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Objective:To investigate the effect of circadian rhythm changes on the expression of retinoic acid-related orphan receptors (RORs) and the RORs agonist SR1078 on corneal epithelial wound repair.Methods:A total of 228 SPF C57BL/6 female mice aged 6-8 weeks old were selected, and 180 mice were divided into the normal circadian rhythm group, full-day group, full-night group, 12-hour reversed circadian rhythm group and 3-week reversed circadian rhythm group, with 36 mice in each group.The remaining 48 mice were randomly divided into phosphate buffered saline (PBS) control group and SR1078 group by random number table method, with 24 mice in each group.According to grouping, the mice were placed in a light box where the light (light intensity of 300 lx) and dark time could be controlled.The light time of the normal circadian rhythm group, the PBS control group and the SR1078 group in the light box was from 7: 00 to 19: 00, and the dark time was from 19: 00 to 7: 00 the next day.According to the Zeitgeber Time method, the starting time of light at 7: 00 was recorded as ZT0, and the time of closing light at 19: 00 was recorded as ZT12.Real-time fluorescence quantitative polymerase chain reaction was used to detect the relative expression levels of RORα and RORγ mRNA at ZT1, ZT5, ZT9, ZT13, ZT17, ZT21 in the five groups.In the PBS control group and SR1078 group, a golf-like knife was used to establish the mouse corneal epithelial injury model, and the model eyes were administered with drugs once every 6 hours according to the grouping.The corneal epithelial defect area was measured with Adobe Photoshop CC2019 software, and the corneal epithelial defect rate was calculated and compared between the two groups.The correlation between the relative expression levels of RORα and RORγ mRNA in mice corneal epithelium of the five groups and corneal epithelial defect rate in the PBS control group and SR1078 group was analyzed.The corneal epithelium repair was observed by whole cornea spreading and immunofluorescence staining, and the number of corneal epithelial dividing cells in the PBS control group and the SR1078 group was calculated and compared.The use and care of animals complied with the ARVO statement.This study protocol was approved by the Laboratory Animal Ethics Committee of Jinan University (No.JN-A-2002-01).Results:Compared with the normal circadian rhythm group, the relative expression levels of RORα/RORγ mRNA in the full-day group, full-night group, 12-hour reversed cirdian rhythym group and 3-week reversed cirdian rhythym group showed an overall decreasing trend.There was a statistically significant difference in the corneal epithelial defect rate between the PBS control group and the SR1078 group at different time points after modeling ( Fgroup=74.01, P<0.001; Ftime=5 171.48, P<0.001). Twelve hours after modeling, the corneal epithelial defect rate in the SR1078 group was significantly lower than that in the PBS control group, and the difference was statistically significant ( P<0.05). The relative expression levels of RORα and RORγ mRNA in corneal tissue was moderately positively correlated with the corneal epithelial defect rate in mice ( r=0.614, 0.537; both at P<0.01); The regression equation of the straight line between the relative expression level of RORα mRNA and the change in corneal epithelial defect rate was Y=33.153X-43.052 ( F=20.58, P<0.001), and the linear regression equation between the relative expression level of RORγ mRNA and the change of corneal epithelial defect rate was Y=2.764X-1.364 ( F=13.11, P<0.001). There was a significant overall difference in the number of corneal epithelial dividing cells at various time points following modeling between the PBS control group and the SR1078 group ( Fgroup=160.55, P<0.001; Ftime=83.57, P<0.001). The number of dividing cells in the SR1078 group was significantly less than that in the PBS control group at 24, 30, and 36 hours following modeling, and the differences were statistically significant (all at P<0.05). Conclusions:Circadian rhythm changes reduce the expression of RORα and RORγ mRNA in the mouse cornea.SR1078 can promote the expression of RORα and RORγ mRNA in corneal epithelium to decrease the number of mouse corneal epithelial dividing cells, and inhibit the repair after corneal trauma.
