Objective:To investigate the effect of tyrosine kinase receptor binding protein B3 (Ephrin-B3) on the expressions of postsynaptic density protein 95 (PSD95), N-methyl-D-aspartic acid (NMDA) receptor subunit 2A and 2B(NR2A/NR2B) and synaptic ultrastructure of hippocampal neurons in the dentate gyrus (DG)area of epileptic rats.Methods:A total of 96 SPF-grade adult male Sprague Dawley(SD) rats were divided into blank group, epilepsy group, lentiviral vectors control group and Ephrin-B3 overexpression group( n=24 in each group) according to the random number table.Another 36 adult male SD rats were randomly divided into recombinant adeno-associated virus control group and Ephrin-B3 suppression group( n=18 in each group). The viruses were stereotaxically injected into bilateral hippocampus DG area of rats and epileptic model was established by intraperitoneal injection of lithium chloride and pilocarpine.Racine standard grading method was used to evaluate seizure rate and the duration and latency of epileptic rats were observed. Immunofluorescence staining was used to observe expression levels of PSD95, NR2A and NR2B protein in hippocampus DG area of rats.PCR and Western blot were used to detect the expression levels of PSD95, NR2A and NR2B mRNA and protein in hippocampus of rats. Transmission electron microscopy was used to observe the ultrastructure of hippocampal neurons in rats. GraphPad Prsim 8 software was used for statistical analysis. Results:(1) The results of Ephrin-B3 overexpression experiment: there were statistically significant differences in the seizure latency and duration among the four groups( F=368.30, 120.00, both P<0.01). The seizure latency in Ephrin-B3 overexpression group was longer than that of the epilepsy group and lentiviral vectors control group((31.32±1.10)d, (24.09±2.02)d, (21.42±0.79)d)(all P<0.01), while the seizure duration in Ephrin-B3 overexpression group was shorter than that of the epilepsy group and lentiviral vectors control group((26.85±1.14)s, (40.40±1.26)s, (36.50±5.50)s)(all P<0.05). Immunofluorescence staining showed the average fluorescence intensity of PSD95, NR2A and NR2B in Ephrin-B3 overexpression group were higher than that in the lentiviral vectors control group(all P<0.01). PCR and Western blot both showed that the Ephrin-B3 overexpression group had higher mRNA expression levels of PSD95, NR2A and NR2B than that in lentiviral vectors control group(all P<0.05). The results of transmission electron microscopy showed the number of synapses in the Ephrin-B3 overexpression group was greater than that in lentiviral vectors control group((9.00±1.00), (6.00±1.00))( P<0.05), synaptic gap was narrower than that in lentiviral vectors control group ((31.60±1.45)nm, (36.43±2.22)nm)(all P<0.05). (2)The results of the Ephrin-B3 suppression experiment: the evaluation of seizures in rats showed that there were statistically significant differences in the seizure latency and duration between recombinant adeno-associated virus control group and Ephrin-B3 suppression groups( t=3.88, 3.93, both P<0.05). The seizure latency in Ephrin-B3 suppression group was shorter than that of recombinant adeno-associated virus control group ((17.10±1.88)d, (23.50±2.15)d)( P<0.05). The seizure duration in Ephrin-B3 suppression group was longer than that of recombinant adeno-associated virus control group((55.16±5.48)s, (42.06±1.83)s)( P<0.05). Western blot results showed that the Ephrin-B3 suppression group had lower protein expression levels of PSD95, NR2A and NR2B than those in recombinant adeno-associated virus control group(all P<0.01). The results of transmission electron microscopy showed the numbers of synapses in the Ephrin-B3 suppression group were less than recombinant adeno-associated virus control group((3.33±0.58), (4.66±0.58))( P<0.05), synaptic gap were narrower than recombinant adeno-associated virus control group((37.27±0.97)nm, (33.33±1.46)nm)( P<0.05). Conclusion:Ephrin-B3 can attenuate seizures and upregulate the synaptic related proteins and glutamate receptor expression, improving synaptic structure.

