Ischemic Neurons Research Articles

Modulation of Ca2+ oscillation following ischemia and nicotinic acetylcholine receptors in primary cortical neurons by high-throughput analysis

Modulation of Ca2+ oscillation following ischemia and nicotinic acetylcholine receptors in primary cortical neurons by high-throughput analysis

Biological Evaluation of Dibenzo-α-Pyrone Derivatives in Neuronal Ischemia by Sciatic Nerve Ligation in Wistar Rats

Biological Evaluation of Dibenzo-α-Pyrone Derivatives in Neuronal Ischemia by Sciatic Nerve Ligation in Wistar Rats

Changes in early endosomes in rat hippocampal CA1 neurons after transient global cerebral ischaemia.

Transient global ischaemia in rodents causes selective loss of hippocampal CA1neurons, but the potential involvement of endocytic pathways has not been fully explored. The aim of this study was to investigate the changes in early endosomes in the CA1 subfield after ischaemia and reperfusion. A four-vessel occlusion (4-VO) model was established in Wistar rats to induce 13 minutes of global cerebral ischaemia. Neuronal death was detected by Fluoro-Jade B (FJ-B) staining at various intervals after reperfusion, and intracellular membrane changes in ischaemic neurons were revealed using DiOC6(3), a lipophilic fluorescent probe. Ras-related protein Rab5 (Rab5) immunostaining was performed to detect changes in early endosomes in ischaemic neurons. Western blot analysis was used to confirm the morphological observations on Rab5 in the CA1 hippocampal subfield. FJ-B staining confirmed progressive neuronal death in the CA1 subfield in ischaemic rats after reperfusion. DiOC6(3) staining revealed abnormally increased membranous components in ischaemic CA1 neurons. Specifically, early endosomes, as labelled by Rab5 immunostaining, significantly increased in number and size in CA1 neurons at 1.5 and 2 days post-reperfusion, followed by rupture at day 3 and a decrease in staining intensity at day 7 post-reperfusion. Western blot analysis confirmed a significant upregulation of Rab5 protein levels at day 2, which returned to near control levels by day 7. Our study revealed significant changes in the dynamics of early endosomes in CA1 neurons after ischaemia-reperfusion injury. The initial increase in the area fraction of early endosomes in CA1 neurons may reflect an upregulation of endocytic activity, whereas the fragmentation and reduction of early endosomes at the later stage may indicate a failure of adaptive mechanisms of ischaemic neurons against ischaemia-induced death. Understanding the temporal dynamics of early endosomes provides critical insights into the cellular mechanisms that govern fate of CA1 hippocampal neuronsl after ischaemia/reperfusion.

Human neural stem cells derived from fetal human brain communicate with each other and rescue ischemic neuronal cells through tunneling nanotubes

Pre-clinical trials have demonstrated the neuroprotective effects of transplanted human neural stem cells (hNSCs) during the post-ischemic phase. However, the exact neuroprotective mechanism remains unclear. Tunneling nanotubes (TNTs) are long plasma membrane bridges that physically connect distant cells, enabling the intercellular transfer of mitochondria and contributing to post-ischemic repair processes. Whether hNSCs communicate through TNTs and their role in post-ischemic neuroprotection remains unknown. In this study, non-immortalized hNSC lines derived from fetal human brain tissues were examined to explore these possibilities and assess the post-ischemic neuroprotection potential of these hNSCs. Using Tau-STED super-resolution confocal microscopy, live cell time-lapse fluorescence microscopy, electron microscopy, and direct or non-contact homotypic co-cultures, we demonstrated that hNSCs generate nestin-positive TNTs in both 3D neurospheres and 2D cultures, through which they transfer functional mitochondria. Co-culturing hNSCs with differentiated SH-SY5Y (dSH-SY5Y) revealed heterotypic TNTs allowing mitochondrial transfer from hNSCs to dSH-SY5Y. To investigate the role of heterotypic TNTs in post-ischemic neuroprotection, dSH-SY5Y were subjected to oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/R) with or without hNSCs in direct or non-contact co-cultures. Compared to normoxia, OGD/R dSH-SY5Y became apoptotic with impaired electrical activity. When OGD/R dSH-SY5Y were co-cultured in direct contact with hNSCs, heterotypic TNTs enabled the transfer of functional mitochondria from hNSCs to OGD/R dSH-SY5Y, rescuing them from apoptosis and restoring the bioelectrical profile toward normoxic dSH-SY5Y. This complete neuroprotection did not occur in the non-contact co-culture. In summary, our data reveal the presence of a functional TNTs network containing nestin within hNSCs, demonstrate the involvement of TNTs in post-ischemic neuroprotection mediated by hNSCs, and highlight the strong efficacy of our hNSC lines in post-ischemic neuroprotection.Human neural stem cells (hNSCs) communicate with each other and rescue ischemic neurons through nestin-positive tunneling nanotubes (TNTs). A Functional mitochondria are exchanged via TNTs between hNSCs. B hNSCs transfer functional mitochondria to ischemic neurons through TNTs, rescuing neurons from ischemia/reperfusion ROS-dependent apoptosis.

Comparative proteomic analysis of retinal hypoxia-ischemia in an acute ocular hypertension model using tandem mass tag-based quantitative proteomics

The main symptom of acute glaucoma is acute ocular hypertension (AOH), which leads to the death of retinal ganglion cells (RGCs) and permanent loss of vision. However, effective treatments for these conditions are lacking. This study aimed to identify major regulators and overall protein changes involved in AOH-induced RGC death. Proteomic patterns of the retinal protein extracts from the AOH and sham groups were analyzed using mass spectrometry (MS), followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.Proteomic analysis revealed 92 proteins in the AOH group compared to the control group; 58 proteins were upregulated and 34 were downregulated. Alterations in fatty acid-binding protein 7 (FABP7) and caveolin-1 (Cav-1), which are related to fatty acid metabolism and ocular inflammatory signaling, were detected using western blotting and biochemical assays. Variations in the expression of galectin-1 (Gal-1), S100 calcium-binding protein A6 (S100a6), and visinin-like protein-1 (VILIP) have been associated with neuronal ischemia. Our investigation demonstrates that neuroinflammation and fatty acid metabolism are involved in retinal impairment following AOH, suggesting a possible treatment approach for acute glaucoma.

N-ethylmaleimide-sensitive factor elicits a neuroprotection against ischemic neuronal injury by restoring autophagic/lysosomal dysfunction

Autophagosome-lysosome fusion defects play a critical role in driving autolysosomal dysfunction, leading to autophagic/lysosomal impairment in neurons following ischemic stroke. However, the mechanisms hindering autophagosome-lysosome fusion remain unclear. Soluble N-ethylmaleimide-sensitive factor (NSF) is an essential ATPase to reactivate STX17 and VAMP8, which are the paired molecules to mediate fusion between autophagosomes and lysosomes. However, NSF is frequently inactivated to inhibit the reactivation of STX17 and VAMP8 in ischemic neurons. Herein, we investigated whether autophagosome-lysosome fusion could be facilitated to alleviate autophagic/lysosomal impairment in ischemic neurons by over-expressing NSF. Rat model of middle cerebral artery occlusion (MCAO) and HT22 neuron ischemia model of oxygen-glucose deprivation (OGD) were prepared, respectively. The results demonstrated that NSF activity was significantly suppressed, accompanied by reduced expressions of STX17 and VAMP8 in penumbral neurons 48 h post-MCAO and in HT22 neurons 2 h post-OGD. Moreover, the attenuated autolysosome formation accompanied by autophagic/lysosomal dysfunction was observed. Thereafter, NSF activity in HT22 neurons was altered by over-expression and siRNA knockdown, respectively. After transfection with recombinant NSF-overexpressing lentiviruses, both STX17 and VAMP8 expressions were concurrently elevated to boost autophagosome-lysosome fusion, as shown by enhanced immunofluorescence intensity co-staining with LC3 and LAMP-1. Consequently, the OGD-created autophagic/lysosomal dysfunction was prominently ameliorated, as reflected by augmented autolysosomal functions and decreased autophagic substrates. By contrast, NSF knockdown conversely aggravated the autophagic/lysosomal impairment, and thereby exacerbated neurological damage. Our study indicates that NSF over-expression induces neuroprotection against ischemic neuronal injury by restoring autophagic/lysosomal dysfunction via the facilitation of autophagosome-lysosome fusion.Over-expression of NSF promotes fusion by reactivating STX17 and VAMP8. Black arrows represent the pathological process after cerebral ischemia, green arrows represent the mechanism of remission after NSF over-expression, and red arrows represent the effect on the pathological process after NSF knockdown.

Microglia through MFG-E8 signaling decrease the density of degenerating neurons and protect the brain from the development of cortical infarction after stroke.

Neuronal loss is a hallmark of stroke and other neurodegenerative diseases, and as such, neuronal loss caused by microglia has been thought to be a contributing factor to disease progression. Here, we show that microglia indeed contribute significantly to neuronal loss in a mouse model of stroke, but this microglial-dependent process of neuronal clearance specifically targets stressed and degenerating neurons in the ischemic cortical region and not healthy non-ischemic neurons. Nonspecific stimulation of microglia decreased the density of neurons in the ischemic cortical region, whereas specific inhibition of MFG-E8 signaling, which is required for microglial phagocytosis of neurons, had the opposite effect. In both scenarios, the effects were microglia specific, as the same treatments had no effect in mice whose microglia were depleted prior to stroke. Finally, even though the inhibition of MFG-E8 signaling increased neuronal density in the ischemic brain region, it substantially exacerbated the development of cortical infarction. In conclusion, microglia through MFG-E8 signaling contribute to the loss of ischemic neurons and, in doing so, minimize the development of cortical infarction after stroke.

Ischemic Neuroprotection by Insulin with Down-Regulation of Divalent Metal Transporter 1 (DMT1) Expression and Ferrous Iron-Dependent Cell Death.

Background: The regulation of divalent metal transporter-1 (DMT1) by insulin has been previously described in Langerhans cells and significant neuroprotection was found by insulin and insulin-like growth factor 1 treatment during experimental cerebral ischemia in acute ischemic stroke patients and in a rat 6-OHDA model of Parkinson's disease, where DMT1 involvement is described. According to the regulation of DMT1, previously described as a target gene of NF-kB in the early phase of post-ischemic neurodegeneration, both in vitro and in vivo, and because insulin controls the NFkB signaling with protection from ischemic cell death in rat cardiomyocytes, we evaluated the role of insulin in relation to DMT1 expression and function during ischemic neurodegeneration. Methods: Insulin neuroprotection is evaluated in differentiated human neuroblastoma cells, SK-N-SH, and in primary mouse cortical neurons exposed to oxygen glucose deprivation (OGD) for 8 h or 3 h, respectively, with or without 300 nM insulin. The insulin neuroprotection during OGD was evaluated in both cellular models in terms of cell death, and in SK-N-SH for DMT1 protein expression and acute ferrous iron treatment, performed in acidic conditions, known to promote the maximum DMT1 uptake as a proton co-transporter; and the transactivation of 1B/DMT1 mouse promoter, already known to be responsive to NF-kB, was analyzed in primary mouse cortical neurons. Results: Insulin neuroprotection during OGD was concomitant to the down-regulation of both DMT1 protein expression and 1B/DMT1 mouse promoter transactivation. We also showed the insulin-dependent protection from cell death after acute ferrous iron treatment. In conclusion, although preliminary, this evaluation highlights the peculiar role of DMT1 as a possible pharmacological target, involved in neuroprotection by insulin during in vitro neuronal ischemia and acute ferrous iron uptake.

IL-17A exacerbates caspase-12-dependent neuronal apoptosis following ischemia through the Src-PLCγ-calpain pathway

Interleukin-17 A (IL-17 A) contributes to inflammation and causes secondary injury in post-stroke patients. However, little is known regarding the mechanisms that IL-17 A is implicated in the processes of neuronal death during ischemia. In this study, the mouse models of middle cerebral artery occlusion/reperfusion (MCAO/R)-induced ischemic stroke and oxygen-glucose deprivation/reoxygenation (OGD/R)-simulated in vitro ischemia in neurons were employed to explore the role of IL-17 A in promoting neuronal apoptosis. Mechanistically, endoplasmic reticulum stress (ERS)-induced neuronal apoptosis was accelerated by IL-17 A activation through the caspase-12-dependent pathway. Blocking calpain or phospholipase Cγ (PLCγ) inhibited IL-17 A-mediated neuronal apoptosis under ERS by inhibiting caspase-12 cleavage. Src and IL-17 A are linked, and PLCγ directly binds to activated Src. This binding causes intracellular Ca2+ flux and activates the calpain-caspase-12 cascade in neurons. The neurological scores showed that intracerebroventricular (ICV) injection of an IL-17 A neutralizing mAb decreased the severity of I/R-induced brain injury and suppressed apoptosis in MCAO mice. Our findings reveal that IL-17 A increases caspase-12-mediated neuronal apoptosis, and IL-17 A suppression may have therapeutic potential for ischemic stroke.

Circulating HMGB1 in acute ischemic stroke and its association with post-stroke cognitive impairment.

Ischemic stroke frequently leads to a condition known as post-stroke cognitive impairment (PSCI). Timely recognition of individuals susceptible to developing PSCI could facilitate the implementation of personalized strategies to mitigate cognitive deterioration. High mobility group box 1 (HMGB1) is a protein released by ischemic neurons and implicated in inflammation after stroke. Circulating levels of HMGB1 could potentially serve as a prognostic indicator for the onset of cognitive impairment following ischemic stroke. To investigate the predictive value of circulating HMGB1 concentrations in the acute phase of ischemic stroke for the development of cognitive dysfunction at the 3-month follow-up. A total of 192 individuals experiencing their initial episode of acute cerebral infarction were prospectively recruited for this longitudinal investigation. Concentrations of circulating HMGB1 were quantified using an enzyme-linked immunosorbent assay (ELISA) technique within the first 24 hours following hospital admission. Patients underwent neurological evaluation including NIHSS scoring. Neuropsychological evaluation was conducted at the 3-month follow-up after the cerebrovascular event, employing the Montreal Cognitive Assessment (MoCA) as the primary tool for assessing cognitive performance. Multivariable logistic regression models were employed to investigate the relationship between circulating HMGB1 concentrations and cognitive dysfunction following stroke, which was operationalized as a MoCA score below 26, while controlling for potential confounders including demographic characteristics, stroke severity, vascular risk factors, and laboratory parameters. Of 192 patients, 84 (44%) developed PSCI. Circulating HMGB1 concentrations were significantly elevated in individuals who developed cognitive dysfunction following stroke compared to those who maintained cognitive integrity (8.4 ±1.2 ng/mL vs 4.6 ±0.5 ng/mL, respectively; p<0.001). The prevalence of PSCI showed a dose-dependent increase with higher HMGB1 quartiles. After controlling for potential confounders such as demographic factors (age, gender, and education), stroke severity, vascular risk factors, and laboratory parameters in a multivariable logistic regression model, circulating HMGB1 concentrations emerged as a significant independent predictor of cognitive dysfunction following stroke (regression coefficient = 0.236, p<0.001). Circulating HMGB1 concentrations quantified within the first 24 hours following acute cerebral infarction are significantly and independently correlated with the likelihood of developing cognitive dysfunction at the 3-month follow-up, even after accounting for potential confounding factors. HMGB1 may be a novel biomarker to identify patients likely to develop post-stroke cognitive impairment for targeted preventive interventions.

Case Report: Traumatic Optic Neuropathy Secondary to Gun Shot Wound

This report discusses a patient with history of a cranial gunshot wound that resulted in Traumatic Optic Neuropathy [TON]. TON is a condition that is caused by direct or indirect trauma to the orbit or head. It is suspected that the mechanical shearing forces endured from such trauma causes diffuse axonal injury, ischemia, necrosis, and/or apoptosis of neurons. Other than observation alone, the management of TON is considered controversial. Various studies investigated the use of high-dose corticosteroid therapy, optic nerve decompression, and intravenous erythropoietin; however, the benefits of each strategy are unclear. The ocular and visual sequelae of TON may present as uniquely as the individual who exhibits them. Treatment and management of TON should be considered on a case-by-case basis.

Adenylate kinase 4 promotes neuronal energy metabolism and mitophagy in early cerebral ischemia via Parkin/PKM2 pathway

Mitochondrial dysfunction is closely related to brain injury and neurological dysfunction in ischemic stroke. Adenylate kinase 4 (AK4) plays a critical role in energy metabolism and mitochondrial homeostasis. However, the underlying mechanisms remain unclear. In the present study, we demonstrated an important role of AK4 in mitochondrial dysfunction in the early cerebral ischemia. Early focal cerebral ischemia induced decrease of AK4 protein expression in ischemic hemispheric brain tissue in mice. Exposure of cultured primary neuron to oxygen-glucose deprivation (OGD) also induced AK4 downregulation. Overexpression of AK4 in neuron using adeno-associated virus (AAV-AK4) in mice promoted neuronal survival reflected by decreased infarction volume and TUNEL staining. AK4 overexpression inhibited mitochondrial decline and downregulation of energy metabolism-associated proteins (p-AMPK and ATP1A3) induced by MCAO. Moreover, AK4 knock-in using lentivirus carried AK4 vector (LV-AK4) induced energy metabolism shift from glycolysis to oxidation in neuron. Using transmission electron microscope and western blot, we revealed that AK4 overexpression promoted mitophagy and mitophagy-associated proteins expression PINK1 and Parkin after MCAO. Mass spectrometry and co-immunoprecipitation revealed an interaction between AK4 and PKM2. Mechanistically, AK4 indirectly decreased PKM2 expression via enhancing its ubiquitination by increasing the interaction between PKM2 and its ubiquitin E3 ligase Parkin, and inhibits Parkin downregulation. In conclusion, our data demonstrate that AK4/ Parkin /PKM axis prevents cerebral ischemia damage via regulation of neuronal energy metabolism model and mitophagy. AK4 was a new target for intervention of early ischemic neuron injury.

4′-O-methylbavachalcone alleviates ischemic stroke injury by inhibiting parthanatos and promoting SIRT3

Cerebral ischemia-reperfusion injury (CIRI) can induce massive death of ischemic penumbra neurons via oxygen burst, exacerbating brain damage. Parthanatos is a form of caspase-independent cell death involving excessive activation of PARP-1, closely associated with intense oxidative stress following CIRI. 4′-O-methylbavachalcone (MeBavaC), an isoprenylated chalcone component in Fructus Psoraleae, has potential neuroprotective effects. This study primarily investigates whether MeBavaC can act on SIRT3 to alleviate parthanatos of ischemic penumbra neurons induced by CIRI. MeBavaC was oral gavaged to the middle cerebral artery occlusion-reperfusion (MCAO/R) rats after occlusion. The effects of MeBavaC on cerebral injury were detected by the neurological deficit score and cerebral infarct volume. In vitro, PC-12 cells were subjected to oxygen and glucose deprivation/reoxygenation (OGD/R), and assessed cell viability and cell injury. Also, the levels of ROS, mitochondrial membrane potential (MMP), and intracellular Ca2+ levels were detected to reflect mitochondrial function. We conducted western blotting analyses of proteins involved in parthanatos and related signaling pathways. Finally, the exact mechanism between the neuroprotection of MeBavaC and parthanatos was explored. Our results indicate that MeBavaC reduces the cerebral infarct volume and neurological deficit scores in MCAO/R rats, and inhibits the decreased viability of PC-12 cells induced by OGD/R. MeBavaC also downregulates the expression of parthanatos-related death proteins PARP-1, PAR, and AIF. However, this inhibitory effect is weakened after the use of a SIRT3 inhibitor. In conclusion, the protective effect of MeBavaC against CIRI may be achieved by inhibiting parthanatos of ischemic penumbra neurons through the SIRT3-PARP-1 axis.

Exploring the neuroprotection of the combination of astragaloside A, chlorogenic acid and scutellarin in treating chronic cerebral ischemia via network analysis and experimental validation

Chronic cerebral ischemia (CCI) primarily causes cognitive dysfunction and other neurological impairments, yet there remains a lack of ideal therapeutic medications. The preparation combination of Astragalus membranaceus (Fisch.) Bunge and Erigeron breviscapus (Vant.) Hand.-Mazz have been utilized to ameliorate neurological dysfunction following cerebral ischemia, but material basis of its synergy remains unclear. The principal active ingredients and their optimal proportions in this combination have been identified through the oxygen and glucose deprivation (OGD) cell model, including astragaloside A, chlorogenic acid and scutellarin (ACS), and its efficacy in enhancing the survival of OGD PC12 cells surpasses that of the combination preparation. Nevertheless, mechanism of ACS against CCI remains elusive. In this study, 63 potential targets of ACS against CCI injury were obtained by network pharmacology, among which AKT1, CASP3 and TNF are the core targets. Subsequent analysis utilizing KEGG and GO suggested that PI3K/AKT pathway may play a crucial role for ACS in ameliorating CCI injury. Then, a right unilateral common carotid artery occlusion (rUCCAO) mouse model and an OGD PC12 cell model were established to replicate the pathological processes of CCI in vivo and in vitro. These models were utilized to explore the anti-CCI effects of ACS and its regulatory mechanisms, particularly focusing on PI3K/AKT pathway. The results showed that ACS facilitated the restoration of cerebral blood flow in CCI mice, enhanced the function of the central cholinergic nervous system, protected against ischemic nerve cell and mitochondrial damage, and improved cognitive function and other neurological impairments. Additionally, ACS upregulated the expression of p-PI3K, p-AKT, p-GSK3β and Bcl-2, and diminished the expression of Cyto-c, cleaved Caspase-3 and Bax significantly. However, the PI3K inhibitor (LY294002) partially reversed the downregulation of Bax, Cyto-c and cleaved Caspase-3 expression as well as the upregulation of p-AKT/AKT, p-GSK3β/GSK3β, and Bcl-2/Bax ratios. These findings suggest that ACS against neuronal damage in cerebral ischemia may be closely related to the activation of PI3K/AKT pathway. These results declared first time ACS may become an ideal candidate drug against CCI due to its neuroprotective effects, which are mediated by the activated PI3K/AKT pathway mitigates mitochondrial damage and prevents cell apoptosis.

Advanced Diagnostic Tools in Hypothermia-Related Fatalities-A Pathological Perspective.

Although classical gross features are known in hypothermia victims, they lack specific diagnosis features. The aim of our study was to reveal specific brain and lung pathological features in a group of hypothermia-related fatalities. The study group comprised 107 cases from our files associated with hypothermia. Routine hematoxylin-eosin (H&E) staining and postmortem immunohistochemistry were performed. The microscopic cerebral exam revealed diffuse perineuronal and perivascular edema, gliosis, mononuclear cell infiltration, acute brain injuries, focal neuronal ischemia, lacunar infarction, and variable hemorrhages. Variable alveolar edema, pulmonary emphysema, intra-alveolar and/or pleural hemorrhage, and bronchopneumonia, as well as other pre-existing lesions, were identified in lung tissue samples. Glial cells displayed S100β expression, while neurons showed moderate Hsp70 immunopositivity. Alveolar basal membranes exhibited diffuse ICAM-1 positive expression, while ICAM-1 and AQP-1 positivity was observed in the alveolar septum vascular endothelium. Statistical analysis revealed a significant correlation between S100β and Hps70 immunoexpression and cerebral pathological features, between ICAM-1 immunoexpression and alveolar edema and pulmonary emphysema, and between AQP-1 immunoexpression and pulmonary emphysema. Our results add supplementary data to brain and lung pathological findings in hypothermia-related fatalities, with potential therapeutic value in hypothermia patients.

Alpha-, Beta-, and Gamma-Secretase, Amyloid Precursor Protein, and Tau Protein Genes in the Hippocampal CA3 Subfield in an Ischemic Model of Alzheimer's Disease with Survival up to 2 Years.

Understanding the phenomena underlying the non-selective susceptibility to ischemia of pyramidal neurons in the CA3 is important from the point of view of elucidating the mechanisms of memory loss and the development of dementia. The aim of the study was to investigate changes in genes expression of amyloid precursor protein, its cleaving enzymes and tau protein in CA3 post-ischemia with survival of 12-24 months. We used an ischemic model of Alzheimer's disease to study the above genes using an RT-PCR protocol. The expression of the amyloid precursor protein gene was above the control values at all times post-ischemia. The expression of the α-secretase gene also exceeded the control values post-ischemia. The expression of the β-secretase gene increased 12 and 24 months post-ischemia, and 18 months was below control values. Presenilin 1 and 2 genes expression was significantly elevated at all times post-ischemia. Also, tau protein gene expression was significantly elevated throughout the observation period, and peak gene expression was present 12 months post-ischemia. The study suggests that the genes studied are involved in the non-amyloidogenic processing of amyloid precursor protein. Additionally data indicate that brain ischemia with long-term survival causes damage and death of pyramidal neurons in the CA3 area of the hippocampus in a modified tau protein-dependent manner. Thus defining a new and important mechanism of pyramidal neuronal death in the CA3 area post-ischemia. In addition expression of tau protein gene modification after brain ischemia is useful in identifying ischemic mechanisms occurring in Alzheimer's disease.

