 4HNE-CD36 signaling facilitates the detection of misfolded proteins upstream of eIF2α phosphorylation, ultimately leading to CHOP-induced apoptosis. We conclude that CD36 deletion modestly, but significantly, improves functional recovery from spinal cord injury by enhancing vascular function and reducing macrophage infiltration. These phenotypes may, in part, stem from reduced ER stress-induced cell death within endothelial and macrophage cells following injury.

The endoplasmic reticulum (ER) stress response (ERSR) is activated to maintain protein homeostasis or induce apoptosis in the ER in response to distinct cellular insults including hypoxia, inflammation, and oxidative damage. Recently, we showed ERSR activation in a mouse model of a contusive spinal cord injury (SCI) and an improved hindlimb locomoter function following SCI when the pro-apoptotic arm of ERSR was genetically inhibited. The objective of the current study was to explore if the pharmacological enhancement of the homeostatic arm of the ERSR pathway can improve the functional outcome after SCI. Salubrinal enhances the homoestatic arm of the ERSR by increasing phosphorylation of eIF2α. Salubrinal significantly enhanced the levels of phosphorylated eIF2α protein and modulated the downstream ERSR effectors assessed at the lesion epicenter 6 hours post-SCI. Hindlimb locomotion showed significant improvement in animals treated with salubrinal. Treadmill-based-gait assessement showed a significant increase in maximum speed of coordinated walking and a decrease in rear stance time and stride length in salubrinal-treated animals. This improved functional recovery corresponded with increased white matter sparing and decreased oligodendrocyte apoptosis. In addition, salubrinal protected cultured mouse oligodendrocyte progenitor cells against the ER stress-inducing toxin tunicamycin. These data suggest that boosting the homeostatic arm of the ERSR reduces oligodendrocyte loss after traumatic SCI and supports the contention that pharmacological targeting of the ERSR after CNS trauma is a therapeutically viable approach.

Ethidium bromide (EB) has been extensively used in the rat as a model of spinal cord demyelination. However, this lesion has not been addressed in the adult mouse, a model with unlimited genetic potential. Here we characterize behavioral function, inflammation, myelin status and axonal viability following bilateral injection of 0.20 mg/mL ethidium bromide or saline into the ventral white matter (VWM) of female C57Bl/6 mice. EB-induced VWM demyelination significantly reduced spared VWM and Basso Mouse Scale (BMS) scores persisting out to 2 months. Chronic hindlimb dysfunction was accompanied by a persistent inflammatory response (demonstrated by CD45(+) immunofluorescence) and axonal loss (demonstrated by NF-M immunofluorescence and electron microscopy; EM). These cellular responses differ from the rat where inflammation resolves by 3-4 weeks and axon loss is minimal following EB demyelination. As these data suggest that EB-injection in the mouse spinal cord is a non-remyelinating lesion, we sought to ask whether wheel running could promote recovery by enhancing plasticity of local lumbar circuitry independent of remyelination. This did not occur as BMS and Treadscan assessment revealed no significant effect of wheel running on recovery. However, this study defines the importance of descending ventral motor pathways to locomotor function in the mouse as VWM loss results in a chronic hindlimb deficit.

This study investigated whether neuronal inhibitor of DNA binding 2 (Id2), a regulator of basic helix-loop-helix (bHLH) transcription factors, can activate the intrinsic neuritogenetic mode of dorsal root ganglion (DRG) neurons in adult mice following spinal cord injury (SCI). First, the Id2 developmental expression profile of DRG neurons, along with the correlated activity of Cdh1-anaphase promoting complex (Cdh1-APC), was characterized. Next, a D-box mutant Id2 (Id2DBM) adenoviral vector, resistant to Cdh1-APC degradation, was developed to enhance neuronal Id2 expression. After the vector was introduced into DRG neurons, the effect of Id2 on neurite outgrowth of cultured DRG neurons and sensory axonal regeneration following spinal cord dorsal hemisection was evaluated. The expression of Id2 in DRG neurons was high in the embryonic stage, downregulated after birth, and significantly reduced in the adult. Expression of Cdh1-APC was opposite to Id2, which may be responsible for Id2 degradation during DRG maturation. Overexpression of Id2DBM in DRG neurons enhanced neuritogenesis on both permissive and inhibitory substrates. Following spinal cord dorsal hemisection, overexpression of Id2DBM reduced axon dieback and increased the number and length of regenerative fibers into the lesion gap. Reprogramming the intrinsic growth status of quiescent adult DRG neurons by enhancing Id2 expression results in active neuritogenesis following SCI. Id2 may be a novel target for enhancing sensory axonal regeneration following injuries to the adult spinal cord.

