Ischemia-induced Changes Research Articles

Cerebral ischemia-induced gene expression changes in diabetic mice from acute to subacute stage

Diabetes is a risk factor for stroke; however, its impact on stroke progression at the genomic level is not well understood. To address this gap, we used transcriptome sequencing to explore the relationship between lncRNA and mRNA expression patterns and the reperfusion duration in the cortex of diabetically induced stroke mice. First, focal ischemia was induced in adult male ob/ob mice, which were then subjected to reperfusion periods of one, three, or seven days. Total RNA was extracted from the ischemic cortical tissue for RNA sequencing, and the resulting reads were aligned to the GRCm38 murine reference genome. A total of 672 mRNAs and 301 lncRNAs were identified as differentially expressed one day post-stroke, 1195 mRNAs and 66 lncRNAs at three days post-stroke, and 1069 mRNAs and 75 lncRNAs at seven days post-stroke. Stage-specific differentially expressed mRNAs were bioinformatically analyzed and found significantly enriched in processes such as apoptosis, angiogenesis, and lipid metabolism at one, three, and seven days post-stroke, respectively. Stage-specific DElncRNA-mRNA cis-regulatory relationships were constructed using these biological processes as examples, revealing the potential roles of four pairs of lncRNA-mRNAs (Gm39787-Lcn2, Gm46111-Drd2, D3300500i16Rik-Fosl1, and Gm41689-Egr1) in apoptosis. Additionally, Gm40237-Tie1 and Gm52352-Pdgfrb are associated with angiogenesis and lipid metabolism, respectively. In conclusion, our study demonstrated that lncRNA and mRNA expression in the cortex of transient focal ischemia-induced diabetic mice undergo extensive alterations, providing insights into complex molecular interactions underlying diabetic stroke.

Extracts from Dendropanax morbifera leaves ameliorates cerebral ischemia-induced hippocampal damage by reducing oxidative damage in gerbil

AimIn this study, we investigated the effects of Dendropanax morbifera extract (DME) on neuroprotection against ischemic damage in gerbils. MethodsDME (100 or 300 mg/kg) was orally administered to gerbils for three weeks, and 2 h after the last DME treatment, transient forebrain ischemia in the common carotid arteries was induced for 5 min. The forebrain ischemia-related cognitive impairments were assessed by spontaneous motor activity and passive avoidance test one and four days after ischemia, respectively. In addition, surviving and degenerating neurons were morphologically confirmed by neuronal nuclei immunohistochemical staining and Fluoro-Jade C staining, respectively, four days after ischemia. Changes of glial morphology were visualized by immunohistochemical staining for each marker such as glial fibrillary acidic protein and ionized calcium-binding protein. Oxidative stress was determined by measurements of dihydroethidium, O2· (formation of formazan) and malondialdehyde two days after ischemia. In addition, glutathione redox system such as reduced glutathione, oxidized glutathione levels, glutathione peroxidase, and glutathione reductase activities were measured two days after ischemia. ResultsSpontaneous motor activity monitoring and passive avoidance tests showed that treatment with 300 mg/kg DME, but not 100 mg/kg, significantly alleviated ischemia-induced memory impairments. In addition, approximately 67 % of mature neurons survived and 29.3 % neurons were degenerated in hippocampal CA1 region four days after ischemia, and ischemia-induced morphological changes in astrocytes and microglia were decreased in the CA1 region after 300 mg/kg DME treatment. Furthermore, treatment with 300 mg/kg DME significantly ameliorated ischemia-induced oxidative stress, such as superoxide formation and lipid peroxidation, two days after ischemia. In addition, ischemia-induced reduction of the glutathione redox system in the hippocampus, assessed two days after the ischemia, was ameliorated by treatment with 300 mg/kg DME. These suggest that DME can potentially reduce ischemia-induced neuronal damage through its antioxidant properties.

Time-Warping Analysis of the T-Wave Peak-to-End Interval to Quantify Ventricular Repolarization Dispersion During Ischemia.

