The Utility of an Artificial Substitute to Improve Corneal Sensitivity in Glaucomatous Patients on Chronic Therapy with Prostaglandin Analogs

Abstract

Purpose:

To evaluate whether a tear substitute can improve corneal subepithelial nerve plexus and corneal sensitivity in glaucomatous patients.

Methods:

This study was prospective, longitudinal, and single arm. Twenty glaucomatous patients were recruited. All the patients were treated with a prostaglandin analog with preservative for at least 1 year. Preservative-free artificial tears (0.5% tamarind seed polysaccharide 0.5^® eye drops single-dose preservative free [Oftagen]) were prescribed thrice per day. The participants were subjected to clinical and instrumental evaluation at baseline (T0), after 1 month (T1) and after 3 months (T3) of treatment. All patients were examined with a digital corneal confocal laser-scanning microscope (HRT II Rostock Cornea Module; Heidelberg Engineering GmbH) and Cochet-Bonnet corneal esthesiometer.

Results:

After the artificial substitute, corneal and conjunctival sensitivity significantly (P<0.001) improved at T1 and T3 compared to the baseline. A significant correlation was found between break-up time and both central corneal sensitivity and the number of fibers.

Conclusion:

The use of a preservative-free artificial substitute in association with a topical therapy with chronic preservative could increase the compliance of patients.

Introduction

Glaucoma is a chronic condition that requires lifelong topical treatment. Irritation and redness of eyes are common symptoms that cause a worsening of quality of life and often a changing in therapy.

Most commercial formulations of antiglaucoma eye drops contain variable concentrations of benzalkonium chloride (BAK). BAK is commonly used as a bactericidal preservative in ophthalmic preparations.¹ Being a quaternary ammonium with high cationic surface action, its germicidal and disinfectant effect is due to detergent action. Many studies^2–7 have elucidated the role of BAK topical eye drops, as the main ingredient responsible for triggering cytotoxic and inflammatory activities.

BAK causes a dose-dependent conjunctival and corneal epithelial cell toxicity, in vivo^8,9 and in vitro^10,11 tear film instability, and corneal epithelial barrier dysfunction.¹² Martone et al.¹³ have reported that patients treated with BAK-preserved glaucoma eye drops have reduced sub-basal nerve density, lower corneal sensitivity, and decreased tear secretion.

The purpose of this study was to evaluate whether a tear substitute can protect corneal subepithelial nerve plexus and corneal sensitivity in glaucomatous. In this study, 0.5% tamarind seed polysaccharide (TS-polysaccharide) was used as a tear substitute because it is a nonionic, neutral branched polysaccharide obtained from tamarind seed and consists of a cellulose-like backbone that carries xylose and galactoxylose substituents leading to a mucin-like molecular structure, similar to transmembrane MUC1,¹⁴ thus conferring optimal mucoadhesive properties and suggesting a role for sustained relief from the symptoms of dry eye disease.¹¹

Methods

This research is part of a prospective, longitudinal, single-arm, and multicenter study already published.¹⁵ Institutional review boards and ethics committees gave their approval to the study. This study followed the principles of the Declaration of Helsinki. Patients joined the study after having signed a written informed consent.

Twenty consecutive patients were recruited according to the following inclusion criteria: age >18 years, best-corrected visual acuity of ≥20/40, a diagnosis of open-angle glaucoma, treatment for at least 1 year with a once-daily BAK-preserved topical prostaglandin analog in monotherapy to control the intraocular pressure (IOP). Target IOP was below 18 mmHg in all the included patients.

Open-angle glaucoma eyes were diagnosed based upon having a reproducible and characteristic visual field defect of 3 nonedge points, all of which were depressed on the pattern deviation plot at P<5%, along with an asymmetrical cupping >0.2 and the presence of a notch on the rim and/or an increased cupping >0.6 when measured as the cup/disc ratio and an open angle at gonioscopy.¹⁶

All subjects underwent at least 2 Swedish Interactive Thresholding Algorithm (SITA) Standard 24-2 perimeters. Reliable tests had fewer than 20% fixation losses and <15% false-negative and false-positive responses.

