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ABSTRACT: Over the past few years, the idea of using intraspinal implantations of olfactory ensheathing cells (OECs) as a therapeutic strategy to enhance recovery after spinal cord injury has quickly moved from experimentation with laboratory mammals to surgical approaches for paralyzed humans. Despite this progression, several important issues have yet to be thoroughly addressed: for instance, which of the many methods currently being used best yields enriched populations of OECs, and how such purity can be empirically tested and validated among different mammalian species, including humans. Here we offer an authoritative review of those methods used to isolate OECs from the olfactory mucosa and/or olfactory bulbs of rats, mice, dogs, pigs, non-human primates, and humans. As well, we assess which biomarkers are currently being utilized to determine the relative proportions of OECs and contaminating cells in these glial cultures. Although there have been numerous review articles regarding OECs in vitro, our review is unique in that it offers a critical assessment of the methods currently being used to generate cultures of mammalian OECs. More specifically, we examine the issue of culture contamination by phenotypically similar Schwann cells. This review is timely because recent clinical usage of OECs has come under intense criticism for a number of reasons, including the reliable identification of cultured human OECs. We believe that once these methodological issues of isolation and characterization of OECs have been resolved, this glial population will offer paralyzed individuals a truly viable cellular strategy for intraspinal therapy.
Journal of neurotrauma 02/2009; 26(2):155-77. · 4.25 Impact Factor
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ABSTRACT: 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 alpha-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 alpha-actin-expressing OECs are capable of facilitating axon remyelination and regeneration.
The Journal of Comparative Neurology 08/2007; 503(2):209-23. · 3.81 Impact Factor
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ABSTRACT: Under normal conditions, expression of the p75 neurotrophin receptor (p75NTR) by sympathetic neurons can increase the affinity of the signaling receptor, trkA, to target-derived nerve growth factor (NGF) at distal axons. We have previously reported that sprouting of sympathetic axons into NGF-rich target tissues is enhanced when p75NTR expression is perturbed, leading to the postulate that p75NTR may restrain sympathetic sprouting in response to elevated NGF levels. These observations were made using mice having a null mutation of the third p75NTR exon, a line that may express a hypomorphic form of this receptor. Since mice carrying a null mutation of the fourth p75NTR exon may not express a similar splice variant, we sought to determine whether these animals possess the same phenotype of enhanced sympathetic sprouting in response to elevated levels of NGF. Both lines of transgenic mice lacking p75NTR displayed similar degrees of sympathetic axonal sprouting into the cerebellum and trigeminal ganglia, two target tissues having elevated levels of NGF protein. Furthermore, the densities of sympathetic axons in both targets were significantly greater than those observed in age-matched NGF transgenic siblings expressing full-length p75NTR. Our new findings provide a comparative analysis of the phenotype in two independent mutations of the same neurotrophin receptor, revealing that p75NTR plays an important role in restricting sympathetic sprouting in response to higher NGF levels.
Experimental Neurology 05/2006; 198(2):416-26. · 4.70 Impact Factor
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ABSTRACT: 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.
Glia 04/2006; 53(4):434-40. · 4.82 Impact Factor
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ABSTRACT: Post-ganglionic sympathetic neurons express the p75 neurotrophin receptor (p75NTR) and brain-derived neurotrophic factor (BDNF), which together have been implicated in controlling the degree of efferent innervation of peripheral organs [Kohn, J., Aloyz, R.S., Toma, J.G., Haak-Frendscho, M., Miller, F.D. 1999. Functionally Antagonistic Interactions between the TrkA and p75 Neurotrophin Receptors Regulate Sympathetic Neuron Growth and Target Innervation. J. Neurosci. 19, 5393-5408]. To examine this concept further, we developed null mutant mice lacking both p75NTR and BDNF, and assessed whether the loss of this receptor-ligand interaction negatively impacts the degree of sympathetic innervation to various target tissues. Between postnatal days 10 and 14, hearts, urinary bladders, kidneys, and submandibular salivary glands were isolated from p75(-/-)/BDNF-/-, p75-/-, BDNF-/-, and wild type siblings. Sympathetic axons were visualized using tyrosine hydroxylase (TH) immunohistochemistry, and TH protein levels were quantified by immunoblotting. Concerning the sympathetic innervation of the heart, urinary bladder and kidneys, no differences were seen in single and double null mutant mice, as compared with their wild type siblings. Sympathetic innervation of the submandibular salivary gland was, however, increased in both p75-/- and p75(-/-)/BDNF-/- mice over control mice. These results reveal that an absence of p75NTR and/or BDNF expression does not perturb the degree of sympathetic innervation of many peripheral tissues during postnatal development, and that a lack of p75NTR expression may actually enhance the density of these efferent fibers in other target tissues, such as the salivary glands.
Autonomic Neuroscience 04/2005; 118(1-2):32-42. · 1.86 Impact Factor
