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Feb 2020

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Cranioplastic Surgery and Acclimation Training for Awake Mouse fMRI
清醒小鼠fMRI的颅成形手术及适应训练   

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Abstract

MRI is a promising tool for translational research to link brain function and structure in animal models of disease to patients with neuropsychiatric disorders. However, given that mouse functional MRI (fMRI) typically relies on anesthetics to suppress head motion and physiological noise, it has been difficult to directly compare brain fMRI in anesthetized mice with that in conscious patients. Here, we developed a new system to acquire fMRI in awake mice, which includes a head positioner and dedicated radio frequency coil. The system was used to investigate functional brain networks in conscious mice, with the goal of enabling future studies to bridge fMRI of disease model animals with human fMRI. Cranioplastic surgery was performed to affix the head mount and the cupped-hand handling method was performed to minimize stress during MRI scanning. Here we describe the new mouse fMRI system, cranioplastic surgery and acclimation protocol.


Graphic abstract:



Awake fMRI system to investigate the neuronal activity in awaked mice.


Keywords: Awake fMRI (觉醒fMRI), Functional MRI (功能性磁共振成), 15q dup mouse (15q dup 小鼠), Autism (自闭症), Diffusion tensor imaging (弥散张量成像), Functional connectivity (功能性连接), Structural connectivity (结构连接)

Background

High-field mouse fMRI is an important translational tool to bridge the gap between invasive research in mouse models of neuropsychiatric diseases and clinical research in patients. However, compared to human fMRI, mouse fMRI studies are typically limited due to anesthesia, which is necessary to suppress body motion and stress during fMRI acquisition. Performing mouse fMRI studies under light anesthesia can ameliorate body motion, but the extent to which functional brain networks and connectivity are influenced by light anesthesia remains unclear. In general, small doses of dexmedetomidine, medetomidine, isoflurane or a mixture of these anesthetics are commonly used to achieve light anesthesia to investigate brain function in rodents (Grandjean et al., 2014; Bukhari et al., 2017; Tsurugizawa et al., 2019 and 2020a). However, these anesthetics induce not only suppression of consciousness (Munglani et al., 1993) but also vasoactive modulation of neurovascular coupling (Tsurugizawa et al., 2010 and 2016), which potentially impacts the relationship between neuronal activation and vascular response. In particular, light sedation alters the BOLD response to physiological stimuli and thus affects the validity of task-based fMRI (Tsurugizawa et al., 2013a). Anesthesia clearly depresses consciousness, even if residual brain function is comparable to an awake state. In addition, it is impossible to perform cognitive tasks under anesthesia. In summary, fMRI under anesthesia, even with light anesthesia, is not comparable to fMRI in a conscious state, and thus rodent fMRI performed in an anesthetized state considerably narrows opportunities to comprehensively characterize brain function and connectivity. Hence, we developed an awake fMRI system for mice that does not necessitate anesthesia, thus enabling mouse fMRI experiments that are analogous to human fMRI.


Previous studies, including our own, have developed fMRI systems and protocols to acquire fMRI in awake mice. These have been used to investigate responses to fear conditioned stimulation (Harris et al., 2015) and optogenetics (Desai et al., 2011) and to investigate resting state functional connectivity (Bergmann et al., 2016; Yoshida et al., 2016; Madularu et al., 2017). Key limitations of the protocols used in these studies include the use of restraint tubes, absence of earplugs to reduce scanner noise (Mowery et al., 2019; Kurioka et al., 2020) and unclear animal handling and acclimation methods. We first developed an awake-mouse fMRI system to investigate the intragastric stimulation of capsaicin (Tsurugizawa et al., 2013b). More recently, we enhanced the system with the addition of a head fixation apparatus and acclimation training. The new system was used to investigate brain function in mice with a chromosome duplication (15q dup) resulting in abnormal behavior resembling ASD symptoms (Nakatani et al., 2009; Tsurugizawa et al., 2020b).


In this report, we describe our fMRI system and protocol for acquiring fMRI in awake mice, including cranioplastic surgery, acclimation training and fMRI acquisition. The protocol enables routine resting-state and task-fMRI mouse studies, where anesthetization of animals introduces a major confound.


