Ischemic Neuronal Damage Research Articles

Neuroprotective effect of pseudoginsenoside-F11 on permanent cerebral ischemia in rats by regulating calpain activity and NR2A submit-mediated AKT-CREB signaling pathways

BackgroundN-methyl-d-aspartate receptors (NMDARs) have been demonstrated to play central roles in stroke pathology and recovery, including dual roles in promoting either neuronal survival or death with their different subtypes and locations. PurposeWe have previously demonstrated that pseudoginsenoside-F11 (PF11) can provide long-term neuroprotective effects on transient and permanent ischemic stroke-induced neuronal damage. However, it is still needed to clarify whether NMDAR-2A (NR2A)-mediated pro-survival signaling pathway is involved in the beneficial effect of PF11 on permanent ischemic stroke. Material and methodsPF11 was administrated in permanent middle cerebral artery occlusion (pMCAO)-operated rats. The effect of PF11 on oxygen-glucose deprivation (OGD)-exposed primary cultured neurons were further evaluated. The regulatory effect of PF11 on NR2A expression and the activation of its downstream AKT-CREB pathway were detected by Western blotting and immunofluorescence in the presence or absence of a specific NR2A antagonist NVP-AAM077 (NVP) both in vivo and in vitro. ResultsPF11 dose- and time-dependently decreased calpain1 (CAPN1) activity and its specific breakdown product α-Fodrin expression, while the expression of Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII-α) was significantly upregulated in the cortex and striatum of rats at 24 h after the onset of pMCAO operation. Moreover, PF11 prevented the downregulation of NR2A, p-AKT/AKT, and p-CREB/CREB in both in vivo and in vitro stroke models. Finally, the results indicated treatment with NVP can abolish the effects of PF11 on alleviating the ischemic injury and activating NR2A-mediated AKT-CREB signaling pathway. ConclusionsOur results demonstrate that PF11 can exert neuroprotective effects on ischemic stroke by inhibiting the activation of CAPN1 and subsequently enhancing the NR2A-medicated activation of AKT-CREB pathway, which provides a mechanistic link between the neuroprotective effect of PF11 against cerebral ischemia and NR2A-associated pro-survival signaling pathway.

Decreased spasticity of Baishaoluoshi Decoction through the BDNF/TrKB-KCC2 pathway on poststroke spasticity rats.

ObjectiveK+–Cl− cotransporter-2 (KCC2), which primarily extrudes chloride in mature neurons, triggers hemiplegia limb spasticity after ischemic stroke by affecting neuronal excitability. Our previous study revealed that the Chinese herb Baishaoluoshi Decoction decreases hemiplegia limb spasticity in poststroke spasticity (PSS) patients. This study aimed at elucidating on the effects of Baishaoluoshi Decoction on the BDNF/TrKB-KCC2 pathway in PSS rat models.MethodsMiddle cerebral artery occlusion (MCAO) was adopted for the establishment of PSS rat models. Muscle tension was evaluated by Modified Ashworth Scale. Nissl staining and transmission electron microscopy were used to measure the protective effects of Baishaoluoshi Decoction on ischemic injury-induced neuronal damage due to MCAO. Expression levels of BDNF, TrKB, and KCC2 in brain tissues around the infarct and brainstem were detected by immunohistochemical staining.ResultsIt was found that Baishaoluoshi Decoction suppressed hemiplegia limb spasticity and alleviated the damage in neurons and synapses in PSS rat models. Importantly, the expression of BDNF, TrKB, and KCC2 in brain tissues around the infarct and brainstem were significantly upregulated after treatment with low-dose and high-dose Baishaoluoshi Decoction.ConclusionSuppression of spasticity by Baishaoluoshi Decoction in PSS rat models may be correlated with upregulated BDNF/TrKB-KCC2 pathway, which may be a complementary therapeutic strategy for PSS.

