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1.  Poly ADP-Ribose Polymerase Inhibition Ameliorates Hind Limb Ischemia Reperfusion Injury in a Murine Model of Type 2 Diabetes 
Annals of surgery  2013;258(6):10.1097/SLA.0b013e31828cced3.
Introduction
Diabetes is known to increase poly-ADP-ribose-polymerase (PARP) activity and posttranslational poly-ADP-ribosylation of several regulatory proteins involved in inflammation and energy metabolism. These experiments test the hypothesis that PARP inhibition will modulate hind limb ischemia reperfusion (IR) in a mouse model of type-II diabetes; ameliorate the ribosylation and the activity/transnuclear localization of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Methods
db/db mice underwent 1.5hrs of hind limb ischemia followed by 1, 7, or 24hrs reperfusion. The treatment group received the PARP inhibitor PJ34 (PJ34) over a 24hrs period; the untreated group received Lactated ringer’s (LR) at the same time points. IR muscles were analyzed for indices of PARP activity, fiber injury, metabolic activity, inflammation, GAPDH activity /intracellular localization and poly-ADP-ribosylation of GAPDH.
Results
PARP activity was significantly lower in the PJ34 treated groups compared to the LR group at 7 and 24 hours reperfusion. There was significantly less muscle fiber injury in the PJ34 treated group compared to LR treated mice at 24 hrs reperfusion. PJ34 lowered levels of select proinflammatory molecules at 7hrs and 24hrs IR. There were significant increases in metabolic activity only at 24 hours IR in the PJ34 group, which temporally correlated with increase in GAPDH activity, decreased GAPDH poly ADP-ribosylation and nuclear translocation of GAPDH.
Conclusions
PJ34 reduced PARP activity, GAPDH ribosylation, GAPDH translocation, ameliorated muscle fiber injury, and increased metabolic activity following hind limb IR injury in a murine model of type-II diabetes. PARP inhibition might be a therapeutic strategy following IR in diabetic humans.
doi:10.1097/SLA.0b013e31828cced3
PMCID: PMC3773522  PMID: 23549425
2.  Reduced hind limb ischemia-reperfusion injury in Toll-like receptor-4 mutant mice is associated with decreased neutrophil extracellular traps 
Journal of vascular surgery  2013;58(6):10.1016/j.jvs.2013.02.241.
Objective
Ischemia-reperfusion (IR) injury is a significant problem in the management of patients with acute limb ischemia (ALI). Despite rapid restoration of blood flow following technically successful open and endovascular revascularization, complications secondary to IR injury continue to occur and limit clinical success. Our aim was to create a murine model of hind limb IR injury to examine the role of Toll-like receptor-4 (TLR4) and to determine whether inactive TLR4 led to a decrease in the detection of neutrophil extracellular traps (NETs), which is known to be highly thrombogenic and may mediate microvascular injury.
Methods
A calibrated tension tourniquet was applied to unilateral hind limb of wild type (WT) and TLR4 receptor mutant (TLR4m) mice for 1.5 hours to induce ischemia and then immediately removed to initiate reperfusion. At the end of 48 hours of reperfusion, mice were sacrificed and hind limb tissue as well as serum specimens were collected for analysis. Hematoxylin and eosin stained sections of hind limb skeletal muscle tissue were examined for fiber injury. For immunohistochemistry, mouse monoclonal anti-histone H2A/H2B/DNA complex antibody to detect NETs and rabbit polyclonal anti-myeloperoxidase (MPO) antibody were used to identify infiltrating cells containing MPO. Muscle ATP levels, nuclear NF-κB activity, IκBα, poly (ADP-ribose) polymerase (PARP) activity and iNOS expression were measured. Systemic levels of KC, MCP-1 and VEGF in the serum samples were also examined.
