UNIVERSIDADE ESTADUAL DE CAMPINAS
Faculdade de Odontologia de Piracicaba
DOUGLAS MASSONI RAMOS
EFEITO DA TOXINA BOTULÍNICA TIPO A SOBRE AS
ESTRUTURAS MUSCULOESQUELÉTICAS DE RATAS
ADULTAS
PIRACICABA 2020
DOUGLAS MASSONI RAMOS
EFEITO DA TOXINA BOTULÍNICA TIPO A SOBRE AS
ESTRUTURAS MUSCULOESQUELÉTICAS DE RATAS
ADULTAS
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutor em Biologia Buco Dental, na Área de concentração em Anatomia.
Orientadora: Profª. Dra. Celia Marisa Rizzatti Barbosa
Este trabalho corresponde à versão final da tese defendida pelo aluno Douglas Massoni Ramos e orientada pelo Profa. Dra. Celia Marisa Rizzatti Barbosa.
PIRACICABA 2020
DEDICATÓRIA
A Deus, minha mãe Madalena Massoni Ramos e minha avó Angélica Joanilli Massoni (in memoriam)
AGRADECIMENTOS ESPECIAIS
Agradeço a Deus, pelo dom da vida e por me permitir a oportunidade da realização de mais um sonho.
A minha mãe, Madalena Massoni, que a ela devo minha vida e tantas outras coisas que me ensinou para que eu pudesse viver da melhor forma, agradeço por ter me ensinado um olhar humilde para o mundo, por ter me ensinado que em tudo o que acontece há uma lição, por ter me ensinado a me resignificar diante daquilo que não posso mudar e me reconciliar comigo mesmo, me livrando da culpa e abrindo o coração para que novas coisas aconteçam, diferentes daquela que desejei e planejei para mim. Agradeço por todo amor, pelo carinho, pela atenção, pelas palavras de apoio e pelas que serviram para me chamar à razão. Agradeço por ter me escolhido como filho e por ser sempre a minha melhor versão.
A minha avó, Angelica Joanilli Massoni (in memoriam) que por mais dias que passem, as pessoas valiosas permanecem no coração, mesmo com a sua partida há alguns meses eu não poderia esquecer da mulher guerreira, carinhosa, gentil e amorosa e que me ensinou tantas coisas sobre a vida. Foi uma honra ter uma avó querida que sempre cuidou de mim com tanto carinho, amor e dedicação em todos os momentos. A senhora me faz muita falta, saudades eternas!
A minha orientadora Profª Drª Célia M. Rizzatti Barboza, primeiro pelo aceite no programa da pós-graduação, em seguida pela marca que deixará em minha vida. Há pessoas que despertam algo em nós, que abrem nossos olhos de modo irreversível e transformam à nossa maneira de ver o mundo. Seus ensinamentos foram cruciais para o meu desenvolvimento. Obrigado pela sua dedicação e paciência durante todo esse processo, e obrigado pelo incentivo e pelas palavras de sabedoria ditas nos momentos mais difíceis.
AGRADECIMENTOS
Ao meu coorientador, Prof. Dr. Marcelo Augusto Marreto Esquisatto, por toda ajuda durante a realização desse projeto, suas contribuições foram cruciais para a conclusão do protocolo experimental, além de toda atenção dada quando necessária.
Aos membros da banca da qualificação e defesa, que gentilmente nos cedeu parte do seu tempo para fazer todas as correções, orientações e contribuições para que a tese possa ficar ainda melhor, que ao final com certeza nos nortearam para a confecção final do trabalho, suas observações são imprescindíveis.
Ao corpo docente do Programa de Pós-Graduação, pelo dom e arte de ensinar, pelos grandes exemplos deixados e por cada e todas as belas lições aprendidas.
Ao Programa de Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), por nos permitir a oportunidade de evoluir ainda mais e por contribuir para o meu
crescimento profissional, proporcionando acima de tudo, o crescimento científico. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior – Brasil (CAPES) - Código de Financiamento 001.
Ao meu companheiro de vida1., Jean Ferreira, que jamais me negou apoio, carinho e incentivo. Obrigado pelas palavras de encorajamento, por acreditar em mim em todos os momentos, por aguentar as crises de estresse e ansiedade e suportar as ausências em alguns momentos. Você é essencial na minha vida!
Agradeço a todos os colegas que de certa forma me ajudaram na realização da pesquisa, em especial a Raira Brito e a Nádia Fávaro que me acompanharam durante todo o processo experimental.
Agradeço imensamente a todos que contribuíram direta ou indiretamente para a realização desse trabalho, sem vocês no meu caminho eu não teria chegado até aqui.
Resumo
A Toxina Botulínica (BoNT-A) tem sido usada na clínica para controlar a dor e a hiperatividade muscular comumente presentes em disfunções temporomandibulares e bruxismo. Quando usada em altas doses pode desenvolver efeitos adversos importantes ao longo do tempo. Nenhum trabalho foi realizado para investigar os efeitos de uma dose única e alta de BoNT-A sobre as fibras musculares, ao longo do tempo. Objetivo: Investigar os efeitos de uma única dose alta de BoNT-A no músculo masseter de ratas Wistar. Material e Métodos: Quarenta e quatro animais adultos foram divididos aleatoriamente em grupo de controle (GC / n=22) e grupo tratado (GT / n=22). O GC recebeu uma única dose de 0,14mL/kg de soro fisiológico no músculo masseter e o TG recebeu um 7U/Kg de BoNT-A no mesmo local. Os animais foram sacrificados no 7° (n=5), 14° (n=5), 21° (n=5), 28° (n=4) e 90° (n=3) dias pós-operatórios, e os tecidos musculares foram analisados pela metodologia histoquímica em relação à área transversal do músculo masseter, quantidade de tecido conjuntivo, diâmetro de miócitos e quantidade de vasos sanguíneos neoformados. Lâminas histológicas foram obtidas a partir do tratamento do tecido com hematoxilina-eosina e analisadas em microscopia óptica; os dados obtidos foram organizados em tabelas para a análise estatística (p=0,05) considerando as variáveis grupos, dias da aplicação BoNT-A e a interação entre eles. Resultados: Os dados demonstraram importantes alterações e piora nos tecidos musculares no GT em relação a todas as variáveis analisadas, com comprometimento crescente das estruturas musculares ao longo do tempo (p<0,05). Conclusão: Uma única dose alta de BoNT-A causa alterações significativas no músculo masseter que pioram ao longo do tempo em relação à área transversal do músculo, quantidade de tecido conjuntivo, diâmetro de miócitos e quantidade de vasos sanguíneos.
Palavras-chave: Toxina Botulínica, Efeitos Adversos, Histoquímica, Ratos, Tecido
Abstract
Botulinum Toxin-A (BoNT-A) has been used in the clinic to control pain and muscle hyperactivity commonly present in temporomandibular dysfunctions and bruxism. When used in high doses it can develop important adverse effects over time. No study was carried out to investigate the effects of a high single dose of BoNT-A on masseter muscle fibers over time.