Contribution of ginsenosides Rg1, Rb1 to the neuroprotective effect of Panax notoginseng in mouse organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation

We previously demonstrated that Panax notoginseng (pNG) root extract treatments exertedneuroprotective effects on brain injuries using middle cerebral artery occlusion in mice. The present study aims to investigate the neuroprotective effects of PNG extract and its ginsenosides Rg1 and Rb1 on ischemic neuronal damage caused by oxygen and glucose deprivation (OGD) in mouse organotypic hippocampal slice cultures (OHSCs). Before the experiments, hippocampal slices collected from 7-day-old Swiss mice were cultured for 7 days. OGD was triggered in OHSCs for 30, 60, or 90 min with the aim of finding the optimal period of OGD for drug testing. PNG extract (10, 30 μg/ml), ginsenosides Rg1 and Rb1 (5, 25 μM), or MK-801 25 μM, a reference drug, was added to the culture medium 24 h before OGD and these treatments were continued for 24 h after the optimum 60-min period of OGD. After 24 h of OGD exposure, the measurement of propidium iodide uptake was analysed in OHSCs to evaluate neuronal cell damage. The results showed that OGD time-dependently increased PI uptake of the OHSCs. PNG 30 μg/ml treatment reduced the OGD-induced neuronal cell damage in OHSCs. Ginsenosides Rg1 25 μM, Rb1 (5, 25 μM), as well as MK-801 (25 μM) significantly inhibited PI uptake 24 h after OGD exposure. However, ginsenoside Rg1 5 μM did not show any significant effects on the OGD-induced neuronal cell damage. These findings indicated that ginsenosides Rg1 and Rb1 contributed to the neuroprotective effects of PNG against ischemic damage in OHSCs and the neuroprotective effect of ginsenoside Rb1 was stronger than that of ginsenoside Rg1.

Neuroprotection of N-benzyl Eicosapentaenamide in Neonatal Mice Following Hypoxic-Ischemic Brain Injury.

Maca (Lepidium meyenii) has emerged as a popular functional plant food because of its medicinal properties and nutritional value. Macamides, as the exclusively active ingredients found in maca, are a unique series of non-polar, long-chain fatty acid N-benzylamides with multiple bioactivities such as antifatigue characteristics and improving reproductive health. In this study, a new kind of macamide, N-benzyl eicosapentaenamide (NB-EPA), was identified from maca. We further explore its potential neuroprotective role in hypoxic–ischemic brain injury. Our findings indicated that treatment with biosynthesized NB-EPA significantly alleviates the size of cerebral infarction and improves neurobehavioral disorders after hypoxic–ischemic brain damage in neonatal mice. NB-EPA inhibited the apoptosis of neuronal cells after ischemic challenge. NB-EPA improved neuronal cell survival and proliferation through the activation of phosphorylated AKT signaling. Of note, the protective property of NB-EPA against ischemic neuronal damage was dependent on suppression of the p53–PUMA pathway. Taken together, these findings suggest that NB-EPA may represent a new neuroprotectant for newborns with hypoxic–ischemic encephalopathy.

Extracellular DJ-1 induces sterile inflammation in the ischemic brain.

Inflammation is implicated in the onset and progression of various diseases, including cerebral pathologies. Here, we report that DJ-1, which plays a role within cells as an antioxidant protein, functions as a damage-associated molecular pattern (DAMP) and triggers inflammation if released from dead cells into the extracellular space. We first found that recombinant DJ-1 protein induces the production of various inflammatory cytokines in bone marrow-derived macrophages (BMMs) and dendritic cells (BMDCs). We further identified a unique peptide sequence in the αG and αH helices of DJ-1 that activates Toll-like receptor 2 (TLR2) and TLR4. In the ischemic brain, DJ-1 is released into the extracellular space from necrotic neurons within 24 h after stroke onset and makes direct contact with TLR2 and TLR4 in infiltrating myeloid cells. Although DJ-1 deficiency in a murine model of middle cerebral artery occlusion did not attenuate neuronal injury, the inflammatory cytokine expression in infiltrating immune cells was significantly decreased. Next, we found that the administration of an antibody to neutralize extracellular DJ-1 suppressed cerebral post-ischemic inflammation and attenuated ischemic neuronal damage. Our results demonstrate a previously unknown function of DJ-1 as a DAMP and suggest that extracellular DJ-1 could be a therapeutic target to prevent inflammation in tissue injuries and neurodegenerative diseases.

RELATIONSHIP BETWEEN LEVEL OF EDUCATION AND POST-STROKE COGNITIVE STATUS IN HOSPITAL-BASED ISCHEMIC STROKE SURVIVORS

Background: Post-ischemic stroke cognitive decline is significantly affecting the quality of life of its survivors. Its prevalence was about 7.5-72% which was mostly determined by the existing of vascular risk factors and cognitive reserve of the subjects. Level of education is one of determinants of cognitive reserve, a factor that affect the susceptibility of subjects to cognitive decline after experiencing ischemic stroke-related neuronal damage. Since level of education is protective for cognitive function, the intervention on it can reduce the occurrence of cognitive decline. Objective: To investigate the relationship between level of education and cognitive status among hospital-based ischemic stroke survivors. Methods: This cross-sectional study involved post-ischemic stroke outpatients in two hospitals. The data collected in this study were demographic data, including level of education, and clinical data as well. The level of education was categorized into ≥12 years and <12 years groups. Cognitive status was assessed using Montreal Cognitive Assessment in Indonesia version (MoCA-Ina) and subjects with score of 26-30 were normal. The relationship between level of education as well as clinical data and cognitive status were analyzed using chi-square test. Results: There were 166 subjects eligible for this study (n=166). The mean age of subjects was 58 years and 68.67% of them were male. Cognitive decline were found 80.12% of subjects (n=133). The level of education was significantly associated with cognitive status of the subjects and hypertension as well. Conclusion: The level of education had significant relationship with cognitive decline in the hospital-based population of ischemic stroke survivors.

Roflumilast prevents ischemic stroke-induced neuronal damage by restricting GSK3β-mediated oxidative stress and IRE1α/TRAF2/JNK pathway

Inhibition of phosphodiesterase 4 (PDE4) protects against neuronal apoptosis induced by cerebral ischemia. However, the exact mechanisms responsible for the protection of PDE4 inhibition have not been completely clarified. Roflumilast (Roflu) is an FDA-approved PDE4 inhibitor for the treatment of chronic obstructive pulmonary disease. The potential protective role of Roflu against ischemic stroke-associated neuronal injury remains unexplored. In this study, we investigated the effect and mechanism of Roflu against ischemic stroke using in vitro oxygen-glucose deprivation reperfusion (OGD/R) and in vivo rat middle cerebral artery occlusion (MCAO) models. We demonstrated that Roflu significantly reduced the apoptosis of HT-22 cells exposed to OGD/R, enhanced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2), and reduced oxidative stress. Treatment with Roflu increased the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3β (GSK3β) but decreased the level of phosphorylated inositol requiring enzyme 1α (IRE1α). Interestingly, constitutively active GSK3β (S9A) mutation abolished the effects of Roflu on oxidative stress and IRE1α phosphorylation. Moreover, Roflu decreased the binding of IRE1α to tumor necrosis factor receptor-associated factor 2 (TRAF2) and attenuated the phosphorylation of c-Jun N-terminal kinase (JNK). We also found that PDE4B knockdown reduced the phosphorylation of both IRE1α and JNK, while overexpression of PDE4B antagonized the role of PDE4B knockdown on the activation of IRE1α and JNK. Besides, the inhibition of PDE4 by Roflu produced similar effects in primary cultured neurons. Finally, Roflu ameliorated MCAO-induced cerebral injury by decreasing infarct volume, restoring neurological score, and reducing the phosphorylation of IRE1α and JNK. Collectively, these data suggest that Roflu protects neurons from cerebral ischemia reperfusion-mediated injury via the activation of GSK3β/Nrf-2 signaling and suppression of the IRE1α/TRAF2/JNK pathway. Roflu has the potential as a protective drug for the treatment of cerebral ischemia.

Comparative evaluation of anesthesia methods for reconstructive carotid surgery

The objective: to assess the impact of total intravenous and combined anesthesia (consisting of regional block and inhalation anesthesia with sevoflurane) on higher mental functions, frequency and nature of postoperative complications in carotid endarterectomy.Subjects and methods. A total of 190 patients with ipsilateral carotid stenosis exceeding 70%, or with the unstable atheromatous plaque were examined. CE was performed in 140 (73.6%) patients, while 50 (26.4%) patients underwent plasty with the autovenous patch. 60 patients had total intravenous anesthesia. The other 60 patients had combination anesthesia (cervical plexus block as per A. Yu. Paschuk + non-inhalation anesthesia with thiopental sodium). In 70 patients, low-flow inhalation anesthesia with sevoran and potentiation of the analgesic effect by bolus administration of fentanyl was performed. The parameters of central hemodynamics, cerebral blood flow, markers of cerebral damage, and state of higher mental functions were assessed.Results. Inhalation anesthesia with sevoflurane was characterized by decrease of total peripheral resistance index by 20% (p = 0.002) and SBP by 13% (p = 0.004) compared to baseline values. Moderate reduction of afterload was accompanied by normalization of cardiac and stroke indices. Optimization of the central hemodynamic parameters led to high levels of the linear velocity of cerebral blood flow and cerebral perfusion pressure which at the main stages of the operation did not differ from the control values. At the end of the operation, a statistically significant insignificant increase in intracranial pressure was noted (on average, by 4 mm Hg exceeded values at the previous stages, p = 0.014) and levels of markers of ischemic neuronal damage: the levels of antibodies to protein S-100 increased by 14% (p = 0.043), and NSE – by 2.8 μg/L. During inhalation anesthesia with sevoflurane, the lowest number of postoperative neurological and cardiac complications was recorded; they developed only in 6 (8.6%) patients and distributed equally. However, more pronounced postoperative cognitive dysfunction was noted when this method of anesthesia was used.Conclusion. Each method of anesthesia provided an unequal level of cerebral protection from ischemic damage which was manifested by a difference in the severity of disorders of higher mental functions, the number and nature of postoperative neurological complications.

