Model Of Ischemic Preconditioning Research Articles

Acidic preconditioning induced intracellular acid adaptation to protect renal injury via dynamic phosphorylation of focal adhesion kinase-dependent activation of sodium hydrogen exchanger 1

BackgroundDisruptions in intracellular pH (pHi) homeostasis, causing deviations from the physiological range, can damage renal epithelial cells. However, the existence of an adaptive mechanism to restore pHi to normalcy remains unclear. Early research identified H+ as a critical mediator of ischemic preconditioning (IPC), leading to the concept of acidic preconditioning (AP). This concept proposes that short-term, repetitive acidic stimulation can enhance a cell’s capacity to withstand subsequent adverse stress. While AP has demonstrated protective effects in various ischemia-reperfusion (I/R) injury models, its application in kidney injury remains largely unexplored.MethodsAn AP model was established in human kidney (HK2) cells by treating them with an acidic medium for 12 h, followed by a recovery period with a normal medium for 6 h. To induce hypoxia/reoxygenation (H/R) injury, HK2 cells were subjected to hypoxia for 24 h and reoxygenation for 1 h. In vivo, a mouse model of IPC was established by clamping the bilateral renal pedicles for 15 min, followed by reperfusion for 4 days. Conversely, the I/R model involved clamping the bilateral renal pedicles for 35 min and reperfusion for 24 h. Western blotting was employed to evaluate the expression levels of cleaved caspase 3, cleaved caspase 9, NHE1, KIM1, FAK, and NOX4. A pH-sensitive fluorescent probe was used to measure pHi, while a Hemin/CNF microelectrode monitored kidney tissue pH. Immunofluorescence staining was performed to visualize the localization of NHE1, NOX4, and FAK, along with the actin cytoskeleton structure in HK2 cells. Cell adhesion and scratch assays were conducted to assess cell motility.ResultsOur findings demonstrated that AP could effectively mitigate H/R injury in HK2 cells. This protective effect and the maintenance of pHi homeostasis by AP involved the upregulation of Na+/H+ exchanger 1 (NHE1) expression and activity. The activity of NHE1 was regulated by dynamic changes in pHi-dependent phosphorylation of Focal Adhesion Kinase (FAK) at Y397. This process was associated with NOX4-mediated reactive oxygen species (ROS) production. Furthermore, AP induced the co-localization of FAK, NOX4, and NHE1 in focal adhesions, promoting cytoskeletal remodeling and enhancing cell adhesion and migration capabilities.ConclusionsThis study provides compelling evidence that AP maintains pHi homeostasis and promotes cytoskeletal remodeling through FAK/NOX4/NHE1 signaling. This signaling pathway ultimately contributes to alleviated H/R injury in HK2 cells.

Chicago sky blue 6B exerts neuroprotective and anti-inflammatory effects on focal cerebral ischemia

Brain ischemia is one of the leading causes of death and long-term disability worldwide. Cessation of the blood supply to the brain directly stimulates many pathological events, including glutamate overload and neuroinflammation. Glial cell activation occurs shortly after ischemia onset, resulting in the release of proinflammatory cytokines and exacerbation of the detrimental effects of neuroinflammation. Proinflammatory signals influence the infiltration of a wide range of immune cells, including neutrophils, T cells and monocytes/macrophages. In this study, we aimed to verify the potential anti-inflammatory effect of Chicago Sky Blue 6B (CSB6B) in a rat model of focal cerebral ischemia (90-minute middle cerebral artery occlusion). CSB6B was administered 2 h before (pretreatment) or 1.5 h after reperfusion onset (posttreatment). A model of ischemic preconditioning was used as the comparator to pretreatment with CSB6B. The results of indicated that posttreatment with CSB6B had profound anti-inflammatory effects that were associated with reduced neurological deficits and a decreased infarct volume. At 24 h, 3 days and 7 days after brain ischemia, CSB6B administration reduced the protein levels of proinflammatory cytokines, such as Il1β, Il6, Il18 and TNFα, in the cerebral cortex and the dorsal striatum. Treatment with CSB6B also limited the scope of microglia and astrocyte activation and the infiltration of immune cells. Taken together, this study shows that compounds such as CSB6B might be promising pharmacological tools; however, further studies on the improvements in the drug-like properties of these compounds must be undertaken.

