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A reliable method to reduce collagen scar formation in the lesioned rat spinal cord
Journal of Neuroscience Methods 110 (2001) 141– 146 www.elsevier.com/locate/jneumeth
A reliable method to reduce collagen scar formation in the lesioned rat spinal cord Susanne Hermanns a, Petra Reiprich b, Hans Werner Mu¨ller a,* a
Molecular Neurobiology Laboratory, Department of Neurology, Heinrich-Heine-Uni6ersity Du¨sseldorf, Moorenstrasse 5, D-40225 Dusseldorf, Germany b Institute of Physiology, Heinrich-Heine-Uni6ersity Du¨sseldorf, Moorenstrasse 5, D-40225 Dusseldorf, Germany Received 18 April 2001; received in revised form 29 June 2001; accepted 3 July 2001
Abstract Following traumatic injury, the formation of a glial scar and deposition of extracellular matrix (ECM) contributes to the regeneration failure in the adult mammalian central nervous system (CNS). Using a postcommissural fornix transection as a brain lesion model in rat, we have previously shown that the collagenous basement membrane (BM) at the lesion site is a major impediment for axon regeneration. Deposition of BM in this lesion model can be delayed by administration of the iron chelator 2,2%-bipyridine (BPY), an inhibitor of prolyl 4-hydroxylase (PH), a key enzyme of collagen biosynthesis. To examine whether this potential therapeutic approach is transferable to other CNS regions, we have chosen the mechanically lesioned rat spinal cord to investigate the effects of BPY administration on BM formation. Due to the close proximity of the lesion zone to meningeal fibroblasts, a cell-type secreting large amounts of collagen IV, BM deposition was much more extensive in the spinal cord than in the brain lesion. Neither immediate injections nor continuous application of BPY resulted in a detectable reduction of BM formation in the spinal cord. Only a combination of anti-scarring treatments including (i) injection of the more potent PH inhibitor [2,2%-bipyridine]-5,5%-dicarboxylic acid (BPY-DCA), (ii) selective inhibition of fibroblast proliferation and ECM production by 8-Br-cAMP, and (iii) continuous application of BPY-DCA, reduced the lesion-induced BM significantly. The present results clearly demonstrate, that the exclusive application of BPY according to a protocol designed for treatment of brain lesions is not sufficient to reduce BM formation in the lesioned adult rat spinal cord. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Spinal cord injury; Collagen scar; 2,2%-Bipyridine; Axonal regeneration; [2,2%-Bipyridine]-5,5%-dicarboxylic acid; Connective tissue growth factor
1. Introduction Worldwide several groups are working on the mechanisms that may inhibit axonal regeneration in the traumatically injured central nervous system (CNS). Myelin constituents (Schnell and Schwab, 1990; Shen et al., 1998), the lack of trophic factors (Novikova et al., 1996; Grill et al., 1997; Bradbury et al., 1999) as well as the presence of a glial scar (Snow et al., 1990; Bovolenta et al., 1997; Moon et al., 2000) have been shown to contribute to the regenerative failure of adult CNS axons. * Corresponding author. Tel.: + 49-211-81-18410; fax: + 49-21181-18411. E-mail address: [email protected] (H.W. Mu¨ller).
Other putative neurite outgrowth inhibitors, especially extracellular matrix (ECM) components become increasingly important in the field of neuroregenerative research. Recent in vitro studies identified increasing numbers of ECM molecules that show inhibitory properties for axonal regeneration (Fitch and Silver, 1997; Fidler et al., 1999; Bovolenta and Fernaud-Espinosa, 2000; Becker et al., 2000). Some of these proteins are associated with the collagenous basement membrane (BM) that develops at the lesion site in the injured CNS (Stichel et al., 1999a). Earlier attempts to remove this collagenous scar tissue in the lesioned spinal cord by means of various protease treatments (Matinian and Andreasian, 1973) could not successfully be reproduced as no histological or functional improvement of the treated animals could
0165-0270/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 7 0 ( 0 1 ) 0 0 4 2 7 - 7
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S. Hermanns et al. / Journal of Neuroscience Methods 110 (2001) 141–146
be observed, probably due to extensive bleeding (Kowalski et al., 1979; Guth et al., 1980). Recently, two novel approaches including pharmacological and immunochemical treatments have been applied successfully to either inhibit collagen biosynthesis or prevent BM deposition following transection of a CNS fiber tract (Stichel et al., 1999b). Blockade of collagen biosynthesis in the transected postcommissural fornix was achieved by inhibition of prolyl 4-hydroxylase (PH), a key enzyme in posttranslational modification of protocollagen mediating proline hydroxylation, through injection of the iron chelator 2,2%-bipyridine (BPY; Stichel et al., 1999b). Fe2 + is an essential co-factor for PH activity (Ikeda et al., 1992; Kivirikko and Myllyla, 1985). Alternatively, assembly of the collagenous BM could be prevented by the application of neutralizing antibodies directed to collagen IV immediately after fornix lesion. Reportedly, both treatments resulted in a transient delay of BM formation that was sufficient to allow axon regrowth across the lesion site and reinnervation of the natural target, the mammillary body (Stichel et al., 1999b). In order to test whether the collagen inhibiting approach could be transferred to other CNS injury models, we have applied BPY and its dicarboxylic acid derivative to the traumatically lesioned spinal cord. 2. Materials and methods
2.1. Animals Adult Wistar rats of both sexes with a weight between 180–300 g were used in this study. The animals were bred under specificated pathogen free conditions at a temperature of 21 °C and a relative humidity of 509 5% and housed under the same conventional conditions. They received dry-food pellets (Altromin) and soured, germ-free water (pH 2) ad libitum. All animals were bred at the Central Animal Facility of the Heinrich-Heine-University, Dusseldorf. All surgical interventions as well as pre/postsurgical animal care were performed in accordance with the German Animal Protection Law.
