The Isolated Mouse Jejunal Afferent Nerve Assay as a Tool to Assess the Effect of Botulinum Neurotoxins in Visceral Nociception

Abstract

For the past two decades, botulinum neurotoxin A (BoNT/A) has been described as a strong candidate in the treatment of pain. With the production of modified toxins and the potential new applications at the visceral level, there is a real need for tools allowing the assessment of these compounds. In this study, we evaluated the jejunal mesenteric afferent nerve assay to investigate BoNT/A effects on visceral nociception. This ex vivo model allowed the continuous recording of neuronal activity in response to various stimuli. BoNT/A was applied intraluminally during three successive distensions, and the jejunum was distended every 15 min for 3 h. Finally, samples were exposed to external capsaicin. BoNT/A intoxication was validated at the molecular level with the presence of cleaved synaptosomal-associated protein of 25 (SNAP25) in nerve terminals in the mucosa and musculosa layers 3 h after treatment. BoNT/A had a progressive inhibitory effect on multiunit discharge frequency induced by jejunal distension, with a significant decrease from 1 h after application without change in jejunal compliance. The capsaicin-induced discharge was also affected by the toxin. This assay allowed the description of an inhibitory effect of BoNT/A on afferent nerve activity in response to distension and capsaicin, suggesting BoNT/A could alleviate visceral nociception.

A Novel Catalytically Inactive Construct of Botulinum Neurotoxin A (BoNT/A) Directly Inhibits Visceral Sensory Signalling

“…Immunohistochemical staining was performed using an avidin-biotin-peroxidase protocol as previously described [18,58]. Sections to be incubated with the anti-SNAP-25 antibody did not receive antigen retrieval, whereas those to be stained with the anticleaved SNAP-25 (anti-cSNAP-25) antibody were immersed in EDTA buffer (pH 9, Sigma-Aldrich, Gillingham, UK, E9884) at 98 • C. All sections were first blocked with 3% hydrogen peroxide (Sigma-Aldrich, Gillingham, UK, 1.07209), followed by 10% serum (Sigma-Aldrich, H1270) in Dako buffer.…”

Intraoperative abobotulinumtoxinA alleviates pain after surgery and improves general wellness in a translational animal model

“…In this study, we confirmed the specificity of our antibody (EF14007; noncommercial antibody) for cleaved SNAP25 42 . Our antibody recognizes cleaved SNAP25 in human and rodent tissues injected with BoNT-A 42,60 . In the pig, cleaved SNAP25 was only detected in aboBoNT-A-injected animals and not in saline-injected controls, suggesting that the antibody detects specifically the cleaved SNAP25 also in pigs.…”