Ischemic Tolerance Research Articles

Peptidylarginine Deiminase 2 Knockout Improves Survival in hemorrhagic shock.

The peptidylarginine deiminase (PAD) family converts arginine into citrulline through protein citrullination. PAD2 and PAD4 inhibitors can improve survival in hemorrhagic shock (HS). However, the impact of isoform-specific PAD inhibition in improving survival has not been studied. In this study, we utilize selective Pad2 knockout mice to elucidate loss of function of PAD2 leads to pro-survival effect in HS. HS: Pad2 and wild-type (WT) mice (n = 5/group) were subjected to lethal HS (55% volume hemorrhage). Survival was monitored over 7 days. Myocardial infarction (MI): Pad2 and WT mice (n = 9/group) were subjected to MI by permanent LAD ligation to examine the effect of ischemia on the heart. After 24 h cardiac function and infarct size were measured. HS: Pad2 mice demonstrated 100% survival compared with 0% for WT mice (P = 0.002). In a sub-lethal HS model, cardiac β-catenin levels were higher in Pad2 compared with WT after 24 h. MI: WT mice demonstrated larger MI (75%) compared with Pad2 (60%) (P < 0.05). Pad2 had significantly higher ejection fraction and fractional shortening compared with WT (P < 0.05). Pad2 improves survival in lethal HS. Possible mechanisms by which loss of PAD2 function improves survival include the activation of cell survival pathways, improved tolerance of cardiac ischemia, and improved cardiac function during ischemia. PAD2 is promising as a future therapeutic target for the treatment of HS and cardiac ischemia.

Remote ischemic postconditioning as well as blood plasma from double-conditioned donor ameliorate reperfusion syndrome in skeletal muscle

The aim of this study was to verify the possibility of preparation and effectiveness of the use of blood plasma containing an effector of ischemic tolerance activated by applying two sublethal stresses to a donor. As sublethal stresses, two periods of 20-minute hindlimb ischemia were used with a two-day interval between them. Active plasma was isolated six hours after the second hindlimb ischemia. The effectiveness of active plasma as well as remote postconditioning was tested after three hours of tourniquet-induced ischemia on the gastrocnemius muscle. The wet/dry ratio of gastrocnemius muscle (degree of tissue oedema), nitroblue tetrazolium reduction (tissue necrosis), and CatWalk test (hind limb functionality) were evaluated 24 h after the end of ischemia. Three hours of ischemia increased muscle oedema and necrosis in comparison to control by 26.72% (p < 0.001) and 41.58% (p < 0.001) respectively. Remote ischemic postconditioning as well as injection of conditioned blood plasma significantly prevented these changes, even when they were applied one or three hours after the end of ischemia. Equally effective double-conditioned plasma appears to have better prospects in life-threatening situations such as stroke and myocardial infarction.

Chronic type 2 but not type 1 diabetes impairs myocardial ischaemic tolerance and preconditioning in C57Bl/6 mice

• What is the central question of this study? What is the impact of chronic adult-onset diabetes on cardiac ischaemic outcomes and preconditioning? • What is the main finding and its importance? Chronic adult-onset type 2 but not type 1 diabetes significantly impairs myocardial ischaemic tolerance and ischaemic preconditioning. Preconditioning may be detrimental in type 2 diabetes, exaggerating nitrosative stress and apoptotic protein expression. Effects of diabetes on myocardial responses to ischaemia-reperfusion (I-R) and cardioprotective stimuli remain contentious, potentially reflecting influences of disease duration and time of onset. Chronic adult-onset type 1 diabetes (T1D) and type 2 diabetes (T2D) were modelled non-genetically in male C57Bl/6 mice via 5×50mgkg-1 daily streptozotocin (STZ) injections + 12weeks' standard chow or 1×75mgkg-1 STZ injection + 12weeks' obesogenic diet (32% calories as fat, 57% carbohydrate, 11% protein), respectively. Systemic outcomes were assessed and myocardial responses to I-R ± ischaemic preconditioning (IPC; 3×5min I-R) determined in Langendorff perfused hearts. Uncontrolled T1D was characterised by pronounced hyperglycaemia (25mm fasting glucose), glucose intolerance and ∼10% body weight loss, whereas T2D mice exhibited moderate hyperglycaemia (15mm), hyperinsulinaemia, glucose intolerance and 17% weight gain. Circulating ghrelin, resistin and noradrenaline were unchanged with T1D, while leptin increased and noradrenaline declined in T2D mice. Ischaemic tolerance and IPC were preserved in T1D hearts. In contrast, T2D worsened post-ischaemic function (∼40% greater diastolic and contractile dysfunction) and cell death (100% higher troponin efflux), and abolished IPC protection. Whereas IPC reduced post-ischaemic nitrotyrosine and pro-apoptotic Bak and Bax levels in non-diabetic hearts, these effects were reduced in T1D and IPC augmented Bax and nitrosylation in T2D hearts. The data demonstrate chronic T1D does not inhibit myocardial I-R tolerance or IPC, whereas metabolic and endocrine disruption in T2D is associated with ischaemic intolerance and inhibition of IPC. Indeed, normally protective IPC may exaggerate damage mechanisms in T2D hearts.