Cathodal bilateral transcranial direct-current stimulation regulates selenium to confer neuroprotection after rat cerebral ischaemia-reperfusion injury.

Non-invasive transcranial direct-current stimulation (tDCS) is a safe ischaemic stroke therapy. Cathodal bilateral tDCS (BtDCS) is a modified tDCS approach established by us recently. Because selenium (Se) plays a crucial role in cerebral ischaemic injury, we investigated whether cathodal BtDCS conferred neuroprotection via regulating Se-dependent signalling in rat cerebral ischaemia-reperfusion (I/R) injury. We first showed that the levels of Se and its transport protein selenoprotein P (SEPP1) were reduced in the rat cortical penumbra following I/R, whereas cathodal BtDCS prevented the reduction of Se and SEPP1. Interestingly, direct-current stimulation (DCS) increased SEPP1 level in cultured astrocytes subjected to oxygen-glucose deprivation reoxygenation (OGD/R) but had no effect on SEPP1 level in OGD/R-insulted neurons, indicating that DCS may increase Se in ischaemic neurons by enhancing the synthesis and secretion of SEPP1 in astrocytes. We then revealed that DCS reduced the number of injured mitochondria in OGD/R-insulted neurons cocultured with astrocytes. DCS and BtDCS prevented the reduction of the mitochondrial quality-control signalling, vesicle-associated membrane protein 2 (VAMP2) and syntaxin-4 (STX4), in OGD/R-insulted neurons cocultured with astrocytes and the ischaemic brain respectively. Under the same experimental conditions, downregulation of SEPP1 blocked DCS- and BtDCS-induced upregulation of VAMP2 and STX4. Finally, we demonstrated that cathodal BtDCS increased Se to reduce infract volume following I/R. Together, the present study uncovered a molecular mechanism by which cathodal BtDCS confers neuroprotection through increasing SEPP1 in astrocytes and subsequent upregulation of SEPP1/VAMP2/STX4 signalling in ischaemic neurons after rat cerebral I/R injury. KEY POINTS: Cathodal bilateral transcranial direct-current stimulation (BtDCS) prevents the reduction of selenium (Se) and selenoprotein P in the ischaemic penumbra. Se plays a crucial role in cerebral ischaemia injury. Direct-current stimulation reduces mitochondria injury and blocks the reduction of vesicle-associated membrane protein 2 (VAMP2) and syntaxin-4 (STX4) in oxygen-glucose deprivation reoxygenation-insulted neurons following coculturing with astrocytes. Cathodal BtDCS regulates Se/VAMP2/STX4 signalling to confer neuroprotection after ischaemia.

Electroacupuncture regulates histone acetylation of Bcl-2 and Caspase-3 genes to improve ischemic stroke injury

BackgroundImbalances between Bcl-2 and caspase-3 are significant evidence of apoptosis, which is considered an influential factor in rapidly occurring neuronal cell death and the decline of neurological function after stroke. Studies have shown that acupuncture can reduce poststroke brain cell damage via either an increase in Bcl-2 or a reduction in caspase-3 exposure. The current study aimed to investigate whether acupuncture could modulate Bcl-2 and caspase-3 expression through histone acetylation modifications, which could potentially serve as a neuroprotective mechanism. MethodsThis study used TTC staining, Nissl staining, Clark neurological system score, and Evans Blue (EB) extravasation to evaluate neurological damage following stroke. The expression of Bcl-2/caspase-3 mRNA was detected by real-time fluorescence quantification of PCR (real-time PCR), whereas the protein expression levels of Bcl-2, Bax, caspase-3, and cleaved caspase-3 were assessed using western blotting. TUNEL staining of the ischemic cortical neurons determined apoptosis in the ischemic cortex. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities, along with the protein performance of AceH3, H3K9ace, and H3K27ace, were detected to evaluate the degree of histone acetylation. The acetylation enrichment levels of H3K9 and K3K27 in the Bcl-2/caspase-3 gene were assessed using Chromatin Immunoprecipitation (ChIP) assay. ResultsOur data demonstrated that electroacupuncture (EA) exerts a significant neuroprotective effect in middle cerebral artery occlusion (MCAO) rats, as evidenced by a reduction in infarct volume, neuronal damage, Blood-Brain Barrier (BBB) disruption, and decreased apoptosis of ischemic cortical neurons. EA treatment can promote the mRNA and protein expression of the Bcl-2 gene in the ischemic brain while reducing the mRNA and protein expression levels of caspase-3 and effectively decreasing the protein expression levels of Bax and cleaved caspase-3. More importantly, EA treatment enhanced the level of histone acetylation, including Ace-H3, H3K9ace, and H3K27ace, significantly enhanced the occupancy of H3K9ace/H3K27ace at the Bcl-2 promoter, and reduced the enrichment of H3K9ace and H3K27ace at the caspase-3 promoter. However, the Histone Acetyltransferase inhibitor (HATi) treatment reversed these effects. ConclusionsOur data demonstrated that EA mediated the expression levels of Bcl-2 and caspase-3 in MCAO rats by regulating the occupancy of acetylated H3K9/H3K27 at the promoters of these two genes, thus exerting a cerebral protective effect in ischemic reperfusion (I/R) injury.

