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A Mouse Model of Preterm Brain Injury after Hypoxia-Ischemia
缺氧缺血后早产儿脑部损伤的小鼠模型   

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Abstract

The rodent hypoxia-ischemia (HI) model, referred to as the Vannucci model, is the most commonly used model for studying perinatal hypoxic-ischemic brain injury (Zhu et al., 2009; Vannucci and Hagberg, 2004). In the Vannucci model, brain injury is acquired by combining a permanent unilateral common carotid artery ligation with subsequent exposure to hypoxia (Rice et al., 1981). The Vannucci model was originally developed in rat pups at postnatal day (PND)7 (Rice et al., 1981), an age at which the development of the rat brain corresponds to that of the human infant at gestational weeks 32-34 (Zhu et al., 2009; Vannucci et al., 2009). The Vannucci HI model has since been adapted to mouse models of perinatal brain injury, and this has allowed the technique to be used with a broader array of genetically modified animals. Mice at PND9 have been the most commonly used, and these correspond to the human near-term infant (Zhu et al., 2009). In the present protocol, the Vannucci model has been adapted to serve as a model of preterm brain injury in C57bl/6J mice at PND5, an age where the development of the mouse brain corresponds to the brain of a human preterm infant (Zhu et al., 2009; Albertsson et al., 2014). The injury acquired with this protocol is characterized by local white matter injury combined with small areas of focal cortical injury and hippocampal atrophy (Albertsson et al., 2014).

Keywords: Mouse(鼠标), Newborn(新生儿), Brain(脑), Hypoxia(缺氧), Ischemia(缺血)

Materials and Reagents

  1. Animals
    C57bl/6J mice (Charles River Laboratories) at PND4-5
    The day of birth is defined as PND1
    The body weight of PND 5 pups should be 2.0 ± 0.2 g
  2. Anesthetics
    1. Isoflurane (Vetflurane, catalog number: 137317 )
    2. Xylocain (local anesthetic) (AstraZeneca, catalog number: 153270 )

Equipment

  1. Suture
    6-0 silk suture attached to a needle for skin closure (Vömel, catalog number: 14719 )
  2. 7-0 silk suture cut into 1.0 cm–1.5 cm pieces for artery ligation (Ethicon, Johnson & Johnson, catalog number: EH7464G )
  3. Needle for skin suture
  4. Micropore surgical tape (Sollentuna, catalog number: 1530-0, 3 M )
  5. Syringe, 1 ml (B. Braun, Omnifix® 100 Solo)
  6. Heating plate (Labotect, model: Hot plate 062 )
  7. Forceps (Figure 1):
    Fine-tipped curved forceps for dissecting the artery (Dumont, tweezer style 7, catalog number: 0302-7-CO )
    Fine-tipped straight forceps for dissecting the artery (Nopa instruments, catalog number: AB 501/05 )
    Hooked forceps for lifting the artery (Dimeda Instrumente GmbH, catalog number: 42.883.07 )
  8. Fine-tipped scissors (sharp) (Dimeda Instrumente GmbH, catalog number: 08.340.09 )
  9. Nasal mask for administering anesthetic gas (Simtec Engineering, catalog number: 10025 )
  10. Anesthetic vaporizer (Ohmeda Isotec Isoflurane Vaporizer)
  11. Surgical microscope (Carl Zeiss, model: S100/OPMI pico )
  12. Water bath with adjustable temperature (Grant, model: W14 )
  13. Hypoxic chamber with controlled temperature, humidified airflow, and variable oxygen level (Figure 2)
    Note: In this protocol, a Dräger transport incubator 5400 and a Gant type W14 water bath (Figure 2A) were used to keep the ambient temperature at 36 °C. A smaller customized incubator, the hypoxia chamber, was kept inside the transport incubator (Figure 2B). Pups were placed inside the hypoxia chamber during the hypoxia procedure. The air and the 10% oxygen were introduced into the bottom of the customized incubator and vented from the top of the incubator. Before being introduced in the hypoxia chamber, the air and the 10% oxygen mixture were humidified and heated to 36 °C with the heated water bath. The customized hypoxia chamber was 19 cm in diameter and 19 cm in length (Figure 2C).


