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Feb 2021

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Transcardiac Perfusion of the Mouse for Brain Tissue Dissection and Fixation
小鼠心脏灌流、脑组织解剖与固定   

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Abstract

Transcardiac perfusion with saline followed by 4% paraformaldehyde (PFA) is widely used to clear blood and preserve brain for immunostaining or in situ hybridization. PFA breaks into formaldehyde in solution, which cross-link protein and DNA molecules to preserve tissue and cell structure. Here we provide a step by step guide for performing this procedure in mouse.

Keywords: Mouse (小鼠), Transcardiac perfusion (经心灌注), Aorta (主动脉), Paraformaldehyde (多聚甲醛), Brain (大脑)

Materials and Reagents

  1. Protective gear (gloves, goggles, masks, lab coat)

  2. Deionized water

  3. Sodium pentobarbital (Sigma, catalog number: P-010)

  4. 1.5% sodium pentobarbital (in saline)

  5. NaCl (Sigma-Aldrich, catalog number: S7653)

  6. Saline (0.9% sodium chloride)

  7. NaH2PO4·2H2O (Sigma-Aldrich, catalog number: 71645)

  8. Na2HPO4·12H2O (Sigma-Aldrich, catalog number: 71663)

  9. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148)

  10. NaOH (Sigma-Aldrich, catalog number: S8045)

  11. Hydrochloric acid (HCl) (Sigma, catalog number: H1758)

  12. Heparin (Sigma, catalog number: H3149)

  13. Sodium azide (Sigma, catalog number: S2002)

  14. 4% paraformaldehyde (PFA, in 0.1 M Phosphate Buffer, pH 7.0-7.4) (see Recipes)

Equipment

  1. Chemical fume hood

  2. Dissecting forceps (RWD, catalog number: F12018-13) (Figure 1)

  3. Ophthalmic forceps (RWD, catalog number: F12006-10) (Figure 1)

  4. Hemostatic forceps (RWD, catalog number: F21002-12) (Figure 1)

  5. Tissue scissors (RWD, catalog number: S13052-12) (Figure 1)

  6. Fine scissors (RWD, catalog number: S12005-10) (Figure 1)

  7. Vascular clamp (RWD, catalog number: R33001-48) (Figure 1)

  8. Micro spatula (Thermo Fisher, catalog number: 21-101-10) (Figure 1)

  9. 1 ml and 20 ml syringe (Figure 1)

  10. 25- or 27-gauge infusion needle (Figure 1)

  11. (Optional) Peristaltic pump (LONGERPUMP, catalog number: BT100-2J)

  12. 2.5 L and 5 L beakers

  13. Thermometer (measuring range:100 °C, division value:1 °C)

  14. Measuring cylinder

  15. Analytical balance (SARTORIUS, catalog number: BSA124S)

  16. Benchtop vacuum

  17. 0.22 μm PVDF filter set (Nalgene, catalog number: 291-4520)

  18. 50 ml conical tube (Thermo Fisher, catalog number: 339652)

  19. Fume hood

  20. Microwave

  21. Magnetic stirrer with heating device (SCILOGEX, catalog number: MS7-H550-Pro)



Figure 1. Surgical instruments

Procedure

  1. Preoperative preparation

    1. Measure and record the body weight to the nearest gram.

    2. Pick the mouse up by its tail, place it on top of a cage rack, and gently pull its tail with one hand while allowing it to grab the iron wire firmly to stabilize it at a steady position.

    3. Press the mouse down around the neck with one hand and hold it by the scruff of the neck. Hold as much skin as possible to have a secure grip so that the mouse cannot move during injection.

    4. Administer 1.5% sodium pentobarbital at a dose of 0.06 ml per 10 g body weight through intraperitoneal injection at the lower left or right quadra of the abdomen (Hou et al., 2021) (Figure 2).

    5. Note: To avoid injuring the internal organ, do not advance the needle too much. Hold the mouse at a head-down position may also help.



