Different Periods Of Reperfusion Research Articles

Hypochlorous acid derived from microglial myeloperoxidase could mediate high-mobility group box 1 release from neurons to amplify brain damage in cerebral ischemia–reperfusion injury

Myeloperoxidase (MPO) plays critical role in the pathology of cerebral ischemia–reperfusion (I/R) injury via producing hypochlorous acid (HOCl) and inducing oxidative modification of proteins. High-mobility group box 1 (HMGB1) oxidation, particularly disulfide HMGB1 formation, facilitates the secretion and release of HMGB1 and activates neuroinflammation, aggravating cerebral I/R injury. However, the cellular sources of MPO/HOCl in ischemic brain injury are unclear yet. Whether HOCl could promote HMGB1 secretion and release remains unknown. In the present study, we investigated the roles of microglia-derived MPO/HOCl in mediating HMGB1 translocation and secretion, and aggravating the brain damage and blood-brain barrier (BBB) disruption in cerebral I/R injury. In vitro, under the co-culture conditions with microglia BV cells but not the single culture conditions, oxygen–glucose deprivation/reoxygenation (OGD/R) significantly increased MPO/HOCl expression in PC12 cells. After the cells were exposed to OGD/R, MPO-containing exosomes derived from BV2 cells were released and transferred to PC12 cells, increasing MPO/HOCl in the PC12 cells. The HOCl promoted disulfide HMGB1 translocation and secretion and aggravated OGD/R-induced apoptosis. In vivo, SD rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) plus different periods of reperfusion. Increased MPO/HOCl production was observed at the reperfusion stage, accomplished with enlarged infarct volume, aggravated BBB disruption and neurological dysfunctions. Treatment of MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH) and HOCl scavenger taurine reversed those changes. HOCl was colocalized with cytoplasm transferred HMGB1, which was blocked by taurine in rat I/R-injured brain. We finally performed a clinical investigation and found that plasma HOCl concentration was positively correlated with infarct volume and neurological deficit scores in ischemic stroke patients. Taken together, we conclude that ischemia/hypoxia could activate microglia to release MPO-containing exosomes that transfer MPO to adjacent cells for HOCl production; Subsequently, the production of HOCl could mediate the translocation and secretion of disulfide HMGB1 that aggravates cerebral I/R injury. Furthermore, plasma HOCl level could be a novel biomarker for indexing brain damage in ischemic stroke patients.

Optimum marker selection of acute liver damage in rats in the experiment

Relevance. Assessment of liver damage and functional state is one of the leading tasks of clinical and laboratory diagnostics. Traditionally used methods for determining the activity of a number of indicator enzymes in blood with relative organ-specificity, such as aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, sorbitol dehydrogenase, alkaline phosphatase, and -glutamyl transferase, have low specificity for liver diseases. In this regard, the determination of the optimal marker of acute liver injury is an urgent problem. Aim. The purpose of the study is to determine the dynamics of changes in liver damage markers in rats at different periods of reperfusion after 20 minutes of ischemia in order to select the indicators that most informatively characterize the state of test-animals under conditions of correction of ischemia-reperfusion syndrome. Materials and methods: the study was performed on 120 white nonlinear male rats weighing 200250 grams. The animals were divided into 8 groups of 15 test-animals; all of them were simulated liver ischemia by clamping the analog of the hepatoduodenal ligament with a vascular clamp for 20 minutes. Then, blood was taken from different groups of rats at different reperfusion times 5, 15, 30, 60, 120, 180 minutes, 8 hours and a day. In the blood plasma of laboratory animals, the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glutathione transferase (GST), and lactate concentration were determined. Results: the results obtained allowed us to characterize two main peaks of indicators: a 5-minute period after restoration of blood flow the maximum activity of glutathione transferase and lactate concentration, increased by 3.94.7 times; 60180 minutes of reperfusion is the peak of aminotransferase activity, a significant increase in the activity of which begins 60 minutes after the restoration of blood flow and reaches its maximum by the 3rd hour of reperfusion, and LDH, the peak of which is recorded already by the 60th minute of revascularization. At the same time, after 8 hours of reperfusion, an obvious tendency for a decrease in all studied parameters was determined, which ends a day after modeling ischemia with a decrease to the level of control values. Conclusion: the assessment of organ damage in the ischemic period and the anti-ischemic effect of metabolic drugs can be carried out with the determination of an increase in lactate concentration and glutathione transferase activity almost immediately after restoration of blood flow. The development of injuries during the reperfusion period is more expedient to assess by determining AST, ALT and LDH after a 3-hour period of blood flow restoration, at which time the maximum values of markers are recorded under the condition of 20-minute total liver ischemia.

