Induce Ischemia-reperfusion Injury Research Articles

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

Extending Vascularized Composite Tissue Survival Using Near-Normothermic Ex Situ Perfusion

Objective/Hypothesis: Currently, vascularized composite allografts (VCAs) are cold preserved (4°C) until transplantation. This process is time limited, as the tissue has to be revascularized within 4 to 6 hours to minimize ischemia reperfusion (IR) injury. Normothermic perfusion was proposed as an alternative method of preservation in solid organ transplantation. This method helps to avoid complications associated with cold preservation and maintains tissue viability without inducing IR injury. Using this method, previous investigators demonstrated its potential to prolong swine forelimb allograft survival up to 24 hours. In this study, we aimed to test this system on human forearm allografts. Material and Methods: Five human forearms were procured from brain-dead adult donors under tourniquet control. Following elbow disarticulation, the brachial artery was cannulated. The limb was flushed with heparinized saline and connected to a temperature-controlled (30°C-33°C) ex situ perfusion system for 24 hours. The perfusate consisted of plasma and red blood cells with a target hemoglobin (Hb) concentration of 4 to 6 g/dL. Muscle biopsies (flexor carpi radialis) were obtained at 0, 12, and 24 hours. Results: Average warm ischemia time was 76 minutes. Average arterial systolic pressure was 93 ± 2 mm Hg, perfusion flow 310 ± 20 mL/min (~6%-8% of the donor’s estimated cardiac output), and vascular resistance 153 ± 16 mm Hg/mL/min. Perfusate had an average pH of 7.43 ± 0.04, pCO2 32 ± 1 mm Hg, pO2 317 ± 18 mm Hg, and Hb 4.8 ± 0.4 g/dL. Electrolytes (sodium, potassium, chloride) remained within a physiologic range. Lactate started to increase steadily throughout the experiment; however, neuromuscular electrical stimulation revealed ongoing contraction throughout the experiment. Hematoxylin-eosin staining showed mild fatty infiltration on some myocytes at 24 hours. There was minimal change in fiber size, likely due to variation in age and gender between donors. Muscle architecture was preserved at the end of 24 hours perfusion. Conclusions: All limbs remained viable after 24 hours of near-normothermic ex situ perfusion as evidenced by ongoing neuromuscular stimulation. While no assumptions can be drawn about the long-term function of the extremity, this approach could help extend VCA transplantation to a wider geographic area. It also has the potential to circumvent complications associated with cold preservation.

Near Normothermic Ex Situ Perfusion of Human Limbs for 24 Hours: N/A - Not a clinical study

