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Objective@#To investigate the relationship between serum glutamate levels and severity of retinopathy of prematurity (ROP) in children and its impact on the prognosis.@*Methods@#A total of 92 children with ROP who were screened and treated at Henan Provincial Eye Hospital from June 2017 to June 2018 and 50 healthy preterm infants screened at Henan Provincial Eye Hospital were selected for clinical control study.Ninety-two children with ROP were divided into the mild ROP group and the severe ROP group according to the stage of lesions, and they were divided into the progressive group and the spontaneous regression group according to whether the disease was progressive.The severity of ROP and prognosis were analyzed by measuring serum glutamate levels in 1 week and 6 weeks after birth.@*Results@#The serum glutamate concentration in the severe ROP group was the highest in the first week after birth[(122.08±14.55) mmol/L] and in the 6th week after birth [(107.13±13.20) mmol/L], followed by the mild ROP group[(98.60±14.48) mmol/L, (85.41±14.49) mmol/L] separately, and the lowest in the control group[(68.52±7.69) mmol/L, (54.97±6.31) mmol/L] separately, and there were significant differences among the 3 groups (all P<0.05). Pearson correlation analysis showed that serum glutamate concentration was positively correlated with the severity of ROP at 1 week and 6 weeks after birth (r=0.869, 0.875, all P<0.05). The levels of serum glutamate at the first week after birth and at the 6th week after birth in the progressive group [(107.18±17.62) mmol/L, (92.94±16.21) mmol/L]were significantly higher than those in the spontaneous regression group[(131.53±10.22) mmol/L, (118.82±8.18) mmol/L], and there were significant differences between the 2 groups (all P<0.05). The area under the curve(AUC) values of serum glutamate concentration at 1 week after birth and 6 weeks after birth were 0.855 and 0.936, respectively, according to the receiver operating characteristic(ROC) curve, while the optimal critical values of serum glutamate concentration at 1 week and 6 weeks after birth were 117.83 mmol/L (sensitivity was 0.909, specificity was 0.728) and 106.69 ng/L (sensitivity was 1.000, specificity was 0.790), respectively.@*Conclusions@#The serum glutamate concentration was positively correlated with the severity of ROP infants in 1 week and 6 weeks after birth.The optimal threshold of serum glutamate concentration in 1 week and 6 weeks after birth was more sensitive and specific in predicting the progression of retinopathy, and had higher value in evaluating the prognosis of the infants.
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Objective To investigate the relationship between serum glutamate levels and severity of retinopathy of prematurity (ROP) in children and its impact on the prognosis.Methods A total of 92 children with ROP who were screened and treated at Henan Provincial Eye Hospital from June 2017 to June 2018 and 50 healthy preterm infants screened at Henan Provincial Eye Hospital were selected for clinical control study.Ninety-two children with ROP were divided into the mild ROP group and the severe ROP group according to the stage of lesions,and they were divided into the progressive group and the spontaneous regression group according to whether the disease was progressive.The severity of ROP and prognosis were analyzed by measuring serum glutamate levels in 1 week and 6 weeks after birth.Results The serum glutamate concentration in the severe ROP group was the highest in the first week after birth [(122.08 ± 14.55) mmol/L] and in the 6th week after birth [(107.13 ± 13.20) mmol/L],followed by the mild ROP group[(98.60 ± 14.48) mmol/L,(85.41 ± 14.49) mmol/L] separately,and the lowest in the control group[(68.52 ±7.69) mmol/L,(54.97 ± 6.31) mmol/L] separately,and there were significant differences among the 3 groups (all P ＜ 0.05).Pearson correlation analysis showed that serum glutamate concentration was positively correlated with the severity of ROP at 1 week and 6 weeks after birth (r =0.869,0.875,all P ＜ O.05).The levels of serum glutamate at the first week after birth and at the 6th week after birth in the progressive group [(107.18 ± 17.62) mmol/L,(92.94 ±16.21) mmol/L] were significantly higher than those in the spontaneous regression group[(131.53 ± 10.22) mmol/L,(118.82 ± 8.18) mmol/L],and there were significant differences between the 2 groups (all P ＜0.05).The area under the curve(AUC) values of serum glutamate concentration at 1 week after birth and 6 weeks after birth were 0.855and 0.936,respectively,according to the receiver operating characteristic(ROC) curve,while the optimal critical valuesof serum glutamate concentration at 1 week and 6 weeks after birth were 117.83 mmol/L (sensitivity was 0.909,specificity was 0.728) and 106.69 ng/L (sensitivity was 1.000,specificity was 0.790),respectively.Conclusions The serum glutamate concentration was positively correlated with the severity of ROP infants in 1 week and 6 weeks after birth.The optimal threshold of serum glutamate concentration in 1 week and 6 weeks after birth was more sensitive and specific in predicting the progression of retinopathy,and had higher value in evaluating the prognosis of the infants.
