Pioneering pain management with botulinum toxin type A: From anti-inflammation to regenerative therapies

Abstract

In the present paper, a comprehensive review was conducted to evaluate the performance of botulinum toxin type A (BTX-A) in managing various types of pain, including myofascial, muscular temporomandibular joint pain, orofacial pain, chronic migraines, and more. Firstly, the mechanism of action and anti-inflammatory effects of BTX-A was introduced. Following this, recent advancements in BTX-A applications were discussed, with an emphasis on emerging combination therapies, regenerative medicine, and personalized treatment strategies. Unlike previous reviews, this study explored a broader spectrum of pain conditions and highlighted BTX-A's versatility and potential as a long-term, minimally invasive pain management option. Additionally, the importance of tailoring BTX-A treatment was emphasized through the integration of biomarkers, genetic factors, and optimized dosing regimens to enhance efficacy and minimize side effects. Novel combinations with regenerative therapies, such as stem cells and tissue engineering, were identified as promising avenues for joint and nerve repair, providing both symptomatic relief and tissue regeneration. Furthermore, digital health tools and artificial intelligence were suggested as innovative approaches to monitor treatment responses and optimize dosing protocols in real-time, advancing personalized pain management. Overall, this review underscores BTX-A's potential in comprehensive and patient-centered pain management and offers recommendations to guide future studies in optimizing BTX-A therapy.

1. Introduction

Botulinum toxin type A (BTX-A), a natural toxin responsible for botulism in humans, was first identified in the 19th century as a foodborne toxin. Initially studied for treating overactive nerve disorders at low doses, BTX-A was found to inhibit acetylcholine release at neuromuscular junctions. In 1986, it was recognized as a pain relief treatment for cervical dystonia [1,2]. Since then, extensive research has explored its applications in pain management and relief, with numerous studies confirming its effectiveness in various pain conditions.

BTX-A has been effectively used in various medical conditions for pain and symptom relief. It reduces migraine frequency with 25 U injections into pericranial muscles [[3], [4], [5]], and is proven effective in treating cervical dystonia [6,7]. BTX-A also manages spasticity in conditions like cerebral palsy, stroke, and multiple sclerosis, with higher doses improving post-stroke lower limb spasticity [8,9], and showing positive outcomes in upper extremity and pediatric spasticity without significant adverse effects [10,11]. Additionally, BTX-A treats hyperhidrosis (excessive sweating) by blocking sweat gland signals, providing relief for both focal and palmar hyperhidrosis safely and effectively [[12], [13], [14], [15]]. Pena et al. [16] found that the temporary side effects of BTX-A treatments are generally mild and self-resolving, including minor injection discomfort, occasional headache, nausea, flu-like symptoms, drooping of eyelids or eyebrows, uneven lower face appearance, and slight fine motor skill impairment after palm injections. Vadoud-Seyedi [17] highlighted the injection procedure as a key factor in BTX-A effectiveness, particularly using the Dermojet technique for treating plantar hyperhidrosis. In patient self-assessments, 7 out of 10 reported satisfactions, and the method proved simple, safe, and effective, with only one minor adverse event, making it a reliable alternative for managing plantar hyperhidrosis. In the case of forehead hyperhidrosis treatment, Trindade de almedia et al. [18] found that BoNTA2 (BOTOX) had greater diffusion in the forehead for hyperhidrosis treatment compared to BoNTA1 (Dysport), yielding better results but with higher risk of side effects, with dose range also being a key factor. Additionally, BTX-A injections have demonstrated potential in alleviating pain associated with temporomandibular joint (TMJ) disorders, collectively referred to as temporomandibular disorders (TMD), which can cause significant jaw, head, and facial pain [19]. While BTX-A has shown promise in managing muscle-related TMD symptoms, earlier studies up to 2015 reported inconsistent findings regarding its therapeutic benefits for intra-articular TMJ pathology [[20], [21], [22]]. Recent evidence suggests that BTX-A may effectively reduce pain linked to muscular hyperactivity around the TMJ, but further research is necessary to confirm its efficacy in addressing joint-specific conditions [23,24]. Myofascial Pain Syndrome is a condition marked by the emergence of trigger points within muscles, causing localized pain and discomfort [25]. BTX-A injections can be targeted at muscle trigger points to release tension, reducing pain, stiffness, and restricted movement [26]. Thambar et al. proved that about TMJ the pain was reduced significantly when utilized BTX-A compared with the placebo group [27]. Another study with more participants confirmed the ability of BTX-A in the pain relief [28]. Kamanli et al. study [29] found BTX-A injection to be a more practical and faster alternative to dry needling with fewer disruptions. Fig. 1 illustrates the diverse applications of BTX-A in managing pain conditions, including chronic migraine (recurring severe headaches), muscle spasms (involuntary muscle contractions), spasticity (increased muscle stiffness and tone), and hyperhidrosis (excessive sweating). It also highlights emerging uses for fibromyalgia (widespread musculoskeletal pain), chronic pelvic pain (persistent pelvic discomfort), and chronic lower back pain. Recent advances have expanded BTX-A's use in personalized therapies based on patient biomarkers, and in combination with regenerative treatments like stem cells. Its integration with digital health tools and AI for real-time monitoring highlights BTX-A's evolving role in comprehensive pain management.

Fig. 1.

The pain relife applications of BTX-A.

This section reviews recent studies up to 2024 on BTX-A's effectiveness in treating pain conditions like myofascial pain, TMJ disorders, and chronic pain. It covers BTX-A's mechanisms, interactions with ion channels, effects on CNS microglial cells, and advancements in neuromodulation and regenerative medicine applications. Future research directions, such as biomarker identification and dosage optimization, are also highlighted.

2. A brief of BTX-A action mechanism

The prolonged efficacy of BTX-A following a single injection is largely attributed to its unique structural and functional properties, which allow it to bind selectively and potently to neuronal targets. This affinity is governed by its protein subunits, each playing a critical role in recognizing specific receptors on neuronal membranes and enabling BTX-A's entry into the cell. The two distinct components, the heavy and light chains, collaborate in this process: the heavy chain is responsible for binding and internalization, while the light chain, once inside the cytoplasm, acts as a protease, cleaving key proteins essential for neurotransmitter release (Fig. 2) [30,31].

Fig. 2.

Mechanisms of BoNT/A(Throughout the manuscript, the abbreviation BTX-A is used to refer to the therapeutic application of botulinum toxin type A) in Modulating Pain Pathways, Derived from Ref. [30] under open access license (CC BY 4.0 Attribution 4.0 International Deed).

One of the primary mechanisms through which BTX-A exerts its therapeutic effects is by inhibiting the SNARE protein complex, particularly through the cleavage of SNAP-25. SNAP-25 is integral to the fusion of synaptic vesicles with the neuronal membrane, a process necessary for the release of neurotransmitters like acetylcholine. By disrupting this mechanism, BTX-A effectively blocks the release of acetylcholine at neuromuscular junctions, leading to temporary muscle paralysis and alleviation of spasticity and associated pain in various clinical applications, such as in cervical dystonia and post-stroke spasticity (1,2) [32]. Additionally, BTX-A modulates the release of various neurotransmitters and neuropeptides involved in pain signaling, such as substance P, calcitonin gene-related peptide (CGRP), and glutamate. These molecules are central to the process of neurogenic inflammation, often linked to chronic pain. By preventing their release, BTX-A helps mitigate inflammatory pathways, thus offering pain relief in cases resistant to conventional therapies [33].

The structural resilience of BTX-A, particularly in evading cellular degradation pathways, allows it to remain active within the cytoplasm for extended periods, thereby sustaining its therapeutic effects over several months. Upon injection, in neutral pH conditions, the non-toxic components of BTX-A rapidly dissociate from the neurotoxin portion, enhancing its efficacy and stability within the cellular environment [34]. This longevity is due to the light chain's capacity to avoid lysosomal degradation, a feature that distinguishes BTX-A from many other neurotoxins. Consequently, the proteolytic activity of the light chain persists within the neuron, continuing to inhibit SNARE complex assembly and neurotransmitter release without necessitating frequent re-administration.

Emerging research has also highlighted BTX-A's influence on ion channels, specifically sodium channels, which play a role in pain perception. By decreasing sodium channel activity in peripheral and central neurons, BTX-A may further reduce pain sensitivity, adding another layer of efficacy in pain management.

