The Role of Botulinum Neurotoxin A in the Conservative Treatment of Fractures: An Experimental Study on Rats.

This paper explores the role of botulinum neurotoxin in aiding fracture recovery through temporary muscle paralysis. Specifically, it investigates the effects of botulinum neurotoxin-induced paralysis of the sternocleidomastoid muscle on clavicle fractures in rats. The research aims to assess safety, effectiveness, and the impact on fracture healing. Healthy male Albino Wistar rats were divided into four groups: clavicle fracture, botulinum neurotoxin injection, both, and control. Surgeries were conducted under anaesthesia, and postoperatively, animals were monitored for 28 days. Euthanasia and radiological assessment followed, examining fracture healing and muscle changes, while tissues were histopathologically evaluated. The modified Lane-Sandhu scoring system was used for the radiographic evaluation of clavicle fractures, and the results varied from complete healing to nonunion. Histopathological examination at 28 days postfracture showed fibrous tissue, mesenchymal cells, and primary callus formation in all groups. Despite varied callus compositions, botulinum neurotoxin administration did not affect clavicle healing, as evidenced by similar scores to the control group. Several studies have explored botulinum neurotoxin applications in fracture recovery. Research suggests its potential to enhance functional recovery in certain types of fractures. Theoretical benefits include managing muscle spasticity, aiding reduction techniques, and preventing nonunion. However, botulinum neurotoxin's transient effect and nonuniversal applications should be considered. The present study found that botulinum toxin had no clear superiority in healing compared to controls, while histological evaluation showed potential adverse effects on muscle tissue. Further research is essential to understand its risk-benefit balance and long-term effects.

Onabotulinumtoxina (craniotomy scar combined with cranial suture line injections) for persistent post craniotomy headache: Case series with long-term follow-up.

BACKGROUND: There is no defined preventive treatment protocol for persistent post-craniotomy headache. In several small case series and individual case reports onabotulinumtoxinA injected into the craniotomy scar has shown possible efficacy. What is lacking is long term follow-up and if focusing on the cranial suture lines along with the craniotomy scar can enhance improvement and provide more sustained benefit. METHODS: Retrospective chart review with case series. RESULTS: Four patients (three women, one man) with ICHD-3 defined persistent post craniotomy headache were treated using a novel onabotulinumtoxinA injection protocol. All the patients presented with continuous head pain of moderate to severe intensity. All had severe allodynia on the side of their craniotomy. All had significant reduction in quality of life. Our application of onabotulinumtoxinA involved injection into both the surgical scar and the transected/irritated cranial suture lines noted on neuroimaging and physical examination. With treatment all patients demonstrated significant benefit including a reduction in daily pain intensity (75%-100%), developing periods of pain freedom (2-7 days per week) and having a dramatic improvement in quality of life (close to 100% in all). The benefit was sustained for at least five years of follow-up. CONCLUSION: From our case series it appears that injection not only along the painful craniotomy scar but into the involved cranial suture lines provides positive efficacy and sustained improvement in patients with persistent post craniotomy headache.

Intradetrusor Versus Suburothelial Onabotulinum Toxin A in Adults with Neurogenic and Non-neurogenic Overactive Bladder Syndrome: A Meta-Analysis.

PURPOSE: This systematic review and meta-analysis aimed to compare the effectiveness and safety of submucosal injection of onabotulinum toxin A (OnabotA) with intradetrusor injection for overactive bladder syndrome (OAB). METHODS: This systematic review is registered with PROSPERO (CRD42021237964). A licensed librarian surveyed Medline, EMBASE, Scopus, and Google Scholar databases to conduct a comprehensive search. Studies comparing suburothelial and intradetrusor techniques of OnabotA injection for OAB were included, along with clinical and urodynamic variables and complications. The studies were assessed for quality on the basis of Cochrane Collaboration guidelines and evaluated using statistical analysis via a random-effect model and I2 statistic. Data extraction and analysis were conducted using Covidence systematic review platform and Review Manager software. RESULTS: Six studies with 299 patients were included in the systematic review, with four reporting that suburothelial injection of OnabotA was as effective as intradetrusor injection and two reporting intradetrusor injection to be more effective. The meta-analysis found no significant difference between the suburothelial and intradetrusor groups for mean daily catheter or voiding frequency (mean difference: 2.12 [95% confidence interval (CI): -1.61, 5.84]) and the mean number of urgency/urge incontinence episodes (mean difference: 0.08 [95% CI: -1.42, 1.57]). However, a significant heterogeneity was found among the studies. Only the mean volume at first detrusor contraction showed a significant difference, being higher for suburothelial injection (mean difference: 33.39 [95% CI: 0.16, 66.63]). No significant difference was noted for mean compliance, mean bladder capacity, and mean maximum detrusor pressure. Urinary tract infections (UTIs) (p = 0.24) and acute urinary retention (p = 0.92) showed no significant difference between the two groups. The risk of bias varied among the studies. CONCLUSIONS: Suburothelial injection of OnabotA is as effective as intradetrusor injection in improving OAB symptoms, and it has similar complication rates. A higher mean volume of the first detrusor contraction was found in a urodynamic study with suburothelial injection.

Design and evaluation of mRNA encoding recombinant neutralizing antibodies for botulinum neurotoxin type B intoxication prophylaxis.

Among all natural and synthetic toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botulinum in an anaerobic environment, are the most toxic polymer proteins. Currently, the most effective modalities for botulism prevention and treatment are vaccination and antitoxin use, respectively. However, these modalities are associated with long response time for active immunization, side effects, and donor limitations. As such, the development of more promising botulism prevention and treatment modalities is warranted. Here, we designed an mRNA encoding B9-hFc - a heavy-chain antibody fused to VHH and human Fc that can neutralize BoNT serotype B (BoNT/B) effectively - and assessed its expression in vitro and in vivo. The results confirmed that our mRNA demonstrates good expression in vitro and in vivo. Moreover, a single mRNA lipid nanoparticle injection effectively prevents BoNT/B intoxication in vivo, with effects comparable to those of protein antibodies. In conclusion, we explored and clarified whether mRNA drugs encoding neutralizing antibodies prevent BoNT/B intoxication. Our results provide an efficient strategy for further research on the prevention and treatment of intoxication by botulinum toxin.

A multicenter, double-blind, randomized, parallel-group, active-controlled, phase 3 clinical trial to compare the effectiveness and safety of two botulinum toxin type A formulations for improving moderate to severe glabellar wrinkles in Asians.

Botulinum toxin type A (BoNT-A) was first isolated in 1946, and since then, several formulations have been developed and widely used to treat wrinkles by inducing muscle paralysis. This multicenter, double-blind, randomized, parallel-group, active-controlled phase 3 clinical trial was designed to evaluate the efficacy and safety of a newly developed BoNT-A formulation, BMI2006, in improving moderate to severe glabellar wrinkles and to compare with existing onabotulinumtoxin A (OBoNT) injections. A total of 276 subjects were enrolled and received 20 units of the randomized material, which was intramuscularly injected into five different locations on the forehead. The primary endpoint, assessed at 4 weeks, showed no statistically significant difference in the improvement rate of glabellar wrinkles between the two groups, with BMI2006 demonstrating non-inferiority to comparator BoNT-A. Secondary endpoints, evaluated by both treating investigators and independent investigators, also exhibited similar improvement rates throughout the study period. Both groups reported high levels of satisfaction with no statistical difference between the two groups. Safety evaluations indicated mild and transient adverse events, with no serious reactions observed. In conclusion, BMI2006 is an effective and safe BoNT-A for treating glabellar wrinkles with an expected duration of action between 8 and 12 weeks.

Positive effect of self-exercise following Botulinum toxin injection on the permanence of the recovery among the patients with HFS and BFS: A clinical trial.

BACKGROUND: Botulinum toxin (BoNT) injection serves as the primary modality for addressing hemifacial spasm (HFS) and blepharospasm (BFS), which are prevalent movement disorders affecting the craniofacial region. However, even though the short-term effectiveness of the botulinum injection may reach over 80%, the long-term effectiveness is still a debatable point Herein, we aim to investigate whether facial self-exercise following the BoNT injection can extend the time period of effectiveness. METHODS: In this study, 51 volunteers who received Onabotilinumtoxin A (BoNTA) treatment for the diagnosis of HFS or BFS, were randomized into 2 groups. A detailed instruction about the self-exercise was given by an experienced physician to the subjects in Group 1. Volunteers were asked to repeat the exercise program afterward and continue to each movement for 5 seconds, to repeat each movement 10 times with a 10-second break, every day, 3 times a week for 1 week. hemifacial spasm grating scale (HSGS) and Jankovic scales were used to assess the efficacy of the treatment. RESULTS: Both groups are similar to each other based on demographic features and the severity of the diseases. According to HSGS and Jankovic scales, at the end of the first month, there was no significant difference between the groups. At the end of the third month, the improvement achieved in the first month remained the same in each parameter in Group 1. On the other hand, in Group 2, most of the values returned to the baseline. CONCLUSION: Facial self-exercise following the botulinum toxin application may extend the period of effectiveness of botulinum toxin treatment the subjects with HFS and BFS.

