OBJECTIVE: To investigate the feasibility of a rat model on hindlimb ischemia induced by embolization from the administration of polyvinyl alcohol (PVA) particles or N-butyl cyanoacrylate (NBCA). MATERIALS AND METHODS: Unilateral hindlimb ischemia was induced by embolization with NBCA (n = 4), PVA (n = 4) or surgical excision (n = 4) in a total of 12 Sprague-Dawley rats. On days 0, 7 and 14, the time-of-flight magnetic resonance angiography (TOF-MRA) and enhanced MRI were obtained as scheduled by using a 3T-MR scanner. The clinical ischemic index, volume change and degree of muscle necrosis observed on the enhanced MRI in the ischemic hindlimb were being compared among three groups using the analysis of variance. Vascular patency on TOF-MRA was evaluated and correlated with angiographic findings when using an inter-rater agreement test. RESULTS: There was a technical success rate of 100% for both the embolization and surgery groups. The clinical ischemic index did not significantly differ. On day 7, the ratios of the muscular infarctions were 0.436, 0.173 and 0 at thigh levels and 0.503, 0.337 and 0 at calf levels for the NBCA, PVA and surgery groups, respectively. In addition, the embolization group presented increased volume and then decreased volume on days 7 and 14, respectively. The surgery group presented a gradual volume decrease. Good correlation was shown between the TOF-MRA and angiographic findings (kappa value of 0.795). CONCLUSION: The examined hindlimb ischemia model using embolization with NBCA and PVA particles in rats is a feasible model for further research, and muscle necrosis was evident as compared with the surgical model.
Animals ; *Disease Models, Animal ; Embolization, Therapeutic/*adverse effects ; Enbucrilate/administration & dosage/*toxicity ; Feasibility Studies ; Hindlimb/*blood supply ; Injections, Intra-Arterial ; Ischemia/*chemically induced/diagnosis ; Magnetic Resonance Angiography/*methods ; Male ; Polyvinyl Alcohol/administration & dosage/*toxicity ; Rats ; Rats, Sprague-Dawley ; Tissue Adhesives/administration & dosage/toxicity

OBJECTIVETo explore the role of myeloperoxidase (MPO) and tumor necrosis factor-α (TNF-α) in myocardial injury induced by hind-limb ischemia-reperfusion (IR) in rats.

METHODSRat models of bilateral hindlimb IR established using a tourniquet were randomized into 9 groups, including a normal control group normal, 2 ischemic groups with hindlimb ischemia for 2 and 4 h, and 6 IR groups with a 4-h ischemia followed by reperfusion for 0.5, 2, 4, 6, 12, and 24 h. The plasma and myocardial levels of MPO and TNF-α in each group were measured, and the myocardial expression of TNF-α was determined with immunohistochemistry.

RESULTSCompared with the normal control group, the rats with a 2-h ischemia showed significantly increased levels of MPO and TNF-α in the plasma and myocardium. Compared with those in rats with a 4-h ischemia, the plasma and myocardial MPO levels increased significantly at 0.5 and 2 h of reperfusion, respectively; the plasma TNF-α level increased significantly at 4 h of reperfusion and myocardial TNF-α level decreased obviously at 12 h; plasma levels of MPO and TNF-α both significantly decreased at 24 h. The plasma MPO and TNF-α and myocardial TNF-α reached the peak levels at 4 h of reperfusion, and the peak myocardial MPO level occurred at 6 h. Immunohistochemistry showed that TNF-α positivity moderately increased after hindlimb ischemia, and further increased at 4 h of reperfusion but obviously reduced at 24 h.

CONCLUSIONThe activation of systemic and local neutrophils and inflammatory cytokines may play an important role in myocardial injury induced by hindlimb IR in rats.

