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Chondroitinase ABC enhances axonal regrowth through Schwann cell-seeded guidance channels after spinal cord injury¹

C. H. CHAU^*,, D. K. Y. SHUM^*,2, H. LI, J. PEI, Y. Y. LUI^*, L. WIRTHLIN^,, Y. S. CHAN and X.-M. XU^,,3

^* Department of Biochemistry, Faculty of Medicine, University of Hong Kong, Hong Kong, China;
Department of Anatomy and Neurobiology, Saint Louis University School of Medicine, Saint Louis, MO 63104, USA;
Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville School of Medicine, Louisville, KY 40292, USA; and
Department of Physiology, Faculty of Medicine, University of Hong Kong, Hong Kong, China

²Correspondence: Department of Biochemistry, Faculty of Medicine, University of Hong Kong, 21 Sassoon Rd., Hong Kong, China. E-mail: shumdkhk{at}hkucc.hku.hk

SPECIFIC AIMS

Grafting of Schwann cell (SC)-seeded channels into hemisected adult rat thoracic spinal cords has been tested as a strategy to bridge the injured spinal cord. Since deposits of chondroitin sulfate (CS) glycoforms at the distal graft-host interface have been implicated as a molecular impediment to regrowing axons, we attempted to diminish the effect by delivery of chondroitinase ABC (Ch^aseABC) close to the interface and examined the resulting tissue for improvements in axonal growth pattern and reactive cell distribution in an environment removed of CS glycoforms.

PRINCIPAL FINDINGS

At the distal graft-host interface
1. Ch^aseABC infusion cleared away CS56-positive deposits
We chose to deliver the CS-degrading enzyme, Ch^aseABC, by intra-parenchymal infusion into the caudal cord close to the graft-host interface immediately after an SC-seeded mini-channel had been implanted into a 3mm hemisection gap of the spinal cord at T8. When examined at the end of the 5^th week of grafting and infusion, a fuzzy, CS56-positive deposit was observed at the distal graft-host interface in cords that received vehicle infusion (Fig. 1A-C ). In cords that received infusion of Ch^aseABC, similar deposits were only barely observable at the distal graft-host interface (Fig. 1E-G ). We further analyzed the extent of action of the infused Ch^aseABC. The activity of the enzyme kept ex vivo in the infusion pump took 3 weeks to decay to half its initial level. At the end of 2 weeks of grafting and enzyme infusion, the distal interface and the adjoining caudal host cord were found CS56-negative but immunopositive for 2B6 which recognizes the 4-sulfated stub on core proteins of CS proteoglycans after Ch^aseABC digestion of the CS moiety. The results indicate that the in vivo activity of the infused enzyme was adequate in the digestive removal of CS glycoforms at the distal graft-host interface and the adjoining caudal host cord tissue.

View larger version (94K): [in this window] [in a new window]   Figure 1. Changes at the distal graft-host interface as a result of ChaseABC infusion. In vehicle-infused controls, a fuzzy CS56-positive zone (region within arrows; A-C and insets) was juxtaposed between an intensely CS56-positive matrix of the graft tissue and a front of GFAP-positive astrocytes in the caudal host cord; ED1-positive macrophages/microglia ( D and inset) were also gathered at the graft-host interface. In recipients of ChaseABC infusion, the fuzzy CS56-positive zone was significantly diminished (region within arrows; E and inset); some GFAP-positive astrocytes apparently left the caudal host front to enter the graft side (; F, G and insets) whereas ED1-positive macrophages/microglia (|aE; H and inset) dispersed into both graft and host sides of the interface. Scale bars, 100µm (A–C, E–G), 50 µm (D, H; insets in A–C, E–G), 12.5 µm (insets in D, H). Digital images were adjusted for brightness and contrast.

2. Axonal re-entry into the caudal host cord was enhanced
At the 4^th week post-treatment, the contralateral intact hemicord was transected to allow unequivocal tracing of biotinylated dextran amine (BDA)-labeled axons from the rostral host cord through the graft into the caudal host cord. Among rats that received Ch^aseABC, 7/12 showed that BDA-labeled axons had successfully re-entered the caudal host cord as far as 5 mm from the interface (mean±SD; 3.18±0.98 mm, n=7). Horizontal sections of the Ch^aseABC-treated cords revealed a mean of 156 regenerating axons (n=3) crossing the distal graft-host interface. In contrast, in vehicle-infused rats (n=12), regenerating axons either skirted the distal graft–host interface or turned back into the graft interior at the interface.

3. Extents of cystic cavitation and macrophage infiltration were reduced
In all thoracic cords grafted with the SC-seeded mini-channel, cavitations were observable in the host tissue at both the proximal and distal graft-host junctions. Caudal host cords that received Ch^aseABC infusion however indicated significant reduction in the extent of cavitation (n=3, P<0.02), estimated at 45% of that which received vehicle infusion. Activated macrophages/microglia indicated by ED1-immunopositivity were found dispersed at and beyond the distal graft–host junction, fewer in cases that received Ch^aseABC infusion (49.25±16.94, n=4; Fig. 1H ) than those that lined up at the interface in cases of the vehicle infusion controls (mean=152; Fig. 1D ). Secondary damage due to invasion of activated macrophages was therefore diminished.

4. Astrocytes crossed the distal graft-host interface
In animals that received vehicle infusion, GFAP-positive astrocytes formed an intensifying front on the host side of the graft-host interface, indicative of intense astrogliosis (Fig. 1B ). In animals that received Ch^aseABC infusion, such a GFAP-positive front was much less intense (Fig. 1 F). In addition, GFAP-positive astrocytes apparently advanced from the distal graft–host interface into the graft tissue (Fig. 1F and G).

