布比卡因致心肌细胞线粒体损伤大鼠模型的建立
|
摘要
目的观察电刺激下布比卡因心肌细胞线粒体形态变化及活性氧(reactive oxygen species, ROS)生成量,探讨建立理想的布比卡因中毒的大鼠心肌细胞模型。方法采用Langendroff装置新鲜分离雄性SD大鼠心肌细胞,细胞计数后将其转移至doff管中随机分为四组:DMEM静置组、DMEM电刺激组、布比卡因静置组、布比卡因电刺激组。实验重复五次。采用透射电镜观察心肌细胞线粒体形态,并使用多功能微孔板检测仪测量ROS生成量。结果 DMEM电刺激组线粒体肿胀程度及ROS生成量与DMEM静置组相比差异无显著性(P> 0.05);而布比卡因电刺激组线粒体肿胀程度明显高于布比卡因静置组(P=0.000),且ROS生成量也明显升高(P<0.05)。结论电刺激下心肌细胞呈节律性收缩,能更好地模拟临床布比卡因中毒时心肌线粒体损伤。
Objective To observe the morphological changes of cardiomyocytic mitochondria and the reactive oxygen species content in bupivacaine-exposed myocardial cells under electrical stimulation, and to establish an ideal myocardial cell model of bupivacaine poisoning. Methods A Langendorff device was used to separate the cardiomyocytes of male SD rats. The cells were divided into four groups randomly: a DMEM static group, DMEM electric stimulation group, bupivacaine static group and bupivacaine plus electric stimulation group. The experiment was repeated for five times. The cardiomyocytic mitochondrial morphology was observed by transmission electron microscopy, and the ROS content was measured with a multifunctional microplate detector. Results The degree of mitochondrial swelling and the ROS content in the DMEM electric stimulation group were not significantly different from those of the DMEM group(P > 0.05), but the mitochondrial swelling in the bupivacaine plus electric stimulation group was significantly higher than that of the bupivacaine group(P=0.000), and the ROS output was also significantly increased(P < 0.05). Conclusions Under electrical stimulation, cardiomyocytes show rhythmic contractions, allowing better simulation of the myocardial mitochondrial injury during clinical bupivacaine poisoning.
Objective To observe the morphological changes of cardiomyocytic mitochondria and the reactive oxygen species content in bupivacaine-exposed myocardial cells under electrical stimulation, and to establish an ideal myocardial cell model of bupivacaine poisoning. Methods A Langendorff device was used to separate the cardiomyocytes of male SD rats. The cells were divided into four groups randomly: a DMEM static group, DMEM electric stimulation group, bupivacaine static group and bupivacaine plus electric stimulation group. The experiment was repeated for five times. The cardiomyocytic mitochondrial morphology was observed by transmission electron microscopy, and the ROS content was measured with a multifunctional microplate detector. Results The degree of mitochondrial swelling and the ROS content in the DMEM electric stimulation group were not significantly different from those of the DMEM group(P > 0.05), but the mitochondrial swelling in the bupivacaine plus electric stimulation group was significantly higher than that of the bupivacaine group(P=0.000), and the ROS output was also significantly increased(P < 0.05). Conclusions Under electrical stimulation, cardiomyocytes show rhythmic contractions, allowing better simulation of the myocardial mitochondrial injury during clinical bupivacaine poisoning.
引文
[1] Tomita A, Satani M, Suzuki K, et al. A case of cardiac arrest following intra-articular administration of levobupivacaine during total knee arthroplasty[J]. Masui, 2016, 65(2): 179-183.
[2] Nelson M, Reens A, Reda L, et al. Profound prolonged bradycardia and hypotension after interscalene brachial plexus block with bupivacaine[J]. J Emerg Med, 2018, 54(3): e41-e43.
[3] Hiller N, Mirtschink P, Merkel C, et al. Myocardial accumulation of bupivacaine and ropivacaine is associated with reversible effects on mitochondria and reduced myocardial function[J]. Anesth Analg, 2013, 116(1):83-92.
[4] Gorelik J, Yang LQ, Zhang Y, et al. A novel Z-groove index characterizing myocardial surface structure[J]. Cardiovasc Res, 2006, 72(3): 422-429.
[5] Joshi MS, Crouser ED, Julian MW, et al. Digital imaging analysis for the study of endotoxin-induced mitochondrial ultrastructure injury[J]. Anal Cell Pathol, 2000, 21(12): 41-48.
[6] Holzerová E, Prokisch H. Mitochondria: Much ado about nothing? How dangerous is reactive oxygen species production? [J]. Int J Biochem Cell Biol, 2015, 63: 16-20.
[7] Wang M, Lu L, Liu Y, et al. FTY720 attenuates hypoxia-reoxygenation-induced apoptosis in cardiomyocytes[J]. Exp Mol Pathol, 2014, 97(2): 218-224.