Inhibition of Vesicular Glutamate Transporters (VGLUTs) with Chicago Sky Blue 6B Before Focal Cerebral Ischemia Offers Neuroprotection

Brain ischemia is one of the leading causes of death and long-term disability in the world. Interruption of the blood supply to the brain is a direct stimulus for many pathological events. The massive vesicular release of glutamate (Glu) after ischemia onset induces excitotoxicity, which is a potent stress on neurons. Loading of presynaptic vesicles with Glu is the first step of glutamatergic neurotransmission. Vesicular glutamate transporters 1, 2, and 3 (VGLUT1, 2, and 3) are the main players involved in filling presynaptic vesicles with Glu. VGLUT1 and VGLUT2 are expressed mainly in glutamatergic neurons. Therefore, the possibility of pharmacological modulation to prevent ischemia-related brain damage is attractive. In this study, we aimed to determine the effect of focal cerebral ischemia on the spatiotemporal expression of VGLUT1 and VGLUT2 in rats. Next, we investigated the influence of VGLUT inhibition with Chicago Sky Blue 6B (CSB6B) on Glu release and stroke outcome. The effect of CSB6B pretreatment on infarct volume and neurological deficit was compared with a reference model of ischemic preconditioning. The results of this study indicate that ischemia upregulated the expression of VGLUT1 in the cerebral cortex and in the dorsal striatum 3 days after ischemia onset. The expression of VGLUT2 was elevated in the dorsal striatum and in the cerebral cortex 24 h and 3 days after ischemia, respectively. Microdialysis revealed that pretreatment with CSB6B significantly reduced the extracellular Glu concentration. Altogether, this study shows that inhibition of VGLUTs might be a promising therapeutic strategy for the future.

Renal Ischemia-reperfusion Injury Attenuated by Exosomes Extracted From Splenic Ischemic Preconditioning Models.

To investigate the protective effects of exosomes extracted from splenic ischemic preconditioning (sIPC) models on renal ischemia-reperfusion injury (IRI). sIPC was conducted on mice before renal IRI, and exosomes derived from sIPC mice were infused into a mouse model of renal IRI. The kidney tissue and serum were collected 24 h later. The morphological changes, inflammation and apoptosis in IR kidneys were determined by hematoxylin-eosin (HE), terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and Ki-67 immunohistochemical staining. In addition, the proinflammatory cytokines in serum and cell supernatant were measured with enzyme-linked immunosorbent assays (ELISAs). Then, we administered exosomes to mouse renal epithelial cells. TUNEL assays and flow cytometry were used to evaluate cell apoptosis. Bax and Bcl-2 levels were measured via Western blotting. HE staining showed that the renal IRI was attenuated after sIPC. TUNEL results showed that renal tissue apoptosis was greatly reduced after sIPC or injection of exosomes. ELISAs showed that the serum creatinine (sCr), tumor necrosis factor alpha, and interleukin-1 β levels induced by IRI decreased with sIPC. In vitro, exosomes extracted from the hypoxia/reoxygenation (H/R) splenic fibroblast model had the same protective effect. TUNEL and flow cytometry results showed that the exosomes reduced apoptosis. ELISAs showed that tumor necrosis factor alpha and interleukin-1 β were significantly increased in the H/R group but decreased due to the exosomes treated with starvation. WB results showed that Bax expression was increased and Bcl-2 expression was decreased in the H/R group. However, exosomes decreased the Bax level and increased the Bcl-2 level. Exosomes extracted from sIPC models exerted a protective effect to attenuate renal IRI.

Abstract WP245: Casein Kinase 2 Preconditions White Matter Against Ischemic Injury

White matter injury (WMI) causes debilitating neurological impairments observed in stroke patients. Ischemic preconditioning (IPC) has been investigated as a protective mechanism against stroke in experimental studies. However, mechanisms of IPC have been mainly studied in gray matter and in vivo rodent models of IPC are not clinically relevant. There is a need to develop clinically-applicable pharmacologically preconditioning approaches that protect the brain against ischemia. Casein kinase 2 (CK2) is upregulated during WMI. Consequently, we have previously reported that application of CX-4945, a CK2 selective inhibitor, protects axon function in young and aged in ex vivo WM stroke model. However, whether CX-4945 preconditioning exerts similar axon function protection and alleviates behavior deficits in ex vivo and in vivo of WMI models remains to be investigated. We hypothesize that pharmacologically preconditioning with CX-4945 protects young and old WM against ischemic injury and alleviates behavioral deficits. Mouse optic nerves (MONs), a pure white matter tract, were used to measure axon function in an ex vivo stroke model. Compound action potentials (CAPs) were evoked and recorded from control and treated MONs. CX-4945 preconditioning did not alter axon function at baseline but protected axon function during recovery against oxygen-glucose deprivation. Behavioral deficits were assessed using cylinder tests and pasta eating tests. At baseline, mice preferentially used both paws; while injured mice showed a biased use of contralateral paw over bilateral paw. Additionally injured mice spent longer time eating pasta and with higher frequency of drops indicating the loss of paw dexterity. Mice were administered orally either CX-4945 (75mg/kg) or vehicle twice/day for 5 days before undergoing WMI. Treated mice showed improved bilateral paw use, and maintained paw dexterity. This behavioral protective effects of CK2 inhibition correlated with survival of oligodendrocyte, preservation of axonal integrity and axonal mitochondrial dynamics. We demonstrated that WM preconditioning with CX-4945 protects axon function, WM integrity, and alleviates behavior deficits in young and age mice.

