The experimental protocol and animal procedures were approved by the Ethical Committee of the State University of Rio de Janeiro, Brazil (H 36/94), in accordance to Guide for the Care and Use of Laboratory Animals .
Experiments were performed on 191 male Syrian hamsters (Mesocricetus auratus, Botucatu, São Paulo, SP, Brazil) weighing 85–138 g (7–10 weeks old). All animals were fed with chow for small rodents commercially available (Nuvilab, Nuvital, Curitiba, Paraná, Brazil) and received tap water ad libitum.
Animals were distributed into 19 groups and treated orally twice a day for 14 consecutive days, at 8:00 a.m. and 5:00 p.m.
Animals treated with physiological saline (n = 11), olive oil (n = 19) and fish oil (Incromega® TG3322, EPA 36 % and DHA 24 %, Croda, East Yorkshire, UK, n = 47) received 0.5 mL/100 g of body weight per day. For evaluation of microvascular diameter, one group was treated with fish oil at 0.2 mL/100 g/day (n = 6). For microvascular permeability, another group was treated with olive oil at 0.1 mL/100 g/day (n = 6).
The dose of fish oil chosen for all experiments was based in a dose-response curve (fish oil × microvascular permeability). Briefly, hamsters were distributed into five groups and treated with different daily doses of fish oil (0.04, 0.08, 0.2, 0.5 and 1.0 mL) and the number of leaks/cm2 for each dose was recorded. The dose-response curve reached a plateau at 0.5 and 1.0 mL daily doses (Fig. 1a). As the same effect was observed with both doses, we chose the smallest one (0.5 mL/day).
We treated olive oil groups with the same daily dose of fish oil in order to warrant that both groups received the same lipid amount. Although the daily dose of olive oil (0.5 mL/day) is considered high, we did not find statistical differences in macromolecular permeability between this dose and a dose five-fold lower (0.1 mL/day) (Fig. 1b).
Sixty-six animals were equally distributed into six groups and three of these groups received EPA TAG (Omégavie® EPA 90 TAG, Polaris, Pleuven, France) and the remaining three groups received DHA TAG (Omégavie® DHA 90 TAG, Polaris, Pleuven, France) with the following doses: 0.004, 0.02 and 0.1 mL per 100 g of body weight per day.
Thirty-six animals were separated into six groups. Three of these groups were treated by EPA EE (Incromega® EPA, Croda, East Yorkshire, UK) and the other three were treated by DHA EE (Incromega® DHA, Croda, South Yorkshire, UK) at different doses: 0.004, 0.02 and 0.1 mL per 100 g of body weight per day.
The I/R Model in the Cheek Pouch Preparation
On the experiment day (one day after the end of treatment), anesthesia was induced by an intraperitoneal injection (0.1–0.2 mL) of sodium pentobarbital (Pentobarbital sodique, Sanofi, Paris, France, 60 mg/mL) and maintained by 100 mg/kg of α-chloralose (Merck, Darmstadt, Germany) administered through a femoral vein catheter also used for fluorescein isothiocyanate (FITC)-dextran (TdB Consultancy, Uppsala, Sweden) or rhodamine-6G (Sigma Chemical Company, St. Louis, MO, USA) administrations.
Throughout surgery and subsequent experiment, the temperature of the animals was kept at 36.5 °C with a heating pad controlled by a rectal thermistor (LTB 750 Thermostat System, Uppsala process data AB, Sweden). A tracheostomy was performed to facilitate spontaneous breathing.