Animals deeply anaesthetized with an intraperitoneal injection of chloral hydrate were shaved on their back and the skin disinfected with 70% ethanol. A dorsal cut was performed to open the skin on a thoracic level, the muscles were exposed and severed blunt. The spine and the vertebral arch at T8 were removed and the dura was opened. The guidance cannula of the wire knife was inserted into the spinal cord and the knife was pulled out (Fig. 1). Then the knife was lifted up and the dorsal CST as well as the dorsal columns were completely transected. The cord had to be pushed down gently with a spatula to prevent its disruption.
2.3. Experimental groups 2.3.1. Group 1 — postcommissural fornix/dorsal corticospinal tract transection These animals were used to compare collagen type IV (Coll IV)-expression in the fornix (n= 5) and the spinal cord lesion (n=25). They received a transection of the postcommissural fornix or the dorsal CST only and were sacrificed 7 days postlesion (pl). 2.3.2. Group 2 — single immediate 2,2 %-bipyridine injections This experimental group was designed to prove whether single immediate injections of the iron chelator BPY as performed after postcommissural fornix transections were sufficient to reduce the lesion-induced BM in spinal cord lesions. The animals received a transection of the dorsal CST followed by a single BPY injection (2 ml, 10 mM) using a fine-tipped glass cannula, connected to a 10 ml Hamilton syringe, directly into the lesion site. After survival times of 7 days pl, the animals were sacrificed by transcardial perfusion with
2.2. Surgeries 2.2.1. Postcommissural fornix lesions The fornix lesions were performed according to the protocol described by Stichel and Mu¨ ller (1994). 2.2.2. Transection of the rat dorsal corticospinal tract The transection of the rat dorsal corticospinal tract (CST) was performed with a scouten wire knife connected to a stereotaxic frame (Small animal adaptor, David Kopf Instruments) under sight of a surgery microscope (SM33SL, Hundt) but not stereotactically.
Fig. 1. Transection of the dorsal CST and the dorsal columns. A wire knife in a guidance cannula is inserted into the spinal cord, then pulled out and lifted up to transect the fiber tracts.
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Fig. 2. Illustration of Elvax preparation. The Elvax/compound solution is prepared between two microscope slides to assure a reproducible thickness of the polymer.
4% paraformaldehyde (PFA) and BM-expression was analyzed immunohistochemically. Control animals received vehicle injections (n =16).
2.3.3. Group 3 — continuous 2,2 %-bipyridine application Animals of this group should demonstrate whether a single BPY injection combined with continuous BPY application (i) by covering the lesion site with BPYsoaked gelfoam or (ii) by osmotic minipumps (Alzet, different models) could achieve a complete reduction of the lesion-induced BM in spinal cord lesions. (1) The dorsal CST was transected. Then the animals received a single BPY injection (2 ml, 10 mM, n = 12; 20 mM, n=6). The lesion site was covered with BPYsoaked gelfoam of corresponding concentrations to ensure a continuous delivery of the chelator. Control animals received vehicle injections in combination with vehicle-soaked gelfoam. (2) After transection of the dorsal CST these animals received a single BPY injection (2 ml, 40 mM). To provide a continuous application BPY-loaded osmotic minipumps of different types were implanted: Alzet 1002, nominal pump-rate: 0.25 ml/h, duration: 14 days, n=7; Alzet 2ML2, nominal pump-rate: 5 ml/h, duration: 14 days, n= 6; Alzet 2001, nominal pump-rate: 1 ml/h, duration: 7 days, n=13. The minipumps were connected to a polyethylene tube with its opening placed directly above the lesion site. The pumps were placed in a skin-pocket at the back of the animals. Control animals received vehicle injections in combination with vehicle-loaded osmotic pumps (n= 8). After survival times of 7 days pl the animals were sacrificed by transcardial perfusion with 4% PFA and BM-expression was analyzed immunohistochemically. 2.3.4. Group 4 — combined treatment
After transection of the dorsal CST animals of this group received six immediate injections of the modified PH inhibitor [2,2%-bipyridine]-5,5%-dicarboxylic acid (BPY-DCA; 0.2 ml each) directly into the lesion site. To reduce fibroblast proliferation and ECM production selectively, solid 8-Br-cAMP was applied to the lesion site with a fine spatula. Finally, the lesion site was covered with a BPY-DCA-containing ethylene vinyl acetate copolymer (Elvax), to provide a continuous application of the substance. Control animals received vehicle injections combined with vehicle-containing Elvax or vehicle injections combined with vehicle-containing Elvax and 8-Br-cAMP to judge the cAMP-mediated effects. After survival times of 7 days pl the animals were sacrificed by transcardial perfusion with 4% PFA and BM-expression was analyzed immunohisto-chemically.
