The antioxidant effect of preischemic dexmedetomidine in a rat model: increased expression of Nrf2/HO-1 via the PKC pathway

Abstract Background The precise underlying mechanism of antioxidant effects of dexmedetomidine-induced neuroprotection against cerebral ischemia has not yet been fully elucidated. Activation of Nuclear factor erythroid 2-related factor (Nrf2) and Heme Oxygenase-1 (HO-1) represents a major antioxidant-defense mechanism. Therefore, we determined whether dexmedetomidine increases Nrf2/HO-1 expression after global transient cerebral ischemia and assessed the involvement of Protein Kinase C (PKC) in the dexmedetomidine-related antioxidant mechanism. Methods Thirty-eight rats were randomly assigned to five groups: sham (n = 6), ischemic (n = 8), chelerythrine (a PKC inhibitor; 5 mg.kg-1 IV administered 30 min before cerebral ischemia) (n = 8), dexmedetomidine (100 µg.kg-1 IP administered 30 min before cerebral ischemia (n = 8), and dexmedetomidine + chelerythrine (n = 8). Global transient cerebral ischemia (10 min) was applied in all groups, except the sham group; histopathologic changes and levels of nuclear Nrf2 and cytoplasmic HO-1 were examined 24 hours after ischemia insult. Results We found fewer necrotic and apoptotic cells in the dexmedetomidine group relative to the ischemic group (p< 0.01) and significantly higher Nrf2 and HO-1 levels in the dexmedetomidine group than in the ischemic group (p< 0.01). Additionally, chelerythrine co-administration with dexmedetomidine attenuated the dexmedetomidine-induced increases in Nrf2 and HO-1 levels (p< 0.05 and p< 0.01, respectively) and diminished its beneficial neuroprotective effects. Conclusion Preischemic dexmedetomidine administration elicited neuroprotection against global transient cerebral ischemia in rats by increasing Nrf2/HO-1 expression partly via PKC signaling, suggesting that this is the antioxidant mechanism underlying dexmedetomidine-mediated neuroprotection.

Infarto cerebral en mujer joven con la enfermedad por el coronavirus del 2019 y sin factores de riesgo cardiovasculares: reporte de caso / Acute stroke in young woman with coronavirus disease 19 and no cardiovascular risk factor: case report

RESUMEN INTRODUCCIÓN: La COVID-19 se acompaña de eventos tromboembólicos venosos y arteriales en pacientes con historia de hipertensión y aterosclerosis. Se reporta el caso de una paciente joven y sin factores de riesgo cardiovascular que adquirió la infección por el SARS-COV2 y luego presentó un accidente cerebrovascular isquémico. CASO: Paciente de 44 años hospitalizada por la covid-19 en tratamiento protocolario con anticoagulación, esteroide y oxígeno suplementario. Al día 7 del inicio de los síntomas respiratorios presentó infarto cerebral por trombosis de arteria carótida derecha, arteria cerebral media derecha e izquierda y edema cerebral hemisférico derecho, tras lo cual fue diagnosticada con muerte encefálica. El estudio vascular no documentó una etiología diferente a la covid-19. DISCUSIÓN: Se plantea que la infección condiciona un estado inflamatorio sistémico, endotelialitis, ulceración de placas ateroscleróticas preexistentes e hipercoagulabilidad. Lo anterior facilita la aparición de trombosis vascular. Es frecuente encontrar elevación de reactantes de fase aguda y de los productos del metabolismo de la fibrina. El accidente cerebrovascular (ACV) isquémico se ha descrito en el 1 al 5 % de los pacientes con la covid-19. En pacientes con factores de riesgo cardiovascular, durante las primeras 2 semanas desde el inicio de los síntomas respiratorios, el compromiso isquémico suele afectar a múltiples territorios vasculares. CONCLUSIÓN: La covid-19 por sí sola es un factor de riesgo para trombosis arterial secundaria a la inflamación sistémica y estados de hipercoagulabilidad, por lo que debe ser tenida en cuenta en el estudio de ACV isquémico en pacientes jóvenes.

ABSTRACT INTRODUCTION: It is known that COVID-19 is associated with venous and arterial thromboembolic events in patients with history of hypertension and atherosclerosis. Herein, it is presented a case of a young woman without known cardiovascular risk, who was infected by SARS-COV 2 and then an ischemic stroke. CASE: 44 years-old woman with moderate COVID-19 in protocolary treatment with anticoagulants, steroid and oxygen. In the seventh day since symptoms onset, she presented acute ischemic stroke due to acute thrombosis of right carotid artery, and right and left medial cerebral arteries, then rapidly developed malignant cerebral edema and brain death. Vascular study failed to report and etiology other than COVID-19. DISCUSSION: It is suggested that the infection produces a systemic inflammatory state, endothelialitis, ulceration of preexisting atherosclerotic plaques, and hypercoagulability that facilitate thrombosis. Ischemic stroke is described in 1 to 5% of COVID-19, it is common to find an elevation of acute phase reactants and products of fibrin metabolism. Multiple vascular territories are usually compromised in patients with known cardiovascular risk factors and 12 days after the onset of symptoms. CONCLUSION: COVID-19 by itself is a risk factor for arterial thrombosis secondary to systemic infection and hypercoagulability states and it should be taken into account in the study of ischemic stroke in young patients.

Fasudil mediates neuroprotection in ischemia/reperfusion by modulating the ROCK-PPARα-NOX axis

Purpose: Cerebral ischemia-reperfusion (I/R) is a neurovascular disorder that leads to brain injury. In mice, Fasudil improves nerve injury induced by I/R. However, it is unclear if this is mediated by increased peroxisome proliferator-activated receptor-α (PPARα) expression and reduced oxidative damage. This study aimed to investigate the neuroprotective mechanism of action of Fasudil. Methods: MCAO (Middle cerebral artery occlusion) was performed in male C57BL/6J wild-type and PPARα KO mice between September 2021 to April 2023. Mice were treated with Fasudil and saline; 2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed to analyze cerebral infarction. PPARα and Rho-associated protein kinase (ROCK) expression were detected using Western blot, and the expression of NADPH subunit Nox2 mRNA was detected using real-time polymerase chain reaction. The NADPH oxidase activity level and reactive oxygen species (ROS) content were also investigated. Results: After cerebral ischemia, the volume of cerebral necrosis was reduced in wild-type mice treated with Fasudil. The expression of PPARα was increased, while ROCK was decreased. Nox2 mRNA expression, NADPH oxidase activity, and ROS content decreased. There were no significant changes in cerebral necrosis volumes, NADPH oxidase activity, and ROS content in the PPARα KO mice treated with Fasudil. Conclusions: In mice, the neuroprotective effect of Fasudil depends on the expression of PPARα induced by ROCK-PPARα-NOX axis-mediated reduction in ROS and associated oxidative damage.

Cerebrovascular mortality: trend and seasonality in Brazilian capitals, 2000-2019 / Mortalidade cerebrovascular: tendência e sazonalidade nas capitais brasileiras, 2000-2019

ABSTRACT OBJECTIVE To evaluate the trend and seasonality of cerebrovascular mortality rates in the adult population of Brazilian capitals from 2000 to 2019. METHODS This is an ecological and descriptive study of a time series of mortality due to cerebrovascular causes in adults (≥ 18 years) living in Brazilian capitals from 2000 to 2019, based on the Brazilian Mortality Information System. Descriptive statistical techniques were applied in the exploratory analysis of data and in the summary of specific, standardized rates and ratios by sociodemographic characteristics. The jointpoint regression model was used to estimate the trend of cerebrovascular mortality rates by gender, age groups, and geographic regions. The seasonal variability of rates by geographic regions was estimated using the generalized additive model by smoothing cubic splines. RESULTS People aged over 60 years comprised 77% of all cerebrovascular deaths. Women (52%), white individuals (47%), single people (59%), and those with low schooling (57%, elementary school) predominated in our sample. Recife (20/1,000 inhab.) and Vitória (16/1,000 inhab.) showed the highest crude mortality rates. Recife (49/10,000 inhab.) and Palmas (47/10,000 inhab.) prevailed after we applied standardized rates. Cerebrovascular mortality rates in Brazil show a favorable declining trend for adults of all genders. Seasonality influenced rate increase from July to August in almost all region capitals, except in the North, which rose in March, April, and May. CONCLUSIONS Deaths due to cerebrovascular causes prevailed in older single adults with low schooling. The trend showed a tendency to decline and winter, the greatest risk. Regional differences can support decision-makers in implementing public policies to reduce cerebrovascular mortality.

RESUMO OBJETIVO Avaliar a tendência e a sazonalidade das taxas de mortalidade cerebrovascular na população adulta das capitais brasileiras de 2000 a 2019. MÉTODOS Estudo ecológico e descritivo de séries temporais de mortalidade por causas cerebrovasculares em adultos (≥ 18 anos) residentes nas capitais do Brasil no período 2000-2019, obtidas do Sistema de Informações sobre Mortalidade. Técnicas de estatística descritiva foram aplicadas na análise exploratória dos dados e no resumo de taxas específicas, padronizadas e razões por características sociodemográficas. A regressão de pontos de junção (jointpoint regression model) estimou a tendência das taxas de mortalidade cerebrovascular por sexo, grupos etários e regiões geográficas. A variabilidade sazonal por regiões geográficas das taxas foi estimada utilizando o modelo aditivo generalizado por meio de splines de suavização cúbica. RESULTADOS As pessoas maiores de 60 anos representaram 77% dos óbitos cerebrovasculares. Predominaram o sexo feminino (52%), a raça branca (47%), os solteiros (59%) e a baixa escolaridade (57%, ensino fundamental). As capitais Recife (20/1.000 hab.) e Vitória (16/1.000 hab.) apresentaram as maiores taxas brutas de mortalidade. Aplicando as taxas padronizadas Recife (49/10.000 hab.) e Palmas (47/10.000 hab.) prevaleceram. As taxas de mortalidade cerebrovascular no Brasil apresentam uma tendência favorável ao declínio em ambos os sexos e em adultos. A sazonalidade mostrou influenciar na elevação das taxas entre os meses de julho a agosto em quase todas as capitais das regiões, exceto na Norte, que se elevaram nos meses de março, abril e maio. CONCLUSÕES Os óbitos por causa cerebrovascular prevaleceram em pessoas idosas, solteiras e com baixa escolaridade. A tendência foi favorável ao declínio, sendo o inverno o período de maior risco. As diferenças regionais permitem subsidiar os tomadores de decisões em relação à implementação de políticas públicas para reduzir a mortalidade cerebrovascular.

Soybean isoflavones alleviate cerebral ischemia/reperfusion injury in rats by inhibiting ferroptosis and inflammatory cascade reaction / 南方医科大学学报

OBJECTIVE@#To explore the mechanism that mediates the effect of soybean isoflavones (SI) against cerebral ischemia/reperfusion (I/R) injury in light of the regulation of regional cerebral blood flow (rCBF), ferroptosis, inflammatory response and blood-brain barrier (BBB) permeability.@*METHODS@#A total of 120 male SD rats were equally randomized into sham-operated group (Sham group), cerebral I/R injury group and SI pretreatment group (SI group). Focal cerebral I/R injury was induced in the latter two groups using a modified monofilament occlusion technique, and the intraoperative changes of real-time cerebral cortex blood flow were monitored using a laser Doppler flowmeter (LDF). The postoperative changes of cerebral pathological morphology and the ultrastructure of the neurons and the BBB were observed with optical and transmission electron microscopy. The neurological deficits of the rats was assessed, and the severities of cerebral infarction, brain edema and BBB disruption were quantified. The contents of Fe2+, GSH, MDA and MPO in the ischemic penumbra were determined with spectrophotometric tests. Serum levels of TNF-α and IL-1βwere analyzed using ELISA, and the expressions of GPX4, MMP-9 and occludin around the lesion were detected with Western blotting and immunohistochemistry.@*RESULTS@#The rCBF was sharply reduced in the rats in I/R group and SI group after successful insertion of the monofilament. Compared with those in Sham group, the rats in I/R group showed significantly increased neurological deficit scores, cerebral infarction volume, brain water content and Evans blue permeability (P < 0.01), decreased Fe2+ level, increased MDA level, decreased GSH content and GPX4 expression (P < 0.01), increased MPO content and serum levels of TNF-α and IL-1β (P < 0.01), increased MMP-9 expression and lowered occludin expression (P < 0.01). All these changes were significantly ameliorated in rats pretreated with IS prior to I/R injury (P < 0.05 or 0.01).@*CONCLUSION@#SI preconditioning reduces cerebral I/R injury in rats possibly by improving rCBF, inhibiting ferroptosis and inflammatory response and protecting the BBB.

Serum lipoprotein-associated phospholipase A2 level is positively correlated with the recurrence risk of acute ischemic cerebral infarction in hypertensive patients / 南方医科大学学报

OBJECTIVE@#To explore the relationship between serum lipoprotein-associated phospholipase A2 (Lp-PLA2) level and the risk of acute ischemic stroke (AIS) recurrence in hypertensive patients.@*METHODS@#This retrospective case-control study was conducted among 211 hypertensive patients with AIS treated in Foshan First People's Hospital, including 35 patients with recurrence of AIS during the 1-year follow-up as confirmed by head CT/MR. In the overall patients, 60 had grade 1 hypertension (including 5 recurrent cases), 76 had grade 2 hypertension (with 11 recurrent cases), and 75 had grade 3 hypertension (with 19 recurrent cases). Univariate analysis, multivariate logistic regression analysis, trend analysis, and smooth curve fitting analysis were performed to explore the correlation between serum Lp-PLA2 level within 24 h after admission and the risk of AIS recurrence. The predictive efficacy of serum Lp-PLA2 level for AIS recurrence in different hypertension grades was evaluated using ROC curve analysis.@*RESULTS@#Serum Lp-PLA2 level, age, NIHSS score at admission, mRS scores at 7 days, homocysteine level and smoking status differed significantly between patients with and without AIS recurrence (P < 0.05). After adjustment for confounding factors, multivariate regression analysis showed that the highest tertile of Lp-PLA2 level was associated with a 4.13-fold increase of AIS recurrence risk compared with the lowest tertile (OR=5.13, 95% CI: 1.35-19.40), and each 1 ng/mL increase of Lp-PLA2 level was associated with a 1% increase of AIS recurrence risk (OR= 1.01, 95% CI: 1.01-1.02). Serum Lp-PLA2 level was shown to positively correlate with AIS recurrence risk, and in patients with grade 3 hypertension, its areas under the ROC curve for predicting AIS recurrence was 0.869 with a specificity of 0.893 and a sensitivity of 0.737.@*CONCLUSION@#Serum Lp-PLA2 concentration is an independent risk factor and potentially an effective predictor for AIS recurrence in patients with grade 3 hypertension.

Forsythiaside B inhibits cerebral ischemia/reperfusion-induced oxidative stress injury in mice via the AMPK/DAF-16/FOXO3 pathway / 南方医科大学学报

OBJECTIVE@#To study the protective effect of forsythiaside B (FB) against cerebral oxidative stress injury induced by cerebral ischemia/reperfusion (I/R) in mice and explore the underlying mechanism.@*METHODS@#Ninety C57BL/6 mice were randomized into sham-operated group, middle cerebral artery occlusion (MCAO) model group, and low-, medium and highdose (10, 20, and 40 mg/kg, respectively) FB groups. The expression levels of MDA, ROS, PCO, 8-OHdG, SOD, GSTα4, CAT and GPx in the brain tissue of the mice were detected using commercial kits, and those of AMPK, P-AMPK, DAF-16, FOXO3 and P-FOXO3 were detected with Western blotting. Compound C (CC), an AMPK inhibitor, was used to verify the role of the AMPK pathway in mediating the therapeutic effect of FB. In another 36 C57BL/6 mice randomized into 4 sham-operated group, MCAO model group, FB (40 mg/kg) treatment group, FB+CC (10 mg/kg) treatment group, TTC staining was used to examine the volume of cerebral infarcts, and the levels of ROS and SOD in the brain were detected; the changes in the protein expressions of AMPK, P-AMPK, DAF-16, FOXO3 and P-FOXO3 in the brain tissue were detected using Western blotting.@*RESULTS@#In mice with cerebral IR injury, treatment with FB significantly reduced the levels of ROS, MDA, PCO and 8-OHdG, increased the activities of antioxidant enzymes SOD, GSTα4, CAT and GPx, and enhanced phosphorylation of AMPK and FOXO3 and DAF-16 protein expression in the brain tissue (P < 0.01). Compared with FB treatment alone, the combined treatment with FB and CC significantly reduced phosphorylation of AMPK and FOXO3, lowered expression of DAF-16 and SOD activity, and increased cerebral infarction volume and ROS level in the brain tissue of the mice (P < 0.01).@*CONCLUSION@#FB inhibits oxidative stress injury caused by cerebral I/R in mice possibly by enhancing AMPK phosphorylation, promoting the downstream DAF-16 protein expression and FOXO3 phosphorylation, increasing the expression of antioxidant enzymes, and reducing ROS level in the brain tissue.

Clinical Effect of Surgical Reconstruction of Extracranial Vertebral Artery / 中国医学科学院学报

Objective To evaluate the effect of surgical reconstruction of extracranial vertebral artery and to summarize the experience. Methods The clinical data of 15 patients undergoing surgical reconstruction of extracranial vertebral artery from September 2018 to June 2022 were collected.The operation methods,operation duration,intraoperative blood loss,operation complications,and relief of symptoms were retrospectively analyzed. Results Eleven patients underwent vertebral artery (V1 segment) to common carotid artery transposition,two patients underwent endarterectomy of V1 segment,two patients underwent V3 segment to external carotid artery bypass or transposition.The operation duration,intraoperative blood loss,and blocking time of common carotid artery varied within 120-340 min,50-300 ml,and 12-25 min,with the medians of 240 min,100 ml,and 16 min,respectively.There was no cardiac accident,cerebral hyperperfusion syndrome,cerebral hemorrhage or lymphatic leakage during the perioperative period.One patient suffered from cerebral infarction and three patients suffered from incomplete Horner's syndrome after the operation.During the follow-up (4-45 months,median of 26 months),there was no anastomotic stenosis,new cerebral infarction or cerebral ischemia. Conclusion Surgical reconstruction of extracranial vertebral artery is safe and effective,and individualized reconstruction strategy should be adopted according to different conditions.

An improved 4-vessel intermittent occlusion method for establishing rat models of global cerebral ischemia-reperfusion injury / 南方医科大学学报

OBJECTIVE@#To improve the classical 4-vessel occlusion (4VO) model established by Pulsinelli and Brierley.@*METHODS@#Thirty-two male SD rats were randomized into sham operation group, I4VO-Con10 group, I4VO-Int10 group and I4VO-Int15 group. The sham surgery group underwent exposure of the bilateral vertebral arteries and carotid arteries without occlusion to block blood flow. The I4VO-Con10 group experienced continuous ischemia by occluding the bilateral vertebral arteries and carotid arteries for 10 minutes followed by reperfusion for 24 hours. The I4VO-Int10 and I4VO-Int15 groups were subjected to intermittent ischemia. The I4VO- Int10 group underwent 5 minutes of ischemia, followed by 5 minutes of reperfusion and another 5 minutes of ischemia, and then reperfusion for 24 hours. The I4VO-Int15 group experienced 5 minutes of ischemia followed by two cycles of 5 minutes of reperfusion and 5 minutes of ischemia, and then reperfusion for 24 hours. The regional cerebral blood flow (rCBF) was monitored with laser Doppler scanning, and survival of the rats was observed. HE staining was used to observe hippocampal pathologies to determine the optimal method for modeling. Another 48 rats were randomized into 6 groups, including a sham operation group and 5 model groups established using the optimal method. The 5 I4VO model groups were further divided based on the reperfusion time points (1, 3, 7, 14, and 28 days) into I4VO-D1, I4VO-D3, I4VO-D7, I4VO- D14, and I4VO- D28 groups. Body weight changes and survival of the rats were recorded. HE staining was used to observe morphological changes in the hippocampal, retinal and optic tract tissues. The Y-maze test and light/dark box test were used to evaluate cognitive and visual functions of the rats in I4VO-D28 group.@*RESULTS@#Occlusion for 5 min for 3 times at the interval of 5 min was the optimal method for 4VO modeling. In the latter 48 rats, the body weight was significantly lower than that of the sham-operated rats at 1, 3, 7, 14 and 28 days after modeling without significant difference in survival rate among the groups. The rats with intermittent vessel occlusion exhibited progressive deterioration of hippocampal neuronal injury and neuronal loss. Cognitive impairment was observed in the rats in I4VO-D28 group, but no obvious ischemic injury of the retina or the optic tract was detected.@*CONCLUSION@#The improved 4VO model can successfully mimic the main pathological processes of global cerebral ischemia-reperfusion injury without causing visual impairment in rats.