There is no financial information to disclose. Currently vascularized composite allografts (VCA) are cold preserved (4°C) until transplantation.1Hartzell T.L. Benhaim P. Imbriglia J.E. Shores J.T. Goitz R.J. Balk M. et al.Surgical and technical aspects of hand transplantation: is it just another replant?.Hand Clin. 2011; 27: 521-530Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar This process is time limited, as the tissue has to be revascularized within 4-6 hours to minimize ischemia reperfusion (IR) injury. Normothermic perfusion was proposed as an alternative method of preservation in solid organ transplantation.2Rojas-Pena A. Reoma J.L. Krause E. Boothman E.L. Padiyar N.P. Cook K.E. et al.Extracorporeal support: improves donor renal graft function after cardiac death.Am J Transpl. 2010; 10: 1365e-1374eCrossref PubMed Scopus (33) Google Scholar This method helps to avoid complications associated with cold preservation and maintains tissue viability without inducing IR injury.3Murata S. Miniati D.N. Kown M.H. Koransky M.L. Lijkwan M.A. Balsam L.B. et al.Superoxide dismutase mimetic m40401 reduces ischemia-reperfusion injury and graft coronary artery disease in rodent cardiac allografts.Transplantation. 2004; 78: 1166-1171Crossref PubMed Scopus (27) Google Scholar Using this method, previous investigators demonstrated its potential to prolong swine forelimb allograft survival up to 24 hours.4Ozer K. Rojas-Pena A. Mendias C.L. Bryner B. Toomasian C. Bartlett R.H. Ex-situ limb perfusion system to extend vascularized composite tissue allograft survival.Transplantation. 2015; 99: 2095-2101Crossref PubMed Scopus (37) Google Scholar, 5Ozer K. Rojas-Pena A. Mendias C.L. Bryner B. Toomasian C. Bartlett R.H. The effect of ex situ perfusion in a swine limb vascularized composite tissue allograft on survival up to 24 hours.J Hand Surg Am. 2016; 41: 3-12Abstract Full Text Full Text PDF Scopus (45) Google Scholar In this study, we aimed to test this system on human limb allografts. Four human forearms were procured from brain-dead adult donors under tourniquet control. Following elbow disarticulation, the brachial artery was cannulated. The limb was flushed with heparinized saline and connected to a temperature controlled (30-33°C) ex situ perfusion system (Figure) for 24 hours. The perfusate consisted of plasma and red blood cells with a target hemoglobin (Hb) concentration of 4-6 g/dL. Muscle biopsies (flexor carpi radialis) were obtained at 0, 12, and 24 hours. Average warm ischemia time was 76 minutes. Average arterial systolic pressure was 98±2 mmHg, perfusion flow 319±18 mL/min (∼6-8% of the donor’s estimated cardiac output), and vascular resistance 158±18 mmHg/mL/min. Perfusate had an average pH of 7.43±0.04, pCO2 32±1 mmHg, pO2 317±18 mmHg, and Hb 4.8±0.4 g/dL. Electrolytes (sodium, potassium, chloride) remained within a physiologic range. Lactate started to increase steadily throughout the experiment; however, neuromuscular electrical stimulation revealed ongoing contraction throughout the experiment and H& E staining showed mild to moderate fatty infiltration on some myocytes at 24 hours. •All limbs appeared viable after 24 hours of near-normothermic ex situ perfusion as evidenced by ongoing neuromuscular stimulation.•While no conclusions can be drawn about the long-term function of the extremity, this approach could help extend VCA transplantation to a wider geographic area.•It also has the potential to circumvent complications associated with cold preservation.

A multidrug cocktail approach attenuates ischemic-type biliary lesions in liver transplantation from non-heart-beating donors

Ischemic-type biliary lesions (ITBL) are the most troublesome biliary complication after liver transplantation (LT) from non-heart-beating donors (NHBD) and frequently result in death or re-transplantation. In transplantation process, warm ischemia (WI) in the donor, cold ischemia and reperfusion injury in the recipient altogether inducing ischemia–reperfusion injury (IRI) is strongly associated with ITBL. This is a cascading injury process, involving in a complex series of inter-connecting events causing variety of cells activation and damage associated with the massive release of inflammatory cytokines and generation of reactive oxygen species (ROS). These damaged cells such as sinusoidal endothelial cells (SECs), Kupffer cells (KCs), hepatocytes and biliary epithelial cells (BECs), coupled with immunological injury and bile salt toxicity altogether contribute to ITBL in NHBD LT. Developed therapeutic strategies to attenuate IRI are essential to improve outcome after LT. Among them, single pharmaceutical interventions blocking a specific pathway of IRI in rodent models play an absolutely dominant role, and show a beneficial effect in some given controlled experiments. But this will likely prove ineffective in complex clinical setting in which more risk parameters are involved. Therefore, we intend to design a multidrug cocktail approach to block different pathways on more than one stage (WI, cold ischemia and reperfusion) of the process of IRI-induced ITBL simultaneously. This multidrug cocktail will include six drugs containing streptokinase, epoprostenol, thiazolidinediones (TZDs), N-Acetylcysteine (NAC), hemin and tauroursodeoxycholic acid (TUDC). These drugs show protective effects by targeting the different key events of IRI, such as anti-inflammatory, anti-fibrosis, anti-oxidation, anti-apoptosis and reduced bile salt toxicity. Ideally, the compounds, dosage, and method of application of drugs included in cocktail should not be definitive. We can consider removing or adding some drugs to the proposed cocktail based on further research. But given the multitude of different combinations, it is extremely difficult to determent which combination is the optimization design. Nevertheless, regardless of the difficulty, our multidrug cocktail approach designed to block different mechanisms on more than one stage of IRI simultaneously may represent a future preventive and therapeutic avenue for ITBL.

