ObjectiveTo explore the light sensitivity and kinetic of the new optogenetics tools Channelrhodopsin-XXM2.0 (XXM2.0) and Channelrhodopsin-PsCatCh2.0 (PsCatCh2.0), and analyze whether they could be used to restore the visual function by optogenetics.MethodsMolecular biology techniques were used to link the gene fragments of XXM2.0 and PsCatCh2.0 to the vector pCIG(c)-msFoxn3 containing ampicillin resistant screening gene and reporter gene to form new plasmid pCIG(c)-msFoxn3-XXM2.0 and pCIG(c)-msFoxn3-PsCatCh2.0. The constructed plasmids were transfected into HEK 293T cells, and light responses were recorded in the whole cell mode with the HEKA patch clamp system. The photocurrent was recorded under three light intensity included 2.7×1016, 4.7×1015, and 6.4×1014 photons/(cm2·s). And then, XXM2.0 and PsCatCh2.0 were stimulated with 2.7×1016 photons/(cm2·s) and fully recovered. The opening and closing time constants were analyzed with Clampfit 10.6 software. At the same light intensity, photocurrents of XXM2.0 and PsCatCh2.0 were recorded by the light pulse stimulating of 2-32 Hz. The current attenuation was analyzed at long intervals of 4000 ms and short intervals of 200 ms after repeated stimulation. Comparisons between groups were performed by independent samples t test.ResultsRestriction endonuclease sites of EcoRⅠ and EcoRⅤ were successfully introduced at XXM2.0 and PsCatCh2.0 sequences. When the digestion was completed, they were ligated by T4 DNA ligase to construct new plasmids pCIG(c)-msFoxn3-XXM2.0 and pCIG (c)-msFoxn3-PsCatCh2.0, and then transfected on HEK 293T cells. The light intensity dependence was showed in XXM2.0 and PsCatCh2.0. The greater light intensity was accompanied by the greater photocurrent. Under the light intensity 6.4×1014 photons/(cm2·s) below the retinal safety threshold, large photocurrent was still generated in XXM2.0 and PsCatCh2.0 with 92.8±142.0 and 13.9±5.6 pA (t=1.24, 1.24; P=0.28, 0.29). The opening time constants of XXM2.0 and PsCatCh2.0 were 23.9±6.7 and 2.4±0.8 ms, and the closing time constants were 5803.0±568.2 and 219.9±25.6 ms. Compared with PsCatCh2.0, the opening and closing time constant of XXM2.0 were both larger than PsCatCh2.0. The differences were statistically significant (t=7.10, 31.60; P=0.00, 0.00). In terms of response frequency, XXM2.0 and PsCatCh2.0 could follow to 32 Hz high-frequency pulsed light stimulation, and all could respond to repeated light stimulation at a long (4000 ms) and a short time (200 ms) interval with the small current decay rate.ConclusionXXM2.0 and PsCatCh2.0 could be activated under light intensity with safety for the retina, and could respond to high frequency (at least 32 Hz) pulsed light stimuli with low current attenuation, which could meet the characteristics of opsins required to restore the visual function by optogenetics.
