Model Of Reperfusion Injury Research Articles

PS208. Edaravone Injected at the Start of Reperfusion Reduces Muscle Injury following Leg Ischemia in Rats

At VAM 2010 & 2011, we have reported that preoperative injection of the free-radical scavenger, edaravone (Radicut®, Mitsubishi Tanabe Pharma Co., Japan) prevented reperfusion injury. In this study, we evaluated whether edaravone can reduce reperfusion injury if injected at the start of reperfusion. Male Lewis rats (525 ± 78 g, n = 10) were subjected to reperfusion injury models by clamping the bilateral common femoral arteries for 5 hours followed by de-clamping. Rats were divided into two groups and intraperitoneally injected with edaravone (group edaravone; 9.0 mg / kg, n = 5) or saline (control group; n = 5) at the same time as removing the clamps. Five hours after de-clamping, the muscles of lower extremity were harvested. Tissues were stained with hematoxylin & eosin (HE), in order to count the viable muscle cells. They were also stained with periodic acid-Schiff (PAS), in order to assess the glycogen storage in muscle cells. The density of viable muscle cells in the group edaravone was significantly greater than that of control group (593±60 cells/mm2 vs. 258±31 cells/mm2, P < .01). The mean percentage of PAS-positive area in the edaravone group was also significantly higher than that in control group (30.1 ± 6.9 % vs. 7.3 ± 2.1%, P < .001, Fig, original 200x). Our results suggest that edaravone injected at the start of reperfusion can also reduce muscle injury following leg ischemia in rats, storing a high level of glycogen in viable muscle cells. Therefore, edaravone might be useful in clinical settings following leg ischemia.

Mycophenolate mofetil inhibits macrophage infiltration and kidney fibrosis in long-term ischemia–reperfusion injury

Immunosuppressants have been widely used in renal transplantation, in which ischemia–reperfusion injury is inevitable. Mycophenolate mofetil (MMF) is a relative novel immunosuppressant and also attenuates ischemia–reperfusion injury in the acute phase, but its long-term effects are still obscure. Unilateral renal ischemia–reperfusion injury model was established in Sprague–Dawley rats and 30mg/kg/day MMF or natural saline was administered a day before the surgery. Renal function was monitored, and histological changes and fibrosis in the kidney were evaluated in both short and long terms. TGF-β1 secretion and MCP-1 expression were determined by immunohistochemistry and real-time PCR respectively. The infiltration of macrophages in renal tissues was also assessed by fluorescence activated cell sorting (FACS). MMF treatment significantly improved renal function in ischemia–reperfusion injury rats in the short and long-term and also effectively prevented interstitial fibrosis. TGF-β1 secretion and MCP-1 expression in the renal tissue of MMF-treated rats were much lower than those in natural saline-treated rats, with much less macrophage infiltration as well. MMF treatment effectively prevented the deterioration of renal function and interstitial fibrosis in ischemia–reperfusion injury rats, which may be associated with decreased TGF-β1, MCP-1 and macrophages. These results provide evidence for the choice of MMF in the renal transplant patients not only for acute renal injury but also for long-term survival of renal allograft.

Abstract 227: Edaravone Injected at the Start of Reperfusion Reduces Muscle Injury Following Leg Ischemia in Rats

Introduction &amp; Hypothesis: It is well known that free radicals cause reperfusion injury after leg ischemia. At ATVB 2010 &amp; 2011, we have reported that preoperative injection of the free-radical scavenger, edaravone (Radicut®, Mitsubishi Tanabe Pharma Co., Japan) prevented reperfusion injury. In this study, we evaluated whether edaravone can reduce reperfusion injury if injected at the start of reperfusion. Methods: Male Lewis rats (525 ± 78 g, n = 10) were subjected to reperfusion injury models by clamping the bilateral common femoral arteries for 5 hours followed by de-clamping. Rats were divided into two groups and intraperitoneally injected with edaravone (group edaravone; 9.0 mg / kg, n = 5) or saline (control group; n = 5) at the same time as removing the clamps (i.e., the start of reperfusion). Five hours after de-clamping, the muscles of lower extremity were harvested. Tissues were stained with hematoxylin &amp; eosin (HE), in order to count the viable muscle cells. They were also stained with periodic acid-Schiff (PAS), in order to assess the glycogen storage in muscle cells. The number of viable muscle cells and the percentages of PAS-positive area were measured on each three different field (original magnification 200x). Results: In the control group, most of the muscle cells were swelling. The density of viable muscle cells in the group edaravone was significantly greater than that of control group (593±60 cells/mm2 vs. 258±31 cells/mm2, p &lt; 0.01). The mean percentage of PAS-positive area in the edaravone group was also significantly higher than that in control group (30.1 ± 6.9 % vs. 7.3 ± 2.1%, p &lt; 0.001, Figure attached). Conclusions: Our results suggest that edaravone injected at the start of reperfusion can also reduce muscle injury following leg ischemia in rats, storing a high level of glycogen in viable muscle cells. Therefore, edaravone might be useful in clinical settings following leg ischemia.

Adipose Derived Stem Cells Protect Skin Flaps Against Ischemia-Reperfusion Injury

Advances in the treatment of ischemia- reperfusion injury have created an opportunity for plastic surgeons to apply these treatments to flaps and implanted tissues. We examined the capability of adipose derived stem cells (ADSCs) to protect tissue against IRI using an extended inferior epigastric artery skin flap as a flap ischemia- reperfusion injury (IRI) model. ADSCs were isolated from Lewis rats and cultured in vitro. Twenty- four rats were randomly divided into three groups. Group I was the sham group and did not undergo ischemic insult; rather, the flap was raised and immediately sutured back (non-ischemic control group). Group II (ischemia control) and group III (ADSCs treatment) underwent 3 h of ischemic insult. During reperfusion group III was treated by intravenous application of ADSCs and group II was left untreated. Five days postoperatively, flap survival and perfusion were assessed. Microvessel density was visualized by immunohistochemistry and semi- quantitative real-time polymerase chain reaction addressed differential gene expression. Treatment with ADSCs significantly increased flap survival (p<0.001) and flap perfusion (p<0.001) when compared to the control group II. Microvessel- density in ADSCs treated group was not significantly increased in any group. No significant differences showed the comparison of the experimental group III and the sham operated control group I. ADSCs treatment (Group III) was accompanied by a significantly enhanced expression of pro-angiogenic and pro-inflammatory genes. Overall, our study demonstrates that ADSCs treatment significantly enhances skin flap survival in the aftermath of ischemia to an extent that almost equals surgical results without ischemia. This effect is accompanied with a pronounced and significant angiogenic response and an improved blood perfusion.

Glucose reintroduction triggers the activation of Nrf2 during experimental ischemia reperfusion

Reperfusion results in a rapid reintroduction of oxygen, glucose, and other restricted components to an ischemic tissue. It brings with it not only the necessary components for cell survival but also a burst of oxidative stress and cellular damage. In this study, our primary aims were to investigate glucose as a determining factor for the activation of the transcription factor NF-E2-related factor 2 (Nrf2) upon reperfusion and the expression of downstream anti-oxidant NADPH-dependent reductases. Exposure of renal epithelial HK-2 cells to oxygen and glucose reintroduction after depletion resulted in an increase in nuclear translocation of Nrf2 protein in a manner dependent upon glucose. This activation and the induction of the Nrf2-dependent gene NAD(P)H dehydrogenase, quinone 1 (NQO1) was observed to be maximum at a concentration of 5 mM glucose. Microarray analysis of mRNA from siRNA targeted cells under these conditions revealed the Nrf2-dependent expression of NADPH-dependent reductase enzymes NQO1, Aldo-keto reductase family 1, members C1-3 and dehydrogenase/reductase (SDR family) member 2 (DHRS2), all genes demonstrated to protect against oxidative stress-mediated cellular injury. In addition, NQO1 and DHRS2 mRNA levels were specifically upregulated on glucose reintroduction and were also increased in an in vivo ischemia reperfusion injury model of murine renal pedicle clamping. In conclusion, we demonstrate that glucose reintroduction after depletion activates Nrf2 and Nrf2 regulated NADPH-dependent reductase expression. We suggest these findings represent a previously unreported mechanism for the activation of Nrf2 as a cytoprotective pathway in IRI.

