FIG. 9.

Lack of Nrf2 increases inflammatory response after FAL. Nrf2^+/+ and Nrf2^−/− mice were subjected to FAL. (A) Representative pictures of the morphology of muscles (caput gastrocnemius—left panel, adductor—right panel), untreated and ischemic (days 3 and 21 after FAL). Hematoxylin/eosin staining. Arrows indicate centrally located nuclei of muscle fibers, which suggest intensive regeneration of tissue. Magnification 1000×(scale bar: 10 μm). (B, C) The lack of Nrf2 tends to increase the degree of T cell infiltration into ischemic muscle. (B) Representative pictures of CD3 antigen staining at day 3 after FAL (caput gastrocnemius—left panel, adductor—right panel). Magnification 1000×(scale bar: 10 μm). Immunohistochemistry. (C) Quantitative analysis of CD-3-positive cells (n_Nrf2+/+=2–3, n_Nrf2−/−=2). (D–F) Preoperatively (untreated) and at days 1 and 3 after FAL the expression of TNF-α and E-sel (D, F respectively, RT-PCR) was examined in caput gastrocnemius (n_Nrf2+/+=10–12; n_Nrf2−/−=10–11), while the protein level of IL-1β was tested in the plasma (E) (Luminex) (n_Nrf2+/+=11; n_Nrf2−/−=10–12). (G, H) Nrf2^+/+ and Nrf2^−/− mice were subjected to FAL and etodolac treatment [10 mg/(kg bw·day⁻¹)]. The ratio of blood flow in the left limb (ischemic) to right limb (non-ischemic) is higher in Nrf2^−/− mice, but tends to be reversed after etodolac treatment. Blood flow was measured 30 min after surgery (day 0) and at days 7, 14, and 20. Laser Doppler perfusion imaging. (G) Representative pictures. (H) Quantitative analysis (n_Nrf2+/+=6, n_Nrf2−/−=5–6); each bar/point represents the mean±SEM. *p<0.05, untreated versus ischemia (appropriate time point) or day 0 versus appropriate time point; ^$p<0.05, control (olive oil) versus etodolac; ^#p<0.05, Nrf2^+/+ versus Nrf2^−/−; ^##p<0.05, Nrf2^+/+ etodolac versus Nrf2^−/− etodolac. E-sel, E-selectin. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars