Geoffrey Raisman

Transplantation of autologous olfactory ensheathing cells in complete human spinal cord injury.

Numerous studies in animals have shown the unique property of olfactory ensheathing cells to stimulate regeneration of lesioned axons in the spinal cord. In a Phase I clinical trial, we assessed the safety and feasibility of transplantation of autologous mucosal olfactory ensheathing cells and olfactory nerve fibroblasts in patients with complete spinal cord injury. Six patients with chronic thoracic paraplegia (American Spinal Injury Association class A-ASIA A) were enrolled for the study. Three patients were operated, and three served as a control group. The trial protocol consisted of pre- and postoperative neurorehabilitation, olfactory mucosal biopsy, culture of olfactory ensheathing cells, and intraspinal cell grafting. Patients clinical state was evaluated by clinical, neurophysiological, and radiological tests. There were no adverse findings related to olfactory mucosa biopsy or transplantation of olfactory ensheathing cells at 1 year after surgery. There was no evidence of neurological deterioration, neuropathic pain, neuroinfection, or tumorigenesis. In one cell-grafted patient, an asymptomatic syringomyelia was observed. Neurological improvement was observed only in transplant recipients. The first two operated patients improved from ASIA A to ASIA C and ASIA B. Diffusion tensor imaging showed restitution of continuity of some white matter tracts throughout the focus of spinal cord injury in these patients. The third operated patient, although remaining ASIA A, showed improved motor and sensory function of the first spinal cords segments below the level of injury. Neurophysiological examinations showed improvement in spinal cord transmission and activity of lower extremity muscles in surgically treated patients but not in patients receiving only neurorehabilitation. Observations at 1 year indicate that the obtaining, culture, and intraspinal transplantation of autologous olfactory ensheathing cells were safe and feasible. The significance of the neurological improvement in the transplant recipients and the extent to which the cell transplants contributed to it will require larger numbers of patients.

Functional regeneration of supraspinal connections in a patient with transected spinal cord following transplantation of bulbar olfactory ensheathing cells with peripheral nerve bridging.

Treatment of patients sustaining a complete spinal cord injury remains an unsolved clinical problem because of the lack of spontaneous regeneration of injured central axons. A 38-year-old man sustained traumatic transection of the thoracic spinal cord at upper vertebral level Th9. At 21 months after injury, the patient presented symptoms of a clinically complete spinal cord injury (American Spinal Injury Association class A-ASIA A). One of the patients olfactory bulbs was removed and used to derive a culture containing olfactory ensheathing cells and olfactory nerve fibroblasts. Following resection of the glial scar, the cultured cells were transplanted into the spinal cord stumps above and below the injury and the 8-mm gap bridged by four strips of autologous sural nerve. The patient underwent an intense pre- and postoperative neurorehabilitation program. No adverse effects were seen at 19 months postoperatively, and unexpectedly, the removal of the olfactory bulb did not lead to persistent unilateral anosmia. The patient improved from ASIA A to ASIA C. There was improved trunk stability, partial recovery of the voluntary movements of the lower extremities, and an increase of the muscle mass in the left thigh, as well as partial recovery of superficial and deep sensation. There was also some indication of improved visceral sensation and improved vascular autoregulation in the left lower limb. The pattern of recovery suggests functional regeneration of both efferent and afferent long-distance fibers. Imaging confirmed that the grafts had bridged the left side of the spinal cord, where the majority of the nerve grafts were implanted, and neurophysiological examinations confirmed the restitution of the integrity of the corticospinal tracts and the voluntary character of recorded muscle contractions. To our knowledge, this is the first clinical indication of the beneficial effects of transplanted autologous bulbar cells.

