## 3 Discussion  
From this review, it seems that BoNT leads to a significant suppression of the severity of many induced disease models by targeting specific biological markers. However, while animal intervention studies still play an important role in human health care research, our findings seem to be in line with current evidence on common pitfalls of animal experiments. For example, [PERSON] et al. (2020) found that in animal studies, diseases are frequently induced with unclear concordance to human conditions [11]. More findings that were also consistent with our results include the great variation in animal characteristics and species studied, diversity of experimental designs, several models for induction of diseases, as well as differences in doses of drugs, timing, and delivery method protocols. Furthermore, the use of inferior models, with smaller dimensions, precluded the application of recommended protocols for BoNT use in humans and approved conditions such as chronic migraine [19; 20]. In line with our study, [PERSON] et al. (2020) also did not find that it the registration of animal studies and to include or adequately report experimental details is yet common practice, including random allocation of animals and/or blinded assessment of the outcomes [11]. We add that there is also a lack of information, whereas allocation to the different groups was reasonably concealed. These shortcomings may result in a lack of internal and external validity, as well as in challenges to conducting meta-analysis and extrapolating animal models to human clinical conditions. Nevertheless, as previously outlined, the authors conducted a systematic review of human studies on the same topic prior to commencing this study [7]. We found that CGRP was the most commonly examined biomarker in human trials on BoNT effects on inflammation-associated chronic conditions. Other biomarkers used in clinical studies included different immune cell classes, as well as several important players in inflammatory processes, serotonin, oxidative stress biomarkers, SNAP25, and SP. These appear to be largely in line with the biomarkers reported in the animal studies from this review. However, while for clinical research the most-studied scenario was the prevention of chronic migraine, with only one study focusing on trigeminal neuralgia, in preclinical research we observed the opposite. Additionally, although we adapted the eligibility  
Figure 5: GRADE assessment [13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 26]. criteria from the systematic review on clinical research, we found within the current study that twelve animal experiments explored other conditions that also fit the inclusion criteria namely, TMJ arthritis such as TMJ osteoarthritis and PHI in TMJ rheumatoid arthritis, anxiety, depression and MDD, hypertrophic scars, atopic dermatitis, and chronic dry skin itch conditions. In addition, findings from this review suggest that several other biomarkers may be implicated in the pathogenesis of the studied chronic conditions, and hence these were considered important to monitor the progression of certain diseases linked to inflammation and the effects of BoNT intervention. Overall, data found that BoNT significantly altered the biomarkers' under/over-expression evoked by the investigated disease models (e.g., IL-1\(\upbeta\), IL-6, c-Fos, TRPA1, dural protein extravasation, collagen (I,III), TGF-\(\upbeta\), \(\upalpha\)-SMA, and myosin II proteins) and had no effect when the levels of these biomarkers were not changed by the induced chronic conditions in animals (e.g., CX3 CR1). Meta-analysis results also revealed a significant effect of BoNT on TNF-\(\upalpha\) levels. However, it is important to note that the evidence contained in our meta-analysis was based on quantitative studies providing sample sizes, which resulted in the exclusion of one study showing that BoNT had no significant effect on TNF-\(\upalpha\) levels in the peri-articular tissues from TMJ and TG.