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Common Carotid Arteries Occlusion Surgery in Adult Rats as a Model of Chronic Cerebral Hypoperfusion
成年大鼠颈总动脉闭塞手术作为慢性脑低灌注模型   

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Molecular Neurobiology
May 2017

Abstract

Chronic cerebral hypoperfusion (CCH) is an important risk factor of vascular dementia (VaD) and Alzheimer’s disease (AD). Hypoxia/ischemia in the whole brain induced by CCH causes serious damage to brain structure and function, which can lead to cognitive impairment. Two-vessel occlusion (2-VO), also known as permanent, bilateral common carotid artery occlusion, is one of the most widely used animal models (e.g., rat) of CCH to investigate the mechanisms of neurodegenerative processes. In this protocol, we present the surgical procedure for 2-VO in rats.

Keywords: Chronic cerebral hypoperfusion (慢性脑灌注不足), Vascular dementia (血管性痴呆), Alzheimer’s disease (阿尔茨海默氏病), Two-vessel occlusion (双血管闭塞), Cerebral blood flow (脑血流量), White matter (白质)

Background

Many neurological and psychiatric illnesses are caused by disorders of the cerebral circulation. A sudden interruption of the blood supply to distinct brain regions can lead to stroke, while a gradual reduction of continuous cerebral blood flow (CBF) impairs memory processes and contributes to the development of dementia (Farkas and Luiten, 2001; Matsuda, 2001; de la Torre, 2002). In the 2-VO rat model, there is a dramatic decrease in CBF to the brain during the acute ischemic phase (2-3 days post-operation) and the cortical and white matter areas have the largest decrease in blood flow, reaching 35%-45% of the control level (Otori et al., 2003). In the chronic ischemic phase (1-3 months post-operation), the CBF values begin to gradually recover at 1 week, but are still significantly lower than the control values 4 weeks after 2-VO induction (Schmidt-Kastner et al., 2001; Otori et al., 2003; Tomimoto et al., 2003). After 8 weeks to 3 months of 2-VO, only a slight reduction or virtually no reduction of flow has been reported (Otori et al., 2003). Finally, after 6 months of 2-VO, the CBF almost returns completely to normal (Choy et al., 2006), because other arterial sources of blood provide compensatory blood flow (via the circle of Willis, Figure 1) to areas that typically are supplied by the common carotid (Farkas et al., 2007). The 2-VO model exhibits characteristic features of human CCH condition, such as cerebral blood flow and metabolic changes (Ohta et al., 1997; Otori et al., 2003), learning and memory disturbances (Farkas and Luiten, 2001; Farkas et al., 2004b; Liu et al., 2005) and the neuropathologic changes (Kreutzberg, 1996; Farkas et al., 2004b; Panickar and Norenberg, 2005; Ohtaki et al., 2006; Eisel et al., 2006). Additionally, this model has been used to study cerebrovascular WM lesions (Wakita et al., 2002; Takizawa et al., 2003; Farkas et al., 2004a).

In the traditional 2-VO experiment, silk suture is used to ligate the bilateral common carotid artery, and an acute phase after the occlusion with dramatic CBF fall ensues. To improve the 2-VO model, researchers have tested some alternative methods. For example, a silicone collar cuff can be placed around the common carotid artery in order to reproduce the inflammatory response caused by atherosclerosis. However, this operation does not cause long-term memory impairment (de Bortoli et al., 2005). A study in which the two common carotid arteries were occluded at intervals of 1 week found that procedure leads to a progressive decrease in brain perfusion, and decreased mortality compared to procedures that occlude both arteries at once (Sarti et al., 2002a and 2002b). But an undesirable feature of these protocols is that the rats must undergo anesthesia twice a week, which can be stressful to the animal. Kitaguchia et al. (2009) use a 30 min delay between carotid arteries in the murine BCAS model to decrease mortality. Other research groups use ameroid constrictors in place of the silk suture. The ameroid constrictors consist of a titanium shell surrounding the hygroscopic casein material with the internal lumen. The casein component gradually absorbs water and thus expands, resulting in narrowing and occlusion of its encased arterial lumen. So the reduction of the CBF could be mild without a sharp drop (Hattori et al., 2015). Here, we described a protocol in detail for performing 2-VO in rat.