Recent data have implicated thrombospondin-1 (TSP-1) signaling in the acute neuropathological events that occur in microvascular endothelial cells (ECs) following spinal cord injury (SCI) (Benton et al., 2008b). We hypothesized that deletion of TSP-1 or its receptor CD47 would reduce these pathological events following SCI. CD47 is expressed in a variety of tissues, including vascular ECs and neutrophils. CD47 binds to TSP-1 and inhibits angiogenesis. CD47 also binds to the signal regulatory protein (SIRP)α and facilitates neutrophil diapedesis across ECs to sites of injury. After contusive SCI, TSP-1(-/-) mice did not show functional improvement compared to wildtype (WT) mice. CD47(-/-) mice, however, exhibited functional locomotor improvements and greater white matter sparing. Whereas targeted deletion of either CD47 or TSP-1 improved acute epicenter vascularity in contused mice, only CD47 deletion reduced neutrophil diapedesis and increased microvascular perfusion. An ex vivo model of the CNS microvasculature revealed that CD47(-/-)-derived microvessels (MVs) prominently exhibit adherent WT or CD47(-/-) neutrophils on the endothelial lumen, whereas WT-derived MVs do not. This implicates a defect in diapedesis mediated by the loss of CD47 expression on ECs. In vitro transmigration assays confirmed the role of SIRPα in neutrophil diapedesis through EC monolayers. We conclude that CD47 deletion modestly, but significantly, improves functional recovery from SCI via an increase in vascular patency and a reduction of SIRPα-mediated neutrophil diapedesis, rather than the abrogation of TSP-1-mediated anti-angiogenic signaling.

Manipulation of Notch signaling has led to significant tumor shrinkage as well as recovery from several traumatic and ischemic injury models indicating its potential clinical application. We have tested both an agonist and antagonist of Notch signaling to study the effects of Notch-mediated angiogenesis on spinal cord vascular pathology following traumatic injury. Initial neonatal retinal vascularization assays showed their respective bioactivities in vivo. Mice were treated with either the antagonist Jagged1-Fc chimera (Jag1-Fc) or agonist Notch1 antibody (N1 Ab) immediately following a mid-thoracic contusive injury through an initial jugular bolus and tail vein injections for 3 days post-injury. After 14 days, activating Notch signaling decreased the overall vascular density within the penumbral gray matter compared to controls while maintaining the density of perfused vessels. Inhibiting Notch signaling did not change the density or perfusion of microvessels within the lesion penumbra. Furthermore, neither activation nor inhibition of Notch signaling significantly altered inflammation, hypoxia, and lesion volume in the epicenter and penumbra. Importantly, neither treatment changed locomotor function. In postnatal retinal vascular assays, administration of Jag1-Fc and N1 Ab increased and decreased both tip cell numbers and branch points in each treatment, respectively. However, these agents did not modulate primary CNS EC proliferation in vitro in spite of sufficient Notch ligand expression. We conclude that Notch signaling, while an important part of developmental angiogenesis, may play a lesser role in mediating vascular recovery following traumatic injury to the CNS.

