The present study was conducted in full compliance with all applicable research ethics and animal welfare regulations, under a general stranding response authorization by the Animal Use Ethics Committee (CEUA) of the Northern Fluminense State University Darcy Ribeiro, under the protocol number: 171/2012, according to brazilian federal law n ° 11794/08.
The Oryctolagus cuniculus rabbits were obtained from Federal University of Viçosa, vivarium sector. The animals were kept at the experimental unit in group cages with no more than 2 rabbits in each space with feeding system ad libitum. The animals were contained manually and received a pre-anesthetic protocol based on acepromazine, at a dose of 0.5 mg/kg/ IM, associated with morphine sulfate at a dose of 4 mg/kg/IM. After obtaining the desired anesthetic effect, trichotomy was performed on the left and right pinna, followed by catheterization of the marginal vein of the left external ear. This access enabled saline 0.9% infusion through syringe pump with a controlled dose of 15 ml/hr. Catheterization of the central artery of the external right ear was performed, enabling the collection of blood samples for hemogasometric and lactate analysis. Syringes for blood collection, were flushed with heparin sodium, and the material was evaluated immediately after collection. Subsequently, the trichotomy of the ventral area of the neck and right thoracic region was performed, enabling, respectively, tracheostomy and surgical access. Finally, a propofol bolus was intravenously administered at a dose of 10 mg/kg, and then tracheostomy and intubation were performed, enabling the realization of manual ventilation. Tracheal tube number 3.0 was connected to an inhalation anesthesia machine through the Baraka system. The animals were maintained under anesthesia with isoflurane inhalation, in a universal vaporizer.
The temperature for conditioning the operating room was 18 °C. All rabbits were subjected to control blood collection (M0) and hemogasometric evaluation, by a portable blood analyzer, to obtain the pH, PaCO2 and HCO3. The same analyzer provided lactate values. A thermal mattress was not used during the procedure. The temperature of the animals was monitored through videothermometry.
The animals were divided into two groups: the treated group and the sham group. The treated group consisted of 10 animals that underwent right lateral thoracotomy in the fourth intercostal space, followed by dissection of anatomical planes to access the chest cavity, and held the dorsal retraction of the lung lobes to expose the caudal mediastinum. After the incision in the caudal mediastinum and careful dissection of the structures, the caudal vena cava, cranial vena cava and azygous vein were identified and individualized, followed by dorsal retraction of the phrenic nerve. The pericardium was incised along the cardiac axis. In sequence, the Satinsky clamps were positioned to respectively occlude the caudal vena cava, the azygous vein and the cranial vena cava. A 30 s period of hyperventilation was performed immediately prior to occlusion. The clamping of vessels by using calipers was maintained for a period of 5 min, and subsequently a blood sample was collected for hemogasometric and lactate evaluation (M5). After 5 min of inflow occlusion, the Satinsky clamps were released, enabling cardiac reperfusion and establishing 5 min of recirculation. After this period, a new blood sample was collected for hemogasometric and lactate concentration evaluation (M10). Then, the rabbits underwent a painless death.
The sham group consisted of 10 animals who underwent the same methodology regarding the surgical access to the animals of the treated group. The pericardial incision along the cardiac axis and careful dissection and identification of the caudal vena cava, cranial vena cava and azygous vein was established in 5 min without any ventilation and circulatory arrest. Subsequently, new blood gas analysis and lactate concentration was performed (M5). Then, the animals were manually ventilated for 5 min, followed by blood sample collection to evaluate the blood gas and lactate concentration (M10). In sequence, the painless death of the rabbits was performed.
For the generation of videothermometric images, we used the MART station, which consists of a computerized work island, linked to an infrared image generator through a lance, which can be adjustable by distance. The distance is important because it enables the patient, lying on a surgical stretcher at a 1 m away to frame the evaluation field. The MART station is placed far from the surgical field, leaving free space for the normal flow of the surgical room, and providing a thermographer (the person performing and editing images through dedicated MART 1.0 software). These professional processes and returns real-time images during surgery. The MART station captures multi and hyperspectral images, performs mapping of multimodal thermal texture, sensor fusion and three-dimensional image. This system is radiation free for reading and medical diagnosis.
The following periods were set for videothermometric analysis regarding the treated group: M0 (time just prior to cardiac arrest); M5 (period of time which the animal was subjected to circulatory arrest); and M10 (period of time which the animal was subjected to reperfusion). The heart temperature was constantly evaluated in each moment. At each period, three temperature values were obtained, and an average of the values were calculated for each moment.
The periods set for videothermometric analysis regarding the sham group were as follows: M0 (period of time immediately before the respiratory arrest); M5 (period of time which the animal was underwent to respiratory arrest); and M10 (period of time which the animal was subjected to the return of ventilation). The heart temperature was constantly evaluated in each moment. Three temperature values, and average values were calculated for each moment.
At the end of the trial, the hearts were collected from animals in both groups, treated and sham, for histopathological analyses to evaluate cell lesions, such as edema, hydropic degeneration, congestion, myocardial infarction and hemorrhage. The histopathological analyses were realized at the Veterinary Anatomopathological Diagnostic Laboratory in the Animal Pathology Sector, which was focused on histological variations among the right atrium, the right ventricle, the left atrium, the left ventricle and the interventricular septum.
Respecting the ethical principles of animal experimentation, immediately after the experiment, the animals were euthanized. The animals were subjected to deep general anesthesia with propofol administration, and subsequently administered 10 ml of 19.1% potassium chloride via intracardiac injection.
In the statistical study of hemogasometry and lactate concentration, statistical analysis within each group was performed by ANOVA, followed by Tukey’s post-test to compare the means between the moments (M0, M5 and M10). The comparisons between the groups were performed by Student’s t-test. The significance level established for the statistical test was (p < 0.05).
In relation to videothermometry, the statistical analysis within each group was performed using Two Way Variance Analysis (ANOVA), followed by Mann-Whitney’s post-test test to compare the means between the moments (M0, M5, and M10). The significance level established for the statistical test was (p < 0.05).