Name: method to obtain pattern of breathing in senescent mice through unrestrained barometric plethysmography
Uploaded: 2020-04-28T13:00:00
Description: Watch this Scientific Journal Video about Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography at JoVE.com

The authors would like to thank Angela Le, Sarah Ruby, and Marisa Mickey for their work maintaining the animal colonies. This work was funded by 1R15 HD076379 (L.R.D.), 3R15 HD076379 (L.R.D. to support CNR), and the McDevitt Undergraduate Research Fellowship in Natural Sciences (BEE).

All procedures were approved by the Le Moyne College Institutional Animal Care and Use Committee. All use of animals was in agreement with the policies described in the Guide for the Care and Use of Laboratory Animals8.
NOTE: (Critical) Prior to experimentation, obtain all necessary approvals and training required for animal use. It is important the experimenters are familiarized with the mouse behaviors and activity levels, including signs of sleep, di...

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The protocol provides information regarding a quiet breathing baseline in mice, as well as collecting data about central apneas and augmented breaths. The representative results show that a 10 min quiet baseline has a similar pattern of breathing when compared to an average of four 15 s bouts for a cohort of old mice. Importantly, the 15 s bouts are not statistically different, nor do these groups have differences in variation from one another using Levene&#8217;s test. These data demonstrate that even one short bout is ...

UBP is a common technique for the assessment of breathing patterns1,2,3,4. In this method, mice are placed in a closed chamber where pressure differences between the main chamber (where the animal is housed) and a reference chamber are filtered through a pneumotachograph to obtain values. The resulting UBP setup is noninvasive and unrestrained and allows for respiratory measures to be assessed without the requirement of anesthesia or surgery. Additionally, this technique is suitable for studies requiring multiple measurements in the same mouse over time. Variables such as breathing frequency, tidal volume, and minute ventilation can be quantified with this method, during a single trial or over several trials. Whole-body UBP also provides measures of peak flows and respiratory cycle duration. Together, these parameters quantify the pattern of breathing. The recorded breathing traces also make it possible to review the data and count the number of central apneas displayed within a given time period. This count can be used alongside an analysis of tidal volume and inspiratory times to gauge other alterations in the pattern of breathing.
While several noninvasive plethysmography techniques exist for the direct assessment of pulmonary physiological parameters, whole-body UBP allows for a way to screen for respiratory function with minimal undue stress to the mouse. Head-out plethysmography, which utilizes tidal midexpiratory flow measures and is also noninvasive, relies on restraint, like many other types of plethysmography (e.g., double-chamber plethysmography). While these methods have been used in rodent models to measure airway responsiveness5, the use of neck collars or small restraint tubes may take mice (vs. other species) longer to acclimate to and return their breathing to resting levels.
Obtaining an optimal air-breathing segment is an important consideration for baseline comparisons. The increased use of commercially available plethysmography systems makes collecting pattern-of-breathing data possible in many laboratories. Importantly, pattern of breathing is variable throughout the collection period, particularly for mice. With that said, it is necessary to standardize baseline analysis as a means of ensuring that the training level of experimenters does not confound results. There are numerous ways to collect an air-breathing segment, serving as one area of variation between experimental designs. One example includes averaging the final 10&#8211;30 min of data following a previously defined set of time within the chamber1, while another method involves waiting until the mouse is visibly calm for 5&#8211;10 min6. The latter can take 2&#8211;3 h to achieve and in some cases, a trial may need to be abandoned if the mouse is not calm for long enough. This concern is an especially important consideration for strains of mice where observed behaviors are more anxious and excitable7. These mice may take longer to adjust to the chamber environment and only remain calm for short bursts of time. Limiting the time devoted to baseline collection standardizes the chamber time for each mouse.
It is crucial that experimenters obtain a suitable baseline that encompasses resting behavior values in the mouse but also occurs in a timely manner. Hence, the goal of this report is to provide a description of methods used to obtain short quiet baseline values for breathing parameters in mice. Moreover, we report that apneas and augmented breaths can be quantified during the first hour in the chamber.

