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ABSTRACT: BACKGROUND:: Autotransplantation of olfactory ensheathing cells (OECs) into the damaged central nervous system is a potential therapeutic strategy for spinal cord and root cord injuries. One limiting factor has been the poor OEC yields from human mucosal biopsies. Previous studies have only commented on their success in obtaining mucosal specimens containing olfactory mucosa, but have not commented on the yield of OECs from those specimens. OBJECTIVE:: To describe a reproducible and safe surgical technique for obtaining human olfactory mucosa and identify patient factors that possibly affect the yield of OEC cultures from the human olfactory mucosa. METHODS:: We obtained mucosal biopsies from 43 consecutive patients using a novel reproducible surgical technique and our laboratory culture protocol. Spearman's rank correlation coefficient was used to assess the relationship between OECs and fibroblast yield with patient characteristics and specimen factors. RESULTS:: A greater yield of OECs was obtained from patients of younger age. In addition, patients with worse mucosal disease yielded poorer cell cultures. Greatest yields were found in patients with absence of mucosal disease. Furthermore, a higher yield of OECs was obtained from specimens harvested from the more caudal portions of the superior turbinate, and OEC yield did not correlate with the ventro-posterior location of the biopsy. CONCLUSION:: We have provided evidence that biopsies closer to the cribriform plate can produce larger yields of OECs, and that patient factors like age and mucosal disease adversely affect the culture yield.
Neurosurgery 11/2012; · 2.79 Impact Factor
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ABSTRACT: As a preliminary to the construction of olfactory ensheathing cells (OECs) bearing scaffold for bridging larger lesions in the spinal cord, we have investigated the response of purified cultured OECs to nanoscale fibers of varying diameter using US FDA-approved, biodegradable poly(lactic-co-glycolic-acid).
Conventional electrospinning produced fibers of approximately 700 nm diameter (nano-700) while nanocomposite electrospinning with quantum dots produced significantly more uniform fibers of a reduced diameter to approximately 237 nm (nano-250). OECs from adult rat were FACS purified, cultured at low density on either a flat surface or a meshwork of randomly orientated nano-700 and nano-250 fibers, and assessed using cytomorphometric analysis of immunofluorescent confocal images and by scanning electron microscopy.
Compared with a flat surface, culture on a nano-700 mesh increases cell attachment. Cells change from rounded to stellate forms in random orientation. Further size reduction to the nano-250 favors bipolarity in cells with unidirectional orientation as observed in the case when transplanted OECs were used to bridge areas of damage in rat spinal cords.
Nanomedicine 05/2012; 7(8):1211-24. · 5.05 Impact Factor
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ABSTRACT: We propose that severed adult CNS axons are intrinsically capable of regeneration and reestablishing lost functions and that the key to repair lies in reconfiguring the scarring response of the astrocytic network. Astrocytes are multifunctional cells with three distinct surfaces: a glia to glial surface, providing the junctions needed to incorporate the astrocytes into the network; a glia to mesodermal surface, at which astrocytes collaborate with the meningeal fibroblasts to maintain the protective covering of the CNS; and a glia to neuronal surface, which provides the routes along which axons travel. After injury, the astrocytes collaborate with the meningeal fibroblasts to form a scar, which provides the necessary defensive sealing of the opened surface of the CNS, but which also has the detrimental effect of closing off the pathways along which axons could regenerate. Incorporation of glial cells transplanted from the olfactory system into a CNS injury causes a re-arrangement of the scarred astrocyte/fibroblast complex so as to produce the alignment of the glia to neuronal surfaces needed to provide a pathway for the regeneration of severed axons. Olfactory ensheathing cells certainly have a direct stimulatory effect on axons, but without concomitant reorganization of the glial scar, this could not in itself lead to regeneration of severed axons to their targets.
Progress in brain research 01/2012; 201:199-218. · 3.04 Impact Factor
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ABSTRACT: 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).
Glia 09/2011; 60(1):13-28. · 4.82 Impact Factor
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ABSTRACT: 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.
Glia 07/2011; 59(11):1658-71. · 4.82 Impact Factor
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ABSTRACT: 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.
