Local Delivery of High-Dose Chondroitinase ABC in the Sub-Acute Stage Promotes Axonal Outgrowth and Functional Recovery after Complete Spinal Cord Transection

Abstract

Chondroitin sulfate proteoglycans (CSPGs) are glial scar-associated molecules considered axonal regeneration inhibitors and can be digested by chondroitinase ABC (ChABC) to promote axonal regeneration after spinal cord injury (SCI). We previously demonstrated that intrathecal delivery of low-dose ChABC (1 U) in the acute stage of SCI promoted axonal regrowth and functional recovery. In this study, high-dose ChABC (50 U) introduced via intrathecal delivery induced subarachnoid hemorrhage and death within 48 h. However, most SCI patients are treated in the sub-acute or chronic stages, when the dense glial scar has formed and is minimally digested by intrathecal delivery of ChABC at the injury site. The present study investigated whether intraparenchymal delivery of ChABC in the sub-acute stage of complete spinal cord transection would promote axonal outgrowth and improve functional recovery. We observed no functional recovery following the low-dose ChABC (1 U or 5 U) treatments. Furthermore, animals treated with high-dose ChABC (50 U or 100 U) showed decreased CSPGs levels. The extent and area of the lesion were also dramatically decreased after ChABC treatment. The outgrowth of the regenerating axons was significantly increased, and some partially crossed the lesion site in the ChABC-treated groups. In addition, retrograde Fluoro-Gold (FG) labeling showed that the outgrowing axons could cross the lesion site and reach several brain stem nuclei involved in sensory and motor functions. The Basso, Beattie and Bresnahan (BBB) open field locomotor scores revealed that the ChABC treatment significantly improved functional recovery compared to the control group at eight weeks after treatment. Our study demonstrates that high-dose ChABC treatment in the sub-acute stage of SCI effectively improves glial scar digestion by reducing the lesion size and increasing axonal regrowth to the related functional nuclei, which promotes locomotor recovery. Thus, our results will aid in the treatment of spinal cord injury.

Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury

“…On average, the lesion area was approximately 45% smaller in animals that received Lenti-Chase (0.44mm 2 in control, 0.24mm 2 in control, p=.013, paired t-test). This reduction in lesion size is consistent with a recent report that chondroitinase treatment reduces lesion volume after spinal contusions, likely by modulating macrophage responses (Bartus et al, 2014; Cheng et al, 2015). Immunohistochemistry for 2B6, the carbohydrate stub produced by chondroitinase-mediate cleavage of GAG chains from CSPGs, confirmed degradation in Lenti-chondroitinase treated animals (Figure 6).…”

“…These data confirm efficient viral transduction of lumbar DRG neurons, and indicate that the majority of dextran-positive DRG axons in the spinal cord (see below) arose from virally transduced cell bodies. To assess viral-mediated degradation of CSPGs, sagittal sections of spinal cord spanning the injury site were prepared and immunohistochemistry performed with 2B6 and C4S antibodies, which recognize carbohydrate stub epitopes generated by chondroitinase’s cleavage of GAG chains (Bartus et al, 2014; Bukhari et al, 2011; Cafferty et al, 2008; Cheng et al, 2015). 2B6 and C4S reactivity were both readily detectable in animals injected with Lenti-Chase, but not vehicle control (Fig.…”

“…CSPGs consist of a protein core adorned with sulphated glycosoaminoglycan (GAG) side chains (Bandtlow and Zimmermann, 2000), and inhibit axon extension in a variety of in vitro assays (Snow et al, 1990; Tom et al, 2004; Usher et al, 2010). Chondroitinase, a bacterial enzyme that cleaves GAG side chains, has been shown repeatedly to reduce CSPG’s inhibitory properties in vitro (Niederost et al, 1999; Snow et al, 1990; Yamada et al, 1997), and to promote axon growth in vivo after spinal injury (Bartus et al, 2012; Bradbury et al, 2002; Cheng et al, 2015; Iseda et al, 2008). Thus chondroitinase-mediated degradation of CSPGs has emerged as an important component of combinatorial treatments to promote axon regeneration (Hunanyan et al, 2013; Kanno et al, 2014; Lee et al, 2013; Steinmetz et al, 2005; Tom et al, 2009; Tropea et al, 2003).…”

Bridging Defects in Chronic Spinal Cord Injury Using Peripheral Nerve Grafts: From Basic Science to Clinical Experience

“…Injected intrathecally in acute injuries at a low dose (1 or 5 IUs), chondroitinase ABC may enhance axonal progression; injected at a high dose (50 IU), it may produce subarachnoid haemorrhage. In subacute or chronic injuries, low-dose injection produces no or limited functional recovery, whilst high-dose injection (50 or 100 IUs) produce lysis of the gliosis [68,69].…”

Novel Medical Management of Spinal Cord Injury

“…Studies have shown that by treating a CNS lesion site with the enzyme chABC both axonal growth and axonal sprouting into and around the lesion are significantly increased [6]. The use of this agent has been shown to reverse CSPG inhibition efficiently and promote axonal sprouting and outgrowth as well as to enable the migration and differentiation of endogenous OPCs [7].…”