Results
IR injury in the hind limb of wild type mice demonstrated significant levels of muscle fiber injury, decreased energy substrates, increased NF-κB activation, decreased I-κBα levels, increased iNOS expression and increased PARP activity levels when compared to the TLR4 knockout mice samples. Additionally, there was marked decrease in the level of neutrophil and monocyte infiltration in the TLR4 mutant mice, which corresponded to similar levels of decreased NETs detection in the interstitial space and in microvascular thrombi. In situ nuclease treatment of wild-type tissue sections significantly diminished the level of NETs immunostaining demonstrating the specificity of our antibody to detect NETs and suggesting a potential role for nuclease treatment in IR injury.
Conclusions
These results suggest a pivotal role for TLR4 in mediating hind limb IR injury and suggest that NETs may contribute to muscle fiber injury.
doi:10.1016/j.jvs.2013.02.241
PMCID: PMC3825746  PMID: 23683381
3.  Divergent Systemic and Local Inflammatory Response to Hind Limb Demand Ischemia in Wild Type And ApoE−/− Mice 
The Journal of surgical research  2013;183(2):952-962.
Introduction
Studies were designed to determine whether the ApoE−/− phenotype modulates the local skeletal muscle and systemic inflammatory (plasma) responses to lower extremity demand ischemia. The ApoE−/− phenotype is an experimental model for atherosclerosis in humans.
Methods
Aged female ApoE −/− and C57BL6 mice underwent femoral artery ligation, then divided into sedentary and demand ischemia (exercise) groups on day 14. Baseline and post exercise limb perfusion and hind limb function were assessed. On day 14, animals in the demand ischemia group underwent daily treadmill exercise through day 28. Sedentary mice were not exercised. On day 28, plasma and skeletal muscle from ischemic limbs were harvested from sedentary and exercised mice. Muscle was assayed for angiogenic and pro-inflammatory proteins, markers of skeletal muscle regeneration, and evidence of skeletal muscle fiber maturation.
Results
Hind limb ischemia was similar in ApoE −/− and C57 mice prior to the onset of exercise. Under sedentary conditions, plasma VEGF, IL-6, but not KC or MIP-2 were higher in ApoE (P<0.0001). Following exercise, plasma levels of VEGF, KC and MIP-2, but not IL-6 were lower in ApoE (P<0.004). The cytokines KC and MIP-2 in muscle was greater in exercised ApoE−/− mice as compared to C57BL6 mice (p=0.01). Increased PAR activity, and mature muscle regeneration was associated with demand ischemia in the C57BL6 mice as compared to the ApoE −/− mice (p=0.01).
Conclusion
Demand limb ischemia in the ApoE−/− phenotype exacerbated the expression of select systemic cytokines in plasma and blunted indices of muscle regeneration.
doi:10.1016/j.jss.2013.02.042
PMCID: PMC3713180  PMID: 23528286
Inflammation; ischemia; exercise; skeletal muscle
4.  Non-thermal, pulsed electric field cell ablation: A novel tool for regenerative medicine and scarless skin regeneration 
Technology  2013;1(1):1-8.
High voltage, short pulsed electric fields (PEF) is a non-thermal ablation method, in which defined PEF irreversibly destabilize cell membranes, while preserving other tissue components such as the extracellular matrix (ECM). In the present report, we show that PEF ablated rat skin retains its microvascular blood supply and ECM structure. Complete regeneration of epidermis, hair follicles, sebaceous glands, and the panniculus carnosusis observed two months after the ablation. Our results clearly indicate that non-thermal PEF has the potential to be a powerful and novel tool for scarless tissue regeneration.
doi:10.1142/S233954781320001X
PMCID: PMC4078877  PMID: 24999487
5.  Differential Effect of Zoledronic Acid on Human Vascular Smooth Muscle Cells 
The Journal of surgical research  2012;182(2):339-346.
