BACKGROUND:Mesenchymal stem cells (MSCs) are promising candidates for immunomodulatory therapy that are currently being tested in corneal allograft rejection. In this study, we tested the effects of Mesenchymal stem cells derived exosomes in the corneal allograft rejection model.METHODS:Mesenchymal stem cells derived exosomes (MSC-exo) were collected and characterized. Wistar-Lewis rat corneal allograft rejection models were established. PKH26 labeled exosomes were used for track experiment. Models were randomly separated into four groups and treated with graded doses of exosomes or same volumn of PBS. Corneal grafts were assessed for rejection degree using slit-lamp biomicroscopy. Grafts were examined histologically using hematoxylin-eosin (H-E) staining and immunohistochemically using antibodies against CD4, CD8 and CD25. A comprehensive graft mRNA gene expression array analysis was conducted and checked by real-time polymerase chain reaction (PCR).RESULTS:The nanovesicles obtained were expressing exosome specific protein markers CD9, CD63, CD81. The labeled exosomes could be detected in both cornea and anterior chamber two hours after injection.The 10 μg exosomes subconjunctival injection can effectively prolong graft survival time (MST 16.3 ± 2.5 days). 10 μg exosomes-treated group can inhibit the infiltration of CD4+ and CD25+ T cells. IFN-γ and CXCL11 levels were significantly decreased in grafts obtained from postoperative exosomes-treated rats when compared with controls.CONCLUSIONS:MSC-exo can cross biological barrier and play better role directly towards target tissue. MSC-exo can effectively prolong grafts survival time. Th1 signaling pathway was significantly inhibited in the exosomes treated group.

An important mechanism involved in dry eye (DE) is the association between tear hyperosmolarity and inflammation severity. Inflammation in DE might be mediated by the NLRP3 inflammasome, which activated by exposure to reactive oxygen species (ROS). A combination of carboxymethylcellulose (CMC) and α-melanocyte stimulating hormone (α-MSH) may influence DE through this mechanism, thus avoiding defects of signal drug. In this study, we assessed whether treatment comprising CMC combined with α-MSH could ameliorate ocular surface function; we found that it promoted tear secretion, reduced the density of fluorescein sodium staining, enhanced the number of conjunctival goblet cells, and reduced the number of corneal apoptotic cells. Investigation of the underlying mechanism suggested that the synergistic effect of combined treatment alleviated DE inflammation through reduction of ROS level and inhibition of the NLRP3 inflammasome in human corneal epithelial cells. These findings indicate that combined CMC + α-MSH treatment could ameliorate lesions and restore ocular surface function in patients with DE through reduction of ROS level and inhibition of NLRP3 signalling.

Background: Mesenchymal stem cells (MSC) are promising candidates for immunomodulatory therapy that are currently being tested in corneal allograft rejection. In this study, we tested the effects of Mesenchymal stem cells derived exosomes in the corneal allograft rejection model.Methods: MSC derived exosomes were collected. Corneal grafts obtained from Wistar rats were transplanted into Lewis rat hosts. Hosts were then randomly separated into four groups and treated with previously harvested exosomes in different doses or PBS. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. Grafts were examined histologically using hematoxylin-eosin (H-E) staining and immunohistochemically using antibodies against CD4. A comprehensive graft mRNA gene expression array analysis was conducted and checked by real-time polymerase chain reaction (PCR).Results: 1μg/ml exosomes subconjunctival injection prolonged graft survival time (MST 16.3±2.5 days). Exosomes-treated groups also showed suppression of inflammatory cell as well as CD4+, CD25+, CD45+ T cells infiltration in the allograft region. IFN-γ and CXCL11 levels were significantly decreased in grafts obtained from postoperative exosomes-treated rats when compared with controls.Conclusions: Subconjunctival injection of MSC derived exosomes in rats was effective in prolonging corneal allograft survival. Th1 signaling pathway was significantly inhibited in the exosomes treated group.