Dexamethasone Improves Neuromuscular Junction Recovery From Ischemia‐Reperfusion Injury Induced by Tourniquet Application in Mouse Hindlimb

Tourniquet‐induced ischemia‐reperfusion (IR) injuries impede long‐term recovery of skeletal muscle contraction. The neuromuscular junction (NMJ) is an important factor to influence skeletal muscle contractile function. Here, we observed the effect of dexamethasone (Dex) on morphological and functional recovery of the NMJ from tourniquet‐induced IR injuries in mouse hindlimb.Unilateral hindlimb of C57/BL6 mice were subjected to 3 hours of ischemia by placing a rubber band and followed different periods of reperfusion (1 week, 2 weeks, 4 weeks, and 6 weeks). Dex treatment (1 mg/kg/day, i.p.) began on the day of IR induction and lasted for 7 days. After 3 hours of ischemia and different periods of reperfusion, NMJs in the gastrocnemius muscle showed significantly the morphological and functional damage. In the gastrocnemius muscle, TNFα and IL‐1β (two inflammatory cytokines) were increased after 1, 2 and 6 weeks of tourniquet‐induced IR. Treatment with Dex inhibited the elevation of TNFα and IL‐1β, mitigated fragmentation of nicotinic acetylcholine receptor (nAChR) clusters, promoted re‐innervation of nAChR clusters, and increased nerve‐stimulated end plate potentials (EPPs).The data suggested that tourniquet‐related IR induced serious skeletal muscle inflammation for a long period. Dex treatment for 1 week inhibits IR‐caused inflammation in the early period of IR, and improves the morphological and functional recovery of the NMJ from tourniquet‐induced IR injuries.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Testicular torsion and reperfusion: Germ cell DNA damage and development.

This study is aimed to analyse the cross-link between cyclin D1, cdk-4, p21, PCNA and DNA damage during different periods of reperfusion following experimental torsion in rats. Thirty mature male Wistar rats (N=6) were used. Following 4hr from torsion induction, the reperfusion was induced. Animals were subdivided into groups, including 4hr torsion-induced (T1), 1hr post-reperfusion (T2), 2hr post-reperfusion (T3), 4hr post-reperfusion (T4) and 8hr post-reperfusion (T5) groups. The seminiferous tubules differentiation (TDI) and spermatogenesis indices were evaluated. The expressions of cyclin D1, cdk-4, p21and PCNA were analysed using Reverse Transcriptase-PCR (RT-PCR). Moreover, the cyclin D1+ , cdk-4+ , p21+ and PCNA+ cell numbers/mm2 of tissue were assessed through immunohistochemical staining. The testicular DNA fragmentation was analysed using TUNEL assay and DNA ladder test. Observations demonstrated that reperfusion significantly increased (p<0.05) up-regulated the expressions of cyclin D1, cdk-4 and PCNA. The animals in T5 group showed diminished expression of p21 and represented diminished DNA fragmentation versus T1 group. In conclusion, minimum 8hr post-reperfusion is needed to re-initiate necessary expressions of cyclin D1, cdk-4 and PCNA to restore cell cycling machinery and ameliorate torsion-induced DNA damage.

Characteristics of nervous tissue after modeling of focal cerebral ischemia in rats at different periods of reperfusion

