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Objective: To establish the rat models of myocardial ischemia reperfusion-related no-reflow under non-artificial ventilator, to evaluate the models by morphology, hematological biochemistry and hemorheology, and to lay the foundation for studying the pathogenesis of cardiovascular diseases and evaluating the pharmacodynamics of drugs of cardiovascular diseases. Methods: Fifty healthy female Wistar rats were randomly divided into sham operation group (n=20) and model group (n=30). The myocardial ischemia reperfusion-related no-reflow rat models in model group were induced by ligation of left anterior descending coronary artery for 2 h and another 2 h for reperfusion. The rats in sham group were only threaded and not ligated. The model was evaluated by detecting the area at risk (AAR), area at infarct (AAD, area at no-reflow (AAN) of myocardium, the activities of creatine kinase MB (CK-MB), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in serum, the whole blood viscosity, the plasma viscosity, the platelet adhesion rate (PAR), the platelet aggregation (PAG) of the rats. Results: Compared with sham operation group, the AAR, AAI, and AAN of myocardium, the activities of CK-MB, LDH, and AST in serum, the whole blood low shear viscosity (20/s), the middle shear viscosity (60/s), the high shear viscosity (120/s), the plasma viscosity, PAR, PAG (1, 3, 5 min) and the maximum platelet aggregation rate (MAPG) of the rats in model group were significantly increased (P<0. 05 or P<0. 01). Conclusion: The rat model of myocardial ischemia reperfusion-related no-reflow under non-artificial ventilator is successfully established. This method is characterized by simple operation, decreased injury to animals, higher stability of model construction, shorter experimental cycle and less cost.
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Objective@#To establish the in vitro porcine gastric model of submucosal eminence lesion and to evaluate its application to endoscopic submucosal dissection(ESD).@*Methods@#Silicone rubber impression materials and steel balls with diameters of 1 cm, 2 cm, and 3 cm were used to make three pairs of spherical cavities. And then raw ground beef was put into spherical cavities and boiled for 20 minutes to make spherical mass models. Six isolated porcine stomach with esophagus and duodenum were selected. The mass models with diameters of 1 cm, 2 cm and 3 cm were imbedded respectively into the submucosa of fundus, body, and antrum of porcine stomach through the incision on serosal layer. The submucosal masses were observed by endoscopy and endoscopic ultrasonography and ESD was performed.@*Results@#A total of 18 mass models were constructed in 6 porcine stomachs, of which 17 models were successfully established and 1 failed. Typical endoscopic characteristics of gastric submucosal eminence lesions were found in 17 models. Endoscopic ultrasonography showed that these models originated from submucosal layer and demonstrated mixed echo. There were no significant differences between mucosa of lesions and that of surrounding areas. ESD was successfully performed in the porcine gastric models of submucosal eminence lesions, and all models were not broken or detached.@*Conclusion@#The in vitro porcine gastric model of submucosal eminence lesions can well replicate disease status and provide a suitable model for study on endoscopic therapy of submucosal eminence lesion and training of endoscopists.
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Models of oxygen-induced retinopathy (OIR) have been built on several animals,including kitten,mouse,rat,rabbit,puppy and zebrafish.These models play an important role in studying the mechanisms and treatments of retinal neovascularization,which is partially mimicking the pathological process of retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR).As the extensive application of transgenic technology,animal models of OIR will provide more evidences on investigating the retinal angiogenic diseases.However,none of these OIR models is perfect to simulate human ROP completely,which still needs more comprehensive and profound exploration.
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Objective To establish a central serous chorioretinopathy (CSC) model on different strains of rabbits by intravenous injection of adrenaline,which may contribute to related researches of CSC.Methods This study was approved by Bioethics Committee of Fourth Military University and complied with Statement for the Use of Animals in Ophthalmic and Visual Research.Fundus fluorescein angiography (FFA) was initially performed on male New Zealand white rabbits (10),Belgium rabbits (5) and Chinchilla rabbits (10) to make sure that the retinas of subjects were normal.For the New Zealand white rabbits,adrenaline was injected via ear vein at a dose of 0.04 mg/kg once per day for the first 8 weeks and followed by a dose of 0.08 mg/kg for the next 4 weeks,while 0.04 mg/kg adrenaline was injected in the same way for 8 weeks in the Belgium rabbits and Chinchilla rabbits.FFA was performed every week after injection of adrenaline to evaluate the fluorescence leakage in ocular fundus.New Zealand white rabbits were sacrificed in 4 (3 rabbits),8 (3 rabbits) and 12 weeks (4 rabbits) after injection respectively,and Belgium rabbits and Chinchilla rabbits were sacrificed in the 8 weeks after injection.The eyeballs of the rabbits were enucleated to prepare the retinal sections for histopathological examination after hematoxylin-eosin staining.The results of FFA and retinal structure were compared among different strains of rabbits.Results No fluorescence leakage was found by FFA in ocular fundus,and the retinal structure was normal in all the 10 New Zealand white rabbits during the experiment.Fluorescence leakage was found by FFA in 2 Belgium rabbits at 1 week and 2 weeks after injection respectively,and retinal detachment and depigmentation of retinal pigment epithelium (RPE) with an enlarged intercellular space were shown by hematoxylin-eosin staining.For the Chinchilla rabbits,fluorescence leakages were found in 7 rabbits throughout the whole period of adrenaline administration.Circumscribed retinal detachment,depigmentation of RPE with enlarged intercellular space were also found in leakage lesions.Conclusions Repeated intravenous injection of adrenaline can induce CSC-like lesions in colored rabbits but not in albino rabbits.
