Search

Highlights • The HSE-RFP reporter detects primarily- and secondarily-damaged neural cells in spinal cord injury. • The HSE-RFP reporter detects primarily- and secondarily-damaged neural cells in sciatic nerve injury. • Reporter-positive secondarily-injured neurons show altered physiological properties., Spinal cord and peripheral nerve injury results in extensive damage to the locally injured cells as well as distant cells that are functionally connected to them. Both primary and secondary damage can cause a broad range of clinical abnormalities, including neuropathic pain and cognitive and memory dysfunction. However, the mechanisms underlying these abnormalities remain unclear, awaiting new methods to identify affected cells to enable examination of their molecular, cellular and physiological characteristics. Here, we report that both primary and secondary damage to cells in mouse models of spinal cord and peripheral nerve injury can be detected in vivo using a novel fluorescent reporter system based on the immediate stress response via activation of Heat Shock Factor 1. We also provide evidence for altered electrophysiological properties of reporter-positive secondarily-injured neurons. The comprehensive identification of injured, but surviving cells located both close and at distant locations from the injury site in vivo will provide a way to study their pathophysiology and possibly prevention of their further deterioration.

Systemic administration of mesenchymal stem cells (MSCs) following cerebral infarction exerts functional improvements. Previous research has suggested potential therapeutic mechanisms that promote neuroprotection and synaptogenesis. These include secretion of neurotrophic factors, remodeling of neural circuits, restoration of the blood brain barrier, reduction of inflammatory infiltration and demyelination, and elevation of trophic factors. In addition to these mechanisms, we hypothesized that restored interhemispheric bilateral motor cortex connectivity might be an additional mechanism of functional recovery. In the present study, we have shown, with both MRI diffusion tensor imaging (DTI) and neuroanatomical tracing techniques using an adeno-associated virus (AAV) expressing GFP, that there was anatomical restoration of cortical interhemispheric connections through the corpus callosum after intravenous infusion of MSCs in a rat middle cerebral artery occlusion (MCAO) stroke model. Moreover, the degree of connectivity was greater in the MSC-treated group than in the vehicle-infused group. In accordance, both the thickness of corpus callosum and synaptic puncta in the contralateral (non-infarcted) motor cortex connected to the corpus callosum were greater in the MSC-treated group than in the vehicle group. Together, these results suggest that distinct preservation of interhemispheric cortical connections through corpus callosum was promoted by intravenous infusion of MSCs. This anatomical preservation of the motor cortex in the contralateral hemisphere may contribute to functional improvements following MSC therapy for cerebral stroke.

Large animal and primate models of spinal cord injury (SCI) are being increasingly utilized for the testing of novel therapies. While these represent intermediary animal species between rodents and humans and offer the opportunity to pose unique research questions prior to clinical trials, the role that such large animal and primate models should play in the translational pipeline is unclear. In this initiative we engaged members of the SCI research community in a questionnaire and round-table focus group discussion around the use of such models. Forty-one SCI researchers from academia, industry, and granting agencies were asked to complete a questionnaire about their opinion regarding the use of large animal and primate models in the context of testing novel therapeutics. The questions centered around how large animal and primate models of SCI would be best utilized in the spectrum of preclinical testing, and how much testing in rodent models was warranted before employing these models. Further questions were posed at a focus group meeting attended by the respondents. The group generally felt that large animal and primate models of SCI serve a potentially useful role in the translational pipeline for novel therapies, and that the rational use of these models would depend on the type of therapy and specific research question being addressed. While testing within these models should not be mandatory, the detection of beneficial effects using these models lends additional support for translating a therapy to humans. These models provides an opportunity to evaluate and refine surgical procedures prior to use in humans, and safety and bio-distribution in a spinal cord more similar in size and anatomy to that of humans. Our results reveal that while many feel that these models are valuable in the testing of novel therapies, important questions remain unanswered about how they should be used and how data derived from them should be interpreted.

Olfactory ensheathing cells (OECs) and Schwann cells (SCs) share many characteristics, including the ability to promote neuronal repair when transplanted directly into spinal cord lesions, but poor survival and migration when transplanted into intact adult spinal cord. Interestingly, transplanted OECs, but not SCs, migrate extensively within the X-irradiated (40 Gy) adult rat spinal cord, suggesting distinct responses to environmental cues [Lankford et al., (2008) GLIA 56:1664–1678]. In this study, GFP-expressing OECs and SCs were transplanted into juvenile rat brains (hippocampus) subjected to a moderate radiation dose (16 Gy). As in the adult spinal cord, OECs, but not SCs, migrated extensively within the irradiated juvenile rat brain. Unbiased stereology revealed that the number of OECs observed within irradiated rat brains three weeks after transplantation was as much as 20 times greater than the number of cells transplanted, and the cells distributed extensively within the brain. In conjunction with the OEC dispersion, the number of activated microglia in OEC-transplanted irradiated brains was reduced. Unlike in the intact adult spinal cord, both OECs and SCs showed some, but limited, migration within nonirradiated rat brains, suggesting that the developing brain may be a more permissive environment for cell migration than the adult CNS. These results show that OECs display unique migratory, proliferative, and microglia interaction properties as compared with SCs when transplanted into the moderately X-irradiated brain. GLIA 2013;62:52–63

Autologous transplantation of olfactory ensheathing cells (OECs) and Schwann cells (SCs) is considered a promising option to promote axonal regrowth and remyelination after spinal cord injury in humans. However, if the experimental data from the rodent model can be directly extrapolated to humans, as widely believed, remains to be established. While limitations of the rodent system have recently been discussed with regard to the distinct organization of the motor systems, the question whether OECs and SCs may display species-specific properties has not been fully addressed. Prompted by recent studies on canine and porcine glia, we performed a detailed analysis of the in vitro and in vivo properties of OECs and SCs and show that rodent but not human, monkey, porcine, and canine glia require mitogens for in vitro expansion, display a complex response to elevated intracellular cAMP, and undergo spontaneous immortalization upon prolonged mitogen stimulation. These data indicate fundamental inter-species differences of the control of cellular proliferation. Whether OECs and SCs from large animals and humans share growth-promoting in vivo properties with their rodent counterpart is not yet clear. Autologous implantation studies in humans did not reveal adverse effects of cell transplantation so far. However, in vivo studies of large animal or human glia and rodent recipients mainly focused on the remyelinating potential of the transplanted cells. Thus, further experimental in vivo studies in large animals are essential to fully define the axonal growth-promoting potential of OECs and SCs. Based on the homology of the in vitro growth control between porcine, canine and human glia, it is concluded that these species may serve as valuable translational models for scaling up human procedures. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