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Objective@#To explore the genotype-phenotype correlation among 3 pedigrees affected with congenital aniridia.@*Methods@#Clinical data and genomic DNA were collected and genetic variations were screened by whole-exome sequencing, with an emphasis on PAX6-related genes.Suspected variations were verified by Sanger sequencing and quantitative polymerase chain reaction (PCR). Written informed consent was obtained from the parents of each propositus prior to entering study cohort.This study protocol was approved by Ethic Committee of Henan Eye Hospital (No.HNEECKY-2017(6)).@*Results@#Genetic analysis identified that a nonsense c. 949 C>T variation and an c. 141+ 1 G>T splicing variation of the PAX6 gene in two of the probands, while the remainder has carried a duplication in 11 p13 (chr11: 31531331-31827959) encompassing the PAX6 and ELP4 genes.Phenotype analysis showed that the probands carrying the nonsense and splicing variations had classical features including complete aniridia, macular hypoplasia, microcornea and nystagmus; the proband carrying the 11p13 duplication had microphthalmos, microcornea, macular dysplasia, iris dysgenesis, and nystagmus.@*Conclusions@#The 11p13 duplication involving the PAX6 gene may have caused over-expression of PAX6 gene, resulting in severe eye abnormalities including microphthalmos and microcornea, macular dysplasia and nystagmus.The relatively mild iris dysgenesis has distinguishing it from classical aniridia due to PAX6 haploinsufficiency.
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Objective To investigate the effect of Tanshinone Ⅱ A on the proliferation and the signaling pathway of human retinal pigment epithelial (RPE) cells in hypoxia.Methods CoCl2(150 μ mol/L) was used to simulate hypoxic condition and the ARPE-19 cells cultured in vitro were divided into blank control group,hypoxia control group,Tanshinone Ⅱ A group and hypoxia-inducible factor-1αt (HIF-1 α) inhibitor group.The different doses of Tanshinone Ⅱ A were used to treat ARPE-19 for 24,48 and 72 hours,respectively.The inhibitory rate of cell proliferation of different groups were detected by MTT after 24,48 and 72 hours of administration cultured,and the apoptosis rate and the cell cycle distribution of cells in the hypoxia were analyzed by flow cytometry.Real-time PCR and Western blot were used to detect the expressions of mRNA and protein of HIF-1α and vascular endothelial growth factor (VEGF).Results MTT assay showed that Tanshinone Ⅱ A could inhibit the proliferation of ARPE-19 cells in a dose-and time-dependent manner,and the proliferation inhibitory rate gradually increased in the 1,5 and 10 mg/L Tanshinone Ⅱ A groups,with significant differences between any two groups (all at P＜0.05).Flow cytometry showed that the apoptosis rate of ARPE-19 in 1,5 and 10 mg/L Tanshinone Ⅱ A groups gradually increased with the elevation of Tanshinone Ⅱ A dosage,with significant differences between any two groups (all at P＜0.05).The cell proportion in the G0/G1 phase gradually increased,while the cell proportion in the S phase gradually decreased along with the elevation of Tanshinone Ⅱ A concentration,significant differences were obtained among the hypoxia control group,1,5 and 10 mg/L Tanshinone Ⅱ A groups (all at P＜0.05).RT-PCR and Western blot showed that the relative expression of VEGF mRNA,HIF-1 α and VEGF protein in the the blank control group,hypoxia control group,1,5 and 10 mg/L Tanshinone Ⅱ A groups and HIF-1α inhibitor group were significantly different (all at P＜0.05).The expression of VEGF mRNA,HIF-1α and VEGF protein decreased successively in the 1,5 and 10 mg/L Tanshinone Ⅱ A groups,with significant differences between them (all at P＜0.05).There were no significant differences between 10 mg/L Tanshinone Ⅱ A and the HIF-1 α inhibitor group of all the test indexes(all at P＞0.05).Conclusions Tanshinone Ⅱ A can inhibit the proliferation of RPE cells,induce apoptosis by arresting cells at G0/G1 phase.The mechanism is related to the suppression of HIF-1 α/VEGF signaling pathway.