A novel mechanism of NALP3 inducing ischemia reperfusion injury by activating MAPK pathway in acute renal failure

Acute renal failure (ARF) is a rapid loss of kidney function. The reasons and mechanism by which this occurs has not been clarified so far thus creating obstacles to management of this disease. Presently, the experimental research using the accepted renal ischemia reperfusion injury (I/R injury) model represented for ARF focuses on several possible relevant factors such as reactive oxygen species, no-reflow phenomenon, apoptosis and extensive inflammatory response. The latter is much talked about currently. Some intracellular danger sensing proteins, such as the nucleotide binding domain leucine rich repeats-containing family proteins known as NLRs, adjust the inflammatory response through the formation of a multi-protein complex known as an inflammasome. The most classic family member of this complex is NALP3 confirmed to serve as a contributor to I/R injury. However, how it contributes to the pathology remains obscure. The extensive inflammatory response is considered to be modulated by the mitogen-activated protein kinases (MAPK) signaling pathway. NOD2, another family member of NLR, which shares similar structure with NALP3, indicated that it induced the activation of MAPK in response to a pathogen, thus we assumed that NALP3 performed the harmful process of I/R injury, resulting probably from the activation of the MAPK signaling pathway. If this hypothesis proves to be correct, it might benefit the management of ARF.

Interleukin-17 expression in murine pressure ulcer tissues

To explore the process of pressure ulcer formation, interleukin (IL)-17 expression levels were observed in a mouse model of pressure ulcers. Twenty mice were divided into experimental and control groups (10 mice per group). A mouse model of pressure ulcers was established by inducing ischemia-reperfusion injury on local tissue in the experimental group. Pressure ulcer tissues in the experimental group and normal mouse tissue in the control group were stained using hematoxylin and eosin (H&E) and observed using light microscopy. The protein and mRNA expression levels of IL-17, in mouse pressure ulcer tissues from the experimental group and in the normal tissue from the control group, were determined using real-time PCR and western blot analysis, respectively. The mRNA and protein expression levels of IL-17 were compared between the two groups. H&E staining indicated that striated muscle was arranged orderly and cellular structure was intact in the control group, whilst inflammatory cell infiltration was observed in the muscle tissue of the experimental group. The expression levels of IL-17 mRNA were 0.307±0.058 ng in the experimental group and 0.112±0.042 ng in the control group (P<0.05). The expression levels of the IL-17 protein were 0.434±0.097 ng in the experimental group and 0.181±0.040 ng in the control group (P<0.05). IL-17 expression levels were increased in pressure ulcers, which suggests that IL-17 may be associated with pressure ulcers.

Refined Qingkailing Protects MCAO Mice from Endoplasmic Reticulum Stress-Induced Apoptosis with a Broad Time Window

In the current study, we are investigating effect of refined QKL on ischemia-reperfusion-induced brain injury in mice. Methods. Mice were employed to induce ischemia-reperfusion injury of brain by middle cerebral artery occlusion (MCAO). RQKL solution was administered with different doses (0, 1.5, 3, and 6 mL/kg body weight) at the same time of onset of ischemia, and with the dose of 1.5 mL/kg at different time points (0, 1.5, 3, 6, and 9 h after MCAO). Neurological function and brain infarction were examined and cell apoptosis and ROS at prefrontal cortex were evaluated 24 h after MCAO, and western blot and intracellular calcium were also researched, respectively. Results. RQKL of all doses can improve neurological function and decrease brain infarction, and it performed significant effect in 0, 1.5, 3, and 6 h groups. Moreover, RQKL was able to reduce apoptotic process by reduction of caspase-3 expression, or restraint of eIF2a phosphorylation and caspase-12 activation. It was also able to reduce ROS and modulate intracellular calcium in the brain. Conclusion. RQKL can prevent ischemic-induced brain injury with a time window of 6 h, and its mechanism might be related to suppress ER stress-mediated apoptotic signaling.