Spinal cord injury (SCI) causes an increase of inhibitory factors that may restrict axonal outgrowth after trauma. During the past decade, the Eph receptors and ephrin ligands have emerged as key repulsive cues known to be involved in neurite outgrowth, synapse formation, and axonal pathfinding during development. Given the non-permissive environment for axonal regeneration after SCI, we questioned whether enhanced-expression of the EphA4 receptor with repulsive activity for axonal outgrowth is potentially responsible for the regenerative failure. To address this possibility, we have examined the expression of EphA4 after SCI in adult rats following a contusion SCI. EphA4 expression studies demonstrated a time-dependent change for EphA4 protein without alterations in beta-actin. EphA4 was downregulated initially and upregulated 7 days after injury. Blockade of EphA4 upregulation with antisense oligonucleotides did not produce an anatomical or physiological response monitored with anterograde tracing studies or transcranial magnetic motor evoked potentials (tcMMEP), respectively. These results demonstrated that upregulation of EphA4 receptors after trauma is not related to axonal regeneration or return of nerve conduction across the injury site.

Spinal cord injury (SCI) is characterized by a total or partial loss of motor and sensory functions due to the inability of neurons to regenerate. This lack of axonal regenerative response has been associated with the induction of inhibitory proteins for regeneration, such as the Eph receptor tyrosine kinases. One member of this family, the EphA4 receptor, coordinates appropriate corticospinal fibers projections during early development and is expressed in spinal commissural interneurons. Its mechanism of action is mediated by repulsive activity after ligand binding, but its role after trauma is unknown. We examined the temporal expression profile of this receptor after spinal cord contusion in adult rats by RT-PCR and immunohistochemistry. SCI induced a biphasic gene expression profile with an initial downregulation at 2 and 4 days post-injury (DPI) followed by a subsequent upregulation. Double labeling studies localized EphA4 immunoreactivity in neurons from the gray matter and astrocytes of the white matter. To test the role of this receptor, we reduced gene upregulation by intrathecal/subdural infusion of EphA4-antisense oligodeoxynucleotide (ODN) and subsequently assessed behavioral outcomes. No locomotor recovery was observed in the rats treated with the EphA4-antisense ODN. Interestingly, reducing EphA4 expression increased mechanical allodynia, as observed by the Von Frey test and decreased exploratory locomotor activity. These results indicate that upregulation of EphA4 receptor after trauma may prevent the development of abnormal pain syndromes and could potentially be exploited as a preventive analgesic mediator to chronic neuropathic pain.

Bone morphogenetic proteins (BMPs) are a large class of secreted factors, which serve as modulators of development in multiple organ systems, including the CNS. Studies investigating the potential of stem cell transplantation for restoration of function and cellular replacement following traumatic spinal cord injury (SCI) have demonstrated that the injured adult spinal cord is not conducive to neurogenesis or oligodendrogenesis of engrafted CNS precursors. In light of recent findings that BMP expression is modulated by SCI, we hypothesized that they may play a role in lineage restriction of multipotent grafts. To test this hypothesis, neural stem or precursor cells were engineered to express noggin, an endogenous antagonist of BMP action, prior to transplantation or in vitro challenge with recombinant BMPs. Adult rats were subjected to both contusion and focal ischemic SCI. One week following injury, the animals were transplanted with either EGFP- or noggin-expressing neural stem or precursor cells. Results demonstrate that noggin expression does not antagonize terminal astroglial differentiation in the engrafted stem cells. Furthermore, neutralizing endogenous BMP in the injured spinal cord significantly increased both the lesion volume and the number of infiltrating macrophages in injured spinal cords receiving noggin-expressing stem cell grafts compared with EGFP controls. These data strongly suggest that endogenous factors in the injured spinal microenvironment other than the BMPs restrict the differentiation of engrafted pluripotent neural stem cells as well as suggest other roles for BMPs in tissue protection in the injured CNS.