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Objective To compare the morphological and functional outcomes of different degrees of ocular burns patients receiving amniotic membrane transplantation (AMT) at different time points after ocular burn.Methods A retrospective analysis was performed.Ninety-two eyes of 76 acute ocular chemical burn patients were enrolled from January 2012 to December 2016 in Henan Eye Hospital.The ocular chemical burns were classified by Dua classifications.According to the operation time of AMT,the patients were divided into within 1 day after injury group,2-6 days after injury group and more than 6 days after injury group.The best corrected visual acuity and limbal stem cell deficiency score were recorded during the at least one year of follow up.The risk factors affecting limbal stem cell deficiency and visual outcome were analyzed.Results Of all the burned eyes,29 eyes (31.5 ％) were result from acid burn,41 eyes(44.6％) were result from alkaline burns and 22 eyes (23.9％) were result from thermal burn.The average burn severity scores of patients with limbal stem cell deficiency score of 0,1 and 2 was 1.86±0.54,3.60±0.94 and 5.35 ± 0.63,respectively,and the overall difference was statistically significant (F =65.532,P ＜0.01).In mild to moderate ocular surface burn patients,the limbal stem cell deficiency score in more than 6 days after injury group was significantly higher than that in within 1 day after injury group and 2-6 days after injury group (Z=-2.21,P=0.03;Z=-2.33,P=0.02).In severe ocular surface burn patients,there was no significant difference in limbal stem cell deficiency score between the groups (P=0.26).At the last follow-up,the average visual acuity of all eyes was 3.19 ± 1.47.COX regression analysis showed that burn grade and operation timing were the main risk factors for visual prognosis (OR =4.925,1.368;both at P＜0.01).Prognostic visual acuity was linearly correlated with the timing of amniotic membrane occlusion and degree of burn (R2 =0.078,0.685;both at P＜0.01),but for the Ⅴ grade and Ⅵ grade eyes,amniotic membrane timing couldn't improve the score of limbal stem cell deficiency.Conclusions Dua classifications is of great significance in evaluating prognosis of ocular burn patients.AMT is an effective adjunctive treatment in the management of acute ocular chemical burns to support epithelial healing and restore ocular surface integrity with potential to improve vision.AMT can't prevent limbal stem cell deficiency or restore vision in eyes with severe burns.
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Objective To investigate the corneal permeability of cyclosprin A (CsA) loaded on polymeric vector after topical application.Methods The grafted copolymer chitosan-graft-cyclodextrin (CS-g-CD) was synthesized,and the physicochemical structures of the polymer were investigated using nuclear magnetic resonance spectroscopy (NMR) and fourier transform infrared spectroscopy (FT-IR).A novel CsA eye drop was prepared using the grafted copolymer as carrier material.The physicochemical properties of eye drop,including drug-loading content,osmotic pressure and viscosity were investigated by high performance liquid chromatography-mass spectrometry (HPLC-MS),osmotic pressure gauge and viscometer,respectively.New Zealand albino rabbits were randomly divided into intact cornea CsA group,epithelium debrided CsA group and epithelium debrided control group.The corneal epithelia of the left eyes was debrided in the cornea epithelium debrided group.Cornea irritation test was performed on New Zealand albino rabbits.The aqueous humor was taken and the corneas were collected at 0.5 hour and 1 hour after instilled.The concentration of CsA was measured by HPLC-MS.Cy5 labeled vector loaded with Coumarin 6 served as model copolymers system,the penetration capabilities of the double fluorescent labeling copolymers system were monitored in vivo using two-photon scanning fluorescence microscopy on murine corneas after topical application.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results The polymer of CS-g-CD was successfully synthesized and confirmed using NMR and FT-IR.The drug loading of CsA in eye drop solution was 0.06 ％;the osmotic pressure was 305 mOsmol/kg and the viscosity was 36.5 cP.The CsA drug delivery system had a reversible temperature-sensitive drug release behavior and had no obvious irritation on the eyes of New Zealand rabbits.One hour after treatment,the concentration of CsA in the cornea and aqueous humor of epithelium debrided CsA group was (5.88 ± 1.46) μg/g and (149.19 ± 3.93) ng/ml,respectively,which was significantly higher than (3.98 ±0.95) μg/g and (30.25± 11.43) ng/ml in epithelium debrided control group (both at P＜0.05);the concentration of CsA in the aqueous humor of intact cornea CsA group was (7.23 ± 1.31)ng/ml,which was significantly lower than that in epithelium debrided CsA group (P＜0.05).Polymer vectors were mainly retained in the corneal epithelium,and coumarin 6 gradually diffused into the deep corneal stroma with time.Conclusions The grafted copolymer can load CsA,and the eye drop can effectively overcome the corneal barrier and increase the corneal permeability of CsA.
