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Background Researches showed that cystic fibrosis transmembrane conductance regulator protein(CFTR) is a channel secreting anion and water,and it plays an important role in tear secreting.Traditional conception thought that CFTR pathway is cAMP-PKA-dependent without the participation of intracellular calcium.However,studies disclosed that elevating intracellular cAMP could not open the CFTR channel.So whether calcium signal is associated with CFTR-related corneal epithelial secretion is controversial.Objective This study was to investigate the association between CFTR secretion and intracellular calcium signaling in rabbit corneal epithelium.Methods Sixteen New Zealand white rabbits were randomized into odd number group and even number group by computer randomized number method.The corneas were obtained under the general anesthesia and placed in Ussing Chamber for the record of short cricuit current (Isc).The right eyes of rabbits in the odd number group were stimulated with ATP and served as ATP stimulating group.The left eyes were pretreated with CFTRinh-172 prior to ATP stimulation and served as CFTRinh-172 pretreated group.In the even number group,the left eyes of rabbits were pretreated with BMPTA/AM before ATP stimulation and served as BMPTA/AM pretreated group,and the right eyes of the rabbits were used to isolate and culture corneal epithelial cells by explant adherent method,the level of intracellular calcium were evaluated using Leica SP5 laser scan confocal microscope.Results The ATP-induced ΔIsc of corneal epithelium was (5.73 ± 1.36),(1.30 ± 0.95) and (2.47 ± 0.55) μA/cm2 in the ATP stimulating group,CFTRinh.172 pretreated group and BMPTA/AM pretreated group,respectively,and the AIsc was significantly reduced in the CFTRinh.172 pretreated group and BMPTA/AM pretreated group compared with ATP stimulating group (t=11.201,5.508,both at P ＜ 0.001).The fluorescence intensity of intracellular calcium release after ATP stimulation was 3.25 folds more than that before ATP stimulation.Conclusions ATP promotes rapid short circuit current of corneal epithelium.CFTRinh-172 depresses the ATP-induced corneal epithelium AIsc,and BMPTA/AM suppresses intracellular calcium release.It is suggested that intracellular calcium signaling secretion probably participates in the functional CFTR activity in rabbit corneal epithelium.
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Background Mooren 's ulcer is an immune-related corneal inflammatory disease,and its pathogenesis remains below understood.Previous studies showed that the imbalance of T helper cell type 1 (Th1) and Th2 cell play important roles in the development of some autoimmune diseases.Thereby the influence of Th1/Th2 cells on the pathogenesis of Mooren's ulcer is being concerned.Objective This study was to investigate the change of Th1 and Th2 subsets in periphery blood of patients with active Mooren's ulcer.Methods Eleven consecutive patients with active Mooren's ulcer and 8 age-and gener-matched healthy controls were included in Zhongshan Ophthalmic Center,Sun Yat-sen University from January 2012 to July 2013 under the approval of Ethic Committee of this hospital and informed consent of each subject.The peripheral blood samples of all the subjects were obtained separately and periphery blood mononuclear cells (PBMCs)were isolated.The percentages of Th1 and Th2 in the PBMCs were assayed by flow cytometer.The relative expressions of T-bet mRNA,GATA-3 mRNA and signal transducer and activator of transcription 5 (Stat5) mRNA in the PBMCs were examined and compared by real-time fluorescence quantitative PCR (RT-qPCR) Results The percentage of Th1 cells in CD4+ T cells and Th1/Th2 value was 0.21％ (0.11％,0.31％) and 8.01 (4.49,12.01) respectively in the Mooren's ulcer group,which were significantly lower than 0.35％ (0.22％,0.71％)and 23.90 (22.49,33.49)in the normal control group,respectively (Z =-2.01,P =0.04 ; Z =-3.06,P =0.00).However,no significant difference was found in the percentage of Th2 between the two groups (Z=-1.98,P＞0.05).The relative expressions of T-bet mRNA and GATA-3 mRNA in PBMCs were significantly lower in the Mooren's ulcer group than those in the normal control group (Z =-3.47,-3.06,both at P=0.00) ;While the relative expression of Stat5 mRNA in PBMCs was insignificant changed between the two groups (Z =-1.05,P =0.33).Conclusions Th1 and Th2 cells are unbalanced in the active Mooren's ulcer patients.In addition,the down-expression of relevant transcription factors in peripheral blood also is seen in these patients.It is inferred that Th1 and Th2 cells may participate in the progress of Mooren's ulcer.
