Activation of moderate autophagy promotes survival of fat graft.

Clinically unpredictable retention following fat grafting remains outstanding problems because of the unrevealed mechanism of grafted fat survival. The role of autophagy, a process to maintain cellular homeostasis through recycling cellular debris, has yet been to be reported in fat grafting. This study aims to improve the survival of fat grafting through the autophagy. First, the relationship between cell death and autophagy in the early stage of fat grafting was evaluated through immunostaining, RNA sequencing, and western blot. Next, rapamycin, an autophagic agonist, was used for the culturing of adipose-derived stem cells and adipocytes during ischemia. Cell death, autophagy, and reactive oxygen species (ROS) were assayed. Finally, rapamycin was used to assist fat grafting in nude mice. The results demonstrated that the peak of cell death at the early stage of fat grafting was accompanied by a decrease in autophagy. In vitro, during ischemia, 25 nM was confirmed as the optimal dose of rapamycin that reduces cell death with enhanced autophagy and mitophagy, improved mitochondrial quality as well as decreased ROS accumulation. In vivo, promoted mitophagy, alleviated oxidative stress, and decreased cell apoptosis of rapamycin-treated fat grafts were observed in the early stage. In addition, rapamycin increased the survival of fat grafts with increased neovascularization and reduced fibrosis. We suggested that moderate autophagy induced by rapamycin contribute to enhanced ischemic tolerance and long term survival of fat grafts through mitochondrial quality control.

Transformation of A1/A2 Astrocytes Participates in Brain Ischemic Tolerance Induced by Cerebral Ischemic Preconditioning via Inhibiting NDRG2.

Cerebral ischemic preconditioning (CIP) has been shown to improve brain ischemic tolerance against subsequent lethal ischemia. Reactive astrocytes play important roles in cerebral ischemia-reperfusion. Recent studies have shown that reactive astrocytes can be polarized into neurotoxic A1 phenotype (C3d) and neuroprotective A2 phenotype (S100A10). However, their role in CIP remains unclear. Here, we focused on the role of N-myc downstream-regulated gene 2 (NDRG2) in regulating the transformation of A1/A2 astrocytes and promoting to brain ischemic tolerance induced by CIP. A Sprague Dawley rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) was used. Rats were divided into the following six groups: (1) sham group; (2) CIP group: left middle cerebral artery was blocked for 10 min; (3) MCAO/R group: left middle cerebral artery was blocked for 90 min; (4) CIP + MCAO/R group: CIP was performed 72 h before MCAO/R; (5) AAV-NDRG2 + CIP + MCAO/R group: adeno-associated virus (AAV) carrying NDRG2 was administered 14 days before CIP + MCAO/R; (6) AAV-Ctrl + CIP + MCAO/R group: empty control group. The rats were subjected to neurological evaluation 24 h after the above treatments, and then were sacrificed for 2, 3, 5-triphenyltetraolium chloride staining, thionin staining, immunofluorescence and western blot analysis. In CIP + MCAO/R group, the neurological deficit scores decreased, infarct volume reduced, and neuronal density increased compared with MCAO/R group. Notably, CIP significantly increased S100A10 expression and the number of S100A10+/GFAP+ cells, and also increased NDRG2 expression. MCAO/R significantly decreased S100A10 expression and the number of S100A10+/GFAP+ cells yet increased C3d expression and the number of C3d+/GFAP+ cells and NDRG2 expression, and these trends were reversed by CIP + MCAO/R. Furthermore, over-expression of NDRG2 before CIP + MCAO/R, the C3d expression and the number of C3d+/GFAP+ cells increased, while S100A10 expression and the number of S100A10+/GFAP+ cells decreased. Meanwhile, over-expression of NDRG2 blocked the CIP-induced brain ischemic tolerance. Taken together, these results suggest that CIP exerts neuroprotective effects against ischemic injury by suppressing A1 astrocyte polarization and promoting A2 astrocyte polarization via inhibiting NDRG2 expression.

Klotho Upregulation via PPARγ Contributes to the Induction of Brain Ischemic Tolerance by Cerebral Ischemic Preconditioning in Rats.

