Effect of bilateral in vivo ischemia/reperfusion on the activities of superoxide dismutase and catalase: Response to a standardized grape suspension

Purpose: Ischemia/reperfusion (I/R) is a major etiological factor in the bladder dysfunctions observed in men with lower tract obstruction, women with postmenopausal incontinence and with aging. A standardized grape suspension protects the rabbit urinary bladder from both the contractile dysfunctions and the morphologic changes mediated by I/R. Using a model of in vivo bilateral ischemia/reperfusion, the current study investigated the effect of this grape suspension on the endogenous antioxidant defense systems. Materials and methods: 24 NZW rabbits were separated into 6 groups of 4. Groups 1–3 were treated by gavage with aqueous grape suspensions; groups 4–6 received sugar-water vehicle. Groups 3 and 6 were controls. Groups 1 and 4 were subjected to bilateral ischemia for 2 h (I). Groups 2 and 5 underwent bilateral ischemia for 2 h and reperfusion for 1 week (I/R). For all rabbit bladders, the muscle and mucosa were separated by blunt dissection and analyzed separately. The effects of the various treatments on bladder antioxidant systems of cytoplasmic superoxide dismutase (Cu-Zn superoxide dismutase; SOD), and catalase (CAT) were evaluated. Results: The standardized grape suspension up-regulated both SOD and CAT activity of bladder muscle and mucosa in control animals. There were few differences in the grape suspension treated animals after ischemia, and in general the activities decreased following I/R. Conclusions: Increases of SOD and CAT activity in control animals as a result of grape suspension suggest a greater antioxidant capacity. This increase in the antioxidant defense system may explain the increased protection of grape suspension in the face of ischemia and I/R. However, the activities of both enzyme systems decreased in the smooth muscle subjected to I/R showing that reperfusion damages these systems probably via oxidation damage to the enzymes themselves.     

The effect of vitamin E on the response of rabbit bladder smooth muscle to hydrogen peroxide

There is increasing evidence that ischemia, reperfusion, and the generation of free radicals are major etiological factors in the progression of bladder dysfunction after partial outlet obstruction. In vitro studies demonstrated that the magnitude of contractile dysfunction following exposure of bladder smooth muscle to hypoxia followed by re-oxygenation was related to the level of lipid peroxidation indicating that membrane lipid peroxidation participated in the contractile failure induced. Recent studies demonstrated that incubation of isolated strips of bladder smooth muscle with hydrogen peroxide (H₂O₂) result in progressive contractile dysfunctions and is associated with progressive increases in MDA (peroxidation product). The current study investigates if feeding rabbits a diet high in vitamin E protects the bladder from the effects of in vitro H₂O₂. Sixty-four male New Zealand White rabbits were separated into two groups: The rabbits in group 1 were fed a normal diet (28 rabbits) whereas the rabbits in group 2 were placed on a diet enriched with -tocopherol (36 rabbits). After 3 weeks on the normal or high E diet, each rabbit was anesthetized and the bladder excised and cut into 6 isolated strips of bladder detrusor. Each strip was mounted in individual 15 ml baths containing oxygenated Tyrode's solution. The contractile responses to field stimulation (FS), carbachol, and KCl were determined. The strips were washed and exposed to one of the following concentrations of hydrogen peroxide (H₂O₂): 0% (control), 0.0625, 0.125, 0.25, 0.5, 1.0 and 3.0% for a period of 1 h. At the end of the hour each strip was washed free of H₂O₂ and a second set of contractile responses were performed and compared to the first set. At the end of the experiment, each strip was frozen and stored at –70°C for analysis of malondialdehyde (MDA) as a measure of peroxidation. In both groups, H₂O₂ produced similar dose dependent decreases in the contractile responses to all forms of stimulation. In the normal-diet group H₂O₂ produced a dose dependent increase in MDA formation, whereas in the high E group there were no increases in MDA at any concentration of H₂O₂. Feeding rabbits a diet high in vitamin E protected the bladder smooth muscle from peroxidation, but had no significant effect on the contractile dysfunctions mediated by direct incubation with H₂O₂.     

