Diagnostic Ultrasound Imaging of Mouse Diaphragm Function
April 21st, 2014
Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.
The overall goal of this procedure is to demonstrate how to accurately evaluate diaphragm contractility in a mouse model. Using ultrasound imaging, the first steps are to anesthetize the mouse and remove its abdominal hair. The next steps are to apply ultrasound gel and resolve the diaphragm image using the ultrasound probes.
Ultimately, the results can show diaphragm excursion and respiratory rate through utilizing the electronic calipers of the software. The main advantage of this technique over existing methods like invasive myography, is that it is not invasive and reduces the number of animals sacrificed. The implications of this technique extend toward the diagnosis and potential therapy of respiratory muscle dysfunction.
Though this method can provide insight into diaphragm contractility in a mouse model, it could also be applied to other systems such as clinical models to evaluate disease progression and therapy efficacy. Set up a clean procedure table with a heated isothermal pad wrapped in a surgical towel. The heated pad should be used to stabilize the animal's core temperature while also reducing potential stress to the animal.
The mouse should be placed in the anesthesia induction chamber with an oxygen flow rate set to 1.5 liters per minute, and with 3.5%vaporized isof fluorine Sedation usually sets within two minutes and is noted by loss of voluntary motor function. Then maintain the anesthetized state by nose cone gas delivery. After anesthetizing the animal apply a small amount of ophthalmic ointment directly to the corneas to reduce eye dryness during the anesthesia.
The mouse should never show the pedal withdrawal reflex. The mucus membranes should always have a pink color and the animal should breathe steadily. Restrain each leg of the anesthetized mouse on the heated procedure table with a removable adhesive, such as surgical tape.
Using an electric razor, remove most of the hair on the ventral body surface between the abdomen and halfway up the thoracic cavity. Then apply hair re removal cream to remove the remaining hair. After two to three minutes, wipe off the cream with a damp gauze pad.
Also, remove the excess hair with water and gauze. Next, clean the shaved region with four scrubs of 70%alcohol or an equivalent antiseptic turn on the ultrasound device. A micro convex array or linear phased array transducer is ideal due to these probes, small footprint and excellent axial resolution.
Adjust the ultrasound frequency to a range of 6.5 to 12 megahertz. Now set the machine's mode, brightness and motion modes together, provide the best imaging of diaphragm contraction. Next, apply a small amount of ultrasound gel to the upper abdomen and massage the gel toward the thoracic cavity.
Place the ultrasound transducer on the gel and angle it upwards towards the heart. Adjust the probe to optimize the image resolution. This is challenging, but will become easier with practice.
It's very important to remember to monitor the animal's respiration while under anesthesia, just to make sure that the results of the examination are not compromised. Press the freeze button to temporarily save the diaphragm images and view the selected contractions. Save the recording as a cyl loop, which allows for later measurements of diaphragmatic excursion and respiration rate.
After imaging, the mouse should completely recover from the anesthesia within 30 minutes. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbent. To analyze the data in M mode, measure the depth and rate of diaphragm movement from relaxation to contraction using the electronic calipers that are part of the ultrasound software.
Alternatively, in B mode, convert the CINA loop file into an MP four file and determine the respiration rate by counting the number of diaphragmatic contractions during the recording period, the electronic calipers may also be used in B mode to determine the depth of diaphragm movement. A mouse's diaphragm, maximal vertical displacement was recorded and the distance was then calculated as described by the protocol using electronic calipers. Such measurements were made on three different mice.
To illustrate, its after converting the CIL loop file into an MP four file. The respiration rate was determined by counting the number of diaphragmatic contractions during a six second recording period. This analysis was performed using B mode.
Alternatively, M mode provided a visualized picture of diaphragm vertical motion as well as respiration rate. There are some potential imaging artifacts which may occur when performing diagnostic ultrasound imaging such as reverberation. Another artifact that can be encountered is the presence of comet tales Once mastered, this technique can take 20 to 30 minutes if performed properly.
After watching this video, you should have good understanding of how to accurately evaluate a diaphragm contractility in the mouse model using a diagnostic ultrasound imaging technique.
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Overview
This article presents a non-invasive ultrasound protocol for evaluating diaphragm contractility in mouse models. The technique provides quantitative insights into respiratory muscle function, which is crucial for diagnosing respiratory diseases.
Key Study Components
Area of Science
- Neuroscience
- Respiratory physiology
- Imaging techniques
Background
- Diagnostic ultrasound imaging is effective for respiratory disease diagnosis.
- The technique is non-invasive, reducing animal sacrifice.
