Objective To explore the mechanism of lncRNA-BC200 (BC200) targeting the ubiquitination of Beta-site APP cleaving enzyme 1 (BACE1) and regulating the repair of nerve cell injury. Methods Mouse hippocampal neuron cell line HT22 was divided into four groups: control group, oxygen-glucose deprivation/reoxygenation(OGD/R) group, OGD/R+si-NC group and OGD/R+si-BC200 group. In order to further explore the relationship between BC200 and BACE1, HT22 cells were divided into four groups: OGD/R group, OGD/R+si-BC200 group, OGD/R+si-BC200+NC group and OGD/R+si-BC200+ BACE1 group. Twenty male C57BL/6J mice were randomly assigned to the following four groups: control group, middle cerebral artery occlusion (MCAO) group, MCAO+si-BC200 group and MCAO+si-BC200+BACE1 group. The mRNA expression levels of BC200 and BACE1 in cells were measured by real-time quantitative reverse transcription polymerase chain reaction. The expressions of c-caspase-3, B-cell lymphoma 2 (Bcl2), Bcl2 associated X protein(BAX) and BACE1 were detected by western blot, and the apoptotic cells were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test. Results Compared with the control group, the activity of HT22 cells in OGD/R group decreased significantly, and the percentage of apoptotic cells increased significantly. Compared with OGD/R+si-NC group, the activity of HT22 cells in OGD/R+si-BC200 group increased significantly, and the percentage of apoptotic cells decreased significantly. Compared with the control group, the expression of BACE1 protein in HT22 cells in OGD/R group was significantly enhanced. Compared with OGD/R+si-NC group, the expression of BACE1 protein in HT22 cells in OGD/R+si-BC200 group decreased significantly. It was observed that after OGD/R treatment, the ubiquitination level of BACE1 decreased significantly and the expression of BACE1 protein increased significantly. After transfection with si-BC200, the ubiquitination level of BACE1 protein increased significantly, while the expression of BACE1 protein decreased significantly. Compared with OGD/R+si-BC200+NC group, the percentage of apoptotic cells, the expression of c-caspase-3 and Bax protein in HT22 cells in OGD/R+si-BC200+BACE1 group increased significantly, and the expression of Bcl2 protein decreased significantly. Compared with the control group, the number of cerebral infarction areas and TUNEL positive cells in MCAO group increased significantly, and the survival number of neurons decreased significantly. Compared with the MCAO group, the number of cerebral infarction areas and TUNEL positive cells in MCAO+si-BC200 group decreased significantly, and the survival number of neurons increased significantly, while the addition of BACE1 reversed the improvement of si-BC200 transfection. Conclusion The combination of BC200 and BACE1 inhibit the ubiquitination of BACE1, and participate in mediating the expression enhancement of BACE1 induced by OGD/R. Specific blocking of BC200/BACE1 axis may be a potential therapeutic target to protect neurons from apoptosis induced by cerebral ischemia/reperfusion.
Animals ; Amyloid Precursor Protein Secretases/genetics* ; RNA, Long Noncoding/physiology* ; Aspartic Acid Endopeptidases/genetics* ; Male ; Neurons/pathology* ; Mice ; Mice, Inbred C57BL ; Apoptosis/genetics* ; Ubiquitination ; Cell Line ; Hippocampus/metabolism* ; bcl-2-Associated X Protein/genetics* ; Caspase 3/genetics* ; Infarction, Middle Cerebral Artery/metabolism*

Brain ; pathology ; Brain Infarction ; diagnosis ; Cerebral Angiography ; Cerebral Arterial Diseases ; diagnosis ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Posterior Cerebral Artery ; pathology

