Določanje posmrtnega intervala z ugotavljanjem viabilnosti hondrocitov v kolenskem sklepu prašičev

Cvetko, Marko (author), Gombač, Mitja (mentor), Alibegović, Armin (comentor)

Namen raziskave je določanje posmrtnega intervala (PMI) z ugotavljanjem viabilnosti hondrocitov v kolenskem sklepu prašičev z metodo s konfokalnim laserskim skenirnim mikroskopom (CLSM) in z metodo s tripanskim modrilom (TM) v različnih letnih časih. Zadnje okončine prašičev smo zakopali v 50 cm globok grob ali pustili na površini. Iz kolenskih sklepov okončin smo odvzeli vzorce hrustanca na dan evtanazije in nato v enodnevnih intervalih naslednje 4 dni ter 8. in 10. dan po evtanaziji. Naredili smo rezine hrustanca, ki smo jih obarvali z barvili kalcein-AM (Ca-AM) in ethidijev homodimer-1 (EthD-1), preostanek hrustanca pa raztopili, izolirali hondrocite in jih obarvali s TM. Postopek smo ponovili v vseh letnih časih. Viabilnost hondrocitov je v vseh letnih časih padala v odvisnosti od časa po evtanaziji in temperature okolice. Padec viabilnosti je bil na začetku majhen in je ustrezal obdobju platoja (obdobje majhnega, statistično neznačilnega padca viabilnosti), sledil je velik in statistično značilen padec, ki je bil največji in je nastopil najhitreje poleti, nekoliko kasneje jeseni in pozimi, najkasneje pa spomladi. Ugotovili smo, da je bil delež viabilnih hondrocitov v vseh obdobjih, z izjemo jesenskega, statistično značilno višji v hrustancih kolenskih sklepov zakopanih okončin. Menimo, da je to posledica izolacijske sposobnosti prsti, ki je v grobovih vzdrževala stabilnejšo, poleti nižjo, v ostalih letnih časih pa višjo temperaturo kot na površju. Tudi med letnimi časi smo ugotovili razliko v deležu viabilnih hondrocitov, ki je najverjetneje posledica spreminjajočih se dejavnikov okolja, predvsem nihajočih temperatur in vpliva žuželk, ki so poleti obžrle mehka tkiva okončin v enem tednu. Ugotovili smo, da je metoda s CLSM zanesljiva metoda za določanje PMI na podlagi viabilnosti hondrocitov v kolenskem sklepu prašičev. V vseh letnih časih, razen poleti, je bila variabilnost deležev viabilnih hondrocitov na posamezen dan višja pri metodi s TM kot pri metodi s CLSM, zato menimo, da je metoda s CLSM natančnejša metoda za ugotavljanje PMI na podlagi deleža viabilnih hondrocitov.

• Language: Slovenian
• Keywords: posmrtni interval – PMI, sklepni hrustanec, prašiči, letni čas, konfokalni laserski skenirni mikroskop – CLSM, tripansko modrilo – TM, viabilnost, žuželke
• Work type: Doctoral dissertation
• Organization: VF - Veterinary Faculty
• Year: 2024
• PID: 20.500.12556/RUL-163061
• Publication date in RUL: 02.10.2024

Secondary language

• Language: English
• Title: Determination of the post-mortem interval by assessing the viability of chondrocytes in the knee joint of pigs

Abstract

The aim of this study was to determine the PMI by assessing the viability of chondrocytes in the knee joints of pigs via the CLSM method and the TM method in different seasons. The hind legs of the pigs were buried in a 50 cm deep grave or left on the surface. Cartilage samples were collected on the day of euthanasia (Day 1), then daily for 4 days and on the 8th and 10th days. The cartilage sections were stained with Ca-AM and EthD-1, the remaining cartilage was dissolved, and the chondrocytes were isolated and stained with TM. The study was repeated in all four seasons. We found a time- and temperature-dependent decrease in chondrocyte viability in all four seasons. Initially, this decrease was small and corresponded to the plateau phase (a period with a small, statistically insignificant decrease in viability). However, a larger and statistically significant decline was subsequently observed, which was the largest and occurred most rapidly in summer, somewhat later in fall and winter, and latest in spring. We found that the proportion of viable chondrocytes in the articular cartilage of the knee was significantly greater in the buried limbs than in the control limbs during all periods except the fall. We hypothesise that this is due to the insulating capacity of the soil, which maintains a more stable temperature in the graves, which is lower in summer and higher in the other seasons than the on surface. We detected a difference in the proportion of viable chondrocytes between seasons, most likely due to changing environmental factors, especially fluctuating temperatures and the influence of insects that consumed the soft tissues of the limbs within a week in summer. We found that the CLSM method is a reliable method for determining the PMI on the basis of the viability of chondrocytes in the knee joints of pigs. In all seasons except summer, the variability of the proportion of viable chondrocytes on a single day was greater with the TM method than with the CLSM method, so we believe that the CLSM method is a more accurate method for determining the PMI on the basis of proportion of viable chondrocytes.

• Keywords: postmortem interval - PMI, articular cartilage, pigs, season, confocal laser scanning microscope – CLSM, trypan blue – TM, viability, insects