Rat Model Of Hindlimb Ischemia Research Articles

MiR-221/222 Promote Endothelial Differentiation of Adipose-Derived Stem Cells by Regulation of PTEN/PI3K/AKT/mTOR Pathway.

Adipose-derived stem cells (ADSCs) are a type of adult mesenchymal stem cell that show a repair effect on ischemic tissues owing to their capacity for endothelial differentiation. MicroRNA-221/222 (miR-221/222) has been extensively studied in endothelial cells (ECs). However, the mechanism that regulates ADSCs differentiation into ECs remains unknown. In this study, we investigated the effects of miR-221/222-overexpression/silence in ADSCs on endothelial differentiation by constructing lentiviral vectors. Differentiation capacity was assessed by measuring the expression of endothelial markers (CD31, CD34, and CD144). In addition, low-density lipoprotein (LDL) uptake and tube-like formation were performed for evaluation of functional characterization. The PTEN/PI3K/AKT/mTOR signaling pathway was investigated using western blotting to clarify the action mechanism of this gene. The revascularization of miR-221/222-transfeted ADSCs was further verified in a rat hind limb ischemia model. The results confirmed that transfection with miR-221/222 promoted the expression of endothelial markers, LDL uptake, and tube-like formation. As expected, the PI3K/AKT signaling pathway was effectively activated when ADSCs showed high expression of miR-221/222 during endothelial differentiation. Furthermore, injection of miR-221/222 transfected ADSCs significantly improved rat hindlimb ischemia, as evidenced by increased blood flow and structural integrity and reduce inflammatory infiltration. The results of this study suggest that miR-221/222 is essential for endothelial differentiation of ADSCs and provides a novel strategy for modulating vascular formation and ischemic tissue regeneration.

Intravenous infusion of bone marrow-derived mesenchymal stem cells improves tissue perfusion in a rat hindlimb ischemia model

Intravenous infusion of stem cells is a minimally invasive cellular delivery method, though a few have been reported in a critical limb-threatening ischemia (CLTI) animal model or patients. In the present study, we hypothesized that intravenous infusion of bone-marrow derived mesenchymal stem cells (MSCs) improves tissue perfusion in a rat hindlimb ischemia model. Hindlimb ischemia was generated in Sprague–Dawley rats by femoral artery removal, then seven days after ischemic induction intravenous infusion of 1 × 106 MSCs (cell group) or vehicle (control group) was performed. As compared with the control, tissue perfusion was significantly increased in the cell group. Histological findings showed that capillary density was significantly increased in the cell group, with infused green fluorescent protein (GFP)-MSCs distributed in the ischemic limb. Furthermore, gene expression of vascular endothelial growth factor (VEGF) was significantly increased in ischemic hindlimb muscle tissues of rats treated with MSC infusion. In conclusion, intravenous infusion of bone-marrow derived MSCs improved tissue perfusion in ischemic hindlimbs through angiogenesis, suggesting that intravenous infusion of MSCs was a promising cell delivery method for treatment of CLTI.

Involvement of endothelial progenitor cells in blood flow recovery through activation of the Wnt/β-catenin signaling pathway and inhibition of high oxidative stress in diabetic hindlimb ischemic rats.