Effect of baicalin on inflammatory response and TLR4/NF-κB signaling pathway of human brain microvascular endothelial cell after hypoxia-reoxygenation injury

In this study, the oxygen-glucose deprivation(OGD) model in the human brain microvascular endothelial cell(HBMEC) was used to simulate the ischemic neuronal damage and observe the inflammatory response, explore the possible mechanisms for treating cerebral ischemia/reperfusion and improving memory impairment from the view point of inhibiting inflammatory response, which is of great reference significance for related Chinese medicine treatment of ischemic diseases. HBMECs were given with drugs at the same time of OGD injury, and reoxygenated for 2 h after 4 h treatment. Cell supernatant was then collected, and the inflammatory factors in cell supernatant were detected. Immunofluorescence assay was used to detect HBMECs morphology and expression of p-nuclear factor kappa-light-chain-enhancer of activated B(p-NF-κB); Western blot was used to detect expression changes of Toll like receptor 4(TLR4), myeloid differentiation primary response 88(MYD88) and p-NF-κB. The results showed that, after OGD modeling, the levels of interleukin 6(IL-6), IL-1α, IL-1β and tumor necrosis factor-α(TNF-α) were significantly increased; baicalin protected HBMEC, inhibited intranuclear transcription of p-NF-κB, significantly decreased HBMEC release of inflammatory factors caused by OGD injury, and inhibited the expression of TLR4, MYD88, and p-NF-κB. The studies suggested that baicalin had obvious protective effect on HBMECs damaged by OGD, and could inhibit inflammatory response. Its protection mechanism may be related to inhibiting TLR4 signaling pathways.

Dietary docosahexaenoic acid inhibits neurodegeneration and prevents stroke.

Stroke severely impairs quality of life and has a high mortality rate. On the other hand, dietary docosahexaenoic acid (DHA) prevents neuronal damage. In this review, we describe the effects of dietary DHA on ischemic stroke-associated neuronal damage and its role in stroke prevention. Recent epidemiological studies have been conducted to analyze stroke prevention through DHA intake. The effects of dietary intake and supply of DHA to neuronal cells, DHA-mediated inhibition of neuronal damage, and its mechanism, including the effects of the DHA metabolite, neuroprotectin D1 (NPD1), were investigated. These studies revealed that DHA intake was associated with a reduced risk of stroke. Moreover, studies have shown that DHA intake may reduce stroke mortality rates. DHA, which is abundant in fish oil, passes through the blood-brain barrier to accumulate as a constituent of phospholipids in the cell membranes of neuronal cells and astrocytes. Astrocytes supply DHA to neuronal cells, and neuronal DHA, in turn, activates Akt and Raf-1 to prevent neuronal death or damage. Therefore, DHA indirectly prevents neuronal damage. Furthermore, NDP1 blocks neuronal apoptosis. DHA, together with NPD1, may block neuronal damage and prevent stroke. The inhibitory effect on neuronal damage is achieved through the antioxidant (via inducing the Nrf2/HO-1 system) and anti-inflammatory effects (via promoting JNK/AP-1 signaling) of DHA.