Introduction
The activation of human vascular smooth muscle cell proliferation, adhesion and migration is essential for intimal hyperplasia formation. These experiments were designed to test whether Zoledronic Acid (ZA) would modulate indices of human smooth muscle cell activation, exert differential effects on proliferating vs. quiescent cells and determine whether these effects were dependent on GTPase binding proteins prenylation. ZA was chosen for testing in these experiments because it is clinically used in humans with cancer, and has been shown to modulate rat smooth muscle cell proliferation and migration.
Methods
Human aortic smooth muscle cells (HASMC) were cultured under either proliferating or growth arrest (quiescent) conditions in the presence or absence of ZA for 48 hours, whereupon the effect of ZA on HASMC proliferation, cellular viability, metabolic activity and membrane integrity were compared. In addition, the effect of ZA on adhesion and migration were assessed in proliferating cells. The effect of increased concentration of ZA on the mevalonate pathway and genomic/cellular stress related poly ADP Ribose polymerase (PARP) enzyme activity were assessed using the relative prenylation of Rap-1A/B protein and the formation of poly ADP- ribosylated proteins (PAR) respectively.
Results
There was a dose dependent inhibition of cellular proliferation, adhesion and migration following ZA treatment. ZA treatment decreased indices of cellular viability and significantly increased membrane injury in proliferating vs. quiescent cells. This was correlated with the appearance of unprenylated Rap-1A protein and dose dependent down regulation of PARP activity.
Conclusions
These data suggest that ZA is effective in inhibiting HASMC proliferation, adhesion and migration which coincide with the appearance of unprenylated RAP-1A/B protein, thereby suggesting that the mevalonate pathway may play a role in the inhibition of HASMC activation.
doi:10.1016/j.jss.2012.10.033
PMCID: PMC3618863  PMID: 23164362
6.  Inhalation of Carbon Monoxide Reduces Skeletal Muscle Injury Following Hind Limb Ischemia Reperfusion Injury in Mice 
American Journal of Surgery  2012;203(4):488-495.
Introduction
The purpose of this study was to determine if inhaled carbon monoxide (CO) can ameliorate skeletal muscle injury, modulate endogenous heme oxygenase-1 (HO) expression, improve indices of tissue integrity and inflammation following hind limb ischemia reperfusion(IR).
Methods
C57BL6 mice inhaling CO (250ppm) or room air were subjected to 1.5 hrs of ischemia followed by limb reperfusion for either 3 or 6 hours (total treatment time of 4.5 or 7.5 hrs). After the initial period of reperfusion, all mice breathed only room air until 24 hours after the onset of ischemia. Mice were sacrificed at either the end of CO treatment or at 24 hours reperfusion. Skeletal muscle was subjected to histologic and biochemical analysis.
Results
CO treatment for 7.5 hours protected skeletal muscle from histologic and structural evidence of skeletal muscle injury. Serum and tissue cytokines were significantly reduced (p<0.05) in mice treated with CO for 7.5 hours. Tubulin, Heme Oxygenase, and ATP levels were higher in CO treated mice.
Conclusions
Inhaled CO protected muscle from structural injury and energy depletion following IR.
doi:10.1016/j.amjsurg.2011.05.005
PMCID: PMC3315834  PMID: 22450026
Carbon Monoxide; Reperfusion Injury; Heme Oxygenase; Skeletal Muscle
7.  Tourniquet-induced acute ischemia-reperfusion injury in mouse skeletal muscles: involvement of superoxide 
European journal of pharmacology  2010;650(1):328-334.
