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, Whole body plethysmography, Respiration
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
Equipment
Software
Procedure
Notes:
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
Acknowledgments
This work was supported by the Ministry of Science and Technology (Most 105-2321-B-001-044) in Taiwan.
References
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