R. Doucette

Defining the role of olfactory ensheathing cells in facilitating axon remyelination following damage to the spinal cord

Olfactory ensheathing cells (OECs) are unique cells that are responsible for the successful regeneration of olfactory axons throughout the life of adult mammals. More than a decade of research has shown that implantation of OECs may be a promising therapy for damage to the nervous system, including spinal cord injury. Based on this research, several clinical trials worldwide have been initiated that use autologous transplantation of olfactory tissue containing OECs into the damaged spinal cord of humans. However, research from several laboratories has challenged the widely held belief that OECs are directly responsible for myelinating axons and promoting axon regeneration. The purpose of this review is to provide a working hypothesis that integrates several current ideas regarding the mechanisms of the beneficial effects of OECs. Specifically, OECs promote axon regeneration and functional recovery indirectly by augmenting the endogenous capacity of host Schwann cells to invade the damaged spinal cord. Together with Schwann cells, OECs create a 3‐dimensional matrix that provides a permissive microenvironment for successful axon regeneration in the adult mammalian central nervous system.—Boyd, J. G., Doucette, R., Kawaja, M. D. Defining the role of olfactory ensheathing cells in facilitating axon remyelination following damage to the spinal cord. FASEB J. 19, 694–703 (2005)

LacZ-expressing olfactory ensheathing cells do not associate with myelinated axons after implantation into the compressed spinal cord

Studies have shown that implanting olfactory ensheathing cells (OECs) may be a promising therapeutic strategy to promote functional recovery after spinal cord injury. Several fundamental questions remain, however, regarding their in vivo interactions in the damaged spinal cord. We have induced a clip compression injury at the T10 level of the spinal cord in adult rats. After a delay of 1 week, OECs isolated from embryonic day 18 rats were implanted into the cystic cavity that had formed at the site of injury. Before implantation, OECs were infected with a LacZ-expressing retrovirus. At 3 weeks after implantation, LacZ-expressing OECs survived the implantation procedure and remained localized to the cystic cavity. At the electron microscopic level, the cystic cavity had clusters of LacZ-expressing OECs and numerous Schwann cells lacking LacZ expression. Although labeled OECs made no direct contact with axons, unlabeled Schwann cells were associated with either a single myelinated axon or multiple unmyelinated axons. Positively labeled OEC processes often enveloped multiple Schwann cell-axon units. These observations suggest that the role of OECs as the primary mediators of the beneficial effects on axon growth, myelination, and functional recovery after spinal cord injury may require re-evaluation.

Proteomic evaluation reveals that olfactory ensheathing cells but not Schwann cells express calponin.

Human clinical trials have begun worldwide that use olfactory ensheathing cells (OECs) to ameliorate the functional deficits following spinal cord injury. These trials have been initiated largely because numerous studies have reported that OECs transform into Schwann Cell (SC)‐like cells that myelinate axons and support new growth in adult rats with spinal injury. This phenomenon is remarkable because OECs do not myelinate olfactory axons in their native environment. Furthermore, these myelinating OECs are morphologically identical to SCs, which can invade the spinal cord after injury. One factor that has contributed to a possible confusion in the identification of these cells is the lack of phenotypic markers to distinguish unequivocally between OECs and SCs. Such markers are required to first assess the degree of SC contamination in OEC cultures before intraspinal implantation, and then to accurately identify grafted OECs and invading SCs in the injured spinal cord. Using two‐dimensional gel electrophoresis, we have identified calponin, an actin binding protein, as the first definitive phenotypic marker that distinguishes between OECs and SCs in vitro and in vivo. We have also provided ultrastructural evidence that calponin‐immunopositive OECs do not transform into myelinating SC‐like cells after intraspinal implantation. Rather, the grafted OECs retain their morphological and neurochemical features. These data yield new insight into the phenotypic characteristics of OECs, which together with invading SCs can enhance regeneration of the injured spinal cord.

Olfactory ensheathing cells express smooth muscle α-actin in vitro and in vivo

One strategy for spinal cord repair after injury that has moved quickly from the research laboratory to the clinic is the implantation of olfactory ensheathing cells (OECs). These unique glial cells of the olfactory system have been associated with axonal remyelination and regeneration after grafting into spinalized animals. Despite these promising observations, there remains a lack of direct empirical evidence of the exact fate of OECs after intraspinal implantation, in large part because of a surprising paucity of defined biomarkers that unequivocally distinguish these cells from phenotypically similar Schwann cells. Here we provide direct neurochemical proof that OECs, both in vitro and in vivo, express smooth muscle α‐actin. That OECs synthesize this contractile protein (and a variety of actin‐binding proteins including caldesmon) provides compelling evidence that these cells are, in fact, quite different from Schwann cells. The identification of several smooth muscle‐related proteins in OECs points to a new appreciation of the structural and functional features of this population of olfactory glia. These biomarkers can now be used to elucidate the fate of OECs after intraspinal implantation, in particular assessing whether smooth muscle α‐actin‐expressing OECs are capable of facilitating axon remyelination and regeneration. J. Comp. Neurol. 503:209–223, 2007.

Differential expression of Hoxa-2 protein along the dorsal-ventral axis of the developing and adult mouse spinal cord.

We have used synthetic oligopeptides derived from the coding sequence of the murine Hoxa‐2 protein to produce polyclonal antibodies that specifically recognize the Hoxa‐2 recombinant protein. Immunohistochemical studies reveal a distinct pattern of spatial and temporal expression of Hoxa‐2 protein within the mouse spinal cord which is concomitant with the cytoarchitectural changes occurring in the developing cord. Hoxa‐2 protein is predominantly detected in the nuclei of cells in the ventral mantle region of 10‐day‐old mouse embryos. Islet‐1, a marker for motor neurons was also shown to be co‐localized with Hoxa‐2 in nuclei of cells in this region. As development progresses from 10‐days to 14‐days of gestation, Hoxa‐2 protein expression gradually extends to the dorsal regions of the mantle layer. The Hoxa‐2 protein expression pattern changes at 16‐days of embryonic development with strong expression visible throughout the dorsal mantle layer. In 18‐day‐old and adult mouse spinal cords, Hoxa‐2 protein was expressed predominantly by cells of the dorsal horn and only by a few cells of the ventral horn. Double labeling studies with an antibody against glial fibrillary acidic protein (GFAP, an astrocyte‐specific intermediate filament protein) showed that within the adult spinal cord, astrocytes rarely expressed the Hoxa‐2 protein. However, Hoxa‐2 and GFAP double‐labeled astrocytes were found in the neopallial cultures, although not all astrocytes expressed Hoxa‐2. Hoxa‐2 expressing oligodendrocyte progenitor cells were also identified after double‐labeling with O4 and Hoxa‐2 antibodies; although cells in this lineage that have begun to develop a more extensive array of cytoplasmic processes were less likely to be Hoxa‐2 positive. The early pattern of Hoxa‐2 protein expression across transverse sections of the neural tube is temporally and spatially modified as each major class of neuron is generated. This congruence in the expression of the Hoxa‐2 protein and the generation of neurons in the cord suggests that the Hoxa‐2 protein may contribute to dorsal‐ventral patterning and/or to the specification of neuronal phenotype. Dev Dyn 1999;216:201–217.

Sensorimotor cortex aspiration: A model for studying Wallerian degeneration-induced glial reactivity along the entire length of a single CNS axonal pathway

Although there are some similarities in the molecular and cellular pattern of Wallerian degeneration in the PNS and CNS, in the CNS the removal of axonal and myelin debris by microglia and astrocytes is not very efficient and occurs over a much longer time frame than seen in a peripheral nerve. Several animal models have been used to study Wallerian degeneration-induced glial reactivity in the CNS and PNS. Although these models have clarified some aspects of the mechanisms underlying the differential glial cell responses in the PNS and CNS, they do not lend themselves easily to deciphering the mechanisms governing the location and extent of Wallerian degeneration-induced CNS glial reactivity. The present study develops a new animal model that entails destruction of the left sensorimotor cortex of adult rats to induce Wallerian degeneration within the total length of a fiber tract (i.e. the dorsal corticospinal tract) that extends all the way from the cerebral cortex to the sacral level of the spinal cord. Since the axonal degeneration in the ventral medulla and dorsal funiculus of the spinal cord would be confined to the corticospinal tract, it was predicted the glial reactivity would also be restricted to this fiber tract. Three distinct proximal-distal levels of this pathway were examined to determine the morphology, distribution and immunophenotype of microglia and astrocytes between 1 day and 16 weeks after sensorimotor cortex aspiration. As expected, there was a proximal to distal gradient in the appearance of glial reactivity along the length of the pathway, with the microglial reactivity being seen as early as 3 weeks in the left pyramid, and by 4 weeks (i.e. at C6) and 6 weeks (i.e. at T11) in the right dorsal corticospinal tract. Astrocytic reactivity lagged behind that of the microglial response at each level of the pathway. The microglial and astrocytic reactivity persisted up to 16 weeks after cortical injury, which was the longest survival time studied. The sensorimotor cortex aspiration model should prove extremely useful in deciphering the molecular mechanisms controlling Wallerian degeneration-induced CNS glial reactivity and in determining the relative role of astrocytes vs microglia in clearance of axonal and myelin debris.