Vasodilatory Effect of the Dissolved Glycine locally applied on Pial Microvessels

By the method of biomicroscopy it was shown that a single application of a dissolved glycine on the parietal region of the rat brain (“open window” technique) leads to a vasodilatation an increase in arteriolar diameter about 1.5-2 times. There were no changes in the microcirculation when saline applied under similar conditions. It was also shown that there were no pH effects on the microvessels. For the testing of the vasodilatory effect of glycine in arteriol spastic conditions the adrenomimetic phenylephrine hydrochloride possessing the vasoconstrictor properties has been used as an agent for ischemic stroke modeling. Further work on the development of an adequate ischemic stroke models is the prospective task for the testing of antiischemic preparations. Research Article Vasodilatory Effect of the Dissolved Glycine locally applied on Pial Microvessels Tyukina ESa*, Sheshegova EV1a, Nartsissov YR2a, Podoprigora GI3a 1PhD in Pharmaceutical Sciences, Russia 2PhD in Physical and Mathematical Sciences, Associate Professor, Russia 3Doctor of Medical Sciences, Professor aInstitute of cytochemistry and molecular pharmacology, 6th Radialnaya str. 24/1, Moscow, Russia Dates: Received: 28 August, 2017; Accepted: 12 October, 2017; Published: 14 October, 2017 *Corresponding author: Tyukina E.S., Institute of cytochemistry and molecular pharmacology, 6th Radialnaya str. 24/1, Moscow, Russia, 115404, Tel: +74953274987; E-mail:


Introduction
In recent years, the increasing prevalence of ischemic diseases affecting the vitally important organs especially those of cardiovascular and central nervous system is observed. Ischemic brain injury is accompanied by the severe neurological disorders, such as cognitive deterioration, dysfunction of motor, verbal and other CNS functions. Stroke ranks fi rst among the causes of persistent disability. Correcting the imbalance of excitatory and inhibitory neurotransmitter systems is one of the most promising areas of neuroprotection and therapeutic target. Attention of researchers is attracted to a role of inhibitory neurotransmitter glycine in the mechanisms of acute cerebral ischemia [1]. The widespread use of the drug "Glycine" in patients with neurological disorders, as well as clinical observation of the positive therapeutic effect of the drug in ischemic stroke [2,3], suggest the existence of one or more fundamental biological mechanisms responsible for the neuroprotective effect of the amino acid at the molecular level. Since one of the important pathogenic mechanisms leading to the development of an ischemic stroke is a disturbance of blood supply to the brain, the effect of any drug that is expected to have clinical benefi t should be assessed in terms of possible normalization of microcirculation. One of the tests used for an assessment of Glycine direct vasodilatory effect is an intravital biomicroscopy of pial microvessels in a brain of laboratory rats. The fi rst studies demonstrated microcirculatory effect of glycine solution on mesenteric microvessels and on pial arterioles in laboratory rats [4][5][6]. A capacity of glycine to restore impaired microcirculation in the arterioles of the rat mesenterium under histamine induced infl ammatory alterations was shown revealing it's great, but little studied yet, antiinfl ammatory potential [5]. Effect of glycine on pial arterioles has been performed by using the original direct biomicroscopy with "open window" technique [6]. The signifi cant vasodilatory effect of glycine on microcirculation in pial microvessels in the cortex of a parietal area of the rat brain was shown. The aim of present work was to study if there is any dependence of Glycine effect on the pH values and the arteriole (blood microvessels) diameter size. The same parameters were used in control study with saline. Also, the possibilities of using the adrenomimetic phenylephrine hydrochloride preparation possessing the vasoconstrictory properties has been tested as a potential model of ischemic stroke and for an assessment of  Figure 1). Permanent control of all the solutions tested at temperature 37ºC (water bath). Control animals were treated with physiological saline. Direct visual monitoring of the microcirculatory bed ( Fig. 1.6) was performed using a digital microscope camera DMC 300. Images of microvessels were obtained with a surface-contact longfocus objective (x10). The method allowed us to obtain images at a 100fold magnifi cation. The state of microcirculation in the pial arterioles of the brain cortex was estimated by the measuring of diameters of arterioles (20 -200 μm) in the program Adobe Photoshop CS5. This parameter used as a major characteristic of blood fl ow in the microvessels (Figure 1).

