Astragaloside IV Alleviates Cerebral Ischemia-Reperfusion Injury through NLRP3 Inflammasome-Mediated Pyroptosis Inhibition via Activating Nrf2

As one of the fundamental components of Astragalus membranaceus, astragaloside IV (AST IV) exerts protective effects against cerebral ischemia-reperfusion injury (CIRI). Nevertheless, the underlying mechanisms have not yet been conclusively elucidated. To do so, here, we report on the regulatory effects of Nrf2 on NLRP3 inflammasome-mediated pyroptosis. CIRI was induced by middle cerebral artery occlusion-reperfusion (MCAO/R) in Sprague Dawley rats and modeled by oxygen and glucose deprivation/reoxygenation (OGD/R) in SH-SY5Y cells. Cerebral infarct volume and neurological deficit score served as indices to evaluate MCAO/R injury. In addition, the CCK-8 assay was used to assess cell viability, the LDH leakage rate was used as a quantitative index, and propidium iodide (PI) staining was used to visualize cells after OGD/R injury. The NLRP3/Caspase-1/GSDMD pathway, which produces the pores in the cell membrane that are central to the pyroptosis process, was assessed to investigate pyroptosis. Nrf2 activation was assessed by detecting Nrf2 protein levels and immunofluorescence analysis. We show that after MCAO/R of rats, the infarct volume and neurological deficit score of rats were strongly increased, and after OGD/R of cell cultures, cell viability was strongly decreased, and the LDH leakage rate and the proportion of PI-positive cells were strongly increased. In turn, MCAO/R and OGD/R enhanced the protein levels of NLRP3, Caspase-1, IL-1β, GSDMD, and GSDMD-N. Moreover, Nrf2 protein levels increased, and Nrf2 translocation was promoted after CIRI. Interestingly, AST IV (i) reduced the cerebral infarct volume and the neurological deficit score in vivo and (ii) increased the cell viability and reduced the LDH leakage rate and the proportion of PI-positive cells in vitro. AST IV reduced the protein levels of NLRP3, Caspase-1, IL-1β, GSDMD, and GSDMD-N, inhibiting NLRP3 inflammasome-mediated pyroptosis. AST IV also increased the protein levels of Nrf2 and promoted the transfer of Nrf2 to the nucleus, accelerating Nrf2 activation. Particularly revealing was that the Nrf2 inhibitor ML385 partly blocked the above effects of AST IV. Taken together, these results demonstrate that AST IV alleviates CIRI through inhibiting NLRP3 inflammasome-mediated pyroptosis via activating Nrf2.

Comparison of Resuscitation Outcomes Between 2‐ or 3‐Stacked Defibrillation Strategies With Minimally Interrupted Chest Compression and the Single Defibrillation Strategy: A Swine Cardiac Arrest Model

Background There is controversy over whether the number and mode of electrical shock are optimal for successful defibrillation. Methods and Results Fifty‐four pigs were randomly assigned to 3 groups. After inducing ventricular fibrillation and a 2‐minute downtime, basic life support was initiated with a 30:2 compression/ventilation ratio for 8 minutes. Subsequently, 20 minutes of advanced life support, including asynchronous ventilation, every 10 chest compressions with 15 L/min of oxygen, was delivered. Animals of the single shock group received a single shock, animals of the 2‐stacked shock group received 2 consecutive shocks, and animals of the 3‐stacked shock group received 3 consecutive shocks. Animals with the return of spontaneous circulation underwent post–cardiac arrest care for 12 hours. The rates of successful defibrillation, return of spontaneous circulation, 24‐hour survival, and 48‐hour survival and neurological deficit score were compared between the groups. Hemodynamic parameters, arterial blood gas profiles, troponin I, and cardiac output were not different between the groups. There was a significant difference in chest compression fraction between the single and 3‐stacked shock groups ( P <0.001), although there was no difference between the single and 2‐stacked shock groups ( P =0.022) or the 2‐stacked and 3‐stacked shock groups ( P =0.040). The rates of successful defibrillation, return of spontaneous circulation, 24‐hour survival, and 48‐hour survival were higher in the 2‐ and 3‐stacked shock groups than in the single shock group ( P =0.021, P =0.015, and P =0.021, respectively). Neurological deficit score at 48 hours was not different between the groups. Conclusions A stacked shock strategy was superior to a single shock strategy for successful defibrillation and better resuscitation outcomes in treating ventricular fibrillation.

