Purpose To evaluate tear neuropeptides (NPs) (vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), substance P (SP), nerve growth factor (NGF)) in chronic ocular topical hypotensive therapy with and without benzalkonium chloride (BAK) preservative.
Methods A comparative, open label, cross-sectional study of patients using antiglaucoma medications for >6 months with BAK (group I), without BAK (group II) and controls was done. Tear NPs (ELISA), ocular surface evaluation tests (tear breakup time (TBUT), Schirmer’s test, corneal and conjunctival staining score) and confocal central corneal subbasal nerve fibre layer (SBNFL) imaging was done.
Results Of 153 eyes evaluated, group 1 (82 eyes (41 patients; mean age 48±14.5 years)) and group 2 (71 eyes (36 patients; mean age 43.11±15 years)) were on therapy for a mean duration of 10.05±2.0 and 9.67±2.3 months, respectively. Tear analysis showed elevated SP and NGF (p<0.01); decreased CGRP (p=0.03), VIP and NPY (p<0.01) compared with controls (n=30, mean age 29.33±5.7 years). Tear NP levels (SP (p=0.1), NGF (p=0.33), CGRP (p=1), VIP (p=0.87), NPY (p=0.83)) and SBNFL (p=0.09) were comparable in both groups. There was no correlation seen between tear NP levels and clinical tests and SBNFL.
Conclusion Our study analysis points towards altered tear NP levels in eyes on chronic topical hypotensive therapy in comparison with controls with no significant difference in tear NP levels and central corneal SBNFL density between the BAK preservative and BAK-free antiglaucoma therapy.
- substance p
- ocular surface
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- substance p
- ocular surface
With about 79.6 million people expected to be affected in 2020,1 glaucoma remains the second-leading cause of blindness in the world. The symptoms experienced with antiglaucoma treatments seem to correlate to the presence of preservatives in the glaucoma medications.2–4 Benzalkonium chloride (BAK), a quaternary ammonium compound commonly used as preservative in topical ophthalmic formulations (0.025%–0.004%)3 causes ocular surface toxicity.4 5 In order to mitigate the ocular surface toxicity with instillation of several BAK-preserved formulations, use of fixed combination of topical antiglaucoma solutions has been advocated aiming to increase compliance and decrease the preservative toxicity. An estimated 48%–59% of patients with ocular hypertension or glaucoma have been observed to have been affected with ocular surface morbidity.6 7 Chronic antiglaucoma medications (AGM) causes a decrease in the number and density of corneal sub-basal nerve fibre bundles.8–10 Recent research points towards a close relationship between corneal nerves and epithelial cells, with the corneal nerves playing a role in promoting epithelial cell proliferation and differentiation.11–13 Corneal sensitivity also seems to correlate with the ocular surface health.11–13
The rich supply of sensory and autonomic nerve fibres of the ocular surface plays a crucial role in maintaining healthy epithelia and also serves as main sources of neurogenic inflammation.14–16 The subbasal nerve plexus along with stromal keratocytes secrete a number of neuropeptides (NPs) which facilitate cell mitogenesis and migration, DNA synthesis, neurite extension and survival, keratocyte proliferation and regulation of epithelial stem cells.17 Besides serving a protective function, corneal nerves also play an important role in regulating corneal epithelial integrity, proliferation and wound healing. The innervation of the corneal epithelial cells and the stroma has an important influence in the corneal trophism and contributes to the maintenance of a healthy corneal surface. Corneal denervation causes impaired wound healing ability of the epithelium and results in dry eye. NPs elaborated by corneal nerves influence corneal epithelial cells and seem to stimulate epithelial growth, proliferation, differentiation and production of collagen type VII.8 9 The ocular surface epithelial cells seem to produce the soluble factors—nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) with a neurotrophic effect.18 Tear levels of NGF have been seen to be increased in patients with dry eye disease.19 Substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) released from ocular surface epithelial cells, lacrimal gland tissues and nerve endings at inflammatory sites seem to modulate the infiltration and activation of the immune cells, causing reflex tearing and ocular discomfort.20 21 Neuromediators are involved in chronic ocular surface diseases such as dry eye with SP and CGRP promoting local inflammation by inducing vasodilatation, extravasation of leucocyte, activation of immune cells and release of several cytokines.22–25 VIP and NPY have anti-inflammatory properties by inhibition of proliferation of T cell and helper T cell type 1 response and modulating the release of cytokines, chemokines and nitric oxide.26–28 Changes in circulating levels of NPs and neurotrophins, as well as the impairment of salivary gland innervation have been documented in patients with Sjogren’s syndrome29 30 with decrease in circulating levels of NPY.31 NGF, a neurotrophin, has a pleiotropic action on the ocular surface, influencing immune reaction, sensitivity, corneal and conjunctival epithelia proliferation and differentiation and stimulation of mucin production by goblet cells,32 and its tear levels have been observed to be increased in patients with dry eye disease.19
Dry eye disease resulting due to ocular surface damage caused by BAK preservatives in chronic topical AGMs can effect alterations in the level of tear NPs in eyes on chronic antiglaucoma therapy. This pilot study was done to evaluate the NP levels—VIP, NPY, CGRP, SP and NGF, in the tears of eyes of patients on long-term AGMs with and without BAK preservatives in comparison with that of normal eyes. The tear levels of NPs in eyes on chronic topical AGMs with and without BAK preservatives was evaluated. Correlation of tear NP levels with the clinical ocular surface evaluation parameters (Schirmer’s test, tear film breakup time (TBUT), ocular surface fluorescein staining, dry eye severity level and corneal subbasal nerve fibre layer density (SBNFLD)) was analysed.