Diabetes mellitus (DM) often causes stenosis and occlusion of hindlimb blood vessels, which are also the main cause for hindlimb ischemia in elderly people. To investigate the therapeutic effect of endothelial progenitor cell (EPC) transplantation on diabetic hindlimb ischemia. Endothelial progenitor cells were separated, labeled with PKH-26 and transplanted into rat models (107 cells/100 g). Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to detect any oxidative stress. Streptozotocin (STZ) was injected to establish a diabetic rat model and hindlimb ischemia model was established via operation. Western blotting was used to detect total β-catenin (T-β-catenin) and non-phospho-β-catenin (NP-β-catenin) levels. The malondialdehyde (MDA), superoxide dismutase (SOD), Wnt3a, Wnt5a and Wnt7a levels were detected using enzyme-linked immunosorbent assay (ELISA). Oxidative stress was measured using DCFH-DA and dihydroethidium (DHE). The endothelial biomarker CD31 was observed to highlight vessels, and PKH-26 to trace migration/adhesion of EPCs. Endothelial progenitor cells were successfully isolated and identified, and diabetic hindlimb ischemic rat models were created. Tempol remarkably improved blood flow in diabetic hindlimb ischemic rats compared to DM+EPCs rats at 14 days (p < 0.001) and 28 days post-operation (p < 0.001). High oxidative stress was observed in diabetic hindlimb ischemic rats. Tempol significantly inhibited oxidative stress levels in diabetic hindlimb ischemic rats. Furthermore, Tempol significantly promoted angiogenesis in diabetic hindlimb ischemic rats compared to DM+EPCs rats. The β-catenin inhibitor, XAV (DM+EPCs+Tempol+XAV group), significantly suppressed blood flow recovery and angiogenesis in diabetic hindlimb ischemic rats when compared to the DM+EPCs+Tempol group at 14 days (p = 0.026) and 28 days (p < 0.001). The XAV remarkably reduced T-β-catenin (p < 0.001) and N-β-catenin (p = 0.030) levels in Tempol-treated diabetic hindlimb ischemic rats, as compared to the DM+EPCs+Tempol group. The Wnt5a participated in the pathology of diabetic hindlimb ischemia. There are high oxidative stress levels in both EPCs in high-glucose environments and diabetic hindlimb ischemia, which can lead to limited blood flow recovery. The high oxidative stress caused the inhibition of Wnt/β-catenin signaling pathway, leading to limited blood flow recovery in diabetic hindlimb ischemia. At the same time, Wnt5a participated in the EPC-mediated blood flow recovery.

Study of the effect of the VEGF receptor blockade on the antiischemic activity of the compound GK-2 in a rat hind limb ischemia model

Earlier, in experiments in vitro and in vivo, it was shown that the TrkA receptor agonist compound GK-2, which was a low molecular weight NGF mimetic, had pronounced angiogenic and antiischemic activity. However, it remained unclear whether this activity was associated with the activation of VEGF-A, since, on the one hand, there are reports that NGF-mediated angiogenesis can be initiated by activating NGF of the vascular endothelial growth factor VEGF-A, and on the other hand, it is shown that a selective antagonist of Flk1 receptors specific for VEGF-A, compound SU-5416 does not affect the angiogenic effect of NGF. Purpose of the investigation. Study of the selective VEGF blockade effect on the antiischemic activity of the TrkA receptor agonist GK-2. Methods. Evaluation of the compound GK-2 antiischemic activity was assessed in model experiments simulating hind limb ischemia in rats. Results. It has been shown that against the background of blockade of VEGF-A binding with specific for it (VEGFR1 / Flt-1 / and VEGFR2 / KDR /) receptors, bevacizumab (2.5 mg / kg, i.p., every 3 days for 14 days) compound GK-2 (1 mg / kg, i.p., daily, for 14 days) realizes its antiischemic activity. Conclusion. The results of these experiments indicate that the selective blockade of VEGF does not significantly affect the anti-ischemic activity of the dipeptide NGF mimetic compound GK-2, which has the properties of a TrkA receptor agonist.

Angiogenic effects of cell therapy within a biomaterial scaffold in a rat hind limb ischemia model

