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.
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 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.
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）
Objective It is difficult to treat chronic osteomyel itis due to the formation of the Staphylococcus aureus biofilms. Liposomal gentamicin-impregnated allogeneic cortical bone can inhibit the formation of the Staphylococcus aureusbiofilms. To explore the treatment of chronic osteomyel itis of rabbit by l iposomal gentamicin-impregnated allogeneic cortical bone. Methods The l iposomal gentamicin, l iposomal gentamicin-impregnated allogeneic cortical bone and gentamicinimpregnated allogeneic cortical bone were produced. Then the chronic Staphylococcus aureus osteomyel itis models of rabbit were made in left lower l imbs of 40 6-month-old rabbits and the right lower l imbs were used as controls. After 2 weeks, the observations of gross and X-ray were done. Four rabbits died within 10 days after the models were made and other 36 rabbits were devided into 6 groups: group A (no antibiotics), group B (intravenous injection of gentamicin), group C (intravenous injection of l i posomal gentamicin), group D (implantation of gentamicin-impregnated allogeneic cortical bone), group E (implantation of l i posomal gentamicin-impregnated allogeneic cortical bone), and group F (implantation of allogeneic cortical bone). After 2 weeks of treatment, the bacterial culture, X-ray and HE staining were done. Results The chronic Staphylococcus aureus osteomyel itis model of rabbit was made successfully. The X-ray showed dissolution of bone and periosteal reaction in groups A, B, C, and F, and no obvious dissolution of bone and periosteal reaction in groups D and E. The Norden scores were (2.5 ± 0.3), (2.1 ± 0.2), (1.5 ± 0.3), (1.5 ± 0.2), (0.9 ± 0.3), and (2.7 ± 0.3) points in groups A-F, respectively; showing significant differences between group A and groups B-E (P lt; 0.05), between groups B, E, F and other groups (P lt; 0.05). The results of blood and marrow cultures for Staphylococcus aureus were positive in groups A and F, and negative in other 4 groups; the results of bone marrow culture for Staphylococcus aureus were positive in 6 rabbits of group B, 4 rabbits of group C and 3 rabitts of group D; and the results were negative in group E. HE staining showed: in groups A and F, abscess and dead bone formed, and no new bone formation were observed; in groups B and C, different degrees of neutrophil accumulation was seen; in group D, some neutrophil accumulation occurred, and osteoprogenitor cells and osteoclasts were seen around implanted bone; and in group E, no neutrophil accumulation was observed, a lot of granulation tissues formed, and osteoprogenitor cells and osteoclasts were seen around implanted bone. Conclusion Implantation of l iposomal gentamicin-impregnated allogeneic cortical bone has remarkly better effect in treating chronic osteomyel itis than intravenous injection of l iposomal gentamicin and implantation of gentamicin-impregnated allogeneic cortical bone.
ObjectiveTo evaluate the effectiveness and safety of the additional catheter-directed thrombolysis (CDT) and conventional treatment (CT) for treatment primary deep vein thrombosis.
MethodsDatabases such as CNKI, WanFang Data, Pubmed, EMBASE.com, Medline, CBM, CSJD, CJFD, and the Cochrane Library were electronically searched from the date of their establishment to 30 June, 2013, and the relevant literatures and conference proceedings were also manually searched to include randomized controlled trials (RCTs) on comparison of additional CDT versus CT for primary deep vein thrombosis. Two reviewers independently screened studies according to the inclusion and exclusion criteria, extracted data, and accessed the methodological quality of the included studies. Then the meta-analysis was performed by using RevMan 5.1 software. Publication bias was assessed by STATA software. A meta-regression model was used to describe between study variability.
ResultsTwo RCTs including 3 literatures contain 224 patients reporting data on safety and efficacy of CDT versus CT were included. There were no publication bias (Begg'S test, Z=1.02. P > 0.05;Egger'S test, t=0.98, P > 0.05). The results of meta-analysis showed that:in 6 months after surgery iliofemoral vein patency rate of CDT group was higher than that of CT group (OR=3.62, 95% CI:1.51-8.64, P=0.004), in 6 months after surgery CDT group with a lower rate than CT group of the iliofemoral vein occlusion and (or)venous reflux rate (OR=0.24, 95% CI:0.11-0.53, P=0.000), and the incidence rate of PTS in 24 months after surgery in CTD group was less than that of CT group (OR=0.55, 95% CI:0.31-0.96, P=0.040). There were no statistically significant of the major complications after surgery between CDT group and CT group (OR=1.34, 95% CI:0.12-15.69, P=0.810). But the incidence rates of minor complications and total complications after surgery in CT group were lower than those of CDT group (OR=13.67, 95% CI:4.08-45.83, P < 0.00 01 and OR=11.67, 95% CI:4.40-30.99, P < 0.000 01).
ConclusionsCDT is a effective and positive way to treat early deep venous thrombosis especially in terms of the patency of vascular and prevent the occlusion and PTS. Due to the limitation of the included studies, large sample size, multicenter, and high quality studies are needed to verify the above conclusion, such as the ATTRACT Study. The intracavitary therapy could be applied to clinic combining individual conditions of patients.
Objective
To prepare cationic Vancomycin hydrochloride multivesicular liposome (MVL) and to inspect its quality.
Methods
Cationic Vancomycin hydrochloride MVLs were prepared by double emulsion method, and the storing solution of Vancomycin was prepared. The analysis method of Vancomycin in vitro was established; the specificity, precision, and resorption rate were estimated. Reverse phase high performance liquid chromatography (RP-HPLC) was used to determine the concentration of Vancomycin, encapsulation efficiency, and release characteristics in vitro. The formulation and pharmaceutical process were optimized by single factor experiments and orthogonal experimental design with the factor of encapsulation efficiency as the criteria. The liposome morphology was observed by optical microscopy and transmission electron microscopy. The particle size and Zeta potential were determined by Malvern instrument. The stability was analyzed by dynamic analysis.