TREM-1-targeting LP17 attenuates cerebral ischemia-induced neuronal injury by inhibiting oxidative stress and pyroptosis

Stroke ranks as the second leading cause of disability and death globally. Trigger receptors expressed on myeloid cells (TREM) −1 are responsible for the activation of the innate immune response and also play a critical role in inflammation. In this study, we reported the contribution of TREM-1 after ischemic damage in a rat middle cerebral artery occlusion (MCAO) model. This study also demonstrated that TREM-1 expression was upregulated following cerebral infarction in rats. TREM-1 inhibition was determined using its selective inhibitor, LP17, which indicated a neuroprotective effect on cerebral infarction damage. The findings revealed that inhibition of TREM-1 by administering LP17 improved cerebral damage and decreased ischemic areas and brain water contents. Moreover, LP17 decreased MCAO-induced microglial activation and neurodegeneration, evidenced by a reduction in the expression of microglial Iba-1 and FJ-B positive cells, and reversed neuronal loss. Besides, the contribution of LP17 to ischemic neuronal damage may be associated with a decrease in the production of pro-inflammatory cytokines, and enhanced production of anti-inflammatory cytokine IL-10. Both in vivo and in vitro studies showed that inhibiting TREM-1 attenuated ROS accumulation, lipid per-oxidation (LPO) contents such as malondialdehyde (MDA) and enhanced the superoxide dismutase (SOD) activity after ischemia. Inhibiting TREM-1 alleviated inflammation and pyroptosis found in MCAO rats. This was achieved through the inhibition of the levels of NLRP3, caspase-1, ASC (an apoptosis-associated speck-like protein containing a CARD) and gasdermin D. These results confirmed that inhibiting TREM-1 protects against ischemia-induced neuronal damage and alleviates microglial mediated neuro-inflammation by reducing oxidative stress and pyroptosis. Therefore, blocking TREM-1 expression provides an effective intervention for improving ischemic stroke.

Pseudoginsenoside-F11 ameliorates ischemic neuron injury by regulating the polarization of neutrophils and macrophages in vitro

Pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, has neuroprotective effects on permanent and transient cerebral ischemia in rats by alleviating autophagic/lysosomal defects and repressing calcium overload, respectively. Ischemic stroke triggers peripheral innate immune cells, mainly neutrophils and macrophages, to infiltrate the damaged brain. The polarization of neutrophils and macrophages after cerebral ischemia is essential for post-stroke damage/recovery. However, it remains elusive whether PF11 ameliorates ischemic neuron injury by regulating the polarization of neutrophils and macrophages. The present study demonstrated for the first time that conditioned media from ischemic neurons induced neutrophils and macrophages to polarize into N1 and M1 phenotypes, respectively. Furthermore, PF11 (30, 100 μM) inhibited the induction of N1 neutrophils by conditioned media from oxygen glucosedeprivation/re-oxygenation (OGD/R)-induced ischemic neurons and promoted the polarization of neutrophils to N2 phenotypes. In addition, PF11 (100 μM) attenuated the exacerbation of N1 neutrophils and facilitated the protection of N2 neutrophils on OGD/R-induced neuronal damage. Similarly, PF11 (100 μM) inhibited the induction of M1 macrophages by conditioned media from ischemic neurons and facilitated the polarization of macrophages to M2 phenotypes. What's more, PF11 (100 μM) attenuated the aggravation of M1 macrophages and promoted the protection of M2 macrophages on OGD/R-induced primary neuron injury. In summary, the present study indicates that PF11 ameliorates ischemic neuron damage by regulating neutrophils and macrophages polarization, suggesting that neutrophils and macrophages may be promising targets for the treatment of cerebral ischemia.

Empagliflozin attenuates transient cerebral ischemia/reperfusion injury in hyperglycemic rats via repressing oxidative-inflammatory-apoptotic pathway.

Hyperglycemia is one of the ischemic neuronal damage triggers that exacerbate the response to oxidative stress, inflammation, and apoptosis induced by cerebral ischemia/reperfusion (I/R) injury. Empagliflozin, a sodium-glucose cotransporter 2 (SGLT 2) inhibitor, was shown to effectively reduce hyperglycemia and glucotoxicity besides improving glycemic control in diabetics. Therefore, the present study was conducted to investigate the neuroprotective effect of empagliflozin against cerebral I/R injury in hyperglycemic rats. Hyperglycemia was induced by streptozotocin (55mg/kg), and transient cerebral I/R was induced by bilateral common carotid occlusion for 30min followed by 24-h reperfusion. Either empagliflozin (10mg/kg; i.p.) or gliclazide (2mg/kg, p.o.) was administered at 1 and 24h after reperfusion. Treatment with empagliflozin showed a significant amelioration of behavioral/neurological functions and histopathological changes observed in brain tissues of hyperglycemic rats subjected to cerebral I/R injury. Comparable to gliclazide, empagliflozin decreased cerebral infarct volume along with suppression of cerebral oxidative stress, inflammatory, and apoptotic markers in brain tissues of hyperglycemic I/R-injured rats. These findings suggested that empagliflozin can significantly alleviate neuronal damage resulting from global I/R injury induced in hyperglycemic rats. The proposed neuroprotective effect of empagliflozin may be attributed to its glycemic control effect and related antioxidant, anti-inflammatory, and antiapoptotic effects.