Purpose: To investigated the effects of a single high dose of BoNT-A in Wistar rats masseter. Material and Methods: Forty-four adult animals were randomly divided into control group
(CG / n=22) and treated group (TG / n=22). The CG received a single dose of 0.14mL/kg of saline in the masseter muscle and the GT received a 7U/Kg of BoNT-A at the same site. The animals were sacrificed on 7th (n=5), 14th (n=5), 21st (n=5), 28th (n=4) and 90th (n=3) days
postoperative, and the masseter muscles were analyzed by histochemical methodology regarding muscle cross-sectional area, amount of connective tissue, diameter of myocytes and new blood vessel score. Histological masseter tissue slides were obtained from hematoxylin-eosin treatment and analyzed in optical microscopy; data were organized into tables for the statistical analysis (p=.05) considering the groups, the days from BoNT-A application, and the interaction among them. Results: The data demonstrated important changes and worsens in muscle tissues of TG regarding all variables analyzed, with increasing impairment of muscle structures over time (p<0.05). Conclusion: A single high dose of BoNT-A causes significant changes that worsen over time in masseter muscle regarding the muscle cross-sectional area, amount of connective tissue, diameter of myocytes and new blood vessel score.
SUMÁRIO
1. INTRODUÇÃO...11
2. ARTIGO: Single High Dose of Botulinum Toxina Chances Masseter Muscle: Study Rats...15
3. DISCUSSÃO...38
4. CONCLUSÃO...42
REFERÊNCIAS ...43
ANEXOS...50
ANEXO 1: Comprovante de Submissão do Artigo...50
ANEXO 2: Verificação de originalidade e prevenção de plágio...51
ANEXO 3: Protocolo de aprovação do Comitê de Ética em Experimentação Animal...52
1 INTRODUÇÃO
Fundamentada na teoria da síndrome dolorosa de disfunção miofascial, a disfunção temporomandibular (DTM) ou desordem craniomandibular é um termo utilizado para reunir um grupo de alterações envolvendo as estruturas do sistema estomatognático (SEG) (Okeson, 1998). De etiologia complexa e multifatorial, envolve cerca de 30 a 60% da população adulta, com alta prevalência em mulheres de meia-idade (Lora et al., 2016; Bagis et al., 2012) forte conotação genética (Sanders et al., 2017; Gui e Rizzatti-Barbosa 2015; Segall et al., 2015; Meloto et al., 2011; Planello et al., 2011; Ribeiro-DaSilva et al., 2009), relaciona-se a pacientes mais sensíveis ao estresse, alterações psicosociais e catastrofismo (Lerman et al., 2018), e frequentemente se associa a outras patologias mutilantes com dor crônica (Pimentel, et al, 2016; Gui et al., 2015; Pimentel et al., 2013; Gui et al., 2013). Pode acometer os músculos mastigatórios, as articulações temporomandibulares e estruturas adjacentes, podendo ser classificada como artrogênica, psicogênica e miogênica (Amantéa et al., 2003).
Os estágios iniciais da DTM costumam comprometer a musculatura mastigatória, principalmente músculos esqueléticos funcionais, como o masseter e o temporal.
A musculatura esquelética constitui um dos maiores tecidos do corpo, possuindo a maior massa celular e maior componente proteico do organismo. Em consonância com o motoneurônio, compõe a unidade motora responsável pela aptidão e autonomia funcional do indivíduo, assim como é responsável pelo seu desempenho físico (Deschenes, 2004). O músculo estriado esquelético, por ser um tecido plástico, apresenta capacidade de adaptação quando submetido a diferentes formas de estímulos (Vechetti Jr. et al., 2010). Para Santos Junior et al. (2010), o tecido muscular é caracterizado por desempenhar contrações, gerando ciclos de encurtamento/estiramento; esse mecanismo contrátil é determinante para a manutenção e preservação das fibras musculares.
Berne et al. (2004) e Minamoto (2005), classificam o músculo estriado esquelético em três principais grupos: as fibras tipo I (fibras vermelhas de contração lenta, oxidativa), as fibras tipo IIA (fibras brancas de contração intermediária, glicolítica e oxidativa) e as fibras tipo IIB (fibras brancas de contração rápida, glicolítica). Alterações na atividade muscular podem conduzir a alterações no tipo de fibra que compõe o músculo, modificando sua dinâmica por um processo nem sempre reversível. De certo modo, a DTM miogênica ou o tipo de intervenção terapêutica que é feita para controlar seus sinais e sintomas podem modificar a estruturação da musculatura envolvida nestes princípios que regem a atividade muscular.
A DTM miogênica é, muitas vezes, considerada uma condição clínica altamente debilitante (Plesh et al., 2011) e, devido a esta etiologia multifatorial, normalmente requer uma
abordagem terapêutica interdisciplinar individualizada, capaz de alicerçar uma proposta de intervenção bem sucedida (Gil et al., 1998 ;Rezende et al., 2012).
De acordo com o quadro clínico, modalidades terapêuticas como aconselhamento (Martins et al., 2016), placas de desoclusão (Zhang et al., 2016) acupuntura (Grillo et al., 2015), fonoaudiologia (Gil et al., 1998), psicologia (Grillo et al., 2015; Gui et al., 2014), terapia medicamentosa (Andrade et al., 2004; Rizzatti-Barbosa et al., 2003; Rizzatti-Barbosa et al., 2003), ultrassom (Rai et al., 2016), laserterapia (Demirkol et al., 2017), analgesia por estimulação elétrica transcutânea (TENS) (Arana et al., 2002), terapia por calor e frio, terapia manual para mobilização de tecidos moles e articulares bem como a liberação miofascial, são procedimentos importantes utilizados até então para melhorar a condição do paciente (Eisensmith, 2007; Chaitow, 2001; Troian, 2005; Matta e Honorato, 2003).
As propostas da medicina integrativa certificam intervenções terapêuticas mais completas e eficientes ao bruxismo e DTM. Dentre as intervenções alternativas para o manejo da DTM miogênica, atualmente encontra-se o uso da toxina botulínica tipo A (BoNT-A) voltada a diminuir a atividade muscular e o quadro álgico presente nestas condições.
Considerada umas das mais potentes neurotoxinas, a BoNT-A é produzida por uma bactéria GRAM POSITIVA, anaeróbia estrita e esporulada, chamada Clostridium botulinum (Benecke, 2012; Silva, 2009).
A BoNT-A promove uma denervação química da célula neuronal inibindo, através da inativação da proteína de membrana SNAP 25, a liberação da acetilcolina (Ach), um neurotransmissor que estimula a contração da fibra muscular nas sinapses neuromusculares (Dressler et al., 2005). Os bloqueios neuromusculares promovidos pela BoNT-A podem aliviar a dor devido à diminuição da hiperatividade muscular (Cahlin el al., 2019). Esta ação sobre os músculos tem favorecido a sua indicação em patologias que envolvem desarranjos musculares, como é o caso das DTM, bruxismo e hipertrofia massetérica.