Critical limb ischemia (CLI) is a life- and limb-threatening condition affecting 1–10% of humans worldwide with peripheral arterial disease. Cellular therapies, such as bone marrow-derived mesenchymal stem cells (MSCs) have been used for the treatment of CLI. However, little information is available regarding the angiogenic potency of MSCs and mast cells (MC) in angiogenesis. The aim of this study was to evaluate the ability of MCs and MSCs to induce angiogenesis in a rat model of ischemic hind limb injury on a background of a tissue engineered hydrogel scaffold. Thirty rats were randomly divided into six control and experimental groups as follows: (a) Control healthy (b) Ischemic positive control with right femoral artery transection, (c) ischemia with hydrogel scaffold, (d) ischemia with hydrogel plus MSC, (e) ischemia with hydrogel plus MC and (f) ischemia with hydrogel plus MSC and MCs. 106 of each cell type, isolated from bone marrow stroma, was injected into the transected artery used to induce hind limb ischemia. The other hind limb served as a non-ischemic control. After 14 days, capillary density, vascular diameter, histomorphometry and immunohistochemistry at the transected location and in gastrocnemius muscles were evaluated. Capillary density and number of blood vessels in the region of the femoral artery transection in animals receiving MSCs and MCs was increased compared to control groups (P < 0.05). Generally the effect of MCs and MSCs was similar although the combined MC/MSC therapy resulted in a reduced, rather than enhanced, effect. In the gastrocnemius muscle, immunohistochemical and histomorphometric observation showed a great ratio of capillaries to muscle fibers in all the cell-receiving groups (P < 0.05). The data indicates that the combination of hydrogel and cell therapy generates a greater angiogenic potential at the ischemic site than cell therapy or hydrogels alone.

Therapeutic potential of mesenchymal stem cells for peripheral artery disease in a rat model of hindlimb ischemia.

Objective(s):Mesenchymal stem cells are viewed as the first choice in regenerative medicine. This study aimed to elucidate the influence of BM-MSCs transplantation on angiogenesis in a rat model of unilateral peripheral vascular disease. Materials and Methods:Twenty-one rats were arbitrarily allocated into three groups (7/group). Group I: control sham-operated rats, Group II: control ischemic group: Rats were subjected to unilateral surgical ligation of the femoral artery, and Group III: ischemia group: Rats were induced as in group II, 24 hr after ligation, they were intramuscularly injected with BM-MSCs. After scarification, gastrocnemius muscle gene expression of stromal cell-derived factor-1 (SDF-1), CXC chemokine receptor 4 (CXCR4), vascular endothelial growth factor receptor 2 (VEGFR2), von Willebrand factor (vWF), and hypoxia-inducible factor-1α (HIF-1α) were analyzed by quantitative real-time PCR. Muscle regeneration and angiogenesis evaluation was assessed through H&E staining of the tissue. Furthermore, Pax3 and Pax7 nuclear expression was immunohistochemically assessed. Results:Rats treated with BM-MSCs showed significantly raised gene expression levels of SDF-1, CXCR4, VEGFR2, and vWF compared with control and ischemia groups. H&E staining of the gastrocnemius showed prominent new vessel formation. Granulation tissue within muscles of the ischemic treated group by BM-MSCs showed cells demonstrating nuclear expression of Pax3 and Pax7. Conclusion:BM-MSCs transplantation has an ameliorating effect on muscle ischemia through promoting angiogenesis, detected by normal muscle architecture restoration and new blood vessel formations observed by H&E, confirmed by increased gene expression levels of SDF-1, CXCR4, VEGFR2, and vWF, decreased HIF-1α gene expression, and increased myogenic Pax7 gene expression.

CD146+Mesenchymal stem cells treatment improves vascularization, muscle contraction and VEGF expression, and reduces apoptosis in rat ischemic hind limb

