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

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’s test. These data demonstrate that even one short bout is sufficient for monitoring quiet breathing. However, it is entirely possible that analyzing individual variation within a mouse at 15 s vs. 10 min may result in different findings, as the 10 min bout could encompass minimal sniffing and grooming activities. However, using Levene’s test for a comparison of individual mouse baseline segments provides a different analysis than the one described in this protocol. Overall, the design of this methodology uses 15 s breathing segments that can be acquired during minutes 60–120 in the chamber, versus having to wait for each mouse to achieve longer durations of quiet baseline.

The shorter duration required for baseline allows for more anxious/agitated strains of mice to be tested for quiet breathing. The use of a longer breathing segment (i.e., 10 or 2 min) lengthens the protocol duration, to a point where a trial may need to be abandoned if the mice do not display a quiet breathing trace within 3 h. Since many experimental designs also incorporate respiratory challenges (i.e., hypoxia), the extended time allotted for other gases highlights the need for baseline collection time to be standardized. The use of a single 15 s bout of quiet breathing helps to relieve the concern of working with mice (and strains of mice) that may be particularly excitable in the chamber. While working with barometric plethysmography, we found that ~10% of mice per study had to be excluded because of their inability to perform as little as 2 min of continuous quiet breathing within the chamber. The implementation of previous familiarization trials was unsuccessful in getting mice to calm down faster when placed in the chamber on the day of experimentation. However, because different strains, sexes, and ages of mice may all react differently to the chamber environment^10,11, it is possible that habituation techniques may be helpful^12,13 for some cohorts. Our familiarization trials consisted of placing the mice in the UBP chamber in the testing room for 1–2 h for several days prior to experimentation. While we observed no changes in animal behavior following this procedure, a previous study has shown that 24 h of habituation was needed to eliminate novelty effects resulting in spontaneous physical activity in mice¹². Additionally, Kabir et al. found that placing plastic cylinders similar in size to the barometric plethysmography chamber in the home cage was advantageous in getting rats to familiarize themselves with the setups prior to experimentation¹³.

This protocol also uncovers possible respiratory dysfunction in mice via the quantification of central apneas, indicative of neural control issues. Thirty minutes of observation prior to the baseline pattern-of-breathing collection showed that all 16 aged mice displayed a high number of apneic episodes and augmented breaths (represented in Figure 1C). The numerous apneas in this aged mouse cohort highlight the ability of this protocol to quantify another important breathing measure without adding additional time to experimentation. It should be noted that age and disease progression (if applicable) can affect the presence and number of apneic episodes.

In order to characterize quiet breathing, it is important to continuously observe the barometric plethysmography chamber and mouse throughout the duration of the protocol. For the quantification of quiet breathing, mice should be awake but not partake in any active behaviors such as sniffing, grooming, or exploring (represented in Figure 1A). Since patterns of breathing during sleep can differ from those in an awake animal^14,15, the collection of calm breathing during the awake state is critical. It is possible that longer segments of quiet breathing could include periods of sleep, which may not be desired depending on the experimental design. In this case, shorter segments of quiet breathing would be ideal to document, as the likelihood of data collection during sleep is reduced when active segments flank the short (15 s) quiet breathing segments. We have observed that longer segments of quiet breathing can be challenging to acquire in the mouse model, as mouse behavior in the chamber seems to be very different compared to that of rats. It is important both to critically observe mouse respiratory flow for appropriate breathing segments and to document animal behavior. In cases of reduced ventilation or unstable breathing, this method can still be utilized. In these instances, it is essential the experimenter is blinded to the cohort when selecting the 15 s segments. The software program should distinguish the breaths, with an acceptance rate of 100% during the 15 s period. We advise taking note of the breathing tracings in addition to ensuring that the animals meet the behavioral criteria for baseline since it is possible that stationary mice may still be anxious. A previous study reported that although rats exhibited calm behavior, they still showed altered breathing patterns (i.e., increased frequency) in response to controlled stimuli within the testing room¹³.