2.1. Anti-inflammatory effects

Various mechanisms by which BTX-A achieves pain relief have been investigated in the literature, with a strong focus on its inhibitory effect on the release of inflammation-associated mediators, such as substance P, CGRP, and glutamate [35]. In terms of anti-inflammatory effects, BTX-A or BoNT in general typically blocks neurotransmitter release by cleaving the SNARE protein SNAP-25, which is composed of 206 amino acids. For each BoNT serotype, this cleavage results in the loss of the intact protein, production of an N-terminal truncated protein, and generation of a small C-terminal peptide. These peptides, by mimicking the C-terminal fragments of SNAP-25, subsequently reduce transmitter release in bovine chromaffin cells and Aplysia buccal ganglion cells. In various systems, this cleavage inhibits the exocytosis of transmitters; however, at specific cholinergic synapses, the inhibitory effect of truncated SNAP-25 is not detectable [36]. Another mechanism by which BTX-A exerts its therapeutic effects is through temporary muscle paralysis, achieved by inhibiting acetylcholine release at neuromuscular junctions. Muscle spasms and tension, which can contribute to localized inflammation and pain, are alleviated by this muscle relaxation, reducing mechanical stress on tissues and thus relieving associated inflammation and discomfort [37]. BTX-A's role in modulating neurogenic inflammation is also significant. This process, a well-documented pathological pathway, involves the release of potent vasoactive neuropeptides mainly CGRP, substance P (SP), and neurokinin A from activated peripheral nociceptive sensory nerve endings, specifically C and A-delta fibers [38]. Moreover, BTX-A can deactivate sodium channel conductance in cell cultures of both central and peripheral neurons. Antonucci et al. [39] suggested that BTX-A's main pain-relieving effects arise from retrograde toxin transport or transcytosis, which inhibits neurotransmitter release onto dorsal horn neurons. Additionally, Ri et al. [35] proposed that beyond altering neuronal function, BTX-A could affect spinal microglial cells.

2.1.1. Structural changes in muscle fibers post BTX-A injection

In addition to its well-documented anti-inflammatory effects, BTX-A has demonstrated notable structural impacts on muscle tissue. Deschrevel et al. [40] conducted a comprehensive study on the histological changes in muscle fibers following BTX-A injections, observing significant reductions in the cross-sectional area (CSA) of muscle fibers. Their findings highlight changes in fiber morphology that appear over several months post-injection, which are essential for understanding how BTX-A alleviates chronic muscle pain and reduces localized inflammation through mechanical and structural adaptation.

According to Deschrevel et al.'s study, BTX-A-induced muscle relaxation is not merely a result of neurotransmitter inhibition at the neuromuscular junction; it also involves physical alterations within the muscle tissue itself. Histological analysis post-BTX-A administration shows increased heterogeneity in muscle fiber size, indicating a remodeling effect. This remodeling includes a reduction in Type II muscle fibers, which are typically associated with fast-twitch, high-force activities. Such changes may contribute to a decrease in muscle spasms and reduce mechanical load on surrounding tissues [41]. This effect supports BTX-A's capacity to alleviate pain in conditions characterized by muscle hyperactivity and spasms, such as dystonia and spasticity.

BTX-A induces structural changes, as illustrated in Fig. 3, including a reduction in cross-sectional area and increased connective tissue between fibers over time. These changes likely enhance BTX-A's ability to reduce mechanical stress, contributing to decreased inflammation-related pain. The observed remodeling effect highlights BTX-A's multifaceted role in addressing both the biochemical and physical aspects of pain relief, preventing the high-force contractions that exacerbate pain in chronic conditions.

Fig. 3.

Representative examples of myosin heavy chain staining of the medial gastrocnemius in a child with CP at baseline (left panel), 3 months (middle panel), and 6 months (right panel) after BoNT-A (Throughout the manuscript, the abbreviation BTX-A is used to refer to the therapeutic application of botulinum toxin type A) injection. Type I fibers are stained in blue; Type IIa in green; and Type IIx and laminin in red. There is considerable variability in fiber size at 3 months post-BoNT-A, which persists to a lesser extent at 6 months, Derived from Ref. [40]under open access license (CC BY 4.0 Attribution 4.0 International Deed).

Quantitative assessment of muscle fiber CSA further illustrates the long-term structural effects of BTX-A treatment. Fig. 4 provides a detailed analysis of these CSA changes, demonstrating a significant reduction in fiber area three months post-BTX-A injection, particularly in Type II fibers. Interestingly, partial recovery in CSA is observed at the six-month mark, suggesting a dynamic remodeling process that balances initial muscle fiber reduction with subsequent adaptive responses over time [42]. This fluctuation in CSA indicates that BTX-A's impact is both immediate and sustained, affecting muscle morphology in a way that may contribute to prolonged symptom relief for patients with chronic muscle-related pain.

Fig. 4.

illustrates the changes in fiber cross-sectional area (ΔfCSAs) across different time intervals following BoNT-A(Throughout the manuscript, the abbreviation BTX-A is used to refer to the therapeutic application of botulinum toxin type A) injection, specifically comparing baseline to 3 months post-treatment (B-3M) and baseline to 6 months post-treatment (B-6M). This figure provides a comparison for all fiber types as well as distinct analyses for Type I, Type IIa, and Type IIx fibers. Each data point represents an individual child, with variations in symbol filling to indicate different dosage levels: open circles for 1U BoNT-A, half-open circles for 2U BoNT-A, and filled circles for 3U BoNT-A, Derived from Ref. [40]under open access license (CC BY 4.0 Attribution 4.0 International Deed).

The results in Fig. 4 indicate that the reduction in CSA, particularly in high-force Type II fibers, reduces the muscle's contraction strength, thereby lowering pain-inducing mechanical stresses. This reduction and partial recovery pattern reflect a remodeling process, where initial muscle fiber shrinkage due to BTX-A treatment is followed by adaptive changes that sustain symptom relief for patients with chronic muscle-related pain. The structural adaptation seen in these figures underscores the dual mechanism of BTX-A in treating chronic pain, where both biochemical and physical changes contribute to long-term therapeutic outcomes.

2.2. Synaptic plasticity modulation by BTX-A

BTX-A has been found to influence synaptic plasticity, which refers to the ability of synapses to strengthen or weaken over time in response to activity levels. Synaptic plasticity plays a critical role in the development and maintenance of chronic pain, where enhanced sensitivity in neural pathways often leads to persistent pain states. BTX-A, through its action on neurotransmitter release and receptor expression, can modulate this plasticity, potentially reducing pain hypersensitivity [43].

Studies suggest that BTX-A may downregulate the expression of genes associated with excitatory neurotransmitters and upregulate inhibitory neurotransmitter pathways [44]. This modulation affects both pre- and post-synaptic structures, reducing excitatory signaling and enhancing inhibitory signals in pain-related pathways. Such changes can lead to long-term decreases in neuronal excitability and may be particularly beneficial in managing chronic pain conditions that involve central sensitization [45].

The capacity of BTX-A to alter synaptic plasticity highlights its potential as a modulator of long-term pain pathways, beyond its immediate effects on neurotransmitter release [46]. Further research is warranted to fully understand the molecular mechanisms underlying these changes and to explore the potential of BTX-A as a therapeutic option in chronic pain syndromes associated with altered synaptic plasticity.

2.3. Potential of BTX-A in combination therapies

The integration of BTX-A with complementary therapeutic methods has demonstrated notable potential to enhance its analgesic effects while potentially lowering the required dose and minimizing adverse effects. Combining BTX-A with specific interventions, such as neuromodulation techniques, anti-inflammatory medications, and physical rehabilitation, can yield synergistic effects that substantially improve patient outcomes, particularly in cases of chronic and refractory pain.

Among the most extensively researched combination approaches is the use of BTX-A alongside neuromodulation techniques, including transcutaneous electrical nerve stimulation (TENS) and spinal cord stimulation (SCS). Neuromodulation techniques modify pain transmission within the nervous system, while BTX-A directly targets neurotransmitter release at the neuromuscular junction. This dual approach addresses both peripheral and central pain pathways, providing a multi-dimensional method for pain management. Recent studies have shown that BTX-A combined with TENS therapy results in significantly greater pain reduction in patients with chronic musculoskeletal pain compared to the effects of either treatment alone [47]. Additionally, BTX-A's combination with pharmacologic agents, particularly anti-inflammatory drugs, has garnered attention for its potential to enhance anti-inflammatory outcomes. While BTX-A inhibits the release of pro-inflammatory neurotransmitters, adjunctive use of anti-inflammatory drugs can further suppress systemic inflammation. For instance, co-administration of corticosteroids or non-steroidal anti-inflammatory drugs (NSAIDs) with BTX-A has been shown to improve outcomes by reducing localized inflammation and alleviating pain in conditions such as rheumatoid arthritis and osteoarthritis [48].

Physical therapy and rehabilitation exercises complement BTX-A by maximizing its muscle relaxation effects, which in turn facilitates physical therapy by reducing muscle spasms and stiffness, enhancing mobility and function. This combination is particularly beneficial for individuals with spasticity or musculoskeletal disorders, as BTX-A-induced muscle relaxation amplifies the therapeutic effects of physical rehabilitation and accelerates recovery [49]. Furthermore, combining BTX-A with analgesic agents, including both opioids and non-opioid analgesics, has demonstrated promising results in enhancing pain relief. The co-administration of BTX-A with low-dose opioids, for instance, may achieve effective pain control while reducing the risks associated with opioid dependency and tolerance. This opioid-sparing effect is especially valuable in managing chronic and severe pain where standard treatments may be insufficient [50]. Additionally, recent research suggests that BTX-A combined with NSAIDs yields synergistic benefits, as BTX-A's modulatory action on neurotransmitter release is supported by the systemic anti-inflammatory effects of NSAIDs. Such combination therapies are particularly advantageous in managing complex pain conditions characterized by inflammation and heightened pain sensitivity, potentially reducing the necessity for higher BTX-A dosages [51,52].

