Antinociceptivni učinak botulinum toksina tipa A u animalnom modelu boli uzrokovane formalinom

master's thesis

• Title (english): Antinociceptive effect of botulinum toxin type A in an animal model of formalin-induced pain
• Author: Matej Mustapić
• Mentor: Lidija Bach-Rojecky (mentor)
• Committee member: Lidija Bach-Rojecky (predsjednik povjerenstva)
• Committee member: Donatella Verbanac (član povjerenstva)
• Committee member: Lana Ganoci (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmacology); Zagreb
• Defense date and country: 2023-07-19, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Botulinum toksin tipa A (BT-A) neurotoksin je kojeg sintetizira bakterija Clostridium botulinum. Njegov specifični
mehanizam djelovanja, koji uključuje proteolitičko kidanje SNAP-25 proteina odgovornog za egzocitozu
neurotransmitera, prepoznat je kao moguće farmakološko rješenje nekih poremećaja autonomnog živčanog sustava,
stanja povezanih s hiperkontraktilnošću mišića i određenih vrsta boli. Antinociceptivni učinak BT-A dugo se vremena
smatrao posljedicom inhibicije perifernog lučenja neurotransmitera, no danas se vjeruje da su u pozadini složeni i još
uvijek nerazjašnjeni mehanizmi unutar središnjeg živčanog sustava (SŽS). Cilj ovog istraživanja bio je ispitati
mogućnost transcitoze kao jednog od mehanizama koji doprinosi kompleksnom središnjem djelovanju BT-A na bol.
BT-A (7 i.j./kg) je unilateralno primijenjen u područje brka Wistar štakora jedan dan prije primjene neutralizirajućeg
antitoksina za BT-A (anti-BT-A) (5 i.j./10 μL) u veliku moždanu komoru. Nakon sedam dana od primjene BT-A,
uzrokovana je bol primjenom formalina (2,5 %) te se kroz period od 45 minuta mjerila učestalost ponašanja povezanih
s boli. Periferno primijenjen BT-A smanjio je bol unutar druge faze formalinskog testa koja odgovara ranoj upalnoj
boli. Anti-BT-A poništio je antinociceptivno djelovanje BT-A, što sugerira središnje učinke BT-A na bol posredovane
transcitozom. Budući da primjena kolhicina, blokatora aksonalnog transporta, u trigeminalni ganglij s kontralateralne
strane nije utjecala na antinociceptivni učinak BT-A, isključena je periferna difuzija toksina i sistemsko širenje na
kontralateralnu stranu. Dodatno, primjenom BT-A sa suprotne strane od mjesta indukcije boli pokazan je
antinociceptivni učinak u upalnoj fazi formalinskog testa, što ukazuje da je kontralateralni učinak BT-A na bol
neovisan o mjestu ozljede i djelovanju toksina na strani primjene. Zaključno, rezultati prikazani u ovom diplomskom
radu doprinose razjašnjenju kompleksnog središnjeg djelovanja BT-A na bol. Po prvi puta se predlaže transcitoza u
senzornom sustavu kao važan čimbenik u antinociceptivnom učinku BT-A, što zahtijeva daljnja detaljna istraživanja.

Abstract (english)

Botulinum toxin type A (BoNT-A) is a neurotoxin synthesised by the bacterium Clostridium botulinum. Its specific
mechanism of action, which involves proteolytic cleavage of SNAP-25, a protein responsible for exocytosis of various
neurotransmitters, was recognized as a possible pharmacological solution for some autonomic disorders, conditions
accompanied by muscle hypercontractility and certain types of pain. Antinociceptive effect of BoNT-A was long
considered to be the consequence of solely peripheral inhibition of neurotransmitter exocytosis, but nowadays it is
believed that more complex and still unresolved mechanisms are at play. The aim of this study was to investigate the
possibility of transcytosis as a mechanism underlying the complex central action of BoNT-A on pain. BoNT-A (7
IU/kg) was unilaterally administered in the facial vibrissae of Wistar rats a day before the injection of antibody against
BoNT-A (5 IU/10μL) inside the cisterna magna. After seven days, pain was caused by formalin application (2.5%) and
the frequency of pain-associated behaviours was measured at 5-minute intervals over a period of 45 minutes.
Peripherally applied BoNT-A reduced pain during the second phase of formalin test, corresponding to early
inflammatory pain. Anti-BoNT-A prevented antinociceptive effect of BoNT-A, proposing central effects, mediated by
transcytosis. Since the application of colchicine, an axonal transport blocker, in the contralateral trigeminal ganglion
did not affect the antinociceptive effect of BoNT-A, possibility of peripheral diffusion of toxin and systemic spread to
the contralateral side was excluded. Additionally, when BT-A was applied on the opposite side of the pain induction
site, an antinociceptive effect was observed during the inflammatory phase of the formalin test, indicating that the
contralateral effect of BT-A on pain is independent of the site of injury and the action of toxin on the site of
application. In conclusion, the results presented in this master’s thesis contribute to the elucidation of the complex
central action of BoNT-A on pain. For the first time, transcytosis in the sensory system is proposed as an important
factor in the antinociceptive effect of BT-A, which requires further detailed investigation.

• Keywords: botulinum toksin; bol; transcitoza; trigeminalni živac; formalin
• Keywords (english): botulinum toxin; pain; transcytosis; trigeminal nerve; formalin
• Language: croatian
• URN:NBN: urn:nbn:hr:163:188134
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2025-07-19
• Created on: 2023-12-07 12:15:25