Peripheral arterial disease (PAD) is an increasingly common narrowing of the peripheral arteries that can lead to lower limb ischemia, muscle weakness and gangrene. Surgical vein or arterial grafts could improve PAD, but may not be suitable in elderly patients, prompting research into less invasive approaches. Mesenchymal stem cells (MSCs) have been proposed as potential therapy, but their effectiveness and underlying mechanisms in limb ischemia are unclear. We tested the hypothesis that treatment with naive MSCs (nMSCs) or MSCs expressing CD146 (CD146+MSCs) could improve vascularity and muscle function in rat model of hind-limb ischemia. Sixteen month old Sprague-Dawley rats were randomly assigned to 4 groups: sham-operated control, ischemia, ischemia + nMSCs and ischemia+CD146+MSCs. After 4 weeks of respective treatment, rat groups were assessed for ischemic clinical score, Tarlov score, muscle capillary density, TUNEL apoptosis assay, contractile force, and vascular endothelial growth factor (VEGF) mRNA expression. CD146+MSCs showed greater CD146 mRNA expression than nMSCs. Treatment with nMSCs or CD146+MSCs improved clinical and Tarlov scores, muscle capillary density, contractile force and VEGF mRNA expression in ischemic limbs as compared to non-treated ischemia group. The improvements in muscle vascularity and function were particularly greater in ischemia+CD146+MSCs than ischemia + nMSCs group. TUNEL positive apoptotic cells were least abundant in ischemia+CD146+MSCs compared with ischemia + nMSCs and non-treated ischemia groups. Thus, MSCs particularly those expressing CD146 improve vascularity, muscle function and VEGF expression and reduce apoptosis in rat ischemic limb, and could represent a promising approach to improve angiogenesis and muscle function in PAD.

Therapeutic angiogenesis using zinc oxide nanoflowers for the treatment of hind limb ischemia in a rat model

Critical limb ischemia (CLI) is a severe type of peripheral artery disease (PAD) which occurs due to an inadequate supply of blood to the limb extremities. Patients with CLI often suffer from extreme cramping pain, impaired wound healing, immobility, cardiovascular complications, amputation of the affected limb and even death. The conventional therapy for treating CLI includes surgical revascularization as well as restoration of angiogenesis using growth factor therapy. However, surgical revascularization is only suitable for a small percentage of CLI patients and is associated with a high perioperative mortality rate. The use of growth factors is also limited in terms of their poor therapeutic angiogenic potential, as observed in earlier clinical studies which could be attributed to their poor bio-availability and non-specificity issues. Therefore, to overcome the aforesaid disadvantages of conventional strategies there is an urgent need for the advancement of new alternative therapeutic biomaterials to treat CLI. In the past few decades, various research groups, including ours, have been involved in developing different pro-angiogenic nanomaterials. Among these, zinc oxide nanoflowers (ZONFs), established in our laboratory, are considered one of the more potent nanoparticles for inducing therapeutic angiogenesis. In our earlier studies we showed that ZONFs promote angiogenesis by inducing the formation of reactive oxygen species and nitric oxide (NO) as well as activating Akt/MAPK/eNOS cell signaling pathways in endothelial cells. Recently, we have also reported the therapeutic potential of ZONFs to treat cerebral ischemia through their neuritogenic and neuroprotective properties, exploiting angio-neural cross-talk. Considering the excellent pro-angiogenic properties of ZONFs and the importance of revascularization for the treatment of CLI, in the present study we comprehensively explore the therapeutic potential of ZONFs in a rat hind limb ischemia model (established by ligating the hind limb femoral artery), an animal model that mimics CLI in humans. The behavioral studies, laser Doppler perfusion imaging, histopathology and immunofluorescence as well as estimation of serum NO level showed that the administration of ZONFs could ameliorate ischemia in rats at a faster rate by promoting therapeutic angiogenesis to the ischemic sites. Altogether, the present study offers an alternative nanomedicine approach employing ZONFs for the treatment of PADs.

Notch signaling-modified mesenchymal stem cells improve tissue perfusion by induction of arteriogenesis in a rat hindlimb ischemia model

Notch signaling-modified human mesenchymal stem cell, SB623 cell, is a promising cell therapy product for ischemic stroke. With the aim to expand indications for their use for critical limb-threatening ischemia (CLTI), we hypothesized that SB623 cells improved tissue perfusion by inducing angiogenesis or arteriogenesis in a hindlimb ischemia model rat. In Sprague–Dawley rats, hindlimb ischemia was generated by femoral artery removal, then seven days after ischemic induction 1 × 105 SB623 cells or PBS was injected into the ischemic adductor muscle. As compared with the PBS group, tissue perfusion was significantly increased in the SB623 group. While capillary density did not vary between the groups, αSMA- and vWF-positive arterioles with a diameter > 15 μm were significantly increased in the SB623 group. Whole transcriptome analysis of endothelial cells co-cultured with SB623 cells showed upregulation of the Notch signaling pathway as well as several other pathways potentially leading to arteriogenesis. Furthermore, rat muscle treated with SB623 cells showed a trend for higher ephrin-B2 and significantly higher EphB4 expression, which are known as arteriogenic markers. In the hindlimb ischemia model, SB623 cells improved tissue perfusion by inducing arteriogenesis, suggesting a promising cell source for treatment of CLTI.