ObjectiveTo explore the light response, retinal inflammation and apoptosis of the retinal ganglion cells (RGCs) 1 year after the new type of channelrhodopsin PsCatCh2.0 was transfected into the retina of rd1 mice. MethodsTwenty-four male rd1 mice were randomly divided into rd1 experimental group and rd1 control group, 12 mice in each group. 1.5 μl of recombinant adeno-associated virus (rAAV)2/2-cytomegalovirus (CMV)-PsCatCh2.0-enhanced green fluorescent protein (EGFP) was injected into the vitreous cavity 1 mm below the corneoscleral limbus of mice in the rd1 experimental group, and the same dose of recombinant virus was injected 2 weeks later at temporal side 1 mm below the corneoscleral limbus. One year after virus injection, the light response of RGCs expressing PsCatCh2.0 was recorded by patch clamp technique; the expression of PsCatCh2.0 in the retina was evaluated by immunofluorescence staining; the transfection efficiency of recombinant virus was evaluated by the transfection efficiency of virus and the number of RGCs. Hematoxylin-eosin staining was performed to measure the inner retinal thickness. Western blotting was used to detect the protein expression of nuclear factor (NF)-κB p65 in retina; real-time quantitative polymerase chain reaction was used to detect the relative expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and Bax mRNA. Terminal deoxynucleotidyl transferase kit was used to observe the apoptosis of retinal cells in each group of mice. ResultsOne year after the intravitreal injection of recombinant virus, PsCatCh2.0-expressing RGCs can still generate 30 pA photocurrent. The virus PsCatCh2.0-EGFP was mainly transfected into RGCs, and partly transfected into amacrine cells, almost no transfection was seen in bipolar and horizontal cells. There were no significant differences in the number of RGCs and thickness of the inner retina between the rd1 experimental group and the rd1 control group (F=14.35, 0.05; P＞0.05), while the rd1 experimental group NF-κB p65 protein expression, TNF-α and IL-6 mRNA quantification were significantly lower than those of rd1 control group (F=4.61, 5.91, 5.78; P＜0.05). The number of red fluorescent apoptotic cells in the retina of mice in the rd1 experimental group was less than that in the rd1 control group, and the Bax mRNA expression was lower than that in the rd1 control group, and the difference was statistically significant (F=7.52, P＜0.01). ConclusionOne year after intravitreal injection of recombinant virus, the PsCatCh2.0 expressing RGCs can still generate photocurrent. Long term transfection and expression of PsCatCh2.0 has no obvious cytotoxic effect on RGCs, nor it increases the inflammatory effect of the retina of rd1 mice with retinal degeneration.
PURPOSE: To explore the pathogenesis of anisometropic and amblyopias. METHODS:To carry out on monocular and binocular atropinized cat models during the developmental period for anisometropia and ametropia ,and measure the cytosomal sectional area and some parameters of the dendric field from the dorsal lateral geniculate nuclei (dLGN)of adult cats by using Golgi-Cox staining. RESULIS:The changes of cytosomal sectional areas and parameters about dendric fields in the dLGN of experimental cats were as following:significant differences between cells of dLGN's A1 lamina by the monocular atropinized eyes and normal ones, binocular atropinized eyea and normal ones;no significant difference between tbat driven by the monoular and binocular atropinized eyes. CONCLUSIONS:There might be resemble pathogenesis between anisomelropic and ametropic amblyopias. （Chin J Ocul Fundus Dis,1996,12:153-156）
ObjectiveTo observe the expression of Rap1, guanosine triphosphate-Rap1 (GTP-Rap1), vascular endothelial growth factor (VEGF) and β-catenin in experimental choroidal neovascularization (CNV).MethodsForty-two brown Norwegian rats were randomly divided into a blank control group (7 rats) and a model group (35 rats). Both eyes were enrolled. The CNV model was established by holmium ion laser photocoagulation in the model group. At 3, 7, 14, 21, and 28 days after photocoagulation, fluorescein fundus angiography (FFA) and choroidal vascular smear were performed to observe the degree of fluorescein leakage and CNV area in rats; Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to detect the expression of Rap1, GTP-Rap1, VEGF, β-catenin and mRNA in CNV.ResultsThe results of FFA examination showed that a large disc-shaped fluorescein leaked in the photo-condensation spot 14 days after photocoagulation. Laser confocal microscopy showed that compared with 7 days after photocoagulation, CNV area increased at 14, 21, 28 days after photocoagulation, and the difference were statistically significant (t=3.725, 5.532, 3.605;P＜0.05). Western blot showed that there was no significant difference in the relative expression of Rap1 protein in CNV at different time points after photocoagulation between the two groups (P=0.156). Compared with the blank control group, the relative expression of GTP-Rap1 protein was significantly decreased, the relative expression of VEGF and β-catenin protein were significantly increased in the model group (P=0.000). The results of RT-PCR showed that there was no significant difference in the relative expression of Rap1 mRNA at different time points after photocoagulation between the two groups (P=0.645), but there were significant difference in the relative expression of β-catenin mRNA (P=0.000). At 7, 14, 21 and 28 days after photocoagulation, there were significant difference in the relative expression of GTP-Rap1 and VEGF mRNA between the two groups (P=0.000).ConclusionsThe expression of GTP-Rap1 in experimental CNV is significantly lower than that in normal rats.