Abstract WP243: Csf1r Antagonism Abolishes Ischemic Preconditioning Mediated Protection In White Matter

Background: Ischemic preconditioning (IPC) is a robust protective phenomenon whereby brief ischemic exposure confers protection against a subsequent prolonged ischemic challenge. IPC has been studied primarily in gray matter predominant models, however, stroke significantly impacts white matter (WM). We have previously reported development of a WM IPC model in the mouse optic nerve (MON), a fully myelinated CNS WM tract. We identified innate immune signaling pathways as required for axonal protection, however the cell type(s) responsible for IPC in WM are unknown. Here we characterize the effects of microglial depletion on IPC-induced protection against OGD-mediated injury to: (i) axonal compound action potential (CAP) recovery, (ii) axonal structural integrity, (iii) oligodendrocyte viability and (iv) nodes of Ranvier (NoR) in MON. Methods: Following microglial depletion by pharmacologic treatment with colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622, MONs were exposed to transient ischemia in vivo, acutely isolated, and subjected to oxygen-glucose deprivation (OGD) ex vivo to simulate a severe ischemic injury. Functional and structural axonal recovery was assessed by electrophysiology with recording of CAP and immunofluorescent/confocal microscopy followed by quantitative stereology. Results: Microglial depletion eliminated IPC-mediated protection of axonal function but intriguingly had no effect on recovery after acute ischemic injury alone (i.e. in the absence of IPC). Microglial depletion abrogated IPC-mediated protective effects on both axonal integrity and the survival of mature (APC+) oligodendrocytes after exposure to OGD. IPC-mediated protection was determined to be independent of retinal injury. Effects on NoR remain under investigation. Conclusions: Based on these findings, we conclude that preconditioned, but not naïve, microglia are critical in the endogenous IPC-induced protective response against ischemic injury that occurs in WM. Thus, preconditioned microglia are a critical cellular target for future therapeutics designed to enhance WM recovery from acute ischemic injury (stroke). Furthermore, our data suggest that IPC-mediated protection in WM is anatomically intrinsic to WM.

CPKCγ-Modulated Autophagy Contributes to Ischemic Preconditioning-Induced Neuroprotection in Mice with Ischemic Stroke via mTOR-ULK1 Pathway.

Neuron-specific conventional protein kinase C (cPKC)γ mediates cerebral hypoxic preconditioning (HPC). In parallel, autophagy plays a prosurvival role in ischemic preconditioning (IPC) against ischemic stroke. However, the effect of cPKCγ on autophagy in IPC still remains to be addressed. In this study, adult and postnatal 1-day-old C57BL/6J wild-type (cPKCγ+/+) and knockout (cPKCγ-/-) mice were used to establish in vivo and in vitro IPC models. The results showed that IPC pretreatment alleviated neuronal damage caused by lethal ischemia, which could be suppressed by autophagy inhibitor 3-MA or bafilomycin A1. Meanwhile, cPKCγ knockout blocked IPC-induced neuroprotection, accompanied by significant increase of LC3-I to LC3-II conversion and Beclin 1 protein level, and a significant decrease in p62 protein level. Immunofluorescent staining results showed a decrease of LC3 puncta numbers in IPC-treated cPKCγ+/+ neurons with fatal ischemia, which was reversed in cPKCγ-/- neurons. In addition, cPKCγ-modulated phosphorylation of mTOR at Ser 2448 and ULK1 at Ser 555, rather than p-Thr-172 AMPK, was detected in IPC-pretreated neurons upon lethal ischemic exposure. The present data demonstrated that cPKCγ-modulated autophagy via the mTOR-ULK1 pathway likely modulated IPC-induced neuroprotection.

Ischemic preconditioning/ischemic postconditioning alleviates anoxia/reoxygenation injury via the Notch1/Hes1/VDAC1 axis.

