Ischemic Incubation Research Articles

Sex-Specific Protection of Endothelial Function after Vascular Ischemia/Reperfusion Injury by the Senomorphic Agent Ruxolitinib.

Ischemia/reperfusion (I/R)-induced endothelial dysfunction occurs in various cardiovascular disorders. I/R injury is partially driven by the release of cytokines. Known for its use in senotherapy, the JAK inhibitor ruxolitinib is able to block the release of cytokines. We investigated the effect of ruxolitinib on the cytokine release and endothelial-dependent vasorelaxation in an in vitro model of I/R. Aortic segments of C57BL/6J mice (N = 12/group) were divided into three groups: control, in vitro I/R (I/R group), and in vitro I/R with ruxolitinib during ischemic incubation (I/R+Ruxo group). We determined cytokine expression. In organ bath chambers, we investigated the maximal endothelial-dependent relaxation to acetylcholine (RmaxACh) and maximal endothelial-independent relaxation to sodium-nitroprusside (RmaxSNP). RmaxACh was decreased in I/R compared to the control (83.6 ± 2.4 vs. 48.6 ± 3.4%; p < 0.05) and I/R+Ruxo (74.4 ± 2.6 vs. 48.6 ± 3.4%; p < 0.05). RmaxSNP was comparable between all groups. IL-10 was detectable only in I/R+Ruxo. CXCL5, CCL2, CCL3, CCL8, CCL11, ICAM-1, IL-1α, IL-7, TNF-α, and G-CSF were decreased or not detectable in I/R+Ruxo. In I/R+Ruxo, ICAM-1 was reduced in rings only from male mice. Treatment of the aorta from mice during in vitro ischemia with the senomorphic agent ruxolitinib reduces cytokine release and protects the endothelium from I/R-mediated dysfunction.

Ischemia and reoxygenation induced amino acid release release and tissue damage in the slices of rat corpus striatum.

Ischemic incubation significantly increased amino acid release from rat striatal slices. Reoxygenation (REO) of the ischemic slices, however, enhanced only taurine and citrulline levels in the medium. Ischemia-induced increases in glutamate, taurine and GABA outputs were accompanied with a similar amount of decline in their tissue levels. Tissue final aspartic acid level, however, was doubled by ischemia. Lactate dehydrogenase (LDH) leakage was not altered by ischemia, but enhanced during REO. Presence of tetrodotoxine (TTX) during ischemic period caused significant decline in ischemia-induced glutamate output, but not altered REO-induced LDH leakage. Although omission of extracellular calcium ions from the medium during ischemic period protected the slices against REO-induced LDH leakage, this treatment failed to alter ischemia-induced glutamate and GABA outputs. The release of other amino acids, however, declined 50% in calcium-free medium. Blockade of the glutamate uptake transporter by L-trans-PDC, on the other hand, doubled ischemia induced glutamate and aspartic acid outputs. These results indicate that more than one mechanisms probably support the ischemia-evoked accumulation of glutamate and other amino acids in the extracellular space. Although LDH leakage enhanced during REO, processes involved in this increment were found to be dependent on extracellular calcium ions during ischemia but not REO period.

Apoptosis and activation of Erkl/2 and Akt in astrocytes postischemia.

We have shown previously that in vitro ischemia could induce apoptosis in primary culture of astrocytes. In this paper we demonstrate that astrocytes in culture could undergo apoptosis during in vitro incubation postischemia. We also measured the changes of phosphorylated Erk1/2 (p-Erk1/2) and phosphorylated Akt (p-Akt) in order to determine whether these two pathways play a role in apoptosis. After 4 h in vitro ischemic incubation of cultured astrocytes, a limited amount of nuclear condensation was demonstrated by Hoechst 33342 staining. When ischemic incubation was halted and the cultures transferred to standard normoxic incubation (postischemia) conditions, DNA fragmentation and apoptosis were demonstrated by TUNEL and DNA laddering analysis. TUNEL-positive astrocytes began to appear at 6 h postischemia and increased in number from 12 h postischemia. Western blot analysis showed that both p-Erk1/2 and p-Akt were elevated in astrocytes subjected to 4 h of ischemia. Elevated p-Erk1/2 levels were sustained during the postischemia incubation for 12 h and decreased significantly afterward, but did not return to the levels in the control cultures that did not experience ischemic insult. In contrast, the p-Akt level continued to increase at 6 and 12 h postischemia before declining significantly. The changes in p-Erk1/2 and p-Akt correlated well with the appearance of apoptotic astrocytes in the culture.

