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Demyelination is associated with a number of neurological disorders including multiple sclerosis (MS), spinal cord injury and nerve compression. MS lesions often show axon loss and therefore reparative therapeutic goals include remyelination and neuroprotection of vulnerable axons. Experimental cellular transplantation has proven successful in a number of demyelination and injury models to remyelinate and improve functional outcome. Here we discuss the remyelination and neuroprotective potential of several myelin-forming cells types and their behavior in different demyelination and injury models. Better understanding of these models and current cell-based strategies for remyelination and neuroprotection offer exciting opportunities to develop strategies for clinical studies.

Intravenous delivery of mesenchymal stem cells (MSCs) prepared from bone marrow (BMSCs) reduces infarction volume and ameliorates functional deficits in a rat cerebral ischemia model. MSC-like multipotent precursor cells (PMSCs) have also been suggested to exist in peripheral blood. To test the hypothesis that treatment with PMSCs may have a therapeutic benefit in stroke, we compared the efficacy of systemic delivery of BMSCs and PMSCs. A permanent middle cerebral artery occlusion (MCAO) in rat was induced by intraluminal vascular occlusion with a microfilament. Rat BMSCs and PMSCs were prepared in culture and intravenously injected into the rats 6 h after MCAO. Lesion size was assessed at 6 h, and 1, 3, and 7 days using MR imaging and histology. The hemodynamic change of cerebral blood perfusion on stroke was assessed the same times using perfusion-weighted image (PWI). Functional outcome was assessed using the treadmill stress test. Both BMSCs and PMSCs treated groups had reduced lesion volume, improved regional cerebral blood flow, and functional improvement compared to the control group. The therapeutic benefits of both MSC-treated groups were similar. These data suggest that PMSCs derived from peripheral blood could be an important cell source of cell therapy for stroke.

Proton magnetic resonance spectroscopy (1-H MRS) has revealed changes of metabolites in acute cerebral infarction. Although the drastic changes of lactate and N-acetyl-aspartate have been reported to be useful indicators of the ischemic damage in both humans and experimental animals, lipid signals are also detected by the short echo time sequence 1-5 days after ischemia. The objective of this study was to find a novel technique to isolate lactate signals from lipid signals in the ischemic brain. First, MRS was used to study the lipid and lactate components of a spherical phantom in vitro, and parameters were established to separate these components in vitro. Then, MR measurements were obtained from the brains of middle cerebral artery occlusion rats. All MR measurements were performed using a 7-T (300 MHz), 18.3-cm-bore superconducting magnet (Oxford Magnet Technologies) interfaced to a Unity INOVA Imaging System (Varian Technologies). T2-weighted images were obtained from a 1.0-mm-thick coronal section using a 3-cm field of view. It is well known that lipid has a shorter and lactate a longer T2 relaxation time. These distinct magnetic characteristics allowed us to separate the lactate signal from the lipid signal. Thus, adjustment of the echo time is essential to analyze the metabolites in acute cerebral infarction, which may be useful in both the clinic and laboratory.

Contusive spinal cord injury (SCI) can result in necrosis of the spinal cord, but often long white matter tracts outside of the central necrotic core are demyelinated. One experimental strategy to improve functional outcome following SCI is to transplant myelin-forming cells to remyelinate these axons and improve conduction. This review focuses on transplantation studies using olfactory ensheathing cell (OEC) to improve functional outcome in experimental models of SCI and demyelination. The biology of the OEC, and recent experimental research and clinical studies using OECs as a potential cell therapy candidate are discussed.

Transplantation of olfactory ensheathing cells (OECs) into injured spinal cord results in improved functional outcome. Mechanisms suggested to account for this functional improvement include axonal regeneration, remyelination and neuroprotection. OECs transplanted into transected peripheral nerve have been shown to modify peripheral axonal regeneration and functional outcome. However, little is known of the detailed integration of OECs at the transplantation site in peripheral nerve. To address this issue, cell populations enriched in OECs were isolated from the olfactory bulbs of adult green fluorescent protein (GFP)-expressing transgenic rats and transplanted into a sciatic nerve crush lesion which transects all axons. Five weeks to 6 months after transplantation, the nerves were studied histologically. GFP-expressing OECs survived in the lesion and distributed longitudinally across the lesion zone. The internodal regions of individual teased fibers distal to the transection site were characterized by GFP expression in the cytoplasmic and nuclear compartments of cells surrounding the axons. Immunoelectron microscopy for GFP indicated that the transplanted OECs formed peripheral type myelin. Immunostaining for sodium channel and Caspr revealed a high density of Na(v)1.6 at the newly formed nodes of Ranvier which were flanked by paranodal Caspr staining. These results indicate that transplanted OECs extensively integrate into transected peripheral nerve and form myelin on regenerated peripheral nerve fibers, and that nodes of Ranvier of these axons display proper sodium channel organization.

Transplantation of mesenchymal stem cells (MSCs) prepared from adult bone marrow (BMSCs) has been reported to ameliorate functional deficits in several CNS diseases in experimental animal models. Bone marrow was enriched in MSCs by selecting for plastic-adherent cells that were grown to confluency in appropriate culture conditions as flattened fibroblast-like cells. Despite the fact that the stem/precursor cells in peripheral blood are widely used for reconstruction in the hematopoietic system, it is not fully understood whether peripheral blood-derived plastic-adherent precursor/stem cells (PMSCs) can differentiate into a neural lineage. To compare the potential of PMSCs and BMSCs for neural differentiation in vitro, BMSCs and PMSCs were prepared from the adult rat and expanded in culture. Although the growth rate of PMSCs was less than BMSCs, immunocytochemical and RT-PCR analyses indicated that both MSC types were successfully induced to nestin-positive neurospheres in the presence of EGF and bFGF. After withdrawal of the mitogens, these cells could differentiate into neurofilament-positive neurons or GFAP-positive glia. Thus, our findings suggest the potential use of PMSCs for a cell therapy in CNS diseases.

Intravenous administration of human mesenchymal stem cells (hMSCs) prepared from adult bone marrow has been reported to ameliorate functional deficits after cerebral artery occlusion in rats. Several hypotheses to account for these therapeutic effects have been suggested, and current thinking is that neuroprotection rather than neurogenesis is responsible. To enhance the therapeutic benefits of hMSCs potentially, we transfected hMSCs with the glial cell line-derived neurotrophic factor (GDNF) gene using a fiber-mutant F/RGD adenovirus vector and investigated whether GDNF gene-modified hMSCs (GDNF-hMSCs) could contribute to functional recovery in a rat permanent middle cerebral artery occlusion (MCAO) model. We induced MCAO by using intraluminal vascular occlusion, and GDNF-hMSCs were intravenously infused into the rats 3 hr later. MRI and behavioral analyses revealed that rats receiving GDNF-hMSCs or hMSCs exhibited increased recovery from ischemia compared with the control group, but the effect was greater in the GDNF-hMSC group. Thus, these results suggest that intravenous administration of hMSCs transfected with the GDNF gene using a fiber-mutant adenovirus vector may be useful in the cerebral ischemia and may represent a new strategy for the treatment of stroke.