Animals ; Disease Models, Animal ; Hindlimb ; blood supply ; Ischemia ; metabolism ; Male ; Myocardium ; metabolism ; Peroxidase ; metabolism ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Animals ; Hindlimb ; blood supply ; Ischemic Preconditioning ; Lung ; metabolism ; Male ; P-Selectin ; metabolism ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism

This study was conducted to investigate the effects of erythropoietin (Epo) on both acute and chronic limb ischemia (ALI and CLI) and to evaluate the differences in mechanisms according to the method of Epo administration. Hindlimb ischemia was made in BALB/c mice with femoral artery ligation. The mice were divided into four groups: Group 1 (control, no treatment), Group 2 (ALI, early multiple doses), Group 3 (ALI, early single high dose), Group 4 (CLI, late multiple doses). Blood flow ratio significantly increased in Group 2 in 4 weeks. Expression of pAkt and Erythropoietin receptor were significantly higher in Group 2 on postoperative day (POD) 7. The number of CD31- and vascular endothelial growth factor-positive cells were significantly higher in Group 2 on POD 7 and 56. Group 3 and 4 showed a tendency of higher cell counts than the control. The early sustained Epo was effective in improving blood flow through angiogenesis. In chronic phase, weekly multiple dosing of Epo induced angiogenesis, however, the blood flow ratio did not increase significantly. The results of this study suggest that Epo administration during the acute phase followed by maintenance for several days may be important for increasing blood flow and angiogenesis.
Acute Disease ; Animals ; Chronic Disease ; Erythropoietin/*pharmacology ; Hindlimb/*blood supply ; Ischemia/*metabolism/pathology ; Laser-Doppler Flowmetry ; Male ; Mice ; Mice, Inbred BALB C ; Neovascularization, Physiologic/drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; Receptors, Erythropoietin/metabolism ; Recombinant Proteins/pharmacology ; Vascular Endothelial Growth Factor A/metabolism

OBJECTIVETo investigate the preventive effects of Salvia miltiorrhiza (SM) on multiple organ edema in the rats which suffered from hind limb ischemia/reperfusion( LI/R).

METHODSTwenty four Wistar rats were randomly divided into 3 groups (n = 8): control group (C group), ischemia/reperfusion group (I/R group ), Salvia miltiorrhiza group (SM group). Referring to Tourniquet method, the model rats which underwent 4 hours ischemia and 4 hours reperfusion of hind limbs were made. Thirty minutes before reperfusion, SM was given to the rats in SM group by tail vein injection at the dose of 5 mL/kg. Accurately weighed one gram of heart, liver, kidney, lung, brain, intestine and skeletal muscle from every animals, weigh these specimens after baking (60 degrees C, 55 hours), calculated the ratio of wet and dry (Wet/Dry,W/D). The levels of interleukin-1 (IL-1) ,interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) in plasma and the contents of Superoxide dismutase (SOD) and malonaldehyde (MDA) were measured. The morphologic changes of skeletal muscle were observed with microscope.

RESULTSIt was found that after suffering from ischemia/reperfusion, the W/D of every specimens increased in different degree (P < 0.05, P < 0.01). In plasma, the values of SOD decreased but MDA increased obviously (P < 0.05, P < 0.01). The levels of IL-1, IL-6 ,TNF-alpha-a in plasma increased (P < 0.05, P <0.01). After LI/R, infiltration of inflammatory cells, broaden interstitial around muscle fiber and disordered arrangement of muscle fibers could be seen under microscope. However, Compared with LI/R group, W/D and levels of serum inflammatory factors in SM group were all lower, the values of SOD in plasma increased but MDA in plasma failed down. Pathological changes in skeletal muscle were improved.

CONCLUSIONLimb ischemia/reperfusion can lead to multiple organ edema, Salvia miltiorrhiza can prevent the edema in some degree by anti-oxidation and anti-inflammation.

Animals ; Cytokines ; blood ; Edema ; pathology ; prevention & control ; Hindlimb ; blood supply ; Male ; Malondialdehyde ; blood ; Rats ; Rats, Wistar ; Reperfusion Injury ; pathology ; prevention & control ; Salvia miltiorrhiza ; Superoxide Dismutase ; blood

OBJECTIVETo investigate the effects of simvastatin on the expression of nuclear factor-κB (NF-κB) and intercellular adhesion molecule-1 (ICAM-1) in the lung tissue of rats with lung injury induced by ischemia-reperfusion (IR) of the hind limbs.