Ischemic preconditioning (IPC), and ischemic postconditioning (IPost) have a significant protective effect on myocardial ischemia/reperfusion (MI/R) injury by alleviating oxidative stress and mitochondrial disturbances, although the underlying molecular mechanisms are unclear. The study was to demonstrate that cardioprotection against anoxia/reoxygenation (A/R) injury is transduced via the Notch1/Hes1/VDAC1 signaling pathway. Using mass spectrometry and tandem affinity purification (TAP), to screen for differentially expressed proteins associated with Hes1, followed by standard bioinformatics analysis. The co-immunoprecipitation (Co-IP) assay confirmed an interaction between Hes1 and VDAC1 proteins. H9c2 cells were transfected with Hes1 adenoviral N-terminal TAP vector (AD-NTAP/Hes1) and Hes1-short hairpin RNA adenoviral vector (AD-Hes1-shRNA) to establish A/R injury, IPC, and IPost models, respectively. The expression of Hes1 and VDAC1 proteins were measured by western blot analysis, while the levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and apoptosis were evaluated by flow cytometry. AD-NTAP/Hes1 can activate the exogenous protein expression of Hes1, thus decreasing creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activity and promoting cell viability. The study found that VDAC1 was a potential target protein for Hes1 and the overexpression of Hes1 protein expression downregulated protein expression levels of VDAC1, reduced ROS production, stabilized ΔΨm, and inhibited apoptosis in H9c2 cells. Additionally, downregulation of Hes1 protein expression also upregulated VDAC1 protein expression, increased ROS production, imbalanced ΔΨm, promoted cell apoptosis, and attenuated the cardioprotection afforded by IPC and IPost. The Notch1/Hes1 signaling pathway activated by IPC/IPost can directly downregulate the protein expression of VDAC1 and consequently relieve A/R injury.

Microglial depletion abolishes ischemic preconditioning in white matter.

Ischemic preconditioning (IPC) is a phenomenon whereby a brief, non-injurious ischemic exposure enhances tolerance to a subsequent ischemic challenge. The mechanism of IPC has mainly been studied in rodent stroke models where gray matter (GM) constitutes about 85% of the cerebrum. In humans, white matter (WM) is 50% of cerebral volume and is a critical component of stroke damage. We developed a novel CNS WM IPC model using the mouse optic nerve (MON) and identified the involved immune signaling pathways. Here we tested the hypothesis that microglia are necessary for WM IPC. Microglia were depleted by treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. MONs were exposed to transient ischemia in vivo, acutely isolated 72 h later, and subjected to oxygen-glucose deprivation (OGD) to simulate a severe ischemic injury (i.e., stroke). Functional and structural axonal recovery was assessed by recording compound action potentials (CAPs) and by microscopy using quantitative stereology. Microglia depletion eliminated IPC-mediated protection. In control mice, CAP recovery was improved in preconditioned MONs compared with non-preconditioned MONs, however, in PLX5622-treated mice, we observed no difference in CAP recovery between preconditioned and non-preconditioned MONs. Microgliadepletion also abolished IPC protective effects on axonal integrity and survival of mature (APC+ ) oligodendrocytes after OGD. IPC-mediated protection was independent of retinal injury suggesting it results from mechanistic processes intrinsic to ischemia-exposed WM. We conclude that preconditioned microglia are critical for IPC in WM. The "preconditioned microglia" phenotype might protect against other CNS pathologies and is a neurotherapeutic horizon worth exploring.

Non-invasive in vivo human model of post-ischaemic skin preconditioning by measurement of flow-mediated 460-nm autofluorescence.

Transient ischaemia and reperfusion (TIAR) induce early ischaemic preconditioning (IPC) in different tissues and organs, including the skin. IPC protects tissues by modifying the mitochondrial function and decreasing the amount of the reduced form of nicotinamide adenine dinucleotide (NADH). Skin 460-nm autofluorescence is proportional to the NADH content and can be non-invasively measured during TIAR. We propose a non-invasive in vivo human model of skin IPC for studying the effects of repeated TIARs on the NADH content. Fifty-one apparently healthy volunteers (36 women) underwent three 100-second forearm ischaemia episodes induced by inflation of brachial pressure cuff to the pressure of 60 mmHg above systolic blood pressure, followed by 500-second long reperfusion episodes. Changes in skin NADH content were measured using 460-nm fluorescence before and during each of the three TIARs. The first two TIARs caused a significant reduction in the skin NADH content before (P = .0065) and during the third ischaemia (P = .0011) and reperfusion (P = .0003) up to 3.0%. During the third TIAR, the increase in skin NADH was 20% lower than during the first ischaemia (P = .0474). The measurement of the 460-nm fluorescence during repeated TIARs allows for a non-invasive in vivo investigation of human skin IPC. Although IPC reduces the overall NADH skin content, the most noticeable NADH reduction appears during ischaemia after earlier TIARs. Studying the skin model of IPC may provide new avenues for in vivo physiological, clinical and pharmacological research on mitochondrial metabolism.