Quando utilizada em dosagem precisa e aplicada através de protocolos corretos, pode ser de muita valia no controle da hiperatividade muscular e dor, principalmente em quadros de cronicidade. Porém, quando utilizada em altas doses, de forma repetida e em intervalos curtos, a BoNT-A é capaz de gerar efeitos adversos em estruturas funcionais importantes para a fisiologia normal dos tecidos (De La Torre et al., 2020). Altas e repetidas doses de BoNT-A tendem a aumentar o risco de desenvolvimento de anticorpos neutralizantes que reduzem sua eficácia (Colhada e Ortega, 2009). A diminuição de força mastigatória após aplicações indevidas, reforçam a necessidade de intervalos entre as doses de aplicação da BoNT-A
(Sposito e Teixeira, 2014). Da mesma forma, doses altas de BoNT-A promovem efeitos indesejáveis sobre a atividade muscular, na estruturação dos tecidos ósseos de origem e inserção muscular, e na eficiência mastigatória, função vital ao organismo (De La Torre et al., 2020).
O número de aplicações e a dosagem de da BoNT-A podem ser fatores determinantes para que a mesma seja indicada corretamente e sem a possibilidade de ocorrência de efeitos adversos ao paciente. A importância de se definir uma dose ideal a ser utilizada mediante indicação pré-estabelecida no diagnóstico pode estabelecer os efeitos benéficos que uma dose única e alta poderiam proporcionar aos quadros de DTM renitentes aos outros tipos de intervenção. Este fato por si, certamente teria que estar dentro do limiar de tolerabilidade das fibras musculares no sentido de evitar danos aos tecidos envolvidos.
No entanto a literatura traz poucas evidências a respeito do uso de uma única dose alta sobre as estruturas dos músculos funcionais, como os envolvidos na atividade mastigatória. Neste caso, particulariza-se o músculo masseter, cuja atividade estabelece relação direta com mastigação.
OBJETIVO
Diante do exposto, o objetivo deste trabalho foi investigar o efeito da toxina botulínica tipo A sobre as estruturas musculoesqueléticas de ratas adultas.
2 ARTICLE: ORIGINAL RESEARCH
SINGLE HIGH DOSE OF BOTULINUM TOXIN-A CHANGES MASSETER MUSCLE: STUDY IN RATS
Article submitted to the Journal of Pain Research (Anexo 1)
Douglas Massoni Ramos1, Raira de Brito Silva1, Marcelo Augusto Marretto Esquisatto2,Nádia
Cristina Fávaro Moreira1, Celia Marisa Rizzatti-Barbosa1.3
1Piracicaba Dental School, University of Campinas, Zip code: 13414-903, Piracicaba-SP,
Brazil
2Araras Dental School, University Center Herminio Ometto, Zip code: 13607339, Araras-SP,
Brazil
3Ingá University Center, Uningá, Zip code: 87035-510, Maringá-PR, Brazil,
Corresponding address:
Celia Marisa Rizzatti-Barbosa Rua Sousa Reis 120, apto 93ª ZIP Code - 05586-080 Vila Indiana - São Paulo - SP Telephone:55 11 999391 2505 Email- rizzatti@unicamp.br
Abstract:
Botulinum Toxin-A (BoNT-A) has been used in the clinic to control pain and muscle hyperactivity commonly present in temporomandibular dysfunctions and bruxism. When used in high doses it can develop important adverse effects over time. No study was carried out to investigate the effects of a high single dose of BoNT-A on masseter muscle fibers over time. Purpose: To investigated the effects of a single high dose of BoNT-A in Wistar rats masseter. Material and Methods: Forty-four adult animals were randomly divided into control group (CG / n=22) and treated group (TG / n=22). The CG received a single dose of 0.14mL/kg of saline in the masseter muscle and the GT received a 7U/Kg of BoNT-A at the same site. The animals were sacrificed on 7th (n=5), 14th (n=5), 21st (n=5), 28th (n=4) and 90th(n=3) days postoperative, and the masseter muscles were analyzed by histochemical methodology regarding muscle cross-sectional area, amount of connective tissue, diameter of myocytes and new blood vessel score. Histological masseter tissue slides were obtained from hematoxylin-eosin treatment and analyzed in optical microscopy; data were organized into tables for the statistical analysis (p=.05) considering the groups, the days from BoNT-A application, and the interaction among them. Results: The data demonstrated important changes and worsens in muscle tissues of TG regarding all variables analyzed, with increasing impairment of muscle structures over time (p<0.05). Conclusion: A single high dose of BoNT-A causes significant changes that worsen over time in masseter muscle regarding the muscle cross-sectional area, amount of connective tissue, diameter of myocytes and new blood vessel score.
Introduction
Temporomandibular dysfunction (TMD) has a high prevalence in the adult population and induces chronic pain in masticatory muscles. Among others interventions, botulinum toxin type A (BoNT-A) may be an important adjunct alternative to control pain and muscle hyperactivity related to TMD and bruxism (Jadhao, 2017, Kim and Yun, 2016). Considered one of the most potent neurotoxins, BoNT-A is produced by Clostridium botulinum, a strict and sporulated GRAM-POSITIVE, anaerobic bacterium (Fredrick and Lin , 2017) that restrain the release of acetylcholine (Ach) into the neuromotor cleft in peripheral cholinergic synapses by cleavage of SNAP-25 on the surface of the neuronal, decreasing muscle activity by chemical denervation (Dressler, Saberi, Barbosa, 2005) relieving pain and muscle hyperactivity (Cahlin, Lindberg, Dahlström, 2019) . The clinical response and duration of the effect of BoNT-A depends on several factors such as age, gender, associated pathology or even antitoxin antibodies production, which influence its efficacy (Bachur et al., 2009). Due to this mechanism of action, BoNT-A has been indicated to control muscle hyperactivity and pain in bruxism and TMD. Because it is dose dependent, its action can last from 45 to 180 days, depending on the dosage (Chagas et al., 2018). Current knowledge states that after a period of three to six months from application, new nerve endings arise, restoring neuronal activity from the original nerve cell, and the affected muscles retrieves their contractile competence (Aoki, 2004). However, this recovering admits controversy, and some adverse effects may occur because of the misuse of BoNT-A protocols. Among the factors that cause problems refers to inappropriate use of high or repeated doses of BoNT-A into the stomatognathic muscle, that causes impairment muscle activity, decreases masticatory competence and changes the bone structure of origin and muscle insertion. (De La Torre et al., 2020).
Study developed in animals showed that muscle fibers injected with repeated high doses of BoNT-A compromise not only the muscle's labor activity, but the mass and composition of muscle fibers type IIa and IIb due to reduced mass function (Tsai and Lin, 2011). Our team also evidenced the deleterious effects of BoNT-A on human muscles when applied once, in high doses (De La Torre et al., 2020). There is clinical evidence of important changes in stomatognathic system structures over the time of toxin action. Possibly, the injected regions present severe changes in morphometry and cell nuclei, with replacement of muscle fibers by connective tissue and an expressive reactional neoformation of blood vessels in the affected sites of the muscle. However, few studies report the real histochemical consequences on muscle fibers injected with only a single high dose BoNT-A over time. Therefore, it is important to investigate this variables, considering the indication of an attack dose to recurrent TMD and
bruxism, if there is no deleterious effect on the musculature, or to contraindicate its use, if deleterious effects on the muscular muscles are proven.