A graded contusion spinal cord injury (SCI) was created in the adult rat spinal cord using the Infinite Horizons (IH) impactor to study the correlation between injury severity and anatomical, behavioral, and electrophysiological outcomes. Adult Fisher rats were equally divided into five groups and received contusion injuries at the ninth thoracic level (T9) with 100, 125, 150, 175, or 200 kdyn impact forces, respectively. Transcranial magnetic motor-evoked potentials (tcMMEPs) and BBB open-field locomotor analyses were performed weekly for 4 weeks postinjury. Our results demonstrated that hindlimb locomotor function decreased in accordance with an increase in injury severity. The locomotor deficits were proportional to the amount of damage to the ventral and lateral white matter (WM). Locomotor function was strongly correlated to the amount of spared WM, which contains the reticulospinal and propriospinal tracts. Normal tcMMEP latencies were recorded in control, all of 100-kdyn-injured and half of 125-kdyn-injured animals. Delayed latency responses were recorded in some of 125-kdyn-injured and all of 150-kdyn-injured animals. No tcMMEP responses were recorded in 175- and 200-kdyn-injured animals. Comparison of tcMMEP responses with areas of WM loss or demyelination identified the medial ventrolateral funiculus (VLF) as the location of the tcMMEP pathway. Immunohistochemical and electromicroscopic (EM) analyses showed the presence of demyelinated axons in WM tracts surrounding the lesion cavities at 28 days postinjury. These data support the notion that widespread WM damage in the ventral and lateral funiculi may be a major cause for locomotor deficits and lack of tcMMEP responses after SCI.

Differentiation of pluripotent neural stem cells engrafted into the adult normal and injured spinal cord is restricted to the glial lineage, suggesting that in vitro induction toward a neuronal lineage prior to transplantation and/or modification of the host environment may be necessary to initiate and increase the differentiation of neurons. In the present study, we investigated the differentiation of neuronal-restricted precursors (NRPs) grafted into the normal and contused adult rat spinal cord. NRPs proliferated through multiple passages in the presence of FGF2 and NT3 and differentiated into only neurons in vitro in the presence of retinoic acid and the absence of FGF2. Differentiated NRPs expressed GABA, glycine, glutamate, and ChAT. Two weeks to 2 months after engraftment of undifferentiated NRPs into adult normal spinal cord, large numbers of surviving cells were seen in all of the animals. The majority differentiated into betaIII-tubulin-positive neurons. Some transplanted NRPs expressed GABA and small numbers were glutamate- and ChAT-positive. NRPs were also transplanted into the epicenter of the contused adult rat spinal cord. Two weeks to 2 months after transplantation, some engrafted NRPs remained undifferentiated nestin-positive cells. Small numbers were MAP2- or betaIII-tubulin-positive neurons. However, the expression of GABA, glutamate, or ChAT was not observed. These results show that NRPs can differentiate into different types of neurons in the normal adult rat spinal cord, but that such differentiation is inhibited in the injured spinal cord. Manipulation of the microenvironment in the injured spinal cord will likely be necessary to facilitate neuronal replacement.

The effects of specific mitogens and substrates on the proliferative capacity and the differentiated phenotypic plasticity of neural precursor cell populations isolated from the adult rat subventricular zone (SVZ) were examined. SVZ cells were grown on uncoated tissue culture plastic, extracellular matrix, or poly-D-ornithine with either laminin or fibronectin. SVZ neural precursor cells could not be generated with platelet-derived growth factor (PDGF), granulocyte macrophage colony stimulating factor, stem cell factor, heparin-binding epidermal growth factor (HB-EGF), granulocyte colony stimulating factor, or ciliary neurotrophic factor (CNTF), but could be with EGF, fibroblast growth factor 2 (FGF2), and FGF2 plus heparin. Varying combinations of substrate and mitogen resulted in very different expansion rates and/or lineage potential. Neurons, oligodendrocytes, and astrocytes differentiated from all cultures, but EGF-generated neural precursor cells were more restricted to an astrocytic lineage and FGF2-generated neural precursor cells had a greater capacity for neuronal differentiation. In both EGF- and FGF2-generated cell populations, CNTF increased the number of differentiated astrocytes, triiodothyronine oligodendrocytes, PDGF neurons, and brain-derived neurotrophic factor neurons only from EGF cells. Electrophysiological analysis of differentiated cells showed three distinct phenotypes, glial, neuronal, and presumed precursor cells, although the neuronal properties were immature. Collectively, these data indicate that CNS neural precursor cell populations isolated with different mitogens and substrates are intrinsically different and their characteristics cannot be directly compared.