Rationale: Corneal transplantation is an effective treatment to corneal blindness. However, the immune rejection imperils corneal allograft survival. An interventional modality is urgently needed to inhibit immune rejection and promote allograft survival. In our previous study, subconjunctival injections of bone marrow-derived mesenchymal stem cells (BM-MSCs) into a rat model of corneal allograft rejection extended allograft survival for 2 d. In this study, we sought to generate IL-10-overexpressing BM-MSCs, aiming to boost the survival-promoting effects of BM-MSCs on corneal allografts and explore the molecular and cellular mechanisms underlying augmented protection. Methods: A population of IL-10-overexpressing BM-MSCs (designated as IL-10-BM-MSCs) were generated by lentivirus transduction and FACS purification. The self-renewal, multi-differentiation, and immunoinhibitory capabilities of IL-10-BM-MSCs were examined by conventional assays. The IL-10-BM-MSCs were subconjunctivally injected into the model of corneal allograft rejection, and the allografts were monitored on a daily basis. The expression profiling of long noncoding RNA (lncRNA) in the allografts was revealed by RNA sequencing and verified by quantitative real-time PCR. The infiltrating immune cell type predominantly upregulating the lncRNA expression was identified by RNAscope in situ hybridization. The function of the upregulated lncRNA was proved by loss- and gain-of-function experiments both in vivo and in vitro. Results: The IL-10-BM-MSCs possessed an enhanced immunoinhibitory capability and unabated self-renewal and multi-differentiation potentials as compared to plain BM-MSCs. The subconjunctivally injected IL-10-BM-MSCs reduced immune cell infiltration and doubled allograft survival time (20 d) as compared to IL-10 protein or plain BM-MSCs in the corneal allograft rejection model. Further, IL-10-BM-MSCs significantly upregulated lncRNA 003946 expression in CD68+ macrophages infiltrating corneal allografts. Silencing and overexpressing lncRNA 003946 in macrophage cultures abolished and mimicked the IL-10-BM-MSCs' suppressing effects on the macrophages' antigen presentation, respectively. In parallel, knocking down and overexpressing the lncRNA in vivo abrogated and simulated the survival-promoting effects of IL-10-BM-MSCs on corneal allografts, respectively. Conclusion: The remarkable protective effects of IL-10-BM-MSCs support further developing them into an effective interventional modality against corneal allograft rejection. IL-10-BM-MSCs promote corneal allograft survival mainly through upregulating a novel lncRNA expression in graft-infiltrating CD68+ macrophages. LncRNA, for the first time, is integrated into an IL-10-BM-MSC-driven immunomodulatory axis against the immune rejection to corneal allograft.

目的 探讨糖尿病与非糖尿病患者中短泪膜破裂时间型(short tear break-up time,sBUT)干眼患者之间眼表状况的不同.方法 收集50 ～ 80岁的糖尿病患者59例(118眼),非糖尿病患者41例(82眼),排除泪液缺乏型干眼患者,用标准干眼评估问卷(standard patient evaluation of eye dryness,SPEED)评分,泪液分泌量,非侵入性泪膜第一秒破裂(BUT1st)和平均破裂时间(BUTavg),睑板腺拍照评分,脂质层厚度(lipid layer thickness,LLT),眨眼频率进行统计分析,进一步分为sBUT干眼患者(糖尿病sBUT组、非糖尿病sBUT组)和非sBUT干眼患者(糖尿病非sBUT组、非糖尿病非sBUT组)后再进行统计分析.结果 相比于非糖尿病组,糖尿病组有更高的SPEED评分和睑板腺拍照评分,更薄的LLT,差异均有统计学意义(均为P＜0.05).糖尿病sBUT组的SPEED评分为(7.40±3.24)分,睑板腺拍照评分为(3.2±1.16)分,眨眼频率(6.78±5.10)次,在4组中最高;而Schirmer值[(5.80±4.74) mm]、BUT1st值[(3.44±1.10)s]、BUTavg值[(5.74±3.17)s]和LLT[(66.40±23.34)nm]在4组中最低.在泪液相对不缺乏的干眼人群中,LLT分别与眨眼频率、SPEED评分之间呈负相关(r=-0.168,P＜0.05;r=-0.298,P＜0.001);眨眼频率与SPEED评分呈正相关(r=0.268,P＜0.001).结论 糖尿病患者睑板腺的短缩和缺失更严重,使LLT变更薄,从而引起泪膜破裂时间变更短,眨眼频率更高,导致糖尿病患者的干眼症状相比于非糖尿病患者更重.