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Background Suture stitching is currently the standard treatment for corneal penetrating injuries.The shortcomings of suture stitching have led to the exploration of non-sutured surgical methods.Tissue adhesive is a promising non-suture replacement procedure.Objective This study was to observe the effect of fibrin glue on large irregular rabbit corneal penetrating injury.Methods Eighteen pure white healthy rabbits were randomly divided into suture group and adhesive group.A total length of 6-mm non-self-sealing corneal penetrating injury in nonpupillary-area of the right eye formed with a 15° corneal puncture knife was repaired with fibrin glue plus temporary suture and therapeutic corneal contact lens (9 eyes) or with 3 to 5 interrupted 10-0 nylon sutures (9 eyes) with the fellow eyes acted as the internal controls respectively.Operative time was compared between the two groups.Clinic observation was performed with slit lamp microscope.Animals were humanely sacrificed for histologic examination at week 1,3,and 8 to evaluate wound healing.Results The average operation time was (3.48±0.48) minutes in the adhesive group,which was significantly lower than that in the suture group ([7.77 ± 1.30] minutes) (t =9.28,P＜ 0.01).The postoperative slit lamp microscope observation indicated corneal wounds were quickly and regularly healed in the glued corneas compared with the stitched ones.The histologic examination revealed that glued corneas had regular healing,mild inflammation,and no corneal neovascularization,while sutured corneas showed irregular fibrin arrays,a large number of inflammatory cells and macrophages infiltration around the suture,heavy inflammatory response.Neovascularization was found at week 3 postoperatively.Conclusions Fibrin glue combined with temporary suture and therapeutic corneal contact lens is an effective treatment in sealing large irregular corneal wounds with considerable advantages over traditional sutures,ircluding simplified operative technique,short surgery time,less postoperative irritation,mild inflammation,more regular wound healing,short healing time,and no corneal neovascularization.
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Objective To compare the repair effect on renal function between different times of bone marrow mesenchymal stem cells (BMSCs) transplant via renal artery route in experimental rats with adriamycininduced nephropathy.Methods Adriamycin-induced nephropathy model was established in 32 rats through injection of adriamycin though the caudal vein.Based on the scheduled times of BMSCs transplant,the experimental rats were randomly and equally divided into M0 group (zero time),M1 group (one time),M2group (2 times) and M3 group (3 times) with 8 rats in each group.Other 8 SD rats were used as normal control group (N group).Single dose of 0.5 rnl BMSC suspension (2×106 cells/ml) was transplanted to the rats of M0 group (zero time),M1 group (one time),M2 group (2 times) and M3 group (3 times),for the rats of the groups not receiving BMSC transplant a single dose of 0.5 ml L-DMEM culture medium,used as a placebo,was adopted to replace BMSC suspension.The transplant interval was one week.Before transplant as well as one and two weeks after last time of transplant,the serum urea nitrogen,serum creatinine,24 h urine protein and 24 h urine microprotein were tested,and one week after last time of transplant pathological sections were made for laser focusing microscope examination to observe renal pathological changes and the distribution of BMSC cells in the kidney.Results The values of serum urea nitrogen,serum creatinine,24 h urine protein and 24 h urine microprotein determined at each observation time point in M0 group,M1 group,M2 group and M3 group were significantly higher than those in N group (P＜0.001).The values of 24 h urine protein and 24 h urine microprotein determined at one week after last time of transplant in M2 group and M3 group were strikingly lower than those in M1 group (P＜0.05),but these differences between M2 group and M3 group were not statistically significant (P=0.063).Conclusion For the treatment of adriamycin-induced nephropathy in experimental rats,two times of using BMSCs transplant via renal artery route can achieve optimal curative effect.
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Background It has not been reported that if the visual cortex M receptor changed during the development of myopia and how it changed if given acupuncture treatment.Objective The aim of this study was to observe the effect of electroacupuncture stimulation on the expression of acetylcholine receptors M1 (AchRM1) in visual cortex of guinea with lens-induced myopia (LIM).Methods Forty-eight three-week-old healthy guinea pigs were randomized into the normal control group,the LIM model group and the LIM electroacupuncture group.The right eyes of the guinea pigs were selected as the experimental eyes.LIM was created by monocularly wearing of-10 D lens for 4 weeks in the right eyes in the LIM model group and LIM electroacupuncture group,and then the acupuncture at the temple and hegu point was performed for 30 minutes per day for consequent 4 weeks,in the LIM electroacupuncture group.The fellow eyes of the guinea pigs were used as the self-control eyes.The refractive power and axial length were examined with retinoscopy and A-type sonography before and 4 weeks after modeling,respectively.The animals were sacrificed by excessive anesthesia at the fourth week after acupuncture and visual vertex tissue was obtained.The expression of M1 receptor mRNA in visual vertex was detected by fluorescence quantitative PCR,and the content of M1 receptor protein in visual vertex was assyed by ELISA.The study protocal was approved by Animal Ethic Committee of Shandong University of Traditional Chinese Medicine,and the use and care complied with Statement of the Association for Research in Vision and Ophthalmology.Results At the fourth week after modeling,the mean diopters were (-3.24±0.28) D and (-3.30±0.45) D in the LIM model group and the LIM eleetroacupuncture group,which were significantly higher than (0.83 ±0.86)D in the normal control group (both at P=0.000),and there was no significant difference in the diopter between the LIM model group and the LIM electroacupuncture group (t =0.200,P =0.659).The mean axial lengths were (8.67 ±0.14) mm and (8.60±0.06) mm in the LIM model group and the LIM electroacupuncture group,which were considerably increased in comparison with (8.33±0.08)mm in the normal control group (both at P＜0.05).The relative expression levels of AchRM1 mRNA in visual cortex were 0.79±0.18,1.36±0.23 and 1.13±0.13 in the normal control group,LIM model group and LIM electroacupuncture group,and the relative expression level of AchRM1 mRNA in the LIM electroacupuncture group was significantly higher than that of the normal control group and lower than that of the LIM model group (both at P＜0.05).In addition,the contents of AchRM1 receptor protein in the visual cortex were 248.00±33.31,455.17±42.40 and 396.17±47.57 in the normal control group,LIM model group and the LIM electroacupuncture group,with a similar pattern among the groups (both at P＜0.05).Conclusions A electroacupuncture stimmulation do not affect the myopic diopter and axial length in LIM model.The AchRM1 and AchRM1 receptor in the visual cortex up-regulate in LIM eyes,infering that electroacupuncture stimmulation can improve vision by decreasing the level of AchRM1 receptor in visual cortex in LIM eyes in guinea pigs.
