Establishment Of An In Vivo Mouse Model For Discogenic Low Back Pain And The Involvement Of VEGF/VEGFR1 Signal In The Pathogenesis Of Discogenic Low Back Pain

Part Ⅰ Establishment and assessment of a mouse model for discogenic low back painBackgroundLow back pain(LBP),which affects over 80% of the adults during their lives,is one of the most common symptoms in clinic.Currently,it is the global leading cause of disability.Although any of the spinal structures could be a source of LBP,the intervertebral disc(IVD)and the associated diseases are thought to be the most common detectable cause.Discogenic low back pain(DLBP)is the chronic intractable pain initiated by the internal disc disruption.Effective treatments are lacking due to DLBP’s unknown pathogenesis.Animal models are essential to understand the mechanism underlying discogenic back pain and screen drugs for this disease.Rat models are usually adopted to study the association between pain and IVD degeneration.Currently,transgenic mice are used extensively in research as models of human disease and play a critical role in studying the mechanisms underlying different medical morbidities.However,there is no in vivo mouse discogenic pain model.Therefore,establishing a mouse model with pain behavior changes is necessary.Objectives1.To establish a reliable mouse DLBP model.2.To explore the possible mechanisms and pain pathways in this model.Experimental procedures1.The disc degeneration was generated by stabbing the lumbar discs(L4/5,L5/6,and L6/S1)with removal of a part of nucleus pulposus using a microscalpel under the microscope.2.Von-Frey was used to evaluate the hindpaw withdrawal threshold to mechanical stimuli after surgery.Response to noxious heat stimuli was determined using hot-plate.Responses to noxious cold stimuli were determined using the acetone test.Burrowing,ambulation,and rearing were used to evaluate the spontaneous behavior changes.3.Micro-CT scanning was used to evaluate the disc height changes at 2,4,8,and 12 weeks post-surgery.Histology staining were used to assess the alteration of extracellular matrix and the degeneration of nucleus pulposus(NP)and annulus fibrosus(AF).4.Immunofluorescence staining was used to assess the expressions of inflammatory cytokines(IL-1β,TNF-α),as well as the expressions of vascular and neural cytokines(VEGF,NGF)after 2,4,8,and 12 weeks of disc injury.PGP9.5 staining was used to evaluate the ingrowth of nociceptive nerve fibers into the disc via the tears in the AF.5.At 2,4,8,and 12 weeks following surgery,immunofluorescence double-staining was used to assess the expressions of different pain-related genes in the dorsal root ganglia(DRG),including CGRP,SP,NGF,Trk A.RT-PCR was performed to verify the differential expression levels of pain-related neurotransmitters between the injury group and the sham group.6.At 2,4,8,and 12 weeks post-surgery,immunofluorescence staining was used to evaluate the activation of astrocytes and microglia cells in the lumbar spinal cord(SC)dorsal horn.Results1.The pain behavior assessments showed that the mechanical withdrawal threshold of the injury group was significantly lower than the sham group from the 2 weeks to 12 weeks after surgery.Compared with the sham group,the injury mice showed the heat and cold hyperalgesia at 8 and 12 weeks post-surgery.The burrowing test demonstrated that the injury group had a burrowing deficit after 4,8,and 12 weeks following disc injury.The activity monitor demonstrated that spontaneous rearing activity was reduced in the DDD group as compared to the sham control group at 12 weeks after disc stabbing.2.The imaging results showed that the disc height decreased continuously in the injury group and the morphology of the end-plate had a degeneration change,including rough edges and osteophytes forming.The disc histology revealed progressively disc degeneration after surgery in which the notochordal cells was decreased,the proteoglycan content was progressively reduced,the AF lamellas were disorganized,and the border between the NP and AF became less distinct.3.The disc immunofluorescence staining demonstrated that the numbers of IL-1β positive and TNF-α positive cells were markedly increased at 2 and 4 weeks(early stage)after surgery,but decreased from 8 weeks to 12 weeks(late stage)in both the NP and AF areas.In contrast,the NGF and VEGF expressions were significantly increased from 2 weeks and maintained at 12 weeks after surgery.From the 4 weeks post-surgery,the peripheral nerve innervation was up-regulated in the AF area,even the nerve fibers ingrowth into the deeper NP area was found at 12 weeks following stabbing surgery.4.At 2,4,8,and 12 weeks,the expressions of CGRP,SP,NGF,Trk A in the injury group was significantly higher than the corresponding expressions in the sham group.Furthermore,the disc injury induced dramatically higher expression levels of pain-related neural peptides in the DRGs at 