Phytosome Loading the Combined Extract of Mulberry Fruit and Ginger Protects against Cerebral Ischemia in Metabolic Syndrome Rats

The prevalence of ischemic stroke in metabolic syndrome (MetS) is continually increasing and produces a great impact on both qualities of life and annual healthcare budget. Due to the efficiency limitation of the current therapeutic strategy, the poor availability of polyphenol substances induced by the first pass effect and the beneficial effects of mulberry fruit and ginger on brain and MetS-related diseases together with the synergistic concept, the neuroprotective effect against ischemic stroke in MetS condition of phytosome containing the combined extract of mulberry fruit and ginger (PMG) has been considered. To explore the neuroprotective effect and possible underlying mechanism of PMG on brain damage in cerebral ischemic rat with MetS, male Wistar rats were induced MetS by high-carbohydrate high-fat diet (HCHF) for 16 weeks and subjected to the cerebral ischemia/reperfusion injury (CIRI) at the right middle cerebral artery (Rt. MCAO). PMG at doses of 50, 100, and 200 mg/kg were orally fed with for 21 days, and they were assessed brain damage, neurological deficit score, and the changes of oxidative stress markers, inflammatory markers, PPARγ expression, and epigenetic modification via DNMT-1 were performed. All doses of PMG significantly improved brain infarction, brain edema, and neurological deficit score. In addition, the reduction in DNMT-1, MDA level, NF-κB, TNFα, and C-reactive protein together with the increase in SOD, CAT, and GPH-Px activities, and PPARγ expression in the lesion brain were also observed. The current data clearly revealed the neuroprotective effect against cerebral ischemia with MetS condition. The possible underlying mechanism might occur partly via the suppression of DNMT-1 giving rise to the improvement of signal transduction via PPARγ resulting in the decreasing of inflammation and oxidative stress. In conclusion, PMG is the potential neuroprotectant candidate against ischemic stroke in the MetS condition. However, the clinical trial is still essential.

Abstract TMP20: The Therapeutic Gas Carbon Monoxide (CO) Attenuates Vasospasm and Improve Neurobehavioral Function After SAH

Introduction: Subarachnoid hemorrhage (SAH) is one of the most devastating forms of hemorrhagic stroke and it is a serious medical condition caused by bleeding of a ruptured aneurysm. The etiopathology of the disease includes early brain injury, vasospasm, and delayed cerebral ischemia. Blood byproducts cause the walls of arteries to constrict and spasm. At physiological concentrations, we and others have reported that carbon monoxide (CO) has functional roles in neuroprotection through anti-inflammation, anti-oxidative stress, and improving blood flow. Hence, this study focuses on the safe noninvasive therapeutic strategy to treat SAH by using CO as therapeutic medical gas. Hypothesis: We propose that CO may prevent SAH by mitigating vasospasm, limiting brain injury and improving functional outcomes. Methods: SAH was induced in adult mice using the endovascular perforation method. Mice were treated either with 250ppm CO or medical Air (at the same flow rate) after 2h of SAH for 1h daily for 7d. Such CO administration protocol keeps carboxyhemoglobin within normal range. Neurobehavioral outcomes such as focal neurological deficit score, open field activity, and motor functions were monitored at day 1 and day 7. Data acquisition and statistical analysis were performed by an investigator blinded to the intervention. Results: At 7d after SAH, CO decreased neurological deficit score (47.4±10.5%, p<0.05), and increased activity (30.0±9.1%, p<0.05) and stereotypic counts (261.5±62.1%, p<0.01) as compared to the air control group. No significant changes in motor function were observed in CO treated mice as compared to SAH at either day 1 or day 7). Of interest, there was a significant increase in CO treated group’s lumen area/wall thickness ratio in the middle cerebral artery (173.5±19.3%, p<0.01) and trended to increase in the anterior cerebral artery (25.5±4.3%, p=0.58) at 7d. Conclusion: This is a first report to demonstrate that CO prevents delayed SAH-induced neurobehavioral deficits, which suggest that post-treatment with CO should be considered a therapeutic strategy against SAH.

