Developing The Strategies Of Promoting Axon Regeneration And Functional Recovery After Central Nervous Injury

Injured central nervous system(CNS)axons are incapable to regenerate after spinal cord injury and stroke in adult mammals.The failure of axon regeneration is due to the inhibitory external environment and lack of intrinsic neruonal regrowth abilities [1].Exogenous axonal inhibitory factors include myelin-associated inhibitory molecules,glial scar formation,inflammation,etc.[2].The endogenous factors of axonal regeneration are developmentally related genes,transcription factors,cytoskeletal regulatory factors,mitochondria,etc.[3].Activation of axonal growth by neutralizing exogenous inhibitory molecules such as NOGO,MAG,CSPG(by chondroitinase ABC),immunoregulation to improve the external environment of axon growth;regulation of signal transduction pathways such as mTOR,STAT3,KLFs,b-RAF and SOX11 together with growth factor BDNF,CNTF,IGF1,can promote CNS axonal regeneration,however,there are few that can be applied to clinical.OPN was a soluble protein and first found in the bone matrix.It was later found to be a cytokine widely present in various organs.OPN binds to integrins or CD44 on the cell surface which transduce intracellular signaling,activate kinase cascades and transcription factors to promote cell adhesion,movement and survival [4].Our previous studies have shown that OPN combined with IGF1 / CNTF can promote axonal regeneration after optic nerve injury [5,6];In addition,potassium channel blockers 4-AP and its derivatives 4-AP-3-Me further promote axonal conduction rates,which achieved signficant improvement of the visual sensitivity after injury [5].However,in SCI and stroke models,whether OPN / IGF1 can promote CST regeneration,and thus promote the recovery of animal function is yet unknown.PD-1 is a type I transmembrane protein which consists of an IgV-like extracellular segment,a transmembrane region,and an intracellular segment.The intracellular fraction of PD-1 is divided into two motifs: ITSM and ITSM [7].PD-1 belongs to one of the members of the CD28 family [8].PD-1 receptor(PD-L1 / PD-L2)is widely expressed in T cells,B cells,NKT cells,macrophages and DC [9,10].In T cells,PD-1 interacts with its receptors to inhibit cell proliferation and cytokine secretion [11,12].In B cells,PD-1 can inhibit B cell activation,proliferation and antibody synthesis [13].In PD-1 knockout mice,it is found that PD-1 is a negative regulator of the immune response and plays an important role in both central and peripheral tolerances [10,14].Study indicated that there is increasing expression of PD-1 on microglia and macrophages after stroke.PD-1 knockout mice have lager infarct volume compared with wild mice [15].Our lab found that the function recovery of PD-1 knockout mice is poorer compared with wild type mice after SCI,PD-1 deletion promotes of microglia/macrophages polarizing to M1 type both in vitro and in vivo.For the first part,we established two models,the pyramidotomy and the photochemical embolization stroke models to study functional recveory after CNS damage.We injected AAV-mcherry and AAV-OPN / IGF1 or AAV-PLAP on the contralateral side of the lesion.Behavior measurement inclucing food retrieval,sticker remove,pasta handling,and irregular ladder walking was conducted every other week.At 14 weeks after injury,4-AP-3-MeOH was injected intraperitoneally,then the performance of the food retrieval and irregular ladder walking were measured.Followed by the elimination of sprouting axons,we continue to carry out the relevant behavioral testing.Finally,the animals were sacrificed at 20 weeks after injury,and immunohistochemical staining was performed to analyze the lateral sprouting of the axons.For the second part,the expression of PD-1 and PD-L1 / PD-L2 was detected by flow cytometry after culturing microglia / macrophages stimulating with LPS + IFN-?.Then,macrophages derived from wild type mice,PD-1 knockout mice,PD-1 overexpressing mice and PD-L1 knockout mice were cultured respectively,and co-cultured with LPS and IFN-? for different times.We used molecular cloning and pharmacology treatment to analyze the molecular mechanism of PD-1 regulation of macrophage polarization.There was no significant behavioral differences between the experimental and the control groups in the pyramidotomy model.However,the injection of the AAV-OPN / IGF1 were significantly more effective than the AAV-PLAP group in the photochemical embolization model with diameter of 4mm.Although the sticker remove showed spontaneous recovery,irregular ladder walking of the experimental group recovered better than the control group.The number of axonal spouting in the experimental group was significantly more than that in the control group in the two injury models.Then we performed a photochemical embolization model with a diameter of 2.5 mm.AAV-OPN / IGF1 or AAV-PLAP was also injected into the contralateral cortex.Food retrieval and irregular ladder walking were performed after injury.Functional recovery of the OPN/IGF1 group was significantly better than the AAV-PLAP group after eighth weeks of injury.After treatment with intraperitoneal injection of potassium channel blocker 4-AP-3-MeOH,the behavior analysis of AAV-PLAP group did not change,while the locomotion recovery of AAV-OPN / IGF1 mice was better than before.There was no difference in the volume of stroke between the AAV-OPN / IGF1 and AAV-PLAP group.However,the axon sprouting number and intensity of the AAV-OPN / IGF1 group were higher than those of the AAV-PLAP group at different levels of the cortex,the C6 cervical and L3 lumbar segments of the spinal cord.The functional recovery of motor function of the AAV-OPN / IGF1 group also disappeared after ablation of neurons that collaterally sprout to the deneravted cercial spinal cord.The number of the sprouting axons was reduced in the cervical,but not lumbar,levels after ablation of neurons.In vitro culture of macrophages or microglia found that under the stimulation of LPS + IFN-?,microglia / macrophages can express PD-1 and its receptor PD-L1,but not PD-L2.The expression of iNOS was much higher in PD-1 or PD-L1 deletion macrophages after LPS and IFN-? treatment for different time points,compared with wild-type macrophages.Meanwhile,the proportion of cells with TNF-? positive expression was also increased.Stat1,p-Stat1 and p-NF-?B were also increased,and AKT /mTOR PD-1 were activated in the PD-1 knockout and PD-L1 knockout macrophages.The expression of Stat1 and NF-?B remained unchanged compared with wild type macrophages,but the activity of S6 K was significantly decreased in the PD-1 overexpression macrophages.The expression of iNOS in PD-1 knockout macrophages was significantly lower than that in the control group after the application of rapamycin.It suggests that PD-1 knockout may promote macrophage polarization to M1 by Akt / mTOR pathway.Through the above experiments,it was suggested that OPN / IGF1 could promote the sprouting of CST after CNS injury,and promote the functional recovery of stroke.It was possible that PD-1 deletion promotes macrophage to M1 polarization by activating Akt / mTOR signaling pathway.Our study provides experimental evidence for the clinical application of OPN / IGF1 to promote axonal regeneration in the CNS injury and to understand the molecular mechanism of PD-1 regulation of macrophage polarization.