Axonal Regeneration And Functional Recovery After Spinal Cord Injury By Combined Chondroitinase And Clenbuterol Treatment

ObjectiveThe adult mammalian central nervous system(CNS)can not normally regenerate after injury.The failure of axons to regenerate after spinal cord injury(SCI)is now attributed to two main obstacles:inhibitors within myelin and a physical barrier presented by the formation of glial scar.In the lesion core,several myelin-associated inhibitors of axonal regeneration,namely myelin-Nogo-A,myelin-associated glycoprotein(MAG),oligodendrocyte myelin glycoprotein(OMgp),and various inhibitory extracellular matrix molecules in the glial scar have been identified to play a crucial role in regeneration failure.Chondroitin sulfate proteoglycans(CSPGs)which produced by reactive astrocytes are one major inhibitory family of extracellular matrix molecules,which includes aggrecan,brecican,neurocan,NG2,phosphacan and versican.Chondroitinase(chABC)was confirmed very effective to remove CSPGs from the lesion site and produce a pro-regenerative environment in vivo by many experiments.Some combinatory strategies involving use of various cellular/acellular implants at the transection site combined with treatments to support axonal regeneration have had some success.Such as,chABC combined with Schwann cell-seeded guidance channels,combining Schwann cell bridges and olfactoryensheathing glia grafts with chABC,combining an autologous peripheral nervous system "bridge" and matrix modification by chondroitinase,and transplantation of a cellular fibroblast bridge(fibroblasts genetically modified to secrete NGF)in a spinal cord lesion site,and so on.A promising strategy to overcome the inhibitory aspect of the injured spinal cord environment is elevation of cyclic adenosine monophosphate(cAMP)levels.The cAMP effect requires activation of the transcription factor cAMP response element binding protein(CREB)to overcome myelin inhibitors.Theβ₂-adrenergic receptor (β₂AR)agonist Clenbuterol(CLEN),which increases neuronal cAMP levels,a defining action ofβ₂ agonists,can improve recovery of locomotor function following spinal cord injury.Electrophoretic mobility shift assays revealed thatβ₂AR agonist CLEN or high levels of cAMP elicited increase in CCAAT/enhancer binding proteinδ(C/EBPδ)binding activity as well as phosphorylated CREB.Here,we determined whether axonal regeneration could be promoted directly by degrading CSPGs enzymatically within the injury site with chABC and combined with CLEN,without a requirement for implantation.MethodsIn C7 hemisection experiment,the objective of this study is to explore the efficacy of Clenbuterol to promote axonal regeneration and recovery of locomotor function after SCI.Wistar rats(n=10-12)received a unilateral hemisection within the dura mater at C7 spinal cord segment.Clenbuterol was administered by adding it to the drinking water at a dose of 9 mg/L.Locomotor function was observed with Cylinder test and Grip strength test.For retrograde axonal tracing,after the predetermined period of observation(6 weeks),rats underwent laminectomy at T2 and 4%Fluorogold was injected into rubrospinal tract(RST).The rats were sacrificed 1 week later.In T10 complete transection experiment,groups of Wistar rats(n=5-8)were completely transected at T10 and received chABC at the transection site(10μL,2.5 units/ml and dorsally in gelfoam)and/or clenbuterol(9 mg/L,drinking H₂O)or were untreated.Locomotor function was determined for 12 weeks post-injury with the 21-point BBB locomotor scale.For retrograde tracing,4%Fluorogold was applied at T13 at 12 weeks postinjury.ResultsIn C7 hemisection experiment,locomotor function progressive recovery started to become apparent at 3 weeks after injury and was significantly greater in Clenbuterol treatment group compared to the untreated controls(final Grip force:403.45±19.82g vs 335.13±13.48g,p＜0.05),and Clenbuterol treated animals used both forelimbs more frequently for vertical exploration(43.39%±5.85%vs 19.42%±6.61%,p＜0.05). Clenbuterol treatment significantly decreased secondary damage after SCI and increased the pCREB positive nuclei throughout the hemisection site.Retrograde axonal tracing revealed Clenbuterol enhanced the regeneration of RST axons.In T10 complete transection experiment,progressive recovery of locomotor function was observed 8-12 weeks postinjury only in the combined chABC and clenbuterol treatment group compared to untreated controls(final BBB score:3.94 vs 1.17,P＜0.05,SNK). Retrograde axonal tracing revealed axonal regeneration,from neurons of the reticular, vestibular,and red nuclei also only in the combined treatment group.RT-97 positive axons ensheathed by Schwann cells were most prominent in the transection site in the combined treatment group.ChABC treatment increased astrocyte number in the proximal and distal stumps aiding blood/cord barrier reformation while decreasing both CSPG and collagen deposition and gap length that are barriers to axon regeneration. Clenbuterol treatment increased numbers of cells with pCREB positive nuclei throughout the transection site which has been associated with neuroprotection and axonal regenerationConclusions1.Clenbuterol promotes the regeneration of axotomized axons and recovery of locomotor function after SCI.2.Chondroitinase can decrease the infiltration of connective tissue after complete spinal cord transaction,reduce the glial scar,and remove the inhibitor CSPGs,produce a proregenerative environment for injured axons.3.ChABC and clenbuterol act synergistically to promote axonal regeneration after complete spinal cord injury.