Name: evaluation of respiratory system mechanics in mice using the forced oscillation technique
Uploaded: 2013-05-15T14:00:00
Duration: 13 min 10 s
Description: Watch this Scientific Journal Video about Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique at JoVE.com

TKMcG is supported by a studentship from the Canadian Thoracic Society.
AUTHORS' CONTRIBUTION
 All authors took part in the conception of the manuscript. In addition, TKMcG initiated the project, collected experimental results, contributed to the writing of the manuscript and its critical review. AR collected and analyzed experimental results, drafted the manuscript and contributed to its critical review. LF collected experimental results and contributed to the critical review of the manuscript. TFS and JGM contributed to the critical review of the manuscript.

The procedures described below were approved by McGill University Institutional Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care (CCAC).
1. Preparatory Steps
<ol>
 <li>Solutions:
 <ol>
 <li>Methacholine: Prepare a stock solution at 50 mg/ml and make serial dilutions (1:1) based on the concentrations to be tested5. Allow the solutions to reach room temperature before nebulizing5.</li>
 <li>Anesthetic agents: Different regimens have been reported in the literature in various strains of mice (Table 1). Note: Regimen 1 was used under the present protocol.</li>
 </ol>
 </li>
 <li>Equipment: The present protocol applies to either of the two flexiVent generations supported by flexiWare 7 software. The software functions are grouped under three modules: Study Definition &amp; Planning, Experimentation Session and Review &amp; Reporting.
 <ol>
 <li>Turn on the system (flexiVent FX only) and/or start the software.</li>
 <li>At the first experimentation session or any time before it, open the Study Definition &amp; Planning module to predefine the study structure.</li>
 <li>Click on the Create a new study button and follow the wizard to create a study, outline the protocol and define the experimental groups and subjects to be studied.</li>
 <li>Initiate an experimentation session by opening the Experimentation Session module and following the start-up sequence for study and template selection.</li>
 <li>Assign a subject to the measurement site and confirm its weight.</li>
 <li>Proceed with the calibration of the system by following the steps described in the operating software. You will be prompted at one point to attach the cannula to be used (step 1.3.3) to the Y-tubing for calibration.</li>
 <li>Critical step. Repeat step 1.2.6 if the calibration values obtained are outside the acceptable range. (Refer to the flexiVent FX or flexiWare 7 user manual for the module specific acceptable ranges of calibration values).</li>
 <li>Cancel prompts to start ventilation and data recording unless ready to start the experiment. These can be initiated at a later point.</li>
 </ol>
 </li>
 <li>Subjects:
 <ol>
 <li>Anesthetize the subject using appropriate doses of anesthetic agents (Table 1).</li>
 <li>Verify that the subject has reached a surgical level of anesthesia. The subject should show no reaction to a toe pinch and its breathing should be regular and not labored.</li>
 <li>Perform a tracheotomy and cannulate the trachea.
 <ol>
 <li>Place the animal on its back and provide a source of heat (e.g. temperature controlled heating blanket or a lamp with a 60 watt bulb situated approximately 45 cm from the mouse to avoid excessive heating).</li>
 <li>Clean the throat area with alcohol and expose the trachea by making a skin incision and gently separating the submaxillary gland and the muscle layer covering it.</li>
 <li>Gently lift the trachea using a pair of micro-forceps and pass suture underneath it.</li>
 <li>Cut between two rings of cartilage nearest the larynx to make a small incision in the trachea without sectioning it.</li>
 <li>Insert the previously calibrated cannula into the incision and advance it gently inside the trachea the length of approximately 5 rings. Note: The present experiments were conducted using a 1.2 cm long metal 18 gauge cannula.</li>
 <li>Critical step. Secure the cannula in place using the suture. The attachment should form an airtight seal around the cannula.</li>
 </ol>
 </li>
 </ol>
 </li>
</ol>
2. Mechanical Ventilation
<ol>
 <li>Bring the animal close to the ventilator.</li>
 <li>Start mechanical ventilation by selecting a predefined or a customized ventilation profile in the ventilation docker.</li>
 <li>Connect the animal to the ventilator via the Y-tubing.</li>
 <li>Critical step. Align the animal to the ventilator and ensure that the tracheal cannula is at the same level as the ventilator to avoid a possible cannula occlusion or tracheal twist.</li>
