| Part one Experimental study on adjacent segmental intervertebral disc degeneration in ovariectomized rats with lumbar fusionObjective: In this study,lumbar fusion was performed in ovariectomized(OVX)rats to establish an animal model of adjacent intervertebral disc degeneration(ASDD)by posterolateral lumbar fusion(PLF).We investigated the pathological condition of intervertebral disc degeneration adjacent to lumbar fusion,and the effect of estrogen reduction caused by ovariectomy on ASDD.Methods: Forty 3-month-old female Sprague-Dawley(SD)rats were randomly divided into the following four groups: Sham group(sham operation group),OVX(bilateral ovariectomized group),PLF group(posterolateral lumbar fusion group)and OVX+PLF group(ovariectomized combined with lumbar fusion group).OVX was performed at the age of 3 months,and PLF was performed 4 weeks after OVX.PLF was established via an intertransverse process fusion using an autologous iliac graft with wire fixation at the L4–L5spinous processes.X-ray scan and imaging score was performed 4 weeks after PLF.Then the rats were killed and retain the L3-6 segment.At the same time,the L3-6 segment assessed palpated manually.The specimens were evaluated by Micro-computed tomography(Micro-CT)and L5-6 specimens were separated and fixed,decalcified,embedded,sliced and stained.HE staining were used to evaluate the ASDD.Results:1 Fusion assessment.1.1 X-ray examination: compared with OVX+PLF group,the new bone formation and radiography density of L4-5 segment in PLF group were increased.At the same time,the imaging score in PLF group was significantly higher than that in OVX+PLF group(P<0.05).1.2 Manual testing: The manual palpation test was performed to reveal no detectable movement at L4-5 segments in the PLF and OVX+PLF groups.2 Micro-CT testing.Compared with Sham group,the BMD,BV/TV,Tb.N and Tb.Th significantly decreased in the OVX and OVX+PLF groups(P< 0.01),while Tb.Sp significantly increased(P< 0.05,P< 0.01).However,there was no significant difference between Sham group and PLF group(P> 0.05).The DHI score in Sham group was significantly higher than that in OVX group,PLF group and OVX+PLF group(P< 0.01).The DHI score in OVX+PLF group was significantly lower than that in OVX group and PLF group(P<0.05,P< 0.01).There was no significant difference between OVX group and PLF group(P> 0.05).3 Histological examinationThe results of HE staining showed that compared with Sham group,nucleus pulposus cells decreased and some of them had myxoid degeneration,fibrous annulus arrangement disorder and endplate calcification in the OVX,PLF and OVX+PLF groups.This change was particularly evident in the OVX+PLF group.The results of histological score showed that compared with Sham group,the scores were significantly higher in the OVX,PLF and OVX+PLF groups(P< 0.05),and the score of OVX+PLF groups was significantly lower than that in the OVX and PLF groups(P< 0.05,P< 0.05).However,there was no significant difference between OVX and OVX+PLF groups(P> 0.05).Part two The study of the potential effects of Denosumab on adjacent segmental intervertebral disc degeneration in ovariectomized rats with lumbar fusionObjective: In this study,posterolateral lumbar fusion was performed to observe the effect of estrogen reduction on adjacent segmental intervertebral disc degeneration in ovariectomized rats,we further investigate the effect of Denosumab(Dmab)on the adjacent segmental intervertebral disc degeneration in ovariectomized rats with lumbar fusion.Methods: Sixty female 3-month-old SD rats were randomly divided into the following five groups: Sham group,OVX group,PLF group,OVX+PLF group and OVX+PLF+Dmab group.The rats were treated with sham operation(Sham group),bilateral ovariectomy(OVX group),posterolateral lumbar fusion(PLF group),ovariectomized with lumbar fusion(OVX+PLF group)and ovariectomized with