Neuro-ophthalmology is a subspecialty of both neurology and ophthalmology concerning visual problems that are related to the nervous system. As the rapid development of computer technology, information technology and radiology, the development of neuro-ophthalmology is in the best period in history. The following important topics are worthy of attention: 1) the diagnosis and therapy of carotid artery stenosis related ophthalmopathy;2)the relationship between papilledema and cranial venous sinus thrombosis; 3) the diagnosis of optic canal fracture with high resolution computer tomography;4)the relationship between optic neuritis and multiple sclerosis.

Objective To construct Nedd4 knockout bone marrow-derived macrophages(BMDM) cell line by CRISPR/Cas9 technology and to provide an effective tool for studying the function and mechanism of Nedd4 in macrophage.Methods First,three high-grade sgRNAs targeting Nedd4 gene exons were screened using the online tool before synthesized sgRNAs were inserted into the PX330 plasmid respectively.Secondly,the recombinant plasmids were transferred into BMDM cells and monoclonal cells were obtained by limiting dilution method.The protein levels of NEDD4 in monoclonal cells were detected by Western blotting.Finally,the DNA sequence of the monoclonal cells was confirmed by sequence analysis.Results One Nedd4 knockout BMDM cell line was obtained.The sequencing result showed that the Nedd4 gene had 16bp deletion mutation in this cell line.Conclusion The Nedd4 knockout BMDM macrophage cell line constructed by CRISPR/Cas9 technology will be a useful tool for studying the function and mechanism of Nedd4 in BMDM cells.

Objective To observe the effects of hyperbaric oxygen (HBO) on the apoptosis expression of intestinal mucosa during the different periods of ischemia-reperfusion injury in order to elucidate the underlying mechanisms. Method Rats were subjected to ischemia for 60 min by clamping superior mesenteric artery, and then had reperfusion for 60 min by unclamping. Rats were randomly divided into four groups: ischemia-reperfusion group (I/R), pre-emptive HBO or HBO treatment before ischemia (HBO-P) group, HBO treatment during ischemia period (HBO-I) group, and HBO treatment during reperfusion (HBO-R) group. After reperfusion for 60 min, samples of small intestine tissue were taken from the end portion of ileum for detecting the levels of ATP by using colorimetric method and the levels of caspase-3 by using immunochemistry. The levels of TNF-α in intestinal tissue were measured by using enzyme-linked immunosorbent assay method ( ELISA). All values were expressed in Mean ± Standard Deviation (x ± s). The different groups were compared among them with SNK- q test of OneWay analysis of variance (One-Way ANOVA plus SNK). Results The levels of TNF-α in HBO-I group were significantly lower than that in HBO-P group ( P ＜ 0.05), and significantly lower in HBO-P group than those in HBO-R or I/R groups ( P ＜ 0.05), and there was no significant difference in TNF-α between HBO-R and I/R group ( P ＞ 0.05). The levels of caspase-3 were significantly lower in HBO-I group than those in HBO-P group ( P ＜ 0. 05), and also significantly lower in HBO-P group than those in I/R or HBO-R groups ( P ＜ O. 05), and no significant difference caspase-3 was found between HBO-R and I/R groups. The ATP levels were significantly lower in HBO-I group than those in HBO- P group ( P ＜ 0. 05), and also significantly lower in HBO- P group than those in I/R or HBO-R group ( P ＜ 0.05), and no significant difference in ATP level between in HBO-R and I/R group. Conclusions There was a connection between HBO and small intestinal I/R injury as well as mucosal cell apoptosis. And HBO maintained ATP and aerobic metabolism, and inhibited the genesis of TNF-α, and thus in turn prevented intestinal mucosa cell from apoptosis. The best result was obtained when HBO was administered during ischemia period, and there was no effect found when HBO was employed during reperfusion period.

