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Thinned-skulled Cranial Window Preparation (Mice)
薄颅骨窗口的制备(小鼠)   

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Abstract

Imaging structural plasticity or activity of neurons in the brain circuit will facilitate understanding the neural mechanisms underlying animal behavior. Here we describe a modified procedure, the polished and reinforced thinned-skull cranial window preparation, by which we can image dendrites and spines in mouse layer I cortex for weeks (Zhang et al., 2016). By this method, we also imaged the glioma initiation in the mouse cortex for two weeks in previous work (Zhang et al., 2012), which included the photographs and video for reference.

Keywords: Neuroscience(神经科学), Mouse brain(小鼠大脑), in vivo imaging(体内成像), Cranial window(颅骨窗口), Structural plasticity(结构可塑性)

Background

Three cranial window procedures are currently available for in vivo imaging, open-skull cranial window (Trachtenberg et al., 2002), thinned-skull cranial window (Yang et al., 2010) and polished and reinforced thinned-skull cranial window (Drew et al., 2010). Each protocol has both advantages and disadvantages. Open-skull has best optical imaging quality, unlimited repetitive imaging times and large field of view, but needs to wait for 2 weeks to recover from surgery and also has the inflammation and glia activation issue; Thinned-skull protocol has minimal disturbance from inflammation and glia activation on the brain, but has limited repetitive imaging times, only 2-5 times for a cranial window, small field of view(< 300 μm in diameter); Polished and reinforced thinned-skull method allows unlimited repetitive imaging, large field view(< 3 mm in diameter) and minimal disturbance, but the optical imaging quality decreases over time because of light diffusion and absorption at the interface with regenerated bone. Researcher may choose an appropriate protocol according to specific study.

Materials and Reagents

  1. Small paper towel
  2. Miniature blade (Surgistar, catalog number: 6900 )
  3. Cotton swabs
  4. #0 cover glass (3 mm diameter) (Warner Instruments)
  5. Custom-made silicone whip (see Drew et al., 2010 supplemental)
  6. Sodium chloride injection (USP)
  7. Cyanoacrylate glue (Loctite)
  8. Mouse, Thy1-YFPH, 6~12 weeks, male
    Note: Younger mouse has faster bone regeneration, which may lead to shorter time window for high quality images.
  9. Ketamine (diluted to 10 mg/ml)
  10. Xylazine (diluted to 1.5 mg/ml)
  11. Carprofen (diluted to 0.05 mg/ml)
  12. Artificial tears ointment (Rugby Laboratories)
  13. Saline
  14. 7.5% Betadine
  15. 70% alcohol
  16. 2% lidocaine
  17. Diamond paste (3.5 micron diamond pastes) (Wicked Edge, catalog number: WE0535SP )
  18. Tin oxide (Lortone, catalog number: 591-038 )
  19. Liner Bond 2V (KURARAY, catalog number: 1921-KA )
  20. Isofluorane (0.2%, 0.4 L/min)

Equipment

  1. Heating blanket
  2. Sterile glass beaker
  3. Scissors
  4. Thumb forceps
  5. Small sterile drape or platform
  6. Custom-made headpiece holder
  7. High-speed micro drill (CellPoint Scientific, model: Ideal Micro Drill Kit )
  8. 0.5 mm-diameter diamond drill bit (Widget Supply, catalog number: D-CM13 )
  9. Hex nut (Small Parts, catalog number: HNX-0090-C )
  10. Dissecting microscope

Software

  1. ImageJ

Procedure

Notes: Work area preparations:

  1.  The aseptic surgical field is the disinfected skin and exposed surgical wound on the dorsal skull.
  2.  The aseptic surgical field is situated on the CLEAN benchtop that is covered with fresh, absorbent paper:
    1. The surgery stage with a heating blanket on its base to maintain a 37 °C body temperature. A small paper towel is placed over the mouse’s neck and back to cover fur;
    2. A sterile instrument resting area, e.g., sterile glass beaker to suspend sterile instrument tips above the clean surgical area.
  1. Weigh the mouse and anesthetize by intraperitoneally (i.p.) injecting ketamine (100 mg/kg) and xylazine (15 mg/kg) at appropriate volume. Carprofen (0.5 mg/kg, s.c.) is used for analgesia. Usually, the surgery will last for 90 min, if the duration of ketamine/xylazine anesthesia needs to be prolonged, ketamine alone, at half of the induction dose (50 mg/kg ketamine) or ketamine with xylazine at one-quarter of the induction dose (25 mg/kg ketamine; ~3 mg/kg xylazine) can be given.
  2. Shave the hair from the dorsal head and extend the furless area caudally to the first cervical vertebrae, making sure to leave lateral margins large enough to prevent hair from entering the incision. Apply ophthalmic ointment to eyes to keep the eyes moist during surgery. Disinfect the exposed head skin with 7.5% Betadine and 70% alcohol.
  3. Full anesthesia is confirmed by absence of a response to a toe pinch and is also monitored frequently during the surgery.
  4. Inject a small volume of 2% lidocaine under the surface of the dorsal scalp to provide additional local analgesia, and then remove a round (along the skull edge and beside the eyes ~1 cm diameter) section of skin from the dorsal scalp sufficient to expose bregma, lambda and the sutures. Use fine forceps and miniature blade to peel and scrape periosteum tissue from the skull.
  5. Use wet cotton swab (dipped in saline) to clean wound and dry cotton swab to absorb extra solution on the skull. Apply a thin layer of cyanoacrylate tissue adhesive along the edges of the skin excision to bond the skin to skull. Wait for 5 min to let the tissue adhesive get dry.
  6. Outline the cortical area of interest with a marker. The interest area should not be over skull suture lines, to avoid damage to underlying blood vessels, and it’s also hard to get good thinned cranial window above sutures because of the heterogeneous bone around sutures.
  7. Treat the skull with the primer and then with the bonding agent equipped in the dental cement kit. Wait for about 1 min until the bonding agent gets dry. Then cover the skull with a thin layer of dental cement (~0.2 mm) except the interest area, making a shallow bowl around the target area which can hold a little solution facilitating subsequent polishing step. To stabilize the mouse head for subsequent surgery and imaging, we embed a sterile hex nut into the dental cement at a distance from the interest area, saving space for imaging objective. After the preparations are all done, treat the dental cement for about 1 min.
    Note: One of modifications of our procedure from the original one (Drew et al., 2010) is the use of light-sensitive dental cement. It has strong bonding and also gives us enough time to prepare the window around the interest area without rush.
  8. Mount the mouse on the stereotaxic stage by screwing a headpiece holder onto the hex nut on the mouse head.
  9. The mouse is ready for the cranial window thinning. Apply one drop of saline onto the target area on the skull, and then hold a high-speed micro drill equipped with 0.5 mm-diameter diamond drill bit to thin the skull under a dissecting microscope. Keep the hand steady and thin evenly in one direction, keep the skull moist to avoid the thermal injury to the underlying brain tissue caused by high speed drill bit. Saline absorbs heat and also helps soften the bone.
  10. The mouse skull comprises two thin layers of compact bone, sandwiching a thick layer of spongy bone. Remove the external layer of compact bone and most of the spongy bone quickly with the drill. Make sure the bone is wet.
  11. After removing the majority of the spongy bone, dry the skull a little bit, you can see the remaining cavities within the spongy bone under the dissecting microscope, indicating that thinning is approaching the internal compact bone layer. At this stage, skull thickness should still be more than 50 μm. Continue the skull thinning carefully until no cavities can be seen, the skull thickness will be ~20 μm, then start the polishing procedure, two polishing steps are in the same way, and the volume ratio of diamond paste to saline or tin oxide to saline is about 1:1. Less or more diamond paste (tin oxide) results in low viscosity or high viscosity, which will cause low polishing quality.
  12. Apply one drop of saline, then add diamond paste (6-15 μm diameter), stir with custom-made silicone whip to get turbid suspension, continue the stirring for approximately 10 min. The diamond-paste polishing serves two purposes. First, it will further thin the skull without applying pressure on the thinned skull, thereby avoiding accidental damage to the cranial window. Second, it serves as an initial polishing step for the thinned skull before the fine-grained tin oxide is used for further skull polishing.
  13. Following the diamond-paste polishing, the thinned skull is polished with tin oxide same as step 12 for another 10 min. Once the skull is polished, the tin oxide is flushed away with saline until the thinned skull appears clean.
  14. Get the skull dry, then apply one drop of clear cyanoacrylate glue on the skull, put the #0 cover glass on the glue, press the glass slightly for ~1 min to make sure the layer of cyanoacrylate glue between the thinned skull and the coverslip as thin as possible. Wait for about 5-10 min to make sure the glue gets dry.
  15. After surgery, inject subcutaneously 1 ml warm sterile saline and provide with supplemental heat to maintain body temperature until fully recovered from anesthesia.
  16. Return the mouse to their home cage, administer ibuprofen in water (2-4 mg/ml) as analgesic, and monitor daily for the first three postsurgical days.
  17. For imaging, mouse is anesthetized with isofluorane on the custom stereotactic frame under the microscope. The cranial window can be used for longitudinal in vivo imaging for weeks.

Data analysis

For image analysis and processing, software ImageJ is recommended. For the dendritic bleb analysis in our previous work (Zhang et al., 2016), we first set up the criteria to identify ‘blebs’, bleb morphology is ‘beads on a string’, which is totally different from regular dendritic spine; the bleb size is above 2 μm2 (area). Then use the ImageJ function of Analyze Particles to quantify the bleb number. For detailed procedures, please see ImageJ manual.

Notes

Steady hand during thinning is the key for a perfect cranial window, which needs a lot of practice. We also set up a custom-constructed three-axis motorized translation stage (unpublished data) to thin the skull automatically, which is also good except that the cranial window is smaller because the skull surface is not flat.

Acknowledgments

Research was supported by NIH/NIDA/IRP. This protocol is adapted or modified from previous work (Zhang et al., 2012; Zhang et al., 2016).

References

  1. Drew, P. J., Shih, A. Y., Driscoll, J. D., Knutsen, P. M., Blinder, P., Davalos, D., Akassoglou, K., Tsai, P. S. and Kleinfeld, D. (2010). Chronic optical access through a polished and reinforced thinned skull. Nat Methods 7(12): 981-984.
  2. Trachtenberg, J. T., Chen, B. E., Knott, G. W., Feng, G., Sanes, J. R., Welker, E. and Svoboda, K. (2002). Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420(6917): 788-794.
  3. Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J. and Gan, W. B. (2010). Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat Protoc 5(2): 201-208.
  4. Zhang, L., Lapierre, A., Roy, B., Lim, M., Zhu, J., Wang, W., Sampson, S. B., Yun, K., Lyons, B., Li, Y. andLin, D. T. (2012). Imaging glioma initiation in vivo through a polished and reinforced thin-skull cranial window. J Vis Exp (69): e4201-e4201.
  5. Zhang, W., Zhang, L., Liang, B., Schroeder, D., Zhang, Z. W., Cox, G. A., Li, Y. and Lin, D. T. (2016). Hyperactive somatostatin interneurons contribute to excitotoxicity in neurodegenerative disorders. Nat Neurosci 19(4): 557-559.

简介

脑电路中神经元的成像结构可塑性或活动将有助于理解动物行为的神经机制。在这里,我们描述了一种改进的方法,该抛光和增强减薄颅骨颅窗制备,通过它我们可以图像树突和在小鼠层予皮质对于周棘(张等人。,2016)。通过这种方法,我们还在以前的工作(Zhang等人,2012)中将小鼠皮层中的胶质瘤起始成像了两周,其中包括照片和视频供参考。

背景 三个颅窗口程序目前可用于体内成像的开放式颅骨颅窗口(特拉亨伯格等人。,2002),间隔剔除颅骨颅窗口(阳等人,2010)和抛光和增强的薄颅骨颅窗(Drew等人,2010)。每个协议都有优缺点。开放头骨具有最佳的光学成像质量,无限次重复成像时间和大视野,但需要等待2周才能从手术中恢复,并且还具有炎症和神经胶质激活问题;瘦骨头协议对脑部炎症和神经胶质细胞活化的影响最小,但重复成像时间有限,颅窗只有2-5倍,视野小(直径<300μm)。抛光和增强减薄头骨方法允许无限重复成像,大视场(<3毫米直径)和最小的干扰,但光学成像质量降低,因为在与再生骨的界面的光漫射和吸收随着时间的推移。研究人员可以根据具体研究选择合适的方案。

关键字:神经科学, 小鼠大脑, 体内成像, 颅骨窗口, 结构可塑性

材料和试剂

  1. 小纸巾
  2. 微型刀片(Surgistar,目录号:6900)
  3. 棉签
  4. #0盖玻璃(3毫米直径)(华纳仪器)
  5. 定制硅胶鞭(参见Drew 等人,2010补充)
  6. 氯化钠注射液(USP)
  7. 氰基丙烯酸酯胶(乐泰)
  8. 小鼠,Thy1-YFPH,6〜12周,男性
    注意:年轻的老鼠有更快的骨骼再生,这可能会导致高质量图像的缩短时间窗口。

