Brazilian Journal of Anesthesiology
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

Downloads: 0
Views: 40


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.

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 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.

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.

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.


Neurotoxicity;  Autophagy;  Local anesthetics;  Apoptosis


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.

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 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.

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.

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.


Neurotoxicidade;  Autofagia;  Anestésicos locais;  Apoptose


1 S. Sakura, Y. Kirihara, T. Muguruma, et al. The comparative neurotoxicity of intrathecal lidocaine and bupivacaine in rats Anesth Analg., 101 (2005), pp. 541-547

2 J. Kato, J. Konishi, H. Yoshida, et al. Cauda equina syndrome following combined spinal and epidural anesthesia: a case report Can J Anaesth., 58 (2011), pp. 638-641

3 T. Komatsu, T. Takenami, Y. Nara, et al. Epinephrine administered with lidocaine solution does not worsen intrathecal lidocaine neurotoxicity in rats Region Anesth Pain M., 38 (2013), pp. 140-144

4 X. Wen, S. Xu, H. Liu, et al. Neurotoxicity induced by bupivacaine via T-type calcium channels in SH-SY5Y cells Plos One., 8 (2013), p. e62942

5 K.F. Hampl, M.C. Schneider, K. Drasner Toxicity of spinal local anaesthetics Curr Opin Anaesthesiol., 12 (1999), pp. 559-564

6 C.H. Jing, L. Wang, P.P. Liu, et al. Autophagy activation is associated with neuroprotection against apoptosis via a mitochondrial pathway in a rat model of subarachnoid hemorrhage Neuroscience., 213 (2012), pp. 144-153

7 V. Nikoletopoulou, M. Markaki, K. Palikaras, et al. Crosstalk between apoptosis, necrosis and autophagy Biochimica Et Biophysica Acta., 1833 (2013), pp. 3448-3459

8 S. Carloni, S. Girelli, C. Scopa, et al. Activation of autophagy and Akt/CREB signaling play an equivalent role in the neuroprotective effect of rapamycin in neonatal hypoxia-ischemia Autophagy., 6 (2010), pp. 366-377

9 N. Mizushima, B. Levine, A.M. Cuervo, et al. Autophagy fights disease through cellular self-digestion Nature., 451 (2008), p. 1069

10 K. Nishida, M. Taneike, K. Otsu The role of autophagic degradation in the heart J Mol Cell Cardiol., 78 (2015), pp. 73-79

11 IA Kochergin, MN. Zakharova The role of autophagy in neurodegenerative diseases Neurochemical Journal., 10 (2016), pp. 7-18

12 KT Jung, KJ. Lim Autophagy: can it be a new experimental research method of neuropathic pain? Korean J Pain., 28 (2015), p. 229

13 H.J. Wu, J.L. Pu, P.R. Krafft, et al. The molecular mechanisms between autophagy and apoptosis: potential role in central nervous system disorders Cell Mol Neurobiol., 35 (2015), pp. 85-99

14 P. Boya, R.A. Gonzálezpolo, N. Casares, et al. Inhibition of macroautophagy triggers apoptosis Cell Mol Biol Lett., 25 (2005), pp. 1025-1040

15 P Codogno, AJ. Meijer Autophagy and signaling: their role in cell survival and cell death Cell Death Differ., 12 (2005), pp. 1509-1518

16 J. Xiong, Q. Kong, L. Dai, et al. Autophagy activated by tuberin/mTOR/p70S6K suppression is a protective mechanism against local anaesthetics neurotoxicity J Cell Mol Med., 21 (2017), pp. 579-587

17 R. Li, H. Ma, X. Zhang, et al. Impaired autophagosome clearance contributes to local anesthetic bupivacaine-induced myotoxicity in mouse myoblasts Anesthesiology., 122 (2015), pp. 595-605

18 DJ Klionsky, SD. Emr Autophagy as a regulated pathway of cellular degradation Science., 290 (2000), pp. 1717-1721

19 Y. Kabeya, N. Mizushima, A. Yamamoto, et al. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation J Cell Sci., 117 (2004), pp. 2805-2812

20 R.U. Jänicke, M.L. Sprengart, M.R. Wati, et al. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis J Biol Chem., 273 (1998), pp. 9357-9360

21 H. Liu, R. Zhu, C. Liu, et al. Evaluation of decalcification techniques for rat femurs using he and immunohistochemical staining Biomed Res Int., 2017 (2017), pp. 1-6

22 P. Formichi, C. Battisti, S. Bianchi, et al. Evidence of apoptosis via TUNEL staining in muscle biopsy from patients with mitochondrial encephaloneuromyopathies J Submicrosc Cytol Pathol., 35 (2003), pp. 29-34

23 S. Hu, W.S. Sheng, J.R. Lokensgard, et al. Morphine induces apoptosis of human microglia and neurons Neuropharmacology., 42 (2002), pp. 829-836

24 C.J. Park, S.A. Park, T.G. Yoon, et al. Bupivacaine induces apoptosis via ROS in the Schwann cell line J Dent Res., 84 (2005), pp. 852-857

25 AG Porter, RU. Jänicke Emerging roles of caspase-3 in apoptosis Cell Death Differ., 6 (1999), pp. 99-104

26 A. Hartmann, S. Hunot, P.P. Michel, et al. Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease Proceedings of the National Academy of Sciences of the United States of America., 97 (2000), pp. 2875-2880

27 C. Liu, Y. Gao, J. Barrett, et al. Autophagy and protein aggregation after brain ischemia J Neurochem., 115 (2010), pp. 68-78

28 T Shintani, DJ. Klionsky Autophagy in Health and Disease: A Double-Edged Sword Science., 306 (2004), pp. 990-995

29 L. Galluzzi, J.M. Vicencio, O. Kepp, et al. To die or not to die: that is the autophagic question Curr Mol Med., 8 (2008), pp. 78-91

5fbfe7940e88251163f5cc93 rba Articles
Links & Downloads

Rev. Bras. Anestesiol.

Share this page
Page Sections