Ocular Surface最新文献

Purpose

Sensory nerve terminals are highly distributed in the cornea, and regulate ocular surface sensation and homeostasis in response to various endogenous and exogenous stimuli. However, little is known about mediators regulating the physiological and pathophysiological activities of corneal sensory nerves. The aim of this study was to investigate the presence of cholinergic regulation in sensory nerves in the cornea.

Methods

Localization of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (vAChT) was evaluated using western blotting and immunohistochemical analysis. The synthesis and liberation of acetylcholine from the cornea were assessed using corneal segments pre-incubated with [³H]choline. The responsiveness of corneal neurons and nerves to cholinergic drugs was explored using calcium imaging with primary cultures of trigeminal ganglion neurons and extracellular recording from corneal preparations in guinea pigs.

Results

ChAT, but not vAChT, was highly distributed in the corneal epithelium. In corneal segments, [³H] acetylcholine was synthesized from [³H]choline, and was also released in response to electrical stimuli. In cultured corneal neurons, the population sensitive to a transient receptor potential melastatin 8 (TRPM8) agonist exhibited high probability of responding to nicotine in a calcium imaging experiment. The firing frequency of cold-sensitive corneal nerves was increased by the application of nicotine, but diminished by an α4 nicotinic acetylcholine receptor antagonist.

Conclusions

The corneal epithelium can synthesize and release acetylcholine. Corneal acetylcholine can excite sensory nerves via nicotinic receptors containing the α4 subunit. Therefore, corneal acetylcholine may be one of the important regulators of corneal nerve activity arranging ocular surface condition and sensation.

Meibomian gland dysfunction (MGD) is a leading cause of dry eye disease and one of the most common ophthalmic conditions encountered in eye clinics worldwide. These holocrine glands are situated in the eyelid, where they produce specialized lipids, or meibum, needed to lubricate the eye surface and slow tear film evaporation – functions which are critical to preserving high-resolution vision. MGD results in tear instability, rapid tear evaporation, changes in local microflora, and dry eye disease, amongst other pathological entities. While studies identifying the mechanisms of MGD have generally focused on gland obstruction, we now know that age is a major risk factor for MGD that is associated with abnormal cell differentiation and renewal. It is also now appreciated that immune-inflammatory disorders, such as certain autoimmune diseases and atopy, may trigger MGD, as demonstrated through a T cell-driven neutrophil response. Here, we independently discuss the underlying roles of gland and immune related factors in MGD, as well as the integration of these two distinct mechanisms into a unified perspective that may aid future studies. From this unique standpoint, we propose a revised model in which glandular dysfunction and immunopathogenic pathways are not primary versus secondary contributors in MGD, but are fluid, interactive, and dynamic, which we likened to the Yin and Yang of MGD.

Purpose

Short chain fatty acids (SCFAs) produced by gut microbiota are known to play primary roles in gut homeostasis by immunomodulation partially through G-protein coupled receptors (GPR) 43. Using mouse models of TLR ligand induced keratitis, we investigated whether SCFAs and GPR43 play any regulatory roles in the pathogenesis of inflammatory responses in the eye.

Methods

Both human and mouse eyes were labeled with a specific antibody for GPR43 and imaged by a laser scanning confocal microscope. Corneal cups from naïve C57BL/6J (B6) and GPR43 knockout (KO) mice were stimulated with TLR ligands in the presence or absence of sodium butyrate overnight and then processed for RT-PCR assay for expression of GPR43 and cytokines. Keratitis was induced by Poly I:C in wild type (WT) B6, GPR43KO and chimeric mice and the disease severity was evaluated by the corneal fluorescein staining test, and infiltrating cell staining and calculating in corneal whole mount.