Intravenous delivery of mesenchymal stem cells (MSCs) prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat cerebral ischaemia models. Placental growth factor (PlGF) is angiogenic to impaired non-neural tissue. To test the hypothesis that PlGF contributes to the therapeutic benefits of MSC delivery in cerebral ischaemia, we compared the efficacy of systemic delivery of human MSCs (hMSCs) and hMSCs transfected with a fibre-mutant F/RGD adenovirus vector with a PlGF gene (PlGF-hMSCs). A permanent middle cerebral artery occlusion (MCAO) was induced by intraluminal vascular occlusion with a microfilament. hMSCs and PlGF-hMSCs were intravenously injected into the rats 3 h after MCAO. Lesion size was assessed at 3 and 6 h, and 1, 3, 4 and 7 days using MR imaging and histology. Functional outcome was assessed using the limb placement test and the treadmill stress test. Both hMSCs and PlGF-hMSCs reduced lesion volume, induced angiogenesis and elicited functional improvement compared with the control sham group, but the effect was greater in the PlGF-hMSC group. Enzyme-linked immunosorbent assay of the infarcted hemisphere revealed an increase in PlGF in both hMSC groups, but a greater increase in the PlGF-hMSC group. These data support the hypothesis that PlGF contributes to neuroprotection and angiogenesis in cerebral ischaemia, and cellular delivery of PlGF to the brain can be achieved by intravenous delivery of hMSCs.

Intravenous infusion of bone marrow cells has demonstrated therapeutic efficacy in animal models of cerebral ischemia and spinal cord injury. We intravenously delivered human mesenchymal stem cells (SH2+, SH3+, CD34-, and CD45-) immortalized with a human-telomerase gene (hTERT-MSCs) and transfected with eGFP or LacZ into rats 12 h after induction of transient middle cerebral artery occlusion (MCAO), to study their potential therapeutic benefit. hTERT-MSCs were delivered at 12 h after lesion induction. Lesion size was assessed using MR imaging and spectroscopy, and histological methods. Functional outcome was assessed using the Morris water maze and a treadmill test. Intravenous delivery of hTERT-MSCs reduced lesion volume and the magnitude of the reduction and functional improvement was positively correlated with the number of cells injected. The reduction of lesion size could be assessed in vivo with MRI and MRS and was correlated with subsequent histological examination of the brain. This work demonstrates that highly purified hTERT-MSCs reduce cerebral infarction volume and improve functional outcome.

Transplantation of olfactory ensheathing cells (OECs) into the damaged rat spinal cord leads to directed elongative axonal regeneration and improved functional outcome. OECs are known to produce a number of neurotrophic molecules. To explore the possibility that OECs are neuroprotective for injured corticospinal tract (CST) neurons, we transplanted OECs into the dorsal transected spinal cord (T9) and examined primary motor cortex (M1) to assess apoptosis and neuronal loss at 1 and 4 weeks post-transplantation. The number of apoptotic cortical neurons was reduced at 1 week, and the extent of neuronal loss was reduced at 4 weeks. Biochemical analysis indicated an increase in BDNF levels in the spinal cord injury zone after OEC transplantation at 1 week. The transplanted OECs associated longitudinally with axons at 4 weeks. Thus, OEC transplantation into the injured spinal cord has distant neuroprotective effects on descending cortical projection neurons.

Myelin-forming glial cells transplanted into the demyelinated spinal cord can form compact myelin and improve conduction properties. However, little is known of the expression and organization of voltage-gated ion channels in the remyelinated central axons or whether the exogenous cells provide appropriate signaling for the maturation of nodes of Ranvier. Here, we transplanted olfactory ensheathing cells from green fluorescent protein (GFP)-expressing donor rats [GFP-olfactory ensheathing cells (OECs)] into a region of spinal cord demyelination and found extensive remyelination, which included the development of mature nodal, paranodal, and juxtaparanodal domains, as assessed by ultrastructural, immunocytochemical, and electrophysiological analyses. In remyelinated axons, Nav1.6 was clustered at nodes, whereas Kv1.2 was aggregated in juxtaparanodal regions, recapitulating the distribution of these channels within mature nodes of uninjured axons. Moreover, the recruitment of Nav and Kv channels to specific membrane domains at remyelinated nodes persisted for at least 8 weeks after GFP-OEC transplantation. In vivo electrophysiological recordings demonstrated enhanced conduction along the GFP-OEC-remyelinated axons. These findings indicate that, in addition to forming myelin, engrafted GFP-OECs provide an environment that supports the development and maturation of nodes of Ranvier and the restoration of impulse conduction in central demyelinated axons.

Transplantation of myelin-forming cells can remyelinate axons, but little is known of the sodium channel organization of axons myelinated by donor cells. Sciatic nerve axons of female wild type mice were transected by a crush injury and Schwann cells (SCs) from green fluorescence protein (GFP)-expressing male mice were transplanted adjacent to the crush site. The male donor cells were identified by GFP fluorescence and fluorescence in situ hybridization (FISH) for Y chromosome. In nerves of GFP-expressing mice, GFP was observed in the axoplasm and in the cytoplasmic compartments of the Schwann cells, but not in the myelin. Following transplantation of GFP-SCs into crushed nerve of wild type mice, immuno-electron microscopic analysis indicated that GFP was observed in the cytoplasmic compartments of engrafted Schwann cells which formed myelin. Nodal and paranodal regions of the axons myelinated by the GFP-SCs were identified by Na(v)1.6 sodium channel and Caspr immunostaining, respectively. Nuclear identification of the Y chromosome by FISH confirmed the donor origin of the myelin-forming cells. These results indicate that engrafted GFP-SCs participate in myelination of regenerated peripheral nerve fibers and that Na(v)1.6 sodium channel, which is the dominant sodium channel at normal nodes, is reconstituted on the regenerated axons.

Peripheral nerve injury results in changes in action potential waveform, ion channel organization, and firing properties of primary afferent neurons. It has been suggested that these changes are the result of reduction in basal trophic support from skin targets. Subcutaneous injections of Fluro-Gold (FG) in the hind limb of the rat were used to identify cutaneous primary afferent neurons. Five days after FG injection, sciatic nerves were ligated and encapsulated in a silicon tube allowing neuroma formation. Green fluorescent protein (GFP)-expressing Schwann cells (SCs) were injected proximal to the cut end of the nerve. Thirteen to 22 days after injury and SC injection, the L4 and L5 dorsal root ganglia (DRG) were prepared for acute culture. Whole cell patch-clamp recordings in current clamp mode were obtained and action potential properties of medium-sized (34-45 microm) FG+ DRG neurons were characterized. In the neuroma group without cell transplantation, action potential duration and spike inflections were reduced as were the amplitude and duration of spike afterhyperpolarizations. These changes were not observed after transection by nerve crush where axons were allowed to regenerate to distal peripheral targets. In the transplantation group, GFP(+)-SCs were extensively distributed throughout the neuroma, and oriented longitudinally along axons proximal to the neuroma. Changes in action potential properties were attenuated in the GFP(+)-SC group. Thus the engrafted SC procedure ameliorated the changes in action potential waveform of cutaneous primary afferents associated with target disconnection and neuroma formation.