Abstract WP261: Dcr1 and Dcr2 Protect Neuron Against Ischemia and Hypoxia Damage via Differential Mechanisms on Ischemia Preconditioning-Induced Neuroprotection

Objective: To investigate the neuroprotective roles and mechanisms of DcR1 and DcR2 under ischemia and hypoxia. Methods: SH-SY5Y cell were used to build to oxygen glucose deprivation model (OGD) and ischemic preconditioning model (IPC) in vitro. CCK-8 and LDH were performed to evaluate cellular survival and cytotoxicity. Overexpressing DcR1, DcR2 and DR5 were induced by lentivirus vectors, knocking down DcR2 was conducted by siRNA technique in SHSY5Y cells. TRAIL Recombiant Human Protein was employed to activate the TRAIL induced extrinsic apoptotic pathway. Western blot was performed to evaluate the protein level of DcR1, DcR2, DR5, DR4, cleaved caspase-8, cleaved caspase-3 and tBid. Co-immunoprecipitation was used to investigate the interaction of DcR1, DcR2, DR5, DR4, TRAIL and caspase-8. Western blot was performed to evaluate the protein level of pAkt, and LY294002 was used to inhibit the activation of PI3K-Akt pathway. Results: We found that IPC ameliorated the cellular apoptosis and cytotoxicity ( P < 0.01), reduced the protein level of DR5 ( P < 0.05) and DR4 ( P < 0.01), and upregulated the protein level of DcR2 ( P < 0.05) after OGD. Besides, overexpressing DcR1 enhanced the protective effect of IPC to cells ( P = 0.045, P = 0.01), and knocking down DcR2 abated the protective effect of IPC ( P = 0.012, P =0.011) against OGD. Additionally, overexpressing DcR1 or knocking down DcR2 regulated expression of extrinsic apoptosis pathway related proteins, including caspase-8, cleaved caspase-3 and tBid. We also found that overexpressing DcR1 competitively combined TRAIL, suppressed DISC from assembling, and subsequently reduced extrinsic cellular apoptosis. Moreover, we discovered that overexpressing DcR2 inhibited the extrinsic apoptotic pathway via interacting with the extracellular domains of DR5, and up-regulated pAkt ( P = 0.0081). Conclution: (1) DcR1 competitively bound with TRAIL, inhibited DISC assembling, and subsequently reduced extrinsic cellular apoptosis. (2) DcR2 inhibited the extrinsic apoptotic pathway via interacting with extracellular domains of DR5. (3) Besides with inhibiting TRAIL, overexpressing DcR2 exerted neuron protection by activating PI3K/Akt pathway.

Ischemic Preconditioning Protects Astrocytes against Oxygen Glucose Deprivation Via the Nuclear Erythroid 2-Related Factor 2 Pathway.

Induction of ischemic preconditioning (IPC) represents a potential therapy against cerebral ischemia by activation of adaptive pathways and modulation of mitochondria to induce ischemic tolerance to various cells and tissues. Mitochondrial dysfunction has been ascribed to contribute to numerous neurodegenerative conditions and cerebral ischemia. Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that has traditionally been involved in upregulating cellular antioxidant systems to combat oxidative stress in the brain; however, the association of Nrf2 with mitochondria in the brain remains unclear. In the present study, we investigated the effects of Nrf2 on (i) IPC-induced protection of astrocytes; (ii) OXPHOS protein expression; and (iii) mitochondrial supercomplex formation.Oxygen-glucose deprivation (OGD) was used as an in vitro model of cerebral ischemia and IPC in cultured rodent astrocytes derived from WT C57Bl/6J and Nrf2-/- mice. OXPHOS proteins were probed via western blotting, and supercomplexes were determined by blue native gel electrophoresis.IPC-induced cytoprotection in wild-type, but not Nrf2-/- mouse astrocyte cultures following a lethal duration of OGD. In addition, our results suggest that Nrf2 localizes to the outer membrane in non-synaptic brain mitochondria, and that a lack of Nrf2 in vivo produces altered supercomplex formation in mitochondria.Our findings support a role of Nrf2 in mediating IPC-induced protection in astrocytes, which can profoundly impact the ischemic tolerance of neurons. In addition, we provide novel evidence for the association of Nrf2 to brain mitochondria and supercomplex formation. These studies offer new targets and pathways of Nrf2, which may be heavily implicated following cerebral ischemia.

