Utjecaj botulinum toksina tipa A na aktivaciju neurona u eksperimentalnoj upalnoj boli

master's thesis

• Title (english): The effect of botulinum toxin type A on neuronal activation in hypothalamus in experimental inflammatory pain
• Author: Karolina Belošević
• Mentor: Lidija Bach-Rojecky (mentor)
• Committee member: Lidija Bach-Rojecky (predsjednik povjerenstva)
• Committee member: Višnja Drinovac (član povjerenstva)
• Committee member: Renata Jurišić Grubešić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmacology); Zagreb
• Defense date and country: 2018-06-05, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Botulinum toksin tipa A (BT-A) je neurotoksin kojeg proizvodi anaerobna bakterija Clostridium botulinum. Smatra se
jednim od najpotentnijih bioloških toksina, no pročišćeni farmakološki pripravak u pikomolarnim dozama koristi se
liječenju različitih kliničkih stanja praćenih povećanom kontraktilnošću mišića. Dugotrajno djelovanje nakon
jednokratne primjene koje traje do nekoliko mjeseci daje mu prednost nad poznatim analgeticima te se sve više koristi
u liječenju određenih tipova kronične boli, primjerice migrene. Brojna istraživanja bave se njegovim
antinociceptivnim djelovanjem s ciljem otkrivanja mehanizma, kako bi se što bolje iskoristio u terapijske svrhe.
Nasuprot općeprihvaćenoj teoriji o djelovanju BT-A na inhibiciju egzocitoze neurotransmitora na perifernim
završecima osjetnih neurona, rezultati brojnih istraživanja ukazuju na središnje mjesto djelovanja, najvjerojatnije na
razini dorzalnog roga kralježnične moždine gdje periferno primijenjen BT-A dolazi retrogradnim aksonalnim
transportom. Ovim radom istraženo je potencijalno mjesto središnjeg djelovanja BT-A injiciranjem toksina u moždane
komore (intracerebroventrikularno, i.c.v.) na modelu formalinom uzrokovane periferne upalne boli, promatrajući
ekspresiju c-Fos proteina kao markera aktivacije neurona. Ispitivanja su provedena na tkivu mozga mužjaka štakora
soja Wistar. BT-A je primijenjen i.c.v. u dozi od 1 i.j./kg, a kontrolnoj je skupini i.c.v. injicirana fiziološka otopina u
odgovarajućem volumenu. Imunohistokemijskom metodom analizirana je ekspresija c-Fos proteina u hipotalamusu,
regiji mozga koja sudjeluje u modulaciji boli. Rezultati kvantitativne analize ekspresije c-Fos proteina očekivano
pokazuju aktivaciju ove regije nakon uzrokovanja upalne boli perifernom injekcijom formalina. Međutim, učinak BTA
nakon primjene u moždane komore (i.c.v.) je izostao, što se očituje podjednakim brojem aktiviranih neurona kod
ispitivanih i kontrolnih uzoraka tkiva. Ovi rezultati u skladu su s hipotezom o središnjem segmentalnom
antinociceptivnom djelovanju BT-A na spinalnoj razini, tj. razini kralježnične moždine na bol perifernog porijekla te
isključuju antinociceptivno djelovanje BT-A na supraspinalnoj razini. Međutim, potrebna su daljna istraživanja kako
bi se otkrilo primarno mjesto središnjeg djelovanja BT-A.

Abstract (english)

Botulinum toxin type A (BTX-A) is a neurotoxin produced by anaerobic bacteria Clostridium botulinum. It is
considered as one of the most potent biological toxins, but purified pharmacological preparation in picomolar doses is
used to treat various clinical conditions followed by increased muscular contractility. Long-term duration of effects
after its single administration represents a special clinical value among known analgesics, so it is widely used in the
treatment of certain types of cronic pain, such as migraine. Numerous researches are based on its antinociceptive
action to find out the mechanism and improve its clinical use. In contrast to generally accepted theory of BTX-A
activity on inhibition of neurotransmitters release on peripheral endings of sensory neurons, the results of numerous
researches point to its central site of action, most likely at the dorsal spinal cord level where peripherally applied BTXA
occurs by retrograde axonal transport. This study investigated the potentially central site of BTX-A action after
supraspinal administration (intracerebroventricular, i.c.v.) in inflammatory pain model induced by formalin injection,
by observing c-Fos protein expression as a marker of neuronal activation. Experiment was performed on brain tissue
of male Wistar rats. BTX-A was injected into the lateral cerebral ventricles (i.c.v.) in a dose of 1 U/kg. Control
animals received 0,9% saline in the appropriate volumes. C-Fos expression was imunohistochemically examined in
hypothalamus, a brain region involved in pain modulation. The results of the quantitative analysis of c-Fos protein
expression show activation of this region because of inflammatory pain induced by peripheal formalin injection.
However, BTX-A application into lateral cerebral ventricles failed to affect pain induced by formalin injection which
is manifested by an equal number of activated neurons in the examined and control tissue samples. These results are
following the hypothesis of the central segmental antinociceptive action of BTX-A at spinal level, i.e. spinal cord level
on the pain of peripheal origin and exclude antinociceptive action of BTX-A on supraspinal level. However, further
research is needed to investigate the primary location of BT-A's central activity.

• Keywords: botulinum toksin tipa A (BT-A); bol; hipotalamus; c-Fos; imunohistokemija; formalinski test; antinociceptivno djelovanje BT-A
• Keywords (english): botulinum toxin type A (BTX-A); pain; hypothalamus; c-Fos; immunohistochemistry; formalin test; antinociceptive action of BTX-A
• Language: croatian
• URN:NBN: urn:nbn:hr:163:379833
• 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
• Created on: 2018-11-28 16:09:54