The effect of pentoxifyllin in a rat model of renal ischemic reperfusion injury, light, transmission, and scanning electron microscopical study

Introduction Renal ischemic/reperfusion injury remains a major problem in renal transplantation and anesthesia. Pentoxifylline (PTX) is commonly used to treat peripheral vascular and cerebrovascular diseases. Aim of the work The aim of the work was to demonstrate the histological effect of ischemia reperfusion on the kidney and to detect the possible protective role of PTX. Materials and methods Adult male rats were divided into three groups. Group I was the control group, in group II, renal ischemia was induced for 30min, followed by reperfusion for 24h, and in group III, the animals received PTX 4mg/rat/day intraperitoneally for 3 days, and then an ischemia–reperfusion procedure was carried out. At the end of the experiment, the kidneys were dissected and processed for light, transmission, and scanning electron microscopical study. Results Group II showed focal affection of the kidney whereas some of the glomeruli appeared small and atrophic. The epithelium of proximal convoluted tubules showed vacuolation of the cytoplasm with a detached apical part and their lumena contained cellular debris. The distal convoluted tubules showed loss of the supranuclear cytoplasm of their lining cells. In the medulla, the collecting tubules were dilated and contained casts in their lumena. Transmission electron microscopy revealed disruption of the glomerular basement membrane. Proximal convoluted tubules and distal convoluted tubules showed cytoplasmic vacuolation, degenerated mitochondria, and destroyed brush borders. Scanning electron microscopy revealed glomeruli with an absence of their covering podocytes and destruction of their glomerular capillaries. The collecting tubules showed complete flattening of their lining cells. In group III, the kidney relatively retained their histological architecture. There was a significant decrease in the mean glomerular surface area in groups II and III in comparison with the control group. However, group III showed a significant increase in comparison with group II. Conclusion The present study concluded that PTX could protect and reduce the severity of renal ischemic reperfusion injury.

Protective Effect of Delayed Remote Limb Ischemic Postconditioning: Role of Mitochondrial KATP Channels in a Rat Model of Focal Cerebral Ischemic Reperfusion Injury

Delayed remote ischemic postconditioning (DRIPost) has been shown to protect the rat brain from ischemic injury. However, extremely short therapeutic time windows hinder its translational use and the mechanism of action remains elusive. Because opening of the mitochondria KATP channel is crucial for cell apoptosis, we hypothesized that the neuroprotective effect of DRIPost may be associated with KATP channels. In the present study, the neuroprotective effects of DRIPost were investigated using adult male Sprague-Dawley rats. Rats were exposed to 90 minutes of middle cerebral artery occlusion followed by 72 hours of reperfusion. Delayed remote ischemic postconditioning was performed with three cycles of bilateral femoral artery occlusion/reperfusion for 5 minutes at 3 or 6 hours after reperfusion. Neurologic deficit scores and infarct volumes were assessed, and cellular apoptosis was monitored by terminal deoxynucleotidyl transferase nick-end labeling. Our results showed that DRIPost applied at 6 hours after reperfusion exerted neuroprotective effects. The KATP opener, diazoxide, protected rat brains from ischemic injury, while the KATP blocker, 5-hydroxydecanote, reversed the neuroprotective effects of DRIPost. These findings indicate that DRIPost reduces focal cerebral ischemic injury and that the neuroprotective effects of DRIPost may be achieved through opening of KATP channels.