Materials and Reagents

  1. Small gauze pad

  2. C57BL6J mice (8-15 weeks)

  3. Super-Bond C & B (Sun medical)

  4. GC UNIFAST Trad (GC Dental Products Corp., Aichi, Japan)

  5. Male (VRSP6) and female (VRF6) Plastic Leur Fitting (Nordson Medical, US)

  6. Earplugs for humans, made of Polyurethane (3M Company, Minnesota)

Equipment

  1. A 4.7T horizontal MRI Avance IIII system (Bruker, Germany)

  2. Mouse head positioner (custom made)

  3. Dedicated mouse volume coil (30 mm diameter) to transmit/receive the radio frequency signal, combined with semicircular acrylic transparent plastic pipe for mouse bed (Takashima Seisakujo Co., Ltd)

  4. Respiration/heart rate monitor system (Model 1025, SA Instruments, Stony Brook, NY, USA)

Procedure

  1. Cranioplastic surgery

    1. Surgery

      1. Anesthetize mice with isoflurane (1.5% with air).

      2. Remove head skin and exposed the cranium.

      3. Carefully polish the surface of the cranium with a small gauze pad containing physiological saline.

      4. Place super-bond (Super-Bond C & B) on the skull.

      5. Mount cranioplastic acrylic cement (GC UNIFAST Trad) on the superbond with a male plastic Luer Fitting (Figure 1).

      6. Mice could recover for more than a week after surgery. Carefully check animal body weight and behavior every day.



        Figure 1. Cranioplastic surgery. A thin layer of super-bond was placed on the skull. The Luer fitting was then rapidly fixed with cranioplastic acrylic cement.


  1. Acclimation training

    1. Mouse handling during recovery

      1. Continue handling during the recovery for at least 1 week. The handler lift the mice with cupped hands and hold the animal for around 30 s (Figure 2).

      2. We confirme that mice did not urinate and did not jump from the cupped hands during handling.

      3. Once body weight increased compared to pre-surgery weight, traine the mice to acclimatize to the awake fMRI conditions before fMRI experiments.



      Figure 2. Handling with cupped hands. Photo shows the handling of a representative mouse with implantation of the Luer fitting to the skull.


    2. Acclimation training

      1. Train mice for 4 days to acclimatize to the awake fMRI conditions before fMRI experiments.  Train them at the same time each day (10:00-18:00) to minimize the effects of circadian rhythm variations.

      2. During the first 2 days, use a pseudo-MRI system consisting of a non-magnet bore and a head positioner (Figure 3 and Figure 4). The acrylic bar is tightly fitted with an elastic tube. Then  train the mice in the MRI bore for the next 2 days.



        Figure 3. Fixation of the head with the custom-made head positioner. Photo of the fixed head (left) and schematic figure of the head fixation system. The head fixation system was custom made.



        Figure 4. Acclimation training with pseudo-MRI head positioner


      3. Anesthetize mice in anesthesia chamber using 2% isoflurane with air.

      4. Once mice are anesthetize, stop isoflurane and rapidly and tightly fixed their head with the non-MRI head fixation system (Figure 3).

      5. The earplug is cut down to fit to the mouse ears and put into the mouse ear canal using tweezers (Figure 5D). The size of dedicated earplugs should be bigger than ear canal so as not to make a gap with the ear canal.

      6. Gently wrap the body with paper towel or gauze so that the mice can move their limbs within the paper towel (or gauze) cover (Figure 4).

      7. In general mice awoke within 10 min following cessation of isoflurane delivery. This was confirmed using electroencephalography (Tsurugizawa et al., 2020b).

      8. Mice remain in the pseudo-MRI apparatus for 30 min on the first day and 90 min on the second day.

      9. Monitor the respiratory rate and heart rate during the training. A small pneumatic pillow, by which respiration is measured, is attached under the animal’s abdomen with surgical tape. The electrodes for electrocardiogram (ECG) are inserted under the skin.

    3. Awake fMRI

      1. Following 2 days training outside of the magnet, start acclimation training in the magnetic bore.

      2. Anesthetize mice in anesthesia chamber with 2% isoflurane with air.

      3. Continue isoflurane anesthesia via the mask (1.5% with air) during the setup.

      4. Fix the animal’s head in the dedicated volume coil with head positioner and the animal’s body is positioned on the bed (Figure 5A). Use the same head positioner as Figure 3. The size of dedicated earplugs should be bigger than ear canal so as not to make a gap with the ear canal.

      5. Insert dedicated earplug (cut down to a 5 mm length) into the mouse ear canal (Figure 5D).



        Figure 5. Dedicated volume coil, head fixation system, and eay plugs. A. Dedicated mouse volume coil and bed. B. Schematic figure of the fixation of mouse head. C. Diagram of the volume coil and bed. D. Left: Earplug for human and (D center) dedicated earplug for mouse. D. Right: The ear of mouse with dedicated earplug. The picture and schematic figure are from Tsurugizawa et al. (2020b).


      6. Gently wrap the body with paper towel or gauze so that they can move their limbs within the paper towel (or gauze) cover.