Although arterial limb tourniquet is one of the first-line treatments to prevent exsanguinating hemorrhage in both civilian pre-hospital and battlefield casualty care, prolonged application of a limb tourniquet can lead to serious ischemia-reperfusion injury. However, the underlying pathomechanisms of tourniquet-induced ischemia-reperfusion injury are still poorly understood. Using a murine model of acute limb ischemia-reperfusion, we investigated if acute limb ischemia-reperfusion injury is mediated by superoxide overproduction and mitochondrial dysfunction. Hind limbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Approximately 40% gastrocnemius muscle suffered infarction in this model. Activities of mitochondrial electron transport chain complexes including complex I, II, III, and IV in gastrocnemius muscle were decreased in the ischemia-reperfusion group compared to sham. Superoxide production was increased while activity of manganese superoxide dismutase (MnSOD, the mitochondria-targeted SOD isoform) was decreased in the ischemia-reperfusion group compared to sham group. Pretreatment with tempol (a SOD mimetic, 50 mg/kg) or co-enzyme Q10 (50 mg/kg) not only decreased the superoxide production, but also reduced the infarct size and normalized mitochondrial dysfunction in the gastrocnemius muscle. Our results suggest that tourniquet-induced skeletal muscle ischemia-reperfusion injuries including infarct size and mitochondrial dysfunction may be mediated via the superoxide over-production and reduced antioxidant activity. In the future, this murine ischemia-reperfusion model can be adapted to mechanistically evaluate anti-ischemic molecules in tourniquet-induced skeletal muscle injury.
doi:10.1016/j.ejphar.2010.10.037
PMCID: PMC3008320  PMID: 21036124
Infarct size; Ischemia-reperfusion injury; Mitochondria; Superoxide; Tourniquet
8.  Post Ischemic PARP Inhibition Reduces Ischemia Reperfusion Injury in a Hind-Limb Ischemia Model 
Surgery  2010;148(1):110-118.
INTRODUCTION
These experiments were designed to determine whether systemic post ischemic administration of PJ34, a Poly ADP-ribose polymerase inhibitor, decreased tissue injury and inflammation following hind limb ischemia reperfusion (I/R).
METHODS
C57BL6 mouse limbs were subjected to 1.5 hrs ischemia followed by 24 hours reperfusion. The treatment group (PJ) received intraperitoneal PJ34 (30 mg/Kg) immediately before, 15 minutes and 2 hours into reperfusion. Control group (CG) received Lactated Ringers alone at the same time intervals as PJ34 administration. Skeletal muscle levels of 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 vs. CG (Absolute ATP: 4.7 ± 0.35 vs. 2.3 ± 0.15 ng/mg tissue, p=0.002). Levels of MIP-2, KC and MPO were lower in PJ vs. CG (MIP-2: 1.4±0.34 vs. 3.67±0.67 pg/mg protein, p=0.014; KC: 4.97±0.97 vs. 12.65±3.05 pg/mg protein, p=0.037, MPO: 46.27±10.53 vs. 107.34±13.58 ng/mg protein, p=0.008). Muscle fiber injury was markedly reduced in PJ vs. CG (4.25±1.9% vs 22.68±3.0% total fibers, p=0.0004).
CONCLUSION
Systemic post ischemic 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.
doi:10.1016/j.surg.2009.12.006
PMCID: PMC2886175  PMID: 20132957
Basic science; skeletal muscle; cytokines; inflammation; vascular disease
9.  Postischemic Treatment With Ethyl Pyruvate Prevents Adenosine Triphosphate Depletion, Ameliorates Inflammation, and Decreases Thrombosis in a Murine Model of Hind-Limb Ischemia and Reperfusion 
The Journal of trauma  2011;70(1):103-110.
Introduction
Experiments were designed to investigate the effects of ethyl pyruvate (EP) in a murine model of hind-limb ischemia-reperfusion (IR) injury.
Methods
C57BL6 mice underwent 90 minutes of unilateral ischemia followed by 24 hours of reperfusion using two treatment protocols. For the preischemic treatment (pre-I) protocol, mice (n = 6) were given 300 mg/kg EP before ischemia, followed by 150 mg/kg of EP just before reperfusion and at 6 hours and 12 hours after reperfusion. In a postischemic treatment (post-I) protocol, mice (n = 7) were treated with 300 mg/kg EP at the end of the ischemic period, then 15 minutes later, and 2 hours after reperfusion and 150 mg/kg of EP at 4 hours, 6 hours, 10 hours, 16 hours, and 22 hours after reperfusion. Controls mice for both protocols were treated with lactated Ringers alone at time intervals identical to EP. Skeletal muscle levels of adenosine triphosphate (ATP), interleukin-1β, keratinocyte chemoattractant protein, and thrombin antithrombin-3 complex were measured. Skeletal muscle architectural integrity was assessed microscopically.
