Chronic Enhancement of the Intrinsic Growth Capacity of Sensory Neurons Combined with the Degradation of Inhibitory Proteoglycans Allows Functional Regeneration of Sensory Axons through the Dorsal Root Entry Zone in the Mammalian Spinal Cord

Abstract

Peripherally conditioned sensory neurons have an increased capacity to regenerate their central processes. However, even conditioned axons struggle in the presence of a hostile CNS environment. We hypothesized that combining an aggressive conditioning strategy with modification of inhibitory reactive astroglial-associated extracellular matrix could enhance regeneration. We screened potential treatments using a model of the dorsal root entry zone (DREZ). In this assay, a gradient of inhibitory chondroitin sulfate proteoglycans (CSPGs) stimulates formation of dystrophic end bulbs on adult sensory axons, which mimics regeneration failure in vivo. Combining inflammation-induced preconditioning of dorsal root ganglia in vivo before harvest, with chondroitinase ABC (ChABC) digestion of proteoglycans in vitro allows for significant regeneration across a once potently inhibitory substrate. We then assessed regeneration through the DREZ after root crush in adult rats receiving the combination treatment, ChABC, or zymosan pretreatment alone or no treatment. Regeneration was never observed in untreated animals, and only minimal regeneration occurred in the ChABC-and zymosanalone groups. However, remarkable regeneration was observed in a majority of animals that received the combination treatment. Regenerated fibers established functional synapses, as demonstrated electrophysiologically by the presence of an H-reflex. Two different postlesion treatment paradigms in which the timing of both zymosan and ChABC administration were varied after injury were ineffective in promoting regeneration. Therefore, zymosan pretreatment, but not posttreatment, of the sensory ganglia, combined with ChABC modification of CSPGs, resulted in robust and functional regeneration of sensory axons through the DREZ after root injury.

Sensory afferents regenerated into dorsal columns after spinal cord injury remain in a chronic pathophysiological state

“…Since the regenerated sensory axons did not form synapses with their targets in the brainstem, it remains unknown how synapse formation will affect the physiology of these axons. After dorsal root crush, sensory fibers have been reported to regenerate into the dorsal horn (Ramer et al 2000;Steinmetz et al 2005). Stimulation of the dorsal root also evokes low amplitude, long latency responses within the dorsal horn.…”

“…Antibody-mediated blockade of growth-inhibitory molecules (Hata et al 2006;Tan et al 2006), enzymatic degradation of growth-inhibitory glycosaminoglycans (GAGs) (Moon et al 2001;Bradbury et al 2002), and pharmacological elevation of cAMP (Qiu et al 2002;Nikulina et al 2004) all promote axon regeneration after spinal cord injury, as does a peripheral nerve conditioning-lesion (Richardson and Issa,1984;Neumann and Woolf, 1999). Combining some of these treatments has resulted in more favorable axon growth patterns (Lu et al 2004;Steinmetz et al 2005;Tan et al 2006), longer fibers (Chau et al 2004;Yin et al 2006), and in some cases, limited behavioral recovery (Pearse et al 2004;Fouad et al 2005).…”

CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure

“…The rapid and long lasting upregulation of proteoglycans within the vicinity of the glial scar has implicated them in the creation of nonpermissive growth environments in the CNS, similar to their role in boundary formation within the CNS during development (Fitch and Silver, 1997b;Grimpe and Silver, 2004;Silver, 1994;Snow et al, 1990). It is now well established that proteoglycans associated with reactive astrocytes clearly inhibit neurite outgrowth in vitro (Bovolenta et al, 1993;Canning et al, 1993;Dou and Levine, 1994;McKeon, et al, 1991;Snow et al, 1990;Tom, et al, 2004b), and these molecules also appear to play a key role in creating an environment that is not appropriate for successful long-distance regeneration of adult neurons after injury in vivo, since their modification allows successful regeneration to occur (Bradbury et al, 2002;Houle et al, 2006;Steinmetz et al, 2005).…”

“…In addition to pro-inflammatory cytokines, macrophages can secrete factors that are growth promoting, such as NGF and NT-3 (Elkabes et al, 1996), thrombospondin (Chamak et al, 1994), and IL-1 (Giulian et al, 1994). Even zymosan, a trigger of robust inflammation, when placed in the vitreous chamber of the eye can stimulate regenerating optic nerve fibers (Leon et al, 2000) and when placed into the DRG before root crush appears essential to the success of DRG regeneration into the spinal cord when combined with modification of the extracellular matrix in the CNS compartment (Steinmetz, et al, 2005). Apparently intense inflammatory states created just outside of the CNS illustrate that in certain circumstances inflammation can promote enhanced regeneration by triggering a conditioning-like effect within the neuron (Calvo et al, 2005;Lund et al, 2002;McQuarrie and Jacob, 1991;Wu et al, 2007a).…”

Biomaterials for spinal cord repair

“…The enzymes chondroitinase (ChABC) (for review see [148] ) and sialidase [149,150] , or a combination of the two were shown to decrease growth inhibition [151] . Many groups have shown that administration of ChABC is associated with sensory axonal regeneration [152] and functional recovery [148,152,153] when experimentally introduced following SCi. When comparing the two, one group found that sialidase increases axonal regeneration and functional recovery [149][150][151] to a greater degree than either ChABC alone or a sialidase/ChABC combination [151] .…”