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Following traumatic injuries of the central nervous system (CNS) a wound healing scar, resembling the molecular structure of a basement membrane and mainly composed of Collagen type IV and associated glycoproteins and proteoglycans, is formed. It is well known that CNS fibers poorly regenerate after traumatic injuries. In this article we summarize data showing that prevention of collagen scar formation enables severed axons in brain and spinal cord to regrow across the lesion site and to elongate in uninjured CNS tissue. We observed that regenerating fibers grow back to their former target where they develop chemical synapses, become remyelinated by resident oligodendrocytes and conduct action potentials.

Traumatic injury of the central nervous system results in formation of a collagenous basement membrane-rich fibrous scar in the lesion centre. Due to accumulation of numerous axon-growth inhibitory molecules the lesion scar is considered a major impediment for axon regeneration. Following transection of the dorsal corticospinal tract (CST) at thoracic level 8 in adult rats, transient suppression of collagenous scarring in the lesion zone by local application of a potent iron chelator and cyclic adenosine monophosphate resulted in the delay of fibrous scarring. Treated animals displayed long-distance growth of CST axons through the lesion area extending for up to 1.5-2 cm into the distal cord. In addition, the treatment showed a strong neuroprotective effect, rescuing cortical motoneurons projecting into the CST that normally die (30%) after thoracic axotomy. Further, anterogradely traced CST axons regenerated through both grey and white matter and developed terminal arborizations in grey matter regions. In contrast to controls, injured animals receiving treatment showed significant functional recovery in the open field, in the horizontal ladder and in CatWalk locomotor tasks. We conclude that the fibrous lesion scar plays a pivotal role as a growth barrier for regenerating axons in adult spinal cord and that a delay in fibrotic scarring by local inhibition of collagen biosynthesis and basement membrane deposition is a promising and unique therapeutic strategy for treating human spinal trauma.

After traumatic injury, adult central nervous system (CNS) axons fail to regenerate. We have previously shown that one major impediment for axon regeneration is the basement membrane (BM) forming at the lesion center, by means of a wound healing collagenous scar, after lesioning the postcommisural fornix of the adult rat [Eur. J. Neurosci. 11 (1999) 632] [6]. This BM consists of a supramolecular network of collagen type IV, laminin (LN), nidogen, and associated proteoglycans [Crit. Rev. Biochem. Mol. Biol. 27 (1992) 93] [5]. Following axotomy, axons of the proximal stump of the transected postcommissural fornix fail to cross the lesion site. This regenerative failure is spatially and temporally highly correlated with the appearance of BM in the lesion site [Restor. Neurol. Neurosci. 15 (1999) 1] [7]. However, if the deposition of BM is prevented, the injured axons: (i) regenerate in their former pathway, (ii) are conductive across and behind the lesion site, and (iii) form chemical synapses in their target area, the mammillary body [Eur. J. Neurosci. 11 (1999) 632]. The developing BM is surrounded by neuropil and can easily be stained immunohistochemically using anti-collagen IV antibodies on fresh frozen sections (10 microm). To examine a clinically more relevant model of traumatic CNS injuries we developed a transection model of the thoracic dorsal corticospinal tract (CST) in the rat spinal cord. In contrast to fornix lesion this transection is performed in close proximity to the meninges. This involves the BM being completely washed out if fresh frozen tissue is used on slides. If the animals are sacrificed by perfusion with aldehydes the collagen IV and the LN antigen are masked by the fixative. To restore the correct immunohistochemical staining pattern (BM, blood vessels) a special protocol including an enzyme digestion is necessary. If thick sections are stained free floating, the tissue is destroyed due to the enzyme treatment. Here we present a method to prevent loss of the lesion-induced BM and to perform the correct immunohistochemical stainings of BM proteins in the traumatically injured spinal cord.

After CNS trauma a sheet-like, collagen type IV (Coll IV) immunopositive basement membrane (BM) develops in the lesion zone as well as at newly formed blood vessels. The basic scaffold of this BM is composed of Coll IV, laminin and nidogen but numerous other proteins some of which are discussed to be inhibitory for axonal regeneration, i.e. chondroitin- and heparansufate-proteoglycans, are associated with BM. This review will focus on the collagenous wound healing scar, discuss its composition and summarize the experimental results that demonstrate its role in the failure of axonal regeneration in the injured mammalian CNS.