Myocardial infarction induces early increased remote ADAM8 expression of rat hearts after cardiac arrest

Background. A disintegrin and metalloproteinase-8 (ADAM8) is a potential surrogate of inflammation which has recently been associated with myocardial infarction. We evaluated in a rat cardiac transplantation model whether ischemia-reperfusion injury alone (IRI) or with early regional myocardial infarction (MI) would suffice to induce inflammatory myocardial remodeling and ADAM8 expression. Material and methods. Isogenic heterotopic cardiac transplantation after cardiac arrest was performed to 48 Fischer 344 rats to induce ischemia-reperfusion injury (IRI), of which 27 rats also underwent ligation of the left anterior coronary artery (LAD) of the heart to yield MI. Histology was performed at 0.5, 24 and 48 h after transplantation. ADAM8 was evaluated by qRT-PCR after graft harvesting. Results. After 0.5 and 48 h respectively, edematous intramyocardial artery nuclei and periadventitial inflammation were more prominent in MI after transplantation, as compared with IRI alone and Controls (57.0 vs 40.0 and 5.0; 1.9 vs 1.1 and 0.9, point score units, p < 0.05, respectively). The expression of ADAM-8 was increased in MI as compared with Controls (1.9 vs 1.0, 1.9 fold increase) at 48 h. In grafts with MI, ADAM8 was localized using immunohistochemistry to the vicinity of the area corresponding to the developing infarction as well as in intramyocardial arteries remote to the infarction area. Conclusions. Remote histopathological changes of ischemic cardiac grafts are associated with increased expression of ADAM8 thus emphasizing a global myocardial impact of MI.

Effects of Ginkgo biloba extract 50 preconditioning on contents of inflammation-related cytokines in myocardium of rats with ischemia-reperfusion injury

To investigate the effects of Ginkgo biloba extract 50 (GBE50) preconditioning on contents of inflammation-related cytokines in rats with myocardial ischemia-reperfusion. Fifty-eight SD rats were divided into sham-operated group, untreated group, Salviae miltiorrhizae (SM) injection group and low-, medium- and high-dose GBE50 groups. After intragastric administration for 7 d, the left anterior descending (LAD) coronary artery was occluded for 30 min followed by 60-min reperfusion to induce ischemia-reperfusion injury. Myocardium histopathologic change was observed by HE staining under a light microscope; myeloperoxidase (MPO) activity in myocardium was measured by colorimetric detection; tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-8 were detected by radioimmunoassay; IL-4 and IL-10 were tested by enzyme-linked immunosorbent assay (ELISA). Compared with untreated group, rats in medium-dose GBE50 group had lower inflammatory reaction and MPO activity (P<0.01). GBE50 also decreased the content of IL-6 (P<0.05, P<0.01) and increased the content of IL-4 in myocardium (P<0.05, P<0.01) as compared with the untreated group. The content of TNF-alpha in myocardium in the medium-dose GBE50 group was lower and IL-10 was higher than those in the untreated group, but without significant differences. GBE50 can decrease the content of IL-6 and increase the content of IL-4 in myocardium after ischemia-reperfusion injury. It suggests that GBE50 can regulate the inflammatory reaction after ischemia-reperfusion injury via inhibiting inflammatory cytokines and promoting anti-inflammatory cytokines.