Results
ATP levels were higher in mice treated with EP compared with controls under the both treatment protocols (p = 0.02). Interleukin-1β, keratinocyte chemoattractant protein, thrombin antithrombin-3 complex (p < 0.05), and the percentage of injured fibers (p < 0.0001) were significantly decreased in treated versus control mice under the both protocols.
Conclusion
Muscle fiber injury and markers of tissue thrombosis and inflammation were reduced, and ATP was preserved with EP in pre-I and post-I protocols. Further investigation of the efficacy of EP to modulate IR injury in a larger animal model of IR injury is warranted.
doi:10.1097/TA.0b013e3182031ccb
PMCID: PMC3056773  PMID: 21217488
Ischemia-reperfusion; Inflammation; Skeletal muscle; cytokines
10.  A Functional Murine Model of Hind Limb Demand Ischemia 
Annals of vascular surgery  2010;24(4):532-537.
Introduction
To date murine models of treadmill exercise have been used to study general exercise physiology and angiogenesis in ischemic hind limbs. The purpose of these experiments was to develop a murine model of demand ischemia in an ischemic limb to mimic claudication in humans. The primary goal was to determine whether treadmill exercise reflected a hemodynamic picture which might be consistent with the hyperemic response observed in humans.
Methods
Aged hypercholesterolemic ApoE null mice ( ApoE−/−, n=13) were subjected to Femoral Artery Ligation (FAL), and allowed to recover from the acute ischemic response. Peripheral perfusion of the hind limbs at rest was determined by serial evaluation using laser Doppler imaging (LDI) on days 0, 7, and 14 following FAL. During the duration of the experiments, the mice were also assessed on an established 5 point clinical ischemic score which assessed the degree of digital amputation, necrosis, and cyanosis as compared to the non ischemic contralateral limb. After stabilization of the LDI ratio (ischemic limb flux/contralateral non ischemic limb flux) and clinical ischemic score, mice underwent two days of treadmill training (10 min @ 10 m/min, incline of 10°) followed by 60 minutes daily treadmill exercise (13 m/min, incline of 10°) through day 25. An evaluation of pre-exercise and post exercise perfusion using LDI was performed on two separate occasions following the onset of daily exercise. During the immediate 15 minute post exercise evaluation, LDI scanning was obtained in quadruplicate, to allow identification of peak flux ratios. Statistical analysis included unpaired t-tests and ANOVA.
Results
After FAL, the LDI Flux ratio reached a nadir between days one and two, then stabilized by day 14 and remained stable through day 25. The clinical ischemic score stabilized at day 7, and remained stable throughout the rest of the experiment. Based on stabilization of both the clinical ischemic score and LDI ratio, exercise training began on day 15. The peak 15 minute post exercise LDI ratio increased significantly as compared to pre-exercise ratio on day 17 (0.48+0.04 vs. 0.34± 0.04, p<0.05) and day 25 (0.37±0.03 vs. 0.27±0.03, p<0.01). Within 2 hours of exercise, the LDI ratio returned to pre-exercise levels on both day 17 and 25.
Conclusions
Clinical and hemodynamic stabilization of limb perfusion is evident by 14 days after FAL. FAL followed by demand ischemia results in a reversible relative hyperemic response similar to those observed in exercising human claudicants. A murine model of FAL associated with demand ischemia may be a useful model to evaluate the metabolic, inflammatory and flow related changes associated with claudication in humans.
doi:10.1016/j.avsg.2009.12.003
PMCID: PMC2909630  PMID: 20363101

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