Objective:To investigate the effect of tyrosine kinase receptor binding protein B3 (Ephrin-B3) on the expressions of postsynaptic density protein 95 (PSD95), N-methyl-D-aspartic acid (NMDA) receptor subunit 2A and 2B(NR2A/NR2B) and synaptic ultrastructure of hippocampal neurons in the dentate gyrus (DG)area of epileptic rats.Methods:A total of 96 SPF-grade adult male Sprague Dawley(SD) rats were divided into blank group, epilepsy group, lentiviral vectors control group and Ephrin-B3 overexpression group( n=24 in each group) according to the random number table.Another 36 adult male SD rats were randomly divided into recombinant adeno-associated virus control group and Ephrin-B3 suppression group( n=18 in each group). The viruses were stereotaxically injected into bilateral hippocampus DG area of rats and epileptic model was established by intraperitoneal injection of lithium chloride and pilocarpine.Racine standard grading method was used to evaluate seizure rate and the duration and latency of epileptic rats were observed. Immunofluorescence staining was used to observe expression levels of PSD95, NR2A and NR2B protein in hippocampus DG area of rats.PCR and Western blot were used to detect the expression levels of PSD95, NR2A and NR2B mRNA and protein in hippocampus of rats. Transmission electron microscopy was used to observe the ultrastructure of hippocampal neurons in rats. GraphPad Prsim 8 software was used for statistical analysis. Results:(1) The results of Ephrin-B3 overexpression experiment: there were statistically significant differences in the seizure latency and duration among the four groups( F=368.30, 120.00, both P<0.01). The seizure latency in Ephrin-B3 overexpression group was longer than that of the epilepsy group and lentiviral vectors control group((31.32±1.10)d, (24.09±2.02)d, (21.42±0.79)d)(all P<0.01), while the seizure duration in Ephrin-B3 overexpression group was shorter than that of the epilepsy group and lentiviral vectors control group((26.85±1.14)s, (40.40±1.26)s, (36.50±5.50)s)(all P<0.05). Immunofluorescence staining showed the average fluorescence intensity of PSD95, NR2A and NR2B in Ephrin-B3 overexpression group were higher than that in the lentiviral vectors control group(all P<0.01). PCR and Western blot both showed that the Ephrin-B3 overexpression group had higher mRNA expression levels of PSD95, NR2A and NR2B than that in lentiviral vectors control group(all P<0.05). The results of transmission electron microscopy showed the number of synapses in the Ephrin-B3 overexpression group was greater than that in lentiviral vectors control group((9.00±1.00), (6.00±1.00))( P<0.05), synaptic gap was narrower than that in lentiviral vectors control group ((31.60±1.45)nm, (36.43±2.22)nm)(all P<0.05). (2)The results of the Ephrin-B3 suppression experiment: the evaluation of seizures in rats showed that there were statistically significant differences in the seizure latency and duration between recombinant adeno-associated virus control group and Ephrin-B3 suppression groups( t=3.88, 3.93, both P<0.05). The seizure latency in Ephrin-B3 suppression group was shorter than that of recombinant adeno-associated virus control group ((17.10±1.88)d, (23.50±2.15)d)( P<0.05). The seizure duration in Ephrin-B3 suppression group was longer than that of recombinant adeno-associated virus control group((55.16±5.48)s, (42.06±1.83)s)( P<0.05). Western blot results showed that the Ephrin-B3 suppression group had lower protein expression levels of PSD95, NR2A and NR2B than those in recombinant adeno-associated virus control group(all P<0.01). The results of transmission electron microscopy showed the numbers of synapses in the Ephrin-B3 suppression group were less than recombinant adeno-associated virus control group((3.33±0.58), (4.66±0.58))( P<0.05), synaptic gap were narrower than recombinant adeno-associated virus control group((37.27±0.97)nm, (33.33±1.46)nm)( P<0.05). Conclusion:Ephrin-B3 can attenuate seizures and upregulate the synaptic related proteins and glutamate receptor expression, improving synaptic structure.