Objective To investigate the inhibitory effect of bromfenac sodium hydrate ophthalmic solution on corneal neovascularization (CNV) induced by alkali burn.Methods A total of 192 specific pathogen free (SPF) degree adult male Sprague-Dawley (SD) rats were used in this study.One hundred and seventy-two rats were chosen to establish CNV model with alkali burn in the right eyes.Following alkali burn,rats were randomly divided into CNV group,model control group,bromfenac sodium group and fluorometholone group,with 43 rats (43 eyes) in each group.Another 20 rats (40 eyes) served as normal control group.One day after modeling,the model control group,bromfenac sodium group and fluorometholone group received phosphate buffer saline (PBS),bromfenac sodium hydrate ophthalmic solution and 0.1％ fluorometholone eye drops,respectively.The state of cornea and anterior chamber and the growth of CNV of rats in each group were observed by slit-lamp microscope every day after modeling.At 1,3,7,14,21 and 28 days after modeling,the anterior segment photos of the experimental eyes were captured,and the percent of cornea areas covered by CNV was calculated.At 7,14 and 28 days after modeling,the eye tissue sections were stained with hematoxylin and eosin staining and immunohistochemistry staining to evaluate the expressions of CD45 and VEGF-A.Real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA)were used to detect the expression of COX-2 and VEGF mRNA and protein level.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology(ARVO).Results Each model group showed corneal edema and opacification 1 day after modeling.The corneal edema was aggravated 7 days after modeling.On the 14th day after modeling,the degree of corneal opacity and edema decreased gradually.On the 28th day after modeling,leucoma was observed in CNV group and model control group,and nebula was observed in bromfenac sodium group and fluorometholone group.At 7,14,21 and 28 days after modeling,the percentages of CNV areas in bromfenac sodium group and fluorometholone group were significantly lower than those in CNV group and model control group (all at P＜0.05).No significant difference was found in the percentage of CNV areas between bromfenac sodium group and fluorometholone group at various time points (all at P＞0.05).On the 7th day after modeling,the thinning of corneal epithelial layer,edema and arrangement disorder of stroma layer were observed,and the expression of VEGF-A was positive in all model groups;a small amount of CD45 positive inflammatory cell infiltrations were observed in CNV group and model control group.On the 14th and 28th day after modeling,CNV was seen in the center of cornea in CNV group and model control group;the epithelial keratosis and reduction of corneal edema were seen in each group,and no inflammatory cell infiltration was observed in each group.On the 7th day after modeling,the expressions of COX-2 and VEGF mRNA in CNV group and model control group were significantly higher than those in normal control group,bromfenac sodium group and fluorometholone group (all at P ＜ 0.05),the expressions of COX-2 and VEGF protein in bromfenac sodium group were significantly lower than those in CNV group (all at P＜0.05).The corneal peroration rate in model control group and bromfenac sodium group was 10％ (1 case in 10 rats).The corneal perforation rate in fluorometholone group was 30％ (3 cases in 10 rats).In each model group,10％ to 30％ rats had hyphema.Conclusions Bromfenac sodium hydrate ophthalmic solution can inhibit the formation and growth of CNV after alkali burn in rats.This effect may be mediated by regulating COX-2 expression,reducing inflammation and inhibiting VEGF production.

PURPOSE:To observe ocular surface changes in Type II diabetic patients with different disease durations and to understand the correlations between clinical parameters and diabetic durations.METHODS:In this cross-sectional, prospective study, 51 healthy controls and 91 patients with Type II diabetes were enrolled. The diabetics were divided into 3 subgroups according to the disease duration, including duration <10 y group, 10 to 20 y group, and ≥21 y group. All subjects underwent clinical ocular examinations, including lipid layer thickness (LLT), blinking rate, tear meniscus height (TMH), noninvasive tear film break-up time (NI-BUT), meibography, superficial punctate keratopathy (SPK) scoring, corneal sensitivity, and Schirmer I test. They were also evaluated using the standard patient evaluation of eye dryness (SPEED) questionnaire.RESULTS:SPEED score, meiboscore, SPK score, LLT, Schirmer I test, and corneal sensitivity differed significantly between the diabetic and healthy control groups. Further, SPEED score, Schirmer I test, corneal sensitivity, meiboscore, and blink rate significantly differed among the 3 diabetic subgroups and the control group. In diabetics, the SPEED score correlated with the SPK score, blink rate, TMH, and LLT; NI-BUT with TMH, LLT, and blink rate; TMH with the SPK score; Schirmer I test with the SPK score; and corneal sensitivity with the meiboscore. More importantly, the Schirmer I test, corneal sensitivity, and SPEED score negatively correlated with diabetic duration.CONCLUSION:Diabetic duration is an important factor that affects functions of the lacrimal functional unit in patients with Type II diabetes. The trends of changes in the ocular parameters vary along the course of diabetes.

