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.
Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.
Recently, diagnostic ultrasound imaging techniques have been applied to mouse models of renovascular hypertension and pancreatic cancer1,2. However, these techniques have not been widely used in rodent respiratory muscle function assay. Therefore, we have developed a diagnostic ultrasound imaging method as a valuable tool for in vivo longitudinal assessments of diaphragm mobility in mice.
There are several advantages to diagnostic ultrasound imaging. For instance, it is noninvasive, safe, portable, and allows for real time measurements at a relatively low cost3. Particularly, certain low frequency ultrasound devices were able to detect air trapping, a clinical characteristic of chronic obstructive pulmonary disease (COPD) with mild to severe airflow limitation4. Thus, diagnostic ultrasound imaging may serve as an easily accessible and reproducible screening method for real-time monitoring of respiratory disorders.
Diagnostic ultrasound imaging techniques are frequently applied to larger animals or human subjects. However, there have been a limited number of ultrasound imaging studies on mouse models, which is likely due to the challenges of performing ultrasound on small-scale subjects. The current protocol outlines a novel procedure for measuring diaphragm function in the mouse. In addition, although there have been several rodent studies on diaphragm function, most of the results were generated by isolating muscle strips directly from the euthanized animal5-7. In contrast, using an di vivo diagnostic ultrasound imaging method for analyzing diaphragm activity would decrease the number of animals sacrificed for experimentation. Furthermore, long-term treatments focused on enhancing diaphragm contractility may be accurately assessed via ultrasound in rodent models without sacrificing animals.
In our lab, we have developed an effective method for visualizing as well as analyzing mouse diaphragm activity using an ultrasound machine, which helps the understanding of diaphragm function in vivo, avoids invasive methods to animals, and aids in the development of therapeutic treatments for respiratory dysfunction.
All procedures involving animal subjects were approved and completed in accordance and compliance with The Ohio State University Institutional Animal Care and Use Committee (IACUC) regulations and guidelines.
1. Mouse Anesthesia
2. Preparing for Diagnostic Ultrasound Imaging Procedure
3. Diagnostic Ultrasound Imaging Protocol
4. Post Anesthesia Animal Recovery
A typical ultrasound image of a mouse diaphragm is shown in Figure 1A. The mouse diaphragm maximal vertical displacement was recorded. This distance was calculated by precisely measuring the depth of diaphragm movement from relaxation to contraction using the electronic calipers that are part of the ultrasound software. Table 1 displays these distance measurements of diaphragmatic contractions from three different mice. After converting the cine loop file into a MPEG file, the respiration rate was determined by counting the number of diaphragmatic contractions during a six second recording period. This analysis can be performed using B-mode. Alternatively, M-mode provides a visualized picture of diaphragm vertical motion as well as respiration rate as shown in Figure 1B. These results demonstrate that this method is effective in accurately observing mouse diaphragm contractions. Furthermore, by recording diaphragm function, this protocol also allows for assessing two important parameters including diaphragm excursion and respiration rate. This imaging method is useful for direct comparison between healthy and diseased diaphragm muscle. However, Figure 2 demonstrates the potential imaging artifacts that may occur when performing diagnostic ultrasound imaging.
Figure 1. A. A representative ultrasound image of a mouse diaphragm muscle (B-mode). The solid and dashed lines depict the diaphragm during contracted and relaxed states, respectively. B. A representative ultrasound image of a contracting mouse diaphragm (M-mode). Please click here to view a larger version of this figure.
Figure 2. An ultrasound image of mouse diaphragm muscle with the presence of possible reverberation (A) and comet-tail (B) artifacts (indicated by the arrows). Please click here to view a larger version of this figure.
1st measurement (mm) | 2nd measurement (mm) | 3rd measurement (mm) | Average (mm) ± SD | |
Mouse 1 | 0.96 | 0.92 | 1.06 | 0.980 ± 0.072 |
Mouse 2 | 0.93 | 0.99 | 1.01 | 0.977 ± 0.042 |
Mouse 3 | 0.91 | 0.93 | 0.89 | 0.910 ± 0.020 |
Table 1. Distance measurements of mouse diaphragm movement. Averages were calculated from three separate recorded values for each individual mouse. Standard deviation is defined as SD. Note, animal variance has no significant effect on the measurement of diaphragm contractions (P = 0.1224).