Measures of frequency, VT, VE, inspiratory and expiratory time, and VE/VCO₂ are all quantified using analyzers and the UBP software and are frequently reported in the literature. Particularly, VT and VE calculations use the Drorbaugh and Fenn equation¹⁶, which requires known values for body temperature, ambient chamber temperature, humidity, and barometric pressure. It is recommended to collect these measures throughout the experiment for the most accurate VT and VE values. Other variables that are calculated by the system should be used with caution. UBP is not a direct measure of pulmonary mechanics; thus, variables related to airway resistance (e.g., enhanced pause [Penh]) should be interpreted with this caveat in mind⁵. Additional components of the UBP setup that can impact variables calculated by the software include flow rates and the general calibration of the system. Confirm seals and gaskets are working properly (no leaks) and ensure proper connection of all equipment to the barometric plethysmography chamber (Figure 5). Flow rates in and out of the chamber should be kept consistent. Required flow rates can differ between UBP setups, so it is important to check these values prior to experimentation. The flow rate into the chamber should be enough to provide fresh air or gas challenges in a timely manner. The flow rate should also be sufficient for allowing the metabolic analyzers to measure O₂ and CO₂ without having CO₂ build up within the chamber environment, which poses the risk of a changing pattern of breathing. Similarly, gas mixer/analyzer calibrations need to be regularly implemented to ensure that the metabolic parameters are accurately measured.

Other considerations for conscious UBP include reducing distractions within the experimental room while animals are being tested. Loud noises, different smells, and the presence of nonessential personnel in the room can all add to anxious behaviors exhibited by mice. Using smaller rooms as testing areas may help, but if this is not possible, cardboard walls (with a small viewing window) can be set up surrounding the chamber to lessen distractions for the mice. Electrical activity within the room should be kept at a minimum to prevent additional noise within the barometric plethysmography tracings. Therefore, it is important to take note of the flow tracings during the 10 min period when the software is collecting data from an empty chamber. These tracings should remain flat, and any interruptions or slight waves are signs of noise and should be addressed. Pressure changes from opening and closing doors or from HVAC functioning can also add to erroneous fluctuations, and ensuring that these actions occur minimally (and noting them when they do occur) is critical. Humidity can also affect the calculated tidal volume and minute ventilation, making it very important to confirm that the chamber and connecting tubes are dried before use. If necessary, the use of Drierite beads in sequence with the flow-in tubes can help remove all condensation in the air prior to chamber entrance. This step would be instituted in cases when the humidity has routinely been higher than levels listed in The Guide for the Care and Use of Animals⁸ (30%–70%, ideally within 10% of setpoint). Humidity can also build up in the chamber due to the presence of the animal. Although some humidity is normal, it may continue to build if the animal is excessively active or placed in the chamber for longer durations. If humidity levels reach maximum levels (99.99%), the chamber may need to be opened and wiped down during experimentation to maintain comparable breathing measures. The software accounts for changes in barometric pressure, ambient temperature, animal temperature, and humidity. Best practice is to maintain the values within a reasonable range so that “apples to apples” are being compared across ages, strains, and sexes. Moreover, the circadian cycle of mice and the time range of testing, as well as specific lighting conditions of the room, are important details to consider^13,17. For instance, we typically test mice in lighting similar to their housing room (either light or dark cycle) and within a 3 h range¹⁸. Experimenters should also remain blinded to the animal groups during data collection and analyses to prevent differences in the selection of a baseline. When possible, the same experimenter should collect all data and/or analyze all tracings in a given experiment. Steps to keep experimenters blinded to the animal groups, as well as randomization and testing during similar times of the day, are crucial to the rigor of investigation. Ultimately, there are extraneous factors that may alter the flow tracings, and these concerns should be considered when performing UBP.

The UBP method is a technique used to characterize the pattern of breathing in mice. Baseline measures can be collected within 2 h when using a 15 s breathing segment. Here we report a method which can be performed with aged mice, who are often more agitated in the chamber than younger mice, suggesting that other anxious or active mouse strains could also be tested with this protocol. The data collected from UBP is noninvasive and allows for testing over multiple time points, which is useful for studies about aging, drug therapy, and disease progression.