Structural basis of glaucoma: The fortified astrocytes of the optic nerve head are the target of raised intraocular pressure

Increased intraocular pressure (IOP) damages the retinal ganglion cell axons as they pass through the optic nerve head (ONH). The massive connective tissue structure of the human lamina cribrosa is generally assumed to be the pressure transducer responsible for the damage. The rat, however, with no lamina cribrosa, suffers the same glaucomatous response to raised IOP. Here, we show that the astrocytes of the rat ONH are “fortified” by extraordinarily dense cytoskeletal filaments that would make them ideal transducers of distorting mechanical forces. The ONH astrocytes are arranged as a fan‐like radial array, firmly attached ventrally to the sheath of the ONH by thick basal processes, but dividing dorsally into progressively more slender processes with only delicate attachments to the sheath. At 1 week after raising the IOP by an injection of magnetic microspheres into the anterior eye chamber, the fine dorsal processes of the ONH astrocytes are torn away from the surrounding sheath. There is no indication of distortion or compression of the axons. Subsequently, despite return of the IOP toward normal levels, the damage to the ONH progresses ventrally through the astrocytic cell bodies, resulting in complete loss of the fortified astrocytes and of the majority of the axons by around 4 weeks. We propose that the dorsal attachments of the astrocytes are the site of initial damage in glaucoma, and that the damage to the axons is not mechanical, but is a consequence oflocalized loss of metabolic support from the astrocytes (Tsacopoulos and Magistretti (1996) J Neurosci 16:877–885).

Transplanted olfactory mucosal cells restore paw reaching function without regeneration of severed corticospinal tract fibres across the lesion

Previous studies from our laboratory reported that transplantation of a mixture of 50% p75+ olfactory ensheathing cells (OECs) and fibroblasts derived from the outer layers of the adult olfactory bulb into unilateral lesions of the rat corticospinal tract (CST) restore function in a directed fore-paw retrieval task and induce regeneration of severed CST axons across the lesion. For future clinical application it would be preferable to obtain reparative cells from an olfactory mucosal biopsy via intranasal endoscopy rather than requiring the more invasive intracranial approach to remove an olfactory bulb. With this purpose, we used our original CST lesion paradigm to examine whether mucosal OEC preparations can provide a similar repair to those from the bulb. We found that, as in the case of bulbar OEC preparations, the mucosal cells also restored directed fore-paw retrieval. Surprisingly, however, there was no evidence of any of the severed CST axons crossing the lesion site, suggesting that the recovery of function is due to some other reaction, such as sprouting of damaged or undamaged fibres. Compared with the previous findings with bulbar cells, the mucosal cell cultures contained only 5% of OECs and a conversely much larger proportion of fibroblasts. These cell preparations showed minimal migratory ability and failed to form complete bridges across the lesions.

Restoration of hand function in a rat model of repair of brachial plexus injury

The incurability of spinal cord injury and subcortical strokes is due to the inability of nerve fibres to regenerate. One of the clearest clinical situations where failure of regeneration leads to a permanent functional deficit is avulsion of the brachial plexus. In current practice, surgical re-implantation of avulsed spinal roots provides a degree of motor recovery, but the patients neither recover sensation nor the use of the hand. In the present rat study, we show that transplantation of cultured adult olfactory ensheathing cells restores the sensory input needed for a complex, goal-directed fore-paw function and re-establishes synaptic transmission to the spinal grey matter and cuneate nucleus by providing a bridge for regeneration of severed dorsal root fibres into the spinal cord. Success in a first application of human olfactory ensheathing cells in clinical brachial plexus injury would open the way to the wider field of brain and spinal cord injuries.

Transplanted olfactory ensheathing cells incorporated into the optic nerve head ensheathe retinal ganglion cell axons: Possible relevance to glaucoma

A mixture of olfactory ensheathing cells and fibroblasts cultured from the adult rat olfactory mucosa was transplanted through a scleral incision into the retina. A major stream of transplanted cells migrated through the stratum opticum and penetrated for up to about 0.5mm into the optic nerve head. This stream of transplanted cells consisted of a mixture of bipolar olfactory ensheathing cells with long processes which give rise to a non-myelinating ensheathment of single retinal ganglion cell axons, and olfactory nerve fibroblasts embedded in a dense fibronectin-positive extracellular matrix. A second stream of ovoid olfactory ensheathing cells with tufted processes and unaccompanied by fibroblasts or matrix migrate into the internal plexiform layer. The incorporation of olfactory ensheathing cells in the optic nerve head may suggest future possibilities for protection of the axons in this vulnerable region from mechanical damage, as in the raised intraocular pressure of glaucoma.