Bradykinin and noradrenaline preconditioning influences level of antioxidant enzymes SOD, CuZn-SOD, Mn-SOD and catalase in the white matter of spinal cord in rabbits after ischemia/reperfusion.

The aim of present work is to assess the effects of bradykinin (Br) or noradrenaline (Nor) preconditioning to the levels of antioxidant enzymes: superoxide dismutase (SOD), copper, zinc superoxide dismutase (CuZn-SOD), manganese superoxide dismutase (Mn-SOD) and catalase in ischemia/reperfusion (I/R) model in the rabbit spinal cord white matter as well as effect on glial fibrillary acidic protein (GFAP) and ubiquitin immunoreaction in glial cells. Rabbits were preconditioned by intraperitoneal single dose of Br or Nor 48 h prior to 20 min of ischemia followed by 24 or 48 h of reperfusion. White matter of L3-L6 spinal cord segments was used for comparison of antioxidant enzyme levels in sham control, ischemic groups and four preconditioned groups. The total SOD level in the Br or Nor preconditioned groups after 48 h of reperfusion was increased vs Br or Nor preconditioned groups after 24 h of reperfusion. The comparison among the ischemic group vs Br preconditioned (P<0.05), and Nor preconditioned (P<0.001) groups after 48 h of reperfusion, showed statistically significant decrease of Mn-SOD activity. Tissue catalase level activity was significantly decreased in the Br preconditioned group after 48 h of reperfusion (P<0.05) and Nor preconditioned groups after 24 h of reperfusion (P<0.001) and also after 48 h of reperfusion (P<0.001), in comparison to ischemic group after 48 h of reperfusion. Significantly decreased tissue catalase activity (P<0.05) in both Nor preconditioned groups after 24 or 48 h of reperfusion was measured vs Br preconditioned group after 48 h of reperfusion. According to our results, in the white matter, activation of stress proteins in glial cells, as well as antioxidant enzymes levels, were influenced by pharmacological preconditioning followed by 20 min of ischemia and 24 or 48 h of reperfusion. These changes contribute to ischemic tolerance acquisition and tissue protection from oxidative stress during reperfusion period.

Mecanismos fisiopatológicos de la tolerancia a la isquemia en el sistema nervioso central

El cerebro es uno de los órganos del cuerpo humano más sensibles a la isquemia, por lo que adquiere mecanismos endógenos para protegerse y recuperarse contra estímulos nocivos. La tolerancia isquémica constituye la adaptación del cerebro a la isquemia cuando se expone por primera vez a cualquier lesión que sea por debajo del umbral del daño celular. En las últimas décadas se han estudiado las vías moleculares y beneficios potenciales del pre y post condicionamiento, representando un objetivo terapéutico para pacientes con lesión cerebral aguda; sin embargo, las estrategias que se han experimentado no han demostrado seguridad en humanos y requieren un seguimiento de investigaciones sistemáticas para abordar su eficacia.

P6299Dietary nitrate restores cardiac ischemic tolerance in type 2 diabetic mice and protects against ischemia-reperfusion injury via soluble guanylate cyclase in red blood cells