METHODSForty-eight male adult SD rats were randomized into 6 equal groups, including a sham-operated group, an IR group, 3 IR+simvastatin groups with intragastric administration of 1, 5, or 10 mg·kg(-1)·d(-1) for 3 days, and a simvastatin control group treated with 10 mg·kg(-1)·d(-1) simvastatin. IR of the hind limbs was induced in the 4 IR groups by occlusion of bilateral femoral arteries for 2 h followed by a 3-h reperfusion. The rats were sacrificed at the end of reperfusion and the arterial blood was taken for blood gas analysis. The lungs were immediately removed for pathological examination and determination of the lung Wet/dry weight ratio (W/D), myeloperoxidase (MPO) activity and polymorphonuclear neutrophil (PMN) counting. The expression of NF-κB p65 mRNA and ICAM-1 protein in the lungs was detected using RT-PCR and Western blotting.

RESULTSAlveolar edema, localized pulmonary atelectasis and large amount of PMN infiltration were found in IR group, and these changes were ameliorated in the 3 simvastatin groups (S(1), S(5), S(10)). Lung W/D, MPO activity and PMN counting were significantly increased in IR group as compared with the sham-operated group (P<0.01). Lung W/D, MPO activity and PMN counting were significantly lowered in the 3 simvastatin groups as compared with IR group (P<0.01). IR-induced decrease in PaO(2) was significantly increased in the 3 simvastatin groups (P<0.01), which also showed significantly lowered expressions of NF-κB p65mRNA and ICAM-1 protein in a dose-dependent manner (P<0.01).

CONCLUSIONSimvastatin attenuates lung injury induced by IR of the hind limbs in rats by suppressing the activation of NF-κB and subsequent accumulation of neutrophils mediated by ICAM-1.

Animals ; Hindlimb ; blood supply ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Ischemia ; physiopathology ; Lung ; metabolism ; Lung Injury ; etiology ; metabolism ; Male ; NF-kappa B ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; complications ; Simvastatin ; pharmacology ; Transcription Factor RelA ; genetics ; metabolism

OBJECTIVETo observe the effects of polyethylene oxide (PEO) on microcirculation of normal rat hindlimb skeletal muscle.

METHODSSixteen male Wistar rats were anesthetized and equally and randomly divided into PEO group (administered with 10 ppm PEO solution) and control group (administered with equal volume of normal saline). The PEO solution or saline was separately injected through the caudal vein at a constant rate of 5 ml/h for 20 minutes. Using short axis view at right mid thigh region, contrast-enhanced ultrasonography was performed before and after the administration of solution. Electrocardiogram, blood pressure, and central venous pressure were also monitored.

RESULTSIn the PEO group, after the administration of PEO, microcirculation capillary volume increased from (20.78±2.63) dB to (22.40±1.94) dB (P=0.023), red blood cell velocity from (0.27±0.08) s-1 to (0.35±0.13) s-1(P=0.010), and capillary blood flow from (5.65±1.81) dB/s to (7.91±3.28) dB/s (P=0.013). In the control group, there were no significant changes in microcirculation capillary volume, red blood cell velocity, and capillary blood flow (all Pþ0.05) after the injection of normal saline. The changes of heart rates, blood pressures and central venous pressure were not significant after the administration of either PEO or saline (all Pþ0.05).

CONCLUSIONPEO can remarkably increase capillary volume, red blood cell velocity, and capillary blood flow in normal rat hindlimb skeletal muscle.