[8] Kimes BW, Brandt BL. Properties of a clonal muscle cell line from rat heart[J]. Exp Cell Res, 1976, 98(2): 367-381.
[9] Sard?o VA, Oliveira PJ, Holy J, et al. Vital imaging of H9c2 myoblasts exposed to tert-butylhydroperoxide-characterization of morphological features of cell death[J]. BMC Cell Biol, 2007, 8: 11.
[10] Ménard C, Pupier S, Mornet D, et al. Modulation of L-type calcium channel expression during retinoic acid-induced differentiation of H9C2 cardiac cells[J]. J Biol Chem, 1999, 274(41): 29063-29070.
[11] Branco AF, Pereira SL, Moreira AC, et al. Isoproterenol cytotoxicity is dependent on the differentiation state of the cardiomyoblast H9c2 cell line[J]. Cardiovasc Toxicol, 2011, 11(3): 191-203.
[12] Powell T, Twist VW. A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to clcium[J]. Biochem Biophys Res Commun, 1976, 72(1): 327-333.
[13] Louch WE, Sheehan KA, Wolska BM. Methods in cardiomyocyte isolation, culture, and gene transfer[J]. J Mol Cell Cardiol, 2011, 51(3): 288-298.
[14] Piper HM, Probst I, Schwartz P, et al. Culturing of calcium stable adult cardiac mocytes[J].J Mol Cell Cardiol, 1982, 14(7): 397-412.
[15] 王影,孙红,范乐明,等. 成年大鼠心肌细胞的分离和培养技术[J]. 徐州医学院学报, 2005, 25(5): 393-396.Wang Y, Sun H, Fan LM, et al. Isolation and culture of adult rat cardiac myocytes [J]. Acta Acad Med Xuzhou, 2005, 25(5): 393-396.
[2] Nelson M, Reens A, Reda L, et al. Profound prolonged bradycardia and hypotension after interscalene brachial plexus block with bupivacaine[J]. J Emerg Med, 2018, 54(3): e41-e43.
[3] Hiller N, Mirtschink P, Merkel C, et al. Myocardial accumulation of bupivacaine and ropivacaine is associated with reversible effects on mitochondria and reduced myocardial function[J]. Anesth Analg, 2013, 116(1):83-92.
[4] Gorelik J, Yang LQ, Zhang Y, et al. A novel Z-groove index characterizing myocardial surface structure[J]. Cardiovasc Res, 2006, 72(3): 422-429.
[5] Joshi MS, Crouser ED, Julian MW, et al. Digital imaging analysis for the study of endotoxin-induced mitochondrial ultrastructure injury[J]. Anal Cell Pathol, 2000, 21(12): 41-48.
[6] Holzerová E, Prokisch H. Mitochondria: Much ado about nothing? How dangerous is reactive oxygen species production? [J]. Int J Biochem Cell Biol, 2015, 63: 16-20.
[7] Wang M, Lu L, Liu Y, et al. FTY720 attenuates hypoxia-reoxygenation-induced apoptosis in cardiomyocytes[J]. Exp Mol Pathol, 2014, 97(2): 218-224.
[8] Kimes BW, Brandt BL. Properties of a clonal muscle cell line from rat heart[J]. Exp Cell Res, 1976, 98(2): 367-381.
[9] Sard?o VA, Oliveira PJ, Holy J, et al. Vital imaging of H9c2 myoblasts exposed to tert-butylhydroperoxide-characterization of morphological features of cell death[J]. BMC Cell Biol, 2007, 8: 11.
[10] Ménard C, Pupier S, Mornet D, et al. Modulation of L-type calcium channel expression during retinoic acid-induced differentiation of H9C2 cardiac cells[J]. J Biol Chem, 1999, 274(41): 29063-29070.
[11] Branco AF, Pereira SL, Moreira AC, et al. Isoproterenol cytotoxicity is dependent on the differentiation state of the cardiomyoblast H9c2 cell line[J]. Cardiovasc Toxicol, 2011, 11(3): 191-203.
[12] Powell T, Twist VW. A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to clcium[J]. Biochem Biophys Res Commun, 1976, 72(1): 327-333.
[13] Louch WE, Sheehan KA, Wolska BM. Methods in cardiomyocyte isolation, culture, and gene transfer[J]. J Mol Cell Cardiol, 2011, 51(3): 288-298.
[14] Piper HM, Probst I, Schwartz P, et al. Culturing of calcium stable adult cardiac mocytes[J].J Mol Cell Cardiol, 1982, 14(7): 397-412.
[15] 王影,孙红,范乐明,等. 成年大鼠心肌细胞的分离和培养技术[J]. 徐州医学院学报, 2005, 25(5): 393-396.Wang Y, Sun H, Fan LM, et al. Isolation and culture of adult rat cardiac myocytes [J]. Acta Acad Med Xuzhou, 2005, 25(5): 393-396.