- Understanding diaphragm kinetics is essential for respiratory health.
- Ultrasound can provide detailed information on muscle function.
Purpose of Study
- To demonstrate a comprehensive ultrasound protocol for diaphragm analysis.
- To evaluate diaphragm contractility in mouse models.
- To highlight the advantages of non-invasive techniques over invasive methods.
Methods Used
- Anesthetizing the mouse and removing abdominal hair.
- Applying ultrasound gel for imaging.
- Using ultrasound probes to capture diaphragm images.
- Analyzing diaphragm excursion and respiratory rate with software.
Main Results
- The protocol successfully visualizes diaphragm movement.
- Quantitative data on respiratory muscle function is obtained.
- The technique shows advantages over invasive myography.
- Results can aid in diagnosing respiratory muscle dysfunction.
Conclusions
- The ultrasound protocol is effective for evaluating diaphragm function.
- This non-invasive method has significant implications for research.
- It can contribute to better understanding and treatment of respiratory issues.
Frequently Asked Questions
Executive Industry Relevance
Non-invasive ultrasound imaging of mouse diaphragm function enables longitudinal monitoring of respiratory muscle contractility without terminal procedures, supporting therapeutic development for respiratory diseases. This approach reduces animal usage while providing quantitative data on diaphragm excursion and respiration rate, aligning with 3R principles and enabling disease progression tracking in preclinical models. The technique offers mechanistic insights into respiratory muscle dysfunction, facilitating target validation and lead identification in pulmonary therapeutic pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses on respiratory muscle function through quantitative assessment of diaphragm contractility in vivo.
- Operational Value: Provides reproducible, non-terminal measurements that support biological de-risking of targets involved in respiratory pathophysiology.
- Predictive Value: Supports portfolio triage by delivering functional readouts that correlate with disease-modifying potential in obstructive lung disease models.
Screening & Assay Development
- Assay Readiness: Generates standardized, quantifiable outputs (diaphragmatic excursion, respiration rate) suitable for high-throughput functional screening of compounds.
- Scalability: The ultrasound-based platform allows repeated measurements in the same animal, reducing variability and increasing statistical power in dose-response studies.
- Cross-functional Utility: Outputs serve as translatable biomarkers for respiratory efficacy, enabling alignment between discovery, preclinical, and clinical teams.
Translational & Preclinical Research
- Disease Relevance: Directly models diaphragm fatigue in obstructive lung diseases, providing a pathophysiologically relevant system for therapeutic evaluation.
- Mechanistic De-risking: Clarifies target engagement effects on respiratory muscle function, reducing ambiguity in mechanism-of-action studies.
- Translational Continuity: Enables longitudinal tracking of therapeutic effects from early discovery through preclinical validation, supporting go/no-go decisions.
Pipeline & Workflow Integration
The method integrates into the discovery continuum by providing functional respiratory readouts after target engagement but prior to lead optimization, enabling mechanism-based compound prioritization.
- Discovery Biology: Supports hypothesis testing on respiratory targets by delivering direct, quantitative measures of diaphragm contractility in intact animals.
- Screening: Delivers assay-ready, reproducible physiological endpoints that facilitate compound evaluation in disease models.
- Analytics: Provides electronic caliper-derived measurements of excursion and rate, enabling statistical comparison across treatment groups.
- Translational Research: Connects to clinical ultrasound applications, offering a bridge for biomarker validation in respiratory therapeutics.
- Enterprise Reuse: Establishes a reusable, non-invasive platform for respiratory function assessment across multiple therapeutic areas and disease models.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing mechanistic ambiguity in respiratory muscle function assays.
- Operational Value: Enhances standardization and reproducibility through non-invasive, repeatable imaging that minimizes inter-animal variability.
- Strategic Value: Improves capital efficiency by enabling longitudinal studies and reducing animal consumption in respiratory drug discovery.
- Portfolio Impact: Informs risk-adjusted advancement decisions by providing early functional data on respiratory safety and efficacy.
Implementation Considerations
- Requires expertise in ultrasound imaging, anesthesia management, and respiratory physiology for accurate data acquisition.
- Dependent on high-frequency microconvex or linear array transducers and software with M-mode and B-mode capabilities for motion analysis.
- Necessitates standardized protocols for animal preparation, including temperature maintenance and hair removal, to ensure image quality.
- Adaptation considerations include transducer frequency adjustment and gel application techniques for different mouse strains and disease states.
- Practical limitations include potential imaging artifacts (reverberation, comet tails) and the need for trained personnel to distinguish true signal from noise.