目的 观察间充质干细胞来源外泌体(mesenchymal stems cells derived exosomes,MSCs-exo)对大鼠角膜移植排斥反应的抑制作用.方法 制作大鼠异体角膜移植排斥反应模型,随机分为4组:空白对照组、MSCs结膜下治疗组、PBS结膜下注射组、MSCs-exo结膜下治疗组.术后第3天开始裂隙灯下观察角膜植片排斥情况,并对MSCs-exo进行染色及示踪观察其结局.结果 空白对照组角膜植片存活时间为(9.67±0.56)d,MSCs结膜下治疗组为(12.33±0.76)d,PBS结膜下注射组为(9.50±0.34)d,MSCs-exo结膜下治疗组为(16.5±1.15)d.PBS结膜下注射组角膜植片存活时间较空白对照组差异无统计学意义(P＞0.05),而MSCs结膜下治疗组与MSCs-exo结膜下治疗组角膜植片存活时间较PBS结膜下注射组均明显延长(均为P＜0.05),MSCs-exo结膜下治疗组植片存活时间长于MSCs结膜下治疗组(P＜0.05).空白对照组大鼠免疫组织化学染色结果显示植片部分厚度比植床角膜厚度显著增厚,上皮层及基质层内见大量CD4+细胞聚集.而MSCs-exo结膜下治疗组植片中CD4+细胞比空白对照组明显减少.MSCs-exo结膜下注射后72 h时,仅有少量外泌体可在结膜下被观测到.进一步将观测时间前推至注射后24 h,发现大量外泌体存在于结膜下,而角膜组织和前房内也可见到外泌体的定位表达.结论 结膜下注射MSCs-exo可以显著延长角膜植片存活时间,外泌体治疗可能成为无细胞治疗的新方法.

Mesenchymal stem cells (MSCs) have been reported to promote long-term cellular and organ transplant acceptance due to their immunotherapeutic characteristics. Previous work from our lab using a rat allograft model has shown that systemic infusion of MSCs inhibited corneal allograft rejection and prolonged graft survival. Here, we further investigated the effects of local MSCs administration in the same animal model.

Objective To investigate the effect of mesenchymal stem cells (MSCs) combined with low-dose cyclosporin A ( CsA) on corneal transplantation rejection and its mechanism. Methods A fully allogeneic rat cornea transplant model (Wistar rats to Lewis rats) was created. Thirty-six model rats were divided into MSCs group, CsA group and MSCs+CsA group randomly by using the random number table method,with 12 rats for each group. In each group,six rats were used for the evalution of symptom observation and six for the assay of immune factor. Three days after corneal transplantation,slit lamp microscope was used to observe and record the vascularization,edema and opacity scores of corneal graft. After 18 days,spleen T helper cell(Th)1 and Th2 cytokines were measured by enzyme linked immunosorbent assay ( ELISA ). The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. This study protocol was approved by Ethic Committee of Tianjin Medical University Eye Hospital (No. TMUaMEC2017028). Results The survival time of corneal grafts in MSCs+CsA group was prolonged in comparison with the MSCs group and CsA group,with significant differences between them([21.3±3.9]days vs. (14.8±2.4)days; [21.3±3.9]days vs. [16.0±1.1]days) (P=0.003,0.004). Eighteen days after corneal transplantation,corneal opacity scores were 3.17±0.17, 3.00±0.00 and 2.17±0.17,corneal neovascularization scores were 2.67 ± 0.21,2.33 ± 0.21 and 1.83 ± 0.21, respectively in the MSCs group, CsA group and MSCs+ CsA group, showing significant differences among the three groups (F=15.500,P<0.01; F=4.524,P=0.029). The corneal opacity scores and corneal neovascularization scores in the MSCs+ CsA group were significantly lower than those in the MSCs group and CsA group(all at P<0.05). Interleukin(IL)-2,IL-4,IL-10,and interferon-γ (IFN-γ) concentrations were all significantly different among the MSCs group,CsA group and MSCs+ CsA group (F=15.000,12.810,10.720,17.960,all at P< 0.01). Compared with the MSCs group and CsA group,Th1 subsets cytokines IFN-γ and IL-2 concentrations in the MSCs+CsA group were significantly lower,and Th2 subsets cytokines IL-4 and IL-10 concentrations were significantly increased in the MSCs+CsA group in comparison with the MSCs group and CsA group ( all at P<0.05 ). Conclusions The combination of MSCs with low-dose CsA can effectively ameliorate immunological rejection by the modulation of the balance of Th1/Th2.

There are well-acknowledged clinical or pre-clinical measurements concerning diabetic peripheral neuropathy (DPN). The current gold standard for diagnosis of diabetic peripheral neuropathy is nerve conduction suitable for detecting large nerve fiber function and intraepidermal nerve fiber density assessment for small fiber damage evaluation [2]. The lack of a sensitive, non-invasive, and repeatable endpoint to measure changes in small nerve fibers is a major factor holding back clinical trials for the treatment of diabetic peripheral neuropathy. As cornea is the most densely innerved tissue, assessing corneal nerves' structure and function will be promising to predict and assess the degree of DPN. In the diabetic micro-environment, damaged corneal nerves lead to decreased corneal sensitivity, both of which resulting in abnormal tear function. According to this theory, the measurements of nerve structure, corneal sensitivity, tear secretion and tear components, to some extent, can reveal and assess the state of corneal neuropathy. This review focuses on summarizing the knowledge of the latest detective methods of diabetic corneal neuropathy, popular in use or possible to further in study and be applied into clinical practice.