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Excision of Visceral Adipose Tissue from Live Mice (VATectomy)
从活小鼠中切除内脏脂肪组织(VAT切除术)   

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Abstract

The visceral adipose tissue (VAT) has been shown to play an important role in various biological functions. It is a storage depot for nutrients and it is an important endocrine organ producing hormones that control systemic metabolism (McGown et al., 2014). Importantly, following diet-induced obesity, VAT accumulates a large number of activated immune cells, which produce cytokines that drive chronic systemic inflammation and promote insulin resistance (Johnson et al., 2012; Wensveen et al., 2015). VAT therefore plays a key role in metabolic, endocrinological and immunological research. To show the importance of this organ in various research models, one may surgically remove the organ in a procedure called VATectomy. This protocol describes the technical procedures required for an efficient VATectomy of the perigonadal fat pads in mice.

Keywords: Visceral Adipose Tissue(内脏脂肪组织), Diabetes(糖尿病), Obesity(肥胖), Inflammation(炎症), Excision(切除)

Materials and Reagents

  1. Male Mouse (e.g., C57BL/6, typically of 6-12 weeks old) (male mice are more severely affected by type 2 diabetes than female mice, and they are used exclusively in diet-induced diabetes studies) (Singer et al., 2015).
  2. Skin disinfectant [e.g., Chlorhexidine gluconate (generic name: Pliva®sept pjenušavi) (PLIVA HRVATSKA, catalog number: 536-02884 )
  3. Non-Steroid anti-inflammatory drugs (NSAIDs) [e.g., Meloxicam (generic name: loxicum) (5 mg/ml) (Norbrook Laboratories, catalog number: SC-200626 )]
  4. Inhalation anesthetics [e.g., Forane (generic name: isofluranum) (Abbott Laboratories, catalog number: B506 ).
  5. Sterile physiological salt solution (0.9% NaCl)
  6. Depilation cream (Depilation) (Afrodita kozmetika, catalog number: 05-506 )
  7. Meloxicam working solution (see Recipes)

Equipment

  1. Inhalation anesthesia equipment (alternatively, one may use intravenous anesthesia)
  2. Basic surgical instruments (e.g., needle holder, tweezers, scissors, scalpel, hemostat, retractor, surgical needle)
  3. (Absorbable) suture materials [e.g., Coated VicrylTM, size 3-0 (Ethicon, model: JB944 ) or stapler [e.g., Reflex, 9 mm applier (AgnTho's, model: 202-1000 )]
  4. Hair trimmer
  5. Scale

Procedure

  1. To reduce the amount of stress experienced by the animals as a result of pain, we recommend injecting animals intraperitoneally with NSAIDs, 30 min before starting the surgical procedure. Weigh mice and apply the dosage per kg of bodyweight as indicated (for meloxicam, use 1 mg/kg dissolved in 0.9% NaCl) (Ramsey, 2014).
  2. Place mice in the anesthetic chamber. Adjust the flow of anesthetics until the animals do not respond to a stimulus that triggers the pain reflex (i.e., pinching the toes). Typically, this requires a 2-4% Isoflurane gas mixture, with 2 L/min of oxygen flow. Visually, one will observe a loss of tail-righting and a reduced breathing rate. Typically it takes approximately 1 min for the mice to be sufficiently anesthetized (Figure 1).


    Figure 1. Anesthesia of animals. Mice are placed in an anesthesia chamber (ideally red transparent) and oxygen/inhalation anesthetic flow is controlled.

  3. When mice are anesthetized, remove the hair from the abdomen (in the umbilical and hypogastric regions along the linea alba) using a trimmer (Figure 2). When working efficiently, one can do this without continuous administration of anesthesia gas, as the animal will take about 60 sec before it regains responsiveness. Alternatively, one can use depilation cream. When using cream, take care to properly clean the animals after application to avoid skin irritation.
  4. Place the animal back in the anesthetic chamber and wait until the animal is again completely anesthetized.
  5. Place the mouse with its back on the operation table and apply the nose cone that provides the inhalation anesthesia to the mouse (Figure 2). Reduce the isoflurane rate to 1.5-2% with 2 L/min of oxygen flow. The operation table should be placed in a biosafety cabinet to ensure a sterile working condition.


    Figure 2. Proper placing of the animal for surgery. Place the animal in such a way that the abdomen is easily accessible, whilst ensuring proper anesthesia. The blue arrow indicates where mice need to be shaved [i.e., in the hypogastric region (down from the umbilicus) along the linea alba].

  6. Disinfect the skin.
  7. Use a scalpel to make a Pfannenstiel incision in the skin along the linea alba in the hypogastric region (down from the umbilicus).
  8. Next, make a cut (0.5 cm) into the muscle and peritoneum (Figure 3).


    Figure 3. Opening of the abdomen. Pull up the skin with tweezers and cut the abdominal wall, making sure to cut skin, muscle and peritoneum, without damaging the underlying internal organs. The red arrow indicates where mice need to be cut.

  9. There are two perigonadal adipose tissues. Pull them out one by one with tweezers and make an incision at the basis of adipose tissue along the epididymis and testis to completely remove the tissue (Figure 4). For sham-operated controls, pull out the fat pads and place them back inside the abdomen one by one (Video 1).

    Video 1. Vasectomy and sham operation


    Figure 4. Removal of the VAT. Gently pull out the abdominal fat pads without touching the gonads and cut the VAT along the gonads.

  10. Sew all three layers (peritoneum, muscle and skin) together (Figure 5). Preferably, use an absorbable fiber. Alternatively, non-absorbable fiber or a surgical suture stapler can be used (Singer et al., 2015; Boyer, 2014.).


    Figure 5. Closing of the abdomen. A. Close the abdomen with one or two stitches. B. sagittal and C. frontal illustration of layers (peritoneum, muscle and skin) and where the sutures should be placed in relation to these layers (Boyer, 2014).

  11. Inject mice with 1 ml of sterile physiological salt solution intraperitoneally to compensate for loss of liquids.
  12. After surgery, mice will be treated twice daily with NSAIDs for three days.
  13. If non-absorbable sewing materials have been used, remove these ten days after surgery.
  14. Fourteen days after surgery, additional experiments can be started. At this time, the site of surgery will have partially been overgrown with new hair.

Notes

  1. All experiments using mice were approved beforehand by your Institutional Animal Care and Use Committee and were in accordance with national and international guidelines.
  2. Laboratory rodents do not have a vomiting reflex. Therefore, fasting prior to start of the surgical procedure (which would be advised for larger animals) is not required.
  3. A skilled surgeon can complete one VATectomy within 5 min. In this period, a warming mat is not required. Unskilled people are advised to use a warming mat to prevent hypothermia of the animals.
  4. We advise that people without prior surgical experience practice the procedure on a euthanized animal before attempting to perform surgery on a living mouse.

Recipes

  1. Meloxicam working solution
  2. Meolxicam (5 mg/ml) 1 ml
  3. Physiological salt solution (0.9% NaCl in H2O) 9 ml

Acknowledgments

When using this protocol, please refer to Wensveen et al. (2015). This work was supported by the European Foundation for the Study of Diabetes (New Horizons Program), the Unity through Knowledge Fund (15/13 to B. P.), the University of Rijeka (13.06.1.1.03 to B. P.), the European Social Fund - ES (HR.3.2.01-0263 to B. P.), the Netherlands Organization for Scientific Research (91614029 to F. M. W.) and the European Commission (PCIG14-GA-2013-630827 to F. M. W.).

References

  1. Boyer, L. (2014). A student guide to wound closure. (On date 4 of Septemer 2015).
  2. Johnson, A. R., Milner, J. J. and Makowski, L. (2012). The inflammation highway: metabolism accelerates inflammatory traffic in obesity. Immunol Rev 249(1): 218-238.
  3. McGown, C., Birerdinc, A. and Younossi, Z. M. (2014). Adipose tissue as an endocrine organ. Clin Liver Dis 18(1): 41-58.
  4. Ramsey, I. (2014). BSAVA Small animal formulary Vol 8th edition. BSAVA, UK.
  5. Singer, K., Maley, N., Mergian, T., DelProposto, J., Cho, K. W., Zamarron, B. F., Martinez-Santibanez, G., Geletka, L., Muir, L., Wachowiak, P., Demirjian, C. and Lumeng, C. N. (2015). Differences in hematopoietic stem cells contribute to sexually dimorphic inflammatory responses to high fat diet-induced obesity. J Biol Chem 290(21): 13250-13262.
  6. Wensveen, F. M., Jelencic, V., Valentic, S., Sestan, M., Wensveen, T. T., Theurich, S., Glasner, A., Mendrila, D., Stimac, D., Wunderlich, F. T., Bruning, J. C., Mandelboim, O. and Polic, B. (2015). NK cells link obesity-induced adipose stress to inflammation and insulin resistance. Nat Immunol 16(4): 376-385.

