| BackgroundRheumatoid arthritis (RA) is a chronic, progressive and multi-systemic, inflammatory autoimmune disease, usually involving polyarthritis. The morbidity of RA is about0.4%in nation and the number in the world is about0.5%to1.0%. At present the mechanism of RA is not fully understood. Studies have shown the pathogenesis of RA is multiple, including the environmental, genetic factors, the immune and hormone levels. Maybe RA has some relationships with special populations, such as smoking crowd, diabetics, obese people who have a higher RA incidence. In addition, the morbility of RA is quite different in different races and genders[2’3]. Thus, RA may be stimulated by a susceptible individual factors directly or indirectly, while B cells, T cells, and a variety of cytokines participate the disease process of joints and systemic organse. Synovitis is the primer pathyologhy change of RA, gradually the cartilage, ligaments, tendons and whole tissue is affected, causing joints pain followed by cartilage damage and joint space becoming narrow. Without effective suppression treatment in early course, the joint destruction rate of RA is70%within three years, leading to joint deformity and disablity. Now, there is no effective drugs can reverse the destruction of bone joints in clinical, and the joint destructions has begun in the early course of RA (within four to twelve months). So it is important to diagnose RA and begin active treatment as soon as possible, which can control and hold back the progression of the disease with a significant reduction in morbidity.Inflammatory cell infiltration, synovial hypertrophy and angiogenesis are characteristics of RA primer pathology. Synovitis is the first step, then, Synovium hyperplasia and inflammatory cell infiltration appear in the joints. Synovial granulation tissue riches in capillaries which is named as pannus. Pannus destructs the cartilage, causing bone erosion and leads to joint deformity and loss of function at last. Obviously, Angiogenesis is an important symbol for generation and maintenance of RA pannus. Pannus enhances the invasiveness and plays an important role in RA erosion and destruction process. Angiogenesis start early in the course of RA and throughout the whole course. Thus, synovial pathology of RA, particularly, the vascularization of synovium is vital for the early diagnosis of RA, activity judgment, observation of drug efficacy and prognosis.2010edition of the American College of Rheumatology/European League Against Rheumatism Association (ACR/EULAR) points out that synovitis is one of the diagnostic criteria for early diagnosis of rheumatoid arthritis (RA).Meanwhile, the inflammatory cells and vascularization can produce a variety of cytokines, including vascular endothelial growth factor(VEGF), tumor necrosis factor-a (tumor necrosis factor-a, TNF-a) and the likes。These cytokines interact in form of net. Not only further promote the formation of new blood vessels, also lead to joints structural damage. VEGF is a multifunctional cytokine having vascular endothelial cell-specific effects,which can promote endothelial cell mitosis, proliferation, migration and result in more angiogenesis [9]. VEGF also increases vascular permeability, promoting substances leaking, which leads to the formation and development of chronic inflammation. So, VEGF is currently considered one of the most important factors in RA and the core of the whole angiogenesis[10].TNF-a is another important immunoregulatory factor in RA, released by mononuclear macrophages. TNF-a takes part in RA by regulating angiogenesis, synovitis and degradation of cartilage matrix. Released early in the immune response, TNF-a produces a large number of inflammatory mediators through autocrine and paracrine activation, stimulating fibroblast proliferation and increase of the release of cytokines, such as IL-6, IL-8[11]. Meanwhile, TNF-a can activate macrophages to promote cytokine production. So, a positive feedback forms between synovial cells and macrophages, which keeps the sustainable development of inflammation, stimulates inflammatory chain reaction[12’13]. Experimental studies in animals show TNF-α is affected by integrin αvβ-3in angiogenesis[14]. Integrins αvβ-3is a member of cell adhesion molecules family, which has low or no expression in the vascular endothelium under normal physiological conditions and high expression in tumor-induced angiogenesis, indicating an important role in the generation of blood vessels[15,16]. Studies have shown integrin may affect many