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Modification and Application of a Commercial Whole-body Plethysmograph to Monitor Respiratory Abnormalities in Neonatal Mice
改造商业全身体积描记器并应用于监测新生小鼠的呼吸异常   

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Abstract

Proper breathing is essential for mammals to acquire oxygen after birth and requires coordinated actions among several tissues, including diaphragm, intercostal muscles, trachea, bronchi, lung and respiration-regulating neurons located in the medulla oblongata. Genetically modified mice that die early postnatally may have respiratory defects caused by maldevelopment of any one of these tissues (Turgeon and Meloche, 2009). Because of the small body size of neonatal pups, whole-body plethysmography can be used to monitor their respiratory activities. In this protocol, we modified the commercial whole-body plethysmograph by increasing metal filters in the pneumotach, connecting an extension tube to the pneumotach and removing the bias flow supply. With these modifications, the sensitivity of this device is significantly increased to enable the detection of rhythmic respiration in neonatal mice as early as postnatal day 1 (P1).

Keywords: Neonatal mice(新生小鼠), Whole body plethysmography(全身体积描记法), Respiration(呼吸)

Background

Several labs have used home-made or custom-built plethysmograph devices to identify respiratory failure in genetically modified neonatal mice (Nsegbe et al., 2004; Crone et al., 2012). However, for researchers who are novices in this field and want to investigate the cause of neonatal lethality in mice, a commercial whole-body plethysmograph (WBP, Buxco system, DSI) is a reasonable choice.
   When we first set up this system to monitor respirations of P0 mice, the respiratory activities in C57BL/6 newborns were most of the time undetectable until mice reached 3 days old. Because the WBP measures the pressure changes within the animal chamber that are due to inspired air humidified and heated by an animal’s lung during breathing, increasing the detection sensitivity of this device was the only way to monitor respiration of C57BL/6 neonates before P3.
   In this protocol, we share our experience in modifying this system to reliably detect rhythmic respiration in C57BL/6 neonatal pups as early as P1 and possibly P0. Once the recorded traces are obtained, several parameters calculated by the FinePointe software are useful for exploring respiratory abnormalities in mice who do not die of cyanosis due to severe respiratory failure right after birth (Lai et al., 2016).

Materials and Reagents

  1. P0 and P3 C57BL/6 litter (C57BL/6) (THE JACKSON LABORATORY, catalog number: 000664 )
  2. ICR foster dams [Crl:CD-1 (ICR), Charles River, strain code: 022]

Equipment

  1. Mouse pup WBP (DATA SCIENCES INTERNATIONAL, Buxco system; Figure 1) with:
    1. Max II Amplifier (DATA SCIENCES INTERNATIONAL, Buxco, model: MAX2275 )
    2. Recording chambers (DATA SCIENCES INTERNATIONAL, Buxco, model: PLY4241 ), ~30 ± 1 °C with built-in heating pad on


      Figure 1. Modified setup for the Buxco mouse-pup whole-body plethysmograph

    3. Flow transducer (DATA SCIENCES INTERNATIONAL, Buxco, model: TRD5700 )
    4. Small rodent bias flow supply (DATA SCIENCES INTERNATIONAL, Buxco, model: B04-BFL0100 , for mouse pup)
    5. Extension tube (length: 14 cm; internal/external diameter: 2.6/3 mm)
    6. Metal filters (DATA SCIENCES INTERNATIONAL, Buxco, model: HDW1514 )
  2. Hybridization oven (GE Healthcare, model: RPN2511E )

Software

  1. FinePointe software (Buxco system, DSI)

Procedure

Notes:

  1. To enhance detection sensitivity, we disassembled the Halcyon pneumotach and increased the number of metal filters from 4 to 7 in both channels of the pneumotach individually connected to animal and reference chambers (Figures 2A and 2B). By doing so, we increased the differential pressure change caused by subtle neonatal respiration. However, this modification also changed the values of some respiratory parameters (Figure 6). Although we contacted Buxco technical services before and after solving the sensitivity problem, we were unable to get any help to properly adjust the instrument settings to maintain the original values.
  2. The aforementioned change increases the detection sensitivity and also the background signal from the Buxco bias flow pump that is usually used with the plethysmograph, so the bias flow supply has to be removed to reduce the noise from the air supply.
  3. We connected a bended extension tube to the Halcyon pneumotach to avoid any disturbed airflow from the upper environment such as the user’s breath (Figure 2C).


