Pain avoidance behavior tests indicated that persistent pain generation occurred on day 7 in the high-dose group. We performed histological assessments on days 0, 1, 3, 5, and 7 after MIA injection to identify biological events around the knee joint in the high-dose group (1.0 mg). Figure
3 presents the cell number changes over time in the perivascular region, IFP body (parenchymal region), and IFP surface (synovial membrane). As shown in this figure, the first sign of the structural change was observed in the perivascular region (Fig.
3a, indicated by the arrowhead). We found that the number of cell nuclei stained by hematoxylin immediately increased after MIA injection on day 1 and then reduced after day 5 in the perivascular region. Cell kinetics was semiquantitatively evaluated and plotted in Fig.
3b. Eosin staining, which visualizes both the cytoplasm and extracellular connective tissues, also indicated the surge of cellularity in the perivascular region as early as day 1 after MIA injection (Fig.
3a). Unlike that of hematoxylin, staining intensity of eosin did not decrease after day 5, suggesting the accumulation of extracellular matrices after the surge of cell numbers in the perivascular region. Cell migration kinetics in the perivascular region seemed to be followed by that in the IFP body (Fig.
3a). The number of the cells gradually increased on day 3 and was continuously high until day 7. Interestingly, staining intensity of eosin dramatically increased between days 5 and 7 without increasing the number of nuclei (stained by hematoxylin), indicating extensive extracellular matrix apposition during this period (Fig.
3a). Hyperplastic changes in the IFP surface (synovial membrane) initiated on day 3 after MIA injection and continued throughout the experimental period (Fig.
3a). These data suggest that the inflammatory response may start around the perivascular region and then move to the IFP surface despite the intra-articular injection of MIA. Representative images of the time course changes in the contralateral side (intra-articular injection of PBS) are shown in Additional file
2: Figure S2A (the top row).
Next, to analyze the changes in inflammatory cell number after intra-articular injection of MIA, immunostaining of IFP by ED-1 antibody, which identifies macrophages, was performed. As presented in Fig.
4, rapid accumulation of macrophages was observed on day 1 in the perivascular region (Fig.
4a, arrows). These macrophages spread throughout the IFP body on day 3 and reached the IFP surface on day 5 (Fig.
4a). Macrophages that infiltrated into the perivascular region and IFP body quickly disappeared after day 5 (Fig.
4a). Semiquantitative analyses also support these observations (Fig.
4b). Representative images of the time course changes in the contralateral side (intra-articular injection of PBS) are shown in Additional file
2: Figure S2 (the second row from the top).
Since HE staining suggested extensive extracellular matrix apposition between days 5 and 7 after MIA injection in the high-dose group, we examined collagen fiber accumulation and its time course changes in the IFP. As presented in Fig.
5, fibrotic changes in the IFP started from the perivascular region on day 1. Then, scattered fibrotic areas in the IFP body increased between days 3 and 5. Interestingly, fibrosis rapidly increased between days 5 and 7, and almost all the adipocytes in the parenchymal region of the IFP disappeared at day 7 (Fig.
5). Representative images of the time course changes in the contralateral side (intra-articular injection of PBS) are shown in Additional file
2: Figure S2 (the bottom row).