Exclusion criteria were the history of active or past ophthalmological diseases different from glaucoma, contraindications to use any of the topical components used in this trial, current use of contact lenses, previous or current use of other ocular medication, including tear substitutes, systemic treatment known to affect tear secretion, any history or slit-lamp evidence of eye surface disorders, a history of ocular surgery or laser treatments, history of ocular trauma, rheumatology and autoimmune diseases, and diabetes, use of systemic steroids, any other systemic medication known to affect the retina, and any neurological condition known to affect the visual field.

The participants of the study were subjected to clinical and instrumental evaluation of the ocular surface by a slit lamp at baseline before the beginning of treatment (T0=baseline), after 1 month (T1), and after 3 months (T3). At the T0 visit, a tear substitute was prescribed to all patients to be used thrice a day, in addition to their usual ophthalmic therapy. In this study, preservative-free 0.5% TS-polysaccharide single-dose eye drops (TSP^®; Oftagen, Pisa, Italy) were evaluated. TS-polysaccharide is a mucin-like polysaccharide extracted from the seeds of the plant Tamarindus Indica with mucoadhesive and pseudoplastic properties and found to be protective for in vitro corneal epithelial cells exposed to damage.^17,18

Conjunctival hyperemia and thickness of the tear meniscus were subjectively evaluated, and the presence of corneal epithelial defects (as assessed by fluorescein staining) and meibomian gland disease was recorded. Conjunctival hyperemia was evaluated at the slit lamp and graded according to the CCLRU grading scale¹⁹ as none, mild, moderate, and severe.

The diagnosis of the meibomian gland disease was made by a clinical examination based on glandular obstruction and meibum quality.^12,20 This grading was obtained by a firm digital pressure over the central third of the upper and lower eyelid while observing the ease excretion and quality of meibum under the slit-lamp biomicroscopy. Meibomian gland obstruction was classified as absent if meibum was clear fluid, easily expressed, and present if a mild to moderate pressure was needed to express cloudy or dense meibomian.

In addition, Schirmer's test (second), break-up time (BUT) (second), and corneal sensitivity have been evaluated. Visual acuity and IOP were also measured for safety and patients. Self-reported subjective symptoms (such as stinging, burning, foreign body sensation, and ocular pain) were recorded at each study visit and used for the analysis.

Esthesiometer

The evaluation of sensitivity was performed on the cornea (central, nasal, and temporal) and conjunctiva (nasal and temporal) by Cochet-Bonnet esthesiometer. The esthesiometer plays an important role in corneal semiology, and the Cochet-Bonnet esthesiometer is still the most commonly used in clinical practice. It is similar to a ballpoint pen from the end of which comes out a nylon thread of 0.12-mm diameter and variable length. While the patient looks at a point straight ahead of him, the examiner places the nylon thread perpendicular to the cornea. The instrument, with a nylon thread to the maximum length (60 mm), is approached slowly to the corneal area to be tested with a strength such as to show it to bend. If the pressure is not sensed by the patient, the dial of the instrument is rotated and the test is repeated with a thread shorter than 5 mm. The instrument is again approached until touching the cornea; the process is repeated until reaching the minimum perceptible length. The thread of nylon string is converted using a conversion table in mg/S or g/m². This value represents the minimum perceptible contact by the corneal area examined. After recording the sensitivity of the central cornea, the sensitivity of the periphery can be tested.

In this study, all measurements were obtained by the same observer (A.V.) in the same room. Patients were laid in the supine position looking straight ahead and they were asked to indicate when the stimulus was felt. The filament was moved toward the cornea smoothly at a perpendicular angle, guided by its corneal reflection. Contact was detected by a slight bending in the filament. If there was no patient response to the first contact, the length of the filament was decreased by 5.0 mm to increase its rigidity and the procedure was repeated until the patient reported the feeling of corneal contact. The mean filament length from a minimum of 3 stimulus applications that elicited a positive response from the patient was considered to be the corneal touch threshold.