Molecular Mechanism of Electroacupuncture Regulating Cerebral Arterial Contractile Protein in Rats with Cerebral Infarction Based on MLCK Pathway / 中国结合医学杂志

OBJECTIVE@#To explore the effect of electroacupuncture (EA) intervention on the vasoconstriction of cerebral artery smooth muscle cells after cerebral infarction.@*METHODS@#Male Wistar rats were randomly divided into 3 groups by a random number table: the model group (n=24), the EA group (n=24), and the normal group (n=6). The model and the EA groups were divided into different time subgroups at 0.5, 1, 3, and 6 h after middle cerebral artery occlusion (MCAO), with 6 rats in each subgroup. MCAO model was established using intraluminal suture occlusion method. The EA group was given EA treatment at acupoint Shuigou (GV 26) instantly after MCAO for 20 min. The contents of cerebrovascular smooth muscle MLCK, the 3 subunits of myosin light chain phosphatase (MLCP) MYPT1, PP1c-δ and M20, as well as myosin-ATPase activity were detected using immunohistochemistry and Western blotting.@*RESULTS@#The overall expression level of the MYPT1 and PP1c-δ in the model group was significantly higher (P<0.01). After EA intervention, the 0.5 h group expression level was close to that of the normal group (P>0.05), and the other subgroups were still significantly higher than the normal group (P<0.01). After EA intervention, the expression level of each subgroup was significantly lower than the corresponding model group. There was a significant difference between the 0.5 and 1 h subgroups (P<0.01), while a difference was also observed between the 3 and 6 h subgroups (P<0.05). The dynamic change rule gradually increased with the prolongation of infarction time within 6 h after infarction.@*CONCLUSION@#EA intervention can inhibit contraction of cerebral vascular smooth muscle cells and regulate smooth muscle relaxation by regulating MLCK pathway.

Chrysin alleviates cerebral ischemia-reperfusion injury by inhibiting ferroptosis in rats / 中国中药杂志

The purpose of this study is to investigate whether chrysin reduces cerebral ischemia-reperfusion injury(CIRI) by inhi-biting ferroptosis in rats. Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups(200, 100, and 50 mg·kg~(-1)), and a positive drug group(Ginaton, 21.6 mg·kg~(-1)). The CIRI model was induced in rats by transient middle cerebral artery occlusion(tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation. The neurological deficit score was used to detect neurological function. The 2,3,5-triphenyl tetrazolium chloride(TTC) staining was used to detect the cerebral infarction area. Hematoxylin-eosin(HE) staining and Nissl staining were used to observe the morphological structure of brain tissues. Prussian blue staining was used to observe the iron accumulation in the brain. Total iron, lipid pero-xide, and malondialdehyde in serum and brain tissues were detected by biochemical reagents. Real-time quantitative polymerase chain reaction(RT-qPCR), immunohistochemistry, and Western blot were used to detect mRNA and protein expression of solute carrier fa-mily 7 member 11(SLC7A11), transferrin receptor 1(TFR1), glutathione peroxidase 4(GPX4), acyl-CoA synthetase long chain family member 4(ACSL4), and prostaglandin-endoperoxide synthase 2(PTGS2) in brain tissues. Compared with the model group, the groups with drug intervention showed restored neurological function, decreased cerebral infarction rate, and alleviated pathological changes. The low-dose chrysin group was selected as the optimal dosing group. Compared with the model group, the chrysin groups showed reduced content of total iron, lipid peroxide, and malondialdehyde in brain tissues and serum, increased mRNA and protein expression levels of SLC7A11 and GPX4, and decreased mRNA and protein expression levels of TFR1, PTGS2, and ACSL4. Chrysin may regulate iron metabolism via regulating the related targets of ferroptosis and inhibit neuronal ferroptosis induced by CIRI.

Bombyx Batryticatus extract promotes microglia polarization to improve neuron injury and behaviors of cerebral ischemia/reperfusion rats / 中国中药杂志

This study aims to investigate the effect of Bombyx Batryticatus extract(BBE) on behaviors of rats with global cerebral ischemia reperfusion(I/R) and the underlying mechanism. The automatic coagulometer was used to detect the four indices of human plasma coagulation after BBE intervention for quality control of the extract. Sixty 4-week-old male SD rats were randomized into sham operation group(equivalent volume of normal saline, ip), model group(equivalent volume of normal saline, ip), positive drug group(900 IU·kg~(-1) heparin, ip), and low-, medium-, and high-dose BBE groups(0.45, 0.9, and 1.8 mg·g~(-1)·d~(-1) BBE, ip). Except the sham operation group, rats were subjected to bilateral common carotid artery occlusion followed by reperfusion(BCCAO/R) to induce I/R. The administration lasted 7 days for all the groups. The behaviors of rats were examined by beam balance test(BBT). Morphological changes of brain tissue were observed based on hematoxylin-eosin(HE) staining. Immunofluorescence method was used to detect common leukocyte antigen(CD45), leukocyte differentiation antigen(CD11b), and arginase-1(Arg-1) in cerebral cortex(CC). The protein expression of interleukin-1β(IL-1β), interleukin-4(IL-4), interleukin-6(IL-6), and interleukin-10(IL-10) was detected by enzyme-linked immunosorbent assay(ELISA). The non-targeted metabonomics was employed to detect the levels of metabolites in plasma and CC of rats after BBE intervention. The results of quality control showed that the BBE prolonged the activated partial thromboplastin time(APTT), prothrombin time(PT), and thrombin time(TT) of human plasma, which was similar to the anticoagulation effect of BBE obtained previously. The results of behavioral test showed that the BBT score of the model group increased compared with that of the sham operation group. Compared with the model group, BBE reduced the BBT score. As for the histomorphological examination, compared with the sham operation group, the model group showed morphological changes of a lot of nerve cells in CC. The nerve cells with abnormal morphology in CC decreased after the intervention of BBE compared with those in the model group. Compared with the sham operation group, the model group had high average fluorescence intensity of CD45 and CD11b in the CC. The average fluorescence intensity of CD11b decreased and the average fluorescence intensity of Arg-1 increased in CC in the low-dose BBE group compared with those in the model group. The average fluorescence intensity of CD45 and CD11b decreased and the average fluorescence intensity of Arg-1 increased in medium-and high-dose BBE groups compared with those in the model group. The expression of IL-1β and IL-6 was higher and the expression of IL-4 and IL-10 was lower in the model group than in the sham operation group. The expression of IL-1β and IL-6 was lower and the expression of IL-4 and IL-10 was higher in the low-dose, medium-dose, and high-dose BBE groups than in the model group. The results of non-targeted metabonomics showed that 809 metabolites of BBE were identified, and 57 new metabolites in rat plasma and 45 new metabolites in rat CC were found. BBE with anticoagulant effect can improve the behaviors of I/R rats, and the mechanism is that it promotes the polarization of microglia to M2 type, enhances its anti-inflammatory and phagocytic functions, and thus alleviates the damage of nerve cells in CC.