Results

GPR43 is expressed in both human and mouse eyes and the expression is bidirectionally regulated by TLR ligands and butyrate. Butyrate significantly inhibited inflammation caused by several TLR ligands such as Poly I:C, Flagellin, and CpG-ODN (TLR-3, 5 and 9 agonists, respectively) in WT, but not GPR43KO, mice. Butyrate inhibition of TLR-induced keratitis is mediated by the GPR43 expressed in tissue but not hematopoietic, cells.

Conclusions

This is the first report to demonstrate of the protective effect of SCFAs on microbial keratitis, and the dynamic expression and anti-inflammatory function of GPR43 in the eye. SCFAs can modulate inflammation and immunity in the eye through GPR43.

This study compared the long-term outcome of different epithelial transplantation techniques to treat limbal stem cell deficiency (LSCD). We conducted a retrospective 15-year comparative systematic cohort study of patients with LSCD who underwent either cultivated limbal epithelial transplantation (CLET), simple limbal epithelial transplantation (SLET), or cultivated oral mucosal epithelial transplantation (COMET). We reviewed the demographic data, etiology, LSCD severity, best-corrected visual acuity, surgical outcomes, and complications. A total of 103 eyes of 94 patients (mean age, 45.0 ± 16.4 years) with LSCD were enrolled. The most common cause of LSCD was chemical injury (42.7 %). The median follow-up time was 75 months. The success rates of CLET, SLET, and COMET were 45.5 %, 77.8 %, and 57.8 %, respectively. The 7-year survival rates after CLET, SLET, and COMET were 50.0 %, 72.2 %, and 53.2 %, respectively. Steven-Johnson syndrome (SJS) had a significantly lower survival rate than other causes (p < 0.001), but SLET had a significantly higher survival rate than CLET (p = 0.018) and COMET (p = 0.047). Visual improvement of more than four Snellen lines was achieved in 53.1 % of successful cases and 28.2 % of failed cases. SJS, Schirmer I test <5 mm, and the presence of postoperative recurrent epithelial defects were significant risk factors for a failed surgery. All epithelial transplantation techniques had favorable long-term surgical outcomes. More than half of the patients achieved a stable ocular surface and visual acuity improvement up to 7 years postoperatively. SLET tends to have a better surgical outcome than CLET and COMET, especially in patients with SJS.

Purpose

To design a novel efficacious scAAV-Gusb viral vector for treating Mucopolysaccharidosis Type VII (MPS VII) caused by a mutation in the β-Glu gene (Gusb allele).

Methods

β-Glu expression of single-stranded AAV-Gusb (ssAAV-Gusb) and self-complementary AAV (scAAV-Gusb) vectors are tested with cultured murine Gusb fibroblasts. The scAAV-Gusb vector was chosen in further studies to prolong the life span and treat corneal pathology of Gusb mice via intrahepatic injection of neonates and intrastromal injection in adults, respectively. Corneal pathology was studied using HRT2 in vivo confocal microscope and histochemistry in mice corneas.

Results

Both ssAAV-Gusb and scAAV-Gusb vectors expressed murine β-Glu in cultured Gusb fibroblasts. The scAAV-Gusb vector had higher transduction efficiency than the ssAAV-Gusb vector. To prolong the life span of Gusb mice, neonates (3 days old) were administered with scAAV-Gusb virus via intrahepatic injection. The treatment improves the survival rate of Gusb mice, prolonging the median survival rate from 22.5 weeks (untreated) to 50 weeks (treated). Thereafter, we determined the efficacy of the scAAV-Gusb virus in ameliorating corneal cloudiness observed in aged Gusb mice. Both corneal cloudiness and stroma thickness decreased, and there was the presence of β-Glu enzyme activity in the Gusb corneas receiving scAAV-Gusb virus associated with morphology change of amoeboid stromal cells in untreated to characteristic dendritic keratocytes morphology after 4–12 weeks of scAAV-Gusb virus injection.

Conclusion

Intrahepatic injection of scAAV-Gusb is efficacious in prolonging the life span of Gusb mice, and intrastromal injection can ameliorate corneal phenotypes. Both strategies can be adapted for treating other MPS.