Imaging of Receptors for Advanced Glycation End Products in Experimental Myocardial Ischemia and Reperfusion Injury

The aim of this study was to image expression of receptor for advanced glycation end products (RAGE) in a mouse model of myocardial reperfusion injury. RAGE and its ligands are implicated in the pathogenesis of ischemia/reperfusion injury and infarction. We hypothesized that RAGE-directed quantitative imaging of myocardial uptake of technetium-99m ((99m)Tc)-anti-RAGE F(ab')(2) in a mouse model of myocardial ischemic injury can detect RAGE expression and show quantitative differences between early (18 to 20 h) and later times (48 h) after reperfusion. Twenty-five wild-type (WT) mice underwent left anterior descending coronary artery occlusion for 30 min. Mice were injected with 19.98 ± 1.78 MBq of (99m)Tc anti-RAGE F(ab')(2) at 2 time points after reperfusion (at 18 to 20 h [n = 8] and at 48 h [n = 12]) and 5 h later with 6.14 ± 2.0 MBq of thallium-201 ((201)Tl). Five WT mice were injected with nonspecific F(ab')(2) and (201)Tl 18 to 20 h after reperfusion. Six WT mice underwent sham operation without coronary intervention. After injection with (201)Tl, all mice immediately underwent dual isotope single-photon emission computed tomography/computed tomography. At completion of imaging, hearts were counted and sectioned. The uptake of (99m)Tc-anti-RAGE F(ab')(2) in the ischemic zone from the scans as mean percentage injected dose was significantly greater at 18 to 20 h (5.7 ± 2.1 × 10(-3)%) as compared with at 48 h (1.4 ± 1.1 × 10(-3)%; p < 0.001) after reperfusion. Disease and antibody controls showed no focal uptake in the infarct. Gamma well counting of the myocardium supported the quantitative scan data. By immunohistochemical staining there was greater caspase-3 and RAGE staining at 18 to 20 h versus at 48 h (p = 0.04 and p = 0.01, respectively). On dual immunofluorescence, RAGE colocalized mainly with injured cardiomyocytes undergoing apoptosis. RAGE expression in myocardial ischemic injury can be imaged in vivo using a novel (99m)Tc-anti-RAGE F(ab')(2). RAGE plays a role in several cardiovascular diseases and is a potential target for clinical imaging.

Neuroprotective Effects of Granulocyte Colony-Stimulating Factor on Ischemia-Reperfusion Injury of the Retina

Purpose: It has been reported that granulocyte colony-stimulating factor (G-CSF) provides neuroprotection in models in which neuronal cell death is induced. This research was designed to investigate the effects of G-CSF on neurodegeneration of the inner retinal layer in a rat model of ischemic reperfusion (I/R) injury. Materials and Methods: Retinal ischemia was induced by increasing the intraocular pressure to 110 mm Hg for 45 min in the left eyes of the rats. A sham operation was carried out on the right eyes. G-CSF (100 µg/kg/day in 0.3 ml saline) or the same volume of saline was intraperitoneally injected just before the operation and continued for 4 consecutive days (a total of 5 consecutive days). Morphological examinations, including the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, were performed 7 days after I/R induction. The expression of phosphorylated AKT in the retina was examined by Western blot analysis and immunohistochemistry. Results: Cell loss in the ganglion cell layer was more significantly reduced in the I/R-induced eyes of the G-CSF-injected rats than in the I/R-induced eyes of the saline-injected rats (20.3 vs. 6.6%). The inner retinal thickness ratios, such as the inner plexiform layer to the inner limiting membrane/outer nuclear layer and the inner nuclear layer/outer nuclear layer, were significantly better preserved in the I/R-induced eyes of the G-CSF-injected rats than in the I/R-induced eyes of the saline-injected rats. TUNEL assays showed fewer apoptotic cells in the retinal sections of the I/R-induced eyes of the G-CSF-injected rats. The phosphorylation of AKT (p-AKT/AKT) was upregulated in the retinas of the I/R-induced eyes of the G-CSF-injected rats. Conclusion: Our results demonstrated that systemic injection of G-CSF can protect retinal ganglion cells and inner retinal layers from I/R injury. The effects could be associated with the activation of AKT.