Iranian brown propolis possesses neuroprotective effect against ischemic neuronal damage in mice

Introduction: Stroke is one of the leading causes of death and disability worldwide. Propolis, a polyphenol-rich resinous product processed by honeybees from a variety of plant sources, has a set of biological activities. We investigated the neuroprotective effect of Iranian brown propolis (IBP) in a mouse model of permanent middle cerebral artery occlusion (MCAO). Methods: Experimentally, water extracts of propolis (WEPs) were obtained from Kerman (KeWEP) and Khorasan Razavi (KhWEP) provinces, Iran. The chemical characterization and total polyphenol content of WEPs were determined using the Folin–Ciocalteu assay and gas chromatography-mass spectrometry (GC-MS). Animals were divided into eight experimental groups including: sham, control, and three groups each of which KeWEP- and KhWEP-treated mice. The drugs were administered at doses of 30, 100 and 200 mg/kg, intraperitoneally (IP), during four different time points. Infarct volume and brain edema were measured at 48 h. Behavioral tests were evaluated at 4, 24 and 48-hour post stroke. Results: The total polyphenol content was 1100 and 1400 mg/L in KhWEP and KeWEP respectively. Compared to the control group, the doses of 100 and 200 mg/kg in both samples decreased infarct volume. Brain edema was also reduced in all treatment groups. The dose of 200 mg/kg in both samples and 100 mg/kg in the KeWEP-treated group significantly increased grasping ability. Sensory-motor function was improved in all groups, too. Conclusion: These results suggest that IBP may reduce ischemic brain injury by its neuroprotective effect on focal cerebral ischemia.

The Cannabinoid Receptor Agonist WIN55,212-2 Ameliorates Hippocampal Neuronal Damage After Chronic Cerebral Hypoperfusion Possibly Through Inhibiting Oxidative Stress and ASK1-p38 Signaling.

Chronic cerebral hypoperfusion (CCH) is a major contributor to cognitive decline and degenerative processes leading to Alzheimer's disease, vascular dementia, and aging. However, the delicate mechanism of CCH-induced neuronal damage, and therefore proper treatment, remains unclear. WIN55,212-2 (WIN) is a nonselective cannabinoid receptor agonist that has been shown to have effects on hippocampal neuron survival. In this study, we investigated the potential roles of WIN, as well as its underlying mechanism in a rat CCH model of bilateral common carotid artery occlusion. Hippocampal morphological changes and mitochondrial ultrastructure were detected using hematoxylin and eosin staining and electron microscopy, respectively. Various biomarkers, such as reactive oxidative species (ROS), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were used to assess the level of oxidative stress in the hippocampus. Furthermore, the expression levels of neuronal nuclei (NeuN), apoptosis signal-regulating kinase 1 (ASK1)-p38 signaling proteins, cleaved Caspase-9 and -3, and cytochrome-c (Cyt-C) were accessed by western blotting. CCH decreased the levels of NeuN, Cyt-C (mitochondrial), SOD, and CAT, and increased the levels of MDA, phosphorylated ASK1 and phosphorylated p38, cleaved Caspase-9 and -3, and Cyt-C (cytoplasm), which were reversed by WIN treatment. Chronic treatment with WIN also improved CCH-induced neuronal degeneration and mitochondrial fragmentation. These findings indicated that WIN may be a potential therapeutic agent for ischemic neuronal damage, involving a mechanism associated with the suppression of oxidative stress and ASK1-p38 signaling.