The stroke-causing problems are extremely important in Ukraine. This makes a heavy burden not only on the health care system, but also on the whole society as a whole. That's why we've studied structural and ultrastructural changes of cortical neurons and striatum of the brain and the development of delayed death of nerve cells after the modeling of the middle cerebral artery occlusion (MCAO) and post ischemic period in rats. We've analyzed the data at different terms after modeling of MCAO. The purpose of the study was to investigate the changes in the nervous tissue in the modeling of focal cerebral ischemia by monofilament occlusion of MCAO in rats at different periods of reperfusion. The statistical processing of primary digital experimental data was carried out using the software Statistica 6.0. It was confirmed that the 60-minute occlusion of the MCAO is an adequate model of focal ischemic brain damage in rats. Changes of locomotor activity and a tactile sensitivity were determined in rats after occlusion and after reperfusion during the post-period period. It was found that in the experimental group with a reperfusion period of 72 hours, a clear increase of the volume of the ischemic area of the brain, accompanied by significant neurological deficiency, was observed. Reduced research activity of the rats was revealed, which was shown in the decrease of the number of squares they crossed, the number of racks, the increase of acts of grooming and the duration of acts of frizings. Following ischemic brain damage, there was also a disbalance of somato-sensory functions, as evidenced by an increase in the time during which the animal took a test stimulus ("Sticky tape") from both the anterior paws when tested for tactile sensitivity (adhesive removal test). An electron microscopic study of the cortex showed that dark wrinkled neurons and enlightened swollen neurons were observed at 72 hours of post-occlusion period, indicating different ways of death of these cells. Changes in striatum were similar to changes in the cortex, which progressed with an increase in the post-occlusion period. The protocol of the serial evaluation of neurological disorders used after MCAO modeling allowed detecting long-term stable functional disorders in laboratory rats. The obtained data indicate significant changes in the structure of the cortex and striatum in the post-ischemic period and the progressive nature of these changes.

Liposomal dexamethasone attenuates tourniquet‐induced ischemia‐reperfusion injury in mouse hindlimb

Tourniquet‐induced ischemia and subsequent release cause serious ischemia‐reperfusion (IR) injury in the skeletal muscle. Here, we observed the effect of liposomal dexamethasone (lipo‐Dex) in tourniquet‐Induced ischemic reperfusion injury (IR) in mouse hindlimb.Unilateral hindlimb of C57/BL6 mice was subjected to 3 hours of ischemia by placing a rubber band and followed different periods of reperfusion (1 week, 2 weeks, 4 weeks, and 6 weeks). Before tourniquet was released, lipo‐Dex (7 mg/kg, equivalent to amg/kg/day of Dex for 1 week) was administered via tail vein injection. After 3 hours of ischemia and different periods of reperfusion, gastrocnemius muscle showed significantly morphological and functional damages. TNFα and IL‐1β were overexpressed in the gastrocnemius muscle after 1, 2, 4, and 6 weeks of tourniquet‐induced IR. Treatment with Lipo‐Dex inhibited the elevation of TNFα and IL‐1β, improved structure of the muscle sarcolemma, reduced fragmentation of nicotinic acetylcholine receptor clusters, and promoted recovery of gastrocnemius muscle contraction.The data suggest that tourniquet‐related IR induces serious skeletal muscle inflammation and degradation. Treatment with lipo‐Dex suppresses IR‐induced inflammation and promotes recovery of skeletal muscle morphology and function from tourniquet‐induced IR injury.Support or Funding InformationUSAMRAA grant from Department of Defense, W81XWH‐17‐1‐0037(PI, Yulong Li).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Morphological regeneration and functional recovery of neuromuscular junction after ischemic/reperfusion injury induced by tourniquet in mouse hind limb

BackgroundTourniquet‐induced ischemia and subsequent reperfusion cause serious acute ischemia‐reperfusion (IR) injury in the neuromuscular junction (NMJ) and skeletal muscle. These injuries are associated with permanent neurological deficiency, and long‐term skeletal muscle contractile dysfunction, even complete limb paralysis. Monitoring structural and functional repair of the NMJ and skeletal muscle after tourniquet‐induced IR is beneficial to exploring the mechanisms responsible for tourniquet‐induced IR injury and finding effective therapeutic interventions. Here, we observed long‐term morphological and functional alterations of the NMJ in tourniquet‐induced hindlimb IR.MethodUnilateral hindlimb of C57/BL6 mice was subjected to 3 hours of ischemia by placing an orthodontic rubber band and followed different periods of reperfusion (1 day, 3 days, 1 weeks, 2 wekks, 4 weeks, and 6 weeks). At each time point of reperfusion, the gastrocnemius muscle was isolated for measurement of the NMJ morphology and function. Motor nerve terminals and nicotinic acetylcholine receptors (nAChRs) in the NMJ were measured by immunofluorescence staining. The end plate potential (EPP) was recorded by the intracellular recording technique.ResultsIn the NMJ of sham mice, the structure of the nAChR clusters remained intact pretzel‐like shape and motor nerve terminals innervated the nAChR clusters to form synapses for signal transmission. The number of fragments in one nAChR cluster was 1.83 ± 0.07 and all nAChR clusters were innervated by motor nerve terminals (100%). The amplitude of EPPs was 29.84 ± 1.02 mV. At 1 day and 3 days of reperfusion, the motor nerve terminals disappeared, the AChR clusters kept normal shape, and no EPPs were recorded. From 1 week to 6 weeks of reperfusion, the motor nerve terminals gradually reinnervated (46.88%, 88.05%, 99.2%, and 100% for 1, 2, 4, and 6 weeks of reperfusion, respectively). However, the AChR clusters were gradually fragmented and the number of fragments in one nAChR cluster increased with longer period of reperfusion (2.92 ± 0.14, 5.50 ± 0.30, 8.94 ± 0.67, and 6.88 ± 0.50 for for 1, 2, 4, and 6 weeks of reperfusion, respectively; p&lt;0.05 vs. sham). The amplitude of EPPs also increased gradually with longer period of reperfusion, but the amplitude of EPPs at 6 weeks of reperfusion didn't restore towards the level seen in sham mice (13.92 ± 1.11 mV, p&lt;0.05 vs. sham).ConclusionTourniquet‐induced ischemia and subsequent reperfusion impaired the NMJ. The morphological change of motor nerve terminals was faster than that of the nAChR clusters. The slow recovery of fragmented nAChR clusters could attenuate neuromuscular transmission.