 <li>Critical Step. Execute a Deep Inflation perturbation by double-clicking on the perturbation name to verify the cannula insertion and attachment. In absence of a leak, the system should be able to hold a pressure of 30 cmH2O over a 3 second period without excessive volume displacement (Figure 1). The recorded volume and pressure traces should also be smooth with no signs of offset or deformation as these could indicate a cannula obstruction or misplacement.</li>
 <li>If needed, connect vital sign transducers for heart rate and body temperature monitoring. Data recording can be initiated either manually or automatically via a script.</li>
</ol>
3. Lung Function Measurements
Measurements or commands (e.g. nebulizer activation, event markers) can be automated using predefined or customized scripts for a highly controlled and repeatable experimental process (Figure 2). Six families of perturbations giving rise to a number of parameters can be used to describe the subject respiratory system mechanics at baseline and following a given challenge (Table 2).
<ol>
 <li>Critical step. When ready to start taking measurements, run a Deep Inflation to recruit closed lung areas and standardize lung volume history.</li>
 <li>Critical step. Verify the absence of spontaneous inspiratory efforts by running a test measurement (e.g. PVs-P or PVs-V). Observe the pressure signal traces in the Selected Dataset view. With stepwise PV curves, pressure plateaus should be well defined with no downwards deflections. A downward swing in pressure would indicate an inspiratory effort from the animal (Figure 3).</li>
 <li>Initiate the selected script by double-clicking on its title. Scripts used in the present study generally included for measurements:
 <ul>
 <li>A sequence of baseline measurements in triplicate.</li>
 <li>Activation of the nebulizer for assessment of airway responsiveness to inhaled methacholine. Note: When prompted by the system, load approximately 100 &mu;l of saline or of a solution of methacholine into the nebulizer. Nebulization will start and stop automatically.</li>
 <li>A sequence of closely spaced measurements (every 10-15 sec) for a period of approximately 3 min following the activation of the nebulizer.</li>
 <li>A prompt to perform another challenge and repeat a sequence of measurements. Note: Drying the inside the nebulizer mount with a swab in between challenges can help prevent droplets or condensation from building-up in the inspiratory line.</li>
 </ul>
 </li>
 <li>At the end of the experiment, stop ventilation and detach the subject.</li>
 <li>Switch to the next subject in the operating software and confirm its weight.</li>
 <li>Critical step. Rinse and dry nebulizer, adaptor, Y-tubing, and cannula between subjects.</li>
 <li>Repeat steps 1.2.6 to 3.6.</li>
 <li>At the end of the day, close the experimental session. Remember to rinse and dry nebulizer, adaptor, Y-tubing, and cannula and to clean the system expiratory valve before leaving the laboratory by following the manufacturer's instructions.</li>
</ol>
4. Data Analysis
The software automatically calculates and displays parameters associated with a perturbation. It also provides a coefficient of determination (COD) which reflects the fit of the mathematical model to the data. Each dataset with an insufficient COD is labelled as excluded by the software. Review of experimental sessions, data re-analysis and creation of export scenarios are done in the Review &amp; Reporting module of the software.
<ol>
 <li>Open the Review &amp; Reporting module and create an export scenario, taking care to only include datasets having a sufficient COD.</li>
 <li>Export as needed parameters, pressure- or flow-volume curve, raw dataset signals or subject information to a spreadsheet application (see Table 3).</li>
 <li>Average baseline measurements for each parameter and plot all measurements as a function of time (see Figure 4). You may then choose to calculate the area under the curve, analyze the general profile of the curves or perform a statistical analysis.</li>
 <li>To express airway responsiveness results as a function of the concentration of methacholine, determine for each subject, parameter and experimental condition either a specific point (e.g. peak) or a specific time after each methacholine challenge. Calculate group averages and report or plot results for each experimental condition (Table 4, Figure 5).</li>
 <li>You may also consider calculating the concentration producing a doubling of a given parameter baseline value (PC200; Figure 5C), applying a normalization (e.g. % baseline) or performing a statistical analysis.</li>
</ol>

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AR, LF, TFS are employed by SCIREQ Scientific Respiratory Equipment Inc. TFS also owns stock. Free access to this article is sponsored by SCIREQ Scientific Respiratory Equipment, Inc.