In a previous report we showed that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) improved functional recovery after contusive spinal cord injury (SCI) in the non-immunosuppressed rat, although the MSCs themselves were not detected at the spinal cord injury (SCI) site [1]. Rather, the MSCs lodged transiently in the lungs for about two days post-infusion. Preliminary studies and a recent report [2] suggest that the effects of intravenous (IV) infusion of MSCs could be mimicked by IV infusion of exosomes isolated from conditioned media of MSC cultures (MSCexos). In this study, we assessed the possible mechanism of MSCexos action on SCI by investigating the tissue distribution and cellular targeting of DiR fluorescent labeled MSCexos at 3 hours and 24 hours after IV infusion in rats with SCI. The IV delivered MSCexos were detected in contused regions of the spinal cord, but not in the noninjured region of the spinal cord, and were also detected in the spleen, which was notably reduced in weight in the SCI rat, compared to control animals. DiR “hotspots” were specifically associated with CD206-expressing M2 macrophages in the spinal cord and this was confirmed by co-localization with anti-CD63 antibodies labeling a tetraspanin characteristically expressed on exosomes. Our findings that MSCexos specifically target M2-type macrophages at the site of SCI, support the idea that extracellular vesicles, released by MSCs, may mediate at least some of the therapeutic effects of IV MSC administration.

Keratinocytes play an important role in the dialog between skin and cutaneous sensory neurons. They are an essential source of cutaneous nerve growth factor (NGF), a neurotrophin that contributes to persistent pain in inflammation and neuropathy. We studied the interaction of human keratinocytes (hKTs) and regenerating afferent nerve fibers by transplanting hKTs into a ligated and transected peripheral nerve. The hKTs self-assembled into a multi-laminar spheroid cellular structure resembling the stratum spinosum of epidermis. Axonal sprouts surrounded the structure although they were excluded from entry. Levels of NGF were elevated at the transplant site. Whole cell patch-clamp recordings from primary afferent neurons whose cut axons were present near the transplanted hKTs displayed extreme hyperexcitability. These neurons generated high frequency trains of action potentials during step depolarization stimuli, and they sometimes showed afterdischarge and fired spontaneously at resting membrane potential. This spontaneous firing originated from subthreshold membrane potential oscillations. The animals with the hKT transplants exhibited spontaneous pain behavior manifest as autotomy. The results demonstrate that an interaction between injured/regenerating nerve fibers and keratinocytes such as may occur during wound healing, results in afferent hyperexcitability and pain. These results have implications for persistent pain associated with burn and traumatic skin injuries.

Transplantation of mesenchymal stem cells (MSCs) derived from bone marrow has been shown to improve functional outcome in spinal cord injury (SCI). We transplanted MSCs derived from human bone marrow (hMSCs) to study their potential therapeutic effect in SCI in the rat. In addition to hMSCs, we used gene-modified hMSCs to secrete brain-derived neurotrophic factor (BDNF-hMSCs). After a dorsal transection lesion was induced at T9, cells were microinjected on each side of the transection site. Fluorogold (FG) was injected into the epicenter of the lesion cavity to identify transected corticospinal tract (CST) neurons. At 5 weeks after transplantation, the animals were perfused. Locomotor recovery improvement was observed for the BDNF-hMSC group, but not in the hMSC group. Structurally there was increased sprouting of injured corticospinal tract and serotonergic projections after hMSC and BDNF-hMSC transplantation. Moreover, an increased number of serotonergic fibers was observed in spinal gray matter including the ventral horn at and below the level of the lesion, indicating increased innervation in the terminal regions of a descending projection important for locomotion. Stereological quantification was performed on the brains to determine neuronal density in primary motor (M1) cortex. The number of FG backfilled cells demonstrated an increased cell survival of CST neurons in M1 cortex in both the hMSC and BDNF-hMSC groups at 5 weeks, but the increase for the BDNF-hMSC group was greater. These results indicate that transplantation of hMSCs hypersecreting BDNF results in structural changes in brain and spinal cord, which are associated with improved functional outcome in acute SCI.

Olfactory ensheathing cells (OECs) are specialized glial cells that guide olfactory receptor axons from the nasal mucosa into the brain where they make synaptic contacts in the olfactory bulb. While a number of studies have demonstrated that in vivo transplantation of OECs into injured spinal cord results in improved functional outcome, precise cellular mechanisms underlying this improvement are not fully understood. Current thinking is that OECs can encourage axonal regeneration, provide trophic support for injured neurons and for angiogenesis, and remyelinate axons. However, Schwann cell (SC) transplantation also results in significant functional improvement in animal models of spinal cord injury. In culture SCs and OECs share a number of phenotypic properties such as expression of the low affinity NGF receptor (p75). An important area of research has been to distinguish potential differences in the in vivo behavior of OECs and SCs to determine if one cell type may offer greater advantage as a cellular therapeutic candidate. In this review we focus on several unique features of OECs when they are transplanted into the spinal cord.

Contusive spinal cord injury (SCI) results in a complex lesion that includes cellular and axonal loss, microglia and macrophage activation, and demyelination. These changes result in permanent neurological deficits in people with SCI and in high financial costs to society. Unlike the peripheral nervous system (PNS), in which axonal regeneration can occur, axonal regeneration in the central nervous system (CNS) is extremely limited. This limited regeneration is thought to result from a lack of a permissive environment and from active inhibitory molecules that are present in the CNS but minimal in the PNS. Currently, cell transplantation approaches are among several experimental strategies being investigated for the treatment of SCI. In the olfactory system, a specialized glial cell called the olfactory ensheathing cell (OEC) has been shown to improve functional outcome when transplanted into rodents with SCI, and clinical studies transplanting OECs into patients with SCI are ongoing in China, Portugal, and other sites. Yet, a number of controversial issues related to OEC biology and transplantation must be addressed to understand the rationale and expectations for OEC cell therapy approaches in SCI. This review provides information on these issues for spinal cord medicine clinicians.

