OBJECTIVE: To observe the effects of electro-scalp acupuncture （ESA） on the expression of microglial markers CD206 and CD32, as well as interleukin (IL)-6, IL-1β, and IL-10 in the ischemic cortex of rats with ischemic stroke, and to explore the mechanisms of ESA on alleviating inflammatory damage of ischemic stroke. METHODS: Sixty 7-week-old male SD rats were randomly selected, with 15 rats assigned to a sham surgery group. The remaining rats were treated with suture method to establish rat model of middle cerebral artery occlusion (MCAO). The rats with successful model were randomly divided into a model group, a VitD₃ group, and an ESA group, with 15 rats in each group. In the ESA group, ESA was performed bilaterally at the "top-temporal anterior oblique line" with disperse-dense wave, a frequency of 2 Hz/100 Hz, and an intensity of 1 mA. Each session lasted for 30 min, once daily, for a total of 7 days. The VitD₃ group were treated with intragastric administration of 1,25-dihydroxyvitamin D₃ (1,25-VitD₃) solution (3 ng/100 g), once daily for 7 days. The neurological deficit scores and neurobehavioral scores were assessed before and after the intervention. After the intervention, the brain infarct volume was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Immunofluorescence double staining was performed to detect the protein expression of CD32 and CD206 in the ischemic cortex. Western blot analysis was conducted to measure the protein expression of IL-6, IL-1β, and IL-10 in the ischemic cortex. RESULTS: Compared with the sham surgery group, the model group showed increased neurological deficit scores and neurobehavioral scores (P<0.01), increased brain infarct volume (P<0.01), increased protein expression of CD32, IL-6, and IL-1β in the ischemic cortex (P<0.01), and decreased protein expression of CD206 and IL-10 in the ischemic cortex (P<0.01). Compared with the model group, both the ESA group and the VitD₃ group showed decreased neurological deficit scores and neurobehavioral scores (P<0.01), reduced brain infarct volume (P<0.01), decreased protein expression of CD32, IL-6, and IL-1β in the ischemic cortex (P<0.01), and increased protein expression of CD206 and IL-10 in the ischemic cortex (P<0.01). Compared with the VitD₃ group, the ESA group had lower neurological deficit score (P<0.05), larger brain infarct volume (P< 0.05), and lower protein expression of CD32, CD206, IL-1β, and IL-10 in the ischemic cortex (P<0.01, P<0.05). CONCLUSION ESA could improve neurological function in MCAO rats, and its mechanism may be related to promoting microglial M1-to-M2 polarization and alleviating inflammatory damage.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; Ischemic Stroke ; Interleukin-10 ; Interleukin-6/genetics* ; Microglia ; Scalp ; Acupuncture Therapy ; Vitamins ; Infarction, Middle Cerebral Artery

This study aims to explore the pharmacodynamic effect of baicalin on rat brain edema induced by cerebral ischemia reperfusion injury and discuss the mechanism from the perspective of inhibiting astrocyte swelling, which is expected to serve as a refe-rence for the treatment of cerebral ischemia with Chinese medicine. To be specific, middle cerebral artery occlusion(suture method) was used to induce cerebral ischemia in rats. Rats were randomized into normal group, model group, high-dose baicalin(20 mg·kg~(-1)) group, and low-dose baicalin(10 mg·kg~(-1)) group. The neurobehavior, brain index, brain water content, and cerebral infarction area of rats were measured 6 h and 24 h after cerebral ischemia. Brain slices were stained with hematoxylin and eosin(HE) for the observation of pathological morphology of cerebral cortex after baicalin treatment. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the content of total L-glutathione(GSH) and glutamic acid(Glu) in brain tissue, Western blot to measure the content of glial fibrillary acidic protein(GFAP), aquaporin-4(AQP4), and transient receptor potential vanilloid type 4(TRPV4), and immunohistochemical staining to observe the expression of GFAP. The low-dose baicalin was used for exploring the mechanism. The experimental results showed that the neurobehavioral scores(6 h and 24 h of cerebral ischemia), brain water content, and cerebral infarction area of the model group were increased, and both high-dose and low-dose baicalin can lower the above three indexes. The content of GSH dropped but the content of Glu raised in brain tissue of rats in the model group. Low-dose baicalin can elevate the content of GSH and lower the content of Glu. According to the immunohistochemical staining result, the model group demonstrated the increase in GFAP expression, and swelling and proliferation of astrocytes, and the low-dose baicalin can significantly improve this situation. The results of Western blot showed that the expression of GFAP, TRPV4, and AQP4 in the cerebral cortex of the model group increased, and the low-dose baicalin reduce their expression. The cerebral cortex of rats in the model group was severely damaged, and the low-dose baicalin can significantly alleviate the damage. The above results indicate that baicalin can effectively relieve the brain edema caused by cerebral ischemia reperfusion injury in rats, possibly by suppressing astrocyte swelling and TRPV4 and AQP4.
Animals ; Aquaporin 4/genetics* ; Astrocytes ; Brain Edema/drug therapy* ; Brain Ischemia/metabolism* ; Flavonoids ; Infarction, Middle Cerebral Artery/drug therapy* ; Rats ; Rats, Sprague-Dawley ; Reperfusion ; TRPV Cation Channels/therapeutic use*