aspects of synovial lesions angiogenesis in rheumatoid arthritis, playing an important regulatory role in angiogenesis.The application of animal models plays an important vole in the study of rheumatoid arthritis. Collagen-induced arthritis (CIA) is one of ideal animal models for rheumatoid arthritis, which is characterized by polyarthritis associated with joint damage[17].Rats become the best choice due to easy feeding and lower experiment costs. A sensitive and effective tool for vivo animal model is crucial, which can provide real-time observation and monitoring RA joint pathological changes, particularly synovial pannus formation and development in the experimental study of RA. Obviously, histopathological examination can reflect joint disease accurately, but the animals have to be killed and continuity of the experiment can not be kept.Specificity of laboratory biochemical parameters is not ideal, for appearing fluctuationssometimes[18]. X-ray has little help for synovium and cartilage in the early RA. Micro-CT image resolution is about100um, and the finger of MRI is500um, which is insufficient to distinguish the structure of rat joints[19’20]. Besides, CT and MRI are suitable for instantaneous image acquisition and can not carry out real-time observation. Lack of ideal imaging methods of bones and joints specifically for small animals (such as rats) is the problem of RA experimental study at present.Ultrasound has high resolution for the soft tissue, especially comprising with liquid. RA synovial capsule has more liquid oozing as synovial hyperplasia and provides a good reflection interface. Synovial morphological changes can be visualized by ultrasonography. Currently, the diagnostic value of high frequency ultrasound for RA has been accepted in clinical[21,23]. Color Doppler, spectral Doppler, and power Doppler ultrasound increase the accuracy and sensitivity of diagnosis in early RA[24,25].Especially, power Doppler imaging (PDI) overcomes angle dependence of color Doppler flow imaging, having high reliability for the detection of synovium low blood flow, and not susceptible to be interferenced [26].However, the report of ultrasound applied in animal studies in rheumatoid is rare. Especially, the application for small animal models of RA, such as rats, has not been reported because clinical US devices is not adapted for rat joints given the small sizeUBM (ultrasound biomicroscopy, UBM) has high resolution provided by the high frequency transducers (up to100MHz), the axial resolution is up to100-20um[27]. As a kind of vivo imaging technique emerging in recent years, UBM is used to observe small animal models and human superficial tissue. The technology has the unique advantage of non-invasive, high-resolution and real-time. Resently, Application of UBM in cardiovascular disease in small animals, embryos and other areas study is becoming a hot[28,29]. However, the application in musculoskeletal joints is still in primary stage[30,31].In this experiment, collagen-induced arthritis (CIA)was induced in SD rat through injection subcutaneously at the base of the tail with100mg of CII emulsified in Freund’s adjuvant[32]. Ultrasonography and Doppler analysis were performed on all rats by ultrasoundbiomicroscope (UBM). The changes of CIA rats were observed in vivo and the flow of synovium were semi-quantitative analyzed. At present, the similar research is rarely reported. The objective of our study was to determine whether UBM analysis could accurately detect arthritic lesions in RA model, named CIA. We thus aim at validating this non-invasive method by comparing it with clinical and histological joint examination, which are the classical methods used until now. Moreover, the change of cytokine expression level, including VEGF, TNF-α, αvβ-3, were analysised, trying to expore the correlation between ultrasonographic changes and RA immune mechanisms. Neovascularization is strongly linked with joint inflammation during arthritis, and is therefore an important parameter of disease evolution. We also evaluated the possibility to estimating vascularization in arthritic joints of CIA rats using power Doppler. By observing the therapeutic effect of different antiangiogenic drugs for RA, we evaluated UBM for efficacy to treatments in RA experimental study.This experiment included the following three parts:Chapter1Ultrasound and Doppler micro-imaging in a model of rheumatoid arthritis in ratsObjective1. To determine whether ultrasonography analysis could accurately detect arthritis lesions in a rat model of