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Purpose: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Materials and methods: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. Results: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. Conclusions: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction. (Mol Cell Biochem 276: 143–148, 2005)     

Effects of L-arginine and L-NAME on chronic partial bladder outlet obstruction in rabbit

Nitric oxide (NO) is synthesized from L-arginine by nitric oxide synthase (NOS). NOS can be inhibited by NG-nitro-L-arginine methyl ester (L-NAME) and stimulated by supplementing the diet with L-arginine. The aim of this study was to investigate the influence of NOS activity on the response of rabbits to chronic partial bladder outlet obstruction (PBOO). Surgical PBOOs (2 and 8 wk) were performed on male New Zealand White rabbits. Before obstruction, one-third of the animals were premedicated for 7 days with L-NAME and another third with L-arginine. The results are summarized as follows. First, bladder weight after 8-wk PBOO was significantly lower in animals treated with L-arginine compared with both untreated and rabbits treated with L-NAME. Second, contractile function decreased progressively with PBOO duration. However, after 8 wk of PBOO, the L-arginine group had significantly greater contractile function compared with the no-treatment group, and the L-NAME group had significantly lower contractile function compared with the no-treatment group. Third, at 8 wk following PBOO, the level of protein oxidation and nitration was lowest for the L-arginine group and highest in the L-NAME group. These studies clearly demonstrated that increasing blood flow by stimulating NOS significantly protected the bladder from PBOO dysfunctions, whereas inhibiting blood flow by L-NAME enhanced the dysfunctions mediated by PBOO.     

Butylated hydroxyanisole inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis and GSH depletion

To evaluate the effects of in vitro ischemia/reperfusion on contractile response to field stimulation (FS), free fatty acid (FFA) content, phospholipid (PL) content, and malondialdehyde (MDA) levels of the rabbit urinary bladder. There is significant evidence that ischemia/reperfusion injury is linked to obstructive bladder dysfunction secondary to men with benign prostatic hyperplasia (BPH). Twelve New Zealand White male rabbits were separated into two groups of six rabbits each. Each rabbit was euthanized, and the bladder was surgically removed intact for whole bladder incubation. The bladders in Group 1 received a 3-h incubation under normal oxygenated physiological conditions. These bladders received electrical field stimulation (32?Hz) after 1 and 3?h. The bladders associated with Group 2 received a 1-h incubation under normal oxygenated physiological conditions. At the end of this 1-h period, the bladders were subjected to FS. After a maximal pressure response was recorded, the stimulation was turned off and the bath medium was changed to one equilibrated with 95% nitrogen, 5% oxygen without glucose (ischemic medium) and incubated for 1?h with field stimulations (32?Hz) occurring at 5-min intervals to represent overactive bladder dysfunction. At the end of this hour of ischemia with repetitive stimulation, the bath was changed to an oxygenated medium with glucose for a 1-h period after which the stimulation was repeated. At the end of the experimental period, each bladder was opened longitudinally and the muscle and mucosa separated by blunt dissection, frozen under liquid nitrogen, and stored at -80°C for biochemical analyses. Each tissue was fractionated by differential centrifugation into nuclear, mitochondrial, synaptosomal, and supernatant (cytosol) components. PL, FFA, and MDA content were analyzed for each fraction using standard biochemical techniques. The bladder contractile responses decreased during the period of in vitro ischemia and returned to only 30% of control after reperfusion. In vitro ischemia/reperfusion showed the following: (1) There was a modest but significant decrease in the FFA content of the microsomes of the muscle and significant increases in the FFA content of the nuclei and mitochondria of the mucosa. (2) There were decreases in the PL content of the homogenate and microsomes of the muscle and decreases in the PL content of the homogenate, microsomes, and supernatant of the mucosa. (3) Significant increases were observed in the MDA levels of the homogenate, mitochondria, and microsomes of both the muscle and mucosa. The significant increases in the lipid peroxidation of the bladder smooth muscle are consistent with the marked decrease in the contractile ability of the bladder following ischemia/reperfusion. The specific increased lipid peroxidation of the mitochondrial and microsomal components is consistent with the specific dysfunctions of the mitochondria and innervations observed following I/R in earlier published studies. The marked increases in lipid peroxidation in the mucosa associated with the loss of PL and FFA from this component are consistent with the significant dysfunction in both the antiadherence and antipermeability properties of the mucosa and may play a major role in the symptomatic nature of I/R-linked diseases of the bladder.     