Patients and methods
This prospective study was conducted in accordance to the tenets of the Declaration of Helsinki. A total of 153 eyes of 77 patients on long-term topical AGMs (6 months or more) and 30 eyes of 30 normal subjects as controls were recruited into the study. Informed written consent pertaining to the collection and storage of tear samples was obtained from all study subjects recruited.
Cases of primary open angle glaucoma diagnosed in the presence of optic nerve head damage (defined by the presence of neuroretinal rim loss of optic nerve head and vertical cup–disc ratio of more than 0.4 or more), on regular follow-up with the glaucoma services of the centre, using topical AGMs of 6 months duration, timolol 0.5% alone/timolol 0.5% with prostaglandin analogue/prostaglandin analogue alone were recruited into the study. Of the 153 eyes of 77 patients using chronic topical AGMs recruited, 82 eyes of 41 patients were on topical AGMs with BAK (the concentration of BAK preservative present in the topical formulations used in our study was 0.005%–0.02%) (group 1) and 71 eyes of 36 patients on topical AGMs without BAK (group 2). Patients with history of intraocular surgery, laser treatment in the last 6 months, contact lens use, autoimmune disease, recent ocular inflammation/injection, eyes with trachomatous changes, dry eye related to other causes, previous or current use of other ocular medications such as artificial tear therapy or other AGMs were excluded from the study.
Demographic and topical glaucoma medications details of the patients were noted on a predesigned proforma. All participants underwent tear sample collection without anaesthesia, followed by ocular surface evaluation and then central corneal confocal microscopy imaging. Comprehensive ocular examination, aided Snellen’s visual acuity, intraocular pressure (Goldman applanation tonometry), ocular surface evaluation tests33 (flourescein TBUT, Schirmer’s I test (without anaesthesia), ocular surface staining score34 and in vivo scanning slit confocal microscopy of the central cornea), dry eye severity (DEWS classification)35 were done. Confocal microscopy was done only for the eyes on antiglaucoma therapy and was not planned in the control eyes due to cost concerns and as most normal subjects were unwilling to undergo cornealconfocal imaging. None of the study participants had any known neurodegenerative diseases. In vivo slit scanning confocal microscopy (ConfoScan 4, NIDEK Technologies, Padova, Italy) of the central cornea was done by an experienced ophthalmic technician, in automatic gain mode using a standard setting of four passes, with a scanning range of 200 µm to image the anterior layers of the cornea that is, epithelium, SBNFL, stromal keratocytes at 40× magnifications.36 If satisfactory images were not obtained, procedure was repeated to get the desired images. Each eye was scanned thrice through its entire depth and the two best images were selected for analysis, of which the best one containing maximum number of SBNFL nerves imaged was selected for analysis. SBNFL image analysis was done in a masked manner (MS) using free downloadable custom NIH Image J software (http://www.imagescience.org/meijering/software/neuronj/). The tracing of subbasal nerves were performed using Neuron J, a semiautomatic Image J plugin to facilitate the tracing and quantification of elongated image structures. Then the total nerve number, total length/frame of subbasal nerves was measured automatically. Nerve branches longer than 50 µm in length were counted as separated nerves. The total number of subbasal nerves was recorded. The mean SBNFLD was calculated as total length of all main nerves and their branches divided by area of standard frame size containing images (460 µm×345 µm, area=0.16 mm²).37
Tear collection, extraction and analysis
Tear samples were collected from all eyes of the subjects recruited in the study without anaesthetic, using glass microcapillary tubes (75 mm×0.5 mm) inserted in the inferior conjuctival fornix of both eyes and the tear samples were immediately transferred into 1.5 mL Eppendorf vial. The vials were then immediately stored at −80°C. ELISA was performed to quantify the amount of NPs in tear samples using specific commercially available kits and following the manufacturer’s instructions (SP, NPY, CGRP and VIP (Phoenix Pharmaceuticals, California, USA) and NGF (Korain Biotech Co., England). The detection limits for the NP levels were NPY 0–100 ng/mL; SP 0–25 ng/mL; CGRP 0–100 ng/mL; VIP 0–25 ng/mL and NGF 5–1200 pg/mL.