The presence of myocardial ischemia can increase ventricular arrhythmic risk due to a raised dispersion of ventricular repolarization. This is reflected on the morphology of the T-wave on the electrocardiogram (ECG), and can be quantified by time-warping based indexes, like dw. However, T-wave morphology can also be affected by ischemia-induced ST segment changes, impacting the accuracy of dw. We hypothesize that ischemia-induced repolarization dispersion can be better quantified by restricting dw to the T-wave peak-to-end (T pe) interval. 101 ECG recordings from patients undergoing a percutaneous coronary intervention (PCI) were analyzed together with their control ECG recordings, acquired before the intervention. A series of dw values was calculated by quantifying the T pe morphological variations between the T-waves from the PCI and a reference derived from the control ECG recordings. We proposed a normalized version of dw, Rd, reflecting relative variations of dw during PCI to control recordings, and compare it with traditional indices behaviour. Values of dw followed a gradually increasing trend as PCI inflation time progressed, reaching a median[range] Rd value of 9.44 [1.01, 80.74] at the occlusion end. During control recordings, dw remained stable, with a low intra-patient standard deviation, range [0.02, 2.09] ms and a median[range] Rd of 1.00 [0.03, 2.93]. Rd at oclussion end was significantly higher than threshold values of 1, 2, 5, and 10 in 94.1%, 85.11%, 64.4% and 48.5% of patients, respectively. The spatial lead-wise analysis of dw showed distinct distributions depending on the occluded artery, suggesting a relation with the ischemia location. The relative variation R with ischemia of index dw (9.4) is greater than for traditional T wave amplitude (7.7), T peak-to-end interval (2.7) and T width (3.0). The index dw, quantifying T pe morphology variations, allows to follow the ischemia induced variations in repolarization in a more robust manner than classical indexes, avoiding ST segment and early T wave distortions not related to repolarization dispersion, and warranting further clinical studies to evaluate its ventricular arrhythmia risk predictive value during ischemia.

Effects of transcranial direct current stimulation on the glutamatergic pathway in the male rat hippocampus after experimental focal cerebral ischemia.

This study aimed to assess the focal cerebral ischemia-induced changes in learning and memory together with glutamatergic pathway in rats and the effects of treatment of the animals with transcranial Direct Current Stimulation (tDCS). One hundred male rats were divided into five groups as sham, tDCS, Ischemia/Reperfusion (IR), IR + tDCS, and IR + E-tDCS groups. Learning, memory, and locomotor activity functions were evaluated by behavioral experiments in rats. Glutamate and glutamine levels, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR1), N-Methyl-D-Aspartate receptors (NMDAR1 and NMDAR2A), vesicular glutamate transporter-1 (VGLUT-1), and excitatory amino acid transporters (EAAT1-3) mRNA expressions in hippocampus tissues were measured. Ischemic areas were analyzed by TTC staining. The increase was observed in IR + tDCS, and IR + E-tDCS groups compared to the IR group while a significant decrease was observed in IR group compared to the sham in the locomotor activity, learning, and memory tests. While glutamate and glutamine levels, AMPAR1, NMDAR1, NMDAR2A, VGLUT1, and EAAT1 mRNA expressions were significantly higher in IR group compared to the sham group, it was found to be significantly lower in IR + tDCS and IR + E-tDCS groups compared to the IR group. EAAT2 and EAAT3 mRNA expressions were significantly higher in IR + tDCS and IR + E-tDCS groups compared to the IR group. Ischemic areas were significantly decreased in IR + tDCS and IR + E-tDCS groups compared to the IR group. Current results suggest that tDCS application after ischemia improves learning and memory disorders and these effects of tDCS may be provided through transporters that regulate glutamate levels.

Global Cerebral Ischemia-induced Depression Accompanies Alteration of Neuronal Excitability in the Infralimbic Cortex Layer 2/3 Pyramidal Neurons.

Cerebral ischemia can lead to a range of sequelae, including depression. The pathogenesis of depression involves neuronal change of the medial prefrontal cortex (mPFC). However, how cerebral ischemia-induced changes manifest across subregions and layers of the mPFC is not well understood. In this study, we induced cerebral ischemia in mice via transient bilateral common carotid artery occlusion (tBCCAO) and observed depressive-like behavior. Using whole-cell patch clamp recording, we identified changes in the excitability of pyramidal neurons in the prelimbic cortex (PL) and infralimbic cortex (IL), the subregions of mPFC. Compared to sham control mice, tBCCAO mice showed significantly reduced neuronal excitability in IL layer 2/3 but not layer 5 pyramidal neurons, accompanied by increased rheobase current and decreased input resistance. In contrast, no changes were observed in the excitability of PL layer 2/3 and layer 5 pyramidal neurons. Our results provide a new direction for studying the pathogenesis of depression following ischemic damage by showing that cerebral ischemia induces subregion- and layer-specific changes in the mPFC pyramidal neurons.

Advanced repeated structuring and learning procedure to detect acute myocardial ischemia in serial 12-lead ECGs

Objectives. Acute myocardial ischemia in the setting of acute coronary syndrome (ACS) may lead to myocardial infarction. Therefore, timely decisions, already in the pre-hospital phase, are crucial to preserving cardiac function as much as possible. Serial electrocardiography, a comparison of the acute electrocardiogram with a previously recorded (reference) ECG of the same patient, aids in identifying ischemia-induced electrocardiographic changes by correcting for interindividual ECG variability. Recently, the combination of deep learning and serial electrocardiography provided promising results in detecting emerging cardiac diseases; thus, the aim of our current study is the application of our novel Advanced Repeated Structuring and Learning Procedure (AdvRS&LP), specifically designed for acute myocardial ischemia detection in the pre-hospital phase by using serial ECG features. Approach. Data belong to the SUBTRACT study, which includes 1425 ECG pairs, 194 (14%) ACS patients, and 1035 (73%) controls. Each ECG pair was characterized by 28 serial features that, with sex and age, constituted the inputs of the AdvRS&LP, an automatic constructive procedure for creating supervised neural networks (NN). We created 100 NNs to compensate for statistical fluctuations due to random data divisions of a limited dataset. We compared the performance of the obtained NNs to a logistic regression (LR) procedure and the Glasgow program (Uni-G) in terms of area-under-the-curve (AUC) of the receiver-operating-characteristic curve, sensitivity (SE), and specificity (SP). Main Results. NNs (median AUC = 83%, median SE = 77%, and median SP = 89%) presented a statistically (P value lower than 0.05) higher testing performance than those presented by LR (median AUC = 80%, median SE = 67%, and median SP = 81%) and by the Uni-G algorithm (median SE = 72% and median SP = 82%). Significance. In conclusion, the positive results underscore the value of serial ECG comparison in ischemia detection, and NNs created by AdvRS&LP seem to be reliable tools in terms of generalization and clinical applicability.

Posttreatment with Ospemifene Attenuates Hypoxia- and Ischemia-Induced Apoptosis in Primary Neuronal Cells via Selective Modulation of Estrogen Receptors

Stroke and perinatal asphyxia have detrimental effects on neuronal cells, causing millions of deaths worldwide each year. Since currently available therapies are insufficient, there is an urgent need for novel neuroprotective strategies to address the effects of cerebrovascular accidents. One such recent approach is based on the neuroprotective properties of estrogen receptors (ERs). However, activation of ERs by estrogens may contribute to the development of endometriosis or hormone-dependent cancers. Therefore, in this study, we utilized ospemifene, a novel selective estrogen receptor modulator (SERM) already used in dyspareunia treatment. Here, we demonstrated that posttreatment with ospemifene in primary neocortical cell cultures subjected to 18 h of hypoxia and/or ischemia followed by 6 h of reoxygenation has robust neuroprotective potential. Ospemifene partially reverses hypoxia- and ischemia-induced changes in LDH release, the degree of neurodegeneration, and metabolic activity. The mechanism of the neuroprotective actions of ospemifene involves the inhibition of apoptosis since the compound decreases caspase-3 overactivity during hypoxia and enhances mitochondrial membrane potential during ischemia. Moreover, in both models, ospemifene decreased the levels of the proapoptotic proteins BAX, FAS, FASL, and GSK3β while increasing the level of the antiapoptotic protein BCL2. Silencing of specific ERs showed that the neuroprotective actions of ospemifene are mediated mainly via ESR1 (during hypoxia and ischemia) and GPER1 (during hypoxia), which is supported by ospemifene-evoked increases in ESR1 protein levels in hypoxic and ischemic neurons. The results identify ospemifene as a promising neuroprotectant, which in the future may be used to treat injuries due to brain hypoxia/ischemia.

The mechanisms of potassium loss in acute myocardial ischemia: New insights from computational simulations.

Acute myocardial ischemia induces hyperkalemia (accumulation of extracellular potassium), a major perpetrator of lethal reentrant ventricular arrhythmias. Despite considerable experimental efforts to explain this pathology in the last decades, the intimate mechanisms behind hyperkalemia remain partially unknown. In order to investigate these mechanisms, we developed a novel computational model of acute myocardial ischemia which couples a) an electrophysiologically detailed human cardiomyocyte model that incorporates modifications to account for ischemia-induced changes in transmembrane currents, with b) a model of cardiac tissue and extracellular K + transport. The resulting model is able to reproduce and explain the triphasic time course of extracellular K + concentration within the ischemic zone, with values of close to 14mmol/L in the central ischemic zone after 30min. In addition, the formation of a border zone of approximately 1.2cm 15minafter the onset of ischemia is predicted by the model. Our results indicate that the primary rising phase of is mainly due to the imbalance between K + efflux, that increases slightly, and K + influx, that follows a reduction of the NaK pump activity by more than 50%. The onset of the plateau phase is caused by the appearance of electrical alternans (a novel mechanism identified by the model), which cause an abrupt reduction in the K + efflux. After the plateau, the secondary rising phase of is caused by a subsequent imbalance between the K + influx, which continues to decrease slowly, and the K + efflux, which remains almost constant. Further, the study shows that the modulation of these mechanisms by the electrotonic coupling is the main responsible for the formation of the ischemic border zone in tissue, with K + transport playing only a minor role. Finally, the results of the model indicate that the injury current established between the healthy and the altered tissue is not sufficient to depolarize non-ischemic cells within the healthy tissue.

Effect of Mucuna pruriens on brain NMDA receptor and tau protein gene expression in cerebral ischemic rats.

Present study aimed to assess effect of pre-treatment with Mucuna pruriens seed extract and its bioactive molecule(s) on NMDAR and Tau protein gene expression in cerebral ischemic rodent model. Methanol extract of M. pruriens seeds was characterized by HPLC, and β-sitosterol was isolated by flash chromatography. In vivo studies to observe the effect of pre-treatment (28 days) with methanol extract of M. pruriens seed and β-sitosterol on the unilateral cerebral ischemic rat model. Cerebral ischemia induced by left common carotid artery occlusion (LCCAO) for 75min (on day 29) followed by reperfusion for 12h. Rats (n = 48) divided into four groups. GroupI (control,Untreated + LCCAO)-No pre-treatment + cerebral ischemia; GroupII(β-sitosterol + Sham)-pre-treatment with β-sitosterol, 10mg/kg/day + sham-operated; GroupIII(β-sitosterol + LCCAO)-pre-treatment with β-sitosterol, 10mg/kg/day + cerebral ischemia; GroupIV(methanol extract + LCCAO)-pre-treatment with methanol extract of M. pruriens seeds, 50mg/kg/day + cerebral ischemia. Neurological deficit score was assessed just before sacrifice. Experimental animals were sacrificed after 12h reperfusion. Brain histopathology was performed. Gene expression of NMDAR and Tau protein of left cerebral hemisphere (occluded side) was performed by RT-PCR. Results revealed that the neurological deficit score was lower in groups III and IV compared to group I. NMDAR and tau protein mRNA expression in left cerebral hemisphere were upregulated in Group I, downregulated in groups III and IV. Histopathology of left cerebral hemisphere (occluded side) in Group I showed features of ischemic brain damage. Groups III and IV, left cerebral hemisphere showed less ischemic damage compared GroupI. Right cerebral hemisphere showed no areas of ischemia-induced brain changes. Pre-treatment with β-sitosterol and methanol extract of M. pruriens seeds may reduce ischemic brain injury following unilateral common carotid artery occlusion in rats.

Oxygen-glucose deprivation-induced glial cell reactivity in the rat primary neuron-glia co-culture.

It has been demonstrated that in vivo brain ischemia induces activation and proliferation of astrocytes and microglia. However, the mechanism underlying the ischemia-induced activation and proliferation of these cells remains to be unclear. Oxygen-glucose deprivation (OGD), an in vitro ischemia mimic, has been extensively used to analyze the hypoxia response of various cell types. This study examined the OGD-induced changes in the expression level of astrocytes and microglia marker proteins and immunoreactivity for Ki-67, a marker protein for cell proliferation, using rat primary hippocampal neuron-glia co-culture (NGC) cells. Furthermore, OGD-induced changes in the expression of M1/M2 microglia phenotype-related genes were also examined. MTT assay indicated that 120 min of OGD decreased cell viability, and immunocytochemistry indicated that 120 min of OGD abolished most microtubule-associated protein 2 (MAP2)-immunopositive neurons. In contrast, glial fibrillary acidic protein (GFAP)-immunopositive astrocytes and ionized calcium-binding adapter protein-1 (Iba-1)-immunopositive microglia, and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase)-immunopositive oligodendrocytes survived OGD. Western blot assays and double-immunofluorescent staining indicated that OGD increased the GFAP expression level and the Ki-67-immunopositive/GFAP-immunopositive cells' ratio. Real-time PCR analysis showed that OGD altered M1 microglia phenotype-related genes. Specifically, OGD decreased the expression level of CD32 and interleukin-1β (IL-1β) genes and increased that of the inducible nitric oxide synthase (iNOS) gene. Therefore, applying OGD to NGC cells could serve as a useful in vitro tool to elucidate the molecular mechanisms underlying brain ischemia-induced changes in GFAP expression, astrocyte proliferation, and M1 microglia phenotype-related gene expression.

Metabolomics Analysis of Electroacupuncture Pretreatment Induced Neuroprotection on Mice with Ischemic Stroke.

The brain metabolic changes caused by the interruption of blood supply are the initial factors of brain injury in ischemic stroke. Electroacupuncture (EA) pretreatment has been shown to protect against ischemic stroke, but whether its neuroprotective mechanism involves metabolic regulation remains unclear. Based on our finding that EA pretreatment significantly alleviated ischemic brain injury in mice by reducing neuronal injury and death, we performed a gas chromatography-time of flight mass spectrometry (GC-TOF/MS) to investigate the metabolic changes in the ischemic brain and whether EA pretreatment influenced these changes. First, we found that some glycolytic metabolites in the normal brain tissues were reduced by EA pretreatment, which may lay the foundation of neuroprotection for EA pretreatment against ischemic stroke. Then, 6[Formula: see text]h of cerebral ischemia-induced brain metabolic changes, especially the enhanced glycolysis, were partially reversed by EA pretreatment, which was manifested by the brain levels of 11 of 35 up-regulated metabolites and 18 of 27 down-regulated metabolites caused by cerebral ischemia significantly decreasing and increasing, respectively, due to EA pretreatment. A further pathway analysis showed that these 11 and 18 markedly changed metabolites were mainly involved in starch and sucrose metabolism, purine metabolism, aspartate metabolism, and the citric acid cycle. Additionally, we found that EA pretreatment raised the levels of neuroprotective metabolites in both normal and ischemic brain tissues. In conclusion, our study revealed that EA pretreatment may attenuate the ischemic brain injury by inhibiting glycolysis and increasing the levels of some neuroprotective metabolites.

Mechanistic study of optic atrophy 1 in ischemia-reperfusion disease.

Mitochondria consist of the inner mitochondrial membrane and the outer mitochondrial membrane, which maintain mitochondrial homeostasis through continuous fission and fusion to ensure a healthy mitochondrial network and thus regulate normal cellular function, namely mitochondrial dynamics. The imbalance between mitochondrial fusion and fission results in abnormal mitochondrial structure and eventually mitochondrial dysfunction, which is involved in the pathological process of ischemia-reperfusion injury (IRI). Optic atrophy 1 (OPA1) is a key protein that regulates mitochondrial inner membrane fusion and ensures normal mitochondrial function by balancing mitochondrial dynamics, participating in various processes such as mitochondrial fusion, oxidative stress, and apoptosis. Ischemia-induced changes in mitochondrial dynamics may be a key factor in limiting the recanalization time window and exacerbating reperfusion injury, and the mechanisms of these changes deserve further attention. Therefore, targeting OPA1-related mitochondrial fusions, thereby balancing mitochondrial dynamics and improving mitochondrial dysfunction, is a promising therapeutic strategy for ischemia-reperfusion diseases. This review will elaborate on the structure and function of OPA1 and the role of OPA1 in IRI to provide promising therapeutic targets for the treatment of ischemia-reperfusion diseases.

Quasi-steady-state amide proton transfer (QUASS APT) MRI enhances pH-weighted imaging of acute stroke.

Chemical exchange saturation transfer (CEST) imaging measurement depends not only on the labile proton concentration and pH-dependent exchange rate but also on experimental conditions, including the relaxation delay and radiofrequency (RF) saturation time. Our study aimed to extend a quasi-steady-state (QUASS) solution to a modified multi-slice CEST MRI sequence and test if it provides enhanced pH imaging after acute stroke. Our study derived the QUASS solution for a modified multislice CEST MRI sequence with an unevenly segmented RF saturation between image readout and signal averaging. Numerical simulation was performed to test if the generalized QUASS solution corrects the impact of insufficiently long relaxation delay, primary and secondary saturation times, and multi-slice readout. In addition, multiparametric MRI scans were obtained after middle cerebral artery occlusion, including relaxation and CEST Z-spectrum, to evaluate the performance of QUASS CEST MRI in a rodent acute stroke model. We also performed Lorentzian fitting to isolate multi-pool CEST contributions. The QUASS analysis enhanced pH-weighted magnetization transfer asymmetry contrast over the routine apparent CEST measurements in both contralateral normal (-3.46% ± 0.62% (apparent) vs. -3.67% ± 0.66% (QUASS), P< 0.05) and ischemic tissue (-5.53% ± 0.68% (apparent) vs. -5.94% ± 0.73% (QUASS), P< 0.05). Lorentzian fitting also showed significant differences between routine and QUASS analysis of ischemia-induced changes in magnetization transfer, amide, amine, guanidyl CEST, and nuclear Overhauser enhancement (-1.6parts per million) effects. Our study demonstrated that generalized QUASS analysis enhanced pH MRI contrast and improved quantification of the underlying CEST contrast mechanism, promising for further in vivo applications.

Trophic factor BDNF inhibits GABAergic signaling by facilitating dendritic enrichment of SUMO E3 ligase PIAS3 and altering gephyrin scaffold

Posttranslational addition of a small ubiquitin-like modifier (SUMO) moiety (SUMOylation) has been implicated in pathologies such as brain ischemia, diabetic peripheral neuropathy, and neurodegeneration. However, nuclear enrichment of SUMO pathway proteins has made it difficult to ascertain how ion channels, proteins that are typically localized to and function at the plasma membrane, and mitochondria are SUMOylated. Here, we report that the trophic factor, brain-derived neurotrophic factor (BDNF) regulates SUMO proteins both spatially and temporally in neurons. We show that BDNF signaling via the receptor tropomyosin-related kinase B facilitates nuclear exodus of SUMO proteins and subsequent enrichment within dendrites. Of the various SUMO E3 ligases, we found that PIAS-3 dendrite enrichment in response to BDNF signaling specifically modulates subsequent ERK1/2 kinase pathway signaling. In addition, we found the PIAS-3 RING and Ser/Thr domains, albeit in opposing manners, functionally inhibit GABA-mediated inhibition. Finally, using oxygen–glucose deprivation as an in vitro model for ischemia, we show that BDNF–tropomyosin-related kinase B signaling negatively impairs clustering of the main scaffolding protein at GABAergic postsynapse, gephyrin, whereby reducing GABAergic neurotransmission postischemia. SUMOylation-defective gephyrin K148R/K724R mutant transgene expression reversed these ischemia-induced changes in gephyrin cluster density. Taken together, these data suggest that BDNF signaling facilitates the temporal relocation of nuclear-enriched SUMO proteins to dendrites to influence postsynaptic protein SUMOylation.

Abstract TP246: The Role Of Pericytes In Ischemic Stroke

Stroke is one of the leading causes of death worldwide. Loss of blood flow, typically from a blood clot that occludes the cerebral vasculature, causes ischemia, tissue death, and long-term cognitive deficits. Following stroke, the blood-brain barrier (BBB) is damaged, causing leakage of ions, molecules, toxins, and immune cells into the brain. Pericytes are a perivascular cell that is important for neurovascular growth and stability, and have been reported to both aid vascular regrowth and transdifferentiate into pro-inflammatory cells after ischemic injury. However, markers used to identify pericytes non-specifically label other cell types, making it unclear whether these studies are specifically identifying pericytes. In order to understand how pericytes adapt to ischemic injury, we modeled stroke in mice with distal middle cerebral artery occlusion (dMCAO) and lineage traced these cells with two novel pericyte markers: TBX18 and Atp13a5, allowing us to track ischemia-induced changes in pericytes and their progeny. We discovered that pericytes in the ischemic core die over the first three days following injury, while surviving pericytes proliferate in response to injury, and investigated how pericyte identity changes with histology and single cell RNA sequencing.