    Figure 1. Surgical tools. Hooked fine-tipped forceps for lifting up the artery (A), fine-tipped straight forceps (B) and curved forceps (C) for dissecting the artery, and fine-tipped scissors for the skin incision (D).



    Figure 2. Equipment used for hypoxia treatment. A. Water bath and transport incubator containing the hypoxia camber. B. The hypoxia chamber inside the transport incubator, and (C) the dimensions of the customized hypoxia chamber.



    Figure 3. Hypoxia-Ischemia procedure time line

Procedure

Note: The duration of hypoxia, the chamber temperature, and the age and strain of the mice are all factors that have significant effects on the brain injury outcome. In this model, we use 70 min of hypoxia (Figure 3). However, this hypoxia time might need to be adjusted empirically for each laboratory’s unique conditions.

  1. Surgery

    Preparation/Notes
    1. Set up the surgery area.
    2. Work on a ventilated area (Figure 4).
    3. Weigh each pup before surgery.
    4. Make sure to keep the pups warm all the time using the heating plate before and after surgery. Minimize the time of surgery to avoid cooling of the pups-this is very important for the outcome of the procedure! Apart from this, no additional attention to animal care is needed. If using normal C57bl/6J pups, the surgery should be done under clean, but not necessarily sterile, conditions. However, this might need to be adjusted depending on the strain of mice used.
    5. The pups are placed in a small clean paper box that has a thin bottom to allow good contact between the box and the heating plate to be sure that the heat can be conducted to the pups lying at the bottom of the box.


    Figure 4. Ventilated area for surgery (A). Heating plate for keeping pups warm (left, B), nasal mask for anesthesia (center, C) and anesthetic vaporizer (right, D).

    Procedure
    1. Anesthetize the pup with isoflurane using a nasal mask (5.0% for induction and 1.5% to 3.0% for maintenance).
    2. Gently fixate the pup on its back using the micropore surgical tape (Figure 5A). Keep the nasal mask with isoflurane in position.
    3. Open the skin with the scissors by making a 0.2 cm–0.3 cm incision in the neck close to the midline (Figure 5B).


      Figure 5. Surgery and artery ligation. A. The fixated pup with nasal mask for anesthesia. B. The midline is indicated by the solid black line, the incision is indicated by the dashed black line, and the top of the sternum is indicated by the black arrow. C. The artery is indicated by arrow 1 and the vein is indicated by arrow 2. D. The dissected artery is lifted up by hooked forceps. E. The suture under the artery is indicated by arrow 1, and the vein without the suture is indicated by arrow 2. F. The ligated artery. G. The sutured wound. H. The application of Xylocain on the wound.

    4. Use fine-tip forceps to carefully push away fat and muscles. Close to the midline there will be a pocket underneath the fat and muscles, and this is where the left common carotid artery will be located.
    5. Very close to the common carotid artery will be a vein. Make sure not to confuse the artery and the vein (Figure 5C).
    6. The vein is more superficial than the artery, and the artery is more medial (to the left) than the vein.
    7. Compared to the vein, the artery is more reddish and will have an obvious pulse.
    8.  Use the fine-tip forceps to carefully dissect the artery free from the vein, the vagal nerve, and other surrounding soft tissues.
    9. When the artery has been dissected, use a curved fine-tip forceps or hooked forceps to isolate and lift up the artery (Figure 5D).
    10. With the other hand, take up the prepared 1 cm-1.5 cm long 7-0 suture using the straight or curved forceps. Grab one end of the suture with the curved or hooked forceps holding up the artery and gently pull the suture under the artery (Figure 5E).
    11. Ligate the artery by making two knots with the suture (Figure 5F).
    12. Close the wound with the 6-0 suture. Make two knots (Figure 5G).
    13.  Infiltrate the wound with local anesthetic by adding one drop of Xylocain onto the wound using the 1 ml syringe (Figure 5H).
    14. Put the pup back on the heating plate and allow the pup to recover from anesthesia. Pups have recovered when they have woken up and started to move again.
    15. When all pups have gone through surgery, take all pups back to their dam to rest for 1 h.

