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Permanent Occlusion of the Left Anterior Coronary Artery in the Rat
大鼠左前冠状动脉的永久性闭塞   

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Abstract

Left ventricular (LV) remodeling occurs in many patients after myocardial infarction (MI). LV remodeling is characterized by progressive ventricular dilatation and contractile dysfunction, consequently to cardiomyocyte hypertrophy and fibrosis. Despite reperfusion therapies, this pathophysiological process is the main cause of cardiac evolution toward heart failure. Moreover, the outcome of patients after MI is largely dependent on the initial cardiac injury. Thus, this is of major clinical interest to develop new pharmacological strategies to limit infarct size and prevent or reverse left ventricular remodeling. Such preclinical cardiovascular treatments are often tested in rodents. The rat model of myocardial infarction is commonly used. In this model, the permanent ligation of the left anterior descending coronary artery is performed (Bousquenaud et al., 2013a).
After being used to this surgical technique and experimented, the operator will need 20 min per rat from the anesthesia to the rat recovering.

Materials and Reagents

  1. Sterile drapes
  2. Non-resorbable silk: Prolene 7.0 (Ethicon, catalog number: F1839 )
  3. Resorbable silk: Vicryl 4.0 (Ethicon, catalog number: V134 )
  4. Syringes (BD Biosciences, catalog number: 309659 )
  5. 2 shoelaces (around 20 cm)
  6. Tracheal tube (Fine Science Tools, catalog number: RSP-ETT1605 )
  7. Endotracheal tube (Kent Scientific Corporation, catalog number: RSP-ETT1605 )
  8. Rat Adult male Wistar rats weighing around 300 g (Charles River Laboratories International)
  9. Dermic Betadine 10%
  10. Antibiotics: Amoxicilline (Clamoxyl 100 mg/kg/24 h)
  11. Trichrome stain (Masson) Kit (Sigma-Aldrich, catalog number: HT15 )
  12. Anti-sarcomeric alpha actinin antibody [EA-53] (Abcam, catalog number: ab9465 )

Equipment

  1. Gaseous anesthesia delivery system (Minerve)
  2. Ventilator (Kent Scientific Corporation, model: TOPO220 )
  3. Fine curved forceps (Fine Science Tools, catalog number: 11272-30 )
  4. Shaver (Kent Scientific Corporation, catalog number: CL8787 )
  5. Rasor blade (Fine Science Tools, catalog number: 10008-13 )
  6. Scalpel (Fine Science Tools, catalog number: 10011-00 )
  7. Needle holders (Fine Science Tools, catalog number: 12001-13 )
  8. Retractor (Fine Science Tools, catalog number: 17012-11 )

Procedure

  1. Anesthesia
    1. Induce a profound anesthesia while providing a mixture of 3.5% isoflurane and 1.5% oxygen in room air. A high percentage of isoflurane is chosen in order to induce a profound anesthesia as a thoracotomy needs to be performed.
    2. Place the rat in the induction chamber. Cover the box with a tissue to put the animal in a dark environment (to reduce the stress).
    3. Set the ventilation system at 50 respiratory cycles per minute. The pressure is set at 30 mmHg and the tidal volume at 2.5/3 ml per cycle; according to the rat weight (0.5 ml/100 g body weight). Pressure must not exceed 2 mmHg. These parameters have been set for use with the Rat.
    4. Plug the tracheal tube to the ventilation system, the pressure inside must not exceed 5 to 6 mmHg, if not clean the tube.
    5. As soon as deep anesthesia is reached (respiratory frequency inferior to 50 cycles per minute and loss of footpad reflex), open the aspiration system and take out the rat from the induction chamber.
      Pull out the rat tongue with a fine curved forceps and with the help of a light find out the trachea aperture. Insert the tracheal tube in the trachea.


      Figure 1. The anatomy of the rat mouth

    6. Quickly plug the tracheal tube to the ventilation system. Be certain that there was no wrong way by checking the lungs swelling. If the esophagus has been intubated, the operator will clearly notice the stomach inflation and must remove the tube immediately.
    7. In case the rat has not been intubated correctly, the operator must let it completely recovering from anesthesia before restarting the procedure from step A1.
    8. Attach the tracheal tube to the tooth of the animal, then to its snout.