Figure 1. Schematic diagram of the circle of Willis in human (A) and rat (B). ACA (anterior cerebral artery); ACOA (anterior communicating artery); AICA (anterior inferior cerebellar artery); ASA (anterior spinal artery); BA (basilar artery); ICA (internal carotid artery); MCA (middle cerebral artery); PCA (posterior cerebral artery); PCOA (posterior communicating artery); SCA (superior cerebellar artery); VA (vertebral artery). This figure is adapted from Eszter Farkas et al. (2001).

Materials and Reagents

  1. Cotton balls (Beijing Sunny Medical Technology Development, catalog number: YG-048 )
  2. Cotton swabs (Beijing Sunny Medical Technology Development, catalog number: YG-053 )
  3. Silk suture (3/0) (Shanghai Pudong Jinhuan Medical Supplies, 1#)
  4. Male adult Sprague Dawley (SD)/Wistar rats at 10-12 weeks of age (or based on experimental needs to determine the age of rats) (Beijing Vital River Laboratory Animal Technology)
  5. 75% ethanol (ANNJET, Q/371402AAJ008)
  6. Iodine tincture (ANNJET, Q/371402AAJ001)
  7. 10% chloral hydrate (300 mg/kg intraperitoneally, Sinopharm Chemical Reagent, catalog number: 30037516 )
  8. 0.9% sodium chloride solution (Shijiazhuang No.4 Pharmaceutical, H13023201)
  9. 10% chloral hydrate (see Recipes)

Equipment

  1. Rat dissection board (BEIJING HELI KECHUANG TECHNOLOGY DEVELOPMENT, model: HL/JPT-2 )
  2. Electronic balance (Shanghai Yoke Instrument, catalog number: YP10002 )
  3. Curved ophthalmic scissors (Shanghai Medical Instruments, catalog number: Y00020 ) (Figure 2D)
  4. Medical suture needle (HANGZHOU HUAWEI MEDICAL APPLIANCE, catalog number: P1531 ) (www.hzhwyl.com.cn)
  5. Operating scissors (Shanghai Medical Instruments, catalog number: J21010 ) (Figure 2B)
  6. Straight ophthalmic scissors (Shanghai Medical Instruments, catalog number: Y00030 ) (Figure 2C)
  7. Hemostatic forceps (Shanghai Medical Instruments, catalog number: J31050 ) (Figure 2E)
  8. Ophthalmic forceps (Shanghai Medical Instruments, catalog number: JD1020 ) (Figure 2F)
  9. Tissue holding forceps (Shanghai Medical Instruments, catalog number: JD2010 ) (Figure 2G)
  10. Needle holder (Shanghai Medical Instruments, catalog number: J32020 ) (Figure 2A)
  11. Sterilizing trays (Shanghai Medical Instruments, catalog number: R0B030 )
  12. Syringe with needle (Shanghai Zhiyu Medical Equipment, 2 ml)


    Figure 2. Surgery tools. Needle holder (A), Operating scissors (B), Straight ophthalmic scissors (C), Curved ophthalmic scissors (D), Hemostatic forceps (E), Ophthalmic forceps (F), Tissue holding forceps (G).

Procedure

Ethical statement: Adult male Sprague-Dawley (SD)/Wistar rats (10-12 weeks) were used in this protocol. All procedures involving animals follow the local animal ethics protocols and standards.