The Effect of Ischemia and Reperfusion on Enteric Glial Cells and Contractile Activity in the Ileum.

We investigated the effects of ischemia followed by different periods of reperfusion (I/R) on immunoreactive S100β-positive glial and Hu-immunoreactive neurons co-expressing the P2X2 receptor in the myenteric plexus of the rat ileum. The ileal artery was occluded for 35 min with an atraumatic vascular clamp. The animals were killed 24 h, 72 h, and 1 week after ischemia. Sham animals were not submitted to ileal artery occlusion. The relative density, size, and co-localization of P2X2 receptor-expressing cells in relation to S100β-immunoreactive glial and Hu-immunoreactive neuronal cells were evaluated. Additionally, we analyzed the effects of I/R on gastrointestinal transit and ileum contractile activity. The cellular density of P2X2 receptor and neuronal Hu immunoreactivity/cm(2) decreased after I/R, whereas glial S100β immunoreactivity/cm(2) increased. No significant differences between sham and I/R groups were observed regarding the perikarya area of Hu-positive neurons. The area of S100β-immunoreactive glial cells increased by 24.1 % 1 week after I/R compared with the 24 h group. Methylene blue progression along the small intestine decreased (P < 0.05) from 24.5 ± 2.3 % in the sham group to 17.2 ± 2.0 % 1 week post-ischemia. We noted a significant (P < 0.05) decrease in the maximal contraction amplitude triggered by electrical field stimulation in the presence of ATP in preparations submitted to 24 h of I/R. Changes in the P2X2 receptor density parallel myenteric neuronal loss following I/R of the rat ileum. This, together with the increase in the activated (oversized) glial cells, may contribute to decreased GI motility after I/R.

Resveratrol prevents CA1 neurons against ischemic injury by parallel modulation of both GSK‐3β and CREB through PI3‐K/Akt pathways

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK-3β) and cAMP response element-binding protein (CREB) through phosphatidylinositol 3-kinase (PI3-K)-dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four-vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK-3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3-K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK-3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3-K, compromised the neuroprotective effect of resveratrol and decreased the level of p-Akt, p-GSK-3β and p-CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro-survival states of Akt, GSK-3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3-K/Akt signaling pathway, subsequently downregulating expression of GSK-3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats.

Ischaemia-induced mucus barrier loss and bacterial penetration are rapidly counteracted by increased goblet cell secretory activity in human and rat colon

ObjectiveColonic ischaemia is frequently observed in clinical practice. This study provides a novel insight into the pathophysiology of colon ischaemia/reperfusion (IR) using a newly developed human and rat experimental model.DesignIn...