Cerebral ischemic preconditioning (CIP)-induced brain ischemic tolerance protects neurons from subsequent lethal ischemic insult. However, the specific mechanisms underlying CIP remain unclear. In the present study, we explored the hypothesis that peroxisome proliferator-activated receptor gamma (PPARγ) participates in the upregulation of Klotho during the induction of brain ischemic tolerance by CIP. First we investigated the expression of Klotho during the brain ischemic tolerance induced by CIP. Lethal ischemia significantly decreased Klotho expression from 6 h to 7 days, while CIP significantly increased Klotho expression from 12 h to 7 days in the hippocampal CA1 region. Inhibition of Klotho expression by its shRNA blocked the neuroprotection induced by CIP. These results indicate that Klotho participates in brain ischemic tolerance by CIP. Furthermore, we tested the role of PPARγ in regulating Klotho expression after CIP. CIP caused PPARγ protein translocation to the nucleus in neurons in the CA1 region of the hippocampus. Pretreatment with GW9962, a PPARγ inhibitor, significantly attenuated the upregulation of Klotho protein and blocked the brain ischemic tolerance induced by CIP. Taken together, it can be concluded that Klotho upregulation via PPARγ contributes to the induction of brain ischemic tolerance by CIP.

Severity of Peripheral Infection Differentially Affects Brain Functions in Mice via Microglia-Dependent and -Independent Mechanisms.

Peripheral infection induces inflammation in peripheral tissues and the brain, impacting brain function. Glial cells are key players in this process. However, the effects of peripheral infection on glial activation and brain function remain unknown. Here, we showed that varying degrees of peripheral infection had different effects on the regulation of brain functions by microglia-dependent and -independent mechanisms. Acute mild infection (one-day LPS challenge: 1LPS) exacerbated middle cerebral artery occlusion (MCAO) injury, and severe infection (four-day LPS challenge: 4LPS) for one week suppressed it. MCAO injury was assessed by triphenyltetrazolium chloride staining. We observed early activation of microglia in the 1LPS and 4LPS groups. Depleting microglia with a colony-stimulating factor-1 receptor (CSF1R) antagonist had no effect on 1LPS-induced brain injury exacerbation but abolished 4LPS-induced protection, indicating microglial independence and dependence, respectively. Microglia-independent exacerbation caused by 1LPS involved peripheral immune cells including macrophages. RNA sequencing analysis of 4LPS-treated microglia revealed increased factors related to anti-inflammatory and neuronal tissue repair, suggesting their association with the protective effect. In conclusion, varying degrees of peripheral inflammation had contradictory effects (exacerbation vs. protection) on MCAO, which may be attributed to microglial dependence. Our findings highlight the significant impact of peripheral infection on brain function, particularly in relation to glial cells.

Ischemic Tolerance Induced by Glial Cells.

Ischemic tolerance is a phenomenon in which resistance to subsequent invasive ischemia is acquired by a preceding noninvasive ischemic application, and is observed in many organs, including the brain, the organ most vulnerable to ischemic insult. To date, much research has been conducted on cerebral ischemic tolerance as a cell-autonomous action of neurons. In this article, we review the essential roles of microglia and astrocytes in the acquisition of ischemic tolerance through neuron-non-autonomous mechanisms, where the two types of glial cells function in a concerted manner to induce ischemic tolerance.

Modifications of gene expression detected in peripheral blood after brain ischemia treated with remote postconditioning.

BACKGROUND: A stroke is an acute damage to a certain area of a nerve tissue of the brain. In developed countries, it ranks second among the most often causes of death and is also the leading cause of disability. Recent findings emphasize the significant neuroprotective effect of conditioning on the course and rate of recovery after ischemic attack; however the molecular mechanism of ischemic tolerance induced by conditioning is still not completely explored. METHODS AND RESULTS: The purpose of this study is an identification of changes in gene expression induced by stimulation of reaction cascades after activation of the neuroprotective mechanism using an experimental rat model of global ischemia. The induction of neuroprotective cascades was stimulated by the application of early and delayed form of remote ischemic postconditioning. The quantitative qRT-PCR method was used to assess the rate of change in ADM, BDNF, CDKN1A, CREB, GADD45G, IL6, nNOS, and TM4SF1 gene expression levels 72 h after ischemic attack. The detected results confirm the neuroprotective effect of both forms of postconditioning. Participation of neuroprotection-related gene expression changes was observed once as an early one (CREB, GADD45G), once as a delayed one (ADM, IL6), or both (BDNF, CDKN1A, nNOS, TM4SF1) postconditioning forms, depending on the particular gene. CONCLUSIONS: Our results characterize impact of ischemic tolerance on the molecular level. We predict ischemic tolerance to be consisted of complex combination of early and delayed remote postconditioning.

P2X7 Receptors in Astrocytes: A Switch for Ischemic Tolerance.