2.4. El6ax preparation The Elvax preparation was performed according to a modified protocol of Smith et al. (1995). Ethylene vinyl acetate copolymer globules were added to dichloromethane (10% w/v) and stirred for 20 min under the hood until the globules were completely resolved. Then a 10× stock solution of BPY-DCA was prepared and 100 ml of this stock was diluted in the Elvax solution. One percent (v/v) fast green was added to visualize the polymer and stirred for another 20 min. Two microscope slides were connected with Leukosilk® and put on dry ice. A Parafilm®-frame was cut and placed onto one slide (thickness of Parafilm®: 100 mm; assures that the polymer sheets are exactly 100-mm thick). A drop of the final solution was put on one slide, then the slides were folded up, fixed with photoclamps and frozen on dry ice and desiccated later (Fig. 2).
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2.5. Tissue processing/immunohistochemistry The perfused tissue was dehydrated in an ascending ethanol line and in methylbenzoate overnight. After an incubation in benzene for 15 min, the tissue was incubated in a benzene/paraffin mixture (1:10, v/v) for 30 min at 57 °C. The paraffin was changed three times (incubation 1 h at 57 °C each) and finally the tissue was incubated in paraffin overnight. After that the tissue was paraffin-embedded and cut parasagittally on a paraffin-microtome (10 mm). Serial parasagittal paraffin sections (10 mm) were consecutively stained with a polyclonal antibody against Coll IV (every fifth section) to judge for BM reduction. Slides were deparaffinized, washed, endogenous peroxidases were inactivated by a H2O2 reaction in methanol, and the Coll IV epitope, masked by the PFA-fixation was retrieved by incubation with protease XXIV (0.05% in Trisbuffer, Sigma) for 8 min at 37 °C. Then the slides were carefully washed and blocked with 3% normal goat serum prior to incubation with the first antibody (1:100, Biogenex) overnight at 4 °C. On the second day, slides were washed again, incubated with the second antibody (biotinylated goat anti-rabbit, 1:150, Vector) for 45 min at room temperature, washed and incubated with the Vectastain ABC-Elite kit. After that, the chromogen reaction was performed using the diaminobenzidine reaction. Slides were embedded with DPX (for detailed protocol, see Hermanns and Mu¨ ller (2001)).
3. Results/discussion
3.1. Application of 2,2 %-bipyridine fails to reduce the lesion-induced basement membrane After transection of the dorsal CST and the dorsal columns with a Scouten wire knife, a massive Coll IV immunopositive BM developed at the lesion site (Fig. 3(B)). This BM is more extensive than the BM develop-
ing after fornix lesions (Fig. 3(A)) probably due to the close proximity of the lesion to the meninges. The lesion is invaded by fibroblasts, a cell-type that is known to secrete Coll IV (Kuhl et al., 1984). Immunohistochemical staining of Coll IV revealed that, in none of the animal groups receiving BPY-treatment reduction of the lesion-induced BM could be detected when compared to vehicle treated control animals (Fig. 4(B)–(E)). Single injections of BPY (Fig. 4(C)) resulted in BM-expression at the lesion site that could not be distinguished from vehicle injections. Continuous application of BPY by covering the lesion with substance-soaked gelfoam only led to a slight reduction of BM sheet formation at the lesion site, but a complete reduction could never be achieved (Fig. 4(D)), probably because gelfoam attracts fibroblasts, the major collagen source in lesions close to the leptomeninges. Continuous application of BPY by osmotic minipumps resulted in a more extensive BM-expression at the lesion site than in gelfoam-treated animals (Fig. 4(E)). This effect may be due to contact of the polyethylene tube with the spinal cord, thus disturbing the tissue and enhancing fibroblast and glial reactions. In addition, low pumprates and opening of the dura causing dilution of the infusion media could prevent BM reduction in this lesion paradigm. It turned out that even the continuous application of the very potent PH inhibitor BPY-DCA alone was not sufficient to reduce the extensive deposition of Coll IV in the lesioned spinal cord (data not shown).
3.2. Application of a combined ‘anti-scarring’ -treatment results in complete basement membrane reduction Only after a combined application of (i) multiple BPY-DCA injections, (ii) inhibition of fibroblast proliferation and ECM production by 8-Br-cAMP, and (iii) the continuous application of BPY-DCA-containing Elvax copolymers, a complete reduction of the lesion-induced BM could be achieved. BM of blood vessels at
Fig. 3. Rostro-caudal extension of the lesion-induced BM in the transected postcommissural fornix (A) and the lesioned spinal cord (B) stained for Coll IV. Due to the spatial vicinity of the spinal cord lesion to meningeal cells, the collagen-expression in the spinal cord is far bigger than in the fornix lesion. Magnification bars: 100 mm.