  1. Fast the rats for 12 h before the experiment.
  2. Anesthetize rats with chloral hydrate (300 mg/kg) by intraperitoneal injection using a 2 ml syringe with a needle.
    Note: Slightly press the paw of the rat, it indicates the rat is in deep anesthesia if there are no reflex actions of hind limb.
  3. Fix the rat on its back on a dissection board. Shave the fur of the ventral neck using curved ophthalmic scissors. Sterilize the shaved skin first with iodine tincture and then with 75% ethanol using cotton swabs (repeat three times) (Figure 3).
    Note: In order to minimize the risk of infections, thoroughly sterilize the dissection board with 75% alcohol before the surgery.


    Figure 3. Rat fixed on a dissection board before the 2-VO surgery

  4. Use operating scissors to make a midventral cervical incision in the middle of the neck in the upper edge of the sternum (about 1-1.5 cm long). Gently remove the submandibular gland using the ophthalmic forceps. This will expose the sternocleidomastoid muscle and sternohyoid muscle (Figure 4).


    Figure 4. Image of the exposed sternocleidomastoid muscle (indicated by the thin arrow) and sternohyoid muscle (indicated by the thick arrow). Cut the skin, remove the tissues, and expose the sternocleidomastoid muscle and sternohyoid muscle.

  5. Carefully separate the common carotid arteries from the adhering tissues using ophthalmic forceps. Be careful and avoid causing any damage to the vagus nerve (Figures 5 and 6, Video 1).
    Note: Gently pull apart the sternocleidomastoid muscle deep in the sternohyoid muscle (until the sternohyoid muscle appears), there will be the common carotid arteria wrapped by fibrous connective tissue, which contains the vagus nerve in its sheath and pulses regularly.


    Figure 5. Isolation of the common carotid artery


    Figure 6. Schematic diagram of the Bilateral carotid artery. The arrows indicate the common carotid artery. This figure is adapted from Eszter Farkas et al. (2001).

    Video 1. Isolation of the common carotid artery

  6. The bilateral common carotid arteries are doubly ligated with 4-0 silk suture, the two sutures are next to each other, then the surgical wounds are sutured back with silk suture. Return the rat back to its cage until it fully recovers from anesthesia (Figure 7, Video 2).
    Notes:
    1. The silk suture won’t be absorbed over time. They remain in place until it is executed.
    2. After surgery, feed the rat with an appropriate amount of penicillin to prevent infection. The rat is kept warm on the heating pad (37.0 ± 0.5 °C) throughout the surgery until the rat is completely awake.


    Figure 7. The common carotid artery ligation and wound closure. The blue arrow indicates the common carotid artery; the black arrow indicates the vagus nerve.

    Video 2. Ligation of the common carotid artery

Data analysis

Cerebral blood flow was tested by ASL in our experiment. Cerebral Blood flow reduced a lot 2-3 days after the 2-VO surgery, reaching 30%-45% of the control level. Two weeks after the surgery, the CBF recovers to 55-65% of the control, and it can reach up to 70%-80% of the control level four weeks after 2-VO surgery.

Notes

  1. All instruments directly in contact with the wound must be sterilized using the dry sterilizer.
  2. Keep the rat warm after surgery.
  3. Do not damage the trachea and vagus nerve during surgery. The trachea is just below the sternal hyoid muscle, and the vagus nerve is accompanied with the common carotid artery in the carotid sheath.
  4. Make sure that the anesthesia is successful because repeated anesthesia can lead to increased rat mortality.
  5. Carry out the operation as quickly as possible. It is better to finish the whole procedure in 15-20 min (not more than 30 min at most). The rat will wake up about half an hour after surgery. Drooping eyelid and small fission indicate a successful operation. After the rate is fully awake, it is fed a normal diet.

Recipes

  1. 10% chloral hydrate
    100 mg/ml chloral hydrate dissolved in 0.9% sodium chloride solution

Acknowledgments

This work was supported by National Natural Science Foundation of China (No. 81271310) and Key project of science and technology program of Beijing Municipal Education Commission (No. KZ201610025021). This protocol was adapted from Farkas et al. (2007). The author(s) declared no potential conflicts of interest with respect to the research, authorship, or publication of this article.