  9. 氯胺酮(稀释至10mg / ml)
  10. 西拉嗪(稀释至1.5 mg / ml)
  11. Carprofen(稀释至0.05 mg / ml)
  12. 人造泪液软膏(Rugby Laboratories)
  13. 盐水
  14. 7.5%Betadine
  15. 70%酒精
  16. 2%利多卡因
  17. 钻石膏(3.5微米金刚石膏)(Wicked Edge,目录号:WE0535SP)
  18. 氧化锡(Lortone,目录号:591-038)
  19. 衬纸2V(KURARAY,目录号:1921-KA)
  20. 异丙烷(0.2%,0.4L / min)

设备

  1. 加热毯
  2. 无菌玻璃烧杯
  3. 剪刀
  4. 拇指钳
  5. 小无菌悬垂或平台
  6. 定制头盔架
  7. 高速微钻(CellPoint Scientific,型号:Ideal Micro Drill Kit)
  8. 0.5毫米直径的金刚石钻头(Widget Supply,目录号:D-CM13)
  9. 六角螺母(小零件,目录号:HNX-0090-C)
  10. 解剖显微镜

软件

  1. ImageJ

程序

注意:工作区准备:

  1. 无菌手术领域是消毒的皮肤,并在背部头骨上暴露手术伤口。
  2. 无菌手术领域位于CLEAN台面,覆盖着新鲜的吸水纸:
    1. 手术阶段采用加热毯在其底部保持37°C体温。
      将小纸巾放在鼠标的脖子上,并覆盖毛皮
    2. 一种无菌仪器休息区,例如无菌玻璃烧杯,以将无菌仪器尖端悬挂在干净的手术区域之上。
  1. 称量小鼠,并以适当的体积通过腹膜内(i.p.)注射氯胺酮(100mg / kg)和赛拉嗪(15mg / kg)麻醉。卡洛芬(0.5mg / kg,s.c)用于止痛。通常手术持续90分钟,如果需要延长氯胺酮/西拉嗪麻醉持续时间,单独使用氯胺酮,诱导剂量的一半(50 mg / kg氯胺酮)或氯胺酮与给予咪达嗪的四分之一的感染可以给予剂量(25mg / kg氯胺酮;〜3mg / kg赛拉嗪)
  2. 从头部剃去头发,将毛皮区域尾部延伸至第一颈椎,确保留下足够大的外侧边缘以防止头发进入切口。在眼睛中涂上眼科软膏,以保持手术中的眼睛湿润。用7.5%Betadine和70%酒精消毒暴露的头皮。
  3. 完全麻醉通过不对脚趾夹紧的反应来确认,并且在手术期间也经常监测。
  4. 在背部头皮表面注射少量2%的利多卡因,以提供额外的局部镇痛,然后从背部头皮中移除一个圆形(沿着颅骨边缘和眼睛旁边〜1厘米直径)的皮肤部分,足以露出普瑞姆,拉姆斯和缝合线。使用细镊子和微型刀片从头骨剥离和刮擦骨膜组织。
  5. 使用湿棉签(浸在盐水中)清洁伤口和干棉签以吸收头骨上的额外溶液。沿着皮肤切除边缘涂一层薄薄的氰基丙烯酸酯组织粘合剂,将皮肤粘结到头骨上。等待5分钟让组织粘合剂变干。
  6. 用标记物概括皮质区域。感兴趣区域不应超过颅骨缝合线,以避免损伤潜在的血管,并且由于缝合线周围的异质骨也难以在缝线上方获得良好的变薄的颅窗。
  7. 用底漆处理头骨,然后用牙科水泥套件中的粘合剂处理。等待约1分钟,直到粘合剂变干。然后用除了兴趣区域之外的一层薄薄的牙科水泥(〜0.2mm)覆盖颅骨,在目标区域周围形成一个浅碗,可以容纳一些溶液,有助于随后的抛光步骤。为了稳定鼠标头以进行后续手术和成像,我们将一个无菌六角螺母嵌入距离兴趣区域一定距离的牙科水泥中,为成像目标节省空间。准备完成后,将牙科牙膏处理约1分钟。
    注意:我们从原来的一个修改过程(Drew et al。,2010)是使用光敏牙科水泥。它具有很强的粘合力,也给我们足够的时间来准备围绕兴趣区域的窗口,而不用急于。
  8. 将鼠标固定在鼠标头上的六角螺母上,将鼠标固定在立体定位台上。
  9. 鼠标准备颅骨变薄。在头骨上的目标区域上涂一滴生理盐水,然后握住装有0.5毫米直径金刚石钻头的高速微型钻头,在解剖显微镜下使头骨变薄。保持手在一个方向上均匀均匀,保持颅骨潮湿,避免高速钻头引起的脑组织的热损伤。盐水吸收热量,也有助于软化骨骼。
  10. 小鼠头骨包括两层紧密骨,夹有厚厚的海绵状骨。用钻头快速移除紧密骨骼和大部分海绵骨的外层。确保骨骼潮湿。
  11. 去除大部分海绵状骨后,将颅骨干一点,您可以在解剖显微镜下看到海绵状骨中剩余的空洞,表明变薄正在接近内部紧密骨层。在这个阶段,颅骨厚度仍应大于50μm。继续颅骨变薄小心,直到没有空洞,颅骨厚度将为〜20μm,然后开始抛光程序,两个抛光步骤以相同的方式,金刚石膏对盐水或氧化锡与盐水的体积比约为1:1。较少或更多的金刚石膏(氧化锡)导致低粘度或高粘度,这将导致低的抛光质量。
  12. 应用一滴盐水,然后加入金刚石膏(直径6-15μm),用定制的硅胶鞭子搅拌,得到混浊的悬浮液,继续搅拌约10分钟。钻石膏抛光有两个目的。首先,它会进一步减薄颅骨,而不会对变薄的头骨施加压力,从而避免意外损坏颅窗。其次,在细粒氧化锡用于进一步颅骨抛光之前,它作为稀释头骨的初步抛光步骤。
  13. 在金刚石膏抛光之后,将稀释的颅骨用与氧化锡相同的步骤12进行抛光另外10分钟。一旦颅骨被抛光,氧化锡用盐水冲洗掉,直到稀疏的头骨看起来清洁
  14. 将颅骨干燥,然后在颅骨上涂一滴清澈的氰基丙烯酸胶,将#0盖玻片放在胶上,轻轻按下玻璃〜1分钟,以确保在稀释的头骨和盖玻片之间的氰基丙烯酸酯胶层尽可能薄。等待约5-10分钟,以确保胶水变干。
  15. 手术后,皮下注射1毫升温暖的无菌盐水,并提供补充的热量以维持体温,直到完全从麻醉中恢复。
  16. 将鼠标放回家中,用水(2-4mg / ml)作为镇痛剂施用布洛芬,并在前三天进行日常监测。
  17. 对于成像,在显微镜下,在定制的立体定向框架上用异氟烷麻醉小鼠。颅窗可用于纵向"体内"成像数周。