ObjectiveTo guide fluid management and evaluate the clinical index through monitoring extravascular lung water index(EVLWI) of patients with pulmonary contusion.MethodsThirtyone severe chest trauma patients with pulmonary contusion were selected,performed with central venous catheterization or femoral artery catheterization,and connected to pulse index continuous cardiac output (PICCO).EVLWI after pulmonary contusion were measured by using thermal dilution.The intake and output of patients were recorded in detail,fluid intake was controlled,furosemide was prescribed,and the changes and correlations of oxygenation index,EVLWI and liquid balance difference at different time points were evaluated.ResultsEVLWI after pulmonary contusion at 1st to 7th d after hospitalization was respectively (9.25 ±0.71),(8.98 ±0.61),(8.61 ±0.59),(7.75±0.53),(6.64 ±0.49),(6.22±0.36),(5.59 ±0.39) ml/kg.Comparing with 1st d after hospitalization,EVLWI declined from 3rd d (P ＜ 0.05).Oxygenation index at Ist to 7th d after hospitallzation was respectively( 145.76±23.61 ),( 144.19±20.24),( 146.67±19.25 ),(159.33±15.42),(177.38±14.25),(199.33±19.04),(213.71±18.51) mm Hg(1 mm Hg =0.133 kPa).Comparing with 1st d,oxygenation index from 4th to 7th d had significant difference (P ＜ 0.05 ).The fluid volume at 1st d to 7th d were all negative balance,that of 3rd d to 6th d had significant difference comparing with 1st d [(-431.43±121.17),(-601.43±127.09),(-629.52±140.69),(-320.01 ±93.71) ml vs.(-213.81±63.91 ) ml](P ＜ 0.05 ).Oxygenation index and EVLWI had negative correlation(r =-0.824,P＜0.01).Liquid balance difference and the changes of oxygenation index and EVLWI had no correlation.ConclusionEVLWI effectively monitoring after pulmonary contusion can not only evaluate the changes of pulmonary vascular permeability of patients with pulmonary contusion,but also have important significance to guide fluid management.

Teaching of Critical Care Medicine faces several challenging issues includingcomprehensive intensive or specialist intensive, approach of curriculum, tralning of team working ability, building of teaching platform and teaching staff. Critical care medical education requires the concept of viewing the discipline as a whole. Under its guidance and with the opportunity of critical care medical subspecialties building, critical care medical education should focus on bothcompre-hensive critical care and specialist critical care, and have rational planning of Critical Care Medicine course. Through the construction and integration of ICU, we should create a comprehen-sive clinical practice platform of critical care medicine to carry out clinical practice and team work tralning. Meanwhile, construction of quality critical care medicine faculty should be based on its pro-fessional features.

Objective To establish a mouse model of acute liver failure induced by lipopolysaccharide /D-galac-tosamine ( LPS/D-GalN) .Methods The optimum dose of LPS/D-GalN was determined by i .p.injection of eight differ-ent doses of LPS and D-GalN into 40 female C57BL/6 mice and observation of their survival time .Then, 32 female C57BL/6 mice were i.p.injected with the optimal dose of LPS/D-GalN and sacrificed at 0, 1, 4, 8 hours after the injec-tion, 8 mice in each group.The control mice received saline injection .Hepatic changes were observed by pathology and se-rum ALT, IL-6, MCP-1 and TNF-αwere measured by biochemistry or flow cytometry .Results LPS (2.5 mg/kg) and D-GalN (0.3 g/kg) were determined as the optimal dose for the establishment of mouse model of acute liver injury .Com-pared with the control group , the hepatocellular damages were progressing in a positive correlation with the time course after LPS/D-GalN administration .The level of serum ALT was significantly increased after LPS/D-GalN administration ( P <0.001).The levels of inflammatory cytokines IL-6, MCP-1 and TNF-αwere increased and reached a peak at one hour after LPS/D-GalN administration and then decreased almost to that of the control group 8 hours later(P<0.001).Conclusions The mouse model of acute liver injury is successfully established by LPS /D-GalN administration , and provide an effective animal model for the study of pathogenic mechanisms of acute liver failure and evaluation of therapeutic drugs .

Objective To obtain Chinese hamster ovary (CHO) cell lines that stably express a targeting complement inhibitor CR2-CD59.Methods The recombinant plasmid PEE14.1-CR2-CD59 was constru-cted by cloning the DNA fragment CR2-CD59 into plasmid PEE14.1,and the obtained plasmid was transfected into CHO cells by FuGENE 6.The clones with stable high expression of target fragment were selected by methionine sulfoximine (MSX),the expression of CR2-CD59 was analyzed by ELISA,SDS-PAGE and Western blotting analysis.Results Several stable expression clones were obtained,and CR2-CD59 was highly expressed in the secret form in CHO cells.SDS-PAGE analysis showed that the molecular weight of the recombined protein CR2-CD59 was consistent with the predicted one.ELISA and Western blotting results revealed that the CR2-CD59 could react with both anti-human CR2 and anti-human CD59 polyclonal antibodies.Compared with serum-containing medium,the protein was highly expressed in serum-free medium (P