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			<td height="21" style="height:21px;width:351px;">Carbon Dioxide Analyzer</td>
			<td style="width:135px;">AEI Technologies</td>
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			<td style="width:135px;">AEI Technologies</td>
			<td style="width:119px;">&#160;P-61B</td>
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			<td height="21" style="height:21px;width:351px;">Computer</td>
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			<td>must be compliant with Ponemah requirements</td>
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			<td height="21" style="height:21px;width:351px;">Drierite beads</td>
			<td style="width:135px;">PermaPure LLC</td>
			<td style="width:119px;">DM-AR</td>
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			<td height="21" style="height:21px;width:351px;">Flow Control</td>
			<td style="width:135px;">AEI Technologies</td>
			<td style="width:119px;">R-1</td>
			<td>vacuum</td>
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			<td>need one per chamber and one for vacuum</td>
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			<td height="21" style="height:21px;width:351px;">Gas Mixer</td>
			<td style="width:135px;">MCQ Instruments</td>
			<td style="width:119px;">GB-103</td>
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			<td height="21" style="height:21px;width:351px;">Gas Tanks</td>
			<td style="width:135px;">Haun</td>
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			<td>100% oxygen, 100% carbon dioxide, 100% nitrogen - food grade, or pre-mixed tanks for nomal room air and gas challenges</td>
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			<td height="21" style="height:21px;width:351px;">Oxygen Analyzer</td>
			<td style="width:135px;">AEI Technologies</td>
			<td style="width:119px;">S-3A</td>
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			<td height="21" style="height:21px;width:351px;">Oxygen Sensor</td>
			<td style="width:135px;">AEI Technologies</td>
			<td style="width:119px;">&#160;N-22M</td>
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			<td height="21" style="height:21px;width:351px;">Ponemah Software</td>
			<td style="width:135px;">DSI</td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:351px;">Small Rodent Chamber</td>
			<td style="width:135px;">Buxco/DSI</td>
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			<td></td>
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			<td height="21" style="height:21px;width:351px;">Thermometer (LifeChip System)</td>
			<td style="width:135px;">Destron-Fearing</td>
			<td style="width:119px;"></td>
			<td>any type of thermometer to take accurate body temperatures is appropriate, but the use of implantable chips allows for minimal disturbance to animal for taking several body temperature measurements while the animal is still in the UBP chamber&#160;</td>
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			<td height="21" style="height:21px;width:351px;">Transducers</td>
			<td style="width:135px;">Validyne</td>
			<td style="width:119px;">DP45</td>
			<td>need one per chamber&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:351px;">Whole Body Plethysmography System&#160;</td>
			<td style="width:135px;">Data Science International (DSI)</td>
			<td style="width:119px;"></td>
			<td>Includes ACQ-7700, pressure/temperature probes, etc.&#160;</td>
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method to obtain pattern of breathing in senescent mice through unrestrained barometric plethysmography

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and minute ventilation are routinely reported. Moreover, information can be collected regarding the neural output of breathing, including the existence of central apneas and augmented breaths. An important consideration for UBP is obtaining a breathing segment with a minimal impact of anxious or active behaviors, to elucidate the response to breathing challenges. Here, we present a protocol that allows for short, quiet baselines to be obtained in aged mice, comparable to waiting for longer bouts of quiet breathing. The use of shorter time segments is valuable, as some strains of mice may be increasingly excitable or anxious, and longer periods of quiet breathing may not be achieved within a reasonable timeframe. We placed 22 month-old mice in a UBP chamber and compared four 15 s quiet breathing segments between minutes 60&#8211;120 to a longer 10 min quiet breathing period that took&#160;2&#8211;3 h to acquire. We also obtained counts of central apneas and augmented breaths prior to the quiet breathing segments, following a 30 min familiarization period. We show that 10 min of quiet breathing is comparable to using a much shorter 15 s duration. Additionally, the time leading up to these 15 s quiet breathing segments can be used to gather data regarding apneas of central origin. This protocol allows investigators to collect pattern-of-breathing data in a set amount of time and makes quiet baseline measures feasible for mice that may exhibit increased amounts of excitable behavior. The UBP methodology itself provides a useful and noninvasive way to collect pattern-of-breathing data and allows for mice to be tested over several time points.

The results of UBP as an evaluation of pattern of breathing in 16 aged (22-month-old) mice performed under normal air gas (20.93% O2 with balanced N2) are reported. The analysis first included a comparison of a longer 10 min quiet breathing segment (which took over 2 h to obtain) versus the average of four short 15 s segments (quantified within minutes 60&#8211;120). A representative flow tracing of quiet breathing, where breathing is consistent with no active breathing behaviors, is provided in <st...

Watch this Scientific Journal Video about Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography at JoVE.com

Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography

Unrestrained barometric plethysmography is used to quantify the pattern of breathing in awake mice. We show that 15 s segments under a standardized protocol display similar values to an extended duration of quiet breathing. This methodology also allows for the quantification of apnea and augmented breaths during the first hour in the chamber.

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and ...

This protocol can be used to measure patterns of breathing in mice. Importantly, it can minimize the impact of active behaviors in genetically modified strains that can confound results. The main advantages of this technique are that it is noninvasive and that it does not require the use of anesthetic or stress-inducing restraints.Begin by making sure that all of the hoses and tubes are connected to the barometric plethysmograph chamber and by connecting a gas inflow tube and a vacuum outflow tube directly to the barometric plethysmography chamber. To calibrate the low flow into the chamber, set the flow into and out of the chamber to zero and remove the flow tube into the chamber. Turn off the vacuum and enter a zero into the low unit cell to record the zero flow in the 7700 amplifier setup of the barometric plethysmograph software.Then reattach the inflow tube and turn on vacuum flow to calibrate the high flow and to allow 20.93%oxygen balanced nitrogen gas to flow through the barometric plethysmography chamber from the gas mixer. Next, convert the inflow value measured from the flow meter from liters per minute into milliliters per second and click the high unit cell to enter the value in milliliters per second. Double-click high cal, change the time to three seconds and click Measure.Then leave the 7700 amplifier setup tab open to calibrate the metabolic analyzers to the barometric plethysmography software. To calibrate the metabolic analyzer, in the gas mixer program, set the gas mixer to 20.93%oxygen and 79.07%nitrogen. And on the metabolic analyzer, set the oxygen calibration level to 20.93%and the carbon dioxide to 0%Turn the dial back to sample once the appropriate values have been entered on the gas analyzers to set the high oxygen and low carbon dioxide percentage.Click on the ABCD 4 tab of the barometric plethysmography software and enter 20.93 under high unit of the C2 line for oxygen. Under high cal, change the time to three seconds and click Measure. Enter zero under low cal of the C3 line for carbon dioxide.Then change the time to three seconds and click Measure under low cal. In the gas mixer program, change the oxygen value to 10%and the carbon dioxide value to 5%and wait several minutes for the gas flow to adjust to these values. On the metabolic analyzers, turn the adjustment knobs to calibrate the carbon dioxide to 5%taking care to turn the dial back to sample once the values have been calibrated.After checking that the analyzer readings are stable, click high unit under C3 and enter five for carbon dioxide. Then change high cal to three seconds and click Measure. Click low unit under the C2 option and enter 10 for oxygen and click low cal, input three seconds and click Measure.Change the gas mixer back to 20.93%oxygen and 79.07%nitrogen and wait several minutes for the chamber to adjust to these values. Routinely run additional calibrations with certified gas tanks to confirm the flow mixer and oxygen-carbon dioxide analyzers are performing properly. When the metabolic analyzers have been calibrated, recheck the flow meters connected to the barometric plethysmograph chamber and adjust the air flow into and out of the chamber to rates appropriate for the experiment.When all of the settings have been applied to the barometric plethysmography software, click Close in the Acquisition tab. Hit Start Acquisition, name the file and click OK to begin recording. For unrestrained barometric plethysmography, record the weight and initial body temperature of the mouse before returning the mouse to its home cage.Wait 10 minutes to collect the background oxygen and carbon dioxide data from the empty chamber before placing the mouse into the chamber. During the first hour, document the behavior of the animal taking detailed notes that include the specific values of the in and outflow of the chamber. At the end of the chamber habituation, watch for segments of quiet breathing for the next 60 minutes taking body temperature measurements every 10 minutes when using an implantable device.Take care to become familiar with general mouse behaviors so that instances of calm breathing with no sniffing, grooming or exploring can be appropriately identified. At the end of the experiment, return the mouse to its cage and thoroughly clean and wipe down the equipment. For a metabolism analysis, open the metabolic panel in the software and obtain the average of the first 10 minutes of the oxygen and carbon dioxide levels from when the chamber was empty.View the flow panel of the barometric plethysmography software and right-click Analyze Attribute to set appropriate parameters. For a pattern of breathing analysis, confirm the times for the 15 seconds of quiet breathing using notes about animal behavior as well as the flow panel tracing. Then enter the times under open parser dialogue from the data parser tab and click Parser View Mode to show only the specific 15-second segments of interest.To analyze apneas and augmented breath, in the open review file, exit the parser view mode and click Setup P3 Setup and Graph Setup to select Page View as the type and five as the number of panes. Enter minus two into the box labeled low and two into the box labeled high for the flow measures in milliliters per second and apply the changes. Then scroll to the 30-minute mark on the flow tracings panel and manually count the apneas and augmented breaths for the 30 to 60-minute period after the mouse was placed in the chamber.For this analysis, periods of suspended breathing lasting longer than or equal to 0.5 seconds are indicative of an apnea. Augmented breaths are indicated by a sharp rise in the breathing trace above 1.25 milliliters per second followed by a sharp decrease below minus 0.75 milliliters per second. This representative flow tracing of quiet breathing in a 22-month-old mouse is typical of a pattern in which the breathing is consistent with no active breathing behaviors.This breathing pattern is from a more active segment during which the mice were exploring the chamber sniffing and/or grooming and is not ideal for the type of breathing collection used for this methodology. The parameter selected for the assessment of possible pattern of breathing differences between the two time points were breathing frequency, tidal volume, minute ventilation, tidal volume inspiratory time ratio, and minute ventilation expelled carbon dioxide ratio. Further analysis was performed with each of the four 15-second baseline segments for frequency, tidal volume, minute ventilation, tidal volume inspiratory time ratio, and minute ventilation expelled carbon dioxide ratio.No significant differences between any of the time points and no differences in the variability between any of the four time segments for any pattern of breathing measure were found. Quantification of the number of apneas and augmented breaths observed for each animal during minutes 30 to 60 of the unrestrained barometric plethysmography protocol revealed that aged animals showcased a high number of apneas and the presence of augmented breaths within a 30-minute span. Researchers can also employ gas challenges such as hypoxia or hypercapnia to document any changes in the breathing pattern resulting from an intervention or therapy.This technique provides a means for characterizing patterns of breathing in small rodents and genetically modified animal strains that may exhibit distinct behavioral differences.

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