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Objective To investigate the corneal permeability of cyclosprin A (CsA) loaded on polymeric vector after topical application. Methods The grafted copolymer chitosan.graft.cyclodextrin ( CS.g.CD ) was synthesized, and the physicochemical structures of the polymer were investigated using nuclear magnetic resonance spectroscopy ( NMR) and fourier transform infrared spectroscopy ( FT.IR) . A novel CsA eye drop was prepared using the grafted copolymer as carrier material. The physicochemical properties of eye drop,including drug.loading content, osmotic pressure and viscosity were investigated by high performance liquid chromatography.mass spectrometry ( HPLC.MS) ,osmotic pressure gauge and viscometer,respectively. New Zealand albino rabbits were randomly divided into intact cornea CsA group, epithelium debrided CsA group and epithelium debrided control group. The corneal epithelia of the left eyes was debrided in the cornea epithelium debrided group. Cornea irritation test was performed on New Zealand albino rabbits. The aqueous humor was taken and the corneas were collected at 0. 5 hour and 1 hour after instilled. The concentration of CsA was measured by HPLC.MS. Cy5 labeled vector loaded with Coumarin 6 served as model copolymers system, the penetration capabilities of the double fluorescent labeling copolymers system were monitored in vivo using two.photon scanning fluorescence microscopy on murine corneas after topical application. The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The polymer of CS.g.CD was successfully synthesized and confirmed using NMR and FT.IR. The drug loading of CsA in eye drop solution was 0. 06 ％;the osmotic pressure was 305 mOsmol/kg and the viscosity was 36. 5 cP. The CsA drug delivery system had a reversible temperature.sensitive drug release behavior and had no obvious irritation on the eyes of New Zealand rabbits. One hour after treatment,the concentration of CsA in the cornea and aqueous humor of epithelium debrided CsA group was (5. 88±1. 46)μg/g and (149. 19±3. 93)ng/ml,respectively,which was significantly higher than (3. 98±0. 95)μg/g and (30. 25±11. 43)ng/ml in epithelium debrided control group (both at P<0. 05);the concentration of CsA in the aqueous humor of intact cornea CsA group was ( 7. 23 ± 1. 31 ) ng/ml, which was significantly lower than that in epithelium debrided CsA group ( P<0. 05 ) . Polymer vectors were mainly retained in the corneal epithelium, and coumarin 6 gradually diffused into the deep corneal stroma with time. Conclusions The grafted copolymer can load CsA,and the eye drop can effectively overcome the corneal barrier and increase the corneal permeability of CsA.
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Objective To analyze the pathogenic gene types and phenotypic characteristics of 6 albinism families.Methods A retrospective series of case studies.Six probands of albinism and 20 family members were recruited for this study,5 probands with clinical manifestations of oculocutaneous albinism (OCA) and 1 proband of ocular albinism (OA).Genomic DNA was extracted from peripheral venous blood which was collected from 6 probands and 20 family members.Genetic variations were screened by whole-exome sequencing or Sanger sequencing and then analyzed the relationship between genotypes and phenotypes.Results Genetic sequencing identified 6 potential pathogenic variants in 4 probands,including 2 compound heterozygous mutations in the 2 genes [TYR (c.1037-7T＞A,c.925_c.926insC),OCA2 (c.2359G＞A,c.587T＞C)] associated with OCA1 and OCA2,and 2 hemizygous mutations in the GPR143[GPR143 (c.11C ＞ G),GPR 143 (c.333 G ＞ A)] as sociated with OA 1,respectively.In which,5 were novel mutations and confirmed by Sanger sequencing.One case was accorded with OCA in clinical phenotype,but genetic diagnosis was OA1,the others were agreement between clinical diagnosis and genetic diagnosis.Conclusion There are 4 families with mutations in 6 families,representative of 3 type of albinism (OCA1,OCA2,OA1).
