Model Of Hindlimb Ischemia-reperfusion Research Articles

Imaging Hydrogen Sulfide in Hypoxic Tissue with [99mTc]Tc-Gluconate.

Hydrogen sulfide (H2S) is the third gasotransmitter and is generated endogenously in hypoxic or inflammatory tissues and various cancers. We have recently demonstrated that endogenous H2S can be imaged with [99mTc]Tc-gluconate. In the present study, we detected H2S generated in hypoxic tissue, both in vitro and in vivo, using [99mTc]Tc-gluconate. In vitro uptake of [99mTc]Tc-gluconate was measured under hypoxic and normoxic conditions, using the colon carcinoma cell line CT26, and was higher in hypoxic cells than that in normoxic cells. An acute hindlimb ischemia-reperfusion model was established in BALB/c mice by exposing the animals to 3 h of ischemia and 3 h of reperfusion prior to in vivo imaging. [99mTc]Tc-gluconate (12.5 MBq) was intravenously injected through the tail vein, and uptake in the lower limb was analyzed by single-photon emission computed tomography/computed tomography (SPECT/CT). SPECT/CT images showed five times higher uptake in the ischemic limb than that in the normal limb. The standard uptake value (SUVmean) of the ischemic limb was 0.39 ± 0.03, while that of the normal limb was 0.07 ± 0.01. [99mTc]Tc-gluconate is a novel imaging agent that can be used both in vitro and in vivo for the detection of endogenous H2S generated in hypoxic tissue.

Resolvin D1 promotes efferocytosis in aging by limiting senescent cell-induced MerTK cleavage.

Inflammation-resolution is mediated by the balance between specialized pro-resolving mediators (SPMs) like resolvin D1 (RvD1) and pro-inflammatory factors, like leukotriene B4 (LTB4). A key cellular process of inflammation-resolution is efferocytosis. Aging is associated with defective inflammation-resolution and the accumulation of pro-inflammatory senescent cells (SCs). Therefore, understanding mechanism(s) that underpin this impairment is a critical gap. Here, using a model of hind limb ischemia-reperfusion (I/R) remote lung injury, we present evidence that aging is associated with heightened inflammation, impaired SPM:LT ratio, defective efferocytosis, and a decrease in MerTK levels in injured lungs. Treatment with RvD1 mitigated I/R lung injury in aging, promoted efferocytosis, and prevented the decrease of MerTK in injured lungs from old mice. Old MerTK cleavage-resistant mice (MerTKCR) exhibited less neutrophils or polymorpho nuclear cells infiltration and had improved efferocytosis compared with old WT controls. Mechanistically, macrophages that were treated with conditioned media (CM) from senescent cells had increased MerTK cleavage, impaired efferocytosis, and a defective RvD1:LTB4 ratio. Macrophages from MerTKCR mice were resistant to CM-induced efferocytosis defects and had an improved RvD1:LTB4 ratio. RvD1-stimulated macrophages prevented CM-induced MerTK cleavage and promoted efferocytosis. Together, these data suggest a new mechanism and a potential therapy to promote inflammation-resolution and efferocytosis in aging.

Effects of fish oil-based lipid emulsion on inflammation and kidney injury in mice subjected to unilateral hind limb ischemia/reperfusion

This study investigated the effects of a fish oil-based lipid emulsion (FO) on local skeletal muscle and remote renal damage at 72 h post-reperfusion in a murine model of hind limb ischemia-reperfusion (IR) injury. Mice were assigned to 1 sham group and 3 IR groups. The IR groups were treated daily with either saline or FO from 3 days prior to limb ischemia till 3 days after reperfusion. Limb IR was induced by applying a 4.5-oz orthodontic rubber band above the left greater trochanter for 120 min followed by band-released reperfusion for 72 h. Mice were then sacrificed to harvest blood, muscle, and kidney for analysis. The results showed that IR injury led to upregulation of pro-inflammatory monocytes in blood, infiltration of leukocytes into injured muscle, and over-expression of pro-inflammatory genes in muscle and kidney tissues. Supplementing FO either before or after IR injury alleviated IR-induced inflammatory gene expressions in muscle and kidney tissues. Furthermore, FO given after IR injury reduced circulating pro-inflammatory monocytes, limited muscle leukocytic infiltration, and improved renal histology. These results suggest that FO may protect the muscles from IR injury. FO given after IR injury can better downregulate the inflammation seen in IR-induced remote kidney injury.