PURPOSE: Patients with superficial middle cerebral artery (MCA) territory infarction may have concomitant lenticulostriate artery (LSA) territory infarction. We investigated the mechanisms thereof and the outcomes of patients with superficial MCA territory infarction according to the presence or absence of LSA involvement. MATERIALS AND METHODS: Consecutive patients with first-ever infarction in the unilateral superficial MCA territory were included in this study. They were divided into the superficial MCA only (SM) group and the superficial MCA plus LSA (SM+L) group. RESULTS: Of the 398 patients, 84 patients (21.1%) had LSA involvement (SM+L group). The SM+L group more frequently had significant stenosis of the proximal MCA or carotid artery and high-risk cardioembolic sources. Stroke severity and outcomes were remarkably different between the groups. The SM+L group showed more severe neurologic deficits (National Institute of Health Stroke Scale score 10.8±7.1 vs. 4.0±5.0, p<0.001) and larger infarct in the superficial MCA territory (40.8±62.6 cm³ vs. 10.8±21.8 cm³, p<0.001) than the SM group. A poor functional outcome (mRS >2) at 3 months was more common in the SM+L group (64.3% vs. 15.9%, p<0.001). During a mean follow-up of 26 months, 67 patients died. All-cause (hazard ratio, 2.246) and stroke (hazard ratio, 9.193) mortalities were higher in the SM+L group than the SM group. In multivariate analyses, LSA involvement was an independent predictor of poor functional outcomes and stroke mortality. CONCLUSION: LSA territory involvement is predictive of poor long-term outcomes in patients with superficial MCA territory infarction.
Carotid Stenosis/mortality/pathology ; Constriction, Pathologic/pathology ; Female ; Humans ; Infarction, Middle Cerebral Artery/mortality/*pathology ; Male ; Middle Cerebral Artery/*pathology ; Multivariate Analysis ; Severity of Illness Index ; Stroke/mortality/pathology

OBJECTIVETo explore the neuroprotective effect and mechanism of picroside II on extracellular regulated protein kinases1/2 (ERK1/2) signal transduction pathway in cerebral ischemia injuryrats. METHODS The middle cerebral artery occlusion (MCAO) model was established by inserting a monofilament into middle cerebral artery. Totally 96 successfully modeled Wistar rats were divided into the modelgroup, the treatment (picroside II) group, the Lipopolysachcaride (LPS) group, and the U0126 group according to random digit table. Each group was further divided into 3 subgroups, i.e. 6, 12, and 24 h sub-groups. Picroside II (20 mg/kg) was peritoneally injected to rats in the treatment group 2 h after ischemia.LPS (20 mg/kg) and Picroside II (20 mg/kg) were peritoneally injected to rats in the LPS group 2 h after ischemia. U0126-EtOH (20 mg/kg)and Picroside II (20 mg/kg) were peritoneally injected to rats in the U0126group 2 h after ischemia. Equal volume of normal saline was peritoneally injected to rats in the control groupand the model group. The neurobehavioral function was evaluated by modified neurological severity score(mNSS) test. The structure of neurons was observed using hematoxylin-eosinstaining (HE) staining. Theapoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression of phosphorylated extracellular signal-regulated protein kinase1,2 (pERK1,2) in cortex was detected using immunohistochemistry (IHC) and Western blot.

RESULTSAfter cerebral ischemia injury neurological impairment score increased, the damage of neuron in the cortical area was aggravated, apoptotic cells increased in the model group as time went by. The expression of pERK1/2 increased more significantly in the model group than in the control group (P <0.05). The damage of neuron in the cortical area was milder, while apoptotic cells decreased, the expression of pERK1f2 obviously decreased more in the treatment group and the U0126 group (P < 0.05). The early damage of neuron in the cortical area was more severe, apoptotic cells and the expression of pERK12 were comparatively higher in early stage of the LPS group, but the expression of pERK1/2 was somewhat decreased in late stage.

CONCLUSIONSActivating ERK12 pathway could mediate apoptosis and inflammatory reactions of neurons after cerebral ischemia injury. Picroside II could protect the nerve system possibly through reducing activation of ERKI2 pathway, inhibiting apoptosis of neurons and inflammation reaction.

Animals ; Apoptosis ; Brain Ischemia ; drug therapy ; Cinnamates ; pharmacology ; Infarction, Middle Cerebral Artery ; drug therapy ; Iridoid Glucosides ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Neurons ; pathology ; Neuroprotective Agents ; pharmacology ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVETo explore a method for combining Fluoro-Jade B (FJB) staining with immunofluorescent staining in rats with focal cortical infarction.

