A liquid chromatography-tandem mass spectrometry method was established and validated for determining the concentrations of costunolide(CO), piperine(PI), agarotetrol(AG), glycyrrhizic acid(GL), vanillic acid(VA), and glycyrrhetinic acid(GA) in rat plasma. This method was then applied to the toxicokinetic study of these six compounds in rats with chronic cerebral ischemia(CCI) following multiple oral doses of Zhachong Shisanwei Pills. Finally, the effects of continuous multiple-dose administration of Zhachong Shisanwei Pills on the liver of CCI rats were investigated. The results showed that after oral administration of different doses of Zhachong Shisanwei Pills, the in vivo exposure of AG, VA, and GA was relatively high, with AUC_(0-∞) values ranging from 604.0-2 494.2, 1 305.4-4 634.5, and 2 177.5-4 045.7 h·ng·mL~(-1), respectively, while the exposure of CO, PI, and GL was relatively low, with AUC_(0-∞) values ranging from 37.8-238.2, 2.4-17.0, and 146.9-408.5 h·ng·mL~(-1), respectively. The C_(max) and AUC_(0-∞) of the six compounds were positively correlated with the administered dose. The T_(max) of PI and AG ranged from 0.3 to 2.0 h, their T_(1/2) ranged from 0.8 to 2.9 h, and their mean residence time(MRT) ranged from 1.0 to 3.7 h. The T_(max) of GL and VA was shorter(0.4-1.9 h), while their T_(1/2)(2.6-5.9 h) and MRT(2.5-8.5 h) were longer. Both CO and GA exhibited a bimodal phenomenon, with T_(max) ranging from 1.6 to 6.6 h, T_(1/2) ranging from 2.8 to 7.7 h, and MRT ranging from 4.1 to 12.9 h. Liver histopathology after 28 days of continuous multiple-dose administration of Zhachong Shisanwei Pills showed that the liver tissue remained normal at a low dose(crude drug 0.8 g·kg~(-1), approximately 5 times the clinical equivalent dose). However, as the dose increased(crude drug 1.1-3.0 g·kg~(-1), 6.9-18.8 times the clinical equivalent dose), varying degrees of liver damage were observed. Blood biochemical tests revealed no significant changes in the serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), and total bile acid(TBA) in CCI rats from administration groups 1 to 3(crude drug 0.8, 1.1, 1.5 g·kg~(-1)). However, ALT, AST, ALP, and TBA levels in groups 4 and 5(crude drug 2.1, 3.0 g·kg~(-1)) showed significant increases. This study preliminarily elucidated the toxicokinetic characteristics of the six compounds in Zhachong Shisanwei Pills and their effects on liver tissue in CCI rats, providing data as a reference for clinical use.
Animals ; Tandem Mass Spectrometry/methods* ; Rats ; Drugs, Chinese Herbal/toxicity* ; Male ; Rats, Sprague-Dawley ; Brain Ischemia/blood* ; Chromatography, Liquid/methods* ; Polyunsaturated Alkamides/blood* ; Piperidines/toxicity* ; Benzodioxoles/toxicity* ; Alkaloids/blood* ; Liquid Chromatography-Mass Spectrometry

Humans ; Blood Pressure/physiology* ; Cohort Studies ; Patient Discharge ; Ischemic Stroke ; Stroke ; Brain Ischemia ; Diabetes Mellitus ; Hypertension

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.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; Matrix Metalloproteinase 9/metabolism* ; Soybeans/metabolism* ; Occludin/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Ferroptosis ; Blood-Brain Barrier/ultrastructure* ; Brain Ischemia/metabolism* ; Cerebral Infarction ; Reperfusion Injury/metabolism* ; Isoflavones/therapeutic use* ; Infarction, Middle Cerebral Artery

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.
Humans ; Vertebral Artery/surgery* ; Blood Loss, Surgical ; Retrospective Studies ; Brain Ischemia ; Cerebral Infarction

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.
Animals ; Rats ; Blood-Brain Barrier/metabolism* ; Brain Ischemia ; Claudin-5/metabolism* ; Infarction, Middle Cerebral Artery/metabolism* ; Ischemic Stroke/metabolism* ; Occludin/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats, Sprague-Dawley ; Receptor, Fibroblast Growth Factor, Type 1/metabolism* ; Reperfusion Injury/metabolism*