ObjectiveTo investigate the incidence and trendency of healthcare-associated infections (HAIs) in a pediatric intensive care unit (ICU) of a hospital, identify the main objectives of infection control, and formulate corresponding preventive and control measures.MethodsA prospective targeted monitoring method was adopted to investigate HAIs in the pediatric ICU of a hospital from January 2013 to December 2018.ResultsFrom January 2013 to December 2018, the number of target ICU patients was 11 898, the number of patient-days was 55 159; 226 HAIs occurred, the HAI case rate was 1.90%, the incidence of HAI per 1 000 patient-days was 4.10‰, and the adjusted incidence of HAI per 1 000 patient-days was 1.21‰. The main infection site was respiratory tract [83 cases (36.7%)], with ventilator-associated pneumonia in 73 cases (32.3%); secondly, 69 patients (30.5%) had bloodstream infection, among which 48 (21.2%) had non-catheter-related bloodstream infection.ConclusionHospital targeted monitoring is helpful to grasp the situation and trend of HAIs, define the main target of infection control, and formulate corresponding preventive and control measures, which can effectively reduce the incidence of HAIs.
ObjectiveTo evaluate the safety and efficacy of second central venous catheterization in tunnel cuffed dialysis catheter (TCC) dysfunction with fibrin sheath.MethodA total of 14 maintenance hemodialysis patients who required second central venous catheterization were enrolled in West China Hospital of Sichuan University from June 2016 to June 2017 and the clinical information and procedure-related complications were recorded.ResultsAll of the 14 patients were successfully performed with second central venous catheterization, of whom 4 cases had superior vena cava cannulation, 7 cases had right brachiocephalic vein cannulation, 2 cases had internal jugular vein cannulation, and 1 case had external jugular vein cannulation. No procedure-related major complication occurred. During the follow-up, catheter malfunction occurred in 2 cases, which improved by urokinase seal and catheter change, respectively. The rest patients’ catheter function remained normal.ConclusionsWith increasing difficult to construction and maintenance of vascular access, preservation of central vein resource is of high importance. For patients with TCC dysfunction with fibrin sheath, second central venous catheterization based on percutaneous brachiocephalic vein or superior vena cava cannulation is a safe and effective method to establish the lifeline for hemodialysis patients.
Objective To make an individualized administration scheme via evidence-based medicine methods, namely adding heparin into the total nutrient admixture (TNA) solution, so as to help a neonate to prevent the occlusion of peripherally inserted central catheter (PICC). Methods After carefully assessing the condition of neonate, this clinical issue was put forward in accordance with the PICO principles. Randomized controlled trials (RCTs) and systematic reviews on neonates’ PICC occlusion were collected from The Cochrane Library, CCTR, DARE, NGC, MEDLINE (Ovid) and CBM from inception to 2011. The clinical intervention scheme was finally made after the assessment of the retrieved evidence and neonate’s physiological condition. Results A total of 4 RCTs and 1 systematic review related to the issues were identified. The following scheme was finally made for the neonate through the assessment of the retrieved evidence and combination of intentions of the patient’s family members: heparin (0.5 U/mL) was added into TNA to prevent PICC occlusion. During the application, blood routine test and blood coagulation were monitored, and the catheter opening time and extubation reason were recorded. Through the above treatment, the neonate successfully completed the treatment before extubation. The time of both PICC detaining and opening was 20 days in total, and there were no PICC occlusion, no catheter thrombosis, and no catheter related bloodstream infection. Moreover, no observation showed thrombopenia and aggravated coagulation disorders resulted from heparin. Conclusion The evidence-based medicine method is an effective way to make reasonable heparin scheme for neonate, so as to prevent PICC occlusion, reduce catheter thrombosis, decrease risks of catheter related blood circulation infection, assure successful completion of treatment, and guarantee the safety of patients.