      Figure 2. Intraperitoneal injection


    6. Place the mouse back to its cage. Wait ~5 min. Assess if the mouse has reached a surgical plane of anesthesia by loss of response to tail or toe pinches (Reference 4).

    7. Note: For neonatal mouse younger than 6 days of age, anesthesia can be achieved through hypothermia (Behringer et al., 2013). Place mouse pup in a conical tube or wrap it with latex glove (cut off the thumb of the glove for this purpose) and bury it up to the neck in crushed ice for 10-15 min to obtain a surgical pane of anesthesia. Analgesia state lasts ~10 min, thereby do not leave the pup on surgical station for too long. Perform the surgery as soon as possible.

    8. (From this step on, please operate in fume hood.) Place the mouse on its back. Firmly tape or pin its four paws to a Styrofoam or wood surgical station so that nothing moves during the perfusion procedure.

    9. Note: If the heart stops beating due to excessive anesthesia, perfusion should be started immediately, preferentially with saline containing 20 units/ml heparin to avoid clotting of blood.

    10. Trim the tip of a 25- or 27-gauge infusion needle to make it slightly blunt (Figure 3) to reduce the risk of puncturing the aorta wall in the following steps (Reference 1). Polish the edges with sandpaper if necessary.


      Figure 3. Needle trimming. A. Untrimmed needle. B. Trimmed needle.


    11. Connect the transfusion needle to a 20 ml syringe filled with saline. Flush the tubing and needle with saline to expel any air.

    12. Fill another 20 ml syringe with 4% PFA solution.


  2. Transcardiac perfusion with saline

    1. Grip the skin on the chest with ophthalmic forceps and make an incision using tissue scissors to expose the xiphoid (a piece of arrowhead-shaped white bone).

    2. Grip the xiphoid with ophthalmic forceps and make lateral incisions beneath the ribcage using tissue scissors to expose the diaphragm and liver.

    3. Carefully make incisions in the diaphragm along the entire length of the rib cage using fine scissors.

    4. Make two cuts through both sides of the rib cage up to the collarbone using tissue scissors. Reflect the sternum up over the head of the mouse with hemostat (or pint it to the surgical station, or cut it off) to fully expose the heart and lung (Figure 4).


      Figure 4. Thoracotomy


    5. Carefully tear off pericardial sac and any other tissues covering the heart using dissecting forceps to provide a clear view of the heart and vessels.

    6. Secure the heart with dissecting forceps at a steady position. Insert the trimmed needle from the tip of the left ventricle at an angle approximately parallel to the midline of the heart (Figure 5A). As the needle tip is blunt, enough pressure needs to be applied to get through the ventricle wall. However, do not push too hard and mind the angle to make sure the needle does not advance into the left atrium or right ventricle. With experience, one will clearly feel the resistance before penetration and the loss of it afterwards. The heart should be beating and one may observe backflow of blood into the needle before perfusion starts.

    7. Steadily advance the needle until it enters the ascending aorta. The tip of the needle is visible through the aorta wall (Figure 5B-5D). No resistance should be encountered if the needle moves in the right direction. Secure the needle in the aorta with vascular clamp or hemostatic forceps at appropriate position (arrowhead in Figure 5C).
      Notes:

      1. If the needle cannot be successfully inserted into the aorta, leave it in the left ventricle (~5 mm in for adult mouse, shorter for younger mouse; adjust wisely depending on the size of the heart) (Figure 5B), but it has to be secured with pins or tape to avoid slipping out of the heart during perfusion (Video 1). For neonatal pups, the aorta may be too narrow to fit the needle tip and too delicate to clamp, in which case leaving needle tip in the ventricle is preferred.
      2. If the needle advanced too much into right ventricle or even pierce through the heart, withdraw and re-insert the needle promptly. If the wound is not too bad, it may not affect perfusion.
    8. Make a small incision on the right atrium using fine scissors. Dark venous blood should flow out immediately. Start the saline perfusion at once at a constant speed of ~1 ml/5 s by manfully pushing the syringe or using a peristaltic pump. As saline flushes out the blood, the liver gradually turns pale (Figures 5E-F).
      Notes:

      1. If the needle tip is in the left ventricle instead of aorta, slower perfusion speed is preferred.
      2. If the needle tip goes into right ventricle, one may observe signs of failed perfusion including inflation of lung (Figure 6), liquid dripping out of nose or mouth and incomplete decolor of liver. 
      Optional: Instead of manual injection, peristaltic pump can be used to better control the perfusion speed.
    9. Stop perfusion when the fluid flowing out is clear of blood. For adult mice, it takes ~20 ml saline.


