Discussion
Using two different methods to measure cell viability, we demonstrated that a short treatment with the antiseptic PI, commonly used prior to ocular surgeries, can irreversibly damage and kill the three main cell types of the conjunctiva: stratified squamous cells, goblet cells and fibroblasts. The effect of PI was comparable to that of the positive control of 30% H2O2 known to damage cells. One or more concentrations of PI and 30% H2O2 significantly decreased cell viability according to both tests, and all concentrations of PI significantly reduced viability across all preparation types according to the Alamar Blue assay. Fibroblasts, however, were significantly killed by fewer concentrations of PI according to the calcein/AM and EH-1 test suggesting a potential fibroblastic resistance to PI. A clinical investigation into a link between PI treatment and postsurgery vernal conjunctivitis caused by an increase in conjunctival fibroblast growth may be warranted. Our results suggest that care must be taken of the conjunctiva after surgical PI treatment to allow it to heal and repopulate the different cell types. The time needed for the cell numbers and proportions to return to pretreatment levels is unknown and likely varies from type to type and individual to individual. This may be particularly deleterious to patients undergoing repeated procedures.
Low concentrations of PI (0.25%) administered repeatedly throughout cataract surgery does significantly reduce ocular bacterial loads without significantly changing corneal endothelial cell densities.22 The current study demonstrates that a single 0.25% PI application for 5 min does not kill a statistically significant number of fibroblasts in vitro but does significantly affect the viability of both goblet cells and mixed populations of epithelial cells suggesting that while the corneal endothelium probably remains intact, the conjunctival epithelium is likely damaged. A single application of 0.05% PI for only 30 s can reduce ocular bacterial load while minimising damage to the ocular surface in humans35 and concentrations of 0.5% and 1% PI applied only once demonstrate significantly less corneal damage than higher PI concentrations in rabbits as measured by fluorescein stain. The effect of repeated low duration, low concentration PI was not evaluated in this study and may provide an alternative to single-application, high-concentration applications that last for minutes.
It should be noted that repeated applications of 5% PI have been demonstrated to be deleterious to the ocular surface as measured by the Schein dry eye questionnaire and measurements of tear osmolarity.36 Indeed, even a single application of 5% PI can significantly decrease the integrity of the corneal epithelium as measured by a sodium fluorescein stain and increase subjective vision complaints as measured by the Schien dry eye test.37
In the present study mixed cultures were composed of on average 46.5%±0.57% goblet cells, 32.1%±0.30% stratified squamous cells, while the remaining cells are believed to be a combination of fibroblasts and undifferentiated cells. The conjunctiva is generally considered to have more stratified squamous cells than goblet cells. The relative percentage of these cells, however, differs depending on the area of the conjunctiva examined. There is a higher stratified squamous cell to goblet cell ratio in the bulbar conjunctiva than in the forniceal conjunctiva which contains more goblet cells and undifferentiated progenitor cells.38 39 Undifferentiated cells form a small population in the conjunctiva with these cells distributed randomly throughout the conjunctiva. The relative population of goblet cells used herein can thus be used to mimic the conjunctival epithelium.
The two types of viability assays used measure different cellular properties. The Alamar Blue assay measures cell metabolic activity and is often used to determine cell number. The live–dead assay in contrast directly measures cell death. When cell number is compared by the two assays, the three different types of cultures were similarly affected by all concentrations of PI. When cell death was measured, the mixed cultures of epithelial cells were similarly resistant to PI treatment when compared with the purified goblet cell and fibroblast cultures. As the mixed culture used in the present study more closely mimics the ocular surface and contains several cell types, the mixture of cells may have different types of cell–cell interactions than either of the purified cultures. The different cell–cell interactions in the mixed cultures do not appear to provide protection to PI treatment.
The use of PI to prevent infections is well established. Several studies have demonstrated the efficacy of PI to reduce ocular infections. In healthy newborns, 1.25% PI was as effective as 2.5% in reducing the number of colony-forming units40 and 2.5% PI decreased adenoviral conjunctivitis.41 Carrim et al demonstrated that a 3 min treatment with PI decreased the number of positive cultures in patients42 and a 10% treatment reduced the number of colony-forming units in patients undergoing cataract surgery.43 The optimum time and concentration of PI to prevent infections is unknown, however, 5% PI decreases bacterial load significantly more than 1% PI in vivo, while in vitro studies indicate the opposite is true with lower PI concentrations appearing to decrease bacterial load more than higher concentrations.44 45 The finding that even 0.25% PI damages and kills conjunctival cells in the present study highlights the need to determine a minimal, but effective amount and time of contact of PI with the ocular surface to prevent infection with the least amount of cell damage. The negative effect of PI on the ocular surface should be included in that determination. Shimada et al demonstrated that repeated irrigation with 0.025% PI is effective at reducing the bacterial infections after cataract surgery.46 It is possible that this technique, which is the only technique known to sterilise the anterior chamber at the conclusion of cataract surgery, may be less toxic to the conjunctiva, while still protecting the eye from bacterial infection.
Conjunctival goblet cells secrete the mucin MUC5AC into the tear film.5 Stratified squamous cells express the mucins MUC4, 16 and 20 that are the constituents of the apically located glycocalyx which lubricates and protects the ocular surface.2 As PI at all concentrations tested in this study is detrimental to the stratified squamous and goblet cells, it is possible that preoperative treatment with PI could lead to the development of dry eye. Indeed, dry eye syndrome post cataract surgery or post LASIK has not received sufficient attention. It is possible that the surgery prep contributes significantly to this condition.
In patients with an iodine allergy, an alternative preoperative antiseptic, chlorhexidine, is available. Like PI, chlorhexidine is used over a wide range of concentrations (0.05%–4%) and has been demonstrated to significantly reduce ocular surface bacterial load.47 While very few patients display an allergy to chlorhexidine, ocular topical use is specifically contraindicated by the manufacturer (https://www.pfizer.com.au/products/chlorhexidine-irrigation-solution). Chlorhexidine concentrations above 2% are known to be toxic to the corneal epithelium and conjunctiva of rabbits.48 However, in humans, a much lower concentration of chlorhexidine (0.05%) has been successfully used as a single-use ocular antiseptic prior to vitrectomy, although in a limited sample.49 The demonstrated toxicity of PI and lack of a demonstrably reliable alternative highlights the importance of including epithelial toxicity as a criterion in the evaluation of ocular antiseptics.
We conclude that the commonly used antiseptic PI significantly reduces the viability and kills conjunctival cells in culture including goblet cells, fibroblasts and mixed cultures of goblet cells and stratified squamous cells. Our findings suggest that ophthalmologists should consider pursuing preoperative antisepsis techniques that decrease conjunctival toxicity. This would not only be less toxic but would be more effective at decreasing bacteria counts, thus lessening the risk of endophthalmitis.