Objective:To investigate the effects of hippocampal injection of tyrosine kinase receptor binding protein B3(Ephrin-B3) agonist on spontaneous seizures and the expression of hippocampal secretory glycoprotein (Reelin) and phosphorylated adaptor protein (p-Dab1) in epileptic model rats.Methods:Seventy-eight rats were randomly divided into control group and model group according to body mass matching with 39 rats in each group.The rats in control group were fed normaly, and the rats in model group were established epilepsy model by intraperitoneal injection of lithium chloride pilocarpine. The hippocampal tissues were taken in the acute phase (7 days), quiescent phase (14 days) and chronic phase (60 days) after the successful induction of status epilepticus. The levels of Reelin protein and p-Dab1 protein in the hippocampal tissues of epileptic model rats and normal rats were detected by immunohistochemistry and Western blot.And thirteen rats were randomly selected at each time point. Another 48 rats were randomly divided into normal Fc-control group, normal EphB3-Fc group, epilepsy Fc-control group and epilepsy EphB3-Fc group, with 12 rats in each group. Rats in the first two groups were fed normally, and those in the latter two groups were established epileptic model. Seven days after modeling, all rats were injected into bilateral hippocampus with EphB3-Fc (Ephrin-B3 agonist) and FC control (control agent of Ephrin-B3 agonist) according to the grouping, once a day for 7 days. After administration, the changes of behavior and EEG were observed within two weeks. At the same time, the expression of Reelin protein and p-Dab1 protein were detected by immunohistochemistry and Western blot. SPSS 21.0 was used for statistical analysis, One-way ANOVA was used for multi group comparison, and Tukey's test was used for pairwise comparison.Results:The results of immunohistochemistry and Western blot showed that compared with the control group, the levels of Reelin and p-Dab1 protein in hippocampus of model group decreased significantly at 7, 14 and 60 days after epilepsy (all P<0.01). The results of immunohistochemistry showed that compared with epilepsy Fc-control group, the levels of p-Dab1 ((0.41±0.04), (0.58±0.06), P<0.05) in epilepsy EphB3-Fc group increased significantly.Western blot result showed that the level of p-Dab1 in epilepsy EphB3-Fc group increased compared with that of epilepsy Fc-control group (1.34±0.04), (2.26±0.10), P<0.01). Compared with epilepsy Fc-control group, epilepsy EphB3-Fc group showed less average seizure duration ((39.00±1.79)s, (26.50±1.87)s; t=23.21, P<0.01), less frequencies ((2.00±0.89), (0.50±0.55); t=2.32, P<0.01) and less latent period ((6.33±1.37)day, (12.50±1.87)day; t=2.52, P<0.01) in spontaneous recurrent seizures. Compared with epilepsy Fc-control group, epilepsy EphB3-Fc group showed lower average amplitude ((37.30±1.21)μV, (29.00±1.41)μV; t=25.14, P<0.01), less average seizure duration ((5.35±0.19)s, (2.35±0.19)s; t=3.13, P<0.01). Conclusion:Ephrin-B3 alleviated spontaneous recurrent seizures by upregulating Reelin and p-Dab1 in temporal lobe epilepsy rat.