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 GFRalpha 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 GFRalphas 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 GFRalphas, 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 GFRalphas but not Ret. GDNF treatment did not activate the Ras/ERK pathway in these cells, but stimulated a GFRalpha1-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 GFRalpha receptors acting in a cell-autonomous manner independently of Ret.

Growth factor-expanded neural precursor cells isolated from the mammalian central nervous system can differentiate into neurons and glia. Although the morphological and neurochemical development of these neural precursor cells has been investigated, little attention has been paid to their electrophysiology. This study examined the electrophysiological properties of neurons and glia derived from neural precursor cells isolated from the adult rat spinal cord (SC) and subventricular zone (SVZ). Cells were cultured in medium containing epidermal growth factor and/or fibroblast growth factor-2. After at least two passages, spheres of neural precursor cells were plated on coated coverslips and maintained in culture for up to 6 weeks. Whole-cell patch recordings were made using standard current clamp techniques. Immature action potentials were observed within hours of plating for both SC and SVZ cells. Input resistance and time constants decreased over the first week after plating and no further changes were found at later times. At similar times following plating, however, SVZ cells had a lower input resistance and shorter time constant compared to SC cells. SVZ cells also had higher resting membrane potentials and smaller after hyperpolarizations than those of SC cells, despite no significant difference in the amplitude of action potentials. Neither the SC nor the SVZ cells were capable of eliciting more than a single action potential in response to injected current. While all SC cells tested were depolarized by glutamate, the response of SVZ cells to glutamate varied considerably. This study revealed that neural precursor cells from SC and SVZ differ in both active and passive membrane properties. It appears also that the electrophysiological development of SC and SVZ precursor-derived neurons is incomplete under the conditions used. These observations suggest that the neural precursor cells from different anatomical locations may be physiologically diverse and may exhibit some differences in commitment toward neuronal or glial phenotypes.

To investigate the structural basis for genetic regulation of the human serotonin transporter gene, a 1.8 kb fragment upstream to the cap site was cloned and sequenced. The promoter possesses a polymorphic repeat region with 16 and 14 repeats, respectively. Both were cloned and characterized. The promoter sequence revealed an internal 379 bp fragment not reported in previous publications. This novel fragment contains consensus sequences for several transcription factors including SpI and GATA. DNA from 48 unrelated individuals was PCR amplified, in this region, to test for allelic variations. All were found to possess the additional 379 bp fragment. The integrity of the promoter was furthermore confirmed by genomic Southern blotting. The promoter activity was analyzed by reporter gene assays in neuronal and non-neuronal serotonergic cell lines. In immortalized serotonergic raphe neurons, RN46A, three cis-acting, cell specific, activating elements and a silencer were located. One of the activators and the silencer are located in the repeat region and one activator is positioned in the novel fragment. A fourth activating element was found to be active in both RN46A cells and in a non-neuronal serotonergic cell line, JAR. A 3.5 kb fragment from intron 1 was cloned and found to possess cell specific activity in JAR cells indicating the presence of an alternative promoter in intron 1.

NMDA receptors in postmortem human spinal cord were analyzed using [3H]MK-801 ligand binding and immunoblotting with NMDA receptor subunit-specific antibodies. The averageKDfor [3H]MK-801 binding was 1.77 nM with aBmaxof 0.103 pmol/mg. The EC50for stimulation of [3H]MK-801 binding with l -glutamate was 0.34 μM. None of these parameters were affected by postmortem intervals up to 72 h. Immunoblotting of native NMDA receptors showed that NR1, NR2A, NR2C, and NR2D subunits could all be found in the human spinal cord of which NR1 was preferentially located to the dorsal half. Immunoprecipitation of solubilized receptors revealed that NR1, NR2C, and NR2D subunits coprecipitated with the NR2A subunit, indicating that native human spinal cord NMDA receptors are heteroligimeric receptors assembled by at least three different receptor subunits. These results provide a basis for the development of drugs selectively aimed at spinal cord NMDA receptors for the future treatment of spinal cord disorders.