Although several studies have shown that Schwann cells (SCs) and olfactory ensheathing cells (OECs) interact differently with central nervous system (CNS) cells in vitro, all classes of adult myelin-forming cells show poor survival and migration after transplantation into normal CNS. X-irradiation of the spinal cord, however, selectively facilitates migration of oligodendrocyte progenitor cells (OPCs), but not SCs, revealing differences in in vivo migratory capabilities that are not apparent in intact tissue. To compare the in vivo migratory properties of OECs and SCs and evaluate the potential of migrating cells to participate in subsequent repair, we first transplanted freshly isolated GFP-expressing adult rat olfactory bulb-derived OECs and SCs into normal and X-irradiated spinal cords. Both OECs and SCs showed limited survival and migration in normal spinal cord at 3 weeks. However, OECs, unlike SCs, migrated extensively in both grey and white matter of the X-irradiated spinal cord, and exhibited a phagocytic phenotype with OX-42 staining on their processes. If a X-irradiated and OEC transplanted spinal cord was then subjected to a focal demyelinating lesion 3 weeks after transplantation, OECs moved into the delayed demyelinated lesion and remyelinated host axons with a peripheral-like pattern of myelin. These results revealed a clear difference between the migratory properties of OECs and SCs in the X-irradiated spinal cord and demonstrated that engrafted OECs can participate in repair of subsequent lesions.

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.

Ectopic discharge generated in injured afferent axons and cell somatain vivocontributes 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 thein vitropreparation, 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 observedin vivo.

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.

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.

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 predominately 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.

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.

In multiple sclerosis (MS), axons of the central nervous system lose their myelin sheaths, and there is also death of oligodendrocytes. Although some demyelinated axons rebuild their membranes so that they can conduct action potentials in the absence of myelin insulation, others do not; and loss of the myelin thus impairs impulse conduction either temporarily or permanently. Mounting evidence also suggests that loss of central myelin may have the secondary consequence of making axons more sensitive to damage or may, in itself, produce changes that impair axonal integrity, thereby leading to cumulative loss of axons that culminates in irreversible neurological deficits (Waxman, 2006). While a number of treatments such as the beta interferons (IFN-β) and glatirimer acetate (GA) are now available for the treatment of MS, and clinical studies using autologous haematopoietic stem cell transplantation (HSCT) and monoclonal antibody interventions in MS patients have shown profound suppression of inflammatory activity in many patients, these interventions were developed in the attempt to mute the immune attack on the nervous system in MS, and not with the goal of repairing demyelination. Thus, even if the immune assault on the nervous system in MS could be halted by a new immuno-modulatory therapy and the subsequent cascade of tissue damage thereby stalled, hundreds of thousands of people harboring MS lesions would still be left with neurological deficits. It is therefore not surprising that myelin repair has become an area of major interest in MS research. Important progress in this respect has come from studies that have examined cell-based approaches to myelin repair. A critical issue for cell-based myelin repair is the choice of cell type for transplantation. The transplanted cell must be able to survive, migrate to demyelinated lesions, remyelinate axons and not be tumorogenic. Oligodendrocyte lineage cells, neural progenitor cells, post-natal Schwann cells, …

Everill, Brian, Marco A. Rizzo, and Jeffery D. Kocsis. Morphologically identified cutaneous afferent DRG neurons express three different potassium currents in varying proportions. J. Neurophysiol. 79: 1814–1824, 1998. Outward K+ currents were recorded using a whole cell patch-clamp configuration, from acutely dissociated adult rat cutaneous afferent dorsal root ganglion (DRG) neurons (L4 and L5) identified by retrograde labeling with Fluoro-gold. Recordings were obtained 16–24 h after dissociation from cells between 39 and 49 mm in diameter with minimal processes. These cells represent medium-sized DRG neurons relative to the entire population, but are large cutaneous afferent neurons giving rise to myelinated axons. Voltage-activated K+ currents were recorded routinely during 300-ms depolarizing test pulses increasing in 10-mV steps from −40 to +50 mV; the currents were preceded by a 500-ms conditioning prepulse of either −120 or −40 mV. Coexpression of at least three components of K+ current was revealed. Separation of these components was achieved on the basis of sensitivities to the K+ channel blockers, 4-aminopyridine (4-AP) and dendrotoxin (DTx), and by the current responses to variation in conditioning voltage. Changing extracellular K+ concentration from 3 to 40 mM resulted in a shift to the right of the I-V curve commensurate with K+ being the principal charge carrier. Presentation of 100 mM 4-AP revealed a rapidly activating K+ current sensitive to low concentrations of 4-AP. High concentrations of 4-AP (6 mM) extinguished all inactivating current, leaving almost pure sustained current ( I K). On the basis of the relative distribution of K+ currents neurons could be separated into three distinct categories: fast inactivating current ( I A), slow inactivating current ( I D), and sustained current ( I K); only I A and I K; and slow inactivating current and I K. However, I K was always the dominant outward current component. These results indicate that considerable variation in K+ currents is present not only in the entire population of DRG neurons, as previously reported, but even within a restricted size and functional group (large cutaneous afferent neurons).

To examine the mechanisms responsible for the more rapid nerve regeneration observed after a previous (conditioning) nerve injury, adult rats were subjected to a midthigh sciatic nerve transection by using one of three protocols designed to facilitate or restrict nerve regeneration: 1) ligation, in which transected axons were prevented from regenerating; 2) cut, in which transected axons were permitted to extend into peripheral target tissue but were separated from the denervated peripheral nerve stump; and 3) crush, in which axons could regenerate normally through the denervated distal nerve tract. The affected dorsal root ganglia (DRG) were subsequently removed, dissociated, and cultured for up to 3 days, and the timing of neurite initiation, rate of outgrowth, and arborization pattern of previously injured neurons were compared with control DRG. Our results indicate that conditioning lesions have at least four distinct and differentially regulated effects on neuronal morphogenesis: 1) conditioning lesions promote earlier neurite initiation, 2) prior nerve injury decreases the ability of neurons to extend long neurites following a second axotomy, 3) exposure to the environment of a denervated peripheral nerve stimulates greater initial rates of neurite outgrowth, and 4) conditioning lesions reduces initial neuritic branching frequency, resulting in straighter neurites whose growth cones extend further distances from their cell bodies. The primary effect of all conditioning lesions on cultured DRG neurons appeared to be to advance the timing of morphogenesis, resulting in conditioning-lesioned neurons that exhibited characteristics consistent with control neurons that had been cultured for an additional day or more. A secondary effect of conditioning lesions on neurite outgrowth rates was dependent on the local environment of the axons prior to culturing.