Objective　To explore dynamic expression of Ephrin-B3 in hippocampus of pilocarpine induced epileptic rats and the role of Ephrin-B3 in the process of epileptogenesis.Methods　We first established pilocarpine induced epileptic rat model and then detected Ephrin-B3 expression at 7,14 and 60 days post status epilepticus (SE) by real time qPCR,immunohistochemistry and Western blot.Results　After pilocarpine injection,rats showed facial muscles,forelimb and tail clonus and ataxic lurching,head bobbing and wet dog shakes.Real time qPCR revealed that Ephrin-B3 mRNA expression in hippocampus decreased during spontaneous seizure period.Compared with controls,epileptic rats showed decreased Ephrin-B3 expression at 7 days,reached the lowest level at 14 days and then increased slightly at 60 days post-SE (P<0.001).Ephrin-B3 protein in hippocampus also decreased at 7,14 and 60 days detected by Western blot (P<0.001).Immunohistochemistry results showed that Ephrin-B3 mainly expressed in granule cells in dentate gyrus.Analysis of immunohistochemistry revealed decreased Ephrin-B3 expression in epileptic rats compared with controls (P<0.001).Conclusion　The dynamic changes of Ephrin-B3 expression in hippocampus of epileptic rats indicated that Ephrin-B3 may participate in the process of spontaneous recurrent seizures.

Objective:To explore the clinical and electrophysiological characteristics of flail arm syndrome (FAS).Methods:Clinical and electrophysiological data were collected on 13 FAS patients and 31 persons with upper limb onset amyotropic lateral sclerosis (UL-ALS), including the amplitude of compound muscle action potential (CMAP) related to median nerve, ulnar nerve, and axillary nerve motor conduction. A split-hand index (SI) was calculated by dividing the CMAP amplitude of abductor pollicis brevis by that of the abductor digiti minimi. Clinical features, the CMAP amplitudes and SIs were compared between the FAS and UL-ALS patients.Results:Compared with UL-ALS patients, the age at onset among the FAS patients was older (averaging 60.9 years). The development to the second stage was longer (24±6 months). The upper limb reflexes of 15% of the FAS patients had disappeared and those of 77% were weakened, while the lower limb reflexes of 54% of the FAS patients were active and 38% were weakened, significantly different from the UL-ALS patients. However, there were no significant differences in the CMAP amplitudes of the median and ulnar nerves, nor in SI between the FAS and UL-ALS patients. The SIs of the FAS patients with upper motor neuron signs were significantly lower than those of FAS patients without such signs. Among the FAS patients, the average CMAP amplitude of the ulnar nerve was the highest, followed by those of the median and axillary nerves. Among the UL-ALS patients, however, the average CMAP amplitude of the ulnar nerve was not significantly different from that of the axillary nerve.Conclusions:FAS patients with upper motor neuron signs are more likely to have slip hand. The CMAP amplitude of their axillary nerve tends to be lower than that of their median and ulnar nerves. FAS seems to be a special type of ALS.

Objective　To explore the value of the three auxiliary examinations of transcranial ultrasound imaging,magnetic sensitivity weighted imaging,and magnetic resonance imaging in the identification of progressive supranuclear palsy and Parkinson’s disease.Methods　Twenty-two clinically diagnosed patients with PSP,33 patients with PD were selected,and 30 cases in the control group underwent TCS,SWI,and MRI examinations and medical records were comprehensive.ROC curve was used to compare and analyze the results of TCS,SWI and MRI.The value of three auxiliary examinations in the identification of progressive supranuclear palsy and Parkinson’s disease was discussed.Results　Compared with the PD group and the control group,the ratio of hyperechoic lenticular nucleus,the ratio of the minimum diameter of the third ventricle more than 10mm,the midbrain area/pontine area,the ratio of midbrain/pontine diameter,magnetic resonance Parkinson’s index,low signal strength of the putamen,and low signal strength of the red nucleus in the PSP group were higher (P<0.01).Compared with the PSP group and the control group,the substantia nigra hyperechoic area,the ratio of total substantia nigra hyperechoic area to total midbrain area in the PD group were higher (P<0.01).According to the ROC curve,it can be observed that the sensitivity of substantia nigra hyperechoic area in TCS was high while the specificity of low signal intensity of putamen in SWI was high as well;When TCS and SWI jointly identified the PD group and PSP group,the sensitivity was 93.9%,the specificity was 81.8%,the area under the curve (AUC) was 0.938,and the value of identification diagnosis was high.The sensitivity and specificity of the MRPI and P/M values in the identification of the PSP group and PD group reached 100%.Conclusion　The value of TCS,SWI,and MRI in the identification of PSP and PD is different.TCS combined with SWI is of higher diagnosis value in the identification of PD group and PSP group,which provides a new method for the identification diagnosis of PSP.The overall value of differential diagnosis of MRI is better than TCS and SWI.