OBJECTIVE: To investigate the role of long non-coding RNA ZEB1-AS1 in cerebral ischemia/reperfusion injury (CI/RI). METHODS: We detected the temporal changes of ZEB1-AS1 and HMGB1 expression using qPCR and Western blotting in SD rats following CI/RI induced by middle cerebral artery occlusion (MCAO). The rat models of CI/RI were subjected to injections of vectors for ZEB1-AS1 overexpression or knockdown into the lateral ventricle, and the changes in cognitive function, brain water content, blood-brain barrier integrity, and IL-1β and TNF-α levels in the cerebrospinal fluid (CSF) and serum were observed. Neuronal loss and cell apoptosis in the cortex of the rat models were detected by FJC and TUNEL methods, and HMGB1 and TLR-4 expressions were analyzed with Western blotting. We also examined the effects of ZEB1-AS1 knockdown on apoptosis and expressions of HMGB1 and TLR-4 in SH-SY5Y cells with oxygen-glucose deprivation/reoxygenation (OGD/R). RESULTS: In CI/RI rats, the expressions of ZEB1-AS1 and HMGB1 in the brain tissue increased progressively with the extension of reperfusion time, reaching the peak levels at 24 h followed by a gradual decline. ZEB1-AS1 overexpression significantly aggravated icognitive impairment and increased brain water content, albumin content in the CSF, and IL-1β and TNF-α levels in the CSF and serum in CI/RI rats (P < 0.05), while ZEB1-AS1 knockdown produced the opposite effects (P < 0.05 or 0.01). ZEB1-AS1 overexpression obviously increased the number of FJC-positive neurons in the cortex and enhanced the expressions of HMGB1 and TLR-4 in the rat models (P < 0.01); ZEB1-AS1 knockdown significantly reduced the number of FJC-positive neurons and lowered HMGB1 and TLR-4 expressions (P < 0.01). In SH-SY5Y cells with OGD/R, ZEB1-AS1 knockdown significantly suppressed cell apoptosis and lowered the expressions of HMGB1 and TLR-4 (P < 0.01). CONCLUSION ZEB1-AS1 overexpression aggravates CI/RI in rats through the HMGB1/TLR-4 signaling axis.
Animals ; Cell Line, Tumor ; Cell Proliferation/genetics* ; HMGB1 Protein/metabolism* ; Humans ; Infarction, Middle Cerebral Artery ; Neuroblastoma ; RNA, Long Noncoding/metabolism* ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha ; Water