References

  1. Choy, M., Ganesan, V., Thomas, D. L., Thornton, J. S., Proctor, E., King, M. D., van der Weerd, L., Gadian, D. G. and Lythgoe, M. F. (2006). The chronic vascular and haemodynamic response after permanent bilateral common carotid occlusion in newborn and adult rats. J Cereb Blood Flow Metab 26: 1066-1075.
  2. De Bortoli, V. C., Tangrossi Junior, H., de Aguiar Correa, F. M., Almeida Sde, S. and de Oliveira, A. M. (2005). Inhibitory avoidance memory retention in the elevated T-maze is impaired after perivascular manipulation of the common carotid arteries. Life Sci 76: 2103-2114.
  3. De la Torre, J. C. (2002). Vascular basis of Alzheimer's pathogenesis. Ann N Y Acad Sci 977: 196-215.
  4. Eisel, U. L., Biber, K., Luiten, P. G. M. (2006). Life and death of nerve cells: therapeutic cytokine signaling pathways. Curr Signal Transduct Ther 1: 133-146.
  5. Farkas, E., Donka, G., de Vos, R. A., Mihaly, A., Bari, F. and Luiten, P. G. (2004a). Experimental cerebral hypoperfusion induces white matter injury and microglial activation in the rat brain. Acta Neuropathol 108(1): 57-64.
  6. Farkas, E., Institoris, A., Domoki, F., Mihaly, A., Luiten, P. G. and Bari, F. (2004b). Diazoxide and dimethyl sulphoxide prevent cerebral hypoperfusion-related learning dysfunction and brain damage after carotid artery occlusion. Brain Res 1008(2): 252-260.
  7. Farkas, E. and Luiten, P. G. (2001). Cerebral microvascular pathology in aging and Alzheimer's disease. Prog Neurobiol 64(6): 575-611.
  8. 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.
  9. Hattori, Y., Enmi, J., Kitamura, A., Yamamoto, Y., Saito, S., Takahashi, Y., Iguchi, S., Tsuji, M., Yamahara, K., Nagatsuka, K., Iida, H. and Ihara, M. (2015). A novel mouse model of subcortical infarcts with dementia. J Neurosci 35(9): 3915-3928.
  10. Kitaguchi, H., Tomimoto, H., Ihara, M., Shibata, M., Uemura, K., Kalaria, R. N., Kihara, T., Asada-Utsugi, M., Kinoshita, A. and Takahashi, R. (2009). Chronic cerebral hypoperfusion accelerates amyloid beta deposition in APPSwInd transgenic mice. Brain Res 1294: 202-210.
  11. Kreutzberg, G. W. (1996). Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19(8): 312-318.
  12. 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.
  13. Matsuda, H. (2001). Cerebral blood flow and metabolic abnormalities in Alzheimer’s disease. Ann Nucl Med 15: 85-92.
  14. 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.
  15. Ohtaki, H., Fujimoto, T., Sato, T., Kishimoto, K., Fujimoto, M., Moriya, M. and Shioda, S. (2006). Progressive expression of vascular endothelial growth factor (VEGF) and angiogenesis after chronic ischemic hypoperfusion in rat. Acta Neurochir Suppl 96: 283-287.
  16. Otori, T., Katsumata, T., Muramatsu, H., Kashiwagi, F., Katayama, Y., Terashi, A. (2003). Long-term measurements of cerebral blood flow and metabolism in a rat chronic hypoperfusion model. Clin Exp Pharmacol Physiol 30: 266-272.
  17. Panickar, K. S. and Norenberg, M. D. (2005). Astrocytes in cerebral ischemic injury: morphological and general considerations. Glia 50(4): 287-298.
  18. Sarti, C., Pantoni, L., Bartolini, L. and Inzitari, D. (2002a). Persistent impairment of gait performances and working memory after bilateral common carotid artery occlusion in the adult Wistar rat. Behav Brain Res 136(1): 13-20.
  19. Sarti, C., Pantoni, L., Bartolini, L. and Inzitari, D. (2002b). Cognitive impairment and chronic cerebral hypoperfusion: what can be learned from experimental models. J Neurol Sci 203-204: 263-266.
  20. Schmidt-Kastner, R., Truettner, J., Lin, B., Zhao, W., Saul, I., Busto, R. and Ginsberg, M. D. (2001). Transient changes of brain-derived neurotrophic factor (BDNF) mRNA expression in hippocampus during moderate ischemia induced by chronic bilateral common carotid artery occlusions in the rat. Brain Res Mol Brain Res 92(1-2): 157-166.
  21. Takizawa, S., Fukuyama, N., Hirabayashi, H., Kohara, S., Kazahari, S., Shinohara, Y. and Nakazawa, H. (2003). Quercetin, a natural flavonoid, attenuates vacuolar formation in the optic tract in rat chronic cerebral hypoperfusion model. Brain Res 980(1): 156-160.
  22. 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.
  23. Wakita, H., Tomimoto, H., Akiguchi, I., Matsuo, A., Lin, J. X., Ihara, M. and McGeer, P. L. (2002). Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat. Brain Res 924(1): 63-70.