Cardiac Effects of Cupping: Myocardial Infarction, Arrhythmias, Heart Rate and Mean Arterial Blood Pressure in the Rat Heart

This study was carried out to determine the effects of cupping on hemodynamic parameters, arrhythmias and infarct size (IS) after myocardial ischemic reperfusion injury in male rats. Rats were randomly subjected to dry or wet cupping. While dry cupping simply involved stimulation of the skin by suction, in wet cupping, scarification of the back skin was also carried out with a surgical blade and 0.5 ml blood was sucked out in each session. For ischemic reperfusion injury, rats were subjected to 30 min of left anterior descending coronary artery occlusion and 120 min of reperfusion. Our results show that cupping did not change the baseline heart rate or mean arterial blood pressure. Ischemic reperfusion injury caused an IS of 50 ± 5%, whereas dry cupping, single and repeated wet cupping significantly reduced IS to 28 ± 3%, 35 ± 3% and 22 ± 2% of area at risk, respectively. The rate of ischemic induced arrhythmias was significantly modified by wet cupping (P < 0.05). These results indicate for the first time in rats that cupping might be cardioprotective in the ischemic reperfusion injury model.

Extracorporeal shock wave treatment protects skin flaps against ischemia–reperfusion injury

Advances in the treatment of ischemia–reperfusion injury have created an opportunity for plastic surgeons to apply these treatments to flaps and implanted tissues. Using an extended inferior epigastric artery skin flap as a flap ischemia–reperfusion injury (IRI) model, we examined the capability of extracorporeal shock wave treatment (ESWT) to protect tissue against IRI in a rat flap model. Twenty-four rats were used and randomly divided into three groups (n=8 for each group). Group I was the sham group and did not undergo ischemic insult; rather, the flap was raised and immediately sutured back (non-ischemic control group). Group II (ischemia control) and Group III (ESWT) underwent 3h of ischemic insult. During reperfusion Group III was treated with ESWT and Group II was left untreated. Histological evaluation was made to investigate treatment induced tissue alterations. Survival areas were assessed at 5d postoperatively. Skin flap survival and perfusion improved significantly in the ischemic animals following ESWT (p<0.001, respectively). The tissue protecting effect of ESWT resulted in flap survival areas and perfusion data equal to non-ischemic, sham operated flaps. In line with the observation of better flap perfusion, tissue from ESWT-treated animals (Group III) revealed a significantly increased frequency of CD31-positive vessels compared to both the ischemic (Group II; p=0.003) and the non-ischemic, sham operated control (Group I; p<0.005) and an enhanced expression of pro-angiogenic genes. This was accompanied by a mild suppression of pro-inflammatory genes. Our study suggests that ESWT improves flap survival in IRI by promoting angiogenesis and inhibiting tissue inflammation. The study identifies ESWT as a low-cost and easy to use technique for surgical techniques that aim at reducing ischemia–reperfusion-induced tissue injury

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non‐competitive allosteric inhibitor

DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [(35) S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys(99) on CXCR1 and the non-conserved residue Asp(293) on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases.

Co-administration of apocynin with lipoic acid enhances neuroprotection in a rat model of ischemia/reperfusion

Apocynin is a well-known NADPH-oxidase inhibitor currently being investigated for its potential therapeutic use in patients with cardiovascular disease, such as occlusive stroke. However, the use of apocynin as a potential neuroprotective agent has come under criticism due to a narrow experimental therapeutic dose range and possible pro-oxidant effects at high doses. Lipoic acid is a powerful antioxidant due to its ability to scavenge free radicals at very low doses and has been demonstrated to enhance the therapeutic value of several other classes of drugs. Therefore, the present study was designed to determine if co-administration of previously determined non-neuroprotective doses of lipoic acid and apocynin in combination could enhance their neuroprotective ability thus extending the therapeutic dose range. We tested the hypothesis in a rat model of stroke and reperfusion injury. The middle cerebral artery (MCA) in male Sprague-Dawley rats was occluded for 30 min followed by 5.5 h of reperfusion. Pre-treatment with several doses of apocynin (0.05, 0.1 and 1.0 mg/kg) in combination with a single dose of lipoic acid (0.005 mg/kg) resulted in a dose-dependent reduction in infarct volume up to ∼50%. These results demonstrate that a non-effective dose of lipoic acid can enhance the neuroprotective ability of apocynin at doses which were previously demonstrated to be non-neuroprotective. Co-administration of apocynin with lipoic acid may overcome the criticisms of the use of apocynin as a neuroprotectant and provide an effective therapy in the prevention of cell death following stroke.

