Protective effect of asiatic acid on blood-retinal barrier in diabetic rats / 中华实验眼科杂志

Objective:To investigate the protective effect of asiatic acid (AA) on blood-retinal barrier (BRB) in diabetic rats and its possible mechanism.Methods:Ninety-six healthy 8-week-old male SD rats were randomly divided into normal control group, diabetes group, low-dose AA group and high-dose AA group, with 24 rats in each group.Intraperitoneal injection of streptozocin (STZ) was used to establish diabetes model.One month after the establishment of the model, the low-dose AA group and the high-dose AA group were given intragastrical administration of 37.5 mg/kg AA and 75.0 mg/kg AA, respectively, once a day according to grouping.The normal control group and the diabetes group were administrated with the same amount of 0.5% sodium carboxymethyl cellulose.The body weight of the rats were weighted at week 0, 1, 2, 3, 4 after intragastrical administration.Blood was taken from the tail vein and the blood glucose level was measured.The retina was obtained one month following the administration.Pathological changes of the rats retina were detected by hematoxylin-eosin (HE) staining.Evan's blue quantitative method was used to detect the damage of blood-retinal barrier (BRB). Immunofluorescence staining was performed to detect the distribution of Occludin, Notch1, Jagged canonical Notch ligand 1 (JAG1) and Delta like canonical Notch ligand 4 (DLL4) in retina.The mRNA and protein expressive levels of Occludin, Notch1, JAG1 and DLL4 were detected by Real-time PCR and Western blot.The study protocol was approved by a Scientific Research and Clinical Trial Ethics Committee of The First Affiliated Hospital of Zhengzhou University (No.2020-KY-228). The use and care of animals complied with the Guide for the Care and Use of Laboratory Animals of National Institutes of Health and the 3R rules.Results:At 4 weeks after intragastrical administration, the body weight of the high-dose AA group was significantly higher than that of the diabetes group, and the blood glucose values were significantly lower in the high-dose AA group and the low-dose AA group in comparison with the diabetes group (all at P<0.05). The cells were arranged orderly with clear layered structure in the normal control group.In the diabetes group, the retina was thicker than that of the normal control group, with a thicker outer nuclear layer, disordered cell arrangement and unclear layered structure.Compared with the diabetes group, the total retinal thickness and structure were obviously improved in the low-dose AA group and the high-dose AA group.Evan's blue leakage in retina was (3.07±1.30), (13.73±3.88), (9.57±2.69) and (6.55±1.61)ng/mg in the normal control group, the diabetes group, the low-dose AA group and the high-dose AA group, respectively.There was a significant difference in leakage of Evan's blue among the four groups ( F=18.50, P<0.01), among which the leakage of Evan's blue dye in the high-dose AA group was significantly lower than that of the diabetes group ( P<0.01). Compared with the diabetes group, there was significantly higher relative expression level of Occludin protein and significantly lower relative expression levels of Notch1, JAG1 and DLL4 proteins in the other three groups (all at P<0.05). The relative expression level of Occludin protein was significantly higher and the relative expression levels of Notch1, JAG1 and DLL4 proteins were significantly lower in the high-dose AA group than those in the low-dose AA group (all at P<0.05). Compared with the normal control group, the Occludin mRNA expression level was significantly decreased and the expression levels of Notch1, JAG1 and DLL4 mRNA were significantly increased in the diabetes group and low-dose AA group (all at P<0.01). The Occludin mRNA expression level was higher and the Notch1 mRNA expression level was lower in the high-dose AA group than those in the diabetes group and the low-dose AA group, and the expression levels of JAG1 and DLL4 mRNA were lower in the high-dose AA group in comparison with the diabetes group, and the differences were statistically significant (all at P<0.05). Conclusions:Asiatic acid might play a protective role on BRB in diabetic rats by inhibiting Notch1 signaling pathway.

Effect of attenuated-dose aflibercept intravitreal injection on retinopathy of prematurity / 中华实验眼科杂志