Dose optimization of decellularized skeletal muscle extracellular matrix hydrogels for improving perfusion and subsequent validation in an aged hindlimb ischemia model.

Peripheral artery disease (PAD) affects more than 27 million individuals in North America and Europe, and current treatment strategies mainly aim to restore blood perfusion. However, many patients are ineligible for existing procedures, and these therapies are often ineffective. Previous studies have demonstrated success of an injectable decellularized skeletal muscle extracellular matrix (ECM) hydrogel in a young rat hindlimb ischemia model of PAD, but further pre-clinical studies are necessary prior to clinical translation. In this study, varying concentrations of a skeletal muscle ECM hydrogel were investigated for material properties and in vivo effects on restoring blood perfusion. Rheological measurements indicated an increase in viscosity and mechanical strength with the higher concentrations of the ECM hydrogels. When injecting dye-labelled ECM hydrogels into a healthy rat, differences were also observed for the spreading and degradation rate of the various concentrations. The three concentrations for the ECM hydrogel were then further examined in a young rat hindlimb ischemia model. The efficacy of the optimal ECM hydrogel concentration was then further confirmed in an aged mouse hindlimb ischemia model. These results further validate the use of decellularized skeletal muscle ECM hydrogels for improving blood perfusion in small animal models of PAD.

Targeted delivery of nitric oxide via a ‘bump-and-hole’-based enzyme–prodrug pair

The spatiotemporal generation of nitric oxide (NO), a versatile endogenous messenger, is precisely controlled. Despite its therapeutic potential for a wide range of diseases, NO-based therapies are limited clinically due to a lack of effective strategies for precisely delivering NO to a specific site. In the present study, we developed a novel NO delivery system via modification of an enzyme-prodrug pair of galactosidase-galactosyl-NONOate using a 'bump-and-hole' strategy. Precise delivery to targeted tissues was clearly demonstrated by an in vivo near-infrared imaging assay. The therapeutic potential was evaluated in both rat hindlimb ischemia and mouse acute kidney injury models. Targeted delivery of NO clearly enhanced its therapeutic efficacy in tissue repair and function recovery and abolished side effects due to the systemic release of NO. The developed protocol holds broad applicability in the targeted delivery of important gaseous signaling molecules and offers a potent tool for the investigation of relevant molecular mechanisms.

Hyperbaric oxygen boosts long noncoding RNA MALAT1 exosome secretion to suppress microRNA-92a expression in therapeutic angiogenesis

BackgroundHyperbaric oxygen (HBO) could improve wound healing by enhancement of angiogenesis. The effect of HBO on metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a proangiogenic long noncoding RNA, and on endothelial cell-derived exosome is unknown. We aim to investigate both whether MALAT1 is altered in human coronary artery endothelial cells (HCAECs)-derived exosomes in response to HBO as well as the molecular regulatory mechanisms of MALAT1 in HCAECs under HBO treatment. Methods and resultsHCAECs were cultured and HBO was applied at 2.5 atmosphere absolute (ATA) in a hyperbaric chamber. Exosomes were extracted from culture media. A rat model of hind-limb ischemia was performed by ligation of the right femoral artery. HBO at 2.5 ATA significantly increased MALAT1 expression in HCAECs and HCAECs-derived exosomes. MALAT1 suppressed miR-92a expression in HCAEC-derived exosomes under HBO. Silencing MALAT1 by MALAT1 siRNA significantly inhibited KLF2 mRNA expression induced by HBO, as did MiR-92a. MiR-92a significantly decreased KLF2 luciferase activity in HCAECs under HBO. HBO and HBO-induced exosomes significantly increased cell proliferation and the capillary-like network formation of HCAECs. MALAT1 siRNA and miR-92a overexpression significantly attenuated the cell proliferation and tube formation caused by HBO-induced exosome. HBO and HBO-induced exosomes significantly improved neovascularization in a rat model of hind-limb ischemia. ConclusionsHBO upregulates MALAT1 to suppress miR-92a expression and counteracts the inhibitory effect of miR-92a on KLF2 expression in HCAECs to enhance neovascularization. HBO-induced derivation of exosomes from HCAECs enhances angiogenesis. Exosomes containing MALAT1 might serve as a valuable therapeutic tool for neovascularization by HBO.

