Profiling ocular surface responses to preserved and non‐preserved topical glaucoma medications: a two‐year randomised evaluation study

BackgroundUse of topical glaucoma medications has been reported to cause ocular surface (OS) discomfort and inflammation. This study explores the profile of inflammatory cytokines and OS symptoms induced in response to preserved and non‐preserved drops.MethodsProspective, randomized evaluation on thirty‐six treatment‐naïve patients over 24 months of three differently preserved glaucoma drop preparations: Preservative‐free (PF), Polyquad (PQ), and Benzalkonium chloride (BAK). Study participants were evaluated at baseline and then at 1, 3, 6, 12 and 24 months whilst on medication. At each visit, participants completed the Ocular Surface disease index (OSDI) questionnaire, had basal tear sampling and impression cytology (IC) of the conjunctival epithelium. Quantitative polymerase chain reaction was performed to measure the gene expression of inflammatory cytokines (Interleukin (IL)‐6, IL‐8, IL‐10, IL‐12A, IL‐12B, IL‐17A, IL‐1β, and tumour necrosis factor (TNF)‐α) in the IC samples. Corresponding protein expression of cytokines in tear samples was assessed by the Becton‐Dickinson cytometric bead arrays.ResultsCompared to PF and PQ groups, mRNA and protein expression of IL‐6, IL‐8, and IL‐1β increased in samples from the BAK group in a time‐dependent fashion, whereas all other cytokines showed a non‐significant increase. In the BAK group, there was a strong correlation between OSDI and the levels of IC/IL‐1β (r=0.832, R squared=0.692 and p=0.040 ); IC/IL‐10 (r=0.925, R squared=0.856 and p=0.008) and tear/IL‐1β (r=0.899, R squared=0.808 and p=0.014 ). ConclusionBAK‐preserved topical drops stimulate a sterile inflammatory response on the OS within 3‐months which is maintained thereafter. Whereas PF‐drops and PQ‐preserved drops showed no significant OS inflammation

Cultivation and characterisation of human peripheral cornea-derived endothelial cells [abstract]

To confirm that human corneal rims left over from DALK/DSEK/PK surgeries could be useful sources for ex vivo endothelial cell expansion. Human corneal rims remaining from DALK/DSEK/PK surgeries were utilized (1:1 sex ratio, age 63+20 years, endothelial cell density >2,500 cells/mm2). The time from death to use varied between 3 days and 1.5 months. Endothelial cells isolated using a two-step, peel-and-digest method, whereby the Descemet’s membrane and endothelial cells were peeled off under a dissecting microscope, followed by digestion in collagenase. The isolated cells were suspended in TrypLE prior to plating onto FNC-coated tissue culture plates. The cells were then cultured in Ham’s F12:M199 (1:1) media supplemented with, ascorbic acid, transferrin, sodium selenite and bFGF. Characterisation of the cultured cells was performed by RT-qPCR and immunofluorescence staining accordingly. The number of isolated endothelial cells was repeatedly low (< 20,000 cells). However, improved techniques allowed to reduce stromal cell contamination. It was observed that endothelial cell proliferation was improved when the culture surface area was reduced. Furthermore, typical endothelial cobble stone morphology was observed when the cell density was high. Cell morphology and growth showed notable difference related to donor age and preservation time. ZO-1, Na/K-ATPase and PITX2 were used to confirm the endothelial phenotype. Preserved human corneal rims can be utilized for ex vivo expansion of corneal endothelial cells but further optimization is needed