Abstract Background Inorganic nitrate has been shown to exert beneficial cardiovascular effects, which are thought to be mediated via sequential reduction of nitrate to nitrite and nitric oxide (NO). We have previously reported that hearts from type 2 diabetic db/db mice have impaired cardiac ischemic tolerance and that this effect involves reduced export of NO-like bioactivity from red blood cells (RBCs). It remains unknown whether nitrate supplementation may affect cardiac ischemic tolerance in diabetes through interference with RBC function. Purpose To test the hypothesis that dietary nitrate supplementation improves cardiac ischemic tolerance of hearts via an effect mediated through RBCs in type 2 diabetes. Methods Type 2 diabetic (db/db) and wild type (WT) mice on nitrate-free chow were treated with vehicle or nitrate (1 mM) in the drinking water for 4 weeks. Hearts were isolated and perfused using the Langendorff technique. After 30 min stabilization, the hearts were subjected to 40 min global ischemia followed by 60 min reperfusion. In protocol 1, isolated hearts from db/db and WT mice given vehicle or nitrate were perfused with buffer. In protocol 2, only hearts from untreated WT mice were used. Washed RBCs from WT or db/db mice treated with vehicle or nitrate were administered to WT hearts at the onset of ischemia with and without the soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ). In both protocols post-ischemic recovery of cardiac function was evaluated by determination of left ventricular developed pressure (LVDP). Results In Protocol 1, post-ischemic recovery of LVDP was impaired in hearts from db/db mice in comparison with hearts from WT mice (Fig. A). Dietary nitrate restored the ischemic tolerance of hearts from db/db mice but did not affect post-ischemic recovery of hearts from WT mice (Fig. A). In Protocol 2, administration of RBCs collected from vehicle-treated db/db mice significantly impaired post-ischemic recovery of hearts from WT mice (Fig. B). Notably, administration of RBCs from nitrate-treated db/db mice completely reversed the impairment of post-ischemic cardiac function induced by diabetic RBCs (Fig. B). Interestingly, post-ischemic cardiac function did not differ between hearts given RBCs from nitrate-treated db/db and WT mice (Fig. B). The protective effect of RBCs from nitrate-treated mice was abolished by pre-incubation of the RBCs with ODQ, an inhibitor of soluble guanylate cyclase (sGC) (Fig. C). By contrast, pretreatment of isolated WT hearts with ODQ failed to block the protective effect of RBCs from nitrate-treated mice (Fig C) indicating that sGC in the RBC but not in the heart is critical for nitrate-induced cardiac protection. Conclusion Dietary nitrate restores cardiac ischemic tolerance in db/db mice and protects the heart against ischemia–reperfusion injury via an RBC NO-sGC pathway.

P1592A cardioprotective effect of dietary nitrate mediated by red blood cells

Abstract Background Reduced bioavailability of nitric oxide (NO) is a key factor behind coronary artery disease and ischemic heart disease. Besides being produced from L-arginine by NO synthase, reduction of nitrate/nitrite to NO is also an important pathway for NO generation, especially during hypoxic/ischemic conditions. Existing evidences suggest that dietary nitrate improves endothelial function and ischemic tolerance via a NO-dependent mechanism. Recent data indicate that red blood cells (RBCs) are an important source of NO bioactivity that protects the heart from ischemia-reperfusion injury. It is unknown whether dietary nitrate exerts cardioprotection by increasing export of RBC NO bioactivity. Purpose To investigate whether dietary nitrate protects the heart against ischemia-reperfusion injury via a mechanism mediated by RBCs. Methods Patients with mild hypertension (systolic blood pressure &gt;130, ≤159 mmHg) were randomly assigned to three groups after a 2-week run-in period on a diet low in nitrate: group 1 received nitrate-rich vegetables (green leafy, ∼150 g/day) plus capsules with placebo salt (KCl), group 2 received low nitrate vegetables (cherry tomato, sweet corn, capsicum, carrot, ∼150 g/day) and capsules with nitrate salt (KNO3, 300mg) and group 3 received low nitrate vegetables and placebo capsules for 5 weeks. The nitrate content of the pills and nitrate-rich vegetables were precisely matched. As a pre-specified substudy, RBCs were collected blindly from 48 subjects before (baseline) and after the 5-week treatment (follow-up). The RBCs were given to isolated Langendorff-perfused rat hearts at the onset of ischemia with and without the soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ). The hearts were subjected to 25 min global ischemia following 60 min reperfusion. Left ventricular developed pressure (LVDP) was recorded as an indicator of cardiac function. Results The recovery in LVDP during reperfusion following global ischemia in hearts given RBCs collected at baseline was similar in the three groups (Fig. A). Of note, post-ischemic recovery of LVDP was significantly improved by administration of RBCs from patients randomized to high nitrate vegetables or nitrate capsule compared to the group randomized to low nitrate and placebo (Fig. B). There was no difference in LVDP recovery between the groups receiving high nitrate vegetables or nitrate capsule (Fig. B). The nitrate-induced improvement in post-ischemic cardiac recovery was totally abolished by the sGC inhibitor ODQ (Fig. C), indicating that the protective effect induced by RBCs from subjects given nitrate is NO-sGC dependent. Conclusion Dietary nitrate in the form of leafy vegetables induces protection against myocardial ischemia-reperfusion injury via a mechanism involving the NO-sGC signaling pathway in RBCs.