N2 neutrophils may participate in spontaneous recovery after transient cerebral ischemia by inhibiting ischemic neuron injury in rats

Neutrophils have been traditionally considered as the major mediators of harmful inflammatory responses in ischemic stroke, whereas accumulating evidence indicates that neutrophils can be polarized into an N2 phenotype. Similar to M2 microglia, N2 neutrophils contribute to resolution of inflammation and may participate in neuroprotection. However, it remains unclear whether N2 neutrophils protect ischemic neurons and whether they are associated with long-term outcomes after transient cerebral ischemia in rats. The present study proved that N2 neutrophils protected against oxygen glucosedeprivation/re-oxygenation (OGD/R)-induced primary cortical neuron injury via brain-derived neurotrophic factor/tropomyosin-related kinase B (BDNF/TrkB) signaling. In addition, in vivo studies revealed that transient middle cerebral artery occlusion (tMCAO)-induced injury exhibited spontaneous recovery over time in rats. Moreover, neutrophils could infiltrate the ipsilateral brain parenchyma from the periphery after transient cerebral ischemia. Pearson’s correlation analysis indicated that the proportion of N2 neutrophils in ipsilateral brain parenchyma was negatively correlated with the number of degenerating neurons, modified Neurological Severity Score (mNSS), brain water content and infarct volume, and positively correlated with the number of surviving neurons and grip strength. In summary, the present study shows that N2 neutrophils likely participate in spontaneous recovery after transient cerebral ischemia by inhibiting ischemic neuron damage in rats, which indicates that N2 neutrophils may represent promising therapeutic target for promoting recovery after ischemic stroke.

Pаthоlоgenic changes in the brain of mice while modeling of ischemia with an associated viral infection

Objective — to investigate structural changes in the brain cortex of mice exposed to fat diet, herpes infection and ischemia.Methods and subjects. We had 194 mice (Balb/c) for the experiment, distributed into 7 groups (6 + 1 control group): 1st group (n = 52) with fat diet; 2nd group (n = 30, 7 died) with HSV1; 3d group (n = 30) with ОGСА; 4th group (n = 20, 10 died) with HSV1 and ОGСА; 5th group (n = 5) with HSV1 and fat diet with cholesterol; 6th group (n = 16, 10 died) with HSV1, fat diet, and ОGСА.The neuronal neocortex density of the parietal, temporal cortex and hippocampus were morphometrically evaluated on microscopic specimens.Results. In the course of experiment, we discovered the main zones and anatomical formations of brain which received the most damage. Changes were mainly found in the neocortex of the parietal, temporal lobes and hippocampus. To assess the type of neuronal brain damage, the main changes were divided into focal and diffuse. Later in the study, we attempted to investigate the dynamics of changes in the density of neurons with degenerative changes in the parietal and/or temporal cortex of the brain, in the group with fat diet, HSV1 and ОGСА.Conclusions. Based on microscopic examination of the temporal and/or parietal cortex in laboratory animals, we established that the combination of factors such as fat diet, neuroinfectious diseases and ischemia resulted in increase in the density of neurons with degenerative changes and on the dynamics of their development. Using morphometric study of the cerebral cortex in laboratory mice, we determined a statistically significant increase in ischemic neuronal damage resulted in condition of an experimental model with infection with HSV‑1 and fat diet, and under the condition of ОGСА of diffuse character. The increase of the degree of neocortex damage in mice with herpes infection and fat diet evidences the interrelation of them.

Ginaton improves neurological function in ischemic stroke rats via inducing autophagy and maintaining mitochondrial homeostasis.

PurposeThe present study was carried out to confirm the protective effect of extract of Ginkgo biloba (Ginaton) against ischemic neuronal damage post-treatment at 24 h after reperfusion in rats with middle cerebral artery occlusion (MCAO) and further reveal its possible mechanisms.MethodsAdult male Sprague-Dawley rats were modeled by MCAO for 2 h. The rats were divided into three groups: sham, model, and Ginaton (50 mg/kg). All animals received treatment once a day for 14 days from 24 h after reperfusion. Modified neurological severity score test was performed in 1, 7 and 14 days after MCAO, and beam walking test was performed only 14 days after MCAO. Hematoxylin-eosin straining was implemented to measure infarct volume and immunohistochemical analysis was performed to calculate the number of neurons in ischemic cortex penumbra. Western blot was used to evaluate the expression of autophagy (Beclin1, LC3, AMPK, mTOR, ULK), mitochondrial dynamic protein (Parkin, DRP1, OPA1) and apoptosis (Bcl-2, Bax).ResultsPost-treatment with Ginaton for 14 days decreased neurological deficit score, promoted the recovery of motor function, and noticeably reduced infarct size. Besides, Ginaton also alleviated the loss of NeuN-positive cells in ischemic cortex penumbra. In ischemic cortex, Ginaton increased the expression of Beclin1 and LC3-Ⅱ, elevated the AMPK, mTOR and ULK1, and induced autophagy. Moreover, Ginaton treatment upregulated Parkin, DRP1, and OPA1, and elevated the ratio of Bcl-2/Bax in 14 days after MCAO reperfusion injury.ConclusionGinaton exhibited obvious neuroprotective effects in MCAO rats with initial administered 24 h after MCAO. The mechanism of Ginaton included induction of autophagy via activation of the AMPK pathway, maintenance of mitochondrial homeostasis and inhibition of apoptosis.

