Ischemic Cellular Damage Research Articles

Puerarin reduces cell damage from cerebral ischemia-reperfusion by inhibiting ferroptosis

This study explores the protective effects of Puerarin, a compound derived from the traditional Chinese herb Pueraria, against cellular damage induced by Oxygen-Glucose Deprivation/Reoxygenation (OGD/R) in PC12 cells. The research focuses on understanding how Puerarin influences the mechanisms of ferroptosis and oxidative stress, key factors in ischemia-reperfusion injury relevant to neurodegenerative diseases. In our in vitro model, we identified the optimal OGD duration to induce significant cell stress and confirmed the non-toxicity of Puerarin up to 100uM. The results reveal that Puerarin substantially mitigates the detrimental effects of OGD/R, including improvements in cell viability, mitochondrial integrity, and reductions in oxidative stress markers like ROS and lipid peroxidation. Notably, Puerarin modulates key proteins in the autophagy process and the Nrf2 pathway, crucial in cellular stress responses. Further, the use of 3-Methyladenine, an autophagy inhibitor, underscores the significance of autophagy in managing OGD/R-induced stress. These findings suggest Puerarin's potential as a therapeutic agent for conditions characterized by ischemic cellular damage, highlighting the need for further clinical exploration.

Intranasal Erythropoietin Protects CA1 Hippocampal Cells, Modulated by Specific Time Pattern Molecular Changes After Ischemic Damage in Rats.

Erythropoietin, a multitarget molecule exhibited neuroprotective properties, especially against cerebral ischemia. However, little effort has been made to determinate both the administration pathway and doses that diminishes neuronal damage. In this study, we investigate the effect on CA1 region of different intranasal doses of rHuEPO (500, 1000 and 2500IU/kg) applied in distinct post-damage times (1, 6, and 24h) against ischemic cellular damage. Furthermore, most effective dose and time were used to evaluate gen and protein expression changes in 3 key molecules (EPO, EPOR, and βcR). We established that CA1-region present histopathological damage in this ischemia model and that rHuEPO protects cells against damage, particularly at 1000IU dose. Molecular data shows that EPO and EPOR gene expression are upregulated in a short term after damage treatment with rHuEPO (1h); oppositely, BcR is upregulated in ischemic and Isc + EPO. Protein expression data displays no changes on EPO expression in evaluated times after treatment, but a tendency to increase 24h after damage; in the opposite way, EPOR is upregulated significantly 6h after treatment and this effect last until 24h. So, our data suggest that a single intranasal dose of rHuEPO (1h post-injury) provides histological neurorestoration in CA1 hippocampal region, even if we did not observe a dose-dependent dose effect, the medium dose evaluated (1000UI/kg of b.w.) was more effective and sufficient for induces molecular changes that provides a platform for neuroprotection.

Early verification of myocardial ischemia with a novel biomarker of acute tissue damage: C-reactive protein fractional forms

BackgroundWe evaluated the utility of an independent biomarker of early ischemic cellular damage—circulating fractional forms of C-reactive protein (fracCRP), to verify the diagnostic relevance of low Troponin I (TnI) values within the context of a workup for Acute Coronary Syndrome (ACS). MethodsOn a semi-preparative scale, the molecular characteristics of fracCRP were established by electron microscopy and Western Blot, using isolates captured from patient serum on phosphorylcholine beads and purified by size exclusion high-pressure liquid chromatography (SE-HPLC). Captured on an analytical scale, the diagnostic utility of fracCRP was evaluated in first-draw plasma specimens (total CRP not exceeding 6mg/l) recovered from 300 cardiac emergency patients with final discharge diagnoses of ACS ruled out (N=132) or ruled in (N=168). ResultsAt a cutoff value chosen for 97.7% test specificity, the test metric (fracCRP×TnI) identified in the first blood draw 39.9% of all emergency patients ultimately diagnosed with ACS, and 17.9% of ultimately diagnosed patients who arrived with TnI values within the normal reference range (0.01–0.04ng/ml). ConclusionsThese findings suggest that the fracCRP test metric could serve as a rule-in test for ACS in a significant proportion of low to moderate risk emergency patients.

