Early And Quantitative Evaluation Of Rheumatoid Arthritis And Subcutaneous Injection Of Nano-Embedded Methylprednisolone Around The Joint By Multimodal Ultrasound

Objective: AIA RA rabbit model was established in this study.Synovial biopsy guided by ultrasound was used to extract the synovium of rabbit knee joint at different stages.Combined with multimodal ultrasound and serological indicators,the pathological inflammatory changes of synovium were studied longitudinally in the first part,and the neovascularization of synovium was studied longitudinally in the second part.The third part evaluates the effect of nano-embedded methylprednisolone on early rheumatoid arthritis by subcutaneous injection around the joint.Methods: Part ?:(1)Thirty male New Zealand rabbits were randomly divided into AIA RA model group(25 rabbits)and control group(5 rabbits).(2)blood samples of AIA group and control group were taken from the 1st to 16 th weeks of modeling.IL-1,CRP,TNF-a and other indicators were detected by ELISA.(3)routine ultrasound and contrast-enhanced ultrasound were used to detect AIA group and control group rabbits at 6th,8th,12 th and 16 th weeks of modeling.Multimodal ultrasound was obtained in bilateral knee joints.The synovium of AIA knee joints was taken by ultrasound-guided synovial biopsy.Pathological scores and CD3 immunohistochemical tests were performed.The correlation between pathological indexes and multimodal ultrasound and serological indexes was analyzed.Part ?: Microscopically,we observed the changes of microvessels in different periods of synovium in the first part,detected the expression of CD31 and VEGF in synovium by immunohistochemistry,and analyzed the correlation between microvessel density(CD31 marker)and VEGF in different periods and multimodal ultrasound indexes.Part ?:(1)Thirty AIA RA rabbits were randomly divided into control group and four treatment groups,including oral thalidomide group(MT1),oral methylprednisolone group(MT2),nano-embedded methylprednisolone subcutaneous injection group(MT3),oral methotrexate group(MT4).Six rabbits in each group were intervened from the first day of the seventh week of modeling for6 weeks.(2)Blood samples of rabbits in each group were taken once a week before and 1 to 6 weeks after intervention,and serological indexes such as IL-1,CRP,TNF-alpha,and VEGF were detected by ELISA;(3)Bilateral knee joints of rabbits in each group were evaluated by two-dimensional ultrasound and Doppler ultrasound before and 2,4 and 6 weeks after intervention,and bilateral knees of rabbits in each group were evaluated by contrast-enhanced ultrasound before and 6weeks after intervention.Multimodal ultrasound indexes were obtained in the lateral knee joint.Fourth,ultrasound-guided synovial biopsy was performed before and 2,4 and 6 weeks after the intervention to collect the synovial tissue of the bilateral knee joints of rabbits in each group,and the pathological scores and CD3,CD31,and VEGF immunohistochemical tests were carried out.The correlation between pathological indexes and multimodal ultrasound indexes and serological indexes was analyzed.Result: Part ?:(1)The success rate of AIA group was 100%.(2)In AIA group,except for synovial interstitial proliferation subitem,the other pathological indexes(synovial pathological score subitem,total score and CD3 expression level)continued to increase with the prolongation of modeling time from 6 to 16 weeks,and the difference between different periods was statistically significant(P < 0.05).(3)In AIA group,synovial thickness and synovial thickness grading increased continuously with the prolongation of modeling time from 6 to 16 weeks(P < 0.05);PDI grading and CEUS grading increased with the prolongation of modeling time from 6 to 8 weeks(P<0.05);PI value and AUC value were synovial > periarticular tissue,PI value increased smoothly from 8 to 16 weeks.AUC value increased at6-8 weeks and decreased at 8-16 weeks(P<0.05);AUC value increased at 8-12 weeks and decreased at 12-16 weeks(P<0.05);there was no significant difference in TTP change rate at different parts and at different times.(4)The overall success rate of ultrasound-guided synovial biopsy was 75.2%(292/383),and the correlation between the success rate of ultrasound-guided synovial biopsy and synovial thickness and synovial thickness grading was higher(r was 0.798,0.812,P<0.01,respectively).(5)The serological indicators TNF-a,CRP and IL-1 in AIA group showed an obvious upward trend from the fifth week to the eighth week,and then entered a relatively stable plateau period.The control group showed a relatively stable low level.There was no significant difference between AIA group and control group in week 1-4(P >0.05),but there was significant difference between week 5-16(P<0.05).(6)There was a high correlation between synovial thickness,synovial thickness grading,CD3 expression and pathological score of synovitis(r values were 0.827,0.789,0.806,P < 0.01).Part ?: With the prolongation of modeling time,the number of synovial vessels in AIA group increased and the lumen gradually increased;the expression levels of MVD(CD31)and VEGF in AIA group increased with the prolongation of 6-12weeks(P < 0.01),and the changes of CEUS in AIA group were more stable during12-16 weeks(P < 0.01);and CEUS in AIA group increased early,and scattered at 6and 8 weeks after modeling.In AIA group,the correlation between PDI grade and synovial MVD(CD31)and VEGF was 0.451 and 0.432;the correlation between CEUS grade and MVD(CD31)and VEGF was 0.586 and 0.557;the correlation between PI and MVD(CD31)and VEGF was 0.557;and the correlation between PI and MVD(CD31)and VEGF was 0.451 and 0.432,respectively.They were0.579 and 0.587,respectively;the above had statistical significance.Part ?:(1)Except for one rabbit in the oral methylprednisolone group(MT2group),one rabbit died at the 4th week of intervention,and one rabbit retired at the5 th week of intervention because of severe infection.The rest rabbits completed the intervention plan.Before intervention,there were no significant differences in ultrasound,serological and pathological indexes among the groups.