Coenzyme Q10 protect against ischemia/reperfusion induced biochemical and functional changes in rabbit urinary bladder

Purpose Ischemia, reperfusion, and free radical generation have been recently implicated in the progressive bladder dysfunction. Coenzyme Q10 (CoQ10) is a pro-vitamin like substance that appears to be efficient for treatment of neurodegenerative disorders and ischemic heart disease. Our goal was to investigate the potential protective effect of CoQ10 in a rabbit model of in vivo bilateral ischemia and ischemia/reperfusion (I/R). Material and Methods Six groups of four male New Zealand White rabbits each were treated with CoQ10 (3 mg/kg body weight/day—dissolved in peanut oil) (groups 1–3) or vehicle (peanut oil) (groups 4–6). Groups 1 and 4 (ischemia-alone groups) had clamped bilateral vesical arteries for 2 h; in groups 2 and 5 (I/R groups), bilateral ischemia was similarly induced and the rabbits were allowed to recover for 2 weeks. Groups 3 and 6 were controls (shams) and were exposed to sham surgery. The effects on contractile responses to various stimulations and biochemical studies such as citrate synthase (CS), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and catalase (CAT) were evaluated. The protein peroxidation indicator, carbonyl group, and nitrotyrosine contents were analyzed by Western blotting. Results Ischemia resulted in significant reductions in the contractile responses to all forms of stimulation in vehicle-fed rabbits, whereas there were no reductions in CoQ10-treated rabbits. Contractile responses were significantly reduced in vehicle-treated I/R groups, but significantly improved in CoQ10-treated rabbits. Protein carbonylation and nitration increased significantly in ischemia-alone and I/R bladders; CoQ10 treatment significantly attenuated protein carbonylation and nitration. CoQ10 up-regulated SOD and CAT activities in control animals; the few differences in CoQ10-treated animal in SOD and CAT after ischemia and in general increase CAT activities following I/R. Conclusions CoQ10 supplementation provides bladder protection against I/R injury. This protection effect improves mitochondrial function during I/R by repleting mitochondrial CoQ10 stores and potentiating their antioxidant properties.     

Expression of stress proteins (HSP-70 and HSP-90) in the rabbit urinary bladder subjected to partial outlet obstruction

Partial obstruction of the rabbit bladder outlet induces a rapid hypertrophy characterized by increased bladder mass, increased smooth muscle content, and increased collagen deposition. In addition, partial outlet obstruction induces decreased contractile responses to both field stimulation and postsynaptic receptor stimulation. Although the morphological and contractile responses to partial outlet obstruction have been well characterized, there is little information on the cellular and molecular mechanisms of these changes. In a previous study, we demonstrated that one of the earliest genes to be expressed following partial outlet obstruction in rabbits was the gene expressing stress protein-70 (HSP-70). In order to further define the genetic and molecular basis of these responses, the expression of stress gene products HSP-70 and HSP-90 in rabbit urinary bladder subjected to partial outlet obstruction has been quantitatively evaluated by Western blot coupled with laser densitometry using anti-HSP-70 and-90 monoclonal antibodies. The data show that stress gene products HSP-70 and HSP-90 are constitutively expressed in control rabbit bladder tissue and transiently increased following partial outlet obstruction. Increased content of HSP-70 was detected at 6 hr after obstruction and reached a maximum (2.7-fold over the control level) at 24 hr. Increased HSP-90 was also detected at 6 hr but reached a maximum (4.5-fold over the control level) at 12 hr. By 7 day post-obstruction, the content of these two proteins returned to the control levels. This study suggests that alterations of stress gene expression resulting in increased HSP-70 and 90 may play an important role in the response of the bladder to partial outlet obstruction.     