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Objective:To provide a standard protocol for the rapid quantitative analysis of neutrophils in inflamed corneas with flow cytometry.Methods:The corneal epithelium layer of 15 C57BL/6 mice (6-8 weeks old) was mechanically scraped off using a golf-like knife to generate a 2 mm wound region.The mouse corneas with intact limbus were cut out at 18 hours after abrasion.After mechanical shredding, the single cell suspension was obtained by collagenase I and DNase digestion.Then, the number of neutrophils in the corneal cells was sorted under the FACSCanto flow cytometer using the gate technique.Another 6 mice were taken and randomized into wounded group and normal group according to a random number table method, with 3 mice in each group.Corneal cell staining was performed using fluorescent-conjugated anti-mouse CD45, Ly6G, and CD11b antibodies.The number of neutrophils in the corneas of the two groups were enumerated and compared.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO). The study protocol was approved by the Animal Ethics Committee of Medical College of Jinan University (No.JN-A-2002-01).Results:A standard procedure for detecting neutrophils in the cornea by flow cytometry was established.The ratio of CD45 + cells in the total corneal tissue cell population was (20.93±1.72)%.The Ly6G + and CD11b + double positive neutrophil population was sorted in the wounded corneal cell population.The ratios of Ly6G + and CD116 + cells in the CD45 + cells were (75.50±3.25)% and (93.40±4.53)%, respectively, and the ratio of the Ly6G + and CD11b + double positive neutrophils in the total number of CD45 + cells was (67.33±2.80)%.In addition, the number of neutrophils recruited to the cornea at 18 hours after corneal abrasion was (151.47±10.82)%, which was higher than (15.36±1.02)% in the normal cornea ( t=21.689, P<0.01). Conclusions:Flow cytometry can quickly and accurately quantitatively analyze the neutrophil population in the wounded cornea.It provides a rapid quantitative analysis method to further evaluate the changes of neutrophils in corneal inflammation caused by different reasons.
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Objective To observe the effect of reversed circadian rhythms on wound healing of mouse corneal epithelium.Methods Ninety male C57BL/6 mice were divided into LD group (12 hours light/12 hours dark) and DL group (12 hours dark/12 hours light) by random number table,and then were placed in circadian rhythm box for 12 days.The circular area was scarped and marked as 2 mm diameter area in the center of the mouse's cornea with a golf-like knife.The dynamics of epithelial healing in the wound area were observed under microscope by fluorescein staining and hematoxylin-eosin staining.Besides,being marked antibodies of anti-Ly6G-FITC,anti-γδT-PE and DAPl,dynamic changes of the dividing cells,neutrophils and γδT cells were also investigated for every 6 hours until 42 hours.All mice were treated in accordance with the Association for Research in Vision and Ophthalmology's Statement for the Use of Animals in Ophthalmology and Vision Research and the guidelines of the Animal Experimental Committee at Jinan University (JN-A-2002-01).Results In LD group,percentage of corneal epithelial defective area were (100.000 ± 0.000) ％,(37.677 ± 5.243) ％,(14.959 ± 1.739) ％ and (0.000 ± 0.000) ％ after wounding 0 hour,6,12,18 and 24 hours.In DL group,percentage of the corneal epithelial defective area were (100.000±0.000) ％,(10.967 ± 1.065 ％) ％,(1.985 ±0.106) ％ and (0.000±0.000) ％ after wounding 0 hour,6,12,18 and 24 hours.The healing rate in DL group was higher than that in LD group,with a significant difference between them (P＜0.05).As with the uninjured corneal,thickness of corneal epithelium was (33.983 ± 1.074)μm in DL group and (33.993±0.904)μm LD group,with no statistically significant difference between them (P＞0.05).After 24 hours,thickness of corneal epithelium in DL group was (19.473 ±0.856) μm,and was more than that in LD group [(17.485±0.718)μm],with a significant difference between them (P＜0.05).Paraffin section of wounded corneal epithelium after 24 hours by hematoxylin and eosin staining showed that corneal epithelium cells arranged loosely and disorderly and were in irregular shape in both groups.The epithelium were mainly basal cells in LD group,while epithelium included basal cell and a few pinacocytes in DL group.After corneal epithelium wounded,the number of cell division,neutrophils and corneal limbus γδT cells in two groups were statistically significant difference,respectively(P＜0.05).Conclusions Reversed circadian rhythms can significantly regulate the wound healing of corneal epithelium.
