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Notes: Summary Highly purified populations of Schwann cells were grafted into lesioned adult rat spinal cord to determine if they promote axonal regeneration. Dorsal spinal cord lesions were created by a photochemical lesioning technique. Schwann cells derived from E16 rat dorsal root ganglia, either elongated and associated with their extracellular matrix or dissociated and without matrix, were rolled in polymerized collagen to form an implant 4–6 mm long which was grafted at 5 or 28 days after lesioning. No immunosuppression was used. Acellular collagen rolls served as controls. At 14, 28 and 90 days and 4 and 6 months after grafting, animals were analysed histologically with silver and Toluidine Blue stains and EM. The grafts often filled the lesion and the host borders they apposed exhibited only limited astrogliosis. By 14 days, bundles of unmyelinated and occasional thinly myelinated axons populated the periphery of Schwann cell implants. By 28 days and thereafter, numerous unmyelinated and myelinated axons were present in most grafts. Silver staining revealed sprouted axons at the implant border at 28 days and long bundles of axons within the implant at 90 days. Photographs of entire 1 μm plastic cross-sections of nine grafted areas were assembled into montages to count the number of myelinated axons at the graft midpoint; the number of myelinated axons ranged from 517–3214. Electron microscopy of implants showed typical Schwann cell ensheathment and myelination, increased myelin thickness by 90 days, and a preponderance of unmyelinated over myelinated axons. Random EM sampling of five Schwann cell grafts snowed that the ratio of unmyelinated to myelinated axons was highest (20∶1) at 28 days. These ratios implied that axons numbered in the thousands at the graft midpoint. Dissociated Schwann cells without matrix promoted axonal ingrowth and longitudinal orientation as effectively as did elongated Schwann cells accompanied by matrix. There was a suggestion that axonal ingrowth was at least as successful, if not more so, when the delay between lesioning and grafting was 28 rather than 5 days. Acellular collagen grafts did not contain axons at 28 days, the only interval assessed. In sum, grafts of Schwann cells in a rolled collagen layer filled the lesion and were well tolerated by the host. The Schwann cells stimulated rapid and abundant growth of axons into grafts and they ensheathed and myelinated these axons in the normal manner.

Notes: Summary The ability to purify and recombine populations of peripheral neurons, Schwann cells and fibroblasts in tissue culture has enabled us to examine the contribution of fibroblasts to Schwann cell basal lamina assembly and ensheathment of unmyelinated rat superior cervical ganglion neuritesin vitro. Purified perinatal superior cervical ganglion neurons were grown in culture either with Schwann cells or with Schwann cells plus fibroblasts derived from either superior cervical ganglion capsule or cranial periosteum. The cultures were maintained for 2–8 weeks on a collagen substratum in a medium known to promote Schwann cell differentiation (myelin, basal lamina formation) in the presence of dorsal root ganglion neurons. The extent of Schwann cell differentiation (ensheathment, basal lamina formation) in the presence of superior cervical ganglion neurons was evaluated in this study using electron microscopy. In superior cervical ganglion neuron plus Schwann cell cultures (without fibroblasts), Schwann cells achieved only a moderate degree of ensheathment; also, Schwann cell basal lamina was discontinuous and extracellular collagen fibrils were sparse. Although only discontinuous basal lamina was demonstrable by electron microscopy in these cultures, surprisingly, Schwann cell/neurite fascicles were uniformly immunostained for laminin, type IV collagen, and heparan sulfate proteoglycan. The addition of fibroblasts to superior cervical ganglion neuron plus Schwann cell cultures increased the deposition of basal lamina around the Schwann cell/neurite units, the number of collagen fibrils, and the extent of neurite ensheathment. We propose that the presence of basal lamina increases the Schwann cell's ability to ensheathe superior cervical ganglion neurites, possibly through an augmentation of specific extracellular matrix components or by increasing in some way the capacity of these components to become organized into basal lamina. We conclude that, unlike dorsal root ganglion neurons, superior cervical ganglion neurons are unable to stimulate full Schwann cell extracellular matrix expression with the result that these Schwann cells require the extraneuronal influence of fibroblasts to deposit basal lamina and attain their mature phenotype in culture.