A sub-lethal ischemic episode (preconditioning [PC]) protects neurons against a subsequent lethal ischemic injury. This phenomenon is known as ischemic tolerance. PC itself does not cause brain damage, but affects glial responses, especially astrocytes, and transforms them into an ischemia-resistant phenotype. P2X7 receptors (P2X7Rs) in astrocytes play essential roles in PC. Although P2X7Rs trigger inflammatory and toxic responses, PC-induced P2X7Rs in astrocytes function as a switch to protect the brain against ischemia. In this review, we focus on P2X7Rs and summarize recent developments on how astrocytes control P2X7Rs and what molecular mechanisms they use to induce ischemic tolerance.

Mechanisms underlying sensitization of P2X7 receptors in astrocytes for induction of ischemic tolerance.

We previously showed that noninvasive mild ischemia (preconditioning; PC) induced ischemic tolerance by upregulation of P2X7 receptors in astrocytes via a hypoxia inducible factor-1α (HIF-1α)-dependent mechanism. The P2X7 receptor is known as a low-sensitivity P2 receptor that requires a high extracellular ATP (eATP) concentration for activation. PC increased the eATP level but was not sufficient to activate P2X7 receptors. Here, we show that astrocytes possess an elaborate mechanism for activation of P2X7 receptors, thus contributing to ischemic tolerance. Nicotinamide adenine dinucleotide (NAD+ ) was shown to increase the sensitivity of P2X7 receptors to eATP via ecto-ADP-ribosyltransferase 2 (ARTC2)-catalyzed ADP-ribosylation in peripheral immune cells. Although ARTC2-positive signals were mostly absent in the naïve brain, they were selectively increased in astrocytes by PC. The spatiotemporal pattern of PC-evoked ARTC2 was well associated with that of P2X7 receptors. In the in vitro experiments, NAD+ increased the sensitivity of P2X7 receptors to ATP, and at higher concentrations, NAD+ itself activated P2X7 receptors without eATP in cultured astrocytes. In the in vivo experiments using middle cerebral artery occlusion model mice, the PC-evoked increase in HIF-1α in astrocytes was abolished by the ARTC2 inhibitor S + 16a. S + 16a also abolished PC-evoked ischemic tolerance. Taken together, the results suggested that P2X7 receptors can be sensitized to ATP by NAD+ /ARTC2-catalyzed ADP-ribosylation, which allows astrocytes to drive P2X7 receptor-mediated ischemic tolerance even though PC only slightly increases the amount of eATP.

Pharmacological preconditioning by TERT inhibitor BIBR1532 confers neuronal ischemic tolerance through TERT-mediated transcriptional reprogramming.

After a sublethal ischemic preconditioning (IPC) stimulus, the brain has a remarkable capability of acquiring tolerance to subsequent ischemic insult by establishing precautionary self-protective mechanism. Understanding this endogenous mechanism would reveal novel and effective neuroprotective targets for ischemic brain injury. Our previous study has implied that telomerase reverse transcriptase (TERT) is associated with IPC-induced tolerance. Here, we investigated the mechanism of TERT-mediated ischemic tolerance. Preconditioning was modeled by oxygen-glucose deprivation (OGD) and by TERT inhibitor BIBR1532 in primary neurons. We found that ischemic tolerance was conferred by BIBR1532 preconditioning. We used the Cleavage-Under-Targets-And-Tagmentation approach, a recently developed method with superior signal-to-noise ratio, to comprehensively map the genomic binding sites of TERT in primary neurons, and showed that more than 50% of TERT-binding sites were located at the promoter regions. Mechanistically, we demonstrated that under normal conditions TERT physically bound to many previously unknown genomic loci in neurons, whereas BIBR1532 preconditioning significantly altered TERT-chromatin-binding profile. Intriguingly, we found that BIBR1532-preconditioned neurons showed significant up-regulation of promoter binding of TERT to the mitochondrial anti-oxidant genes, which were correlated with their elevated expression. Functional analysis further indicated that BIBR1532-preconditioning significantly reduced ROS levels and enhanced tolerance to severe ischemia-induced mitochondrial oxidative stress in neurons in a TERT-dependent manner. Together, these results demonstrate that BIBR1532 confers neuronal ischemic tolerance through TERT-mediated transcriptional reprogramming for up-regulation of mitochondrial anti-oxidation gene expression, suggesting the translational potential of BIBR1532 as a therapeutic agent for the treatment of cerebral ischemic injury and oxidative stress-induced neurological disorders.

In vivo real-time ATP imaging in zebrafish hearts reveals G0s2 induces ischemic tolerance.