Chlorogenic acid regulates the expression of protein phosphatase 2A subunit B in the cerebral cortex of a rat stroke model and glutamate-exposed neurons

BackgroundIschemic stroke is a serious neurological disorder caused by blockages in cerebral artery. Protein phosphatase 2A (PP2A) is a phosphatase that performs a critical role in cell signaling and growth. PP2A subunit B acts as a neuroprotective agent in the nerve system. Chlorogenic acid, which is mainly found in roasted coffee, has antioxidant, anti-inflammatory, and anti-apoptotic effects. We hypothesized that chlorogenic acid modulates PP2A subunit B expression in ischemic stroke models and glutamate-mediated neurons. Middle artery occlusion (MCAO) surgery was operated and chlorogenic acid (30 mg/kg) or phosphate buffer saline was treated 2 h after MCAO. The cerebral cortex was collected 24 h after surgery and the change of PP2A subunit B expression was analyzed. Glutamate and/or chlorogenic acid were treated in cultured neurons, further study was performed.ResultsA decrease in PP2A subunit B expression in MCAO animals was identified. Chlorogenic acid alleviated this decrease due to ischemic injury. Moreover, the number of PP2A subunit B-positive cells in the ischemic cerebral cortex was significantly decreased, chlorogenic acid alleviated this decrease. We also found protective effects of chlorogenic acid in neurons exposed to glutamate. Glutamate decreased the expression of PP2A subunit B and chlorogenic acid mitigated this decrease. Our results elucidated that chlorogenic acid performs neuroprotective functions and attenuates the reduction of PP2A subunit B by brain damage and glutamate-mediated excitotoxicity.ConclusionsWe showed that chlorogenic acid attenuated the decrease of PP2A subunit B in ischemic injury and neurons exposed to glutamate. Since PP2A subunit B contributes to the protection of brain tissue, we can suggest that chlorogenic acid preserves neurons by modulating PP2A subunit B during ischemic damage.

Kallistatin deficiency exacerbates neuronal damage after cardiac arrest

The purpose of study was to evaluate that kallistatin deficiency causes excessive production of reactive oxygen species and exacerbates neuronal injury after cardiac arrest. For in vitro study, kallistatin knockdown human neuronal cells were given ischemia–reperfusion injury, and the oxidative stress and apoptosis were evaluated. For clinical study, cardiac arrest survivors admitted to the ICU were divided into the good (CPC 1–2) and poor (CPC 3–5) 6-month neurological outcome groups. The serum level of kallistatin, Nox-1, H2O2 were measured. Nox-1 and H2O2 levels were increased in the kallistatin knockdown human neuronal cells with ischemia–reperfusion injury (p < 0.001) and caspase-3 was elevated and apoptosis was promoted (SERPINA4 siRNA: p < 0.01). Among a total of 62 cardiac arrest survivors (16 good, 46 poor), serum kallistatin were lower, and Nox-1 were higher in the poor neurological group at all time points after admission to the ICU (p = 0.013 at admission; p = 0.020 at 24 h; p = 0.011 at 72 h). At 72 h, H2O2 were higher in the poor neurological group (p = 0.038). Kallistatin deficiency exacerbates neuronal ischemia–reperfusion injury and low serum kallistatin levels were associated with poor neurological outcomes in cardiac arrest survivors.