A 2-Min Transient Ischemia Confers Cerebral Ischemic Tolerance in Non-Obese Gerbils, but Results in Neuronal Death in Obese Gerbils by Increasing Abnormal mTOR Activation-Mediated Oxidative Stress and Neuroinflammation.

A brief episode of transient ischemia (TI) can confer cerebral ischemic tolerance against a subsequent severer TI under standard condition. The brain under obesity’s conditions is more sensitive to ischemic injury. However, the impact of a brief episode of TI under obesity’s conditions has not been fully addressed yet. Thus, the objective of this study was to determine the effect of a brief TI in the hippocampus of high-fat diet (HFD)-induced obese gerbils and related mechanisms. Gerbils were maintained on HFD or normal diet (ND) for 12 weeks and subjected to 2 min TI. HFD gerbils were heavier, with higher blood glucose, serum total cholesterol, triglycerides, and leptin levels. Massive loss of pyramidal neurons occurred in the hippocampal cornu ammonis 1 (CA1) field of HFD animals at 5 days after 2 min of TI, but 2 min of TI did not elicit death of pyramidal neurons in ND gerbils. The HFD group showed significantly increased levels of oxidative stress indicators (dihydroethidium and 4-hydroxynonenal) and proinflammatory cytokines (tumor necrosis factor-α and interleukin-1β) and microglial activation in pre- and/or post-ischemic phases compared to the ND group. Levels of mammalian target of rapamycin (mTOR) and phosphorylated-mTOR in the CA1 field of the HFD group were also significantly higher than the ND group. On the other hand, inhibition of mTOR activation by rapamycin (an allosteric mTOR inhibitor) significantly attenuated neuronal death induced by HFD, showing reduction of HFD-induced increases of oxidative stress indicators and proinflammatory cytokines, and microglia activation. Taken together, a brief episode of TI can evoke neuronal death under obesity’s conditions. It might be closely associated with an abnormal increase of mTOR activation-mediated, severe oxidative stress and neuroinflammation in pre- and/or post-ischemic phases.

Pre-arrest hypothermia improved cardiac function of rats by ameliorating the myocardial mitochondrial injury after cardiac arrest.

This study investigated the effects of hypothermia induced before cardiac arrest or after return of spontaneous circulation (ROSC) on cardiac function and myocardial mitochondrial injury after ROSC in a rat cardiac arrest model. Sixty healthy, male Wistar rats were randomly divided into the Normothermia group, pre-arrest hypothermia (Pre-HT) group, and post-resuscitation hypothermia (Post-HT) group. The rats underwent 8 min of untreated ventricular fibrillation followed by cardiopulmonary resuscitation. Twelve rats in each group were used to evaluate the left ventricular ejection fraction before ventricular fibrillation and 4 h after ROSC. Survival was determined at 24 h after ROSC. The remaining eight rats in each group were used to detect for heart malondialdehyde, reduced glutathione, adenosine triphosphate levels and mitochondrial histology. Oxygen consumption rate and mitochondrial membrane potential were evaluated 4 h after ROSC; 10 of 12 rats in Pre-HT group, 5 of 12 in Post-HT group, and 6 of 12 in normothermia group were successfully resuscitated. The survival rate of each group was 66.7%, 33.3%, and 25%, respectively. Rats in the Pre-HT group showed less alteration of the mitochondrial ultrastructure and oxidative stress injury, better maintenance of adenine nucleotides, and more preservation of the mitochondrial membrane potential and respiratory function when compared with rats in the Post-HT and normothermia groups. Transient hypothermia is an effective preconditioning stimulus to induce ischemic tolerance in a cardiac arrest model and worthy of further evaluation for potential clinical use. Impact statement In this paper, we investigated the effects of hypothermia induced before ischemia or after ROSC on cardiac function, oxidative stress damage, and myocardial mitochondrial ischemia–reperfusion injury after cardiac arrest in a rat model with VF. We demonstrated that pre-arrest hypothermia conferred greater cardio-protective benefits than delayed post-resuscitation hypothermia, reduced the number of defibrillations required and dosages of epinephrine during CPR, decreased oxidative stress, ameliorated mitochondrial dysfunction, and subsequently improved survival rate.