Hydroxysafflor Yellow A Alleviates Ischemic Stroke in Rats via HIF-1[Formula: see text], BNIP3, and Notch1-Mediated Inhibition of Autophagy.

Stroke has become a major cause of death and disability worldwide. The cellular recycling pathway autophagy has been implicated in ischemia-induced neuronal changes, but whether autophagy plays a beneficial or detrimental role is controversial. Hydroxysafflor Yellow A (HSYA), a popular herbal medicine, is an extract of Carthamus tinctorius and is used to treat ischemic stroke (IS) in China. HSYA has been shown to prevent cardiovascular and cerebral ischemia/reperfusion injury in animal models. However, the specific active ingredients and molecular mechanisms of HSYA in IS remain unclear. Here, we investigated the effect of HSYA treatment on autophagy in a rat model of IS. IS was induced in rats by middle cerebral artery occlusion. Rats were treated once daily for 3 days with saline, HYSA, or the neuroprotective agent Edaravone. Neurobehavioral testing was performed on days 1, 2, and 3 post-surgery. Brains were removed on day 3 post-surgery for histological evaluation of infarct area, morphology, and for qRT-PCR and western blot analysis of the expression of the autophagy factor LC3 and the signaling molecules HIF-1[Formula: see text], BNIP3, and Notch1. Molecular docking studies were performed in silico to predict potential interactions between HSYA and LC3, HIF-1[Formula: see text], BNIP3, and Notch1 proteins. The result showed that HSYA treatment markedly alleviated IS-induced neurobehavioral deficits and reduced brain infarct area and tissue damage. HSYA also significantly reduced hippocampal expression levels of LC3, HIF-1[Formula: see text], BNIP3, and Notch1. The beneficial effect of HSYA was generally superior to that of Edaravone. Molecular modeling suggested that HSYA may bind strongly to HIF-1[Formula: see text], BNIP3, and Notch1 but weakly to LC3. In conclusion, HSYA inhibits post-IS autophagy induction in the brain, possibly by suppressing HIF-1[Formula: see text], BNIP3 and Notch1. HSYA may have utility as a post-IS neuroprotective agent.

Different changes in pre- and postsynaptic components in the hippocampal CA1 subfield after transient global cerebral ischemia.

To date, ischemia-induced damage to dendritic spines has attracted considerable attention, while the possible effects of ischemia on presynaptic components has received relatively less attention. To further examine ischemia-induced changes in pre- and postsynaptic specializations in the hippocampal CA1 subfield, we modeled global cerebral ischemia with two-stage 4-vessel-occlusion in rats, and found that three postsynaptic markers, microtubule-associated protein 2 (MAP2), postsynaptic density protein 95 (PSD95), and filamentous F-actin (F-actin), were all substantially decreased in the CA1 subfield after ischemia/reperfusion (I/R). Although no significant change was detected in synapsin I, a presynaptic marker, in the CA1 subfield at the protein level, confocal microscopy revealed that the number and size of synapsin I puncta were significantly changed in the CA1 stratum radiatum after I/R. The size of synapsin I puncta became slightly, but significantly reduced on Day 1.5 after I/R. From Days 2 to 7 after I/R, the number of synapsin I puncta became moderately decreased, while the size of synapsin I puncta was significantly increased. Interestingly, some enlarged puncta of synapsin I were observed in close proximity to the dendritic shafts of CA1 pyramidal cells. Due to the more substantial decrease in the number of F-actin puncta, the ratio of synapsin I/F-actin puncta was significantly increased after I/R. The decrease in synapsin I puncta size in the early stage of I/R may be the result of excessive neurotransmitter release due to I/R-induced hyperexcitability in CA3 pyramidal cells, while the increase in synapsin I puncta in the later stage of I/R may reflect a disability of synaptic vesicle release due to the loss of postsynaptic contacts.