The exploration of BTX-A in combination therapies offers an innovative trajectory for both research and clinical practice. Future studies are essential to determine the optimal combination, dosage, and timing parameters for co-administering BTX-A with other therapies. The ultimate objective is to maximize therapeutic efficacy while minimizing adverse effects, thereby establishing a comprehensive and patient-centered approach to pain management.

2.4. Effects of BTX-A on immune cells and microglia

Beyond its established effects on neuronal transmission, BTX-A has demonstrated significant immunomodulatory properties, particularly through its impact on microglial cells within the central nervous system (CNS). Microglia, as shown in Fig. 5, are the CNS's resident immune cells and act as principal mediators of neuroinflammation. Upon activation, these cells release pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) which contribute to increased pain sensitivity via mechanisms of central sensitization. Modulating microglial activity through BTX-A offers an alternative avenue for pain relief, particularly in chronic and refractory pain conditions where CNS inflammation is a driving factor [53].

Fig. 5.

This illustration, informed by previous research [54], suggests that inflammation triggers the release of interleukin-6 (IL-6) and the shedding of soluble IL-6 receptors (sIL6R) from microglia. This process may then stimulate astrocytes, leading them to adopt a neurotoxic-reactive phenotype, potentially damaging neurons. The activation of glucocorticoid receptors is proposed to curb this inflammation-driven cascade of events, Derived from Ref. [55]under open access license (CC BY 4.0 Attribution 4.0 International Deed).

BTX-A's potential to inhibit microglial activation has been shown to reduce levels of inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6), as depicted in Fig. 6. These cytokines are closely linked to pain facilitation through neuronal sensitization and sustained inflammatory signaling. Moreover, BTX-A may upregulate anti-inflammatory markers in microglia, such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β), encouraging a shift from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, which is critical for reducing central sensitization. The M2 microglial phenotype plays a role in neuroprotection and repair within the CNS, offering a promising therapeutic approach for managing chronic pain. By promoting a shift from the pro-inflammatory M1 state to the anti-inflammatory M2 state, BTX-A aids in reducing central sensitization, which is crucial for alleviating chronic pain [56].

Fig. 6.

This graphical overview depicts the biological effects triggered by stress and inflammation. As outlined in Sections 2, 3, both inflammation and psychological stress activate the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system, resulting in increased glutamate levels and reduced inhibition by gamma-aminobutyric acid (GABA). These changes contribute to elevated levels of circulating corticosteroids, catecholamines, increased anxiety, physical and psychological symptoms, and reduced cognitive function. Additionally, both stress and inflammation impact the function of chloride transporters, specifically Na-K-Cl-cotransporter-1 (NKCC1) and K-Cl-cotransporter-2 (KCC2), and initiate IL-6 trans-signaling. Although stress and inflammation may cause similar effects, they could vary in the intensity of individual responses.

Legend: BP = blood pressure, CS = corticosterone, HR = heart rate, NA = noradrenaline, ns = nervous system, PKC = protein kinase C, sIL6R = soluble IL-6 receptor, Derived from Ref. [55] under open access license (CC BY 4.0 Attribution 4.0 International Deed).

Further extending BTX-A's immunomodulatory role, recent research highlights its interaction with Toll-like receptors (TLRs), specifically TLR4, which are central to microglial activation [57]. By inhibiting TLR4 signaling, BTX-A may attenuate neuroinflammatory responses, thereby limiting the release of inflammatory cytokines that contribute to the amplification of pain in chronic conditions such as neuropathic pain and fibromyalgia. This modulation of TLR signaling represents a novel pathway by which BTX-A can influence pain at the cellular level [58].

Emerging evidence also suggests that BTX-A affects neuron-microglia interactions by reducing excitatory signaling from neurons that would otherwise activate microglia. Through this mechanism, BTX-A can potentially interrupt the cycle of neuroinflammation and neuronal sensitization, offering longer-lasting relief for conditions involving central sensitization [59].

BTX-A's capacity to modulate immune activity within the CNS introduces new possibilities for therapeutic applications beyond its established role in neurotransmitter inhibition. Continued research into the specific molecular mechanisms through which BTX-A regulates microglial behavior could deepen our understanding of its potential in managing neuroinflammatory-related chronic pain [60]. These findings may ultimately guide the development of optimized treatment protocols, potentially combining BTX-A with other anti-inflammatory therapies to enhance efficacy and broaden its applicability in chronic pain management.

2.5. Interaction of BTX-A with ion channels, including T-type calcium channels, in sensory pain management

BTX-A's interaction with various ion channels, including sodium, calcium, and potassium channels, represents a crucial aspect of its mechanism for modulating pain sensitivity and prolonging therapeutic effects. Recent studies indicate that BTX-A can inhibit sodium channels on peripheral neurons, which reduces excitability and dampens pain signals transmitted to the central nervous system. This interaction with sodium channels is particularly beneficial in decreasing hyperexcitability associated with chronic pain [61]. Furthermore, BTX-A has been shown to affect calcium channels, particularly voltage-gated calcium channels (VGCCs), which play a critical role in neurotransmitter release. By modulating VGCCs, BTX-A may reduce the influx of calcium into the neuron, thereby lowering the release of pain-associated neurotransmitters such as substance P and glutamate [62]. This effect not only aids in immediate pain relief but also contributes to a decrease in central sensitization over time [63].

BTX-A also exerts significant effects on potassium channels, which are vital for maintaining the resting membrane potential and regulating neuronal excitability. Research shows that modulation of these channels by BTX-A promotes membrane hyperpolarization, which suppresses neuronal firing and enhances the duration of its analgesic effects. Potassium channels, especially voltage-gated channels like Kv7 (M channels), play a crucial role in stabilizing the resting potential of nociceptive neurons, effectively reducing overexcitability that characterizes many chronic pain states [64]. By enhancing the activity of these channels, BTX-A can help maintain neurons in a hyperpolarized state, which lowers their responsiveness to pain-inducing stimuli. This hyperpolarization effect interrupts the nociceptive signaling pathway and decreases pain transmission, potentially offering extended relief in conditions involving hyperexcitability and inflammatory pain. Additionally, targeting potassium channels has emerged as a promising approach in analgesic drug design, as it addresses peripheral overexcitability without entirely blocking sensory signal transmission, thus minimizing side effects commonly associated with broader nervous system suppression [65].

These interactions with ion channels underscore BTX-A's multifaceted approach to pain management, making it a potent option for treating chronic and resistant pain conditions. Further research on the precise molecular mechanisms by which BTX-A modulates these channels could lead to more targeted and effective applications in pain therapy.

BTX-A has traditionally been known for its impact on SNARE protein complex and neurotransmitter release inhibition, yet recent research points to a promising role for BTX-A in modulating ion channels, particularly T-type calcium channels, as a mechanism for sensory pain management. T-type calcium channels, particularly the Cav3.2 subtype, are predominantly expressed in peripheral sensory neurons, where they contribute to the depolarization and excitability of nociceptive neurons [66]. This subtype is critically involved in the initiation and maintenance of pain signals, particularly in cases of inflammatory and neuropathic pain, where increased expression of Cav3.2 has been observed [67].

BTX-A may inhibit the activity of T-type calcium channels through indirect mechanisms that reduce channel phosphorylation and suppress calcium influx in sensory neurons. By decreasing calcium entry via T-type channels, BTX-A can attenuate neuronal excitability, which reduces spontaneous nociceptor firing and prevents the potentiation of sensory transmission, especially under inflammatory conditions where calcium channel upregulation exacerbates pain hypersensitivity [68]. Additionally, T-type calcium channels are involved in the development of central sensitization by facilitating calcium-dependent signaling cascades that enhance synaptic transmission in the spinal dorsal horn. BTX-A's modulation of these channels may inhibit such processes, providing relief from hyperalgesia and allodynia observed in chronic pain conditions. This effect on T-type calcium channels aligns BTX-A with emerging therapeutic approaches that target peripheral and central mechanisms of sensory and inflammatory pain [69].

The potential of BTX-A to influence T-type calcium channel activity introduces an innovative approach to pain management, particularly for patients with heightened nociceptor sensitivity or inflammatory pain. Future studies focusing on the specific molecular pathways through which BTX-A interacts with T-type calcium channels, including potential effects on Cav3.2 phosphorylation and downstream calcium-dependent signaling, could unlock further therapeutic applications, advancing BTX-A's role in precision pain management strategies [70].

3. Recent advances in the field of BTX-A and pain relief

Recent advancements in the application of BTX-A for pain relief are presented in Table 1. Using the keywords "BTX-A″ and "pain relief," numerous studies published after 2019 were identified; however, 19 key papers have been summarized in Table 1 for focused analysis. Additionally, certain highly debated studies have been addressed separately due to their unique findings and relevance to ongoing discussions in the field.

Table 1.

The recent advances in the field of BTX-A and its efficiency in pain relief.