Denervation of CNS neurons and peripheral organs is a consequence of traumatic SCI. Intraspinal transplantation of embryonic CNS neurons is a potential strategy for reinnervating these targets. Neural progenitor cell lines are being investigated as alternates to embryonic CNS neurons. RN33B is an immortalized neural progenitor cell line derived from embryonic rat raphe nuclei following infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T-antigen. Transplantation studies have shown that local epigenetic signals in intact or partially neuron-depleted adult rat hippocampal formation or striatum direct RN33B cell differentiation to complex multipolar morphologies resembling endogenous neurons. After transplantation into neuron-depleted regions of the hippocampal formation or striatum, RN33B cells were relatively undifferentiated or differentiated with bipolar morphologies. The present study examines RN33B cell differentiation after transplantation into normal spinal cord and under different lesion conditions. Adult rats underwent either unilateral lesion of lumbar spinal neurons by intraspinal injection of kainic acid or complete transection at the T10 spinal segment. Neonatal rats underwent either unilateral lesion of lumbar motoneurons by sciatic nerve crush or complete transection at the T10 segment. At 2 or 6-7 wk postinjury, lacZ-labeled RN33B cells were transplanted into the lumbar enlargement of injured and age-matched normal rats. At 2 wk posttransplantation, bipolar and some multipolar RN33B cells were found throughout normal rat gray matter. In contrast, only bipolar RN33B cells were seen in gray matter of kainic acid lesioned, sciatic nerve crush, or transection rats. These observations suggest that RN33B cell multipolar morphological differentiation in normal adult spinal cord is mediated by direct cell-cell interaction through surface molecules on endogenous neurons and may be suppressed by molecules released after SCI. They also indicate that the fate of immortalized neural progenitor cell lines in injured CNS must be stringently characterized.

Neurotrophic factors appear to be crucial for the survival and potential regeneration of injured neurons. We have previously demonstrated that contusive spinal cord injury (SCI) increases the levels of mRNA for basic fibroblast growth factor (FGF2). To determine whether FGF2 protein levels also increase, Western blot analysis was performed on extracts of spinal cord tissue after a standardized SCI and compared to laminectomy controls. In spinal cord extracts, a monoclonal antibody to FGF2 recognized various molecular forms of FGF2 (18–24 kDa) and some characteristic proteolytic fragments. Extracts of spinal cords 1 day after SCI showed a slight increase in the levels of these polypeptides. By 4 days, a significant increase (twofold) was detected in the levels of the 18-kDa and higher molecular weight forms as well as the proteolytic fragments. Immunohistochemical analyses on spinal cord tissue sections confirmed an increased cellular (glial) FGF2 as well as interstitial immunoreactivity surrounding neurons and along blood vessels. Heparin-purified spinal cord extracts from tissue 4 days after SCI showed increased biological activity as indicated by their ability to (i) increase [3H]thymidine incorporation in cultures of Balb/c 3T3 cells and (ii) induce phosphorylation ofsuc-associated neurotrophic factor-induced tyrosine-phosphorylated target, a FGF2 target protein. These data suggest that SCI induces increased FGF2 expression and support the hypothesis that FGF2 may play a role in the partial recovery of function seen following SCI.

The expression of chemotactic receptors in the central nervous system is largely unexplored. In this study, we examined human astrocytes and microglia as well as the conditionally immortalized human astrocyte cell line HSC2 for expression of the C5a-anaphylatoxin receptor (C5aR), the interleukin-8 receptor (IL-8R) and the f-Met-Leu-Phe receptor (FMLPR). Using flow cytometry, indirect immunofluorescence and RT-PCR analysis, we demonstrated that astrocytes, microglia and HSC2 cells contain specific RNA and express surface protein for all three chemotactic receptors. These are the first studies to demonstrate definitively the expression of these chemotactic receptors on astrocytes and microglia, thereby expanding the types of cells known to express chemotactic receptors. Moreover, these data suggest that these chemotactic receptors may play an important role in mediating the inflammatory response and perhaps other yet undescribed biological phenomena in the central nervous system.