S. Hermanns et al. / Journal of Neuroscience Methods 110 (2001) 141–146
Fig. 4. (A) Normal distribution of collagen immunopositive blood vessels in the uninjured spinal cord (g, grey matter; w, white matter). ((B)–(E)) Application of BPY does not reduce BM formation in the mechanically lesioned spinal cord ((B) vehicle injection, (C) immediate injection of BPY 10 mM, (D) immediate injection of BPY 10 mM plus topical application of BPY-soaked gelfoam, (E) immediate injection of BPY 40 mM plus continuous application of BPY 40 mM by an osmotic minipump. (F) The combined application of BPY-DCA immediate injections 30 mM, solid 8-Br-cAMP and Elvax copolymers containing BPY-DCA 90 mM leads to a significant BM reduction in the mechanically lesioned spinal cord. Magnification bar: 100 mm (in (A)–(F)).
the lesion site was immunohistochemically detectable (Fig. 4(F)). This does not mean that the blood–brain barrier (BBB) properties have been restored. There is evidence that the extent as well as the duration of BBB-breakdown are more extensive in the spinal cord compared to brain injuries (Schnell et al., 1999) and it is unlikely that these pathophysiological changes are affected by the treatment. Obviously BPY alone is not potent enough to reduce BM formation in traumatic spinal cord lesions in close proximity to meningeal cells. Compared to postcommissural fornix lesions performed with a Scouten wire knife where the rostro-caudal extension of the BM is : 50 mm, the extension of BM deposition in the spinal cord wire knife lesion is : 1200 mm (Fig. 3). In contrast to the dicarboxylated derivative BPY-DCA with an IC50 of 0.185 mM, BPY shows an IC50 of 34.2 mM (Hales and Beattie, 1993). This implies that BPY-DCA is about 180-fold more potent inhibiting PH than BPY. However, even BPY-DCA is not sufficient to reduce the lesion-induced BM formation in the spinal cord when
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applied alone. The supporting action of 8-Br-cAMP possibly through reduction of fibroblast proliferation and ECM production is indispensable in this experimental approach. One of the major sources of collagen synthesis in topical spinal lesions are invading fibroblasts that secrete large amounts of Coll IV (Kuhl et al., 1984). Fibroblast proliferation and ECM production are mediated by connective tissue growth factor (CTGF) via an autocrine mechanism (Frazier et al., 1996). Due to an inflammatory response after traumatic injuries TGFb is secreted by inflammatory and glial cells at the lesion site. Since the CTGF-promoter contains a TGFb regulatory element (TPRE; Grotendorst, 1997) binding of TGFb leads to transcription of the CTGF-gene and hence to stimulation of fibroblast proliferation and ECM production. Therefore, one possibility to selectively reduce fibroblast ECM production could be achieved through increasing their intracellular cAMPlevel by local administration of the membrane-permeable analogue 8-Br-cAMP. Elevation of the cAMP level results in blockade of the TPRE and, in consequence, in the inhibition of the CTGF-mediated fibroblast proliferation and ECM production (Duncan et al., 1999).
3.3. Anti-fibrotic agents in spinal lesions Our results indicate that the application of antifibrotic agents in the mechanically lesioned spinal cord leads to a significant reduction of the lesion-induced BM. Apparently, the iron chelator BPY is not potent enough to reduce the large amount of Coll IV that is deposited in topical spinal lesions. Therefore, it is not surprising that application of BPY in the partially transected spinal cord did not affect the transected dorsal CST fibers as described before (Weidner et al., 1999). Thus far, only multiple injections plus the continuous application of a more potent anti-fibrotic agent, such as BPY-DCA in combination with a fibroblast anti-proliferative treatment resulted in complete reduction of the lesion-induced BM. The interpretation that ‘anti-scarring’ treatment has no effects on CST regeneration (Weidner et al., 1999) is thus misleading, since it is based on the assumption that BPY treatment alone might be sufficient to reduce BM formation in spinal cord lesions, which is clearly not the case as shown in this investigation. The effect of a complete BM depleted scar on axonal regeneration is in focus of ongoing research in our laboratory. Nevertheless, application of a novel combination of potent anti-fibrotic drugs as described here will be helpful in achieving a reliable and complete reduction of the lesion-induced BM and, possibly, of putative BM-associated axon growth inhibitors.
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Acknowledgements The authors are grateful for stimulating discussions with F. Lausberg and the excellent technical help of M. Gasis. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 194/B5) and is part of the Ph.D. thesis of S.H. at the Faculty of Mathematics and Natural Sciences, Heinrich-Heine-University, Dusseldorf.