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Objective To observe the clinical effect of minimally invasive vitreoretinal (MIV) surgery combined with a modified suprachoroidal drainage surgery for retinal detachment associated with choroidal detachment (RRDCD).Methods A prospective clinical study.A total of 27 patients (27eyes) diagnosed as RRDCD were recruited in this study.There were 16 males and 11 females,with an average of (53.67± 14.82) years.The mean intraocular pressure (IOP) was (8.2± 2.1) mmHg (1 mmHg=0.133 kPa) and best corrected visual acuity (BCVA) of minimum resolution angle logarithm (logMAR) was 1.87±0.58.All subjects underwent 23G MIV combined a modified suprachoroidal drainage surgery,which 23G stab knife and 1 ml syringe needle were used for surgery.The visual outcome,IOP,rate of retinal reattachment and complications were comparatively analyzed preoperatively and postoperatively.Results At 1 day,10 days,1 month and 3 months after surgery,the average of logMAR BCVA were 1.62 ± 0.67,1.51 ± 0.63,1.39 ± 0.54,1.32± 0.56 and the mean of IOP were (13.47 ± 5.06),(14.43 ± 4.09),(14.89 ± 4.30),(15.38 ± 3.37) mmHg,respectively.There were significant differences of logMAR BCVA and IOP between before and after surgery (F=6.19,15.21;P＜0.05).Retinal reattachments were achieved in 27 eyes (100％) at 1 day and 10 days after surgery.At 1 month and 3 months after surgery,the rate of retinal reattachment were 88.89％ (24 eyes) and 85.19％ (23 eyes),respectively.No severe complications such as endophthalmitis and choroidal hemorrhage were found at follow-up visits.Conclusion MIV combined with a modified suprachoroidal drainage surgery is an effective and safe treatment for RRDCD,which can promote retina tear closure,improve visual acuity.
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Objective To screen the disease-causing genes in an autosomal dominant (AD)Weill-Marchesani syndrome (WMS) family from Henan province in China,and to analyze the relationship between genotypes and phenotypes of the AD WMS.Methods A family with suspected WMS was collected and studied in Henan Eye Hospital from September 2016 to July 2017.Clinical data and genomic DNA of the families were analyzed and genetic variations were screened by whole-exome sequencing (WES) The candidate genes related to ectopia lentis (FBN1,ADAMTSL2,ADAMTSL4,TGFBR2,CBS,ADAMTS10,ADAMTS17) were analyzed,and multiplex ligation dependent probe amplification (MLPA) was applied.Novel variants were further evaluated by sequencing 96 normal individuals.The previous reports with similar genetic characteristics were reviewed and the mutation types and clinical features were summarized.Written informed consent was obtained from the participants or their guardians before the collection of their venous blood and clinical data.Ethical approval was obtained from the Institutional Review Board of Henan Eye Institute.Results The suspicious mutation of the c.5260G＞A was detected in exon 42 of the FBN1 by WES in this family,which was predicted to be pathogenic and cosegregated with the disease;the clinical futures of the patients in the family included proportionate short stature,brachydactyly,joint stiffness,and the ocular problems included microspherophakia,moderate myopia,secondary glaucoma.Four mutations of FBN1 that related to WMS were reported in previous literature,and three of them were located in 41-42 exons and the others were the deletion of exons 9-11.All patients had typical clinical features of microspherophakia,short stature,brachydactyly,joint stiffness.In addition,thick skin was common,heart defects were occasional,protuberant abdomen and umbilical hernia were rarely reported.Conclusions The affected members in this family are in according with the clinical and genetic diagnosis of WMS.A novel mutation (c.5260G＞A) in FBN1 is discovered,which increases the spectrum of WMS mutation.The 41-42 exons of the FBN1 are hotspot of mutation in WMS.