Enhanced chemiluminescent (ECL) immunoblotting was used to quantitatively assess the initial changes in jun family transcription factor protein levels in adult rat lumbar dorsal root ganglia (DRG) after peripheral axotomy and dorsal root transection, and to study the effects of neurotrophic factor administration on these changes. Transection of central (dorsal root) or peripheral (spinal nerve) branches of DRG neurons resulted in rapid elevation of c-jun protein levels, which was transient after dorsal root transection but sustained, though slightly attenuated, after spinal nerve transection. These results suggest that injury-induced c-jun elevation is biphasic, consisting of an early, transient, injury-initiated phase and a more prolonged secondary phase specific to peripheral target disconnection. c-jun protein changes were not modulated by administration of NGF or BDNF. Immunohistochemistry was used to localize c-jun protein induction to DRG neurons. Using ECL immunoblotting, we also observed temporally regulated increases in junD protein levels after both injuries. A transient up-regulation of junB was detected by immunoblotting 5 days after peripheral axotomy, coincident with a slight decrease in c-jun protein levels.

Research over the past decade has demonstrated that, under some circumstances, structural reorganization of the CNS, including the spinal cord, can occur after injury, raising hopes that spinal cord repair associated with functional recovery, although a daunting goal, may not be an unreachable one. This brief review dis cusses recent approaches to this problem: use of neurotrophins and the rerouting of axons within the transected spinal cord from white matter to gray matter through nerve grafts, and the transplantation of exogenous myelin-forming glial cells to spinal cord tracts in which myelin has been lost. Results available to date indicate that, in models mimicking some aspects of human spinal cord injury, these approaches may yield anatomical repair that is associated with partial restoration of physiological and behavioral func tion. Many important questions remain unanswered. Nevertheless, although the clinical goal of repairing spinal cords in humans is a very challenging one, results in animal models suggest that spinal cord repair is a realistic objective and provide a glimpse of what is likely to be a period of rapid progress. NEURO SCIENTIST 3:263-269, 1997

A basic strategy for the pharmacological treatment of epilepsy is to develop drugs that reduce the excitability of CNS neurons at times preceding or during the onset of seizure discharge with minimal effects on normal electrical activity. Several antiepileptic drugs currently in use exert their action by modulating sodium channels or receptors of the abundant inhibitory neurotransmitter, GABA. These approaches, which are often successful in reducing the number or severity of seizures, have some effects that limit their clinical use. More recently, a new class of antiepileptic drugs such as vigabatrin, which blocks GABA degradation enzymes, have been developed as effective antiepileptics and are associated with minimal side effects. Although these drugs do not display agonist or antagonist properties at GABA receptor sites, they do appear to interact with brain GABA systems because NMR spectroscopy studies indicate that subjects given these drugs have elevated brain GABA levels, and in vitro electrophysiological studies on CNS tissue reveal elevated GABA release. The precise cellular mechanisms of antiepileptic action of these GABA metabolic modulators are not clear, but current work on the cellular effects of these drugs suggests a model that may explain their action.

1. Whole cell patch-clamp recordings were obtained from identified cutaneous and muscle afferent neurons (33-60 microns diam) in dissociated L4 and L5 dorsal root ganglia (DRGs) from normal rats and from rats 2-3 wk after sciatic nerve ligation or crush injury. gamma-Aminobutyric acid (GABA)-induced conductance was compared in normal and injured neurons from both functional classes of sensory neurons. 2. Control cutaneous afferent neurons had a peak GABA-mediated conductance of 287 +/- 27 (SE) nS compared with 457 +/- 42 nS for control muscle afferent neurons. 3. An inflection on the downslope of the action potential was observed in 47% of cutaneous afferent neurons compared with 20% of muscle afferent neurons. 4. After ligation and transection of the sciatic nerve there was no change in the GABA-mediated conductance of muscle afferent neurons or in the action potential waveform (23% inflected). However, the cutaneous afferent neurons displayed a greater than two-fold increase in their GABA-mediated conductance and displayed a prominent reduction in the number of neurons with inflected action potentials (13% inflected). Input resistance was similar in cutaneous and muscle afferent neurons and decreased after ligation in cutaneous but not muscle afferents. Resting potential averaged from -50 to -56 mV in normal and ligated groups for both cutaneous and muscle afferent neurons. 5. After crush injury in cutaneous afferent neurons where the transected axons were allowed to regenerate into the distal nerve stump, GABAA-receptor-mediated conductance was elevated compared with controls. However, action potential waveform was not altered by crush injury, suggesting a differential regulation of these two properties in cutaneous afferent neurons. 6. These data indicate that injury-induced plasticity of GABAA-receptor-mediated conductance and action potential waveform occurs in cutaneous but not muscle afferent DRG neurons. It appears that peripherally derived influences are critical in maintaining the electrophysiological phenotype of cutaneous afferent neurons but not muscle afferent neurons.

