EVALUATION OF PLATELET ACTIVATION IN PLATELET-RICH PLASMA FROM WISTAR RATS

Abstract

Platelets are anucleated cell fragments contained in the blood plasma of animals, which are extremely important physiologically because they release proteins and growth factors that influence the cellular regeneration of soft and bone tissues. Their importance in veterinary and human medicine has led to the study of biotechnologies that seek to reproduce and intensify their function in the body, such as platelet-rich plasma (PRP), which concentrates a number of platelets 3 to 5 times greater than the initial volume. There are several methods for preparing PRP, which differ in the number of times the plasma is centrifuged, the number of rotations per minute, and, consequently, there are variations in the results obtained, such as platelet concentration and morphology. This study aimed to compare two low-cost and easy-to-prepare PRP protocols to analyze the morphology and integrity of platelets in albino Wistar rats in order to verify possible changes resulting from manipulation. To this end, blood samples were collected by cardiac puncture with hypodermic needles and disposable syringes containing CPDA-1 anticoagulant from 12 adult male albino Wistar rats previously anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg). The samples were aliquoted and stored in three tubes: 500 µL (whole blood) and 1500 µL in each of the two tubes to obtain PRP by PRPS (Sonnleitner et al. (2000) modified) and PRPV (Vendramin et al. (2006) modified) protocols. After collection, the PRP samples were diluted in V-52D DILUENT, and then platelet counts were performed on the whole blood and PRP samples using a BC-5000 Vet® automatic blood cell counter (Sherzhen Mindray Bio-Medical Electronics Co. Ltd. Shenzhen, Guangdong, China), as well as the PRP samples. For the morphological evaluation of the platelets, smears of the whole blood and PRP samples were stained with Romanin B (1:1000) and stained with Romanin B (1:1000). Guangdong China, as were the PRP samples. For morphological evaluation of platelets, smears of whole blood and PRP samples were stained with Romanowsky stain for optical microscope readings; and for evaluation of the platelet activation marker, the ELISA method was used to measure platelet factor 4. Statistical analysis was performed using the Jamovi program (version 2.4.11), with analysis of variance (ANOVA), followed by the Shapiro-Wilk test. The platelet count results revealed that platelet concentrations were in accordance with those proposed by Marx et al. (1998). The morphological evaluation of platelets from ST (68±22%), PRPS (74±18%), and PRPV (75±14%) smears did not differ between groups. Similarly, the amount of platelet factor 4 did not differ between groups.