The cheek pouch preparation was dissected as previously described [16, 17] and mounted in an experimental chamber where it was continuously superfused, at a rate of 4.6 mL/min by a HEPES supported HCO3
−-buffered saline solution (composition in mM: 110.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 18.0 NaHCO3, and 15.39 HEPES Na+-salt) bubbled with 5 % CO2 -95 % N2 to keep the superfusate at pH 7.40 and pO2 at 12–15 mmHg. The temperature of the superfusion solution was maintained at 36.0 ± 0.5 °C with a circulating bath (Polyscience, model 8005, Polyscience, Niles, Illinois, USA). After 30 min of stabilization (resting period), if the preparation presented a brisk blood flow in all parts of the vascular bed including larger veins (where erythrocytes should not be discernible in the image of the blood stream), no spontaneous plasma leakage and few rolling and sticking leukocytes, a local ischemia of 30 min was performed. Ischemia in cheek pouch preparations was induced by means of a cuff, made of thin latex tubing, mounted around the proximal part of the everted pouch . The cuff was placed without any visible interference of local blood flow. The intracuff pressure could be quickly increased by air compression using a syringe and rapidly decreased when required. An intracuff pressure of 200–220 mmHg resulted in a complete arrest of microvascular blood flow within a few seconds.
Before and after ischemia (reperfusion), changes in arteriolar and venular diameters, the number of rolling and adherent leukocytes and the number of leaky sites in post-capillary venules were assessed and at the end of all experiments hamsters were euthanized, under anesthesia, by an intravenous injection of potassium chloride (KCl 3 M).
Observation of Arteriolar and Venular Diameters by Intravital Microscopy
For each cheek pouch preparation, three observation fields containing one arteriole and one venule were chosen for measurements of arteriolar and venular diameters (total magnification of 400×). The selected fields were evaluated by taking 1-min videotape recordings before ischemia, at onset, 15, 30 and 45 min of reperfusion. From videotape recordings, registration of the internal diameter of each arteriole and venule was obtained by means of an image-shearing monitor (Vista Electronics, San Diego, CA, USA, model 908). Changes in arteriolar and venular diameters were expressed as percentages, considering measurements taken before ischemia as 100 %.
Observation of Rolling and Adherent Leukocytes by Intravital Microscopy
Circulating leukocytes were labeled by rhodamine 6G administered by an intravenous injection of 0.4 mL (0.1 mg/mL) immediately prior to observations and followed by a continuous infusion (10 μL/min) of the fluorescent dye thereafter (Syringe Pump, model 55-2222, Harvard Apparatus, Hollister, MA, USA). Fluorescent leukocytes were observed with a UV-light microscope (Leica DM LS, Leica, Wetzlar, Germany) with a set of filters (Excitation BP 546-12/Emission LP 590, Leica, Wetzlar, Germany) coupled to a closed circuit TV system (445× magnification). In each preparation, two venules (with diameters and lengths ranging from 10 to 15 μm and from 100 to 400 μm, respectively) were selected taking into account the possibility to return exactly to the same site (proximity of fat cells and bifurcations) for consecutive measurements. Experiments were performed by taking a 1-min videotape recordings of selected microvessel fields in initial control conditions (before ischemia) and subsequently at onset, 15, 30, 45 min of reperfusion.
Rolling and sticking leukocytes in post-capillary venules were counted using videotape recordings and frame-by-frame analysis. A leukocyte was considered as rolling when it was in contact with the venular wall and had a lower velocity than circulating erythrocytes and as adherent when it was immobilized at one position for at least 30 s .
Macromolecular Permeability Assessment
Microvascular permeability for large molecules was quantified as the number of leaky sites (=leaks) in the area of observation. Briefly, fluorescein isothiocyanate-dextran (FITC-Dextran, MW 150,000, 50 mg/mL) was given intravenously to hamsters at 25 mg/100 mg body weight, just after the resting period. Leaks of labeled dextran were defined as visible extravascular spots (diameter > 40 µm) in post-capillary venules (internal diameter ranging from 9 to 16 μm) seen under fluorescent light using an UV-light intravital microscope (optical magnification 40×).
The number of leaks in post-capillary venules was manually scanned and counted before ischemia and during reperfusion. Maximal response to I/R occurs at 10 min after the onset of reperfusion and for this reason, this is the value reported for each experiment.
Results were expressed as means ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test and repeated measures by two-way ANOVA followed by Bonferroni’s post hoc test when appropriate (Graph Pad Prism 5.0 software, Graph Pad Software Inc., San Diego, CA, USA). A P value of less than 0.05 was considered significant.