ELISA protocol for NPY, SP, CGRP and VIP
The tear samples were diluted 1:5 with 1× phosphate-buffered saline buffer. Fifty microlitres of standards, samples and positive controls were added to the immunoplate wells. Then 25 µL of primary antibody and biotinylated peptide were each added and the immunoplate was incubated at room temperature (20°C–30°C) for 2 hours. The immunoplates were washed four times with 350 µL/well of 1× assay buffer. Hundred microlitres of streptavidin-horseradish peroxidase solution were added and incubated at room temperature (20°C–30°C) for 1 hour. The immunoplates were washed four times with 350 µL/well of 1× assay buffer. Hundred microlitres of 3,3′,5,5′-tetramethylbenzidine substrate solution was added and incubated at room temperature (20°C–30°C) for 1 hour. The reaction was terminated with 100 µL of 2N HCL. The absorbance optical density was read at 450 nm and results were calculated.
ELISA protocol for NGF
The samples and standards were prepared and then added to the immunoplate. The microplate was incubated at room temperature (37°C) for 1 hour and then washed five times with wash buffer. The substrate solutions A and B were added to the immunoplate and incubated for 10 min at 37°C for colour development. The stop solution was then added to terminate the reaction. The absorbance optical density was read at 450 nm by a microplate enzyme-linked immunoassay reader (Sunrise; Tecan Systems, San Jose, California, USA). All samples were evaluated in duplicate and results were calculated.
Data were analysed by Stata V.14 and mean (SD), median (range) and frequency (%), categorical variable were compared among groups by χ2/Fisher’s exact test. Continuous variable followed by normal distribution were compared among the groups by t-test (two groups) and one-way analysis of variance followed by multiple comparison using Bonferroni Correction. While data following non-normal distribution were compared by a Wilcoxon Rank sum and Kruskal-Wallis test followed by multiple comparison using Dunn’s test with Bonferroni test. P value less than 0.5 considered as statistical significance
The demographic details of the study subjects are detailed in table 1 . Among patients on topical AGMs (figure 1), 22 eyes of 11 patients were on topical timolol 0.5% with BAK preservative and 14 eyes of 7 patients were on topical timolol 0.5% without BAK preservative. Among those using combination of timolol 0.5% and prostaglandin analogue therapy, 36 eyes of 18 patients were on topical timolol 0.5% and prostaglandin analogue with BAK preservative and 13 eyes of 7 patients were on topical timolol 0.5%+prostaglandin analogue without BAK preservative. Among the prostaglandin analogue users, 24 eyes of 12 patients were on topical prostaglandin analogue with BAK preservative, while 44 eyes of 22 patients were on topical prostaglandin analogue without BAK preservative. Best-corrected visual acuity, intraocular pressure and ocular surface evaluation test parameters are shown in table 2 . Dry eye severity level in AGMs with BAK preservative group noted was level 2 and while that in AGMs without BAK preservative group was noted to be level 1. While the ocular surface evaluation tests of TBUT, Schirmer’s test and staining scores showed significant difference between the study groups and the controls, no significant difference was seen between the two groups (table 2). The central corneal SBNFL analysis for the number of nerves, nerve length and density between the study and control eyes did not show any significant difference (table 3).
The NP levels in tears of the study and control eyes are tabulated in table 3 . Of the 82 eyes of 41 patients (mean age 48±14.5 years) who were on topical AGMs with BAK preservatives over a mean therapy duration of 10.05±2 months and 71 eyes of 36 patients (mean age 43.11±15 years) on topical AGMs without BAK preservatives over a mean therapy duration of 9.67±2.3 months, SP and NGF levels (p<0.01), were noted to be significantly elevated while CGRP (p=0.03), VIP, NPY (p<0.01) were observed to be significantly decreased as compared with that of the controls (30 eyes of 30 controls of mean age 29.33±5.7 years) (figures 2 and 3).