The role of serotonin in ischemic cellular damage and the infarct size-reducing effect of sarpogrelate, a 5-hydroxytryptamine-2 receptor blocker, in rabbit hearts

ObjectivesWe aimed to clarify the relation between sarpogrelate (SG), a 5-hydroxytryptamine (5-HT)-2 receptor blocker, and myocardial interstitial serotonin or infarct size during ischemia and reperfusion. BackgroundIn cardiac tissues serotonin is rich in vascular platelets, mast cells, sympathetic nerve endings, and the receptors are present in platelets and cardiomyocytes. MethodsThe myocardial interstitial serotonin levels were measured using a microdialysis technique during 30-min ischemia with and without SG in in vivo as well as isolated rabbit hearts. Other rabbits underwent 30 min of ischemia and 48 h of reperfusion, and the effect of SG on the infarct size was investigated in the absence and presence of a selective protein kinase C (PKC) inhibitor, chelerythrine (5 mg/kg, intravenously), or a mitochondrial adenosine triphosphate sensitive potassium (KATP) channel blocker, 5-hydroxydecanoate (5-HD) (5 mg/kg, intravenously). In another series, the effect of SG on PKC isoforms in cytosol and membrane fraction was assessed after a 20-min global ischemia in isolated rabbit hearts. ResultsInterstitial serotonin levels were markedly increased during 30-min ischemia in in vivo and isolated hearts, and the increases were inhibited by SG in each. The infarct size was reduced by SG (27 ± 2% vs. 40 ± 3% of control). This effect was blocked by chelerythrine and 5-HD, respectively. Sarpogrelate further enhanced the ischemia-induced translocation of PKC-ϵ to the membrane fraction. ConclusionsSarpogrelate reduces the myocardial infarct size by inhibiting the serotonin release followed by enhancement of PKC-ϵ translocation and opening of the mitochondrial KATP channel in ischemic myocytes.

Hydrogen Peroxide Stimulates c-Src-mediated Big Mitogen-activated Protein Kinase 1 (BMK1) and the MEF2C Signaling Pathway in PC12 Cells: POTENTIAL ROLE IN CELL SURVIVAL FOLLOWING OXIDATIVE INSULTS

Reactive oxygen species, generated by reduction-oxidation (redox) reactions, have been recognized as one of the major mediators of ischemia and reperfusion injury in the brain. Reactive oxygen species-induced cerebral events are attributable, in part, to the change in intracellular signaling molecules including mitogen-activated protein (MAP) kinases. Big MAP kinase 1 (BMK1), also known as ERK5, is a newly identified member of the MAP kinase family and has been reported to be sensitive to oxidative stress. In the present study, we examined the effect of H(2)O(2) on BMK1 activity in PC12 cells, and we investigated the pathophysiological implication of BMK1. Findings showed that BMK1 was rapidly and significantly activated by H(2)O(2) in a concentration-dependent manner in PC12 cells. BMK1 activation by H(2)O(2) was inhibited by both PD98059 and U0126, which were reported to inhibit MEK5 as well as MEK1/2. c-Src was suggested to be involved in BMK1 activation from the experiments with herbimycin A and PP2, specific inhibitors of Src family kinases. Transfection of kinase-inactive Src also inhibited H(2)O(2)-induced BMK1 activation. In addition, H(2)O(2) treatment of cells induced an enhancement of DNA binding activity of MEF2C, a downstream transcription factor of BMK1 in PC12 cells. Finally, pretreatment of cells with PD98059 and U0126 resulted in an increase in cell death including apoptosis by H(2)O(2) in ERK1/2 down-regulated cells as well as in intact PC12 cells. These findings suggest that c-Src mediated BMK1 activation by H(2)O(2) may counteract ischemic cellular damage probably through the activation of MEF2C transcription factor.

