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May 2017
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A Rodent Model for Chronic Brain Hypoperfusion Related Diseases: Permanent Bilateral Occlusion of the Common Carotid Arteries (2VO) in Rats
慢性脑灌注不足相关疾病的啮齿动物模型:大鼠双侧颈总动脉永久性闭塞(2VO)   

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Abstract

Permanent occlusion of bilateral common carotid arteries (2VO) in rat is considered as a suitable animal model to mimic chronic brain hypoperfusion status, which is proved to be a risk factor to precede the Alzheimer’s disease or/and vascular dementia. In this protocol, we describe how to successfully ligate the bilateral common carotid arteries covered by anterior cervical muscle group, and provide the details for understanding the surgical procedures of 2VO.

Keywords: Permanent occlusion of bilateral common carotid arteries (双侧颈总动脉永久性闭塞), Chronic brain hypoperfusion (慢性脑灌注不足), Rat (大鼠)

Background

Currently, chronic brain hypoperfusion (CBH) is considered as a preclinical condition of mild cognitive impairment, which is thought to precede dementia (Ruitenberg et al., 2005; Gorelick et al., 2011). However, how CBH produces dementia is largely unknown.

The method of permanent occlusion of bilateral common carotid arteries (2VO) in rat was first established in the 1970s (Eklof and Siesjo, 1972). Since 2VO in rats provokes CBH without motor dysfunction, it is considered as a suitable animal model to elicit CBH status (Tanaka et al., 1996). After the 2VO procedures, three phases are theoretically determined by the degree of cerebral blood flow (CBF) and metabolic changes of rats (Farkas et al., 2007). After initiation of 2VO procedures, the first phase of acute ischemia lasts for about 2-3 days (Ohta et al., 1997; Otori et al., 2003; Tomimoto et al., 2003). During this period, the CBF drops dramatically and remains at a significantly low level in the following 4 weeks with reduced glucose utilization (Otori et al., 2003), sudden depletion of ATP and phosphocreatine (Plaschke, 2005). The second phase lasts for 8 to 12 weeks and corresponds well with the CBH in aging and dementia, and a slight reduction of the CBF with restored ATP as well as remaining low level of phosphocreatine. However, in the final phase, the CBF recovers to the baseline and the metabolic changes gradually cease after 6 months of 2VO (Ohta et al., 1997; Otori et al., 2003).

We also noted that numerous studies using 2VO rat model also reveal lots of pathological characteristics, including impaired learning and memory evaluated by both Morris water maze or eight-arm radial maze, where the 2VO rats display longer escape latencies to find hiding platform in Morris water maze (Liu et al., 2005) and commit more errors to enter a never-baited arm than the rats from sham group in the eight-arm radial maze (Sopala and Danysz, 2001); the neuronal cell death in hippocampus (Farkas et al., 2004); reduced dendritic arborizations (Chen et al., 2017); astrocytic reactions (Panickar and Norenberg, 2005) and microglial activation (Abraham and Lazar, 2000), etc. All these phenomena provide the sounded evidence that 2VO rat model is a valuable animal model to study the molecular mechanism of CBH associated diseases. In this protocol, we describe the detailed procedures of how to successfully establish a rodent model of CBH by permanently ligating the bilateral common carotid arteries of rat. And this protocol was based on and modified from the previously published paper: Kumaran et al., 2008.