Objective　To explore the three auxiliary examinations of magnetic resonance imaging（MRI），anal sphincter electromyography（EAS-EMG） and transcranial ultrasound imaging（TCS） for multi-system atrophy of P-type（MSA-P） and Parkinson’s disease（PD） differential diagnostic value. Methods　Twenty-one patients with clinically diagnosed MSA-P and 33 patients with PD were enrolled. All of the patients underwent MRI，EAS-EMG，and TCS，and the medical records were comprehensive and preserved. The MRI，EAS-EMG and TCS results were compared and analyzed by the receiver operating characteristic（ROC） curves. Results　Slit-like hyperintensity in the posterolateral margin of the putamen，MUP mean time，mean amplitude，percentage of polyphase wave，incidence of spontaneous generation and satellite potential were higher in the MSA-P group than in the PD group. However，the high echogenic area of the substantia nigra，the ratio of the total area of the substantia nigra and the total area of the midbrain were lower than those of the PD group（both P<0.01）. The ROC curve showed that the slit-like hyperintensity in the posterolateral margin of the putamen had the highest specificity（97.0%） for the differential diagnosis of the two，the highest sensitivity of the hyperechoic area of the substantia nigra（81.0%），and the highest AUC of the MUP mean time（0.781）. The sensitivity of the three indicators combined to the differential diagnosis was 95.2%，and the AUC was 0.939.The differential diagnosis value was extremely high. Conclusion　The diagnostic value of MRI，EAS-EMG and TCS in MSA-P and PD is different and complement each other，and the combination of the three indicators has good diagnostic value.

Objective: To study the value of unmethylated cytosine guanine dinucleotide oligodeoxynucleotide (DSP30) and IL-2 in the conventional cytogenetic (CA) detection of the chromosomal aberrations in chronic lymphocytic leukemia (CLL) . Methods: Bone marrow or peripheral blood cells of CLL patients were cultured with DSP30 plus IL-2 for 72 h, following which R-banding analysis was conducted. Fluorescence in situ hybridization (FISH) was performed in 85 patients. CA results were compared with data obtained by FISH. Results: Among 89 CLL patients, the success rate of chromosome analysis was 94.38% (84/89) . Clonal aberrations were detected in 51 patients (51/84, 60.71%) . Of them, 27 (27/51, 52.94%) were complex karyotype. Among 85 CLL patients tested by FISH, chromosomal abnormalities were detected in 74 (74/85, 87.06%) patients, of which 2 (2/74) patients were complex karyotypes, accounting for 2.70%. Of the 85 CLL patients examined by FISH, 50 had abnormal karyotype analysis, 30 had normal karyotype, 5 failed to have chromosome analysis. Among them, 25 cases showed clonal aberrations by FISH assay but normal by CA, and 4 cases were normal by FISH but displayed aberrations in chromosome analysis, and totally 78 (91.76%) cases with abnormality detected by the combination of the two methods. The frequency of 13q- abnormality detected by FISH was significantly higher than that by CA analysis (69.41%vs 16.67%, P<0.001) , while the frequency of 11q-,+12 and 17p- detected by two methods showed no significant difference (P>0.05) . The detection rate of complex abnormalities in conventional karyotype analysis was higher than that in FISH (50.98%vs 2.70%) . In addition, 11 low-risk and 9 intermediate-risk patients according to FISH results showed complex karyotype by cytogenetics, and were classified into high-risk cytogenetic subgroup. Conclusion DSP30 and IL-2 are effective in improving the detection rate of CA in CLL patients (60.71%) and CA is more effective to detect complex karyotype. However, FISH had a higher overall abnormality detection rate (87.06%) than CA, especially for 13q-. The combination of CA and FISH not only enhanced the detection rate of clonal aberrations to 91.76%, but also provided more precise prognosis stratification for CLL patients, thus to provide more information for clinical implication.