  2. Hypoxia

    Preparation/Notes
    1. The temperature in the chamber should be kept strictly at 36 °C. It is important that this temperature does not change because the pups are not able to maintain their body temperature. A 1-2 °C drop in temperature (hypothermia) can result in reduced brain injury, and increased temperature can result in increased brain injury.
    2. To keep the chamber temperature strictly at 36 °C, the air and 10% oxygen must be warmed and humidified before flowing through the chamber.
    3. The airflow and the 10% oxygen flow through the chamber are both 4 L/min.
    4. Under normal situation in our lab, the hypoxia-ischemia treatment causes less than 10% mortality. The experimenter needs to pay attention to increases in mortality that might be indicating problems with the equipment or the procedure.

    Procedure
    1. Start the flow of warmed and humidified air through the chamber 5 min prior to putting the pups in the chamber.
    2. After the pups have recovered from surgery for 1 h with their dam, put the pups in the chamber and maintain the airflow through the chamber for 10 min.
    3. After 10 min of airflow, change the flow from air to 10% oxygen (by mixing air and nitrogen) for 70 min.
    4. Check the temperature regularly during the 10% oxygen incubation to make sure that the temperature is stable at 36 °C.
    5. After 70 min in 10% oxygen, change the flow from 10% oxygen to air for 10 min.
    6. After 10 min in air, take the pups from the chamber and return them to their dam.

Representative data


Figure 6. Representative data. Representative photomicrograph of a thionin/acid fuchsin-stained brain coronal section shows the degree of injury in the ipsilateral hemisphere (the right side) at 3 days after HI at PND5 with 70 min of hypoxia. Arrows indicate focal subcortical white-matter injury and the loss of hippocampus in the ipsilateral hemisphere of the mouse brain.

Notes

HI should be performed during the daytime when the dam is less active. This makes it more likely that the dam stays in the nest to keep the pups warm when they return after the surgery/HI. Otherwise, the pups might suffer from hypothermia that would influence the degree of brain injury degree and contribute to variation in the results.

Acknowledgments

This protocol was adapted from the previously published study of Albertsson et al. (2014). XW and AM research is supported by the Swedish Medical Research Council (VR 2008-2286 and VR 2013-2475 to XW), the Bill & Melinda Gates Foundation Grand Challenge Explorations and Global Health (OPP1036135 to XW), Swedish governmental grants to researchers in the public health service (ALFGBG-367051 and ALFGBG-367051 to XW), VINNMER-Marie Curie international qualification (VINNOVA, 2011-03458 to XW), and Wilhelm and Martina Lundgren (37/2013 to XW and vet2-26/2013 to AM).

References

  1. Albertsson, A. M., Bi, D., Duan, L., Zhang, X., Leavenworth, J. W., Qiao, L., Zhu, C., Cardell, S., Cantor, H., Hagberg, H., Mallard, C. and Wang, X. (2014). The immune response after hypoxia-ischemia in a mouse model of preterm brain injury. J Neuroinflammation 11(1): 153.
  2. Hedtjarn, M., Leverin, A. L., Eriksson, K., Blomgren, K., Mallard, C. and Hagberg, H. (2002). Interleukin-18 involvement in hypoxic-ischemic brain injury. J Neurosci 22(14): 5910-5919.
  3. Rice, J. E., 3rd, Vannucci, R. C. and Brierley, J. B. (1981). The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 9(2): 131-141.
  4. Sheldon, R. A., Sedik, C. and Ferriero, D. M. (1998). Strain-related brain injury in neonatal mice subjected to hypoxia-ischemia. Brain Res 810(1-2): 114-122.
  5. Vannucci, R. C., Connor, J. R., Mauger, D. T., Palmer, C., Smith, M. B., Towfighi, J. and Vannucci, S. J. (1999). Rat model of perinatal hypoxic-ischemic brain damage. J Neurosci Res 55(2): 158-163.
  6. Vannucci, S. J. and Hagberg, H. (2004). Hypoxia-ischemia in the immature brain. J Exp Biol 207(Pt 18): 3149-3154.
  7. Zhu, C., Wang, X. and Blomgren, K. (2009). Cerebral Hypoxia-Ischemia in neonatal rats or mice: A model of perinatal brain injury, in animal models of acute neurological injuries. In: Chen, J. et al. (eds). Humana Press, 221-230.

简介

啮齿动物缺氧缺血(HI)模型,称为Vannucci模型,是研究围产期缺氧缺血性脑损伤的最常用的模型(Zhu等人,2009; Vannucci和Hagberg, 2004)。在Vannucci模型中,通过将永久性单侧颈总动脉结扎与随后暴露于缺氧来获得脑损伤(Rice等人,1981)。 Vannucci模型最初是在出生后(PND)7(Rice等人,1981)的大鼠幼仔中开发的,大鼠大脑的发育对应于人类婴儿的发育的年龄妊娠周32-34(Zhu等人,2009; Vannucci等人,2009)。 Vannucci HI模型已经适应于围产期脑损伤的小鼠模型,并且这允许该技术与更广泛的基因修饰动物一起使用。 PND9的小鼠是最常用的,并且这些对应于人近期婴儿(Zhu等人,2009)。在本协议中,Vannucci模型已经适应作为在PND5的C57bl/6J小鼠中的早产性脑损伤的模型,PND5是小鼠脑的发育对应于人早产婴儿的大脑的年龄(Zhu et al。,2009; Albertsson et al。,2014)。使用该方案获得的损伤的特征在于局部白质损伤与小面积的局灶性皮层损伤和海马萎缩(Albertsson等人,2014)。

关键字:鼠标, 新生儿, 脑, 缺氧, 缺血

材料和试剂

  1. 动物
    C57bl/6J小鼠(Charles River Laboratories),在PND4-5
    出生日定义为PND1
    PND 5只幼仔的体重应为2.0±0.2 g
  2. 美学
    1. 异氟醚(Vetflurane,目录号:137317)
    2. Xylocain(局部麻醉剂)(AstraZeneca,目录号:153270)

设备

  1. 缝合
    6-0丝缝合针连接到皮肤闭合针(Vömel,目录号:14719)
  2. 7-0丝缝合切成1.0cm-1.5cm用于动脉结扎(Ethicon,Johnson& Johnson,目录号:EH7464G)
  3. 皮肤缝合针
  4. 微孔手术胶带(Sollentuna,目录号:1530-0,3M)
  5. 注射器,1ml(B.Braun,Omnifix 100 Solo)
  6. 加热板(Labotect,型号:Hot plate 062)
  7. 镊子(图1):
    用于解剖动脉的细尖弯曲钳(Dumont,镊子样式7,目录号:0302-7-CO)
    用于解剖动脉的细尖直钳(Nopa instruments,目录号:AB 501/05)
    用于提升动脉的钩形钳(Dimeda Instrumente GmbH,目录号:42.883.07)
  8. 细尖剪刀(锋利)(Dimeda Instrumente GmbH,目录号:08.340.09)
  9. 用于给予麻醉气体的鼻罩(Simtec Engineering,目录号:10025)
  10. 麻醉蒸发器(Ohmeda Isotec Isoflurane Vaporizer)
  11. 手术显微镜(Carl Zeiss,型号:S100/OPMI pico)
  12. 水温可调(格兰特,型号:W14)
  13. 缺氧室,温度受控,加湿气流和可变氧水平(图2)
    注意:在该方案中,使用Dräger运输培养箱5400和Gant型W14水浴(图2A)将环境温度保持在36℃。将较小的定制培养箱(缺氧室)保持在运输培养箱内(图2B)。在缺氧程序期间将小狗放置在缺氧室内。将空气和10%氧气引入定制培养箱的底部,并从培养箱的顶部排出。在被引入缺氧室之前,将空气和10%氧气的混合物加湿,并用热水浴加热至36℃。定制的缺氧室直径为19cm,长度为19cm(图2C)。