Subclinical effects of botulinum toxin A and microwave thermolysis for axillary hyperhidrosis: A descriptive study with line-field confocal optical coherence tomography and histology.

Botulinum toxin A (BTX) and microwave thermolysis (MWT) are standard axillary hyperhidrosis treatments, but comparison of their subclinical effects is lacking. Line-field confocal optical coherence tomography (LC-OCT) is a promising non-invasive imaging tool for visualizing tissue-interactions. This study aimed to describe subclinical effects of BTX and MWT for axillary hyperhidrosis with LC-OCT-imaging compared to histology. This study derived from an intra-individual, randomized, controlled trial, treating axillary hyperhidrosis with BTX versus MWT. Subclinical effects based on LC-OCT images from baseline and 6-month follow-up (n = 8 patients) were evaluated and compared to corresponding histological samples. At baseline, LC-OCT visualized eccrine pores at the skin surface and ducts in the upper dermis (500 µm), but not deeper-lying sweat glands. Histology identified entire sweat glands. Six months post-treatment, LC-OCT revealed no detectable morphology changes in any BTX-treated axillae (100%), while recognizing obstructed eccrine pores and atrophy of eccrine ducts in most MWT-treated axillae (75%). Histology corroborated LC-OCT findings, while also showing substantial changes to entire sweat glands. LC-OCT enabled visualization of subclinical alterations of superficial eccrine ducts after MWT and unchanged morphology after BTX. LC-OCT is a promising tool for non-invasive assessment of treatment-specific tissue-interactions that can be complementary to histology.

Transition of endochondral bone formation at the normal and botulinum-treated mandibular condyle of growing juvenile rat.

OBJECTIVE: The aim of this study was to understand the temporal and spatial distribution of canonical endochondral ossification (CEO) and non-canonical endochondral ossification (NCEO) of the normal growing rat condyle, and to evaluate their histomorphological changes following the simultaneous hypotrophy of the unilateral masticatory closing muscles with botulinum toxin (BTX). DESIGN: 46 rats at postnatal 4 weeks were used for the experiment and euthanized at postnatal 4, 8, and 16 weeks. The right masticatory muscles of rats in experimental group were injected with BTX, the left being injected with saline as a control. The samples were evaluated using 3D morphometric, histological, and immunohistochemical analysis with three-dimensional regional mapping of endochondral ossifications. RESULTS: The results showed that condylar endochondral ossification changed from CEO to NCEO at the main articulating surface during the experimental period and that the BTX-treated condyle presented a retroclined smaller condyle with an anteriorly-shifted narrower articulating surface. This articulating region showed a thinner layer of the endochondral cells, and a compact distribution of flattened cells. These were related to the load concentration, decreased cellular proliferation with thin cellular layers, reduced extracellular matrix, increased cellular differentiation toward the osteoblastic bone formation, and accelerated transition of the ossification types from CEO to NCEO. CONCLUSION: The results suggest that endochondral ossification under loading tended to show more NCEO, and that masticatory muscular hypofunction by BTX had deleterious effects on endochondral bone formation and changed the condylar growth vector, resulting in a retroclined, smaller, asymmetrical, and deformed condyle with thin cartilage.

Trends in American Plastic Surgery Procedures (2005-2019): Observing the Growth Rate of Maxillofacial Surgery and Cosmetic Minimally Invasive Procedures in ASPS Data.

BACKGROUND: This study explores the dynamic changes in plastic surgery practices, focusing on the growth rates across different subcategories, particularly highlighting the trends in the United States. OBJECTIVES: To analyze evolving trends in plastic surgery using statistical methods, providing insights into the changes and growth patterns in various segments over time. MATERIALS AND METHODS: Utilizing data from 2005 to 2019 from the American Society of Plastic Surgeons, the authors, conducted a comprehensive statistical analysis using the SPSS program to investigate trends in plastic surgery procedures. RESULTS: Our analysis revealed no significant differences in year-on-year growth rates between cosmetic and reconstructive surgery. However, in reconstructive surgery subcategories, maxillofacial surgery showed the highest mean growth rate at 11.61%, though this was not statistically significant. In cosmetic minimally invasive procedures, soft tissue filler injections and botulinum toxin A injections experienced notably higher growth trends compared with microdermabrasion. CONCLUSION: The findings suggest that practitioners considering a career in plastic surgery should not focus solely on choosing between reconstructive and cosmetic surgery. Recognizing substantial growth in cosmetic minimally invasive procedures, especially botulinum toxin A injections and fillers, is vital for making informed career decisions.

