Vpliv ketamina in midazolama na viabilnost astrocitov možganske skorje podgane

Faganeli, Dan (avtor), Lipnik Štangelj, Metoda (mentor)

Namen: Ketamin in midazolam sta anestetika, pogosto uporabljena za dnevne kirurške posege in indukcijo v anestezijo. V zadnjih letih se po eni strani poroča o njuni toksičnosti, vidni po anesteziji, po drugi strani pa o možnosti zaščitnega delovanja pri možganski kapi. Sočasno se vloga astrocitov izkazuje kot ključnega pomena v homeostazi možganov in vzdrževanju celotne možganske funkcije. Viabilnost astrocitov je zato neposredno povezana z ustrezno funkcijo celotnega centralnega živčevja. Namen te naloge je in vitro preveriti in primerjati vpliv obeh anestetikov na viabilnost astrocitov, postavljenih v simulirane fiziološke oziroma patološke pogoje. Metode: Primarno celično kulturo astrocitov možganske skorje podgane smo uporabili za osnovo eksperimentalnega modela. Celice smo razdelili v dve skupini, pri čemer smo drugo skupino pred tretiranjem poškodovali s simulirano možgansko ishemijo. Obe skupini celic smo nato za 24 ur izpostavili naraščajočim koncentracijam ketamina in midazolama, vključno s tipičnimi koncentracijami, prisotnimi v plazmi tekom anestezije. Za določitev viabilnosti predhodno nepoškodovanih astrocitov smo s pretočno citometrijo merili apoptozo in nekroptozo, kot vrsti celične smrti. V skupini ishemično poškodovanih astrocitov smo na viabilnost sklepali z merjenjem mitohondrijskega membranskega potenciala z metodo pretočne citometrije ter metabolne aktivnosti celic, ki smo jo določili s testom AlamarBlue® s čitalcem za mikrotitrske plošče. Rezultati: Terapevtske koncentracije ketamina in midazolama niso vplivale na viabilnost in indukcijo celične smrti nepoškodovanih astrocitov. Viabilnost se je zmanjšala šele pri 100-krat višjih koncentracijah predvsem na račun apoptoze pri ketaminu ter pri 400-krat višjih na račun nekroze pri midazolamu. Ishemično poškodovanim astrocitom je v terapevstkih koncentracijah (0,05 mM) ketamin stabiliziral mitohondrijski membranski potencial in povečal njihovo metabolno aktivnost. Nasprotno, v terapevtskih koncetracijah midazolam ni pokazal nobenega vpliva na viabilnost ishemično poškodovanih astrocitov. Sklep: V terapevtskih koncentracijah ketamin in midazolam in-vitro ne zmanjšujeta viabilnosti in ne delujeta toksično na astrocite v fizioloških pogojih. V patoloških pogojih, značilnih za možgansko ishemijo, terapevtske koncentracije ketamina, ne pa midazolama, in vitro povečujejo viabilnost in nakazujejo zaščitni učinek do ishemično poškodovanih astrocitov.

• Jezik: Slovenski jezik
• Ključne besede: ketamin, midazolam, viabilnost, astrociti, možganska ishemija
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-147204
• Datum objave v RUL: 25.06.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: The effect of ketamine and midazolam on rat cortical astrocyte viability

Izvleček

Aim: Ketamine and midazolam are two anaesthetics frequently used in anesthesia for day surgeries and induction of general anesthesia. In recent years, several reports have emerged reporting their toxicity following anesthesia use. On the other hand, both are being investigated as possible cytoprotective agents after brain injury. Simultaneously, the role of astrocytes has been shown to be crucial in preserving brain homeostasis and function. The viability of astrocytes directly affects the function of the entire central nervous system. The objective of this study is to examine and compare, in vitro, the effect of both anaesthetics on the viability of astrocytes placed in simulated physiological or pathological conditions. Methods: Primary cell cultures of rat cortical astrocytes were used as the basis for the experimental model. The cells were divided into two groups: one group was damaged with simulated brain ischemia before treatment, and the other group was not. Both cell groups were then exposed for 24 hours to increasing concentrations of ketamine and midazolam, including clinically observed plasma concentrations during anesthesia. To assess the viability of previously undamaged astrocytes, flow cytometric analysis was used to measure apoptosis and necroptosis as two types of regulated cell death. The viability of the ischemically damaged cell group was determined by measuring mitochondrial membrane potential with flow cytometry and cell metabolic activity with AlamarBlue® test, using a microplate reader. Results: Therapeutic concentrations of ketamine and midazolam did not affect the viability or induce cell death in previously undamaged astrocytes. A decrease in cell viability was observed only at concentrations 100-times higher due to apoptosis in ketamine and 400-times higher due to necrosis in midazolam. Ketamine stabilized the mitochondrial membrane potential of ischemically damaged astrocytes and increased their metabolic activity at therapeutic concentrations (0,05 mM). However, such a protective effect on ischemically damaged astrocytes was not observed with midazolam at therapeutic concentrations. Conclusions: Ketamine and midazolam do not show toxicity and do not decrease astrocyte viability in simulated physiological conditions in vitro. In pathological conditions, such as brain ischemia, therapeutic concentrations of ketamine, but not midazolam, increase astrocyte viability in vitro and indicate a protective effect towards ischemically damaged astrocytes.

• Ključne besede: ketamine, midazolam, viability, astrocytes, brain ischemia