  2. Surgery
    1. Place the rat in dorsolateral recumbency and place the light on top of it.
    2. Attach the left anterior foot of the animal in order to stretch it.
    3. Shave the left part of the thorax and sternum.
    4. Disinfect the skin with Betadine.
    5. Cut the skin at an angle of 45° going from the sternum.
    6. Using a round-tip tweezer, detach the skin from the more superficial muscle layer (pectoralis major), and then detach it from the deeper muscle layer (serratus anterior).
    7. Strongly hold the 6th rib and cut the intercostal muscle in between the 6th and the 5th rib, inducing thoracotomy. Insert the retractor between the 6th and 5th rib.
    8. Proceed to pericardiotomy with the fine curved forceps: start from the apical part of the pericardium to make free the left ventricle.
    9. Using the permanent silk, proceed to the ligation of the left anterior coronary artery. Go in the ventricular muscle with a rotation movement, this allowing staying inside the muscle and avoiding penetrating inside the ventricle.
    10. Decrease the isoflurane flux to 1.5%.
    11. Remove the retractor and put the drain inside the cardiac cavity.
    12. Stop the anesthesia.
    13. Close the thorax: Join the 2 ribs with the non-permanent silk. Then close the muscle layer. Close the second muscle layer and then the skin.
    14. Drain the cavity and clean the skin with Betadine.

  3. Post-surgical care and recovery
    1. Administrate the antibiotic by intramuscular injection (200 mg/kg) every 48 h and during one week.
    2. Make the animal left foot free and remove the tracheal tube.
    3. Stop the ventilation, but check the recovery of the animal. If it stops breathing, immediately put on back the ventilation. Do it until the animal has completely recovered a strong breathing and starts to show discomfort with the tube.
    4. Remove the tube and at the same time aspirate the mucus with the syringe.
    5. Place the rat on dorsal recumbency under heather and check it until it is completely awake.
    6. Administrate antibiotic 24 h and 48 h later.
    7. Mortality is very low after 48 h.

Representative data


Figure 2. Illustration of the ex vivo characterization of an infarcted and a healthy rat heart; described by in vivo positron emission tomography (PET). 18F-fluorodeoxyglucose (FDG)-PET is a reference cardiac imaging technique in man and rat, because of its ability to accurately differentiate healthy from necrotic myocardium based on cardiomyocytes metabolic activity. (1) Vertical long-axis slices (upper half) recorded in the same rat in vivo by Fluorodeoxyglucose-PET and ex vivo by µIMAGERTM. (2) Immunohistochemical data (lower half) showing fibrosis development (blue color at Masson trichrome) and the decrease in cytoskeleton elements (alpha-sarco actinin staining in brown color) within the infarcted segments. Pictures from a rat with large MI and a sham-operated rat are shown. Magnification *40. (Bousquenaud et al., 2012)

Notes

  1. By its shape and diameter, the anatomy of the rat left anterior coronary artery displays a strong inter-individual variability. Thus, regardless of the accuracy and level of experience of the manipulator, this surgical model provides a variable extent of myocardial infarct. This will have to be taken into account when planning the number of animals to use in order to get significant results.
  2. The survival of the animals is very dependent on the speed of recovery after surgery. It is recommended to start decreasing the isoflurane flux when starting to stitch up the muscles, and stop the anesthesia as soon as the skin wound is closed. This can be performed only when the experimenter works rapidly, since the recovery from isoflurane anesthesia is fast.

Acknowledgments

This work was supported by grants from the National Funds of Research, the Society for Research on Cardiovascular Diseases, the Ministry of Culture, Higher Education and Research of Luxembourg, and the ‘‘Fondation de France’’.

References

  1. Bousquenaud, M., Maskali, F., Poussier, S., Marie, P. Y., Boutley, H., Karcher, G., Wagner, D. R. and Devaux, Y. (2012). Acipimox-enhanced (1)(8)F-fluorodeoxyglucose positron emission tomography for characterizing and predicting early remodeling in the rat infarct model. Int J Cardiovasc Imaging 28(6): 1407-1415.
  2. Bousquenaud, M., Maskali, F., Poussier, S., Zangrando, J., Marie, P. Y., Boutley, H., Fay, R., Karcher, G., Wagner, D. R. and Devaux, Y. (2013a). Cardioprotective effects of adenosine within the border and remote areas of myocardial infarction. EJNMMI Res 3(1): 65.
  3. Bousquenaud, M., Wagner, D. R., Maskali, F., Marie, P. Y. and Devaux, Y. (2013b). Long-term survival after a massive left ventricular infarction evidenced by FDG-PET and leaving intact only the septal wall. Int J Clin Exp Med 6(1): 84-85.

简介

左心室(LV)重塑在心肌梗死(MI)后发生在许多患者中。 LV重塑的特征在于进行性心室扩张和收缩功能障碍,从而导致心肌细胞肥大和纤维化。尽管再灌注治疗,这种病理生理过程是心脏进入心力衰竭的主要原因。此外,MI后患者的结果主要取决于初始心脏损伤。因此,这是开发新的药理策略以限制梗死面积和防止或逆转左心室重塑的重大临床兴趣。这种临床前心血管治疗通常在啮齿动物中进行测试。通常使用大鼠心肌梗死模型。在该模型中,进行左前降支冠状动脉的永久性结扎(Bousquenaud等人,2013a)。
在用于这种手术技术和实验之后,操作者将需要每只大鼠从麻醉到大鼠恢复20分钟。

材料和试剂

  1. 无菌罩
  2. 不可再吸收丝:Prolene 7.0(Ethicon,目录号:F1839)
  3. 可再吸收丝:Vicryl 4.0(Ethicon,目录号:V134)
  4. Syringues(BD Biosciences,目录号:309659)
  5. 2条鞋带(约20厘米)
  6. 气管导管(Fine Science Tools,目录号:RSP-ETT1605)
  7. 气管内管(Kent Scientific Corporation,目录号:RSP-ETT1605)
  8. 大鼠成年雄性Wistar大鼠加权约300g(Charles River Laboratories International)
  9. 皮肤Betadine 10%
  10. 抗生素:阿莫西林(Clamoxyl 100 mg/kg/24 h)
  11. 三色染料(Masson)试剂盒(Sigma-Aldrich,目录号:HT15)
  12. 抗肌节α肌动蛋白抗体[EA-53](Abcam,目录号:ab9465)

设备

  1. 气体麻醉输送系统(Minerve)
  2. 通风器(Kent Scientific Corporation,型号:TOPO220)
  3. 精细弯曲钳(Fine Science Tools,目录号:11272-30)
  4. Shaver(Kent Scientific Corporation,目录号:CL8787)
  5. Rasor刀片(Fine Science Tools,目录号:10008-13)
  6. Scalpel(Fine Science Tools,目录号:10011-00)
  7. 针架(Fine Science Tools,目录号:12001-13)
  8. 牵开器(Fine Science Tools,目录号:17012-11)

程序

  1. 麻醉
    1. 诱导严重麻醉,同时提供3.5% 异氟烷和1.5%氧气。高百分比的异氟烷 以便诱导作为开胸术的深度麻醉 需要执行。
    2. 将大鼠放入诱导室。 用纸巾盖住盒子,将动物放在黑暗的环境中 减少压力)。
    3. 将通气系统设置为50呼吸 ?循环/分钟。压力设置为30mmHg和潮气量 每个循环为2.5/3ml;根据大鼠体重(0.5ml/100g体重 重量)。压力不得超过2 mmHg。这些参数已设置 ?与鼠一起使用。
    4. 将气管导管插入通气系统,如果不清洁导管,内部压力不得超过5至6 mmHg。
    5. 一旦达到深度麻醉(呼吸频率 低于每分钟50个周期和脚垫反射损失),打开 抽吸系统,并从诱导室中取出老鼠。
      用细弯曲的镊子拉出鼠舌,并在a的帮助下 ?光找出气管孔径。将气管插入 气管

      图1.老鼠嘴的解剖

    6. 快速插入气管插管 通风系统。确保没有错误的方式通过检查 肺肿胀。如果食管已经插管,操作者 将清楚地注意到胃膨胀并且必须移除管 立即。
    7. 在大鼠没有正确插管的情况下, 操作者必须让其从麻醉完全恢复 重新启动从步骤A1开始的过程
    8. 将气管管连接到动物的牙齿,然后到其鼻子。