Objective To investigate the effect of thyroid hormone(T3)on the expressions of cytochrome c (CytC) and apoptosis?inducing factor(AIF) after cerebral ischemia reperfusion injury in rats and its mechanism. Methods SD male rats were randomly divided into four groups: sham operation group(sham1),sham operation group + T3(sham2) ,ischemia?reperfusion group (IR) ,and thyroid hormone treatment group(T3). A rat model of cerebral ischemia?reperfusion injury was established by right middle cerebral artery occlusion for 2 h,followed by reperfusion for 24 h. Thyroid hormones (10μg/100 g) or normal saline were given at 1 h after onset of ischemia and 6 h after reperfusionby intraperitoneal injection. Neurological deficit scores were evaluated 24 h after reperfusion. Cerebral infarction volume was evaluated by TTC staining. Histological changes was observed by HE staining. Expressions and mRNA levels of CytC and AIF in ischemic brain tissue were evaluated by immunohistochemistry and real?time fluorescent quantitative RT?PCR. Results Ascompared with those in sham operation groups ,the expressions and mRNA levels of CytC and AIFincreased significantlyin IR groups. As compared with those in IR groups ,the indexes were remarkably decreasedin T3 groups (P < 0.01). Nerve function was markedly improved andinfarction area narrowed.Conclusions Thyroid hormone plays a certain role in protection of cerebral ischemia?reperfusion injury in rats,whose mechanism may be associated with inhibition of the expression of apoptosis factors?CytC and AIF.

Objective To investigate the effect of thyroid hormone on the expression of NF-κB and TNF-oα in the ischemic cortex of rats after focal cerebral ischemia-reperfusion.Methods 96 male SD rats were randomly divided into sham-operation group,sham-operation+T3 group,IR group and IR+T3 group.Using suture legal method to establish a rat model of middle cerebral artery occlusion for 2 h followed by reperfusion.In sham-operation+T3 group and IR+T3 group,T3 was given 3 days before ischemia and 1 hour after ischemia,respectively,intraperitoneal injection T3 10 μg/100 g for rats.Rats in other groups were given the same volume normal saline at the same time.The infarct size was determined by TTC staining at 24 h after reperfusion.HE staining was used to observe the morphological and structural changes of brain tissue.Using Real-time PCR method and immunohistochemical staining method to detect the expression of NF-κB mRNA,TNF-α mRNA and protein in ischemic cortex of rats.Results Compared with sham-operation group and sham-operation+T3 group,the pathological damage of brain tissue in IR group was obvious,while the pathologic damage of IR +T3 group was less than that in IR group.Immunohistochemistry assay showed that the expression of NF-κB was(49.19±5.55)in sham-operation group,(45.75±2.12) in sham-operation+T3 group,(56.88±2.23)in IR group and(50.25±1.67)in IR +T3 group,the expression of TNF-α was (22.50±3.07) in sham-operation group,(24.13±2.03) in sham-operation+T3 group,(37.25±2.82) in IR group and (30.25±1.67) in IR +T3 group,and the NF-κB,TNF-α in IR group were obviously higher than that in sham-operation group and sham-operation+T3 group(P＜0.05),while IR+T3 group were lower than that in IR group(P＜0.05).Real-time PCR showed that NF-κB mRNA,TNF-α mRNA level in IR group was the highest,which was higher than that of sham-operation group and sham-operation+T3 group(P＜0.05),and the NF-κB mRNA,TNF-oα mRNA expression in IR+T3 group were significantly decreased compared with that in IR group(P＜0.05).Conclusion Thyroid hormone has a protective effect on cerebral ischenia reperfusion injury,which may be achieved by reducing the expression of inflammatory factor NF-κB and TNF-oα.