Effects Of Degrading Up-expressed CSPGs With Chondrointinase ABC On Axonal Regeneration And Survival Of Rats Retinal Ganglion Cells In Vivo Following Optic Nerve Crush

ObjectivesThe failure of adult mammalian central nervous system to support axonal regeneration has been attributed to an abundance of inhibitory molecules. Traumatic injury of the CNS causes robust glial reactions. The cell surface and extracellular matrix molecules produced by reactive glia may be inhibitory to axonal regeneration. As one of the putative components of the Extracellular matrix (ECM), Chondrointin sulfate proteoglycans(CSPGs) are particularly abundant in the nervous system and are highly expressed in CNS injury. In present study, we tested the expression of CSPGs after optic nerve crush and the effect of degrading up-expressed CSPGs on axonal regeneration as well as survival of RGC in vivo by means of intrathecal treatment of chondrointinase ABC. Based on these observation, we suggested that their manipulation will be useful for treatment of human optic nerve injury.Methods1. To evaluate the expression of CSPGs after optic nerve injury, we use CS-56 antibody which recognizes the chondrointin sulfate carbohydrate moiety to stain CSPGs.2. To test the effect of degrading up-expressed CSPGs on axonal regeneration as well as survival of RGC in vivo by means of intrathecal treatment of chondrointinase chABC, we use flurogold retrograde tracing to stain RGCs and actived microglia, we also use WGA anterograde tracing and electron microscopy to evaluate axonal regeneration of RGCs.Results1. Immunocytochemical labeling of CSPGs after optic nerve crush show that CSPGs is upregulated at the injury site and the adjacent area, peaks at 5d, then 7d after injury expression begin gradually deline. Two weeks after injury CSPGs expression reduced to nomal level. At high resolution, we found that CSPGs is highly colocalized with surfaceof actived cells and the boundary of cavities.2. Degrading CSPGs with treatment of chondrointinase ABC can rescue some RGCs , and the survival RGC significantly increased from 79% lw post-injury, 36% 2w post-injury, 24% 3w post-injury to 89%, 48%, 28% respectively compared to control eyes(p<0.05).3. Treatment of chondrointinase ABC can reduce glial response to injury, decrease the population of labeled microglia and retard activation of microglia, with the consequence of reducing non-selective phagocytosis of degenerating but still surviving RGCs.4. Treatment of chondrointinase chABC can break down inhibitory molecules at the lesion site, thereby clearing the path for axons to sprout and improving more axons to regenerate unhindered.Conclusions1. CSPGs is upregulated in optic nerve after injury which contribute to inhibiting axon regeneration.2. Degrading CSPGs with treatment of chondrointinase ABC can rescue some RGCs and reduce activation of microglia. Furthermore it can clearing the path for axons to sprout and improving more axons to regenerate, but it is necessary to note that combination of blocking the inhibitory signaling pathways and enhancing the intrinsic neuronal regenerative capacities will result in synergistic effects in stimulating axon regeneration.