lumbar fusion and Dmab intervention(OVX+PLF+Dmab group).Seventy-two hours after lumbar fusion,the rats in the OVX+PLF+Dmab group were subcutaneously injected with Dmab at a dose of 0.25 mg/kg/day,five days a week,while the rats in the other groups were subcutaneously injected with the same volume of normal saline.After 4weeks of Dmab intervention,the animals were euthanized and the L3-6segments were preserved and assessed palpated manually.At the same time,Micro-computed tomography(Micro-CT)testing was performed.The m RNA level of matrix metalloproteinase-13(MMP-13),Aggrecan(Agg)and metalloproteinase with thrombospondin motifs-4(ADAMTS-4)were assessed in L3-4 intervertebral disc by RT-PCR.The L5-6 segments were decalcified,embedded,sectioned and stained.In addition,immunohistochemical staining was used to detect the expression of MMP-13,Agg and ADAMTS-4 in L5-6intervertebral disc.Results:1 Manual testing: The manual palpation test was performed to reveal no detectable movement at L4-5 segments in the PLF and OVX+PLF groups.2.Micro-CT testing.Compared with Sham group,the BMD,BV/TV,and Tb.N significantly decreased in the OVX and OVX+PLF groups(P< 0.05,P< 0.01),while Tb.Sp significantly increased(P< 0.01).Compared with OVX+PLF group,the BMD,BV/TV,Tb.N and Tb.Th increased significantly in the OVX+PLF+Dmab group(P< 0.01).3 Immunohistochemical staining.In the Sham group,the NP showed strong immunoreactivity for aggrecan and weak immunoreactivity for MMP-13 and ADAMTS-4.In contrast,the NP showed weak Aggrecan(P< 0.01)immunostaining and strong MMP-13(P<0.01)and ADAMTS-4(P< 0.05,P< 0.05,P< 0.01)immunoreactivity in the OVX,PLF,and OVX+PLF groups,especially in the OVX+PLF group.However,the OVX+PLF+Dmab group showed stronger immunostaining for Aggrecan(P< 0.01)and weaker immunostaining for MMP-13(P< 0.01)and ADAMTS-4(P< 0.01)compared to the OVX+PLF group.4 RT-PCR testingCompared to the Sham group,the OVX,PLF,and OVX+PLF groups showed lower aggrecan level(P< 0.01)but higher MMP-13(P< 0.01)and ADAMTS-4(P< 0.01)m RNA level.The OVX+PLF+Dmab group showed higher Aggrecan(P< 0.01)level and lower MMP-13(P< 0.01)and ADAMTS-4(P< 0.01)m RNA level compared with the OVX+PLF group.Part three The mechanism of the potential effects of Denosumab on adjacent segmental intervertebral disc degeneration in ovariectomized rats with lumbar fusionObjective: This study is based on Denosumab can delay the adjacent segmental intervertebral disc degeneration(ASDD),Although adjacent segmental intervertebral disc degeneration(ASDD)is one of the most common complications after lumbar fusion,but its underlying potential mechanism has not been elucidated.In this part,to investigate the mechanism of Dmab on ASDD,we used an ASDD model with early intervention of Dmab by subcutaneous injection in ovariectomized rats with lumbar fusion.Methods: Three-month-old female Sprague-Dawley rats that underwent L4–L5 posterolateral lumbar fusion(PLF)with spinous-process wire fixation four weeks after bilateral ovariectomy(OVX)surgery were given Dmab four weeks after PLF surgery(OVX+PLF+Dmab group).In addition,the following control groups were defined: Sham,OVX,PLF,and OVX+PLF group(n=12).Manual palpation was used to evaluate the state of lumbar fusion.The bone microstructure in the endplate of L6 vertebra were evaluated by Micro-CT.The characteristic alterations of ASDD were identified via Safranin-O green staining.The expression level of SOX-9(Sry related HMG box-9,SOX-9)and CD24(Cluster of differentiation 24,CD24)in the intervertebral disc were detected by immunohistochemistry.To detect changes in the mechanical properties of adjacent vertebrae,we performed compression tests on L6 segments.Results:1 Fusion assessment.During manual palpation,the wire on the L4-L5 spinous processes of all rats who underwent PLF surgery were found to be well connected to the spinous processes,and