PGC-1α Downregulation in Steatotic Liver Enhances Ischemia-Reperfusion Injury and Impairs Ischemic Preconditioning.

Liver steatosis is associated with mitochondrial dysfunction and elevated reactive oxygen species (ROS) levels together with enhanced sensitivity to ischemia-reperfusion (IR) injury and limited response to preconditioning protocols. Here, we sought to determine whether the downregulation in the steatotic liver of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), a master regulator of mitochondrial metabolism and ROS that is known to play a role in liver metabolic control, could be responsible for the sensitivity of the steatotic liver to ischemic damage. PGC-1α was induced in normal liver after exposure to an IR protocol, which was concomitant with an increase in the levels of antioxidant proteins. By contrast, its induction was severely blunted in the steatotic liver, resulting in a modest induction of antioxidant proteins. Livers of PGC-1α-/- mice on a chow diet were normal, but they exhibited an enhanced sensitivity to IR injury and also a lack of response to ischemic preconditioning (IPC), a phenotype that recapitulated the features of the steatotic liver in terms of liver damage, although the inflammatory response differed between both models. Utilizing an in vitro model of IPC, we found that PGC-1α expression was downregulated in hepatic cells cultured at 1% O2; whereas it was induced after reoxygenation (3% O2), and it was responsible for the recovery of antioxidant gene expression after the ischemic period. Innovation & Conclusion: PGC-1α plays an important role in the protection against IR injury in the liver, which is likely associated with its capacity to induce antioxidant gene expression. Antioxid. Redox Signal. 27, 1332-1346.

Intervention action of total flavonoids from root of Ilex pubescens in cerebral ischemic tolerance with blood stasis

The aim of this study was to explore the action characters of total flavonoids from MDQ on cerebral ischemic tolerance with blood stasis. Fully understanding the mechanism of action of total flavonoids from MDQ is helpful for the development of new drugs and the utilization of resources. Male Wistar rat model of blood stasis was established by injecting dexamethasone into the intramuscular side of the thigh. Then they were given related drugs via an intragastric administration for a successive 10 days. After 7 days, the following occurred: firstly, the method of blocking the bilateral common carotid artery (CCA) was used for 10 min, followed by a restoration of perfusion. After 72 h, we performed a temporary occlusion of the rat’s middle cerebral artery for 2 h with an intraluminal thread method. This was followed by reperfusion for 24 h, respectively, to establish the rat model of cerebral ischemic tolerance with blood stasis. Viscosity of the whole blood was measured after the last administration was given blood. Brain was removed, and then the activity of ATP enzyme and T-SOD was determined. To observe the pathological changes of the hippocampus area by HE staining, and the expression of Bcl-2 and Bax were observed by immunohistochemical method. The rat model of cerebral ischemic tolerance with blood stasis was copied successfully. The whole blood viscosity, the activity of NOS, the content of Gluin in the ischemic brain in the IPC model group and the ischemia–reperfusion group were increased significantly. The activity of ATPase was decreased significantly. Compared with the ischemia–reperfusion model group, the activity of ATPase and the whole blood viscosity in the ischemic preconditioning (IPC) group were increased significantly. The activity of NOS and the content of Gluin were decreased significantly. The degree of pathological injury of the brain tissue was also relieved significantly. Total flavonoids of MDQ were used, improving blood circulation, improving energy metabolism, activating endogenous anti-oxidative capability, enhancing the antiapoptotic effect, and relieving the injury of the nerve cell. Hence, the use of MDQ flavonoids improves the tolerance ability of cerebral ischemia.

Ischemic preconditioning inhibits over-expression of arginyl-tRNA synthetase gene Rars in ischemia-injured neurons.

The expression changes of Rars gene in ischemia-injured neurons were investigated by detecting its translational product arginyl-tRNA synthetase (ArgRS), and the inhibitory effects of ischemic preconditioning (IPC) on Rars gene were explored. Both IPC model and prolonged ischemia (PI) model were established by using the classic oxygen glucose deprivation (OGD) method. The primary cultured neurons were assigned into the following groups: the experimental group (IPC+PI group), undergoing PI after a short period of IPC; the conditional control group (PI control group), subjected to PI without IPC; blank control group, the normally cultured neurons. The Rars transcriptional activities and ArgRS expression levels were measured at different time points after re-oxygenation (3 h/6 h/12 h/24 h). Data were collected and statistically analyzed. Compared to the blank control group, the Rars activities and ArgRS levels were significantly increased in PI control group, peaking at the time point of 6 h after re-oxygenation. Rars activities and ArgRS levels were significantly lower in the experimental group than in the PI control group at different time points after re-oxygenation. PI insult can induce an escalating activity of Rars and lead to ArgRS over-expression in primary cultured neurons. IPC can inhibit the increased Rars activity and down-regulate ArgRS expression of ischemia-insulted neurons. This mechanism may confer ischemic tolerance on neurons.