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Background To understand the distribution and development of corneal nerve in animal or human has an important significance for clinical and basic research of corneal diseases.At present,some studies on cornea nerve development and location have been performed.However,the quantified study on innervation and distribution of corneal nerve fibers as embryonic development has not been reported.Objective This study attempted to understand the distribution of corneal nerve fibers in the development of chick embryo,and to evaluate the changes of the length and density of corneal nerve fibers with aging of chick embryo.Methods Whole chick corneas with limbus were obtained from chick embryo aged 6-20 days (E6-E20),and corneal nerve fiber was labeled using immunofluorescence technique by anti-neuron-specific β-Ⅲ tubulin antibody.The corneas were radially cut into 4 parts,and the integrate corneal flat mounts were prepared with the upward epithelium and mounting with anti-fade fluorescent quenching buffer glycerin containing DAPI.Fluorescence microscope was used to capture the nerve fiber images in cornea,and cornea area and the number of nerve fiber bundles were exhibited by using Photoshop CS4.Cornea nerve fiber density and total length were measured by Imaris x64 7.4.2 software.Results Total cornea flat mounts showed that the nerve bundles grew from temporal scleral forward cornea limbus at E6-E8,and the nerve fibers formed the ring surrounding by limbus during E9-E10.Then the fibers extended forward the central cornea in E11 to E15 and developed into nerve fiber plexus on the whole cornea in E16 to E20.During the period of E6-E20,the corneal surface area,the length and density of corneal nerve fibers were gradually increased with the aging of chick embryo,showing statistically significant differences among different time points (F =127.007,227.051,67.748,all at P＜0.01).The increase of the corneal area of the chick embryo presented a strong positive correlation with the extending of length of the corneal nerve fiber (r =0.863,P＜0.01).Chick corneal nerve fiber bundle appeared at E13,with a number of (59.00 ± 1.14)/mm2 and then increased to a peak of (576.75 ±29.16)/mm2 at E 18 and reduced to (299.67± 25.46)/mm2 at E20,with a significant difference among them (F =13.759,P=0.000).Conclusions Corneal nerve starts to develop in E9 of chick embryo,and the corneal surface area,the total length of the corneal nerve fibers and the density rapidly increase concurrently with the development of chick embryo.
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To find out the clinical characteristics and risk factors for deep venous thrombosis [DVT] after gynecological surgery. Four hundred and ninety-eight patients treated surgically in the department of gynecology of our hospital from July 2012 to May 2014 were reviewed retrospectively. The data including patient age, gender, medical history, hospital stay, anesthesia type, operation time, occupation type, operative or postoperative medicine, perioperative bleeding, postoperative activity time, mortality rate and so on, were collected. Among 498 patients, 58 were included in the thrombosis group, 423 patients in the non-thrombosis group and 17 patients were excluded. The incidence of deep venous thrombosis was 11.6%. In 58 cases with deep venous thrombosis, 6 cases developed pulmonary embolism and two patients died, the mortality rate for pulmonary embolism is 33.3%. In multivariate analysis, age, malignant tumor, cardiovascular comorbidity and postoperative hemostatics dose are independent risk factors, physical labour and minimally invasive surgery are protective factors for DVT. The patients with elder age, malignant tumor, cardiovascular comorbidity or large postoperative hemostatics dose should be paid high attention to and the minimally invasive surgery are optimal treatment in preventing DVT
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Humans , Female , Gynecologic Surgical Procedures , Risk Factors , Retrospective StudiesABSTRACT
[ ABSTRACT] AIM: To observe the effect of circadian rhythms on wound healing of mouse corneal epithelium. METHODS:The C57BL/6 male mice were used in the study.A part of corneal epithelium (2 mm in diameter) was struck off by a golf-like knife to form a round wound area.The dynamics of epithelial healing in the wound area were ob-served under microscope with fluorescein staining.In addition, with related antibodies and DAPI, the dynamic changes of the neutrophils, platelets and dividing cells were also investigated.RESULTS:The healing rates in LL group (12 h light/12 h light) and DD group (12 h dark/12 h dark) were obviously slower than that in LD group (12 h light/12 h dark), mainly showing delayed re-epithelialization, decreased epithelial cells, increased diameter of blood vessel, and delayed re-cruitment of neutrophils and platelets, but more cell number.CONCLUSION:Disruption of circadian rhythms significant-ly inhibits the wound healing of corneal epithelium, mainly through delaying the inflammation and re-epithelialization, but aggravating the inflammatory responses.