Purpose

Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo.

Methods

Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies.

Results

The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 10⁷ copies of DCN and 2.31 × 10⁷ copies of PEDF genes per μg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea.

Conclusion

The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.

Myoepithelial cells (MECs) are a unique subset of epithelial cells that possess several smooth muscle cell characteristics, such as a high number of actin-myosin filaments and the ability to contract. These cells are primarily located around the secretory cells of exocrine glands, including the salivary, mammary, lacrimal, and sweat glands. Their primary functions involve the construction of the basement membrane and help with secretion of gland products through contraction. So far, no comparative analysis of MECs in different exocrine glands had ever evaluated their differences. In this review, we took advantage of the various publicly available scRNAseq data from mouse exocrine glands to identify their shared and unique characteristics.

The aim of this review is to compare the role of MECs in maintaining healthy glandular function, their involvement in disease states, and their regenerative capacity, with a particular emphasis on the latest research findings in these areas.

Purpose

While changes in meibum quality are correlated with severity of meibomian gland dysfunction (MGD) and dry eye disease, little is known regarding the mechanics of meibum secretion. The purpose of this study was to develop a finite element model of meibum secretion and evaluate the effect of various factors that might impact meibum delivery to the ocular surface.

Methods

A finite element analysis in COMSOL 6.0 was used to simulate the flow of meibum within the gland's terminal excretory duct. Historical normal human meibum rheology data taken over the meibum melting range from fluid (35–40 °C) to solid (25–30 °C) were then used to calculate the minimum yield stress and plastic viscosity of meibum. The effects of meibum melting state, eyelid pressure and terminal duct diameter on meibum flow rates were then systematically investigated.

Results

The melting state of meibum from liquid to solid was associated with an increase in the minimum yield stress and plastic viscosity that caused an exponential decrease in meibum flow. Modeling also established that there was a linear correlation between meibum flow rate and eyelid pressure needed to express meibum and the 4th power of the terminal duct radius.

Conclusions

Our results suggest that changes in the melting state of meibum from fluid to solid, as well as changes in the radius of the terminal excretory duct and the force exerted by the eyelid can lead to dramatic decreases in the flow of meibum. Together these findings suggest alternative mechanisms for meibomian gland obstruction.

Purpose

Herpes simplex keratitis (HSK), caused by type 1 herpes simplex virus (HSV) reactivation, is a severe infectious disease that leads to vision loss. HSV can trigger metabolic reprogramming in the host cell and change the extracellular vesicles (EV) cargos; however, little is known about the EV metabolic signatures during ocular HSV infection. Here, we aimed to depict the EV-associated metabolic landscape in HSK patients’ tears.

Methods

We collected 82 samples from 41 participants with unilateral HSK (contralateral unaffected tears were set as negative control), including subtype cohorts of 13 epithelial, 20 stromal, and 8 endothelial HSK. We isolated tear EVs via our previously established platform and conducted metabolic analysis using LC-MS/MS. The metabolic signatures for recognizing HSK and subtypes were assessed through differential analysis and machine learning algorithms.

Results

Hypopsia and increased extracellular CD63 levels were observed in affected eyes. We identified 339 metabolites based on sEVs isolated from tears. Differential analysis revealed alterations in energy and amino acid metabolism, as well as the infectious microenvironment. Furthermore, we observed dysregulated metabolite such as methyldopa, which is associated with inappropriate neovascularization and corneal sensation loss, contributing to the HSK severity particularly in the stromal subtype. Moreover, machine learning classification also suggested a set of EV metabolic signatures that have potential for pan-keratitis detection.

Conclusions

Our findings demonstrate that tear EV metabolites can serve as valuable indicators for comprehending the underlying pathological mechanisms. This knowledge is expected to facilitate the development of liquid biopsy means and therapeutic target discovery.