ObjectiveTo analyze the effect of mitochondrial ultrastructural changes caused by morphine toxicity on abnormal discharge of cat cerebral cortex, and to explore the possible mechanism of brain function damage caused by morphine dependence.MethodsTwelve domestic cats were divided into control group (3 cats) and morphine exposed group (9 cats) according to the method of random number table. After the model was successfully established by the method of dose increasing, the changes of mitochondrial ultrastructure of cortical neurons were observed under the electron microscope.ResultsElectroencephalogram (EEG) monitoring in morphine exposed group showed that the cortical EEG was widely abnormal, physiological waves were reduced, and abnormal discharges were frequent. And the electron microscopy showed that the number, morphology, internal membrane structure and the inclusion body in the matrix of neurons changed in various aspects. The EEG and electron microscopy of the control group were normal.ConclusionMorphine can damage neurons in the cerebral cortex and lead to abnormal discharge, which is closely related to the ultrastructural changes of neuron mitochondria. The toxicity of morphine mitochondria can be the initial mechanism of energy metabolism dysfunction of brain cells and eventually lead to the disorder of brain electrophysiological function.
ObjectivesTo systematically evaluate the efficacy and safety of dabigatran vs. rivaroxaban for perioperative anticoagulation in the ablation of nonvalvular atrial fibrillation. MethodsPubMed, EMbase, Web of Science, The Cochrane Library, WanFang Data, CNKI and VIP databases were electronically searched to collect cohort studies on the efficacy and safety of dabigatran vs. rivaroxaban for perioperative anticoagulation in the ablation of nonvalvular atrial fibrillation from inception to July 1st, 2018. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included studies, then, meta-analysis was performed by RevMan 5.3 and Stata 12.0 software. ResultsA total of 12 cohort studies involving 4 051 patients were included. The results of meta-analysis showed that: there were no differences in the rate of thromboembolic (OR=0.92, 95%CI 0.36 to 2.35, P=0.86), ischemic stroke (OR=1.15, 95%CI 0.22 to 6.07, P=0.87), major bleeding (OR=0.84, 95%CI 0.43 to 1.66, P=0.61), minor bleeding (OR=0.90, 95%CI 0.60 to 1.34, P=0.60) and pericardial tamponade (OR=1.05, 95%CI 0.45 to 2.47, P=0.90) between dabigatran and rivaroxaban groups. ConclusionsCurrent evidence shows that the efficacy and safety of dabigatran vs. rivaroxaban for perioperative anticoagulation in the ablation of nonvalvular atrial fibrillation are similar. Due to limited quality and quantity of the included studies, more high quality studies are required to verify the above conclusion.
ObjectiveTo investigate the diagnostic value of simultaneous culture of central venous catheter (CVC) blood and peripheral blood for catheter-related bloodstream infections (CRBSI). MethodsNon-septic patients who were treated with CVC for 1 to 7 days were enrolled from February 2011 to February 2015 in the First Hospital of Wuhan City. Blood were collected from both peripheral vein and CVC for bacterial culture once a day. The CVCs were removed from patients who got CRBSI from the first to sixth day and who did not by the end of the seventh day for semi-quantitative catheter culture, quantitative catheter culture, CVC culture and catheter exit-site pus culture. The diagnosis of CRBSI were based on 4 methods as follows:A, both peripheral and CVC blood were positive and the time of CVC blood positive were 2 hours earlier than peripheral blood; B, the colonies of semi-quantitative catheter cultures were ≥15 CFU and the microorganisms in both CVC and peripheral blood were the same; C, the colonies ratio of CVC and peripheral blood cultures were ≥5:1; D, the microorganisms in both the peripheral blood and catheter exit-site pus were the same. The diagnostic value of the four methods was compared. ResultsA total of 1 086 patients were finally included. From 1 to 7 days, 64 patients were peripheral blood positive, 79 were CVC blood positive. The patients diagnosed as CRBSI using A, B, C, and D methods were 58, 55, 51, and 36, respectively. Sixty patients were diagnosed as CRBSI based on the clinical and laboratory methods. For the number of patients diagnosed with CRBSI, there was no significant difference between A and B (P>0.05), as well as A and C (P>0.05), however, significant difference was found between A and D (P<0.05). In the diagnostic value of CRBSI, A is similar to B (sensitivity:93.33% vs. 91.67%, specificity:99.81% vs. 100%, Youden index:0.93 vs. 0.92). A, B and C had almost similar specificity (all >99%), however, A had higher sensitivity (93.33% vs. 76.67%, 58.33%) and Youden index (0.93 vs. 0.76, 0.58). ConclusionSimultaneous culture of CVC blood and peripheral blood has a good diagnostic value for CRBSI.