Thus, this study aimed to evaluate the effects of a single high dose of BoNT-A on masseter muscle of rats over time. Morphometric effects, percentage analysis of substituting connective tissue, analysis of myocytes nuclei and count of newformed blood vessels in muscle were considered. Null hypothesis was that BoNT-A does not alter the musculoskeletal structures of the masseter muscle.
Methodology
Forty-four Wistar rats aged 60 days were used. The animals were acquired in the Central Bioterium of the University of Campinas (Unicamp) and kept in the Bioterium of the Dental School of Piracicaba at controlled room temperature, in a 12-hour light-dark controlled lighting cycle, with feed and water ad libitum throughout the experiment. Five animals were allocated in each polyethylene cage. The body mass of all animals was quantified daily from day 0 (before the intervention) to the end of the experiment (90 days). This project was approved by the Ethics Committee on the Use of Animals of Unicamp under protocol no. 4554-1/2017.
Experimental design
Sample size was determined by analyzing the relevant literature, considering 3 to 5 animals for each subgroup. The animals were randomly divided into 2 groups: the control group (CG) (n=22), that was injected with 14 mL of sodium chloride at 0.9% in the masseter muscle, and the treated group (GT) (n=22), that was injected in the same region with 7U/Kg of BoNT-A (Botox®, BoNT-Allergam, Inc. Irvine, CBoNT-A, USBoNT-A; 100 units were reconstituted in 2mL of 0.9% saline solution (Lora et al., 2017). The two groups were divided in five subgroups for sacrifice at the experimental days from BoNT-A application (day 0), becoming subgroups of 7th day
(n=5), 14th day (n=5), 21st day (n=5), 28th day (n=4) and 90th day (n=3). All animals and cages
were identified according to their respective subgroups.
Tissue extraction
Euthanasia of the animals was made to remove the tissues for analyse, according to the experimental design. Briefly, the animals were anesthetized with intraperitoneal injection of a mixture of Dopalen® (50 mg/mL) and Rompun® (2g/100mL), in a 1:1 ratio, at a dose of 0.3 mL/100 g body mass. After presenting signs of general anesthesia, the animals were infused with 60 mL of PBS solution (cardiac perfusion). The masseter muscles of animals were
removed, dissected, weighed, and divided into two proportional transverse parts intended for analysis in light microscopy.
Histology and morphometric analysis
The muscles slides were prepared for histological and morphometric analysis according to the predefined experimental the days of BoNT-A application. Briefly, tissue masseter samples were fixed in 20 volumes of 10% formaldehyde in Millonig buffer (pH 7.4) for 24h at 24ºC and processed for inclusion of Paraplast™. Histological slides of cross-sections of the samples (5μm) were stained by the hematoxylin-eosin method for structural observation and morphometric analysis. The following morphometric parameters were evaluated: area of muscle fibers and presence of connective tissue (% per 10,000 μm2), number of newformed
blood vessels, number of myocyte nuclei (n per 10,000 μm2), and average diameter of muscle
fibers (µm). Five fields of each of the five sections obtained from the mean region of the tissue collected in the animals of each experimental subgroup/days from BoNT-A application were documented. The mean diameter of the fibers was measured using 75 fibers of each experimental group/time (n=5 / 25 fibers per animal). All images were analyzed in a Leica DM2000 photomicroscope. The measurements were made from the captured images, using the Software Image Leica Measure and Sigma Scan Pro 5.0.
Statistical analysis
Descriptive and exploratory analyses of the data were performed. Exploratory analyses indicated that the data did not meet the assumptions of variance analysis (ANOVA). Then, generalized linear models were used.
Subgroups and days from BoNT-A application (day 0) were considered for the statistical analyses, as well as the interaction among them. The analyses were carried out with resources from the R Core Team program (2019) (Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria).
Results
The numerical results were organized and expressed in tables containing Means (standard deviations / SD) and Medians (minimum and maximum / min.; max.), and expressed in tables and graphics. Subjective results of the analysis of muscle fibers and the presence of connective tissue were expressed in comparative figures of groups and subgroups. It was observed that there was significant interaction among the study factors in the subgroups and the
days at evaluation (p<0.05) considering the values of the cross-sectional area of the masseter muscle (5μm) (Table 1, Graphic 1, and Figure 1).
In all the days of BoNT-A application analyzed, the area and values were significantly lower in the TG compared to the CG (p<0.05). In the CG there was a significant increase in the cross-sectional area of the muscle, with the perceptible results from day 7th to day 28th , and
the animals of the 90th day subgroup presented a much larger area than those of 7th , 14th and
21st days. However, among TG subgroups animals there was a significant decrease in the
cross-sectional area of muscle fibers, with a significant decrease when considering the days of BoNT-A application factor during the experimental period (p<0.05). But there was no significant alterations when the days of BoNT-A application factor was considered for subgroups of 7th ,
14th and 21st days (p>0.05).
Table 1. Cross-sectional area of the masseter muscle (5μm) in function of the group and the
days from BoNT-A application
Days Group
CG TG
Mean (SD) Median (min.; max.) Mean (SD) Median (min.; max.) 7 75,86 (1,91) Ac 76,06 (72,87; 78,15) 68,71 (1,14) Ba 69,00 (66,89; 69,86) 14 78.79 (1.51) Ab 78,23 (77,36; 80,92) 60,34 (2,66) Bb 61,08 (56,64; 63,20) 21 80.62 (0.69) Ab 80,78 (79,70; 81,52) 54,95 (2,04) Bc 55,34 (52,77; 56,94) 28 82,29 (0,85) Aab 82,27 (81,39; 83,25) 49,41 (1,26) Bd 49,30 (48,32; 50,74) 90 84,39 (0,14) Aa 84,43 (84,23; 84,50) 48,99 (1,00) Bd 48,64 (48,22; 50,12) Means followed by distinct letters (uppercase comparing horizontally and lowercase comparing vertically) differ from each other (p≤0.05); p(groups)<0.0001; p(days from BoNT-A application)<0.0001; p(interaction)<0.0001.
Graphic 1. Box plot of the cross-sectional area of the masseter muscle (5μm) considering the
subgroup and the days from BoNT-A application
CG
TG
5µm 5µm
Figure 1A: Fibers in cross sections of the masseter muscle (HE staining). CG: control group,
TG: treated group with BoNT-A; microscopic enlargement: 40x (line = 5μm). The arrows indicate a muscle fiber of the group 7th day. The difference in the size of the muscle fibers in the
CG
TG
Figure 1C: Fibers in cross sections of the masseter muscle (HE staining). CG: control group, TG:
treated group with BoNT-A; microscopic enlargement: 40x (line = 5μm). The arrows indicate a muscle fiber of the group 21st day. The difference in the size of the muscle fibers in the arrows
pointed out is noted. .