CiteSpace knowledge map analysis of Angong Niuhuang Pills in recent twenty years / 中国中药杂志

Angong Niuhuang Pills, a classical formula in traditional Chinese medicine, are lauded as one of the "three treasures of febrile diseases" and have been widely used in the treatment of diverse disorders with definite efficacy. However, there is still a lack of bibliometric analysis of research progress and development trend regarding Angong Niuhuang Pills. Research articles on Angong Niuhuang Pills in China and abroad(2000-2022) were retrieved from CNKI and Web of Science. CiteSpace 6.1 was used to visualize the key contents of the research articles. In addition, the research status of Angong Niuhuang Pills was analyzed by information extraction to allow insight into the research trends and hotspots about Angong Niuhuang Pills. A total of 460 Chinese articles and 41 English articles were included. Beijing University of Chinese Medicine and Sun Yat-Sen University were the research institutions that have published the largest amount of research articles in Chinese and English. The keyword analysis showed that the Chinese articles focused on cerebral hemorrhage, stroke, neurological function, coma, cerebral infarction, craniocerebral injury, and clinical application, while the English articles focused on the mechanisms of cerebral ischemia, stroke, heavy metal, blood-brain barrier, and oxidative stress. Stroke, blood-brain barrier, and oxidative stress were presumably the research hotspots in the future. At present, the research on Angong Niuhuang Pills is still in the developing stage. It is necessary to highlight the in-depth research on the active components and mechanism of action and carry out large-scale randomized controlled clinical trials to provide references for the further development and application of Angong Niuhuang Pills.

Protective effects of three kinds of borneol on different brain regions in acute cerebral ischemia/reperfusion model rats / 中国中药杂志

This study compared the ameliorating effects of L-borneol, natural borneol, and synthetic borneol on the injury of different brain regions in the rat model of acute phase of cerebral ischemia/reperfusion(I/R) for the first time, which provides a reference for guiding the rational application of borneol in the early treatment of ischemic stroke and has important academic and application values. Healthy specific pathogen-free(SPF)-grade SD male rats were randomly assigned into 13 groups: a sham-operation group, a model group, a Tween model group, a positive drug(nimodipine) group, and high-, medium-, and low-dose(0.2, 0.1, and 0.05 g·kg~(-1), respectively) groups of L-borneol, natural borneol, and synthetic borneol according to body weight. After 3 days of pre-administration, the rat model of I/R was established by suture-occluded method and confirmed by laser speckle imaging. The corresponding agents in different groups were then administered for 1 day. The body temperature was monitored regularly before pre-administration, days 1, 2, and 3 of pre-administration, 2 h after model awakening, and 1 d after model establishment. Neurological function was evaluated based on Zea-Longa score and modified neurological severity score(mNSS) 2 h and next day after awakening. The rats were anesthetized 30 min after the last administration, and blood was collected from the abdominal aorta. Enzyme-linked immunoassay assay(ELISA) was employed to determine the serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-4, and transforming growth factor-beta1(TGF-β1). The brain tissues were stained with triphenyltetrazolium chloride(TTC) for the calculation of cerebral infarction rate, and hematoxylin-eosin(HE) staining was used for observing and semi-quantitatively evaluating the pathological damage in different brain regions. Immunohistochemistry was employed to detect the expression of ionized calcium binding adapter molecule 1(IBA1) in microglia. q-PCR was carried out to determine the mRNA levels of iNOS and arginase 1(Arg1), markers of polarization phenotype M1 and M2 in microglia. Compared with the sham-operation group, the model group and the Tween model group showed significantly elevated body temperature, Zea-Longa score, mNSS, and cerebral infarction rate, severely damaged cortex, hippocampus, and striatum, increased serum levels of IL-6 and TNF-α, and decreased serum levels of IL-4 and TGF-β1. The three borneol products had a tendency to reduce the body temperature of rats 1 day after modeling. Synthetic borneol at the doses of 0.2 and 0.05 g·kg~(-1), as well as L-borneol of 0.1 g·kg~(-1), significantly reduced Zea-Longa score and mNSS. The three borneol products at the dose of 0.2 g·kg~(-1) significantly reduced the cerebral infarction rate. L-borneol at the doses of 0.2 and 0.1 g·kg~(-1) and natural borneol at the dose of 0.1 g·kg~(-1) significantly reduced the pathological damage of the cortex. L-borneol and natural borneol at the dose of 0.1 g·kg~(-1) attenuated the pathological damage of hippocampus, and 0.2 g·kg~(-1) L-borneol attenuated the damage of striatum. The 0.2 g·kg~(-1) L-borneol and the three doses of natural borneol and synthetic borneol significantly reduced the serum level of TNF-α, and the 0.1 g·kg~(-1) synthetic borneol reduced the level of IL-6. L-borneol and synthetic borneol at the dose of 0.2 g·kg~(-1) significantly inhibited the activation of cortical microglia, and 0.2 g·kg~(-1) L-borneol up-regulated the expression of Arg1 and down-regulated the expression level of iNOS. In conclusion, the three borneol products may alleviate inflammation to ameliorate the pathological damage of brain regions of rats in the acute phase of I/R by inhibiting the activation of microglia and promoting the polarization of microglia from M1 type to M2 type. The protective effect on brain followed a trend of L-borneol > synthetic borneol > natural borneol. We suggest L-borneol the first choice for the treatment of I/R in the acute phase.

Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury / 中国中药杂志

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.

Impact of statin use on major adverse cardiovascular events in patients with normal stress myocardial perfusion imaging and elevated coronary artery calcium score

INTRODUCTION@#Normal stress myocardial perfusion imaging (MPI) carries a favourable prognosis. Conversely, elevated coronary artery calcium (CAC) is associated with increased major adverse cardiovascular events (MACE). There is limited information on the prognosis and management of patients with elevated CAC and normal MPI. We aimed to assess the outcomes of patients with elevated CAC and normal MPI in relation to post-MPI statin use.@*METHODS@#A retrospective review of normal MPI with CAC score >300 was performed between 1 March 2016 and 31 January 2017 in a Singapore tertiary hospital. Patients with known atherosclerotic cardiovascular disease or left ventricular ejection fraction <50% on MPI were excluded. Patient demographics, prescriptions and MACE (cardiac death, nonfatal myocardial infarction and/or ischaemic stroke) at 24 months after MPI were traced using electronic records. Binary logistic regression was used to evaluate for independent predictors of MACE.@*RESULTS@#We included 311 patients (median age 71 years, 56.3% male), of whom 65.0% were on moderate to high-intensity statins (MHIS) after MPI. MACE was significantly lower in the post-MPI MHIS group (3.5% vs. 9.2%, P = 0.035). On univariate binary logistic regression, post-MPI MHIS use was the only significant predictor for MACE (odds ratio [OR] 0.355 [95% confidence interval (CI) 0.131-0.962], P = 0.042), even after multivariate adjustment (adjusted OR 0.363, 95% confidence interval 0.134-0.984, P = 0.046).@*CONCLUSION@#Post-MPI MHIS use is associated with lower MACE and is an independent negative predictor for 24-month MACE among patients with normal MPI and CAC >300.

Endogenous FGF21 attenuates blood-brain barrier disruption in penumbra after delayed recanalization in MCAO rats through FGFR1/PI3K/Akt pathway / 中南大学学报(医学版)

OBJECTIVES@#Restoration of blood circulation within "time window" is the principal treating goal for treating acute ischemic stroke. Previous studies revealed that delayed recanalization might cause serious ischemia/reperfusion injury. However, plenty of evidences showed delayed recanalization improved neurological outcomes in acute ischemic stroke. This study aims to explore the role of delayed recanalization on blood-brain barrier (BBB) in the penumbra (surrounding ischemic core) and neurological outcomes after middle cerebral artery occlusion (MCAO).@*METHODS@#Recanalization was performed on the 3rd day after MCAO. BBB disruption was tested by Western blotting, Evans blue dye, and immunofluorescence staining. Infarct volume and neurological outcomes were evaluated on the 7th day after MCAO. The expression of fibroblast growth factor 21 (FGF21), fibroblast growth factor receptor 1 (FGFR1), phosphatidylinositol-3-kinase (PI3K), and serine/threonine kinase (Akt) in the penumbra were observed by immunofluorescence staining and/or Western blotting.@*RESULTS@#The extraversion of Evans blue, IgG, and albumin increased surrounding ischemic core after MCAO, but significantly decreased after recanalization. The expression of Claudin-5, Occludin, and zona occludens 1 (ZO-1) decreased surrounding ischemic core after MCAO, but significantly increased after recanalization. Infarct volume reduced and neurological outcomes improved following recanalization (on the 7th day after MCAO). The expressions of Claudin-5, Occludin, and ZO-1 decreased surrounding ischemic core following MCAO, which were up-regulated corresponding to the increases of FGF21, p-FGFR1, PI3K, and p-Akt after recanalization. Intra-cerebroventricular injection of FGFR1 inhibitor SU5402 down-regulated the expression of PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 in the penumbra, which weakened the beneficial effects of recanalization on neurological outcomes after MCAO.@*CONCLUSIONS@#Delayed recanalization on the 3rd day after MCAO increases endogenous FGF21 in the penumbra and activates FGFR1/PI3K/Akt pathway, which attenuates BBB disruption in the penumbra and improves neurobehavior in MCAO rats.