Preactivation of Notch1 in remote ischemic preconditioning reduces cerebral ischemia-reperfusion injury through crosstalk with the NF-\u03baB pathway

BackgroundRemote ischemic preconditioning (RIPC) initiates endogenous protective pathways in the brain from a distance and represents a new, promising paradigm in neuroprotection against cerebral ischemia-reperfusion (I/R) injury. However, the underlying mechanism of RIPC-mediated cerebral ischemia tolerance is complicated and not well understood. We reported previously that preactivation of Notch1 mediated the neuroprotective effects of cerebral ischemic preconditioning in rats subjected to cerebral I/R injury. The present study seeks to further explore the role of crosstalk between the Notch1 and NF-κB signaling pathways in the process of RIPC-induced neuroprotection.MethodsMiddle cerebral artery occlusion and reperfusion (MCAO/R) in adult male rats and oxygen-glucose deprivation and reoxygenation (OGD/R) in primary hippocampal neurons were used as models of I/R injury in vivo and in vitro, respectively. RIPC was induced by a 3-day procedure with 4 cycles of 5 min of left hind limb ischemia followed by 5 min of reperfusion each day before MCAO/R. Intracerebroventricular DAPT injection and sh-Notch1 lentivirus interference were used to inhibit the Notch1 signaling pathway in vivo and in vitro, respectively. After 24 h of reperfusion, neurological deficit scores, infarct volume, neuronal apoptosis, and cell viability were assessed. The protein expression levels of NICD, Hes1, Phospho-IKKα/β (p-IKK α/β), Phospho-NF-κB p65 (p-NF-κB p65), Bcl-2, and Bax were assessed by Western blotting.ResultsRIPC significantly improved neurological scores and reduced infarct volume and neuronal apoptosis in rats subjected to I/R injury. OGD preconditioning significantly reduced neuronal apoptosis and improved cell viability after I/R injury on days 3 and 7 after OGD/R. However, the neuroprotective effect was reversed by DAPT in vivo and attenuated by Notch1-RNAi in vitro. RIPC significantly upregulated the expression of proteins related to the Notch1 and NF-κB pathways. NF-κB signaling pathway activity was suppressed by a Notch1 signaling pathway inhibitor and Notch1-RNAi.ConclusionsThe neuroprotective effect of RIPC against cerebral I/R injury was associated with preactivation of the Notch1 and NF-κB pathways in neurons. The NF-κB pathway is a downstream target of the Notch1 pathway in RIPC and helps protect focal cerebral I/R injury.

Endogenous neuroprotective potential due to preconditioning exercise in stroke.

Stroke is a leading cause of serious long-term physical disability due to insufficient neurorepair mechanisms. In general, physical activity is an important modifiable risk factor, particularly for stroke and cardiovascular diseases. Physical exercise has shown to be neuroprotective in both animal experiments and clinical settings. Exercise can be considered a mild stressor and follows the prototypical preconditioning stimulus. It has beneficial effects on brain health and cognitive function. Preconditioning exercise, which is prophylactic exercise prior to ischemia, can protect the brain from subsequent serious injury through promotion of angiogenesis, mediation of inflammatory responses, inhibition of glutamate over-activation, protection of the blood-brain barrier, and inhibition of apoptosis. Preconditioning exercise appears to induce brain ischemic tolerance and it has been shown to exert beneficial effects. It is clinically safe and feasible and represents an exciting new paradigm in endogenous neuroprotection for patients with acute stroke. In this review, we describe the neuroprotective potential of preconditioning exercise and clinical applications in patients with acute ischemic stroke.