Materials and Reagents

  1. 75% alcohol cotton ball (Bettering, catalog number: BY ACB )
  2. 22 mm (3/8 circle) surgical needle with suture (Foosin Medical Supplies, Weigao, model: 300 series ) (Figure 1B)
  3. 1 ml syringe for anesthetizing (Xi’an Sentansha Medical Investment Management, catalog number: STS-SY003-#0037 ) (Figure 1H)
  4. 3-0 silk suture (Jinhuan, model: 3-0 ) (Figure 1A)
  5. Medical absorbent cotton (Zhushi Parmaceutical Group, model: ZS-HM019 )
  6. Animals
    Male Sprague-Dawley rats (weight 280-300 g, usually 4-5 months old, obtained from the Animal Center of the Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China) were housed at 23 ± 1 °C with 55 ± 5% of humidity and maintained on 12 h dark/light artificial cycle (lights on at 7:00 AM) with food and water available ad libitum. All procedures regarding the animal experiments were approved by the Institutional Animal Care and Use Committee at Harbin Medical University (No. HMUIRB-2008-06) and the Institute of Laboratory Animal Science of China (A5655-01)
  7. 0.9% sodium chloride (Qingdao Fraken International Trading, model: High Quality 0.9% Compound Sodium Chloride Injection )
  8. Chloral hydrate (Aladdin, catalog number: C104202 )
  9. Gentamycin sulfate, 50 mg/ml solution, sterile (Sangon Biotech, catalog number: B540724 )
  10. 10% chloral hydrate solution (see Recipes)
  11. 20 mg/ml gentamycin sulfate solution (see Recipes)

Equipment

  1. Glass dish (Canfort Laboratory and Education Supplies, catalog number: LG075 ) (Figure 1C)
  2. Needle holder (Sklar Surgery Instrument, model: 5-1/4”, catalog number: 21-8001 ) (Figure 1D)
  3. Forceps (Fine Science Tools, catalog number: 11009-13 ) (Figure 1E)
  4. Artery clips (Nut Link International, model: Approximator Clamps ) (Figure 1F)
  5. Scissors (Fine Science Tools, catalog number: 14068-12 ) (Figure 1G)
  6. Glass hook (Figure 1I)
  7. Electric animal shaver (Yiwu Kemei Electric Appliances, model: KM-970 ) (Figure 1J)
  8. Fiber optic illuminator (Mineralogical Research, catalog number: MA312101 ) (Figure 1K)
  9. Electric heating pad for pet (Dongxiyi, catalog number: wi95919 ) (Figure 1L)


    Figure 1. The materials and equipment. A. 3-0 silk suture; B. Surgical needle with suture; C. Glass dish with the sterile 0.9% sodium chloride; D. Needle holder; E. Forceps; F. Artery clips; G. Scissors; H. 1 ml syringe for anesthetizing; I. Glass hook; J. Electric animal shaver; K. Fiber optic illuminator; L. Electric heating pad.
    Notes: Before the surgery, all the metal instruments should be sterilized at high temperature, 200 °C.

Procedure

  1. Anesthesia
    After fasting 12 h, the rat is anesthetized with 10% chloral hydrate (300 mg/kg, see Recipes) by intraperitoneal injection and the rat should reach surgical anesthesia within 5-10 min.
    Note: Depth of anesthesia is assessed by corneal blink and tail-pinch reflexes. Surgery should not be operated until the rat has reached full anesthesia. Anesthesia should be maintained at least for 1 h to complete all surgical procedures.
  2. Expose the common carotid artery of rat (Video 1).

    Video 1. Expose the common carotid artery of rat. This video shows the exact surgical procedures of how to expose common carotid artery covered by omohyoideus.
    Note: To maintain body temperature, it is recommended to place the rat on a heating pad (37 °C) during procedures.

    1. Mount the rat onto an electric heating pad and clean the fur around neck using an electric shaver. (Figure 2A)
    2. Sterilize neck skin using 75% alcohol cotton ball and then cut a 2-cm incision above the manubrium along the anterior midline of the neck. (Figure 2B)
    3. Carefully blunted dissect hypoderm and expose the sternohyoid muscle. (Figure 2C)
    4. Carefully blunted dissect the fascia in order to expose the carotid triangle muscle structure that is consist of posterior belly of digastric muscle, omohyoideus and sternocleidomastoid, and then vertically separate omohyoideus to finally expose the sheath for crucial vessels and nerves. (Figure 2D)
      Note: Within the exposed triangle area, two important vessels and nerve trunks could be identified, including internal vein and common carotid artery with their tributaries, and Vagus and aortic depressor nerve (50-80 μm varying upon the age and gender) that commonly located between carotid artery and Vagus. The dark red pulse-free internal vein mostly covered by sternocleidomastoid muscle is located outside the red pulsing common carotid artery. So they can be easily distinguished by the color and pulse. Importantly, Careful attention would be necessary for successful surgery during the blunted dissection to avoid damaging both nerves.
  3. Separate the common carotid artery from the Vagus and aortic depressor nerve. (Video 2)

