Postischemic poly (ADP-ribose) polymerase (PARP) inhibition reduces ischemia reperfusion injury in a hind-limb ischemia model.

BACKGROUND: Several experiments were designed to determine whether the systemic, postischemic administration of PJ34,which is a poly-adenosine diphosphate (ADP)-ribose polymerase inhibitor, decreased tissue injury and inflammation after hind-limb ischemia reperfusion (I/R). METHODS: C57BL6 mouse limbs were subjected to 1.5 h ischemia followed by 24-h reperfusion. The treatment group (PJ) received intraperitoneal PJ34 (30 mg/kg) immediately before reperfusion, as well as 15 min and 2 h into reperfusion. The control group (CG) received lactated Ringer's alone at the same time intervals as PJ34 administration. The skeletal muscle levels of adenosine triphosphate (ATP), macrophage inflammatory protein-2 (MIP-2), keratinocyte derived chemokine (KC), and myeloperoxidase (MPO) were measured. Quantitative measurement of skeletal muscle tissue injury was assessed by microscopic analysis of fiber injury. RESULTS: ATP levels were higher in limbs of PJ versus CG mice (absolute ATP: 4.7 +/- 0.35 vs 2.3 +/- 0.15-ng/mg tissue, P = .002). The levels of MIP-2, KC, and MPO were lower in PJ versus CG mice (MIP-2: 1.4 +/- 0.34 vs 3.67 +/- 0.67-pg/mg protein, P = .014; KC: 4.97 +/- 0.97 vs 12.65 +/- 3.05-pg/mg protein, P = .037; MPO: 46.27 +/- 10.53 vs 107.34 +/- 13.58-ng/mg protein, P = .008). Muscle fiber injury was markedly reduced in PJ versus CG mice (4.25 +/- 1.9% vs 22.68 +/- 3.0% total fibers, P = .0004). CONCLUSION: Systemic postischemic administration of PJ34 preserved skeletal muscle energy levels, decreased inflammatory markers, and preserved tissue viability post-I/R. These results support PARP inhibition as a viable treatment for skeletal muscle I/R in a clinically relevant post hoc scenario.

Apolipoprotein E-/- mice have delayed skeletal muscle healing after hind limb ischemia-reperfusion.

INTRODUCTION: Classic studies of limb ischemia-reperfusion injury have been performed using young healthy mice. However, patients with peripheral vascular disease are older and often exhibit metabolic derangements that may delay healing after revascularization. Mice with genetic deletion of apolipoprotein E (ApoE(-/-)) have been used as a model in various experimental scenarios of hypercholesterolemia. These experiments evaluated the inflammatory response and changes in skeletal muscle morphology during the acute and chronic phases of limb ischemia-reperfusion injury in aged ApoE(-/-) mice. METHODS: Age-matched ApoE(-/-) and wild-type (Wt) mice underwent 1.5 hours of unilateral hind limb ischemia, followed by 1, 7, or 14 days of reperfusion (DR). Histologic analysis of skeletal muscle fiber injury was assessed at 1DR. Morphologic evidence of muscular fiber maturation was assessed at 14DR. Levels of MyoD and myogenin, markers of skeletal muscle differentiation, were assessed at 7 and 14DR using Western blots. Markers of inflammation, including myeloperoxidase, macrophage inflammatory protein-2 (MIP-2), monocyte chemotactic protein-1 (MCP-1), and osteopontin, were assayed using enzyme-linked immunosorbent assay and chemokine (C-C motif) receptor 2 (CCR2) using Western blots at 1, 7, and 14DR. After 1DR, tissue adenosine 5'-triphosphate (ATP) levels were measured to assess metabolic activity. Unpaired t test and Mann-Whitney test were used for comparisons. RESULTS: Histologic evaluation of skeletal muscle after 1DR showed no difference in the degree of injury between Wt and ApoE(-/-) mice. However, at 14DR, ApoE(-/-) mice had higher percentage of immature muscle fibers than Wt mice. Myogenin level was lower in the ApoE(-/-) mice at 7DR. Injured skeletal muscle of ApoE(-/-) mice had lower levels of myeloperoxidase than Wt mice at 7 DR and higher levels of MCP-1 at 14DR. There was no difference in the levels of tissue ATP, MIP-2, osteopontin, or CCR2 at all experimental intervals. CONCLUSION: Although there was no difference between the injured muscle of Wt and ApoE(-/-) mice during the acute phase of reperfusion, ApoE(-/-) mice showed delay in skeletal muscle healing during the chronic phase of reperfusion. This lag in muscle regeneration was associated with lower levels of myogenin at 7DR and an increased level of MCP-1 at 14DR in the ApoE(-/-) mice. The delay in skeletal muscle healing in the ApoE(-/-) mice may have broader implications for poor tissue healing and functional recovery in elderly patients who have vascular risk factors such as hypercholesterolemia.

A novel model of acute murine hindlimb ischemia.

The McGivney hemorrhoidal ligator (MHL), a band designed to cause tissue necrosis, is the preferred experimental tool to create hindlimb ischemia-reperfusion (I/R) injury in rodents. This report defines and compares the ex vivo band tension exerted by MHL and orthodontic rubber bands (ORBs) along with select in vivo characteristics of I/R. As to method, ex vivo band tension was measured over relevant diameters using a tensiometer. In vivo assessment of murine limb perfusion during ischemia with ORB and MHL was compared using laser Doppler imaging and measurement of wet weight-to-dry weight ratio. Neuromuscular scoring and histological extent of muscle fiber injury after I/R with MHL and ORB were also compared. A dose-response curve, between the duration of ORB-induced I/R with both mitochondrial activity (methyl-thiazol-tetrazolium) or tail perfusion [laser Doppler imaging (LDI)], was generated. As a results, ex vivo measurements showed that ORB exerted significantly less force than the MHL. Despite less tension in ORB, in vivo testing of the ORB confirmed complete ischemia by both LDI and wet weight-to-dry weight ratio. After I/R, caused by ORB, there was significantly less neuromuscular dysfunction. Histological assessment confirmed similar degrees of muscle fiber injury after I/R with either the MHL or ORB. Increasing durations of ischemia created by the ORB followed by reperfusion significantly decreased mitochondrial activity and tail perfusion after 24 h of ischemia. In conclusions, ORB produced similar levels of tissue ischemia in murine models of limb I/R with fewer levels of nonspecific injury. ORB may be the preferred model for selected studies of limb I/R.