Confocal imaging of Schwann-cell migration along muscle-vein combined grafts used to bridge nerve defects in the rat

Università degli Studi di Torino, Torino, Piedmont, Italy
Microsurgery (Impact Factor: 2.42). 01/2001; 21(4):153-5. DOI: 10.1002/micr.1029
Source: PubMed

ABSTRACT Schwann cells guide axonal regrowth during peripheral nerve repair. In a case of a nerve lesion with substance loss, a graft conduit is necessary to enable axons to reach the distal nerve stump. If a non-nervous autograft is used, the question arises as to the presence and origin of Schwann cells along the grafted tube. We addressed this issue using a tubulization technique based on the use of an autologous vein filled with fresh skeletal muscle for the repair of sciatic nerve defects in the rat. We showed that both ends of the graft were early and progressively colonized by a number of glial fibrillar acid protein-immunopositive and S-100 immunonegative cells, an immunocytochemical pattern typical of immature Schwann cells. These cells, which were located in the interstice between grafted skeletal muscle fibers, are mainly organized into long chains oriented along the main axis of the graft and progressively colonize all the graft. Schwann cells coming from the distal nerve end are suitable for being responsible for guiding regeneration of nerve fibers along the graft toward the correct periphery (tissue specificity).

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    • "Axonal regeneration in ANAs has been demonstrated to be similar to that in autografts across short gaps (Moore et al., 2011a, 2011b; Whitlock et al., 2009), but is reduced across longer defects (Whitlock et al., 2009), due to a poorly-understood mechanism. Following nerve repair with ANAs, there is early and progressive migration of SCs from both the proximal and especially the distal nerve stumps (Fornaro et al., 2001; Hayashi et al., 2007; Tseng et al., 2003; Whitlock et al., 2010a, 2010b). Host SCs provide the environment necessary for axonal regeneration in ANAs (Hall, 1986a, 1986b) through synthesis of neurotrophic factors (Bunge, 1993), adhesion molecules (Bixby et al., 1988), and axonal myelination (Bunge, 1993; Levi et al., 1994, 1997) and organization(Fansa et al., 2001). "
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    ABSTRACT: Repair of large nerve defects with acellular nerve allografts (ANAs) is an appealing alternative to autografting and allotransplantation. ANAs have been shown to be similar to autografts in supporting axonal regeneration across short gaps, but fail in larger defects due to a poorly-understood mechanism. ANAs depend on proliferating Schwann cells (SCs) from host tissue to support axonal regeneration. Populating longer ANAs places a greater proliferative demand on host SCs that may stress host SCs, resulting in senescence. In this study, we investigated axonal regeneration across increasing isograft and ANA lengths. We also evaluated the presence of senescent SCs within both graft type. A sciatic nerve graft model in rats was used to evaluate regeneration across increasing isograft (~autograft) and ANA lengths (20, 40, and 60mm). Axonal regeneration and functional recovery decreased with increased graft length and the performance of the isograft was superior to ANAs at all lengths. Transgenic Thy1-GFP rats and qRT-PCR demonstrated that failure of the regenerating axonal front in ANAs was associated with increased levels of senescence related markers in the graft (senescence associated β-galactosidase, p16(INK4A), and IL6). Lastly, electron microscopy (EM) was used to qualitatively assess senescence-associated changes in chromatin of SCs in each graft type. EM demonstrated an increase in the presence of SCs with abnormal chromatin in isografts and ANAs of increasing graft length. These results are the first to suggest that SC senescence plays a role in limited axonal regeneration across nerve grafts of increasing gap lengths.
    Experimental Neurology 05/2013; 247. DOI:10.1016/j.expneurol.2013.04.011 · 4.62 Impact Factor
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    • "During development, the axon-based ligand Type III Neuregulin-1 (NRG-1) binds ErbB family receptors to provide a critical maturation signal to neural crest cells (Garratt, et al., 2000,Jessen and Mirsky, 2005) that is also instrumental in SC proliferation, survival (Dong, et al., 1995), migration (Lyons, et al., 2005), and myelin regulation (Taveggia, et al., 2005) (Michailov, et al., 2004). Both intact and injured axons rely upon the paracrine trophic support afforded them by local SCs (Hayworth, et al., 2006,Mirsky, et al., 2002,Yin, et al., 2001,Zhu, et al., 2003), but following traumatic nerve injury, axons can traverse short distances through acellular synthetic conduits (Ahmed, et al., 2003,Hadlock, et al., 1998,Mosahebi, et al., 2003), vein grafts (Chiu and Strauch, 1990,Tseng, et al., 2003,Ulkur, et al., 2003,Weber and Mackinnon, 2005), and muscle tissue without clear evidence of SC support (Fornaro, et al., 2001,Varejao, et al., 2003). Moreover, denervated but mature SCs can survive for prolonged periods without axon-based signals before reinnervation occurs (Dedkov, et al., 2002,Sulaiman and Gordon, 2002,Sulaiman, et al., 2002). "