    Video 2. Separate the common carotid artery from the Vagus and aortic depressor nerve

    1. Carefully peel off the sheath around the common carotid artery. (Figure 2E)
    2. Slightly lift up the common carotid artery with one forceps and gently separate vagal nerve from the common carotid artery using the glass hook. (Figure 2F)
      Note: More attention should be paid to avoid the damage of the Vagus as well as aortic depressor never, as the damage can induce vegetative nerve functional disturbance and increase mortality rate.


      Figure 2. The Procedures 2-6 of 2VO rat model. A. Mount the rat to the heating pad; B. Cut a 2.0 cm incision above the manubrium; C. Expose the sternohyoid muscle; D. Expose the carotid triangle muscle structures that are consist of posterior belly of digastric muscle, omohyoideus and sternocleidomastoid; E. Carefully peel off the carotid sheath; F. Separate the Vagus from the common carotid artery; G. Permanently ligate the bilateral common carotid artery; H. Schematic diagram of permanent ligation of bilateral common carotid artery; I. Cut off the common carotid artery in between the two ligated silk sutures; J. Schematic diagram of cutting off the common carotid artery in between the two ligated silk sutures; K. Use 20 mg/ml gentamycin sulfate solution to avoid potential postoperative infection; L. Close the wounds.

  4. Permanently ligate or cut off the common carotid artery. (Video 3)

    Video 3. Permanently ligate or cut off the common carotid artery

    1. Clamp the common carotid artery using two artery clips at the heart and head side each to avoid bleeding. (Figure 2G)
    2. Two 3-0 silk sutures (immersed in the 0.9% sterile sodium chloride) are placed beneath the common carotid artery with the help of forceps and right in between the clips (Figures 2G and 2H). For the permanent ligation of the common carotid artery, silk sutures are tightened up and then the common carotid artery is cut off in between the two ligated silk sutures (Figures 2I and 2J).
    3. Gently take off the artery clips.
    4. During the surgical procedures, the medical absorbent cotton would be necessary to remove any potential bleeding and exudates to reduce the chance of postoperative infection maximally.
  5. Perform the same procedures on the contralateral common carotid artery. (Video 4)

    Video 4. Permanently ligate the contralateral carotid artery

  6. Postoperative caring
    1. After the surgical procedures, all the anterior cervical muscles should be back to their original location.
    2. To avoid potential postoperative infection, the wounds should be washed before closing by 20 mg/ml gentamycin sulfate solution and the redundant solution has to be removed completely. (Figure 2K) (Video 5)

      Video 5. Protect the wounds from infection

    3. Close the wounds. (Figure 2L)
    4. Place the rat back to the cage, and use a warm illuminant to keep a stable temperature until the rat recovers from anesthesia that may take at least 1-2 h after the surgery.

Notes

  1. Timing: All the surgical procedures need to be finished within 5 min after the rat is completely relaxed, which will significantly reduce the chance of infection.
  2. Selection of animals: Rats are considered to be the suitable rodent species for the 2VO operation because rats can survive from the severe ischemia by their complete circle of Willis, which provides constant reduction of blood flow after the 2VO. In other words, the Willis’ circle in gerbils or most strains of mice are under developed. It is worth mentioning that, in our group, SD rather than Wistar rats have been selected to establish the 2VO animal model simply because of a higher mortality while using Wistar compared with SD rats that have relatively large circle of Willis. Additionally, the body weight of rat is a significant factor. In our experience, an ideal body weight is between 280-300 g, and it will definitely increase the risk of mortality during and after procedures if the body weight is lower than 280 g or higher than 300 g.
  3. Selection of silk suture size: The most suitable size of suture is 3-0 to 5-0, because the diameter of silk suture less than 3-0 may damage the artery while ligation resulting in bleeding and the diameter of silk suture larger than 5-0 may cause incompletely ligation or re-perfusion due largely to the loosen of ligated sutures and finally result in the failure to induce a CBH status.
  4. Protection of Vagus and aortic depressor nerve: The damage of vagal and aortic depressor nerve during operation will induce vegetative nerve functional disturbance of rats and increase the possibility of death.
  5. Gentle performance: Gentle and quick performance is essential here. A rough operation can easily induce severe bleeding and result in the death of rats.