Most eukaryotic cells generate adenosine triphosphate (ATP) through the oxidative phosphorylation system (OXPHOS) to support cellular activities. In cultured cell-based experiments, we recently identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS, and showed that G0s2 protects cultured cardiomyocytes from hypoxia. In this study, we examined the in vivo protective role of G0s2 against hypoxia by generating both loss-of-function and gain-of-function models of g0s2 in zebrafish. Zebrafish harboring transcription activator-like effector nuclease (TALEN)-mediated knockout of g0s2 lost hypoxic tolerance. Conversely, cardiomyocyte-specific transgenic zebrafish hearts exhibited strong tolerance against hypoxia. To clarify the mechanism by which G0s2 protects cardiac function under hypoxia, we introduced a mitochondrially targeted FRET-based ATP biosensor into zebrafish heart to visualize ATP dynamics in in vivo beating hearts. In addition, we employed a mosaic overexpression model of g0s2 to compare the contraction and ATP dynamics between g0s2-expressing and non-expressing cardiomyocytes, side-by-side within the same heart. These techniques revealed that g0s2-expressing cardiomyocyte populations exhibited preserved contractility coupled with maintained intra-mitochondrial ATP concentrations even under hypoxic condition. Collectively, these results demonstrate that G0s2 provides ischemic tolerance in vivo by maintaining ATP production, and therefore represents a promising therapeutic target for hypoxia-related diseases.

Refocusing the Brain: New Approaches in Neuroprotection Against Ischemic Injury.

A new era for neuroprotective strategies is emerging in ischemia/reperfusion. This has forced to review the studies existing to date based in neuroprotection against oxidative stress, which have undoubtedly contributed to clarify the brain endogenous mechanisms, as well as to identify possible therapeutic targets or biomarkers in stroke and other neurological diseases. The efficacy of exogenous administration of neuroprotective compounds has been shown in different studies so far. However, something must be missing to get these treatments successfully applied in the clinical environment. Here, the mechanisms involved in neuronal protection against physiological level of ROS and the main neuroprotective signaling pathways induced by excitotoxic and ischemic stimuli are reviewed. Also, the endogenous ischemic tolerance in terms of brain self-protection mechanisms against subsequent cerebral ischemia is revisited to highlight how the preconditioning has emerged as a powerful tool to understand these phenomena. A better understanding of endogenous defense against exacerbated ROS and metabolism in nervous cells will therefore aid to design pharmacological antioxidants targeted specifically against oxidative damage induced by ischemic injury, but also might be very valuable for translational medicine.

Motor evoked potential monitoring can evaluate ischemic tolerance to carotid artery occlusion during surgery.

Balloon test occlusion (BTO) is a useful examination for evaluating ischemic tolerance to internal carotid artery (ICA) occlusion. The aim of this study was to investigate the relationships between intraoperative motor evoked potential (MEP) monitoring and the results of preoperative BTO. Between 2013 and 2017, 32 patients undergoing surgery under general anesthesia with intraoperative MEP monitoring, in whom preoperative BTO was performed, were identified. A receiver operator characteristic (ROC) analysis was performed to determine the appropriate cutoff value of MEP amplitude for BTO-positive. Furthermore, the accuracy of MEP monitoring for BTO-positive was compared with electroencephalogram (EEG) and somatosensory evoked potential (SEP) monitoring. Four of 32 (12.5%) patients were BTO-positive. The cutoff value of MEP amplitude for BTO-positive was a > 80% reduction from the baseline level, which showed sensitivity of 100% and specificity of 100%. Thus, the sensitivity and specificity for BTO-positive were significantly higher for MEP than for EEG (100% and 72.0%, p = 0.02) in 28 patients, but they were not significantly different compared with SEP (33.3% and 100%, p = 0.48) in 21 patients. MEP monitoring might be one of the alternatives for evaluating ischemic tolerance to ICA occlusion during surgery. The cutoff value of MEP amplitude was a > 80% reduction.

Preconditioning-Activated AKT Controls Neuronal Tolerance to Ischemia through the MDM2-p53 Pathway.