Pulsed acoustic cellular expression as a protective therapy against I/R injury in a cremaster muscle flap model

BackgroundTissue ischemia and reperfusion (I/R) affects blood flow restoration and oxygen delivery to the damaged tissues contributing to tissue morbidity and microcirculatory compromise. Pulsed acoustic cellular expression (PACE) technology is known to support tissue neovascularization. The aim of this study was to test PACE conditioning mechanism of action on microcirculatory hemodynamics in ischemia–reperfusion injury model. Methods34 rat cremaster muscle flaps were monitored under intravital microscopy system in 4 experimental groups: 1) non-ischemic controls (n=10), 2) 5h ischemia without conditioning (n=8), 3) pre-ischemic (5h) PACE conditioning (n=8), 4) post-ischemic (5h) PACE conditioning (n=8). Standard microcirculatory hemodynamics of RBC velocity, vessel diameters and functional capillary perfusion were recorded for 2h after I/R. Immunohistochemistry assessed expression of proangiogenic factors: VEGF and vWF, whereas real-time PCR assessed proangiogenic (VEGF, eNOS) and proinflammatory factors (iNOS; chemokines: CCL2, CXCL5 and chemokine receptor CCR2). ResultsPre-ischemic PACE conditioning (group 3) resulted in increased RBC velocity of second (A-2) and third order arterioles (A-3) and venule (V-1) by 40%, 15% and 24% respectively comparing to ischemic group without conditioning (p<0.05). Post-ischemic PACE conditioning (group 4) revealed: 1) increase in RBC velocity in second (A-2) and third order arterioles (A-3) by 65% and 31% respectively comparing to ischemia without conditioning (group 2), 2) 33% increase in first order arterioles diameter (A-1) (p<0.05) compared to ischemic controls, 3) 21% increase in number of functional capillaries compared to ischemia without conditioning (group 2) (P<0.05). Immunostaining assays showed that PACE postconditioning up-regulated proangiogenic factors vWF and VEGF protein expression. This correlated with increased gene expression of VEGF (up to 180%). In contrast, gene expression of proinflammatory factors (iNOS, CCL2, CXCL5) decreased compared to ischemic controls. Pre-ischemic PACE conditioning decreased gene expression of proinflammatory chemokines (CCL2 and CXCL5), compared to ischemic controls without conditioning. ConclusionsAs expected 5h ischemia resulted in deterioration of microcirculatory hemodynamics confirmed by decreased vessels diameters and RBC velocities. This was alleviated by pre- and post-ischemic PACE conditioning which improved functional capillary density and stimulated angiogenesis as confirmed by up-regulated VEGF expression. Furthermore, post-ischemic PACE conditioning correlated with decreased expression of early proinflammatory factors (iNOS, CCL2, CXCL5). Both types of PACE conditioning ameliorated deleterious effect of ischemia–reperfusion injury on microcirculatory hemodynamics of muscle flaps.

Protective perioperative strategy using a third generation hydroxyethyl starch during surgery in a murine model of liver reperfusion injury

To investigate whether a third generation colloid, hydroxyethyl starch (HES 130/0.4), used for perioperative fluid therapy, protects the rat liver against the late-phase response of ischemia/reperfusion injury (IRI) and if inhibition of neutrophil hepatic infiltration plays a part in this mechanism. Wistar rats were used (8 in each group). Three groups had IRI induced by lobar vascular occlusion (60 minutes) and reperfusion (24 hours) and received HES (13 mL/kg iv), 7.5% saline (HS) (13 mL/kg iv) or no fluid. Three other groups were sham-operated and received the same fluid as the test groups. After 24 hours of reperfusion, blood was drawn for alanine aminotransferase (ALT) quantification and ischemic liver samples were taken for histological study (hematoxylin and eosin and chloroacetate staining of neutrophils). HES treatment attenuated the elevation in serum ALT (P=0.001) and reduced the extent of hepatocellular necrosis (P<0.01) compared with the IRI controls. HES-mediated cytoprotection was associated with a decrease of infiltration of neutrophils in the necrotic areas (P<0.05) compared with the untreated IRI rats, but not with the volume control IRI rats (P>0.05). Hydroxyethyl starch suppresses inflammatory response and ameliorates the late-phase response of hepatic ischemia/reperfusion injury.

