Daqing Li

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.

Olfactory ensheathing cells: ripples of an incoming tide?

Until now, brain and spinal cord injuries that sever nerve fibres have resulted in a degree of incurable functional loss. An incoming tide of research is now beginning to challenge this as yet unbreached sea wall. One of the most promising approaches involves a recently discovered type of cell, the olfactory ensheathing cell, which can be obtained from the adult nasal lining. In animal models transplantation of cultured olfactory ensheathing cells into an injured spinal cord induces regeneration, remyelination of severed spinal nerve fibres, and functional recovery. Although several clinical centres worldwide have shown an interest in applying this approach to patients with spinal cord injury, there is no agreement on cell technology, and claims of beneficial results lack independent confirmation. Important aspects still need to be worked out at the laboratory level. Overall, the outlook is optimistic, but there is still some way to go.

Transplanted Schwann cells, not olfactory ensheathing cells, myelinate optic nerve fibres

In a previous study we found that olfactory ensheathing cells transplanted into complete retrobulbar transections of the rat optic nerve mediated regeneration of severed retinal ganglion cell axons through the graft region. Although the regenerating axons were ensheathed by the transplanted cells, none of the regenerating axons became myelinated by either central or peripheral type myelin. In the present study we used the same operative procedure but transplanted Schwann cells instead of olfactory ensheathing cells. As with the olfactory ensheathing cell transplants the Schwann cells transplants also induced regeneration of the severed retinal ganglion cell axons into the graft region. In contrast to the situation with the olfactory ensheathing cell transplants, however, a considerable number of the regenerating axons became myelinated by peripheral type myelin produced by the transplanted Schwann cells. This observation identifies a further distinction between these two cell types which are phenotypically similar in many ways, but which have been shown to have major functional differences with regard to regeneration in spinal cord lesions.

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.

A prospective observational study of the yield of olfactory ensheathing cells cultured from biopsies of septal nasal mucosa

OBJECTIVE We sought to study the yield of olfactory ensheathing cells from biopsies of the mucosa of the nasal septum. These specialized cells encourage regeneration of nerves of the central nervous system and may be of value for spinal cord and nerve injuries. METHODS We undertook a prospective observational study of biopsies of nasal mucosa by endonasal dissection of the mucosa of the nasal septum during the approach for routine transsphenoidal surgeries. Samples were cultured in the laboratory, and the yield of olfactory ensheathing cells was compared as to the location, size, and weight of the biopsies and the age of the patients. RESULTS A better yield of olfactory ensheathing cells was obtained from areas of the septum that were more superior and posterior in position. The yield was not related to the size of the biopsy or the patients age. CONCLUSION Septal mucosa is a possible source of olfactory ensheathing cells, although the yield may be smaller than that which may be obtained from mucosa of the lateral nasal cavity and superior turbinate.

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.

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.