To investigate the effects of salt-inducible kinase 2 (SIK2) on energy metabolism in rats with cerebral ischemia-reperfusion. Adult SD male rats were divided into 5 groups: sham group, ischemia group, reperfusion group, adenovirus no-load group, and SIK2 overexpression group with 5 animals in each group. The middle cerebral artery occlusion (MCAO) was induced with the modified Zea-Longa line thrombus method to establish the cerebral ischemia reperfusion model. Eight days before the MCAO, SIK2 overexpression was induced by injecting 7 μL adenovirus in the right ventricle, then MCAO was performed for followed by reperfusion HE staining was used to observe the pathological changes of cerebral tissue in rats; TTC staining was used to observe the volume of cerebral infarct. The levels of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) in rat brain tissue were detected by ELISA; the levels of SIK2 and hypoxia-inducible factor 1α (HIF-1α) in the rat brain tissues were detected by RT-qPCR and Western blotting. Compared with the sham group, SIK2 level was decreased in the ischemia group, and it was further declined in the reperfusion group (<0.05). Compared with the sham group and ischemic group, the pathological injury in reperfusion group were more severe, and the infarct size was larger; compared with the reperfusion group and adenovirus no-load group, the pathological injury of the SIK2 overexpression group was milder, and the infarct size is less. Compared with the sharn group, HIF-1α was increased in both ischemia group and reperfusion group, especially in ischemia group (all <0.05); HIF-1α level in the SIK2 overexpression group was higher than that in the reperfusion group and adenovirus no-load group (all <0.05). ATP level in ischemia group and reperfusion group was lower than that in the sham group, and the reperfusion group decreased more significantly than the ischemia group (<0.05); ADP content was increased in the ischemia and reperfusion group, and the ADP content in reperfusion group was significantly higher than that in the ischemia group (<0.05). ATP level in the SIK2 overexpression group was higher than that in the reperfusion group and adenovirus no-load group (all <0.05), and ADP was decreased in the SIK2 overexpression group (all <0.05). SIK2 can up-regulate the ATP level and down-regulate the ADP level in rat brain tissue and alleviate cerebral ischemia-reperfusion injury by increase the level of HIF-1α.
Animals ; Brain Ischemia ; Energy Metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Infarction, Middle Cerebral Artery ; Male ; Protein-Serine-Threonine Kinases ; Rats ; Rats, Sprague-Dawley ; Reperfusion ; Reperfusion Injury

Danshen-Chuanxiongqin Injection (DCI) is a commonly used traditional Chinese medicine for the treatment of cerebral ischemic stroke in China. However, its underlying mechanisms remain completely understood. The current study was designed to explore the protective mechanisms of DCI against cerebral ischemic stroke through integrating whole-transcriptome sequencing coupled with network pharmacology analysis. First, using a mouse model of cerebral ischemic stroke by transient middle cerebral artery occlusion (tMCAO), we found that DCI (4.10 mL·kg
Brain Ischemia/genetics* ; Drugs, Chinese Herbal ; Humans ; Infarction, Middle Cerebral Artery/genetics* ; Ischemic Stroke ; Myeloid Differentiation Factor 88/genetics* ; NF-kappa B/metabolism* ; Stroke/genetics* ; Toll-Like Receptor 2 ; Toll-Like Receptor 4/metabolism*

BACKGROUNDGinsenoside Rd (GSRd), one of the main active ingredients in traditional Chinese herbal Panax ginseng, has been found to have therapeutic effects on ischemic stroke. However, the molecular mechanisms of GSRd's neuroprotective function remain unclear. Ischemic stroke-induced oxidative stress results in DNA damage, which triggers cell death and contributes to poor prognosis. Oxidative DNA damage is primarily processed by the base excision repair (BER) pathway. Three of the five major DNA glycosylases that initiate the BER pathway in the event of DNA damage from oxidation are the endonuclease VIII-like (NEIL) proteins. This study aimed to investigate the effect of GSRd on the expression of DNA glycosylases NEILs in a rat model of focal cerebral ischemia.

METHODSNEIL expression patterns were evaluated by quantitative real-time polymerase chain reaction in both normal and middle cerebral artery occlusion (MCAO) rat models. Survival rate and Zea-Longa neurological scores were used to assess the effect of GSRd administration on MCAO rats. Mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damages were evaluated by the way of real-time analysis of mutation frequency. NEIL expressions were measured in both messenger RNA (mRNA) and protein levels by quantitative polymerase chain reaction and Western blotting analysis. Apoptosis level was quantitated by the expression of cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay.

RESULTSWe found that GSRd administration reduced mtDNA and nDNA damages, which contributed to an improvement in survival rate and neurological function; significantly up-regulated NEIL1 and NEIL3 expressions in both mRNA and protein levels of MCAO rats; and reduced cell apoptosis and the expression of cleaved caspase-3 in rats at 7 days after MCAO.

