Influence of acute and intermittent hyperbaric oxygenation on the reactivity of the blood vessels in healthy Sprague-Dawley rats

Cilj: Cilj ove doktorske disertacije bio je utvrditi kako hiperbarična terapija (HBO2) utječe na vaskularnu reaktivnost provodnih krvnih žila u zdravih štakora ovisno o dužini izlaganja hiperbaričnom kisiku. Uvod: Hiperbarična oksigenacija (HBO2) koristi se kao dodatna terapija u stanjima gdje je smanjena isporuka kisika u tkiva. Poznato je da promjene u p02 mogu utjecati na sintezu metabolita arahidonske kiseline i time utjecati na vaskularnu reaktivnost u zdravlju i bolesti, uključivanjem proizvodnje CYP450 metabolita. Općenito, kod zdravih životinja relaksacijski odgovor na hipoksiju je posredovan prostaciklinom (PGI2), dok je relaksacija kao odgovor na acetilkolin (ACh) uglavnom posredovan NO putom. Podatci o tome kako je HIF-1α uključen u mehanizam relaksacije vrlo su rijetki, nedostatni i oprečni. Materijali i metode: Zdravi Sprague-Dawley štakori od 9-12 tjedana podijeljeni su u četiri eksperimentalne skupine (1. kontrola, 2.grupa akutno izložena HBO2 (A-HBO2), 3. Grupa analizirana 24 sata nakon akutne izloženosti (24H-HBO2); i 4. grupa koja je izlagana HBO2 kroz četiri uzastopna dana (intermitentna, 4D-HBO2). Reakcija na ACh i smanjeni p02 ispitivana je u noradrenalinom prekontrahiranim aortalnim prstenima u odsutnosti ili u prisustvu L-NAME-a, INDO, MS-PPOH, hvatača superoksida TEMPOL-a i LW6 HIF-inhibitora u tkivnoj kupelji. Indikatori oksidacijskog stresa mjereni su metodama TBARS i FRAP i izravnim mjerenjem proizvodnje endotelnog superoksida. Određen je izražaj antioksidativnih gena i gena ciljanih enzima pomoću RT PCR i proteina pomoću Western blota. Rezultati: U A-HBO2 skupini, HBO2 dovodi do značajno smanjene vazorelaksacije na ACh i hipoksiju, koju TEMPOL in vitro obnavlja na razine slične kontrolnoj skupini. ACh inducirana vazorelaksacija posredovana je uglavnom s NO, uz doprinos metabolita CYP450 u HBO2 skupinama. Hipoksična relaksacija je djelomično blokirana s oba inhibitora, INDO ili MS-PPOH, dok L-NAME nije imao utjecaja na relaksaciju u hipoksičnim uvjetima. HIF-inhibitor na funkcionalnoj razini smanjuje relaksaciju u 24H-HBO2 skupini. TBARS i produkcija endotelnog superoksida povećani su u A-HBO2 grupi u usporedbi s drugim testiranim skupinama. Zaključak: Akutno izlaganje HBO2 uzrokuje prolaznu i reverzibilnu poremećenu relaksaciju aortnih prstenova, koja je najvjerojatnije izazvana povećanom endotelnom produkcijom superoksida i sveukupno povećanim oksidativnim stresom. Intermitentno izlaganje uzrokuje poboljšanu relaksaciju najvjerojatnije aktiviranjem CYP450 epoksigenaznog puta metaboliziranja arahidonske kiseline i povećanjem stvaranja i osjetljivosti na EETs.Aim: The aim of this study was to determine the mechanisms of vasorelaxation in aortic rings in response to ACh and/or reduced pO2 and the role of oxidative stress and HIF-1α in acute and intermittent HBO2 protocols compared to untreated animals. Introduction: Hyperbaric oxygenation (HBO2) is used as adjuvant therapy in condition where tissue oxygen delivery is impaired. It is known that changes in pO2 may affect the synthesis of metabolites of arachidonic acid and thus vascular reactivity in health and disease, by involving CYP450 metabolites production. Generally, in healthy animals, vasorelaxationin response to hypoxia is mediated by prostacyclin (PGI2), while vasorelaxation in response to acetylcholine (ACh) is mainly mediated by NO pathway. Data on how is HIF-1α involved in relaxation mechanism are very scarce, inadequate and contradictory. Methods: Healthy Sprague-Dawley 9-12 weeks old rats were divided in four experimental groups (1. healthy control; 2. acute HBO2 group exposed to HBO2 (A-HBO2); 3. 24 hour after acute exposure (24H-HBO2); and 4. four subsequent days of HBO2 exposure (intermittent, 4D-HBO2). Reactivity to ACh and reduced pO2was tested in norepinephrine-precontracted aortic rings in the absence or in the presence of L-NAME, INDO, MS-PPOH, superoxide scavenger TEMPOL and LW6 HIF-1α inhibitor in tissue bath. The indicators of oxidative stress were measured by TBARS and FRAP methods and by direct measurement of endothelial superoxide production. Expression of antioxidative genes and enzyme-targeted genes was performed using RT PCR and proteins expression measurement was done by the Western blot. Results: In A-HBO2 group, HBO2 leads to impaired vasorelaxation response to both ACh and hypoxia, restored by TEMPOL in vitro to levels similar to control group vasorelaxation. ACh-induced vasorelaxation was mediated mainly by NO, with the contribution of CYP450 vasodilator metabolites in HBO2 groups respectively. Relaxation in response to reduced pO2 was partially blocked by both, INDO or MS-PPOH and not affected by L-NAME in all groups. HIF-inhibitor on functional level affected only relaxation in 24H-HBO2 group. TBARS and endothelial superoxide production were increased in A-HBO2 group compared to other tested groups. Conclusion: Acute exposure to HBO2 causes transient and reversible impaired relaxation of aortic rings, which is most likely caused by increased endothelial production of superoxide and overall increased oxidative stress. Intermitent exposure causes improved relaxation most likely by the activation of the CYP450 epoxygenase pathway of arachidonic acid metabolism and by increasing the generation and sensitivity to EETs