Peroxisome proliferator‐activated receptor gamma participates in the acquisition of brain ischemic tolerance induced by ischemic preconditioning via glial glutamate transporter 1 in vivo and in vitro

Glial glutamate transporter 1 (GLT-1) plays a vital role in the induction of brain ischemic tolerance (BIT) by ischemic preconditioning (IPC). However, the mechanism still needs to be further explained. The aim of this study was to investigate whether peroxisome proliferator-activated receptor gamma (PPARγ) participates in regulating GLT-1 during the acquisition of BIT induced by IPC. Initially, cerebral IPC induced BIT and enhanced PPARγ and GLT-1 expression in the CA1 hippocampus in rats. The ratio of nuclear/cytoplasmic PPARγ was also increased. At the same time, the up-regulation of PPARγ expression in astrocytes in the CA1 hippocampus was revealed by double immunofluorescence for PPARγ and glial fibrillary acidic protein. Then, the mechanism by which PPARγ regulates GLT-1 was studied in rat cortical astrocyte-neuron cocultures. We found that IPC [45min of oxygen glucose deprivation (OGD)] protected neuronal survival after lethal OGD (4h of OGD), which usually leads to neuronal death. The activation of PPARγ occurred earlier than the up-regulation of GLT-1 in astrocytes after IPC, as determined by western blot and immunofluorescence. Moreover, the preadministration of the PPARγ antagonist T0070907 or PPARγ siRNA significantly attenuated GLT-1 up-regulation and the neuroprotective effects induced by IPC in vitro. Finally, the effect of the PPARγ antagonist on GLT-1 expression and BIT was verified in vivo. We observed that the preadministration of T0070907 by intracerebroventricular injection dose-dependently attenuated the up-regulation of GLT-1 and BIT induced by cerebral IPC in rats. In conclusion, PPARγ participates in regulating GLT-1 during the acquisition of BIT induced by IPC. Cover Image for this issue: doi: 10.1111/jnc.14532. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Hypercapnic hypoxia as a potential means to extend life expectancy and improve physiological activity in mice

The application of combined hypoxia and hypercapnia (hypercapnic hypoxia) during respiratory exercises results in a maximum increase in resistance to acute hypoxia and ischemic tolerance of the brain. The results of those researches allow the assumption that hypercapnic hypoxia is a promising method for prophylaxis, treatment, and rehabilitation, as well as a means to increase life expectancy. The study was conducted to verify the hypothesis that it is possible to extend the life span through regular courses of respiratory exercises with hypercapnic hypoxia. In the present experimental research carried out on mice, the geroprotective effect of regular hypercapnic-hypoxic exercises (PO2-90mm Hg and PCO2-50mm Hg) was assessed in the context of the average life expectancy and the main criteria of its quality (reproductive function, muscle strength, and behavior). Results suggest that with regular training, life span is extended significantly by 16%. This result was accompanied by improved reproductive and cognitive functions, increased motor and search activities, and physical stamina in old age mices. This important phenomenon is accompanied by improved reproductive and cognitive functions, high motor function and search activity, as well as better physical stamina in old-aged mices. Recurring respiratory training under combined hypoxia and hypercapnia (hypercapnic hypoxia) during the lifetime significantly extended the life span of mice in the experiments.

The End Effector of Ischemic Tolerance Present in Blood Plasma from Double Conditioned Donors Ameliorates Trimethyltin Provoked Damage in Brain

Background: Hundreds of experiments have been done to demonstrate robust ischemic tolerance efficiency using mostly young and healthy animals. The translation of these results to usually elderly and sick patients moreover taking many various medicines has to date been disappointing. 3-methyltin poisoning, as well as short-term transient cerebral ischemia, causes severe damage, especially to selectively vulnerable brain regions such as hippocampal CA1. Methods: Using dual conditioning, we activated the full ischemic tolerance the products of which is located to blood plasma. As sublethal stresses, two periods of 20-minute hindlimb ischemia was used with a two-day interval between them. Active plasma was isolated 6 hours after the second hindlimb ischemia. Brain damage was detected by visualizing dead neurons by Fluorojade B staining. Neuronal survival by NeuN immunoreaction. The functional capabilities of surviving neurons were monitored by Morris water maze. Results: The use of activated plasma dramatically reduced the hippocampal degenerative CA1 neurons from 56.59% to 7.51%. NeuN positivity increased from 23.82% to 78.67%. Time spent searching for hidden island has been shortened from 48.46 to 21.63 seconds. Conclusions: By using double conditioning it is possible to provoke the synthesis of substances present in the blood plasma, providing the organism with significant protection against acute degenerative processes.