In a previous study, we have shown that microtransplanted Schwann cell suspensions foster structural recovery of the acutely transected postcommissural fornix. The emphasis of the present study was to examine whether subacutely and chronically injured axons also demonstrate significant responsiveness to implanted Schwann cells. Microinjected suspensions of cultured Schwann cells i) elicited a growth response and attracted axons in a subacute and chronic traumatic lesion but ii) failed to stimulate regrowth of the postcommissural fornix projection at any nonacute postlesion stage. In conclusion, the single intervention strategy of Schwann cell microimplantation is not sufficient to ensure regeneration of the subacutally or chronically transected postcommissural fornix. The use of Schwann cells as stimulators of axon regrowth depends on the neuronal cell type and the appropriate postinjury time point., (Copyright 1999 Wiley-Liss, Inc.)

Scarring impedes axon regrowth across the lesion site and is one major extrinsic constraint to effective regeneration in the adult mammalian central nervous system. In the present study we determined whether specific biochemical or immunochemical modulation of one major component of the scar, the basal membrane (BM), would provide a means to stimulate axon regeneration in the mechanically transected postcommissural fornix of the adult rat. Basal membrane developed within the first 2 weeks after transection in spatiotemporal coincidence with the abrupt growth arrest of spontaneously regrowing axons. Local injection of anticollagen IV antibodies or alpha, alpha'-dipyridyl, an inhibitor of collagen triple helix formation and synthesis, significantly reduced lesion-induced BM deposition. This treatment allowed massive axon elongation across the lesion site. Anterograde tracing provided unequivocal evidence that regenerating axons follow their original pathway, reinnervate the appropriate target, the mammillary body, and become remyelinated with compact myelin. Presynaptic electrophysiological recordings of regenerated fibre tracts showed recovery to nearly normal conduction properties. Our results indicate that lesion-induced BM is an impediment for successful axonal regeneration and its reduction is a prerequisite and sufficient condition for regrowing axons to cross the lesion site.

After injury of the adult mammalian CNS axonal regeneration across or around the lesion scar is negligible. Previously, we have shown that the lesion-induced basal membrane (BM) within the lesion center participates in a growth barrier for axon regeneration and that its reduction by means of pharmacological or immunochemical treatment is a prerequisite and sufficient condition for regrowing axons to cross the lesion site. The present study was designed to further investigate this observation by analyzing the effect of a delayed treatment on the regeneration of both subacutely and chronically lesioned axons.Adult rats underwent unilateral transection of the postcommissural fornix. At one to five days after transection one group of animals received a local injection of 2, 2'-dipyridyl (DPY), an inhibitor of collagen triple helix formation and synthesis. Another group received a second transection within the former lesion site followed by an immediate DPY-injection at five days or 4 weeks after transection. Six weeks after the last surgery BM deposition and axonal regeneration were analysed using immunocytochemical methods.A local injection of DPY clearly reduced the lesion-induced BM deposition when applied within the first 3 days after transection. Under these conditions regrowing axons still crossed the former impermeable lesion site and regenerated within their normal pathway up to their former target, the mammillary body. However, in late subacute (5 d) and chronic stages (4 w) the double transection+injection paradigm failed to reduce BM deposition and, in consequence, also to induce axonal regeneration.These results demonstrate the potential of the collagen IV-reducing strategy to promote axonal regeneration across the lesion scar not only in acute but also in early subacute traumatic injuries.

Previous studies have shown that Schwann cell implantation offers a potential therapeutic approach to a variety of neurodegenerative disorders and traumatic injuries. In a clinically relevant paradigm, however, the implantation of autologous Schwann cells is problematic and the use of heterogenetic Schwann cells will be required. In the present study we addressed this important issue and analysed the immunogenicity and survival of allogeneic and xenogeneic Schwann cell suspension grafts in a prelesioned CNS fiber tract, the transected postcommissural fornix of the adult Wistar rat. Cultured Schwann cells from Wistar rat or human peripheral nerve were injected either immediately or after a delay into the transection site and the spatio-temporal pattern of leukocyte infiltration and of major histocompatibility antigen expression was characterized and semiquantified with immunocytochemical methods. Our main findings are that (1) invasive cerebral lesions induce the expression of MHC class I and II antigens, but only sparse infiltration of T-lymphocytes, (2) both allogeneic and xenogeneic discordant Schwann cell suspension grafts, from either neonatal or adult peripheral nerve, survive without any overt signs of rejection for up to 10 weeks after implantation; and (3) delayed implantation procedures have no effect on immune responses to allogeneic Schwann cell grafts. These results demonstrate that there is no marked ongoing immune reactions to heterogenetic Schwann cell suspension grafts and that long-term survival of cross-species Schwann cell grafts can be achieved in the absence of any immunsuppressive treatment. Thus the conditions for functional transplantation of Schwann cells across immunological barriers seem to be favourable and will have implications for future cross-species studies, and possibly also for clinical application.