Recipes

  1. 10% chloral hydrate solution
    1. 5 g chloral hydrate is dissolved in 50 ml physiological saline solution
    2. Store at 37 °C
  2. 20 mg/ml gentamycin sulfate solution
    1. 4 ml gentamycin sulfate (50 mg/ml) is diluted with sterile sodium chloride (0.9%) up to 10 ml
    2. Store at -20 °C

Acknowledgments

Thanks for the support of the Natural Science Foundation of China (81271207, 81070882, 81471115, 81671052). This protocol was based on and modified from the previously published paper: Kumaran et al. (2008, Neuroscience, 155: 626-639). We want to thank Shuai Zhang for shooting the videos and Biddyut Das for words editing.

References

  1. Abraham, H. and Lazar, G. (2000). Early microglial reaction following mild forebrain ischemia induced by common carotid artery occlusion in rats. Brain Res 862(1-2): 63-73.
  2. Chen, X., Jiang, X. M., Zhao, L. J., Sun, L. L., Yan, M. L., Tian, Y., Zhang, S., Duan, M. J., Zhao, H. M., Li, W. R., Hao, Y. Y., Wang, L. B., Xiong, Q. J. and Ai, J. (2017). MicroRNA-195 prevents dendritic degeneration and neuron death in rats following chronic brain hypoperfusion. Cell Death Dis 8(6): e2850.
  3. Eklof, B. and Siesjo, B. K. (1972). The effect of bilateral carotid artery ligation upon the blood flow and the energy state of the rat brain. Acta Physiol Scand 86(2): 155-165.
  4. Farkas, E., Institoris, A., Domoki, F., Mihaly, A., Luiten, P. G. and Bari, F. (2004). Diazoxide and dimethyl sulphoxide prevent cerebral hypoperfusion-related learning dysfunction and brain damage after carotid artery occlusion. Brain Res 1008(2): 252-260.
  5. Farkas, E., Luiten, P. G. and Bari, F. (2007). Permanent, bilateral common carotid artery occlusion in the rat: a model for chronic cerebral hypoperfusion-related neurodegenerative diseases. Brain Res Rev 54(1): 162-180.
  6. Gorelick, P. B., Scuteri, A., Black, S. E., Decarli, C., Greenberg, S. M., Iadecola, C., Launer, L. J., Laurent, S., Lopez, O. L., Nyenhuis, D., Petersen, R. C., Schneider, J. A., Tzourio, C., Arnett, D. K., Bennett, D. A., Chui, H. C., Higashida, R. T., Lindquist, R., Nilsson, P. M. and Roman, G. C., et al. (2011). Vascular contributions to cognitive impairment and dementia: a statement for healthcare professionals from the american heart association/american stroke association. Stroke 42(9): 2672-2713.
  7. Kumaran, D., Udayabanu, M., Kumar, M., Aneja, R. and Katyal, A. (2008). Involvement of angiotensin converting enzyme in cerebral hypoperfusion induced anterograde memory impairment and cholinergic dysfunction in rats. Neuroscience 155(3): 626-639.
  8. Liu, H. X., Zhang, J. J., Zheng, P. and Zhang, Y. (2005). Altered expression of MAP-2, GAP-43, and synaptophysin in the hippocampus of rats with chronic cerebral hypoperfusion correlates with cognitive impairment. Brain Res Mol Brain Res 139(1): 169-177.
  9. Ohta, H., Nishikawa, H., Kimura, H., Anayama, H. and Miyamoto, M. (1997). Chronic cerebral hypoperfusion by permanent internal carotid ligation produces learning impairment without brain damage in rats. Neuroscience 79(4): 1039-1050.
  10. Otori, T., Katsumata, T., Muramatsu, H., Kashiwagi, F., Katayama, Y. and Terashi, A. (2003). Long-term measurement of cerebral blood flow and metabolism in a rat chronic hypoperfusion model. Clin Exp Pharmacol Physiol 30(4): 266-272.
  11. Panickar, K .S. and Norenberg, M. D. (2005). Astrocytes in cerebral ischemic injury: morphological and general considerations. Glia 50(4): 287-98.
  12. Plaschke, K. (2005). Aspects of ageing in chronic cerebral oligaemia. Mechanisms of degeneration and compensation in rat models. J Neural Transm (Vienna) 112(3): 393-413.
  13. Ruitenberg, A., den Heijer, T., Bakker, S. L., van Swieten, J. C., Koudstaal, P. J., Hofman, A. and Breteler, M. M. (2005). Cerebral hypoperfusion and clinical onset of dementia: the Rotterdam Study. Ann Neurol 57(6): 789-794.
  14. Sopala, M. and Danysz, W. (2001). Chronic cerebral hypoperfusion in the rat enhances age-related deficits in spatial memory. J Neural Transm (Vienna) 108(12): 1445-56.
  15. Tanaka, K., Ogawa, N., Asanuma, M., Kondo, Y. and Nomura, M. (1996). Relationship between cholinergic dysfunction and discrimination learning disabilities in Wistar rats following chronic cerebral hypoperfusion. Brain Res 729(1): 55-65.
  16. Tomimoto, H., Ihara, M., Wakita, H., Ohtani, R., Lin, J. X., Akiguchi, I., Kinoshita, M. and Shibasaki, H. (2003). Chronic cerebral hypoperfusion induces white matter lesions and loss of oligodendroglia with DNA fragmentation in the rat. Acta Neuropathol 106(6): 527-534.

