Djelovanje dušik (II) oksida na propusnost krvno-moždane barijere u šarana (Cyprinus carpio L.) [Role of nitric oxide on blood-brain permeability in common carp (Cyprinus carpio L.)]

The aim of this study was to determine the effect of nitric oxide (NO) on blood-brain barrier permeability in vivo in common carp by using glyceryltrinitrate (GTN) as an NO donor. We have established the reversibility and duration of increased permeability of the blood-brain barrier; we have observed, described and determined the duration of brain edema formation, and defined the similarities of the mechanism of NO regulated increase of the blood-brain barrier permeability in lower vertebrates and the species investigated so far. We experimentally treated two-year-old carp (Cyprinus carpio L.) with 1 mg glyceryltrinitrate/ kg body weight. Total nitrate and nitrite concentrations in the carp sera were determined 15 minutes, 1 hour, 3 hours, 6 hours, 8 hours, 12 hours, and 24 hours after treatment by using the commercial kit. In both control and treated groups blood-brain barrier permeability was analyzed at 6, 12, and 24 hours after GTN application based on Evans blue leakage in the brain specimens; the specimens were also observed under a fluorescent microscope. Brain edema was determined by using the wet/dry method and described on hemalaun-eosin stained preparations under a light microscope 6 and 24 hours after GTN treatment. The total concentration of nitrite/nitrate showed a biphasic pattern with maximum concentrations 15 minutes after GTN application, mean values ± SEM: 188.1 ± 23.82 μmol/l, and after 6 hours (236.4 ± 64.67 μmol/l) and 8 hours (255.2 ± 25.81 μmol/l). The dynamic changes of nitrate concentrations followed that of total nitrite/nitrate concentrations. Maximum values were observed 15 minutes after GTN application (180.2 ± 25.76 μmol/l), and after 6 (232.4 ± 63.84 μmol/l) and 8 hours (236.9 ± 89.12 μmol/l). Nitrite concentrations were considerably lower than nitrate concentrations, and maximum values were achieved 3 hours (14.28 ± 9.63 μmol/l) and 8 hours (16.17 ± 5.94 μmol/l) following NO donor application, although those values were not statistically different as compared to control group. All parts of the carp brain (forebrain, midbrain, hindbrain, little brain, spinal cord) showed coloration score »3« in the group analyzed 6 hours after GTN application. Corresponding parts of the brains in the control group and in the groups analyzed 12 and 24 hours following GTN application showed no coloration and/or mild coloration in the areas of circumventricular organs. These findings indicate increased blood-brain barrier permeability 6 hours after NO donor application, while no increased permeability was observed in the control group. The blood-brain barrier in the groups analyzed 12 and 24 hours after GTN application was again impermeable. The results were confirmed by the method of Evans blue leakage analyzed under a fluorescent microscope. The degree of brain edema was evaluated using the wet-dry weight method. The control group brains contained M = 74.2% (IQR 25 - 75% = 72.2% - 76.1%) water, while the brains of both groups of the animals treated with GTN showed signs of edema with 78.1% (77.0% – 80.2%) water observed 6 hours after treatment, and 78.5% (77.5% – 80.4%) water observed 24 hours after treatment. These findings were histologically confirmed with hemalaun-eosin staining of the preparations and analyzed under a light microscope. This investigation has shown that application of NO donor glyceryltrinitrate induced endogenous synthesis of nitric oxide which is up-regulated 6 and 8 hours after application. Increased NO synthesis is associated with increased permeability of the blood-brain barrier and develops at the same time interval of 6 hours following NO donor application. Although 12 and especially 24 hours after treatment the blood-brain barrier had completely restituted, brain edema still persisted 24 hours following GTN treatment. Our research demonstrated a statistically significant positive correlations between total nitrate and nitrite concentrations in sera and blood-brain barrier permeability, and between total nitrate and nitrite concentrations and brain edema presence. From these results we can conclude that NO donor application causes reversible blood-brain barrier opening and brain edema formation in common carp, a phenomenon which has so far been proven on some mammal species