Changes of ATP and ADP in cultured astrocytes under and after in vitro ischemia.

A very large body of evidence from in vivo studies has been accumulated on a link between the change of energy and cell survival/apoptosis. Using an in vitro ischemia model, we have previously shown that ischemia could induce apoptosis in astrocytes. In this study, we utilized the same in vitro model to investigate changes in ATP and ADP levels in cultured astrocytes and attempted to demonstrate an energy-cell death linkage. Astrocytes remained unaltered after 2 hr of ischemia but were moderately or severely damaged after 4 or 6-8 hr, respectively. The astrocytes that survived various lengths of in vitro ischemic incubation retained their ability to produce ATP after ischemia. Both ATP and ADP levels were increased in astrocytes that remained alive under in vitro ischemia for over 6 hr. The largest decline in the percent of viable astrocytes during ischemia corresponded well to the reduction in ATP and ADP levels in these cultures.

Establishment of neuronal in vitro models of ischemia in 96-well microtiter strip-plates that result in acute, progressive and delayed neuronal death

Using 96-well microtiter strip-plates we established in vitro ischemia models with acute, progressive and delayed neuronal death onset. In vitro ischemia was induced by washing neuronal cultures with a balanced salt solution with (acute/delayed models) or without (progressive model) 25 mM 2-deoxy- D-glucose and incubating in an anaerobic chamber. Reperfusion was performed by removing cultures from the anaerobic chamber and washing and/or adding Dulbecco’s modified Eagle medium containing N2 supplement. Acute neuronal death resulted in cell swelling during in vitro ischemic incubation with the majority of neurons appearing swollen and necrotic within 3 h post-insult. Progressive neuronal death was characterized by cell shrinkage during and immediately following in vitro ischemia with increasing neuronal degeneration resembling both necrosis and apoptosis over a 24-h period post- in vitro ischemia. Delayed neuronal death was induced by glutamate-receptor blockade during in vitro ischemia. Neurons appeared morphologically normal immediately following and up to 6 h after in vitro ischemia and then started to degenerate over the next 42 h by a process resembling apoptosis. We monitored oxygen consumption during in vitro ischemia and found it to be similar for the three models and have shown that plastic culture wells store oxygen. The establishment of acute, progressive and delayed in vitro models of ischemia using 96-well microtiter strip-plates will provide useful tools to further investigate ischemic neuronal death/survival mechanisms and provide a high-throughput system to evaluate potential neuroprotective agents. Oxygen storage in plastic culture wells is likely to contribute to the extended oxygen- and oxygen–glucose-deprivation times required to induce significant neuronal injury in vitro.

Ischemia-induced apoptosis in primary cultures of astrocytes.

Astrocytes participate in a wide variety of important physiological processes and pathological insults, including ischemia. Information on the mechanism of astroglial injury and death during ischemic insult, however, is scarce. In this study, we investigated the mode of astrocytic cell death using an in vitro ischemic model. Cultured astrocytes exhibited several distinct morphological and biochemical features of apoptosis under ischemia. At 4 h of ischemia, Annexin V staining demonstrated an early commitment of some astrocytes to apoptosis. Condensed nuclei became visible from 4 h and the number increased with ischemic incubation time. Electron microscopy showed compacted and segregated chromatin along the edges of nuclear membranes. The number of TUNEL-positive nuclei and the degree of DNA laddering increased with ischemic incubation. Caspase-3, but not caspase-1, activity was increased in ischemia-injured astrocytes. Swollen mitochondria and vacuoles found in some cells with chromatin condensation indicated that these apoptotic-like cells might die of necrosis. The results imply that astrocytes are capable of undergoing apoptosis without the presence of other cell types, such as neurons. Ischemia can induce apoptosis in astrocytes contributing to the pathogenesis of ischemic injury in the CNS.