Relationship Between Ischemia/Reperfusion Injury and the Stimulus of Fibrogenesis in an Experimental Model: Comparison Among Different Preservation Solutions

Background and Aims Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis. Materials and Methods Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW + SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-β1 and angiotensin II AT1 receptor. Results AST was significantly lower during cold storage with UW than with the older media ( P = .001); ALT was lower in the FBP group ( P = .023) and LDH was lower in the FBP and SNAC groups ( P = .007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups ( P = .008 and P = .006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 ( P = .004). Renin levels were significantly lower in the FBP group ( P = .022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-β1 or AT1 receptor. Conclusion In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW.

Heme oxygenase-1 could mediate the protective effects of hyperbaric oxygen preconditioning against hepatic ischemia-reperfusion injury in rats

1. Heme oxygenase 1 (HO-1) has been shown to play a pivotal role in the maintenance of cellular homeostasis when the liver undergoes sublethal stress, such as ischaemia-reperfusion (I/R) injury. In the present study, we investigated the protective role of HO-1 in hyperbaric oxygen (HBO) preconditioning against liver injury after I/R. 2. A total hepatic ischaemia (30 min) and reperfusion (60 min) injury model in rats was used in the present study. Preconditioned groups were exposed to HBO 24 h prior to the induction of I/R injury. Other groups were injected with zinc protoporphyrin IX (ZnPP) intraperitoneally 1 h before I/R to inhibit HO-1 activity. At the end of the reperfusion period, blood and liver samples were collected for the analysis of liver injury markers, morphological changes, and HO-1 expression and activity in the liver. 3. In untreated rats, I/R induced an increase in hepatic injury markers, such as plasma transaminases, inflammatory cytokines (tumour necrosis factor-α and interleukin-1β), and tissue malondialdehyde. However, HBO preconditioning attenuated the I/R-induced increases in these hepatic injury markers, and prevented both the necrosis and apoptosis of hepatocytes induced by I/R injury. Furthermore, HBO preconditioning significantly increased HO-1 mRNA and protein levels in the liver. In rats in which HO-1 activity had been inhibited with ZnPP pretreatment, the protective effects of HBO preconditioning against I/R injury were abolished. 4. In conclusion, HBO preconditioning can protect the liver against I/R injury and it appears that this effect might be mediated by the induction of HO-1.

Transient ischemia-induced paresis and complete paraplegia displayed distinct reactions of microglia and macrophages

In this study, we perform a detailed analysis of the microglial and macrophage responses in a model of spinal cord ischemia and reperfusion (SCI/R) injury in Wistar rats. The rats underwent occlusion across the descending aorta for 13 min, causing paraplegia or paresis of varying severity. They were divided into four groups based on neurological assessment: sham, mild paresis, moderate paresis, and severe (complete) paraplegia. To examine the origin of microglia and macrophages in the ischemic lesion, bone marrow from rats expressing green fluorescent protein (GFP) was transplanted into test subjects one month before performing SCI/R. Many GFP +/CD68 + microglia and macrophages were present 7 d after SCI/R. Resident (GFP −/Iba1 +/CD68 −) microglia and bone marrow-derived macrophages (BMDMs; GFP +/Iba1 +/CD68 +) colocalized in the mild group 7 d after SCI/R. In the moderate group, BMDMs outnumbered resident microglia. A greater accumulation of BMDMs expressing insulin-like growth factor-1 (IGF-1) was observed in lesions in the severe group, relative to the moderate group. BMDMs in the severe group strongly expressed tumor necrosis factor α, interleukin-1β, and inducible nitric oxide synthase, in addition to IGF-1. A robust accumulation of BMDMs occupying the entire ischemic gray matter was observed only in the severe group. These results demonstrate that the magnitude of the microglial and BMDM responses varies considerably, and that it correlates with the severity of the neurological dysfunction. Remarkably, BMDMs appear to have a beneficial effect on the spinal cord in paresis. In contrast, BMDMs seem to exhibit both beneficial and harmful effects in severe paraplegia.