Potentiating a non-neuronal cardiac cholinergic system reinforces the functional integrity of the blood brain barrier associated with systemic anti-inflammatory responses

We previously reported that the heart-specific choline acetyltransferase (ChAT) gene overexpressing mice (ChAT tg) show specific phenotypes including ischemic tolerance and the CNS stress tolerance. In the current study, we focused on molecular mechanisms responsible for systemic and localized anti-inflammatory phenotypes of ChAT tg. ChAT tg were resistant to systemic inflammation induced by lipopolysaccharides due to an attenuated cytokine response. In addition, ChAT tg, originally equipped with less reactive Kupffer cells, were refractory to brain cold injury, with decreased blood brain barrier (BBB) permeability and reduced inflammation. This is because ChAT tg brain endothelial cells expressed more claudin-5, and their astrocytes were less reactive, causing decreased hypertrophy. Moreover, reconstruction of the BBB integrity in vitro confirmed the consolidation of ChAT tg. ChAT tg were also resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neuronal toxicity due to lower mortality rate and neuronal loss of substantia nigra. Additionally, ChAT tg subjected to MPTP showed attenuated BBB disruption, as evident from reduced sodium fluorescein levels in the brain parenchyma. The activated central cholinergic pathway of ChAT tg lead to anti-convulsive effects like vagus nerve stimulation. However, DSP-4, a noradrenergic neuron-selective neurotoxin against the CNS including the locus ceruleus, abrogated the beneficial phenotype and vagotomy attenuated expression of claudin-5, suggesting the link between the cholinergic pathway and BBB function. Altogether, these findings indicate that ChAT tg possess an anti-inflammatory response potential, associated with upregulated claudin-5, leading to the consolidation of BBB integrity. These characteristics protect ChAT tg against systemic and localized inflammatory pathological disorders, which targets the CNS.

The Key Role of Warm and Cold Ischemia in Uterus Transplantation: A Review.

Introduction: Uterus transplantation (UTx) is a promising treatment for uterine infertility that has resulted in several births since 2014. Ischemia is a key step in organ transplantation because it may lead to changes jeopardizing graft viability. Method: We performed a systematic review of animal and human studies relating to uterine ischemia. Results: We retained 64 studies published since 2000. There were 35 studies in animals, 24 in humans, and five literature reviews. Modest preliminary results in large animals and humans are limited but encouraging. In small animals, pregnancies have been reported to occur after 24 h of cold ischemia (CI). In ewes, uterine contractions have been detected after 24 h of CI. Furthermore, it has been shown in animals that uterine tolerance to CI and to warm ischemia (WI) can be increased by pharmacological products. In women, mean CI time in studies of births from uteri obtained from live donors was between 2 h 47 min and 6 h 20 min from a deceased donor; with only one birth in this case. Muscle contractions have also been demonstrated in myometrial samples from women, after six or more hours of CI. Conclusion: The uterus seems to be able to tolerate a prolonged period of CI, of at least six hours. Studies of the ischemia tolerance of the uterus and ways to improve it are essential for the development of UTx, particularly for procedures using grafts from deceased donors.

Hippocampal sub-regional differences in the microRNA response to forebrain ischemia

Transient forebrain ischemia, as occurs with cardiac arrest and resuscitation, results in impaired cognitive function secondary to delayed neuronal cell death in hippocampal cornu ammonis-1 (CA1). Comparatively, hippocampal neurons in the adjacent dentate gyrus (DG) survive, suggesting that elucidating the molecular mechanisms underpinning hippocampal sub-regional differences in ischemic tolerance could be central in the development of novel interventions to improve outcome in survivors of forebrain ischemia. MicroRNAs (miRNAs) are non-coding RNAs that modulate the translation of target genes and have been established as an effective therapeutic target for other models of injury. The objective of the present study was to assess and compare post-injury miRNA profiles between CA1 and DG using a rat model of forebrain ischemia. CA1 and DG sub-regions were dissected from rat hippocampi following 10 min of forebrain ischemia at three time points (3 h, 24 h, and 72 h) and at baseline. Pronounced differences between CA1 and DG were observed for several select miRNAs, including miR-181a-5p, a known regulator of cerebral ischemic injury. We complexed fluorescent in situ hybridization with immunohistochemistry to observe cell-type specific and temporal differences in mir-181a-5p expression between CA1 and DG in response to injury. Using established miRNA-mRNA prediction algorithms, we extended our observations in CA1 miRNA dysregulation to identify key functional pathways as potential modulators of CA1 ischemic vulnerability. In summary, our observations support a central role for miRNAs in selective CA1 ischemic vulnerability and suggest that cell-specific miRNA targeting could be a viable clinical approach to preserve CA1 neurons and improve cognitive outcomes for survivors of transient forebrain ischemia.