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Background p21 is a cyclin-dependent kinase inhibitor,and it can prevent cells from going through the G1/S phase checkpoint and inhibit cell proliferation.Stuies determined that the expression level of p21 WAF1/CIP1 is associated with proliferative diseases.Traumatic proliferative vitreoretinopathy (PVR) is a proliferative response of eye.Understaining the relationship of dynamic expression levels of p21 WAF1/CIP1 in PVR is of significance for the prevention and management of PVR.Objective This study was to investigate the expression of p21 WAF1/CIP1 during the course of experimental traumatic PVR in rabbits.Methods Fifty-four pigmented rabbits were randomized into the normal control group and different experimerital groups,and one lateral eye of each rabbit served as experimental eye.PVR models were established by intravitreal injection of human platelet-rich plasma (PRP) (0.4 ml)combined with cryotherapy for 5 seconds,and vitreous and retinas were examined with B type sonography.The rabbits were sacrificed in 7,14,21 and 28 days after operation,and histopathological examination of the retinas was performed by haematoxylin and eosin stain.The expression levels of p21WAF1/CIP1 protein and gene were detected by immunohistochemistry,Western blot and reverse transcription-PCR (RT-PCR).The use and care of the rabbits complied with Statement of ARVO.Results B type sonography showed that the retinal morphology was normal in the normal control group.However,the proliferative membrane was gradually thickened 1 to 7 days after operation.Retinal folds of rabbits were seen in 7 days,and tractional retinal detachment was found in 14 days and 28 days after operation.The histopathological examination of the retinas showed epiretinal membrane and infiltration of inflammatory cells 7 days and fixed ruffle 28 days after operation.The p21WAF1/CIP1 was strongly expressed in the cell nucleus of retinal ganglion cell layer (GCL) and inner nuclear layer (INL) in the normal control group,and the expression was gradually weakened after modeling,with the weakest expression in the retinas in 14 days after modeling.The relative expression levels of p21 WAF1/CIP1 protein was 0.74±0.08,0.60±0.05,0.56±0.03,0.74±0.02 and 0.65 ±0.04 in the normal control group,postoperative 7-day group,postoperative 14-day group,postoperative 21-day group and postoperative 28-day group,respectively,showing a significant difference among the groups (F =20.55,P =0.00),and the expression levels of p21WAF1/CIP1 protein were significantly lower in the postoperative 7-day group and postoperative 14-day group than those of the normal control group,postoperative 21-day group and postoperative 28-day group (all at P＜0.05).The relative expression levels of p21 WAF1/CIP1 mRNA was 0.65 ± 0.09,0.57 ± 0.05,0.45 ±0.04,0.46±0.02 and 0.47±0.04 in the normal control group,postoperative 7-day group,postoperative 14-day group,postoperative 21-day group and postoperative 28-day group,respectively,with a significant difference among the groups (F =18.06,P =0.00),and the expression levels were significantly lower in the postoperative 14-day group,postoperative 21-day group and postoperative 28-day group than those of the normal control group and postoperative 7-day group (all at P＜0.05).Conclusions The dynamic expression of p21WAF1/CIP1 in the retinas is consistant with the prograssion of traumatic PVR,and the reduce tendency of p21 WAF1/CIP1expression is similar to cell prolieration change,indicating that reduce of p21WAF1/CIP1 expression in the retinas may promote the development of traumatic PVR.
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Objective To observe the effects of Yougui Pills (YP) intervention on SREBP pathway related factors of sterol regulatory element binding protein-1c ( SREBP-1c) , cholesteryl ester transfer protein ( CETP) and fatty acid synthase ( FAS) protein and gene expression in kidney yang deficiency hyperlipidemia rat model. Methods Sixty SD rats were divided into normal control group, model group (in the dosage of 2.43 g/kg), and YP group. Rat model of kidney yang deficiency hyperlipidemia was induced with intramuscular injection of hydrocortisone. Normal control group and model group were given normal saline, and YP group was given YP suspension intragastrically. After treatment, the hepatic pathomorphology of rats in the three groups was examined, and the contents of serum lipids were examined. Hepatic SREBP-1c, CETP and FAS protein expression was detected by Western-blotting method, and their mRNA expression was detected by real-time polymerase chain reaction ( RT-PCR) . Results Compared with the normal control group, serum triglyceride ( TG ) , total cholesterol ( TC ) , low-density lipoprotein cholesterol ( LDL-C ) contents were significantly increased (P<0.01), high-density lipoprotein cholesterol (HDL-C) was significantly decreased (P<0.01), and hepatic pathomorphological changes of hyperlipoidemia were obvious in the model group. Compared with the model group, TG, TC, LDL-C contents were significantly decreased ( P<0.01) , and HDL-C was significantly increased (P<0.01) in YP group. Model group had higher SREBP-1c, CETP and FAS mRNA and protein expression than the normal control group ( P<0.05) , while YP group had lower SREBP-1c, CETP and FAS mRNA and protein expression than the model group ( P<0.05) . Conclusion YP can decrease the blood lipid levels probably by down-regulating the expression levels of SREBP-1c, CETP and FAS gene and protein related to the the SREBP pathway in rats of kidney yang deficiency hyperlipidemia induced by intramuscular injection of hydrocortisone.