简介

大鼠双侧颈总动脉(2VO)的永久闭塞被认为是模拟慢性脑灌注不足状态的合适动物模型,其被证明是阿尔茨海默病或/和血管性痴呆之前的危险因素。 在此协议中,我们描述了如何成功结扎颈前肌群覆盖的双侧颈总动脉,并提供了解2VO手术的详细信息。

【背景】目前,慢性脑灌注不足(CBH)被认为是轻度认知障碍的临床前状态,认为其在痴呆之前(Ruitenberg等人,2005; Gorelick等人 >,2011)。然而,CBH如何产生痴呆在很大程度上是未知的。

20世纪70年代首次建立了双侧颈总动脉(2VO)永久闭塞的方法(Eklof和Siesjo,1972)。由于大鼠中的2VO引起无运动功能障碍的CBH,因此被认为是引起CBH状态的合适的动物模型(Tanaka等人,1996)。在2VO程序之后,三个阶段在理论上由脑血流量(CBF)和大鼠代谢变化决定(Farkas等人,2007)。在开始2VO过程之后,急性局部缺血的第一阶段持续约2-3天(Ohta等人,1997; Otori等人,2003; Tomimoto等人,等人,2003)。在此期间,CBF急剧下降,并在接下来的4周内保持在显着低水平,葡萄糖利用率降低(Otori等人,2003),ATP和磷酸肌酸的突然消耗(Plaschke,2005 )。第二阶段持续8-12周,与衰老和痴呆的CBH相当,CBF轻度减少,ATP恢复,磷酸肌酸水平保持低水平。然而,在最后阶段,CBF恢复到基线并且在2VO 6个月后代谢变化逐渐停止(Ohta等人,1997; Otori等人 ,2003)。