Objective:To analyze the clinical features and prognosis factors of aquaporin 4 (AQP4) antibody-positive neuromyelitis optica spectrum disorders related optic neuritis (NMOSD-ON).Methods:An ambidirectional cohort study. From June 1, 2015 to June 1, 2019, 103 patients with AQP4 antibody-positive NMOSD-ON in Department of Neuro-ophthalmology, The First Medical Center of PLA General Hospital were included. All patients of followed-up period were ≥24 months. According to the best corrected visual acuity (BCVA) at the last follow-up, the affected eyes were divided into the low vision group [log of minimum resolution angle (logMAR) BCVA≥1.0] and the non-low vision group (logMAR BCVA<1.0), 66 and 37 cases, respectively. The two groups of patients were compared the genernal clinical characteristics, and the logistic regression model and COX proportional hazard model were used to analyze the relevant factors affecting the patient's visual prognosis and recurrence.Results:Among the 103 cases, 96 cases (93.2%, 96/103) were female; 94 cases (91.3%, 94/103) had unilateral disease; 48 cases (46.6%, 48/103) were the first onset; 85 cases (82.5%, 85/103) were effected by eye pain or orbital pain; 21 cases (20.4%, 21/103) had optic disc edema; 51 cases (49.5%, 51/103) serologically autoimmune antibody test were positive. Orbital magnetic resonance imaging (MRI) was performed in 101 cases. There was no obvious abnormal signal in visual pathways except for 5 cases (5.0%, 5/101); 96 cases (95.0%, 96/101) had abnormal signal in the visual path, and the optic nerve was found in the orbit; 52 cases had abnormal optic nerve in orbital segment (51.5%, 52/101); 37 cases (35.9%, 37/103) recurred within 24 months. The recovery of logMAR BCVA after the first onset and the logMAR BCVA at the first onset, at 6 months of follow-up in two groups were 1.4±1.0, 0.3±0.4, 1.9±0.7 and 0.4±0.5, 2.1±0.6, 0.3±0.4, respectively; and there were statistically significant differences between the two groups of patients at different times ( Z=-4.967,-7.603,-8.027; P<0.001). Logistic regression multivariate analysis showed that recovery of BCVA≥1.0 logMAR after the first onset [odds ratio ( OR)=226.276, P<0.001 ] and the number of attacks ( OR=8.554, P=0.003) were independent risk factors for low vision. Multivariate analysis of the Cox proportional hazards model showed the higher the MRI score [hazard ratio ( HR)=0.588, P=0.007] and plasma exchange ( HR=0.124, P=0.049) in the acute phase were protective factors for recurrence. Conclusions:Vision loss accompanied by eye pain or orbital pain is the main symptom of onset AQP4 antibody-positive NMOSD-ON, a small number of patients have disc edema, 49.5% patients serologically autoimmune antibody test are positive. Abnormal optic nerve signals can be seen in 95.0% of patients in orbital MRI, and 51.5% patients have abnormalities in the orbital optic nerve. The worse the recovery of BCVA after the first onset and the greater the number of attacks are unfavorable factors affecting the prognosis of vision. High MRI scores and plasma exchange in the acute phase are favorable factors to prevent the recurrence of the disease.

OBJECTIVETo construct a dual-reporter gene system that will be applicable for use as a tool to screen and evaluate therapeutic drug compounds that inhibit transcription of the gene encoding collagen I, chain at1 (COL1A1).

METHODSThe full-length eDNA of transforming growth factor beta1 (TGFbeta1) was cloned by RT-PCR and inserted into two vectors, pcDNA3.1 and pJW4303, for construction of two eukaryotic expression vectors, pcDNA3.1-TGFbeta1 and pJW4303-TGFbeta1.Next, the promoter region of COL1A1, cloned by PCR using human genome DNA as template, was inserted into the vector pGL4.29 to construct the reporter gene vector, pGL4.29-COL1A1 promoter.All three recombinant vectors were verified by restriction enzyme digestion and DNA sequencing.Either the pcDNA3.1-TGFbeta1 or pJW4303-TGFbeta1 vector along with the pGL4.29-COL1A1 promoter vector or a pRL-null, control reporter, vector were co-transfected into the LX-2 human hepatic stellate cells to establish the transcription-activated dualreporter gene system.This system was used as a cell model for screening anti-liver fibrosis compounds that inhibit the transcription of COL1A1.Dexamethasone, a model drug that is known to inhibit the expression of COL1A1, was used as a control to validate the dual-reporter gene system.