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Background Researches showed that CD4+CD25+ natural regulatory T cells (nTregs) play an important role in maintaining peripheral immune tolerance, while immunotherapy using in vitro-expanded induced regulatory T cells (iTregs) suppresses allograft rejection in multiple organ transplantation.The inducing method of iTregs still needs to be optimized.Furthermore,the effect of iTregs on grafts of keratoplasty is unclear.Objective This study was to investigate the inducing and expansion method of iTregs and explore its inhibitory effects on corneal allograft rejection.Methods Bone marrow-derived dendritic cells (BMDCs) were isolated from C57BL/6 mice femora and cultured.CD4+ CD25+ T cells and CD4+ CD25-T cells were isolated from mouse spleen and separated using flow cytometry.The CD4+CD25-T cells were divided into negative control group (CD4+CD25-T cells), CD3/ 28 antibody bead group (CD4+CD25-T cells+CD3/28 antibody bead) ,2.5 ng/ml transforming growth factor (TGF)-β1 induced group and 10.0 ng/ml TGF-β1 induced group.The iTregs was formed after induction of different concentrations of TGF-β1 and CD3/CD28 antibody bead (1 : 1).CD3/CD28 antibody bead (1 : 2) , interleukin-2 (IL-2) and TGF-β1 were used to expand iTregs.The phenotype and proliferation of iTregs were assayed by flow cytometry,and the inhibitory effect of iTregs on effector T cells (Teffs) was analyzed by mixed lymphocyte reaction.Allogenic keratoplasty model (C57BL/6→BALB/c) was build,and 0.1 ml iTregs or nTregs suspension or PBS was injected via posterior venous plexus of fellow eyes to assess the graft survival time.The use and care of the mice followed the ARVO statement.Results The CD4+CD25+ T cell proportions were (6±3)％ ,(91±4)％ ,(91±3)％ and (86± 6) ％ in the negative control group,CD3/CD28 antibody bead group, 2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1induced group, showing significant increases in the CD3/CD28 antibody bead group, 2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1 induced group compared with the negative control group (all at P＜0.01).The Foxp3+ T cell proportions of the CD3/CD28 antibody bead group,2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1 induced group were (1.18 ±0.20) ％ , (8.70± 1.80) ％ and (21.80±3.36) ％ , showing significant increases in the 2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1 induced group compared with the CD3/CD28 antibody bead group (both at P＜0.01).Compared with the nTregs, the expression of CD69 was lower, and the expressions of PD-1 and Foxp3 were raised in the iTregs (all at P＜0.01).The proliferation of Teffs were decreased when cocultured with iTregs in comparison with nTregs at 1 : 1,1 : 2,1 : 4,1 : 8,1 : 16 Tregs/Teffs rations (all at P＜ 0.01).The survival time of mouse corneal grafts was 4 weeks with the permanent tolerance of 50％ in the iTregs injected group,which was superior to the 3 weeks survival time and 17％ permanent tolerance in the nTregs injected group(P＜0.05).Conclusions TGF-β1 can induce CD4+ CD25-T cells to form iTregs, which highly express Foxp3.iTregs show a stronger inhibitory effect on the growth of lymphocytes than nTregs, and therefore suppress the graft rejection after keratoplasty.
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Background Flicker light can induce myopia,but its mechanism remains unclear.As one of immediate early genes,early growth response-1 (Egr-1) gene can generate rapid response to visual stimulation,however,its effect on the formation and development of myopia is below understood.Objective This study was to investigate the dynamic expression of Egr-1 gene in retinas of flicker light-induced eyes (FL) and compare the results with form deprived eyes (FD).Methods One hundred and fifty 28-day-old C57BL/6J mice were randomly assigned to the normal control group,FD group and FL group.The right eyes of mice were occluded with a semitransparent hemispherical thin plastic shell for 2 weeks in the FD group,and the right eyes of mice were stimulated by 2 Hz flicker light for 2 weeks in the FL group,and then the mice were fed in the normal light environment for 1 week.The refractive state and axial length of the model eyes were measured by murine-specific eccentric infrared photorefraction and A-scan ultrasonography before modeling and 1 hour,I day,1 week,2 weeks after modeling as well as 1 week after termination,respectively.The mice were sacrificed in above-mentioned time points to isolate the retinas.The expressions and location of Egr-1 protein and mRNA in the retinas were detected by Western blot,and reverse transcription PCR (RT-PCR) and immunochemistry.The expressions of Egr-1 markers,neuron and protein kinase C (PKC)-α,in the retinas were assayed by using immunofluorescence.The care and use of the animals followed the administration regulations for experimental animals of Jiangsu Province.Results Two weeks after modeling,the refraction of the FL group was (0.32±0.14) D,which was significantly lower than (-0.66±0.43)D in the FD group (t=6.78,P=0.00).One hour after modeling,The expression levels of Egr-1 mRNA in mouse retinas were 0.626±0.044 and 0.695±0.058 in the FD group and FL group,which were significantly declined in comparison with 1.009±0.089 of the normal group (t=14.81,P=0.01;t=9.15,P=0.03).In 2 weeks after modeling,the expression levels of Egr-1 mRNA were still lower in the FD group and F:L group compared with the normal group (all at P＜0.05).However,the expression levels were significantly elevated in the FD group and FL group compared with the normal group (t=4.13,P=0.01;t=4.26,P=0.01) at 1 week after termination.Western blot showed a dynamic decrease in the expressions of Egr-1 protein with lapse of time in the FD group and FL group with the lowest expressing level in the second week after modeling.In I week after termination of modeling,the expressing level was raised in the FD group or the FL group,but it was still lower than that ir the normal group (t =6.32,P=0.00;t =5.45,P=0.01).Egr-1 protein was mainly expressed in the retinal ganglion cell (RGC) layer,inner nuclear layer and photoreceptor layer in the normal mice,and the expression intensity was obviously weaker in the FD mice and FL mice 2 weeks after modeling.Htowever,the expression was enhanced in 1 week after termination of modeling.Neuron and PKC-α were strongly expressed in the RGCs and bipolar cells in the normal mice.Conclusions The eyes show a myopic trend after induce of flicker light in B6 mice.The expression level of Egr-1 gene in the retina down-regulates with the reduce of refraction in FL eyes,and its dynamic expressing change is consistent between the FD eyes and FL eyes.