A flexible millimeter ruler was used to know the exact location of the nasal conjunctiva (2 mm from limbus), nasal cornea (2 mm from limbus), temporal cornea (2 mm from limbus), and temporal conjunctiva (2 mm from limbus).

Corneal confocal microscopy

All patients were examined with a digital corneal confocal laser-scanning microscope (HRT II Rostock Cornea Module; Heidelberg Engineering GmbH, Heidelberg, Germany), a laser scanning in vivo confocal microscopy that uses a 670-nm red wavelength helium neon diode laser source.²¹ The confocal scanning laser device uses a 60X objective water immersion lens and a working distance of 0–3 mm from the applanating cap. The images measure 400×400 μm, and the manufacturer specifies an optical section thickness of 4 μm. The module uses an entirely digital capture system.

Confocal microscopy was performed in the center of the cornea, in the temporal and nasal periphery of the cornea, and in the temporal and nasal conjunctive by the same operator. The examination was carried out under topical anesthesia instilled in the lower conjunctival fornix just before the examination. Proper alignment and positioning of the head were maintained with the help of a dedicated target mobile red fixation light for the contralateral eye. A digital camera mounted on a side arm provided a lateral view of the eye and objective lens to monitor the position of the objective lens on the surface of the eye. At least 20 images in the central area of the corneal epithelium, sub-basal plexus, and stroma were obtained for each eye. The procedure lasted 2–5 min. The cornea was then examined by a slit lamp to ensure its integrity.

The confocal images were evaluated in a masked manner at the end of the study; the investigator was unaware of when the images were taken. The best focused and most representative images were selected for each time patient and stored in a digital format; the mean of 3–5 images for each parameter was considered for statistical analysis.

The following corneal parameters were evaluated.

• Number of sub-basal nerves: this parameter is defined as the sum of the nerve branches present in 1 field.

• Sub-basal nerve tortuosity and reflectivity: the grade of tortuosity and reflectivity were classified in 4 grades according to a pre-existing scale.

Statistical analysis

The data were evaluated by a descriptive analysis. When the distribution of the data was normal, a 2-tailed paired t-test was used; when the distribution of the data was non-normal, a Mann–Whitney test was used. Pearson's coefficient was used to analyze the correlation among all the parameters, while Spearman's coefficient was used when the distribution of the data was not normal. A P-value<0.01 was considered statistically significant.

Results

Twenty patients (13 M, 7 F) aged between 35 and 80 years with primary open-angle glaucoma in therapy with prostaglandin analogs were selected.

After additional treatment with TS-polysaccharide 0.5% TID, conjunctival hyperemia and thickness of the tear meniscus improved at T1 (0.56±0.34) and T3 (0.48±0.24). Corneal epithelial defects assessed by fluorescein staining disappeared by time T1 and T3. The BUT significantly improved (t-test) (Table 1). In addition, Schirmer's test improved after 3 months of treatment.

Table 1.

Clinical Sign Assessment

				P-value
	Baseline	T1	T3	Baseline vs. T1	T1 vs. T3	Baseline vs. T3
Tear meniscus (mm)	0.6 (0.38)	0.56 (0.34)	0.48 (0.24)	n.s.	n.s.	n.s.
Schirmer test (mm)	6.3 (1.90)	7.88 (2.12)	7.82 (1.62)	n.s.	0.0052	0.0094
Break-up time (second)	5.9 (1.42)	6.45 (0.98)	9.45 (1.92)	0.0015	<0.001	<0.001

n.s., not significant; T1, after 1 month; T3, after 3 months.

Central corneal and conjunctival sensitivity measured by the Cochet-Bonnet esthesiometer significantly (t-test) improved at T1 (P<0.001) and T3 (P<0.001) compared to baseline (Table 2).