Objective	Study Population	Treatment Duration	Pain Type	Main Findings	Side effects	Ref.
Investigating the efficacy of BTX-A dose in the case of persistent myofascial pain (MFP)	100 female subjects with persistent MFP	Up to 24 weeks after treatment	Myofascial Pain	-Reducing pain intensity as well as increased pressure pain threshold	-Some doses have adverse impacts	[72]
				-There were no considerable differences among BTX-A and OA (similar results in Ref. [71])	-Needing to use of low BTX-A doses
Evaluate the BTX-A effectiveness in the case of painful TMJ	-Randomized controlled trials (RCTs)	1, 6, and 12 months	Painful TMJ	BTX-A has been determined to be more effective than a placebo, providing at least one month of significant pain reduction	The risk of adverse events was not increased significantly	[73]
	−10 databases were used
-Concerning the refractory hemiplegic shoulder pain	38 patients with refractory HSP	12 weeks treatment after BTX-A	Hemiplegic Shoulder Pain (HSP)	A considerable improvement in pain scores at rest and in the process of arm passive abduction	-Without any collateral impacts reported -Hyperglycemia in 5 patients after corticosteroid injection, lasting for 3 days	[74]
-Evaluating the effectiveness of ultrasound-guided subacromial-subdeltoid (SASD) bursa injections with BTX-A compared to steroids
-Determining the topographical site of the auriculotemporal nerve (ATN)	36 hemifaces along with 25 Korean cadavers	–	Chronic Migraine (CM)	Optimal injection points identified in the temporal region, 2 cm anterior and 3 cm superior to the tragus	–	[75]
- Evaluating the injection points for treating chronic migraine (CM) with BTX-A
To investigate the mechanisms of BTX-A on neuropathic pain and its effect on glycine transporter 2 (GlyT2) expression in the spinal cord of rats with chronic constriction injury (CCI)	Male Sprague-Dawley rats Weighing 220–250 g	–	Neuropathic Pain	The use of BTX-A led to reducing CCI-induced mechanical allodynia and downregulated the expression of GlyT2 in the spinal cord	–	[76]
To compare the effectiveness of extranormal and intraoral approaches on the injection of BTX-A in the case of the lateral pterygoid muscle (LPM) in patients with anterior disc displacement with reduction (ADDWR)	14 patients	–	ADDWR	A good improvement in TMJ clicking from after 1 weak from injection with no group difference	–	[77]
Compare the effectiveness of BTX-A and corticosteroid in hypertrophic scar and keloid	Patients with hypertrophic scar and keloid	The post-injection of 8 and 12 weeks without statistical difference	Hypertrophic scar and keloid pains	Intralesional BTX-A was determined to be more effective compared to corticosteroid or placebo in treating hypertrophic scar and keloid	Not highlighted	[78]
Impact of BTX-A on pain relief and opioid use in vasospasm, Role of	20 patients with ischemic pain	-Administration resulted in rapid pain relief (within 20 min)	Ischemic pain from vasospasm	-Rapid and sustained pain relief for months -Opioid use was reduced -Disability was improved for 6 months -PPG testing helped BTX-A use	Not highlighted	[79]
(photoplethysmograph) testing in treatment decisions		- Several months for full results
-The calcitonin gene-related peptide (CGRP) plasma levels were investigated in classical trigeminal neuralgia (TN) patients	45 patients with classical TN, 30 healthy controls	Not specified	Classical trigeminal neuralgia	Plasma CGRP concentrations significantly decreased after BTX-A treatment. In responders, CGRP levels were significantly lower after treatment, while non-responders showed no significant change	Nonresponders. with no significant differences	[80]
- BTX-A effects were also evaluated
The efficacy of botulinum toxin injection was concerned with muscular TMD treatment	25 patients with muscular dysfunction of the origin	6 months for 100 % therapy	Muscular TMD	Successful step-by-step BTX-A injection in 9 out of 25 patients who received the drug, drug-physical therapy, and occlusal splint therapy	No side effects were observed during a 6-month follow-up	[81]
Analyze the clinical outcome of intramuscular BTX injections in TMD patients	68 patients with TMD symptoms who received intramuscular BTX injections	63 patients	TMD	87 % of patients reported favorable outcomes: 13 % not beneficial, 24 % beneficial, 63 % highly beneficial	Females responded better to BTX therapy	[82]
To assay cortical excitability and plasticity in patients with CM treated by BTX-A utilizing transcranial magnetic stimulation (TMS)	11 episodic migraine patients	Four cycles of BTX-A treatment over one year	CM	-BTX-A treatment partially reduced cortical excitability in patients with CM	–	[83]
	and 11 chronic migraine patients with chronic migraine			-BTX-A treatment led to a significant decrease in the Migraine Disability Assessment Score (MIDAS) in patients with CM
The effects of BTX-A injection in trigeminal neuralgia, myofascial temporomandibular disorders, and oromandibular dystonia patients	28 patients with trigeminal neuralgia	12 weeks for 100 % recovery	Trigeminal neuralgia, myofascial temporomandibular disorders, Oromandibular dystonia	Visual analog scale, pain frequency, and pain scales significantly	No significant adverse effects were reported during injections in all patients	[84]
Compare effects of BTX-A and Acupuncture on TMD	54 women with TMD	Effective after 1 month	Myofascial TMD	Both therapies reduced pain, and BTX-A improved PPT values	-Temporary regional weakness	[85]
					-Mild tenderness at injection sites -minor discomfort during chewing
					-The acupuncture group proved any dramatic PPT improvements
Assess BTX-A effect on diabetic neuropathy	32 patients with type 2 DM	Effective after 3 months	Diabetic neuropathy	-Reducing BTX-A neuropathic pain	–	[86]
				-Enhancing the quality of life as well as sleep in diabetic patients
Manage masticatory myofascial pain	50 patients	–	Masticatory myofascial pain	Successful in the treatment of masticatory myofascial pain	–	[87]
The efficacy of a BTX-A single injection in the knee joint cavity to relieve pain	30 participants, elderly people	4 weeks	Knee osteoarthritis	- BTX-A was found to be an effective initial treatment	Patients reported to have minimum adverse effects and increased satisfaction	[88]
				- Clear clinical benefits were demonstrated, and minimal adverse effects were observed
Investigate BTX-A efficacy and safety in TN patients	104 patients with classical TN	Effective results 12 months	Medically refractory	BTX-A local injection showed 83.7 % success	16.3 % reported mild side effects; mild and transient side effects observed	[89]
Investigating the BTX-A efficacy in intractable chronic migraine patients non-responsive to previous pharmacological management and with largely no pain-free time, including those with new-onset daily persistent headache	33 patients with severe HIT-6 scores	BTX-A injection for each 3-month (maximum 33-month period) as per Phase III PRE-EMPT protocol over a specified duration	Chronic migraine without pain-free time, including new onset daily persistent headache	A considerable decrease in HIT-6 scores	–	[90]

Based on Table 1, comprehensive information has been provided regarding the use of BTX-A for pain relief. Notably, many different pains including orofacial joint, muscular, chronic migraine, diabetic neuropathy, etc., and the effect of BTX-A on pain treatment and relief were concerns based on the studies published after 2019. In the broader context, these findings collectively underscore the potential of BTX-A as a versatile and effective pain relief option. The prolonged duration of pain relief, coupled with the minimal side effects observed, bolsters its standing as a viable therapeutic avenue across various pain-related conditions. By amassing such comprehensive evidence, these studies lay the groundwork for future research and open the door to exploring BTX-A's potential in broader pain management strategies. Machado et al. [73]demonstrated effective pain reduction in temporomandibular joint dysfunction, with no notable reported side effects during the treatment period. Furthermore, Dhaliwal et al. [78] highlighted the superior efficacy of BTX-A compared to steroids in treating keloid and hypertrophic pains, and no significant side effects were reported. Treatment with BTX-A also exhibited substantial improvement in conditions such as masticatory myofascial pain [87] and diabetic neuropathy [86] with minimal or rare adverse effects observed. Torre Canales et al. [72]have utilized the BTX-A to alleviate persistent myofascial pain (MFP) considering 24 weeks' post-injection time for 100 female subjects with persistent MFP and highlighted BTX-A's ability to effectively reduce pain intensity. However, they found that some patients are sensitive to high and low doses. BTX-A is a new approach but different traditional methods of pain relief and treatment (e.g., Physical rehabilitation therapy including Extracorporeal shock wave therapy, Hyperthermia, phototherapy and magnetic therapy, Manipulation, stretching, and kinesiology tape, etc.). Therefore, the use of BTX-A injection along with other methods is very important with the classification of patients and can compensate them in cases of low doses of BTX-A. Moreover, patients should eliminate the fear of the disease, and appropriately cooperate with medical care and the trigger points of pain through the above comprehensive treatment. Good living habits and exercise have been introduced as key elements in the early recovery of MPS [90]. Ramos-Herrada et al. [91]stated that low doses of BTX-A can be effective in the treatment of MPS and TMD disorders, simultaneously. Hence, the dosage can be an important parameter. More important than the dose, researchers raise the fact that it is not always possible to have a specific pain, and in some cases, samples with different pains should be concerned (e.g., myofascial pain associated with temporomandibular disorders). Therefore, the lack of selectivity and reaching a general condition can be very important. Wu et al [74].have focused on refractory hemiplegic shoulder pain. They utilized both BTX-A and corticosteroid groups for 12 weeks and demonstrated significant pain relief at rest and during arm passive abduction. Furthermore, Atraszkiewicz et al. [90] also proved the great efficiency of BTX-A in addressing chronic migraine. In Sipahi Calis's study [81], sixteen patients were treated with drug-physical therapy-occlusal splint therapy and BTX-A treatment, and in the case of nine patients appropriate results were provided. No side effects were observed at 6 months follow-up. They stated that 100 % therapy after 6 months was demonstrated and determined as an outstanding method. Shebl [92]focused on investigating the potential anti-cancer effects of BTX-A and focused on its role as a therapeutic agent for cancer treatment, particularly in comparison to captopril. They evaluated its cytotoxicity against colon (HCT116) and prostate cancer (DU145) cells while considering its impact on normal Vero cells. The study also assessed BTX-A's influence on cancer cell proliferation, migration, and apoptosis induction. They have demonstrated that BTX-A displayed cytotoxic effects on cancer cells without causing harm to normal cells. Briefly, they have proved the anti-cancer potential of BTX-A.