Objective:To observe the effect of attenuated-dose aflibercept in the treatment of retinopathy of prematurity(ROP).Methods:A non-randomized controlled study was conducted, and 76 eyes of 38 ROP pediatric patients treated in First Affiliated Hospital of Zhengzhou University from December 2018 to May 2020 were enrolled.According to the requirements of their guardians, the patients were divided into ranibizumab group with 42 eyes of 21 cases and attenuated-dose aflibercept group with 34 eyes of 17 cases, and received intravitreal injection of ranibizumab 0.025 ml (0.25 mg) or aflibercept 0.012 5 ml (0.5 mg) according to grouping respectively.Retcam fundus photography was used to observe the treatment response at 1 week, 2, 4 weeks and 2, 3, and 6 months after treatment, and the effective rate at the end of follow-up was calculated.The intraocular pressure was measured with Icare PRO magnetic rebound tonometer at 1 minute, 10, and 30 minutes after injection. The ocular and systemic complications were observed during the 6-month follow-up period.All the guardians signed the informed consent prior to treatment.This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No.2020-KY-228).Results:The effective rates of single ranibizumab and attenuated-dose aflibercept were 90.5% (38/42) and 88.2% (30/34), respectively, with no significant difference between the two groups ( χ2=0.10, P=0.75). The intraocular pressure of the ranibizumab group at 1 minute and 10 minutes after the operation were higher than those of the attenuated-dose aflibercept group, and the difference was statistically significant (both at P<0.01). The intraocular pressure recovered to the baseline level at 30 minutes after the operation.In the ranibizumab group, 4 eyes were ineffective after a single injection, among which 2 eyes were effective after second intravitreal injection of ranibizumab and 1 eye was effective after retinal laser photocoagulation treatment and 1 eye underwent vitrectomy due to the progress of retinal detachment one week after intravitreal injection, and the posterior retina reattached well.In the attenuated-dose aflibercept group, 4 eyes did not respond to treatment, of which 3 eyes were effective after second intravitreal injection of aflibercept, and 1 eye was effective after retinal laser photocoagulation.No ocular or systemic complications were observed during the followed-up period. Conclusions:Reduced dose of aflibercept is safe and effective in the treatment of ROP, and has little influence on intraocular pressure.

Genetic analysis of a Chinese family with congenital aniridia and detection of PAX6 mutation locus / 中华实验眼科杂志

Background Congenital aniridia is a rare bilateral hereditary ophthalmopathy which impact panocular.Researches showed that congenital aniridia can be caused by different mutation locus of PAX6 genes,and the mutations are multifarious.Objective This study was to detect and anaiyze the mutations of a Chinese family with congenital aniridia by using targeted sequence capture sequencing and direct Sanger sequencing.Methods This study was approved by Ethic Committee of the First Affiliated Hospital of Zhengzhou University and followed Declaration of Helsinki.Written informed consent was obtained from subjects or their custodians before any related medical examination.A cross-sectional study was performed.A Chinese congenital aniridia family was included at the First Affiliated Hospital of Zhengzhou University in March,2016.All the family members received systemic medical examinations including nervous system and oral glucose tolerance test and then the ocular examinations were carried out.The periphery blood of 10 ml was collected from the members for genomic DNA extraction.Targeted sequence capture sequencing was performed on the DNA of proband to screen out the suspicious mutant locus.The mutation was verified by comparing the Sanger direct sequencing results from all family members.Results A total of 3 generations of 9 members were included in this congenital aniridia pedigree,and the Ⅰ 1 was dead without eye abnormality.Three patients (Ⅱ2 and her children Ⅲ1,Ⅲ2) and 5 normal family members were determined,showing an autosomal dominant inheritance pattern.No abnormal signs were found in nervous system and oral glucose tolerance test in the families.The reduce of visual acuity,ocular hypertension (21 mmHg),absence of biocular iris,opacification of corneal stroma,horizontal nystagmus,hapoplasia of fovea were found in all the sufferers.In addition,the ptosis of the left eye,congenital cataract of the right eye in Ⅱ 2 patient as well as biocular cataract and subluxation of lenses also were exhibited.The c.183C＞A mutation of the PAX6 gene was screened out to be a possible pathogenic mutation.The result of Sanger direct sequencing in the families verified a co-segregation of this mutation with mutant phenotypes.Conclusions PAX6 gene c.183C ＞A,a rare mutation in Chinese population,is a virulence mutation site in this aniridia family.

Analysis of GJA3 mutation associated with a Chinese family with autosomal dominant congenital cataract by whole-exome sequencing / 中华实验眼科杂志

