Mechanical Release of Dinucleotides to the Tear Film

Abstract

Abstract: : Purpose:Investigate under which stimuli dinucleotides are released to the tear film. Methods:Tear secretion was measured in both eyes using the Schirmer's test in normal conditions and after application to the center of the cornea with a gas esthesiometer the following stimuli:gas pulses of 170ml/min and 260ml/min(mechanical),gas pulses of 80%CO₂ in air(chemical)and cooled gas pulses decreasing temperature in -1°C and -4.5°C(cold).Stimulation consisted in 3 pulses of 3 sec duration,applied sequentially.Inmediately after the stimulus,the Schirmer strip was applied for 5 min.Blinking experiments: individuals were invited to blink 0,12,30 and 60 times/min with the Schirmer strip placed in the lower lid during 5 min.Patientes with dry eye related symptoms,were submitted to Schirmer for 5 min.All the Schirmer strips were processed as follows:strips were placed in 500µL of ultrapure water and stirred.Tubes were placed at 100°C for 10 min,centrifuged at 13,000 rpm for 10 min,the supernatant was taken and submitted to ionicexchange chromatografy through SEP-PAKs Accell QMA(Waters).The elution of the retained dinucleotides was carried out with a mixture of 0.2NKCl,0.1NHCl(500µL).This eluate was injected in the HPLC for analysis.The HPLC mobile phase consisted of 0.1mMKH₂PO₄, 2mM TBA,15%acetonitrile,pH:7.5. Results: Of the entire stimulus applied to the corneal surface with the esthesiometer, only the mechanical one produced a statistically significant increase of the release of diadenosine polyphosphates. Ap₄A presence in tear was 3.22±0.25µmol(control value 2.17±0.20µmol) and Ap₅A was 1.86±0.26µmol(c.v:1.14±0.16µmol).Increasing the blinking rate of healthy individuals carried out mechanical stimulation of the cornea. Starting from 0 blinks(2.76±0.15µmol Ap₄A and 1.05±0.12µmol Ap₅A)we observed a gradual increase of both dinucleotides the maximal value being 71.76±3.51µmol for Ap₄A and 72.04±5.27µmol for Ap₅A measured at 60 blinks/min.Individuals presenting symptoms of dry eye showed variable concentrations of both dinucleotides. Patients with low tear production show values of Ap₄A and Ap₅A of 36.34±1.87µmol and 42.36±2.89µmol respectively.These patients presented a higher frequency in their blinking rate as described by many authors. Conclusions: From the presented data we can conclude that dinucleotides are released to the tear film due to a mechanical stimulation of the corneal surface.

Keywords: cornea: epithelium • cornea: basic science • lacrimal gland