Role of age-related alterations of the cerebral venous circulation in the pathogenesis of vascular cognitive impairment.

There has been an increasing appreciation of the role of vascular contributions to cognitive impairment and dementia (VCID) associated with old age. Strong preclinical and translational evidence links age-related dysfunction and structural alterations of the cerebral arteries, arterioles, and capillaries to the pathogenesis of many types of dementia in the elderly, including Alzheimer's disease. The low-pressure, low-velocity, and large-volume venous circulation of the brain also plays critical roles in the maintenance of homeostasis in the central nervous system. Despite its physiological importance, the role of age-related alterations of the brain venous circulation in the pathogenesis of vascular cognitive impairment and dementia is much less understood. This overview discusses the role of cerebral veins in the pathogenesis of VCID. Pathophysiological consequences of age-related dysregulation of the cerebral venous circulation are explored, including blood-brain barrier disruption, neuroinflammation, exacerbation of neurodegeneration, development of cerebral microhemorrhages of venous origin, altered production of cerebrospinal fluid, impaired function of the glymphatics system, dysregulation of cerebral blood flow, and ischemic neuronal dysfunction and damage. Understanding the age-related functional and phenotypic alterations of the cerebral venous circulation is critical for developing new preventive, diagnostic, and therapeutic approaches to preserve brain health in older individuals.

Upregulation of miR-216a exerts neuroprotective effects against ischemic injury through negatively regulating JAK2/STAT3-involved apoptosis and inflammatory pathways.

Ischemic stroke remains a significant cause of death and disability in industrialized nations. Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) of the JAK2/STAT3 pathway play important roles in the downstream signal pathway regulation of ischemic stroke-related inflammatory neuronal damage. Recently, microRNAs (miRNAs) have emerged as major regulators in cerebral ischemic injury; therefore, the authors aimed to investigate the underlying molecular mechanism between miRNAs and ischemic stroke, which may provide potential therapeutic targets for ischemic stroke. The JAK2- and JAK3-related miRNA (miR-135, miR-216a, and miR-433) expression levels were detected by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis in both oxygen-glucose deprivation (OGD)-treated primary cultured neuronal cells and mouse brain with middle cerebral artery occlusion (MCAO)-induced ischemic stroke. The miR-135, miR-216a, and miR-433 were determined by bioinformatics analysis that may target JAK2, and miR-216a was further confirmed by 3' untranslated region (3'UTR) dual-luciferase assay. The study further detected cell apoptosis, the level of lactate dehydrogenase, and inflammatory mediators (inducible nitric oxide synthase [iNOS], matrix metalloproteinase-9 [MMP-9], tumor necrosis factor-α [TNF-α], and interleukin-1β [IL-1β]) after cells were transfected with miR-NC (miRNA negative control) or miR-216a mimics and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) damage with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V-FITC/PI, Western blots, and enzyme-linked immunosorbent assay detection. Furthermore, neurological deficit detection and neurological behavior grading were performed to determine the infarction area and neurological deficits. JAK2 showed its highest level while miR-216a showed its lowest level at day 1 after ischemic reperfusion. However, miR-135 and miR-433 had no obvious change during the process. The luciferase assay data further confirmed that miR-216a can directly target the 3'UTR of JAK2, and overexpression of miR-216a repressed JAK2 protein levels in OGD/R-treated neuronal cells as well as in the MCAO model ischemic region. In addition, overexpression of miR-216a mitigated cell apoptosis both in vitro and in vivo, which was consistent with the effect of knockdown of JAK2. Furthermore, the study found that miR-216a obviously inhibited the inflammatory mediators after OGD/R, including inflammatory enzymes (iNOS and MMP-9) and cytokines (TNF-α and IL-1β). Upregulating miR-216a levels reduced ischemic infarction and improved neurological deficit. These findings suggest that upregulation of miR-216a, which targets JAK2, could induce neuroprotection against ischemic injury in vitro and in vivo, which provides a potential therapeutic target for ischemic stroke.