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    ABSTRACT: We propose that double-transgenic thy1-CFP(23)/S100-GFP mice whose Schwann cells constitutively express green fluorescent protein (GFP) and axons express cyan fluorescent protein (CFP) can be used to serially evaluate the temporal relationship between nerve regeneration and Schwann cell migration through acellular nerve grafts. Thy1-CFP(23)/S100-GFP and S100-GFP mice received non-fluorescing cold preserved nerve allografts from immunologically disparate donors. In vivo fluorescent imaging of these grafts was then performed at multiple points. The transected sciatic nerve was reconstructed with a 1-cm nerve allograft harvested from a Balb-C mouse and acellularized via 7 weeks of cold preservation prior to transplantation. The presence of regenerated axons and migrating Schwann cells was confirmed with confocal and electron microscopy on fixed tissue. Schwann cells migrated into the acellular graft (163+/-15 intensity units) from both proximal and distal stumps, and bridged the whole graft within 10 days (388+/-107 intensity units in the central 4-6 mm segment). Nerve regeneration lagged behind Schwann cell migration with 5 or 6 axons imaged traversing the proximal 4 mm of the graft under confocal microcopy within 10 days, and up to 21 labeled axons crossing the distal coaptation site by 15 days. Corroborative electron and light microscopy 5 mm into the graft demonstrated relatively narrow diameter myelinated (431+/-31) and unmyelinated (64+/-9) axons by 28 but not 10 days. Live imaging of the double-transgenic thy1-CFP(23)/S100-GFP murine line enabled serial assessment of Schwann cell-axonal relationships in traumatic nerve injuries reconstructed with acellular nerve allografts.
    Experimental Neurology 10/2007; 207(1):128-38. DOI:10.1016/j.expneurol.2007.06.004 · 4.62 Impact Factor
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    • "Independently of the type of non-nervous conduit employed to bridge a nerve gap, it was soon clear to researchers that the availability of enough SCs along the whole graft length to support axon regeneration was the critical point, as judged by the eventual success of the nerve repair. In fact, when non-nervous guides are used, SCs that are required for successful axon regeneration inside the tube originate from bilateral migration from both severed nerve stumps (Williams et al., 1983; Dubovy and Svizenska, 1992, 1994; Torigoe, 1997; Dubovy, 1998; Dubovy et al., 2001; Fornaro et al., 2001). SC migration from the proximal nerve stump is slower and accompanies axon regrowth, whereas SC migration from the distal stump is faster and is thought to guide axon regeneration, contributing to the exertion of the distal neurotropic lure (Dubovy, 1998; Tos et al., 2000, 2004). "
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    ABSTRACT: Schwann cells play a critical role in peripheral nerve regeneration. When a non-nervous conduit is used to bridge a nerve defect, the conduit is soon colonized by a number of Schwann cells that make a pathway for regrowing axons. By using electron microscopy, immunohistochemistry, and reverse transcriptase-polymerase chain reaction analysis, we have investigated the behavior of migratory glial cells along a particular type of autologous tissue-engineered conduit made of a vein filled with fresh skeletal muscle, using the rat sciatic nerve model. With this particular type of autograft, our data show that many Schwann cells soon take up a close relationship with grafted muscle fibers, and especially with their basal lamina, which appears to serve as a migration pathway for them. The early and massive colonization of the conduit is sustained by both Schwann cell migration and proliferation, as demonstrated by PCNA immunostaining. Later, as they meet regenerating axons, Schwann cells become closely associated with them and eventually lose their connections with grafted muscle fibers because of the formation of perineurial envelopes. Because previous studies showed that alpha(2a-2b) NRG1 is overexpressed at early stages along the muscle-vein combined tubes, we have also investigated mRNA expression of its two receptors, erbB2 and erbB3. Both messengers are overexpressed, although with different time courses. Overall, our results provide some morphological and biochemical bases for explaining the effectiveness of fresh muscle-vein combined nerve guides and throw an interesting light on the possible role of alpha(2a-2b) NRG1 through the erbB2/erbB3 heterodimer receptor for nerve regeneration inside non-nervous conduits.
    The Journal of Comparative Neurology 08/2005; 489(2):249-59. DOI:10.1002/cne.20625 · 3.51 Impact Factor
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