A Comparative Study About Three Kinds Of Rat Models Of Knee Osteoarthritis

On the basis of clinical research,our research group planned to carry out“the mechanism of warming acupuncture on cartilage degeneration of knee osteoarthritis in rats”,then we found there were many methods in literature for making KOA model with the different choice and dosage of drugs,location of the operation and the time of model formation,which brought more problems for the basic research on osteoarthritis.Objective:In this experiment,three kinds of modeling methods were selected,we choose a relatively large number of the drug injection method in the literature,in which 4%papain were mixed with 0.03mol/L L-cysteine with the proportion of 2:1,and the knee joint cavities were injected with the amount of 0.1mL/kg;in the operation methods,we used the way reported by the literature—anterior cruciate ligament cutting method;in the immobilization methods,we selected the flexion position.To compare the effect of three models of osteoarthritis of knee joint in SD rats and define the time of formation of osteoarthritis in each group of models,and to select a more ideal animal model,and to provide experimental evidence for the application of animal models in the study of the pathogenesis and therapeutic effect of osteoarthritis of the knee.Methods:128 SPF-level healthy male SD rats were randomLy divided into blank group?group A?,immobilization group?group B?,papain injection group?group C?,and operation group?group D?,each group with 32 rats and building models on the right knee of rats.In group B,the knee joints of the rats were continuously immobiled in the flexion position;In group C,4%papain be mixed with 0.03 mol/L L-cysteine by 2:1 blending ratio,and the right knee joint cavities were injected with the amount of 0.1mL/kg on the first,fourth,and seventh days of the experiment;The operation group adopts ACLT method which is anterior cruciate ligament amputation.The rats in group A were only grabed.The general state of rats in each group was recorded daily,and on the last day of 2,4,6 and 8 weeks of the experiment,four groups of rats were treated in four batches,8 rats each time.To conduct behavioral observation,the rats were graded according to Lequesne MG method.The rat pain threshold was measured with the WAGNER tenderizer.Image observation was performed on the right hind limb of rat.The right knee joints of the rats were taken and the cartilage surface was grossly observed,then HE staining was performed on tissue of the cartilage which would be observed with light microscope.Results:1.Comparison in the group:?1?The rats of group A performed normally during the whole experiment.The cartilage surface of the right knee joint was smooth and transparent.There was no swelling in the right knee joint at all time points.There was no obvious pain reaction when the rats were pressed.The rats ran powerfully in normal gait.The range of motion of the knee joint was normal and no significant changes were observed during the entire experiment.There was no significant difference in the pain threshold at 2^th,4^th,6^th,and 8^thweek?P>0.05?,indicating that there was no evidence of osteoarthritis in the blank group.?2?The rat cartilage generally observed score,LequesneMG score,pain threshold of group B were significantly different at 2^th,4^th,and 6^thweek?P<0.05?,and there were significant differences while comparing 8^thh week with 2^th,4^thh week?P<0.05?,there was no significant difference between the 6^thh week and 8^thh week?P>0.05?.?3?The scores of Lequesne MG and pain thresholds in group C were significantly different at 2^th,4^th,6^thh and 8^thh week?P<0.05?.The gross observation scores of cartilage at6^thh and 8^thh week were not significantly different?P>0.05?,the rest data were significantly different to each other?P<0.05?.?4?The rat cartilage generally observed score,LequesneMG score,pain threshold of Group D were not significantly different at 4^th,6 ^thh week?P>0.05?,the rest data were significantly different to each other?P<0.05?.2.Comparison between groups:?1?Comparison of cartilage generally observed score:the score of group B,C,D had statistically significant differences with group A at 2^th,4^th,6^thand 8^thweek?P=0.000<0.05?;the score of group B at 2^th,4^thh week were higher than the other two groups;the score difference of 3 groups at 6 week was not significant and group D was slightly higher;the B and D groups were higher than the group C at 8 week.?2?Comparison of Lequesne MG score:The score of group B,C,D had statistically significant differences with group A at 2^th,4^th,6^thh and 8^th?P=0.000<0.05?;the score of group D at 2^th,4^thweek were higher than the other two groups;compared