      Figure 5. Needle insertion. A. Position and direction of needle insertion. B. The needle tip can be left in the left ventricle halfway through the heart. Mind the trajectory so that the needle does not pierce through the septum (open arrowhead) into the right ventricles. C. Advance the needle into the aorta and clamp it at appropriate position (closed arrowhead). D. The needle tip is visible through the aortic wall (closed arrowhead). E. Make a small incision in the right atrium (closed arrowhead). F. The liver is filled with blood and appears red before perfusion. G. The liver loses blood and becomes pale after perfusion. H. Improper perfusion leads to accumulation of liquid in the lung and pulmonary edema (black arrowhead). 

      Video 1. Transcardiac Perfusion of the Mouse.

  3. Transcardiac perfusion with 4% PFA (Video 1)

    1. Switch from saline to 4% PFA. Make sure no bubble gets into the perfusion system during the switch (Gage et al., 2012).

    2. Steadily perfuse 4% PFA at a constant speed of ~1 ml/5 s. As PFA goes into circulation, one can observe signs including body twitching, tail flicking and head moving.

    3. Perfuse ~15-20 ml 4% PFA for adult mouse. The whole mouse body should be very stiff and the liver may turns slightly pale brownish (a little bit darker color than it was before 4% PFA perfusion).

    4. Remove the needle and unpin the mouse from the surgical station.


  4. Brain dissection (Video 1)

    1. Decapitate the mouse with tissue dissecting scissors (Figure 6, horizontal line). Remove the skin by first making an incision along the midline from the neck to the nose (Figure 6, vertical line) and then reflect the two flaps of skin rostrally and laterally to expose the skull (Figure 6B).

    2. Hold the head tightly with one hand. Trim off bones at the caudal end with dissecting scissors (Figure 6B, dashed line). Clear off any residual muscle on the skull with forceps or scissors. If the 4% PFA was done properly, the brain will shrink a little bit and there will be some space between the brain and the bones forming the foramen magnum.

    3. Slide one blade of the fine scissors into the foramen magnum (underneath the brain) with the sharp side facing the bone on one side. Make a lateral cut. Repeat the cut on the contralateral side. Carefully remove the skull covering the cerebellum with ophthalmic forceps.

    4. At the rostral end of the mid-sagittal suture, insert the tip of one blade of the fine scissors between the foramen magnum and the brain with the sharp side facing the bone. Rapidly slide and cut the cranium along the mid-sagittal suture. Carefully remove the skull with forceps to expose the brain. An additional cut may be necessary to expose the olfactory bulb (Figure 6C).

    5. Note: Be careful not to damage the brain during the operation. Peel off any meninges attaching to the skull before removing the bone chips; otherwise it may slice the soft brain tissue.



      Figure 6. Brain dissection. A. Cut the head off, cut the fur from the neck towards the tip of the nose (the dotted line), and expose the skull. B-C. Cut off the skull below the brainstem along the dotted line, and then remove the skull covering the cerebellum and cerebrum in turn to expose the brain tissue.


    6. Using a micro spatula (or other flat and blunt tool) to sever the nerve bundle on the ventral surface of the brain and scoop the brain out.
      Notes:

      1. Compared to freshly dissected brain (Figure 7A, very soft and full of blood), successfully perfused and fixed brain will shrink a little bit, become harder and appear slightly pale brownish (Figure 7C). Unsuccessful perfusion may leave blood in the brain (Figure 7B) and make it appear pinkish. Post-fix the brain in 4% PFA overnight at 4 °C. 
      2. Postfix will further shrink, harden and darken the brain (Figure 7D).