Neuroprotection mediated by remote preconditioning is associated with a decrease in systemic oxidative stress and changes in brain and blood glutamate concentration

It has been shown that ischemia of remote organs can generate resistance to ischemic conditions within sensitive brain tissues. However, only limited information about its mechanism is available. In the present paper, we used hind-limb ischemia by tourniquet to generate early remote ischemic tolerance in rats. The main objective was to investigate the role of glutamate in the process of neuroprotection and discover parameters that are affected in the blood of ischemia-affected animals. Our results showed that pretreatment with a hind-limb tourniquet caused a decrease in neurodegeneration by about 30%. However, we did not observe neurological deficit recovery. When compared to ischemia, glutamate concentration decreased in all observed brain regions (cortex, CA1 and dentate gyrus of hippocampus), regardless of their sensitivity to blood restrictions. In contrast to this, the blood levels raised significantly from 26% to 29% during the first four days of postischemic reperfusion. Pretreatment of animals reduced systemic oxidative stress—as represented by lymphocytic DNA damage—by about 80%, while changes in blood antioxidant enzymes (catalase, superoxide dismutase) were not detected.With these data we can further hypothesize that hind-limb-tourniquet preconditioning could accelerate brain-to-blood efflux of glutamate which could positively impact neuronal survival of ischemia-affected brain regions. Moreover, remote preconditioning improved systemic oxidative stress and did not seem to be affected by enzymatic antioxidant defenses in the blood.

Predictors for basal ganglia viability after mechanical thrombectomy in proximal middle cerebral artery occlusion

Background/purposeIn acute proximal middle cerebral artery (MCA) occlusion covering the lenticulostriate arteries (LSA), ischemic tolerance of basal ganglia is limited due to supposed lack of collateral supply. However, in several patients, basal ganglia (BG) infarction was absent after successful mechanical thrombectomy (MTE). Purpose of our study was to evaluate predictors for BG viability in stroke patients despite prolonged MCA mainstem occlusion. Material/methodsWe retrospectively reviewed all stroke patients from our local registry with MCA mainstem occlusion after mechanical thrombectomy between November 2009 and October 2016. All patients underwent non-enhanced post-interventional cranial CT imaging (NCCT) and were classified according to 6 patterns of BG viability: complete: putamen (P) and globus pallidus (GP); partial: P or GP, and combination of complete or partial BG and/or adjacent white matter/cortical (WMC) viability. We compared viability patterns with respect to pre-interventional stroke imaging including NCCT, CT-angiography (CTA), CT perfusion (CTP); demographics, pre- and intra-procedural data and occurrence of post-procedural intracerebral hematoma (ICH). CTP imaging of the affected and contralateral BG-territories were obtained separately and CTA-collateral score (CS) was assessed. ResultsA significant correlation between higher collateral score and viability of GP (OR = 1.949; p = .011), P (OR = 2.039, p = .011), and the combination of GP, P and WMC (OR = 2.767, p = .007) was revealed. Higher relative CBV ratio (rCBVR) was significantly associated with viability of the pattern GP + WMC (univariate: OR = 3.160, p = .014; multivariate: OR = 6.058; p = .021). ConclusionCTA collateral score and rCBVR were predictive for BG viability in stroke patients after successful MTE in prolonged complete MCA-mainstem and LSA occlusion.

Developmental and sex differences in cardiac tolerance to ischemia-reperfusion injury: the role of mitochondria 1.

Age and sex play an essential role in the cardiac tolerance to ischemia-reperfusion injury: cardiac resistance significantly decreases during postnatal maturation and the female heart is more tolerant than the male myocardium. It is widely accepted that mitochondrial dysfunction, and particularly mitochondrial permeability transition pore (MPTP) opening, plays a major role in determining the extent of cardiac ischemia-reperfusion injury. We have observed that the MPTP sensitivity to the calcium load differs in mitochondria isolated from neonatal and adult myocardium, as well as from adult male and female hearts. Neonatal and female mitochondria are more resistant both in the extent and in the rate of mitochondrial swelling induced by high calcium concentration. Our data further suggest that age- and sex-dependent specificity of the MPTP is not the result of different amounts of ATP synthase and cyclophilin D: neonatal and adult hearts, similarly as the male and female hearts, contain comparable amounts of MPTP and its regulatory protein cyclophilin D. We can speculate that the lower sensitivity of MPTP to the calcium-induced swelling may be related to the higher ischemic tolerance of both neonatal and female myocardium.