Objective To explore the central mechanism of acupuncture at Baihui (GV20) for memory impairment after stroke. Methods 32 stroke patients were randomized to receive acupuncture at Baihui (GV20) (as observation group) and acupuncture at Yanglingquan (GB34) (as control group) for 8 weeks. At the meantime, all participants received routine treatment including physical and occupational therapies. They were scanned with resting-state functional magnetic resonance imaging (fMRI) to find functional connectivity and assessed with Wechsler Memory Scale (WMS) before and after treatment. The results of WMS and functional connectivity were analyzed with Pearson's correlation. Results The memory scores and memory quotient improved significantly after treatment in the observation group (P<0.05). The functional connectivity significantly increased occurred in the left hippocampus to right inferior frontal gyrus, right middle frontal gyrus and left inferior frontal gyrus; and right hippocampus to left middle frontal gyrus, left superior frontal gyrus and left parietal lobe. Significant correlations were found between memory quotient and functional connectivity of hippocampus to frontal lobe and left parietal lobe in the observation group. There was no statistical significance in memory scores and memory quotient in the control group. The functional connectivity significantly decreased in left hippocampus to right middle occipital gyrus, and right hippocampus to right superior temporal gyrus and right posterior lobe of cerebellum. There was no statistical correlation between functional connectivity and results of WMS. Conclusion The acupuncture at Baihui can improves memory ability of stroke patients, which may associate with the increase of functional connectivity of hippocampus with frontal lobes and parietal lobes.

ObjectiveTo investigate the clinical efficacy of wrist-ankle acupuncture plus continuous exercise therapy for post-stroke shoulder pain.MethodsEighty patients with post-stroke shoulder pain were randomly allocated to treatment and control groups, 40 cases each. The control group received continuous exercise training with an upper limb multi-joint rehabilitation training instrument and the treatment group, wrist-ankle acupuncture in addition. The affected limb SEP N9 and N13 latency values, shoulder pain severity (the VAS score) and the upper limb motor function score were observed in the two groups before and after treatment. The clinical therapeutic effects were compared between the two groups.ResultsThe total efficacy rate was 90.0% in the treatment group and 75.0% in the control group; there was a statistically significant difference between the two groups (P<0.05). There was a statistically significant pre-/post-treatment difference in SEP N9-N13 wave interval on the affected side in the two groups (P<0.01). There was a statistically significant post-treatment difference in SEP N9-N13 wave interval on the affected side between the treatment and control groups (P<0.05). There were statistically significant pre-/post-treatment differences in the VAS score and the Fugl-Meyer score in the two groups (P<0.01). There were statistically significant post-treatment differences in the VAS score and the Fugl-Meyer score between the treatment and control groups (P<0.01).ConclusionsWrist-ankle acupuncture plus continuous exercise therapy is an effective way to treat post-stroke shoulder pain.

Objective To investigate the effects of high-calorie diet and age on brain function of ApoE-/-mice.Methods A total of 20 adult ApoE mice(8 months old)and elderly ApoE-/-mice(18 months old)were randomly divided into normal diet adult group,normal diet elderly group,high-calorie diet adult group,and high-calorie diet elderly group,with 5 animals in each group.The mice were fed with corresponding standard diet and high-fat diet for 8 weeks.Their body mass was monitored,and blood glucose was detected with glucose tolerance test.The relative contents of NAA and Cho in the hippocampus and hypothalamus were detected by Magnetic resonance spectroscopy.Y-Maze and open field tests were performed to detect cognitive function,and West-ern blotting was applied to detect the expression of synaptic associated protein 25(SNAP-25),synaptophysin,postsynaptic dense protein-95(PSD-95),iNOS and IL-1β.Results Compared with the normal diet adult group,the NAA content in the hippocampus,Cho and NAA contents in the hypothalamus,spontaneous alternation rate,and expression levels of SNAP-25,synaptophysin and PSD-95 in brain tissue(P＜0.05,P＜0.01)were decreased,and the expression of iNOS and IL-1β(P＜0.01)was increased in the high-calorie diet adult group.The normal diet elderly group had reduced contents of NAA in the hippocampus and Cho in the hypothalamus,and expression levels of SNAP-25,synaptophysin and PSD-95(P＜0.05,P＜0.01),and elevated expression of iNOS and IL-1β(P＜0.01)when compared with the normal diet adult group.Compared with the normal diet elderly group,high-calorie diet resulted in decreased Cho and NAA in both hippocampus and hy-pothalamus,central distance/total distance and down-regulation of SNAP-25,synaptophysin and PSD-95(P＜0.05,P＜0.01),and enhanced expression of iNOS and IL-1β(P＜0.01)in the elderly mice.Compared with the high-calorie diet adult group,the high-calorie diet elderly group had reduced NAA in hippocampus,central distance/total distance and average speed,and decreased expression of synaptophysin(P＜0.05,P＜0.01),and increased expression of iNOS and IL-1β(1.61±0.10 vs 1.35±0.13,2.04±0.08 vs 1.54±0.11,P＜0.05,P＜0.01).Conclusion High-calorie diet results in metabolic disorders and neuroinflammation,inhibits the expression of syn-aptic proteins,and thus leads to cognitive dysfunction in ApoE mice.Long-term high-calorie diet and ageing promote the decline of brain function in ApoE mice.