Abstract WP317: Neutrophil Subtypes and Nets Changes in Diabetes With Acute Ischemic Stroke

Objective: Ischemic stroke (AIS) is a globally high-risk cerebrovascular disease. Diabetes can aggravate ischemic stroke, while its mechanism is still unknown, Neutrophils are the first peripheral immune cells that break through the blood-brain barrier and infiltrate the brain after AIS. They have two polarization states of N1 and N2 subtypes. N1 subtype neutrophils can release neutrophil extracellular traps (NETs) containing a large number of pro-inflammatory factors and exacerbate the neuroinflammatory response. This study will explore the immunological mechanisms of diabetes exacerbating ischemic stroke. Methods: Patients with acute ischemic stroke who were admitted to hospital from Jan 2012 to Dec 2018 were collected, compared blood neutrophil count and neutrophil/lymphocyte ratio in diabetic and non-diabetic AIS patients at 1 day; Middle cerebral artery infarction (MCAO) mice was used for animal model of AIS, high-fat diet and STZ-induced type 2 diabetes model. The neurological deficit score, TTC staining, flow cytometry and immunofluorescence staining were used to observe the infarct volume, neutrophil subtypes and NETs changes between AIS with and without diabetes. Results: The neutrophil count of AIS patients with diabetes was higher than that of non-diabetic AIS patients. Animal experiments found that AIS with diabetes increase neurological deficit score and infarct volume. Flow cytometry and immunofluorescence staining found that AIS with diabetes mice can promote neutrophils infiltration into the brain and promote the polarization of neutrophils to N1 subtype and increase NETs formation. Conclusion: Diabetes can promote the neutrophil polarization of N1 subtype which leads to increased NETs formation and exacerbate ischemic stroke.

Flurbiprofen axetil attenuates cerebral ischemia/reperfusion injury by reducing inflammation in a rat model of transient global cerebral ischemia/reperfusion

Ischemic stroke has been ranked as the second cause of death in patients worldwide. Inflammation which is activated during cerebral ischemia/reperfusion (I/R) is an important mechanism leading to brain injury. The present study aimed to investigate the effect of flurbiprofen axetil on cerebral I/R injury and the role of inflammation in this process. Rats were subjected to sham operation or global cerebral I/R with or without flurbiprofen axetil (5 or 10 mg/kg). Global cerebral ischemia was achieved by occlusion of bilateral common carotid arteries combined with hypotension for 20 min followed by reperfusion for 72 h. Then the neurological deficit score, hippocampal cell apoptosis, levels of aquaporin (AQP) 4, AQP9, intercellular cell adhesion molecule-1 (ICAM-1), nuclear factor-κB (NF-κB), tumor necrosis factor (TNF-α), interleukin-1 β (IL-1β), thromboxane B2 (TXB2), and 6-keto-PGI1α were assessed. After reperfusion, neurological deficit score was significantly increased accompanied by severe neuronal damage (exacerbated morphological deficit, increased terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL)-positive cells and cleaved caspase-3 protein expression in hippocampal CA1 region). Cerebral I/R injury also enhanced expressions of TNF-α, IL-1β, NF-κB, AQP4 and AQP9 as well as TXB2 and TXB2/6-keto-PGI1α. All these changes were reversed by pretreatment with flurbiprofen axetil. Flurbiprofen axetil protects the brain from cerebral I/R injury through reducing inflammation and brain edema.

Injectable Caltrop Fruit Saponin Protects Against Ischemia-Reperfusion Injury in Rat Brain

The present study aimed to investigate the protective effects of injectable caltrop fruit saponin preparation (ICFSP) on ischemia-reperfusion injury in rat brain. Rats were injected with ICFSP and then subjected to cerebral ischemia-reperfusion injury induced by middle cerebral artery occlusion. Then the neurological deficit score was evaluated by Bederson's method. The infarct size was assessed by TTC staining. The content of malondialdehyde (MDA) and nitric oxide (NO), and the activity of superoxide dismutase (SOD) in rat cerebrum were measured with kits, and the content of 6 K prostaglandin F1α (6-K-PGF 1α), thromboxane B2 (TXB2) and endothelin (ET) in blood plasma was measured by radioimmunoassay. The results demonstrated that ICFSP led to a decrease in infarct size (p < 0.01), neurological deficit score (p < 0.05) and plasma content of TXB2 and ET (p < 0.05), and an increase of the plasma level of 6-K-PGF 1α (p < 0.05) and SOD activity in cerebrum, where the MDA and NO content were decreased. The treatment improved forelimb function. ICFSP showed a similar potency compared to that of Ligustrazine hydrochloride parenteral solution (LHPS) and nimodipine (Nim). We concluded that ICFSP protects the brain damage caused by ischemia-reperfusion injury in rats, and this may be closely related to the regulation of reactive oxygen species (MDA and SOD activity) and NO levels in the rat cerebrum, as well as vasoactive factors in the plasma (6-K-PGF 1α, TXB2 and ET).