简介

慢性脑低灌注(CCH)是血管性痴呆(VaD)和阿尔茨海默病(AD)的重要危险因素。 CCH诱导的全脑缺氧/缺血对脑结构和功能造成严重损伤,导致认知损害。 双血管闭塞(2-VO)也称为永久性双侧颈总动脉闭塞,是CCH最广泛使用的动物模型之一( eg ,大鼠) 神经退化过程。 在这个协议中,我们介绍了2-VO在大鼠的手术过程。

【背景】许多神经和精神疾病是由脑循环障碍引起的。脑血流突然中断可导致中风,而连续性脑血流量(CBF)逐渐减少则损害记忆过程,并导致痴呆的发生(Farkas and Luiten,2001; Matsuda,2001; de拉托雷,2002年)。在2-VO大鼠模型中,在急性缺血期(术后2-3天)脑CBF显着降低,皮质和白质区血流减少最多,达35% -45%的控制水平(Otori等人,2003年)。在慢性缺血期(术后1-3个月),CBF值在1周开始逐渐恢复,但在2-VO诱导后4周仍显着低于对照值(Schmidt-Kastner等,et al 2001; Otori等人,2003; Tomimoto等人,2003)。已经报道了8周至3个月的2-VO之后,只有轻微的减少或几乎没有减少的流量(Otori等人,2003)。最后,在2-VO 6个月后,CBF几乎完全恢复正常(Choy et al。,2006),因为其他动脉血源提供补偿血流(通过Willis循环,图1)到通常由颈总动脉提供的区域(Farkas et al。,2007)。 2-VO模型表现出人类CCH状况的特征,例如脑血流量和代谢变化(Ohta et al。,1997; Otori et al。,2003) ,学习和记忆障碍(Farkas和Luiten,2001; Farkas等人,2004b; Liu等人,2005)和神经病理学变化(Kreutzberg,1996; Farkas等人,2004b; Panickar和Norenberg,2005; Ohtaki等人,2006; Eisel等人,2006)。此外,该模型已被用于研究脑血管性WM病变(Wakita et al。,2002; Takizawa et al。,2003; Farkas et al。/ em >,2004a)。