3.1. The current state of BTX-A in pain management

In exploring the application of BTX-A injections for pain management, recent research underscores its multifaceted efficacy across a range of pain-related conditions, including orofacial joint pain, muscular pain, chronic migraines, and diabetic neuropathy. Numerous studies have demonstrated BTX-A's capability to significantly diminish pain intensity, thereby enhancing patients' quality of life. For instance, a pilot study by Blanco-Rueda et al. [93] on patients with temporomandibular disorders (TMDs) highlighted that 85 % of participants experienced a reduction in pain intensity during mouth opening, while 90 % reported relief in pain associated with mastication, showcasing BTX-A's potential in alleviating TMD-specific symptoms [94]. These findings are consistent with broader evidence suggesting that BTX-A not only reduces pain but also provides prolonged relief with minimal side effects, affirming its role as a reliable, safe therapeutic option for a variety of myofascial conditions.

The study's detailed findings are further supported by visual data, which provide critical insights into the methodology and efficacy of BTX-A administration. For instance, Fig. 7 provides a detailed anatomical guide illustrating the specific injection points within the masseter, temporalis, and lateral pterygoid muscles, as well as the TMJ, which are critical target sites for BTX-A administration in managing TMD-related pain. The figure not only offers a precise mapping of these muscle and joint regions but also underscores the necessity of strategic injection placement to optimize the localized action of BTX-A. Targeted points within the masseter (1, 2, and 3) focus on reducing hyperactivity in the primary masticatory muscle, whereas points in the lateral pterygoid (4 and 5) aim to relieve tension impacting mandibular function. The inclusion of TMJ (point 6) and anterior fibers of the temporalis (points 7 and 8) addresses joint and accessory muscle involvement in TMD pain pathways. This mapping serves as a critical reference for clinical application, emphasizing the importance of anatomical precision in injection techniques to ensure consistent, reproducible results across treatments. By carefully targeting each injection point, practitioners can maximize BTX-A's therapeutic effects, enhancing pain relief while minimizing adverse reactions. Additionally, this localization approach contributes to standardizing clinical procedures, enabling practitioners to reliably reproduce injection protocols that have demonstrated efficacy in clinical studies, thus fostering better patient outcomes and promoting safe, effective pain management practices.

Fig. 7.

Designated sites for injection: Points 1, 2, and 3 correspond to specific areas within the masseter muscle, optimizing localized relief. Point 4 targets the lateral pterygoid muscle through an extra-oral approach, while Point 5 accesses the same muscle intra-orally. Point 6 represents the temporomandibular joint (TMJ), and Points 7 and 8 mark the anterior fibers of the temporalis muscle, Derived from Ref. [93] under open access license (CC BY 4.0 Attribution 4.0 International Deed).

Furthermore, Fig. 8 presents a detailed quantitative assessment of pain intensity scores, measured using the Visual Analog Scale (VAS), comparing baseline pain levels with scores recorded six weeks following BTX-A treatment. This figure effectively demonstrates the therapeutic efficacy of BTX-A, showing a statistically significant decrease in pain across all targeted craniofacial regions, including key areas such as the masseter, temporalis, and pterygoid muscles. The substantial reduction in pain scores post-treatment provides strong empirical evidence of BTX-A's analgesic potential, particularly in clinical settings for patients suffering from TMD and related myofascial pain disorders. The use of VAS as a standardized metric enhances the reliability and comparability of these findings, allowing for precise quantification of pain relief across time. This figure not only validates BTX-A's capacity to alleviate pain but also underscores its sustained effectiveness over several weeks, highlighting its potential as a long-term solution in chronic pain management. Such robust data support the use of BTX-A as a viable, evidence-based intervention in clinical practice, guiding practitioners in implementing effective pain management protocols for craniofacial disorders.

Fig. 8.

Measurement of pain levels in cervicofacial muscles using the Visual Analog Scale (VAS) conducted before and six weeks following intramuscular BTX-A injection. This figure displays a comparison of pain intensity, highlighting the reduction in discomfort across the treated regions after the intervention, Derived from Ref. [93] under open access license (CC BY 4.0 Attribution 4.0 International Deed).

This review aims to synthesize such key findings to present a holistic view of BTX-A's therapeutic applications, with an emphasis on its anti-inflammatory mechanisms, pain-relief efficacy, and adaptability to personalized treatment plans. By consolidating essential studies on BTX-A and discussing recent advancements, this work aspires to establish a comprehensive framework for future research, directing attention toward optimal application techniques and potential integrations with multidisciplinary pain management approaches.

The therapeutic applications of BTX-A span a diverse range of pain conditions, with varying dosages, injection techniques, and combination therapies tailored to each indication. Table 2 summarizes these aspects, providing an overview of BTX-A's role in managing chronic pain, spasticity, myofascial disorders, and hyperhidrosis. While certain conditions, such as migraine and TMD, are discussed in detail within this manuscript, other indications reflect broader findings from the literature, underscoring BTX-A's expanding versatility in clinical practice.

Table 2.

Summary of BTX-A dosage, techniques, and combination therapies across pain conditions.

Condition	Dose(U)	Technique	Combination theraphy	Outcome	REF
Chronic Migraine	25–50 U	Intramuscular (Pericranial)	NSAIDs, TENS	Reduced migraine frequency	[95]
Temporomandibular Disorders (TMD)	20–40 U	Intramuscular (Masseter, Pterygoid, Temporal)	Dry needling, Physical therapy	Improved jaw function, reduced pain	[96]
Spasticity (post-stroke)	200–400 U	Intramuscular (Targeted Muscles)	Physical rehabilitation	Reduced muscle stiffness, enhanced mobility	[97]
Myofascial Pain	40–100 U	Trigger Point Injection	Massage therapy, Acupuncture	Reduced pain, increased muscle relaxation	[98]
Hyperhidrosis	50–200 U	Intradermal (Palmar, Axilla)	Antiperspirants	Reduced sweating, improved quality of life	[99]
Pelvic Pain	35–80 U	Pelvic Floor Injection	Muscle relaxants, CBT	Relief from chronic pelvic pain, reduced muscle tension	[100]

3.1.1. Optimizing dosage and targeted injection sites

Recent studies have focused on determining optimal dosages and precise injection sites to enhance the effectiveness and safety of BTX-A in pain management. By examining various doses and specific target muscles, researchers aim to maximize pain relief while minimizing potential side effects. For instance, targeted injections into precise muscle groups, such as the masseter, temporalis, and lateral pterygoid muscles for orofacial pain, have shown promise in reducing hyperactivity and pain with minimal adverse effects [101]. Moreover, studies suggest that lower doses of BTX-A can be effective in managing chronic pain when administered to specific trigger points associated with pain pathways, reducing the need for higher doses and potentially diminishing associated risks. This approach not only improves the therapeutic outcomes of BTX-A but also provides a more personalized treatment plan for patients with chronic and localized pain conditions [55].

The ongoing research in optimizing dosage and injection sites underscores the importance of individualized treatment protocols and highlights BTX-A's versatility in addressing diverse pain conditions. Future research focusing on these parameters may further establish standardized guidelines for BTX-A application, making it a safer and more effective option in pain management.

3.1.2. Role of BTX-A in pain treatment hierarchy

BTX-A occupies a unique position in the pain treatment hierarchy, bridging the gap between conventional pharmacological treatments (such as NSAIDs, opioids, and corticosteroids) and invasive interventions (e.g., nerve blocks and surgical procedures). While first-line therapies often include oral medications and physical rehabilitation, BTX-A is typically considered when these approaches prove ineffective, particularly for chronic or refractory pain conditions. In the pain management ladder, BTX-A is generally categorized as a second or third-line treatment, introduced after failure to achieve adequate relief through less invasive modalities. Its long-lasting effects and minimal systemic side effects make it an attractive alternative to prolonged opioid use, reducing the risk of addiction and medication-related complications. For conditions such as chronic migraine, myofascial pain, spasticity, and TMD, BTX-A is often used in combination with physical therapy, neuromodulation, and behavioral interventions, ensuring comprehensive pain management. This multimodal approach targets pain at multiple levels, providing relief through both muscle relaxation and neurotransmitter modulation [102,103].

3.1.3. Addressing adverse effects of BTX-A on muscle and bone tissues

While BTX-A has gained widespread recognition for its efficacy in managing chronic pain conditions such as migraines, spasticity, and TMD), the potential adverse effects on muscle and bone tissues necessitate careful consideration in clinical practice. Addressing these effects is critical for optimizing therapeutic outcomes, enhancing patient safety, and ensuring long-term treatment efficacy.