我们还注意到许多使用2VO大鼠模型的研究还揭示了许多病理学特征,包括由Morris水迷宫或八臂径向迷宫评估的学习和记忆受损,其中2VO大鼠显示较长的逃避潜伏期以在Morris水中找到隐藏平台迷宫(Liu等人,2005),并且在八臂放射状迷宫中比在假手术组的大鼠进入一个从未诱饵的手臂更多的错误(Sopala和Danysz,2001)。海马中的神经元细胞死亡(Farkas等人,2004);减少树突状枝化(Chen等人,2017);星形细胞反应(Panickar和Norenberg,2005)和小胶质细胞激活(Abraham和Lazar,2000),等等。所有这些现象提供了有力的证据,2VO大鼠模型是研究CBH相关疾病的分子机制的有价值的动物模型。在这个协议中,我们描述了如何通过永久结扎大鼠的双侧颈总动脉来成功建立CBH的啮齿动物模型的详细步骤。这个协议是基于和修改了以前发表的文章:Kumaran 等。,2008。

关键字:双侧颈总动脉永久性闭塞, 慢性脑灌注不足, 大鼠

材料和试剂

  1. 75%酒精棉球(Bettering,目录号:BY ACB)
  2. 22毫米(3/8圈)带缝线的手术针(Foosin Medical Supplies,Weigao,型号:300系列)(图1B)
  3. 1 ml麻醉注射器(西安圣坦医疗投资管理公司,产品目录编号:STS-SY003-#0037)(图1H)
  4. 3-0丝线(金环,型号:3-0)(图1A)
  5. 医用脱脂棉(竹石药业集团,型号:ZS-HM019)
  6. 动物
    哈尔滨医科大学附属第二医院动物中心,哈尔滨黑龙江省,雄性Sprague-Dawley大鼠(体重280-300g,通常为4-5个月龄)分别饲养于23±1℃, 55±5%的湿度,并保持12小时黑暗/光线人工周期(早上7点开灯),随意提供食物和水。所有有关动物实验的程序均经哈尔滨医科大学动物保护与利用委员会(HMUIRB-2008-06)和中国实验动物科学研究院(A5655-01)的批准。
  7. 0.9%氯化钠(青岛福斯肯国际贸易公司,型号:优质0.9%复方氯化钠注射液)
  8. 水合氯醛(阿拉丁,目录号:C104202)
  9. 硫酸庆大霉素,50mg / ml溶液,无菌(Sangon Biotech,目录号:B540724)
  10. 10%水合氯醛溶液(见食谱)
  11. 20 mg / ml硫酸庆大霉素溶液(见食谱)

设备

  1. 玻璃盘(Canfort实验室和教育用品,目录号:LG075)(图1C)
  2. 持针器(Sklar手术器械,型号:5-1 / 4“,目录号:21-8001)(图1D)
  3. 镊子(精细科学工具,目录号:11009-13)(图1E)
  4. 动脉剪辑(螺母国际,模型:Approximator夹)(图1F)
  5. 剪刀(精细科学工具,目录号:14068-12)(图1G)
  6. 玻璃挂钩(图1I)
  7. 电动剃须刀(义乌科美电器,型号:KM-970)(图1J)
  8. 光纤照明器(Mineralogical Research,目录号:MA312101)(图1K)
  9. 宠物用电热垫(东西易,产品编号:wi95919)(图1L)


    图1.材料和设备A. 3-0丝线缝合; B.缝合的手术针; C.用无菌0.9%氯化钠的玻璃皿; D.持针器; E.钳子; F.动脉夹; G.剪刀; H. 1毫升麻醉注射器; I.玻璃钩; J.电动剃须刀; K.光纤照明器; L.电热垫。
    注意:在手术之前,所有的金属器械应该在200°C的高温下进行消毒。

程序

  1. 麻醉
    禁食12小时后,用10%水合氯醛(300mg / kg,参见食谱)通过腹膜内注射麻醉大鼠,大鼠在5-10分钟内达到手术麻醉。
    注:麻醉深度由角膜眨眼和尾部反射评估。在大鼠达到完全麻醉之前,手术不应该进行。麻醉应至少维持1小时,以完成所有手术过程。
  2. 暴露大鼠颈总动脉(视频1)。