Within the graft or regenerated tissue cable
5. Ch^aseABC infusion did not clear CS56 immunopositivity in the graft
Infused Ch^aseABC had limited access to the CS56-positive matrix of the graft tissue. This contrasted with the Ch^aseABC-susceptible, CS56-positive tissue at the distal graft-host interface and the adjoining caudal cord tissue (Fig. 1A and C). To find if the Matrigel (MG) matrix limited access of the enzyme to CS56-positive components of the graft, Western blot analysis of MG was performed. Indeed heterodisperse CS56-positive components (50-85 kDa) were not completely digestible by Ch^aseABC, possibly a result of MG gelation at 37°C. We found that acidification and subsequent neutralization of MG prevented gelation with no loss in CS56 epitopes. The CS56 epitopes in the resultant liquid form were then completely accessible to digestion by Ch^aseABC. The results suggest supramolecular interactions in the gel form limited access of the enzyme to CS56-positive components of the graft tissue.

6. Axonal growth and myelination within the regenerated tissue cable were enhanced
Cross sections of the regenerated tissue cable that formed within the channel showed widespread fascicles of myelinated axons in recipients of Ch^aseABC infusion. EMs of such sections revealed that the regenerated axons were either myelinated or ensheathed by SCs and that these axon-SC units were arranged in fascicles subtended by cell layers in perineurium-like arrangement. This tissue organization was evidently supportive of the abundance of myelinated axons (1,500 per tissue cable, n=5) that coursed through the cable. In contrast, cross sections of tissue cables of the vehicle-infused control group showed loose, fascicular organization with significantly fewer myelinated axons (400 per tissue cable, n=5, P<0.002). The fibers varied in axonal profile and extent of myelination. As both vascularization and cross-sectional area of the tissue cables did not differ between enzyme and vehicle infusion, these aspects were apparently unrelated to the enzyme-induced tissue organization of the cable.

CONCLUSIONS AND SIGNIFICANCE

We present evidence that CS moieties deposited in the gliotic front of the distal graft-host interface constituted a molecular barrier to axonal advancement into the caudal host cord and that its removal by the action of infused Ch^aseABC in vivo was a means to diminish the barrier effect (Fig. 2 ). The timely removal of CS glycoforms not only improved the interface environment for axonal exit from the regenerated tissue cable but also supported tissue reorganization within the cable.

View larger version (76K): [in this window] [in a new window]   Figure 2. Schematic diagram showing changes at the distal graft–host interface as a result of ChaseABC infusion. In vehicle-infused controls, regrowing axons skirt the distal interface where deposits of CS56-positive glycoforms were found to build up among ED1-positive macrophages/microglia against a front of reactive astrocytes. As ChaseABC infusion clears away CS56-positive glycoforms, macrophages/microglia disperse, astrocytes migrate into the regenerating tissue cable, and regrowing axons can negotiate across the interface into the caudal host cord. The dashed outline marks the boundary of a CS56-negative but 2B6-positive zone corresponding to the CS56-positive deposit in the vehicle-infused controls.

At the distal graft–host interface in vehicle-infused animals, we observed a fuzzy CS56-positive zone alongside the buildup of reactive astrocytes. Production of CS proteoglycans could have been increased when astrocytes interfaced with SCs. Macrophages that later infiltrated into the distal graft–host interface may well secrete soluble factors to trigger withdrawal and exile of the pioneer astrocytes. In addition, macrophages and oligodendrocyte precursor cells have been reported to contribute CS proteoglycans in the injured spinal cord. The relative importance of the different core protein domains and their CS substituents to the axon-restrictive property of the gliotic front remains unresolved. Our results however bear out that CS glycoforms produced at the gliotic front constitute a molecular impediment to axonal advancement.

Our intra-parenchymal infusion of Ch^aseABC into the caudal host cord close to the distal graft–host interface demonstrated accessibility of CS56-positive deposits at the interface to in vivo actions of the enzyme. This micro-regulated delivery paradigm represents an effective alternative to repeated injections or gelfoam administration of Ch^aseABC to target CS glycoforms in the injured CNS. However, we observed that the enzyme was limited in its access to CS56 epitopes contributed largely by Matrigel in the graft tissue. Nevertheless, regrowing axons that were ensheathed or myelinated by the grafted SCs made the way through the CS-enriched graft environment. We reasoned that regrowing axons that skirted the CS barrier in our vehicle-infused animals were unaccompanied by SCs as they negotiated the CS barrier at the distal graft–host interface. Our observations of BDA-labeled regrowing axons in the caudal host cord indicated that regrowing axons could advance through diminished barrier effects at the interface as a result of Ch^aseABC treatment.

The diminished CS barrier at the gliotic front of Ch^aseABC-infused animals apparently supported astrocyte entry into the graft and macrophage dispersal from the distal graft-host interface. Our results further suggested that astrocyte entry into the regenerating tissue cable supported preservation of SC-myelinated axons and fascicular organization of the cable. The dispersal of ED1-immunopositive cells at the distal graft–host interface, in correlation with the decreased extent of cavitation, suggests subdued secondary injury on regrowing axons that approached and crossed the interface. We expect growing ends of ascending fibers that reach the interface following digestive removal of the CS barrier can also cross the distal interface into the transplant and thus contribute to the changed tissue organization at both the interface and the regenerating tissue cable.

Altogether, Ch^aseABC infusion into the distal graft-host interface promises to advance prospects of the therapeutic use of SC-seeded guidance channels in bridging axonal regrowth across the site of traumatic injury in the spinal cord.

FOOTNOTES

³ Correspondence: Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville School of Medicine, 511 South Floyd St., Louisville, KY 40292, USA. E-mail: xmxu0001@gwise.louisville.edu

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0196fje;

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