Ultrashort wave alleviates oxygen -glucose deprivation/reoxygenation injury via up -regulation of SPCA1 expression in N2a cells / 中南大学学报(医学版)

OBJECTIVES@#Application of ultrashort wave (USW) to rats with cerebral ischemia and reperfusion injury could inhibit the decrease of expression of secretory pathway Ca2+-ATPase 1 (SPCA1), an important participant in Golgi stress, reduce the damage of Golgi apparatus and the apoptosis of neuronal cells, thereby alleviating cerebral ischemia-reperfusion injury. This study aims to investigate the effect of USW on oxygen-glucose deprivation/reperfusion (OGD/R) injury and the expression of SPCA1 at the cellular level.@*METHODS@#N2a cells were randomly divided into a control (Con) group, an OGD/R group, and an USW group. The cells in the Con group were cultured without exposure to OGD. The cells in the OGD/R group were treated with OGD/R. The cells in the USW group were treated with USW after OGD/R. Cell morphology was observed under the inverted phase-contrast optical microscope, cell activity was detected by cell counting kit-8 (CCK-8), apoptosis was detected by flow cytometry, and SPCA1 expression was detected by Western blotting.@*RESULTS@#Most of the cells in the Con group showed spindle shape with a clear outline and good adhesion. In the OGD/R group, cells were wrinkled, with blurred outline, poor adhesion, and lots of suspended dead cells appeared; compared with the OGD/R group, the cell morphology and adherence were improved, with clearer outlines and fewer dead cells in the USW group. Compared with the Con group, the OGD/R group showed decreased cell activity, increased apoptotic rate, and down-regulating SPCA1 expression with significant differences (all P<0.001); compared with the OGD/R group, the USW group showed increased cell activity, decreased apoptotic rate, and up-regulating SPCA1 expression with significant differences (P<0.01 or P<0.001).@*CONCLUSIONS@#USW alleviates the injury of cellular OGD/R, and its protective effect may be related to its up-regulation of SPCA1 expression.

Effect of Xiaoxuming Decoction on synaptic plasticity following acute cerebral ischemia-reperfusion in rats / 中国中药杂志

This study aims to explore the effect of Xiaoxuming Decoction on synaptic plasticity in rats with acute cerebral ischemia-reperfusion. A rat model of cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion(MCAO). Rats were randomly assigned into a sham group, a MCAO group, and a Xiaoxuming Decoction(60 g·kg~(-1)·d~(-1)) group. The Longa score was rated to assess the neurological function of rats with cerebral ischemia for 1.5 h and reperfusion for 24 h. The 2,3,5-triphenyltetrazolium chloride(TTC) staining and hematoxylin-eosin(HE) staining were employed to observe the cerebral infarction and the pathological changes of brain tissue after cerebral ischemia, respectively. Transmission electron microscopy was employed to detect the structural changes of neurons and synapses in the ischemic penumbra, and immunofluorescence, Western blot to determine the expression of synaptophysin(SYN), neuronal nuclei(NEUN), and postsynaptic density 95(PSD95) in the ischemic penumbra. The experimental results showed that the modeling increased the Longa score and led to cerebral infarction after 24 h of ischemia-reperfusion. Compared with the model group, Xiaoxuming Decoction intervention significantly decreased the Longa score and reduced the formation of cerebral infarction area. The modeling led to the shrinking and vacuolar changes of nuclei in the brain tissue, disordered cell arrangement, and severe cortical ischemia-reperfusion injury, while the pathological damage in the Xiaoxuming Decoction group was mild. The modeling blurred the synaptic boundaries and broadened the synaptic gap, while such changes were recovered in the Xiaoxuming Decoction group. The modeling decreased the fluorescence intensity of NEUN and SYN, while the intensity in Xiaoxuming Decoction group was significantly higher than that in the model group. The expression of SYN and PSD95 in the ischemic penumbra was down-regulated in the model group, while such down-regulation can be alleviated by Xiaoxuming Decoction. In summary, Xiaoxuming Decoction may improve the synaptic plasticity of ischemic penumbra during acute cerebral ischemia-reperfusion by up-regulating the expression of SYN and PSD95.

Applicability of [18F]FDG/PET for investigating rosmarinic acid preconditioning efficacy in a global stroke model in mice

ABSTRACT Positron emission tomography (PET) is a non-invasive nuclear imaging technique that uses radiotracers to track cell activity. The radiopharmaceutical 18F-fluoro-2-deoxyglucose ([18F] FDG) is most commonly used in nuclear medicine for the diagnosis of various diseases, including stroke. A stroke is a serious condition with high mortality and morbidity rates. Rosmarinic acid (RA) is a promising therapeutic agent that exerts neuroprotective effects against various neurological diseases. Therefore, this study aimed to evaluate the applicability of [18F]FDG/PET for investigating the neuroprotective effects of RA in case of a global stroke model in mice. The [18F]FDG/PET technique facilitates the observation of ischemia and reperfusion injuries in the brain. Moreover, the recovery of glucose metabolism in three specific brain regions, the striatum, superior colliculus, and inferior colliculus, was observed after preconditioning with RA. It was concluded that the [18F]FDG/PET technique may be useful for stroke diagnosis and the assessment of treatment response. In addition, a long-term longitudinal study using biochemical analysis in conjunction with functional imaging may provide further conclusive results regarding the effect of RA on cerebral ischemia.