This project was aimed to investigate the role and the underlying mechanism of microglia polarization on blood-brain barrier (BBB) during cerebral ischemia-reperfusion. After construction of the mouse model of cerebral ischemia-reperfusion, upregulated IL-6 and TNF-α in peripheral blood and increased IL-6 and iNOS in ischemia tissues were confirmed. The supernatant expression of TNF-α and IL-6, as well as IL-6, iNOS and CD86 mRNA, was significantly increased in the of Bv-2 cells after oxygen-glucose deprivation/reoxygenation (OGD/R) model was constructed in vitro. For further understanding the expression pattern of RNAs, the next-generation RNA sequencing was performed and upregulation of Robo4 (roundabout guidance receptor 4) was found both in M1-polarized and OGD/R treated Bv-2 cells, which was also confirmed by RT-qPCR. Extracellular soluble Robo4 (sRobo4) protein also increased in the supernatant of M1-polarized and OGD/R treated Bv-2 cells. Treating bEND3 cells with the Robo4 recombinant protein, M1-polarized Bv-2 cell supernatant or OGD/R Bv-2 cell supernatant decreased trans-endothelial electrical resistance (TEER), suggesting the injury of BBB. In addition, Robo4 was also highly expressed in the serum of patients who experienced acute ischemia stroke and mechanical thrombectomy operation. All the results suggest that increased secretion of Robo4 by M1-polarized-microglia during cerebral ischemia-reperfusion is most likely one of the causes of BBB injury, and Robo4 may be one of the therapeutic targets for BBB functional protection.
Animals ; Blood-Brain Barrier/metabolism* ; Brain Ischemia/drug therapy* ; Glucose/metabolism* ; Interleukin-6/metabolism* ; Mice ; Microglia/metabolism* ; Oxygen/metabolism* ; Receptors, Cell Surface/metabolism* ; Reperfusion ; Reperfusion Injury/drug therapy* ; Tumor Necrosis Factor-alpha/metabolism*

This study aims to observe the therapeutic effect of salidroside on cerebral ischemia-reperfusion(I/R) model rats, and to specifically explore the protection of salidroside on endothelial cell barrier after I/R and the mechanism. In the experiment, SD rats were randomized into sham group, model group, and high-, medium-, and low-dose(10, 5, and 2.5 mg·kg~(-1)) salidroside groups. The suture method was used to induce I/R in rats. The infarct area, neurobehavioral evaluation, and brain water content were used to evaluate the efficacy of salidroside. As for the experiment on the mechanism, high-dose and low-dose salidroside groups were designed. The pathological morphology was observed based on hematoxylin and eosin(HE) staining, and ultrastructure of vascular endothelial cells based on transmission electron microscopy. The content of nitric oxide(NO) in serum, four indexes of blood coagulation, and the content of von Willebrand factor(vWF) in plasma were measured. Western blot(WB) and immunofluorescence(IF) were employed to determine the expression of tight junction proteins(ZO-1, occluding, and claudin-1) and matrix metalloproteinase 9(MMP-9) in the cortex. The results showed that the model group had obvious neurological deficit, obvious infarct in the right brain tissue, and significant increase in water content in brain tissue compared with the sham group. Compared with the model group, high-dose and low-dose salidroside groups showed decrease in neurobehavioral score, and the high-, medium-, and low-dose salidroside groups demonstrated obviously small infarct area and significant decrease in water content in brain tissue. The results of HE staining and transmission electron microscopy showed that rats had necrosis of neurons, damage of original physiological structure of endothelial cells, and disintegration of the tight junction between endothelial cells after I/R compared with the sham group. Compared with the model group, the high-dose and low-dose salidroside groups showed alleviation of neuron injury and intact physiological structure of endothelial cells. The model group had significantly lower serum level of NO, significantly higher plasma levels of vWF and fibrinogen(FIB), and significantly shorter thrombin time(TT) and prothrombin time(PT) than the sham group. Compared with model group, the high-dose and low-dose salidroside groups increased the serum content of NO in serum, decreased the plasma levels of FIB and vWF, and significantly prolonged TT and PT. WB and IF results showed that the model group had significantly lower levels of ZO-1, occluding, and claudin-1 among endothelial cells and significantly higher level of MMP-9 than the sham group. Compared with the model group, high-dose and low-dose salidroside significantly increased the levels of ZO-1, occluding, and claudin-1 in the cortex. The above experimental results show that salidroside has clear therapeutic effect on I/R rats and protects the brain. To be specific, it alleviates the damage of endothelial cells by increasing NO synthesis in endothelial cells, inhibiting coagulation reaction and MMP-9 expression, up-regulating the expression of ZO-1, occludin, and claudin-1, thereby protecting the brain.
Animals ; Rats ; Matrix Metalloproteinase 9/metabolism* ; Endothelial Cells/metabolism* ; Reperfusion Injury/metabolism* ; Blood-Brain Barrier ; Claudin-1/therapeutic use* ; von Willebrand Factor/therapeutic use* ; Rats, Sprague-Dawley ; Brain Ischemia/metabolism* ; Cerebral Infarction ; Reperfusion ; Water/metabolism*

