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）
Objective To study the effects of long term application of cathartics on electromyography of rat colon, and to explore the role of interstitial cells of Cajal (ICC) in it. Methods Colonic slow waves of the rat was examined after 3-month feeding of phenolphthalein, and ICC in myenteric plexus was observed by ZIO method, and ultrastructure changes of nerves and ICC was observed. Results The frequency of slow waves of cathartic colon was reduced significantly(Ｐ＜0.05). The distribution of ICC in myenteric plexus was uneven, and the processes were mussily connected each other. Vacuolar degeneration of axon and ICC-like cells was revealed by electron microscope in myenteric plexus of cathartic colon. Conclusion Long term application of phenolphthalein could reduce the frequency of colonic slow waves, and the possible mechanism was degeneration of ICC and myenteric plexus nerves.
ObjectiveTo observe the application value with color Doppler flow imaging (CDFI) in the detection of macular diseases in senile cataract.MethodsA cross-sectional descriptive study. From December 1 to December 30, 2017, 720 senile cataract patients (1380 eyes) diagnosed in Beijing Tongren Eye Center were included in this study. There were 330 males (629 eyes) and 390 females (751 eyes), with the mean age of 70.35±19.05 years. There were 716 right eyes and 664 left eyes, with the visual acuity 0.05-0.8. The fundus of all the patients can be observed. All patients were examined by OCT using Cirrus HD-Model 5000 produced by German Carl Zeiss company and CDFI using MyLab90 color Doppler ultrasound system produced by ESAOTE S.P.A. Comparing the detection rate of macular lesions between the two methods by using χ2 test. Using statistical methods to evaluate diagnostic tests, a crosstabulation was made to compare the consistency of frequency-domain OCT and CDFI in the diagnosis of macular lesions. Logistic regression analysis of correlation between detection of CDFI macular lesions and central foveal thickness (CFT), average thickness (AT), with or without macular anterior membrane, retinal folds, retinal cystoid edema, hard exudation, macular hole or lamellar macular hole, superficial detachment of neuroepithelium, detachment of pigment epithelium, vitreous macular traction.ResultsTwo hundreds and thirty-nine eyes (17.3%) showed macular disease by OCT, and 161 eyes (11.7%) showed the abnormal echo in the macular region by CDFI. The detection rate of the macular disease between two methods were statistically significant (χ2=851.661, P＜0.001), however, the consistency was well (κ=0.766, P=0.000). Logistic analysis showed that the detection rate of macular lesions was related to retinal folds, cysts in retina and CFT (r=1.396, 1.041, 0.12; P＜0.01).ConclusionCDFI can effectively detect macular lesions that affect the visual prognosis of senile cataract patients.
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)
Objective To observe the effect of cationic liposomal ceftazidime (CLC) combined with nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) in the treatment of chronic osteomyelitis of rabbits. Methods Thirty healthy New Zealand white rabbits (4-6 months old; weighing, 2-3 kg) were selected to prepare the chronic osteomyelitis models. After 4 weeks, the gross observation, X-ray examination, and bacteriological and histopathological examinations were done; the models were made successfully in 27 rabbits. Of 27 rabbits, 24 were randomly divided into 4 groups (n=6): only debridement was performed in group A; ceftazidime was given (90 mg/kg), twice a day for 8 weeks after debridement in group B; ceftazidime and n-HA/β-TC were implanted after debridement in group C; and CLC and n-HA/β-TCP were implanted after debridement in group D. Before and after treatments, X-ray examination was done, and Norden score was recorded. At 8 weeks after treatment, the specimens were harvested for gross observation and for gross bone pathological score (GBPS) using Rissing standard; half of the specimens was used for histological observation and Smeltzer scoring, the other half for bacteriological examination and calculation of the positive rate of bacteria culture. Results At 8 weeks after treatment, Norden score of group D was significantly lower than that of groups A, B, and C (P lt; 0.05), but no significant difference was found among groups A, B, and C (P gt; 0.05). At 8 weeks after treatment, sinus healed in groups C and D, but sinus was observed in groups A and B; the GBPS scores of groups C and D were significantly lower than those of groups A and B (P lt; 0.05). The Smeltzer scores of groups C and D were significantly lower than those of groups A and B (P lt; 0.05). The positive rates of bacteria culture of groups C (0) and D (0) were significantly lower than those of group A (25.0%) and group B (16.7%) (P lt; 0.05). Conclusion CLC combined with n-HA/β-TCP has good effect in treating chronic osteomyelitis of rabbits, and it has better effect in treating chronic osteomyelitis of rabbits than ceftazidime with n-HA/β-TCP.
ObjectiveTo explore etiopathogenesis of slow transit constipation(STC). MethodsThe model of rat with “cathartic colon” was established, and the changes of colonic electromyography of the rat was examined. ResultsThe frequency and amplitude of slow wave in vivo of rats with cathartic colon was decreased markedly. Postmeal spike potential and duration was decreased, showing that gastrocolonic reflex of rats colon was decreased.ConclusionLong term abuse of stimulant laxuatives could damage enteric nervous system and accelerate the pathological changes of STC.
Objective To explore the risk factors of catheter-associated bloodstream infections ( CRBSI) in intensive care unit ( ICU) of primary hospital. Methods A total of 623 patients with central venous catheters were recruited in the study. 60 of themsuffered fromCRBSI served as an observation group and other 563 cases without CRBSI served as control. Univariate analysis was used to scan possible risk factors. Then logistic regression analysis was used to exclude the confounding factors. Results The overall incidence rate of CRBSI was 9. 63% ( 60 /623) . There were significant differences in APACHE score, type of catheter, location of catheter, duration of central venous catheter, intravenous nutrition, use of steroid, times of intubation, urgent intubations, nutritional status, diabetes, and MODS between the two groups. Logistic regression analysis revealed that higher APACHE score, double-lumen catheter, femoral vein catheter, catheter indwelling more than two weeks, intravenous nutrition, intubation more than 2 times, and emergency intubation were risk factors of CRBSI. Conclusions Higher APACHE score, double-lumen catheter, femoral vein catheter, catheter indwelling more than two weeks, intravenous nutrition, intubation more than 2 times, and emergency intubation were major risk factors of CRBSI in ICU of primary hospital.
Diabetic retinopathy (DR) is a common ocular complication in diabetic patients, which is chronic and progressive and seriously impairs visual acuity. The rapid occurrence and progress of cataract in diabetic patients is also one of the important reasons for visual impairment in DR patients. Compared with non-diabetic patients, diabetic patients have higher risk of complications after cataract surgery. Studies have shown that anti-vascular endothelial growth factor (VEGF) therapy after cataract surgery can prevent the aggravation of diabetic macular edema in DR patients. However, due to the lack of systematic review of the clinical effect of anti-VEGF drugs in DR patients undergoing cataract surgery, the use of anti-VEGF drugs is relatively conservative in clinic. It is believed that with the deepening of research and the progress of clinical trials, the wide application of anti-VEGF drugs in clinical practice is expected to provide more accurate and effective treatment for DR patients in the future.