rheumatoid arthritis, summarize sonographic features of CIA and normal rats by UBM.2. To study the relationship of sonographic feature and pathological changes in joint of CIA rats by quantitative analysis of synovial thickness, PDI blood and synovial angiogenesis, evaluate the role of UBM in study synovial hyperplasia and angiogenesis in CIA.Materials and Methods1.110female SD rats were randomly divided into two groups according to the random number table:the blank control group,10and CIA group,100.2. CIA group rats were induced by immunized with native bovine collagen type Ⅱ (CII). Every rat was injected subcutaneously at the base of the tail with0.2ml of CⅡ emulsified in Freund’s adjuvant. On the15th day, rats were boosted with a subcutaneous injection of CII in incomplete Freund’s adjuvant,wjile the control group rat was given0.2ml saline injections subcutaneously at the base of the tail at the same time.3. Before and after injection the modeling drug, the clinical features of the experimental rats were observed. The weight of the rats and the paw volume were observed, and arthritis index(AI) was calculated the swelling of joint and every five days.4. Joints examinations were performed using UBM (VEVO770, Visualsonics) both in B mode and power Doppler imaging(PDI) on the day of0,15and35. Synovial fluid and synovial hypertrophy intra-articular were recessed, and Synovial flow at each joint was evaluated by PDI. The number of joint synovial thickening detected and the number of intraarticular joint appearing flow signal in knee, ankle, and feet joint were recorded.5. Parts of CIA rats were killed, their legs were dissected and processed for histological studies. The lesions were evaluated in each joint as previously described using five point scale (0-4, where0is normal and4severe) reflecting synovitis:synovial cells arranged in neat rows, no proliferation, less than three layers.0points;synovial hyperplasia to5-6layers no significant infiltration of lymphocytes, score1; synovial hyperplasia more than six layers, lymphatic cell infiltration, score2;synovial hyperplasia was significantly associated with pannus formation, score3; cartilage and bone erosion, score4. Single rats articular synovial pathological score is the highest score in various pathological lesions. Number of pathological articular is the summary of joint, which synovium pathology score≥1point.6. SPSS17.0statistical package was appliced for statistical analysis. The experimental data and measurement data were indicated by means士standard. The data between the two groups were compared using independent samples T-test and paired samples T-test. The data comparison among several groups was made by means of one-way ANOVA. LSD method was used when test for homogeneity of variance. when test of homogeneity of variance, multiple comparisons between the groups using; If heterogeneity of variance is adopted, Welch approximate variance analysis and multiple comparisons were used between groups. Pearson and Sperman correlation analysis were used for relevance comparison. Enumeration data was test by X2. A value of P<0.05was considered statistically significant.Results1.72rats were successful set up in CIA group, modeling success rate was72%. The clinical and pathology performance of72successful modeling rat of the CIA group was significantly. There was a significant difference between unsuccessful CIA group and control group (P<0.05).2. Positive detection rate of CIA rats used by UBM was88%, the finger of AI was 44%, the difference is significant (P<0.01).3. Counts of joints with synovial hypertrophy signal, joints with PDI signal, Synovial thickness, synovial UBM grade, PDI grade increase with the progression in CIA successful group. The difference of rats diseased joints was statistically significant on the day of15and35after injection modeling drug(P<0.01).4. Count of pathology joint and synovial pathology scores in successful CIA rat were higher than the unsuccessful group, with significant difference(P<0.01).5. Synovial thickness was positively correlated respectively with synovial pathology score, PDI classification and UBM grade in CIA successful group (r=0.649,0.528,0.619, P<0.01). PDI grade was positively correlated with pathology score and UBM grade respectively (r,0.610,0.690, P<0.01), with significant difference.Conclusions1. RA rat model of CIA was successfully established。