there was no detectable movement at the fusion level.These results revealed adequate lumbar fusion of rats after PLF.2 L5–L6 caudal endplates and DHI testingMicro-CT parameters of L5–L6 caudal endplates were examined.Compared to the Sham group,the OVX and OVX+PLF groups exhibited significantly lower closed pores number(P< 0.05,P< 0.01),but significantly higher open porosity(P< 0.05,P< 0.01)and total pore volume(P< 0.01).However,compared to the OVX+PLF group,the OVX+PLF+Dmab group had a significantly higher number of closed pores(P< 0.01),as well as lower open porosity and total pore volume(P< 0.01).3 Histological examinationSafranin-O green staining showed that the structure of the intervertebral disc was intact in the sham group.Specifically,nucleus pulposus(NP)contained abundant notochordal cells accompanied by rich gel-like tissue(extracellular matrix),and annulus fibrosus(AF)annulus was arranged regularly and tightly.The boundary between the NP and AF was clear.Hyaline cartilage of the endplate(AF)contained chondrocytes.In contrast,intervertebral disc degeneration occurred in the OVX group,PLF group,and OVX+PLF group to varying degrees.The degenerative changes were the most obvious in the OVX+PLF group.Compared to the OVX+PLF group,pathological changes were effectively retarded in the OVX+PLF+Dmab group.The results of histological score showed that compared with Sham group,the scores were significantly higher in the OVX,PLF and OVX+PLF groups(P<0.05),and the score of OVX+PLF group was significantly higher than that in the PLF group(P< 0.05).The score of OVX+PLF+Dmab group was significantly lower than that in the OVX+PLF group(P< 0.05).4 The compression tests on L6 vertebra.Compared to the Sham group,maximum load,yield stress,maximum stress,and elastic modulus values were significantly lower than that in the OVX,PLF,and OVX+PLF groups(P< 0.01).However,Dmab treatment significantly improved the mechanical parameters in the OVX+PLF rats(P<0.01).5 Immunohistochemical staining.Compared with the Sham group,the protein expression of SOX-9 was substantially higher than that in OVX group,PLF group and OVX+PLF group(P< 0.01).The protein expression of SOX-9 in the OVX+PLF group was significantly higher than that in OVX group and PLF group(P< 0.01).Compared with the OVX+PLF group,the protein expression level of SOX-9markedly lower than that in OVX+PLF+Dmab group(P< 0.01).Compared with the Sham group,the protein expression of CD24 was substantially lower than that in OVX group,PLF group and OVX+PLF group(P< 0.01).The protein expression of CD24 in the OVX+PLF group was significantly lower than that in OVX group and PLF group(P< 0.05).Compared with the OVX+PLF group,the protein expression level of CD24 markedly higher than that in OVX+PLF+Dmab group(P < 0.01).Conclusions:1.Lumbar fusion with autogenous iliac bone graft and spinous process wire fixation in ovariectomized rats can lead to ASDD.This model provides a good basis for elaborating the pathological changes of ASDD and subsequent drug intervention.2.Osteoporosis induced by estrogen reduction can aggravate adjacent segmental intervertebral disc degeneration in rats,suggesting that estrogen deficiency is an important risk factor in the pathological progression of ASDD after lumbar fusion.In the animal model of ASDD after ovariectomy with lumbar fusion,Dmab intervention early can improve vertebral osteoporosis and delay ASDD,suggesting that Dmab may be a potential preventive drug for ASDD.3.Dmab can not only effectively maintain the bone mineral density of vertebra,but also improve the biomechanical properties of vertebra and endplate trabecular microstructure by promoting osteogenesis,at the same time protect the integrity of intervertebral disc structure and function,and then delay the pathological changes of ASDD. |
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