    视频1
    注意:为保持体温,建议在操作过程中将老鼠放在加热垫(37°C)上。

    1. 将鼠放在电热垫上,用电动剃须刀清洁颈部周围的毛皮。 (图2A)
    2. 用75%的酒精棉球消毒颈部皮肤,然后沿着颈部前中线在柄上方切一个2厘米的切口。 (图2B)
    3. 仔细钝化解剖皮下组织并暴露胸骨舌骨肌。 (图2C)
    4. 仔细解剖筋膜,以暴露由二腹肌后腹,躯干肌和胸锁乳突肌组成的颈总动脉三角肌结构,然后垂直分离肌腱膜,最后暴露关键血管鞘神经。 (图2D)
      注意:在暴露的三角区内,可以识别出两个重要血管和神经干,包括内部静脉和颈总动脉及其支流,迷走神经和主动脉抑制神经(年龄和性别各不相同)通常位于颈动脉和迷走神经之间。主要由胸锁乳突肌覆盖的深红色无脉内静脉位于红色脉动颈总动脉外。所以他们可以很容易地通过颜色和脉冲来区分。重要的是,在平滑的解剖过程中,为了成功的手术,小心的注意是必要的,以避免损伤两个神经。
  3. 将颈总动脉与迷走神经和主动脉抑制神经分开。 (视频2)

    视频2

    1. 小心剥离颈总动脉周围的鞘。 (图2E)
    2. 用一只镊子稍微抬起颈总动脉,用玻璃钩轻轻地将颈总动脉中的迷走神经分开。 (图2F)
      注意:注意避免迷走神经和主动脉压迫者的损伤,因为这种损伤可能会引起植物神经功能紊乱,增加死亡率。


      图2. 2VO大鼠模型的程序2-6。 :一种。将老鼠装到加热垫上; B.剪下一个2.0厘米的切口, C.暴露胸骨舌骨肌; D,揭示由二腹肌后腹,嗅鞘细胞和胸锁乳突肌组成的颈总动脉三角肌结构; E.小心剥离颈动脉鞘; F.将迷走神经与颈总动脉分开; G.永久性结扎双侧颈总动脉; H.双侧颈总动脉永久性结扎示意图; I.切断两结扎丝线缝合线之间的颈总动脉; J.切断两结扎丝线之间的颈总动脉的示意图; K.使用20 mg / ml硫酸庆大霉素溶液避免潜在的术后感染; L.关闭伤口。

  4. 永久结扎或切断颈总动脉。 (视频3)

    视频3

    1. 使用两个动脉夹夹住颈总动脉夹在心脏和头侧每个避免流血。 (图2G)
    2. 将两根3-0丝线(浸入0.9%无菌氯化钠中)置于颈总动脉的下面,用钳子夹住右侧(图2G和2H)。为了颈总动脉的永久性结扎,收紧丝线,然后在两根结扎的丝线之间切断颈总动脉(图2I和2J)。
    3. 轻轻地取下动脉夹子。
    4. 在手术过程中,医用脱脂棉将有必要去除任何潜在的出血和渗出物,以最大限度地减少术后感染的机会。
  5. 在对侧颈总动脉上进行同样的手术。 (视频4)

    视频4

  6. 术后护理
    1. 手术后,所有的颈前肌都应该回到原来的位置。
    2. 为避免潜在的术后感染,伤口应在关闭前用20 mg / ml硫酸庆大霉素溶液清洗,多余的溶液必须彻底清除。 (图2K)(视频5)