      7. Adjust basic frequency, magnetic field homogeneity, reference pulse gain and receiver gain. This adjustment is automatically performed by Bruker system.

      8. Acquire an anatomical image with rapid acquisition with relaxation enhancement (RARE) sequence following cessation of isoflurane using the following parameters: time of repetition, 2,500 ms; effective echo time, 60 ms; RARE factor, 8; acquisition matrix, 128 × 128; field of view, 16 mm × 16 mm; slice thickness, 1 mm; 15 slices and four averages.

      9. Start to acquire fMRI data 10 min after cessation of anesthesia using the following parameters: time of repetition, 2,000 ms; echo time, 21 ms; acquisition matrix, 80 × 80; field of view, 16 mm × 16 mm; slice thickness, 1 mm; 15 slices and four averages.

      10. Monitor the respiratory rate and heart rate during the MRI experiment (Model 1025, SA Instrument).

      11. Perform fMRI training (3rd and 4th days) with the same protocol as the fMRI experiment.

      12. Complet the acclimation training when the heart rate and respiratory rate are reduced to nominal levels (Figure 6). If they do not decrease, we continued acclimation training.



        Figure 6. Heart rate and respiratory rate during acclimation training. (A) Heart rate and (B) respiratory rate reduced to nominal levels at the end of the training (n = 15). *P < 0.05 by Tukey-Kramer multiple comparison test. Data are from Tsurugizawa et al. (2020b).

Acknowledgments

This work is based on our previous work published in Science Advances (Tsurugizawa et al., 2020b). This work was supported by KAKENHI Grant-in-Aid for Young Scientists, Scientific Research (S), and Scientific Research on Innovative Areas (16H06316, 16H06463, 24700380), JST CREST, and the Takeda Science Foundation and Smoking Science Foundation, the Australian National Health and Medical Research Council (NHMRC) Senior Research Fellowship B (ID: 1136649).

Competing interests

The authors declare no competing interests.

Ethics

All animal experimental procedures in the present study were approved by the institutional review board of animal ethical committee who followed institutional guidelines in Ethical Committee of AIST (2020-0365-A) and the Ethics Committee of RIKEN Brain Science Institute (W2019-2-042).

References

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简介

[摘要] MRI是一种有希望的转化研究工具,可将疾病动物模型中的脑功能和结构与神经精神疾病患者联系起来。但是,鉴于小鼠功能性MRI(fMRI)通常依赖于麻醉剂来抑制头部运动和生理噪声,因此很难将麻醉后的小鼠中的脑fMRI与有意识的患者中的脑fMRI直接进行比较。在这里,我们开发了一种新的系统来在清醒的小鼠中采集功能磁共振成像,其中包括一个头部定位器和专用射频线圈。该系统用于研究清醒小鼠的功能性大脑网络,目的是使未来的研究能够将疾病模型动物的功能磁共振成像与人功能磁共振成像联系起来。进行颅骨整形手术以固定头部支架,并执行杯形手处理方法以最小化MRI扫描过程中的压力。在这里,我们描述了新的小鼠功能磁共振成像系统,颅骨手术和适应协议。

图形摘要:


唤醒功能磁共振成像系统以研究唤醒小鼠的神经元活动。



[背景技术]高场磁共振成像鼠标是填补在神经精神性疾病和临床研究的患者的小鼠模型中研究侵入之间的间隙的一个重要工具的平移。但是,与人功能磁共振成像相比,小鼠功能磁共振成像研究通常由于麻醉而受到限制,而麻醉是抑制功能磁共振成像采集期间身体运动和压力所必需的。在轻度麻醉下进行小鼠fMRI研究可改善人体运动,但轻度麻醉对功能性大脑网络和连接性的影响程度尚不清楚。通常,小剂量右美托咪定,美托咪定,异氟烷或这些麻醉剂的混合物通常用于实现轻度麻醉,以研究啮齿动物的脑功能(Grandjean等人,2014 ; Bukhari等人,2017 ; Tsurugizawa等人, 2019和2020a )。但是,这些麻醉剂不仅会导致意识抑制(Munglani等,1993),而且还会引起神经血管耦合的血管活性调节(Tsrugizawa等,2010和2016),这可能会影响神经元激活与血管反应之间的关系。特别是,轻度镇静会降低BOLD对生理刺激的反应,从而影响基于任务的功能性核磁共振成像的有效性(Tsrugizawa等人,2013a)。即使残留的脑功能可与清醒状态相媲美,麻醉也明显会降低意识。另外,不可能在麻醉下执行认知任务。总之,即使在轻度麻醉下,麻醉下的功能磁共振成像也无法与意识状态下的功能磁共振成像相提并论,因此,在麻醉状态下进行的啮齿动物功能磁共振成像大大缩小了全面表征脑功能和连通性的机会。因此,我们为小鼠开发了无需麻醉的清醒fMRI系统,从而实现了类似于人类fMRI的小鼠fMRI实验。

