Olfactory Ensheathing Glia Transplantation Research Articles

Tissue sparing, behavioral recovery, supraspinal axonal sparing/regeneration following sub-acute glial transplantation in a model of spinal cord contusion

BackgroundIt has been shown that olfactory ensheathing glia (OEG) and Schwann cell (SCs) transplantation are beneficial as cellular treatments for spinal cord injury (SCI), especially acute and sub-acute time points. In this study, we transplanted DsRED transduced adult OEG and SCs sub-acutely (14 days) following a T10 moderate spinal cord contusion injury in the rat. Behaviour was measured by open field (BBB) and horizontal ladder walking tests to ascertain improvements in locomotor function. Fluorogold staining was injected into the distal spinal cord to determine the extent of supraspinal and propriospinal axonal sparing/regeneration at 4 months post injection time point. The purpose of this study was to investigate if OEG and SCs cells injected sub acutely (14 days after injury) could: (i) improve behavioral outcomes, (ii) induce sparing/regeneration of propriospinal and supraspinal projections, and (iii) reduce tissue loss.ResultsOEG and SCs transplanted rats showed significant increased locomotion when compared to control injury only in the open field tests (BBB). However, the ladder walk test did not show statistically significant differences between treatment and control groups. Fluorogold retrograde tracing showed a statistically significant increase in the number of supraspinal nuclei projecting into the distal spinal cord in both OEG and SCs transplanted rats. These included the raphe, reticular and vestibular systems. Further pairwise multiple comparison tests also showed a statistically significant increase in raphe projecting neurons in OEG transplanted rats when compared to SCs transplanted animals. Immunohistochemistry of spinal cord sections short term (2 weeks) and long term (4 months) showed differences in host glial activity, migration and proteoglycan deposits between the two cell types. Histochemical staining revealed that the volume of tissue remaining at the lesion site had increased in all OEG and SCs treated groups. Significant tissue sparing was observed at both time points following glial SCs transplantation. In addition, OEG transplants showed significantly decreased chondroitin proteoglycan synthesis in the lesion site, suggesting a more CNS tolerant graft.ConclusionsThese results show that transplantation of OEG and SCs in a sub-acute phase can improve anatomical outcomes after a contusion injury to the spinal cord, by increasing the number of spared/regenerated supraspinal fibers, reducing cavitation and enhancing tissue integrity. This provides important information on the time window of glial transplantation for the repair of the spinal cord.

Clinical Observation of Fetal Olfactory Ensheathing Glia Transplantation (OEGT) in Patients with Complete Chronic Spinal Cord Injury

This study was designed to observe the functional changes after fetal olfactory ensheathing glia transplantation (OEGT) into the spinal cord of patients with chronic spinal cord injury (SCI). Patients whose recovery had plateaued for longer than 6 months were enrolled. Six thoracic patients were tested for safety and five cervical patients for efficacy. OEGT was performed according to the method developed by Huang. Average follow-up was 14 months (range 1.0-1.5 years). Sensation improved moderately (light touch 14.2, pin prick 13.6); as did spasticity (1-2 modified Ashworth scale down). Locomotion recovery was minimal (1.6). Useful reticular formation functions were observed, but due to a lack of appropriate outcome measure, they were not recorded and reported.

Axon Regeneration Can Facilitate or Suppress Hindlimb Function after Olfactory Ensheathing Glia Transplantation

Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function.