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Background High myopia is seriously harmful to visual function.To explore the mechanism of high myopia is significant for the prevention and treatment.Complement proteins C1q and C3 have emerged as critical mediators of synaptic refinement and plasticity,however,their effects in retina on myopia remain elusive.Objective This study was to investigate the expressions of complement factors C1q and C3 in the retina with negative lensdefocused myopia in guinea pigs.Methods The use and care of animals complied with the application of Peking Union Medical College Hospital Iaboratory Animal Welfare Ethics Committee.Twelve 3-day-old pigmented guinea pigs were assigned randomly to the lens-defocused group and the normal control group.The left eyes were covered by -10 D PMMA lens for 4 weeks as the defocused eye group,and the right eyes were covered using 0 D PMMA lens in the same way as the fellow control eye group.The right eyes of the normal guinea pigs were used as the normal control eye group.The refractive diopter of guinea pigs was examined by retinoscopy.The animals were sacrificed at the fourth week and the expressions of complement C1q and C3 in the retinas of guinea pigs were detected by Western blot.Results The diptors were (-1.21±0.71)D,(+2.46±0.75)D and (+1.75±0.50)Din the defocused eye group,normal eye group and the fellow eye group at the fourth week,showing a significant difference among the groups (F=51.55,P=0.69),and diopters were insignificantly different between the normal eye group and fellow eye group (q =2.62,P=0.08).However,the diopters of the defocused eye group were significantly higher than those of the fellow eye group (q =10.92,P＜0.01).The expressions of C1q and C3 proteins in the retinas were significantly different among the defocused eye group,fellow eye group and normal eye group (C1 q:F=8.810,P =0.003;C3:F =14.490,P＜0.001),and expression levels of C1q and C3 proteins in the defocused eye group were significant higher than those of the fellow eye group (C1q:q=4.14,P=0.01 ;C3:q=4.71,P=0.005) ;while no significant differences were found between the fellow eye group and the normal eye group (C1q:q =1.61,P =0.27; C3:q =2.82,P =0.070).Conclusions The expression levels of C1q and C3 up-regulate in the retinas with lens-defocused myopic animal model.Excessive complement activation in retinas may be involved in the development of myopia.
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Background Retina fixed flat-mount perfused by Evans blue (EB) is a common method for the evaluation of blood-retinal barrier (BRB).However,previous method is inconvenient for some laboratories because the retinal specimen can not be observed by gereral microscope rather than confocal laser scanning microscope after the fixation.Objective This study was to modify the preparing way of flat-mounted retina in order to obtain transparent specimen for the observation of rat retinal vessels and the evaluation of leakage under the ordinary fluorescence microscope.Methods Forty male SD rats were divided into the control group,diabetes mellitus (DM) 1-month group,DM 3-month group and DM 6-month group according to the random number table.Streptozotocinum (STZ) of 2％ dissolved in 0.05 mmol/L sodium citrate-hydrochloric acid buffer was intraperitoneally injected in SD rats to establish DM models,and the equal volume of solvent was injected in the same way in the control rats.One month,three months and six months after injection,EB of 30 g/L was injected via rat femoral vein in the dose of 45 mg/kg.Fifteen minutes after injection of EB,the rats were sacrificed and the retinas were isolated and cut radially to prepare the flat-mounted retinas in PBS immediately and then were dried till the specimens were transparent.The specimens were examined under the fluorescence microscope.The percentage of EB leakage was quantitatively calculated by IPP 6.0 software.All procedures were performed following approval of the institutional animal care and use committee of Tianjin Medical University.Results The retina morphology was normal in the control group,and EB filled the vessels,exhibiting the red fluorescence under the fluorescence microscope.Compared with the control group,retinal background fluorescence was enhanced slightly in the DM 1-month group,and focal leakage of the EB from capillaries and focal dilated vessels were found in the DM 3-month group,further,vascular caliber inequality,retinal hypoperfusion area and a larger number of hyperfluorescence areas were seen in the DM 6-month group.The percentage of leakage area was (0.05 ±0.02) ％,(0.27 ±0.06) ％,(1.17 ±0.18)％ and (4.77 ±0.66)％ in the control group,DM 1-month group,DM 3-month group and DM 6-month group,respectively,showing a significant difference among the four groups (F =795.800,P＜0.001),and the leakage area was obviously larger in the DM 3-month group and DM 6-month group than that in thecontrol group (q'=10.338,q'=43.475,both at P＜0.001).Conclusions Modified EB-perfused retinal wholemount method is easy and helpful for clear visualization of retinal vessel leakage induced by BRB breakdown in the diabetic rats under the common fluorescence microscope.