Animals ; Hindlimb ; blood supply ; Male ; Microcirculation ; drug effects ; Muscle, Skeletal ; blood supply ; Polyethylene Glycols ; pharmacology ; Rats ; Rats, Wistar

Animals ; Hindlimb ; blood supply ; Intestine, Small ; metabolism ; Ischemia ; metabolism ; prevention & control ; Ischemic Preconditioning ; Lung Injury ; metabolism ; Male ; Muscle, Skeletal ; metabolism ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism

The aim of study was to explore the effect of mesenchymal stem cells (MSC) transfected with recombinant adenovirus-mediated human vascular endothelium growth factor 165 (ad-vegf-165) on treating ischemic necrosis limbs. Adult SD rats were selected for study. Limb ischemic necrosis model was established by right femoral artery ligation in SD rats. 7 days after ligation, MSC, ad-h-vegf-165-MSC and ad-LacZ-MSC labelled by DAPI were injected into ischemic necrosis limb in rats respectively. One week after injection, the expression of VEGF in ischemic necrosis limbs was detected by Western blot. And at 1, 2 or 4 weeks after injection, the expressions of FVIII and myosin on MSC were evaluated by immunohistochemistry. The results indicated that the MSC labelled by DAPI could be found in the transplantation site of ischemic necrosis limbs under fluorescent microscope. And the number of MSC in MSC-vegf group was more than that in MSC and MSC-LacZ groups. The VEGF expression in MSC-vegf group was higher than that in MSC and MSC-LacZ groups. More importantly, the number of endothelial cells demonstrated characteristic FVIII positive MSC in MSC-vegf group was more than that in MSC and MSC-LacZ group after injections of 1, 2 and 4 weeks. However, the number of myosin positive MSC among MSC-vegf, MSC and MSC-LacZ groups showed no significant difference. It is concluded that MSC transfected with Ad-vegf promotes angiogenesis to repair ischemic necrosis limbs through the increased expression of VEGF.
Adenoviridae ; genetics ; Animals ; Hindlimb ; blood supply ; Humans ; Ischemia ; pathology ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; Necrosis ; Neovascularization, Physiologic ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection ; Vascular Endothelial Growth Factor A ; genetics

OBJECTIVETo explore whether overexpression of P-selectin glycoprotein ligand-1 (PSGL-1) promotes the adhesive ability of endothelial progenitor cells and functionally facilitates neovascularization in mouse model of hindlimb ischemia.

METHODSRat endothelial progenitor cells were transfected with recombinant adenovirus vector encoding human PSGL-1. The mRNA and protein expression levels of PSGL-1 were measured by RT-PCR and Western blot, respectively. The effect of overexpression of PSGL-1 in endothelial progenitor cells was analyzed by adherence assay. Histological examination of skeletal muscle sections retrieved from the mouse ischemic hindlimbs was performed, and the hindlimb blood flow was measured by laser Doppler flow meter.

RESULTSAdenovirus vector expressing of PSGL-1 gene was successfully constructed with high titer of 3.1 × 10¹¹ pfu/ml. After transfection, PSGL-1 gene was highly expressed in the transfected endothelial progenitor cells. In vitro assay showed that overexpression of PSGL-1 enhanced the adhesive properties of endothelial progenitor cells. When the transfected endothelial progenitor cells were transplanted into the ischemic hindlimb of nude mice, the number of new capillary vessels was (41.0 ± 2.2)/HPF compared to that of (21.0 ± 2.5)/HPF in the negative control group and (10.0 ± 1.6)/HPF in the blank control group (P < 0.01). Furthermore, the blood flow was increased in the experimental group (119.1% ± 7.0%), whereas in the negative control group, it was (93.3% ± 3.0%) and in the blank control group it was (76.3% ± 12.0%), P < 0.01.

CONCLUSIONSOverexpression of PSGL-1 enhances the adhesive and angiogenic properties of endothelial progenitor cells. The approach may provide an effective therapeutic strategy to improve the efficiency of cell-based proangiogenic therapy.

Adenoviridae ; genetics ; Animals ; Cell Adhesion ; Cells, Cultured ; Endothelial Cells ; cytology ; Hindlimb ; blood supply ; Humans ; Ischemia ; therapy ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; Mice, Nude ; Neovascularization, Physiologic ; physiology ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Stem Cell Transplantation ; Stem Cells ; cytology ; metabolism ; Transfection