Utjecaj botulinum toksina tipa A na aktivaciju neurona leđne moždine u modelu upalne boli

master's thesis

• Title (english): The effect of botulinum toxin type A on c-fos activation in spinal cord in inflammatory pain model
• Author: Valentina Meglić
• Mentor: Lidija Bach-Rojecky (mentor)
• Committee member: Lidija Bach-Rojecky (predsjednik povjerenstva)
• Committee member: Toma Keser (član povjerenstva)
• Committee member: Višnja Drinovac Vlah (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmacology); Zagreb
• Defense date and country: 2022-07-14, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Botulinum toksin tipa A (BT-A) više od 30 godina koristi se u terapiji različitih kliničkih stanja praćenih mišićnom spastičnošću. Na različitim eksperimentalnim modelima upalne i neuropatske boli uočeno je njegovo dugotrajno antinociceptivno djelovanje nakon jednokratne periferne primjene, što ga uz malen broj nuspojava razlikuje od ostalih sistemskih analgetika u trenutnoj primjeni. Dosad su provedena brojna pretklinička i klinička istraživanja s ciljem razjašnjavanja točnog mehanizma i mjesta djelovanja toksina, što bi omogućilo proširenje njegovih indikacija na brojna bolna stanja. Usprkos općeprihvaćenoj teoriji o perifernom inhibitornom djelovanju BT-A na egzocitozu neurotransmitora na završetcima senzornih neurona, novija istraživanja ukazuju na središnje djelovanje toksina, najvjerojatnije na razini dorzalnog roga leđne moždine kamo periferno primijenjen BT-A dolazi retrogradnim aksonalnim transportom. Također postoje dokazi o bilateralnom antinociceptivnom djelovanju BT-A nakon njegove unilateralne periferne primjene. Cilj ovog diplomskog rada bio je potvrditi ove navode na modelu bilateralne upalne boli uzrokovane injiciranjem karagenana u stražnje šape mužjaka štakora soja Wistar. Jedna je skupina životinja prethodno tretirana s BT-A u dozi 7 i.j./kg u stražnju desnu šapu, dok je druga skupina uz BT-A primila i antitoksin za BT-A (anti-BT-A). Kontrolnim skupinama injicirana je fiziološka otopina umjesto BT-A, a u jednoj od njih primjenom fiziološke otopine umjesto karagenana izbjegnuto je izazivanje upale. Imunohistokemijski je praćena ekspresija markera neuronalne aktivacije c-Fosa u poprečnim L4/L5 presjecima leđne moždine štakora. Rezultati pokazuju kako, sukladno očekivanjima, karagenan dovodi do bilateralne aktivacije neurona u leđnoj moždini dok unilateralna primjena BT-A smanjuje istu na obje strane leđne moždine. Pritom je statistički značajno smanjenje ekspresije c-Fosa zabilježeno u odnosu na kontrolnu skupinu i na skupinu tretiranu s anti-BT-A na ipsilateralnoj, ali i na kontralateralnoj strani leđne moždine. Ovo je još jedan u nizu dokaza o središnjem mjestu antinociceptivnog djelovanja BT-A, pri čemu se ne može isključiti mogući transinaptički prijenos BT-A u druge neurone/stanice uključene u proces nocicepcije na razini leđne moždine.

Abstract (english)

Botulinum toxin type A (BTX-A) has been used to treat numerous clinical conditions characterised by muscle spasticity for years. Long-term antinociceptive effect of BTX-A after its single peripheal application has been reported in various experimental models of inflammatory and neuropathic pain. This, combined with its low side effects frequency differs BT-A from other currently used systemic painkillers. Until now, many preclinical and clinical studies have been conducted with the goal of discovering BT-A's exact mechanism and site of action in mind, which would allow for the expansion of current indications to many other pain conditions. In contrast to generally accepted theory of BTX-A's peripheral suppression of neurotransmitters release from sensory nerve endings, the results of several recent researches suggest its central site of action, most likely at the dorsal horn of spinal cord level, where peripherally applied BTX-A is delivered by retrograde axonal transport. There is also evidence that BTX-A has bilateral antinociceptive effect after its unilateral peripheral application. The goal of this master's thesis was to confirm these statements using inflammatory pain model where pain is inflicted by carrageenan injection into hindpaws of male Wistar rats. One group of animals was pretreated with a BTX-A dose of 7 i.u./kg, while another group was treated with BTX-A antitoxin (anti-BTX-A) alongside BTX-A. Control groups have received saline injection (0,9 % NaCl) instead of BTX-A and one of them was treated with saline instead of carrageenan in an attempt to avoid inflammation. Expression of c-Fos (neuronal activity marker), which was used as indirect pain indicator, was imunohistochemically examined in L4/L5 cross sections of rat spinal cord. The results show that, in line with our expectations, carrageenan is enabling bilateral activation of spinal neurons while BTX-A reduced this activation on both sides of the spinal cord. Statistically significant reduction of c-Fos expression was recorded in comparison with control group and with experimental group treated with anti-BTX-A on both the ipsilateral and contralateral side of spinal cord. This is just another proof that BTX-A is exerting its antinociceptive effects centrally, with both sides of spinal cord as its site of action.

• Keywords: botulinum toksin tipa A; dorzalni rog; c-Fos; bol; karagenanski model upalne boli; imunohistokemija
• Keywords (english): botulinum toxin type A; dorsal horn; c-Fos; pain; carrageenan inflammatory pain model; immunohistochemistry
• Language: croatian
• URN:NBN: urn:nbn:hr:163:580284
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2024-07-14
• Created on: 2022-07-20 11:34:43