Myocardial protection by ischemic preconditioning and δ-opioid receptor activation in the isolated working rat heart

Objective: δ-Opioid receptors are involved in the cardioprotective effect of ischemic preconditioning. This study was designed (1) to assess the protective capacities of ischemic preconditioning and the synthetic δ-opioid receptor agonist D-Ala2-D-Leu5 enkephalin (DADLE) in a functionally oriented experimental model of ischemia and reperfusion and (2) to assess whether the effects of both protective measures are similarly blocked by naloxone, a nonspecific δ-opioid receptor antagonist. Methods: Sixty-four isolated working rat hearts were subjected to 45 minutes of hypothermic ischemia at 30°C followed by 25 minutes of normothermic reperfusion. Rats were pretreated with DADLE (1 mg/kg body weight intravenously), naloxone (3 mg/kg body weight intravenously), or a combination thereof within 60 minutes before onset of isolated heart perfusion. During the preischemic perfusion period, 8 hearts per group were preconditioned by one cycle of 5 minutes of normothermic global ischemia and subsequent reperfusion whereas another 8 served as nonpreconditioned controls. The postischemic functional recovery of hearts and their creatine kinase leakage were determined. Results: Pretreatment with DADLE and ischemic preconditioning improved the postischemic recovery of aortic flow when compared with nonpreconditioning (57.7% ± 4.0% and 60.8% ± 4.3% vs 40.0% ± 4.2% of preischemic baseline value, P ||.001). Combined pretreatment with DADLE before ischemic preconditioning afforded additional aortic flow recovery compared with pretreatment with DADLE alone (68.6% ± 3.3% vs 57.7% ± 4.0% of preischemic baseline value; P =.038). With combined pretreatment, early postischemic creatine kinase release was lower than control in hearts without pretreatment (0.48 ± 0.11 vs 0.80 ± 0.12 IU/5 minutes per heart; P =.001). Naloxone abolished the beneficial functional effects of pretreatment with DADLE and ischemic preconditioning. Conclusions: Pharmacologic activation of δ-opioid receptors affords improvement of functional protection in isolated working rat hearts similar to that conferred by classic ischemic preconditioning. The combination of both pretreatments reduces ischemic cellular damage and further adds to postischemic functional recovery. These changes are reversed by naloxone, an observation providing evidence that ischemic preconditioning involves signaling through opioid receptors.J Thorac Cardiovasc Surg 2001;122:986-92

The role of directly applied hypothermia in spinal cord injury.

