Botulinum toxin type A (BoNT-A) is a subtype of a potent neurotoxin secreted by the bacterium Clostridium botulinum. BoNT-A penetrates nerve endings and cleaves synaptosomal-associated protein 25 (SNAP25), one of the proteins of the SNARE complex (soluble N-ethylmaleimide-sensitive factor-binding protein receptor). By neutralizing the SNAP-25 protein, BoNT-A inhibits the release of acetylcholine from the axon terminals of neurons at the neuromuscular junction, resulting in temporary paralysis of the affected muscle (Muñoz Lora et al., 2019, Dutra et al., 2016).

The impact of BoNT/A in various autonomic and movement disorders is well-established, and its application in treating painful conditions has expanded (Matak et al., 2019). The pain-relieving effects of BoNT-A have been shown in experimental pain models and are separate from its muscle-relaxing properties (Favre-Guilmard et al., 2017, Bach-Rojecky and Lacković, 2005, Clark et al., 2007). Alongside inhibiting acetylcholine release, BoNT-A is thought to hinder the release of pain neurotransmitters like glutamate, substance P, and calcitonin gene-related peptide (Morris, 2003, Cui et al., 2004, Aoki, 2005, Morris et al., 2001). However, the pain-relieving effect of BoNT-A may not solely rely on peripheral nerve endings (Lackovic, 2021). Recent preclinical findings suggest that botulinum toxin may have a central action within the nervous system (Matak et al., 2011, Bomba-Warczak et al., 2016, Drinovac Vlah et al., 2018, Matak et al., 2019). Drinovac Vlah et al. (2018) observed axonal transport of BoNT/A and potential transcytosis, indicating that pain alleviation might involve interactions between the toxin and the central endogenous opioid system. Bomba-Warczak et al. (2016) explored the potential distant effects of BoNT/A using central neurons cultured in microfluidic devices, showing that the toxin can undergo cell-to-cell transfer to exert its distant effects. Despite compelling preclinical evidence suggesting that BoNT/A relieves peripheral and central sensitization, further research is necessary to comprehend the biological and molecular mechanisms underpinning its role in pain management.

Lately, the application of BoNT-A into the masticatory muscles has been employed to treat myofascial pain syndrome (including bruxism), temporomandibular joint disorders (TMD), tension headache, and chronic migraine headache (Guarda-Nardini et al., 2008, Schwartz and Freund, 2002, Dutra et al., 2016). The prevalence of myofascial pain associated with temporomandibular disorders varies between 10.3 % and 50 % in the populations studied (Liu and Steinkeler, 2013, Bertoli et al., 2018). BoNT-A could be an option for treating myofascial pain, mainly because its antinociceptive effect is more effective with a mild risk of adverse effects compared to other available therapies. However, clinical trials investigating the effects of BoNT-A on myofascial pain have shown conflicting results (Ernberg et al., 2011, Patel et al., 2017, Pihut et al., 2016).

Despite clinical reports and experimental studies, there needs to be more information in the literature about how BoNT-A induces antinociception. Studies evaluating the effect of analgesic or anti-inflammatory drugs on nociception have traditionally been performed by infiltrating carrageenan into the hind paws of rats and mice (Cui et al., 2004; Bach-Rojecky & Lacković, 2005; Favre-Guilmard et al., 2009; Favre-Guilmard et al., 2017), although other anatomical sites can be used. Pro-nociceptive stimuli affect tissues differently, as they vary in structure and innervation (Ren, 1999). Bagüés et al. (2017) demonstrated in rats that the injection of carrageenan into the masseter, a masticatory muscle innervated by the trigeminal ganglion, caused longer-lasting hyperalgesia when compared to carrageenan infiltration into the gastrocnemius muscle, a large muscle on the posterior compartment of the leg supplied by spinal nerves.

Some studies have demonstrated that botulinum toxin can alter muscle structure, affecting contractile function. One of the principal histopathological findings is muscle atrophy (Pingel et al., 2017, Rafferty et al., 2012, Botzenhart et al., 2020, Dutra and Yadav, 2019). These investigations have assessed the impact of elevated doses of botulinum toxin on the masseter muscle in healthy animals, as well as the effects of standard doses in rodents with genetic muscle diseases, such as Duchenne muscular dystrophy (Pingel et al., 2017, Botzenhart et al., 2020). Nevertheless, no study has examined the influence of intramuscular botulinum toxin on the histological and exudative changes induced by carrageenan in the masseter muscle.

Based on an experimental pain model we adapted for the masseter muscle (De Souza et al., 2024), the present study investigated the effects of BoNT-A compared to ibuprofen in a carrageenan-induced myofascial pain model. We assessed the influence of BoNT-A on mechanical hyperalgesia induced by the injection of carrageenan into the masseter muscle of rats using the Von Frey test. Ibuprofen is one of the most frequently prescribed non-steroidal anti-inflammatory drugs in dental practice for addressing pain and swelling. The effect of ibuprofen on the inflammation induced by carrageenan has been well-documented (Favre-Guilmard et al., 2009, Daif, 2012), allowing its use as a gold standard for analyzing the efficacy of therapies for musculoskeletal pain. Since histological changes are known to occur during inflammation provoked by carrageenan, we also performed a histopathological study of the masseters to verify the influence of BoNT-A on the injured tissue.