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Hind Limb Ischemia Reperfusion Accelerates Vascular Injury in Susceptible Vessels

Virendra I Patel¹, Hassan Albadawi¹, Thomas A Abbruzzese¹, Jeanwan Kang¹, Hyung-Jin Yoo¹, Christian Ferran², Richard P Cambria¹, Michael T Watkins¹ ∙
¹Massachusetts General Hospital, Boston, MA; ²Beth Israel Deaconess Medical Center, Boston, MA

Objectives: Patients with vascular disease have synchronous atherosclerotic lesions in many vascular beds. Some of these beds (heart, brain, kidney) are sites where fatal and devastating complications are observed following acute limb ischemia-reperfusion injury (IR). These studies were designed to evaluate the effect of hind limb IR on the systemic inflammatory response and remote vascular injury in susceptible vessels (carotid blood flow cessation model) in apolipoprotein E-deficient (ApoE -/-) mice.

Methods: Apo E -/- mice and C57BL6/J control mice underwent 3 hours of hind limb ischemia followed by 24 or 96 hours of reperfusion. Systemic inflammation was assessed by measuring serum levels of Interleukin-6 (IL-6) and monocyte chemottractant protein 1 (MCP-1). Only ApoE -/- mice underwent ligation of the left common carotid artery bifurcation, and at 72hrs, were subjected to either 3 hours of limb ischemia or anesthesia alone. At 7 and 28 days following ligation, the carotid arteries were formalin fixed, embedded and serially sectioned. 7-day vessels were analyzed for BrdU incorporation by immunohistochemistry. The intima to media (I/M) ratio was analyzed in sections of 28-day vessels. Mortality was assessed using Fisher’s exact test. Cytokine and I/M ratios were compared with unpaired t-tests.

Results: Mortality in ApoE -/- mice following IR was higher than the control mice (48% vs. 4%, p= 0.001). Systemic IL-6 levels were elevated in ApoE -/- (n=6) compared to controls (n=8) at 24h (262±30 vs. 154±20 pg/ml; p<0.001) and 96h (37±6; n=6 vs. 20±3 pg/ml; n=8, p=0.001). No difference in MCP-1 level was detected. The I/M ratio in ApoE -/- mice undergoing carotid ligation and 3h IR was greater than that observed following carotid ligation alone (1.3±0.4; n=9 vs. 0.4±0.3; n=10, p<0.005). Mice with ligation and IR had increased proliferation of medial smooth muscle cells as detected by BrdU staining.

Conclusions: Compared to control mice, ischemia reperfusion in mice with susceptible vessels (Apo E -/-) is associated with an increased systemic inflammatory response, increased mortality and accelerated remote arterial injury. Remote arterial injury following limb IR may contribute to cardiovascular, renal and cerebrovascular morbidity.