Ischemic preconditioning in rat heart: No correlation between glycogen content and return of function

We tested the hypothesis that glycogen levels at the beginning of ischemia affect lactate production during ischemia and postischemic contractile function.Isolated working rat hearts were perfused at physiological workload with bicarbonate buffer containing glucose (10 mmol/L). Hearts were subjected to four different preconditioning protocols, and cardiac function was assessed on reperfusion. Ischemic preconditioning was induced by either one cycle of 5 min ischemia followed by 5, 10, or 20 min of reperfusion (PC5/5, PC5/10, PC5/20), or three cycles of 5 min ischemia followed by 5 min of reperfusion (PC3 × 5/5). All hearts were subjected to 15 min total, global ischemia, followed by 30 min of reperfusion. We measured lactate release, timed the return of aortic flow, compared postischemic to preischemic power, and determined tissue metabolites at selected time points.Compared with preischemic function, cardiac power during reperfusion improved in groups PC5/10 and PC5/20, but was not different from control in groups PC5/5 and PC3 × 5/5. There was no correlation between preischemic glycogen levels and recovery of function during reperfusion. There was also no correlation between glycogen breakdown (or resynthesis) and recovery of function. Lactate accumulation during ischemia was lowest in group PC5/20 and highest in the group with three cycles of preconditioning (PC3 × 5/5). Lactate release during reperfusion was significantly higher in the groups with low recovery of power than in the groups with high recovery of power.In glucose-perfused rat heart recovery of function is independent from both pre- and postischemic myocardial glycogen content over a wide range of glycogen levels. The ability to utilize lactate during reperfusion is an indicator for postischemic return of contractile function.     

The relationship between erythropoietin pretreatment with blood-brain barrier and lipid peroxidation after ischemia/reperfusion in rats

Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including ischemia and some neurodegenerative disorders. In recent years, erythropoietin (EPO) has been shown to exert neuroprotection in many pathological conditions including ischemia in the brain. This study aimed to investigate the effects of EPO on BBB integrity, infarct size and lipid peroxidation following global brain ischemia/reperfusion in rats. Wistar male rats were divided into four groups (each group n=8); Group I; control group (sham-operated), Group II; ischemia/reperfusion group, Group III; EPO treated group (24 h before decapitation--000 U/kg r-Hu EPO i.p.), Group IV; EPO+ ischemia/reperfusion group (24 h before ischemia/reperfusion--3000 U/kg r-Hu EPO i.p.). Global brain ischemia was produced by the combination of bilateral common carotid arteries occlusion and hemorrhagic hypotension. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage was observed for BBB integrity. Infarct size was calculated based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Lipid peroxidation in the brain tissue was determined as the concentration of thiobarbituric acid-reactive substances (TBARS) for each group. Ischemic insult caused bilateral and regional BBB breakdown (hippocampus, cortex, corpus striatum, midbrain, brain stem and thalamus). EPO pretreatment reduced BBB disruption, infarct size and lipid peroxide levels in brain tissue with 20 min ischemia and 20 min reperfusion. These results suggest that EPO plays an important role in protecting against brain ischemia/reperfusion through inhibiting lipid peroxidation and decreasing BBB disruption.     

Myocardial contractile function during postischemic low-flow reperfusion: critical thresholds of NADH and O2 delivery

The degree of myocardial oxygen delivery (Do2) that is necessary to reestablish functional contractile activity after short-term global ischemia in heart is not known. To determine the relationship between Do2 and recovery of contractile and metabolic functions, we used tissue NADH fluorometric changes to characterize adequacy of reperfusion flow. Isolated perfused rat hearts were subjected to global ischemia and were reperfused at variable flow rates that ranged from 1 to 100% of baseline flow. Myocardial function and tissue NADH changes were continuously measured. NADH fluorescence rapidly increased and plateaued during ischemia. A strong inverse logarithmic correlation between NADH fluorescence and reperfusion Do2 was demonstrated (r = -0.952). Left ventricular function (rate-pressure product) was inversely related to NADH fluorescence at reperfusion flows from 25 to 100% of baseline (r = -0.922) but not at lower reperfusion flow levels. An apparent reperfusion threshold of 25% of baseline Do2 was necessary to resume contractile function. At very low reperfusion flows (1% of baseline), another threshold flow was identified at which NADH levels increased beyond that observed during global ischemia (3.4 +/- 3.0%, means +/- SE, n = 9), which suggests further reduction of the cellular redox state. This NADH increase at 1% of baseline reperfusion flow was blocked by removing glucose from the perfusate. NADH fluorescence is a sensitive indicator of myocardial cellular oxygen utilization over a wide range of reperfusion Do2 values. Although oxygen is utilized at very low flow rates, as indicated by changes in NADH, a critical threshold of approximately 25% of baseline Do2 is necessary to restore contractile function after short-term global ischemia.     

