## 3 Discussion
### Limitations  
One of the primary limitations was the high heterogeneity of data from several animal experiments subjected to bias. Most studies favoured male animals over female or failed to disclose the sex of the animals investigated, underestimating sex-related physiologic differences confounding experimental results owning to distinct disease predisposition, prognosis, and response to BoNT (e.g., effect of sex hormones on nociceptive thresholds and biomarker levels). In contrast, with the advancements in clinical research reporting and designs, the gender bias was prevalent. Considering the important sex differences in varied aspects of brain functioning, data obtained exclusively from male animals limits the possibility of female-specific therapeutics advancing and translation. The evident paradox is that women are more vulnerable to the highly prevalent neurologic and psychiatric disorders reported in the included studies. Besides the susceptibility to develop certain conditions, sex differences extend to the presentation, the course of the illness and/or treatment effectiveness. Similarly, when reported, there were differences in age among the animals studied. Considering that the disease phenotype and response to therapies can be affected by the age, this contributes to increased heterogeneity of the studies. Another important source of bias was the variations in study design and methods. For example, we cannot exclude the overestimation of treatment effect, which may result from the lack of information on randomization, allocation concealment, or blinded outcomes assessment. Moreover, although studies showed significant results, indicating a sufficient sample size, they are based on male inferior animals, which limits the recommendations for clinical trials. Specifically, there was a preponderance of male rodents, and although these have been widely used in preclinical research, whether rats and mice are reliable models to study chronic inflammation remains controversial. Bearing in mind that publication bias in the systematic reviews of animal studies may severely affect its validity, we have attempted to conduct a comprehensive and systematic search. For that, we have defined and prespecified a research question focusing on inflammation-associated chronic conditions, and we set the eligibility criteria to exclude studies in disorders related to muscles and glands hyperactivity. However, the pathophysiology of many conditions to which BoNT treatment has been proposed is complex and we may have excluded diseases of interest and able to amplify the results. For example, we have excluded movement disorders of the nervous system such as [PERSON]'s disease. However, recent studies have suggested that this progressive neurodegenerative disease is most likely the result of inflammatory pathways malfunction linked to genetic vulnerability and immune deregulation caused by ageing and the activation of glia from neuronal insult [42]. Thus, a possible effective treatment of [PERSON]'s disease through BoNT anti-neuroinflammatory properties rather than only its actions at the typical associated motor symptoms of tremors, bradykinesia, and rigidity via transient paralysis of the treated muscles is hinted at [43]. Moreover, we have narrowed the searches to head and neck conditions in an attempt to limit the heterogeneity of chronic conditions and biological sampling across studies to enhance interpretability and obtain a meaningful summary effect. However, we may have excluded relevant disease models (e.g., psoriasis) illustrated in the Supplementary File S2, Table S1. Finally, most of the included studies did not present results in tabular or numerical formats but instead relied on graphical plots. To extract data from these plots, ImageJ software (1.54k 15 September 2024) was used to estimate numerical values. While we acknowledge that the extracted data may not be perfectly precise, we believe the impact of this limitation on accuracy is negligible due to the reliability of ImageJ.