One of the most documented adverse effects of BTX-A is muscle atrophy, which often results from prolonged paralysis induced by repeated high-dose injections. Research demonstrates that high-dose BTX-A can significantly reduce muscle mass, with studies in animal models reporting up to a 45 % decrease in muscle wet weight following administration. This reduction occurs due to disuse atrophy, where the targeted muscle remains in a paralyzed state, leading to gradual deterioration in muscle fibers and structural integrity. Consequently, patients may experience localized weakness, reduced mobility, and compromised functionality, particularly in cases where critical muscle groups are repeatedly targeted [104].

In addition to muscle atrophy, growing evidence highlights the detrimental impact of BTX-A on bone tissues. Muscle-bone interactions play a pivotal role in maintaining skeletal integrity, and disruption of this dynamic through muscle paralysis can accelerate bone resorption. Preclinical studies indicate that BTX-A-induced muscle paralysis increases osteoclastic activity, resulting in focal bone loss and reduced bone density. This effect is particularly concerning in patients receiving BTX-A for TMD, as mandibular bone thinning and cortical degradation have been observed following injections into masticatory muscles. The extent of bone loss correlates with both the dosage and frequency of BTX-A administration, underscoring the importance of tailored dosing strategies to minimize skeletal side effects [105,106].

Mitigating these risks involves careful dose titration, individualized treatment planning, and appropriate spacing between injection sessions. Lower doses of BTX-A, when combined with adjunct therapies such as physical rehabilitation and neuromodulation, can achieve significant pain relief while minimizing the likelihood of adverse musculoskeletal effects. Additionally, long-term monitoring of muscle and bone health in patients undergoing repeated BTX-A treatments is essential to detect early signs of atrophy or bone loss, allowing for timely intervention [107,108].

Future research should focus on refining injection techniques, optimizing dosing protocols, and exploring novel formulations that preserve the therapeutic benefits of BTX-A while mitigating adverse effects. Emerging approaches, such as combining BTX-A with regenerative therapies like stem cells or bone growth factors, hold promise in enhancing muscle recovery and maintaining bone density. By addressing these challenges, clinicians can harness the full potential of BTX-A in pain management while safeguarding patient outcomes.

3.2. Future potential research and applications of BTX-A

In the previous section, we highlighted key research gaps that warrant further investigation. Future research should prioritize identifying factors that contribute to the variability in BTX-A efficacy across different pain conditions. An important direction involves exploring biomarkers and genetic factors that could predict patient responsiveness, advancing a precision medicine approach to BTX-A treatment. Furthermore, combination therapy remains a critical area for further study. While combining BTX-A with physical rehabilitation, lifestyle adjustments, and complementary therapies may yield synergistic effects, investigating newer adjunct therapies such as nerve growth factor inhibitors, advanced neuromodulation techniques, and regenerative medicine could reveal powerful synergies that enhance patient outcomes [109].

While BTX-A's primary mechanism revolves around muscle relaxation and inhibition of neurotransmitter release, the management of chronic pain often necessitates a combination of therapies. In clinical practice, BTX-A is frequently administered alongside physical rehabilitation, neuromodulation techniques (such as Transcutaneous Electrical Nerve Stimulation (TENS)), and anti-inflammatory medications to optimize pain relief. This multimodal approach targets not only muscular hyperactivity but also peripheral and central sensitization pathways, addressing pain at multiple levels [110,111]. Future research could explore these synergistic effects more extensively to enhance the efficacy and duration of BTX-A treatments, ultimately contributing to more comprehensive pain management strategies.

Long-term and real-world studies are also essential to establish a comprehensive understanding of BTX-A's safety, efficacy, and cost-effectiveness across diverse patient populations and treatment cycles. Such studies would clarify BTX-A's sustained impact and side-effect profile, thus supporting evidence-based clinical guidelines. Standardizing dosing and administration protocols is also crucial to optimize BTX-A's use across various pain types and severities. Developing precise dosing algorithms tailored to individual patient characteristics and specific pain profiles could improve both therapeutic outcomes and patient safety [108].

Additionally, the potential of BTX-A in managing pain associated with psychiatric conditions, such as fibromyalgia or somatic symptom disorder, represents an emerging and promising field. Given the strong link between chronic pain and mental health, focused research in this area is warranted. Finally, integrating digital health tools presents a valuable opportunity. Technologies like wearable pain monitors and mobile applications could enable real-time tracking of treatment responses, generating valuable data on BTX-A's effects and facilitating more personalized, patient-specific adjustments [112,113]. Together, these future directions offer significant potential to deepen our understanding of BTX-A in pain management, paving the way for optimized, individualized, and multidisciplinary therapeutic strategies.

3.2.1. Personalized medicine and biomarker-guided treatment

One promising direction for future research in BTX-A therapy is the application of personalized medicine, particularly through the use of biomarkers and genetic data to predict patient response. Biomarker-guided treatment can enhance BTX-A's effectiveness by identifying patients who are likely to respond positively to the therapy, thereby improving the overall success rate of pain management interventions. For instance, specific genetic markers related to neurotransmitter regulation, inflammatory pathways, and cellular response mechanisms may help clinicians tailor BTX-A treatment to individual patient profiles [114].

Recent studies suggest that patients with particular biomarker profiles, such as genetic variations in genes associated with pain sensitivity, neurotransmitter release, or inflammatory response, may experience more pronounced benefits from BTX-A injections. For instance, genes involved in the regulation of neurotransmitters like serotonin and dopamine, which influence pain perception and emotional response, could play a significant role in determining patient sensitivity to BTX-A. Variants in these genes may indicate whether a patient is more likely to achieve significant pain relief or experience prolonged effects from BTX-A treatment [115]. Additionally, genes related to inflammatory pathways, such as those encoding cytokines or other immune modulators, can impact the body's inflammatory response to pain and, consequently, BTX-A's effectiveness in reducing pain sensitivity [116].

By incorporating genetic testing and biomarker analysis into the treatment planning process, clinicians can identify specific markers that predict positive responses to BTX-A. This approach can enable practitioners to adjust doses or treatment frequencies according to each patient's biological profile, maximizing the therapeutic potential of BTX-A while minimizing the likelihood of adverse effects that can arise from non-optimized dosing [117].

The integration of biomarker data into BTX-A treatment protocols marks a shift toward precision medicine in pain management. Research in this area could lead to the development of standardized biomarker panels, where patients would undergo testing for certain markers before treatment. Based on these results, personalized dosing strategies could be established, helping clinicians determine the ideal concentration, injection sites, and frequency for each patient. Genetic screening tools that analyze a patient's overall pain response profile and tolerance levels to BTX-A may also enhance patient stratification, allowing for more accurate selection of candidates who are likely to benefit from this therapy [118].

As more research is conducted on biomarkers and genetic profiles, this personalized approach to BTX-A therapy could become the standard in pain management, offering a tailored treatment option that addresses individual differences in pain perception and sensitivity. Such advancements could transform BTX-A therapy into a predictable and highly effective method for a diverse range of chronic pain conditions, optimizing patient outcomes and reducing healthcare costs by minimizing trial-and-error approaches and potential adverse effects.

3.2.2. Potential of BTX-A in combination with neurogenetic and optogenetic techniques

The integration of BTX-A with advanced neurogenetic and optogenetic techniques is a promising area of research that may enhance the precision and efficacy of chronic pain management. Neurogenetics allows for targeted manipulation of specific genes within neuronal pathways, enabling researchers to better understand the genetic underpinnings of pain and to modulate pain pathways with higher specificity [119]. Similarly, optogenetics, which involves the use of light-sensitive proteins to control neural activity, enables precise control over neuron firing patterns in targeted regions of the brain and spinal cord [120]. By combining BTX-A with these technologies, it may be possible to achieve more refined modulation of pain pathways, particularly in conditions involving central sensitization and neuropathic pain.

Recent studies suggest that optogenetic tools can precisely activate or inhibit specific neurons involved in pain perception, potentially enhancing the effects of BTX-A. For instance, optogenetic control of inhibitory interneurons could amplify BTX-A's analgesic effects by further reducing excitability in pain pathways. When administered in combination with optogenetics, BTX-A may maintain its role in blocking neurotransmitter release at neuromuscular junctions, while optogenetic modulation provides an additional layer of control, allowing for dynamic adjustment of neuronal activity based on patient response. This approach could offer immediate and on-demand pain relief, especially valuable in treatment-resistant chronic pain conditions [121].

In the context of neurogenetic applications, the use of BTX-A in genetically modified models allows for investigation into specific receptors and neurotransmitters involved in chronic pain. This combination of BTX-A and neurogenetic manipulation enables researchers to pinpoint the molecular targets that enhance or inhibit pain perception, which could lead to the development of highly personalized treatment protocols based on each patient's genetic profile [122].

As research progresses, the potential to combine BTX-A with neurogenetic and optogenetic interventions could revolutionize chronic pain management. These techniques provide a more tailored and controlled approach, offering the possibility of real-time pain modulation with minimal side effects. Future clinical trials and preclinical studies are necessary to explore the safety and efficacy of these combined treatments, but early findings indicate that the synergy between BTX-A and these advanced neurotechnologies could pave the way for innovative, targeted therapies in pain management.

