A comprehensive approach to clinical safety assessment of neuroretinoprotectors administered in various routes
Introduction. In the present article we proved the possibility of toxicological safety assessment for neuroretinoprotective drugs, using the complex approach to evaluation of intracellular metabolic functional parameters in different ophthalmic structures (retina, optic nerve and anterior corneal epithelium), by the example of ademol administered in various routes. The physiological normal ranges have been determined for neuronal specific enolase activity, cell cycle metrics based on flow cytometry data, oxidative and nitrosative stress markers (malondialdehyde level, carbonyl groups of protein, glutathioneperoxidase activity, nitrite and nitrate balance and adenosine triphosphoric acid content as an indicator of energy processes).
The goal - to establish physiological standards neyronspetsyfichnoyi enolazy, nuclear DNA content of cells in the retina and anterior corneal epithelium, biochemical indices of oxidative stress, energy balance and metabolism of nitrogen monoxide. To prove the use of biochemical and toxicological cytometric diagnostic impact assessment tsytoprotektoriv the retina, cornea and optic nerve for example modulator activity of NMDA-receptors - Ademolu.
Materials and methods. Experiments conducted on rabbits breed Chinchilla weight 3,0-3,9 kg. All animals are kept in vivarium Vinnitsa National Pirogov Medical University (VNMU) on a standard water-diet with natural light and free access to water and feed. Animals were in under 12 hours of darkness, 12 hours of illumination.
To determine the presence of functional changes of intracellular metabolism in the retina, optic nerve and cornea of rabbits without eye pathology on the background of a course of Ademolu one group of animals injected it intramuscularly (w / m) dose of 2 mg / kg, and in another instylyuvaly con ' yuktyvalnyy bag made Ex tempore 0,5% aqueous solution Ademolu with 1.0% solution every 12 hours for 30 days.
Served as a comparison group without oftalmopatolohiyi intact animals, which have not entered any pharmacological agents.
Euthanasia of animals performing in terms propofolovoho anesthesia (60 mg / kg intravenously (v/v) ("FreseniusKabi", Austria)) [Khodakivskiy, 2014].
The evaluation process neyroretynodestruktsiyi (neuron-specific activity enolazy (NSE)) in the blood serum of animals was measured by ELISA using a set NSE ELISA KIT (DAI, USA) appliance company "Hipson" (Czech Republic) [Khodakivskiy, 2014].
The energy and carbohydrate metabolism in the retina evaluated the content of adenosine triphosphate (ATP). ATP content was determined in bezbilkovomu trichloroacetic extract retina 1: 5 (10% solution of trichloroacetic acid) chromatographic method [Prokhorov, 1982]. The intensity of oxidative stress in the retina determined by malondialdehyde (MDA) and oxidative modification of protein index (OMB).
State antioxidant defense system in the retina evaluated the activity of glutathione peroxidase (GPO). MDA content was determined by reaction with acid tiobarbiturovoyu [Vladimirov, Archakov 1972] PSC - the reaction of 2,4-dynitrofenilhidrazynom [Zaichko, 2003]. The total content of nitrites and nitrates was determined by reaction with a reagent Hrissa [Korenman, 1975]. The activity of glutathione peroxidase was determined spectrophotometric method [Vlasov et al., 1990].
The content of DNA in the nuclei of rat retinal cells was determined by flow cytometry. In the process of making nuclear suspensions using disposable filters CellTrics 50 microns (Partec, Germany). Flow analysis was performed on a multipurpose research flow cytometer "Partec PAS" (Partec, Germany).
Results. The research activity marker Nero destructive processes - NSE showed that its background value in venous blood is 0,35±0,021 ng / ml, which is matched with indicators of activity that received by other researchers, and indicates no fracture membrane processes neyrotsytiv [Dunker et al., 2001; Quintyn et al., 2005; Yee et al., 2012].
Topical (eye drops) or parenteral administration Ademol not cause and is not accompanied by the development neydestruktyvnyh changes in the retina, and demonstrates the lack of action neyroretynotoksychnoyi with prolonged use.
Research on energy metabolism macro level (ATP) showed that the contents intact animals in the retina was 50,40±1,50 nmol/mg protein, indicating the presence of aerobic glycolysis and points of sufficient energy retina. In intact animals no data on the development nitrozatyvnoho stress and imbalance in available nitrogen monoxide metabolism: the level of nitrites and nitrates averaged 5,42±0,21 nmol/mg protein.
Ademol daily intramuscular injection for 30 days, and its use as eye drops, does not cause metabolic changes in the retina. The content of lipid peroxidation products, stress levels of stable metabolites of nitrogen monoxide, ATP is not significantly different from the performance of intact animals.
We also identified the changes of mentioned above parameters on the background of neuroretinoprotective drug ademol.
The other researches may use the physiological normal ranges determined by us for comparative assessment of metabolic processes in the retinal cells under different pathological conditions.
Installed physiological levels and limits the activity of biochemical and DNA markers cytometric cell homeostasis retina, optic nerve and anterior corneal epithelium of rabbits. NSE activity was 0,35 ± 0,021 ng / ml; the level of malondialdehyde, protein carbonyl groups, adenosine triphosphate, nitrites and nitrates, and glutathione peroxidase activity in the retina respectively 3,40 ± 0,13; 1,10 ± 0,04; 50,40 ± 1,50; 5,42 ± 0,21 nmol / mg protein and 5,71 ± 0,19 mmol / min. 1 mg protein.
Investigation of course daily intramuscular administration rabbits without oftalmopatolohiyi Ademolu 1.0% solution (2 mg / kg) or instillation into the eyes of its 0.5% solution showed no negative effect of such therapy on DNA content (cell cycle, DNA frahmenatsiya , ploidy) and biochemical homeostasis of cells of the retina, optic nerve and anterior corneal epithelium by criteria neyromarkeriv activity, flow cytometric analysis and metabolism (energy metabolism and oxidant-antioxidant balance).
The methods for toxicological evaluation of the functional state of the metabolism of the retina, optic nerve and cornea epithelium given physiological levels and defined the boundaries of the proposed activity markers can be recommended both for preclinical evaluation of new neyroretynoprotektornoyi activity of biological substances and to determine their possible toxic effects on eyesight.