Brazilian Journal of Anesthesiology
https://bjan-sba.org/article/doi/10.1016/j.bjane.2020.11.001
Brazilian Journal of Anesthesiology
Experimental Trials

Autophagy activation attenuates the neurotoxicity of local anaesthetics by decreasing caspase-3 activity in rats

A ativação autofágica atenua a neurotoxicidade dos anestésicos locais ao diminuir a atividade da caspase-3 em ratos

Xing Xue, Ying Lv, Yufang Leng, Yan Zhang

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Abstract

Background and objectives
The mechanisms by which local anaesthetics cause neurotoxicity are very complicated. Apoptosis and autophagy are highly coordinated mechanisms that maintain cellular homeostasis against stress. Studies have shown that autophagy activation serves as a protective mechanism in vitro. However, whether it also plays the same role in vivo is unclear. The aim of this study was to explore the role of autophagy in local anaesthetic-induced neurotoxicity and to elucidate the mechanism of neurotoxicity in an intrathecally injected rat model.

Methods
Eighteen healthy adult male Sprague-Dawley rats were randomly divided into three groups. Before receiving an intrathecal injection of 1% bupivacaine, each rat received an intraperitoneal injection of vehicle or rapamycin (1 mg.kg-1) once a day for 3 days. The pathological changes were examined by Haematoxylin and Eosin (HE) staining. Apoptosis was analysed by TdT-mediated dUTP Nick-End Labelling (TUNEL) staining. Caspase-3, Beclin1 and LC3 expression was examined by Immunohistochemical (IHC) staining. Beclin1 and LC3 expression and the LC3-II/LC3-I ratio were detected by western blot analysis.

Results
After bupivacaine was injected intrathecally, pathological damage occurred in spinal cord neurons, and the levels of apoptosis and caspase-3 increased. Enhancement of autophagy with rapamycin markedly alleviated the pathological changes and decreased the levels of apoptosis and caspase-3 while increasing the expression of LC3 and Beclin1 and the ratio of LC3-II to LC3-I.

Conclusions
Enhancement of autophagy decreases caspase-3-dependent apoptosis and improves neuronal survivalin vivo. Activation of autophagy may be a potential therapeutic strategy for local anaesthetic-induced neurotoxicity.

Keywords

Neurotoxicity;  Autophagy;  Local anesthetics;  Apoptosis

Resumo

Introdução e objetivos
Os mecanismos de neurotoxicidade dos anestésicos locais são complexos. A apoptose e a autofagia são mecanismos altamente organizados que mantêm a homeostase celular durante o estresse. Estudos revelam que a ativação da autofagia atua como mecanismo de proteção in vitro. Não está claro se a autofagia também desempenha essa função in vivo. O objetivo deste estudo foi analisar o papel da autofagia na neurotoxicidade induzida por anestésico local e esclarecer o mecanismo dessa neurotoxicidade utilizando um modelo de injeção intratecal em ratos.

Métodos
Dezoito ratos Sprague-Dawley machos adultos saudáveis ​​foram divididos aleatoriamente em três grupos. Antes de receber a injeção intratecal de bupivacaína a 1%, cada rato recebeu injeção intraperitoneal de veículo ou rapamicina (1 mg.kg-1) uma vez ao dia durante 3 dias. As alterações patológicas foram examinadas por coloração com Hematoxilina e Eosina (HE). A apoptose foi analisada por coloração com o método dUTP Nick-End Labeling (TUNEL) mediado por TdT. A expressão de caspase-3, Beclin1 e LC3 foram examinadas por coloração Imunohistoquímica (IHQ). A expressão de Beclin1 e LC3 e a razão LC3-II/LC3-I foram detectadas por análise de western blot.

Resultados
Após a injeção intratecal de bupivacaína, ocorreu lesão patológica nos neurônios da medula espinhal e os níveis de apoptose e caspase-3 aumentaram. A ativação da autofagia causada pela rapamicina mitigou de forma expressiva as alterações patológicas e diminuiu os níveis de apoptose e caspase-3, aumentando a expressão de LC3 e Beclin1 e a razão LC3-II/LC3-I.

Conclusões
O aumento da autofagia diminui a apoptose dependente da caspase-3 e melhora a sobrevivência neuronalin vivo. A ativação da autofagia pode ser uma estratégia terapêutica potencial para a neurotoxicidade induzida por anestésicos locais.

Palavras-chave

Neurotoxicidade;  Autofagia;  Anestésicos locais;  Apoptose

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