The clinical and pathology performance of CIA rats were in accordance with RA.2. UBM can display Synovial hyperplasia, angiogenesis and joint effusion. So UBM maybe an ideal imaging methods for early diagnosis and severity assessment of small animal models of RA.3. Synovial thickness and PDI grade can be used as an objective indicators for diagnose of early RA and the extent of synovial proliferation and angiogenesis. Chapter2Correlation of ultrasonographic features and level of VEGF, TNF-α, αvβ-3expression in early RA experimental studyObjective1. To explore the role of VEGF, TNF-α, αvβ-3in synovial inflammation and angiogenesis by observing the expression level in serum and synovial tissue of CIA rats, which can provide experimental basis for RA vascular targeting therapy2. To study the correlation between ultrasonographic findings and level of VEGF, TNF-α, αvβ-3in serum and synovial tissue and explore the possibility of whether synovial thickness or PDI grade can be used as sonographic markers to assess synovial hyperplasia and pannus formation in early RA.Materials and Methods1.110female SD rats were randomly divided into two groups, the blank control group,10and CIA group,100.2. CIA group rats were immune-induced by bovine type Ⅱ of collagen to make RA model, named CIA rat.3. Before and on the15th and35th day after injection modeling drug, the VEGF, TNF-α and αvβ-3level in serum of the experimental rats were detected by By enzyme-linked immunosorbent assay (ELISA).4. Joints examinations were performed using UBM both in B mode and PDI on the day of before and after injection modeling drug15and35days respectively. Joint effusion and synovial proliferation within intra-articular were recessed. The flow of synovium at each joint was evaluated by PDI. Recorded the number of joint synovial thickening detected and the number of intraarticular joint appearing flow signal in knee, ankle, and feet joint.5. All of the blank group and parts of CIA rats were killed on the35th day. Take Pathology and immunohistochemical examination for experimental rats joint. Synovial pathological damage was assessed by with the former Semi-quantitative scoring criteria.6. VEGF, TNF-α, αvβ-3expression level in synovium was measured using mouse αvβ-3, VEGF, TNF-a immunohistochemical kit in strict accordance with the operating instructions for the joints which synovial pathology score≥1. The expression of αvβ-3, VEGF, TNF-a was determined by semi-quantitative integral method:positive cells<5%, score0;6%-25%, score1;26%-50%, score2;51%-75%, score3;>75%, score4. The score of stain:no specific staining, score0; yellow staining, score1; light brown, score2; brown, score3. To multiply the score marks, the scores were defined as flows:0-1, negative(-),2-4, weekly positive(+);5-8, middle positive(++);and9-12, strong positive(+++).7. Statistical methods were same as the former.Results1. The levels of serum VEGF, TNF-α, αvβ-3in CIA successful group rats increased with time after modeling injection, the levels of the35th day compared with the15th day and the0day with a significant difference (P<0.05).2. After injection modeling drug35days, the VEGF, TNF-α, αyβ-3level in serum of CIA successful group rats were:98.29±13.86pg/ml,27.37±5.10pg/ml,2.15±0.58pg/ml, and the difference compared with the blank control group and the unsuccessful CIA group was statistically significant (P<0.05).3. There had no significant difference between the VEGF, TNF-α, αvβ-3level in serum of CIA unsuccessful group between control group (P>0.05).4. On the35th day after injection modeling drug, the levels of VEGF, TNF-α, αvβ-3in synovium of CIA successful group rats were2.23±0.92,1.91±0.97,1.81±0.91respectively, and the difference compared with the blank control group and CIA unsuccessful group was statistically significant (P<0.05).5. In CIA successful group, synovium thickness and VEGF, TNF-α, αvβ-3expression levels were positively correlated respectively, r was0.713,0.749and0.548, P<0.01, with statistical significance.6. In CIA successful group, synovial PDI grade were positively correlated with VEGF, TNF-α, αvβ-3expression level respectively, r was0.576,0.635and0.789, P<0.01, with statistical significance.Conclusions1. The expression of VEGF, TNF-α and αvβ-3in serum of CIA rats increase with the progression and consistent with the clinical and pathological joint score. The expression level of VEGF, TNF-α, and αvβ-3in serum may reflect activity in rheumatoid progression.2. VEGF, TNF-α and αvβ-3in synovium showed high expression level in CIA successful group, which were closely related with synovial lesions and angiogenesis in RA.3. Synovium thickness and PDI grade were positively correlated with synovial VEGF, TNF-α, αvβ-3expression respectively. UBM is an effective tool for noninvasive evaluation of synovial proliferation and angiogenesis. Chapter3Evaluation of UBM in anti-angiogenic therapy for early RA in experimental studyObjective1. To observe effect of Bevacizumab and etanercept treatment in rheumatoid arthritis model rats and provide anti-angiogenic therapy experimental basis. 