包括我们自己在内的先前研究已经开发了fMRI系统和协议,以在清醒的小鼠中获取fMRI。这些已被用于研究响应恐惧条件刺激(哈里斯等人,2015)和光遗传学(德赛等人,2011)和研究静息状态的功能连接(贝格曼等人,2016 ;吉田等人,2016 ; Madularu et al。,2017)。这些研究中使用的方案的主要局限性包括使用约束管,不使用耳塞以降低扫描仪噪音(Mowery等,2019; Kurioka等,2020)和不清楚的动物处理和驯化方法。我们首先开发了唤醒小鼠功能磁共振成像系统,以研究辣椒素对胃内的刺激作用(Tsurugizawa等人,2013b )。最近,我们增加了头部固定装置和适应训练,从而增强了系统。该新系统用于研究具有染色体重复(15q dup )的小鼠的脑功能,该小鼠具有类似于ASD症状的异常行为(Nakatani等,2009; Tsrugizawa等,2020b )。

在本报告中,我们描述了用于在清醒小鼠中进行功能磁共振成像的功能磁共振成像系统和方案,包括颅骨整形手术,适应训练和功能磁共振成像采集。该协议可以进行常规的静息状态和任务功能磁共振成像小鼠研究,其中动物的麻醉引入了一个主要的困惑。

关键字:觉醒fMRI, 功能性磁共振成, 15q dup 小鼠, 自闭症, 弥散张量成像, 功能性连接, 结构连接



材料和试剂


1.小纱布垫     

2. C57BL6J小鼠(8-15周)     

3.超级债券C&B(Sun医疗)     

4. GC UNIFAST Trad(日本爱知县GC牙科产品公司)     

5.公头(VRSP 6)和母头(VRF6)塑料Leur接头(美国诺信医疗公司)     

6.聚氨酯制人用耳塞(明尼苏达州3M公司)     



设备


4.7T水平MRI Avance IIII系统(德国布鲁克)
鼠标头定位器(定制)
用于发射/接收射频信号的专用鼠标体积线圈(直径为30 mm),与用于鼠标床的半圆形丙烯酸透明塑料管相结合(高岛制作所有限公司)
呼吸/心率监测系统(美国纽约州石溪的SA Instruments,M odel 1025)


程序


颅骨整形手术
外科手术
我们用异氟烷(1.5%的空气)麻醉小鼠。
我们去除了头皮并暴露了颅骨。
我们用一个装有生理盐水的小纱布垫小心地抛光了颅骨的表面。
我们在头骨上放置了一个高级债券(超级债券C和B)。
我们安装Ç ranioplastic丙烯酸水泥(GC UNIFAST岛)上superbond有阳塑料路厄配件(图URE 1)。
小鼠在手术后可能会恢复一周以上的时间。我们仔细地检查一天天nimal体重和行为。






图1 。颅骨整形手术。在颅骨上放置一层薄薄的超级粘结层。然后将Luer接头用颅塑料丙烯酸水泥快速固定。


适应训练
恢复期间的鼠标处理
在恢复过程中,我们继续处理了至少1周。该处理程序与解除杯状手小鼠和大约30秒(图举行的动物URE 2)。
我们确认老鼠在搬运过程中没有小便,也没有从杯形手跳下。
一旦体重与手术前体重相比增加,我们将在功能磁共振成像实验之前训练小鼠适应清醒的功能磁共振成像条件。




图2 。用手心处理。照片显示了将Luer配件植入颅骨中的代表性鼠标的处理方式。


适应训练
在功能磁共振成像实验之前,我们训练小鼠4天以适应清醒的功能磁共振成像条件。我们每天(10:00-18:00)在同一时间训练他们,以最大程度地降低昼夜节律变化的影响。
在最初的2天中,我们使用了由非磁铁孔和头部定位器组成的伪MRI系统(图3和图4)。丙烯酸棒紧密地装有弹性管。然后我们在接下来的2天中在MRI孔中训练了小鼠。