CONCLUSIONSOur results indicated that the neuroprotective function of GSRd for acute ischemic stroke might be partially explained by the up-regulation of NEIL1 and NEIL3 expressions.

Animals ; Blotting, Western ; Brain Ischemia ; drug therapy ; enzymology ; DNA Damage ; drug effects ; DNA Glycosylases ; genetics ; metabolism ; Ginsenosides ; therapeutic use ; Infarction, Middle Cerebral Artery ; drug therapy ; enzymology ; Male ; N-Glycosyl Hydrolases ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

To study the protective effect of Danqi Piantan capsule ( DPC) and its antelope horn substitution (DPCAS) on the cerebral ischemia, in order to preliminary study the possibility of replacing antelope horn with artificial bezoar. In this study, the left middle cerebral artery occlusion (MCAO) was adopted. Totally 150 SD rats were randomly divided into 5 groups: the sham operation group, the model group, the Danqi Piantan capsule (DPC) group (0.246 g x kg(-1) x d(-1)), the Danqi Piantan capsule without antelope horn (DPCRA) group (0.246 g x kg(-1) x d(-1)), the Danqi Piantan capsule without antelope horn and with double artificial bezoar (DPCDB) group (0.246 g x kg(-1) x d(-1)). The MCAO model was prepared 1 h later after the administration on the 5th day. At 24 h after the operation, the inner canthus blood was collected to determine the serum superoxide dismutase (SOD) activity and the endothelin (ET) content. At 72 h after the operation, the cerebral infarct size and the cerebral index were determined by TTC-staining. The fluorescent quantitative PCR method was used to detect brain Bcl-2, Caspase-3, IL-1β, P-selectin, E-selectin, ICAM-1 mRNA expressions. The mmunohistochemical method was used to detect ICAM-1, IL-1β, TNF-α, IL-6 expressions in ischemic penumbra. According to the results, compared with the model group, DPCDB and DPC groups showed almost consistent results, indicating both of the two group can significantly improved cerebral infarction index and cerebral index (P < 0.05), increase the serum SOD activity (P < 0.05), decrease the serum ET level and Caspase-3 expression, IL-1β, P-selectin, E-selectin, ICAM-1 mRNA expressions in brain tissues (P < 0.05) and expressions of ICAM-1, IL-1,6, TNF-α, IL-6 positive cells in ischemic penumbra (P < 0.05) and increase the Bcl-2 expression (P < 0.05). The DPCRA group showed much lower impacts on indexes than DPCDB and DPC groups. This suggests that DPCDB and DPC reveal similar efficacies and antelope horn in Danqi Piantan capsule can be substitutes by artificial bezoar.
Animals ; Antelopes ; Bile ; chemistry ; Biological Factors ; administration & dosage ; chemical synthesis ; chemistry ; Brain ; drug effects ; metabolism ; Caspase 3 ; genetics ; metabolism ; Drug Compounding ; Horns ; chemistry ; Humans ; Infarction, Middle Cerebral Artery ; drug therapy ; genetics ; metabolism ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Interleukin-1beta ; genetics ; metabolism ; Male ; Medicine, Chinese Traditional ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; blood ; genetics ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo study optical intrinsic signal (OIS) imaging of peri-infarct depolarizations (PIDs) in mice and investigate the influence of knockout of glial fibrillary acidic protein and vimentin on PIDs.

METHODSGFAP(⁺/⁺)Vim(⁺/⁺) mice and GFAP(⁺/⁺)Vim(⁺/⁺) mice were subjected to MCAO by standard intraluminal filament method. The main characteristics of PIDs in 4 h were studied by 4-wavelength OIS imaging technique.

RESULTSPIDs were identified as consistent, red and blue interaction waves in the cortical reflectance that slowly propagated peripherally from the origin site. There were 5 patterns of PID propagation, namely rostro-caudal, latero-medial, caudo-rostral, contralateral and medial-lateral. No significant differences were found in PID frequency, propagation patterns, velocity or duration time between the two groups (P>0.05).

CONCLUSIONThe 4-wavelength OIS system allows acquisition of high temporal-spatial resolution color images for analyzing temporal-spatial characteristics of PIDs in detail. Knockout of GFAP and vimentin do not affect PIDs in 4 h following middle cerebral artery occlusion.