The continued study of airway dysfunction as it relates to asthma and other lung disorders remains paramount to the understanding of underlying mechanisms of disease and the development of treatment options. The use of mice to model airway disease has been essential in gaining understanding into these disease mechanisms. When considering evaluating airway dysfunction in a subject as small as a mouse, having reliable and accurate tools by which to measure lung function is critical. Furthermore, having tools capable of providing insights on the location of airway dysfunction or therapeutic effect is invaluable. The FOT technique combines all these attributes and provides a powerful, integrative and translational approach to evaluate physiological changes.
In order to succeed with this type of measurement in mice, special attention should be given to a few steps, namely the calibration of the system, the resistance of the endotracheal cannula, the type of nebulizer (as well as its operating settings) the positioning of the animal and the standardization of lung volume history. In addition, it is imperative in order to obtain valid datasets that the subject's respiratory system remains passive during measurements. This can be achieved by the administration of a muscle paralyzing agent, working at a deep plane of anesthesia or by hyperventilating the subject to induce apnea (see Table 1). Investigators can start by mastering the system and its operating software, if desired, with test loads, while acquiring the necessary skills for measurements in mice. It would then be logical to generate reproducible results in na&iuml;ve animals before moving to disease models or treated mice. Since an important proportion of disease models in respiratory research involve exposing animals to agents such as allergens, toxins, pollutants, cigarette smoke or gases, variability in the results obtained with the measurement technique described in this article could therefore be influenced by the exposure procedure used. Standardization of key experimental processes (e.g. using computer-controlled exposure and measurement systems6, 13, 14) could potentially have a significant impact in reducing variability.
Examples presented in this article represent a selection of typical results from na&iuml;ve and chlorine-exposed mice experiments while highlighting the strengths as well as the limitations of the technique. As can be seen for example in Figure 6, the technique is capable of generating reproducible lung function measurements. While similar baseline resistance values were reported between mouse strains, differences in elastance were however observed15. Substantial changes are also to be expected between infant and adult mice16. As for other in vivo physiological assessment, high precision results, such as those generated by the FOT, come with a concession as to the natural state of the subjects. This principle, which is referred to as the phenotyping uncertainty principle1, applies to the present protocol in the sense that measurements need to be made in anesthetized, tracheotomised (or orally intubated) and mechanically ventilated subjects. Another limitation of the technique is observable in Figure 5D-5F where data are not available at the highest concentrations for the chlorine-exposed group because the fit of the Constant Phase Model to the data is poor above moderate levels of bronchoconstriction. However, severely bronchoconstricted animals could be assessed by analyzing Zrs directly15, or by using third party post-analysis software to fit more complex mathematical models, e.g. taking into account the heterogeneity of mechanical function17. Excluded datasets can also be observed if the animal's airways are not sufficiently passive or if the resistance of the cannula is too high. As a rule of thumb, the resistance of the cannula should not exceed the animal's resistance at baseline. Working with a cannula of larger inner diameter and/or shorter length will help reduce the cannula resistance. Finally, the present demonstration of FOT measurements in mice may be perceived as a time-consuming and therefore less efficient methodology or less applicable to longitudinal studies compared to less invasive techniques. However the latter are associated with a great deal of uncertainty as to the basis of their outcomes and are viewed by many as being flawed1. Repeated invasive measurements are possible in orally intubated animals, although technically more challenging17.
From the examples provided, results demonstrated the equivalence of the two generations of the flexiVent system at producing measurements of respiratory mechanics, as well as airway hyperreactivity and hypersensitivity to inhaled methacholine following chlorine-exposure in mice. When used to characterize or understand physiological changes or disease models, the detailed measurement aspect related to the technique can contribute to extend the current state of knowledge.

