Gale Tang, MD, David N. Charo, Rong Wang, PhD, Israel F

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CCR2-/- knockout mice revascularize normally in response to severe hindlimb ischemia Gale Tang, MD, David N. Charo, Rong Wang, PhD, Israel F. Charo, MD, PhD, Louis Messina, MD Journal of Vascular Surgery Volume 40, Issue 4, Pages 786-795 (October 2004) DOI: 10.1016/j.jvs.2004.07.012 Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

Fig 1 Mouse hindlimb ischemia models. A, Severe hindlimb ischemia was induced by femoral artery excision as diagrammed. Dashed line indicates level at which muscle sections for the thigh were harvested for MOMA-2 immunohistochemistry. B, Mild hindlimb ischemia was induced by femoral artery ligation as shown by simplified diagram. Journal of Vascular Surgery 2004 40, 786-795DOI: (10.1016/j.jvs.2004.07.012) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

Fig 2 CCR2-/- mice recover blood flow normally following induction of mild or severe hindlimb ischemia. A, Digital picture showing anesthetized mouse positioned for laser Doppler perfusion imaging (left) and representative laser Doppler perfusion scanned images at postoperative day 31 in wild-type and CCR2-/- mice with footpad (red) and calf (yellow) regions of interest outlined. B, Footpad (left panel) and calf (right panel) recovery of blood flow by wild type (solid squares, n = 16) and CCR2-/- mice (open circles, n = 16) were equivalent over 31 days after induction of severe hindlimb ischemia as measured by laser Doppler perfusion imaging under isoflurane anesthesia (P = .78 and P = .38, respectively). Journal of Vascular Surgery 2004 40, 786-795DOI: (10.1016/j.jvs.2004.07.012) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

Fig 3 Angioscores of wild-type and CCR2-/- mice are equivalent at postoperative days 14 and 31. A, Representative angiograms of wild-type and CCR2-/- mice at postoperative day 31. B, Angioscores of wild-type (n = 3) or CCR2-/- mice (n = 4) at postoperative day 14 and at postoperative day 31 (n = 7 per group, right panel) were not statistically different (4.37 ± 0.46 vs 5.08 ± 0.73, P = .46, and 6.35 ± 0.0.66 vs 5.97 ± 0.58, P = .67, respectively) Journal of Vascular Surgery 2004 40, 786-795DOI: (10.1016/j.jvs.2004.07.012) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

Fig 4 CCR2-/- hindlimb skeletal muscle develops increased edema after induction of hindlimb ischemia. Tibialis anterior (A) and gastrocnemius muscle mass (B), presented as the ratio between left (operated) and right (unoperated) muscle mass, is significantly increased secondary to increased skeletal muscle edema over wild-type muscle mass at postoperative day 7 (n = 6 per group, 1.81 ± 0.20 vs 0.78 ± 0.08, P = .009, and 1.44 ± 0.20 vs 0.78 ± 0.02, P = .03, respectively). Muscle mass recovery at postoperative days 14 and 31 is equivalent between the 2 groups (n = 3 wild-type and n = 4 CCR2-/- mice, 0.97 ± 0.04 vs 1.05 ± 0.03, P = .22, and 0.89 ± 0.10 vs 0.96 ± 0.03, P = .51, for postoperative day 14; n = 7 wild-type and n = 4 CCR2-/- mice, 1.00 ± 0.08 vs 1.12 ± 0.09, P = .35, and 0.91 ± 0.14 vs 1.00 ± 0.08, P = .64, for postoperative day 31). Representative photomicrographs of gastrocnemius muscle stained with hematoxylin and eosin demonstrate increased edema in CCR2-/- muscle at postoperative day 7 (C). Journal of Vascular Surgery 2004 40, 786-795DOI: (10.1016/j.jvs.2004.07.012) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

Fig 5 Ischemia-induced monocyte/macrophage recruitment is decreased in CCR2-/- mice. Wild type (A, C, E, G, I) and CCR2-/- (B, D, F, H, J) muscle sections stained for the monocyte/macrophage marker MOMA-2. Arrows indicate representative intraluminal MOMA-2+ cells. Arrowheads indicate representative perivascular MOMA-2+ cells. A, B, Unoperated gastrocnemius muscle. C, D, Right gastrocnemius muscle. E, F, Left gastrocnemius muscle. G, H, Right thigh muscle. I, J, Left thigh muscle. K, Total MOMA-2 positive staining cells per high-powered field were significantly higher in the ischemic calf and distal thigh of wild-type mice 7 days after left femoral artery excision than in the ischemic calf and distal thigh of CCR2-/- mice or unoperated mice (184.8 ± 19.9 vs 110.6 ± 10.3 in CCR2-/- mice vs 40 ± 15.6 in unoperated wild-type mice, and 116.5 ± 20.4 vs 85 ± 9.5 in CCR2-/- mice vs 27.3 ± 4.2 in unoperated wild-type mice, respectively, *P < .05 vs unoperated control; #P < .05 vs CCR2-/- mice). L, Perivascular MOMA-2 positive staining cells were significantly higher in the ischemic calf of wild-type mice 7 days after left femoral artery excision than in the ischemic calf of either CCR2-/- mice or unoperated mice (10.0 ± 1.7 vs 7.9 ± 0.3 or 5.3 ± 2.5, respectively; *P < .05 vs unoperated control; #P < .05 vs CCR2-/- mice). Journal of Vascular Surgery 2004 40, 786-795DOI: (10.1016/j.jvs.2004.07.012) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions