Sural nerve defects after nerve biopsy or nerve transfer as a sensory regeneration model for peripheral nerve conduit implantation.

Nerve repair after injury can be effectively accomplished by direct suture approximation of the proximal and distal segments. This is more successful if coadaptation can be achieved without tension. Currently, the gold standard repair of larger deficits is the transplantation of an autologous sensory sural nerve graft. However, a significant disadvantage of this technique is the inevitable donor morbidity (sensory loss, neuroma and scar formation) after harvesting of the sural nerve. Moreover, limitation of autologous donor nerve length and fixed diameter of the available sural nerve are major drawbacks of current autograft treatment. Another approach that was introduced for nerve repair is the implantation of alloplastic nerve tubes made of, for example, poly-L-lactide. In these, nerve stumps of the transected nerves are surgically bridged using the biosynthetic conduit. A number of experimental studies, primarily in rodents, indicate axonal regeneration and remyelination after implantation of various conduits. However, only limited clinical studies with conduit implantation have been performed in acute peripheral nerve injuries particularly on digital nerves. Clinical transfer of animal studies, which can be carefully calibrated for site and extent of injury, to humans is difficult to interpret due to the intrinsic variability in human nerve injuries. This prevents effective quantification of improvement and induces bias in the study. Therefore, standardization of lesion/repair in human studies is warranted. Here we propose to use sural nerve defects, induced due to nerve graft harvesting or from diagnostic nerve biopsies as a model site to enable standardization of nerve conduit implantation. This would help better with the characterization of the implants and its effectiveness in axonal regeneration and remyelination. Nerve regeneration can be assessed, for example, by recovery of sensation, measured non-invasively by threshold to von Frey filaments and cold allodynia. Moreover, the implantation of nerve conduits may not only serve as a model to examine nerve repair, but it could also prevent neuroma formation, which is a major morbidity of sural nerve extraction.

Sciatic nerve regeneration is not inhibited by anti-NGF antibody treatment in the adult rat.

Elevated nerve growth factor (NGF) is believed to play a role in many types of pain. An NGF-blocking antibody (muMab 911) has been shown to reduce pain and hyperalgesia in pain models, suggesting a novel therapeutic approach for pain management. Since NGF also plays important roles in peripheral nervous system development and sensory nerve outgrowth, we asked whether anti-NGF antibodies would adversely impact peripheral nerve regeneration. Adult rats underwent a unilateral sciatic nerve crush to transect axons and were subcutaneously dosed weekly for 8weeks with muMab 911 or vehicle beginning 1day prior to injury. Plasma levels of muMab 911 were assessed from blood samples and foot print analysis was used to assess functional recovery. At 8-weeks post-nerve injury, sciatic nerves were prepared for light and electron microscopy. In a separate group, Fluro-Gold was injected subcutaneously at the ankle prior to perfusion, and counts and sizes of retrogradely labeled and unlabeled dorsal root ganglion neurons were obtained. There was no difference in the time course of gait recovery in antibody-treated and vehicle-treated animals. The number of myelinated and nonmyelinated axons was the same in the muMab 911-treated crushed nerves and intact nerves, consistent with observed complete recovery. Treatment with muMab 911 did however result in a small decrease in average cell body size on both the intact and injured sides. These results indicate that muMab 911 did not impair functional recovery or nerve regeneration after nerve injury in adult rats.

Peripheral glial cell differentiation from neurospheres derived from adipose mesenchymal stem cells.

Mesenchymal stem cells derived from bone marrow and adipose tissue are being considered for use in neural repair because they can differentiate after appropriate induction in culture into neurons and glia. The question we asked was if neurospheres could be harvested from adipose-derived stem cells and if they then could differentiate in culture to peripheral glial-like cells. Here, we demonstrate that adipose-derived mesenchymal stem cells can form nestin-positive non-adherent neurosphere cellular aggregates when cultured with basic fibroblast growth factor and epidermal growth factor. Dissociation of these neurospheres and removal of mitogens results in expression of the characteristic Schwann cell markers S100 and p75 nerve growth factor receptor and GFAP. The simultaneous expression of these glia markers are characteristic features of Schwann cells and olfactory ensheathing cells which have unique properties regarding remyelination and enhancement of axonal regeneration. When co-cultured with dorsal root ganglion neurons, the peripheral glial-like cells derived from adipose mesenchymal stem cells aligned with neuritis and stimulated neuritic outgrowth. These results indicate that neurospheres can be generated from adipose-derived mesenchymal stem cells, and upon mitogen withdrawal can differentiate into peripheral glial cells with neurotrophic effects.

Demyelinating diseases and potential repair strategies.

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.

Neuroprotection by PlGF gene-modified human mesenchymal stem cells after cerebral ischaemia.

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 immortalized human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat.

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.

I.V. infusion of brain-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat.

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.

Hepatocyte growth factor is a mitogen for olfactory ensheathing cells.

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.

Sodium currents of large (Abeta-type) adult cutaneous afferent dorsal root ganglion neurons display rapid recovery from inactivation before and after axotomy.

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.

[The role of transplanted astrocytes for the regeneration of CNS axons].

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.

Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons.

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.

Transplantation of cryopreserved adult human Schwann cells enhances axonal conduction in demyelinated spinal cord.

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.

Transplantation of clonal neural precursor cells derived from adult human brain establishes functional peripheral myelin in the rat spinal cord.

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.

Nerve growth factor maintains potassium conductance after nerve injury in adult cutaneous afferent dorsal root ganglion neurons.

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.

Xenotransplantation of transgenic pig olfactory ensheathing cells promotes axonal regeneration in rat spinal cord.

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.

[Characteristic improvement of the function following Schwann cell transplantation for demyelinated 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.

[Comparison of myelin-forming cells as candidates for therapeutic transplantation in demyelinated CNS axons].

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.

Transplantation of human olfactory ensheathing cells elicits remyelination of demyelinated rat spinal cord.

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.

Voltage-gated calcium currents in axotomized adult rat cutaneous afferent neurons.

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.

Transplantation of olfactory ensheathing cells or Schwann cells restores rapid and secure conduction across the transected spinal cord.

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.