METHODPermanent distal middle cerebral artery occlusion (dMCAO) was induced in rats by electrocoagulation. The rat models were randomized into two groups, and frozen sections of the brain tissues from each group were stained with FJB followed by immunofluorescent staining or in the reverse order.

RESULTSFJB staining followed by immunofluorescence staining clearly visualized both FJB-positive and immunofluorescence-positive cells in the frozen sections, but the staining protocol in the reverse sequence failed to clearly show the immunofluorescence-positive cells.

CONCLUSIONFJB staining prior to immunofluorescence staining does not affect the staining effect of protein immunofluorescent staining and better visualizes the positive cells.

Animals ; Brain ; pathology ; Fluoresceins ; chemistry ; Fluorescent Antibody Technique ; methods ; Fluorescent Dyes ; chemistry ; Infarction, Middle Cerebral Artery ; Rats ; Staining and Labeling ; methods

The purpose of this study was to identify time-related changes in clinical, MRI, histopathologic, and immunohistochemical findings associated with ischemic stroke in dogs. Additionally, the association of cerebrospinal fluid (CSF) and tissue levels of interleukin (IL)-6 with clinical prognosis was assessed. Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAO) in nine healthy experimental dogs. The dogs were divided into three groups according to survival time and duration of the experimental period: group A (survived only 1 day), group B (1-week experimental period), and group C (2-week experimental period). Neurologic status was evaluated daily. Magnetic resonance imaging (MRI) was performed according to a predetermined schedule. Concentration of IL-6 in CSF was measured serially after ischemic stroke. Postmortem examination was performed for all experimental dogs. During histopathological examination, variable degrees of cavitation and necrosis due to neuronal cytopathic effects, such as pyknotic nuclei and cytoplasmic shrinkage, were observed on the affected side of the cerebral cortex in all dogs. Immunohistochemistry specific for IL-6 showed increased expression in the ischemic lesions. CSF IL-6 concentrations and ischemic lesion volumes 1 day after ischemic stroke were significantly higher in group A compared to groups B and C.
Animals ; Brain Ischemia/*etiology ; Dogs ; Female ; *Immunohistochemistry ; *Infarction, Middle Cerebral Artery ; *Magnetic Resonance Imaging ; Male ; Stroke/*pathology

OBJECTIVETo observe the neurological protective effects of progesterone (PROG) on focal cerebral ischemia/reperfusion injury in rats and to explore its possible mechanism.

METHODSOne handred and twenty male SD rats were divided into three groups randomly: sham-operated group, middle cerebral artery occlusion ( MCAO ) group and PROG + MCAO group( n = 40). The right temporary MCAO model was established by the line-embolism method. The PROG + MCAO group rats were according to 8 mg/kg intraperitoneal injection PROG, after that 30 min, the rats were suffered ischemia/reperfusion. After rats were suffered ischemia for 2 h and reperfusion 0, 24, 48, 72 h stress, the nervous functional defect degree were evaluated by longe scoring, and the expression of two-pore domain K channel 3 (TASK3) mRNA in brain tissue were detected by the real-time PCR.

RESULTSPROG (8 mg/kg) could significantly reduced the nervous functional defect degree in rats after ischemia/reperfusion 24, 48, 72 h (P < 0.05). The results of real-time PCR showed that the TASK3 mRNA expression in the brain tissue at all time points significantly decreased in MCAO group compared with sham-operated group (P < 0.05). However, compared with MCAO group, the expression of TASK3 mRNA in brain tissue at all time points dramatically increased in PROG + MCAO group (P < 0.05).

CONCLUSIONPROG can improve the nervous functional defect degree after focal cerebral ischemia/reperfusion injury in rats, and the mechanism might be associated with up-regulating the expression of TASK3 mRNA in brain tissue.

Animals ; Brain ; drug effects ; pathology ; Brain Ischemia ; drug therapy ; Infarction, Middle Cerebral Artery ; Male ; Neuroprotective Agents ; pharmacology ; Potassium Channels, Tandem Pore Domain ; metabolism ; Progesterone ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Reperfusion Injury ; drug therapy

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 evaluate the thrombus length on CT perfusion imaging and to assess its predictive value for recanalization and clinical outcome after intravenous thrombolysis therapy (IVT).