    7. Transfer the brain into PBS containing 0.01% sodium azide for long term storage at 4°C. Alternatively, transfer the brain into 30% sucrose solution, wait until it sinks to the bottom, and store at minus 20-80 °C (Hou et al., 2021).



      Figure 7. Representative images of brains dissected out under different conditions. A. Freshly dissected brain is full of blood and very soft. B. Unsuccessfully perfused brain retains some blood. C. Successfully perfused and fixed brain before post-fixation. D. Brain after post-fixation. Scale bar: 4 mm.

    Notes

    Table 1. Troubleshooting

    Recipes

    1. 4% paraformaldehyde (Table 2)

      Precaution! Wear protective gear including goggles, mask, gloves and lab coat in the whole process.
      1. Wash the measuring cylinder and beaker with deionized water.

      2. Weigh 12.481 g NaH2PO4·2H2O and 114.605 g Na2HPO4·12H2O with an analytical balance.

      3. Dissolve the salts in 2 L deionized water in a 2.5 L beaker with magnetic stirrer until the solution became clear.

      4. Heat up the phosphate buffer to ~60 °C using microwave or heater.

      5. Weigh 160 g PFA powder with analytical balance. Carefully add the PFA powder into the bottom of a 5 L beaker placed on a magnetic stirrer with heating device in a chemical fume hood.

      6. Slowly pour the preheated phosphate buffer into the beaker with PFA while stirring. Adjust the heat setting to keep the buffer at a temperature between 50 and 60 °C. Stir the solution until PFA is fully dissolved which may take several hours. Use a thermometer to monitor the temperature throughout the whole process.

      7. Note: 5 mol/L(N) NaOH solution can be added dropwise to raise the pH and help dissolving PFA, but equimolar HCl should be added to bring back the pH. Prepare 5 N NaOH solution: Weigh 2 g NaOH in a 50ml conical tube and add 10 ml deionized water. Gently shake the tube to fully dissolve NaOH. Do not close the cap or shake the tube too vigorously to avoid explosion of the tube or splashing of the solution due to sudden accumulation of large amount of heat during this process.
      8. Add deionized water to bring up the volume to 4 L.

      9. Connect the 0.22 μm PVDF bottle top filter set with a benchtop vacuum and a collecting bottle to filter the PFA solution.

      10. For long term storage, aliquot the filtered solution into 50 ml centrifuge tube and store at -20 °C. Melt at 4 °C or room temperature and mix well before use.

      Note: Aliquot no more than 45 ml per tube as the volume will expand when the liquid freezes.


      Table 2. Recipe of 4% PFA

      Acknowledgments

      The present protocol is modified from the original version of our article (Hou et al., 2021). This work is supported by funds from Shanghai Municipal Science and Technology Major Project (No.2018SHZDZX01) and ZJLab.

      Competing interests

      The authors declare no conflict of interest or competing interest.

      Ethics

      Experimental protocols and the use of animals were approved by the Institutional Animal Care and Use Committee at Fudan University and conducted in accordance with institutional guidelines of Institutes of Brain Science (IOBS), Fudan University, China.

      References

      1. Adult Mouse Transcardiac Perfusion. https://www.mbl.edu/bie/files/2015/01/mouse_transcard_perf11.pdf.

      2. Behringer, R., Gertsenstein, M., Nagy, K. and Nagy, A. (2013). Manipulating the mouse embryo: a laboratory manual. 4th edition. Cold Spring Harbor Laboratory Press.

      3. Gage, G. J., Kipke, D. R. and Shain, W. (2012). Whole animal perfusion fixation for rodents.J Vis Exp 65(65).

      4. Hou, Y., Qi, Z., Hongzhi, L., Jinyun, W., Shi, Y., Yanqing, Q., Mengmeng, S., Zhenggang, Y., Jiangteng, L., Zhuhao, W., Ling, G. and Miao, H. (2021). Topographical organization of mammillary neurogenesis and efferent projections in the mouse brain. Cell Rep 34(6).