Ectopic discharge generated in injured afferent axons and cell somata in vivo contributes significantly to chronic neuropathic dysesthesia and pain after nerve trauma. Progress has been made toward understanding the processes responsible for this discharge using a preparation consisting of whole excised dorsal root ganglia (DRGs) with the cut nerve attached. In the in vitro preparation, however, spike activity originates in the DRG cell soma but rarely in the axon. We have now overcome this impediment to understanding the overall electrogenic processes in soma and axon, including the resulting discharge patterns, by modifying the bath medium in which recordings are made. At both sites, bursts can be triggered by subthreshold oscillations, a phasic stimulus, or spikes arising elsewhere in the neuron. In the soma, once triggered, bursts are maintained by depolarizing afterpotentials, whereas in the axon, an additional process also plays a role, delayed depolarizing potentials. This alternative process appears to be involved in "clock-like" bursting, a discharge pattern much more common in axons than somata. Ectopic spikes arise alternatively in the soma, the injured axon end (neuroma), and the region of the axonal T-junction. Discharge sequences, and even individual multiplet bursts, may be a mosaic of action potentials that originate at these alternative electrogenic sites within the neuron. Correspondingly, discharge generated at these alternative sites may interact, explaining the sometimes-complex firing patterns observed in vivo.

I.V. delivery of mesenchymal stem cells prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat cerebral ischemia models. Administration of the brain-derived neurotrophic factor to the infarction site has also been demonstrated to be neuroprotective. To test the hypothesis that brain-derived neurotrophic factor contributes to the therapeutic benefits of mesenchymal stem cell delivery, we compared the efficacy of systemic delivery of human mesenchymal stem cells and human mesenchymal stem cells transfected with a fiber-mutant F/RGD adenovirus vector with a brain-derived neurotrophic factor gene (brain-derived neurotrophic factor-human mesenchymal stem cells). A permanent middle cerebral artery occlusion was induced by intraluminal vascular occlusion with a microfilament. Human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells were i.v. injected into the rats 6 h after middle cerebral artery occlusion. Lesion size was assessed at 6 h, 1, 3 and 7 days using MR imaging, and histological methods. Functional outcome was assessed using the treadmill stress test. Both human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells reduced lesion volume and elicited functional improvement compared with the control sham group, but the effect was greater in the brain-derived neurotrophic factor-human mesenchymal stem cell group. ELISA analysis of the infarcted hemisphere revealed an increase in brain-derived neurotrophic factor in the human mesenchymal stem cell groups, but a greater increase in the brain-derived neurotrophic factor-human mesenchymal stem cell group. These data support the hypothesis that brain-derived neurotrophic factor contributes to neuroprotection in cerebral ischemia and cellular delivery of brain-derived neurotrophic factor can be achieved by i.v. delivery of human mesenchymal stem cells.

The objective of this study was to establish if neural precursor cells could safely be developed from biopsy of the subventricular zone (SVZ) in the non-human primate (marmoset), and to determine their myelinating potential after autologous transplantation into a demyelinated lesion. Small amounts of tissue were safely collected from the subventricular-subependymal zone of the adult primate brain under ultrasonography without any neurological deficit. Neural precursor cells were isolated and expanded in the presence of mitogen in vitro. The dorsal columns of the adult marmoset spinal cord were demyelinated by X-irradiation and intraspinal injections of ethidium bromide in the center of the radiation field. Cell suspensions of the neural precursors were microinjected through a micropipette into the demyelinated lesion site in the spinal cord. Lesions were histologically examined 3 weeks after transplantation. Light and electron microscopic examination of plastic embedded sections revealed a significant number of myelinating profiles in the transplantation zone; no myelination was observed in control lesions. The myelinated axons had predominantly peripheral patterns of myelination. These results demonstrate that autologous transplantation of neural precursor cells in the adult nonhuman primate can remyelinate demyelinated central nervous system (CNS) axons, thus suggesting the potential utility of such an approach in demyelinating lesions in humans.

Olfactory ensheathing cells (OECs) prepared from the olfactory bulbs of adult transgenic Sprague Dawley (SD) rats expressing green fluorescent protein (GFP) were transplanted into a dorsal spinal cord transection lesion of SD rats. Five weeks after transplantation, the cells survived within the lesion zone and oriented longitudinally along axons that bridged the transection site. Although the highest density of GFP cells was within the lesion zone, some cells distributed longitudinally outside of the lesion area. Myelinated axons spanning the lesion were observed in discrete bundles encapsulated by a cellular element. Electron micrographs of spinal cords immunostained with an anti-GFP antibody indicated that a majority of the peripheral-like myelinated axons were derived from donor OECs. Open-field locomotor behavior was significantly improved in the OEC transplantation group. Thus, transplanted OECs derived from the adult olfactory bulb can survive and orient longitudinally across a spinal cord transection site and form myelin. This pattern of repair is associated with improved locomotion.

The primary objective of this study was to test the hypothesis that intravenous administration of autologous bone marrow cells could improve functional recovery after middle cerebral artery occlusion (MCAO) for 45 min in the rat and to determine specific time windows for efficacy. Mononuclear cells from autologous bone marrow were transfected with the LacZ reporter gene, and injected intravenously into rats at 3-72 h after induction of MCAO. Histological analysis of the ischemic lesion at 14 days after transplantation revealed reduced ischemic lesion volume. Lesion volume was 250+/-45 mm(3) (n=6) after MCAO without cell transplantation. Lesions were minimally detected by absence of 2,3,5-triphenyltetrazolium chloride (TTC) staining when bone marrow cells were infused 3 h after lesion induction. Lesions were clearly detected beginning with the 6-h postlesion group and became progressively larger at 12, 24 and 72 h (80+/-25, 140+/-18, and 180+/-22 mm(3), respectively; n=6 for each group). Transplanted LacZ(+) bone marrow cells accumulated extensively in and around the ischemic lesions, and immunohistochemistry suggests some neuronal and glial lineage differentiation. Behavioral testing (Morris water maze and Treadmill stress test) indicated greater functional recovery in the treated group. These findings suggest that early intervention with intravenous administration of autologous mononuclear cells from bone marrow can reduce lesion size in the MCAO model in the rat, and improve functional outcome.

Schwann cells and neural precursor cells derived from adult human brain (subventricular zone) and from bone marrow were studied anatomically and physiologically after transplantation into the demyelinated rat spinal cord. All cell types formed myelin and restored conduction velocity. Following transection of the dorsal funiculus, Schwann cells and olfactory ensheathing cells facilitated axonal regeneration and restoration of conduction across the lesion site. There is discussion on the challenges of cell type selection and preparation for a potential clinical cell therapy study in human demyelinating diseases.

