Preparing a rat brain tissue samples for acetylcholinesterase activity measurement - the MM method

Sonja Marinković; Đorđe Đukanović; Nebojša Mandić-Kovačević; Snežana Uletilović; Tanja Cvjetković; Žana M Maksimović

doi:10.5937/scriptamed52-35485

Sonja Marinković Center for Biomedical Research, Faculty of Medicine, University of Banja Luka
Đorđe Đukanović Center for Biomedical Research, Faculty of Medicine, University of Banja Luka
Nebojša Mandić-Kovačević Center for Biomedical Research, Faculty of Medicine, University of Banja Luka
Snežana Uletilović Center for Biomedical Research, Faculty of Medicine, University of Banja Luka
Tanja Cvjetković Center for Biomedical Research, Faculty of Medicine, University of Banja Luka
Žana M Maksimović Center for Biomedical Research, Faculty of Medicine, University of Banja Luka

DOI: https://doi.org/10.5937/scriptamed52-35485

Keywords: Organophosphorus compounds, oximes, acetylcholinesterase, enzyme activity, rat brain

Abstract

Background/Aim: Organophosphorus compounds (OP) bind to acetylcholinesterase (AChE) causing an irreversible inhibition of the enzyme. When doing in vivo studies of OP intoxication, to precisely measure AChE activity in the brain tissue it is necessary to remove as much blood from the brain as possible. By doing so, interference of the OPs present in the blood is avoided. Usually this demands expensive equipment, therefore, the aim of this study was to find a simple and economical method to eliminate the blood from brain blood vessels.

Methods: Wistar albino rats were divided into four groups named Control (C), Control washout (CW), Paraoxon (Pox) and Paraoxon washout (PoxW) group. Rats in Pox and PoxW were treated with 0.25 mg/kg paraoxon subcutaneously (sc), while C and CW received 1 mL/kg sc saline instead. The “Marinković-Maksimović” (“MM”) method was performed in rats from PoxW and CW groups. Activity of AChE was measured both in erythrocyte lysate and in brain tissue using spectrophotometry.

Results: Macroscopic examination revealed that the elimination of blood was achieved in CW and PoxW groups. Activity of AChE in homogenised brain tissue was expectedly lower in the Pox and PoxW group, when compared to C and CW group, respectively. The CW group had a lower value of AChE activity in the brain tissue compared to C group, while activity of AChE in the PoxW group was statistically higher than in the Pox group (p = 0.044).

Conclusion: The MM method provides good elimination of blood from the brain. Together with blood, present confounding factors that interfere with analysis in homogenised brain tissue, were also eliminated.