Protection against ischemic injury in primary cultured astrocytes of mouse cerebral cortex by bis(7)-tacrine, a novel anti-Alzheimer's agent

The effects of bis(7)-tacrine, a novel acetylcholinesterase inhibitor, on ischemia-induced cell death and apoptosis were investigated in primary cerebral cortical astrocytes of mice. Following a 6 h in vitro ischemic incubation of the cultures, a marked decrease in the percentage of viable cells was observed by lactate dehydrogenase (LDH) release assay. Furthermore, using bisbenzimide staining, we determined that approximately 65% of the cells underwent apoptosis. Treatment with bis(7)-tacrine (1–10 nM) during ischemic incubation effectively inhibited the ischemia-induced apoptosis, as demonstrated by morphological and biochemical tests. Our results demonstrated that bis(7)-tacrine could protect astrocytes against ischemia-induced cell injury, indicating that the drug might be beneficial for the treatment of vascular dementia, in addition to Alzheimer's disease.

Phosphorylation State of hsp27 and p38 MAPK During Preconditioning and Protein Phosphatase Inhibitor Protection of Rabbit Cardiomyocytes

Small heat shock proteins (hsp) have been implicated in mediation of classic preconditioning in the rabbit. Hsp27 is a terminal substrate of the p38 MAPK cascade. One and 2D gel electrophoresis and immunoblotting of cell fractions was used to determine p38 MAPK and hsp27 phosphorylation levels, respectively, duringin vitroischemia in control, calyculin A (Cal A)-treated (protein phosphatase inhibitor), SB203580-treated (p38MAPK inhibitor) and preconditioned (IPC) isolated adult rabbit cardiomyocytes. The dual phosphorylation of p38 MAPK was increased by early ischemia (30–60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by IPC. Cal A strongly activated dual phosphorylation of p38 MAPK in oxygenated cells and this was maintained into early ischemia. SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents. Hsp27 phosphorylation was quantitated as the fraction of diphosphorylated hsp27, based on IEF mobility shifts of hsp27 phosphorylation isoforms. In oxygenated control cells, cytosolic and cytoskeletal hsp27 was highly phosphorylated. After 90 min ischemia, cytoskeletal hsp27 was markedly dephosphorylated. Cal A slightly increased control cytoskeletal hsp27 phosphorylation. During ischemic incubation, Cal A blocked ischemic dephosphorylation. SB203580 accelerated ischemic hsp27 dephosphorylation and injury. IPC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Phosphorylation is regulated by both kinase and phosphatase activities. IPC protection was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged (>60–90 min) ischemia.

Preconditioning of Isolated Rabbit Cardiomyocytes: No Evident Separation of Induction, Memory and Protection

Cardiomyocytes isolated from rabbit hearts were preconditionedin vitroby 10 min of ischemia or treatment with 100μmadenosine. Protection was assessed as average integrated mortality following osmotic swelling and determination of viability by trypan blue exclusion over 60–180 min ischemia. Repetitive sub-maximal stimulations with 1μmadenosine amplified the protective response. Treatment with adenosine only at the onset of prolonged ischemia afforded a dose-dependent protection. The PKC inhibitor calphostin C (500 nm) blocked preconditioning and, when added during ischemic incubation of non-preconditioned cells, significantly increased injury. The memory of adenosine-induced preconditioning decayed over a 60 min post-incubation period. Light activation of calphostin C initially added to preconditioned ischemic cells in the dark indicated that a 10 min period of PKC activity at the onset of ischemia affords full protection. The reversible PKC inhibitors chelerythrine (5μm) or staurosporine (100 nm) added only to bracket induction of ischemia, reduced but did not abolish protection. Protection was abolished when either drug was present during induction and a subsequent 30 min post-incubation period. Staurosporine included during initiation and post-incubation but washed out in the final 5 min of post-incubation allowed significant protection to occur. It is concluded that a single adenosine receptor-stimulation induces protection as it preconditions, and PKC activity appears to be required for both induction and protection. Memory may reside in post-receptor amplification of an initial protective response.