5µm 5µm
5µm 5µm
Figure 1B: Fibers in cross sections of the masseter muscle (HE staining). CG: control group, TG:
treated group with BoNT-A; microscopic enlargement: 40x (line = 5μm). The arrows indicate a muscle fiber of the group 14th day. The difference in the size of the muscle fibers in the arrows
Results of the amount of connective tissue (% in 10,000 μm2) show that the interaction between the factors was also significant for this variable (p<0.05) (Table 2, Graphic 2 and Figure 2). At all days analyzed, the amount of tissue was significantly higher in the BoNT-A subgroups than in the control subgroups (p<0.05). Among the control subgroups there was a
Figure 1D: Fibers in cross sections of the masseter muscle (HE staining). CG: control group, TG:
treated group with BoNT-A; Microscopic enlargement: 40x (line = 5μm). The arrows indicate a muscle fiber of the group 28th day. The difference in the size of the muscle fibers in the arrows
pointed out is noted.
CG
TG
5µm 5µm
5µm 5µm
CG
TG
Figure 1E: Fibers in cross sections of the masseter muscle (HE staining). CG: control group, TG:
treated group with BoNT-A; microscopic enlargement: 40x (line = 5μm). The arrows indicate a muscle fiber of the group 90th day. The difference in the size of the muscle fibers in the arrows pointed
significant decrease in the amount of tissue over time (p<0.05). In the 90th day subgroup the
amount of tissue was significantly lower than in the other subgroups (p<0.05). For the treated subgroups there was a significant increase in the amount of tissue over time, up to 28th day
(p<0.05). The subgroups with 28th and 90th day presented a higher amount of tissues than the
other subgroups (p<0.05), with no significant difference between them (p>0.05).
Table 2. Amount of connective tissue (% in 10,000 μm2) considering the groups the days from
BoNT-A application
Days Group
CG TG
Mean (SD) Median (min; max) Mean (SD) Median (min; max) 7 24,39 (2,13) Ba 24,42 (21,42; 27,09) 31.05 (1.41) 31,00 (29,43; 33,11) 14 21,17 (1,48) Bb 21,77 (19,08; 22,48) 39,61 (2,63) Ac 38,92 (36,80; 43,40) 21 19,37 (0,67) Bc 19,18 (18,48; 20,22) 45.00 (2.08) Ab 44,50 (42,90; 47,23) 28 17,71 (0,85) Bd 17,74 (16,75; 18,61) 50,60 (1,29) Aa 50,70 (49,26; 51,76) 90 15,61 (0,18) Be 15,57 (15,46; 15,81) 51,03 (1,02) Aa 51,40 (49,88; 51,81) Means followed by distinct letters (uppercase comparing horizontally and lowercase comparing vertically) differ from each other (p≤0.05). p(groups)<0.0001; p(days from BoNT-A application)<0.0001; p(interaction)<0.0001.
Graphic 2. Box Plot of the amount of connective tissue (% in 10,000 μm2) considering the
Figure 2A: Connective tissue of the masseter muscle stained with HE. CG: control group;
GT: treated group with BoNT-A; microscopic enlargement: 40x (% at 10,000 μm2). Arrows
indicate connective tissue of the subgroup 7th day. The difference in the amount of connective tissue in the pointed arrows is noted.
Figure 2B: Connective tissue of the masseter muscle stained with HE. CG: control group;
GT: treated group with BoNT-A; microscopic enlargement: 40x (% at 10,000 μm2). Arrows
indicate connective tissue of the subgroup 14th day. The difference in the amount of connective
tissue in the pointed arrows is noted.
CG
TG
Figure 2C: Connective tissue of the masseter muscle stained with HE. CG: control group;
GT: treated group with BoNT-A; microscopic enlargement: 40x (% at 10,000 μm2). Arrows
indicate connective tissue of the subgroup 21st day. The difference in the amount of connective
tissue in the pointed arrows is noted.
Figure 2D: Connective tissue of the masseter muscle stained with HE. CG: control group;
GT: treated group with BoNT-A; microscopic enlargement: 40x (% at 10,000 μm2). Arrows
indicate connective tissue of the subgroup 28th day. The difference in the amount of connective
tissue in the pointed arrows is noted.
Figure 2E: Connective tissue of the masseter muscle stained with HE. Group: CG: control
group; TG: treated group with BoNT-A; microscopic enlargement: 40x (% at 10,000 μm2).
Arrows indicate connective tissue of the subgroup 90th day. The difference in the amount of
connective tissue in the pointed arrows is noted.
Table 3 and Figures 3 shows that the interaction between subgroups and day from BoNT-A application factors was also significant for myocyte count. In the 7th day, there was
no significant difference between the CG and TG (p>0.05). From 14th day on, the treated
subgroups presented higher myocyte counts than the control groups, when compared at the same times (p<0.05). Among the control groups, there was a decrease in the count in the subgroup of 14th day in relation to 7th das, remaining up to 28th days and with a subsequent
decrease in the subgroup of 90th day (p<0.05). Among the treated subgroups there was an
increase with 21st day, remaining up to 90th day (p<0.05).
Table 3. Myocyte count (10,000 μm2) considering group and BoNT-A action days
Days Group
GC GT
Mean (SD) Median (min; max) Mean (SD) Median (min; max) 7 4,78 (0,74) Aa 4,84 (3,64; 5,52) 5.24 (0.47) Ab 5,44 (4,64; 5,80) 14 3,69 (0,23) Bbc 3,56 (3,48; 4,04) 5.39 (0.68) Ab 5,36 (4,76; 6,52) 21 4,07 (0,21) Bb 4,04 (3,80; 4,28) 6,73 (0,27) Aa 6,68 (6,40; 7,08) 28 4,05 (0,18) Bb 3,98 (3,92; 4,32) 6,45 (0,35) Aa 6,40 (6,08; 6,92) 90 3,28 (0,11) Bc 3,32 (3,16; 3,36) 6,08 (0,59) Aa 6,36 (5,40; 6,48) Means followed by distinct letters (uppercase comparing horizontally and lowercase comparing vertically) differ from each other (p≤0.05). p(subgroups)<0.0001; p(days of BoNT-A application)=0.0009; p(interaction)<0.0001.
Graphic 3. Box plot, amount of myocytes (10,000 μm2) according to the subgroups and the
days from BoNT-A application.
For blood vessel counts (Table 4 and Graphic 4) there was no significant interaction among study factors (p>0.05). Regardless of time, the BoNT-A subgroups presented higher counting than the control subgroups (p<0.05). Regardless of the subgroup, there was an increase in the number of blood vessels up to 21st days (p<0.05).
Table 4. Blood vessel count (10,000 μm2) considering the group and the days from BoNT-A
application
Days Group Comparisons
GC GT
Mean (SD) Median (min; max) Mean (SD) Median (min; max)
7 1,31 (0,26) 1,44 (0,96; 1,52) 1,40 (0,06) 1,40 (1,32; 1,48) c 14 1,29 (0,14) 1,24 (1,16; 1,52) 1,54 (0,17) 1,60 (1,36; 1,72) bc 21 1,61 (0,26) 1,52 (1,32; 1,96) 1,72 (0,37) 1,52 (1,40; 2,16) a 28 1,36 (0,17) 1,30 (1,24; 1,60) 1,87 (0,19) 1,92 (1,60; 2,04) ab 90 1,55 (0,12) 1,52 (1,44; 1,68) 1,95 (0,49) 1,84 (1,52; 2,48) a Comparisons B A
Distinct letters (uppercase comparing horizontally and lowercase comparing vertically) indicate significant differences (p≤0.05). (subgroups)=0.0002; p(days of BoNT-A application)=0.0020; p(interaction)=0.3813.