Protective functions of recombinant protein targeted at RANKL against hepatic ischemia/reperfusion injury transfected by retrovirus in mice

To explore the protective functions of recombinant protein RANK-Fc against hepatic ischemia/reperfusion injury and clarify its possible mechanism. Sixty male Balb/c mice were randomly divided into 3 groups according to different treatments: serum-free medium control (Sham) group, target gene retrovirus (RANK-Fc) group and empty vector retrovirus (eGFP) group. All mice were injected with 2.5 ml solution (with or without retrovirus) within 6 seconds via tail vein. After 3 days, the model of 70% hepatic ischemia/reperfusion was induced under warm conditions for 90 minutes after different periods of reperfusion in RANK-Fc and eGFP groups; Sham group underwent the same procedure without the occlusion of blood supply. Blood and liver samples were obtained at different time points (1, 3, 6 and 24 h; n = 5 in each). Reverse transcription-polymerase chain reaction (RT-PCR) was used for the evaluation of eGFP mRNA expression. RANK-Fc was assessed by Western blot. Liver transaminases and histopathological changes were used for the evaluation of hepatic injury. The activity of NF-κB in liver nucleus was analyzed by Western blot and immunohistochemistry. The activation level of JNK was also assessed by Western blot. Liver homogenate levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were detected by enzyme-linked immunosorbent assay (ELISA). Apoptosis was identified by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. The differences between three treatment groups at each time point were detected by the one-way ANOVA. Statistical analysis for inter-comparison was performed by Student's t test. RANK-Fc and eGFP were successfully expressed in liver after hydrodynamics-based transfection. Compared with eGFP group, RANK-Fc significantly improved liver functions at the same time point (P < 0.01), decreased NF-κB nuclear translocation (t = 6.726, P < 0.01)and JNK phosphorylation (t = 6.713, P < 0.01)and obviously suppressed the release of pro-inflammatory cytokine TNF-α (t = 4.779, P < 0.01) and IL-6 (t = 5.482, P < 0.01). Morphological injuries were markedly alleviated while the expressions of immunohistochemical positive cells and TUNEL staining positive cells decreased in RANF-Fc group. RANK-Fc has protective functions against hepatic ischemia/reperfusion injury in mice. Its mechanism is at least partially related with the suppressions of proinflammatory NF-κB and proapoptotic JNK signaling pathways.

Resveratrol prevents oxidative stress and inhibition of Na +K +-ATPase activity induced by transient global cerebral ischemia in rats

Increased oxidative stress and energy metabolism deficit have been regarded as an important underlying cause for neuronal damage induced by cerebral ischemia/reperfusion (I/R) injury. In this study, we investigated the oxidative mechanisms underlying the neuroprotective effects of resveratrol, a potent polyphenol antioxidant found in grapes, on structural and biochemical abnormalities in rats subjected to global cerebral ischemia. Experimental model of transient global cerebral ischemia was induced in Wistar rats by the four vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl and fluoro jade C stained indicated extensive neuronal death at 7 days after I/R. These findings were preceded by a rapid increase in the generation of reactive oxygen species (ROS), nitric oxide (NO), lipid peroxidation, as well as by a decrease in Na +K +-ATPase activity and disrupted antioxidant defenses (enzymatic and non-enzymatic) in hippocampus and cortex. Administrating resveratrol 7 days prior to ischemia by intraperitoneal injections (30 mg/kg) significantly attenuated neuronal death in both studied structures, as well as decreased the generation of ROS, lipid peroxidation and NO content. Furthermore, resveratrol brought antioxidant and Na +K +-ATPase activity in cortex and hippocampus back to normal levels. These results support that resveratrol could be used as a preventive, or therapeutic, agent in global cerebral ischemia and suggest that scavenging of ROS contributes, at least in part, to resveratrol-induced neuroprotection.

Brain regional and cellular localization of gelatinase activity in rat that have undergone transient middle cerebral artery occlusion

Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of ischemic stroke. In particular, the gelatinases MMP-2 and MMP-9 contribute to disruption of the blood–brain barrier and hemorrhagic transformation following ischemic injury. In addition to extracellular matrix degradation, MMPs may directly regulate neuronal cell death through mechanisms that are not completely understood. Here we describe the spatio-temporal distribution of activated MMP-2 and MMP-9 in the brain of rats subjected to 2 h middle cerebral artery occlusion (MCAo) followed by different periods of reperfusion (15 min, 2 h, 6 h and 22 h). By in situ zymography we have observed that gelatinases become activated 15 min and 2 h after the beginning of reperfusion in the ischemic core and penumbra, respectively. In situ zymography signal broadly co-localized with NeuN-positive cells, thus suggesting that proteolysis mainly occurs in neurons. Gelatinolytic activity was mainly detected in cell nuclei, marginally appearing in the cytosol only at later stages following the insult; we did not detect variations in gelatinolysis in the extracellular matrix. Finally, we report that pharmacological inhibition of MMPs by N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpenthanoyl]- l-tryptophan methylamide (GM6001) significantly reduces brain infarct volume induced by transient MCAo. Taken together our data underscore the crucial role of gelatinases during the early stages of reperfusion and further extend previous observations documenting the detrimental role of these enzymes in the pathophysiology of brain ischemia.