References

1. Stojiljković MP. Nerve agents – a clear and present danger to mankind. Scr Med 2019;50(3):109-11.
2. Eddleston M, Phillips MR. Self poisoning with pesticides. BMJ 2004 Jan 3;328(7430):42-4.
3. Worek F, Diepold C, Eyer P. Dimethylphosphoryl-inhibited human cholinesterases: inhibition, reactivation, and aging kinetics. Arch Toxicol 1999 Feb;73(1):7-14.
4. Maksimović ŽM, Jović-Stošić J, Vučinić S, Perković-Vukčević N, Vuković-Ercegović G, Škrbić R, et al. Acute organophosphate and carbamate pesticide poisonings - a five-year survey from the National Poison Control Centre of Serbia. Drug Chem Toxicol 2021 Dec 7:1-9. doi: 10.1080/01480545.2021.2012481.
5. Vale JA. Toxicokinetic and toxicodynamic aspects of organophosphorus (OP) insecticide poisoning. Toxicol Lett 1998 Dec 28;102-103:649-52.
6. Tsatsakis AM, Manousakis A, Anastasaki M, Tzatzarakis M, Katsanoulas K, Delaki C, et al. Clinical and toxicological data in fenthion and omethoate acute poisoning. J Environ Sci Health B 1998 Nov;33(6):657-70.
7. Norrrahim MNF, Razakb MAIU, Shahc NAA, Kasimc H, Yusoffc WYW, Halim NA. Recent developments on oximes to improve the blood brain barrier penetration for the treatment of organophosphorus poisoning: a review. RSC Adv 2020;10:4465-89.
8. Rickett DL, Glenn JF, Beers ET. Central respiratory effects versus neuromuscular actions of nerve agents. Neurotoxicology 1986 Spring;7(1):225-36.
9. Škrbić R, Stojiljković MP, Ćetković SS, Dobrić S, Jeremić D, Vulović M. Naloxone antagonizes soman-induced central respiratory depression in rats. Basic Clin Pharmacol Toxicol 2017 Jun;120(6):615-20.
10. Kalász H, Nurulain SM, Veress G, Antus S, Darvas F, Adeghate E, et al. Mini review on blood-brain barrier penetration of pyridinium aldoximes. J Appl Toxicol 2015 Feb;35(2):116-23.
11. Orct T, Jurasović J, Micek V, Karaica D, Sabolić I. Macro- and microelements in the rat liver, kidneys, and brain tissues; sex differences and effect of blood removal by perfusion in vivo. J Trace Elem Med Biol 2017 Mar;40:104-11.
12. Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961 Jul;7:88-95.
13. Maksimović ŽM, Duka D, Bednarčuk N, Škrbić R, Stojiljković MP. Onset rate and intensity of signs of organophosphate poisoning related to paraoxon dose and survival in rats. Scr Med 2021 Mar;52(1):49-58.
14. Wei Z, Liu YQ, Wang YA, Li WH, Zhou XB, Zhao J, et al. Novel nonquaternary reactivators showing reactivation efficiency for soman-inhibited human acetylcholinesterase. Toxicol Lett 2016 Mar 30;246:1-6.
15. Sakurada K, Matsubara K, Shimizu K, Shiono H, Seto Y, Tsuge K, et al. Pralidoxime iodide (2-PAM) penetrates across the blood-brain barrier. Neurochem Res 2003 Sep;28(9):1401-7.
16. Firemark H, Barlow CF, Roth LJ. The penetration of 2-PAM-c14 into brain and the effect of cholinesterase inhibitors on its transport. J Pharmacol Exp Ther 1964 Aug;145:252-65.
17. Petroianu G, Toomes LM, Petroianu A, Bergler W, Rufer R. Control of blood pressure, heart rate and haematocrit during high dose intravenous paraoxon exposure in mini pigs. J Appl Toxicol 1998;18:293–8.
18. Eddleston M, Eyer P, Worek F, Mohamed F, Senarathna L, von Meyer L, et al. Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study. Lancet 2005 Oct 22-28;366(9495):1452-9.
19. Namba T, Nolte CT, Jackrel J, Grob D. Poisoning due to organophosphate insecticides. Acute and chronic manifestations. Am J Med 1971;50(4):475–92.
20. Ballantyne B, Marrs TC. Overview of the biological and clinical aspects of organophosphates and carbamates. In: Ballantyne B, Marrs TC, eds. Clinical and experimental toxicology of organophosphates and carbamates. Oxford: Butterworth-Heinemann; 1992. p. 3–14.
21. Houze P, Pronzola L, Kayouka M, Villa A, Debray M, Baud FJ. Ventilatory effects of low-dose paraoxon result from central muscarinic effects. Toxicol Appl Pharmacol 2008 Dec 1;233(2):186-92.
22. Dube SN, Ghosh AK, Jeevarathinam K, Kumar D, Das Gupta S, Pant BP, et al. Studies on the efficacy of diethyxime as an antidote against organophosphorus intoxication in rats. Jpn J Pharmacol 1986;41:267–71.
23. de Koning MC, van Grol M, Noort D. Peripheral site ligand conjugation to a non-quaternary oxime enhances reactivation of nerve agent-inhibited human acetylcholinesterase. Toxicol Lett 2011 Sep 25;206(1):54-9.
24. Zorbaz T, Malinak D, Maraković N, Maček Hrvat N, Zandona A, Novotny M, et al. Pyridinium oximes with ortho-positioned chlorine moiety exhibit improved physicochemical properties and efficient reactivation of human acetylcholinesterase inhibited by several nerve agents. J Med Chem 2018 Dec 13;61(23):10753-66.
25. Musilek K, Dolezal M, Gunn-Moore F, Kuca K. Design, evaluation and structure-activity relationship studies of the AChE reactivators against organophosphorus pesticides. Med Res Rev 2011 Jul;31(4):548-75.
26. Kuca K, Jun D, Junova L, Musilek K, Hrabinova M, da Silva JAV, et al. Synthesis, biological evaluation, and docking studies of novel bisquaternary aldoxime reactivators on acetylcholinesterase and butyrylcholinesterase inhibited by paraoxon. Molecules 2018 May 7;23(5):1103. doi: 10.3390/molecules23051103.