Figure 1.
Definition of Lanes 1, 2 and 3.
Knee joints were sliced along the sagittal axis (a). Lane 1 is the section in which the central region of the medial meniscus was continuous (b, arrow). Lane 2 and Lane 3 are 100 µm and 200 µm lateral to Lane 1, respectively (c, d).
Figure 2.
Histological analysis of the IPFP of mice fed a HFD and a normal diet.
Sections of articular cartilage from control (a) and HFD (b) mice stained with HE. Note advanced angiogenesis (arrows) and hypertrophic adipocytes in (b). (c); HFD mice weighed 10% more than normal diet mice by four weeks, 20% more by eight weeks and 50% more by twelve weeks. (d–f); Histological quantification of the IPFP area (d), average individual adipocyte area (e) and total vascular area (f) in IPFP. * = P<0.05 using the Mann-Whitney U test.
Figure 3.
High magnification of the anterior edge of the tibial plateau in the HFD and the control diet mice at the indicated weeks after diet initiation.
Representative HE stained sections (a–f) from control mice (a–c) and mice fed the HFD diet (d–f). g, h, i are neighbor sections in Safranin-O staining of d, e, f respectively. Increased synovial cell aggregation was observed at the anterior edge of the tibial plateau and synovium in the HFD (asterisk) mice beginning at week eight. Simultaneously, angiogenesis was activated within the IPFP (arrows) in the HFD group. Osteophytes were first noted at week eight of the HFD (triangles).
Figure 4.
Quantification of osteophytes and real-time RT-PCR analysis of the IPFP.
a; The mean osteophyte volume in the control and the HFD group at indicated weeks after onset of the diet. Osteophyte volume was significantly increased in HFD mice beginning at week eight. Reported values are the mean ±1 standard deviation (SD). b; Histological changes in the OA joints were assigned cartilage destruction scores. Values reported include the mean ±1 SD of five to ten mice per group. * = P<0.05 by Mann-Whitney U test. (c–h); mRNA expression of IPFP Leptin, Nampt, TNF-α, VEGF, TGF-β1 and CD68 for both the HFD and the control group. The expression levels of these cytokines were elevated in the HFD mice at week eight. Values are the mean ±1 SD of five mice per group. * = P<0.05 with the Mann-Whitney U test.
Figure 5.
Correlation between adipokines and inflammatory cytokines or CD68 and immunohistological evaluation of knee joints.
(a); Correlation between mRNA expression of adipokines, such as Leptin and mRNA expression of Nampt, and CD68 or inflammatory cytokines. The expression of Leptin and Nampt were positively correlated with TNF-α, VEGF and TGF-β, Nampt expression was strongly correlated with the macrophage marker CD68. (b); Immunostaining for Nampt and PCNA in the IPFP from week twelve of the HFD and in controls. Nampt protein expression was increased at the IPFP in the HFD. PCNA positive cells were abundantly observed at the peripheral region of osteophyte formation in the HFD group. (c, d); Analysis of apoptosis in TUNEL-stained knee joint sections from HFD and control mice. The number of TUNEL-positive cells was increased in knee joint cartilage from mice in the HFD group especially at the areas with osteophytes (triangle) and the superficial layer of articular cartilage (arrows). (e, f); The number of TUNEL-positive cells per section in the superficial layer of the articular cartilage was determined under fluorescence microscopy. Reported values are the mean ±1 SD of five mice per group. * = P<0.05 by Mann-Whitney U test.