Hypoxia-inducible Factor-1 Mediates Neuronal Expression of the Receptor for Advanced Glycation End Products following Hypoxia/Ischemia

Activation of the receptor for advanced glycation endproducts (RAGE) by its multiple ligands can trigger diverse signaling pathways with injurious or pro-survival consequences. In this study, we show that Rage mRNA and protein levels were stimulated in the mouse brain after experimental stroke and systemic hypoxia. In both cases, RAGE expression was primarily associated with neurons. Activation of RAGE-dependent pathway(s) post-ischemia appears to have a neuroprotective role because mice genetically deficient for RAGE exhibited increased infarct size 24 h after injury. Up-regulation of RAGE expression was also observed in primary neurons subjected to hypoxia or oxygen-glucose deprivation, an in vitro model of ischemia. Treatment of neurons with low concentrations of S100B decreased neuronal death after oxygen-glucose deprivation, and this effect was abolished by a neutralizing antibody against RAGE. Conversely, high concentrations of exogenous S100B had a cytotoxic effect that seems to be RAGE-independent. As an important novel finding, we demonstrate that hypoxic stimulation of RAGE expression is mediated by the transcription factor hypoxia-inducible factor-1. This conclusion is supported by the finding that HIF-1alpha down-regulation by Cre-mediated excision drastically decreased RAGE induction by hypoxia or desferrioxamine. In addition, we showed that the mouse RAGE promoter region contains at least one functional HIF-1 binding site, located upstream of the proposed transcription start site. A luciferase reporter construct containing this RAGE promoter fragment was activated by hypoxia, and mutation at the potential HIF-1 binding site decreased hypoxia-dependent promoter activation. Specific binding of HIF-1 to this putative HRE in hypoxic cells was detected by chromatin immunoprecipitation assay.

Alanyl-glutamine dipeptide inhibits hepatic ischemia-reperfusion injury in rats

To investigate the protective effect and mechanism of alanyl-glutamine dipeptide (Ala-Gln) against hepatic ischemia-reperfusion injury in rats. Rats were divided into group C as normal control Group (n=16) and group G as alanyl-glutamine pretreatment (n=16). Rats were intravenously infused with 0.9% saline solution in group C and Ala-Gln -enriched (2% glutamine) 0.9% saline solution in group G via central venous catheter for three days. Then all rats underwent hepatic warm ischemia for 30 min followed by different periods of reperfusion. Changes in biochemical parameters,the content of glutathione (GSH) and the activity of superoxide dismutase (SOD) in liver tissue, Bcl-2 and Bax protein expression and morphological changes of liver tissue were compared between both groups. One hour after reperfusion, the levels of liver enzymes in group G were significantly lower than those in group C (P<0.05).Twenty-four hours after reperfusion, the levels of liver enzymes in both groups were markedly recovered and the levels of liver enzyme in group G were also significantly lower than those in group C (P<0.01). One and 24 h after reperfusion, GSH content in group G was significantly higher than that in group C (P<0.05). There was no statistical difference in activities of SOD between the two groups. One and 24 h after reperfusion, the positive expression rate of Bcl-2 protein was higher in group G than in group C (P<0.05) and the positive expression rate of Bax protein was lower in group G than in group C (P<0.05). Histological and ultrastructural changes of liver tissue were inhibited in group C compared to group G. Our results suggest that Ala-Gln pretreatment provides the rat liver with significant tolerance to warm ischemia-reperfusion injury,which may be mediated partially by enhancing GSH content and regulating the expression of Bcl-2 and Bax proteins in the liver tissue.