    图1.手术工具。用于抬起动脉(A),细尖的直镊子(B)和弯曲的钳子(C)用于解剖动脉的钩状细尖镊子,以及细尖的用于皮肤切口的剪刀(D)。



    图2.用于缺氧治疗的设备 A.含有缺氧外倾的水浴和运输孵化器。 B.运输培养箱内的缺氧室,和(C)定制的缺氧室的尺寸


    图3.缺氧 - 缺血程序时间线

程序

注意:缺氧的持续时间,室温度,以及小鼠的年龄和品系都是对脑损伤结果具有显着影响的因素。在这个模型中,我们使用70分钟的缺氧(图3)。然而,这种缺氧时间可能需要根据每个实验室的独特条件经验调整

  1. 手术

    准备/备注
    1. 设置手术区。
    2. 在通风处工作(图4)。
    3. 在手术前称每只小狗。
    4. 确保使用加热板保持小狗温暖 手术前后。 尽量减少手术时间,避免冷却 的小狗 - 这对于程序的结果非常重要! 除此之外,不需要额外关注动物护理。 如果 使用正常的C57bl/6J幼仔,手术应在清洁下进行,但是 不一定无菌,条件。 但是,这可能需要 根据所使用的小鼠的应变进行调整。
    5. 放置小狗   在一个小的干净的纸盒有一个薄的底部允许良好的接触   之间的箱子和加热板,以确保热量可以 进行到位于箱子底部的幼仔。


    用于保持瞳孔温暖的加热板(左,B),用于麻醉的鼻罩(中心,C)和麻醉蒸发器(右,D)。

    过程
    1. 使用鼻罩(5.0%用于诱导和1.5%至3.0%用于维护)用异氟烷麻醉小鼠。
    2. 使用微孔手术胶带轻轻固定小狗的背部 (图5A)。保持鼻罩与异氟烷的位置。
    3. 用剪刀打开皮肤,在靠近中线的颈部做一个0.2厘米-0.3厘米的切口(图5B)。


      图  5.手术和动脉结扎 A.用鼻罩固定的小狗 麻醉。中线用黑色实线表示 切口由黑色虚线表示,并且顶部 胸骨由黑色箭头指示。动脉表示为 箭头1和静脉由箭头2指示。D.切开的动脉是  由钩状钳子举起。 E.在动脉下的缝线 如箭头1所示,并且没有缝合线的静脉由 箭头2. F.结扎的动脉。 G.缝合伤口。 H.申请   的Xylloc在伤口上
    4. 使用细尖镊子小心推开脂肪和肌肉。 相近 中线会有一个口袋下面的脂肪和肌肉,和 这是左颈总动脉将位于的位置。
    5. 非常接近颈总动脉将是一条静脉。 确保不要混淆动脉和静脉(图5C)。
    6. 静脉比动脉更为表面,动脉比静脉更内侧(向左)。
    7. 与静脉相比,动脉更红,并且将具有明显的脉搏。
    8.  使用细尖镊子仔细解剖动脉 静脉,迷走神经和其他周围软组织。
    9. 什么时候 动脉已经解剖,使用弯曲的细尖镊子或钩 镊子隔离和提起动脉(图5D)。
    10. 有了 另一只手,拿起准备好的1 cm-1.5 cm长的7-0缝线 直或弯钳。 抓住缝合线的一端与弯曲 或钩状镊子抱住动脉,轻轻拉动缝合线   动脉(图5E)。
    11. 通过用缝合线制造两个结来缝合动脉(图5F)。
    12. 用6-0缝线闭合伤口。 做两个结(图5G)。
    13.  用局部麻醉剂通过加入一滴渗透伤口 使用1ml注射器将Xyllocin涂到伤口上(图5H)。
    14. 放 小狗回到加热板上并允许小狗从中恢复 麻醉。 小狗已经恢复,当他们醒了,开始 再次移动。
    15. 当所有的小狗都经过手术,所有的小狗回到他们的大坝休息1小时。

