Pathogenic Role of Endoplasmic Reticulum Stress in Diabetic Corneal Endothelial Dysfunction.

PURPOSE: Progressive corneal edema and endothelial cell loss represent the major corneal complications observed in diabetic patients after intraocular surgery. However, the underlying pathogenesis and potential treatment remain incompletely understood. METHODS: We used streptozotocin-induced type 1 diabetic mice and db/db type 2 diabetic mice as diabetic animal models. These mice were treated with the endoplasmic reticulum (ER) stress agonist thapsigargin; 60-mmHg intraocular pressure (IOP) with the ER stress antagonist 4-phenylbutyric acid (4-PBA); mitochondria-targeted antioxidant SkQ1; or reactive oxygen species scavenger N-acetyl-l-cysteine (NAC). Corneal thickness and endothelial cell density were measured before and after treatment. Human corneal endothelial cells were treated with high glucose with or without 4-PBA. The expression of corneal endothelial- and ER stress-related genes was detected by western blot and immunofluorescence staining. Mitochondrial bioenergetics were measured with an Agilent Seahorse XFp Analyzer. RESULTS: In diabetic mice, the appearance of ER stress preceded morphological changes in the corneal endothelium. The persistent ER stress directly caused corneal edema and endothelial cell loss in normal mice. Pharmacological inhibition of ER stress was sufficient to mitigate corneal edema and endothelial cell loss in both diabetic mice after high IOP treatment. Mechanistically, inhibiting ER stress ameliorated the hyperglycemia-induced mitochondrial bioenergetic deficits and improved the barrier and pump functional recovery of the corneal endothelium. When compared with NAC, 4-PBA and SkQ1 exhibited better improvement of corneal edema and endothelial cell loss in diabetic mice. CONCLUSIONS: Hyperglycemia-induced ER stress contributes to the dysfunction of diabetic corneal endothelium, and inhibiting ER stress may offer therapeutic potential by improving mitochondrial bioenergetics.

The advanced glycation end-products (AGEs)/ROS/NLRP3 inflammasome axis contributes to delayed diabetic corneal wound healing and nerve regeneration.

Diabetic keratopathy (DK) is an important diabetic complication at the ocular surface. Chronic low-grade inflammation mediated by the NLRP3 inflammasome promotes pathogenesis of diabetes and its complications. However, the effect of the NLRP3 inflammasome on DK pathogenesis remains elusive. Wild-type (WT) and Nlrp3 knockout (KO) C57 mice were used to establish a type I diabetes model by intraperitoneal injection of streptozotocin. The effect of the NLRP3 inflammasome on diabetic corneal wound healing and never regeneration was examined by a corneal epithelial abrasion model. Western blot, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and pharmacological treatment were performed to investigate the regulatory mechanism of advanced glycation end products (AGEs) on NLRP3 inflammasome activation and corneal wound healing in vivo. The cultured mouse corneal epithelial cells (TKE2) were used to evaluate the effect and mechanism of AGEs on NLRP3 inflammasome activation in vitro. We revealed that NLRP3 inflammasome-mediated inflammation and pyroptosis contributed to DK pathogenesis. Under physiological conditions, the NLRP3 inflammasome was required for corneal wound healing and nerve regeneration. However, under a diabetic scenario, sustained activation of the NLRP3 inflammasome resulted in postponed corneal wound healing and impaired nerve regeneration. Mechanistically, the accumulated AGEs promoted hyperactivation of the NLRP3 inflammasome through ROS production. Moreover, genetically and pharmacologically blocking the AGEs/ROS/NLRP3 inflammasome axis significantly expedited diabetic corneal epithelial wound closure and nerve regeneration. Our results revealed that AGEs-induced hyperactivation of the NLRP3 inflammasome resulted in delayed diabetic corneal wound healing and impaired nerve regeneration, which further highlighted the NLRP3 inflammasome as a promising target for DK treatment.

Hyperglycemia induces corneal endothelial dysfunction through attenuating mitophagy.

Hyperglycemia increases the risk of corneal endothelial dysfunction, resulting in damage to corneal endothelial structure and function. However, the effect and mechanism of hyperglycemia-induced corneal endothelial damage remain elusive. In this study, we demonstrated that hyperglycemia reduced the expression of pump-related protein Na+/K+ ATPase and barrier-related protein ZO-1. Moreover, we found hyperglycemia caused abnormal changes of morphological mitochondria and dynamics in vitro. In addition, the decreased levels of mitophagy were further confirmed Western blotting and LC3B-Mitotracker Immunofluorescence. Exogenous application of mitophagy agonist carbonyl cyanide m-chlorophenyl hydrazine (CCCP) increases the expression of Na+/K+ ATPase and ZO-1 in corneal endothelial cells through up-regulated mitophagy in vitro. In addition, CCCP effectively reverses the phenomenon of corneal opacity and increased corneal thickness in diabetic mice. Therefore, our demonstrated the novel function of mitophagy in the pathogenesis of diabetic cornea endothelial dysfunction, and provide potential approach for treating diabetic corneal endothelial dysfunction.

Periplocin ameliorates mouse age-related meibomian gland dysfunction through up-regulation of Na/K-ATPase via SRC pathway.

Age-related meibomian gland dysfunction (MGD) is the main cause of evaporative dry eye disease in an aging population. Decreased meibocyte cell renewal and lipid synthesis are associated with age-related MGD. Here, we found an obvious decline of Ki67, ΔNp63, and Na+/K+ ATPase expression in aged meibomian glands. Potential Na+/K+ ATPase agonist periplocin, a naturally occurring compound extracted from the traditional herbal medicine cortex periplocae, could promote the proliferation and stem cell activity of meibocyte cells in vitro. Moreover, we observed that periplocin treatment effectively increased the expression of Na+ /K+ ATPase, accompanied with the enhanced expression of Ki67 and ΔNp63 in aged meibomian glands, indicating that periplocin may accelerate meibocyte cell renewal in aged mice. LipidTox staining showed increased lipid accumulation after periplocin treatment in cultured meibomian gland cells and aged meibomian glands. Furthermore, we demonstrated that the SRC pathway was inhibited in aged meibomian glands; however, it was activated by periplocin. Accordingly, the inhibition of the SRC signaling pathway by saracatinib blocked periplocin-induced proliferation and lipid accumulation in meibomian gland cells. In sum, we suggest periplocin-ameliorated meibocyte cell renewal and lipid synthesis in aged meibomian glands via the SRC pathway, which could be a promising candidate for age-related MGD.

Efficacy of rhNGF-loaded amniotic membrane transplantation for rabbit corneal epithelial and nerve regeneration.

AIM: To evaluate the efficacy of recombinant human nerve growth factor-loaded amniotic membrane (rhNGF-AM) on corneal epithelial and nerve regeneration in rabbit model. METHODS: Freshly prepared human amniotic membrane (AM) were immersed into PBS buffer containing 100 or 500 µg/mL rhNGF for 15, 30, and 60min at 4°C. The in vitro release kinetics of rhNGF was measured with ELISA. For in vivo evaluation, the AM were immersed with 500 µg/mL rhNGF for 30min. Fifty-seven rabbits were selected to establish corneal epithelial defect model. In addition to the 19 rabbits in control group, 38 rabbits received AM transplantation with or without rhNGF after the removal of central epithelium. Corneal epithelial defect area, sub-epithelial nerve fiber density, corneal sensitivity, rhNGF contents in resident AM and corneas were measured after the surgery. RESULTS: rhNGF was sustained release from the AM within 14d in vitro, with the positive correlation with initial immersion concentration. The immersion of AM in 500 µg/mL rhNGF for 30min achieved the most stable release within 14d. After transplantation in rabbit cornea, a high concentration of rhNGF in resident rhNGF-AM and cornea was maintained within 8d. Corneal epithelial healing, nerve fiber regeneration and the recovery of corneal sensitivity were significantly accelerated after the rhNGF-AM transplantation when compared to simple AM transplantation (all P<0.05). CONCLUSION: Simple immersion of AM achieves the sustained release of rhNGF, and promotes corneal epithelial wound healing and nerve regeneration, as well as the recovery of corneal sensitivity in rabbit.

Lipidomic analysis of meibomian glands from type-1 diabetes mouse model and preliminary studies of potential mechanism.

Diabetes is a significant risk factor for meibomian gland dysfunction (MGD), but its mechanism is poorly understood. The main function of the meibomian glands (MGs) is to synthesize, store, and secrete lipids. In this study, we found that the amount of lipids in the meibomian acini in STZ-induced type 1 diabetic mice decreased, and the lipid droplets became larger and irregular. In all, 31 lipid subclasses were identified in the mouse MGs, which contained 1378 lipid species in total through lipidomics analysis based on LC-MS/MS. Diabetes caused a significant increase in the content of ceramides (Cer) in the MGs but a significant decrease in the ration of sphingomyelin to ceramides (SM/Cer). The quantity of meibocytes in diabetic mice was dramatically decreased, and the proliferation activity was alleviated, which may be associated with cell cycle arrest caused by diabetes-induced abnormal Cer metabolism in MGs. We found an increase in macrophage and neutrophils infiltration in the diabetic MGs, which may be related to the significant reduction of AcCa in diabetic MGs. Taken together, the results of the present study demonstrated that diabetes induced disruption of lipid homeostasis in MGs, which may mediate the decreased cell proliferation and increased inflammation caused by diabetes in MGs.