  2. 手术
    1. 将大鼠在背外侧卧,并将光线放在它的顶部
    2. 附加动物的左前脚,以拉伸它。
    3. 剃掉胸部和胸骨的左侧部分。
    4. 用Betadine消毒皮肤。
    5. 切割皮肤与45度角从胸骨。
    6. 使用圆头镊子,从更表面的皮肤分离 ?肌肉层(胸大肌),然后从更深的分离 肌肉层(serratus anterior)。
    7. 牢牢握住6 th 肋 切割在第6 和第5 肋之间的肋间肌,诱导 胸廓切开术。在6 th 和5 th 之间插入卷收器。
    8. 进行心包切开术用精细弯曲的钳子:从 使心包的心尖部分释放左心室。
    9. 使用永久性丝,继续左前结扎 冠状动脉。进入心室肌与旋转运动, 这允许停留在肌肉内部并避免穿透内部 心室。
    10. 减少异氟烷通量至1.5%。
    11. 取出牵开器,将排水管放入心腔。
    12. 停止麻醉。
    13. 关闭胸部:用非永久性丝加入2个肋。然后 ?关闭肌肉层。关闭第二肌肉层,然后 皮肤。
    14. 排空腔,用Betadine清洁皮肤。

  3. 手术后护理和康复
    1. 通过每48小时和在一周内肌内注射(200mg/kg)来施用抗生素。
    2. 使动物左脚自由,并删除气管管。
    3. 停止通风,但检查动物的恢复。如果它 停止呼吸,立即放回通风。直到 动物完全恢复了强烈的呼吸并开始 显示管不适。
    4. 取出试管,同时用注射器吸出粘液
    5. 将大鼠在背部躺在石南花下,检查它,直到它完全清醒。
    6. 24小时和48小时后给予抗生素。
    7. 48小时后死亡率非常低。

代表数据


图2.梗塞和健康大鼠心脏的体外表征的示意图; (PET)描述。 18F-氟脱氧葡萄糖(FDG)-PET是人和大鼠中的参考心脏成像技术,因为其能够准确地区分健康的坏死心肌基于心肌细胞的代谢活性。 (1)通过Fluorodeoxyglucose-PET在体内记录在相同大鼠中的垂直长轴切片(上半部分)和通过μIMAGER TM 体外记录的垂直长轴切片。 (2)免疫组织化学数据(下半部分)显示梗塞区段内的纤维化发展(Masson三色的蓝色)和细胞骨架元件的减少(棕色的α-肌质肌动蛋白染色)。显示来自具有大MI的大鼠和假手术大鼠的图片。放大率* 40。 (Bousquenaud et al。,2012)

笔记

  1. 通过其形状和直径,大鼠左前冠状动脉的解剖学显示出强烈的个体间变异性。因此,不管操纵器的经验的准确性和水平如何,该外科模型提供了可变程度的心肌梗塞。在规划使用的动物数量以获得显着结果时,必须考虑这一点。
  2. 动物的存活率非常依赖于手术后的恢复速度。建议在开始缝合肌肉时开始减少异氟烷通量,并在皮肤伤口闭合后立即停止麻醉。这只能在实验者快速工作时才能进行,因为从异氟烷麻醉中的恢复快。

致谢

这项工作得到了国家研究基金,心血管疾病研究学会,卢森堡文化,高等教育和研究部以及"法国基金会"的资助。

参考文献

  1. Bousquenaud,M.,Maskali,F.,Poussier,S.,Marie,P.Y.,Boutley,H.,Karcher,G.,Wagner,D.R。和Devaux,Y。 阿昔单抗增强(1)(8)F-氟脱氧葡萄糖正电子发射断层扫描术表征和预测早期重建在大鼠梗塞模型中。 Int J Cardiovasc Imaging 28(6):1407-1415。
  2. Bousquenaud,M.,Maskali,F.,Poussier,S.,Zangrando,J.,Marie,PY,Boutley,H.,Fay,R.,Karcher,G.,Wagner,DRand Devaux, 。 腺苷在心肌梗死边界和偏远地区的心脏保护作用 EJNMMI Res 3(1):65.
  3. Bousquenaud,M.,Wagner,D.R.,Maskali,F.,Marie,P.Y.and Devaux,Y。(2013b)。 由FDG-PET证实的大量左心室梗死后的长期生存,并且仅保留完整的隔膜。 Int J Clin Exp Med 6(1):84-85。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bousquenaud, M., Marie, P. and Devaux, Y. (2015). Permanent Occlusion of the Left Anterior Coronary Artery in the Rat. Bio-protocol 5(22): e1663. DOI: 10.21769/BioProtoc.1663.
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