      5. Standard Operating Procedures (Transcardiac Perfusion). https://www.mcgill.ca/research/files/research/305-_transcardiac_perfusion_-_jan_2018_1.pdf.

简介

[摘要]经盐水灌注心内膜灌注4%多聚甲醛(PFA )被广泛用于清除血液和保存脑部以进行免疫染色或原位杂交。PFA在溶液中分解成甲醛,甲醛使蛋白质和DNA分子交联以保留组织和细胞结构。在这里,我们提供了在鼠标中执行此过程的逐步指南。

关键字:小鼠, 经心灌注, 主动脉, 多聚甲醛, 大脑

材料和试剂
防护装备(手套,护目镜,口罩,实验服)
去离子水
戊巴比妥钠(西格玛(Sigma),目录号:P-010 )
1.5%戊巴比妥钠(在盐水中)
Ñ ACL (Sigma-Aldrich公司,目录号:S7653 )
盐水(0.9%氯化钠)
NaH 2 PO 4 ·2H 2 O(Sigma-Aldrich,目录号:71645)
Na 2 HPO 4 ·12H 2 O(Sigma-Aldrich,目录号:71663)
多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
NaOH(Sigma-Aldrich ,目录号:S8045 )
盐酸(HCl)(Sigma,目录号:H1758)
肝素(Sigma ,目录号:H3149 )
钠叠氮化物(Sigma公司,目录号:S2002)
4%多聚甲醛(PFA,在0.1 M磷酸盐缓冲液中,pH 7.0-7.4)(参见配方)

设备
化学通风柜
解剖钳(RWD,目录号:F12018-13 )(图1)
眼科钳(RWD ,目录号:F12006-10 )(图1)
止血钳(RWD ,目录号:F21002-12 )(图1)
组织剪刀(RWD ,目录号:S13052-12 )(图1)
细剪刀(RWD ,目录号:S12005-10 )(图1)
血管钳(RWD ,目录号:R33001-48)(图1)
微型刮铲(Thermo Fisher ,目录号:21-101-10)(图1)
1 ml和20 ml注射器(图1)
25号或27号输液针(图1)
(可选)蠕动泵(LONGERPUMP,目录号:BT100-2J )
2.5 L和5 L烧杯
温度计(测量范围:100 °C,分度值:1°C)
量筒
分析天平(SARTORIUS,目录号:BSA124S )
台式真空
0.22 μ米PVDF过滤器集(的Nalgene,目录号:291-4520 )
50 ml锥形管(Thermo Fisher,目录号:339652 )
通风柜
微波
带有加热装置的磁力搅拌器(SCILOGEX ,目录号:MS7-H550-Pro )

图1.手术器械

程序
术前准备
测量并记录体重,精确到克。
抓住鼠标尾巴,将其放在笼子架的顶部,然后用一只手轻轻拉动它的尾巴,同时让它牢牢抓住铁线,以将其稳定在稳定的位置。
用一只手将鼠标向下压在脖子上,并抓住其颈背。尽可能握住皮肤以牢牢握住鼠标,以便在注射过程中鼠标不能移动。
通过在腹腔左下或右腹腔腹膜内注射,以每10克体重0.06毫升的剂量使用1.5%的戊巴比妥钠(图2)。
注意:为避免伤害内部器官,请勿将针头推得太多。将鼠标保持在头朝下的位置也可能有帮助。

图2.腹腔注射

将鼠标放回其笼中。等待〜5分钟。通过失去对尾巴或脚趾捏的反应来评估鼠标是否已到达手术麻醉平面(参考文献4)。
注意:对于小于6天的新生小鼠, 麻醉可以通过体温过低来实现(Behringer等,2013 )。将小鼠幼崽放在锥形管中,或用乳胶手套将其包裹(为此目的,切掉手套的拇指),然后将其埋入碎冰中直至颈部,静置10-15分钟,以获得手术用麻醉玻璃。镇痛状态持续约10分钟,因此不要让幼犬在手术台上停留太长时间。尽快进行手术。