Objective:To investigate the role and mechanism of astrocyte (AS) derived exosomes in protecting brain microvascular endothelial cells (bEnd. 3) from cerebral ischemia reperfusion injury by establishing an oxygen-glucose deprivation/re-oxygenation (OGD/R) model in vitro. Methods:(1) Dual-luciferase reporter gene assay was used to confirm the regulating effect of miR-193b-3p on transient receptor potential cation channel, subfamily M, member 2 (TRPM2). OGD/R model was established by OGD 8 h followed by reoxygenation 24 h in bEnd. 3 cells after being transfected miR-193b-3p mimics/negative sequence (OGD/R+miR-193b-3p mimics group or OGD/R+miR-193b-3p negative sequence group); real-time quantitative PCR (RT-qPCR) was used to detect the miR-193b-3p expression, Western blotting (WB) was used to detect the expressions of TRPM2, cleaved caspase-3, Bax, Bcl-2, ZO-1 and Claudin-5, and flow cytometry was used to detect the apoptosis. (2) AS was extracted from the cerebral cortex of C57BL/6 suckling mice and identified; modeling time was determined by CCK-8 and AS-derived exosomes were extracted by ultracentrifugation from cell supernatant and identified by electron microscopy, particle size analysis, and WB for marker proteins. RT-qPCR was used to detect the miR-193b-3p expression in AS and AS-derived exosomes. Low-expressed miR-193b-3p exosomes were extracted from AS after being transfected miR-193b-3p inhibitory sequence and co-incubated with OGD/R bEnd. 3 cells (group of OGD/R+AS-derived inhibitory sequence exosomes); exosomes were extracted from AS transfecting with miR-193b-3p negative sequence, and co-incubated with OGD/R bEnd.3 cells (group of OGD/R+AS-derived negative sequence exosomes); and normal exosomes were co-incubated with OGD/R bEnd. 3 cells (group of OGD/R+AS-derived exosomes). The miR-193b-3p expression in these 3 groups was detected by RT-qPCR, expressions of TRPM2, cleaved-caspase-3, Bax, Bcl-2, ZO-1, and Claudin-5 were detected by WB, cell apoptosis was detected by flow cytometry, and cell migration ability was detected by scratch test.Results:(1) Dual-luciferase reporter gene assay showed that miR-193b-3p could bind to TRPM2 mRNA. The miR-193b-3p can improve the TRPM2-mediated apoptosis and tight junction reduction in bEnd.3 cells during OGD/R: compared with OGD/R group, OGD/R+miR-193b-3p mimics group had significantly decreased TRPM2, cleaved-caspase-3, and Bax protein expressions, and statistically increased Bcl-2, ZO-1 and Claudin-5 protein expressions ( P<0.05). Flow cytometry further verified the above results: compared with OGD/R group, OGD/R+miR-193b-3p mimics group had significantly decreased cell apoptosis rate (21.34% vs. 13.93%, P<0.05). (2) The extracted exosomes exhibited lipid bilayer cup-like structure with particle size of 126.5 nm (exosome marker proteins: negative Cal and positive CD81 and SG101), indicating successful exosome extraction. After modeling, miR-193b-3p expression in AS and AS-derived exosomes was significantly decreased compared with that in the Control goup ( P<0.05). Compared with the OGD/R group, the group of OGD/R+AS-derived exosomes and group of OGD/R+AS-derived negative sequence exosomes had significantly increased miR-193b-3p expression, statistically decreased TRPM2, cleaved-caspase-3 and Bax protein expressions, and significantly increased ZO-1, Claudin-5 and Bcl-2 expressions ( P<0.05); while compared with those in the OGD/R group, no significant changes in the above protein expressions in group of OGD/R+AS-derived inhibitory sequence exosomes were noted ( P>0.05). Compared