3.2.3. Digital tools and artificial intelligence for optimizing BTX-A therapy

The integration of digital health tools and artificial intelligence (AI) into BTX-A therapy represents a transformative advancement in personalized pain management. Wearable devices, mobile applications, and AI-powered algorithms enable real-time monitoring of patient responses to BTX-A treatment, providing valuable data on effectiveness, duration, and any side effects experienced. This continuous feedback can be used to adjust treatment plans dynamically, ensuring that patients receive the optimal dosage and treatment frequency based on their unique pain patterns and therapeutic responses [123].

Wearable pain monitors, for instance, allow clinicians to track fluctuations in pain levels and correlate them with BTX-A injection points and dosage. By analyzing this data, healthcare providers can identify specific trends and tailor future treatments to achieve better pain control. Additionally, mobile applications can empower patients to log their symptoms, side effects, and pain relief levels, creating a comprehensive data set that both patients and clinicians can access to make informed decisions about treatment adjustments [124].

Artificial intelligence further enhances the potential of BTX-A therapy by enabling predictive analytics and personalized treatment algorithms. Machine learning models can analyze vast amounts of patient data to predict optimal dosing schedules, assess risk factors for adverse effects, and identify the most effective injection sites for individual patients. By utilizing AI, clinicians can create adaptive treatment protocols that evolve based on patient responses, leading to more consistent and effective outcomes [125].

The development of digital tools and AI applications for BTX-A therapy also promotes the standardization of treatment protocols across various healthcare settings. With real-time data on patient outcomes and AI-driven insights, healthcare providers can implement evidence-based practices with greater precision and reliability. This approach not only enhances the quality of care but also reduces trial-and-error methods, minimizing patient discomfort and optimizing resource utilization in clinical practice [126].

Incorporating digital health and AI technologies into BTX-A therapy marks a significant step toward precision medicine in pain management. As research in this field progresses, these tools have the potential to revolutionize BTX-A treatment, making it more accessible, efficient, and tailored to each patient's unique needs and pain profile.

3.2.4. Advanced animal models for precise evaluation of BTX-A effects

Understanding the full range of effects of BTX-A, especially in long-term therapeutic applications, requires in-depth investigation using advanced animal models. Traditional approaches primarily focused on the immediate muscle relaxation and neurotransmission-blocking effects of BTX-A. However, recent studies indicate that the toxin's impacts may extend beyond these short-term outcomes, affecting muscle mechanics, structural integrity, and pain pathways over prolonged periods. Advanced animal models provide a controlled and precise method for studying these effects, allowing researchers to analyze how different doses, injection sites, and durations of exposure to BTX-A influence both local and systemic responses [127].

These models not only facilitate a deeper exploration into the molecular and mechanical interactions of BTX-A but also allow for the identification of potential risks, such as altered muscle function or structural damage in high-dose applications. By examining BTX-A's interactions with ion channels and its effects on neurogenetic factors within animal models, researchers gain valuable insights into optimizing its clinical applications for managing chronic pain, spasticity, and other neurological conditions [128].

Kaya et al. [127], the long-term impacts of BTX-A on muscle mechanics were evaluated in a rat model. The findings demonstrated that BTX-A injections led to significant decreases in active forces within the tibialis anterior muscle, increases in passive forces, and changes in collagen content. Additionally, the study showed that high-dose intramuscular injections could impair muscle function and lead to structural damage in both fibrillar and non-fibrillar muscle components. These results highlight that while BTX-A has effective therapeutic properties, it may also induce unintended mechanical and structural changes that persist over time, affecting both injected and adjacent muscles. This underscores the importance of careful dose management to avoid potential adverse effects on muscle function. In a similar vein, Yılmaz et al. [129], explored the impact of BTX-A on bi-articular muscles. This study revealed that prolonged exposure to BTX-A compromised the functional capacity of these muscles, as indicated by a reduced range of active force exertion and increased passive forces. Such findings underscore the potential limitations of BTX-A in spasticity management, suggesting that long-term treatment may necessitate monitoring and adjustments to mitigate adverse effects on muscle function.

Pingel et al. [130] conducted an in-depth study to examine how high doses of BTX-A affect muscle tissue in rats, focusing on microstructural changes, gait patterns, gene expression, and muscle atrophy. By employing Synchrotron Radiation X-ray Tomographic Microscopy (SRXTM), the researchers captured high-resolution images of muscle tissue post-BTX-A injection, providing valuable insights into the cellular and structural impacts of the treatment.

To provide a comprehensive illustration of BTX-A's impact, Fig. 9A and B in the study present a detailed three-dimensional reconstruction of muscle tissue post-BTX-A administration. This visualization reveals extensive microstructural changes, including disruption of fibrillar organization and increased presence of non-fibrillar tissue, indicating significant tissue remodeling and muscle degeneration. The SRXTM technique used to create this image captures the extent of structural compromise, with a clear loss of the linear alignment of muscle fibers, replaced by a more isotropic (random) arrangement. These changes underscore the potential for high BTX-A doses to induce lasting damage within muscle tissues, particularly when injection dosage and site selection are not carefully optimized. This visual evidence is essential in understanding how BTX-A affects cellular structures, especially in high-dose applications that can lead to muscle atrophy and altered gait patterns, as seen in the animal models used in this research.

Fig. 9.

The 3D visualizations derived from SRXTM tomograms reveal the fibrillar (in red) and non-fibrillar (in yellow) components of muscle tissue in both the BTX-A injected leg (A) and the control leg (B) of a rat, observed three weeks after an injection totaling 6 U of BTX-A (administered in three doses of 20 pg each). The SRXTM results demonstrate that BTX-A injection leads to structural and organizational damage within the muscle tissue, affecting its overall integrity and arrangement, Derived from Ref. [130] under open access license (CC BY 4.0 Attribution 4.0 International Deed).

In addition to microstructural changes, Fig. 10A and B demonstrate the functional implications of BTX-A through gait analysis. This figure shows specific alterations in stride length, foot angle, and other gait parameters that reflect the influence of BTX-A on motor function. Such changes underscore the potential motor consequences of BTX-A treatment in higher doses, providing clinical insights into how BTX-A affects movement and emphasizing the importance of dose adjustment to optimize therapeutic outcomes.

Fig. 10.

In the footstep analysis, the rats' hind paws were dipped in ink, allowing them to run across graph paper to record their steps at two points: (A) baseline (2 days before injection) and (B) 21 days after BTX-A injection into the left triceps surae muscle. The injection dosage was 6 U of BTX-A, administered in three doses of approximately 20 pg each (totaling around 60 pg). The study measured stride length, foot angle, and foot length, all of which showed significant changes in the BTX-A injected leg compared to both the baseline measurements and the control leg (p < 0.05),Derived from Ref. [130] under open access license (CC BY 4.0 Attribution 4.0 International Deed).

The use of these advanced animal models has provided invaluable insights into the dose-dependent and duration-dependent effects of BTX-A, underscoring the importance of precise dosing and targeted application. As this research continues, animal models will play a critical role in refining BTX-A treatment protocols, enhancing its efficacy and safety in clinical applications, and paving the way for more targeted approaches in managing chronic pain and muscle spasticity.

3.2.5. New molecular combinations of BTX-A for enhanced therapeutic effects

To further enhance the therapeutic potential of BTX-A, recent research has focused on developing new molecular combinations that increase its efficacy while reducing the required dosage. These combinations aim to amplify BTX-A's effects, potentially lowering the risk of adverse reactions associated with higher doses and extending its duration of action.

One promising approach involves conjugating BTX-A with biocompatible polymers or nanoparticles, such as polyethylene glycol (PEG), poly (lactic-co-glycolic acid) (PLGA), and chitosan, which can facilitate controlled release and improve toxin stability within the target tissue [[131], [132], [133]]. For instance, BTX-A encapsulated in nanoparticles can demonstrat sustained release profiles, leading to prolonged therapeutic effects and enhanced bioavailability at the injection site. This slow-release mechanism allows for a more gradual release of BTX-A, potentially reducing the frequency of injections required in chronic pain management and minimizing fluctuations in therapeutic levels [134]. Additionally, combining BTX-A with other biologically active molecules, such as growth factors or anti-inflammatory agents, has shown potential in enhancing its efficacy for specific medical conditions. For example, conjugation with anti-inflammatory peptides or cytokine inhibitors may complement BTX-A's neuromodulatory effects, addressing both neuronal and inflammatory components of pain simultaneously. This dual-action approach could be particularly beneficial in managing complex pain syndromes with inflammatory involvement, such as arthritis or neuropathic pain [135,136].

Another promising direction is the engineering of BTX-A variants with modified protein structures that selectively target specific neuronal subtypes or receptors. These engineered molecules can provide targeted modulation of pain pathways with minimal impact on surrounding tissues, allowing for more precise and effective pain relief [137].

These innovative molecular combinations and engineered variants of BTX-A represent a significant advancement in its therapeutic applications. Future research on optimizing these combinations, evaluating their safety profiles, and conducting clinical trials could pave the way for next-generation BTX-A formulations that offer increased efficacy, extended action, and a reduction in adverse effects, establishing BTX-A as a cornerstone in personalized pain management strategies.