with the other two groups,group B was relatively higher at 6^th,8^thh week.?3?Comparison of pain threshold:?1?After 2 weeks,pain threshold of group B,C,D were significantly lower than group A?P<0.05?;group C,D compared with group A,no statistical significance?P>0.05?;group C compared with group D,there was statistically significant differences?P<0.05?.?2?After 4 weeks,pain threshold of group B,C,D were significantly lower than group A?P<0.05?,and there was a statistical difference;B,C and D groups compared with each other,there was not statistically significant?P>0.05?.?3?After6 weeks,the pain threshold of B,C and D was significantly lower than group A?P<0.05?;comparing group C,D with group B,there was not statistically significant?P>0.05?;group C compared with group D,there was statistically significant differences?P<0.05?.?4?After8 weeks,the pain threshold of B,C and D was significantly lower than group A,there was a statistical difference?P<0.05?;B,C and D groups compared with each other,there was not statistically significant?P>0.05?.3.X-ray of the rat knee showed:?1?Group A:there was no obvious abnormality in knee joints X films at 2^th,4^th,6^thh and8^thh week.?2?Group B:there was no obvious abnormality at 2^thweek;at 4^thweek,there was slight hyperplasia in the intercondylar eminence;at 6 ^thh week,the intercondylar eminence and the medial border of the tibia were hyperplastic and the articular surface was rough,the joint space was slightly narrowed;at 8^thweek,the medial and lateral joint space was inconsistent and slightly narrowed,the articular surface was not smooth,the intercondylar ridge became sharp,an the articular cartilage was dense.?3?Group C:there was no obvious abnormality at 2^thweek;at 4^thweek,there was slight hyperplasia in the intercondylar region;at 6v week,the intercondylar eminence and the medial border of the tibia were hyperplastic,and the articular surface contour was not Smooth,slightly narrowed joint space;at 8^thweek,the medial and lateral joint space was inconsistent and narrowed,the articular surface was not smooth,the intercondylar axilla was sharpened,and the articular cartilage was dense.?4?Group D:there was no obvious abnormality at 2^thweek;at 4^th,6^thweek,there was slight hyperplasia in the intercondylar region,and the joints inside and outside were the same,no obvious stenosis was found,and the bony articular surface was not smooth,articular surface contour clear and complete;at 8^thweek,the medial and lateral joint space was inconsistent and narrowed,the articular surface was not smooth,the intercondylar axilla was sharpened,and the articular cartilage was dense.4.The result of HE staining in cartilage tissue:?1?Group A:there was no obvious abnormality at 2^th,4^th,6^thand 8^th.?2?Group B:there was no obvious abnormality at 2 ^thh week;the pathological changes of early osteoarthritis could be seen at 4^thh week,which showed rough surface layer of cartilage,diffuse cell hypertrophy and normal HE Stainning;the pathological changes of intermediate osteoarthritis could be found at 6^th,8^thh week,which expressed fissuration into surface layer of cartilage,disorder arrangement of chondrocyte,chondrocyte decreasing,light HE staining.?3?Group C:there was no obvious abnormality at 2 week;the pathological changes of early osteoarthritis could be seen at 4^thh week,which showed rough surface layer of cartilage,diffuse cell hypertrophy and normal HE Stainning;the pathological changes of intermediate osteoarthritis could be found at 6^th,8^thh week,which expressed fissuration into surface layer of cartilage,disorder arrangement of chondrocyte,chondrocyte decreasing,light HE staining.?4?Group D:there was no obvious abnormality at 2 week;the pathological changes of early osteoarthritis could be seen at 4^th,6^thh week,which showed rough surface layer of cartilage,diffuse cell hypertrophy and normal HE Stainning;the pathological changes of intermediate osteoarthritis could be found at 8^thh week,which expressed fissuration into surface layer of cartilage,disorder arrangement of chondrocyte,chondrocyte decreasing,light HE staining.Conclusion:?1?The knee osteoarthritis model can be successfully established by immobilization,intra-articular injection of papain and anterior cruciate ligament cutting.?2?After immobilizating,early stage changes occur at 4 ^thh week,metaphase at 6 ^thh and 8^thweek;papain group is the same as the immobilization group;after ACLT,early stage changes occur at 4^th,6^thweek,metaphase at 8^thweek.?3?Injecting papain into joint cavity is easy to operate,and the animal's quality of life is relatively high,4 ^thh week is the early stage of KOA,6 ^thh and 8 ^thh week are the middle period.It is an ideal method for modeling.