Delayed Applications of L1 and Chondroitinase ABC Promote Recovery after Spinal Cord Injury

Abstract

The inhibitory environment of the injured spinal cord is an obstacle to functional recovery and axonal regeneration in adult mammals. We had previously shown that injection of adeno-associated virus (AAV) encoding the L1 cell adhesion molecule (AAV-L1) at the time of acute thoracic compression injury of adult mice promotes locomotor recovery, which is associated with ameliorated astrogliosis and improved axonal regeneration in the lumbar spinal cord. In the present study, we investigated whether delayed injection of AAV-L1, chondroitinase ABC (Chase), or the combination of the two agents into the mouse spinal cord 3 weeks after injury would also lead to improved recovery. The Basso Mouse Scale showed enhanced locomotor recovery 12 weeks after application of the agents in all treatment groups compared to the control group that was injected with AAV encoding green fluorescent protein (AAV-GFP). Investigation of hindlimb function using single-frame motion analysis revealed, however, that L1 overexpression, but not injection of Chase, improved voluntary movements without body weight support, whereas injection of Chase, but not L1 overexpression, enhanced body weight support during stepping. Mice with the combined application of AAV-L1 and Chase showed improvement in both parameters. Enhanced motor recovery after combined application correlated with increased densities of cholinergic and GABAergic terminals at motoneuronal cell bodies, and of lamina-specific glutamatergic sensory afferents 15 weeks after injury, indicating enhanced synaptic rearrangements in the lumbar spinal cord below the lesion site. These findings suggest that L1 overexpression combined with Chase application may contribute to the treatment of sub-chronic spinal cord injury.

Extrinsic and intrinsic regulation of axon regeneration at a crossroads

“…Chondroitinase ABC (chABC), a bacterial enzyme that digests the GAG chains, abrogates CSPG-dependent neurite outgrowth inhibition in vitro and improves neurite sprouting and functional recovery after SCI (Bradbury et al, 2002). The efficacy of chABC alone and in combination with other approaches in promoting recovery after SCI has been independently confirmed in multiple laboratories and is a promising approach for future translation (Alilain et al, 2011; García-Alías et al, 2011; Lee et al, 2012). Mechanistically, contrary to the view that CSPGs repel growth cones, recent studies from the Silver group suggest that CSPGs in fact tightly adhere to and confine growth cones (Filous et al, 2014; Lang et al, 2015).…”

Transgenic overexpression of the cell adhesion molecule L1 in neurons facilitates recovery after mouse spinal cord injury

“…We thus propose that these additional effects, generally accepted to be beneficial for regeneration, were due to ectopic L1 expression by astrocytes via AAV. Interestingly, when the same L1 carrying virus was applied to the spinal cord 3 weeks after SCI, L1 promoted functional recovery, but did not alter GFAP scar formation when compared to application of AAV-GFP (Lee et al, 2012). It thus appears that, both, the cell types expressing L1 and the timing of expression after SCI, are critical for the effect of L1 on glial scar formation.…”

“…Spaced serial 25-lm-thick transverse spinal cord sections 250 lm apart were immunostained for CGRP in the lumbar spinal cord. We estimated the area of the immunopositive structures normalized to the total image area using ImageJ software as described (Lee et al, 2012) using images obtained on an LSM 510 confocal microscope (Zeiss) with a 40Â oil-immersion objective and a digital resolution of 1024 Â 1024 pixels. Images of the CGRP immunostained area in the dorsal horns were taken from three consecutive transverse sections (i.e.…”

“…To investigate expression of GFAP rostral to the lesion site we estimated the area of the immunopositive structures normalized to the total image area using ImageJ software, as described previously (Lee et al, 2012;Wu et al, 2012). This estimation was performed on images obtained on an LSM 510 confocal microscope (Zeiss) using a 40Â oil-immersion objective and a digital resolution of 1024 Â 1024 pixels.…”

“…First evidence that L1 could promote regeneration of the adult mammalian central nervous system (CNS) was obtained by using human recombinant extracellular domain of L1-Fc fusion protein to improve recovery after spinal cord injury (SCI) in rats (Roonprapunt et al, 2003). When L1 was applied acutely or 3 weeks after injury via adeno-associated virus (AAV-L1) to mouse spinal cord, leading to overexpression of L1 in neurons and ectopic expression in astrocytes, motor recovery was improved compared to control virus injected mice Lee et al, 2012). In another mode of delivery, L1 overexpressing embryonic stem cells (Chen et al, 2005), stem cell-derived neural aggregates , Schwann cells (Lavdas et al, 2010) and radial glia were shown to promote recovery when compared to control conditions without L1 overexpression.…”