Table 2.

Confocal Microscopy Assessment and Sensitivity

				P-value
	Baseline	T1	T3	Baseline vs. T1	T1 vs. T3	Baseline vs. T3
Number of fibers	6.15 (1.46)	7.28 (1.94)	8.75 (2.41)	<0.001	<0.001	<0.001
Tortuosity	1.79 (0.85)	2.14 (0.42)	2.03 (0.69)	0.036	n.s.	0.057
Reflectivity	2.18 (0.72)	2.34 (0.63)	2.42 (0.50)	n.s.	n.s.	0.036
T Conj.	1.55 (0.69)	2.05 (0.89)	2.21 (0.72)	0.004	n.s.	<0.001
TC	2.51 (0.89)	3.16 (0.94)	3.66 (0.81)	<0.001	n.s.	<0.001
CC	4.26 (0.79)	4.79 (0.77)	5.58 (0.52)	<0.001	<0.001	<0.001
NC	2.76 (0.86)	3.27 (1.00)	3.42 (1.01)	0.0135	n.s.	0.0023
N Conj.	1.73 (0.75)	2.26 (0.91)	2.14 (0.84)	<0.001	n.s.	0.01

Mean (standard deviation).

CC, central corneal sensitivity; NC, nasal corneal sensitivity; N Conj., nasal conjunctival sensitivity; T Conj., temporal conjunctival sensitivity; TC, temporal corneal sensitivity.

With confocal microscopy, a significant (Mann–Whitney) increase of sub-basal nerve fibers was observed after 1 month (P<0.001) and 3 months (P<0.001) of treatment, while the tortuosity and reflectivity of the fibers remained unchanged (P=0.109 and P=0.06, respectively) (Table 2) (Fig. 1).

FIG. 1.

Confocal microscope images. (A) Typical confocal scan image of a cornea after 1 year of drops with preservatives (T0); (B) typical confocal scan image after 3 months of treatment with an artificial substitute (T3). There is an improvement of the number of subepithelial nerve fibers at T3.

No significant correlation (Spearman) was found between the sensitivity data and confocal microscopy. However, a significant correlation was found between central corneal sensitivity and BUT; at baseline, the correlation was not significant (r = −0.13), while after 3 months the Pearson's coefficient improved to 0.42. A significant correlation was found between BUT and the number of fibers, with a Pearson coefficient of 0.33 at the baseline and 0.50 after 3 months of follow-up.

Discussion

The cornea is the body tissue with the richest innervation.²² In the human cornea, the corneal sensory innervation comes mainly from the afferent ophthalmic branch of the trigeminal ganglion, through the long ciliary nerves. The 2 or 3 long ciliary nerves across the bulbar wall are divided into 12–16 nervous trunks, which penetrate the stroma in different parts of the corneal periphery forming, together with the short ciliary nerves, the scleral plexus at the level of the limbus.

Many nerve branches, about 60–80, starting from the limbus, enter radially in the thickness of the corneal stroma and anastomose and divide to form the deep plexus stromal. From the stromal plexus, fibers go toward the corneal surface; after entering Bowman's membrane, the fibers are divided into thinner branches, which run parallel with the corneal surface, forming the subepithelial plexus. From this plexus, nerve fibers that come through the epithelial basement membrane are distributed among the basal cells. The corneal sensitivity by the Cochet-Bonnet esthesiometer is a relevant parameter in the evaluation of anatomical and functional characteristics of the corneal tissue.

Many studies have already shown that corneal sensitivity is reduced in patients taking chronic glaucoma medications with preservatives.²³ The purpose of this study was to analyze through clinical tests and in vivo confocal microscopy, the effect of a TS-polysaccharide tear substitute on the ocular surface in patients treated with prostaglandins analogs.