The RN46A cell line was derived from Embryonic Day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen. This cell line is neuronally restricted and constitutively differentiates following a shift to nonpermissive temperature. Undifferentiated RN46A cells express low levels of tryptophan hydroxylase (TPH), low-affinity neurotrophin receptor (p75NTR), and trkB immunoreactivities, but no detectable levels of serotonin (5HT) immunoreactivity. TrkB, p75NTR, and TPH, but not 5HT, expressions increase with differentiation and treatment with brain-derived neurotrophic factor (BDNF). 5HT synthesis in RN46A cells requires initial treatment with BDNF, followed by growth under partial membrane depolarizing conditions. Embryonic raphe cultures treated similarly with BDNF and partial depolarizing conditions also demonstrate increased 5HT synthesis. The sodium-dependent transporter for 5HT reuptake is present in undifferentiated RN46A cells, and the apparent Km and Bmax are unchanged by differentiation or BDNF treatment and membrane depolarization. The high-affinity 5HT1A receptor is present in both undifferentiated and differentiated RN46A cells, and while the Kd is unaffected by differentiation or BDNF/membrane depolarization, the Bmax increases 20-fold after differentiation and 3.5-fold further with BDNF under depolarizing conditions. The expression of the synaptic vesicular monoamine transporter, as determined by the binding of [125I]iodovinyltetrabenazine, also increases in RN46A cells with differentiation. However, 5HT release is constitutive and is independent of acute membrane depolarization. Collectively these data indicate that distinct aspects of serotonin metabolism are differentially regulated during development and suggest that 5HT may function as a developmental signal in an autocrine loop during early serotonergic differentiation.

Two neuronal cell lines, RN33B and RN46A, were derived from E12 rat medullary raphe neuroepithelial cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen (T-ag). At permissive temperature (33°C), both cell lines express nuclear T-ag immunoreactivity, low levels of neuron-specific enolase (NSE), and low (NF-L) and medium (NFM), but not high (NF-H), MW neurofilament proteins. Once differentiated, both cell lines cease dividing, take on a neuronal morphology, and express enhanced levels of NSE and all three NF proteins. The complex interaction of variables which mediates the differentiation of these neuronal cell lines is discussed. RN33B and RN46A cells show both similar and different physiological properties, and those features are highlighted to demonstrate the utility of these cell lines in studying neuronal differentiation, both in vitro and in vivo . For example, the expression of NF-L and NF-M in differentiating RN33B and RN46A cells can be potentiated by activation of protein kinase A. RN33B cells are glutamatergic while RN46A cells are serotonergic. 5-HT uptake and synthesis in RN46A cells are differentially regulated, as synthesis is maximal only in differentiated RN46A cells which have been treated with BDNF followed by partial depolarization. 5-HT uptake is seen at similar levels in both undifferentiated and differentiated RN46A cells and is independent of neurotrophin regulation. Following transplantation into the CNS, RN33B cells survive ≥6 months and differentiate according to local environmental signals; RN46A cells survive ≤2 months and do not show similar plasticity in vivo . These data are modeled to provide a conceptual framework as to the generation and differentiation capacity of neuronal cell lines derived by retroviral transduction.

A clonal, neuronally-differentiating cell line, RN33B, was previously developed by retroviral infection of neural tissue derived from embryonic Sprague-Dawley raphe nuclei with a retrovirus encoding the temperature-sensitive allele of SV40 large T-antigen. In the present study, RN33B cells were transplanted into two target areas of the raphe nuclei, the spinal cord and hippocampal formation, of adult allogeneic hosts. Prior to transplantation, RN33B cells were infected in vitro with a retroviral vector carrying the Escherichia coli lacZ reporter gene and were visualized in vivo using a βgalactosidase immunohistochemical technique. RN33B cells were seen throughout the spinal cord and hippocampal formation of the adult hosts at 15 days post-transplantation. T-antigen-immunoreactive nuclei were detected where RN33B cells were observed, but in much greater numbers than β-galactosidase-immunoreactive cells. Bipolar RN33B cells were found in the spinal cord grey matter. RN33B cells with multipolar morphologies were visualized in the hippocampal and subicular pyramidal cell layers, and also in the dentate gyrus granule cell and polymorph layers, while bipolar RN33B cells were seen in the remainder of the hippocampal formation. The results suggest that immortalized neural cell lines of CNS origin can differentiate in the adult CNS with their ultimate morphology being determined by local tissue signals. We speculate that endogenous neutrophins may significantly influence RN33B cell differentiation in vivo.

Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen, a clonal cell line, RN33B, was isolated by serial dilution. At 33 degrees C, RN33B cells divide with a doubling time of 48 h and show T antigen, vimentin, nestin, diffuse neuron-specific enolase, and low and medium molecular weight neurofilament immunoreactivities. RN33B cells are immortal, but not transformed, as they will not grow in soft agar. At non-permissive temperature (38.5 degrees C), T antigen expression is markedly decreased and RN33B cells cease mitotic activity and differentiate with phase bright cell bodies and 'neuritic-like' processes. Differentiated RN33B cells express enhanced neuronal-specific protein expression but do not synthesize astrocytic or oligodendrocytic-specific proteins. Moreover, differentiated RN33B cells returned to 33 degrees C re-express T antigen, but do not de-differentiate or begin dividing. Co-culture with embryonic hippocampus and cerebral cortex, but not medullary raphe or spinal cord, resulted in significantly greater survival, more complex neuronal morphology, and enhanced expression of neuronal-specific antigens. Immunohistochemical and Northern blot analysis revealed high levels of low affinity NGF receptor protein and mRNA in differentiated RN33B cells. PCR analysis demonstrated the presence of trkB, but not trkA or trkC, mRNA in both undifferentiated and differentiated RN33B cells. These data suggest that the observed target regulation of RN33B cell neuronal differentiation in co-culture may be mediated by neurotrophin(s).

In normal spinal cord, acidic fibroblast growth factor (aFGF) immunoreactivity was localized in the cytoplasm of ventral motor neurons and sensory fibers in the dorsal columns. Basic FGF (bFGF) immunoreactivity was restricted to astrocyte nuclei and the cytoplasm of a few neurons in the intermediate gray matter. Spinal cord lesions resulted in complete destruction of the dorsal columns at T8. Two days postlesion, aFGF immunoreactivity was increased in ventral motor neurons and was now seen in intermediate gray matter neurons. Acidic FGF was not detected in the lesioned fasciculus gracilis at T4-5, but markedly increased in the fasciculus cuneatus. At L1-2, aFGF-immunoreactive fibers in the fasciculus gracilis also increased. This aFGF immunostaining was maintained 5 and 12 days postlesion. A lesion-induced loss of aFGF immunoreactivity in the nucleus gracilis suggests that aFGF is anterogradely transported in ascending sensory fibers. Two days postlesion, glial fibrillary acidic protein immunoreactivity increased at the lesion site, as well as at T4-5 and L1-2, with no change in bFGF staining. Five days postlesion, increased bFGF immunoreactivity appeared at the edge of the cystic cavity and the dorsal columns at T4-5 in both the nucleus and the cytoplasm of reactive astrocytes, and was increased at 12 days postlesion. The differential cellular, temporal, and spatial expression of aFGF and bFGF following spinal cord lesion suggest they subserve distinct roles in the response to CNS injury.

Tissue from neonatal, adult and aged Sprague-Dawley rat brain contained low levels of survival promoting activity for embryonic neurons from various rat brain regions. This basal neuronotrophic activity was 2-fold higher in adult and 4-fold higher in aged rat brain, with respect to that in neonatal brain. Tissue extracts also contained 4-fold higher levels of neuronotoxic activity in adult and aged than in neonatal brain. Ablation of the occipital/entorhinal cortex caused a 3-fold increase in neuronotrophic activity in the tissue surrounding the wound in neonates and 4-fold in adult and aged brain, with respect to the basal levels. Maximal activities occurred at 3 days, 6 days and 15 days postlesion in neonatal, adult and aged tissue respectively, and subsequently returned to basal levels. Neuronotoxic activity was not induced following injury. The slower neuronotrophic response to injury in older animals may be one of the determinant factors of the slower recovery from brain damage observed in aging.