(从此步骤开始,请在通风橱中操作。)将鼠标放在其背面。用力将四个爪子用胶带固定或固定在泡沫聚苯乙烯或木材手术台上,以免在灌注过程中任何东西移动。
注意:如果由于过度麻醉而使心脏停止跳动,应立即开始灌注,优先使用含20单位/ ml肝素的生理盐水以避免血液凝结。

修剪25或27规格输注针的尖端,使其稍钝(图3),以减少在以下步骤中刺破主动脉壁的风险(参考文献1)。如有必要,用砂纸擦拭边缘。

图3.修针。A.未修剪的针。B.修剪过的针。
将输液针连接到装有盐水的20 ml注射器上。用盐水冲洗管道和针头以排出空气。
用4%PFA溶液填充另一个20 ml注射器。
经盐水灌注心脏
用眼科镊子握住胸部的皮肤,并用组织剪刀切开切口,以露出剑突(一块箭头形的白骨)。
用眼科镊子握住剑突,并使用组织剪刀在横纹肌下方的侧切口切开the肌和肝脏。
用细剪刀小心地沿着肋骨笼的整个长度在隔膜上切开切口。
用组织剪刀从肋骨的两侧切开直至锁骨。用止血器将胸骨向上反射到小鼠的头上(或将其钉在手术台上或切断),以完全暴露心脏和肺部(图4)。

图4.开胸手术
小心撕下心包和任何其它组织小号使用解剖钳,以提供心脏和血管的清晰视图覆盖心脏。
用解剖钳将心脏固定在稳定的位置。从左心室的尖端以大约平行于心脏中线的角度插入修剪好的针头(图5A)。由于针尖变钝,需要施加足够的压力才能穿过心室壁。但是,请勿过分用力并注意角度以确保针头不会进入左心房或右心室。随着经验的积累,人会清楚地感觉到渗入之前的阻力和在它之后的损失。心脏应该跳动,在灌注开始之前,可以观察到血液回流到针头中。
稳步推进针直到进入升主动脉。可以通过主动脉壁看到针尖(图5B - 5 D )。如果针向正确方向移动,则不会遇到阻力。用血管钳或止血钳将针头固定在主动脉中的适当位置(图5C中的箭头)。
笔记:
如果针不能被成功地插入到主动脉中,升屋檐它在左心室(〜5毫米为成年小鼠,更短的年轻小鼠;调整明智地取决于心脏的尺寸)(图5B),但它具有用大头针或胶带固定,以免在灌注过程中滑出心脏。对于新生幼崽,主动脉可能太窄而无法配合针尖,而又太脆弱而无法夹紧,在这种情况下,最好将针尖留在心室中。
如果针头伸入右心室过多,甚至刺穿心脏,请立即拔出并重新插入针头。如果伤口不太糟,则可能不会影响灌注。
用细剪刀在右心房做一个小切口。深色静脉血应立即流出。手动推动注射器或使用蠕动泵以约1 ml / 5 s的恒定速度 立即开始盐水灌注。盐水冲洗出来的血液,肝脏逐渐变成浅(图小号5E - F)。
笔记:
如果针尖位于左心室而不是主动脉,则较慢的灌注速度是首选。
如果针尖进入右心室,则可能会观察到灌注失败的迹象,包括肺膨胀(图6),液体从鼻子或嘴中滴落以及肝脏不完全脱色。
可选:代替手动注射,可以使用蠕动泵更好地控制灌注速度。

当流出的液体中没有血液时,停止灌注。对于成年小鼠,需要约20 ml生理盐水。

图5.针插入。A.针插入的位置和方向。B.针尖可以留在心脏中间的左心室。注意轨迹,以免针刺穿隔垫(打开的箭头)进入右心室。C.将针头插入主动脉并将其夹在适当的位置(闭合的箭头)。D.可以从主动脉壁(闭合的箭头)看到针尖。E.在右心房做一个小切口(闭合的箭头)。F.肝脏充满血液,在灌注前显示为红色。G.肝LO小号ES血液,成为小号灌注后脸色苍白。H.灌注不当会导致液体在肺和肺水肿中积聚(黑色箭头)。