British Journal of Neurosurgery 10/2010; 25(1):16-27. · 0.88 Impact Factor
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ABSTRACT: Transplants of olfactory ensheathing cells (OECs) cultured from the olfactory bulb are able to induce structural regeneration of severed central axons and return of function in rat models. For clinical purposes it would be preferable to obtain the cells from the more accessible olfactory mucosa in the nasal lining. However, in our laboratory preparations cultures from mucosal samples yielded around 5% of OECs compared with the 50% obtained from samples cultured from the bulb, and when transplanted these mucosal cell preparations were less effective at repair. There are a number of manipulations which may increase the OEC content and the effectiveness of mucosal preparations, but in vivo transplantation would be a highly labour intensive method for evaluating them. As a candidate for a high throughput assay to screen for beneficial effects of modifications to mucosal cells we here report the effects of co-culture of the cells with retinal explants. Both bulbar and mucosal cell preparations prolong the survival of the explants. Counts of the surviving retinal ganglion cells, identified by beta-III-tubulin immunohistochemistry and by their axon trajectory, show that the bulbar cell preparations have around twice the potency of those from the mucosa. This in vitro system, therefore, provides a bioassay that discriminates bulbar and mucosal cell preparations, and a useful tool for evaluating the functional effects of manipulations of cultured mucosal preparations.
Brain research 10/2010; 1354:40-6. · 2.46 Impact Factor
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ABSTRACT: The failure of cut axons to grow along fibre tracts in the adult CNS contrasts with their ability to do so in development. Organotypic slices culture of a number of areas enables the time of failure to be pinpointed to around the second week of postnatal life in the rat. 'Heterochronic' co-culture of slices above and below this age shows that the failure is due to the inability of the older axons to grow into either the same age or younger targets. Using hippocampo-septal slices the present experiments show that this failure is due to an inability to recognise the glial pathway of the fimbria, even when this is of a younger age. However, the older hippocampal neurons retain the ability to grow axons into septal target tissue when they are placed in direct contact with it. This exactly mirrors the inability of cut central axons to regenerate along their previous fibre pathways while they retain their ability to reinnervate neuropil.
European Journal of Neuroscience 04/2010; 31(8):1352-8. · 3.63 Impact Factor
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ABSTRACT: 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.
Brain research 09/2009; 1303:26-31. · 2.46 Impact Factor
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ABSTRACT: 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.
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.
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 patient's age.
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.
Neurosurgery 06/2008; 62(5):1140-4; discussion 1144-5. · 2.79 Impact Factor
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ABSTRACT: 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.
Glia 03/2007; 55(3):312-6. · 4.82 Impact Factor
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ABSTRACT: 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.
The Lancet Neurology 06/2006; 5(5):453-7. · 23.46 Impact Factor
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ABSTRACT: Transplantation of cultured adult olfactory ensheathing cells has been shown to induce anatomical and functional repair of lesions of the adult rat spinal cord and spinal roots. Histological analysis of olfactory ensheathing cells, both in their normal location in the olfactory nerves and also after transplantation into spinal cord lesions, shows that they provide channels for the growth of regenerating nerve fibres. These channels have an outer, basal lamina-lined surface apposed by fibroblasts, and an inner, naked surface in contact with the nerve fibres. A crucial property of olfactory ensheathing cells, in which they differ from Schwann cells, is their superior ability to interact with astrocytes. When confronted with olfactory ensheathing cells the superficial astrocytic processes, which form the glial scar after lesions, change their configuration so that their outer pial surfaces are reflected in continuity with the outer surfaces of the olfactory ensheathing cells. The effect is to open a door into the central nervous system. We propose that this formation of a bridging pathway may be the crucial event by which transplanted olfactory ensheathing cells allow the innate growth capacity of severed adult axons to be translated into regeneration across a lesion so that functionally valuable connections can be established.
Journal of Neurocytology 10/2005; 34(3-5):343-51. · 1.94 Impact Factor
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ABSTRACT: Transplantation of olfactory ensheathing cells into spinal cord lesions promotes regeneration of cut axons into terminal fields and functional recovery. This repair involves the formation of a peripheral nerve-like bridge in which perineurial-like fibroblasts are organized into a longitudinal stack of parallel tubular channels, some of which contain regenerating axons enwrapped by Schwann-like olfactory ensheathing cells. The present study examines whether cut retinal ganglion cell axons will also respond to these cells, and if so, whether they form the same type of arrangement. In adult rats, the optic nerve was completely severed behind the optic disc, and a matrix containing cultured olfactory ensheathing cells was inserted between the proximal and distal stumps. After 6 months, the transplanted cells had migrated for up to 10 mm into the distal stump. Anterograde labeling with cholera toxin B showed that cut retinal ganglion cell axons had regenerated through the transplants, entered the distal stump, and elongated for 10 mm together with the transplanted cells. Electron microscopy showed that a peripheral nerve-like tissue had been formed, similar to that seen in the spinal cord transplants. However, in contrast to the spinal cord, the axons did not reach the terminal fields, but terminated in large vesicle-filled expansions beyond which the distal optic nerve stump was reduced to a densely interwoven mass of astrocytic processes.
Journal of Neuroscience 09/2003; 23(21):7783-8. · 7.11 Impact Factor