简介

内脏脂肪组织(VAT)已显示在各种生物功能中发挥重要作用。 它是营养物质的储存库,它是产生控制系统代谢的激素的重要内分泌器官(McGown等人,2014)。 重要的是,在饮食诱发的肥胖症之后,VAT累积大量的活化免疫细胞,其产生驱动慢性全身炎症和促进胰岛素抵抗的细胞因子(Johnson等人,2012; Wensveen等人 al。,2015)。 因此,增值剂在代谢,内分泌和免疫学研究中起着关键作用。 为了显示该器官在各种研究模型中的重要性,可以在称为VATectomy的手术中手术切除器官。 该协议描述了有效的VAT切除小鼠的perigonadal脂肪垫所需的技术程序。

关键字:内脏脂肪组织, 糖尿病, 肥胖, 炎症, 切除

材料和试剂

  1. 雄性小鼠(例如 C57BL/6,通常6-12周龄)(雄性小鼠比雌性小鼠更严重地受2型糖尿病的影响,并且它们仅在饮食诱导的糖尿病研究中使用) (Singer等人,2015)。
  2. 皮肤消毒剂[例如葡萄糖酸氯己定(通用名:Pliva septpjenu?avi)(PLIVA HRVATSKA,目录号:536-02884)
  3. 非类固醇抗炎药(NSAID)[例如美洛昔康(通用名:loxicum)(5mg/ml)(Norbrook Laboratories,目录号:SC-200626)]
  4. 吸入麻醉剂[例如Forane(通用名称:isofluranum)(Abbott Laboratories,目录号:B506)。
  5. 无菌生理盐溶液(0.9%NaCl)
  6. 脱毛膏(脱毛)(Afrodita kozmetika,目录号:05-506)
  7. 美洛昔康工作溶液(参见配方)

设备

  1. 吸入麻醉设备(或者,可以使用静脉麻醉)
  2. 基本手术器械(例如:持针器,镊子,剪刀,手术刀,止血钳,牵开器,手术针)
  3. (可吸收)缝合材料[例如覆盖Vicryl TM ,尺寸3-0(Ethicon,型号:JB944)或吻合器[例如 Reflex,9 mm应用器(AgnTho's,型号:202-1000)]
  4. 头发修剪器
  5. 缩放

程序

  1. 为了减少动物由于疼痛而经受的应激量,我们建议将动物腹膜内注射NSAID,30分钟。在开始外科手术之前。称量小鼠并应用所示的每kg体重的剂量(对于美洛昔康,使用溶解在0.9%NaCl中的1mg/kg)(Ramsey,2014)。
  2. 将小鼠放在麻醉室。调整麻醉剂的流量,直到动物对触发疼痛反射的刺激没有反应(捏趾)。通常,这需要2-4%的异氟烷??气体混合物,具有2L/min的氧气流。在视觉上,人们将观察到尾部纠正的损失和降低的呼吸速率。通常需要约1分钟使小鼠充分麻醉(图1)。


    图1.将动物麻醉放置在麻醉室(理想地红色透明)中,并控制氧气/吸入麻醉剂流动。

  3. 当小鼠麻醉时,使用修剪器从腹部(沿脐带和沿胃线的下腹部)移除毛发(图2)。当有效地工作时,可以在不连续给予麻醉气体的情况下这样做,因为动物将在大约60秒后恢复响应性。或者,可以使用脱毛霜。使用奶油时,在使用后小心妥善清洁动物,以避免皮肤刺激。
  4. 将动物放回麻醉室,等待动物再次完全麻醉。
  5. 将鼠标与它的背部放在手术台上,应用提供吸入麻醉的小鼠鼻锥(图2)。用2 L/min的氧气流量将异氟烷速率降低到1.5-2%。操作表应放置在生物安全柜中,以确保无菌工作条件。


    图2.正确放置动物进行手术。将动物放置在方便腹部的位置,同时确保适当的麻醉。蓝色箭头表示需要在沿着线alba的下腹部区域(从肚脐下方)剃刮[/em]。

  6. 消毒皮肤。
  7. 使用手术刀在沿腹线的下腹部区域(从脐部)的皮肤中做Pfannenstiel切口。
  8. 接下来,对肌肉和腹膜进行切割(0.5厘米)(图3)