One of the most important mechanisms of preconditioning-mediated neuroprotection is the attenuation of cell apoptosis, inducing brain tolerance after a subsequent injurious ischemia. In this context, the antiapoptotic PI3K/AKT signaling pathway plays a key role by regulating cell differentiation and survival. Active AKT is known to increase the expression of murine double minute-2 (MDM2), an E3-ubiquitin ligase that destabilizes p53 to promote the survival of cancer cells. In neurons, we recently showed that the MDM2-p53 interaction is potentiated by pharmacological preconditioning, based on subtoxic stimulation of NMDA glutamate receptor, which prevents ischemia-induced neuronal apoptosis. However, whether this mechanism contributes to the neuronal tolerance during ischemic preconditioning (IPC) is unknown. Here, we show that IPC induced PI3K-mediated phosphorylation of AKT at Ser473, which in turn phosphorylated MDM2 at Ser166. This phosphorylation triggered the nuclear stabilization of MDM2, leading to p53 destabilization, thus preventing neuronal apoptosis upon an ischemic insult. Inhibition of the PI3K/AKT pathway with wortmannin or by AKT silencing induced the accumulation of cytosolic MDM2, abrogating IPC-induced neuroprotection. Thus, IPC enhances the activation of PI3K/AKT signaling pathway and promotes neuronal tolerance by controlling the MDM2-p53 interaction. Our findings provide a new mechanistic pathway involved in IPC-induced neuroprotection via modulation of AKT signaling, suggesting that AKT is a potential therapeutic target against ischemic injury.

Ischemic Preconditioning of the Kidney.

The phenomenon of ischemic preconditioning was discovered in 1986 in experiments with the heart, and then it was observed in almost all organs, the kidneys included. This phenomenon is underlain by conditioning of the tissues with short ischemia/reperfusion cycles intended for subsequent exposure to pathological ischemia. Despite the kidneys are not viewed as so vital organs as the brain or the heart, the acute ischemic injury to kidneys is a widespread pathology responsible for the yearly death of almost 2 million patients, while the number of patients with chronic kidney disease is estimated as hundreds of millions or nearly 10% adult population the world over. Currently, it is believed that adaptation of the kidneys to ischemia by preconditioning is the most effective way to prevent the development of acute kidney injury, so deep insight into its molecular mechanisms will be a launch pad for creating the nephroprotective therapy by elevating renal tolerance to oxygen deficiency. This review focuses on the key signaling pathways of kidney ischemic preconditioning, the potential pharmacological mimetics of its key elements, and the limitations of this therapeutic avenue associated with age-related decline of ischemic tolerance of the kidneys.

Hypoxic preconditioning attenuates necroptotic neuronal death induced by global cerebral ischemia via Drp1-dependent signaling pathway mediated by CaMKIIα inactivation in adult rats.

Hypoxic preconditioning (HPC) alleviates the selective and delayed neuronal death in the hippocampal CA1 region induced by transient global cerebral ischemia (tGCI). This type of cell death may include different programmed cell death mechanisms, namely, apoptosis and necroptosis. Although apoptotic signaling is well defined, the mechanisms that underlie neuronal necroptosis are yet to be fully elucidated. In this study, we investigated whether HPC protects neurons from cerebral ischemia-induced necroptosis. We observed that tGCI up-regulated the expression of receptor-interacting protein (RIP) 3 and increased the interaction of RIP1-RIP3 in CA1 at the early stage of reperfusion. The pretreatment with HPC or necrostatin-1 decreased the expression of RIP3 and the formation of RIP1-RIP3 after tGCI. We also found that HPC decreased the expression and the activity of caspase-8 in CA1 after tGCI, and notably, the pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, did not trigger necroptosis but attenuated the tGCI-induced neuronal damage. Furthermore, we demonstrated that HPC decreased the activation of calcium-calmodulin kinase (CaMK) IIα and the interaction of RIP1 and CaMKIIα induced by tGCI. Intriguingly, the pretreatment with a CaMKs inhibitor KN-93 before tGCI resulted in significantly reduced RIP1-3 interaction and tGCI-induced neuronal damage. Finally, we ascertained that HPC prevented the dephosphorylation of dynamin-related protein 1 (Drp1)-Ser637 (serine 637) and inhibited the translocation of Drp1 to mitochondria induced by tGCI. Importantly, the treatment with a Drp1 inhibitor Mdivi-1 or necrostatin-1 before tGCI also abolished Drp1 dephosphorylation at Ser637 and mitochondrial translocation. Taken together, our results highlight that HPC attenuates necroptotic neuronal death induced by tGCI via Drp1-dependent mitochondrial signaling pathways mediated by CaMKIIα inactivation.-Zhan, L., Lu, Z., Zhu, X., Xu, W., Li, L., Li, X., Chen, S., Sun, W., Xu, E. Hypoxic preconditioning attenuates necroptotic neuronal death induced by global cerebral ischemia via Drp1-dependent signaling pathway mediated by CaMKIIα inactivation in adult rats.