3.2.6. Emerging applications of BTX-A in complex and less common pain syndromes

The therapeutic potential of BTX-A extends beyond traditional pain conditions, with recent research exploring its efficacy in managing more complex and less common pain syndromes such as fibromyalgia and complex regional pain syndrome (CRPS). These conditions, characterized by chronic pain, heightened sensitivity, and often debilitating symptoms, present challenges for conventional pain management approaches. BTX-A's unique mechanisms, particularly its effects on neurotransmitter release and neuromodulation, offer promising avenues for providing relief in these complex pain syndromes [138].

Fibromyalgia is a chronic pain condition marked by widespread musculoskeletal pain, fatigue, and cognitive disturbances. Patients with fibromyalgia often experience heightened pain sensitivity, possibly due to dysregulated pain pathways and neurotransmitter imbalances. Recent studies have indicated that BTX-A injections, particularly when targeted at specific trigger points, may help reduce pain intensity and muscle stiffness in fibromyalgia patients. By modulating the release of pain-associated neurotransmitters and reducing muscle hyperactivity, BTX-A offers a potential treatment option that targets the underlying neurochemical imbalances in fibromyalgia, thus alleviating both local and systemic pain symptoms [139].

CRPS is another debilitating condition involving persistent pain, typically following an injury or surgery. CRPS is associated with autonomic and inflammatory changes, making it resistant to many standard pain therapies. Research has shown that BTX-A, when administered in specific areas affected by CRPS, can alleviate localized pain by blocking abnormal neurotransmitter release at sensory nerve endings. Additionally, BTX-A's anti-inflammatory effects may play a role in reducing the localized inflammation that often exacerbates CRPS symptoms, providing a multifaceted approach to pain management in this complex condition [140].

These emerging applications highlight the versatility of BTX-A in addressing pain conditions that are otherwise difficult to manage. By exploring BTX-A's impact on complex and atypical pain syndromes, future studies may establish optimized protocols for its use in these challenging cases, offering patients an alternative or adjunctive treatment option. Continued research into the specific mechanisms of BTX-A in fibromyalgia and CRPS could not only enhance our understanding of these conditions but also expand the therapeutic utility of BTX-A in the field of pain management.

3.2.7. Potential of combining BTX-A with regenerative therapies for joint and nerve repair

The integration of BTX-A with regenerative therapies, such as stem cell therapy and tissue engineering, represents a promising new direction in the management of joint and nerve injuries. While BTX-A is widely recognized for its neuromodulatory and muscle-relaxant effects, its combination with regenerative treatments could offer enhanced benefits by addressing both symptomatic pain relief and tissue repair simultaneously [141].

Stem cell therapy has shown potential in promoting tissue regeneration and repair, particularly in cases of cartilage damage, joint degeneration, and peripheral nerve injuries. Combining BTX-A with stem cell injections may create a supportive environment for stem cells by reducing inflammation, muscle spasms, and abnormal nerve activity, which are common in patients with joint and nerve injuries. This reduction in local stress can improve the efficacy of stem cell therapy, allowing cells to regenerate damaged tissues more effectively. Studies indicate that BTX-A may modulate local immune responses, further enhancing the success of stem cell-based approaches by minimizing the inflammatory environment that often inhibits regenerative processes [142,143].

Tissue engineering and scaffold-based therapies offer additional avenues for combining BTX-A with regenerative approaches. In cases of extensive tissue damage or joint degeneration, scaffold-based techniques can provide structural support while promoting cellular growth and tissue repair. When used in conjunction with BTX-A, scaffolds designed to deliver both regenerative cells and BTX-A locally could enable controlled pain management alongside tissue reconstruction. This combination may be particularly useful in chronic joint injuries, where BTX-A can reduce joint stress and spasticity, thereby aiding in the structural integration and effectiveness of tissue-engineered scaffolds [144].

The use of BTX-A in combination with regenerative therapies not only expands its therapeutic potential but also highlights a new frontier in precision medicine for pain management and tissue repair. Future studies focusing on the optimal dosage, timing, and delivery mechanisms for BTX-A when combined with regenerative treatments could pave the way for novel protocols that integrate pain management with tissue healing. Such approaches could revolutionize treatment options for patients with joint and nerve injuries, offering a more comprehensive, restorative solution.

3.3. Economic considerations of BTX-A

BTX-A remains a significant factor affecting its widespread adoption in pain management. Compared to conventional analgesics such as NSAIDs, corticosteroids, or opioids, BTX-A involves a higher upfront expense. However, its prolonged therapeutic effects present economic benefits that extend beyond the initial cost.

Beyond economic considerations, BTX-A offers therapeutic advantages that extend to reducing neurogenic inflammation, modulating ion channels, and preventing pain progression in chronic conditions. These multifaceted mechanisms allow BTX-A to address pain at different levels (peripheral and central), providing a more comprehensive and long-lasting approach to pain relief compared to short-acting analgesics. This dual benefit, economic savings and enhanced clinical outcomes, positions BTX-A as a valuable option for patients with refractory or complex pain who have not responded to conventional therapies [145].

BTX-A can provide sustained pain relief for 3–6 months following a single injection, reducing the need for frequent medication and follow-up visits. In chronic pain conditions such as migraine, spasticity, and myofascial pain, this extended relief can substantially lower healthcare utilization by minimizing repeat consultations, hospital visits, and additional pharmacological interventions [146,147].

Moreover, BTX-A's targeted mechanism of action decreases the risk of systemic side effects commonly associated with long-term Non-Steroidal Anti-Inflammatory Drug (NSAID) or opioid use, including gastrointestinal, cardiovascular, and renal complications [148]. By mitigating these adverse effects, BTX-A not only enhances patients' quality of life but also reduces indirect costs related to managing secondary conditions and medication-induced complications [149].

Cost-effectiveness studies in patients with chronic migraine and spasticity have shown that although BTX-A may initially appear more expensive, its ability to reduce disability, enhance productivity, and decrease absenteeism results in favorable long-term economic outcomes [150,151]. Additionally, BTX-A has demonstrated potential to prevent disease progression in conditions such as cervical dystonia and TMD, potentially reducing the need for invasive procedures [95].

Despite these benefits, it is crucial to identify patients most likely to respond to BTX-A through personalized treatment approaches. The use of biomarkers and predictive models can improve treatment precision, enhancing cost-effectiveness by ensuring that resources are allocated efficiently and avoiding unnecessary interventions for non-responders.

4. Conclusion and future directions

The focus of this work was placed on the recent advances in the field of BTX-A usages to manage different types of pain. Based on the previous studies, the optimization of BTX-A dosing and injection techniques (such as specific injection points) was identified as a critical factor that can greatly enhance pain relief efficiency. Extending the follow-up duration beyond specified periods has shown potential in assessing the prolonged benefits of BTX-A and whether it can offer a sustained solution for chronic pain management. Although BTX-A has demonstrated high efficacy in pain relief, side effects remain a concern, particularly at higher doses. The integration of traditional therapies and medications alongside BTX-A may provide further insight into its comparative effectiveness, helping BTX-A find its place within the broader pain management treatment hierarchy and guiding clinicians and patients in making informed decisions.

The personalization of BTX-A treatment by investigating patient-specific factors—such as age, gender, pain history, and underlying medical conditions—has also emerged as essential for optimized outcomes. Although these factors have been examined in several studies, comprehensive exploration on a larger scale remains limited. Tailoring treatment strategies based on these patient characteristics could lead to more customized and effective pain relief approaches.

Furthermore, recent advances highlight the potential of BTX-A when combined with regenerative therapies like stem cells and tissue engineering, particularly in cases involving joint or nerve injuries. Such combinations not only alleviate pain but also promote tissue repair, representing a promising frontier for integrating pain management with regenerative medicine. This multi-faceted approach addresses both symptomatic relief and the underlying pathology, positioning BTX-A as a unique component in advanced therapeutic strategies.

Incorporating BTX-A with non-invasive traditional approaches, including physical rehabilitation, acupuncture, behavioral therapies, or lifestyle modifications, offers additional avenues for synergistic effects in pain management. Using low-dose BTX-A in combination with these methods may maximize pain relief while minimizing adverse effects, offering patients a comprehensive and holistic approach to pain management. Although many studies have reported minimal side effects associated with BTX-A, further research with larger patient populations is needed to solidify its risk-benefit profile.

Moreover, addressing the diverse presentations of pain within a generalized treatment approach remains a challenge. Developing algorithms or guidelines that tailor BTX-A treatment based on specific pain characteristics, such as type, intensity, and underlying mechanisms, could improve treatment precision and effectiveness. Emerging technologies, including digital health tools and artificial intelligence, also offer promising support in monitoring patient responses and optimizing BTX-A dosing in real-time, contributing to a more dynamic and patient-centered approach in chronic pain management.

CRediT authorship contribution statement

Hamta Rahmatipour: Writing – original draft. Salar Mohammadi Shabestari: Writing – original draft, Resources. Soheila Zamanlui Benisi: Writing – review & editing, Visualization, Supervision, Conceptualization. Hamidreza Samadikhah: Writing – review & editing.

Data and code availability statement

Data included in the article is referenced in the article.

Declaration of competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.