Neutrophil elastase inhibitor, sivelestat sodium hydrate prevents ischemia–reperfusion injury in the rat bladder

In the present study, we evaluated the effect of neutrophil elastase inhibitor, sivelestat sodium hydrate on ischemia-reperfusion injury in the rat bladder. Rat abdominal aorta was clamping with a small clip to induce ischemia-reperfusion injury in the bladder. Eight-week-old male Sprague Dawley rats were divided into four groups; sham-operated control rats, 30 min ischemia-60 min reperfusion (IR) rats, and IR rats treated with 15 or 60 mg/kg of sivelestat sodium hydrate. Sixty minutes prior to induction of ischemia, sivelestat sodium hydrate was administrated intraperitoneally. Real-time monitoring of blood flow and nitric oxide (NO) release were measured simultaneously with a laser Doppler flowmeter and an NO-selective electrode, respectively. The NO2-NO3 and malonaldehyde (MDA) concentrations were measured in the experimental urinary bladders. Clamping of the abdominal aorta, blood flow was rapidly decreased and NO release was gradually increased. After removing the clip, blood flow was rapidly increased and NO release was gradually returned to the basal level. These movements of blood flow and NO release were inhibited by treatment with sivelestat sodium hydrate in a dose-dependent manner. Both NO2-NO3 and MDA concentrations in the bladder were increased by induction of IR, and NO2-NO3 and MDA concentrations were decreased by treatment with high dose of sivelestat sodium hydrate significantly. Our data indicated that sivelestat sodium hydrate could inhibit increasing NO2-NO3 and MDA concentrations by IR, and it has potentiality protective effects on IR injury in the rat urinary bladder.

Abstract 782: Fra2 Mediates Oxygen-induced TGFbeta-1 mRNA Expression In Adult Cardiac Fibroblasts: Significance In Myocardial Fibrosis Following Ischemia-reperfusion Injury

Background . Transforming growth factor beta-1 (TGFbeta-1) is a key cytokine implicated in the development of cardiac fibrosis following ischemia-reperfusion (IR) injury. The profibrotic effects of TGFbeta-1 are primarily attributable to the differentiation of cardiac fibroblasts (CF) to myofibroblasts. Previously, we have reported perceived hyperoxia (Circ Res 92:264 –71), sub-lethal reoxygenation shock during IR, induces differentiation of CF to myofibroblasts at the infarct site. The mechanisms underlying oxygen-sensitive induction of TGFbeta-1 mRNA remain to be characterized. Hypothesis . Fra2 mediates oxygen-induced TGFbeta-1 mRNA expression in adult cardiac fibroblasts. Methods. TGFbeta-1 mRNA expression in infarct tissue was investigated in an IR injury model. The left anterior descending coronary artery of mice was transiently occluded for 60 minutes followed by reperfusion to induce IR injury. Spatially resolved infarct and non-infarct tissues were collected at 0, 1, 3, 5, and 7 days post-IR using laser capture microdissection. TGFbeta-1 mRNA levels were measured using real-time PCR. To investigate the role of oxygen in the regulation of TGFbeta-1, we used our previously reported model of perceived hyperoxia where CF (from 5wks old mice) after isolation were cultured at 5%O 2 (physiological pO 2 ) followed by transferring them to 20%O 2 to induce hyperoxic insult. Results &amp; Conclusions. In vivo, a significant increase (p&lt;0.01; n=5) in TGFbeta-1 mRNA was observed at the infarct site already at day 1 post-IR. The levels continued to increase until day 7 post-IR. In vitro, exposure of CF to 20%O 2 hyperoxic insult induced TGFbeta-1 mRNA (p&lt;0.001; n=4) and protein (p&lt;0.01; n=4) expression. Using a TGFbeta-1 promoter-luciferase reporter and DNA binding assays, we collected first evidence that AP-1 and its component Fra2 as major mediators of oxygen-induced TGFbeta-1 expression. Exposure to 20%O 2 resulted in increased localization of Fra2 in nucleus. siRNA-dependent Fra-2 knock-down completely abrogated oxygen-induced TGFbeta1 expression. In conclusion, this study presents first evidence that Fra-2 is involved in inducible TGFbeta1 expression in CF. Fra2 was noted as being central in regulating oxygen-induced TGFbeta-1 expression.s

Beneficial effect of preconditioning on ischemia–reperfusion injury in the rat bladder in vivo