Results and Discussion
The study performed revealed signifi cant vasodilatory activity of glycine solution applied to the surface of the pial microvessels. These results were in full compliance with early reported data [6]. At the same time the mechanisms of the glycine vasodilatory infl uence are still poor understood. Dacey Jr. [7], showed that the isolated rat cerebral blood vessels may respond to changes in the pH of saline solution in which they are placed.
Our study showed no dependence on pH value of saline solution (in the range of 2 to 9.7) applied to the surface of the cerebral membrane. No change of the arteriole' diameters due to this fact was observed. Thus, the diameter of the pial arterioles doesn't depend on the pH value of the locally applied solution. Vascular reaction was observed 1 -3 min after application of glycine ( Figure 2). The pronounced dilation of the arterioles has been registered. Their diameter increased by 150 -250%. The diameter of dilated microvessels returned to an initial size 10 -15 min after application of glycine. The diameter of venules at the same conditions remained unchanged. Also, the infl uence of glycine solution with the different pH in the range of 2 to 10 on the diameter of blood microvessels was investigated. The changes were the same as in the previous experiments, regardless of the pH of the solution. ( Figure. 2) To study the action of the amino acid glycine in vasospastic conditions we tested phenylephrine hydrochloride the synthetic adrenomimetic drug stimulating the -adrenergic receptors with a little effect on -receptors of the heart. It causes constriction of arterioles and increases blood pressure. According to Shigehiko Ogoh et al. [8], when administered intravenously to healthy adult, phenylephrine hydrochloride was reported to induce vasoconstriction of the arteries of the brain.
The series of experiments for selection of the concentration of phenylephrine hydrochloride have been performed aiming to obtain the maximum arteriolar spasm. For this purpose the solutions phenylephrine hydrochloride with concentrations of 0.015 mM, 0.03 mM, 0.06 mM, 0.3 mM, 0.6 mM and 0.9 mM have been tested.
It was shown that the phenylephrine hydrochloride solution at low concentrations didn't lead to signifi cant reduction in the diameter of arterioles ( Figure 3). The effect was not expressed enough to be used as a model for ischemic stroke. Moreover, an application of the solution of phenylephrine hydrochloride with higher concentrations even caused an increase in arteriolar diameters in 1.5 -2 times (Figure 4). Just after fl ushing the reagent with saline some reduction of the arteriole diameter in 1.5 -2 times was observed. It was comparable to the initial state of the vessels used as a model of vasoconstriction of the cerebral blood vessels (Figures 3,4).
An experiment was carried out to fi nd out the infl uence of a 1 M solution of the amino acid glycine on the diameter of pre-spastic blood vessels. During this experiment, solution of phenylephrine hydrochloride with concentration of 0.3 mM was applied to the surface of the rat brain; this solution was maintained for 10 minutes, and then fl ushed with saline. After 1. 2.

5.
6.    Of especial importance is the adequate models for reproduction of microcirculatory disturbances linked with brain tissue ischemia and stroke for testing of candidate preparation for its potential microcirculatory activity. The technique of ligation of carotid arterias (one side or bilateral) did not give a reproducible results concerning pronounced alteration in pial microcirculation according to other investigators including our experience. The most reliable technique recommended is [12]. However, the method appeared to be a diffi cult enough for routine reproducing. Selection of an adequate biomodels for ischemic stroke along with visualization of focal for experimental therapy purposes is of prospective task of our research.

Conclusions
Nowadays it is important to search for new potential substances for the treatment of ischemic stroke and preclinical models for its investigation.
The study performed revealed signifi cant vasodilatory activity of glycine solution applied to the surface of the pial microvessels, and it is correlated with early reported data [4,6]. But previously some conditions that could distort the results of the study were not taken into account. Thus, the study of the effect of saline with pH values from 2.0 to 9.7 on microcirculation in pial microvessels of the rat's brain showed that there were no pH effects on microvessels. Also, this study allowed us to obtain new data about the effect of glycine on microcirculation in the spastic microvessels of the brain. Its application was followed by pronounced dilatation of arterioles ( Figure 2), their diameter increased by 150 -200%. Effect was observed 1 -3 minutes after application of the amino acid solution. The diameter of microvessels returned to normal 10 -15 min after application of glycine. Repetitive application of glycine led to a similar increase in the diameter of blood microvessels. This effect remains when glycine was applied to spastic arterioles. Further development and application of the laboratory biomodel of local cerebral ischemia based on chemically or physically induced vasoconstrictory effect followed by microcirculation alteration may be one of the prospective therapeutic targets and value for preclinical testing and evaluation of new microcirculatory active drugs.