Congenital cataract is one of the important reasons for the blindness of children,and most congenital cataracts are genetic.At present,thirty-nine genes have been identified relating to autosomal dominant congenital cataract (ADCC).Objective This study was to identify and analyze the virulence gene of a Chinese family pedigree with ADCC by whole-exome sequencing.Methods A Chinese ADCC family was recruited in Affiliated First Hospital of Zhengzhou University from August to September in 2014.The family disease history and clinical data were recorded.The peripheral venous blood of 10 ml was collected in 14 patients with congenital cataract and 14 families with normal phenotype,and the peripheral blood samples were obtained from 100 healthy examined people as controls.The genomic DNA was extracted form all subjects using standard phenol chlorum method,and proband DNA was screened by whole-exome sequencing.Then mutation locus of the candidate gene was selected after compared with the information of database in the proband.The mutation locus of the candidate gene from 14 normal families and 100 healthy controls were amplified and sequenced by PCR technique based on the primer sequence of mutation locus of proband to verify the pathogenic gene of this ADCC family.This study protocol was approved by Ethic Committee of Affiliated First Hospital of Zhengzhou University and complied with Helsinki Declaration.Written informed consent was obtained from subjects or custodian before any medical examination.Results The family had a total of 5 generations of 68 members,in which 20 subjects were found with congenital cataract.The inheritance mode consisted with autosomal dominant inheritance.Cortical cataract was found in both eyes in the patients.Whole-exome sequencing showed that the 143rd ribonucleotide A of exon 2 explicit factor of chromosome 13 GJA3 gene mutated into G (c.143A＞G) in the proband,which resulted in the 48th amino acids changed from glutamate into glycine (p.E48G).PCR amplification product sequencing displayed that the same mutation of DNA appeared in all the patients of this family,while not the same mutation was seen in the candidate genes of normal phenotype families and 100 healthy controls.Conclusions GJA3 gene c.143A＞G is a virulence mutation site in this ADCC family,it is a supplement of the mutation spectrum of GJA3 gene.

A novel PAX6 mutation in a Chinese family with congenital aniridia / 中华实验眼科杂志

Background Aniridia is a rare congenital hereditary eye disease.Studies determined that PAX6 gene mutation is closely associated with congenital aniridia,but the mutation locus are varied.Objective This study was to identify virulence mutation locus of PAX6 gene of a Chinese family pedigree with autosomal dominant aniridia.Methods A Chinese family affected with autosomal dominant aniridia was collected and examined in Affiliated First Hospital of Zhengzhou University in August 2014.Periphery blood of 10 ml was collected from all the families and 100 unrelated health controls.The genomic DNA was extracted by standardized phenol-chloroform method,and all exons and splicing junctions of PAX6 were amplified by PCR.Real-time fluorescence quantitative PCR was performed to examine the relative expression of PAX6 mRNA in patients and normal phenotype families and heahh controls.This study protocol was approved by Ethic Committee of Affiliated First Hospital of Zhengzhou University and complied with Helsinki Declaration.Written informed consent was obtained from subjects or custodian before any medical examination.Results This Chinese family inclued 3 generations and 9 members,with a classic autosomal dominant inheritance mode.Five patients were found,showing the absence of iris and cataract in 3 adult patients and only absence of the iris in 2 children,and other 4 members showed the normal phenotype.A novel heterozygous PAX6 deletion mutation c.796 del G (p.A266 fs) (GenBank ID:KP255960) in exon 10 was exclusively found in all affected individuals but not in any of the unaffected families or unrelated health controls.PAX6 mRNA level in lymphocytes was about 50％ lower in aniridia patients than in unaffected family members,indicating that this mutation caused nonsense-mediated mRNA decay.Conclusions A novel deletion mutation in PAX6 gene results in an abnormal PAX6 COOH-terminal extension in the Chinese aniridia family.This finding expands the mutation spectrum of PAX6 gene.

Indocyanine green-assisted macular epiretinal membrane combined internal limiting membrane peeling for idiopathic macular epiretinal membrane / 中华实验眼科杂志

Background Idiopathic macular epiretinal membrane (IMEM) occurs probably along with vitreous macular traction syndrome (VMTS),persudo macular hole (PMH) and lamellar macular hole (LMH).Removing posterior hyaloid and completely peeling IMEM are the key to the treatment.Objective This study was to investigate the effectiveness of indocyanine green (ICG)-assisted macular epiretinal membrane combined internal limiting membrane (ILM) peeling for IMEM.Methods Twenty nine eyes of 29 patients with IMEM were collected in Affiliated First Hospital of Zhengzhou University from June 2010 to September 2012,including 16 eyes with simple macular epiretinal membrane,6 eyes with both IMEM and VMTS,3 eyes with IMEM and PMH,4 eyes with IMEM and LMH.A standard three-port pars plana vitrectomy was performed.After removal of posterior hyaloid,0.25％ ICG was used to assist IMEM and ILM peeling.The process and results were recored.Results After staining,the free boundary of the IMEM became obvious and IMEM was peeled directly in 17 of the 29 eyes (58％).In the others (42％),a free petal of ILM was made,IMEM and ILM were peeled together.In all the 29 eyes,the peeled zone could be easily recognized.No serious intraoperative complication was found.The mean postoperative follow-up was (9.65 ±7.58)months (ranged,1 to 28 months).Visual acuity was improved in 20 eyes (69％).The LogMAR vision was significantly improved in postoperation in comprison with preoperation (0.62 ±0.56 versus 0.72 ±0.67) (t =2.370,P=0.025).No IMEM recurred during the following-up duration.Conclusions ICG-assisted ILM peeling can make the surgery of IMEM safer and prevent recurrence.