Effect of atorvastatin on oxidative damage and inflammation in experimental hindlimb ischemia–reperfusion model

Ischemia–reperfusion is defined as cellular damage after the reperfusion of ischemic tissue, and it is likely to occur in relation to various diseases and surgical procedures. The purpose of this study was to evaluate the capability of atorvastatin to prevent oxidative damage and modulate the release of proinflammatory cytokines in rat hindlimb during ischemia–reperfusion injury. The animals were divided into 4 groups (ischemia–reperfusion + vehicle, ischemia–reperfusion + atorvastatin, sham, and healthy controls) with 15 rats per group. The animals were exposed to ischemia for 6 h, followed by 24 h, 7 days, and 14 days of reperfusion. Atorvastatin was administered by gavage 14 days before ischemia–reperfusion induction. We then measured the serum concentrations and mRNA transcript levels of TNF-α, IL-1β, IL-6, IL-10, SOD2, and CAT. Hematoxylin and eosin stain were performed for histological analyses. Animals subjected to ischemia–reperfusion showed increased serum and transcript levels of TNF-α, IL-1β, IL-6, and IL-10 expressions with a concurrent increase in mRNA transcripts levels compared with sham and healthy controls. Groups treated with atorvastatin showed a significant CAT increase in the first 24 h, but CAT levels decreased at 7 and 14 days. SOD2 enzyme increased in serum without significant changes in mRNA expression. Histological analysis showed inflammatory infiltrate, microhemorrhages, and distortion of the tissue architecture in the first 7 days. At 14 days, the tissue showed loss and damage to myocytes. However, animals treated with atorvastatin showed few histological changes and a decrease in inflammatory cytokines. No significant changes in NO2, NO3, or 8-OHdG were observed. Atorvastatin showed a protective effect on the inflammation and tissue damage induced by ischemia–reperfusion in the hindlimb. The antioxidant effect of atorvastatin in the hindlimb is already unclear, and further research is needed to elucidate the molecular mechanism of this drug in the extremities.

Correction to: Apocynin and dimethyl sulfoxide synergistically protect against ischemia-reperfusion injury in a rat hind limb ischemia-reperfusion model

Correction to: Apocynin and dimethyl sulfoxide synergistically protect against ischemia-reperfusion injury in a rat hind limb ischemia-reperfusion model

Apocynin and dimethyl sulfoxide synergistically protect against ischemia-reperfusion injury in a rat hind limb ischemia-reperfusion model

Acute ischemia and reperfusion in extremities ignite a complicated inflammatory cycle and lead to damage in the extremities and target tissues. Rats were randomly divided into four groups. Each experimental group contained eight rats. Group 1 was the sham surgery group. Group 2 was the control I/R group with intraperitoneal serum physiologic (SP) treatment. Group 3 was the I/R group with vehicle treatment (8% DMSO). Group 4 was the I/R group with 20 mg/kg apocynin (vehicle 8% DMSO). The potential protective effects of apocynin and vehicle (DMSO) in the rat hind limb ischemia-reperfusion model were evaluated by measuring the levels of plasma pro-inflammatory cytokines and oxidative stress markers in muscle tissue. The results revealed that both apocynin and vehicle (DMSO) alone increased the antioxidative capacity of ischemic tissue and reduced the levels of plasma proinflammatory cytokines compared to control I/R group. There was a statically more significant effect in the apocynin (vehicle 8% DMSO)-treated group 4. We observed that apocynin and DMSO synergistically prevent the I/R injury in rat hind limb ischemia-reperfusion injury model and meaningfully decrease the inflammatory parameters. Level of Evidence: Not ratable.

Role of angiotensin-converting enzyme (ACE and ACE2) imbalance on tourniquet-induced remote kidney injury in a mouse hindlimb ischemia–reperfusion model

In this study, the relationship between the local imbalance of angiotensin converting enzymes ACE and ACE2 as well as Ang II and Ang (1–7) and renal injury was observed in the different genotypes mice subjected to tourniquet-induced ischemia–reperfusion on hind limbs. In wild-type mice, renal ACE expression increased while renal ACE2 expression decreased significantly after reperfusion, accompanied by elevated serum angiotensin II (Ang II) level and lowered serum angiotensin (1–7) (Ang (1–7)) level. However, renal Ang (1–7) also increased markedly while renal Ang II was elevated. Renal injury became evident after limb reperfusion, with increased malondialdehyde (MDA), decreased super-oxide dismutase (SOD) activity and increased serum blood urea nitrogen (BUN) and creatinine (Cr), compared to control mice. These mice also developed severe renal pathology including infiltration of inflammatory cells in the renal interstitium and degeneration of tubule epithelial cells. In ACE2 knock-out mice with ACE up-regulation, tourniquet-induced renal injury was significantly aggravated as shown by increased levels of MDA, BUN and Cr, decreased SOD activity, more severe renal pathology, and decreased survival rate, compared with tourniquet-treated wild-type mice. Conversely, ACE2 transgenic mice with normal ACE expression were more resistant to tourniquet challenge as evidenced by decreased levels of MDA, BUN and Cr, increased SOD activity, attenuated renal pathological changes and increased survival rate. Our results suggest that the deregulation of ACE and ACE2 plays an important role in tourniquet-induced renal injury and that ACE2 up-regulation to restore the proper ACE/ACE2 balance is a potential therapeutic strategy for kidney injury.