在传统的2-VO实验中,使用丝线缝合双侧颈总动脉,发生严重CBF下坠的急性期。为了改进2-VO模型,研究人员已经测试了一些替代方法。例如,可以在颈总动脉周围放置硅胶套箍以重现由动脉粥样硬化引起的炎症反应。然而,这种手术不会造成长期的记忆障碍(de Bortoli et al。,2005)。两个颈总动脉闭塞间隔1周的研究发现,与一次闭塞两个动脉的程序相比,程序导致脑灌注逐渐减少,死亡率降低(Sarti等人 >,2002a和2002b)。但这些方案的一个不合要求的特点是,大鼠必须每周进行两次麻醉,这对动物可能是有压力的。 Kitaguchia 等。 (2009)使用小鼠BCAS模型中颈动脉之间30分钟的延迟来降低死亡率。其他研究小组使用ameroid缩小器代替丝线缝合。 ameroid缩小器由一个围绕吸湿酪蛋白材料与内腔的钛壳组成。酪蛋白组分逐渐吸收水分并因此膨胀,导致其封闭的动脉腔变窄和堵塞。所以CBF的减少可以是温和的而不会急剧下降(Hattori et al。,2015)。在这里,我们详细描述了在大鼠中进行2-VO的方案。

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图1人类(A)和大鼠(B)中Willis环的示意图。ACA(大脑前动脉); ACOA(前交通动脉); AICA(小脑前下动脉); ASA(脊髓前动脉); BA(基底动脉); ICA(颈内动脉); MCA(大脑中动脉); PCA(大脑后动脉); PCOA(后交通动脉); SCA(小脑上动脉); VA(椎动脉)。这个数字是根据Eszter Farkas et al 改编的。 (2001年)。

关键字:慢性脑灌注不足, 血管性痴呆, 阿尔茨海默氏病, 双血管闭塞, 脑血流量, 白质

材料和试剂

  1. 棉球(北京阳光医疗科技发展有限公司,产品编号:YG-048)
  2. 棉签(北京阳光医疗科技发展有限公司,产品编号:YG-053)
  3. 丝绸缝合(3/0)(上海浦东金环医疗用品,1#)
  4. 雄性成年Sprague Dawley(SD)/ Wistar大鼠10-12周龄(或根据实验需要确定大鼠的年龄)(北京Vital River Laboratory Animal Technology)
  5. 75%乙醇(ANNJET,Q / 371402AAJ008)
  6. 碘酊(ANNJET,Q / 371402AAJ001)
  7. 10%水合氯醛(300 mg / kg腹腔注射,国药集团化学试剂,产品目录号:30037516)
  8. 0.9%氯化钠溶液(石家庄四药,H13023201)
  9. 10%的水合氯醛(见食谱)

设备

  1. 鼠解剖板(北京和立科技开发有限公司,型号:HL / JPT-2)
  2. 电子天平(上海Yoke仪器,目录号:YP10002)
  3. 弯曲的眼科剪(上海医疗器械,目录号:Y00020)(图2D)
  4. 医用缝合针(HANGZHOU HUAWEI MEDICAL APPLIANCE,产品目录编号:P1531)( www.hzhwyl.com.cn
  5. 经营剪刀(上海医疗器械,目录编号:J21010)(图2B)
  6. 直式眼科剪(上海医疗器械,产品编号:Y00030)(图2C)
  7. 止血钳(上海医疗器械,目录号:J31050)(图2E)
  8. 眼科钳(上海医疗器械,产品编号:JD1020)(图2F)
  9. 组织保持钳(上海医疗器械,目录号:JD2010)(图2G)
  10. 针头(上海医疗器械,产品编号:J32020)(图2A)
  11. 消毒托盘(上海医疗器械,产品目录号:R0B030)
  12. 带针注射器(上海致郁医疗器械,2毫升)