Combined rehabilitation therapy with botulinum toxin to the upper limbs for acute spinal cord injury: A case report.

INTRODUCTION: Patients with spinal cord injury (SCI) and incomplete paralysis may experience muscle immobilization due to functional impairment of agonist and antagonist muscles. This can induce spasticity and pain, with the risk of contracture increasing as the period of immobilization increases. This could be aggravated by pain, which interferes with rehabilitation. There is no established treatment for pain and joint contractures caused by acute SCI. Botulinum therapy plays a role in relieving muscle tension. Here, we report a case of acute SCI in which botulinum therapy was administered. CASE PRESENTATION: The patient experienced a C5-cervical SCI due to a road traffic accident, with marked paralysis in the upper and lower limbs below the C5 level. The patient had persistent elbow flexion since the SCI and complained of excruciating pain, making adequate rehabilitation difficult. Botulinum toxin was administered to the biceps and brachialis muscles 15 days after the SCI. After administration, the patient experienced a reduction in pain with relaxation of the elbow flexor muscles, and rehabilitation treatment was resumed. This patient's contracture did not worsen, his pain decreased, and he was able to initiate self-feeding training. DISCUSSION: In this case, occupational and physical therapy was administered with botulinum therapy. Together, they successfully reduced pain. Botulinum therapy provides analgesia and muscle relaxation and may lead to functional recovery, and we believe that treatment can be considered even in the acute phase.

The impact of the migraine treatment onabotulinumtoxinA on inflammatory and pain responses: Insights from an animal model.

OBJECTIVE: Migraine, a prevalent and debilitating disease, involves complex pathophysiology possibly including inflammation and heightened pain sensitivity. The current study utilized the complete Freund's adjuvant (CFA) model of inflammation, with onabotulinumtoxinA (BoNT/A) as a treatment of interest due to its use in clinical migraine management. Using an animal model, the study sought to investigate the role of BoNT/A in modulating CFA-induced inflammation, alterations in pain sensitivity, and the regulation of calcitonin gene-related peptide (CGRP) release. Further, we aimed to assess the changes in SNAP-25 through western blot analysis to gain insights into the mechanistic action of BoNT/A. METHODS: BoNT/A or control was administered subcutaneously at the periorbital region of rats 3 days before the induction of inflammation using CFA. Periorbital mechanical sensitivity was assessed post-inflammation, and alterations in CGRP release were evaluated. Changes in SNAP-25 levels were determined using western blot analysis. RESULTS: Upon CFA-induced inflammation, there was a marked increase in periorbital mechanical sensitivity, with the inflammation side showing increased sensitivity compared to other periorbital areas. BoNT/A did decrease the withdrawal thresholds in the electronic von Frey test. Despite not being able to observe differences in pain thresholds or CGRP release, BoNT/A reduced baseline release under CFA inflamed conditions. Analysis of SNAP-25 levels in the trigeminal ganglion revealed both intact and cleaved forms that were notably elevated in BoNT/A-treated animals. These findings, derived from western blot analysis, suggest an effect on neurotransmitter release. CONCLUSION: Our investigation highlights the role of BoNT/A in reducing baseline CGRP in the context of inflammation and its involvement in SNAP-25 cleavage. In contrast, BoNT/A did not appear to alter facial pain sensitivity induced by inflammation, suggesting that mechanisms other than baseline CGRP could be implicated in the elevated thresholds in the CFA model.

Sequential administration of peripheral nerve blocks and onabotulinumtoxinA for the treatment of chronic migraine and other headache disorders-A retrospective tolerability and safety study.