Effects of the hyperbaric oxygen treatment on the Na +,K +-ATPase and superoxide dismutase activities in the optic nerves of global cerebral ischemia-exposed rats

The effects of hyperbaric oxygen (HBO) treatment on the Na +,K +-ATPase and superoxide dismutase (SOD) activities were examined in the optic nerves of the rats exposed to global cerebral ischemia. Animals were exposed to global cerebral ischemia of 20-min duration and were either sacrificed or exposed to the first HBO treatment immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na +,K +-ATPase activities measurement) or 2, 24, 48 or 168 h after ischemia (for SOD activities measurement). HBO procedure was repeated for 7 consecutive days. It was found that global cerebral ischemia induced a statistically significant decrease in the Na +,K +-ATPase activity of the optic nerves, starting from 0.5 to 168 h of reperfusion. Maximal enzymatic inhibition was registered 24 h after the ischemic damage. The decline in the Na +,K +-ATPase activity was prevented in the animals exposed to HBO treatment within the first 6 h of reperfusion. The results of the presented experiments demonstrated also a statistically significant increase in the SOD activity after 24, 48 and 168 h of reperfusion in the optic nerves of non-HBO-treated ischemic animals as well as in the ischemic animals treated with HBO. Our results indicate that global cerebral ischemia induced a significant alterations in the Na +,K +-ATPase and SOD activities in the optic nerves during different periods of reperfusion. HBO treatment, started within the first 6 h of reperfusion, prevented ischemia-induced changes in the Na +,K +-ATPase activity, while the level of the SOD activity in the ischemic animals was not changed after HBO administration.

Hyperbaric oxygen treatment: the influence on the hippocampal superoxide dismutase and Na +,K +-ATPase activities in global cerebral ischemia-exposed rats

The influence of hyperbaric oxygen (HBO) treatment on the activities of superoxide dismutase (SOD) and Na +,K +-ATPase was determined during different time periods of reperfusion in rats exposed to global cerebral ischemia. Ischemic animals were either sacrificed or exposed to the first HBO treatment 2, 24, 48 or 168 h after ischemic insult (for SOD activities measurement) or immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na +,K +-ATPase activities measurement). Hyperbaric oxygenation procedure was repeated for seven consecutive days. The results of presented experiments demonstrated the statistically significant increase in the hippocampal SOD activity 24 and 48 h after global cerebral ischemia followed by a decrease in the enzymatic activity 168 h after ischemic insult. In the ischemic rats treated with HBO the level of hippocampal SOD activity was significantly higher after 168 h of reperfusion in comparison to the ischemic, non HBO-treated animals. In addition, it was found that global cerebral ischemia induced a statistically significant decrease of the hippocampal Na +,K +-ATPase activity starting from 1 to 168 h of reperfusion. Maximal enzymatic inhibition was obtained 24 h after the ischemic damage. Decline in Na +,K +-ATPase activity was prevented in the animals exposed to HBO treatment within the first 24 h of reperfusion. Our results suggest that global cerebral ischemia induces significant alterations in the hippocampal SOD and Na +,K +-ATPase activities during different periods of reperfusion. Enhanced SOD activity and preserved Na +,K +-ATPase activity within particular periods of reperfusion, could be indicators of a possible benefitial role of HBO treatment in severe brain ischemia.

Middle cerebral artery function after stroke: the threshold duration of reperfusion for myogenic activity.

Myogenic activity of the cerebral arteries is an important contributor to autoregulation of cerebral blood flow. Previous studies have demonstrated that increasing periods of ischemia diminished the amount of myogenic tone in cerebral arteries. In the present study, we investigated the effect of different periods of postischemic reperfusion on the myogenic behavior of middle cerebral arteries (MCAs). We measured both the amount of spontaneous myogenic tone that developed at 75 mm Hg and the contractile response to increased transmural pressure (TMP), ie, myogenic reactivity. The MCA occlusion model was used in male Wistar rats (n=45) to induce 30 minutes of temporary ischemia, followed by different periods of reperfusion (0 or sham; 30 minutes; and 6, 12, 18, 20, and 22 hours), confirmed by laser Doppler flowmetry. MCAs were studied in vitro using an arteriograph system that allowed control of TMP and measurement of lumen diameter. After equilibration for 1 hour at 75 mm Hg, TMP was increased stepwise in 25-mm Hg increments to 125 mm Hg and lumen diameter measured at each pressure. The amount of spontaneous myogenic tone was determined in both ischemic and contralateral arteries for each reperfusion period and compared with the right and left MCAs in the sham group. Arteries were then fixed with 10% formalin pressurized in the arteriograph bath and stained for filamentous (F)-actin with fluorescently labeled phalloidin, a specific probe for F-actin. The amount of F-actin was quantified using confocal microscopy. MCAs from the sham-operated control group possessed considerable myogenic tone (35%). However, the amount of tone in ischemic MCAs progressively diminished as the reperfusion duration increased. In addition, sham-operated control arteries responded myogenically to increases in TMP, decreasing diameter as pressure increased. There was a similar response in arteries exposed to 30 minutes and 6 hours of reperfusion, all producing a negative slope on the pressure-diameter curve; however, myogenic reactivity was diminished at the longer periods of reperfusion, producing a positive slope of the graph. The slopes of the pressure-diameter curves were as follows: -0.10+/--0.06 (sham), -0.07+/--0.12 (30 minutes), -0.08+/--0.11 (6 hours), +0.09+/-0.09 (12 hours), +0.25+/-0.16 (18 hours), +0.38+/-0.09 (20 hours), and +0.57+/-0.09 (22 hours). F-actin content was significantly less only in ischemic MCAs at 6 and 12 hours of reperfusion. These results demonstrate that longer periods of reperfusion significantly diminish myogenic activity of MCAs. Understanding how different periods of ischemia and reperfusion affect the function of the cerebral circulation may promote more effective treatment of ischemic stroke.