Olfactory ensheathing cells (OECs) have been shown to mediate remyelination and to stimulate axonal regeneration in a number of in vivo rodent spinal cord studies. However, whether OECs display similar properties in the primate model has not been tested so far. In the present study, we thus transplanted highly-purified OECs isolated from transgenic pigs expressing the alpha1,2 fucosyltransferase gene (H-transferase or HT) gene into a demyelinated lesion of the African green monkey spinal cord. Four weeks posttransplantation, robust remyelination was found in 62.5% of the lesion sites, whereas there was virtually no remyelination in the nontransplanted controls. This together with the immunohistochemical demonstration of the grafted cells within the lesioned area confirmed that remyelination was indeed achieved by OECs. Additional in vitro assays demonstrated 1) that the applied cell suspension consisted of >98% OECs, 2) that the majority of the cells expressed the transgene, and 3) that expression of the HT gene reduced complement activation more than twofold compared with the nontransgenic control. This is the first demonstration that xenotransplantation of characterized OECs into the primate spinal cord results in remyelination.

Transplantation of cell suspensions containing olfactory ensheathing cells (OECs) has been reported to remyelinate demyelinated axons in the spinal cord with a Schwann cell (SC)-like pattern of myelination. However, questions have been raised recently as to whether OECs can form SC-like myelin. To address this issue we prepared SCs and OECs from transgenic rats in which a marker gene, human placental alkaline phosphatase (hPAP), is linked to the ubiquitously active promoter of the R26 gene. SCs were prepared from the sciatic nerve and OECs from the outer nerve-fiber layer of the olfactory bulb. Positive S100 and p75 immunostaining indicated that >95% of cells in culture displayed either SC or OEC phenotypes. Suspensions of either SCs or OECs were transplanted into an X-irradiation/ethidium bromide demyelinating lesion in the spinal cord. We observed extensive SC-like remyelination following either SC or OEC transplantation 3 weeks after injection of the cells. Alkaline phosphatase (ALP) chromagen reaction product was associated clearly with the myelin-forming cells. Thus, cell suspensions that are enriched in either SCs or OECs result in peripheral-like myelin when transplanted in vivo.

Cocaethylene is a naturally occurring cocaine derivative that has been used as a tool in both clinical studies of cocaine reward and as a potential model compound for agonist substitution therapy in cocaine dependence. It is equipotent to cocaine at inhibiting dopamine uptake in-vitro and in-vivo. Because it has been reported that local anesthetic properties may influence the reinforcing effects of dopamine uptake inhibitors, we investigated the local anesthetic properties of cocaethylene as well as isopropylcocaine, another potential pharmacological tool in studies of cocaine reward and agonist substitution therapy. We compared the efficacy of nerve impulse blockade by lidocaine, cocaine, cocaethylene and isopropylcocaine using rat sciatic nerves and dorsal roots (DRs). Nerves were placed in a modified sucrose gap chamber and repetitively stimulated at high frequency. The amplitude of compound action potentials (CAPs) at the beginning and end of each stimulus train was measured before and after exposure to each compound. All compounds produced concentration-dependent and use-dependent decrements in CAP amplitude, but cocaethylene and isopropylcocaine at medium to high concentration (0.375-1.875 mM) showed a more prolonged block after washout relative to cocaine or lidocaine. Patch clamp studies on dorsal root ganglion (DRG) neurons indicated a use-dependent blockade of sodium channels. These studies provide a more complete understanding of the pharmaocology of potential agonist treatment candidates, and suggest a mechanism whereby cocaethylene produces a decreased euphoria in humans compared to cocaine.

The remyelinating potential of autologous bone marrow cells was studied after direct injection and following intravenous injection into rats with a demyelinated lesion in the spinal cord. Both focal and intravenous injections of acutely isolated mononuclear bone marrow cell fractions resulted in varying degrees of remyelination. Suspensions of bone marrow cells collected from the same rat were delivered at varied concentrations (10(2) to 10(5) for direct injection and 10(4) to 10(7) for i.v. injections). The lesions were examined histologically 3 weeks after transplantation. Light microscopic examination revealed remyelination in the dorsal funiculus with both injection protocols, but the extent of remyelination was proportional to the number of injected cells. To attain the same relative density of remyelination achieved by direct injection, intravenous administration of cells required delivery of substantially more cells (two orders of magnitude). However, the availability of autologous bone marrow cells in large number and the potential for systemically delivering cells to target lesion areas without neurosurgical intervention suggest the potential utility of intravenous cell delivery as a prospective therapeutic approach in demyelinating disease., (Copyright 2003 Wiley-Liss, Inc.)

A noninactivating, persistent sodium current has been demonstrated previously in dorsal root ganglia neurons and in rat optic nerve. We report here that Na(+) channel blockade with tetrodotoxin (TTX) in isolated dorsal and ventral roots elicits membrane hyperpolarization, suggesting the presence of a persistent Na(+) current in peripheral axons. We used a modified sucrose-gap chamber to monitor resting and action potentials and observed a hyperpolarizing shift in the nerve potential of rat dorsal and ventral roots by TTX. The block of transient inward Na(+) currents was confirmed by the abolition of compound action potentials (CAPs). Moreover, depolarization of nerve roots by elevating extracellular K(+) concentrations to 40 mM eliminated CAPs but did not significantly alter TTX-induced hyperpolarizations, indicating that the persistent Na(+) currents in nerve roots are not voltage-dependent. Tetrodotoxin-sensitive persistent inward Na(+) currents are present in both dorsal and ventral root axons at rest and may contribute to axonal excitability.

Peripheral nerve transection in the rat alters the spinal cord dorsal horn central projections from both small and large DRG neurons. Injured neurons with C-fibers exhibit transganglionic degeneration of their terminations within lamina II of the spinal cord dorsal horn, while peripheral nerve injury of medium to large neurons induces collateral sprouting of myelinated A-fibers from lamina I and III/IV into lamina II in rats, cats, and primates. To date, it is not known what sequelae are responsible for the collateral sprouting of A-fibers after peripheral nerve injury, although target-derived factors are thought to play an important role. To determine whether target-derived factors are necessary for changes in A-fiber laminar terminations in rat spinal cord dorsal horn, we unilaterally transected the sciatic nerve and ensheathed the proximal nerve stump in a silicone cap. Three days before sacrifice of rat, the injured sciatic nerve was injected with cholera toxin beta-subunit conjugated to horseradish peroxidase (betaHRP) that effectively labels both peripheral and central A-fiber axons. The effect of the ligature, axotomy, and silicone cap treatment was evaluated by analyzing the extent of betaHRP-, Substance P-(SP-), and isolectin B4- (IB4-) immunoreactive (ir) fibers in the somatotopically appropriate spinal cord dorsal horn regions. In all animals, 2-5 weeks after nerve transection (treated or otherwise), IB4- and SP-ir is absent from lamina II. Animals without nerve cap treatment exhibited robust fiber sprouting into lamina II at 2 weeks. In sharp contrast, animals treated with silicone caps did not exhibit betaHRP-ir fibers in lamina II at 2 weeks. This observation was extended up to 5 weeks postinjury. These results suggest that axotomy-induced expansion of betaHRP-ir primary afferent central terminations in the spinal cord dorsal horn is dependent on factors produced in the injury site milieu. While our understanding of local repair mechanisms of injured peripheral nerves is incomplete, it is clear that the time-dependent production of growth factors in the nerve injury microenvironment favor nerve fiber outgrowth, both peripherally and centrally.