Protective effects of hydrogen-rich saline against hind limb ischemia reperfusion injury and associated remote lung injury

Objective To explore the protective effects of hydrogen-rich saline against hind limb ischemia reperfusion injury and associated remote lung injury in rats. Methods Thirty Sprague-Dawley rats were randomly divided into three groups: sham group ( group S) ; saline control group (group C) , undergoing intravenous infusion of saline (1 ml/100 g) at 10 min before reperfusion; hydrogen-treated group ( group H) , undergoing intravenous infusion of hydrogen-rich saline (1 ml/100 g) at 10 min before reperfusion. Hind limb ischemia reperfusion model was established by 4 h of the abdominal aorta ligation followed by a 4 h of reperfusion. Rats were killed at the end of reperfusion, and blood samples were collected for determination of serum levels of tumor necrosis factor-α ( TNF-α) , interleukin-6 (IL-6) and malonyldialdehyde ( MDA). Specimens of lung and gastrocnemius muscle were collected for measurement of wet-todry weight (W/D) ratio, light microscopic examination and detection of plasma TNF-α, IL-6 and MDA.Results The pathological changes were milder in group H than in group C. The lung (4. 47 ±0. 23) and gastrocnemius muscle (4. 04 ±0. 22) W/D ratio in group H was significantly lower than in group C (4. 67 ±0.27 vs4. 67 ±0.27, P＜0.05). The serum [(2.08 ± 1. 27) μmol/L, (528. 22 ±221. 17) , (41.14± 13. 01) ng/L, respectively] and homogenate levels [(3. 58 ± 1. 62) μmol/L, ( 1. 17 ±0. 15) , (0. 79±0.29) ng/L in the lung; (8.91 ±3. 13) μmol/L, (3. 13 ±0. 41) ,(3. 07 ± 1. 69) ng/L in the gastrocnemius muscle, respectively] of MDA, IL-6 and TNF-a were also significantly lower in group H than in group C (P ＜0. 05) , but there was no significant difference from group S. Conclusion Infusion of hydrogen-rich saline before reperfsion significantly alleviates the hind limb ischemia reperfusion injury and associated remote lung injury. Key words: Hydrogen-rich saline; Lung; Skeletal muscle; Reperfusion injury; Free radicals

Postischemic Treatment With Ethyl Pyruvate Prevents Adenosine Triphosphate Depletion, Ameliorates Inflammation, and Decreases Thrombosis in a Murine Model of Hind-Limb Ischemia and Reperfusion

Experiments were designed to investigate the effects of ethyl pyruvate (EP) in a murine model of hind-limb ischemia-reperfusion (IR) injury. 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. 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. 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.

Effect of Acoustic Power on In Vivo Molecular Imaging with Targeted Microbubbles: Implications for Low-Mechanical Index Real-Time Imaging

The aim of this study was to evaluate the influence of acoustic power on ultrasound molecular imaging data with targeted microbubbles. Imaging was performed with a contrast-specific multipulse method at mechanical indexes (MIs) of 0.18 and 0.97. In vitro imaging was used to measure concentration-intensity relationships and to assess whether damping from microbubble attachment to cultured endothelial cells affected signal enhancement. Power-related differences in signal enhancement were evaluated in vivo by P-selectin-targeted and control microbubble imaging in a murine model of hind-limb ischemia-reperfusion injury. During in vitro experiments, there was minimal acoustic damping from microbubble-cell attachment at either MI. Signal enhancement in the in vitro and in vivo experiments was 2-fold to 3-fold higher for high-MI imaging compared with low-MI imaging, which was due to greater pixel intensity, the detection of a greater number of retained microbubbles, and increased point-spread function. Yet there was a linear relationship between high-MI and low-MI data indicating that the relative degree of enhancement was similar. During molecular imaging, high-MI protocols produce more robust targeted signal enhancement than low-MI protocols, although differences in relative enhancement caused by condition or agent are similar.