The effect of intense local hypothermia was evaluated in a precision model of spinal canal narrowing and spinal cord injury in rats. The spinal cord injury was cooled with a custom cooling well used over the epidural surface. Basso, Beattie, and Bresnahan (BBB) motor scores and transcranial magnetic motor-evoked potential (tcMMEP) responses were used after injury to accurately evaluate neurologic recovery. This study was undertaken to determine whether the prognosis for neurologic recovery in a standardized rat spinal cord injury model is altered by the direct application of precisely controlled hypothermia to the area of injury. The role of hypothermia in the treatment of spinal cord injuries with neurologic deficits remains undefined. Hypothermia may decrease an area of spinal cord injury and limit secondary damage, therefore improving neurologic recovery. However, it has been difficult to consistently apply localized cooling to an area of spinal cord injury, and the use of systemic hypothermia is fraught with complications. This fact, along with the unavailability of a precise spinal cord injury model, has resulted in inconsistent results, both clinically and in the laboratory. In a rat model of spinal cord injury, 37 C and 19 C temperatures were used to study the role of hypothermia on neurologic recovery. Male Spraque-Dawley rats (n = 52; weight, 277.7 g) were anesthetized with pentobarbital and subjected to laminectomy at T10. The rats were divided into three groups: 1) placement of a 50% spacer in the epidural space (16 rats), 2) severe (25 g/cm) spinal cord injury (16 rats), 3) 50% spacer in combination with spinal cord injury (16 rats). Eight rats in each group were tested at two temperatures: normothermic (37 C) and hypothermic (19 C). With the use of a specially designed hypothermic pool placed directly over the spinal cord for 2 hours, epidural heating to 37 C, and epidural cooling to 19 C was accomplished. Simultaneous measurements of spinal cord and body temperatures were performed. The rats underwent behavior testing using the BBB motor scores and serial tcMMEPs for 5 weeks. Statistical methods consisted of Student's t tests, one-way analysis of variance, Tukey post hoc t tests and chi2 tests. There was a significant improvement in motor scores in rats subjected to hypothermia compared with those that were normothermic after insertion of a 50% spacer. This improvement was observed during the 5-week duration of follow-up. In the severe spinal cord injury group and the spinal cord injury-spacer groups, no significant improvement in motor scores were obtained when the spinal cord was exposed to hypothermia. The results demonstrate that there is a statistically significant (P < 0.05) improvement in neurologic function in rats subjected to hypothermia (19 C) after insertion of a spacer that induced an ischemic spinal cord injury. This indicates that directly applied hypothermia may be beneficial in preventing injury secondary to ischemic cellular damage. The data demonstrated minimal therapeutic benefit of hypothermia (19 C) after a severe spinal cord injury.

Identification of cerebral ischemic lesions in rat using eigenimage filtered magnetic resonance imaging

An accurate noninvasive time-independent identification of an ischemic cerebral lesion is an important objective of magnetic resonance imaging (MRI). This study describes a novel application of a multiparameter MRI analysis algorithm, the Eigenimage (EI) filter, to experimental stroke. The EI is a linear filter that maximizes the projection of a desired tissue (ischemic tissue) while it minimizes the projection of undesired tissues (nonischemic tissue) onto a composite image called an eigenimage. Rats ( n=26) were subjected to permanent middle cerebral artery occlusion. T2- and T1-weighted coronal MRI were acquired on separate groups of animals. The animals were immediately sacrificed after each imaging session for histopathological analysis of tissue at 4–8 h, 16–24 h, and 48–168 h after stroke onset. Lesion areas from MRI were defined using EI. The EI defined lesion areas were coregistered and warped to the corresponding histopathological sections. The ischemic lesion as defined by EI exhibited ischemic cell damage ranging from scattered acute cell damage to pan necrosis. Ischemic cellular damage was not detected in homologous contralateral hemisphere regions. EI lesion areas overlaid on histopathological sections were significantly correlated ( r=0.92, p<0.05) acutely, ( r=0.98, p<0.05) subacutely, and ( r=0.99, p<0.05) chronically. These data indicate that EI methodology can accurately segment ischemic damage after MCA occlusion from 4–168 h after stroke.

Effect of ethanol on pancreatic interstitial pH and blood flow in cats with chronic pancreatitis.

The objective of this study was to investigate the effects of ethanol on pancreatic blood flow and interstitial pH in chronic pancreatitis. Ethanol is known to contribute to the development of both acute and chronic pancreatitis. However, it is unclear how ethanol precipitates episodes of acute pancreatic inflammation in the setting of chronic pancreatitis. In a model of chronic pancreatitis in cats, it is known that pancreatic blood flow is abnormally low and decreases further after ethanol ingestion. Because it is possible that this reduction in blood flow might be damaging to the pancreas, we investigated the effects of ethanol on pancreatic interstitial pH, an index of pancreatic ischemia. In normal cats and cats with obstructive chronic pancreatitis, pancreatic blood flow and interstitial pH were measured using the hydrogen gas clearance technique and pH microelectrode, respectively. In normal cats, intragastric, but not intravenous, ethanol reduced both pancreatic blood flow by 62% (p < 0.05) and interstitial pH (7.38 +/- 0.03 to 7.20 +/- 0.03, p < 0.05). In cats with chronic pancreatitis in which basal pancreatic blood flow was already only 60% of normal flow, both intragastric and intravenous ethanol decreased both pancreatic blood flow (intragastric, 40% decrease, p < 0.05; intravenous, 34% decrease, p < 0.05) and interstitial pH (intragastric, 7.24 +/- 0.04 to 7.08 +/- 0.04, p < 0.05; intravenous 7.20 +/- 0.08 to 7.07 +/- 0.07, p < 0.05). This profound decrease in pH, lasting up to 2 hours after ethanol exposure in the chronic pancreatitis animals, suggests the possibility of ischemic cellular damage to the pancreas. These findings may explain the pathogenesis of bouts of acute pancreatic inflammation after ethanol ingestion in the setting of chronic disease.