The role of interleukin-6, endothelins, and apoptotic genes in small bowel transplantation, in a swine model of ischemia and reperfusion injury

IRI is closely related to sepsis in ITx setting. Complete understanding of the mechanisms involved in IRI development may improve outcomes. Ortothopic ITx without immunosuppression was performed in order to characterize IRI-associated mucosal damage. Twenty pigs underwent ITx. Two groups were assigned to different CI times: G1: 90 min and, G2: 180 min. Euro-Collins was used as preservation solution. Jejunal fragments were collected at donor laparotomy, 30 min, and 3 days after reperfusion. IRI assessment involved: histopathologic analysis, quantification of MPO-positive cells through immunohistochemical studies, quantification of epithelial apoptotic cells using TUNEL staining, and quantification of IL-6, ET-1, Bak, and Bcl-XL genes expression by RT-PCR. Neutrophilic infiltration increased in a similar fashion in both groups, but lasted longer in G2. Apoptosis detected by TUNEL staining increased and anti-apoptotic gene Bcl-XL expression decreased significantly in G1, 3 days after surgery. Endothelin-1 and IL-6 genes expression increased 30 min after the procedure and returned to baseline 3 days after surgery. In conclusion, IL-6 and ET-1 are involved precociously in the development of intestinal IRI. Apoptosis was more frequently detected in G1 grafts by TUNEL-staining and by RT-PCR.

The hest interventional time of α7nAChR agonist postconditioning attenuating myocardial ischemia/reperfusion injury in rats

Objective To determine the best interventional time of α7nAChR agonist postconditioning for attenuating myocardial ischemia reperfusion injury in rat in vivo model. Methods Sixty SD rats were randomly divided into six groups(n=10):sham group (S group), ischemia/reperfusion group (I/R group ), α7nAChR agonist postconditioning at onset of reperfusion group( P0group), α7nAChR agonist postconditioning at 30, 60 and 90 min of reperfusion group(P30, P60 and P90 groups). Serum concentrations of TnI, TNF-α and HMGB1 were assayed at 180 min after reperfusion. At the end of experiment, infarction sizes were assessed from excised hearts by Evans blue and triphenyltetrazolium chloride(TTC) staining. Results The infarct sizes(IS％) were (78±16)％ in IR group, (60±7)％ in P0 group, (49±17)％ in P30 group, (54±12)％ in P60 group and (64±15)％ in P90 group, respectively. Serum concentrations of TnI were ( 1.016±0.121 ), (0.168±0.037), (0.156±0.019), (0.194±0.041) and(0.138±0.029) μg/L in these groups respectively. As compared to the S group, serum concentrations of TNF-α in the I/R and P30 groups were significantly increased,TNF-α in the P0 group was significantly reduced. Serum concentration of HMGB1 was significantly higher in the I/R group than that in the S group, but that of all α7nAChR agonist postconditioning groups was significantly lower. As compared with the I/R group, infarct size and serum concentrations of TnI, TNF-α and HMGB1 in all α7nAChR agonist postconditioning groups were significantly reduced. As compared with the P0group, serum concentrations of TNF-α and HMGB1 in the P30, P60 and P90 groups were significantly increased. As compared with the P90 group, in the P30 group, serum concentrations of TNF-α and HMGB1 were significantly increased and IS％ significantly decreased. Conclusion In rat in vivo models of myocardial ischemia reperfusion injury, α7nAChR agonist postconditioning at 30 min of reperfusion could produce the strongest cardioprotection. Key words: Ischemia/reperfusion injury; α7nAChR; Inflammation; Pharmacological postconditioning