Olfactory Ensheathing Glia Transplantation Combined with LASERPONCTURE® in Human Spinal Cord Injury: Results Measured by Electromyography Monitoring

Preliminary results were measured by electromyography monitoring (electromyoscan) on three subjects suffering from spinal cord injury and who underwent a double therapy. The aim of this study was to evaluate regained voluntary activity below the injury in subjects who received a double therapy: 1) an olfactory ensheathing glia (OEG) transplantation using procedures developed by Dr. Hongyun Huang at the Xishan Hospital and Rehabilitation Centre, Beijing, China, and 2) LASERPONCTURE developed by Albert Bohbot, Laboratoire de Recherches sur le LASERPONCTURE, La Chapelle Montlinard, France. Materials uses were the LASERPONCTURE device developed by Albert Bohbot; the PROCOMP5 equipment with softwares BIOGRAPH INFINITI 5 and REHAB SUITE; the sensors MYOSCAN-PRO EMG (SA9401M-50) to record muscle activity, and FLEX/PRO-SA9309M to record skin conductance were fixed on the skin. An infrared laser, whose frequencies and power settings cannot be disclosed due to its proprietary nature, was applied after an OEG injection performed according to Dr. Hongyun Huang's procedures. Three cases, two males and one female, were selected for this study. Presentation and comments of the graphs recordings of voluntary muscle activity below the injury are provided. This preliminary study suggests that the double therapy restores some voluntary muscle activity as measured by electromyography monitoring.

Noradrenergic innervation of the rat spinal cord caudal to a complete spinal cord transection: Effects of olfactory ensheathing glia

Transplantation of olfactory bulb-derived olfactory ensheathing glia (OEG) combined with step training improves hindlimb locomotion in adult rats with a complete spinal cord transection. Spinal cord injury studies use the presence of noradrenergic (NA) axons caudal to the injury site as evidence of axonal regeneration and we previously found more NA axons just caudal to the transection in OEG- than media-injected spinal rats. We therefore hypothesized that OEG transplantation promotes descending coeruleospinal regeneration that contributes to the recovery of hindlimb locomotion. Now we report that NA axons are present throughout the caudal stump of both media- and OEG-injected spinal rats and they enter the spinal cord from the periphery via dorsal and ventral roots and along large penetrating blood vessels. These results indicate that the presence of NA fibers in the caudal spinal cord is not a reliable indicator of coeruleospinal regeneration. We then asked if NA axons appose cholinergic neurons associated with motor functions, i.e., central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more NA varicosities adjacent to central canal cluster cells, partition cells, and SMNs in the lumbar enlargement of OEG- than media-injected rats. As non-synaptic release of NA is common in the spinal cord, more associations between NA varicosities and motor-associated cholinergic neurons in the lumbar spinal cord may contribute to the improved treadmill stepping observed in OEG-injected spinal rats. This effect could be mediated through direct association with SMNs and/or indirectly via cholinergic interneurons.

Analysis of Olfactory Ensheathing Glia Transplantation-Induced Repair of Spinal Cord Injury by Electrophysiological, Behavioral, and Histochemical Methods in Rats

This study examined the efficacy of transplanting olfactory ensheathing glia (OEG) in repairing spinal cord injury (SCI) using behavioral tests, retrograde labeling, as well as somatosensory and motor evoked potentials in rats. One week after surgery, motor function in OEG-treated rats was significantly superior to untreated controls (P < 0.05). Also, we found that up to 8 weeks following surgery to induce SCI, somatosensory and motor evoked potentials were found in the OEG-treated groups, but not in the transplantation and damage control groups. Retrograde labeling from the area distal to the SCI produced a higher number of labeled neurons in the ventrolateral division of red nucleus and motor cortex of OEG-treated rats compared to controls, which showed no retrograde labeling (P < 0.05). We believe that this study has important implications for characterizing the mechanisms of OEG transplantation as a treatment for SCI.

Slow‐ and fast‐twitch rat hind limb skeletal muscle phenotypes 8 months after spinal cord transection and olfactory ensheathing glia transplantation