    Figure 2. Modification of the Halcyon pneumotach and recording chamber. A. The number of metal filters in both pneumotach channels was increased to 7. B. Loading metal filters in the Halcyon pneumotach; C. The modified pneumotach assembled onto the recording chamber was connected with an extension tube (arrow) facing downward to avoid any airflow disturbance.

  1. Transfer the P0 C57BL/6 litter to an ICR foster dam for care until recording.
  2. Incubate a cup of water in an oven at 34 ± 1 °C for humidification. Retrieve all P1 pups and keep them warm in the humidified oven for at least 20 min before plethysmographic recording.
  3. Calibrate the device by following the instructions in the FinePointe wizard (Figure 3).
    1. Click the calibrate icon (Figure 3A).


      Figure 3. Calibration of the chamber. A-H. Follow the instructions in the FinePointe wizard, including zeroing the amplifier, balancing the transducer and injecting air into the chamber.

    2. Click the wrench icon to calibrate each chamber (Figure 3B).
    3. Set the gain level to 3.5 (Figure 3C).
    4. Switch the amplifier to the direct current position (DC).
    5. Turn the balance screw until the chart reads 0 volt (Figures 3D and 3E).
    6. Inject 200 μl air into the chamber (Figure 3F).
    7. Ensure the injected airflow is completely contained within the highlighted purple region and that the red cursor is on 0 (Figure 3G).
    8. Switch the amplifier to the alternating current position (AC) position and push the ‘next’ button for re-zeroing.
    9. Click the ‘finish’ button to complete the calibration (Figure 3H).
  4. Place a P1 pup in the animal chamber gently.
  5. Close the chamber and let the pup habituate for 1 min.
  6. Start the measurement. Record each pup for three 3-min sessions. To avoid the accumulation of CO2 and humidity in the absence of bias flow air supply, open the chamber at the end of each session for 1 min.
  7. Remove the pup from the animal chamber.
  8. Save the recording traces (Figure 4) and proceed to data analysis (Figure 5).


    Figure 4. Representative breathing traces from P0, P1 and P3 neonatal mice. A and B. Respiratory flow of P3 mice by using the original and modified recording chamber. C and D. Respiratory flow of P0 mice by using the original and modified recording chamber. Arrows indicate the inspiratory peaks with peak flow which is > 0.01 ml/sec. E. Respiratory flow of P1 CPEB2-knockout mice by using the modified recording chamber. Bracket indicates the apnea.

Data analysis

Because breathing movements are measured noninvasively in non-anesthetized pups, only signals recorded from the pup at resting state are analyzed. However, FinePointe software (Buxco system, DSI) calculates all rhythmic signals, including those generated from body movements or respiration during movement. Thus, to analyze real signals from resting respiration, we selected the raw data for all respiratory parameters from the table (Figure 5, arrow) and copied and pasted them into an Excel spreadsheet. We also closely observed and marked the times when the pup was not at rest during plethysmographic recording to manually exclude these problematic data points due to body movement. Only the data from resting respiratory activities were averaged to derive respiratory frequency, tidal volume, inspiratory time, expiratory time, peak inspiratory flow and peak expiratory flow. Respiratory frequency of P0 mice was manually analyzed by counting the number of inspiratory peaks with peak flow > 0.01 ml/sec (Figure 4D, arrows). An apneic episode is defined when a ventilatory pause is twice longer than the preceding breath duration (Figure 4E), which is scored manually for the occurring number per minute and duration.


Figure 5. Extracted raw data for all respiratory parameters from the trend data table. Selected data points were copied and pasted into an Excel spreadsheet.

Notes

  1. If the genetically modified mice do not die immediately after birth, we suggest transferring pups to an ICR dam for 18-24 h until measurement because C57BL/6 dams have poor maternal instincts and may not rear pups with respiratory deficiencies. In our modified WBP, P1 mice show more rhythmic and stronger respiratory movements than P0 mice, so analyzing respiratory parameters from their recording traces is more reliable.
  2. Due to subtle respiratory signals of P0 mice, we only analyze the respiratory frequency by manually counting the number of inspiratory peaks > 0.01 ml/sec (Figure 4D, arrows).
  3. The values of tidal volume, peak inspiratory flow (PIF) and peak expiratory flow (PEF) are amplified by using the modified WBP, so they are compared between the control and experimental groups. In contrast, the values of respiratory frequency, inspiratory time (Ti) and expiratory time (Te) are unaffected (Figure 6).


    Figure 6. Comparison of respiratory parameters recorded in mice at P3 by using the original and modified recording chambers. Ti, inspiratory time; Te, expiratory time; PIF, peak inspiratory flow; PEF, peak expiratory flow. Data are mean ± SEM (n = 3). *P < 0.05, **P < 0.01, Student’s t-test.

  4. Lights are on from 8 AM to 8 PM in our mouse room. We usually perform the respiratory recording between 10 AM to 5 PM. In our experience, neonatal mice appeared more active when recording was performed after 5 PM, likely due to the circadian rhythm.

Acknowledgments

This work was supported by the Ministry of Science and Technology (Most 105-2321-B-001-044) in Taiwan.

References

  1. Crone, S. A., Viemari, J. C., Droho, S., Mrejeru, A., Ramirez, J. M. and Sharma, K. (2012). Irregular breathing in mice following genetic ablation of V2a neurons. J Neurosci 32(23): 7895-7906.
  2. Lai, Y. T., Su, C. K., Jiang, S. T., Chang, Y. J., Lai, A. C. and Huang, Y. S. (2016). Deficiency of CPEB2-confined choline acetyltransferase expression in the dorsal motor nucleus of vagus causes hyperactivated parasympathetic signaling-associated bronchoconstriction. J Neurosci 36(50): 12661-12676.
  3. Nsegbe, E., Wallen-Mackenzie, A., Dauger, S., Roux, J. C., Shvarev, Y., Lagercrantz, H., Perlmann, T. and Herlenius, E. (2004). Congenital hypoventilation and impaired hypoxic response in Nurr1 mutant mice. J Physiol 556(Pt 1): 43-59.
  4. Turgeon, B. and Meloche, S. (2009). Interpreting neonatal lethal phenotypes in mouse mutants: insights into gene function and human diseases. Physiol Rev 89(1): 1-26.

简介

正确的呼吸对于哺乳动物在出生后获得氧气是必需的,并且需要在位于延髓中的几个组织之间的协调作用,包括隔膜,肋间肌肉,气管,支气管,肺和呼吸调节神经元。 在死后早期死亡的转基因小鼠可能由于这些组织中的任何一种发育不良而引起呼吸道缺陷(Turgeon和Meloche,2009)。 由于新生儿小体型体积小,全身体积描记法可用于监测其呼吸活动。 在本协议中,我们通过增加冲击中的金属过滤器来修改商业全身体积描记器,将延伸管连接到喘振器并除去偏流装置。 通过这些修改,该装置的敏感性显着增加,以便能够在出生后第1天(P1)之前检测新生小鼠的节律性呼吸。
【背景】几个实验室使用自制或定制的体积描记器装置来鉴定转基因新生小鼠的呼吸衰竭(Nsegbe等人,2004; Crone等,2012)。然而,对于这一领域的新手和想要调查小鼠新生儿致死原因的研究人员来说,商业全身体积描记器(WBP,Buxco系统,DSI)是一个合理的选择。
当我们第一次建立这个系统来监测P0小鼠的呼吸时,C57BL / 6新生儿的呼吸活动大部分时间都不能检测到,直到小鼠达到3天龄。因为WBP测量动物室内的压力变化,这是由于呼吸过程中被动物肺吸收和加热的灵感空气引起的,因此增加该装置的检测灵敏度是在P3之前监测C57BL / 6新生儿呼吸的唯一方法。
在本协议中,我们分享了我们在修改该系统方面的经验,可以早在P1和P0可靠地检测C57BL / 6新生儿幼仔的节奏呼吸。一旦获得记录的痕迹,由FinePointe软件计算的几个参数可用于探索出生后由于严重的呼吸衰竭而不死于紫in的小鼠的呼吸异常(Lai et al。,2016)。

关键字:新生小鼠, 全身体积描记法, 呼吸

材料和试剂

  1. P0和P3 C57BL/6垫料(C57BL/6)(JACKSON LABORATORY,目录号:000664)
  2. ICR养殖水坝[Crl:CD-1(ICR),Charles River,菌株编号:022]

设备

  1. 鼠标小狗WBP(数据科学国际,Buxco系统;图1)与:
    1. Max II放大器(DATA SCIENCES INTERNATIONAL,Buxco,型号:MAX2275)
    2. 记录室(DATA SCIENCES INTERNATIONAL,Buxco,型号:PLY4241),〜30±1°C,内置加热垫,