We investigated the effect of preconditioning on ischemia–reperfusion injury in the rat bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia–reperfusion injury in the bladder. Twelve-week-old male SD rats were divided into three groups; sham-operated control (Cont), 30 min ischemia–60 min reperfusion (IR) and three times of 5 min ischemia and then 30 min ischemia–60 min reperfusion (PC) groups. The bladder functions were estimated by cystometric and functional studies. Contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype selective muscarinic antagonists, i.e. atropine (non-selective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-DAMP (M1/M3 selective). We also measured tissue levels of malonaldehyde (MDA) and examined possible histological changes in these rats' bladders. Preconditioning partially prevented the reduction of bladder dysfunction induced by ischemia–reperfusion. Estimation of the pA2 values for atropine, pirenzepine, methoctramine, and 4-DAMP indicates that the carbachol-induced contractile response in bladder dome is mediated through the M3 receptor subtype in all groups. The MDA concentration in the IR group was significantly larger than that of the control group, and preconditioning significantly reduced MDA production in the bladder. In histological studies, the ischemia–reperfusion with or without preconditioning caused infiltration of leukocytes and rupture of microcirculation in the regions of submucosa and smooth muscle without a corresponding sloughing of mucosal cells. Our data indicate that preconditioning has a beneficial effect on ischemia–reperfusion injury in the rat bladder.

Effects of Ischemic Liver Preconditioning on Hepatic Ischemia/Reperfusion Injury in the Rat

To minimize bleeding during major liver resections or liver transplantation, surgical measures have been adopted that induce ischemia-reperfusion injury (I/R) which may significantly contribute to morbidity and mortality of partial liver resections. Several methods have sought to minimize I/R hepatic lesions. The present project assessed the protective role of ischemic preconditioning (IPC) in rat livers. The IPC was accomplished by clamping the hepatic pedicle for 5 minutes, followed by a 5-minute reperfusion (R) period before a 2-hour ischemia. Thereafter, reperfusions of 1, 3, and 24 hours were compared among IPC and control groups without IPC. Liver biopsy and blood samples were measured for mitochondrial respiratory control ratio (RCR), serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT). IPC protected liver mitochondrial function. Serum aminotransferase levels were significantly lower among animals undergoing IPC compared with groups without IPC. Thus, we verified the effects of IPC for hepatocellular protection against I/R lesions.

Expression of HSP 70 and its mRNAS during ischemia-reperfusion in the rat bladder

HSP 70 is an important protein that repairs damaged tissue after injury. In the present study, we investigated the expression of HSP 70 and its mRNAs during ischemia-reperfusion in the rat bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia-reperfusion injury in the bladder dome. Male Wistar rats, 8 weeks old, were divided into six groups: controls, 30-min ischemia, 30-min ischemia and 30-, 60-minute, 1- and 7-day reperfusion, groups A, B, C, D, E, and F, respectively. In functional studies, contractile responses to carbachol were measured in these groups. The expression of HSP 70-1/2 mRNAs was quantified using a real-time PCR method, and that of HSP 70 proteins was measured using ELISA in the bladders. In the functional study, Emax values of carbachol to bladders in the A, B, C, D, E and F groups were 9.3 ± 1.3, 7.9 ± 1.7, 4.3 ± 0.8, 4.2 ± 0.7, 4.5 ± 0.6, and 8.1 ± 1.2 g/mm 2, respectively. In the control group, the expression of HSP 70-1/2 mRNA was detected, and the expression of HSP 70-1 mRNAs was significantly higher than that of HSP 70-2 mRNAs in each group. The expression of HSP 70-1 mRNA increased in groups B and C, but decreased in groups D, E, and F. The expression of HSP 70-2 mRNA in group C was significantly higher than that of groups A, D, E, and F. The expression of HSP 70-1/2 mRNAs after 1 day or 1 week of reperfusion was similar to control levels. The expression of HSP 70 proteins was increased shortly after the expression of their mRNAs. The expression of HSP 70 after 1 day or 1 week of reperfusion was almost identical to control levels. Our data indicate that contractile responses of the bladder were decreased by ischemia reperfusion, and that expression of HSP 70 and its mRNAs appeared to increase after a short period of the insult.