Paresthesiae, dysesthesiae, and hyperesthesiae (‘positive symptoms’) result from ectopic nerve impulses secondary to inappropriate membrane excitability which develops in the setting of chronic sensor

Dendntes are the primary site of synaptic input to neurons in the CNS. They are imbued with specialized structures, such as dendritic spines, which receive much of the synaptic inputs from other neurons. They have long been considered electrically passive in that they receive nonregenerative synaptic inputs, both excitatory and inhibitory, and, through summation of passive electrotonic spread of current, they convey information in the form of membrane potential changes to the cell body and the initial axon segment, which are specialized to initiate action potentials. More than 40 years ago, this view was challenged by electrophysiological studies suggesting that dendrites can also generate action po tentials. It was proposed that these regenerative dendritic action potentials can serve as "booster" sites to assure the fidelity of synaptic signaling to the distant initial axon seg ment where nerve impulses are initiated for propagation to their terminals for signal trans mission to other neurons. Two recent articles in Science reporting the use of simultaneous patch clamp recordings from a dendrite and the cell body of rat hippocampal neurons have rekindled interest in dendrites as active generators of action potentials. In this Up date, these articles are reviewed and discussed in the context of the functional implica tions of active dendritic electrogenesis. The Neuroscientist 1:312-316, 1995

The effects of axotomy on the electrophysiologic properties of adult rat dorsal root ganglion (DRG) neurons were studied to understand the changes in excitability induced by traumatic nerve injury. Nerve injury was induced in vivo by sciatic nerve ligation with distal nerve transection. Two to four weeks after nerve ligation, a time when a neuroma forms, lumbar (L4 and L5) DRG neurons were removed and placed in short-term tissue culture. Whole cell patch-clamp recordings were made 5–24 h after plating.DRG neurons were grouped into large (43–65 μm)-, medium (34–42 μm)-, and small (20–32 pm)- sized classes. Large neurons had short duration action potentials with ∼60% having inflections on the falling phase of their action potentials. In contrast, action potentials of medium and small neurons were longer in duration and ∼68% had inflections.Pressure microejection of γ-aminobutyric acid (GABA, 100 μM) or muscimol (100 μM) onto voltage-clamped DRG neurons elicited a rapidly desensitizing inward current that was blocked by 200 μM bicuculline. To measure the peak conductance induced by GABA or muscimol, neurons were voltage-clamped at a holding potential of -60 mV, and pulses to -80 mV and -100 mV were applied at a rate of 2.5 or 5 Hz during drug application. Slope conductances were calculated from plots of whole cell current measured at each of these potentials.GABA-induced currents and conductances of control DRG neurons increased progressively with cell diameter. The mean GABA conductance was 36 ± 10 nS (mean ± SE) in small neurons, 124 ± 21 nS in medium neurons, and 527 ± 65 nS in large neurons.After axotomy, medium neurons had significantly larger GABA-induced conductances compared with medium control neurons (390 ± 50 vs. 124 ± 21; P < 0.001). The increase in GABA conductance of medium neurons was associated with a decrease in duration of action potentials. In contrast, small neurons had no change in GABA conductance or action potential duration after ligation. The GABA conductance of large control neurons was highly variable, and ligation resulted in an increase that was significant only for neurons >50 μm. The mean action potential duration in large neurons was not significantly changed, but neurons with inflections on the falling phase of the action potential were less common after ligation. There was no difference in resting potential or input resistance between control and ligated groups, except that the resting potential was less negative in small cells after axotomy.Histograms of neuronal diameter distributions were constructed for control and ligated groups. The cell diameter distribution of control and ligated neurons were similar, but the ligated group had a decrease in the proportion of large neurons and a 27% increase in medium neurons, with no change in the relative proportion of other size classes. The 27% increase in the number of medium neurons was unlikely to be solely responsible for the 314% increase in GABA conductance seen in medium neurons after ligation, although some of the observed change could be attributed to a shift of large neurons (and their associated electrophysiologic properties) into the medium group.These results indicate that axotomy resulted in a significant increase in GABAA receptor-mediated conductance in specific size classes of sensory neurons. We hypothesize that this selective increase in GABA conductance results from an injury-induced increase in the density of GABAA receptors on the soma or a change in the expression of specific GABAA receptor subtypes with different single channel conductances.

Voltage-dependent Na+ conductances were studied in small (18–25 μm diam) adult rat dorsal root ganglion (DRG) neurons with the use of the whole cell patch-clamp technique. Na+ currents were also recorded from larger (44–50 μm diam) neurons and compared with those of the small neurons.The predominant Na+ conductance in the small neurons was selective over tetramethylammonium by at least 10-fold and was resistant to 1 μM external tetrodotoxin (TTX). Na+ conductances in many larger DRG neurons were kinetically faster and, in contrast, were blocked by 1 μM TTX.The Na+ conductance in the small neurons was kinetically slow. Activation half-times were voltage dependent and ranged from 2 ms at −20 mV to 0.7 ms at +50 mV. Approximately 50% of the activation half-time was comprised of an initial delay. Inactivation half-times were voltage dependent and ranged from 11 ms at −20 mV to 2 ms at +50 mV.Peak slow Na+ conductances were near maximal with conditioning potentials negative to −120 mV and were significantly reduced or eliminated with conditioning potentials positive to −40 mV. The slow Na+ conductance increased gradually with test potentials extending from −40 to +40 mV. In some cells the conductance could be saturated at +10 mV. Peak conductance/voltage relationships, although stable in a given neuron, revealed marked variability among neurons, spanning >20- and 50-mV domains for steady-state activation and inactivation (current availability), respectively.Kinetics remained stable within a given neuron over the course of an experiment. However, considerable kinetic variation was exhibited from neuron to neuron, such that the half-times of activation and of inactivation spanned an order of magnitude. In all small neurons studied there appeared to be a singular kinetic component of the current, based on sensitivity to the conditioning potential, voltage dependence of activation, and inactivation half-time.Unique closing properties were exhibited by Na+ channels of the small neurons. Hyperpolarization following a depolarization-induced fully inactivated state resulted in tail currents that appeared to be the consequence of reactivation of the slow Na+ conductance. Tail currents recorded at various times during a fixed level of depolarization revealed that the underlying channels accumulated into a volatile inactivated state over the course of the preceding depolarization.Larger neurons had a different repertoire of Na+ conductances, with either only a TTX-sensitive, kinetically fast type, or a combination of fast TTX-sensitive and slow currents. In larger neurons the kinetically separable fast current had a greater sensitivity to the conditioning potential, i.e., a left-shifted steady-state inactivation curve.The different properties of the slow Na+ conductance in different neurons is likely to reflect heterogeneity of the structure of the underlying channel molecule. Although consistent with what others have found in equivalent preparations, this heterogeneity is far broader in scope than what has so far been described. We suggest that biosynthetic constraints within a given small neuron maintain ion channel uniformity.