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Objective To evaluate the use of amniotic membrane patches for mild or moderate eye burns at the stage of acute burns and the involved microsurgical techniques.Methods Thirty-four eyes with corneal burns of Ⅱ to Ⅲ degree in which may have partial limbal necrosis were accepted amniotic membrane patching (21 eyes) or pharmaceutical treatment (13 eyes).Four amniotic patches were procured and examined by transmitting electronic microscope when the exposed corneal surface became re-epithelialization after amniotic membrane inching.Results Amniotic patches became partially melting or prolapsed 5-10 (11± 2) days after surgery.The uncovered corneal surface showed quickly re-epithehalized.There were a few thin fibrovascular membrane invaded onto corneal surface in the patients with more than a half limbal necrosis.Postoperative visual acuity increased 1 to 6 (3.3 ± 1.2) lines.The electronic findings showed that most of the infiltrated polymorphonuclear neutrophils in the anmiotic patches became apoptosis.Corneal melting presented in four of 13 eyes who received only drug treatment and lamellar keratoplasty was then performed.The other eyes manifested pseudo-pterygium or symblepharon at different extent.Conclusion Amniotic,patches may reduce the inflammation of burned cornea and corneal neovascularization,accelerate re-epithelialization of corneal surface,even improve the rehabilitation of burned limbal stem cells.
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<p><b>OBJECTIVE</b>To investigate the effects of fresh and preserved amniotic membrane on polymorphonuclear neutrophils (PMNs) so as to understand the anti-inflammatory mechanism of amniotic membrane transplantation.</p><p><b>METHODS</b>Conditioned medium was collected 48 hours after fresh or preserved amnions were cultured in DMEM and 5% CO(2) at 37 degrees C. Then, polymorphonuclear cells were cultured in conditioned culture or DMEM. Fluorescent microscopy with 4',6-diamidino-2-phenylindole (DAPI) staining and cytometry were performed 6, 9, 12, and 15 hours later.</p><p><b>RESULTS</b>Apoptotic neutrophils were found in each group at different time points. The percentage of apoptotic cells at 6, 9, 12, and 15 hours after culture in the fresh and preserved amnion groups and the control group was 17.3%, 24.4%, 29.8%, 37.1%, and 16.2%, 20.1%, 23.7%, 27.7%, and 10.2%, 13.7%, 21.1%, 26.4%, respectively (t test, P(1) < 0.01, P(2) < 0.01 and P(3) < 0.01).</p><p><b>CONCLUSION</b>Amniotic membrane can accelerate apoptosis of polymorphonuclear neutrophils, reduce inflammation, and prevent ocular surface collagen from resolution, indicating that fresh amnion might have a stronger effect than preserved amnion.</p>
Subject(s)
Amnion , Physiology , Apoptosis , Cells, Cultured , Neutrophils , Cell Biology , Allergy and ImmunologyABSTRACT
Objective To investigate the long-term results of amniotic membrane transplantation for conjunctival surface reconstruction and analyze the involved microsurgical techniques and associated factors. Methods Fifty-one consecutive cases (55 eyes) with symblepharon at degree due to eye burns or Stevens-Johnson syndrome were accepted amniotic membrane transplantation after lysis of symblepharon. Results Observation time varied from 36 to 44 months (mean value, 38?4 months). Fifty-six point four percent(31/55) eyes got enough conjunctival fornix and resolution of eye movement restrict. About sixteen percent of them (9/55) recurred less symblepharon. Fifteen-five eyes of them(27.3%) recurred moderate symblepharon. The results of AMT for those patients with symblepharon at different degree and between those patients who were performed in different time after eye burns had significant difference statistically ( ? 2 test, P