The correlation analysis of tear NP levels with ocular surface evaluation tests with the tear NP levels in the eyes with glaucoma medications and control eyes (table 4) showed no correlation in both groups. The correlation analysis between the central corneal SBNFLD with the tear NP levels in the glaucoma eyes is given in table 4.
BAK preservative in topical ophthalmic solutions can result in dry eye disease. This gets further compounded by the twice daily dosing that is required in patients with long-term glaucoma. Decreased ocular surface toxicity has been noted with use of antiglaucoma ophthalmic combination solutions that did not contain BAK preservatives.38 In patients on chronic topical hypotensive therapy, both the hypotensive drugs and their preservatives (especially BAK preservative) have been credited to result in increased inflammatory cellular and fibroblasts infiltration of conjunctiva with decrease in goblet cells, leading to ocular surface changes manifested clinically as dry eye.10 39–43 Altered ocular surface neurobiology contribute to the mechanisms responsible for the symptoms of dry eye. The association between corneal subbasal nerve density and corneal sensitivity has been established in dry eye.44 45 SP is released by the corneal sensory nerve fibres which stimulates corneal epithelial cell growth12 46 and promotes corneal cell migration47 48 and its metabolites tend to induce ocular surface neurogenic inflammation that causes chronicity of dry eye disease.49 Both SP and CGRP serve as key neurotransmitters in the transmission of ocular sensations at the human ocular surface50 and are also released during local inflammation.49 They have been implicated to play a key role in corneal wound healing influencing the corneal epithelial cell renewal and repair.46 51 As both these NPs are present in normal human tears,52–55 changes in their tear levels can serve as an indicator of the corneal health and nerve function.56 CGRP is reduced in tears of dry eyes,52 while SP levels decrease in corneal hypaesthesia.57 Decreased tear levels of SP and NGF and increased tear levels of CGRP, NPY and VIP have been reported in dry eye conditions.52–57 This study explores the relationship between tear NP levels, ocular surface signs and central corneal SBNFLD morphology in ocular surfaces exposed to long-term topical antiglaucoma therapy. Our study showed statistically significant elevated SP level and decreased VIP, NPY level of NP in ocular hypotensive therapy group (both with and without BAK preservative) compared with controls, signifying local inflammation by triggering activation of different immune cells in dry eyes concurring with the results of Lambiase et al’s study.52 Of interest, increase in the level of NGF has also been documented in dry eyes.
A recent study by Golebiowski et al on nerve density, tear CGRP and corneal sensitivity in contact lens wearers showed that the markers of corneal neurobiology and sensory function were not altered in contact lens wear with minimal symptoms despite altered tear osmolarity and stability in lens wearers. Our study, a cross-sectional, open label comparative single-visit study involving 77 participants on long-term antiglaucoma topical therapy is the first attempting to analyse the levels of tear NPs on ocular surfaces exposed to the toxicity of BAK preservative in the topical AGMs and compare them with those on therapy without BAK preservative. The study population was evaluated in two groups with group I comprising of eyes with AGMs containing BAK preservative and group II comprising of eyes with AGMs without BAK preservative and compared with normal eyes as controls. The ocular surface evaluation tests (TBUT and Schirmer’s I test) values were significantly decreased in both groups compared with the control eyes, while they were not significantly different between the BAK preservative group I and BAK-free group II. The central corneal SBNFL evaluation did not reveal any significant differences between the eyes on chronic antiglaucoma therapy with BAK preservative and without BAK preservative. Tear levels of SP and NGF were observed to be significantly increased in the eyes on chronic antiglaucoma therapy (both the BAK and BAK-free groups) compared with the control eyes, while tear levels of CGRP, VIP and NPY were significantly decreased in the eyes on chronic topical antiglaucoma therapy compared with the control eyes (table 3). No significant differences in the levels of NPs were observed between group I and group II. Spearman’s correlation analysis did not reveal any correlation between the ocular surface evaluation tests and central corneal SBNFL characteristics with the tear NP levels in the eyes of both the study population and the controls. Both the study groups were age matched and the mean duration of antiglaucoma therapy was also similar. Though use of chronic ocular hypotensive ophthalmic solutions containing BAK preservative detected higher dry eye disease levels in comparison with the dry eye disease in those using BAK-free therapy for over 6 months, the ocular surface evaluation test, corneal SBNFLD and tear NPs did not seem to be affected differentially as compared with normal controls (tables 2 and 3). All participants in our AGM groups (with and without BAK) had evidence of dry eye disease of severity level 1 in those using BAK-free medications and severity level 2 in those using BAK preservative medications. There was no statistically significant difference between the ocular surface evaluation tests between the two AGM groups. Altered NP levels in eyes with AGM with or without BAK in comparison with normal eyes could be either due to the use of AGM or the occurrence of dry eye. This along with the lesser duration of AGM perhaps explains the lack of correlation between the clinical ocular surface evaluation tests and the levels of tear NPs. The duration of topical antiglaucoma therapy that is required to reflect changes in tear NP levels is also not clear. The lack of difference in the central corneal SBNFLD between the two AGM groups probably signifies that the severity level of dry eye disease did not differ significantly in both the AGM groups.