Baclofen does not protect against cerebral ischemia in rats.

Presynaptic release of glutamate into the extracellular compartment and activation of receptor-operated calcium channels may contribute to ischemic neuronal damage. We evaluated the effect of baclofen, a selective inhibitor of presynaptic glutamate release, on mortality, working memory, and light microscopic hippocampal and cortical damage in the four-vessel occlusion model of cerebral ischemia using 64 male Wistar rats. Baclofen (10 mg/kg i.p.) given 1 hour before and 30-60 minutes after 20 minutes of global ischemia did not lessen mortality, prevent ischemic cellular damage, or significantly improve working memory compared with no treatment. We conclude that preischemic and postischemic administration of baclofen does not protect neurons from ischemic injury.

Plasma Level of the Creatine Phosphokinase BB Isoenzyme During Experimental Intestinal Ischemia

Changes in the serum of CPK-BB isoenzyme were investigated in dogs with intestinal ischemia in order to determine their use as an enzymatic diagnostic test for bowel infarction. The study consisted of 12 mongrel dogs. Group I (n = 6) was assigned as a control and was subjected to lumen obstruction of a jejunal segment 50-60 cm in length. Group II (n = 6) had an intestinal infarction created by ligation of the arteries of a jejunal segment 50-60 cm in length. A catheter was placed into the right atrium through the left jugular vein for blood sampling and monitoring. Blood samples were withdrawn every three hours for biochemical assays. CPK-BB measurements were performed by electrophoresis in agarose gel. CPK-BB remained unchanged in Group I during the course of the study (median range 17 to 20 IU) showing no significant difference from normal levels. Significant elevation of CPK-BB occurred three hours after establishing the ischemia in Group II (median 50 IU) as compared to Group I (p less than 0.05). The highest levels were determined to be 96 and 80 IU at the sixth and ninth hour, respectively. From hour 15 until completion of the experiment, no significant difference was detected between Groups I and II. We conclude that CPK-BB elevation in serum signifies its release from the smooth muscles of the bowel due to ischemic cellular damage.

Ischemia aggravating effects of platelet-activating factor in acute myocardial ischemia.

The effect of platelet-activating factor (PAF) was studied during the acute phase of myocardial ischemia in cats. PAF infusion (0.75 micrograms/kg/h for 4.5h) in anesthetized, open-chest cat decreased arterial blood pressure, but did not influence heart rate or biochemical indices of cell integrity. The same dose of PAF, however, started 30 min after coronary ligation, resulted in a significantly higher elevation of plasma creatine phosphokinase (CK) activity and a reduced CK content in the region of the ischemic myocardium. Treatment with the thromboxane A2 synthetase inhibitor, CGS-13080, significantly attenuated the PAF-aggravated ischemic cellular damage. These experiments suggest that hypoxia-generated PAF may contribute to the aggravation of myocardial ischemia, part of which appears to be due to PAF-induced release of thromboxane A2.

Protective effect of coenzyme Q10 against hypoxic cellular damage.