with the OGD/R group (18.22%), group of OGD/R+AS-derived exosomes and group of OGD/R+AS-derived negative sequence exosomes had significantly decreased apoptosis rate (14.09% and 13.79%, P<0.05), while group of OGD/R+AS-derived inhibitory sequence exosomes had no significant change (18.41%, P>0.05). Compared with the OGD/R group (13.55%), group of OGD/R+AS-derived exosomes and group of OGD/R+AS-derived negative sequence exosomes had significantly increased migration ability (43.01% and 40.59%, P<0.05), while group of OGD/R+AS-derived inhibitory sequence exosomes had no significant change (16.26%, P>0.05). Conclusion:AS-derived exosomes can suppress TRPM2 protein expression in brain microvascular endothelial cells distantly by miR-193b-3p to improve the brain microvascular endothelial cell injury caused by OGD/R, and then improve OGD/R injury.

Objective:To investigate the mechanism of learning and memory impairment of apolipoprotein E -/- ( ApoE-/-) mice after transient global cerebral ischemia by diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS). Methods:Ten-week-old C57BL/6(WT) mice and ApoE-/- mice were randomly divided into WT sham-operated group ( n=8), WT 7 d group ( n=12) and WT 30 d group ( n=12), and ApoE-/- sham-operated ( n=8), ApoE-/- 7 d group ( n=12) and ApoE-/- 30 d group ( n=12). The mice in the WT 7 d group, WT 30 d group, ApoE-/- 7 d group, and ApoE-/- 30 d group received bilateral common carotid artery ischemia-reperfusion injury, while mice in the WT sham-operated group and ApoE sham-operated group only stripped the blood vessels without ligation. On the 7 th and 30 th d of modeling, Morrris water maze test was employed to detect the learning and memory abilities of these mice; DTI was used to detect the fractional anisotropy (FA) values in the bilateral hippocampus of mice, and MRS was used to detect the contents of choline complex (Cho) and N-acetylaspartate (NAA) in the bilateral hippocampus of mice. Results:(1) On 2 nd, 3 rd, and 4 th d of water maze experiment, the escape latency of mice in ApoE-/- 7 d group was significantly prolonged as compared with that in the ApoE-/- sham-operated group and WT 7 d group ( P<0.05); since the 3 rd d of water maze experiment, the escape latency of mice in ApoE-/- 30 d group was significantly prolonged as compared with that in WT 30 d group ( P<0.05). The number of times crossing platform in ApoE-/- 7 d group was significantly smaller than that in WT 7 d group, and the residence time in the third quadrant was significantly shorter ( P<0.05); the number of times crossing platform in ApoE-/- 30 d group were significantly smaller as compared with that in the WT 30 d group, and the residence time in the third quadrant was significantly shortened ( P<0.05). (2) DTI results showed that there was no significant difference in bilateral hippocampal FA values between ApoE-/- 7 d group and WT 7 d group ( P>0.05); the bilateral hippocampal FA values of mice in the ApoE-/- 30 d group were statistically lower than those in WT 30 d group ( P<0.05). (3) MRS results showed that the relative contents of hippocampal Cho and NAA in the ApoE-/- 7 d group were significantly lower than those in the WT 7 d group, and the relative contents of hippocampal Cho and NAA in the ApoE-/- 30 d group were significantly lower than those in the WT 30 d group ( P<0.05). Conclusion:ApoE-/- mice have poor learning and memory abilities after transient global cerebral ischemic injury, whose mechanism is closely related to the damage of hippocampal white matter fibers and abnormal metabolism of nerve cells.