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Objective To investigate the sore-throat-relieving, anti-inflammation and antitussive actions of Qingyan mixture on the animal models. Methods Sore-throat-relieving action of Qingyan mixture was observed in SD rats with acute pharyngitis induced by spray of ammonia water at volume fraction of 15%. Anti-inflammation action of Qingyan mixture was observed on mice model of xylene-induced ear edema, and antitussive action of Qingyan mixture was carried out in mice with cough induced by concentrated ammonia spray. Results (1) The results of sore-throat-relieving action of Qingyan mixture showed that the infiltration of inflammatory cells did not happen in the blank control group, but moderate/severe inflammatory cell infiltration was shown in the model group. In the positive group, 4 rats had slight inflammatory cell infiltration while the left 6 rats had no inflammatory cell infiltration. Low-dose Qingyan mixture group had no inflammatory cell infiltration in one rat, and had slight infiltration in 9 rats. In middle-and high-dose Qingyan mixture groups, 4 rats had no infiltration and 6 had slight infiltration, inflammatory cell infiltration was markedly relieved ( P0.05). ( 2) The anti-inflammation rate was 69.8% in the positive control group, and was 27.3%, 60.4%, 60.0% in low-, middle- and high-dose Qingyan mixture groups respectively. The inhibitory effect of middle-and high-dose Qingyan mixture on xylene-induced mice ear edema was similar to that of the positive control group ( P>0.05) . ( 3) The cough-relieving rate was 42.6%, 139.0%, 64.3%, 104.0% in positive control group, and low-, middle-and high-dose Qingyan mixture groups respectively. Mice cough latent was obviously prolonged and cough frequency was decreased in the three Qingyan mixture groups, the differences being significant compared with the model group ( P<0.05 or P<0.01) . Conclusion Qingyan mixture has certain sore-throat-relieving, anti-inflammation and antitussive actions, which can be used for the treatment of patients with actue/chironic laryngitis and the complication of cough.
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Background It has been proved that as an important adhesion protein of extracellular matrix,osteopontion (OPN) can affect tumor neovascularization.Some new researches showed that anti-OPN antibody plays a role in regulating the neovascular vessel formation.Choroidal neovascularization (CNV) has the same structure with tumor neovascularization,but whether anti-OPN antibody restricts new vessel formation is unclear.Objective This study was to investigate the inhibitory effect of anti-OPN antibody on CNV.Methods Laser-induced CNV models were created in 40 eyes of 40 male SPF C57BL/6J mice by Argon laser photocoagulation of retinas,with the wavelength 514 nm.Thirty-six successful models were randomly divided into anti-OPN antibody group,mouse-IgG group and PBS group by the randomized number table.On the second day after photocoagulation,anti-OPN antibody of 400 μg was intraperitoneally injected in the anti-OPN antibody group,and the equivalent amount of mouse IgG and PBS were used in the same way in the mouse IgG group and PBS group.The CNV was evaluated by fundus fluorescein angiography (FFA) on the seventh days after photocoagulation.The mice were immediately sacrificed and the eyeballs were enucleated on the fourteenth day after photocoagulation,and 4 eyeballs in each group were used to observe the areas of CNV on the retinal pigmental epithelium-choroid-sclera fiat mounts,and the other 8 eyeballs of each groups were used to analyze the expression levels of OPN mRNA and vascular endothelial growth factor(VEGF) mRNA using quantitative fluorescence-PCR (QF-PCR).Results FFA showed fluorescein leakage areas around laser spots 7 days after photocoagulation,indicating that CNV appeared.The CNV areas were ([16.98±0.70] × 103) μm2,([27.13 ± 0.81] × 103) μm2 and ([35.39±2.14] ×103) μm2 respectively in the anti-OPN antibody group,mouse IgG group and PBS group,with a significant difference among the 3 groups (F =533.76,P =0.00),and the CNV area was significantly smaller in the anti-OPN antibody group compared with those of the mouse IgG group and PBS group (q =-3.95,-4.40,both at P＜0.05).No significant difference was found in the OPN mRNA expression between the antiOPN antibody group and mouse IgG group (t =-5.26,P =0.66).However,the expression of VEGF mRNA in choroidal tissue was significantly declined in the anti-OPN antibody group than that in the mouse IgG group (t =-6.74,P＜0.01).Conclusions Anti-OPN antibody suppresses the formation of CNV in laser-induced mouse model by down-regulating VEGF.
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Background Dry eye is increasing gradually recently,but its etiology and manifestation are very diverse.Studies showed that menopause of adult females was one of the risk factors of dry eye.In addition,some inflammatory factors also participate in the pathogenesis and development.But the study on the relationship of sex hormone with inflammation and ocular surface damage is still below understanding.Objective This study was to investigate the expressing changes of interleukin (IL) and tumor necrosis factor-α (TNF-α) in conjunctiva and the manifestation of ocular surface in ovariectomized rat model.Methods Twenty clear female SD rats were randomized into the ovariectomized group and the sham operative group according to randomized number table.Ovariectomy was performed in the ovariectomized group,and abdominal myotomy without ovariectomy was performed in the sham operative group.Serum estrogen and androgen levels were detected by radiation immunoassay 3 months after operation.Schirmer Ⅰ test (S Ⅰ t) and corneal fluorescence staining were carried out in the rats before operation and 1 month,2 and 3 months after operation.The morphology of conjunctival epithelial cells was examined by hematoxylin & eosin staining at the 3rd month after operation.The expressions of IL-17A,IL-1 β,IL-6 and TNF-α in conjunctiva were semi-quantitative analyzed by immunohistochemistry and Western blot.The use and care of the animals complied with State Science and Technology Commission Regulations for the Administration of Affair Concerning Experimental Animals.Results Serum estrogen levels were (23.53 ± 1.65) pg/L and (47.89 ± 1.05) pg/L 3 months after surgery in the ovariectomized group and the sham operative group,respectively; the serum androgen levels were (1.84±0.09) ng/L and (2.47±0.12)ng/L in the ovariectomized group and the sham operative group,respectively,showing a significant decline of serum estrogen and androgen levels in the ovariectomized group compared with the sham operative group (t=-35.37,-12.13,both at P＜0.01).No significant differences were seen in S Ⅰ t between the two groups among various time points (Fgroup =0.38,P =0.55 ; Ftime =0.13,P =0.72 ; Finteraction =0.39,P =0.76).No obvious fluorescence staining was seen in the cornea of both the ovariectomized group and the sham operative group.The histopathological examination showed that the layers of rat conjunctival epithelial cells increased with the disordered arrangement in the ovariectomized group.Immunochemistry showed that the expressions of IL-17A,IL-1β,IL-6 and TNF-α (A values) were significantly higher in the ovariectomized group than those in the sham operative group (IL-17A:t=8.22,P＜0.01 ;IL-1β:t=16.43,P＜0.01 ;IL-6:t=13.44,P＜0.01 ;TNF-α:t=16.26,P＜0.01).Western blot assay showed the similar results (IL-17A:t=19.41,P＜0.01 ;IL-1β:t=12.63,P＜0.01 ;IL-6:t=17.17,P＜0.01 ;TNF-α:t=15.19,P＜0.01).Conclusions Serum estrogen and androgen levels drop obviously,and there is an up-regulation of IL and TNF-α expression in conjunctiva tissue in the ovariectromized SD rats.However,no obvious dry eye-related sign occurs.