Topical calcitriol application promotes diabetic corneal wound healing and reinnervation through inhibiting NLRP3 inflammasome activation.

Vitamin D (VD) deficiency delays corneal wound healing in those with diabetes, which cannot be rescued with supplemental diet. Here, we employed topical calcitriol application to evaluate its efficiency in corneal wound healing and reinnervation in diabetic mice. Type 1 diabetic mice were topically administrated calcitriol, or subconjunctivally injected with NLRP3 antagonist MCC950 or IL-1ß blocking antibody after epithelial debridement. Serum VD levels, corneal epithelial defect, corneal sensation and nerve density, NLRP3 inflammasome activation, neutrophil infiltration, macrophage phenotypes, and gene expressions were examined. Compared with those of normal mice, diabetic mice showed reduced serum VD levels. Topical calcitriol application promoted corneal wound healing and nerve regeneration, as well as sensation recovery in diabetic mice. Moreover, calcitriol ameliorated neutrophil infiltration and promoted the M1-to-M2 macrophage transition, accompanied by suppressed overactivation of the NLRP3 inflammasome. Treatment with NLRP3 antagonist or IL-1ß blockage demonstrated similar improvements as those of topical calcitriol application. Additionally, calcitriol administration upregulated desmosomal and hemidesmosomal gene expression in the diabetic cornea. In conclusion, topical calcitriol application promotes corneal wound healing and reinnervation during diabetes, which may be related to the suppression of the overactivation of NLRP3 inflammasome.

A retrospective survey of strabismus surgery in a tertiary eye center in northern China, 2014-2019.

BACKGROUND: To evaluate the distribution pattern and changes of strabismus surgery in northern China. METHODS: The records of strabismus patients at Qingdao Eye Hospital from January 2014 to December 2019 were reviewed retrospectively. The characteristics analyzed included gender, regional distribution, constituent ratio of age and type of strabismus. Changes during the periods 2014-2016 and 2017-2019 were compared and analyzed. RESULTS: A total of 5746 strabismus patients were recruited. The number of strabismus patients was relatively stable each year from 2014 to 2016 but gradually increased each year from 2017 to 2019. Of these, 51.7% (2968/5746) were male, and 48.3% (2778/5746) were female. The majority (89.8%, 5159/5746) of the patients were from Shandong Province. The statistical results of the constituent ratio of age showed that 32.4% (1860/5746) were 7-12 years old (primary school level). Patients under 12 years of age (preschool and primary school level) accounted for 60.0% (3447/5746) of all the patients. In terms of the types of strabismus, exotropia accounted for 63.5% (3650/5746), followed by esotropia and vertical rotational strabismus at 13.2% (758/5746) and 9.7% (555/5746), respectively. Intermittent exotropia was the most common type among the exotropia patients, accounting for 71.3% (2604/3650). Among the patients with intermittent exotropia, 62.5% (1627/2604) were children aged 4-12 years, and the basic type of intermittent exotropia was the main type. Four percent (231/5746) of the patients, of which adult patients comprised the main population, required reoperation. CONCLUSIONS: Patients with strabismus at primary school level comprised the largest group of strabismus patients in north China. Exotropia was the most common type of strabismus, and intermittent exotropia was the most common type of exotropia. The rate of exotropia to esotropia was 5:1.

The effects of simultaneous operation on dissociated vertical deviation with horizontal and torsional strabismus.

AIM: To investigate the therapeutic effects of simultaneous horizontal and vertical operations on dissociated vertical deviation (DVD) associated with other deviations. METHODS: Forty-five cases of DVD with horizontal and torsional strabismus underwent combined operation were collected retrospectively. All clinical records were analyzed. All patients were followed up for 6 to 24mo. Wilcoxon signed-ranks test was performed to evaluate the changes of vertical and horizontal deviation. χ 2 test was used to evaluate the changes of binocular visual function. RESULTS: Forty-five cases included 36 patients with intermittent exotropia and binocular inferior oblique overaction (IOOA), 5 patients with concomitant esotropia and binocular IOOA, 4 patients with intermittent exotropia and monocular superior oblique palsy. The superior rectus recession (SRR) combined with horizontal rectus recession and the myectomy of inferior oblique or anterior transposition were operated simultaneously to correct all types of strabismus. There were 43 cases who achieved normal eye position in vertical direction, while 2 cases were with undercorrection of 5Δ to 6Δ. In patients with horizontal strabismus, 2 cases of exotropia were with overcorrection of 6Δ to 8Δ, 1 case of esotropia was with undercorrection of 6Δ, and 1 case of monocular superior oblique palsy with compensatory head posture was not significantly improved. The binocular visual function of most patients recovered after operation. The difference of the binocular visual function and eye position were significant compared with that before operation (P<0.05). CONCLUSION: The simultaneous operation on DVD with horizontal and torsional strabismus is successful.