Bone marrow contains a population of pluripotent cells that can differentiate into a variety of cell lineages, including neural cells. When injected directly into the demyelinated spinal cord they can elicit remyelination. Recent work has shown that following systemic delivery of bone marrow cells functional improvement occurs in contusive spinal cord injury and stroke models in rat. We report here that secondary to intravenous introduction of an acutely isolated bone marrow cell fraction (mononuclear fraction) from adult rat femoral bones separated on a density gradient, ultrastructurally defined remyelination occurs throughout a focal demyelinated spinal cord lesion. The anatomical pattern of remyelination was characteristic of both oligodendrocyte and Schwann cell myelination; conduction velocity improved in the remyelinated axons. When the injected bone marrow cells were transfected to express LacZ, beta-galactosidase reaction product was observed in some myelin-forming cells in the spinal cord. Intravenous injection of other myelin-forming cells (Schwann cells and olfactory ensheathing cells) or the residual cell fraction of the gradient did not result in remyelination, suggesting that remyelination was specific to the delivery of the mononuclear fraction. While the precise mechanism of the repair, myelination by the bone marrow cells or facilitation of an endogenous repair process, cannot be fully determined, the results demonstrate an unprecedented level of myelin repair by systemic delivery of the mononuclear cells., (Copyright 2002 Wiley-Liss, Inc.)

Bone marrow contains a population of stem-like cells that can differentiate into neurons or glia. Stromal cells from green fluorescent protein (GFP)-expressing mice were isolated by initial separation on a density gradient and then cultured as adherent cells on plastic that proliferated in culture to confluency with a typical flattened elongative morphology. The large majority of the isolated stromal cells were GFP expressing and immunopositive for collagen type I, fibronectin, and CD44. Transplantation of these cells by direct microinjection into the demyelinated spinal cord of the immunosuppressed rat resulted in remyelination. The remyelinated axons showed characteristics of both central and peripheral myelination as observed by electron microscopy; conduction velocity of the axons was improved. GFP-positive cells and myelin profiles were observed in the remyelinated spinal cord region, indicating that the donor-isolated stromal cells were responsible for the formation of the new myelin. The GFP-positive cells were colocalized with myelin basic protein-positive and P0-positive cellular elements. These findings indicate that cells contained within the stromal cell fraction of the mononuclear cell layer of bone marrow can form functional myelin during transplantation into demyelinated spinal cord.

Whole cell patch-clamp recordings were obtained from dissociated mouse lumbar dorsal root ganglion (DRG) neurons. Recordings were made from control neurons and neurons axotomized by transection of the corresponding spinal nerve 1-2 days prior to dissociation. Medium to large muscle and cutaneous afferent neurons were identified by retrograde transport of True Blue or Fluoro-Gold injected into the corresponding peripheral tissue. Action potentials were classified as non-inflected spikes (A(0)) and inflected spikes (A(inf)). High-frequency, low-amplitude subthreshold membrane potential oscillations were observed in 8% of control A(0) neurons, but their incidence increased to 31% in the nerve injury group. Fifty percent of axotomized muscle afferent A(0) cells displayed oscillations, while 26% of axotomized cutaneous afferents exhibited oscillations. Lower-frequency oscillations were also observed in a small fraction (4%) of A(inf) neurons on strong depolarization. Their numbers were increased after the nerve injury, but the difference was not statistically significant. The oscillations often triggered burst firing in distinct patterns of action potential activity. These results indicate that injury-induced membrane oscillations of DRG neurons, previously observed in whole DRG of rats, are present in dissociated DRG neurons of the adult mouse. Moreover, these observations indicate that both muscle and cutaneous afferents in the A(beta) size range give rise to injury-induced membrane oscillations, with muscle afferents being more prone to develop oscillations.

Quantitative morphometric techniques were used to assess the extent and pattern of remyelination produced by transplanting allogenic Schwann cells into demyelinated lesions in adult rat spinal cords. The effects of donor age, prior culturing of donor cells, prior lesioning of donor nerves, and host immunosuppression were evaluated by transplanting suspensions of 30,000 acutely dissociated or cultured Schwann cells from neonatal, young adult, or aged adult rat sciatic nerves into X-irradiation and ethidium bromide-induced demyelinated dorsal column lesions, with or without co-transplantation of neonatal optic nerve astrocytes. Three weeks after transplantation, spinal cords were processed for histological analysis. Under all Schwann cell transplant protocols, large areas containing many Schwann cell-like myelinated axon profiles could be readily observed throughout most of the lesion length. Within these "myelin-rich" regions, the vast majority of detectable axons showed a peripheral-like pattern of myelination. However, interaxonal spacing also increased, resulting in densities of myelinated axons that were more similar to peripheral nerve than intact dorsal columns. Freshly isolated Schwann cells remyelinated more axonal length than cultured Schwann cells, and cells from younger donors remyelinated slightly more axon length than cells from older donors, but all Schwann cell transplant protocols remyelinated tens of thousands of millimeters of axon length and remyelinated axons at similar densities. These results indicate that Schwann cells prepared under a variety of conditions are capable of eliciting remyelination, but that the density of remyelinated axons is much lower than the myelinated axon density in intact spinal cords., (Copyright 2002 Wiley-Liss, Inc.)

Ectopic spike activity, generated at low levels in intact sensory dorsal root ganglia and intensified following axotomy, is an important cause of neuropathic pain. The spikes are triggered by subthreshold membrane potential oscillations. The depolarizing phase of oscillation sinusoids is due to a phasic voltage-sensitive Na(+) conductance (gNa(+)). Here we examine the repolarizing phase for which K(+) conductance (gK(+)) is implicated. In vivo, gK(+) blockers have excitatory effects inconsistent with the elimination of oscillations. Indeed, using excised dorsal root ganglia in vitro, we found that gK(+) block does not eliminate oscillations; on the contrary, it has a variety of facilitatory effects. However, oscillations were eliminated by shifting the K(+) reversal potential so as to neutralize voltage-insensitive K(+) leak channels. Based on these data, we propose a novel oscillatory model: oscillation sinusoids are due to reciprocation between a phasically activating voltage-dependent, tetrodotoxin-sensitive Na(+) conductance and passive, voltage-independent K(+) leak. In drug-free media, voltage-sensitive K(+) channels act to suppress oscillations and increase their frequency. Numerical simulations support this model and account for the effects of gK(+) block. Oscillations in dorsal root ganglia neurones appear to be based on the simplest possible configuration of ionic conductances compatible with sustained high frequency oscillatory behaviour. The oscillatory mechanism might be exploited in the search for novel analgesic drugs.

Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes, and it has multi-functional effects in a variety of cells in various organs. HGF stimulates DNA synthesis and promotes cell migration and morphogenesis in several cell types including the olfactory system. To characterize the potential mitogenic activity of HGF that might contribute to olfactory ensheathing cell (OEC) proliferation, we tested the ability of HGF to stimulate OEC division in vitro. OECs were obtained from adult rat olfactory bulbs and cultured in serum-free medium, and were identified by double immunostaining for p75 and S-100 antibodies. DNA synthesis assayed by pulsing BrdU for 24 hr showed that HGF at the concentration of 5-100 ng/ml elicited a 5-10-fold increase of OEC proliferation. By immunocytochemical analysis, we demonstrated that c-Met-immunoreactivity was present in cultured OECs, and c-Met anti-serum significantly sequestered the activity of HGF on OECs proliferation, suggesting that HGF-induced proliferation of OECs is mediated by the c-Met receptor. The mitogenic activity of HGF was potentiated by addition of heregulin (HRG), but inhibited by addition of forskolin. These results demonstrate that HGF is a novel mitogen for rat OECs in vitro, and HGF/c-Met system is involved in regulating OECs growth and development., (Copyright 2001 Wiley-Liss, Inc.)