含4%PFA的经心灌注
从盐水切换到4%PFA。确保在切换过程中没有气泡进入灌注系统(Gage等,2012 )。
以约1 ml / 5 s的恒定速度稳定地灌注4%PFA。随着PFA的流通,人们可以观察到包括身体抽搐,甩尾和甩头的迹象。
为成年小鼠灌注〜15-20 ml 4%PFA。整个老鼠的身体应该非常僵硬,肝脏可能会变成浅淡的淡褐色(比4%PFA灌注之前的颜色要暗一些)。
取下针头,然后从手术台上松开鼠标。
脑解剖
用组织解剖剪刀使小鼠脱垂(图6,水平线)。首先从颈部到鼻子的中线切开一个切口(图6,垂直线),然后从侧面和侧面反射皮肤的两个瓣以露出头骨(图6B),以去除皮肤。
用一只手紧紧抓住头部。用解剖剪刀修剪尾端的骨头(图6B,虚线)。用镊子或剪刀清除颅骨上的残留肌肉。如果正确地完成4%PFA,大脑将稍微收缩,并且大脑和骨头之间将形成一定的空间,形成大孔。
将一把细剪刀的刀片滑入大孔中(在大脑下方),使锋利的一面朝向骨头。进行横向切割。在对侧重复切割。小心地用眼科镊子取下覆盖小脑的颅骨。
在矢状缝合线的弓形末端,将一把细剪刀的刀片的尖端插入大孔与大脑之间,其尖锐的一面朝向骨头。迅速滑动并沿矢状中线切开颅骨。用镊子小心地去除头骨,露出大脑。可能需要额外切割才能露出嗅球(图6C)。
注意:要小心,不要在操作过程中损害大脑。剥离任何meninge小号附接到颅骨去除骨前芯片; 否则可能会切碎大脑软组织。

图6.脑解剖。A.砍掉头,从脖子到鼻尖(虚线)切开毛皮,然后露出头骨。公元前。沿虚线切下脑干下方的颅骨,然后依次去除覆盖小脑和大脑的颅骨以暴露脑组织。

使用微型刮铲(或其他平钝工具)切断大脑腹侧表面的神经束,并将大脑sc出。
注意事项:
与刚解剖过的大脑(图7A ,非常柔软且充满血液)相比,成功地灌注并固定的大脑会稍微收缩,变硬,并显得略带淡褐色(图7C )。不成功的灌注可能会在大脑中留下血液(图7B )并使其看起来呈粉红色。将大脑在4%PFA中固定在4 °C下过夜。
后缀将使大脑进一步收缩,变硬和变暗(图7D )。
将大脑转移到含有0.01%叠氮化钠的PBS中,以便在4 °C下长期保存。或者,将大脑转移到30%的蔗糖溶液中,等待其沉到底部,然后在-20-80 °C下储存。

图7 。在不同条件下解剖出的代表性大脑图像。答:新鲜解剖的大脑充满血液,非常柔软。B.灌注失败的大脑保留了一些血液。C.固定后成功地灌注并固定了大脑。D.固定后的大脑。小号Cale的栏:为4mm。



ñ OTES



表1.故障排除


观察到问题


可能的原因





流体从右心房以外的位置流出。


针pierc ING通过心脏主动脉或



拔出针头并以正确的角度和深度重新插入


肺扩张;液体从口鼻流出来


针进入右心室


拔出针头并以正确的角度和深度重新插入


灌注后大脑中的血液;肝不会变苍白


术前因心脏骤停导致盐水灌注无效




血液凝结



灌注系统中的气泡


麻醉时注意不要过量使用鼠标


练习以避免操作过程中不必要的延迟


用含20单位/毫升肝素的盐水灌注


避免在灌注系统中出现气泡


大脑苍白但灌注后变软


由于灌注系统中的气泡或针头放置不当,导致固定剂灌注无效



低质量的4%PFA(包含未溶解的颗粒或已过期)