RESULTSThe two TGFbeta1-expressing vectors and the reporter gene vector containing the promoter region of COL1A1 were successfully constructed.The results of a dual-reporter gene assay showed that TGFbeta1 co-expression increased the activity of the COL1A1 promoter by above 200-fold (t =21.78, P =0.0001), whereas in the absence of TGF31 co-expression the activity was below 2-fold (t =3.396, P =0.0274).The transcriptionactivated dual-reporter gene system was successfully established.The model drug, dexamethasone, effectively inhibited the activity of the COL 1A1 promoter in dose-dependent manner; the activity decreased 29.6% with 10 mumol/L dexamethasone (t =4.140, P =0.0144) and 53.9% with 100 mumol/L (t =6.193, P =0.0035).

CONCLUSIONThe dual-luciferase reporter system of TGFbeta1 and COL1A1 co-expression developed here can be used as a cell model to screen and evaluate anti-liver fibrosis compounds that inhibit activity of the COL1A1.

Base Sequence ; Collagen Type I ; genetics ; Drug Evaluation, Preclinical ; Genes, Reporter ; Genetic Vectors ; Humans ; Liver Cirrhosis ; drug therapy ; Luciferases ; Promoter Regions, Genetic ; Transcriptional Activation ; drug effects ; Transfection ; Transforming Growth Factor beta1

Objective To evaluate the efficacy and safety of repeated treatments with low-dose rituximab for relapsing neuromyelitis optica spectrum disorder (NMOSD).Methods A perspective study.21 patients who were diagnosed with NMOSD one year ago were recruited for rituximab treatment.Of 21 patients,one was male,20 were females.Onset age was 10-51 years,the mean onset age was (26.2± 12.0) years.Duration of disease was 2.3-25.8 years,the mean duration was (9.2 ± 5.9) years.Best corrected vision activity (BCVA),expanded disability status scale (EDSS),annualized relapsing rate (ARR) were valued to investigate the efficacy and safety of repeated treatments with low-dose rituximab.The BCVA was examined using Snellen chart,and converted to logMAR.The mean BCVA was 1.13 ± 1.09,the mean BCVA in better eyes was 0.4±0.68,the mean BCVA in latter eyes was 1.87±0.90.The mean EDSS was 3.09±0.70.The mean ARR was 1.04± 0.65.All patients underwent two cycles of RTX treatment.The annually induction treatment was RTX 100 mg per week for 4 weeks.Of 21 patients,12 patients had treatment within one month after attack.The mean follow-up period was (28.4±4.9) months.The side effects were recorded,BCVA,EDSS,ARR were valued to investigate the efficacy and safety of repeated treatments with low-dose rituximab.Paired t test,independent sample t test and Chi-squared test were used.Results The mean BCVA at last follow-up was 0.62 ± 0.91,the mean BCVA in better eye was 0.62±0.91,the BCVA in latter eye was 1.0± 1.01.The mean EDSS was 2.26± 1.07.The mean ARR was 0.21 ± 0.3.After the treatment,patient had significant improvement on BCVA in worst eye (t=4.256),ARR (t=2.900),EDSS (t=4.620) with the significant differences (P＜0.05).Thirteen relapses in 9 patients were observed.B lymph cells were more than 0.01％ in all relapses.There was no significant difference on the BCVA in better eye (t=1.840,P＞0.05).There were 9 patients had relapse,13 times in total.Of 13 relapses,B lymph cell count was performed in 12 relapses,and the counts were 0.01％-0.14％.There were no significant difference between relapsed patients and non-relapsed patients on onset age (t=0.67,P=0.51),whether underwent plasma exchange treatment (x2=1.61,P＞ 0.05),with/without auto-immune antibody ratio (x2=1.61,P＞ 0.05).Of 21 patients,8 patients had side effects,including 5 patients with infection,4 patients with chest congestion,3 patients with hair losing,2 patients with skin rashes,headache and short of breath,1 patient with tinnitus,palpitation and fatigue.Four patients had more than one symptom.Of all patients who had side effects,slowing down the infusion speed of RTX or infusing 5 mg of dexamethasone could relieve the discomfort.Conclusion Lose-dose rituximab reduces the frequency of NMOSD relapses and is well tolerated.