图3.用定制的头部定位器固定头部。固定头的照片(左)和头固定系统的示意图。头部固定系统是定制的。




图4 。伪MRI头部定位器进行适应训练


我们使用2%异氟烷与空气在麻醉室内麻醉小鼠。
一旦小鼠麻醉,我们停止异氟醚和迅速,紧紧固定其与非MRI头部固定系统(图头URE 3)。
切下耳塞以适合老鼠的耳朵,并用镊子将其插入老鼠的耳道(图5D)。专用耳塞的尺寸应大于耳道,以免与耳道形成间隙。
我们用纸巾或纱布轻轻包裹身体,以便小鼠可以在纸巾(或纱布)盖内移动其四肢(图4)。
通常,小鼠在停止异氟烷递送后10分钟内醒来。使用脑电图证实了这一点(Tsurugizawa等,2020b )。
小鼠在第一天停留在伪MRI设备中30分钟,第二天停留90分钟。
在训练过程中,我们监测了呼吸频率和心率。用手术胶带将一个小的气动枕头连接到动物的腹部,以此来测量呼吸。心电图电极(ECG)插入皮肤下。
唤醒功能磁共振成像
在磁铁外进行了2天的训练后,我们开始了对磁孔的适应训练。
我们用2%异氟烷与空气在麻醉室内麻醉小鼠。
在安装过程中,我们通过面罩(空气中1.5%)继续进行异氟烷麻醉。
我们用头部定位器将动物的头部固定在专用的体积线圈中,然后将动物的身体放置在床上(图5A)。我们使用了与图3相同的头部定位器。专用耳塞的尺寸应大于耳道,以免与耳道形成间隙。
我们将专用的耳塞(切成5毫米长)插入了鼠标耳道(图5D)。




图5.专用音量线圈,头部固定系统和eay插头。一。专用鼠标音量线圈和床。乙。鼠标头固定示意图。Ç 。容积线圈和床的示意图。d 。左:用于人类的耳塞和(用于D中心的)专用于鼠标的耳塞。d 。右:带有专用耳塞的鼠标耳。图片和示意图来自Tsurugizawa等人。(2020b )。


我们用纸巾或纱布轻轻包裹身体,以便它们的四肢在纸巾(或纱布)盖内移动。
我们调整了基本频率,磁场均匀性,参考脉冲增益和接收器增益。该调整是由布鲁克系统自动执行的。
我们获取与快速采集弛豫增强(RARE)序列的解剖学图像以下使用以下参数的异氟醚的停止:重复的时间,2 ,500毫秒; 有效回波时间,60毫秒;稀有系数8; 采集矩阵128×128; 视野16毫米×16毫米; 切片厚度1毫米;15片和4个平均值。
我们开始获取fMRI数据使用以下参数麻醉停止后10分钟:重复的时间,2 ,000毫秒; 回声时间21毫秒; 采集矩阵,80×80;视野16毫米×16毫米; 切片厚度1毫米;15片和4个平均值。
我们在MRI实验(1025型,SA仪器)中监测了呼吸频率和心率。
我们进行的fMRI训练(3次和第4次使用相同的协议作为功能磁共振成像实验天)。
我们完成牛逼,他的适应训练时,心脏和呼吸率分别降至标称水平(图URE 6)。如果它们没有减少,我们将继续进行适应训练。




图6.适应训练期间的心率和呼吸率。(A)心率和(B)呼吸率在训练结束时降至名义水平(n = 15)。* P <0.05,通过Tukey-Kramer多重比较测试。数据来自Tsurugizawa等人。(2020b )。


致谢


这项工作是根据我们以前发表在工作科学进展(Tsurugizawa等人湖年,2020年b )。这项工作得到了KAKENHI青年科学家,科学研究和创新领域科学研究的资助(16H06316、16H06463、24700380),JST CREST以及澳大利亚武田科学基金会和吸烟科学基金会的支持国家卫生与医学研究理事会(NHMRC)高级研究奖学金B(ID:1136649)。


利益争夺


作者宣称没有利益冲突。


伦理


在本研究中的所有动物实验过程由动物伦理委员会的机构审查委员会谁跟随机构准则批准AIST(2020-0365-A)的伦理委员会和RIKEN脑科学研究所的伦理委员会(W2019-2-042 )。


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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tsurugizawa, T., Tamada, K., Debacker, C., Zalesky, A. and Takumi, T. (2021). Cranioplastic Surgery and Acclimation Training for Awake Mouse fMRI. Bio-protocol 11(7): e3972. DOI: 10.21769/BioProtoc.3972.
  2. Tsurugizawa, T., Tamada, K., Ono, N., Karakawa, S., Kodama, Y., Debacker, C., Hata, J., Okano, H., Kitamura, A., Zalesky, A. and Takumi, T. (2020b). Awake functional MRI detects neural circuit dysfunction in a mouse model of autism. Sci Adv 6(6): eaav4520.
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