GDF11 Improves Angiogenic Function of EPCs in Diabetic Limb Ischemia.

Growth differentiation factor 11 (GDF11) has been shown to promote stem cell activity and rejuvenate the function of multiple organs in old mice, but little is known about the functions of GDF11 in the diabetic rat model of hindlimb ischemia. In this study, we found that systematic replenishment of GDF11 rescues angiogenic function of endothelial progenitor cells (EPCs) and subsequently improves vascularization and increases blood flow in diabetic rats with hindlimb ischemia. Conversely, anti-GDF11 monoclonal antibody treatment caused impairment of vascularization and thus, decreased blood flow. In vitro treatment of EPCs with recombinant GDF11 attenuated EPC dysfunction and apoptosis. Mechanistically, the GDF11-mediated positive effects could be attributed to the activation of the transforming growth factor-β/Smad2/3 and protein kinase B/hypoxia-inducible factor 1α pathways. These findings suggest that GDF11 repletion may enhance EPC resistance to diabetes-induced damage, improve angiogenesis, and thus, increase blood flow. This benefit of GDF11 may lead to a new therapeutic approach for diabetic hindlimb ischemia.

A modified rat model of hindlimb ischemia for augmentation and functional measurement of arteriogenesis.

Arteriogenesis (collateral artery development) is an adaptive pathway critical for salvage of tissue in the setting of arterial occlusion. Rodent models of arteriogenesis typically involve an experimental occlusion (ligation) of a hindlimb artery and then rely on indirect measures such as laser Doppler perfusion imaging to assess blood flow recovery. Unfortunately, the more commonly utilized measures of distal tissue perfusion at rest are unable to account for hemodynamic and vasoactive variables and thus provide an incomplete assessment of collateral network capacity. We provide a detailed description of modifications to the commonly used model of femoral artery ligation. These serve to alter and then directly assess collateral network’s hemodynamic capacity. By incorporating an arteriovenous fistula distal to the arterial ligation, arterial growth is maximized. Hindlimb perfusion may be isolated to measure minimum resistance of flow around the arterial occlusion, which provides a direct measure of collateral network capacity. Our results reinforce that arteriogenesis is driven by hemodynamic variables, and it can be reliably augmented and measured in absolute terms. Using these modifications to a widely used model, functional arteriogenesis may be more directly studied.

Antiangiogenic Effects of Nerve Growth Factor Loop 4 Monomeric Dipeptide Mimetic.

The effects of GK-1, a monomeric dipeptide mimetic of nerve growth factor (NGF) loop 4, on angiogenesis were studied in vitro and in vivo. Experiments on human umbilical vein endothelial cells HUVEC showed that the test compound did not affect tubulogenesis (initial stage of angiogenesis) and prevented realization of the angiogenic effect of NGF and its dimeric dipeptide mimetic GK-2. Experiments on rat hind limb ischemia model demonstrated that GK-1 (1 mg/kg/day intraperitoneally over 14 days) significantly reduced the density of the capillary network in ischemic tissue and increased the number and area of Zenker necrosis in comparison with the control. These data suggest that GK-1 exhibits a pronounced antiangiogenic activity.

Transplantation of Rat Mesenchymal Stem Cells Overexpressing Hypoxia-Inducible Factor 2α Improves Blood Perfusion and Arteriogenesis in a Rat Hindlimb Ischemia Model.