Adequate characterization of the mechanical properties of the lungs in small animals has become essential since the burgeoning of murine models in respiratory science. When performed using the forced oscillation technique (FOT), a technique also used in human subjects, these measurements provide a powerful, integrative and translational approach to study meaningful physiological changes. FOT measurements are typically obtained by analyzing pressure and volume signals acquired in reaction to a predefined, small amplitude, oscillatory airflow waveform (also referred to as perturbation or input signal) applied at the subject's airway opening1. In its simplest form, a FOT perturbation would be a single sinusoidal waveform at a well-defined frequency. More complex perturbations typically consist of a superposition of a selection of specific (mutually prime) frequency waveforms covering a broad spectrum. The decomposition of the multi-frequency input and output signals into their constituents using the Fourier transform allows the calculation of respiratory system input impedance (Zrs), i.e. the transfer function between the input and output signals, at every frequency included in the perturbation2. Therefore, FOT permits the simultaneous assessment of respiratory mechanics over a range of frequencies in a single maneuver2. Fitting advanced mathematical models (e.g. the Constant Phase Model3) to the impedance data then permit a partitioning of the response into airway (central and peripheral) and parenchymal lung tissue dependent parameters1, 3. Because many factors influencing the physiological response (e.g. breathing frequency, tidal volume, lung volume, upper airways, spontaneous breathing efforts, timing of measurements) are controlled and standardized by the measurement system and experimental procedures,1 the technique is capable of generating precise and reproducible measurements provided that it is performed correctly. The objective of this article is to provide a detailed, chronological description of the procedure needed in order to execute such measurements in mice. The protocol consists of four parts: preparatory steps (reagents, equipment and subjects), mechanical ventilation, lung function measurements, and data analysis. Examples of representative results of respiratory system mechanics generated using a computer-controlled piston ventilator (flexiVent, SCIREQ Inc, Montreal, Qc, Canada) are provided. These were obtained from na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage characterized by airway inflammation, epithelial cell damage and increased airway responsiveness to inhaled aerosolized methacholine4. While this protocol is often used to assess airway responsiveness to inhaled methacholine, it extends to other outcomes and various pathologies including asthma, chronic obstructive pulmonary disease (COPD), emphysema, lung fibrosis, lung injury as well as to transgenic mouse models of pathologies similar to human disease. Research findings using this tool can contribute to a better characterization and understanding of physiological changes or disease models as well as to expansion into new research areas.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Acetyl-&beta;-methylcholine chloride </td>
 <td>Sigma-Aldrich</td>
 <td>A-2251</td>
 <td>Methacholine</td>
 </tr>
 <tr>
 <td>Micro-Adson forceps, serrated 12 cm</td>
 <td>Fine Science Tools</td>
 <td>11018-12</td>
 <td></td>
 </tr>
 <tr>
 <td>Moria MC31 forceps, serrated-curved</td>
 <td>Fine Science Tools</td>
 <td>11370-31</td>
 <td></td>
 </tr>
 <tr>
 <td>Iris scissors-tough cut, straight 11.5 cm</td>
 <td>Fine Science Tools</td>
 <td>14058-11</td>
 <td></td>
 </tr>
 <tr>
 <td>Spring scissors-2.5 mm blades, straight</td>
 <td>Fine Science Tools</td>
 <td>15000-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Non-sterile blunt needle (18g x &frac12;")</td>
 <td>Brico Medical Supplies Inc.</td>
 <td>BN1805</td>
 <td>Endotracheal cannula</td>
 </tr>
 <tr>
 <td>Non-sterile 5-0 silk suture</td>
 <td>Seraflex</td>
 <td>IDI58000</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered solution</td>
 <td>Gibco</td>
 <td>14190-144</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml conical tubes</td>
 <td>Starstedt</td>
 <td>SS-4001</td>
 <td></td>
 </tr>
 <tr>
 <td>1 ml TB syringes</td>
 <td>Becton Dickinson</td>
 <td>309626</td>
 <td></td>
 </tr>
 <tr>
 <td>200 &mu;l filter tips</td>
 <td>Biosphere</td>
 <td>70.760.211</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>flexiVent FX</td>
 <td>SCIREQ Inc. </td>
 <td>sales@scireq.com</td>
 <td><a href="http://www.scireq.com/" target="_blank">www.scireq.com</a></td>
 </tr>
 <tr>
 <td>Aerogen Aeroneb nebulizer</td>
 <td>SCIREQ Inc.</td>
 <td>sales@scireq.com</td>
 <td><a href="http://www.scireq.com/" target="_blank">www.scireq.com</a></td>
 </tr>
 </tbody>
 </table>

evaluation of respiratory system mechanics in mice using the forced oscillation technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