  2. 缺氧

    准备/备注
    1. 腔室中的温度应严格保持在36℃。 重要的是,这个温度不会改变,因为幼仔 不能保持其体温。 1-2°C下降 温度(低体温)可以导致减少的脑损伤,和 增加的温度可导致增加的脑损伤。
    2. 为了将室温严格保持在36℃,空气和10% 氧气必须在流过室之前被加热和加湿。
    3. 气流和通过室的10%氧气流量均为4L/min。
    4. 在我们实验室的正常情况下,缺氧缺血治疗 导致小于10%的死亡率。 实验者需要注意 增加死亡率,可能是指示问题 设备或程序。

    过程
    1. 在将幼仔放入腔室之前5分钟开始加热和加湿的空气流过腔室。
    2. 小狗从手术中恢复了1小时,他们的大坝,放 小室中并且保持通过室的气流   10分钟。
    3. 在10分钟的气流之后,将气流从空气变为10%氧气(通过混合空气和氮气)70分钟。
    4. 在10%氧气孵育期间定期检查温度,以确保温度稳定在36°C。
    5. 在10%氧气中70分钟后,将流量从10%氧气改变为空气10分钟。
    6. 在空气中10分钟后,从小室里的小狗,并返回他们的大坝。

代表数据


图6。 代表数据。硫素/酸性品红染色的脑冠状切片的代表性显微照片显示3天同侧半球(右侧)的损伤程度在PND5的HI后70分钟的缺氧。箭头表示局部皮质下白质损伤和小鼠大脑同侧半球的海马损失。

笔记

HI应该在大坝活动较少的白天进行。这使得更可能的是,水坝停留在巢中,以保持小狗在手术后/HI返回时温暖。否则,幼崽可能遭受低温,这将影响脑损伤程度,并有助于结果的变化。

致谢

该协议改编自先前发表的Albertsson等人的研究(2014)。 XW和AM研究由瑞典医学研究委员会(VR 2008-2286和VR 2013-2475至XW),比尔& Melinda Gates基金会大挑战探索与全球健康(OPP1036135至XW),瑞典政府资助给公共卫生服务研究人员(ALFGBG-367051和ALFGBG-367051至XW),VINNMER-Marie Curie国际资格(VINNOVA,2011-03458至XW),Wilhelm和Martina Lundgren(37/2013至XW和vet2-26/2013至AM)。

参考文献

  1. Albertsson,AM,Bi,D.,Duan,L.,Zhang,X.,Leavenworth,JW,Qiao,L.,Zhu,C.,Cardell,S.,Cantor,H.,Hagberg, C.和Wang,X。(2014)。 早产性脑损伤小鼠模型缺氧缺血后的免疫反应。 em> J Neuroinflammation 11(1):153
  2. Hedtjarn,M.,Leverin,A.L.,Eriksson,K.,Blomgren,K.,Mallard,C。和Hagberg,H。(2002)。 白细胞介素-18参与缺氧缺血性脑损伤 J Neurosci 22(14):5910-5919。
  3. Rice,J.E.,3rd,Vannucci,R.C。和Brierley,J.B。(1981)。 不成熟对大鼠缺氧缺血性脑损伤的影响 Ann Neurol 9(2):131-141。
  4. Sheldon,R.A.,Sedik,C.and Ferriero,D.M。(1998)。 在缺氧缺血的新生小鼠中的应变相关的脑损伤。 脑Res 810(1-2):114-122。
  5. Vannucci,R.C.,Connor,J.R.,Mauger,D.T.,Palmer,C.,Smith,M.B.,Towfighi,J。和Vannucci,S.J。(1999)。 围产期缺氧缺血性脑损伤的大鼠模型 J Neurosci Res 55(2):158-163。
  6. Vannucci,S.J.and Hagberg,H。(2004)。 未成熟大脑中的缺氧缺血 J Exp Biol 207(Pt 18):3149-3154。
  7. Zhu,C.,Wang,X。和Blomgren,K。(2009)。 脑缺氧 - 新生大鼠或小鼠中的缺血:围产期脑损伤的模型,在急性神经损伤的动物模型中。 参见:Chen,J.et al。(eds)。 Humana Press,221-230。
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引用:Albertsson, A. and Wang, X. (2015). A Mouse Model of Preterm Brain Injury after Hypoxia-Ischemia. Bio-protocol 5(13): e1526. DOI: 10.21769/BioProtoc.1526.
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