Human recombinant erythropoietin protects the striated muscle microcirculation of the dorsal skinfold from postischemic injury in mice

Erythropoietin (EPO) has been proposed as a novel cytoprotectant in ischemia-reperfusion (I/R) injury of the brain, heart, and kidney. However, whether EPO exerts its protection by prevention of postischemic microcirculatory deterioration is unknown. We have investigated the effect of EPO on I/R-induced microcirculatory dysfunctions. We used the mouse dorsal skinfold chamber preparation to study nutritive microcirculation and leukocyte-endothelial cell interaction in striated muscle of the dorsal skinfold by in vivo fluorescence microscopy before 3 h of ischemia and during 5 days of reperfusion. Animals were pretreated with EPO (5,000 U/kg body wt) 1 or 24 h before ischemia. Vehicle-treated I/R-injured animals served as controls. Additional animals underwent sham operation only or were pretreated with EPO but not subjected to I/R. I/R significantly (P < 0.05) reduced functional capillary density, increased microvascular permeability, and enhanced venular leukocyte-endothelial cell interaction during early reperfusion. These findings were associated with pronounced (P < 0.05) arteriolar constriction and diminution of blood flow during late reperfusion. Pretreatment with EPO induced EPO receptor and endothelial nitric oxide synthase expression at 6 h of reperfusion (P < 0.05). In parallel, EPO significantly (P < 0.05) reduced capillary perfusion failure and microvascular hyperpermeability during early reperfusion and arteriolar constriction and flow during late reperfusion. EPO pretreatment substantially (P < 0.05) diminished I/R-induced leukocytic inflammation by reducing the number of rolling and firmly adhering leukocytes in postcapillary venules. EPO applied 1 h before ischemia induced angiogenic budding and sprouting at 1 and 3 days of reperfusion and formation of new capillary networks at 5 days of reperfusion. Thus our study demonstrates for the first time that EPO effectively attenuates I/R injury by preserving nutritive perfusion, reducing leukocytic inflammation, and inducing new vessel formation.     

Rb1、Rg1对肾缺血／再灌注血清诱导HK-2细胞凋亡中Bcl-2、Bax表达的影响

目的：探讨人参皂甙Rb1、Rg1在肾缺血／再灌注血清诱导HK-2细胞凋亡中对Bol-2、Bax表达的影响。方法：制备家兔肾缺血／再灌注血清（SIR）和对照组血清（SC）用于HK-2细胞培养,TUNEL法检测细胞凋亡。实验分组：对照组、缺血／再灌注组、Rb1干预组、Rg1干预组,培养24h后免疫细胞化学法检测Bcl-2、Bax的表达。结果：与缺血／再灌注组比较,Rb1干预组和Rg1干预组Bax的表达明显下降（P〈0．01）,Bcl-2／Bax比值增大。结论：人参皂甙Rb1、Rg1对肾缺血／再灌注血清诱导HK-2细胞凋亡具有保护作用。     

Effect of acute and chronic simvastatin treatment on post-ischemic contractile dysfunction in isolated rat heart

Statins are powerful lipid-lowering drugs, widely used in patients with hyperlipidemia and coronary artery disease. It was found, however, that statins appear to have a pleiotropic effect beyond their lipid-lowering ability. They exert anti-inflammatory, antithrombotic and antioxidant effects, increase nitric oxide production and improve endothelial dysfunction. The aim of our study was to examine the effect of chronic and acute treatment with simvastatin on the contractile function of the isolated perfused rat heart after ischemia/reperfusion injury. Contractile function was measured on isolated rat hearts, perfused according to Langendorff under constant pressure. The hearts were subjected to 20 min of global ischemia, followed by 40 min of reperfusion. To investigate the acute effect, simvastatin at a concentration of 10 micromol/l was added to the perfusion solution during reperfusion. In chronic experiments the rats were fed simvastatin at a concentration of 10 mg/kg for two weeks before the measurement of the contractile function. Acute simvastatin administration significantly increased reparation of the peak of pressure development [(+dP/dt)(max)] (52.9+/-8.2 %) after global ischemia, as compared with the control group (28.8+/-5.2 %). Similar differences were also observed in the time course of the recovery of [(+dP/dt)(max)]. Chronic simvastatin was without any protective effect. Our results reveal that the acute administration of simvastatin during reperfusion, unlike the chronic treatment, significantly reduced contractile dysfunction induced by ischemia/reperfusion injury. This supports the idea of possible cardioprotective effect of statin administration in the first-line therapy of the acute coronary syndrome.     