Animals ; Glial Fibrillary Acidic Protein ; Infarction, Middle Cerebral Artery ; pathology ; Mice ; Mice, Knockout ; Nerve Tissue Proteins ; genetics ; Optical Imaging ; Vimentin ; genetics

OBJECTIVETo discuss the protective effect of Mailuoning injection on ischemia/reperfusion (I/R) injury in rats and its mechanism.

METHODHealthy male adult Sprague-Dawley (SD) rats were randomly divided into the sham operation group, the model group, the edaravone (3 mg x kg(-1)) control group, and Mailuoning high, middle and low-dose groups (4, 2, 1 mL x kg(-1)), with 10 rats in each group, and administered with drugs through tail intravenous injection. The middle cerebral artery occlusion (MCAO) was adopted to establish the rat ischemia/reperfusion model. After the ischemia for 2 h and reperfusion for 24 h, the pathological changes in neurovascular units (NVU) of brain tissues at the ischemia side was observed by HE staining. The expressions of glialfibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Ibal) were detected by the immunohistochemical method. The expressions of tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were detected by the western blotting technique.

RESULTMailuoning injection could significantly improve the pathological changes in cortical penumbra brain tissue UVN of (I/R) rats, reduce the number of GFAP and Ibal positive cells, and significantly decrease the expressions of TNF-alpha, IL-1beta, VCAM-1 and ICAM-1 of brain tissues of I/R rats.

CONCLUSIONMailuoning injection shows an obvious protective effect on UVN of I/R rats. Its mechanism may involve the inhibition of the activation of astrocyte and microglia and the secretion and expression of various inflammatory factors.

Animals ; Brain ; drug effects ; metabolism ; Brain Ischemia ; surgery ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Infarction, Middle Cerebral Artery ; genetics ; metabolism ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Male ; Protective Agents ; administration & dosage ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; genetics ; metabolism ; prevention & control ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; Vascular Cell Adhesion Molecule-1 ; genetics ; metabolism

Neural stem cells (NSCs) have been suggested as a groundbreaking solution for stroke patients because they have the potential for self-renewal and differentiation into neurons. The differentiation of NSCs into neurons is integral for increasing the therapeutic efficiency of NSCs during inflammation. Apoptosis signal-regulating kinase 1 (ASK1) is preferentially activated by oxidative stress and inflammation, which is the fundamental pathology of brain damage in stroke. ASK1 may be involved in the early inflammation response after stroke and may be related to the differentiation of NSCs because of the relationship between ASK1 and the p38 mitogen-activated protein kinase pathway. Therefore, we investigated whether ASK1 is linked to the differentiation of NSCs under the context of inflammation. On the basis of the results of a microarray analysis, we performed the following experiments: western blot analysis to confirm ASK1, DCX, MAP2, phospho-p38 expression; fluorescence-activated cell sorting assay to estimate cell death; and immunocytochemistry to visualize and confirm the differentiation of cells in brain tissue. Neurosphere size and cell survival were highly maintained in ASK1-suppressed, lipopolysaccharide (LPS)-treated brains compared with only LPS-treated brains. The number of positive cells for MAP2, a neuronal marker, was lower in the ASK1-suppressed group than in the control group. According to our microarray data, phospho-p38 expression was inversely linked to ASK1 suppression, and our immunohistochemistry data showed that slight upregulation of ASK1 by LPS promoted the differentiation of endogenous, neuronal stem cells into neurons, but highly increased ASK1 levels after cerebral ischemic damage led to high levels of cell death. We conclude that ASK1 is regulated in response to the early inflammation phase and regulates the differentiation of NSCs after inflammatory-inducing events, such as ischemic stroke.
Animals ; Cell Death ; Infarction, Middle Cerebral Artery/*metabolism ; Lipopolysaccharides/pharmacology ; MAP Kinase Kinase Kinase 5/genetics/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Microtubule-Associated Proteins/genetics/metabolism ; Neural Stem Cells/cytology/drug effects/*metabolism ; *Neurogenesis ; Neuropeptides/genetics/metabolism ; p38 Mitogen-Activated Protein Kinases/genetics/metabolism