Respiratory system mechanics measurements. Table 4 shows typical results from na&iuml;ve A/J mice obtained at baseline and following methacholine-induced bronchoconstriction (12.5 mg/ml) using either of the two flexiVent generations supported by flexiWare 7 software. Mechanics of the respiratory system, i.e. under closed chest conditions, were assessed by alternating perturbations of the single and broadband frequency forced oscillation families in a closely spaced manner (SnapShot-150, Quick Prime-3, respectively). Since ventilation is paused during measurements, the Quick Prime-3, which covers a similar frequency range as the Prime-8 but has a shorter duration (3 vs 8 sec), was selected in order to shorten the apneic period, minimize the effect of the perturbation on blood gases and provide a better resolution of the response. Parameters associated with each perturbation were calculated automatically by the operating software. Results illustrate that the two generations of the flexiVent system produced equivalent measurements of respiratory mechanics.
 Site of lung response. Distinguishing the site of lung response allows the investigator to further pinpoint affected regions as well as to identify potential points of pharmacological intervention6. For example, na&iuml;ve A/J mice show an increase of baseline resistance when the end expiratory pressure against which measurements are made is increased from 3 to 9 cmH2O (Figure 6A, SnapShot-150). In the present example, the use of broadband FOT measurements (Quick Prime-3) provided details to clarify the basis for the change in resistance: The change in end expiratory pressure resulted in a decrease in the airway resistance (RN) consistent with the bronchodilating effects of a higher lung volume and the larger inflation pressure (Figure 6D) and an increase in tissue damping (G; Figure 6E), a parameter closely related to tissue resistance that reflects tissue viscoelasticity and possibly the resistance of the small airways7. The latter is known to increase with increasing lung volume.
 Airway hyperresponsiveness. Following chlorine gas exposure, airway responsiveness to inhaled methacholine increases compared to air exposure in Balb/c mice as a result of airway damage4 (Figure 2). Chlorine is known to induce oxidative stress, leading to destruction of structural cells in the airways, in particular epithelial cells, and inducing recruitment of inflammatory cells. As represented in Figure 5, changes in all parameters describing respiratory system mechanics can be seen in response to increasing methacholine challenges. In comparison to air-exposed mice, mice exposed to chlorine gas displayed greater maximal responses at all FOT parameters (Figure 5A, 5B, 5D-5F) as well as a statistically significant leftward shift of the concentration-response curve exemplified by a reduction of the concentration of methacholine required to cause a doubling in resistance and elastance (PC200; Figure 5C). Those results illustrate, respectively, airway hyperresponsiveness and hypersensitivity to inhaled methacholine following exposure to chlorine gas. 
 Other measurements. In addition to FOT, the flexiVent system can also be used to collect other types of lung function8-10 or cardiovascular11 measurements. Figure 7 displays a representative stepwise, pressure-driven pressure-volume curve in na&iuml;ve A/J mice under baseline conditions. The upper portion of the deflation limb of the curve is fit to the Salazar-Knowles equation12 and parameters are automatically calculated by the software.
<img src="/files/ftp_upload/50172/50172table1.jpg" alt="Table 1" fo:src="/files/ftp_upload/50172/50172table1highres.jpg" fo:content-width="6in" /> 
Table 1. Examples of anesthetic regimens used in mice. <a href="https://www.jove.com/files/ftp_upload/50172/50172table1large.jpg" target="_blank"> Click here to view larger table</a>.
 <img src="/files/ftp_upload/50172/50172table2.jpg" alt="Table 2" fo:src="/files/ftp_upload/50172/50172table2highres.jpg" fo:content-width="5in" /> 
Table 2. Perturbations used for lung function measurements in mice. *Extension required to the system. The subject also needs to be in a closed plethysmograph chamber during measurements. <a href="https://www.jove.com/files/ftp_upload/50172/50172table2large.jpg" target="_blank"> Click here to view larger table</a>.
<img src="/files/ftp_upload/50172/50172table3.jpg" alt="Table 3" fo:src="/files/ftp_upload/50172/50172table3highres.jpg" fo:content-width="6in" /> 
Table 3. Example of exported parameters from the single and broadband frequency forced oscillation perturbation families. <a href="https://www.jove.com/files/ftp_upload/50172/50172table3large.jpg" target="_blank"> Click here to view larger table</a>.