缺血后处理对兔脊髓缺血再灌注微循环损伤的影响

探讨缺血后处理对兔脊髓缺血再灌注微循环损伤的影响.成年新西兰大白兔24只随机分为假手术组(C组),缺血再灌注损伤组(IR组),缺血后处理组(P组).IR组和P组采用Zivin改进法制备脊髓缺血再灌注模型,P组在缺血30 min后行复灌1 min/缺血1 min相同处理3次.采用激光多普勒检测缺血前,缺血时及再灌注各时点血流量值,在再灌注24 h时取兔脊髓组织作HE染色观察病理形态学,比色法检测脊髓组织一氧化氮(Nitric oxide,NO)的含量,放免法检测内皮素-1(Endothelin-1,ET-1)及免疫组化法检测血红素氧合酶(Hemeoxygenase-1,HO-1)的表达.研究发现与缺血前基础值相比,再灌注10 min时IR组与P组血流量均有增高,在再灌注30、60、120 min,IR组血流量值有不同程度的降低;与IR组相比,P组血流量值在再灌注各时点均有不同程度的增高.与IR组相比,P组NO含量与HO-1表达均有增加,ET-1含量明显减少,NO/ET-1显著高于IR组(P<0.05或0.01),且P组脊髓病理学损伤轻于IR组.结果表明缺血后处理可减轻兔脊髓缺血再灌注微循环损伤,改善脊髓血流量,...     

Alterations in the contractile phenotype of the bladder: lessons for understanding physiological and pathological remodelling of smooth muscle

The contractile properties of the urinary bladder are changed by the conditions of normal development and partial bladder outlet obstruction. This change in the contractile phenotype is accompanied by changes in the regulatory cascades and filaments that regulate contractility. This review focuses on such changes during the course of normal development and in response to obstruction. Our goal is to discuss the experimental evidence that has accumulated from work in animal models and correlate these findings with the human voiding phenotype.     

The Early Activation of Toll-Like Receptor (TLR)-3 Initiates Kidney Injury after Ischemia and Reperfusion

Acute kidney injury (AKI) is one of the most important complications in hospitalized patients and its pathomechanisms are not completely elucidated. We hypothesize that signaling via toll-like receptor (TLR)-3, a receptor that is activated upon binding of double-stranded nucleotides, might play a crucial role in the pathogenesis of AKI following ischemia and reperfusion (IR). Male adult C57Bl6 wild-type (wt) mice and TLR-3 knock-out (-/-) mice were subjected to 30 minutes bilateral selective clamping of the renal artery followed by reperfusion for 30 min 2.5h and 23.5 hours or subjected to sham procedures. TLR-3 down-stream signaling was activated already within 3 h of ischemia and reperfusion in post-ischemic kidneys of wt mice lead to impaired blood perfusion followed by a strong pro-inflammatory response with significant neutrophil invasion. In contrast, this effect was absent in TLR-3-/- mice. Moreover, the quick TLR-3 activation resulted in kidney damage that was histomorphologically associated with significantly increased apoptosis and necrosis rates in renal tubules of wt mice. This finding was confirmed by increased kidney injury marker NGAL in wt mice and a better preserved renal perfusion after IR in TLR-3-/- mice than wt mice. Overall, the absence of TLR-3 is associated with lower cumulative kidney damage and maintained renal blood perfusion within the first 24 hours of reperfusion. Thus, we conclude that TLR-3 seems to participate in the pathogenesis of early acute kidney injury.     

