Changes And Contributions Of Inflammation Reaction In Acute Traumatic Deep Venous Thrombosis

Objective To dynamically detect femoral vein gene expression changes in a rat model of acute traumatic deep vein thrombosis (TDVT) and to screen differential expression genes involved in this pathological process. Then to mainly analyze the expression changes of inflammmation-associated genes; combined with variations of inflammatory cells infiltration in venous wall, blood cell count, variation of TNF-α, IL-1β, IL-6, IL-10 in serum and a correlation analysis between these factors and thrombus weight, to explore the changes and contributions to acute TDVT.Methods 1．Directly clamping bilateral femoral veins combined with hip spica fixation was performed to establish acute TDVT rat models. According to different observation phases and gross observation results, 150 SD rats were divided into 7 groups: the control(group A),post-traumatic instant (group B), initial period of thrombogenesis (group C), thrombogenesis at thrombotic crest-time (group D), thrombusi resolution (group E), thrombus insolution (group F) and nonthrombogenesis in post-traumatic 7d (group G). 2．At each phases, bilateral femoral veins were cut, in which, 0．5cm long vein tissune was for HE staining and histological obervation in order to ensure grouping and observe inflammatory cell infiltration in venous wall; the rest 3～4cm long vein tissue from same groups were mixed and total RNAs of them were extracted respectively through TRIzol one-step method. After the qualities of RNA samples were confirmed by 3% agarose gel electrophoresis (AGE) and Lab-on quality determination system, the RNAs expression levels in femoral vein were detected through Affymetrix genechips. 3．Combined with Fold Change analysis, to screen differential expression genes from the genechips data of each group and to perform GO classification. 4．Through an index word inflammation, the inflammation-associated genes were selected from the genechip data including 15 864 genes and analyzed through Fold Change analysis. 5． Cluster analysis was performed in these genes through Cluster software 3．0．6．RT-RCR were applied to detect expression levels of IL-1α, IL-1β, IL-6, Cinc2, IL-10．And to compare the results of genechips and RT-PCR. 7．Another 150 rats were modeled and grouped through the above-mentioned method, and 0．5ml blood samples of them were drawn from heart for white blood cell differential count; blood serum from another 1．5ml blood samples were used for ELISA determinations of TNF-α, IL-1β, IL-6, IL-10．8．Meanwhile, the femoral veins from inguinal ligament to the affluxion point of great saphenous vein were cut for length and weight measurement so as to calculate thrombus weight. And the correlation between thrombus weight and the expression quantities of TNF-α, IL-1β, IL-6, IL-10 in serum was analyzed through Pearson correlation analysis.Results 1．In this rat model, no thrombogenesis in A, B groups; to post-traumatic 2．5 h, thrombi were observed in 3% femoral veins; to post-traumatic 25 h, thrombi were observed in 60% femoral veins of group D. After that, there was 3% femoral vein with thrombogenesis occurrence, but most of thrombi solved gradually. To post-traumatic 72h, the rate of thrombi solution was 60% and the rate of thrombi insolution was 40%. In the whole process, there were 47% rats without thrombogenesis. 2．The femoral vein macroscopic observation results of the 7 groups were coherent to the histological observation results. 3．Compared with A, B, E, F groups, the venous wall infiltrating inflammatory cells counts were much higher in C, D, F groups. In B, C groups, neutrophilic granulocyte was the main inflammatory cell; in E, F, G groups, lymphocyte was the main ones. 4．Meanwhile, 2458 genes presented differential expression in this pathological process; in which, 1146 genes up-regulated (Log Ratio≥1 and Change was signed asⅠ), 1312 genes down-regulated (Log Ratio≤-1 and Change was signed as D). Most of them were unknown genes. And the genes with known functions were mainly involved in DNA dependent transcription regulation, inflammation, immunity, infra-or inter-cellular signal transmission, fibrolysis-antifibrolysis, blood coagulation and anti-coagulation, etc. 5．Fifty-five inflammation-associated genes were screened through Affymetrix Rat 230A genechips; in which, 24 presented differential expression in this process. 6． The RT-PCR and genechip detecting results of IL-1α, IL-1β, IL-6, Cinc2, IL-10 presented a similar trends, however, RT-PCR was more sensitive (Z=-3．785,P=0．00<0．05) . 7．The result of Cluster analysis showed that the 55 inflammation-associated genes could be divided into three clusters: the first cluster genes, including IL-1, IL-6, Cinc2, etc., presented a similar expression trend which coincided to the trend of thrombi genesis and evolution; the second cluster genes, including IL-10, IL-2,etc, presented upregulation following the first cluster genes. However, in thrombi solution/insolution groups, the variational trends of these genes were opposite to the first ones. The third cluster genes with disunity patterns, including TNF, CD34, etc., could make an assistant effects to the biological funtion of the aforementioned two cluster genes. 8．Compared with A, E, G groups, the white blood cell counts in B, C, D, F groups increased much more. 9．The expression variaties and transcriptional levels of TNF-α, IL-1β, IL-6,IL-10 were unequal. And there was no correlation btween TNF-α, IL-1β, IL-6, IL-10 and thrombi weight (Pearson correlation coefficients were 0．14, 0．4, 0．39, 0．22 respectively, p>0．05) ; thrombi weight was positively correlated to a ratio between IL-1βand IL-10 (Pearson r=0．87, p=0．01<0．05) .Conclusions 1 .An acute TDVT animal model can be established through direct clamping combined with hip spica fixation; the modeling method is convenient, the rate of thrombogenesis is higher and the pathological process in this model is similar to the direct injury of vessel in clinical practice. Most of the thrombi in this model are occlusive, which is profit for feasibility and reliability of grouping. 2．The TDVT pathological process was also followed with a change from acute to chronic inflammation. 3．Acute TDVT is a multigenic disorder, which involved inflammation, immunity fibrolysis/anti-fibrolysis, blood coagulation/anticoagulation, etc.. 4．There is a close relathionship between post-traumatic inflammatory reaction and TDVT. Inflammatory mediators such as IL-1α, IL-1β, IL-6 could enhance thrombogenesis, however, anti-inflammatory mediators sunch as IL-10, IL-2 could inhibit thrombogenesis. 5．The ratio between IL-1βand Il-10 can indirectly reflect the different states and prognosis of TDVT. 6．From chronologicaol order, the close relationship between thrombogenesis and the changes of genes and proteins related to inflammatory reaction and the contributions of TNF-α, IL-1, IL-6 to blood coagulation and fibrolysis systems, the results of this study supports that inflammation is a reason of thrombogenesis, which needs a further study.