The role of BAK topical eye drops as the main responsible for triggering cytotoxic and inflammatory activities is well known.^2–7 TS-polysaccharide (0.5%) should be a nonionic, neutral, branched polysaccharide tear substitute,¹⁴ suggesting a role for sustained relief from the symptoms of dry eye disease.¹¹ Different authors have shown that the lack of BAK in the drops can improve the ocular surface of glaucomatous patients.^15,24,25

Our results suggest that a single-dose tear supplement may reduce the damaging effects of the preservative BAK on conjunctival cells, on the tear film, and on sensory nerve fibers with alteration of the threshold of excitability, which follows a reduced sensitivity. Since corneal sensitivity and the number of nerve fibers in the cornea and BUT improved, a tear film supplement might be associated to a better corneal function.

The explanation for this effect could be either a wash-out effect on the preservative or a protective effect of the ocular surface as a result of supplementary tear film. Chronic treatment with its side effects is one of the major factors that can compromise the quality of life of patients. Thus, the instillation of a tear substitute improving the compliance/adherence could make the treatment more efficient, even if the patient had to put a different drop in the eye. The positive effect on the ocular surface and the improvement of the symptoms might help the patient to use one more drop.

Although both corneal and conjunctival sensitivity improved during 3 months of tear supplementation together with the number of nerve fibers in the cornea, no significant correlation was found between the 2 parameters. The lack of correlation is probably due to the different scale used to measure the corneal sensitivity and the number of nerve fibers.

Furthermore, there are some issues to consider before drawing any final conclusion such as the limited number of patients, the absence of a control group, and the lack of a double-masked analysis. Another disadvantage could be that the measurements of in vivo confocal microscopy were made only in the center of the cornea and in the temporal and nasal peripheral area; different results could be in different corneal areas.

In conclusion, these data might motivate the use of a preservative-free artificial substitute in association with a topical therapy with chronic preservative to increase the adherence and ultimately the efficacy of the IOP-lowering treatment. Furthermore, in this study, TS-polysaccharide was shown to be able to reduce or eliminate the toxicity of BAK.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Valente

, Iester

, Corsi

, and Rolando

Symptoms and signs of tear film dysfunction in glaucomatous patients. J. Ocul. Pharmacol. Ther., 27:281–285, 2011.

Valente

, and Iester

Impact of glaucoma medication on ocular tissue. Expert Rev. Ophthalmol., 5:405–412, 2010.

Baudouin

, Hamard

, Liang

, et al. Conjunctival epithelial cell expression of interleukins and inflammatory markers in glaucoma patients treated over the long term. Ophthalmology. 111:2186–2192, 2004.

Pisella

P.J.

, Debbasch

, Hamard

, et al. Conjunctival proinflammatory and proapoptotic effects of latanoprost and preserved and unpreserved timolol: an ex vivo and in vitro study. Invest. Ophthalmol. Vis. Sci., 45:1360–1368, 2004.

Liang

, Pauly

, Riancho

, et al. Toxicological evaluation of preservative-containing and preservative-free topical prostaglandin analogues on a three-dimensional-reconstituted corneal epithelium system. Br. J. Ophthalmol., 95:869–875, 2011.

Walker

T.D.

Benzalkonium toxicity. Clin. Exp. Ophthalmol., 32:180–184, 2004.

Noecker

, and Miller

K.V.

Benzalkonium chloride in glaucoma medications. Ocul. Surf., 9:159–162, 2011.

European Glaucoma Society. Terminology and Guidelines for Glaucoma, European Glaucoma Society. 4th ed. Savona, Italy: Publicomm; 2014; chapter 3.

European Glaucoma Society. Terminology and Guidelines for Glaucoma, European Glaucoma Society. 3rd ed. Savona, Italy: Dogma; 2008; chapter 1.

10.

Raimondi

, Lodovici

, Guglielmi

, et al. The polysaccharide from Tamarindus indica (TS-polysaccharide) protects cultured corneal-derived cells (SIRC cells) from ultraviolet rays. J. Pharm. Pharmacol., 55:333–338, 2003.

11.

