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.)]

Kovačić, Sanja (2011) 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.)]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

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.

Abstract in Croatian

Cilj ovoga istraživanja bio je utvrditi djelovanje dušik (II) oksida (NO) na propusnost krvno-moždane barijere in vivo u šarana korištenjem gliceriltrinitrata (GTN) kao donora NO. Ustanovili smo i vremenski odredili reverzibilnost procesa povećane propusnosti krvnomoždane barijere, pratili, opisali i vremenski odredili razvoj moždanog edema te utvrdili sličnost mehanizma povećanja propusnosti krvno-moždane barijere u kojem sudjeluje NO na nižim kralježnjacima u odnosu na do sada istraživane vrste. Pokus je izveden na dvogodišnjoj šaranskoj mlađi (Cyprinus carpio L.) tretiranoj gliceriltrinitratom u dozi od 1 mg/kg tjelesne mase. U serumu životinja u vremenskim je razmacima od 15 minuta te 1 sat, 3 sata, 6 sati, 8 sati, 12 sati i 24 sata nakon tretmana određena koncentracija ukupnih nitrita i nitrata komercijalnim test-paketom. U kontrolnoj skupini i u tretiranim skupinama analizirana je propusnost krvno-moždane barijere 6 sati, 12 sati i 24 sata nakon primjene GTN na temelju istjecanja Evans plavila na makroskopskom uzorku mozga i mikroskopskim preparatima s pomoću fluorescentnog mikroskopa. U vremenskim razmacima od 6 sati i 24 sata nakon tretmana gravimetrijskom metodom odredili smo pojavu moždanog edema, koji smo opisali na hemalaun-eozin obojenim preparatima mozga pod svjetlosnim mikroskopom. Ukupna koncentracija nitrata i nitrita pokazala je bifazičan obrazac promjene s najvišim koncentracijama 15 minuta nakon primjene GTN, srednja vrijednost ± SEM: 188,1 ± 23,82 μmol/l te nakon 6 sati (236,4 ± 64,67 μmol/l) i 8 sati (255,2 ± 25,81 μmol/l). Dinamika promjene koncentracije nitrata odgovara ukupnoj koncentraciji nitrata i nitrita. Najviše su vrijednosti 15 minuta nakon primjene GTN (180,2 ± 25,76 μmol/l) te 6 sati (232,4 ± 63,84 μmol/l) i 8 sati nakon primjene (236,9 ± 89,12 μmol/l). Koncentracije nitrita su značajno niže od koncentracije nitrata, a maksimalne vrijednosti pokazuju nakon 3 sata (14,28 ± 9,63 μmol/l) i 8 sati (16,17 ± 5,94 μmol/l) od primjene donora NO, iako se te vrijednosti statistički značajno ne razlikuju u odnosu na kontrolnu skupinu. Svi dijelovi mozga životinja (prednji mozak, međumozak, srednji mozak, mali mozak i leđna moždina) u skupini koja je analizirana 6 sati nakon primjene GTN pokazale su stupanj obojenja »3«. Odgovarajući dijelovi mozga u kontrolnoj skupini i u skupinama koje su analizirane 12 sati i 24 sata nakon primjene donora NO pokazali su neobojenje, odnosno blago obojenje u područjima cirkumventrikularnih organa. Nalaz upućuje na povećano propusnu krvno-moždanu barijeru 6 sati nakon primjene donorskog sredstva NO, dok u kontrolnoj skupini nije došlo do otvaranja krvno-moždane barijere, a u skupinama koje su analizirane 12 sati i 24 sata nakon primjene ponovno je došlo do njezinog zatvaranja. Navedeni rezultati potvrđeni su metodom istjecanja Evans plavila koja se izvodi analiziranjem pod fluorescentnim mikroskopom. Ispitivanje moždanog edema gravimetrijskom metodom pokazalo je da mozak kontrolne skupine životinja ima M = 74,2% (IQR 25-75% = 72,2% – 76,1%), dok mozak skupina tretiranih gliceriltrinitratom pokazuje znakove edema te nakon 6 sati ima 78,1% (77,0% – 80,2%) vode, a nakon 24 sata 78,5% (77,5% – 80,4%) vode, što je u obje tretirane skupine statistički značajno više u odnosu na kontrolnu skupinu. Ovi nalazi potvrđeni su i histološki opisani na preparatima obojenima hemalaun-eozinom te analizirani pod svjetlosnim mikroskopom. Istraživanje je pokazalo da je primjena gliceriltrinitrata kao donora dušik (II) oksida inducirala endogenu sintezu NO, koja je vidljiva nakon 6 sati i 8 sati od primjene. Povećana sinteza NO povezana je s povećanom propusnošću krvno-moždane barijere u šarana u istom vremenskom intervalu od 6 sati nakon primjene donorskog sredstva. Nakon 12 sati, a poglavito nakon 24 sata, krvno-moždana barijera vraća se u prvotno stanje, no i dalje ostaje moždani edem koji je jasno vidljiv i 24 sata nakon primjene gliceriltrinitrata. Pokazali smo da postoji statistički značajna pozitivna povezanost između koncentracije ukupnih serumskih nitrata i nitrita i propusnosti krvno-moždane barijere te između koncentracije ukupnih nitrata i nitrita i postojanja moždanog edema. Iz rezultata navedenih istraživanja možemo zaključiti da primjena donora NO dovodi do reverzibilnog otvaranja krvno-moždane barijere i pojave moždanog edema u šarana, što je do sada dokazano i na nekim vrstama sisavaca.

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