The permeability of non-N-methyl-D-aspartate (non-NMDA) glutamate channels to divalent cations and specifically the entry of Ca2+ and subsequent elevations in cytoplasmic and nuclear Ca2+ signals were investigated in cultured neonatal rat retinal ganglion cells using the whole cell patch-clamp technique and Ca2+ imaging with confocal microscopy. In addition, divalent-permeable non-NMDA receptor channels were studied in retinal slices using a Co2+ staining technique.Using Ca2+ (2.5 mM) as the only permeable cation in the external solution, stimulation with 100 μM kainate produced nondesensitizing, nonselective cation currents with either low or high Ca2+ permeability. Both currents were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Neurons with the low divalent-permeable currents (type 1) had reversal potentials of −41.5 ± 4.4 mV (mean ± SD), and neurons with the high divalent-permeable currents (type 2) had reversal potentials of −22.6 ± 5.5 mV. The permeability ratio PCa/PCs was 3.3 for the type 1 currents and 8.5 for the type 2 currents, indicating a 2.5-fold greater permeability to Ca2+ for the type 2 non-NMDA glutamate channels.Both types of non-NMDA glutamate channels showed relatively little selectivity between Ca2+ and Co2+. The type 1 neurons had a slightly higher permeability to Co2+ than to Ca2+, whereas the type 2 neurons were equally permeable to both divalent cations. The type 2 neurons had a much higher permeability for both divalent cations compared with the type 1 neurons.Staining for Co2+ uptake through kainate-stimulated non-NMDA glutamate channels in retinal slices provided additional evidence for the presence of the two ganglion cell populations. Activation of the neurons by kainate in conditions isolating the non-NMDA glutamate channel caused differential uptake of Co2+. In contrast, depolarization in the presence of the non-NMDA antagonist CNQX failed to cause Co2+ influx.Imaging experiments using confocal microscopy showed that kainate stimulation induced an increase in intracellular Ca2+ in both types of retinal ganglion cells, but only the type 2 neurons showed a substantial increase in cytoplasmic and nuclear Ca2+ signals. Kainate-induced Ca2+ signals in the type 2 neurons were almost nine times greater than those of the type 1 neurons.When intracellular Ca2+ stores were depleted by brief treatment with thapsigargin, kainate-induced Ca2+ signals in the type 1 neurons were unchanged. However, in the type 2 neurons kainate no longer induced large Ca2+ signals in the cytoplasm and nucleus, despite normal influx of Ca2+. After thapsigargin treatment, kainate-induced Ca2+ signals in the type 2 neurons were reduced to the levels of the treated or untreated type 1 neurons. Calcium influx via the type 2 receptor channels therefore appears capable of triggering the release of intracellular Ca2+ stores.The two types of non-NMDA glutamate channels described here are likely to contribute to a variety of Ca2+-dependent intracellular processes. Glutamate may differentially affect intracellular Ca2+ in retinal ganglion cells. In particular, it appears that Ca2+ entry via the high divalent-permeable type 2 non-NMDA glutamate channel can trigger Ca2+-induced Ca2+ release and thereby amplify intracellular Ca2+ signals.

1. In situ hybridization with subtype-specific probes was used to ask whether there is a change in the types of sodium channels that are expressed in dorsal root ganglion (DRG) neurons after axotomy. 2. Types I and II sodium channel mRNA are expressed at moderate-to-high levels in control DRG neurons of adult rat, but type III sodium channel mRNA is not detectable. 3. When adult rat DRG neurons are examined by in situ hybridization 7–9 days following axotomy, type III sodium channel mRNA is expressed at moderate-to-high levels, in addition to types I and II mRNA that are present at relatively high levels. 4. To determine whether the expression of type III sodium channel mRNA following axotomy represents up-regulation of a gene that had been expressed at earlier developmental stages, we also studied DRG neurons from embryonic (E17) rats. In these embryonic DRG neurons, type I sodium channel mRNA is expressed at low levels, type II mRNA at high levels, and type III at high levels. 5. These results demonstrate altered expression of sodium channel mRNA in DRG neurons following axotomy, and suggest that in at least some DRG neurons, there is a de-differentiation after axotomy that includes a reversion to an embryonic mode of sodium channel expression. Different channel characteristics, as well as an altered spatial distribution of sodium channels, may contribute to the electrophysiological changes that are observed in axotomized neurons.

Confocal microscopy and the Ca 2+ -sensitive fluorescent dye fluo-3 were used to study subcellular Ca 2+ signals in embryonic, neonatal, and adult dorsal root ganglion (DRG) neurons in excised dorsal rooot ganglia. Optical images obtained from isolated' whole embryonic and neonatal ganglia revealed a marked variability in the resting Ca 2+ signals of different neurons as compared to signals in adult neurons which were uniformly faint. Many of the embryonic and neonatal neurons displayed nuclear Ca 2+ signals at rest which were larger than those in the cytoplasm. Embryonic DRG neurons showed a significant increase in nuclear and cytoplasmic fluorescence in response to depolarization with elevated extracellular potassium or electrical stimulation. A single brief electrical stimulus was sufficient to elicit nuclear Ca 2+ signals in a subset of the embryonic neurons. The depolarization-induced Ca 2+ signals were blocked by removal of extracellular Ca 2+ , but not by treatment with 2,5-di ( tert -butyl)- 1,4 benzohydroquinone (DTBHQ), a compound which depletes intracellular Ca 2+ stores. The intensity of the depolarization-induced Ca 2+ signals declined significantly between the late embryonic (E18–E20) and early postnatal time periods (P0–P1). The nuclear and cytoplasmic Ca 2+ signals of the embryonic DRG neurons in the excised tissue preparation occur at a time of intense target innervation, suggesting a role for Ca 2+ signals in the development and maturation of rat DRG neurons.