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bipyridine and its 5,5%-dicarboxylic acid derivatives. J Med Chem 1993;36:3853 – 8. Hermanns S, Mu¨ ller HW. Preservation and detection of lesion-induced collagenous scar in the CNS depend on the method of tissue processing. Brain Res Brain Res Protoc 2001;7:162 – 7. Ikeda H, Wu GY, Wu CH. Evidence that an iron chelator regulates collagen synthesis by decreasing the stability of procollagen mRNA. Hepatology 1992;15:282 – 7. Kivirikko KI, Myllyla R. Post-translational processing of procollagens. Ann NY Acad Sci 1985;460:187 – 201. Kowalski TF, Vahlsing HL, Feringa ER. Lidase treatment of spinal cord transected rats. Ann Neurol 1979;6:78 – 9. Kuhl U, Ocalan M, Timpl R, Mayne R, Hay E, von der MK. Role of muscle fibroblasts in the deposition of type-IV collagen in the basal lamina of myotubes. Differentiation 1984;28:164 – 72. Matinian LA, Andreasian AS. Enzyme therapy in organic lesions of the spinal cord. Akademia Nauk Armenian SSR 1973;94 (English translation: Brain Information Service, Los Angeles, University of California, 1976, 156 pp.). Moon LD, Brecknell JE, Franklin RJ, Dunnett SB, Fawcett JW. Robust regeneration of CNS axons through a track depleted of CNS glia. Exp Neurol 2000;161:49 – 66. Novikova L, Novikov L, Kellerth JO. Brain-derived neurotrophic factor reduces necrotic zone and supports neuronal survival after spinal cord hemisection in adult rats. Neurosci Lett 1996;220:203 – 6. Schnell L, Schwab ME. Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors. Nature 1990;343:269 – 72. Schnell L, Fearn S, Klassen H, Schwab ME, Perry VH. Acute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord. Eur J Neurosci 1999;11:3648 – 58. Shen YJ, DeBellard ME, Salzer JL, Roder J, Filbin MT. Myelin-associated glycoprotein in myelin and expressed by Schwann cells inhibits axonal regeneration and branching. Mol Cell Neurosci 1998;12:79 – 91. Smith AL, Cordery PM, Thompson ID. Manufacture and release characteristics of Elvax polymers containing glutamate receptor antagonists. J Neurosci Methods 1995;60:211 – 7. Snow DM, Lemmon V, Carrino DA, Caplan AI, Silver J. Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro. Exp Neurol 1990;109:111 – 30. Stichel CC, Mu¨ ller HW. Extensive and long-lasting changes of glial cells following transection of the postcommissural fornix in the adult rat. Glia 1994;10:89 – 100. Stichel CC, Niermann H, D’Urso D, Lausberg F, Hermanns S, Muller HW. Basal membrane-depleted scar in lesioned CNS: characteristics and relationships with regenerating axons. Neuroscience 1999a;93:321 – 33. Stichel CC, Hermanns S, Luhmann HJ, Lausberg F, Niermann H, D’Urso D, et al. Inhibition of collagen IV deposition promotes regeneration of injured CNS axons. Eur J Neurosci 1999b;11:632 – 46. Weidner N, Grill RJ, Tuszynski MH. Elimination of basal lamina and the collagen ‘‘scar’’ after spinal cord injury fails to augment corticospinal tract regeneration. Exp Neurol 1999;160:40 –50.
A reliable method to reduce collagen scar formation in the lesioned rat spinal cord Susanne Hermanns a, Petra Reiprich b, Hans Werner Mu¨ller a,* a
Molecular Neurobiology Laboratory, Department of Neurology, Heinrich-Heine-Uni6ersity Du¨sseldorf, Moorenstrasse 5, D-40225 Dusseldorf, Germany b Institute of Physiology, Heinrich-Heine-Uni6ersity Du¨sseldorf, Moorenstrasse 5, D-40225 Dusseldorf, Germany Received 18 April 2001; received in revised form 29 June 2001; accepted 3 July 2001
Abstract Following traumatic injury, the formation of a glial scar and deposition of extracellular matrix (ECM) contributes to the regeneration failure in the adult mammalian central nervous system (CNS). Using a postcommissural fornix transection as a brain lesion model in rat, we have previously shown that the collagenous basement membrane (BM) at the lesion site is a major impediment for axon regeneration. Deposition of BM in this lesion model can be delayed by administration of the iron chelator 2,2%-bipyridine (BPY), an inhibitor of prolyl 4-hydroxylase (PH), a key enzyme of collagen biosynthesis. To examine whether this potential therapeutic approach is transferable to other CNS regions, we have chosen the mechanically lesioned rat spinal cord to investigate the effects of BPY administration on BM formation. Due to the close proximity of the lesion zone to meningeal fibroblasts, a cell-type secreting large amounts of collagen IV, BM deposition was much more extensive in the spinal cord than in the brain lesion. Neither immediate injections nor continuous application of BPY resulted in a detectable reduction of BM formation in the spinal cord. Only a combination of anti-scarring treatments including (i) injection of the more potent PH inhibitor [2,2%-bipyridine]-5,5%-dicarboxylic acid (BPY-DCA), (ii) selective inhibition of fibroblast proliferation and ECM production by 8-Br-cAMP, and (iii) continuous application of BPY-DCA, reduced the lesion-induced BM significantly. The present results clearly demonstrate, that the exclusive application of BPY according to a protocol designed for treatment of brain lesions is not sufficient to reduce BM formation in the lesioned adult rat spinal cord. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Spinal cord injury; Collagen scar; 2,2%-Bipyridine; Axonal regeneration; [2,2%-Bipyridine]-5,5%-dicarboxylic acid; Connective tissue growth factor
1. Introduction Worldwide several groups are working on the mechanisms that may inhibit axonal regeneration in the traumatically injured central nervous system (CNS). Myelin constituents (Schnell and Schwab, 1990; Shen et al., 1998), the lack of trophic factors (Novikova et al., 1996; Grill et al., 1997; Bradbury et al., 1999) as well as the presence of a glial scar (Snow et al., 1990; Bovolenta et al., 1997; Moon et al., 2000) have been shown to contribute to the regenerative failure of adult CNS axons. * Corresponding author. Tel.: + 49-211-81-18410; fax: + 49-21181-18411. E-mail address: [email protected] (H.W. Mu¨ller).