Mesenchymal stem cells (MSCs) have been increasingly tested in cell-based therapy to treat numerous diseases. Genetic modification to improve MSC behavior may enhance posttransplantation outcome. This study aims to test the potential therapeutic benefits of rat bone marrow MSCs overexpressing hypoxia-inducible factor 2α (rMSCsHIF-2α) in a rat hindlimb ischemia model. PBS, rMSCs, or rMSCsHIF-2α were injected into rat ischemic hindlimb. Compared with the injection of PBS or rMSCs, transplantation of rMSCsHIF-2α significantly improved blood perfusion, increased the number of vessel branches in the muscle of the ischemic hindlimb, and improved the foot mobility of the ischemic hindlimb (all P < 0.05). rMSCHIF-2α transplantation also markedly increased the expression of proangiogenic factors VEGF, bFGF, and SDF1 and Notch signaling proteins including DII4, NICD, Hey1, and Hes1, whereas it reduced the expression of proapoptotic factor Bax in the muscle of the ischemic hindlimb. Overexpression of HIF-2α did not affect rMSC stemness and proliferation under normoxia but significantly increased rMSC migration and tube formation in matrigel under hypoxia (all P < 0.05). RMSCsHIF-2α stimulated endothelial cell invasion under hypoxia significantly (P < 0.05). Genetic modification of rMSCs via overexpression of HIF-2α improves posttransplantation outcomes in a rat hindlimb ischemia model possibly by stimulating proangiogenic growth factors and cytokines.

Colchicine protects rat skeletal muscle from ischemia/reperfusion injury by suppressing oxidative stress and inflammation.

Neutrophils play an important role in ischemia/reperfusion (IR) induced skeletal muscle injury. Microtubules are required for neutrophil activation in response to various stimuli. This study aimed to investigate the effects of colchicine, a microtubule-disrupting agent, on skeletal muscle IR injury in a rat hindlimb ischemia model. Twenty-one Sprague-Dawley rats were randomly allocated into three groups IR group, colchicine treated-IR (CO) group and sham operation (SM) group. Rats of both the IR and CO groups were subjected to 3 hr of ischemia by clamping the right femoral artery followed by 2 hr of reperfusion. Colchicine (1 mg/kg) was administrated intraperitoneally prior to hindlimb ischemia in the CO group. After 2 hr of reperfusion, we measured superoxide dismutase (SOD) and myeloperoxidase (MPO) activities, and malondialdehyde (MDA), tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels in the muscle samples. Plasma creatinine kinase (CK) and lactate dehydrogenase (LDH) levels were measured. We also evaluated the histological damage score and wet/dry weight (W/D) ratio. The histological damage score, W/D ratio, MPO activity, MDA, TNF-α and IL-1β levels in muscle tissues were significantly increased, SOD activity was decreased, and plasma CK and LDH levels were remarkably elevated in both the IR and CO groups compared to the SM group (P<0.05). Colchicine treatment significantly reduced muscle damage and edema, oxidative stress and levels of the inflammatory parameters in the CO group compared to the IR group (P<0.05). Colchicine attenuates IR-induced skeletal muscle injury in rats.

Abstract 125: Vitamin D3-Induced Arterial Calcification Decreases Functional Recovery and Limb Perfusion in a Murine Model of Hindlimb Ischemia