DDAH-1 via HIF-1 target genes improves cerebral ischemic tolerance after hypoxic preconditioning and middle cerebral artery occlusion-reperfusion.

Dimethylarginine dimethylamino hydrolase-1 (DDAH-1) as an indirect regulator of nitric oxide (NO) metabolism, its role in hypoxic preconditioning (HPC) and ischemic tolerance (IT) of ischemic stroke has still been unknown and needs to be elucidated. Herein, DDAH-1 knock-out (KO) and wild-type (WT) rats underwent HPC and middle cerebral artery occlusion/reperfusion (MCAO/R) model. After 24 h, neurological severity scores, TTC staining and TUNEL assay were used to evaluate neurological damages. To explore the mechanism, the expression of hypoxia inducible factor (HIF-1α) and its target genes were assessed by Western blot and RT-qPCR. NO and ADMA contents were also tested. In addition, supplementation of l-arginine to DDAH-1 KO rats was used to explore the role of DDAH-1 in regulating NO. After HPC the ischemic outcome improved in both KO and WT rats, while KO rats showed attenuated IT exhibiting less expression of HIF-1α and its target genes, lower NO but higher ADMA content. The supplement of l-arginine to KO rats partly alleviated neurological damages accompanied with higher expression of HIF-1α. To sum up, DDAH-1 could regulate the level of NO and enhance IT following HPC and MCAO model via activating the expression of HIF-1α and its target genes.

Neuroprotective Effect of Chronic Intracranial Toxoplasma gondii Infection in a Mouse Cerebral Ischemia Model.

Toxoplasma gondii is an obligate intracellular protozoan parasite that can invade various organs in the host body, including the central nervous system. Chronic intracranial T. gondii is known to be associated with neuroprotection against neurodegenerative diseases through interaction with host brain cells in various ways. The present study investigated the neuroprotective effects of chronic T. gondii infection in mice with cerebral ischemia experimentally produced by middle cerebral artery occlusion (MCAO) surgery. The neurobehavioral effects of cerebral ischemia were assessed by measurement of Garcia score and Rotarod behavior tests. The volume of brain ischemia was measured by triphenyltetrazolium chloride staining. The expression levels of related genes and proteins were determined. After cerebral ischemia, corrected infarction volume was significantly reduced in T. gondii infected mice, and their neurobehavioral function was significantly better than that of the uninfection control group. Chronic T. gondii infection induced the expression of hypoxia-inducible factor 1-alpha (HIF-1α) in the brain before MCAO. T. gondii infection also increased the expression of vascular endothelial growth factor after the cerebral ischemia. It is suggested that chronic intracerebral infection of T. gondii may be a potential preconditioning strategy to reduce neural deficits associated with cerebral ischemia and induce brain ischemic tolerance through the regulation of HIF-1α expression.

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 [45 min of oxygen glucose deprivation (OGD)] protected neuronal survival after lethal OGD (4 h 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/.

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.

Brain ischemic preconditioning protects against moderate, not severe, transient global cerebral ischemic injury.

Ischemic preconditioning (IPC) in the brain increases ischemic tolerance to subsequent ischemic insults. In this study, we examined whether IPC protects neurons and attenuates microgliosis or not in the hippocampus following severe transient global cerebral ischemia (TCI) in gerbils. Gerbils were assigned to 8 groups; 5- and 15-min sham operated groups, 5-min and 15-min TCI operated groups, IPC plus 5- and 15-min sham operated groups, and IPC plus 5- and 15-min TCI operated groups. IPC was induced by subjecting animals to 2-min transient ischemia 1 day before 5-min TCI for a typical transient ischemia and 15-min TCI for severe transient ischemia. Neuronal damage was examined by cresyl violet staining and Fluoro-Jade B histofluorescence staining. In addition, microglial activation was examined using immunohistochemistry for Iba-1 (a marker for microglia). Delayed neuronal death and microgliosis was found in the CA1 alone in the 5-min TCI operated group at 5 days post-ischemia, and, in the 15-min TCI operated group, neuronal death and microgliosis was shown in all CA areas (CA1-3) and the dentate gyrus. IPC displayed neuroprotection and attenuated microglial activation in the 5-min TCI operated group. However, in the 15-min TCI operated group, IPC did not show neuroprotection and not attenuate microglial activation. Our present findings indicate that IPC hardly protect against severe transient cerebral ischemic injury.