Hypertension plays an important role in the pathogenesis of stroke,which,however,is only known at the blood pressure level.The relationship between circadian rhythm of blood pressure(especially the circadian rhythm disorder of blood pressure)and stroke has been a hot research topic.This article reviews the concept of circadian rhythm of blood pressure,classification of circadian rhythm disorder of blood pressure,and the relationship of circadian rhythm disorder of blood pressure with ischemic stroke.
Blood Pressure ; Brain Ischemia/complications* ; Chronobiology Disorders/complications* ; Circadian Rhythm ; Humans ; Hypertension/complications* ; Ischemic Stroke/complications*

OBJECTIVE: To evaluate the therapeutic effect of the combined treatment with balance acupuncture therapy and exercise re-learning rehabilitation therapy and the impact on serum cAMP and cGMP in the patients with hemiplegia of cerebral ischemic stroke. METHODS: A total of 90 patients of hemiplegia of cerebral ischemic stroke were randomized into an observation group and a control group, 45 cases in each one. All of the patients in the two groups received health education, diet guidance, routine symptomatic treatment as well as exercise re-learning rehabilitation therapy. Additionally, in the observation group, balance acupuncture therapy was applied, in which, the acupoints on the aspect of the human body, on the governor vessel and bladder meridian were adopted in the morning and those on the aspect of the human body, on the conception vessel and kidney meridian were stimulated in the afternoon. In the control group, the regular acupuncture was given. In the two groups, both acupuncture and rehabilitation therapies were given 5 days a week, 2 week-treatment as one course and totally 2 courses were required. Separately, before and after treatment, the score of Fugl-Meyer assessment (FMA) and the score of Chinese stroke scale (CSS) were recorded, the levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) detected in serum and the clinical therapeutic effect were evaluated in the two groups. RESULTS: After treatment, FMA score was increased in the patients of either of the groups as compared with that before treatment (<0.01) and CSS score decreased as compared with that before treatment (<0.01). After treatment, FMA score in the observation group was higher than that in the control group (<0.01) and CSS score was lower than the control group (<0.01). After treatment, the level of serum cAMP of the patients in either of the groups was increased as compared with that before treatment (<0.01) and that of cGMP decreased as compared with that before treatment (<0.01). After treatment, the level of cAMP in the observation group was higher than that in the control group (<0.01) and that of cGMP was lower than the control group (<0.01). The total effective rate was 93.3% (42/45) in the observation group, better than 73.3% (33/45) in the control group (<0.01). CONCLUSION The balance acupuncture therapy combined with exercise re-learning rehabilitation effectively improves the motor function of the affected limb, relieves injury and regulate the levels of serum cAMP and cGMP in the patients with hemiplegia of ischemic stroke.
Acupuncture Points ; Acupuncture Therapy ; Brain Ischemia ; therapy ; Cyclic AMP ; blood ; Cyclic GMP ; blood ; Hemiplegia ; therapy ; Humans ; Stroke ; therapy ; Stroke Rehabilitation ; Treatment Outcome

The complement cascade is a central component of innate immunity which plays a critical role in brain inflammation. Complement C3a receptor (C3aR) is a key mediator of post-ischemic cerebral injury, and pharmacological antagonism of the C3a receptor is neuroprotective in stroke. Cerebral ischemia injures brain endothelial cells, causing blood brain barrier (BBB) disruption which further exacerbates ischemic neuronal injury. In this study, we used an in vitro model of ischemia (oxygen glucose deprivation; OGD) to investigate the protective effect of a C3aR antagonist (C3aRA, SB290157) on brain endothelial cells (bEnd.3). Following 24 hours of reperfusion, OGD-induced cell death was assessed by TUNEL and Caspase-3 staining. Western blot and immunocytochemistry were utilized to demonstrate that OGD upregulates inflammatory, oxidative stress and antioxidant markers (ICAM-1, Cox-2, Nox-2 and MnSOD) in endothelial cells and that C3aRA treatment significantly attenuate these markers. We also found that C3aRA administration restored the expression level of the tight junction protein occludin in endothelial cells following OGD. Interestingly, OGD/reperfusion injury increased the phosphorylation of ERK1/2 and C3aR inhibition significantly reduced the activation of ERK suggesting that endothelial C3aR may act via ERK signaling. Furthermore, exogenous C3a administration stimulates these same inflammatory mechanisms both with and without OGD, and C3aRA suppresses these C3a-mediated responses, supporting an antagonist role for C3aRA. Based on these results, we conclude that C3aRA administration attenuates inflammation, oxidative stress, ERK activation, and protects brain endothelial cells following experimental brain ischemia.
Blood-Brain Barrier ; Blotting, Western ; Brain Ischemia ; Brain ; Caspase 3 ; Cell Death ; Complement C3a ; Complement System Proteins ; Encephalitis ; Endothelial Cells ; Glucose ; Immunity, Innate ; Immunohistochemistry ; In Situ Nick-End Labeling ; In Vitro Techniques ; Inflammation ; Ischemia ; Neurons ; Occludin ; Oxidative Stress ; Phosphorylation ; Reperfusion ; Stroke ; Tight Junctions