Other putative neurite outgrowth inhibitors, especially extracellular matrix (ECM) components become increasingly important in the field of neuroregenerative research. Recent in vitro studies identified increasing numbers of ECM molecules that show inhibitory properties for axonal regeneration (Fitch and Silver, 1997; Fidler et al., 1999; Bovolenta and Fernaud-Espinosa, 2000; Becker et al., 2000). Some of these proteins are associated with the collagenous basement membrane (BM) that develops at the lesion site in the injured CNS (Stichel et al., 1999a). Earlier attempts to remove this collagenous scar tissue in the lesioned spinal cord by means of various protease treatments (Matinian and Andreasian, 1973) could not successfully be reproduced as no histological or functional improvement of the treated animals could
0165-0270/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 7 0 ( 0 1 ) 0 0 4 2 7 - 7
142
S. Hermanns et al. / Journal of Neuroscience Methods 110 (2001) 141–146
be observed, probably due to extensive bleeding (Kowalski et al., 1979; Guth et al., 1980). Recently, two novel approaches including pharmacological and immunochemical treatments have been applied successfully to either inhibit collagen biosynthesis or prevent BM deposition following transection of a CNS fiber tract (Stichel et al., 1999b). Blockade of collagen biosynthesis in the transected postcommissural fornix was achieved by inhibition of prolyl 4-hydroxylase (PH), a key enzyme in posttranslational modification of protocollagen mediating proline hydroxylation, through injection of the iron chelator 2,2%-bipyridine (BPY; Stichel et al., 1999b). Fe2 + is an essential co-factor for PH activity (Ikeda et al., 1992; Kivirikko and Myllyla, 1985). Alternatively, assembly of the collagenous BM could be prevented by the application of neutralizing antibodies directed to collagen IV immediately after fornix lesion. Reportedly, both treatments resulted in a transient delay of BM formation that was sufficient to allow axon regrowth across the lesion site and reinnervation of the natural target, the mammillary body (Stichel et al., 1999b). In order to test whether the collagen inhibiting approach could be transferred to other CNS injury models, we have applied BPY and its dicarboxylic acid derivative to the traumatically lesioned spinal cord. 2. Materials and methods
2.1. Animals Adult Wistar rats of both sexes with a weight between 180–300 g were used in this study. The animals were bred under specificated pathogen free conditions at a temperature of 21 °C and a relative humidity of 509 5% and housed under the same conventional conditions. They received dry-food pellets (Altromin) and soured, germ-free water (pH 2) ad libitum. All animals were bred at the Central Animal Facility of the Heinrich-Heine-University, Dusseldorf. All surgical interventions as well as pre/postsurgical animal care were performed in accordance with the German Animal Protection Law.
Animals deeply anaesthetized with an intraperitoneal injection of chloral hydrate were shaved on their back and the skin disinfected with 70% ethanol. A dorsal cut was performed to open the skin on a thoracic level, the muscles were exposed and severed blunt. The spine and the vertebral arch at T8 were removed and the dura was opened. The guidance cannula of the wire knife was inserted into the spinal cord and the knife was pulled out (Fig. 1). Then the knife was lifted up and the dorsal CST as well as the dorsal columns were completely transected. The cord had to be pushed down gently with a spatula to prevent its disruption.
2.3. Experimental groups 2.3.1. Group 1 — postcommissural fornix/dorsal corticospinal tract transection These animals were used to compare collagen type IV (Coll IV)-expression in the fornix (n= 5) and the spinal cord lesion (n=25). They received a transection of the postcommissural fornix or the dorsal CST only and were sacrificed 7 days postlesion (pl). 2.3.2. Group 2 — single immediate 2,2 %-bipyridine injections This experimental group was designed to prove whether single immediate injections of the iron chelator BPY as performed after postcommissural fornix transections were sufficient to reduce the lesion-induced BM in spinal cord lesions. The animals received a transection of the dorsal CST followed by a single BPY injection (2 ml, 10 mM) using a fine-tipped glass cannula, connected to a 10 ml Hamilton syringe, directly into the lesion site. After survival times of 7 days pl, the animals were sacrificed by transcardial perfusion with
2.2. Surgeries 2.2.1. Postcommissural fornix lesions The fornix lesions were performed according to the protocol described by Stichel and Mu¨ ller (1994). 2.2.2. Transection of the rat dorsal corticospinal tract The transection of the rat dorsal corticospinal tract (CST) was performed with a scouten wire knife connected to a stereotaxic frame (Small animal adaptor, David Kopf Instruments) under sight of a surgery microscope (SM33SL, Hundt) but not stereotactically.
Fig. 1. Transection of the dorsal CST and the dorsal columns. A wire knife in a guidance cannula is inserted into the spinal cord, then pulled out and lifted up to transect the fiber tracts.
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Fig. 2. Illustration of Elvax preparation. The Elvax/compound solution is prepared between two microscope slides to assure a reproducible thickness of the polymer.
4% paraformaldehyde (PFA) and BM-expression was analyzed immunohistochemically. Control animals received vehicle injections (n =16).