(2)After intervention,the synovial thickness,PDI and CEUS of the control group were decreased in the oral methylprednisolone group(MT2 group),the nano-embedded methylprednisolone subcutaneous injection group(MT3 group)and the oral methotrexate group(MT4 group),while those of the oral methylprednisolone group were decreased.The synovial thickness,PDI grade and CEUS grade of thalidomide group(MT1 group)increased,the difference was statistically significant,RI value did not change significantly;PI value of control group and oral thalidomide group(MT1 group)increased,while that of oral methylprednisolone group(MT2 group)and nano-embedded methylprednisolone subcutaneous injection group(MT group).The PI values of the three groups and the MT4 group decreased,and the differences were statistically significant.There was no significant difference in AUC and TTP between the three groups.(3)The level of serum TNF-alpha in the control group increased slowly after 3 weeks of intervention,while the levels of CRP,IL-1 and VEGF were relatively stable;the levels of serum TNF-alpha,CRP,IL-1 and VEGF in the oral thalidomide group(MT1 group)increased gradually with the increase of intervention time,and increased more significantly after 3weeks,compared with the control group,the difference was statistically significant.Significance: After the intervention of oral methylprednisolone group(MT2 group),nano-embedding methylprednisolone subcutaneous injection group(MT3 group)and oral methotrexate group(MT4 group),the changes of serum CRP,TNF-a,IL-1and VEGF were relatively stable,with no significant difference.(4)After intervention,the scores of synovial pathological sub-items and total scores,CD3,MVD(CD31)and expression of vascular endothelial growth factor increased in oral thalidomide group(MT1 group);the scores and total scores of synovial pathological sub-items in oral methylprednisolone group(MT2 group),nano-embedded methylprednisolone subcutaneous injection group(MT3 group)and oral methotrexate group(MT4 group).The expression levels of CD3,MVD(CD31)and vascular endothelial growth factor were significantly lower than those of control group and oral thalidomide group(MT1 group).(5)The correlations between synovial pathological inflammation score and synovial thickness,PDI grade,PI,CD3 and CD31 were 0.669,0.556,0.601,0.715 and 0.524,respectively.The correlations between synovial MVD(CD31)and PDI grade,CEUS grade and PI were 0.463,0.512,0.571,P < 0.01.Conclusion: Part ?:(1)AIA rabbit model can well simulate the course of RA.Ultrasound-guided synovial biopsy suggests that the 4th-8th week of the model is equivalent to the early stage of RA(inflammatory stage),the 8th-12 th week is equivalent to the middle stage of RA(pannus formation stage),and the 12th-16 th week is equivalent to the late stage of RA(fibrosis stage).(2)Ultrasound-guided 18 G semi-automatic synovial biopsy has a high success rate.The quality and quantity of biopsy tissue strips obtained can meet the needs of histopathology and immunohistochemical CD3 detection,and can meet the clinical needs.(3)Among the multimodal ultrasound indexes,synovial thickness and synovial thickness grading can best reflect the pathological changes of synovitis in AIA RA rabbit model.(4)In the early stage of AIA RA rabbit model,the synovial thickness grading and PDI grading were mainly grade 1.With the progress of the disease,the above indexes gradually increased.In the late stage of AIA RA rabbit model,the synovial thickness grading and PDI grading were mainly grade 3.(5)The expression level of CD3,an immunohistochemical marker of synovium in AIA RA rabbit model,increased continuously with the prolongation of modeling time,which may play an important role in the progress of disease and the development of pathological inflammation.Part ?:(1)In AIA rabbit model,the expression of CD31 and vascular endothelial growth factor increased in the early stage(inflammation stage),higher in the middle stage(pannus formation stage)than in the inflammation stage,and more stable in the late stage(fibrosis stage).(2)Among the multimodal ultrasound indicators,PDI,CEUS and PI are the best indicators to reflect the degree of synovial vascularization;CEUS enhanced early vascular morphological characteristics have a certain value for the early diagnosis of RA.(3)Multimodal ultrasound,especially combined with the expression level of CD31 in synovial biopsy,can accurately evaluate the degree of synovial vascularization in AIA rabbit model at an early stage.Part ?:(1)In AIA rabbit model,early intervention with nano-embedding methylprednisolone,oral methylprednisolone and oral methotrexate at the early stage can effectively inhibit the progress of arthritis.(2)Subcutaneous injection of nano-embedding methylprednisolone around the joint to intervene in early synovitis has less toxic side effects than oral methylprednisolone,faster onset than oral methotrexate,and longer duration of low inflammatory activity after intervention.It is a potential new strategy for early intervention of RA.(3)On the pathological level,ultrasound can reflect the effect of early RA intervention earlier than serological indicators;synovial thickness,PDI grading and CEUS quantitative parameter PI can better reflect the pathological inflammatory status of synovium after intervention;PDI grading,CEUS grading and PI can better reflect the status of synovial neovascularization after intervention.(4)Ultrasound-guided 18 G semi-automatic synovial biopsy can obtain synovial tissue of knee joint of AIA rabbit model after intervention.CD3 can reflect pathological inflammatory changes after intervention,and CD31 can reflect angiogenesis changes after intervention.