Objective To investigate the association of silent information regulator 1 (SIRT1) polymorphisms with intracerebral hemorrhage (ICH) susceptibility.Methods From September 1,2013 to May 30,2017,Han ethnic 201 ICH patients and 203 controls from South China were enrolled in this study.Genotyping and sequencing ofSIRT1 polymorphisms (rs7069102,rs2273773 and rs7895833) were performed by PCR-restriction fragment length polymorphism (PCR-RFLP).The correlation of SIRT1 polymorphisms with ICH was analyzed.Results (1) The rs7895833 A allele frequency distribution was significantly higher and the rs7895833 GG+AG gene frequency distribution was significantly lower in the ICH group than those in the control group (P＜0.05);the rs7069102 C allele frequency distribution was lower and the GG+CG gene frequency distribution was higher in the ICH group than those in the control group,without significant differences (P＞0.05).(2) Logistic regression analysis indicated rs7895833 AA genotype carriers had increased risk for ICH (OR:1.57,95％CI:1.14-2.18,P=0.006).(3) As compared with patients with rs2273773 TT genotype,patients with CC and CT genotypes had significantly higher high-density lipoprotein cholesterol level (P＜0.05);there were no associations between rs2273773/rs7069102 and ICH.Conclusion SIRT1 rs7895833 is significantly associated with ICH susceptibility;rs2273773 genotypes affect plasma high-density lipoprotein cholesterol level in the Chinese Han ethnic population.

ObjectiveTo investigate the mechanism of Zexie Decoction （泽泻汤） in inhibiting neuroinflammation and improving cognitive impairment mediated by high-calorie diet. MethodsTwenty seven C57BL/J mice were randomly divided into control group （n = 9）， model group （n = 9） and Zexie Decoction group （n = 9）. The mice in the model group and the Zexie Decoction group were fed with high-calorie diet to establish the model of cognitive impairment. Meanwhile， the mice in Zexie Decoction group were also fed with 0.36 g/（kg·d）Zexie Decoction， and the mice in the control group and model group were fed with the same volume of normal saline for 8 weeks. The body weight of mice was recorded at the same time every week； after intervention， oral glucose tolerance test （OGTT） and insulin tolerance test（ITT） commenced； the cognitive level of mice was detected by Morris water maze， open field test， new object recognition test and Y maze； magnetic resonance spectroscopy （MRS） was used to detect the expression of N-acetylaspartate （NAA）， choline （CHO）， lactic acid （Lac）， creatine （Cr）， lipid （Lip）， and myoInositol （mI） in left hippocampus， hypothalamus and cortex. Western blotting was used to detect the expression of synaptophysin （SYN）， synaptosome associated protein-25 （SNAP-25）， postsynaptic dense protein-95 （PSD-95）， tumor necrosis factor-α （TNF-α）， nuclear factor kappa B （NF-κB p65） and its phosphorylated form （P-NF-B p65） in mouse brain； Nissl's staining was used to detect the morphological changes of hippocampal neurons. ResultsCompared with the control group， body mass， blood glucose in oral glucose tolerance test， and blood glucose in insulin tolerance test increased in the model group； in the Morris water maze experiment， the total distance travelled and escape latency of the model group mice increased， the time spent in the platform area and the number of times traversing the platform decreased on days 3 and 4； in the open-field experiment， the number of times the model group mice entered the central area， the ratio of the time in the central area to the total time， and the ratio of the distance travelled in the central area to the total distance significantly decreased； in the new object recognition test， the frequency of new object recognition and recognition index were significantly lower in the model group mice； in the Y-maze test， the spontaneous alternation rate of mice in the model group was significantly lower （P＜0.05 or P＜0.01）； in the left hippocampus， hypothalamus， and cortex of mice in the model group， the CHO/Cr， NAA/Cr significantly decreased， and the mI/Cr， Lac/Cr and Lip/Cr significantly increased； SYN/β-actin， SNAP-25/β-actin and PSD-95/β-actin values significantly decreased， and p-NF-κB p65/NF-κB p65 and TNF-α/β-actin values significantly increased in brain tissue （P＜0.05 or P＜0.01）. Compared with the model group， the above indexes of mice in the Zexie Decoction group significantly improved （P＜0.05 or P＜0.01）. The results of Nissl staining showed that compared with the control group， the neurons in the dentate gyrus of the hippocampus in the model group were scattered and sparsely arranged， the density was significantly reduced， the nuclei of the cells had consolidation and shrinkage， the number of Nissl vesicles was reduced， and the staining became lighter； compared with the model group， the density of neurons in the hippocampal dentate gyrus region of the Zexie Decoction group increased， the wrinkling of nuclei improved， the cell gap narrowed， and the arrangement was slightly tight. Concusion The ameliorative effect of Zexie Decoction on cognitive function in mice with high-calorie diet-induced cognitive impairment may relate to the restructuring of glucose metabolism homeostasis， inhibition of neuroinflammation， reduction of neuronal damage， and enhancement of synaptic plasticity.