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Background Fungal corneal ulcer is a visual-threatening eye disease,and drug therapy has a limiting efficacy.Corneal transplantation or eye enucleation sometimes is necessary to the severe patients.Corneal collagen cross-linking (CXL) is an effective method for some corneal diseases,but the study on CXL for fungal corneal ulcer is lack.Objective This study was to evaluate the clinical effectiveness and safety CXL for fungal corneal ulcer.Methods Fifteen 8-week-old healthy New Zealand white rabbits were used in this study and other 5 rabbits served as normal controls.Fungal corneal ulcer models were established in the right eyes of other 10 rabbits by infecting sickle bacteria liquid after corneal scratching and removing corneal epithelium,then decellularized ostrich corneal patch covered the defected cornea.The models were randomly divided into the non-treatment group and the CXL treatment group.Corneal lesions were examined under the slit lamp microscope every day,and cornea was pictured by laser scanning confocal microscope on the 3rd,7th,14th,21st and 28th day individually after CXL.All rabbits were sacrificed and corneal tissues were obtained 4 weeks after treatment,and the collagen fiber diameter and fibrocytes were observed under the scanning electron microscope.Results Fungal corneal ulcer models were successfully established by corneal scratching and decellularized ostrich cornea covering.The gray ulcer lesions and hypbae like bean pod were seen by slit lamp microscope and laser scanning confocal microscope 3 days after modeling.Corneal ulcer deepened and expanded 1 week later,and there were a large number of spore and hyphae criss-crossing as short rod in shallow stroma.Inflammatory cells were observed in corneal endothelial cells and ocular anterior chamber.In the CXL treatment group,the range of corneal epithelial deficiency was less than that in the nontreatment group on the 3rd,7th,14th,and 21st (all at P＜ 0.05).The diameters of collagen fibers were (24.6± 1.8) nm,(24.9 ± 1.9) nm and (43.0 ± 7.4) nm in the normal control group,non-treatment group and CXL treatment group,showing a significant difference among the 3 groups (F =27.05,P =0.00),and the collagen diameters were thicker in the CXL treatment group than those in the normal control group and non-treatment group (t =5.40,-5.30,both at P＜0.05),and fibrocytes were seen among the collagen fibers.No significant difference was found in the collagen diameters between the non-treatment group and normal control group,and the fibrocytes were less in the non-treatment group.Conclusions CXL therapy can treat fungal corneal ulcer by enhancing collagen,promoting fibrocytes proliferation,suppressing fungus and inflammatory response and accelerating tissue repair.
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Background Most anti-inflammation eyedrops are limited in clinical application owing to multiple adverse effects.A novel peptide GC31 derived from human thrombomodulin has a natural anti-inflammatory activity.Compared with conventional anti-inflammatory eyedrops,GC31 possesses more advantages and potential clinical transforming value.However,relevant study is still lack.Objective The purpose of this study was to evaluate the anti-inflammatory effect of GC31 and the possible mechanisms.Methods Sixty SPF male Wistar rats aged 8-10 weeks were randomized into 6 groups using randomized number table.Non-specific keratitis models were established in 40 rats by intrastromal injection of 10 μl of lipopolysaccharide (LPS) dissolved in PBS.Different doses of GC31 (125 μg or 250 μg) or dexamethason soluble in PBS were sunconjunctically injected in the experimental eyes respectively in the low dose GC31 group,high dose of GC31 group and the dexamethason group,and 10 μl of PBS was used in the same way in the PBS control group.No drug was injected in the model group,and the normal rats were employed as the blank control group.The corneas were examined by slit lamp microscope and were scored based on the criteria of Anand 24 hours after injection.Then the corneas were collected for histopathological examination.Expression of nuclear factor-κB (NF-κB) p65 in the corneas was detected using immunochemistry.Expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) proteins were assayed using ELISA.Real-time PCR was used to detect the expressions of IL-6 mRNA and TNF-α mRNA.The use and care of the experimental animals followed Regulation for the Administration of Affair Concerning Experiment animals by State Science and Techonology Commission.Results A significant difference was seen in the ocular inflammatory scores among the six groups (F =301.238,P =0.000).The inflammatory scores were significantly lower in the high dose of GC31 group than those in the model group (1.85 ± 0.36 versus 2.90± 0.43) (t' =-5.144,P =0.000) ; and the scores in the dexamethason group was lower than those in the high dose of GC31 group(t' =-3.931,P=0.000).Infiltration of inflammatory cells in corneal tissue was milder in the high dose of GC31 and the dexamethason group compared with the model group.The positive response for NF-κB p65 was obviously weaker in the rat corneas in the low and high dose of GC31 groups and the dexamethason group in comparison with the model group.The contents of IL-6 and TNF-α proteins in the corneas were significantly reduced in the low and high dose of GC31 group and the dexamethason group compared with the model group (low dose group:t=-2.626,P=0.009;t'=-2.310,P=0.017.high dose group:t =-3.361,P=0.001 ;t'=-3.151,P=0.002),and the contents of IL-6 and TNF-α proteins in the dexamethason group were lower than those in the high dose of GC31 group (t=-3.361,P=0.001;t'=-3.360,P=0.000).In addition,the expression trend and compared results of IL-6 mRNA and TNF-α mRNA among the groups were similar to those of the IL-6 and TNF-α proteins (all at P＜0.01).Conclusions GC31 suppresses LPS-induced corneal inflammation response by downregulating the expression of inflammatory eytokines.The effect is more dominant in the doses of 250 μg than that in the doses of 125 μg.