The limitation of recruiting patients of similar topical ophthalmic formulations precluded inclusion of a higher sample size in our study. The shorter mean duration of AGM perhaps also precluded the occurrence of more significant effects on the ocular surface in our study. The availability and affordability of BAK-free topical ophthalmic formulations for the patient profiles under our centre’s care further was another limiting factor as well. Hence, the statistically relevant analysis between the three drug schedules: timolol 0.5% only, timolol 0.5% with prostaglandin analogue, prostaglandin analogue only in BAK and BAK-free ophthalmic formulations could not be further elaborated.
Altered levels of tear NPs (elevated SP and NGF levels and decreased levels of CGRP, VIP and NPY) has been documented in dry eye disease. Increase in SP and NGF levels occurs in dry eye disease, which was noted in our study as well. Tear NP levels of GCRP, VIP and NPY were significantly decreased in all the eyes on chronic antiglaucoma therapy (both with and without BAK preservative) which also corroborates with the reported changes in dry eye disease. While BAK preservatives in chronic ocular hypotensive therapy has been implicated to cause ocular surface toxicity, our study was not able to find a difference in levels of tear NP biomarkers of dry eye disease between the BAK and BAK free eyes. Our study highlights equivocal results between the BAK preservative and BAK-free group. This study explores the levels of NPs in eye with chronic AGM and has found them to be similar to that reported in patients with dry eye disease. Whether these changes are because of chronic AGM or the use of BAK or because of the dry eye needs further studies. While this 6-month study shows no changes, long-term longitudinal studies would perhaps clearly establish the relationship between tear NPs, BAK preservatives and antiglaucoma therapies. It also remains to be seen if the BAK preservatives implicated to be the ocular surface toxicity causing agents in chronic AGMs are really not as harmful as it is being considered to be and perhaps the drug molecule itself also contributes to toxicity-induced damage of the ocular surface. Our study results in agreement with those of reported studies in terms of altered NP levels in other dry eye disease states.
In summary, our study group comprising of eyes on chronic topical antiglaucoma therapy with BAK (group I) and without BAK (group II), eyes were observed to have elevated SP, NGF (p<0.01), decreased CGRP (p=0.03) and decreased VIP and NPY (p<0.01) in comparison with controls. There was no significant difference in the tear NP levels between the BAK preservative and BAK-free chronic topical antiglaucoma therapy. There was also no statistically significant difference in TBUT, Schirmer’s, staining scores and SBNFLD between the two groups. No correlation was seen between tear NP levels with the clinical ocular surface evaluation tests (TBUT, Schirmer’s, staining scores) and central corneal SBNFLD in both groups.
We acknowledge the inputs of Dr Talvir Sidhu, Dr Shikha Gupta, Mr Pawan Kumar (Senior Ophthalmic Technician) and Mr Ashish Upadhyay (Department of Biostatistics).
Contributors VM contributed to the conception of the work, critical revision and accountable for all aspects of the work. VM, RD, MS and SV contributed to the analysis and interpretation of data for the work.
RD, MS, VG, TD and SV contributed to data acquisition. VG and TD contributed to the interpretation of data for the work. All authors contributed to the manuscript drafting and final approval of the version.
Funding This study was supported by Extramural Research Funding from the Indian Council of Medical Research (Project ID: 5/4/6/7/Oph/12-NCD-II).
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval All India Institute of Medical Sciences Ethics Committee Institute Ethics Approval (Ref No:- IEC/NP-241/2012).
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.