Graphic 4. Box plot of blood vessel count (10,000 μm2)considering the subgroup and the days
from BoNT-A application
There was significant interaction in the diameter of myocytes (Table 5 and Figure 5) (p<0.05). For all subgroups, the diameter was significantly smaller in the BoNT-A subgroups (p<0.05). There was an increase to CG in the 14th day subgroup at the 7th day, remaining up to
28th day (p>0.05), with a subsequent increase at 90th day (p<0.05). In TG, there was a significant
decrease up to 28th day (p<0.05), remaining up to 90th day (p>0.05).
Table 5. Diameter of myocytes (μm) considering on the group and the days from BoNT-A
application.
Days Group
GC GT
Mean (SD) Median (min; max) Mean (SD) Median (min; max) 7 9,50 (0,25) Ac 9,32 (9,32; 9,86) 8,09 (0,41) Ba 7,80 (7,80; 8,67) 14 10.37 (0.06) Ab 10,34 (10,34; 10,47) 7,20 (0,23) Bb 7,07 (7,07; 7,60) 21 10.66 (0.17) Ab 10,54 (10,54; 10,90) 6,64 (0,34) Bc 6,41 (6,41; 7,16) 28 11.00 (0.14) Ab 10,97 (10,88; 11,16) 6,06 (0,22) Bd 5,99 (5,89; 6,36) 90 13,71 (2,69) Aa 14,03 (10,88; 16,23) 6,01 (0,15) Bd 5,92 (5,92; 6,18)
Means followed by distinct letters (uppercase comparing horizontally and lowercase comparing vertically) differ from each other (p≤0.05). p(subgroups)<0.0001; p(days of BoNT-A application)=0.0013; p(interaction)<0.0001.
Graphic 5. Box plot of the diameter of myocytes (μm) considering the groups and the days
from BoNT-A application.
Discussion
This study aimed to investigate the morphometric alterations that occurred in musculoskeletal tissue after application of a single high dose of BoNT-A and its consequences on masseter muscle over time.
It is known biological tissues have the ability to adapt through numerous functional demands, and muscle tissue is one of the most affected when submitted to stimuli (Aquino, Viana and Fonseca, 2005). Musculoskeletal tissue consists in elongated cells with cytoplasmic filament numbers, in which myofibrils are responsible for generating cell contraction. When changing, their biochemical, mechanical and structural functions develop muscle atrophy, where the reduction of myofibers is what characterizes the decrease in muscle mass (Ferreira and Neuparth, 2004).
After statistical analysis, we observed that the area of the cross-section of masseter muscle (5μm) was significantly smaller for TG than CG (p<0.05) on all days evaluation. These findings corroborate with the results of Lee et al., (2017) when investigated soft and hard tissue changes after BoNT-A administration in humans. They observed that repeated application of BoNT-A may induce changes in musculoskeletal tissues (Lee et al., 2017).
There is evidence that the average thickness and areas of the cross section of the masseter decrease on average from 18 to 20% in thickness after BoNT-A injection. (Park and Ahn, 2003, Kim and Jeon, 2000). The natural tendency of the musculature, when not altered by syndromes, pathological processes or drugs, is to increase over time during the stage of tissue
development, and then to decline due to aging. The animals of our experiment were in adulthood and in the still developing phase. The data observed in tables, graphics and figures reveal that the cross-sectional area of the masseter muscle (5μm) increased over time CG. There was a significant increase with 14th days in relation to 7th days. Also at 90th subgroup when
compared with 7th, 14th and 21st (p<0.05). However, when we applied a single high dose of
BoNT-A into masseter muscle of the TG, the cross-sectional area of the muscle, that expresses the size of the muscle fiber, decreased appreciably over time, until the 28th postoperative day
(p<0.05). Analyzing this consequence of a single high dose used in our study, we consider that must be careful in some clinical protocols that indicate higher or repeated doses to control TMD, bruxism and masseter muscle hypertrophy. It is also observed in literature (Jadhao et al., 2017; Fedorowicz and Van Zuuren, 2013; Kim and Chung, 2005; Choe et al., 2005; Lindern, 2001). This suggestion should also be observed in treatments of facial muscles with essentially aesthetic purpose. In this case, high doses of BoNT-A are used in weaker and thinner muscles than the masseter, and certainly these adverse effects will also be assimilated by those muscles over time. In a blind and randomized clinical trial, our team investigated the adverse effects of a single high dosage on the consequent effects on masticatory, muscle and bone physiology (De La Torre et al., 2020), where we verified significant changes in masticatory competence, muscle activity and important bone changes.
When used in rats, the dosage of the present study (7U/Kg) demonstrated efficacy in controlling nociception, as well as in reducing substance P, CGRP and IL1ß, all pain promoting agents (Lora et al., 2017). Comparing both studies, there was a need to prove how much this dosage, considered high, could interfere in the physiology of the structures involved in chewing, even though it is beneficial to control the pain present in clinical conditions associated with bruxism and TMD. Mainly because half of BoNT-A dose (3.5U /Kg) inhibits glutamate production in rats sensitized for TMD pain in the same way as a high dose (7U/Kg) (Cui, Khanijou and Aoki, 2004).
Our study allowed to verify that a high dose, even if applied once, leads to significant changes in muscle structure that can cause highly deleterious effects on the tissue physiology. Interestingly, in addition to the changes in muscle, there was also a gradual and methodical replacement of muscle fibers by fibrous connective tissue, demonstrating the possibly irreversibility of the situation. The large infiltrate of connective tissue replacing muscle tissue in the TG increased significantly over time (p<0.05) throughout all experimental period, suggesting the presence of fibrosis and, consequently, muscle disfunction. This was not
observed in CG, which presented the quantitative and gradual decrease of connective tissue and increase in muscle fiber size over time (p<0.05).
The proliferation of intramuscular connective tissue after application of BoNT-A is related to decreased muscle activity. It is considered a powerful blocker of neuromuscular junction Ach releasing, which induces muscle paralysis. Loss of function leads to progressive muscle atrophy (Hwang et al., 2006). These observations have a strong relationship with the biochemistry of the muscle cell that precedes this effect. When there is no muscle activity, important changes occur in mitochondria and substances related to myocyte physiology, such as the mRNA level of Atrogin-1/MAFbx, muscle RING finger 1 (MuRF-1), Muscle atrophy F-box (MuRF-1), and Myogenin (Myo) (Balanta-Melo et al., 2018); that may explain what happens in the chemical denervation promoted by BoNT-A. In this way, the decrease in muscle mechanical tension alters growth production and collagen synthesis, while increasing the proliferation of connective tissue. Thus, fibroblasts, which are subject to muscle tensions, both active and passive, have altered their composition and metabolism (Hwang et al., 2006).