    (A),手术剪(B),直式眼科剪(C),弯曲眼科剪(D),止血钳(E),眼科钳(F) ,组织夹持钳(G)。

程序

道德声明:在该方案中使用成年雄性Sprague-Dawley(SD)/ Wistar大鼠(10-12周)。涉及动物的所有程序都遵循当地的动物伦理规范和标准。


  1. 在实验前将大鼠禁食12小时
  2. 麻醉大鼠水合氯醛(300毫克/千克)腹腔注射使用2毫升注射器针。
    注意:轻轻按下鼠爪,表明大鼠在后肢没有反射作用的情况下处于深度麻醉状态。
  3. 将老鼠固定在解剖板上。用弯曲的眼科剪刀刮除腹侧颈部的毛皮。先用碘酒消毒剃光的皮肤,然后用棉签擦拭75%的乙醇(重复三次)(图3)。
    注意:为了尽量减少感染的风险,手术前用75%的酒精彻底消毒解剖板。


    图3.大鼠在2-VO手术前固定在解剖板上

  4. 使用手术剪在胸骨上缘(大约1-1.5厘米长)颈部中间做一个中间颈椎切口。用眼科钳轻轻地去除颌下腺。这将暴露胸锁乳突肌和胸骨舌骨肌(图4)。


    图4.暴露的胸锁乳突肌(用细箭头表示)和胸骨舌骨肌(用粗箭头表示)切开皮肤,取出组织,暴露胸锁乳突肌和胸骨舌骨肌。

  5. 使用眼科钳将颈总动脉与粘附的组织仔细分开。小心避免损伤迷走神经(图5和6,视频1)。
    注意:轻轻拉开胸锁乳突肌至胸骨舌骨肌深处(直至出现胸骨舌骨肌),颈总动脉由纤维结缔组织包裹,鞘内含有迷走神经,并有规律地脉冲。 /


    图5.颈总动脉的分离


    图6.双侧颈总动脉示意图箭头表示颈总动脉。这个数字是根据Eszter Farkas et al 改编的。 (2001)。

    视频1

  6. 双侧颈总动脉用4-0丝线双重结扎,两根缝线相邻,再用丝线缝合手术创面。将老鼠放回笼中,直到完全从麻醉中恢复(图7,视频2)。
    注意:
    1. 丝线不会随着时间的推移被吸收。它们保持在位,直到它被执行。
    2. 手术后,给大鼠喂适量的青霉素以预防感染。在整个手术过程中,大鼠在加热垫上保持温热(37.0±0.5℃),直到大鼠完全清醒。


    图7.颈总动脉结扎和伤口闭合。蓝色箭头表示颈总动脉。黑色箭头表示迷走神经。

    视频2

数据分析

在我们的实验中,通过ASL测试脑血流量。 2-VO手术后2〜3天脑血流减少,达到对照水平的30%〜45%。术后2周,CBF恢复至对照组的55-65%,2-VO手术后4周可达对照水平的70%-80%。

笔记

  1. 直接接触伤口的所有器械都必须使用干燥消毒器进行消毒。

  2. 手术后让老鼠保持温暖
  3. 手术过程中不要损伤气管和迷走神经。气管正位于胸骨舌骨肌下面,迷走神经伴有颈总动脉的颈总动脉。
  4. 确保麻醉是成功的,因为反复麻醉会导致鼠死亡率增加。
  5. 尽可能快地进行操作。最好在15-20分钟内完成整个过程(最多不超过30分钟)。大鼠手术后半小时左右醒来。下垂的眼睑和小的裂变表明手术成功。饮食完全清醒后,喂食正常饮食。

食谱

  1. 10%的水合氯醛
    100 mg / ml水合氯醛溶于0.9%氯化钠溶液

致谢

这项工作得到了国家自然科学基金(No.81271310)和北京市教委科技计划重点项目(No.ZZ201610025021)的支持。该协议是从Farkas等人改编而来的。 (2007年)。作者声明对本文的研究,作者或出版物没有潜在的利益冲突。

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引用:Cao, D., Bai, Y. and Li, L. (2018). Common Carotid Arteries Occlusion Surgery in Adult Rats as a Model of Chronic Cerebral Hypoperfusion. Bio-protocol 8(2): e2704. DOI: 10.21769/BioProtoc.2704.
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