EFFECT OF HYPERBARIC OXYGEN THERAPY ON MODULATION OF MACROVASCULAR REACTIVITY IN FEMALE RATS WITH TYPE 1 DIABETES MELLITUS

Cilj: Cilj predložene doktorske disertacije bio je utvrditi kako HBOT utječe na funkciju krvnih žila te identificirati specifične mehanizme kojima HBOT utječe na relaksaciju aortnih prstenova potaknutu acetilkolinom kod zdravih štakorica i dijabetičkih štakorica. Dodatni cilj bio je rasvijetliti ulogu 20-HETE u makrovaskularnoj reaktivnosti ovisnoj o endotelu kod ženki dijabetičkih štakora. Uvod: U stanjima smanjene dopreme kisika tkivu kao dopunsku terapiju koristimo hiperbaričnu oksigenaciju (HBOT). Promjene parcijalnog tlaka kisika (pO2) mogu djelovati na sintezu metabolita arahidonske kiseline i mogu mijenjati vaskularnu reaktivnost u zdravih i bolesnih pacijenata proizvodnjom CYP450 metabolita. U zdravih životinja relaksacijski odgovor na hipoksiju posredovan je prostaciklinom (PGI2), a relaksacija kao odgovor na acetilkolin (ACh) posredovana je uglavnom NO putem. Materijali i metode: U ovoj studiji koristio sam Sprague-Dawley (SD) štakorice u dobi od 12 tjedana starosti u vrijeme pokusa, trajanje dijabetesa 6 tjedana. Životinje su bile podijeljene u 4 skupine: skupina 1 (CTR) – zdrave neliječene štakorice, skupina 2 (DM) – štakorice kojima je izazvan dijabetes melitus tip 1 pomoću streptozocina, skupina 3 (DM + HBOT) – štakorice s DM-om izložene djelovanju hiperbaričnog kisika u barokomori, skupina 4 (CTR + HBOT) – zdrave štakorice izložene djelovanju hiperbaričnog kisika u barokomori. Dodatno su uvedene još dvije skupine ovarijektomiranih zdravih (CTR + OVX) i dijabetičkih (DM + OVX) štakorica. Vaskularni odgovor na kumulativne koncentracije acetilkolina (10-10 – 10-5 M) u prekontrahiranim aortnim prstenovima mjerio se s/bez indometacina, L-NAME, MS-PPOH te HET0016. Odvojeno su se provodili pokusi s natrij-nitroprusidom (SNP). U svrhu utvrđivanja utjecaja HBOT-a na izražajnost enzima koji stvaraju metabolite odgovorne za vaskularnu reaktivnost u odgovoru na ACh, određivao sam mRNA za eNOS, iNOS, CYP450-ω hidroksilazu (izoforme), COX 1, 2; TXA2 sintazu i PGI2 sintazu te vaskularne izoforme CYP450 epoksigenaze u sve 4 skupine štakorica. Rezultati: Nije bilo značajne razlike u AChIR izoliranih aortnih prstenova štakorica među skupinama. U CTR i DM skupini štakorica analiza osjetljivosti aortnog prstena na ACh pokazala je da je osjetljivost na ACh u prisutnosti L-NAME i u prisutnosti MSPPOH znatno smanjena u usporedbi s osnovnim odgovorom ili odgovorom na ACh u prisutnosti indometacina. U CTR + HBOT i DM + HBOT skupini samo prisutnost L-NAME, ali ne i MS-PPOH ili INDO znatno je smanjila AChIR izoliranih aortnih prstenova štakorica. Ekspresija mRNA za iNOS u aortnom tkivu štakorica znatno je povećana u DM skupini štakorica u usporedbi s CTR skupinom štakorica. Ekspresija mRNA za eNOS, COX-1, COX-2, TBXAS1, CYP2J3, CYP4A1 i CYP4A3 u tkivu torakalnih aorta nije bila znatno različita među eksperimentalnim skupinama štakorica. Ova studija pokazuje da trajanje DM-a tipa 1 od 6 tjedana slabi reaktivnost aortnih prstenova na ACh samo u OVX štakorica, ali ne i kod ne-OVX SD štakorica. Zaključak: HBOT i DM mijenjaju mehanizme vazorelaksacije prema NO ovisnom putu. Trajanje DM-a tipa 1 od 6 tjedana slabi reaktivnost aortnih prstenova na ACh samo u OVX štakorica, ali ne i kod ne-OVX SD štakorica, što ukazuje na to da ženski spolni hormoni mogu imati zaštitni učinak (barem privremeno) na vaskularnu relaksaciju kod štakorica s DM-om tipa 1. Primjena HBOT-a u strogo definiranim uvjetima pokusa (2 atm, 120 minuta, 4 dana uzastopno) ne povećava oksidativni stres. Rezultati ove studije potvrdili su prethodna otkrića o zaštitnom učinku estrogena na vaskularnu i endotelnu funkciju jer su i OVX kontrole i štakorice s dijabetesom tipa 1 pokazale smanjenu vazorelaksaciju na ACh u odnosu na ne-OVX-CTR i ne-OVX-DM štakorice.Objective: The objective of the proposed dissertation was to determine how HBOT affects vascular function and to identify specific mechanisms through which HBOT affects acetylcholine-induced relaxation of aortic rings in healthy and diabetic female rats. Additional objective was to clarify the role of 20-HETE in endothelium-dependent macrovascular reactivity in diabetic female rats. Introduction: Hyperbaric oxygen therapy (HBOT) is used as complementary treatment in cases of hypoxia. Changes in partial pressure of oxygen (pO2) may affect the synthesis of arachidonic acid metabolites, and change vascular reactivity in health and disease by producing CYP450 metabolite. In healthy animals, relaxation response to hypoxia is mediated by prostacyclin (PGI2), and relaxation response to acetylcholine (ACh) is mediated mainly by NO pathway. Materials and Methods: Sprague-Dawley (SD) female rats used in this study were 12 weeks old at the time of the experiment, with diabetes duration of 6 weeks. The animals were divided into 4 groups: group 1 (CTR) – healthy untreated female rats, group 2 (DM) – female rats with streptozotocin-induced type 1 diabetes mellitus, group 3 (DM + HBOT) – female rats with DM exposed to hyperbaric oxygen in a hyperbaric chamber, group 4 (CTR + HBOT) – healthy female rats exposed to hyperbaric oxygen in a hyperbaric chamber. In addition, two more groups of healthy ovariectomized female rats (CTR+OVX) and ovariectomized diabetic female rats (DM + OVX) were included in the study. Vascular response to cumulative acetylcholine concentrations (10-10 – 10-5 M) in precontracted aortic rings was measured in the presence/absence of indomethacin, L-NAME, MS-PPOH and HET0016. Experiments with sodium nitroprusside (SNP) were conducted separately. For the purpose of determining the effect of HBOT on the expression of enzymes producing metabolites responsible for vascular reactivity in response to ACh, mRNA was determined for eNOS, iNOS, CYP450-ω hydroxylase (isoforms), COX 1,2; TXA2 synthase and PGI2 synthase, and vascular isoforms of CYP450 epoxygenases in all 4 groups of female rats. Results: There were no significant differences in AChIR of isolated aortic rings between different rat groups. In the CTR and DM groups of rats, sensitivity analysis of aortic ring to ACh has shown that sensitivity to ACh in the presence of L-NAME and MS-PPOH is significantly reduced compared to the basic response or response to ACh in the presence of indomethacin. In the CTR + HBOT and DM + HBOT groups, only the presence of L-NAME significantly reduced the AChIR of isolated aortic rings of rats, whereas the presence of MS-PPOH or indomethacin had no such effect. mRNA expression of iNOS in rat aortic tissue in the DM group was significantly increased in comparison with the CTR group. mRNA expression of eNOS, COX-1, COX-2, TBXAS1, CYP2J3, CYP4A1 and CYP4A3 in thoracic aorta tissue was not significantly different between the experimental groups of female rats. This study has shown that 6-week T1DM reduces the reactivity of aortic rings to ACh only in the OVX female SD rats. Conclusion: HBOT and DM modify the vasorelaxation mechanisms according to NO-dependent pathway. 6-week T1DM reduces the reactivity of aortic rings to ACh only in the OVX female SD rats, but not in the non-OVX ones, which indicates that female sex hormones can have a protective effect (at least temporarily) on vascular relaxation in female rats with T1DM. Application of HBOT under strictly defined experimental conditions (2 atm, 120 minutes, 4 consecutive days) does not increase oxidative stress level. Results of this study confirmed previous discoveries on the protective effect of estrogen on vascular and endothelial function, since both the OVX controls and the T1DM female rats exhibited reduced vasorelaxation to ACh in comparison with the non-OVX-CTR and non-OVX-DM groups of female rats