Coronary ischemia/reperfusion increases proliferating cell nuclear antigen in vascular endothelial cells in rat hearts.

To elucidate the sequence of early events in the increase in capillarity caused by reperfusion following transient coronary occlusion, the time course of expression of proliferating cell nuclear antigen (PCNA) was studied immunohistochemically in rat hearts subjected to different periods of reperfusion after a 3 minute occlusion. Twenty-one male Wistar rats were killed after different periods of reperfusion following occlusion of the coronary artery for 3 minutes. The left ventricles were removed and paired serial sections were treated immunohistochemically for PCNA or stained to show the enzymes characteristic of the arteriolar, intermediate and venular portions of the capillary bed. The time course of PCNA expression, and its distribution in relation to the different portions, was examined. An increase in PCNA-expressing nuclei was found at 24 hours after the start of reperfusion; numbers reached a maximum between 72 and 96 hours, and at 168 hours had decreased again. The majority of PCNA-expressing elements were localized to the dipeptidylpeptidase IV-reactive (i.e. venular) capillary portions, with some in the intermediate and alkaline phosphatase-reactive (i.e. arteriolar) portions. The distribution of PCNA in the early reperfusion period suggests that angiogenesis after transient ischemia occurs mainly from the venular side of the capillary bed, with some contribution from intermediate and arteriolar capillary portions.

Kallikrein-like amidase activity in renal ischemia and reperfusion.

We assessed a kallikrein-like amidase activity probably related to the kallikrein-kinin system, as well as the participation of leukocyte infiltration in renal ischemia and reperfusion. Male C57BL/KSJmdb mice were subjected to 20 or 60 min of ischemia and to different periods of reperfusion. A control group consisted of sham-operated mice, under similar conditions, except for ischemia induction. Kallikrein-like amidase activity, Evans blue extravasation and myeloperoxidase activity were measured in kidney homogenates, previously perfused with 0.9% NaCl. Plasma creatinine concentration increased only in the 60-min ischemic group. After 20 min of ischemia and 1 or 24 h of reperfusion, no change in kallikrein-like amidase activity or Evans blue extravasation was observed. In the mice subjected to 20 min of ischemia, edema was evident at 1 h of reperfusion, but kidney water content returned to basal levels after 24 h of reperfusion. In the 60-min ischemic group, kallikrein-like amidase activity and Evans blue extravasation showed a similar significant increase along reperfusion time. Kallikrein-like amidase activity increased from 4 nmol PNA mg protein-1 min-1 in the basal condition to 15 nmol PNA mg protein-1 min-1 at 10 h of reperfusion. For dye extravasation the concentration measured was near 200 microg of Evans blue/g dry tissue in the basal condition and 1750 microg of Evans blue/g dry tissue at 10 h of reperfusion. No variation could be detected in the control group. A significant increase from 5 to 40 units of DeltaAbs 655 nm g wet tissue-1 min-1 in the activity of the enzyme myeloperoxidase was observed in the 60-min ischemic group, when it was evaluated after 24 h of reperfusion. Histological analysis of the kidneys showed migration of polymorphonuclear leukocytes from the vascular bed to the interstitial tissue in the 60-min ischemic group after 24 h of reperfusion. We conclude that the duration of ischemia is critical for the development of damage during reperfusion and that the increase in renal cortex kallikrein-like amidase activity probably released from both the kidney and leukocytes may be responsible, at least in part, for the observed effects, probably through direct induction of increased vascular permeability.