DNA Content in Extracellular Vesicles Isolated from Porcine Coronary Venous Blood Directly after Myocardial Ischemic Preconditioning

BackgroundExtracellular vesicles (EV) are nano-sized membranous structures released from most cells. They have the capacity to carry bioactive molecules and gene expression signals between cells, thus mediating intercellular communication. It is believed that EV confer protection after ischemic preconditioning (IPC). We hypothesize that myocardial ischemic preconditioning will lead to rapid alteration of EV DNA content in EV collected from coronary venous effluent.Materials and MethodsIn a porcine myocardial ischemic preconditioning model, EV were isolated from coronary venous blood before and after IPC by differential centrifugation steps culminating in preparative ultracentrifugation combined with density gradient ultracentrifugation. The EV preparation was validated, the DNA was extracted and further characterized by DNA sequencing followed by bioinformatics analysis.ResultsPorcine genomic DNA fragments representing each chromosome, including mitochondrial DNA sequences, were detected in EV isolated before and after IPC. There was no difference detected in the number of sequenced gene fragments (reads) or in the genomic coverage of the sequenced DNA fragments in EV isolated before and after IPC. Gene ontology analysis showed an enrichment of genes coding for ion channels, enzymes and proteins for basal metabolism and vesicle biogenesis and specific cardiac proteins.ConclusionsThis study demonstrates that porcine EV isolated from coronary venous blood plasma contain fragments of DNA from the entire genome, including the mitochondria. In this model we did not find specific qualitative or quantitative changes of the DNA content in EV collected immediately after an in vivo myocardial IPC provocation. This does not rule out the possibility that EV DNA content changes in response to myocardial IPC which could occur in a later time frame.

Effects of circulating microvesicles derived from myocardial ischemic preconditioning on myocardial ischemia/reperfusion injury in rats

To investigate the effects of circulating microvesicles (MVs) derived from ischemic preconditioning (IPC) on myocardial ischemia/reperfusion (I/R) injury in rats and explore the underlying mechanism. To establish the IPC model, the rats were subjected to brief cycles of left anterior descending (LAD) coronary occlusion and reperfusion. The blood was drawn from abdominal aorta once the operation was finished. IPC-MVs were isolated by ultracentrifugation from the peripheral blood and characterized by flow cytometry. The myocardial I/R model of rats was established in vivo. Rats were injected via the femoral vein with IPC-MVs at 7 mg/kg. Morphological changes of myocardium were observed microscopically after HE staining. Apoptosis of myocardial cells was detected with TUNEL assay. Myocardial infarct size was detected by TTC staining. Moreover, activity of plasma lactate dehydrogenase (LDH) was tested by colorimetry. The activity of caspase 3 in myocardium was assayed with spectrophotometry. Expression levels of Bcl-2 and Bax protein were examined with Western blot. The concentration of IPC-MVs, which was detected by flow cytometry, was 4380±745 cells/μl. Compared with I/R group, IPC-MVs alleviated the damage of tissues in I/R injured rats significantly. The myocardial infarct size and the cardiomyocyte apoptotic index were obviously decreased after IPC-MVs treatment (P<0.01, respectively). The activity of plasma LDH was significantly decreased in IPC-MVs treated rats (P<0.01). Moreover, the activity of caspase 3 was markedly decreased after IPC-MVs treatment (P<0.01). In addition, the expression of Bcl-2 was increased (P<0.01), the expression of Bax was decreased (P<0.01), the ratio of Bcl-2/Bax was significantly increased after IPC-MVs treatment (P<0.01). IPC-MVs protected myocardial against I/R injury by up-regulating the expression of Bcl-2 protein, down-regulating the expression of Bax protein, increasing the ratio of Bcl-2/Bax and decreasing cleavage of caspase 3.