METHODSFifty-six consecutive acute ischemic stroke patients with proximal middle cerebral artery (M1 segment) occlusion underwent CT perfusion imaging examination before IVT between June 2009 and May 2015. The onset-to needle time was (214.3 ± 82.0) min, and the pretreatment NIHSS score of patients was 13 (IQR 8-17). The thrombus length was determined as the distance between the proximal and distal thrombus end delineated on dynamic angiography, which was reconstructed from CT perfusion source images. Recanalization was evaluated according to Arterial Occlusive Lesion (AOL) scale, and functional outcome was based on modified Rankin scale (mRS) 3 months after IVT. Logistic regression model was used to investigate the relationship between thrombus length and recanalization, and the optimal cut-off points were determined by receiver operating characteristic curve (ROC).

RESULTSAmong 56 patients, 42 (75%) achieved recanalization 24 h after IVT with mean thrombus length of (9.0 ± 4.7) mm; and 14 (25%) patients remained occlusion with mean thrombus length of (10.0 ± 5.4) mm. Logistic regression analysis demonstrated that thrombus length was an independent predictor for both recanalization (OR=0.869; 95% CI:0.764-0.987; P=0.031) and unfavorable outcome (OR=1.180;95% CI:1.023-1.362; P=0.023). Thrombus length of 11.3 mm was identified as the optimal cut-off value for recanalization (AUC=0.697, sensitivity 71.4%, specificity 76.2%), while thrombus length of 9.9 mm was the optimal cut-off value for unfavorable functional outcome (AUC=0.689, sensitivity 64.7%, specificity 71.4%).

CONCLUSIONThe thrombus length evaluated on CT perfusion imaging is an effective predictor for recanalization and unfavorable outcome after IVT in acute ischemic stroke patients with middle cerebral artery occlusion.

Angiography ; Humans ; Infarction, Middle Cerebral Artery ; pathology ; Logistic Models ; Perfusion Imaging ; Sensitivity and Specificity ; Stroke ; diagnosis ; drug therapy ; Thrombolytic Therapy ; Thrombosis ; diagnosis ; drug therapy ; Tomography, X-Ray Computed

BACKGROUNDMotor dysfunction is common in stroke patients. Clinical electrophysiological studies suggest that transsynaptic degeneration occurred in the lower motor neurons, while pathological evidence is lacked. This study aimed to combine the electrophysiological and pathological results to prove the existence of transsynaptic degeneration in the motor system after stroke.

METHODSModified neurologic severity score, electrophysiological, and pathological assessments were evaluated in rats before middle cerebral artery occlusion (MCAO), and at 24 hours, 7 days, and 14 days after MCAO. Paired and independent-sample t-tests were applied to assess the changes of electrophysiological and pathological data.

RESULTSCompound motor action potential amplitude in the paretic side was significantly lower than the nonparetic side at both 24 hours (61.9 ± 10.4 vs. 66.6 ± 8.9, P < 0.05) and 7 days (60.9 ± 8.4 vs. 67.3 ± 9.6, P < 0.05) after MCAO. Motor unit number estimation of the paretic side was significantly less than the nonparetic side (379.0 ± 84.6 vs. 445.0 ± 89.5, P < 0.05) at 7 days after MCAO. Until 14 days after stroke, the pathological loss of motor neurons was detected. Motor neurons in 14-day MCAO group were significantly decreased, compared with control group (5.3 ± 0.7 vs. 7.3 ± 1.8, P < 0.05).

CONCLUSIONSBoth electrophysiological and pathological studies showed transsynaptic degeneration after stroke. This study identified the asynchronization in changes of electrophysiology and pathology. The abnormal physiological changes and function impairment can be detected in the early stage and recovered quickly, while the pathological loss of motor neuron can be detected only in a later stage.

Animals ; Electrophysiology ; Infarction, Middle Cerebral Artery ; pathology ; physiopathology ; Male ; Motor Neurons ; pathology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; pathology ; physiopathology