      图1. Buxco鼠标全身体积描记器的修改设置

    3. 流量传感器(DATA SCIENCES INTERNATIONAL,Buxco,型号:TRD5700)
    4. 小型啮齿动物偏流供应(DATA SCIENCES INTERNATIONAL,Buxco,型号:B04-BFL0100,用于小狗)
    5. 延长管(长度:14厘米;内/外径:2.6/3毫米)
    6. 金属过滤器(DATA SCIENCES INTERNATIONAL,Buxco,型号:HDW1514)
  2. 杂交炉(GE Healthcare,型号:RPN2511E)

软件

  1. FinePointe软件(Buxco系统,DSI)

程序

注意:

  1. 为了提高检测灵敏度,我们拆卸了Halcyon气垫车,并将金属过滤器的数量从4个增加到7个,这两个通道分别连接到动物和参考室(图2A和2B)。通过这样做,我们增加了由微妙的新生儿呼吸引起的压力差异变化。然而,这种修改也改变了一些呼吸参数的值(图6)。尽管我们在解决灵敏度问题之前和之后联系了Buxco技术服务,但我们无法获得任何帮助,以正确调整仪器设置以维持原始值。
  2. 上述改变提高了检测灵敏度,也增加了通常用于体积描记器的Buxco偏流泵的背景信号,因此必须去除偏流装置,以减少空气源的噪音。 >

  3. 我们将弯曲的延长管连接到Halcyon pneumotach,以避免任何来自上层环境的干扰气流(如用户呼吸)(图2C)。

    图2. Halcyon气冲击和记录室的修改.A。两个气动通道中的金属过滤器数量增加到7. B.在Halcyon气动装置中装载金属过滤器; C.组装到记录室上的改进型气动马达与面向下的延长管(箭头)连接,以避免任何气流扰动。

  1. 将P0 C57BL/6垃圾转移到ICR养殖大坝,直到记录。
  2. 将一杯水在烘箱中在34±1℃下孵育,以加湿。检索所有P1幼仔,并保持他们在加湿的烤箱中保持温暖至少20分钟,然后进行体积描记录。
  3. 按照FinePointe向导中的说明来校准设备(图3)。
    1. 单击校准图标(图3A)。


      图3.室的校准。 A-H。按照FinePointe向导中的说明进行操作,包括调零放大器,平衡传感器并将空气注入到室内。

    2. 单击扳手图标以校准每个室(图3B)。
    3. 将增益级别设置为3.5(图3C)。
    4. 将放大器切换到直流位置(DC)。
    5. 转动平衡螺丝,直到图表读取0伏(图3D和3E)。
    6. 在室内注入200μl空气(图3F)。
    7. 确保注入的气流完全包含在突出显示的紫色区域,红色光标处于0(图3G)。
    8. 将放大器切换到交流电源位置,然后按下"下一个"按钮进行重新调零。
    9. 点击"完成"按钮完成校准(图3H)。
  4. 轻轻地将P1小狗放在动物室内。
  5. 关闭房间,让小狗习惯1分钟。
  6. 开始测量记录每只小狗三个三分钟的会话。为避免在不存在偏流气源的情况下CO 2 2和湿度的积累,在每个会话结束时打开腔室1分钟。
  7. 从动物室中取出小狗。
  8. 保存记录轨迹(图4)并进行数据分析(图5)

    图4.来自P0,P1和P3新生小鼠的代表性呼吸痕迹.A和B.使用原始和改良记录室的P3小鼠呼吸流量。 C和D.使用原始和改进的记录室的P0小鼠的呼吸流量。箭头表示峰值流量的吸气峰值> 0.01毫升/秒。 E.使用改良记录室对P1 CPEB2敲除小鼠进行呼吸流量测定。括号表示呼吸暂停。