Paralysed skeletal muscle of rats with spinal cord injury (SCI) undergoes atrophy and a switch in gene expression pattern which leads to faster, more fatigable phenotypes. Olfactory ensheathing glia (OEG) transplants have been reported to promote axonal regeneration and to restore sensory-motor function in animals with SCI. We hypothesized that OEG transplants could attenuate skeletal muscle phenotypic deterioration and that this effect could underlie the functional recovery observed in behavioural tests. A variety of morphological, metabolic and molecular markers were assessed in soleus (SOL) and extensor digitorum longus (EDL) muscles of spinal cord transected (SCT), OEG-transplanted rats 8 months after the intervention and compared with non-transplanted SCT rats and sham-operated (without SCT) controls (C). A multivariate analysis encompassing all the parameters indicated that OEG-transplanted rats displayed skeletal muscle phenotypes intermediate between non-transplanted and sham-operated controls, but different from both. A high correlation was observed between behaviourally tested sensory-motor functional capacity and expression level of slow- and fast-twitch hind limb skeletal muscle phenotypic markers, particularly the histochemical glycerol-3-phosphate dehydrogenase activity (-0.843, P < 0.0001) and the fraction of variant 2s of the slow regulatory myosin light chain isoform (0.848, P < 0.0001) in SOL. Despite the mean overall effect of OEG transplants in patterning skeletal muscle protein expression towards normal, in 6 out of 9 animals they appeared insufficient to overcome fibre type switching and to support a consistent and generalized long-term maintenance of normal skeletal muscle characteristics. The interplay of OEG and exercise-mediated neurotrophic actions is a plausible mechanism underlying OEG transplantation effects on paralysed skeletal muscle.

OEG implantation and step training enhance hindlimb-stepping ability in adult spinal transected rats

Numerous treatment strategies for spinal cord injury seek to maximize recovery of function and two strategies that show substantial promise are olfactory bulb-derived olfactory ensheathing glia (OEG) transplantation and treadmill step training. In this study we re-examined the issue of the effectiveness of OEG implantation but used objective, quantitative measures of motor performance to test if there is a complementary effect of long-term step training and olfactory bulb-derived OEG implantation. We studied complete mid-thoracic spinal cord transected adult female rats and compared four experimental groups: media-untrained, media-trained, OEG-untrained and OEG-trained. To assess the extent of hindlimb locomotor recovery at 4 and 7 months post-transection we used three quantitative measures of stepping ability: plantar stepping performance until failure, joint movement shape and movement frequency compared to sham controls. OEG transplantation alone significantly increased the number of plantar steps performed at 7 months post-transection, while training alone had no effect at either time point. Only OEG-injected rats plantar placed their hindpaws for more than two steps by the 7-month endpoint of the study. OEG transplantation combined with training resulted in the highest percentage of spinal rats per group that plantar stepped, and was the only group to significantly improve its stepping abilities between the 4- and 7-month evaluations. Additionally, OEG transplantation promoted tissue sparing at the transection site, regeneration of noradrenergic axons and serotonergic axons spanning the injury site. Interestingly, the caudal stump of media- and OEG-injected rats contained a similar density of serotonergic axons and occasional serotonin-labelled interneurons. These data demonstrate that olfactory bulb-derived OEG transplantation improves hindlimb stepping in paraplegic rats and further suggest that task-specific training may enhance this OEG effect.

NT-3 expression from engineered olfactory ensheathing glia promotes spinal sparing and regeneration

Adenoviral (AdV) vectors encoding neurotrophin-3 (AdV-NT-3) or the bacterial marker enzyme beta-galactosidase (LacZ gene) were used to transduce olfactory ensheathing glia (OEG) cultures. AdV vector-transduced OEG expressed high levels of recombinant neurotrophin as shown by in situ hybridization and enzyme-linked immunosorbent assay techniques. The biological activity of vector-derived NT-3 was determined in a dorsal root ganglia neurite outgrowth assay. Engineered cell suspensions were then injected into adult Fischer 344 rat spinal cord immediately after unilateral cervical (C4) corticospinal tract (CST) transection. Transplanted animals received a total of 200,000 cells; either non-transduced OEG or OEG transduced with AdV vectors encoding NT-3 or LacZ, respectively. At 3 months after injury, lesion volumes were significantly smaller in all OEG-transplanted rats when compared with control (medium-injected) rats. Anterograde tracing of the lesioned CST projection, originating from the contralateral sensorimotor cortex, showed a significantly greater number of distal CST axons only in OEG-NT-3-transplanted rats. Behavioural analysis was performed on all rats using open field locomotion scoring, and a forelimb reaching task with Eshkol-Wachman movement notation. Analysis of behavioural tests revealed no significant differences in recovery between experimental groups, although movement analysis indicated that possible compensatory mechanisms were occurring after OEG implantation. The results demonstrate that OEG transplantation per se can promote tissue sparing after injury, but, after appropriate genetic modification, these olfactory-derived cells become far more effective in promoting long-distance maintenance/regeneration of lesioned adult CST axons.