The potential of bone marrow cells to differentiate into myelin-forming cells and to repair the demyelinated rat spinal cord in vivo was studied using cell transplantation techniques. The dorsal funiculus of the spinal cord was demyelinated by x-irradiation treatment, followed by microinjection of ethidium bromide. Suspensions of a bone marrow cell fraction acutely isolated from femoral bones in LacZ transgenic mice were prepared by centrifugation on a density gradient (Ficoll-Paque) to remove erythrocytes, platelets, and debris. The isolated cell fraction contained hematopoietic and nonhematopoietic stem and precursor cells and lymphocytes. The cells were transplanted into the demyelinated dorsal column lesions of immunosuppressed rats. An intense blue beta-galactosidase reaction was observed in the transplantation zone. The genetically labeled bone marrow cells remyelinated the spinal cord with predominately a peripheral pattern of myelination reminiscent of Schwann cell myelination. Transplantation of CD34(+) hematopoietic stem cells survived in the lesion, but did not form myelin. These results indicate that bone marrow cells can differentiate in vivo into myelin-forming cells and repair demyelinated CNS., (Copyright 2001 Wiley-Liss, Inc.)

Long tract axons in the mammalian CNS do not normally regenerate for appreciable distance after they transected. But we reported transplantation of Schwann cells(SCs) or olfactory ensheathing cells induced regeneration of transected rat dorsal column (DC) axons and improved the conduction. Scar formation(gliosis), for which astrocytes(ACs) play an important role, may be one of strong and physical barriers for the regeneration of CNS axon. Oligodendrocyte and myelin associated protein or products also inhibit the regeneration of the axons, as chemical barriers. To investigate how effective the promotion or the reduction of scar or myelin formation may be for axonal regeneration, we transplanted AC into transected DCs, or radiated(X-ray) the DCs, and compared to normal DCs or regenerated DCs following by SC transplantation. DCs of adult rats were transected at Th 11 and transplanted with SCs(6 x 10(4)) of adult rats or ACs(6 x 10(4)) of neonatal rats. Five to six weeks later, the spinal cords were removed and pinned in a recording chamber, and compound action potentials (CAPs) along the DC through the transected lesion were recorded, to investigate conduction properties(conduction velocity and response after high frequency stimulations). Following transplantation of SCs or ACs, histological examination revealed regenerated axons with SC-like patterns of remyelination in transected DCs. X-ray irradiation did not enhance the regeneration of DC axons. SC transplantation improved the conduction properties of transected DCs and increased the number of regenerated axons, compared to transected DCs without cell transplantation. AC transplantation resulted in improvement of the conduction properties, but the number of regenerated axons was similar to that of transected DCs without the transplantation. X-ray irradiation (40 Gy) three days before DC transection and AC transplantation prevented the electrophysiological continuity of axons through the transected lesion. This evidence revealed that AC transplantation secondarily enhanced the regeneration of axons, probably endogeneous SCs of dorsal roots migrated into the transected lesion and enhanced the axonal regeneration.

Schwann cells derived from human sural nerve may provide a valuable source of tissue for a cell-based therapy in multiple sclerosis. However, it is essential to show that transplanted human Schwann cells can remyelinate axons in adult CNS and improve axonal conduction. Sections of sural nerve were removed from amputated legs of patients with vascular disease or diabetes, and Schwann cells were isolated and cryopreserved. Suspensions of reconstituted cells were transplanted into the X-irradiation/ethidium bromide lesioned dorsal columns of immunosuppressed Wistar rat. After 3-5 weeks of extensive remyelination, a typical Schwann cell pattern was observed in the lesion zone. Many cells in the lesion were immunopositive for an anti-human nuclei monoclonal antibody. The dorsal columns were removed and maintained in an in vitro recording chamber; the conduction properties were studied using field potential and intra-axonal recording techniques. The transplanted dorsal columns displayed improved conduction velocity and frequency-response properties, and action potentials conducted over a greater distance into the lesion, suggesting that conduction block was overcome. These data support the conclusion that transplantation of human Schwann cells results in functional remyelination of a dorsal column lesion.

We examined the myelin repair potential of transplanted neural precursor cells derived from the adult human brain from tissue removed during surgery. Sections of removed brain indicated that nestin-positive cells were found predominantly in the subventricular zone around the anterior horns of the lateral ventricle and in the dentate nucleus. Neurospheres were established and the nestin-positive cells were clonally expanded in EGF and bFGF. Upon mitogen withdrawal in vitro, the cells differentiated into neuron- and glia-like cells as distinguished by antigenic profiles; the majority of cells in culture showed neuronal and astrocytic properties with a small number of cells showing properties of oligodendrocytes and Schwann cells. When transplanted into the demyelinated adult rat spinal cord immediately upon mitogen withdrawal, the cells elicited extensive remyelination with a peripheral myelin pattern similar to Schwann cell myelination characterized by large cytoplasmic and nuclear regions, a basement membrane, and P0 immunoreactivity. The remyelinated axons conducted impulses at near normal conduction velocities. This suggests that a common neural progenitor cell for CNS and PNS previously described for embryonic neuroepithelial cells may be present in the adult human brain and that transplantation of these cells into the demyelinated spinal cord results in functional remyelination.

Voltage-dependent Na-currents were studied, using whole cell voltage clamp, in acutely dissociated, large (mostly Abeta-fiber type) cutaneous afferent dorsal root ganglia neurons (L(4) and L(5)) from the adult rat. Cells were dissociated 14-17 days after axotomy. Control and axotomized neurons were identified via the retrograde marker hydroxy-stilbamide (fluorogold) which was injected into the lateral and plantar region of the skin of the foot and were studied using whole cell patch clamp techniques within 12-20 h of dissociation and plating. Cells were dissociated 14-17 days after injury. Both control and axotomized neurons displayed complex Na-currents composed of components with distinct kinetic and pharmacological properties. The large (48-50 microm diameter) control cutaneous afferent neurons, many of which likely give rise to myelinated Abeta-fibers, exhibited Na-currents with both slow and fast inactivating kinetics. The fast inactivating current in large cutaneous afferent dorsal root ganglion neurons was tetrodotoxin-sensitive and recovered from inactivation approximately four-fold faster at -60 mV (P<0.001) and approximately six-fold faster at -70 mV (P<0.001) than the tetrodotoxin-sensitive current in small (<30 microm diameter) neurons. Further, while the tetrodotoxin-sensitive currents in smaller dorsal root ganglion neurons (mainly C-fiber type) reprime approximately four-fold faster following peripheral axotomy, repriming of the fast inactivating current in larger cutaneous afferent neurons was not significantly altered following axotomy. However, while 77% of control large neurons were observed to express the slower inactivating, tetrodotoxin-resistant current, only 45% of these large neurons did after axotomy. These results indicate that large adult cutaneous afferent dorsal root ganglion neurons (Abeta-type) express tetrodotoxin-sensitive Na-currents, which have much faster repriming than Na-currents in small (C-type) neurons, both before, and after axotomy. Like small neurons, the majority of large neurons downregulate the tetrodotoxin-resistant current following sciatic nerve section.