Olfactory ensheathing cells (OECs) can remyelinate demyelinated spinal cord axons when transplanted into chemically induced demyelinated lesions. Cell transplantation is typically performed within a few days after lesion induction, i.e. during active demyelination when myelin debris, cytokine level increases and macrophage/microglia activation is extensive. Inflammatory signaling has been suggested to facilitate remyelination in cell transplant studies. In this review we discuss the migration and remyelination properties of OECs transplanted into various demyelinating lesion environments including conditions when inflammation is active and when it is largely subsided. While sharing many common properties, comparisons of the in vivo fate between OECs and SCs suggest unique properties of OECs as compared to SCs. A complicating factor in the assessment of experimental remyelination by transplantation of myelin-forming cells in general is the rapidity of endogenous myelin repair in most rodent models of demyelination. Alternative persistent demyelination models are discussed as potential tools to study both the competency of chronic demyelinated axons for remyelination and the remyelination potential of cells such as human progenitors that require longer times to mobilize and remyelinate axons. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

GABA ($\gamma $-aminobutyric acid) is a major inhibitory synaptic neurotransmitter with widespread distribution in the central nervous system (CNS). GABA can also modulate axonal excitability by activation of GABA$_{\text{A}}$ receptors in CNS white matter regions where synapses and neuronal cell bodies are not present. Studies on cultured glia cells have revealed the synthesis of GABA in rat optic nerve O-2A progenitor cells that give rise to oligodendrocytes and type 2 astrocytes in vitro. We report here that: (i) GABA is detected by immuno-electron microscopy in intact rat optic nerve and is localized to glia and pre-myelinated axons during the first few weeks of postnatal development, but is markedly reduced or absent in the adult; and (ii) neonatal optic nerve is depolarized by GABA$_{\text{A}}$ receptor agonists or by the inhibition of GABA uptake. These results demonstrate the presence of functional GABA$_{\text{A}}$ receptors, and GABA uptake and release mechanisms in developing rat optic nerve, and suggest that excitability of developing axons can be modulated by endogenous neurotransmitter at non-synaptic sites.

BACKGROUND:Progenitor cells isolated from adult brain tissue are important tools for experimental studies of remyelination. Cells harvested from neurogenic regions in the adult brain such as the subependymal zone have demonstrated remyelination potential. Multipotent cells from the progenitor fraction have been isolated from the adult olfactory bulb (OB) but their potential to remyelinate has not been studied. METHODOLOGY/PRINCIPAL FINDINGS:We used the buoyant density gradient centrifugation method to isolate the progenitor fraction and harvest self-renewing multipotent neural cells grown in monolayers from the adult green-fluorescent protein (GFP) transgenic rat OB. OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient. The progenitor fraction was isolated and these cells were plated in growth media with serum for 24 hrs. Cells were then propagated in N2 supplemented serum-free media containing b-FGF. Cells at passage 4 (P4) were introduced into a demyelinated spinal cord lesion. The GFP(+) cells survived and integrated into the lesion, and extensive remyelination was observed in plastic sections. Immunohistochemistry revealed GFP(+) cells in the spinal cord to be glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and neurofilament negative. The GFP(+) cells were found among primarily P0(+) myelin profiles, although some myelin basic protein (MBP) profiles were present. Immuno-electron microscopy for GFP revealed GFP(+) cell bodies adjacent to and surrounding peripheral-type myelin rings. CONCLUSIONS/SIGNIFICANCE:We report that neural cells from the progenitor fraction of the adult rat OB grown in monolayers can be expanded for several passages in culture and that upon transplantation into a demyelinated spinal cord lesion provide extensive remyelination without ectopic neuronal differentiation.

Peripheral nerve injury is a common clinical problem, and the development of novel strategies to enhance peripheral nerve regeneration is important. Traumatic events, including motor vehicle accidents, sports-related injuries, violence, and falls, lead to significant numbers of peripheral nerve lesions. Traumatic nerve injuries are often associated with life-threatening injuries, which must be treated first. During the delay in nerve repair, the transected nerves undergo Wallerian degeneration. Therefore, delay before surgical treatment is critical, but care must also be taken to ensure that nerve reapposition is performed in a manner that will result in a therapeutic benefit. Peripheral nerve repair after transection injury combined with transplantation of myelin-forming glia cells, for example, Schwann cells (SCs) or olfactory ensheathing cells (OECs), may facilitate the regenerative process. Cell-based therapies are being considered in clinical trials for a number of neurological diseases, including multiple sclerosis, spinal cord injury, Parkinson's disease, and stroke. The rationale is that transplanted cells may provide neuroprotection by production of chemokines and neurotrophins or could serve as a replacement therapy. A number of cells derived from adult peripheral tissues for cell therapies are also being actively investigated. These cells include SCs from peripheral nerve, olfactory OECs from the olfactory system, and stromal cells from bone marrow (mesenchymal stem cells, MSCs). In principle, these cells could be derived autologously, and used acutely or expanded in culture and used for cell-based therapies. Here, we review experimental work demonstrating the potential of one of these cells, the OEC, as an experimental tool for promoting recovery in peripheral nerve injury.

Monitoring pathology/regeneration in experimental models of de-/remyelination requires an accurate measure not only of functional changes but also of the amount of myelin. We tested whether X-ray diffraction (XRD), which measures periodicity in unfixed myelin, can assess the structural integrity of myelin in fixed tissue. From laboratories involved in spinal cord injury research and in studying the aging primate brain, we solicited "blind" samples and used an electronic detector to record rapidly the diffraction patterns (30 min each pattern) from them. We assessed myelin integrity by measuring its periodicity and relative amount. Fixation of tissue itself introduced +/-10% variation in periodicity and +/-40% variation in relative amount of myelin. For samples having the most native-like periods, the relative amounts of myelin detected allowed distinctions to be made between normal and demyelinating segments, between motor and sensory tracts within the spinal cord, and between aged and young primate CNS. Different periodicities also allowed distinctions to be made between samples from spinal cord and nerve roots and between well-fixed and poorly fixed samples. Our findings suggest that, in addition to evaluating the effectiveness of different fixatives, XRD could also be used as a robust and rapid technique for quantitating the relative amount of myelin among spinal cords and other CNS tissue samples from experimental models of de- and remyelination.