2.3.3. Group 3 — continuous 2,2 %-bipyridine application Animals of this group should demonstrate whether a single BPY injection combined with continuous BPY application (i) by covering the lesion site with BPYsoaked gelfoam or (ii) by osmotic minipumps (Alzet, different models) could achieve a complete reduction of the lesion-induced BM in spinal cord lesions. (1) The dorsal CST was transected. Then the animals received a single BPY injection (2 ml, 10 mM, n = 12; 20 mM, n=6). The lesion site was covered with BPYsoaked gelfoam of corresponding concentrations to ensure a continuous delivery of the chelator. Control animals received vehicle injections in combination with vehicle-soaked gelfoam. (2) After transection of the dorsal CST these animals received a single BPY injection (2 ml, 40 mM). To provide a continuous application BPY-loaded osmotic minipumps of different types were implanted: Alzet 1002, nominal pump-rate: 0.25 ml/h, duration: 14 days, n=7; Alzet 2ML2, nominal pump-rate: 5 ml/h, duration: 14 days, n= 6; Alzet 2001, nominal pump-rate: 1 ml/h, duration: 7 days, n=13. The minipumps were connected to a polyethylene tube with its opening placed directly above the lesion site. The pumps were placed in a skin-pocket at the back of the animals. Control animals received vehicle injections in combination with vehicle-loaded osmotic pumps (n= 8). After survival times of 7 days pl the animals were sacrificed by transcardial perfusion with 4% PFA and BM-expression was analyzed immunohistochemically. 2.3.4. Group 4 — combined treatment
After transection of the dorsal CST animals of this group received six immediate injections of the modified PH inhibitor [2,2%-bipyridine]-5,5%-dicarboxylic acid (BPY-DCA; 0.2 ml each) directly into the lesion site. To reduce fibroblast proliferation and ECM production selectively, solid 8-Br-cAMP was applied to the lesion site with a fine spatula. Finally, the lesion site was covered with a BPY-DCA-containing ethylene vinyl acetate copolymer (Elvax), to provide a continuous application of the substance. Control animals received vehicle injections combined with vehicle-containing Elvax or vehicle injections combined with vehicle-containing Elvax and 8-Br-cAMP to judge the cAMP-mediated effects. After survival times of 7 days pl the animals were sacrificed by transcardial perfusion with 4% PFA and BM-expression was analyzed immunohisto-chemically.
2.4. El6ax preparation The Elvax preparation was performed according to a modified protocol of Smith et al. (1995). Ethylene vinyl acetate copolymer globules were added to dichloromethane (10% w/v) and stirred for 20 min under the hood until the globules were completely resolved. Then a 10× stock solution of BPY-DCA was prepared and 100 ml of this stock was diluted in the Elvax solution. One percent (v/v) fast green was added to visualize the polymer and stirred for another 20 min. Two microscope slides were connected with Leukosilk® and put on dry ice. A Parafilm®-frame was cut and placed onto one slide (thickness of Parafilm®: 100 mm; assures that the polymer sheets are exactly 100-mm thick). A drop of the final solution was put on one slide, then the slides were folded up, fixed with photoclamps and frozen on dry ice and desiccated later (Fig. 2).
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2.5. Tissue processing/immunohistochemistry The perfused tissue was dehydrated in an ascending ethanol line and in methylbenzoate overnight. After an incubation in benzene for 15 min, the tissue was incubated in a benzene/paraffin mixture (1:10, v/v) for 30 min at 57 °C. The paraffin was changed three times (incubation 1 h at 57 °C each) and finally the tissue was incubated in paraffin overnight. After that the tissue was paraffin-embedded and cut parasagittally on a paraffin-microtome (10 mm). Serial parasagittal paraffin sections (10 mm) were consecutively stained with a polyclonal antibody against Coll IV (every fifth section) to judge for BM reduction. Slides were deparaffinized, washed, endogenous peroxidases were inactivated by a H2O2 reaction in methanol, and the Coll IV epitope, masked by the PFA-fixation was retrieved by incubation with protease XXIV (0.05% in Trisbuffer, Sigma) for 8 min at 37 °C. Then the slides were carefully washed and blocked with 3% normal goat serum prior to incubation with the first antibody (1:100, Biogenex) overnight at 4 °C. On the second day, slides were washed again, incubated with the second antibody (biotinylated goat anti-rabbit, 1:150, Vector) for 45 min at room temperature, washed and incubated with the Vectastain ABC-Elite kit. After that, the chromogen reaction was performed using the diaminobenzidine reaction. Slides were embedded with DPX (for detailed protocol, see Hermanns and Mu¨ ller (2001)).
3. Results/discussion
3.1. Application of 2,2 %-bipyridine fails to reduce the lesion-induced basement membrane After transection of the dorsal CST and the dorsal columns with a Scouten wire knife, a massive Coll IV immunopositive BM developed at the lesion site (Fig. 3(B)). This BM is more extensive than the BM develop-
ing after fornix lesions (Fig. 3(A)) probably due to the close proximity of the lesion to the meninges. The lesion is invaded by fibroblasts, a cell-type that is known to secrete Coll IV (Kuhl et al., 1984). Immunohistochemical staining of Coll IV revealed that, in none of the animal groups receiving BPY-treatment reduction of the lesion-induced BM could be detected when compared to vehicle treated control animals (Fig. 4(B)–(E)). Single injections of BPY (Fig. 4(C)) resulted in BM-expression at the lesion site that could not be distinguished from vehicle injections. Continuous application of BPY by covering the lesion with substance-soaked gelfoam only led to a slight reduction of BM sheet formation at the lesion site, but a complete reduction could never be achieved (Fig. 4(D)), probably because gelfoam attracts fibroblasts, the major collagen source in lesions close to the leptomeninges. Continuous application of BPY by osmotic minipumps resulted in a more extensive BM-expression at the lesion site than in gelfoam-treated animals (Fig. 4(E)). This effect may be due to contact of the polyethylene tube with the spinal cord, thus disturbing the tissue and enhancing fibroblast and glial reactions. In addition, low pumprates and opening of the dura causing dilution of the infusion media could prevent BM reduction in this lesion paradigm. It turned out that even the continuous application of the very potent PH inhibitor BPY-DCA alone was not sufficient to reduce the extensive deposition of Coll IV in the lesioned spinal cord (data not shown).