数据分析

对于图像分析和处理,推荐使用ImageJ软件。对于我们以前的工作中的树突状泡泡分析(Zhang等人,2016年),我们首先建立了识别"气泡"的标准,水泡形态是"串上的珠子",这完全是不同于常规树突棘;气泡尺寸大于2μm<2(面积)。然后使用分析粒子的ImageJ函数来量化泡泡数。详细步骤请参见ImageJ手册。

笔记

在变薄期间稳定的手是完美的颅窗的关键,需要大量的练习。我们还建立了一个定制的三轴电动平移舞台(未发表的数据),以自动细化颅骨,这也是很好的,除了颅骨窗口较小,因为颅骨表面不平坦。

致谢

研究由NIH / NIDA / IRP支持。该协议是从以前的工作中适应或修改的(Zhang等人,2012; Zhang等人,2016)。

参考文献

  1. Drew,PJ,Shih,AY,Driscoll,JD,Knutsen,PM,Blinder,P.,Davalos,D.,Akassoglou,K.,Tsai,PS和Kleinfeld,D。(2010)。通过抛光和增强的薄化头骨进行慢性光学访问。方法 7(12):981-984。
  2. Trachtenberg,JT,Chen,BE,Knott,GW,Feng,G.,Sanes,JR,Welker,E。和Svoboda,K。(2002)。长期在体内成像经验依赖的突触可塑性成像皮层。 >自然 420(6917):788-794。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, L., Liang, B., Li, Y. and Lin, D. (2017). Thinned-skulled Cranial Window Preparation (Mice). Bio-protocol 7(5): e2158. DOI: 10.21769/BioProtoc.2158.
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