Olfactory ensheathing glia transplantation: a therapy to promote repair in the mammalian central nervous system.

A therapy to treat injuries to the central nervous system (CNS) is, to date, a major clinical challenge. The devastating functional consequences they cause in human patients have encouraged many scientists to search, in animal models, for a repair strategy that could, in the future, be applied to humans. However, although several experimental approaches have obtained some degree of success, very few have been translated into clinical trials. Traumatic and demyelinating lesions of the spinal cord have attracted several groups with the same aim: to find a way to promote axonal regeneration, remyelination, and functional recovery, by using a simple, safe, effective, and viable procedure. During the past decade, olfactory ensheathing glia (OEG) transplantation has emerged as a very promising experimental therapy to promote repair of spinal cords, after different types of injuries. Transplants of these cells promoted axonal regeneration and functional recovery after partial and complete spinal cord lesions. Moreover, olfactory ensheathing glia were able to form myelin sheaths around demyelinated axons. In this article, we review these recent advances and discuss to what extent olfactory ensheathing glia transplantation might have a future as a therapy for different spinal cord affections in humans.

Transplantation strategies to promote repair of the injured spinal cord

This review describes the results of the transplantation of Schwann cells and olfactory ensheathing glia in combination with other interventions. The complete transection injury model was used to test the combination of Schwann cell bridges with methylprednisolone, neurotrophins, or olfactory ensheathing glia. The contusion injury model was used to compare Schwann cell and olfactory ensheathing glia transplantation and to examine the results of combining Schwann cell transplants with elevated levels of cyclic adenosine monophosphate. The combination strategies were more effective than cell transplantation alone. The improved regeneration response usually involved a reduction in secondary tissue loss, axonal regeneration from brainstem neurons, an increase in myelinated fibers in the transplant, the exit of regenerated fibers from the transplant into the contiguous cord, and an improvement in locomotor function.

Schwann cell but not olfactory ensheathing glia transplants improve hindlimb locomotor performance in the moderately contused adult rat thoracic spinal cord.

Cultured adult rat Schwann cells (SCs) or olfactory ensheathing glia (OEG), or both, were transplanted in the adult Fischer rat thoracic (T9) spinal cord 1 week after a moderate contusion (10 gm, 12.5 mm, NYU impactor). Rats received either a total of 2 x 10(6) cells suspended in culture medium or culture medium only (controls). At 12 weeks after injury, all grafted animals exhibited diminished cavitation. Although in medium-injected rats 33% of spinal tissue within a 5-mm-long segment of cord centered at the injury site was spared, significantly more tissue was spared in SC (51%), OEG (43%), and SC/OEG (44%) grafted animals. All three types of glial grafts were filled with axons, primarily of spinal origin. SC grafts contained more myelinated axons than SC/OEG and OEG grafts. Both types of SC-containing grafts expressed more intense staining for glial fibrillary acidic protein and chondroitin sulfate proteoglycan compared with OEG-only grafts. Retrograde tracing demonstrated that the number of propriospinal and brainstem axons reaching 5-6 mm beyond the grafted area was significantly higher with SC and SC/OEG grafts but not with OEG-only grafts compared with controls. Corticospinal fibers terminated closer to the lesion epicenter in all grafted animals than in controls. With SC-only grafts, a modest but statistically significant improvement in hindlimb locomotor performance was detected at 8-11 weeks after injury. Thus, in addition to this functional improvement, our results show that an SC graft is more effective in promoting axonal sparing/regeneration than an SC/OEG or OEG graft in the moderately contused adult rat thoracic spinal cord.

