Susanne Hermanns

Inhibition of collagen IV deposition promotes regeneration of injured CNS axons

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 α, α′‐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.

Suppression of fibrous scarring in spinal cord injury of rat promotes long-distance regeneration of corticospinal tract axons, rescue of primary motoneurons in somatosensory cortex and significant functional recovery

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.

Basal membrane-depleted scar in lesioned CNS: characteristics and relationships with regenerating axons

The lesion scar formed after CNS injury is an impediment to axonal regeneration and leads to growth arrest or misrouting of sprouting axons. Our previous study showed that pharmacological reduction of basal membrane formation within the scar can overcome this scar impermeability [Stichel C. C. et al. (1999) Eur. J. Neurosci. 11, 632-646]. The aim of the present study was to characterize the basal membrane-depleted scar and to analyse its relationships with penetrating axons. The experiments comprised two groups of animals in which the left postcommissural fornix was transected; in addition, one group received a local immediate injection of the collagen IV-reducing agent dipyridyl, while the other group received an injection of phosphate-buffered saline. Immunohistochemical methods were used to characterize scar formation and scar-axon relationships. Animals receiving dipyridyl showed reduction of collagen IV-immunopositive basal membrane in the lesion center, which was accompanied by: (i) a decrease in laminin, as well as chondroitin and heparan sulfate proteoglycan, deposition in the lesion center; (ii) an increase in chondroitin and keratan sulfate proteoglycan expression in the perilesional area; (iii) a typical activation pattern of astrocytes and microglia/macrophages; (iv) axons regenerating through this modified scar were closely associated with various glial cell types and crossed a prominent chondroitin/keratan sulfate proteoglycan matrix. Our results suggest that neither the formation of a reactive astroglial network nor the accumulation of microglia/macrophages or the deposition of chondroitin and keratan sulfate proteoglycans in the perilesional area represent a barrier to regrowing axons. The present approach demonstrates that the lesion-induced basal membrane itself is the primary determinant of scar impermeability.

Lack of immune responses to immediate or delayed implanted allogeneic and xenogeneic Schwann cell suspensions

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. GLIA 21:299–314, 1997.

Preservation and detection of lesion-induced collagenous scar in the CNS depend on the method of tissue processing

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.

Effects of Schwann cell suspension grafts on axon regeneration in subacute and chronic CNS traumatic injuries

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. GLIA 28:156–165, 1999.

Local administration of DFO-loaded lipid particles improves recovery after end-to-end reconstruction of rat median nerve.

PURPOSE The improvement of regeneration and functional recovery after peripheral nerve injury is a major challenge in neurosurgery. Although microsurgical techniques for nerve reconstruction have seen great advancements over the last years, the clinical outcome with patients is often unsatisfactory. The aim of the present study was to investigate if administration of the iron chelator Deferroxamine (DFO), can improve postoperative outcome in the rat median nerve reconstruction model. METHODS After complete transection, the right median nerve was repaired by end-to-end neurorrhaphy. The suture site was wrapped by a 1-cm-long external jugular vein segment, either empty or filled with DFO-loaded lipid particles (Perineurin or with a vehicle (unloaded lipid particles) alone. Functional testing was carried out weekly by means of the grasping test. At the time of withdrawal, 12 weeks post-operatively, muscle tropism recovery was assessed by weighing flexor digitorum sublimis muscle that is innervated by the median nerve only. Before harvesting of the nerve specimens electrophysiological analyses were performed with measuring the latency, the threshold and the conduction velocity. Finally, the repaired nerves were withdrawn for immunocytochemistry with a neurofilament antibody and axon quantitative morphology. RESULTS The comparison between the groups showed that intraoperative application of the DFO-loaded lipid particles at the neurorrhaphy site led to a significant increase in the density of regenerating axons as well as to an accelerated recovery of both muscle tropism and motor function. The electrophysiological results demonstrated a decrease of the threshold, a lower latency, and a higher conduction velocity in the Perineurin-treated animals. CONCLUSIONS The results of the present study suggest that local administration of Perineurin might have a therapeutic potential for improving the postoperative outcome after microsurgical nerve reconstruction in patients.