 <img src="/files/ftp_upload/50172/50172table4.jpg" alt="Table 4" fo:src="/files/ftp_upload/50172/50172table4highres.jpg" fo:content-width="6in" /> 
Table 4. System Comparison. Comparison of lung mechanics parameters collected using the two generations of the flexiVent system operated by flexiWare 7 software. Results were generated in na&iuml;ve A/J mice (n=5/group) at baseline and following methacholine-induced bronchoconstriction (Mch 12.5 mg/ml). *Groups were compared using a two-way ANOVA for repeated measurements and the log10 of individual responses for homogeneity of variances (GraphPad Prism, version 5.03; GraphPad Software, San Diego, USA).
<img src="/files/ftp_upload/50172/50172fig1.jpg" alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50172/50172fig1highres.jpg"/> Figure 1. Screenshot of a deep lung inflation. The upper panel shows the volume displaced by the piston of the ventilator (red trace) and the volume delivered to the subject (grey trace). The lower panel displays the cylinder pressure increasing to a set pressure of 30 cmH2O over a period of 3 seconds and held constant for the same time period.
 <img src="/files/ftp_upload/50172/50172fig2.jpg" alt="Figure 2" /> 
Figure 2. Example of a typical script used to assess respiratory system mechanics at baseline.
<img src="/files/ftp_upload/50172/50172fig3.jpg" alt="Figure 3" /> 
Figure 3. Spontaneous inspiratory efforts during the execution of a stepwise pressure-volume curve.
<img src="/files/ftp_upload/50172/50172fig4.jpg" alt="Figure 4" fo:content-width="2.6in" fo:src="/files/ftp_upload/50172/50172fig4highres.jpg"/> 
Figure 4. Time-course response following increasing inhaled methacholine challenges. Results are expressed as the mean (&plusmn; standard deviation) of a group of 5 na&iuml;ve spontaneously hyperresponsive A/J mice. <a href="https://www.jove.com/files/ftp_upload/50172/50172fig4large.jpg" target="_blank">Click here to view larger figure</a>. 
<img src="/files/ftp_upload/50172/50172fig5.jpg" alt="Figure 5" fo:content-width="6in" fo:src="/files/ftp_upload/50172/50172fig5highres.jpg"/> 
Figure 5. Changes in respiratory system mechanics following increasing methacholine challenges in chlorine- and air-exposed Balb/c mice. Peak value was identified for each parameter in every subject and experimental condition. Group averages were then calculated (mean &plusmn; standard deviation; n=4-6). Differences between groups were assessed by analysis of variance using the log10 of individual responses for homogeneity of variances. The concentration of methacholine producing a doubling of baseline (PC200) was obtained by fitting a second order polynomial to individual dose-response curves and interpolation of the fitted curve. Data points are missing in D, E and F in the chlorine-exposed mice at the two highest methacholine concentrations due to insufficiently high coefficients of determination reflecting a poor fit of the mathematical model to the data. <a href="https://www.jove.com/files/ftp_upload/50172/50172fig5large.jpg" target="_blank">Click here to view larger figure</a>.
 <img src="/files/ftp_upload/50172/50172fig6.jpg" alt="Figure 6" fo:content-width="6in" fo:src="/files/ftp_upload/50172/50172fig6highres.jpg"/> 
 Figure 6. Partitioning of the respiratory response into airway and lung tissue mechanics. Experimental trace from a na&iuml;ve A/J mice illustrating single (2.5 Hz) and broadband (1-20.5Hz) frequency forced oscillation measurements of respiratory mechanics in triplicate at two different end expiratory pressures (3 &amp; 9 cmH2O). <a href="https://www.jove.com/files/ftp_upload/50172/50172fig6large.jpg" target="_blank">Click here to view larger figure</a>.
 <img src="/files/ftp_upload/50172/50172fig7.jpg" alt="Figure 7" fo:content-width="4in" fo:src="/files/ftp_upload/50172/50172fig7highres.jpg"/> 
 Figure 7. Pressure-volume curve in na&iuml;ve A/J mice under baseline conditions. Pressure-volume curves were generated using a stepwise pressure-driven perturbation (PVs-P) to ensure that each mouse lungs were inflated to the same pressure, independently of their condition. Salazar-Knowles equation parameters extracted from individual pressure-volume curves were also averaged and reported in a table format. Results are expressed as the mean &plusmn; standard deviation (n=6).

Watch this Scientific Journal Video about Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique at JoVE.com

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

evaluation-respiratory-system-mechanics-mice-using-forced-oscillation

Research

JoVE Journal

Biology

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...