Novel strategies in brachial plexus repair after traumatic avulsion

Clinical trials in spinal cord injury (SCI) can be affected by many confounding variables including spontaneous recovery, variation in the lesion type and extend. However, the clinical need and the paucity of effective therapies has spawned a large number of animal studies and clinical trials for SCI. In this review, we suggest that brachial plexus avulsion injury, a longitudinal spinal cord lesion, is a simpler model to test methods of spinal cord repair. We explore reconstructive techniques currently explored for the repair of brachial plexus avulsion and focus on the use of olfactory ensheathing cell transplantation as an adjunct treatment in brachial plexus repair.

Comparison of Bulbar and Mucosal Olfactory Ensheathing Cells Using FACS and Simultaneous Antigenic Bivariate Cell Cycle Analysis

Transplantation of olfactory ensheathing cells (OECs) is a promising route for CNS repair. There have, however, been major discrepancies between the results from different groups. Part of this can be attributed to variations in cell sources and culture protocols. Accurate estimation of the proportions of OECs and their associated fibroblasts (ONFs) and their evolution with time in culture is an essential baseline for establishing the reparative properties of transplants. In this study, we compare the evolution of cultures from the superficial layers of the olfactory bulb with tissue from the olfactory mucosa, both whole and split into lamina propria and epithelial layer. We used FACS based on p75 and Thy1 to provide a robust and objective numerical estimate of the numbers of OECs and ONFs, respectively in the cultures. A novel four color simultaneous antigenic bivariate cell cycle analysis shows that proliferation of OECs is time‐limited, and is unable to prevent an overall loss of OECs with time. Overall, the numbers of OECs in the cultures were inversely correlated with the deposition of fibronectin (FN). Further, culture of the cells purified by flow cytometry shows that, whereas the Thy1 population is terminally differentiated, the p75 population from the mucosal samples generates subpopulations with different antigenic phenotypes, including the reappearance of a subpopulation of p75 cells expressing FN. Culturing epithelial samples at high density reveals an unexpected transient stem cell‐like population of rapidly proliferating p75 positive cells.

Clinical prospects for transplantation of OECs in the repair of brachial and lumbosacral plexus injuries: Opening a door

The reparative effects of olfactory ensheathing cells have largely been examined in lesions entirely within the CNS. There is, however, evidence that they can induce the ingrowth of severed dorsal root axons and increase the outgrowth of severed ventral root axons. The ingrowth of dorsal root axons results in reinnervation of appropriate regions in the spinal cord and dorsal column nuclei with restoration of electrical transmission and muscular control. This article discusses the further possibilities of these observations in rat studies and their potential translation to clinical injuries. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Comparison of olfactory bulbar and mucosal cultures in a rat rhizotomy model.

In an ongoing clinical trial, a spinal injured patient who received a transplant of autologous cells cultured from the olfactory bulb is showing greater functional benefit than three previous patients with transplants of mucosal origin. Previous laboratory studies of transplantation into rat spinal cord injuries show that the superior reparative benefits of bulbar over mucosal cultures are associated with regeneration of severed corticospinal tract fibers over a bridge of olfactory ensheathing cells (OECs) formed across the injury site. In a rat rhizotomy paradigm, we reported that transplantation of bulbar cell cultures also enables severed axons of the C6–T1 dorsal roots to regenerate across a bridge of OECs into the spinal cord and restore electrophysiological transmission and forepaw grasping during a climbing test. We now report a repeat of the same rhizotomy procedure in 25 rats receiving cells cultured from olfactory mucosal biopsies. In no case did the transplanted cells form a bridging pathway. No axons crossed from the severed roots to the spinal cord, and there was no restoration of forepaw grasping. This suggests that the superior clinical benefit in the patient receiving bulbar cell transplants is due to regeneration of severed fibers across the injury site, and this correlates with imaging and the pattern of functional recovery. Using present culture protocols, the yield of OECs from bulbar biopsies is around 50%, but that from mucosal biopsies is less than 5%. Improving the yield of OECs from mucosal biopsies might avoid the necessity for the intracranial approach to obtain bulbar cells.