Here we describe transplantation of olfactory ensheathing cells (OECs) or Schwann cells derived from transgenic pigs expressing the human complement inhibitory protein, CD59 (hCD59), into transected dorsal column lesions of the spinal cord of the immunosuppressed rat to induce axonal regeneration. Non-transplanted lesion-controlled rats exhibited no impulse conduction across the transection site, whereas in animals receiving transgenic pig OECs or Schwann cells impulse conduction was restored across and beyond the lesion site for more than a centimeter. Cell labeling indicated that the donor cells migrated into the denervated host tract. Conduction velocity measurements showed that the regenerated axons conducted impulses faster than normal axons. By morphological analysis, the axons seemed thickly myelinated with a peripheral pattern of myelin expected from the donor cell type. These results indicate that xenotranplantation of myelin-forming cells from pigs genetically altered to reduce the hyperacute response in humans are able to induce elongative axonal regeneration and remyelination and restore impulse conduction across the transected spinal cord.

Transplantation of Schwann cells (SCs) induced remyelination of demyelinated rat dorsal column (DC) axons and improved conduction. To investigate the difference between oligodendrocyte (OL) and SC myelination in conductive functions of axons, we compared normal DCs, demyelinated DCs, demyelinated DCs remyelinated by SC transplantation, and normal dorsal roots. All of the axons was originated from dorsal root ganglion neurons. Dorsal roots of adult rats were demyelinated at T11 by X-ray irradiation and ethidium bromide, and transplanted with SCs (3 x 10(4)) of adult rats. Three weeks later, the spinal cord was removed and pinned in a recording chamber and compound action potentials (CAPs) were recorded, to investigate conduction properties (conduction velocity and response after high frequency stimulation). Normal DCs or dorsal roots were recorded in same manner. Following transplantation of SCs, histological examination revealed SC-like patterns of remyelination in demyelinated DCs. SC transplantation improved significantly conduction properties compared to demyelinated axons, but less than normal DC. Moreover, remyelinated axons by SC transplantation showed as low amplitude of CAP as dorsal roots, but lower conduction velocity than dorsal roots. Though anatomical difference and/or time after transplantation influenced the conduction, these result suggested that SC myelination resulted in lower amplitude of CAP than OL, and SC remyelination might be insufficient for conduction velocity.

Demyelination of axons resulted in distinct reduction of conduction velocity or block of conduction. Remyelination by transplantation of myelin-forming cells may provide a therapeutic approach for demyelinated diseases. However, which cell type will be the most appropriate candidate for such a cell therapy is not established. To investigate how effective grafted neonatal brain cell (BC) (including oligodendrocyte and astrocyte) isolated from neonatal fronto-temporal lobes, adult olfactory ensheathing cell (OEC) or adult Schwann cell (SC) may be for demyelinated CNS axons in vivo, dorsal columns(DCs) of adult rat spinal cord were demyelinated at Th 11 by X-ray irradiation (day 0) and the injection of ethidium bromide (day 3), and transplanted 5 x 10(4) of BCs, 3 x 10(4) of OECs, or 3 x 10(4) of SCs into the lesion (day 6). Day 28-31, spinal cord were removed and transferred an in vitro recording chamber to record field potentials using glass micropipettes, to investigate conduction properties at 36 degrees. Normal DCs were recorded in same manner. Histological examination revealed that OECs and SCs resulted in substantial SC-like patterns of remyelination to equal degree, BC transplantation resulted in less myelination. The conduction velocities were significantly improved to 4.2 +/- 2.4 m/s(BC, n = 5), 8.5 +/- 3.3 m/s(OEC, n = 6) and 7.7 +/- 1.5 m/s(SC, n = 5), compared to demyelinated axons(1.2 +/- 0.4 m/s, n = 7). A 600 Hz 0.5 sec stimulus train led to an amplitude decrement of 7.1 +/- 7.5% (n = 7) in demyelinated axons. Following transplantation, the amplitude decreased in 31.3 +/- 18.7% (BC, n = 5), 49.9 +/- 19.9% (OEC, n = 6) and 66.2 +/- 11.9% (SC, n = 5). Transplanted OECs and SCs enhanced the remyelination of demyelinated CNS axons, and improved conduction properties were similar, and more effective than that induced from isolated CNS tissue which included oligodendrocyte.

Human olfactory ensheathing cells (OECs) were prepared from adult human olfactory nerves, which were removed during surgery for frontal base tumors, and were transplanted into the demyelinated spinal cord of immunosuppressed adult rats. Extensive remyelination was observed in the lesion site: In situ hybridization using a human DNA probe (COT-1) indicated a similar number of COT-1-positive cells and OEC nuclei within the repaired lesion. The myelination was of a peripheral type with large nuclei and cytoplasmic regions surrounding the axons, characteristic of Schwann cell and OEC remyelination. These results provide evidence that adult human OECs are able to produce Schwann cell-like myelin sheaths around demyelinated axons in the adult mammalian CNS in vivo.

The effect of sciatic nerve injury on the somatic expression of voltage-gated calcium currents in adult rat cutaneous afferent dorsal root ganglion (DRG) neurons identified via retrograde Fluoro-gold labeling was studied using whole cell patch-clamp techniques. Two weeks after a unilateral ligation and transection of the sciatic nerve, the L(4)-L(5) DRG were dissociated and barium currents were recorded from cells 3-10 h later. Cutaneous afferents (35-50 microm diam) were classified as type 1 (possessing only high-voltage-activated currents; HVA) or type 2 (having both high- and low-voltage-activated currents). Axotomy did not change the percentage of neurons exhibiting a type 2 phenotype or the properties of low-threshold T-type current found in type 2 neurons. However, in type 1 neurons the peak density of HVA current available at a holding potential of -60 mV was reduced in axotomized neurons (83.9 +/- 5.6 pA/pF, n = 53) as compared with control cells (108.7 +/- 6.9 pA/pF, n = 58, P < 0.01, unpaired t-test). A similar reduction was observed at more negative holding potentials, suggesting differences in steady-state inactivation are not responsible for the effect. Separation of the type 1 cells into different size classes indicates that the reduction in voltage-gated barium current occurs selectively in the larger (capacitance >80 pF) cutaneous afferents (control: 112.4 +/- 10.6 pA/pF, n = 30; ligated: 72.6 +/- 5.0 pA/pF, n = 36; P < 0.001); no change was observed in cells with capacitances of 45-80 pF. Isolation of the N- and P¿Q-type components of the HVA current in the large neurons using omega-conotoxin GVIA and omega-agatoxin TK suggests a selective reduction in N-type barium current after nerve injury, as the density of omega-CgTx GVIA-sensitive current decreased from 56.9 +/- 6.6 pA/pF in control cells (n = 13) to 31.3 +/- 4.6 pA/pF in the ligated group (n = 12; P < 0.005). The HVA barium current of large cutaneous afferents also demonstrates a depolarizing shift in the voltage dependence of inactivation after axotomy. Injured type 1 cells exhibited faster inactivation kinetics than control neurons, although the rate of recovery from inactivation was similar in the two groups. The present results indicate that nerve injury leads to a reorganization of the HVA calcium current properties in a subset of cutaneous afferent neurons.