Brain ischemia decreases phosphatidylcholine-phospholipase D but not phosphatidylinositol-phospholipase C in rats.

Phosphatidylcholine (PC)-phospholipase D (PLD) is an important intracellular signaling pathway in response to a variety of agonists, but little is known about the effects of brain ischemia on the PC-PLD system. We thus have examined the effects of global cerebral ischemia on PLD in rats. We have examined the effects of global ischemia (decapitation or four-vessel occlusion) on PLD and PLC activity in the membrane fraction of rat brains. We measured the PLD and PLC activity in detergent-mixed micelle assay systems using 3H-labeled exogenous substrate. The results demonstrate that basal PLD activity showed a gradual decrease with increased duration (5 to 30 minutes) of ischemia by decapitation in the hippocampus; after 30 minutes of ischemia, PLD activity was significantly decreased compared with the control. Lineweaver-Burk plots showed that the apparent Vmax value of PLD in ischemia was one half of that in the control without changes in Km value. Ischemia by decapitation significantly decreased PLD activity in the brain stem as well as the hippocampus, whereas in four-vessel occlusion study, ischemia significantly decreased PLD activity in the hippocampus but not in the brain stem. Lowered temperature (30 degrees C and 22 degrees C) during ischemic incubation did not reverse the ischemia-induced PLD activity decrease. In contrast to PLD, ischemia by decapitation had no effect on basal phosphatidylinositol-phospholipase C activity or the amount of phospholipase C beta 1 in the membrane fractions from 30-minute ischemic hippocampus by immunoblots probed with the antibody. These results suggest that PC-PLD is one of the target enzymes of ischemia; its decrease may cause a perturbation of PC hydrolysis and/or disorders of intracellular transduction of signals or choline metabolism for acetylcholine formation in brain.

Flow cytometric analysis of isolated adult cardiomyocytes: Vinculin and tubulin fluorescence during metabolic inhibition and ischemia

Immunofluorescence and quantitative flow cytometry was used to determine if alterations in cytoskeletal proteins (vinculin and tubulin) occur during metabolic inhibition and ischemic incubation of isolated adult rat cardiomyocytes. Effects of cell shape changes on fluorescence, were controlled for by the contractile inhibitor, butanedione monoxime (BDM) and gated analysis. Flow cytometry differentiated rod- and round-shaped myocytes on the basis of forward and side scattering. Severe contracture of metabolically inhibited (iodoacetic acid and amytal) myocytes caused an artefactual increase in fluorescence intensity and a redistribution of tubulin into microblebs on the cell surface, which tended to mask specific losses of fluorescence. Fluorescence microscopy showed that round cells stained intensely for vinculin, but not for tubulin and that vinculin redistributed into coarse patches between 60 and 90 min, times which corresponded to small rebounds of fluorescence. With gated analysis, to exclude severely contracted round and squared cells, and with BDM inhibition of contracture, both metabolically inhibited and ischemic pelleted myocytes showed an early decrease in specific immunofluorescence staining for tubulin and vinculin, which preceded loss of cell viability, as determined by trypan blue staining. In both ischemic and metabolically inhibited cells, decreases of vinculin fluorescence preceded or coincided with increasing osmotic fragility. It is concluded that early cytoskeletal alterations of vinculin in ischemic and anoxic injury correlate with the development of osmotic fragility and irreversible myocyte injury.

Quantitative changes in the phosphorus fractions of transplanted brain tumors during complete ischemic incubation.

Glukose, Glykogen, Milchsaure, ATP und 5 phosphathaltige Fraktionen wurden an experimentellen, anarobisch-inkubierten Hirntumoren bestimmt. Die Mengen anorganischen Phosphats und Milchsaure waren grosser als die vom Katabolismus niedermolekularer Substanzen erwarteten. Die Moglichkeit besteht, dass die Pentose aus Nukleinsauren und saureloslichen Oligonukleotiden als Quelle der uberschussigen Milchsaure entstammt. Dies wurde unter anarobischen Bedingungen eine weitere Energiereserve fur Tumorzellen darstellen.