OBJECTIVE: To determine the tolerability and safety of concurrent peripheral nerve blocks and onabotulinumtoxinA treatment during a single outpatient clinic procedure visit. BACKGROUND: Procedural interventions are available for the treatment of headache disorders. OnabotulinumtoxinA and peripheral nerve blocks are used as alternatives or in addition to oral therapies to reduce the frequency and intensity of migraine attacks. There is currently a lack of safety data focusing on the sequential administration of local anesthetic via peripheral nerve blocks and onabotulinumtoxinA during a single clinical encounter for the treatment of headache. The primary aim of the study was to determine the safety and tolerability of concurrent peripheral nerve blockade and onabotulinumtoxinA injections during a single outpatient clinic procedure visit. We hypothesized that the dual intervention would be safe and well tolerated by patients with chronic migraine and other headache disorders. METHODS: A retrospective chart review was performed using clinical data from patients seen by multiple providers over a 16-month timeframe at one outpatient headache clinic. Patients were identified by procedure codes and those receiving peripheral nerve block(s) and onabotulinumtoxinA injections during a single encounter within the study period were eligible for inclusion. Inclusion criteria were (1) patients 18 years and older who were (2) receiving both peripheral nerve blocks and onabotulinumtoxinA injections for the treatment of chronic migraine. Patients were excluded if they were under age 18, received their procedure outside of the clinic (emergency room, inpatient ward), or were receiving sphenopalatine ganglion blocks. Age- and sex-matched patients who received one procedure, either peripheral nerve blocks or onabotulinumtoxinA, were used for control. The primary outcome of this safety study was the number of adverse events that occurred in the dual intervention group compared to the single intervention control arms. Information regarding adverse events was gathered via retrospective chart review. If an adverse event was recorded, it was then graded by the reviewer utilizing the Common Terminology Criteria for Adverse Events ranging from Grade 1 Mild Event to Grade 5 Death. Additionally, it was noted whether the adverse event led to treatment discontinuation. RESULTS: In total, 375 patients were considered eligible for inclusion in the study. After age and sex matching of controls, 131 patients receiving dual intervention were able to be compared to 131 patients receiving onabotulinumtoxinA alone and 104 patients receiving dual intervention were able to be compared to 104 patients receiving peripheral nerve block(s) alone. The primary endpoint analysis showed no significant difference in total adverse events between dual intervention compared to nerve blocks alone or onabotulinumtoxinA alone. The number of adverse events that led to treatment discontinuation approached but did not reach statistical significance for those receiving dual intervention versus onabotulinumtoxinA alone in the number of adverse events that led to treatment termination (4.6%, 6/131 vs. 0.8%, 1/131, p = 0.065); however, the number of patients who discontinued therapy was not significantly different between those groups (2.3%, 3/131 vs. 0.8%, 1/131; p = 0.314; odds ratio 0.3 [0-3.2]; p = 0.338). CONCLUSIONS: In this retrospective chart review, there was no significant difference in adverse events or therapy discontinuation between patients receiving sequential peripheral nerve block(s) and onabotulinumtoxinA injections versus those receiving either peripheral nerve block(s) or onabotulinumtoxinA injections alone. As a result, we concluded that the combination procedure is likely safe and well tolerated in routine clinical practice.

Botulinum toxin and hyperhidrosis of the amputation stump in war amputees.

INTRODUCTION: Stump hyperhidrosis is a common condition after lower limb amputation. It affects the prosthesis use, and the quality of life of patients. Several case reports tried to prove benefit of using Botulinum toxin in its treatment. AIM: This study was to conduct a larger workforce clinical trial and to demonstrate benefits of botulinum toxin injection in the treatment of stump hyperhidrosis. METHODS: A prospective study was conducted. War amputees who complained of annoying excessive sweating of the stump were included. They received intradermal injection of botulinum toxin A in the residual limb area in contact with prosthetic socket. Abundance of sweating and degree of functional discomfort associated with it were assessed before, after 3 weeks, 6 and 12 months. RESULTS: Seventeen male patients, followed for post-traumatic limb amputation were included in the study. Discomfort and bothersome in relation to Hyperhidrosis did decrease after treatment (p<0,001). Reported satisfaction after 3 weeks was 73,33%. Improvement of prothesis loosening up after 3 weeks was 72,5% [±15,6]. Mean injection-induced pain on the visual analogue scale was 5.17/10 (±1.58). The mean interval after the onset of improvement was 5.13 days [min:3, max:8]. The mean time of improvement was 10.4 months after the injection [min:6, max:12]. No major adverse events were reported following treatment. CONCLUSION: Intradermal injections of botulinum toxin in the symptomatic treatment of stump hyperhidrosis are effective and have few adverse effects. It improves the quality of life of our patients thanks to a better tolerance of the prosthesis.