Heart cells from mouse embryos were cultured in either 5% CO2 and 95% air or 5% CO2 and 95% N2. Single myocardial cells showed frequent irregular beating under both conditions. Using this experimental system, we studied the effect of coenzyme Q10 (CoQ10) on the cellular functions of myocardial cells. Under the hypoxic conditions, CoQ10 decreased the irregular beating frequency without changing the beating rate. CoQ10 increased the cardiac adenosine 3', 5'-cyclic monophosphate (cAMP) level under the normal conditions and tended to decrease cardiac cAMP level under the hypoxic conditions. After incubation of deuterium-labeled CoQ10 (CoQ10-d5), the presence and levels of endogenous CoQ9, CoQ10 and exogenous CoQ10 were determined. Endogenous CoQ9 and CoQ10 were found in the mitochondrial fraction. Exogenous CoQ10 was found in the plasma membrane, cytosol and mitochondrial fractions. These findings confirm the protective effect of CoQ10 in ischemic heart disease. The exogeneous CoQ10 may provide protection against ischemic cellular damage by modulating cell functions.

Transmural cellular damage and blood flow distribution in early ischemia in pig hearts.

Transmural histological changes were determined morphometrically in the left ventricular free wall of 20 pigs after ligation of distal left anterior descending coronary artery for 10, 20, 40, and 120 minutes. Hemodynamics were recorded and regional blood flow distribution was measured in the ischemic zone. Coronary occlusion produced regional transmural ischemia without producing significant systemic hemodynamic change. The microsphere blood flow technique revealed that blood flow was less than 0.05 ml/min per g in all layers of the ischemic zone, i.e., inner, middle, and outer thirds. Ischemic cellular damage was classified and quantified from grade 0 to grade 5 (0 being normal and 5 being the most severe damage) with light microscopy and confirmed by electron microscopy. Layers of 200 micrometers immediately beneath the endocardium and epicardium showed minimal ischemic damage of less than grade 1.4 regardless of duration of ischemia in all hearts. In the ischemic left ventricular wall, except for the above layers, a definite transmural gradient of the cellular damage existed from the inner third (grade 2.3 +/- 0.1) to the outer third (grade 1.3 +/- 0.2) at 20 minutes of ischemia and at 40 minutes of ischemia (grades 3.6 +/- 0.1 and 1.9 +/- 0.3, respectively). The transmural ischemic damage gradient disappeared at 120 minutes of ischemia, where the inner and outer third ischemic grades were both 5.0 +/- 0.1. The data suggest that the limited ischemic damage which occurs in the few cell layers beneath endocardium and epicardium may be explained by regional collateral blood flow. An early ischemic damage wavefront phenomenon does exist in the pig myocardium and is independent of myocardial blood flow and its distribution. The transmural cell damage gradient may be the result of transmural gradients of wall stress and intramyocardial pressure in vivo. Therefore, it appears that factors other than blood flow are the major determinants of ischemic cellular damage in the left ventricular wall of hearts lacking a collateral blood supply.

Ultrastructural evaluation of the effects of global ischemia and reperfusion on human myocardium.

During open-heart surgery, myocardial biopsies were taken from 31 patients undergoing aortic valve replacement on total cardiopulmonary bypass. The first needle biopsy was taken before the induction of cardiac arrest (Kirsch cardioplegia), the second at the end of global ischemia, and the third during the reperfusion period. The tissue was investigated by electron microscopy using a semiquantitative scoring system for changes in both myocytes and blood vessels. Mitochondrial volume and surface density were determined by morphometry. Reversible ischemic injury of moderate to severe degree occurred in cardiac cells and in small blood vessels. On reperfusion, signs of damage regressed earlier in myocardial than in vascular tissue. Morphometry revealed significant mitochondrial swelling during the reperfusion phase, but this was not present after ischemia alone. It is concluded that Kirsch cardioplegia as applied here, is unable to protect the heart from ischemic cellular damage.