The animal research ethics committee of the Federal University of Minas Gerais approved this study.
Animals
Four mongrel male animals of each species were used (dogs and cats), and all of the subjects were clinically healthy at the time of blood collection.
Preparation of autologous platelet concentrates to obtain a fibrin clot
Blood was collected by puncturing the jugular vein in the cats and the saphenous vein in the dogs with a butterfly catheter 21 G (Blood Collection Set, Becton Dickinson and Company Vacutainer, New Jersey, USA). Blood samples were placed in an 8.5-mL tube with 1.5 mL ACD-A (trisodium citrate 22 g/L, citric acid 8 g/L and dextrose 24.5 g/L) (Becton Dickinson and Company Vacutainer, New Jersey, USA). To obtain PC, the feline blood samples were centrifuged (Hettich Rotofix 32A, Tuttlingen, Germany) at 85 g, and the canine blood samples were spun at 191 g for 6 minutes (SIGMA 3 K30, Germany).
Activation of platelet concentrates
Before activation, PC samples were analyzed using an automated counting device by volumetric impedance (Abacus Junior Vet, Austria). Each sample was analyzed in triplicate. Next, a micropipette with a fixed volume of 1000 μL was used to collect (approximately) the first 100 μL of the red portion and the first 900 μL of plasma below and above the blood-plasma interface, respectively. Samples of each species were divided into two aliquots of 500 μL each, thus forming two groups. The PC samples from one group were activated with 50 μL of calcium gluconate 10% (Ropsohn Therapeutics Ltda, Bogotá, Colombia), and the samples of the other group were activated with 50 μL of batroxobin (Plateltex, Praha, Czech Republic) and reconstituted with 1 mL of calcium gluconate 10%.
Fibrin clot (platelet gel) preparation for transmission electron microscopy
Two hours after activation, the supernatant was removed from each sample, and the clots were fixed with a “Karnovsky - modified” primary solution (equal parts glutaraldehyde 2.5% and paraformaldehyde 2%) and post-fixed with osmium tetroxide 2% buffered. Subsequently, the fibrin clots were dehydrated in successive passes of 15 minutes in ethanol at concentrations of 50%, 70% (2 times), 85% (2 times), 95% (2 times), and 100% (three times). Finally, they were placed in acetone for 15 minutes (2 times). The fibrin clots were embedded in resin-acetone (1:2) for 1 hour, resin-acetone (1:1) for 1 hour, resin-acetone (2:1) for 12 hours at room temperature, and pure resin for 1 hour, followed by incorporation into molds and storage in a 40°C oven for 1 hour and a 60°C oven for 48 hours. The polymerized samples were used to obtain ultrathin sections of approximately 60 nm, taken once every 300 nm (approximately). The samples were stained for contrast with uranyl acetate and lead citrate. The plates were viewed on a transmission electron microscope (Tecnai G2 Spirit - FEI - 2006, 120 kV). One plate of each sample was evaluated on the transmission electron microscope.
Evaluation of the micrographs
Microscopic analysis of the 25 micrographs was performed for each plate by taking 100 photomicrographs for each activating substance for each species (200 total for each species). Morphological changes in the platelets of the fibrin clots were initially scored for both species using a scale from 0 to III [17], where 0: unstimulated platelet, slightly oval-shaped, with evenly dispersed organelles in the cytoplasm; I: platelet in uncertain state, rounded profile shape, non-centralized organelles; II: Platelet fully activated, irregular oval with centralization of organelles and extension of pseudopodia; III: damaged platelet, with total lysis and loss of normal architecture.
In addition to the initial classification used for both species, some platelet and fibrin characteristics were independently analyzed for each species. The parameters considered in cats were individual platelet area (μm2), percentage of platelet area, fibrin fiber area (μm2), percentage of fibrin fiber area, the ratio between the minor and major axes, the ratio between the major and minor axes of platelets, and the number of α-granules found within each platelet (n = 100 platelets for each activating substance).
For the dog samples, the intercellular area (μm2), percentage of intercellular space, fibrin fiber area (μm2), and percentage of fibrin fiber area were analyzed. This last evaluation was performed in 30 fibrin fibers, including those just over 0.5 mm in length, of which the width was measured in the middle third of each fiber, perpendicular to its central axis. The analyses were performed with a magnification of 18,000 X. The measurements were made with the digital analysis software Image J (Image Processing and Analysis in Java, National Institutes of Health, Maryland, USA).
Data analysis
For all of the data, a Shapiro-Wilk normality test was performed. The data from the digital analysis software were compared using a Student’s t- test (t) for paired samples, and the number of α-granules was compared using the Wilcoxon test. The threshold for a statistically significant difference was P ≤ 0.01 for all tests.