Sucrose gap and intra-axonal recording techniques were used to identify the types of ion channels and inward rectification that are present in regenerated axons of adult (greater than 8 weeks) rat sciatic nerve after crush injury. In sucrose gap recordings, 4-aminopyridine (4-AP) led to slight broadening of the compound action potential (CAP) in normal nerve, and a greater broadening in regenerated nerves. By 12 days after sciatic nerve crush, regenerated nerves manifested an afterhyperpolarization (AHP) lasting up to 250 ms that was sensitive to tetraethylammonium (TEA). A similar TEA-sensitive AHP could be elicited with repetitive stimulation. Hyperpolarizing constant current steps (0.1 to 0.5 mA; 600-900 ms duration) applied across the sucrose gap through regenerated axons evoked membrane hyperpolarizations with a depolarizing, Cs(+)-sensitive relaxation in the response to hyperpolarization, which is characteristic of inward rectification, occurring after about 70 ms. The relaxation was present as early as 21 days after nerve crush. Intra-axonal recordings showed burst firing in 4-AP that was terminated by an AHP that temporally correlated with the TEA-sensitive AHP, and a relaxation in the response to hyperpolarizing current, similar to that of whole nerve recordings. The results demonstrate that in addition to voltage-sensitive sodium channels and 4-AP-sensitive potassium channels, there are TEA-sensitive and inwardly rectifying channels on mammalian regenerated peripheral nerve axons.

Myelinated primary afferent fibers have both peripheral and central nervous system components. As the fibers course through peripheral nerve and dorsal roots they are myelinated by Schwann cells, but after they invade the spinal cord they become myelinated by oligodendrocytes and have associations with astrocytes. This presents the opportunity to compare the pathophysiology of PNS (Schwann cell-associated) vs. CNS (oligodendrocyte/astrocyte-associated) portions of the same axonal trunk located in the dorsal roots and dorsal columns, respectively. Dorsal spinal roots and slices of dorsal columns isolated from adult rats were studied in a sucrose gap chamber from which compound action potential and membrane potential changes could be recorded. The results indicate that the peripheral component of the afferent fibers is resistant to hypoxia as evidenced by stable action and membrane potential when O2 in the bathing medium was completely replaced with N2 for periods up to 2 h. In contrast, the axons become sensitive to hypoxia as they project through the dorsal columns as evidenced by rapid reduction in action potential amplitude accompanied by membrane depolarization when O2 is replaced by N2. This differential response to hypoxia, observed on the same axon branches but over CNS vs. PNS trajectories, suggests that differences related to the extracellular environment or in axo-glial organization in dorsal root vs. dorsal column may confer different degrees of susceptibility to anoxia.

Demyelinating diseases can arise in a variety of forms in the central and as well as in the peripheral nervous system, and loss of myelin results in conduction abnormalities and functional deficits. Remyelination can occur from activation of endogenous progenitor cells and by exogenously transplanted myelin-forming cells. Here we discuss conduction abnormalities associated with demyelination and myelin repair by transplanted myelin-forming cells. Axonal repair and proper impulse conduction following remyelination also requires appropriate ion channel organization on the remyelinated axon. Axonal sodium and potassium channel distribution on remyelinated axons is discussed. Axon damage and transection is another prominent pathological feature of demyelinated lesions in multiple sclerosis (MS). This chapter also discusses potential neuroprotective effects of transplantation of myelin-forming cells into sites of axonal transection.

1. Rat optic nerves were studied using intra-axonal and whole-nerve recording techniques in a sucrose-gap chamber. Constant-current pulses were applied across the outer compartments of the chamber to achieve a current clamp. 2. The nerves displayed a prominent time-dependent conductance increase elicited by a hyperpolarizing constant-current pulse, as evidenced by a relaxation or 'sag' in membrane potential towards resting potential. The inward current began at about 80 ms and reached a steady level over the next 100-200 ms. Its magnitude progressively increased with increasing levels of hyperpolarization. 3. The inward current elicited by hyperpolarization was reduced, but not abolished, when Na+ was reduced from the normal bath concentration of 151 mM to 0 mM. In Na(+)-free solutions the bath K+ concentration, [K+]o, was varied between 0 and 5 mM; the inward current was greatest when [K+]o was 5 mM and was abolished when [K+]o was zero. 4. The inward current was not abolished by tetrodotoxin (TTX), tetraethylammonium (TEA) or 4-aminopyridine (4-AP) suggesting that conventional voltage-dependent sodium and potassium channels do not underlie the time-dependent conductance increase. Low concentrations of Cs+ completely blocked the inward current, and Ba2+ induced a partial block. External application of divalent cations (Cd2+ and Mg2+) did not block the inward current. These properties are similar to the inwardly rectifying conductance observed in a central nervous system neurone. 5. Stimulus-response curves obtained during the hyperpolarization pulse, before and during the conductance increase, indicate that excitability is increased during the conductance increase. This along with the intra-axonal recordings demonstrates that the origin of the increased conductance is axonal and not glial. 6. It is concluded that central nervous system myelinated fibres in rat optic nerve display a prominent time-dependent conductance increase in response to hyperpolarization that depends on both Na+ and K+ and is blocked by Cs+. This conductance is similar to an inward rectifier described for a variety of neurone types. The increased axonal excitability observed during the conductance increase suggests that its functional role may be to maintain or stabilize axonal excitability during periods of intense action potential activity.

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.

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 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.

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 Navand Kvchannels to specific membrane domains at remyelinated nodes persisted for at least 8 weeks after GFP-OEC transplantation.In vivoelectrophysiological 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.

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 μm) 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.

—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 developing nervous system is replete with excessive synaptic connections, many of which will be lost during development. This process of pruning leads to a more precise pattern of synaptic connectivity of the mature nervous system. To sculpt away inappro priate synaptic connections is considered a crucial determinant in establishing the pre cision of synaptic interactions in the nervous system. Much evidence suggests that the pattern of nerve impulse activity in the afferent fiber component of the synapse plays a primary role in loss of superfluous synaptic connections during development. Perhaps the best studied example of this developmental phenomenon is at the neuromuscular junction of the neonatal mouse where multiple motor neurons innervate a given muscle fiber; the multiple synapses are usually located on the same motor endplate. However, over a few weeks all but one motor neuron are eliminated so that a given muscle fiber receives its innervation from only one motor neuron (Fig. 1). The mechanisms for this retraction of inappropriate or redundant synaptic connections have broad implications for the under standing of the development of the synaptic organization of the nervous system as well as for other forms of neuronal plasticity, including those that may underlie learning and memory processes in the adult nervous system. A novel approach by Balice-Gordon and Lichtman (Nature 1994;519-524) to understanding the mechanisms of the loss and pres ervation of synapses in the developing and regenerating mammalian nervous system is reviewed in this Update. The Neuroscientist 1:185-187, 1995