DIRECT DETECTION OF NITRIC OXIDE IN RAT URINARY BLADDER DURING ISCHEMIA-REPERFUSION

We attempted to investigate the role of nitric oxide (NO) in ischemia-reperfusion injury in the rat bladder. Rat abdominal aorta were clamped with a small clip to induce ischemia-reperfusion injury in the rat bladder dome. To investigate the role of NO in ischemia-reperfusion injury, N(G)-nitro-L-arginine methylester (L-NAME, 30 mg./kg.) was injected intraperitoneally to measure carbachol-induced contractions of 60 minutes ischemia-30 minutes reperfusion in the rat bladder dome. In vivo real-time blood flow and NO release were measured in the rat bladder with a laser Doppler flowmeter and an NO-selective electrode, respectively. Moreover, participation of NO synthase subtypes was investigated by immunohistochemical staining of bladder sections with anti-endothelial I and anti-inducible NO synthase subtype antibodies. Treatment with L-NAME (30 mg./kg., i.p.) partially prevented the reduction of bladder strips in contraction induced by the ischemia-reperfusion. Clamping of the aorta decreased blood flow to 10% of the basal level measured before the clamping. Administration of L-NAME reduced the blood flow to the bladder by 65% compared to the control level during ischemia-reperfusion. Real-time monitoring of NO in the bladder revealed that ischemia increased NO release, which, in turn, reached a plateau 30-40 minutes after the induction of ischemia. Immediately after the reperfusion, NO release returned to the basal level. In the control rat bladder, the endothelial subtype (eNOS) was observed in the mucosa. After the ischemia-reperfusion, iNOS was detected in the infiltrated neutrophils in the muscular and submucosal regions. Our data indicate that NO from leukocytes may participate in cell/tissue injury during ischemia-reperfusion of the rat bladder and that the damage may be preventable by treatment with L-NAME.

Effect of ischemia-reperfusion on contractile function of rat urinary bladder: Possible role of nitric oxide

Because there are increasing evidences that nitric oxide (NO) plays important roles in ischemia-reperfusion injury in several systems, we investigated the role of NO in ischemiareperfusion injury of the rat urinary bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia-reperfusion injury in the rat bladder dome. In functional studies, contractile responses to carbachol were cumulatively measured after the urinary bladder was treated with various duration (0, 30, 60, and 90 min) of ischemia. The injury of rat bladder functioning was dependent on ischemic periods. Significant decreases in the E max (maximum contractile response) values were observed in the bladder subjected to 60 or 90 min ischemia. Furthermore, the subsequent 30 min reperfusion caused additional damages of the contractile response in bladder muscles. To investigate the role of NO in the ischemia (30 min)-reperfusion (30 min) injury, N G-nitro-L-arginine methylester (L-NAME) was injected intraperitoneally 30 min before the ischemia. Treatment of L-NAME (30 and 100 mg kg ) partly but significantly prevented the reduction contractile responses to carbachol of the rat bladder dome. In histological studies, the ischemia-reperfusion caused infiltration of leukocytes and rupture of microcirculation in the regions of submucosa and smooth muscle without a corresponding sloughing of mucosal cells. The histological damages were also prevented by treatment with L-NAME. Therefore, these data suggested that ischemia-reperfusion of the urinary bladder may result in dysfunction of the contractile response to autonomic nervous system and that nitric oxide may act as a cell/tissue damaging agent in ischemia-reperfusion injury.

Free Radicals Induce Ischemia-Reperfusion Injury and Histamine Release in the Isolated Guinea Pig Heart

Free radicals are produced by perfusion of isolated guinea pig heart with FeCl3/ADP (10 microM/100 microM) and/or occlusion and opening of the left anterior descending coronary artery. Cardiac histopathology was correlated with histamine and lactate dehydrogenase release and with malondialdehyde production. A differential release of histamine and lactate dehydrogenase in the perfusate was detected, showing a preferential liberation of histamine in the reperfusion phase. The increase in lipid peroxidation product in left ventricular tissues after left coronary artery occlusion was maximal at the end of ischemia.