The Effect of Hyperbaric Oxygenation on Ischemic/Reperfusion Brain Injury in Diabetic Female Rats.

Cilj istraživanja: Utvrditi optimalno trajanje tranzitorne okluzije srednje moždane arterije (t- MCAO) u modelu moždanog udara kod dijabetičkih SD (Sprague Dawley) štakorica. Utvrditi učinke jednokratne i višekratne hiperbarične oksigenacije (HBO2), 20-HETE inhibicije s N- hidroksi-N'-(4-butil-2metilfenil)-formamidinom (HET0016) na ishod eksperimentalnog moždanog udara. Materijali i metode: Štakorice s streptozotocinom induciranim dijabetesom tip-1 (N=25, 12 tjedana starosti, 5 skupina; N=5/skupini), su izložene različitom trajanju t-MCAO (20 minuta (’), 30’, 45’, 60’ i 90’) i reperfuziji. Kontrolna skupina štakorica bez dijabetesa (N=5) je izložena 30’ t-MCAO i reperfuziji. U drugom pokusu su dijabetičke štakorice (N=42, 12 tjedana starosti, N=7/skupini), izložene t-MCAO tijekom 30’ te reperfuziji, a potom podijeljene u slijedeće skupine; a) kontrolna skupina, bez liječenje; i skupine izložene; b) HBO2; c) višekratnim HBO2 (prva odmah, a druga 12h nakon t-MCAO); d) HET0016 predtretmanu (1mg/kg/3 dana prije t-MCAO) kombiniranom s HBO2 nakon t-MCAO; e) HET0016 liječenju (1h prije, tijekom t-MCAO te tijekom 6 sljedećih sati lmg/kg/h); i f) HET0016 liječenju kombiniranim s HBO2 nakon t-MCAO. 24h nakon reperfuzije, volumen infarkta mozga je analiziran 2,3,5-trifeniltetrazolij kloridom (TTC). Rezultati: 20’ t-MCAO rezultiralo je malim infarktom (3±5% ishemične hemisfere), a 30’ značajno većim infarktom (46±6%). 45’ i 60’ ishemija su stvorile infarkte 52±5% i 59±3% ishemične hemisfere, a 90’ t-MCAO masivni infarktom mozga (89±6% ishemične hemisfere (cijelog striatuma (22±3%) i skoro cijelog korteksa opskrbljenog s MCA (67±6%)). 30’ t - MCAO nije rezultiralo infarktom u kontrolnoj skupini. U drugom pokusu liječenje s HET0016 i HBO2 jednako su značajno smanjili kortikalni i ukupni infarkt mozga, u usporedbi s kontrolom. Kombinirano liječenje s HET0016 i HBO2 nije bilo učinkovitije u usporedbi s liječenjem samo s HET0016 bez obzira na način primjene. Cyp2J3 mRNA bila je značajno izraženija u svim ispitivanim skupinama, aCyp2C11 mRNA značajno izraženija u skupinama liječenim višekratnom HBO2 i HET0016 predtretman+HBO2 skupini, u usporedbi s kontrolom. 65 Zaključak: Model moždanog udara kod SD dijabetičkih štakorica treba biti drugačiji nego kod nedijabetičkih, s kraćim trajanjem ishemije, optimalno na 30', radi njihove visoke senzitivnosti na ishemiju mozga. Kod dijabetičkig SD štakorica i HBO2 i HET0016 izrazito su učinkoviti načini liječenja moždanog udara, što sugrerira sudjelovanje CYP450 metabolita u ovom učinku.Objectives: To determine optimal duration of transient middle cerebral artery occlusion (t- MCAO) for stroke model in female diabetic Sprague-Dawley (SD) rats. To evaluate effects of acute and repetitive hyperbaric oxygenation (HBO2) as well as 20- HETE inhibition by N-hydroxy-N'-(4-butyl-2methylphenyl)-formamidine (HET0016) on experimental stroke outcomes. Material and Methods: 25 streptozotocin-induced type-1 diabetic SD female rats (N=25, 12 weeks old, 5 groups; N=5 per group), were subjected to different duration of t-MCAO (20 minutes (’), 30’, 45’, 60’ and 90’) followed by reperfusion. Control group of rats without diabetes (N=5) was subjected to 30’ of t-MCAO followed by reperfusion. 42 streptozotocin- induced type-1 diabetic Sprague-Dawley female rats (N=42, 12 weeks old, N=7/group), were subjected to transient middle cerebral artery occlusion (t-MCAO) for 30’ followed by reperfusion and divided in following groups; a) control group, without treatment; and groups exposed to; b) HBO2; c) multiple HBO2 (first HBO2 immediately and second 12h after t- MCAO); d) HET0016 pretreatment (1mg/kg/3 days prior to t-MCAO) combined with HBO2 after t-MCAO; e) HET0016 treatment (1h before, during and for 6 hours after t-MCAO); and f) HET0016 treatment followed by HBO2 after t-MCAO. 24h after reperfusion infarct volumes were evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining. mRNA expression of CYP2J3, CYP3C11 was determined by real-time qPCR. Results: Intra-ischemic reductions of regional cerebral blood flow (rCBF) were similar in all groups (68%-75% of baseline values). Reperfusion was significantly impaired in 90’ ischemia group (56-62% vs. 80-125% in other groups). 20’ t-MCAO induced small infarct (3±5% of ischemic hemisphere). 30’ ischemia produced significantly larger infarct (46±6%). In 45’ and 60’ ischemia infarct was 52±5% and 59±3% of ischemic hemisphere, respectively. Ischemia of 90’ led to massive stroke (89±6% of ischemic hemisphere encompassing the whole striatum (22±3%) and almost the whole MCA irrigated cortex area (67±6%)). 30’ t -MCAO did not produce stroke in the control group. HBO2 and HET0016 treatment equally significantly reduced size brain infarct compared to control. Combined treatment with HET0016 and HBO2 provided no significant additive effect compared to HET0016 treatment only, regardless of administration method. mRNA of Cyp2J3 was significantly increased in all study groups compared to control, and mRNA of Cyp2C11 was significantly increased in multiple HBO2 and HET0016 pretreatmen+HBO2 groups compared to control. Conclusion: Diabetic rat stroke model should be different from non-diabetic, because female type-1 diabetic SD rats are highly sensitive to brain ischemia and it is necessary to significantly shorten duration of t-MCAO, optimally to 30’. In diabetic female SD rats HBO2 and HET0016 are highly effective stroke treatments, suggesting involvement of CYP450 metabolites in this therapeutic effect