Electrophysiological recordings were obtained from rat dorsal roots in a sucrose gap chamber to study changes in Na(+) currents following nerve injury. Application of 4-aminopyridine unmasks a prominent and well-characterized depolarization (delayed depolarization) following the action potential. In our previous studies, this potential, which is only present in cutaneous afferent axons, has been shown to correlate with activation of a slow Na(+) current. The delayed depolarization in the dorsal root was reduced 1 week after sciatic nerve ligation, suggesting a reduction in the kinetically slow Na(+) currents on dorsal root axons [control: 44. 2+/-7.3% (n=5); injury: 7.3+/-4.7% (n=5), P<0.001]. The refractory period of the action potential was reduced following nerve injury, in agreement with biophysical studies indicating faster "repriming" of fast Na(+) currents on cutaneous afferent cell bodies. Dorsal root ligation near the spinal cord also results in a reduction in the delayed depolarization. These results indicate that changes in Na(+) channel organization occur on dorsal root axons following either central or peripheral target disconnection, suggesting trophic support can be derived from either the CNS or the PNS.

Intracellular recording and extracellular field potential (FP) recordings were obtained from spinal cord dorsal horn neurons (laminae I-IV) in a rat transverse slice preparation with attached dorsal roots. To study changes in synaptic inputs after neuroma formation, the sciatic nerve was sectioned and ligated 3 weeks before in vitro electrophysiological analysis. Horseradish peroxidase labeling of dorsal root axons indicated that Abeta fibers sprouted into laminae I-II from deeper laminae after sciatic nerve section. FP recordings from dorsal horns of normal spinal cord slices revealed long-latency synaptic responses in lamina II and short-latency responses in lamina III. The latencies of synaptic FPs recorded in lamina II of the dorsal horn after sciatic nerve section were reduced. The majority of monosynaptic EPSPs recorded with intracellular microelectrodes from lamina II neurons in control slices were elicited by high-threshold nerve stimulation, whereas the majority of monosynaptic EPSPs recorded in lamina III were elicited by low-threshold nerve stimulation. After sciatic nerve section, 31 of 57 (54%) EPSPs recorded in lamina II were elicited by low-threshold stimulation. The majority of low-threshold EPSPs in lamina II neurons after axotomy displayed properties similar to low-threshold EPSPs in lamina III of control slices. These results indicate that reoccupation of lamina II synapses by sprouting Abeta fibers normally terminating in lamina III occurs after sciatic nerve neuroma formation. Furthermore, these observations indicate that the lamina II neurons receive inappropriate sensory information from low-threshold mechanoreceptor after sciatic nerve neuroma formation.

Olfactory ensheathing cells (OECs) or Schwann cells were transplanted into the transected dorsal columns of the rat spinal cord to induce axonal regeneration. Electrophysiological recordings were obtained in an isolated spinal cord preparation. Without transplantation of cells, no impulse conduction was observed across the transection site; but following cell transplantation, impulse conduction was observed for over a centimeter beyond the lesion. Cell labelling indicated that the regenerated axons were derived from the appropriate neuronal source, and that donor cells migrated into the denervated host tract. As reported in previous studies, the number of regenerated axons was limited. Conduction velocity measurements and morphology indicated that the regenerated axons were myelinated, but conducted faster and had larger axon areas than normal axons. These results indicate that the regenerated spinal cord axons induced by cell transplantation provide a quantitatively limited but rapidly conducting new pathway across the transection site.

Whole-cell patch-clamp techniques were used to study the effects of nerve growth factor on voltage-dependent potassium conductance in normal and axotomized identified large cutaneous afferent dorsal root ganglion neurons (48-50 micrometer diameter) many of which probably give rise to myelinated Abeta fibers. K-currents were isolated by blocking Na- and Ca-currents with appropriate ion replacement and channel blockers. Separation of current components was achieved on the basis of response to variation in conditioning voltage. Cutaneous afferents were labeled by the retrograde marker hydroxy-stilbamide (FluoroGold) which was injected into the skin of the foot. The sciatic nerve was either ligated or crushed with fine forceps five to seven days later. Neurons were dissociated 14-17 days after injury. The cut ends of the sciatic nerves were positioned into polyethylene tubes, which were connected to mini-osmotic pumps filled with either nerve growth factor or sterile saline. Control neurons displayed a prominent sustained K-current and the transient potassium currents "A" and "D". Nerve ligation, which blocks target reconnection resulted in near 50% reduction of total outward current; isolated sustained K-current and transient A-current were reduced by a comparable amount. Nerve crush, which allows regeneration to peripheral targets and exposure of the regenerating nerve to the distal nerve segment, resulted in a small reduction in sustained K-current but no reduction in transient A-current compared to controls. Levels of transient A-current and sustained K-current were maintained at control levels after nerve growth factor treatment. These results indicate that the large reduction in transient A-current, and in sustained K-current, observed in cutaneous afferent cell bodies after nerve ligation is prevented by application of nerve growth factor.

Transplantation of myelin-forming cells into the demyelinated spinal cord results in remyelination. This paper reviews the electrophysiological properties of demyelinated axons remyelinated by transplantation of myelin-forming cells. Conduction velocity and frequency-response properties of the remyelinated axons are restored to near normal values. Moreover, conduction block can be overcome by remyelination, and no abnormal firing is observed. There is discussion of the challenges of a potential cell therapy approach in human demyelinating disease.

Potassium currents have an important role in modulating neuronal excitability. We have investigated the effects of axotomy on three voltage-activated K(+) currents, one sustained and two transient, in cutaneous afferent dorsal root ganglion (DRG) neurons. Fourteen to 21 days after axotomy, L(4) and L(5) DRG neurons were acutely dissociated and were studied 2-8 h after plating. Whole cell patch-clamp recordings were obtained from identified cutaneous afferent neurons (46-50 microm diam); K(+) currents were isolated by blocking Na(+) and Ca(2+) currents with appropriate ion replacement and channel blockers. Separation of the current components was achieved on the basis of sensitivity to dendrotoxin or 4-aminopyridine and by the response to variation in conditioning voltage. Both control and injured neurons displayed qualitatively similar complex K(+) currents composed of distinct kinetic and pharmacological components. Three distinct K(+) current components, a sustained (I(K)) and two transient (I(A) and I(D)), were identified in variable proportions. However, total peak current was reduced by 52% in the axotomized cells when compared with control cells. Two current components were reduced after ligation, I(A) by 60%, I(K) by over 65%, compared with control cells. I(D) appeared unaffected after acute ligation. These results indicate a large reduction in overall K(+) current, resulting from reductions in I(K) and I(A), on large cutaneous afferent neurons after nerve ligation and have implications for excitability changes of injured primary afferent neurons.