      视频5

    3. 关闭伤口。 (图2L)
    4. 将老鼠放回笼子,并使用温暖的光源保持稳定的温度,直到大鼠从麻醉中恢复,手术后可能需要至少1-2小时。

笔记

  1. 所有的手术过程都需要在大鼠完全放松后5分钟内完成,这将大大减少感染的机会。
  2. 动物的选择:大鼠被认为是适合2VO操作的啮齿动物物种,因为大鼠能够通过其完整的Willis环而从严重局部缺血存活,其在2VO之后不断减少血流。换句话说,Willis在沙鼠或大多数小鼠品系中的圈子正在开发中。值得一提的是,在我们的研究组中,选择SD而不是Wistar大鼠建立2VO动物模型仅仅是因为使用Wistar的死亡率高于具有相对大的Willis环的SD大鼠。另外,大鼠的体重是重要的因素。根据我们的经验,理想的体重在280-300克之间,如果体重低于280克或高于300克,肯定会增加手术过程中和手术后的死亡风险。
  3. 丝线缝合尺寸的选择:缝合线的最佳尺寸为3-0至5-0,因为丝线缝合线的直径小于3-0可能会损伤动脉,而结扎会导致出血。直径大于5-0的丝线可能导致不完全的结扎或再灌注,主要是由于结扎缝线的松动,最终导致不能诱导CBH状态。
  4. 迷走神经和主动脉抑制神经的保护:迷走神经和主动脉压迫神经在手术过程中的损伤会引起大鼠的植物神经功能紊乱,增加死亡的可能性。
  5. 温柔的表演:温柔而快速的表演在这里是必不可少的。粗略的手术很容易导致严重的出血,导致大鼠死亡。

食谱

  1. 10%水合氯醛溶液
    1. 5克水合氯醛溶于50毫升生理盐水中
    2. 在37°C储存
  2. 20mg / ml硫酸庆大霉素溶液
    1. 4毫升硫酸庆大霉素(50毫克/毫升)用无菌氯化钠(0.9%)稀释至10毫升。
    2. 在-20°C储存

致谢

感谢中国自然科学基金的支持(81271207,81070882,81471115,81671052)。该协议基于并修改了以前发表的文章:Kumaran等人(2008,Neuroscience,155:626-639)。我们要感谢帅帅拍摄的视频和Biddyut达斯的文字编辑。

参考

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  2. Chen X,Jiang,XM,Zhao,LJ,Sun,LL,Yan,ML,Tian,Y.,Zhang,S,段,MJ,Zhao,HM,Li,WR,Hao,YY,Wang,LB ,Xiong,QJ和Ai,J。(2017)。 MicroRNA-195可预防慢性脑灌注不足时的树突变性和神经元死亡。 < em> Cell Death Dis 8(6):e2850。
  3. Eklof,B。和Siesjo,B.K。(1972)。 双侧颈总动脉结扎对大鼠脑血流量和能量状态的影响< / Phys> Scand 86(2):155-165。
  4. Farkas,E.,Institoris,A.,Domoki,F.,Mihaly,A.,Luiten,P.G。和Bari,F。(2004)。 二氮嗪和二甲基亚砜可预防颈动脉闭塞后的脑低灌注相关学习功能障碍和脑损伤。 (Brain Res) 1008(2):252-260。
  5. Farkas,E.,Luiten,P.G。和Bari,F。(2007)。 大鼠永久,双侧颈总动脉闭塞:慢性脑低灌注相关性神经退行性疾病模型。 Brain Res Rev 54(1):162-180。
  6. Gorelick,PB,Scuteri,A.,Black,SE,Decarli,C.,Greenberg,SM,Iadecola,C.,Launer,LJ,Laurent,S.,Lopez,OL,Nyenhuis,D.,Petersen,RC,Schneider ,JA,Tzourio,C.,Arnett,DK,Bennett,DA,Chui,HC,Higashida,RT,Lindquist,R.,Nilsson,PM和Roman,GC,等人。 (2011年)。 血管对认知功能障碍和痴呆的作用:美国心脏协会/美国中风协会。 Stroke 42(9):2672-2713。
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引用:Yan, M. and Ai, J. (2018). A Rodent Model for Chronic Brain Hypoperfusion Related Diseases: Permanent Bilateral Occlusion of the Common Carotid Arteries (2VO) in Rats. Bio-protocol 8(1): e2668. DOI: 10.21769/BioProtoc.2668.
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