Abstract 75: Ischemic Heart Failure is Exacerbated in CD39-null Mice

Background: CD39 (ectonucleoside triphosphate diphosphohydrolase) is a nucleotidase expressed on endothelial cells, vascular smooth muscles cells, and leukocytes. CD39 plays a key role in vascular homeostasis, hydrolyzing extracellular ATP and ADP. CD39 has been shown to be important in models of ischemic preconditioning and cardiac ischemia reperfusion. However, the effect of CD39 activity on functional recovery of heart after myocardial infarction (MI) has not been evaluated. Hypothesis: Genetic ablation of CD39 expression exacerbates post-myocardial infarction cardiac function and fibrosis. Methods: Wild-type (WT) and CD39-null mice were subjected to coronary artery ligation. Cardiac function and protein evaluation of fibrotic markers was performed at day 28 post-MI. Results: Evaluation at Day 28 post-MI revealed that while mice of both genotypes had similarly reduced ejection fraction and equally compromised contractile function (dP/dtmax), there was a more pronounced negative effect on lusitropy (dP/dtmin) and increased left ventricular end-diastolic pressure in CD39-null mice. Therefore, cd39 gene ablation associates with the development of worsening cardiac performance. Histological analysis revealed increased collagen deposition and abundance of alpha-smooth muscle actin (αSMA) positive interstitial cells in the CD39-null hearts compared to WT hearts. To quantify these findings immunoblot analysis for collagen and αSMA was performed. We found that collagen and αSMA were increased at Day 28 post-MI, in CD39-null hearts compared to WT hearts. Conclusion: CD39 ablation has detrimental effects on post-MI recovery, resulting in diminished cardiac performance and increased fibrosis.

Myocardial ischemic preconditioning in a porcine model leads to rapid changes in cardiac extracellular vesicle messenger RNA content

BackgroundExtracellular vesicles (EVs) are thought to exert protective effects after ischemic and remote ischemic preconditioning. It is not well understood which EV content factors are most relevant for protective effects. We hypothesize that ischemic preconditioning leads to qualitative changes in EV mRNA content and quantitative changes in EV size and number. MethodsUsing an in vivo porcine ischemic preconditioning model, EVs were collected from coronary venous blood, and isolated by differential ultracentrifugations. The presence and purity of EV were verified by electron microscopy and Western blot, and EV number was assessed by nanoparticle tracking analysis. The mRNA EV was identified by microarray. ResultsGene ontology analysis showed enrichment of EV mRNA coding for proteins associated with regulation of transcription, translation, extracellular matrix, morphogenic development and feeding behavior. There were 11,678 different mRNA transcripts detected in EV, where a total of 1103 was significantly increased or decreased after preconditioning, of which 638 mRNA sequences were up-regulated and/or emerged due to preconditioning. Several of them have known association with ischemic preconditioning. There was no significant difference in EV quantity or size before and after preconditioning. ConclusionsThese findings demonstrate in an in vivo model that myocardial ischemic preconditioning influences the composition of mRNA in EV, including gene transcripts for proteins associated with the protective effect of ischemic preconditioning. The finding that preconditioned parental cells release EV containing mRNA that is qualitatively different from those released by non-preconditioned cells shows the importance of the external milieu on parental cell EV production.

The 4th Window: A Novel, Clinically Relevant Model of Delayed Ischemic Preconditioning in Conscious, Chronically Instrumented Pigs

Most studies on cardiac ischemic preconditioning (IPC) focused on the 1st or 2nd window, where the preconditioning ischemic stimulus was induced briefly and the cardiac protection all occurred within a few hours or days. More recently 3rd window models have been described with IPC induced over several days. The goal of this investigation was to determine if IPC could be extended for a longer time, making it more clinically relevant. In this new IPC model, which we named the 4th window, pigs, chronically instrumented with coronary blood flow transducers, hydraulic occluders, and ultrasonic wall thickness crystals implanted in potentially ischemic and non‐ischemic zones, underwent IPC induced by 6 episodes of 90 min coronary stenosis (40% reduction in blood flow but no necrosis) every 12 hours over 3 days. 60 min coronary artery occlusion (CAO) followed by 3 days of coronary reperfusion, induced one week after IPC, resulted in infarct size, as a fraction of the area at risk, of 11±2.5%, less (p&lt;0.05) than in the absence of IPC (42±3.9%). Most genes expressed in this model were novel, compared to prior IPC models, included genes mediating angiogenesis. To confirm angiogenesis, we found increased capillary density adjacent to the infarct in the 4th window by 154% (p&lt;0.05), and 38% increased, p&lt;0.05, blood flow (measured with microspheres) during the 60min CAO, compared in the same samples with brief CAO before IPC. In summary, this novel model of 4th window IPC induces angiogenesis and markedly different gene regulation than previous IPC models.