The Effect of Timolol on the Iris Vascular Tone in Rats

1 Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, „Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 2 Department of Physiology, Faculty of Pharmacy, „Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 3 Ambulatory Care Center, „Dr. Carol Davila” Central Military Emergency University Hospital, Bucharest, Romania Corresponding author: Horia Paunescu, Faculty of Medicine, 8th Eroilor Sanitari Boulevard, 5th District, Bucharest, Romania. E-mail: phpaunescu@yahoo.com Abstract


INTRODUCTION
Vascular tone is generally controlled by both the humoral component and the neuro-vegetative component.Regarding the second one, catecholaminergic sympathetic innervation of blood vessels is almost a rule in the body 1,2 .
It is known that the iris is sympathetically innervated, both at the level of the iris dilator muscle 1,2 and at the level of iris blood vessels 3 .Cathecolamines are responsible for vasoconstriction and, in some vascular beds, vasodilation.Stimulation of neuropeptide Y positive nerve fi bers, belonging to sympathetic postganglionic neurons from the upper cervical node caused a decrease in the iris blood fl ow 4

(vasoconstriction).
It is known that neuropeptide Y is a cotransmitter of noradrenaline.Regarding the possibility of a catecholaminergic vasodilator tone in iris vessels, the results are contradictory.Propranolol administered conjunctivally, in vivo, causes iris vessels constriction in rabbits 5 , and timolol, in vitro, antagonized the possible vasodilator eff ect of isoprenaline, quantifi ed by the decrease of permeability to carbon particles, in rats' iris vessels 6 .Opposite to those mentioned above, timolol in conjunctival instillations produces, in vivo, dilation of iris vessels, in rhesus monkeys 7 .
Th e aim of this study was to evaluate in vivo, in rats, the possible existence of a catecholaminergic vasodilator tone produced by  2 adrenergic receptors, in iris vessels.
posteriorly at the intersection, was considered a vein.We measured vessel diameters on grayscale images (see fi gure 1) using ImageJ 1.51j8 software with the plugin named Diameter 8 .At each moment, four arterial measurements and vein measurements were made for each evaluated eye.For each determination and each moment, the percent variation of the vascular diameter, relative to intial diameter at moment t 0 was calculated using the formula: where D rel is the percentage change in diameter in relation to moment t 0 , D x is the vessel's diameter in pixels at the moment of measurement and D 0 is the diameter in pixels of the vessel at moment t 0 .For each moment, the relative diameter, in percent, and standard error were calculated.Subsequently, the statistical significance of the diff erences between each moment of the determination and the moment t 0 was determined.Th e statistical signifi cance was calculated using t-student test, the paired variant (each analyzed eye was his own control).Diff erences were considered statistically signifi cant, if p <0.05.

RESULTS
Th e evolution in time of the relative diameter (in percent) of the iris vessels is shown in Figure 2, Figure 3 and Table 1.Th e administration of 2.5 mM timolol after distilled water produced a decrease in the iris arteries diameters by 8.25% +/-2.46% at moment t 7 , by 9.22% +/-2.82% at moment t 8 , by 9.26% +/-2.53% at moment t 10 , and by 10.58% +/-3.50% at moment t 11 .Th ese values are statistically signifi cant in relation to moment t 0 , p<0.05.
Administration of the 2.5 mM timolol solution did not cause signifi cant changes in the relative diameter of iris veins.Distilled water did not cause statistically signifi cant changes of the relative diameters of iris arteries or veins.

DISCUSSIONS
For standardisation purpose, the terms iris arteries and veins, instead of arterioles and venules were used.Th e 2.5 mM timolol concentration was chosen, because at this molar concentration, we obtained pertinent results in experiments investigating the role of histamine and serotonin in regulating the iris vascular motricity (unpublished results).