Protective Effect of Astragaloside on Focal Cerebral Ischemia/Reperfusion Injury in Rats

This study was to observe the neurological protective effects of astragalosides (AST) on focal cerebral ischemia-reperfusion (I/R) injury in rats and to explore its possible mechanism. Male SD rats received right middle cerebral artery occlusion for 120 min and AST (40 mg/kg) was orally administered. The rats were decapitated 1, 3, 7, and 14 days after reperfusion. The neurological deficit score, infarct volume and water content of brain were measured; the activity of superoxide dismutase (SOD), lactate dehydrogenase (LDH) and nitric oxide synthase (NOS), and the content of malondialdehyde (MDA), lactate (LD) and nitric oxide (NO) of brain tissue were detected too. The expression of inducible nitric synthase (iNOS), nerve growth factor (NGF) and tropomyosin receptor kinase A (TrkA) mRNA were measured by RT-PCR or real-time PCR. AST could significantly reduce the neurological deficit score; infract volume and water content, increase SOD and LDH activities, decrease NOS activity and MDA, LD and NO content. AST treatment could down-regulate expression of iNOS mRNA, while, NGF and TrkA mRNA were up-regulated. Our data suggest that AST have the protective effects on focal cerebral ischemia in rats at the different reperfusion time points, the mechanism may be related to the antioxidation, regulated the expressions of iNOS, NGF and TrkA mRNA.

Neurochemical Mechanism of Electroacupuncture: Anti-Injury Effect on Cerebral Function after Focal Cerebral Ischemia in Rats

We explored the neurochemical mechanism of electroacupuncture's (EA) protective effect on brain function in focal cerebral ischemia rats, using cerebral ischemia/reperfusion rats established by the middle cerebral artery occlusion (MCAO) method. Adult male Sprague–Dawley rats were randomly divided into four groups: Sham, Sham+EA, MCAO and MCAO+EA. The rats in Sham+EA and MCAO+EA were accepted EA treatment at ‘GV26’ and ‘GV20’ acupoints for 30 min. Electric stimulation was produced by a G-6805 generator and neurological deficit scores were recorded. Mitochondria respiratory function and the activities of respiratory enzymes were measured by a computer-aided Clark oxygen electrode system. Results showed that EA treatment might reduce the neurological deficit score, and significantly improve respiratory control ratio (RCR), the index of mitochondrial respiratory function, and increase the activities of succinic dehydrogenase, NADH dehydrogenase and cytochrome C oxidase in the MCAO rats. Results suggest that EA might markedly decrease the neurological deficit score, promote the activities of respiratory enzymes and reduce the generation of reactive oxygen species (ROS), resulting in improvement of respiratory chain function and anti-oxidative capability of brain tissues in the infarct penumbra zone. This be a mechanism of EA's anti-injury effect on brain function in MCAO rats.

Ferulic Acid Reduces Cerebral Infarct Through Its Antioxidative and Anti-Inflammatory Effects Following Transient Focal Cerebral Ischemia in Rats

Both Angelica sinensis (Oliv.) Diels (AS) and Ligusticum chuanxiong Hort. (LC) have been used to treat stroke in traditional Chinese medicine for centuries. Ferulic acid (FA), a component in both AS and LC, plays a role in neuroprotection. The purpose of this study was to investigate the effects of FA on cerebral infarct and the involvement of neuroprotective pathway. Rats underwent 2 hours and 24 hours of reperfusion after 90 min middle cerebral artery occlusion (MCAo). The cerebral infarct and neurological deficits were measured after 24 hours of reperfusion. Furthermore, the expression of superoxide radicals, intercellular adhesion molecule-1 (ICAM-1), myeloperoxidase (MPO), nuclear factor-κB (NF-κB) immunoreactive cells were assessed after 2 hours and 24 hours of reperfusion. Administration of 80 and 100 mg/kg of FA at the beginning of MCAo significantly reduced cerebral infarct and neurological deficit-score, similar results were obtained by 100 mg/kg of FA administered 30 min after MCAo. FA treatment (100 mg/kg i.v.) effectively suppressed superoxide radicals in the parenchyma lesion, and ICAM-1 immunoreactive vessels in the ischemic striatum after 2 hours of reperfusion. FA (100 mg/kg i.v.) reduced the expression of ICAM-1 and NF-κB in the ischemic cortex and striatum, also down-regulated MPO immunoreactive cells in the ischemic cortex after 24 hours of reperfusion. These results showed that the effect of FA on reducing cerebral infarct area and neurological deficit-score were at least partially attributed to the inhibition of superoxide radicals, ICAM-1 and NF-κB expression in transient MCAo rats.