## 1 Introduction  
In recent years, botulinum toxin (BoNT) has been suggested as an alternative treatment to a few chronic conditions characterised by inflammatory states [1; 2]. The unexpected discovery that the on-label cosmetic protocol for BoNT to ameliorate glabellar lines was beneficial in chronic migraine and has the potential to improve mood led to extensive research in the realm of headaches, pain conditions, and psychological illness [3; 4]. Given the limitations of the few available pharmacotherapeutic options for the treatment of some of these chronic conditions, more attention has been paid to the promising long-lasting clinical effects and safety profile of BoNT [5]. The efficacy of BoNT therapy could not be unequivocally proven in some conditions or patient subpopulations [5]. Nevertheless, a trend for positive responses has been observed for many chronic conditions that have been linked to inflammation [6]. The lack of strong clinical evidence, to some extent, may be explained by methodological limitations of the studies being performed [7]. In addition, there is still fundamental unknown information on BoNT mechanisms that may preclude high-quality studies evaluating efficacy and immediate clinical translation [6]. At present, the mechanisms that mediate the non-motor effects of BoNT are not fully understood and constitute a field of active investigation [6]. Several factors related to BoNT mechanisms of action (e.g., protease longevity inside neurons and specificity for SNARE proteins, repeatability and reversibility of neuroparalysis, segmental actions in the sensory nucleus/spinal cord dorsal horn, specificity for hyperactive neurons, and selectivity for determined sensory neurons) may impact the therapeutic success of BoNT in certain clinical settings [6]. Hence, there are efforts taking place to uncover the potential effects of BoNT in the peripheral sensory nerves and central nervous system [1]. In parallel, the identification and application of biomarkers has gained increasing attention in the research and clinical fields [8; 9]. Among the numerous advantages of biomarkers is their potential for evaluating the mechanistic actions of drugs through a precise measurement, providing less bias and an objective assessment [10]. In addition, a recent systematic review found limited studies in humans evaluating BoNT key effects on biochemical biomarkers, mostly calcitonin gene-related peptide (CGRP), and concluded that BoNT may influence some biomarkers found in head and neck chronic inflammatory conditions [7]. Therefore, deeper understanding of this topic, likely to broaden the horizon of BoNT research and clinical practice, is needed. Despite challenges and controversies, preclinical experimentation is thought to play a crucial role in research aiming to develop health care, providing unique insights into the search for new drug targets and biomarkers [11]. Moreover, exploratory animal studies may offer a broader range of possibilities to assess the drug's pharmacokinetic and pharmacodynamic parameters [11]. In this direction, there has been growing awareness of the value of systematic reviews of animal studies to evaluate the quality of the preclinical evidence [12]. However, there are currently no systematic reviews of animal studies summarizing the key effects of BoNT and the relationship with biochemical biomarkers in head and neck chronic conditions characterised by inflammatory states. Understanding the effects of BoNT on the biomarkers that have been used in preclinical studies may offer an important step forward in future research, by encouraging a more consistent approach for measuring the therapeutic effects of BoNT. Moreover, preclinical research should focus on preferential targeting of BoNTsto relevant pathways for improving chronic inflammatory conditions, which may allow a clearer insight into the neurobiological mechanisms involved, as well as expedite the development of new strategies (e.g., novel engineered BoNT) with the potential to improve the safety and clinical efficacy.