Objectives: Clinical studies demonstrate associations between arterial calcification and adverse outcomes in patients with peripheral arterial disease. The causal relationship between calcification and ischemia, however, remains unclear. This study aims to evaluate the effects of arterial calcification on functional recovery and limb perfusion following acute limb ischemia in a murine model. Methods: Arterial calcification was induced in 64 Sprague-Dawley rats using Vitamin D 3 . A control group of 32 rats received vehicle only. After 2 weeks, to allow for calcification, unilateral hindlimb ischemia was induced by ligation and excision of the superficial femoral artery. At weekly intervals, functional and ischemia status were evaluated in a blinded manner and laser Doppler perfusion imaging was performed. Results: Calcium assay demonstrated greater arterial calcification in the superficial femoral arteries (0.1 vs. 17.0, P &lt; .01) of Vitamin D 3 injected animals. Rats injected with Vitamin D3 showed significantly worse Tarlov, Ischemia, Modified Ischemia, and Total Ischemia scores at each post-operative time point (Week 1: 16.6 vs. 14.5 P &lt;.01, Week 2: 17.1 vs. 14.5 P &lt;.01, Week 3 17.5 vs. 15.0 P &lt; .01). The control group regained normal functional status by week 4; however Vitamin D3-injected rats did not return to baseline by the conclusion of the study (17.9 vs. 16.6, P &lt; .01). Laser Doppler perfusion revealed decreased raw perfusion values pre-operatively (616 vs. 428, P &lt; .01) and at weekly intervals beginning at week 2 in both the ischemic and non-ischemic limbs among animals with calcified arteries as compared to controls (Table 1). Conclusions: Vitamin D3 induced calcification is associated with decreased perfusion and worse functional recovery in a rat model of hindlimb ischemia. Future research aimed at understanding the relationship between vitamin D 3 , arterial calcification, and lower extremity ischemia may help to improve outcomes in patients with PAD.

Priming with erythropoietin enhances cell survival and angiogenic effect of mesenchymal stem cell implantation in rat limb ischemia

IntroductionBone marrow mesenchymal stem cells (BMMSCs) ameliorate tissue damage after ischemic injury. Erythropoietin (Epo) has pleiotropic effects in addition to hematopoietic activity. The aim of this study was to investigate whether Epo enhanced cell survival and angiogenic effect of BMMSC implantation in rat limb ischemia model.Methods and resultsMSCs were isolated from BM in GFP-transgenic rats. In a culture study, Epo promoted BMMSC proliferation in normoxia and enhanced cell survival under the culture condition mimicking ischemia (1% oxygen and nutrient deprivation). BMMSCs with and without 48 h of pretreatment by Epo (80 IU/ml) were locally administered to rat hindlimb ischemia models in vivo. At 3 days after implantation, BMMSC engraftment in the perivascular area of the injured muscle was significantly higher in the cells preconditioned with Epo than in the cells without preconditioning. Stromal derived factor-1α and fibroblast growth factor-2 expressions were detected in the engrafted BMMSCs. At 14 days after implantation, the Epo-preconditioned BMMSCs significantly promoted blood perfusion and capillary growth compared to the controls in laser Doppler and histological studies. In addition to promoting neovascularization, the Epo-preconditioned BMMSCs significantly inhibited macrophage infiltration in the perivascular area.ConclusionEpo elicited pro-survival potential in the BMMSCs. Pharmacological cell modification with Epo before implantation may become a feasible and promising strategy for improving current therapeutic angiogenesis with BMMSCs.

Effects of alpha lipoic acid on ishemia/reperfusion injury in rat hindlimb ischemia model

This study was performed to evaluate the effect of alpha lipoic acid (ALA) on prevention of ischemia-reperfusion (IR) injury in rat hindlimb ischemia model. Forty male Sprague Dawley rats weighing between 250 and 300 g were divided into 4 groups of 10 rats. Hindlimb composite island flaps were raised in all rats. Clamps were applied to femoral vessels of all subjects, but immediately released without causing ischemia in Group 1. In Group 2, after 4 hours of ischemia, 24 hours of reperfusion was performed. Following 4 hours of ischemia, saline was administered to rats in Group 3 and flaps were reperfused for 24 hours. In Group 4, ALA was administered intraperitoneally after 4 hours of ischemia and flaps were reperfused for 24 hours. In Group 4, there was a significant decrease of liver malondialdehyde compared to Group 2 and decrease of muscle tumor necrosis factor-alpha compared to Group 3. There was also increase in levels of glutathione in erythrocytes compared to Group 3 and increase of plasma vitamin C compared to all groups. ALA was found to be effective in prevention of ischemia-reperfusion injury. Further studies are needed before clinical application.