Results
An RP-HPLC method was established for the assay of Vancomycin. The analysis method was precise, simple, and reliable for the quality control of Vancomycin. Vancomycin hydrochloride MVLs were round and well-distributed. The average particle size and the encapsulation efficiency were 3.3 μm and 24.9%, respectively. Zeta potential was 24.53 mV, and 90.5% of Vancomycin hydrochloride was released after 264 hours in normal saline under 37℃. Cationic Vancomycin MVLs
were stored for 1 month at 4 ℃, which mantained good stability.
Conclusion
Cationic Vancomycin hydrochloride MVLs have
good appearance, high encapsulation efficiency, good stability, and significant sustained release properties.
Objective
To investigate the effects of phacoemulsification on macula in diabetics．
Methods
Thirty eyes of cataract in diabetics were chosen randomly for measurement of the thickness of fovea of retina using OCT before phacoemulsification and 1 month after surgery ． The other eyes in these patients and 30 eyes of cataract in nondiabetic pati ents with phacoemulsification were as control．
Results
In 30 eyes of diabetics with phacoemulsification, the mean fovea thickness were (148．5plusmn;27．7) mu;m preoperatively and (219．4plusmn;68．23) mu;m postoperatively, and the difference was significant (Plt;0．05)． In 30 eyes of diabetics without surgery, the mean foveal thickness were (147.4plusmn;27.5) mu;m preoperatively and (148.2plusmn;27.3) mu;m postoperatively and the difference was not significant (Pgt;0．05)． In 30 eyes of cataract in nondiabetic patients, the mean fovea thickness were (142．37plusmn;12．7) mu;m preoperatively and (151．9plusmn;23．7) mu;m postoperatively and the difference was not significant (Pgt;0．05)． In 30 eyes of diabetic s with phacoemulsification, 11 eyes had new macula edema after surgery and 3 eye s had significant retinal thickening． In 6 eyes with macular edema before surgery, the macular edema were aggravated in 3 eyes after surgery． The macular stru ctural changes were not found in two control groups．
Conclusion
The thickness of retina is inreased after phacoemulsification in deabetics,and morbidity and its severity of postopevative macular edema are increas ed as well．
(Chin J Ocul Fundus Dis, 2001,17:175-177)
Abstract： Objective To analyze clinical outcomes of interventional therapy for common congenital heart diseases （CHD）. Methods We retrospectively analyzed the clinical records of 57 patients with CHD who underwent catheter interventional therapy in People’s Hospital of Deyang City between March 2009 and January 2012. There were 31 male patients and 26 female patients with their mean age of 29.4±3.5 years（ranging from 1.5 to 75.0 years）. There were 21 patients with patent ductus arteriosus （PDA）, 12 patients with ventricular septal defect （VSD）, and 20 patients with atrial septal defect（ASD）；and 2 patients with VSD plus ASD, 1 patient with VSD plus PDA, and 1 patient with ASD plus PDA. Occluder and supplying system made in China were used in the interventional therapy. For the patients with ASD plus PDA, PDA was blocked before ASD；for the patients with VSD plus PDA, PDA was blocked before VSD；and for the patients with VSD plus ASD, VSD was blocked before ASD. Results The duration of interventional therapy ranged from 30 to 90 minutes. The success rate of operation was 98.3%（56/57）. Operation was given up in an old female patient with ASD and chronic obstructive pulmonary disease because of pulmonary hypertension. The hospital stay was 3 -7 days. All the patients were followed up at the outpatient department for 1 month to 2 years by color Doppler echocardiogram, chest X-ray and electrocardiograph, and no complication occurred during follow-up. Conclusion With complete understanding of surgical indications and strict compliance with procedures, catheter interventional therapy is safe, minimal invasive, and effective in CHD treatment.
Objective Healthcare risk has become a popular topic in medical research field all over the world. With the increasing of healthcare safety adverse events and medical conflict cases every year, publications about this topic have been rapidly increased in China, but research on the essentials and categories of healthcare risk are still lacked. This paper aims to summarize the essentials and categories of healthcare risk research systematically through analyzing domestic studies in healthcare risk over the past decade, so as to provide decision-making evidences and policy suggenstions on healthcare risk management in China. Methods We searched the China Academic Journal Network Publishing Database of CNKI (1999-2010), in which those papers’ title containing the phrase of healthcare risk had been brought into our research, then we summerized the domestic concepts and contents in healthcare risk by essential analysis and frequency statistics. Results 255 papers were included, including 26 Class B papers (accounting for 10.20%), six Class C papers (accounting for 2.35%) and 223 Class D papers (accounting for 87.45%); but there were only 79 papers (accounting for 35.11% ) which had a clear definition of healthcare risk. From these papers, we found the essentials of healthcare risk included object, consequences, process and causes, and the research category of healthcare risk included prevention and treatment of risk, causes and effects of factors, risk sharing mechanisms, healthcare risk of special diseases, concept and characteristics, education and training, management method, risks of medical appliances, the species and object of healthcare risk, the category of healthcare risk, the research methods of healthcare risk etc. Conclusion At present, the study of healthcare risk focuses on the risk control and how to release risks from the results, besides, systematic studies are lacked, and the unified definition of healthcare risk has not been formed, Therefore, we recommend that systematic study of the healthcare risks should be strengthened, the definition of healthcare risk should be unified, and the early warning, monitoring, evaluating, and identifying methods should be positively developed, providing decision-making evidences to establish healthcare risk management system in China.