The effect of partial outlet obstruction on prostaglandin generation in the rabbit urinary bladder

Partial outlet obstruction of the urinary bladder has been demonstrated to induce specific dysfunctions in cellular and sub-cellular membrane structures within the bladder's smooth muscle and mucosal compartments. Recent studies have linked these membrane dysfunctions to alterations in phospholipid metabolism leading to mobilization of free arachidonic acid, the precursor for synthesis of prostaglandins (PG). The purpose of this study was to determine if partial outlet obstruction of the urinary bladder induces changes in the capacity of bladder smooth muscle and mucosa to generate PG. PG were isolated from control and partially obstructed urinary bladder smooth muscle and mucosa of male New Zealand White (NZW) rabbits. PG concentrations (PGE2, PGF2alpha and PGI2, as its stable metabolite 6-keto-PGF1alpha) were determined after 30 minute incubations using enzyme-linked immunoassay (ELISA) kits. In both control and obstructed rabbit urinary bladders, PG generation was significantly higher in isolated mucosa than muscle tissues. A significantly higher concentration of PGF2alpha, and 6-keto-PGF1alpha was measured in obstructed muscle tissue relative to controls. The concentration of 6-keto-PGF1alpha was also significantly higher than the concentrations measured for PGE2 and PGF2alpha in both control and obstructed smooth muscle samples. The generation of PGE2 was significantly higher in rabbit urinary bladder mucosa than either PGF2alpha or 6-keto-PGF1alpha in both control and obstructed samples. The capacity of obstructed mucosal tissue to generate 6-keto-PGF1alpha was significantly higher than control tissue, while no significant differences in PGE or PGF2alpha generation were noted. These data suggest obstruction of the urinary bladder induce specific elevations in PG in both smooth muscle and mucosal tissues.     

Protective effects of leptin on ischemia/reperfusion injury in rat bladder

The aim of the study was to evaluate protective effects of exogenous leptin on ischemia/reperfusion (I/R)-induced injuries to the urinary bladder tissue and to investigate the effect on tumor necrosis factor alpha (TNF-alpha) levels and apoptotic cells during I/R injury. Bladder I/R injury was induced by abdominal aorta occlusion by ischemia for 45 min, followed by 60 min of reperfusion in rats. The rats were divided into three groups: control (n = 8 + 8), I/R (n = 8 + 8) and I/R+leptin group (n = 8 + 8). The rats in the I/R+leptin group were treated intraperitoneally with leptin (10 microg/kg) 60 min prior to ischemia induction. At the end of the reperfusion period, urinary bladders of the first eight rats from each group were removed for TUNEL staining processing while the others were removed for biochemical analyses for MDA and TNF-alpha levels. In the I/R group, the ratios of TUNEL-positive nuclei were higher than the control and the I/R+leptin groups. The MDA and TNF-alpha levels of the bladder tissue in the I/R group were higher than the control and leptin-treated groups. TUNEL-staining and biochemical studies revealed that leptin has a protective effect on urinary bladder I/R injury.     

Ischemic preconditioning-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contraction-induced myocardial injury

Dystrophin is an integral membrane protein involved in the stabilization of the sarcolemmal membrane in cardiac muscle. We hypothesized that the loss of membrane dystrophin during ischemia and reperfusion is responsible for contractile force-induced myocardial injury and that cardioprotection afforded by ischemic preconditioning (IPC) is related to the preservation of membrane dystrophin. Isolated and perfused rat hearts were subjected to 30 min of global ischemia, followed by reperfusion with or without the contractile blocker 2,3-butanedione monoxime (BDM). IPC was introduced by three cycles of 5-min ischemia and 5-min reperfusion before the global ischemia. Dystrophin was distributed exclusively in the membrane of myocytes in the normally perfused heart but was redistributed to the myofibril fraction after 30 min of ischemia and was lost from both of these compartments during reperfusion in the presence or absence of BDM. The loss of dystrophin preceded uptake of the membrane-impermeable Evans blue dye by myocytes that occurred after the withdrawal of BDM and was associated with creatine kinase release and the development of contracture. Although IPC did not alter the redistribution of membrane dystrophin induced by 30 min of ischemia, it facilitated the restoration of membrane dystrophin during reperfusion. Also, myocyte necrosis was not observed when BDM was withdrawn after complete restoration of membrane dystrophin. These results demonstrate that IPC-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contractile force-induced myocardial injury and suggest that the cardioprotection conferred by IPC can be enhanced by the temporary blockade of contractile activity until restoration of membrane dystrophin during reperfusion.