Paradoxical masseteric bulging after botulinum neurotoxin type A injection.

Paradoxical masseteric bulging refers to an unexpected occurrence of masseter muscle bulging or protrusion following the administration of botulinum toxin injections, contrary to the anticipated muscle weakening effect. It may occur secondary to toxin failing to diffuse through the entire masseter muscle due to the presence of an inferior tendon structure within the superficial masseter that divides it into a superficial and deep belly. We report a clinical case of paradoxical masseteric bulging in a female in her late 40s who developed this adverse effect within a week of her masseter botulinum neurotoxin type A injections. We also describe the masseter two-site injection technique for the management of this complication.

Isolation and characterization of Hc-targeting chimeric heavy chain antibodies neutralizing botulinum neurotoxin type B.

Background: Botulinum neurotoxin (BoNT) produced by Clostridium botulinum is one of the most potent known toxins. Moreover, BoNT is classified as one of the most important biological warfare agents that threatens the biosafety of the world. Currently, the approved treatment for botulism in humans is the use of polyvalent horse serum antitoxins. However, they are greatly limited because of insufficient supply and adverse reactions. Thus, treatment of human botulism requires the development of effective toxin-neutralizing antibodies. Considering their advantages, neutralizing nanobodies will play an increasing role as BoNTs therapeutics. Methods: Herein, neutralizing nanobodies binding to the heavy chain (Hc) domain of BoNT/B (BHc) were screened from a phage display library. Then, BoNT/B-specific clones were identified and fused with the human Fc fragment (hFc) to form chimeric heavy chain antibodies. Finally, the affinity, specificity, and neutralizing activity of antibodies against BoNT/B in vivo were evaluated. Results: The B5-hFc, B9-hFc and B12-hFc antibodies demonstrated high affinity for BHc in the nanomolar range. The three antibodies were proven to have potent neutralizing activity against BoNT/B in vivo. Conclusion: The results demonstrate that inhibiting toxin binding to the host receptor is an efficient strategy and the three antibodies could be used as candidates for the further development of drugs to prevent and treat botulism.

Beyond Pain: The Effects of OnabotulinumtoxinA Therapy on Sensitization and Interictal Symptoms in Chronic Migraine.

Chronic migraine is a disease with a high burden on patients from both a working and quality of life point of view. The pathophysiology of this subtype of migraine is due to several factors, such as medication overuse. Nevertheless, the detrimental recurring of headache attacks with central and peripheral sensitization plays a central role and explains some additional symptoms complained about by these patients even in the interictal phase. OnabotulinumtoxinA is a therapy indicated for chronic migraine since it has proven to reduce peripheral sensitization, showing even efficacy on central symptoms. The aim of this narrative review is to present the current evidence regarding the effect of OnabotulinumtoxinA on sensitization and interictal symptoms.

Intramuscular Botulinum Neurotoxin Serotypes E and A Elicit Distinct Effects on SNAP25 Protein Fragments, Muscular Histology, Spread and Neuronal Transport: An Integrated Histology-Based Study in the Rat.

Botulinum neurotoxins E (BoNT/E) and A (BoNT/A) act by cleaving Synaptosome-Associated Protein 25 (SNAP25) at two different C-terminal sites, but they display very distinct durations of action, BoNT/E being short acting and BoNT/A long acting. We investigated the duration of action, spread and neuronal transport of BoNT/E (6.5 ng/kg) and BoNT/A (125 pg/kg) after single intramuscular administrations of high equivalent efficacious doses, in rats, over a 30- or 75-day periods, respectively. To achieve this, we used (i) digit abduction score assay, (ii) immunohistochemistry for SNAP25 (N-ter part; SNAP25N-ter and C-ter part; SNAP25C-ter) and its cleavage sites (cleaved SNAP25; c-SNAP25E and c-SNAP25A) and (iii) muscular changes in histopathology evaluation. Combined in vivo observation and immunohistochemistry analysis revealed that, compared to BoNT/A, BoNT/E induces minimal muscular changes, possesses a lower duration of action, a reduced ability to spread and a decreased capacity to be transported to the lumbar spinal cord. Interestingly, SNAP25C-ter completely disappeared for both toxins during the peak of efficacy, suggesting that the persistence of toxin effects is driven by the persistence of proteases in tissues. These data unveil some new molecular mechanisms of action of the short-acting BoNT/E and long-acting BoNT/A, and reinforce their overall safety profiles.