The function of musculoskeletal structures depends on the balance among mechanical, neural, and physiological factors, so that it exercises its function and dynamics in the best possible way. knowing the structural and morphological changes in tissues, especially muscle, is essential to prevent adverse effects. Controlling the inactivity of the musculoskeletal structure through the correct use of BoNT-A, the professional will have greater capacity to evaluate and conduce treatment according to the biological individualities of the patients.
Many myoprogenitor cells merge and form several peripheral nuclei to form new cells, which gives them great exposure capacity of contrite proteins to form myofibers (Shenkman et al., 2010). Skeletal striated muscle fibers are multinucleated and the nuclei are located in the peripheral region of the fiber, below the plasma membrane (Silva and Carvalho, 2007). Changes in the metabolism of these cells induce changes in muscle regeneration and proliferation, as well as in the amount and size of cell nuclei (Addison et al.,2019; Ito, Higa and Goto, 2018). In this study, it was observed, that there was no significant difference among the groups regarding myocytes count at 7th day (p>0.05). However, the TG presented significantly higher
counts than the CG on 14th day (p<0.05), as well as the diameter of myocytes were significantly
lower in the TG (p<0.05), for all evaluated days. The amount of myocytes decreased significantly in TG over time (p<0.05). In CG there was a significant decrease in the days of BoNT-A of 14th day in relation to 7th day, and then on 90th day in relation to 7th , 21st and 28th
significant increase in the 14th day when compared to 7th day (p<0.05), and in the 90th day when
compared to the other days (p<0.05).
The amount of myocytes increased over time on TG. There was a significant increase in the days of BoNT-A of 21st day in relation to 7th and 14th day, remaining until the days of
BoNT-A of 90th day (p>0.05), and the diameter of myocytes decreased significantly over time
up to 28th days (p<0.05). The muscle tissue has great capacity for regeneration, even when
subjected to several cycles of aggression (Zammit, Partridge and Yablonka-Reuveni, 2006). There is evidence that these cells proliferate and divide asymmetrically, generating other cells, forming a stock of mother cells and daughter cells by differentiation. This condition merge to repair or replace damaged muscle fibers due to some change in their conformation due to some trauma (Broek et al., 2010; Zammit et al., 2004; Rantanen et al., 1995). The inactivation promoted by the high dose of BoNT-A probably constituted a factor of cellular aggression, and we observed an evident attempt to repair the inactivated cell tissues by increasing the number and myocytes and their union, forming a cluster of cells, which was evidenced over time. This fact, when transposed to the clinic, provides support to consider that the muscle alterations promoted by BoNT-A can be considered as an adverse effect, since they may be configured as an irreversible process.
Interestingly, this fact probably motivated a substantial compensatory increase in vascular neo formation that occurred in TG, both by sprouting and intussusception, up to 21st day (p<0.05). The endothelial vascular growth factor (VEGF) is a cytokine of great
relevance for the microvasculature of muscle tissue, so its high expression favors the regeneration of muscle cells (Prior, Yang and Terjung, 1985). In order to stabilize vessels in muscle regeneration, endothelial cells interact with myogenic precursors and promote the process of myogenesis and angiogenesis (Yin, Price and Rudnicki, 2013; Arsic et al., 2004). This probably happened as an attempt of tissue repair, since it is a functional muscle involved in mastication, a vital activity necessary for the survival of the animal. In a previous study developed by our team, we found that, in humans, there is a classic decrease in the myoelectric activity of the mastication muscles when submitted to high doses of BoNT-A, which sensitively impairs of chewing (De La Torre et al., 2020).
In this study, there was a need to grind the feed of TG animals to continue feeding, and in order to preserve their lives until the end of the experiment. Because of this event, one animal of the 28th day and two animals of the 90th day belonging to the TG did not resist changes in
their masticatory competence. So it was necessary to reduce the animals equivalently to the CG as well. We did not consider this an experimental bias because the sample calculation allowed
us to quantify the samples from 3 to 5 animals per day subgroup, without prejudice the dependent variables data.
These evident muscle injuries need to be considered when in the indication of muscle treatment by BoNT-A taking into account the need for high doses, as well as the frequency of applications. It is also necessary to consider this possibility of muscles alteration when the purposed treatments are essentially for aesthetic applications in mimic muscles, because in the same way that changes occur in functional muscles, they certainly happen in facial expression muscles. Therefore, its indiscriminate use needs to be reconsidered, since it may lead to deleterious effects over time.
Finally, we believe that randomized and blind clinical trials should be conducted in humans, both investigating functional muscles and mimic muscles of the face, in order to confirm whether these findings are repeated, considering that the facial muscles have smaller volume and are thinner than the functional ones. Myoelectric activity and muscle thickness by ultrasound imaging can be investigated considering the dose of BoNT-A over time.
It is also recommended to develop complementary studies to verify the effects of these substances at the cellular level as to axonal budding, as well as biochemical changes that may be present in the affected cells before the use of BoNT-A.
Conclusion
A single high dose of BoNT-A alters the cross-sectional area of the masseter muscles, promotes local new vascularization, increases connective tissue formation, as well as increments the number and diameter of myocytes over time.
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3 DISCUSSÃO
O equilíbrio muscular está essencialmente relacionado com a capacidade de modificação muscular e de novas condições a que se submete, uma vez que as alterações anátomo-funcionais baseiam-se em repetidos redirecionamentos do fluxo de energia de acordo com a disponibilidade de substratos metabólicos. Nesta, a plasticidade metabólica das fibras musculares possibilita ajustes constantes e lineares buscando adaptar-se a tríade atividade/demanda/disponibilidade de substratos (Durigan et al., 2005).
As manifestações clínicas comumente associadas ao uso de toxina botulínica-A (BoNT-A) são as alterações morfofuncionais que ocorrem nos tecidos. As propriedades mecânicas das estruturas ficam comprometidas, e, desta forma, diminuem a capacidade de produção de força em diferentes comprimentos e velocidades, podendo assim influenciar de maneira significativa a vida dos indivíduos (Lieber; Fridén, 2000).
A análise estatística deste estudo demonstrou que houve uma diminuição significativa da área de secção transversa do músculo masseter (5μm) (p<0,05) no grupo que recebeu aplicação de BoNT-A. Esses achados reforçam os que Lee et al. (2017), que observaram as alterações de tecidos moles e duros após a administração de injeção de BoNT-A em músculo masseter com hipertrofia. Os autores utilizaram tomografia computadorizada tridimensional para observar as alterações estruturais e constataram que, quando administradas em intervalos curtos e doses não controladas, a substância proporciona alterações significativas nos músculos. Há evidência científica demonstrando que a inatividade muscular gera atrofia nas suas estruturas e, consequentemente, diminuem sua área de secção transversa, assim como Herrera et al. (2001) observaram em seus grupos experimentais ao avaliarem o músculo estriado esquelético de ratos após inatividade muscular.
Tanaka et al. (2004) associaram os tipos de fibras musculares com o grau de atrofia muscular e verificaram em seus estudos que as fibras do tipo I tem maior propensão em reduzir sua estruturação física durante a inativação muscular, decorrente da diminuição de estímulos às fibras durante condições de movimento.