Dihydrocapsaicin effectively mitigates cerebral ischemia-induced pathological changes in vivo, partly via antioxidant and anti-apoptotic pathways

AimsIschemic stroke occurs when there is a sudden blockage of cerebral blood flow. This condition is a major cause of mortality, especially in low-income countries, and its incidence is dramatically increasing. Therapeutic strategies against stroke are therefore required. The present study explored the effects of dihydrocapsaicin on neuronal loss, brain infarct volume, and antioxidants in a rat model of permanent occlusion of the right middle cerebral artery (Rt.MCAO). Main methodsMale Wistar rats received dihydrocapsaicin intraperitoneally for 7 days after permanent occlusion of their right middle cerebral artery (Rt.MCAO). Then, the brain infarct volume, neuronal density, and antioxidant and anti-apoptotic activities in the cortex and hippocampus were determined at the end of the study. Key findingDihydrocapsaicin treatment was found to significantly improve neuronal density, decrease infarct volume, reduce MDA elevation, improve CAT and SOD activities, decrease the density ratio of Bax and caspase-3, and increase the density ratio of Bcl-XL to β-actin in the cerebral cortex and hippocampus. SignificanceThe present study suggests that dihydrocapsaicin effectively mitigates cerebral ischemia-induced pathological changes in vivo, partly via antioxidant and anti-apoptotic pathways.

Ischemia Regulates Endoglin Expression in a Pig Skin Flap Model

Endoglin is a transmembrane protein that binds transforming growth factor b (TGF-b) with high affinity. It is thought to modulate TGF-b signaling and responses through interactions on cell surfaces with TGF-b receptors. It is highly expressed on endothelial cells of tissues undergoing angiogenesis. Since ischemia-induced changes such as angiogenesis are in part regulated by TGF-b, it is important to elucidate the precise mechanism by which they occur if we are to influence the effects of ischemia on tissue. Since endoglin is one of the receptors involved in regulating TGF-b signaling, we tested whether endoglin expression will increase in response to ischemic injury. A pig skin flap model was developed and used in our laboratory to study the effect of ischemia on the expression of endoglin and its ligand TGF-b1 using immunohistochemistry. We observed an increased expression of endoglin and TGF-b1 in the epidermal layer, blood vessels, and fibroblasts in ischemic skin flaps as compared to non-ischemic control skin flaps. Given the role of TGF-b and endoglin in angiogenesis, this may represent a mechanism by which ischemia stimulates angiogenesis. Much work remains to be done to delineate the exact mechanisms by which TGF-b acts and the exact role of endoglin in TGF-b signaling. Understanding how to regulate the actions of TGF-b by manipulating the expression of endoglin may provide a therapeutic avenue given the importance of TGF-b in tumour angiogenesis.

Metabolomics reveals the impact of Type 2 diabetes on local muscle and vascular responses to ischemic stress.

Type 2 diabetes mellitus (T2DM) reduces exercise capacity, but the mechanisms are incompletely understood. We probed the impact of ischemic stress on skeletal muscle metabolite signatures and T2DM-related vascular dysfunction. we recruited 38 subjects (18 healthy, 20 T2DM), placed an antecubital intravenous catheter, and performed ipsilateral brachial artery reactivity testing. Blood samples for plasma metabolite profiling were obtained at baseline and immediately upon cuff release after 5 min of ischemia. Brachial artery diameter was measured at baseline and 1 min after cuff release. as expected, flow-mediated vasodilation was attenuated in subjects with T2DM (P<0.01). We confirmed known T2DM-associated baseline differences in plasma metabolites, including homocysteine, dimethylguanidino valeric acid and β-alanine (all P<0.05). Ischemia-induced metabolite changes that differed between groups included 5-hydroxyindoleacetic acid (healthy: -27%; DM +14%), orotic acid (healthy: +5%; DM -7%), trimethylamine-N-oxide (healthy: -51%; DM +0.2%), and glyoxylic acid (healthy: +19%; DM -6%) (all P<0.05). Levels of serine, betaine, β-aminoisobutyric acid and anthranilic acid were associated with vessel diameter at baseline, but only in T2DM (all P<0.05). Metabolite responses to ischemia were significantly associated with vasodilation extent, but primarily observed in T2DM, and included enrichment in phospholipid metabolism (P<0.05). our study highlights impairments in muscle and vascular signaling at rest and during ischemic stress in T2DM. While metabolites change in both healthy and T2DM subjects in response to ischemia, the relationship between muscle metabolism and vascular function is modified in T2DM, suggesting that dysregulated muscle metabolism in T2DM may have direct effects on vascular function.