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Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that zonula occludens-1 (ZO-1) can inhibit pathological angiogenesis through physical barrier formed by tight junction structure.However,whether ZO-1 plays a role in CNV is unclear.Objective The aim of this study was to explore the effect of ZO-1,a tight junction protein on experimental CNV.Methods The CNV models were established in the left eyes of 24 clear male BALB/c mice aged 7-8 weeks by putting NaOH filter paper in the center of corneas for 15 seconds (15 s group) or 40 seconds (40 s group).CNV was examined and evaluated under the slit lamp microscope,and the expression of ZO-1 mRNA in the corneas were detected and compared by reverse transcription PCR (RT-PCR) between the two groups 2 weeks after modeling.In addition,54 models created by the same method were assigned to 3 groups according to randomized number table,0.2％ hyaluronic acid (HA),antiZO-1 neutralizing antibody (10 mg/L) +0.2％ HA and mouse hypoxia inducible factor-1α (HIF-1α) recombinant protein (5 mg/L)+0.2％ HA were topically administrated in the mice three times a day for 1 week after modeling respectively.The corneas were extracted 2 weeks after application of the drugs.Expression of CD31 in the CNV was assayed to calculate the number and the area of CNV by immunohistochemistry.The expression of VEGF mRNA in the corneas was detected by RT-PCR.The percentages of macrophage-specific F4/80 positive cells and neutrophilsspecific Ly-6G positive cells were calculated to evaluate the infiltrations of inflammatory cells in the corneas by flow cytometry.Results In 2 weeks after alkali burn of corneas,the number of severe CNV was more in the 40 s group than that in the 15 s group (x2 =6.032,P=0.049),and the expression level of ZO-1 mRNA was lower in the 40 s group than that in the 15 s group (1.15±0.08 versus 1.53±0.04) (t=4.157,P=0.014).CD31 positive cell number was more and the staining area was larger in the ZO-1 antibody group and HIF-1α positive control group than those in the 0.2％ HA group (cells:t=-129.590,-226.820,both at P=0.000;area:t =-5.310,-8.840,both at P=0.000).The relative expressions level of vascular endothelial growth factor (VEGF) mRNA was 1.33±0.10 and 1.46±0.11 in the ZO-1 antibody group and HIF-1 α positive control group respectively,which were significantly higher than 0.93±0.06 of the 0.2％ HA group (t =-5.820,-7.284,both at P =0.000).The percentages of positive cells in the ZO-1 antibody group and HIF-1α positive control group were significantly increased in comparison with the 0.2％ HA group for F4/80 (t =-16.750,-17.480,both at P =0.000) and for Ly-6G (t =-21.450,-27.680,both at P=0.000).Conclusions Alkali burn induced CNV downregulates the expression of ZO-1 mRNA.Administration of ZO-1 antibody causes the rise of VEGF mRNA in CNV and the infiltration inflammation cells,which suggests that the influence of ZO-1 on CNV is associated with the expression of VEGF.
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Background Minocycline possesses neuroprotective effect in a variety of animal models and clinical trials of central nervous system,but whether it works on optic nerve injury remains unclear.Objective This study aimed to observe the protective effects of minocycline on retinal ganglion cells (RGCs) in the early stage of optic nerve crush and explore its mechanism.Methods One hundred and thirty-six clean C57BL/6J mice were randomly divided into normal control group,normal saline solution group and minocycline group.The optic nerve crush injury models were induced in the left eyes of the mice in the normal saline solution group and minocycline group by a cross-action forceps for 3 seconds.Minocycline was injected intraperitoneally in the minocycline group firstly 45 mg/kg(0.4 ml) and followed by 22.5 mg/kg per day after 24 hours until sacrifice of the animals,and the equivalent volume of normal saline solution was injected in the same way in the normal saline solution group.The mice were euthanized at 4,7,11,14 days postoperatively and the left eyeballs were collected.Retinal flat mounts and DAPI staining was used to observe and compare the change of RGCs density among different groups and various time points.Apoptosis of mice RGCs were assessed by TdT-mediated dUTP nick end labeling (TUNEL).Real-time polymerase chain reaction (real-time PCR) was used to detect the expression of CD11b mRNA in retinal microglials.Results DAPI staining in retinal flat mounts showed that the average RGCs density was (77.50±2.38)/0.01 mm2 and (70.00±2.94) /0.01 mm2 in the 4th and 7th day after modeling in the normal saline solution group,and those in the minocycline group were (88.75 ± 2.36) /0.01 mm2 and (81.00 ± 3.92)/0.01 mm2,with significant differences between the two groups (t4d =-6.708,P＜0.01 ;t7d =--4.491,P＜0.01).The apoptotic RGCs were (12±1)/mm and (4±1)/mm in the normal saline solution,which were significantly more than (4±1)/mm and (1±0)/mm in the minocycline group (t4 d =12.832,P＜0.01 ; t7d =3.455,P =0.026).However,no significant difference was found in apoptotic RGCs in postoperative 11 days and 14 days between the normal saline solution group and the minocycline group (P =0.708,0.777).The expressing levels of CD11 b mRNA in the retinal microglials were significantly higher in the 4th and 7th day in the normal saline solution group than those in the minocycline group (t4 d =8.312,P＜0.01 ;t7d=5.407,P＜0.01),but were not significantly different in the 11st and 14th day after modeling between the two groups (P=0.055,0.170).Conclusions Minocycline can play a neuroprotective effect on RGCs in the early stage of optic nerve crush in mice by inhibiting microglia activation and decreasing RGCs apoptosis.