Satoh et al. (2001), após aplicarem a BoNT-A no músculo masseter, analisaram a atividade muscular, o metabolismo e a composição das fibras usando eletromiografia (EMG), ressonância magnética (P-MRS) e análise histoquímica. A EMG não apontou hiperatividade muscular, e a P-MRS mostrou aspecto normal. No entanto, a composição das fibras musculares apontou grande diferença entre as que receberam BoNT-A e o músculo sadio, demonstrando perda das fibras do tipo IIB, aumento das fibras do tipo IIA, e do tipo IM e II, e uma diminuição das fibras do tipo I, assim como observaram Tanaka et al. (2004).
Em um processo natural de maturação muscular, o músculo modifica suas microestruturas e aumenta a sua conformidade, quando não existem síndromes, patologias ou sob efeito de drogas miorrelaxantes. O aumento progressivo do tecido ocorre com o fator idade, e ao longo do desenvolvimento, e espera-se uma diminuição natural devido aos efeitos fisiológicos do envelhecimento. Pelo fato de os animais do grupo controle estarem em processo de desenvolvimento próximo à fase adulta, a musculatura permaneceu no seu percurso natural de desenvolvimento e apresentou um aumento das suas fibras, e consequente aumento da área de secção transversa (5μm). Em contrapartida, no grupo que recebeu aplicação de toxina, observou-se uma diminuição significativa (p<0,05) com o aumento do tempo até 28 dias pós operatórios, como pode ser observado nas análises de dados.
Investigando os desfechos da aplicação de uma única e alta dose de toxina durante o desenvolvimento do projeto, ressalta-se a importância dos respaldos de boas bases cientificas que garantam eficiência no uso dessa substância. O cuidado ao considerar alguns protocolos clínicos que indicam doses maiores ou repetidas, ou aqueles que não respeitam o tempo adequado de recuperação das estruturas envolvidas deve ser tido como norma (Jadhao et al., 2017; Fedorowicz e Van Zuuren, 2013; Kim e Chung, 2005; Choe et al., 2005; Lindern, 2001). Sabe-se que a função das estruturas musculoesqueléticas depende do equilíbrio entre fatores mecânicos, neurais e fisiológicos para que exista uma dinâmica funcional diante de sua melhor performance (Piovesan, 2010). A diminuição da tensão mecânica muscular causada pela administração de uma única dose de BoNT-A altera a proliferação do tecido conjuntivo, seu crescimento e a síntese de colágeno. Os fibroblastos, que estão sujeitos as tensões musculares, tanto ativas como passivas, têm a sua composição e metabolismo alterados, assim como a densidade de área de tecido conjuntivo intramuscular (Hwang et al., 2006).
Com a análise estatística, foi possível observar que, ao longo do tempo após a aplicação, houve aumento gradual e significativo do tecido conjuntivo (p<0,05) no grupo que recebeu a toxina, sinalizando a presença de fibrose em substituição à fibra muscular. Quando analisado o grupo controle, não houve aumento do tecido conjuntivo, mas sim sua diminuição progressiva (p<0,05) à medida que a musculatura foi se desenvolvendo ao longo do tempo.
Durante o processo de miogênese os mióctios, que se apresentam moninucleados, se juntam para formar miotubos. No animal em desenvolvimento normal, o músculo estriado esquelético se torna um tecido estável e caracterizado por fibras musculares nucleadas (Schmalbruch & Lewis, 2000; Decary et al., 1997). Esta estruturação das fibras musculares não foi observada nos animais dos subgrupos tratados, particularmente naqueles de maior tempo de inatividade muscular.
Mudanças nas composições do metabolismo dessas estruturas sugerem alterações na regeneração e proliferação muscular, bem como no tamanho e quantidade das células (Addison et al., 2019; Ito, Higa e Goto, 2018). Neste estudo, observou-se que houve alterações significativa entre os grupos tratado e controle no que se refere à contagem de miócitos. Os subgrupos tratados apresentaram contagens maiores do que os seus respectivos controles (p<0,05), bem como os diâmetros dos miócitos foram significativamente menores nos subgrupos do GT para todos os períodos investigados (p<0,05). Nos subgrupos controles houve uma diminuição em número (p<0,05) e aumento em diâmetro dos miócitos ao longo do tempo (p<0,05).
Há evidências de que essas células se proliferam e se dividem assimetricamente, gerando outras células, formando então um aglomerado de células para diferenciação, que acabam por se juntar para reparar ou substituir as fibras musculares que foram submetidas a alguma mudança na sua conformação devido a traumas. (Broek, Grefte e Von Den Hoff, 2010; Zammit et al., 2004; Rantanen et al., 1995). Em função da alta dose de BoNT-A, sugere-se que houve uma agressão celular significativa nas estruturas musculares, revelada pela tentativa de reparo tecidual.
Este fato pode ter motivado a neoformação vascular, tanto por brotamento quanto por intussuscepção, apresentada no grupo tratado. Este achado corrobora com Yin, Price e Rudnicki (2013) e Arsic et al. (2004) quando descrevem que, durante a regeneração muscular, para a estabilização dos vasos, as células endoteliais interagem com precursores miogênicos e promovem o processo de miogênese e angiogênese, sendo que o fator de crescimento vascular endotelial (VEGF) tem relevância na formação microvasculatura do tecido muscular, e sua alta expressão favorece a regeneração de células musculares.
Ocorreu neste estudo, a necessidade de modificar a alimentação dos animais do grupo toxina, devido à falta de ativação muscular e deficiência na competência mastigatória que os animais apresentaram. Isto foi necessário para mantê-los vivos até o término do experimento. Diante disso, ocorreu a perda de alguns animais no decorrer do projeto, o que levou à redução no número dos animais do grupo tratado, e, para equivalência estatística, do grupo controle, nos grupos de 28° e 90° dias. Porém, isto não comprometeu a amostragem experimental e não sucedeu prejuízos dos dados analisados, uma vez que o cálculo amostral previa de 3 a 5 animais por subgrupo. Ainda, seguiu-se à risca todas as recomendações do comitê de ética em pesquisa em experimentação animal quanto à alimentação, hidratação, controle do ambiente, temperatura e ciclo claro-escuro.
Diante do exposto, considera-se que as lesões estruturais do músculo precisam ser levadas em consideração quando há indicação de alguma intervenção utilizando BoNT-A, principalmente no que diz respeito às dosagens altas e frequência das aplicações, seja para controle de dores orofaciais ou aplicações estéticas em músculos da mímica. Seu uso imoderado precisa ser repensado, pois pode causar alterações significativas ao tecido muscular ao longo do tempo.
Recomenda-se desenvolver estudos complementares para verificar os efeitos dessas substâncias no nível celular quanto ao brotamento axonal, bem como alterações bioquímicas que podem estar presentes nas células afetadas pela BoNT-A.
4 CONCLUSÃO
Uma dose única alta de BoNT-A altera a área transversal dos músculos masseter, promove a neovascularização local, aumenta a formação de tecido conjuntivo, além de aumentar o número e o diâmetro dos miócitos ao longo do tempo.
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2 De acordo com as normas da UNICAMP/FOP, baseadas na padronização do International Committee of Medical Journal