MATERIALS AND METHODS
We used one group of 6 adult albino male rats weighting between 280 and 320 grams, brought in the laboratory with 4 days before the experiment and with access ad libitum to water and food.Th e experiments were performed between 9:00 and 17:00.Th e animals were used once and the recordings were made at one eye, the right one.Th e experiments were carried out with the approval of the Local Ethics Committee of "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania.
Experimental procedure: Animals were anesthetized with 10% ketamine (0.2 ml/100 g weight) and after 15 minutes they were placed in left lateral decubitus for optimal visualization of the right eyeball, and the eyelids were maintained opened by manual traction.Th e right eye was viewed at 400 × maximum magnifi cation, with an optical system made of NIKON objective lens and an adapter (NAVITAR 1X Adapter 1-6015) connected to a CCD camera (TOSHIBA-IK642E) and a Logilink video-grabber analog-to-digital converter USB 2.0.For illumination, we used a ring-shaped (circular) cold light source (Dolan-Jenner Industries Inc.FiberLite Series 180).Magnifi cation and lighting conditions were not altered during the recordings.Th e recording of the eyes lasted 11 minutes.For each analyzed iris, image captures were made, and arteries and veins diameters were measured at the following times: t 0 = 0 seconds, t 2 = 120 seconds, t 3 = 180 seconds, t 4 = 210 seconds, t 5 = 300 seconds, t 7 = 420 seconds, t 8 = 480 seconds, t 9 = 510 seconds, t 10 = 600 seconds, t11 = 630 seconds after the start of the recording.At t 1 = 30 seconds, distilled water was administered, and at t 6 = 330 seconds, the 2.5 mM timolol solution was administered.Under these conditions, the diameters of the arteries and veins were practically measured at the begining of the recording, before instillation of distilled water, then at 90 seconds, 150 seconds, 180 seconds, 270 seconds after the administration of distilled water, and at 90 seconds, 150 seconds, 180 seconds, 270 seconds and 300 seconds after the administration of 2.5 mM timolol.Measurements were made in pixels at the intersection between an artery and a vein, the smaller sized vessel located anteriorly at the intersection was considered an artery, and the larger vessel, located Some mentions are needed to be made.Our aim was to verify the existence of a tonic regulation, not a phasic regulation of iris vessels motricity.Th ere are two conditions for the tonic control of a body structure function: the existence in permanence or, in any case, over long periods of time in that structure, of a minimal endogenous biologically active substance concentration, as well as an adequate density of the receptors for that substance, which acts as an agonist.Administration of an antagonist of those receptors results in opposite eff ects to those of the agonists, if there is tonic control.Th e phasic control supposes that the antagonist produces no eff ect, but it will be able to prevent the physio-logical agonist action, if the agonist will be present in that structure 9 .
Our results show that administration of timolol produces statistically signifi cant vasoconstriction in the iris arteries, without statistically signifi cant eff ect in iris veins.Th us, we can state that there is a vasodilatory catecholaminergic tone in iris arteries.No such vasodilator catecholaminergic tone could be observed in iris veins.
We supposed that vasoconstriction produced by timolol in the arterial territory of the iris could be produced by blocking the vascular adrenergic  2 receptors.vasoconstriction observed with timolol.But, there is a mention to be made: the fact that we didn't fi nd a cathecolaminergic vasodilatatory tone in iris veins could be determined by two factors: there aren't  2 adrenergic receptors in veins or there is no agonist for these receptor.Malmfors T. found that, in rat, only the arteries appear to be innervated 3 , meaning that noradrenaline may not be present in veins, but could be present in arteries.

CONCLUSIONS
Th e use of 2.5 mM timolol in conjunctival instillations, in rats, reduced the diameter of iris arteries, without It is plausible to have vascular  2 adrenergic receptors in the iris arteries.Th e tonic stimulation of these  2 adrenergic receptors can be achieved either by adrenaline or noradrenaline as endogenous biologically active substances.Considering that adrenaline has greater affi nity (about 6 times higher) for  2 adrenergic receptors than noradrenaline 11 , and that we have used ketamine as a general anesthetic, a substance that increases, globally, the adrenergic tone of the body and causes the release of large amounts of adrenaline from the adrenal medulla in the blood 11 , adrenaline may had been the major contributer to this cathecolaminergic tone.Blocking the action of adrenaline on  2 adrenergic receptors in iris arteries, probably, determined the Figure 3.Time evolution of iris veins relative diameters after administration of distilled water at moment t 1 and subsequently of 2.5 mM timolol at moment t 6 .On the horizontal are the moments at which determinations were made, the height of the co lu mns represents the percentage of change in the vascular relative diameter.There were no statistically signifi cant chan ges.Table 3.Time evolution of iris arteries and veins relative diameters after administration of distilled water at moment t 1 and, subsequently, of 2.5 mM timolol at t 6 .For each moment of the determination, it is highlighted the mean relative diameter +/ -standard error, in percent.

Iris arteries Iris veins
The moment of measurement

Compliance with ethics requirements:
Th e authors declare no confl ict of interest regarding this article.Th e authors declare that all the procedures and experiments of this study respect the ethical standards in the Helsinki Declaration of 1975, as revised in 2008(5), as well as the national law.Informed consent was obtained from all the patients included in the study.
having an eff ect on the diameter of iris veins.Our results suggest that there is a catecholaminergic vasodilator tonus present in iris arteries and not in iris veins, which, in our experimental conditions (ketamine anesthesia), is probably achieved through  2 -adrenergic receptors.

Figure 1 .
Figure 1.Grayscale images captured from the 11 minutes recording of the same eye, at moment t 0 and t 8 .

Figure 2 .
Figure 2. Time evolution of iris artery rela ti ve diameters after administration of dis til led water at moment t 1 and, subse quently, of 2.5 mM timolol at moment t 6 .On the horizontal are the moments at which determinations were made, the height of the columns represents the percen tage of change in the relative vas cular dia meter ( ** p <0.05).