    图3.打开腹部。用镊子拉起皮肤,切开腹壁,确保切割皮肤,肌肉和腹膜,而不损坏下面的内脏。红色箭头表示需要切割小鼠的位置。

  9. 有两个perigonadal脂肪组织。用镊子一个一个地拉出,并在沿着epydidimidis和睾丸的脂肪组织切口,以完全去除组织(图4)。对于假手术的控制,拉出脂肪垫,将它们放回腹部一个一个(视频1)。

    视频1. Vatectomy和假手术
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    获取Adobe Flash Player

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    图4.去除增值税。轻轻地拉出腹部脂肪垫,而不触及性腺,沿着性腺切割增值税。

  10. 缝合所有三层(腹膜,肌肉和皮肤)在一起(图5)。优选地,使用可吸收纤维。或者,可以使用非吸收性纤维或手术缝合钉器(Singer等人,2015; Boyer,2014)。


    图5.关闭腹部。 A.用一个或两个针头闭合腹部。 B.矢状面和C.正面图层(腹膜,肌肉和皮肤),缝线应放置在这些层之间(Boyer,2014)。

  11. 用1ml无菌生理盐溶液腹腔注射小鼠以补偿液体的损失。
  12. 手术后,小鼠每日用NSAID治疗三天。
  13. 如果使用不可吸收的缝合材料,请在手术后十天取出。
  14. 手术后14天,可以开始另外的实验。此时,手术部位将部分地长满新毛发。

笔记

  1. 所有使用小鼠的实验都由您的机构动物护理和使用委员会事先批准,并且符合国家和国际指南
  2. 实验室啮齿类动物没有呕吐反射。因此,不需要在开始外科手术之前禁食(这将被建议用于较大的动物)。
  3. 有经验的外科医生可以在5分钟内完成一次VAT切除术。在此期间,不需要加热垫。不熟练的人建议使用暖垫,以防止动物低温。
  4. 我们建议没有事先手术经验的人在尝试对活的小鼠进行手术之前,对无痛致死动物实施手术。

食谱

  1. 美洛昔康工作溶液
  2. 美洛昔康(5mg/ml)1ml
  3. 生理盐溶液(0.9%NaCl在H 2 O中)9ml

致谢

使用此协议时,请参阅Wensveen等人(2015)。这项工作得到了欧洲糖尿病研究基金会(新视野计划),通过知识基金统一(15/13至BP),里耶卡大学(13.06.1.1.03至BP),欧洲社会基金 - ES(HR.3.2.01-0263至BP),荷兰科学研究组织(91614029至FMW)和欧洲委员会(PCIG14-GA-2013-630827至FMW)。

参考文献

  1. Boyer,L.(2014)。 伤口闭合的学生指南(2015年9月4日第4日)。
  2. Johnson,A.R.,Milner,J.J.and Makowski,L。(2012)。 炎症性高血压:代谢加速肥胖中的炎症性交通。 Immunol Rev 249(1):218-238。
  3. McGown,C.,Birerdinc,A.和Younossi,Z.M。(2014)。 将脂肪组织作为内分泌器官。 Clin Liver Dis 18(1):41-58。
  4. Ramsey,I.(2014)。 BSAVA小动物制剂第8版。 BSAVA,UK。
  5. Singer,K.,Maley,N.,Mergian,T.,DelProposto,J.,Cho,KW,Zamarron,BF,Martinez-Santibanez,G.,Geletka,L.,Muir,L.,Wachowiak, Demirjian,C。和Lumeng,CN(2015)。 造血干细胞中的差异导致对高脂肪饮食诱导的肥胖的性别二态性炎症反应。 a> J Biol Chem 290(21):13250-13262。
  6. Wensveen,FM,Jelencic,V.,Valentic,S.,Sestan,M.,Wensveen,TT,Theurich,S.,Glasner,A.,Mendrila,D.,Stimac,D.,Wunderlich,FT,Bruning,JC ,Mandelboim,O.和Polic,B。(2015)。 NK细胞将肥胖诱导的脂肪应激与炎症和胰岛素抵抗联系起来。 Nat Immunol 16(4):376-385。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Šestan, M., Wensveen, F. M. and Polić, B. (2015). Excision of Visceral Adipose Tissue from Live Mice (VATectomy). Bio-protocol 5(23): e1668. DOI: 10.21769/BioProtoc.1668.
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