2. To evaluate the use of UBM, including quantitative Doppler analysis of synovial vascularisation, before and after anti-angiogenic treatment with Bevacizumab and etanercept in rheumatoid arthritis model rats.Materials and Methods1.50CIA rats were divided into three groups:bevacizumab group,20; etanercept treatment group according to the number and extent of arthritic joints,20; control group,10. Drug according to the weight was given once a week in14days.2. Before and after drug intervention, the clinical features of the experimental rats were observed. The weight of the rats and the arthritis index scores were measured.3. Joints examinations were performed by UBM just as previously described.4. Before and after drug intervention, the VEGF, TNF-α and αvβ-3level in serum of the experimental rats were detected by ELISA.5. Synovial VEGF, TNF-α, αvβ-3expression level were measured using mouse VEGF, TNF-a, avP-3immunohistochemistry kit in strict accordance with the operating instructions for the joints which synovial pathology score≥1. The method was same as previously described.6. SPSS17.0statistical package was appliced for statistical analysis. The experimental data and measurement data were indicated by means±standard. The data comparison among several groups was made by means of one-way ANOVA. LSD method was used when test for homogeneity of variance. If heterogeneity of variance was adopted, Welch approximate variance analysis and multiple comparisons were used between groups. Pearson and Sperman correlation analysis were used for relevance comparison. Enumeration data was test by X2. A value of P<0.05was considered statistically significant.Results1. Before drug intervention, there was no significant difference for Sonographic Ultrasonographic features in three groups (P>0.05). After treatment, comparied with control group, the count of joint appearing flow and flow PDI grade reduced in both bevacizumab group and etanercept group, and there were significant differences (P <0.05); Meanwhile, the count of joint appearing flow and flow PDI grade had no difference between bevacizumab group and etanercept group(P>0.05).2. Before drug intervention, serum VEGF, TNF, αvβ-3levels had no differences in the three groups(P>0.05); while a significant difference appeared after treatment(P <0.01). Comparied with control group, the level of bevacizumab group and etanercept group declined, there was significant differences (P<0.01). There was no significant difference the between bevacizumab group and etanercept group(P>0.05).3. After bevacizumab and etanercept treatment, AI and Ultrasonographic features decreased with significant differences (P<0.05).4. In control group, compared with before treatment, synovial thickness and PDI grade increased with statistically significant difference(P<0.05). Before and after treatment, AI, atthritis joint count detected by UBM, the number of joints with blood and UBM grade didn’t change significantly(P>0.05).5. After drug intervention, there was no significant difference for pathological joint count among the three groups(P>0.05); Synovial VEGF, TNF-a, αvβ-3expression and pathological scores were significantly different (P<0.05) among the three groups; Blank control group were higher than the indicators of bevacizumab group and the etanercept group, there was a significant difference(P<0.05);There was no significant difference between bevacizumab and etanercept group (P>0.05).6. Synovial thickness decreased after drug intervention, PDI and synovial pathology grading score showed a positive correlation VEGF, TNF-α, αvβ-3expression levels respectively(P<0.05). Conclusions1. Bevacizumab and etanercept can suppress early RA synovial hyperplasia, decrease synovial angiogenesis and lower synovial pathology score. There was no difference between the treatment of the two drugs.2. Synovial thickness, PDI grade can be used as sonographic markers to assess synovial hyperplasia and pannus formation in early RA, and value response to treatment. compared with synovial thickness, PDI is more sensitive in anti-angiogenic therapy.3. UBM has application value for RA drug trials and efficacy. |
PDF Full Text Request