12 weeks after disc injury.5.At all time points,the number of astrocytes were significantly and progressively increased after surgery in mice with disc injury.The microglial cells,however,were dramatically elevated and reached the highest level at 2 weeks after surgery,and then gradually decreased.Conclusion1.The mice displayed hypersensitivity of the hindpaw to mechanical-,heat-,and cold-induced hyperalgesia along with decreased burrowing and rearing behaviors after disc injury,indicating that the animals exhibited discomfort and a painful state.The injury also induced a progressively disc degeneration which is similar to the clinical condition.These findings suggested that the disc stabbing with NP removal using a microscalpel established a reliable and reproducible mouse discogenic pain model.2.The cellular and molecular results showed that the disc injury also produced a progressive increase in neuropeptides in DRG and glial cells in the SC,as well as in deep nerve ingrowth,and a persistent increase in neurovascular mediators in the degenerated discs.Therefore,the pain pathway from this model could be used to investigate the mechanisms underlying pathologic processes related to development of discogenic pain,and was also benefit for screening the possible therapeutic drugs for DLBP.Part Ⅱ The involvement of VEGF/VEGFR1 signal in the pathogenesis of discogenic low back painBackgroundNew vascular vessel formation and high VEGF expression are associated with disc degeneration.VEGF receptor 1(VEGFR1)has been found involved in cancer pain.Our model showed that the expressions of VEGF and VEGFR1 were induced after disc injury,which suggests that VEGF/VEGFR1 signal may be involved in the development of discogenic pain.However,limited studies have examined the role VEGF/VEGFR1 plays in the pathogenesis of discogenic low back pain.ObjectiveTo investigate the role of VEGF/VEGFR1 signal in the pathogenesis of discogenic pain.Experimental procedures1.Establishment of discogenic pain model in the VEGFR1-TK-/-mice and Wt mice using procedures described in part I.2.Mechanical hyperalgesia,heat hyperalgesia,and burrowing behavior were used to compare the pain behavior changes between the VEGFR1-TK-/-and Wt mice.3.Micro-CT and histology were used to evaluate the disc degeneration at 12 weeks post-surgery.Immunofluorescence staining was used to assess the differential expressions of NGF in disc and pain neuropeptides in DRG,as well as activation of glial cells in SC.4.NP and AF cells were cultured in vitro and stimulated with VEGF and/or si RNA-VEGFRs for 24 hours.RT-PCR was used to detect the expressions of NGF and catabolic genes,including Adamts-5 and MMP-13.5.DRG neurons,DRG and SC organs from the VEGFR1-TK-/-and Wt mice were cultured in vitro and stimulated with VEGF.Cell immunofluorescence staining and RT-PCR were used to explore the differential expressions of neuropeptides(CGRP,NGF,Transient receptor potential vanilloid type-1(TRPV1),and Transient receptor potential ankyrin-1(TRPA1))in DRG neurons.RT-PCR was used to detect the gene differential expressions in the spinal cord,including glial fibrillary acidic protein(GFAP)and CD11 b.Results1.The mechanical and thermal withdrawal thresholds,as well as burrowing grams in the VEGFR1-TK-/-mice were significantly higher than that in the Wt mice after disc surgery.2.At 12 weeks following surgery,the disc in the VEGFR1-TK-/-mice and Wt mice had a severe degeneration.However,the difference was not statistically significant.Compared to the Wt mice,the disc injury in the VEGFR1-TK-/-mice induced a lower NGF expression in the disc,a deceased neuropeptides in the DRG,and less activation of astrocytes in the SC.3.VEGF stimulation dramatically induced the expression of NGF in both the NP and AF cells,while the NGF induction was significantly attenuated by the si RNA-VEGFR1 transfection.VEGF treatments also markedly induced the expressions of Adamts-5 and MMP-13 in the NP and AF cells,while the increased of these two genes were reversed by the si RNA-VEGFR2 transfection.4.In the DRG of Wt mice,the expressions of pain-related neuropeptides in the protein and m RNA levels were significantly increased by the VEGF stimulation,while VEGF has no effect on the DRG of VEGFR1-TK-/-mice.In the SC of Wt mice,VEGF treatment significantly increased the expressions of GFAP and CD11 b,while no effect on the SC of VEGFR1-TK-/-mice.ConclusionThe mutation of VEGFR1 tyrosine kinase relieved the pain induction caused by disc injury.Possible underlying mechanisms include a much lower extent of the NGF induction in disc,the neuropeptides induction in DRG,and the activation of astrocytes in SC.Findings from this study suggest that VEGF could induce the NGF expression in disc cells and mediate pain signal transduction in the DRG and SC via VEGFR1.These findings indicate that the VEGF/ VEGFR1 signal play an essential role in the pathogenesis of discogenic pain.