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Objective To evaluate ocular surface changes following minimal vitreoretinal surgery in postmenopausal women patients with proliferative diabetic retinopathy (PDR).Methods Sixty-one women PDR patients (61 eyes) underwent vitreous microsurgery were recruited in this prospective study,including 31 postmenopausal women (PMW group) and 30 non-postmenopausal women (non-PMW group).The contralateral eyes were considered as the control group.Corneal fluorescein (FL) staining,tear break-up time (TBUT),Schirmer I test (SIT),central corneal sensitivity and ocular surface disease index (OSDI) were estimated.All tests were carried out 1 day preoperatively and 1 day,10 days,1 month and 3 months postoperatively.The student's t test or Mann-Whitney U and ANOVA for repeat measurements test were used.Results Preoperatively,TBUT of surgery and non-surgery eyes in PMW were shorter than non-PMW (t=-2.115,-2.035;P＜0.05),but higher OSDI scores were found in PMW (t=2.482,2.208;P＜ 0.05).TBUT reduction rate (Z=-2.771,-1.993;P＜0.05) and OSDI rising rate (Z=2.539,2.157;P＜0.05) of surgery eyes in PMW were higher than non-PMW 1 day and 10 days postoperatively.The lower SIT of surgery eyes in PMW were observed at 1 day and 10 days (t=-2.403,-2.029;P＜0.05) after surgery.At 10 days after surgery,FL and OSDI scores of surgery eyes in non-PMW returned to preoperative level (Z=-0.447,-0.513;P＞0.05),but in PMW,the recovery process experienced 1 month (Z=-1.500,-0.853;P＞0.05).TBUT and SIT of surgery eyes in two groups both reached preoperative level at 1 month following surgery (Z=-0.715,-1.266,-1.531,-0.522;P＞ 0.05).Conclnsions PMW with PDR had ocular surface dysfunction,which resulted in aggravated dry eye after minimal vitreoretinal surgery.
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Background Local medical treatment of refractory immunologic keratitis is unsuccessful,and systemic steroids and immunosuppressive agents could cause severe side-effects.Tacrolimus is a potent immunosuppressive drug,it has been proved that topical application of tacrolimus could reduce immunologic inflammation.The safety and efficacy of 0.05％ tacrolimus eye drops for refractory immunologic keratitis has not been described.Objective This study was to evaluate efficacy and safety of 0.05％ tacrolimus eye drops for refractory ulcerative keratitis.Methods A retrospective study was performed.Twenty-one eyes of 17 patients with refractory immunologic keratitis,which had uncontrolled inflammation despite initially treatment including topical steroids and 1％ cyclosporine A,were enrolled,including 11 males and 6 females,with the mean ages of 52 years.Infectious ulcer was excluded by laboratory tests.No systemic disease was found in 11 patients,and Wegener's granulomatosis,rheumatoid arthritis and ulcerative colitis were seen in 1 patient,4 patients and 1 patient respectively before presentation and they were all in remission under conventional systemic therapy.Four patients got binocularly involved and thirteen patients were monocularly involved.Of the 21 eyes,2 eyes with ulcer were ≥ 3 quarters of the limbus,and 19 eyes with ulcer were ≤ 2 quarters.All patients were treated with 0.05％ tacrolimus eyedrops after discontinuing cyclosporine A.The dosage was adjusted according to the severity of inflammation and was gradually tapered when improvement occurred.The corneal lesions were examined under the slit lamp microscope and Heidelberg HRT3 Rostock Cornea Module regularly,and inflammatory cell infiltrations were analyzed with Cell Count(R) software (Heidelberg Engineering GmbH).The safety variables were monitored regularly,including adverse response of eye,tacrolimus blood concentrations measured by chemiluminescent microparticle immunoassay (CMIA) and laboratory examinations of blood routine,blood glucose level,liver and kidney function.Results The patients were treated and followed-up for a mean duration of 18.1 months (range,8-24 months).Corneal ulcer area was obviously reduced 1 month after treatment in 19 eyes,and 2 eyes of 2 cases received anterior lamellar keratoplasty due to progressive corneal destruction despite of tacrolimus therapy.Corneal ulcer was cured 3 months after treatment,and stromal edema and infiltration disappeared 6 months after treatment under the slit lamp microscope.The inflammatory cell densities at lesion zone were (958±329),(858±339),(459±261),(192±124),(98±52),(44±24) and (3±2)/mm2 before treatment and 1 week,1 month as well as 3,6,12,24 months following treatment,respectively,showing a gradually decline as time lapse (F =125.439,P =0.000),and the inflammatory cells were significantly decreased in 1,3,6,12 and 24 months following the administration of 0.05％ tacrolimus eye drops in comparison with that before treatment (all at P =0.000).The therapy duration was 12 months in 9 eyes and 24 months in 12 eyes.Transient irritation sensation occurred in 4 eyes during the treating period.Blood concentrations of tacrolimus were below 1.0 ng/ml in all of the patients.No abnormality was found in laboratory tests.Conclusions The use of 0.05％ tacrolimus eye drops is a safe and effective approach to refractory immunologic keratitis.