Antinociceptive effect of botulinum toxin type A on CGRP release in the trigeminal ganglion in an animal model of formalin-induced pain

Abstract

Botulinum toksin tipa A (BT-A) potentan je neurotoksin kojeg proizvodi bakterija Clostridium botulinum. Mehanizam djelovanja mu se temelji na sprječavanju otpuštanja neurotransmitera u sinaptičku pukotinu proteolitičkim kidanjem SNARE proteina zaduženih za njihovu egzocitozu. Dugo se vremena smatralo da je djelovanje BT-A ograničeno samo na periferna područja u kojima je i primijenjen, zbog čega mu je terapijska primjena bila ograničena na liječenje autonomnih poremećaja, lokalizirane spastičnosti i hiperkinetičkih poremećaja pokreta. S vremenom je bilo ustanovljeno kako BT-A ima izraženo antinociceptivno djelovanje i na središnjoj razini uz minimalne nuspojave, dugotrajan učinak i izostanak tolerancije za razliku od ostalih analgetika sa sistemskim djelovanjem što ga čini izuzetno privlačnim kandidatom za daljnja istraživanja, posebice u vidu liječenja boli kroničnog tijeka. Cilj ovog diplomskog rada bio je istražiti utjecaj BT-A na ekspresiju CGRP-a, neuropeptidnog medijatora boli i upale, u području trigeminalnog ganglija, u modelu upalne orofacijalne boli uzrokovane primjenom formalina u štakora. BT-A (7 i.j./kg) bio je unilateralno primijenjen u područje desnog brka (ipsilateralno) Wistar štakora. Šest dana kasnije u područje lijevog brka (kontralateralno) uzrokovana je bol primjenom 50 μL 2,5 % formalina nakon čega su životinje žrtvovane te su analizirani prerezi njihovih trigeminalnih ganglija. U negativnoj kontrolnoj skupini se umjesto BT-A koristila fiziološka otopina primijenjena bilateralno, dok se u pozitivnoj kontrolnoj skupini fiziološka otopina primijenila ipsilateralno, a 50 μL 2,5 % formalina kontralateralno. Imunohistokemijskom analizom i statističkom obradom praćen je i kvantificiran intenzitet signala CGRP-a u poprečnim prerezima trigeminalnih ganglija. U eksperimentalnoj skupini primijećeno je statistički značajno smanjenje ekspresije CGRP-a u odnosnu na kontrolnu skupinu. Dobiveni rezultati sugeriraju da BT-A djeluje i na suprotnoj strani od mjesta primjene, što upućuje na njegovu mogućnost aksonalnog transporta i potencijalnog među-sinaptičkog prijenosa (transcitoze). Zaključno, ovaj diplomski rad pokazuje da BT-A može djelovati ne samo na lokalnoj razini, već i na razini središnjeg živčanog sustava putem moguće transcitoze što otvara prostor za daljnja istraživanja njegovog središnjeg antinociceptivnog djelovanja.Botulinum toxin type A (BT-A) is a potent neurotoxin produced by the bacterium Clostridium botulinum. Its mechanism of action is based on preventing the release of neurotransmitters into the synaptic cleft by proteolytically cleaving SNARE proteins responsible for their exocytosis. For a long time, it was believed that the effects of BT-A were limited to the peripheral areas where it was applied, restricting its therapeutic use to treating autonomic disorders, localized spasticity, and hyperkinetic movement disorders. Over time, it was established that BT-A has significant antinociceptive effects at the central level, with minimal side effects, long-lasting impact, and no development of tolerance compared to other systemically acting analgesics, making it an exceptionally attractive candidate for further research, particularly in the treatment of chronic pain. The aim of this thesis was to investigate the influence of BT-A on the expression of CGRP, a neuropeptide mediator of pain and inflammation, in the trigeminal ganglion in a model of inflammatory orofacial pain induced by formalin application in rats. BT-A (7 i.j./kg) was unilaterally administered to the area of the right facial vibrissae (ipsilateral) in Wistar rats. Six days later, pain was induced in the area of the left facial vibrissae (contralateral) by applying 50 μL of 2.5% formalin, after which the animals were sacrificed and sections of their trigeminal ganglia were analyzed. In the negative control group, saline was used instead of BT-A, administered bilaterally, while in the positive control group, saline was administered ipsilaterally, and 50 μL of 2.5% formalin was applied contralaterally. Immunohistochemical analysis and statistical processing were used to monitor and quantify the intensity of CGRP signals in transverse sections of the trigeminal ganglia. In the experimental group, a statistically significant reduction in CGRP expression was observed compared to the control group. The obtained results suggest that BT-A also acts on the opposite side of the application site, indicating its potential for axonal transport and possible trans-synaptic transfer (transcytosis). In conclusion, this thesis demonstrates that BT-A can act not only at the local level but also at the central nervous system level through potential transcytosis, opening up the possibility for further research into its central antinociceptive effects