Chapter 19 Bridging the transected or contused adult rat spinal cord with Schwann cell and olfactory ensheathing glia transplants

Publisher Summary This chapter compares new findings from studies of cell transplantation in a rat contusion model with those utilizing a complete transection/cellular bridge paradigm. In both cases, Schwann cells (SCs) support substantial axonal regeneration and myelination in the graft. In neither case, brainstem or cortical axons appear to enter the graft. Studying transplantation in a contusion model is clinically relevant; however, there is the challenge of distinguishing spared and sprouted fibers from fibers that have regenerated. Therefore, the chapter uses a complete transection model to obtain unambiguous results by placing a cellular bridge between the stumps of completely transected adult Fischer rat thoracic spinal cord.

Functional Recovery of Paraplegic Rats and Motor Axon Regeneration in Their Spinal Cords by Olfactory Ensheathing Glia

Axonal regeneration in the lesioned mammalian central nervous system is abortive, and this causes permanent disabilities in individuals with spinal cord injuries. In adult rats, olfactory ensheathing glia (OEG) transplants successfully led to functional and structural recovery after complete spinal cord transection. From 3 to 7 months post surgery, all OEG-transplanted animals recovered locomotor functions and sensorimotor reflexes. They presented voluntary hindlimb movements, they supported their body weight, and their hindlimbs responded to light skin contact and proprioceptive stimuli. In addition, relevant motor axons (corticospinal, raphespinal, and coeruleospinal) regenerated for long distances within caudal cord stumps. Therefore, OEG transplantation provides a useful repair strategy in adult mammals with traumatic spinal cord injuries. Our results with these cells could lead to new therapies for the treatment of spinal cord lesions in humans.

Olfactory ensheathing glia transplantation into the injured spinal cord

Olfactory ensheathing glia transplantation into the injured spinal cord

Long-Distance Axonal Regeneration in the Transected Adult Rat Spinal Cord Is Promoted by Olfactory Ensheathing Glia Transplants

The lack of axonal regeneration in the injured adult mammalian spinal cord leads to permanent functional impairment. To induce axonal regeneration in the transected adult rat spinal cord, we have used the axonal growth-promoting properties of adult olfactory bulb ensheathing glia (EG). Schwann cell (SC)-filled guidance channels were grafted to bridge both cord stumps, and suspensions of pure (98%) Hoechst-labeled EG were stereotaxically injected into the midline of both stumps, 1 mm from the edges of the channel. In EG-transplanted animals, numerous neurofilament-, GAP-43-, anti-calcitonin gene-related peptide (CGRP)-, and serotonin-immunoreactive fibers traversed the glial scars formed at both cord-graft interfaces. Supraspinal serotonergic axons crossed the transection gap through connective tissue bridges formed on the exterior of the channels, avoiding the channel interior. Strikingly, after crossing the distal glial scar, these fibers elongated in white and periaqueductal gray matter, reaching the farthest distance analyzed (1.5 cm). Tracer-labeled axons present in SC grafts were found to extend across the distal interface and up to 800 microm beyond in the distal cord. Long-distance regeneration (at least 2.5 cm) of injured ascending propriospinal axons was observed in the rostral spinal cord. Transplanted EG migrated longitudinally and laterally from the injection sites, reaching the farthest distance analyzed (1.5 cm). They moved through white matter tracts, gray matter, and glial scars, overcoming the inhibitory nature of the CNS environment, and invaded SC and connective tissue bridges and the dorsal and ventral roots adjacent to the transection site. Transplanted EG and regenerating axons were found in the same locations. Because EG seem to provide injured spinal axons with appropriate factors for long-distance elongation, these cells offer new possibilities for treatment of CNS conditions that require axonal regeneration.