3.2. Application of a combined ‘anti-scarring’ -treatment results in complete basement membrane reduction Only after a combined application of (i) multiple BPY-DCA injections, (ii) inhibition of fibroblast proliferation and ECM production by 8-Br-cAMP, and (iii) the continuous application of BPY-DCA-containing Elvax copolymers, a complete reduction of the lesion-induced BM could be achieved. BM of blood vessels at
Fig. 3. Rostro-caudal extension of the lesion-induced BM in the transected postcommissural fornix (A) and the lesioned spinal cord (B) stained for Coll IV. Due to the spatial vicinity of the spinal cord lesion to meningeal cells, the collagen-expression in the spinal cord is far bigger than in the fornix lesion. Magnification bars: 100 mm.
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Fig. 4. (A) Normal distribution of collagen immunopositive blood vessels in the uninjured spinal cord (g, grey matter; w, white matter). ((B)–(E)) Application of BPY does not reduce BM formation in the mechanically lesioned spinal cord ((B) vehicle injection, (C) immediate injection of BPY 10 mM, (D) immediate injection of BPY 10 mM plus topical application of BPY-soaked gelfoam, (E) immediate injection of BPY 40 mM plus continuous application of BPY 40 mM by an osmotic minipump. (F) The combined application of BPY-DCA immediate injections 30 mM, solid 8-Br-cAMP and Elvax copolymers containing BPY-DCA 90 mM leads to a significant BM reduction in the mechanically lesioned spinal cord. Magnification bar: 100 mm (in (A)–(F)).
the lesion site was immunohistochemically detectable (Fig. 4(F)). This does not mean that the blood–brain barrier (BBB) properties have been restored. There is evidence that the extent as well as the duration of BBB-breakdown are more extensive in the spinal cord compared to brain injuries (Schnell et al., 1999) and it is unlikely that these pathophysiological changes are affected by the treatment. Obviously BPY alone is not potent enough to reduce BM formation in traumatic spinal cord lesions in close proximity to meningeal cells. Compared to postcommissural fornix lesions performed with a Scouten wire knife where the rostro-caudal extension of the BM is : 50 mm, the extension of BM deposition in the spinal cord wire knife lesion is : 1200 mm (Fig. 3). In contrast to the dicarboxylated derivative BPY-DCA with an IC50 of 0.185 mM, BPY shows an IC50 of 34.2 mM (Hales and Beattie, 1993). This implies that BPY-DCA is about 180-fold more potent inhibiting PH than BPY. However, even BPY-DCA is not sufficient to reduce the lesion-induced BM formation in the spinal cord when
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applied alone. The supporting action of 8-Br-cAMP possibly through reduction of fibroblast proliferation and ECM production is indispensable in this experimental approach. One of the major sources of collagen synthesis in topical spinal lesions are invading fibroblasts that secrete large amounts of Coll IV (Kuhl et al., 1984). Fibroblast proliferation and ECM production are mediated by connective tissue growth factor (CTGF) via an autocrine mechanism (Frazier et al., 1996). Due to an inflammatory response after traumatic injuries TGFb is secreted by inflammatory and glial cells at the lesion site. Since the CTGF-promoter contains a TGFb regulatory element (TPRE; Grotendorst, 1997) binding of TGFb leads to transcription of the CTGF-gene and hence to stimulation of fibroblast proliferation and ECM production. Therefore, one possibility to selectively reduce fibroblast ECM production could be achieved through increasing their intracellular cAMPlevel by local administration of the membrane-permeable analogue 8-Br-cAMP. Elevation of the cAMP level results in blockade of the TPRE and, in consequence, in the inhibition of the CTGF-mediated fibroblast proliferation and ECM production (Duncan et al., 1999).
3.3. Anti-fibrotic agents in spinal lesions Our results indicate that the application of antifibrotic agents in the mechanically lesioned spinal cord leads to a significant reduction of the lesion-induced BM. Apparently, the iron chelator BPY is not potent enough to reduce the large amount of Coll IV that is deposited in topical spinal lesions. Therefore, it is not surprising that application of BPY in the partially transected spinal cord did not affect the transected dorsal CST fibers as described before (Weidner et al., 1999). Thus far, only multiple injections plus the continuous application of a more potent anti-fibrotic agent, such as BPY-DCA in combination with a fibroblast anti-proliferative treatment resulted in complete reduction of the lesion-induced BM. The interpretation that ‘anti-scarring’ treatment has no effects on CST regeneration (Weidner et al., 1999) is thus misleading, since it is based on the assumption that BPY treatment alone might be sufficient to reduce BM formation in spinal cord lesions, which is clearly not the case as shown in this investigation. The effect of a complete BM depleted scar on axonal regeneration is in focus of ongoing research in our laboratory. Nevertheless, application of a novel combination of potent anti-fibrotic drugs as described here will be helpful in achieving a reliable and complete reduction of the lesion-induced BM and, possibly, of putative BM-associated axon growth inhibitors.
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Acknowledgements The authors are grateful for stimulating discussions with F. Lausberg and the excellent technical help of M. Gasis. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 194/B5) and is part of the Ph.D. thesis of S.H. at the Faculty of Mathematics and Natural Sciences, Heinrich-Heine-University, Dusseldorf.
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