The molecular mechanisms contributing to posttraumatic ankle osteoarthritis (PTAOA) remain unclear. Here, we developed a rat model of PTAOA induced by joint instability and evaluated both pain and pathological changes. We paid special attention to the synovial tissue, which acts as a critical, pain-generating structure in OA. We induced ankle instability by resection of the anterior talofibular ligament (ATFL) and calcaneofibular ligament (CFL) (RAC group). Safranin-O and hematoxylin & eosin staining were conducted to evaluate histological changes. Pain-related behavior was also assessed. Bulk RNA sequencing for dorsal root ganglia (DRG) and single-cell RNA sequencing for synovial tissues were performed to examine molecular changes in PTAOA. Cartilage loss (Mean Difference (MD)=0.52, 95%CI: -0.019 to 1.060) and synovitis (MD=2.24, 95% CI: 1.473 to 3.009) were observed in the ankle joints of RAC rats (n=8), compared to the Sham control (n=8). Pain also developed in PTAOA rats. RNA sequencing revealed that the TGF-β-MAPK/PI3K-Akt-CTGF signaling axis and the THBS2-FN1 axis were activated in PTAOA synovial tissue. In particular, suppressing THBS2 expression decreased fibrosis marker expression in human fibroblast culture. Lastly, systemic administration of meloxicam reduced pain in PTAOA rats compared with the placebo group (MD=50.4, 95% CI: 4.8 to 96.1) (n=8 per group). This study sheds light on the pathological changes in synovial tissue and identifies key pathways involved in PTAOA. The findings suggest novel therapeutic targets for managing PTAOA. Additionally, the PTAOA model developed in this research can be used as a preclinical model to test potential treatments.

Next-generation sequencing can identify previously uncharacterized gene expression patterns in disease. Beyond differentially expressed gene (DEG) analysis, we investigated the ability of within-population diversity (α-diversity) of the transcriptome to reveal additional biological information in alcohol-associated liver disease (ALD), comparing differential Shannon diversity (DSD) to transcriptome heterogeneity changes. RNA sequencing data from normal livers and patients with early ALD and severe AH were analyzed. α-Diversity indices and percentage Shannon diversity of a gene, which refers to this gene’s contribution to total Shannon entropy, were calculated. Ingenuity pathway analysis identified canonical pathways determined by DEG and DSD approaches. ALD significantly decreased hepatic transcriptome α-diversity, correlating with increased relative contribution of select genes. These changes were driven by lower-abundance gene expression loss. DEG and DSD analyses showed overlapping genes and canonical pathways, but DSD also identified additional genes and pathways not highlighted by DEGs, including fatty acid oxidation, extracellular matrix degradation, and cholesterol metabolism pathways that may represent additional therapeutic targets. Importantly, DSD more effectively identified differences between ASH and AH. Overall, α-diversity analysis revealed that ALD progressively reduces transcriptome heterogeneity, and that DSD provides complementary insights into disease mechanisms missed by standard approaches.