数据分析

因为呼吸运动在非麻醉的小狗中非侵入性地测量,所以仅分析从休息状态下从小狗记录的信号。然而,FinePointe软件(Buxco系统,DSI)计算所有节律信号,包括在运动过程中由身体运动或呼吸产生的信号。因此,为了分析来自静息呼吸的实际信号,我们从表中选择了所有呼吸参数的原始数据(图5,箭头),并将其复制并粘贴到Excel电子表格中。我们还仔细观察并标记了在体积描记录期间小狗不休息的时间,以手动排除由于身体运动引起的这些有问题的数据点。平均静息呼吸活动数据得出呼吸频率,潮气量,吸气时间,呼气时间,吸气峰值峰值和呼气峰值流量。通过对峰值流量吸气峰数进行计数手动分析P0小鼠的呼吸频率。 0.01ml/sec(图4D,箭头)。当呼吸暂停比前一次呼吸持续时间长两倍时,定义呼吸暂停发作(图4E),手动分时针对每分钟和持续时间发生的次数进行评分。


图5.从趋势数据表中提取所有呼吸参数的原始数据。将所选数据点复制并粘贴到Excel电子表格中。

笔记

  1. 如果遗传修饰的小鼠在出生后不立即死亡,我们建议将小狗转移到ICR大坝18-24小时,直到测量为止,因为C57BL/6水坝具有差的母体本能,并且不能具有呼吸缺陷的后排小狗。在我们修改的WBP中,P1小鼠比P0小鼠显示出更多的节奏和更强的呼吸运动,因此从其记录轨迹分析呼吸参数更可靠。
  2. 由于P0小鼠的微妙呼吸信号,我们仅通过手动计数吸气峰数量来分析呼吸频率> 0.01毫升/秒(图4D,箭头)
  3. 通过使用改良的WBP扩增潮气量,峰值吸气流量(PIF)和呼气峰值流量(PEF)的值,因此在对照组和实验组之间进行比较。相比之下,呼吸频率,吸气时间(Ti)和呼气时间(Te)的值不受影响(图6)。


    图6.使用原始和修改的记录室在P3记录的小鼠中呼吸参数的比较。
    Ti,吸气时间; Te,呼气时间PIF,吸气峰值流量; PEF,呼气峰值流量。数据为平均值±SEM(n = 3)。 * 0.05,** P < 0.01,Student's t -test。

  4. 灯在我们的鼠标室上午8点到晚上8点。我们通常在10 AM到5 PM之间进行呼吸记录。根据我们的经验,新生儿小鼠在下午5点之后进行记录时可能会更加活跃,这可能是由于昼夜节律。

致谢

这项工作得到了台湾科技部(105-2321-B-001-044)的支持。

参考

  1. Crone,SA,Viemari,JC,Droho,S.,Mrejeru,A.,Ramirez,JM和Sharma,K。(2012)。< a class ="ke-insertfile"href ="http: ncbi.nlm.nih.gov/pubmed/22674265"target ="_ blank"> V2a神经元的基因消融后小鼠不规则呼吸。 Neurosci 32(23):7895-7906 。
  2. Lai,YT,Su,CK,Jiang,ST,Chang,YJ,Lai,AC and Huang,YS(2016)。  迷走神经背侧运动细胞核中CPEB2限制性胆碱乙酰转移酶表达缺乏导致过度活化的副交感神经信号相关性支气管收缩。 Neurosci 36(50):12661-12676。
  3. Nsegbe,E.,Wallen-Mackenzie,A.,Dauger,S.,Roux,JC,Shvarev,Y.,Lagercrantz,H.,Perlmann,T.and Herlenius,E。(2004)。 Nurr1突变小鼠先天性通气不足和缺氧缺氧反应。 J Physiol 556(Pt 1):43-59。
  4. Turgeon,B.和Meloche,S。(2009)。解释小鼠突变体中的新生儿致死表型:对基因功能和人类疾病的了解生物化学版89(1):1-26。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 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. Lai, Y. and Huang, Y. (2017). Modification and Application of a Commercial Whole-body Plethysmograph to Monitor Respiratory Abnormalities in Neonatal Mice. Bio-protocol 7(12): e2343. DOI: 10.21769/BioProtoc.2343.
  2. Lai, Y. T., Su, C. K., Jiang, S. T., Chang, Y. J., Lai, A. C. and Huang, Y. S. (2016). Deficiency of CPEB2-confined choline acetyltransferase expression in the dorsal motor nucleus of vagus causes hyperactivated parasympathetic signaling-associated bronchoconstriction. J Neurosci 36(50): 12661-12676.
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