Antibodies against the NG2 Proteoglycan Promote the Regeneration of Sensory Axons within the Dorsal Columns of the Spinal Cord

Abstract

The NG2 chondroitin sulfate proteoglycan inhibits axon growth in vitro. Levels of NG2 increase rapidly in the glial scars that form at sites of CNS injury, suggesting that NG2 may inhibit axon regeneration. To determine the functions of NG2, we infused mixtures of neutralizing or non-neutralizing anti-NG2 monoclonal antibodies into the dorsally transected adult rat spinal cord and analyzed the regeneration of ascending mechanosensory axons anatomically. At 1 week after injury, ascending sensory axons in control animals terminated caudal to the lesion within an area containing dense deposits of NG2 immunoreactivity. In animals treated with the neutralizing anti-NG2 antibodies, labeled axons penetrated the caudal border of the lesion and grew into and beyond the lesion center. The low intrinsic growth capacity of adult neurons may also limit the ability of damaged axons to regenerate. To enhance growth, we combined antibody treatment with a peripheral nerve conditioning lesion. After a conditioning lesion and treatment with control, non-neutralizing antibodies, many sensory axons grew into the lesion core. These axons did not grow past the rostral border of the lesion; rather, they grew along the dorsal surface of the spinal cord and within any remaining pieces of the dorsal roots. In contrast, combining a peripheral nerve conditioning lesion with neutralizing anti-NG2 antibodies resulted in sensory axon regeneration past the glial scar and into the white matter rostral to the injury site. The combinatorial approach used here that neutralizes extrinsic inhibition and increases intrinsic growth results in anatomically correct axon regeneration, a prerequisite for functional recovery.

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

“…We showed previously that transected large and medium diameter sensory axons can regenerate past the glial scar when animals are given a combinatorial treatment consisting of a prior peripheral nerve conditioning lesion and the infusion of the spinal cord lesion site with monoclonal antibodies that neutralize the growth inhibitory properties of the NG2 chondroitin sulfate proteoglycan (Tan et al 2006). A peripheral nerve conditioning lesion alone is sufficient to promote some axonal regeneration; however, those axons grow in ectopic locations, mostly along the dorsal surface of the spinal cord.…”

“…Adult Sprague Dawley rats (∼150-175g) were anesthetized with a mixture of ketamine and xylazine and subjected to a peripheral nerve conditioning-lesion 1 week prior to spinal injury and antibody application. The spinal cord injury model and antibodies used here have been described previously (Tan et al 2006). Following a dorsal laminectomy at thoracic level 8, the dura was resected to expose the spinal cord.…”

“…Prior work demonstrated 2 populations of regenerating dorsal column axons; those that regenerated on the surface of the cord, and those whose regeneration through the dorsal column is dependent on neutralizing anti-NG2 antibodies treatment (Tan et al 2006). Rostral to the injury, the stimulation electrode was placed at the coordinates (provided by results of the stimulation grid) that yielded the largest CAP from the "deep" regenerated axons.…”

“…Immunofluorescence staining was carried out as described previously (Tan et al 2006) using the following antibodies. Primary antibodies used were: rat anti-MBP (Serotec 1:100), goat anti-CTB antibody (List Biological Laboratories, 1:5000), and rabbit anti-GFAP (DAKO, 1:2000).…”

“…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).…”