ObjectiveTo study the effect of Wulingsan on cerebral edema after intracerebral hemorrhage （ICH） in mice and explore the treatment mechanism. MethodsThe mouse model of ICH was established by injection of collagenase into the caudate nucleus. Mice were randomly assigned into the following groups： sham， ICH， intervention before modeling with low-dose and high-dose （3.69， 11.07 g·kg^-1， respectively） Wulingsan， and intervention after modeling with high-dose Wulingsan. The modified neurological severity score （mNSS） was recorded， and the small animal MRI T2 sequential scanning was performed to measure the volume of cerebral hemorrhage after the modeling of ICH in each group. The Y-maze test， open field test， and Morris water maze test were conducted to evaluate the neurological behaviors of mice in each group. Hematoxylin-eosin staining was employed to observe the pathological changes in the brain tissue. Immunohistochemistry was employed to observe the expression of aquaporin 4 （AQP4）， neuronal nuclei （NeuN）， and glial fibrillary acidic protein （GFAP） in the brain tissue. Western blot was employed to determine the protein levels of AQP4， Claudin-5， and zonula occludens-1 （ZO-1） in the hematoma area. ResultsCompared with the sham group， the ICH group showed increases in the mNSS， the cerebral hemorrhage volume， and the escape latency in the Morris water maze test （P<0.01）， decreases in the times of touching the platform and times of entering the quadrant where the platform was located in the Morris water maze test， and reductions in the spontaneous alternation rate in the Y-maze test and the ratio of distance of center travel to total travel distance in the open field test （P<0.01）. Moreover， pathological changes such as cell disarrangement， cell space enlargement， and cell swelling were observed in the ICH group. Immunohistochemistry results showed that the ICH group had higher proportions of AQP4- and GFAP-positive cells and lower proportion of NeuN-positive cells than the sham group （P<0.01）. Compared with the sham group， the ICH group showed an up-regulated protein level of AQP4 and down-regulated protein levels of Claudin-5 and ZO-1 （P<0.01）. Compared with the ICH group， the intervention with Wulingsan decreased the mNSS， the volume of cerebral hemorrhage， and the escape latency in the Morris water maze test （P<0.05， P<0.01）， while increasing the times of touching the platform and times of entering the quadrant where the platform was located in the Morris water maze test， the spontaneous alternation rate in the Y-maze test， and the ratio of distance of center travel to total travel distance in the open field test （P<0.05， P<0.01）. Furthermore， the intervention with Wulingsan alleviated the pathological changes in the brain tissue after ICH， decreased the proportion of AQP4- and GFAP-positive cells （P<0.01）， increased the proportion of NeuN-positive cells （P<0.01）， down-regulated the protein level of AQP4 （P<0.01）， and up-regulated the protein levels of Claudin-5 and ZO-1 （P<0.01）. ConclusionThe intervention with Wulingsan could reduce the neural function score and the cerebral hemorrhage volume， up-regulate the expression of Claudin-5 and ZO-1， and down-regulate the expression of AQP4 to ameliorate the neurological function defect and cerebral edema after ICH， thereby protecting the brain.