小心不要在灌注系统引入气泡时改变从盐水到PFA; 调整针的位置


过滤PFA与0.22微米PVDF; 使用新鲜制备的PFA或正确存储的PFA(有关更多详细信息,请参阅附录)



菜谱



4%多聚甲醛(表2 )
预防!在整个过程中,应穿戴防护装备,包括护目镜,口罩,手套和实验服。


用去离子水清洗量筒和烧杯。
称重12.481 g NaH 2 PO 4 · 2H 2 O和114.605 g Na 2 HPO 4 · 12H 2 O ,具有分析天平。
用磁力搅拌器在2.5 L的烧杯中将盐溶解在2 L的去离子水中,直到溶液变澄清为止。
使用微波炉或加热器将磷酸盐缓冲液加热至〜60 °C 。
称量具有分析天平的160 g PFA粉末。将PFA粉末小心地放入5 L烧杯的底部,该烧杯放置在带有化学通风橱中带有加热装置的磁力搅拌器上。
在搅拌的同时,将预热的磷酸盐缓冲液与PFA缓慢倒入烧杯中。调整加热设置,以将缓冲液保持在50至60 °C之间。搅拌解u Ñ直到PFA完全溶解可能需要几个小时。使用温度计监测整个过程中的温度。
注意:可以逐滴添加5 mol / L(N)NaOH溶液以提高pH值并有助于溶解PFA,但应添加等摩尔的HCl以恢复pH值。准备5 N NaOH溶液:在50ml锥形管中称取2 g NaOH,然后加入10 ml去离子水。轻轻摇动试管以完全溶解NaOH。在此过程中,请勿过度拧紧盖子或剧烈摇动管子,以免引起管子爆炸或溶液飞溅,因为溶液会突然积聚大量热量。


加入去离子水使体积达到4L。
连接0.22微米PVDF瓶顶过滤器组与一个台式真空和收集瓶过滤PFA溶液。
为了长期保存,将滤过的溶液等分到50 ml离心管中,并保存在-20 °C下。使用前在4 °C或室温下融化并充分混合。
注意:每支试管的等分试样数量不得超过45毫升,因为当液体冻结时体积会扩大。



表2 。配方为4%PFA


化学制品


分子


重量


加工


浓度


1升(克)


2升(克)


4升(克)


5升(克)


NaH 2 PO 4 · 2H 2 O


156.01


0.02牛


3.1202


6.2404


12.4808


15.601


Na 2 HPO 4 · 12H 2 O


358.14


0.08牛


28.651


57.302


114.605


143.256


多聚甲醛


30.03


4%


40


80


160


200





致谢



这项工作得到了上海市科技重大专项(No.2018SHZDZX01)和ZJLab的资助。



利益争夺



作者声明没有利益冲突或竞争利益。



伦理



实验方案和动物的使用已得到复旦大学机构动物护理和使用委员会的批准,并根据中国复旦大学脑科学研究所(IOBS)的机构指南进行。



参考



                                          成年小鼠经心灌注。https://www.mbl.edu/bie/files/2015/01/mouse_transcard_perf11.pdf。
              Behringer,R.,Gertsenstein ,M.,Nagy,K.和Nagy,A.(2013)。操纵小鼠胚胎:实验室手册。第四版。冷泉港实验室出版社。
Gage,GJ,Kipke,DR和Shain ,W。(2012)。啮齿动物的全动物灌注固定。J Vis Exp 65(65)。
                                          标准操作程序(经心灌注)。https://www.mcgill.ca/research/files/research/305-_transcardiac_perfusion_-_jan_2018_1.pdf。
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引用:Wu, J., cai, Y., Wu, X., Ying, Y., Tai, Y. and He, M. (2021). Transcardiac Perfusion of the Mouse for Brain Tissue Dissection and Fixation. Bio-protocol 11(5): e3988. DOI: 10.21769/BioProtoc.3988.
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