Rolando

, and Valente

Establishing the tolerability and performance of tamarind seed polysaccharide (TSP) in treating dry eye syndrome: results of a clinical study. BMC Ophthalmol., 7:5, 2007.

12.

Martone

G.L.

, Frezzotti

, Tosi

G.M.

, et al. An in vivo confocal microscopy analysis of effects of topical antiglaucoma therapy with preservative on corneal innervation and morphology. Am. J. Ophthalmol., 147:725–735, 2009.

13.

Heijl

, Leske

M.C.

, Bengtsson

, et al. Reduction of intraocular pressure and glaucoma progression: results from the early manifest glaucoma trial. Arch. Ophthalmol., 120:1268–1279, 2002.

14.

Gidley

M.J.

, Lillford

P.J.

, Rowlands

D.W.

, et al. Structure and solution properties of tamarind-seed polysaccharide. Carbohydr. Res., 214:299–314, 1991.

15.

Iester

, Oddone

, Fogagnolo

, Frezzotti

, Figus

; Confocal Microscopy Study Group. Changes in the morphological and functional patterns of the ocular surface in patients treated with prostaglandin analogues after the use of TSP 0.5% preservative-free eyedrop: a prospective, multicenter study. Ophthalmic. Res., 51:146–152, 2014.

16.

Labbé

, Pauly

, Liang

, et al. Comparison of toxicological profiles of benzalkonium chloride and polyquaternium-1: an experimental study. J. Ocul. Pharmacol. Ther., 22:267–278, 2006.

17.

Buron

, Micheau

, Cathelin

, et al. Differential mechanisms of conjunctival cell death induction by ultraviolet irradiation and benzalkonium chloride. Invest. Ophthalmol. Vis. Sci., 47:4221–4230, 2006.

18.

Zhou

, Liu

, Zhou

, et al. Modulation of the canonical Wnt pathway by benzalkonium chloride in corneal epithelium. Exp Eye Res., 93:355–362, 2011.

19.

Georgiev

G.A.

, Yokoi

, Koev

, et al. Surface chemistry study of the interactions of benzalkonium chloride with films of meibum, corneal cells lipids, and whole tears. Invest. Ophthalmol. Vis. Sci., 52:4645–4654, 2011.

20.

Ziai

, Dolan

J.W.

, Kacere

R.D.

, and Brubake

R.F.

The effects on aqueous dynamics of PhXA41, a new prostaglandin F2 alpha analogue, after topical application in normal and ocular hypertensive human eyes. Arch. Ophthalmol., 111:1351–1358, 1993.

21.

Uusitalo

, Pillunat

L.E.

, and Ropo

Efficacy and safety of tafluprost 0.0015% versus latanoprost 0.005% eye drops in open-angle glaucoma and ocular hypertension: 24 month results of a randomized, double-masked phase III study. Acta. Ophthalmol., 88:12–19, 2010.

22.

Yee

R.W.

The effect of drop vehicle on the efficacy and side effects of topical glaucoma therapy: a review. Curr. Opin. Ophthalmol., 18:134–139, 2007.

23.

Pisella

P.J.

, Pouliquen

, and Baudouin

Prevalence of ocular symptoms and signs with preserved and preservative-free glaucoma medication. Br. J. Ophthalmol., 86:418–423, 2002.

24.

Frezzotti

, Fogagnolo

, Haka

, Motolese

, Iester

, Bagaglia

S.A.

, Mittica

, Menicacci

, Rossetti

, and Motolese

In vivo confocal microscopy of conjunctiva in preservative-free timolol 0.1% gel formulation therapy for glaucoma. Acta. Ophthalmol., 92:e133–e140, 2014.

25.

Iester

, Telani

, Frezzotti

, Motolese

, Figus

, Fogagnolo

, and Perdicchi

Ocular surface changes in glaucomatous patients treated with and without preservatives beta-blockers. J. Ocul. Pharmacol. Ther., 30:476–481, 2014.