Cyclooxygenase-2 inhibitor celecoxib in a rat model of hindlimb ischemia reperfusion

Acute ischemia-reperfusion (IR) of the limbs initiates both local and systemic injuries by triggering a systemic inflammatory response. Cyclooxygenase-2 (COX-2), an endogenous inducible enzyme, rises in response to inflammation. The aim of this work is to investigate the role of celecoxib, a selective COX-2 inhibitor, in abrogating remote organ dysfunction after hindlimb IR in rats by comparing it with a standard hemorrheologic drug (pentoxifylline). Rats were divided into 4 groups (n = 6 each), group I (sham control, received saline and was kept under anaesthetic for 7 h). Group II (IR, subjected to 1 h of hindlimb tourniquet ischemia and 6 h reperfusion). Group III and IV (pretreated with 200 mg/kg pentoxifylline or 10 mg/kg celecoxib, respectively, intragastrically for 7 days before IR induction). Administration of pentoxifylline or celecoxib produced significant reduction in SGPT, serum creatinine, malondialdehyde, and tumor necrosis factor-alpha and significant increase in blood pH, blood adenosine triphosphate, and reduced glutathione compared with the IR group (p < 0.05). There was no significant difference among the pretreated groups. Histopathologic findings of the IR lung showed alveolar destruction, inflammatory cells infiltration, mast cell degranulation, and necrosis of the gastrocnemius muscle fibres. These changes were attenuated in the pretreated groups. In conclusion, celecoxib can ameliorate IR induced remote organ injury to a similar extent as pentoxifylline through its antiinflammatory and antioxidant action.

Protective roles of adenosine A1, A2A, and A3receptors in skeletal muscle ischemia and reperfusion injury

Although adenosine exerts cardio-and vasculoprotective effects, the roles and signaling mechanisms of different adenosine receptors in mediating skeletal muscle protection are not well understood. We used a mouse hindlimb ischemia-reperfusion model to delineate the function of three adenosine receptor subtypes. Adenosine A(3) receptor-selective agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IBMECA; 0.07 mg/kg ip) reduced skeletal muscle injury with a significant decrease in both Evans blue dye staining (5.4 +/- 2.6%, n = 8 mice vs. vehicle-treated 28 +/- 6%, n = 7 mice, P < 0.05) and serum creatine kinase level (1,840 +/- 910 U/l, n = 13 vs. vehicle-treated 12,600 +/- 3,300 U/l, n = 14, P < 0.05), an effect that was selectively blocked by an A(3) receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191; 0.05 mg/kg). The adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 0.05 mg/kg) also exerted a cytoprotective effect, which was selectively blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.2 mg/kg). The adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.07 mg/kg)-induced decrease in skeletal muscle injury was selectively blocked by the A(2A) antagonist 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e] [1,2,4]triazolo[1,5-C]pyrimidin-5-amine (SCH-442416; 0.017 mg/kg). The protection induced by the A(3) receptor was abrogated in phospholipase C-beta2/beta3 null mice, but the protection mediated by the A(1) or A(2A) receptor remained unaffected in these animals. The adenosine A(3) receptor is a novel cytoprotective receptor that signals selectively via phospholipase C-beta and represents a new target for ameliorating skeletal muscle injury.

Microcirculatory perfusion deficits are not essential for remote parenchymal injury within the liver.

A normotensive model of hindlimb ischemia-reperfusion in Wistar rats was used to test the hypothesis that microvascular perfusion deficits contribute to the initiation of remote hepatic injury during a systemic inflammatory response. Animals were randomly assigned to one of three groups: 4 h of ischemia with 6 h of reperfusion (I/R-6; n = 4), 4 h of ischemia with 3 h of reperfusion (I/R-3; n = 5), or no ischemia (naive; n = 5). With intravital fluorescence microscopy, propidium iodide (PI; 0.05 mg/100 g body wt) was injected for the in vivo labeling of lethally injured hepatocytes (number/10(-1) mm(3)). PI-positive hepatocytes increased progressively over the 6-h period (naive 32.9 +/- 7.8 vs. I/R-3 92.8 +/- 11.5 vs. I/R-6 232 +/- 39.2), with no difference between periportal and pericentral regions of the lobule. Additionally, a significant decrease in continuously perfused sinusoids (naive 70.0 +/- 1.5 vs. I/R-3 65.0 +/- 1.0 vs. I/R-6 48.8 +/- 0.9%) was measured. Regional sinusoidal perfusion differences were only observed after 3 h of limb reperfusion. Indirect measures of hepatocellular injury using alanine transaminase levels support the progressive nature of hepatic parenchymal injury (0 h 57.8 +/- 6.5 vs. 3 h 115.3 +/- 20.7 vs. 6 h 125.6 +/- 19.5 U/l). Evidence from this study suggests that remote hepatic parenchymal injury occurs early and progresses after the induction of a systemic inflammatory response and that microvascular perfusion deficits are not essential for the initiation of such injury.