The current experimental protocol develops diagnostic ultrasound imaging techniques specific to the diaphragm activity in a mouse model via a non-invasive, in vivo approach. The anesthesia apparatus settings are approximated values, which may be slightly adjusted for each animal since individual mice may respond differently to anesthesia. To prevent improper anesthesia administration, it is important to regularly monitor the mouse's vital signs including the heart rate, respiration rate, and body temperature. Furthermore, hair must be removed prior to ultrasound scanning because unshaved hair may blur the image and prevent accurate visualization of the diaphragm contractions.
We used B-mode ultrasound imaging to provide a two-dimensional (2D) cross sectional image of the mouse diaphragm and used M-mode to monitor diaphragm motion kinetics. It is important to use a micro-convex array transducer for 2D images because it provides improved axial resolution of superficially located anatomical structures compared to traditional linear array transducers10. It is also important to note that electronic focusing of the image degrades as the focal length is extended9. In addition, an adequate amount of ultrasound gel should be applied to the mouse's abdomen for acquiring distinct images. The gel limits the possibility of air pockets between the skin and the transducer to produce a high resolution image11.
Although the application of diagnostic ultrasound imaging is promising, there are limitations to using this technique as a research tool. For instance, it is critical that the end user is well-trained in obtaining accurate and reproducible images and is able to interpret diaphragm activity consistently. Furthermore, mirror image artifacts occur when one anatomical structure, such as the diaphragm, is displayed twice on the monitor. This is considered a propagation artifact and results in the reflection being improperly located within the ultrasound operating system12,13. For example, ultrasound transducers can produce several off-axis beams which can reflect off an anatomical structure that is not in the pathway of the main beam12,14. Moreover, due to refraction, the beam may not always travel in a straight line from the reflector. Since the ultrasound can only process the signal that is returned to the transducer and cannot determine the timing of the refraction, the monitor may likely display the same anatomical structure twice at different distances and thus exhibit a mirror image artifact.
An additional propagation artifact that is encountered is reverberation. This artifact displays evenly spaced parallel lines that are generally positioned perpendicularly to the ultrasound beam (Figure 2A)15. This type of artifact may disrupt a true field of vision and mask specific anatomical structures of interest. Thus, caution should be taken when analyzing the data. A subset of reverberation artifact is the comet-tail or B-lines. These are a type of reverberation artifact that forms a vertical trajectory of dense echoes, extending from the diaphragm to the edge of the ultrasound screen16 (as illustrated in Figure 2B). These artifacts are produced by multiple reflections that are occurring between or within a structure and the transducer13, which can be minimized by angling the transducer to avoid a perpendicular contact with the specular objects17. Despite these limitations, diagnostic ultrasound imaging enables a safe, sensitive, and rapid analysis of diaphragm function in a mouse model, which can potentially evaluate rodent diaphragm dysfunction and help develop novel pre-clinical therapies for respiratory disorders.
The authors have nothing to disclose.
This work is supported by grants of OU General Fund G110 and Research Excellence Fund of Biomedical Research and OSU-HRS Fund 013000. The authors would like to thank Lauren Chen for her assistance in preparing this manuscript.
Veterinary digital ultrasonic diagnostic imaging system | Edan | DUS 3 VET | Ultrasound parameters include: frequency of 6.5 MHz, Depth of 29 mm. Note: An equivalent ultrasound machine may be used for this protocol |
Micro-convex array transducer | Edan | C611 | Or equivalent |
GE Logiq i hand-carried unit (HCU) | GE Healthcare | GE Logiq i hand-carried unit (HCU) | Or equivalent |
GE 12 MHz linear array probe | GE Healthcare | 12L-RS | Or equivalent |
Veterinary anesthetic vaporizer | Webster Veterinary | Serial #: W422021 | Isoflurane was exclusively used with this vaporizer (or equivalent). A custom made induction chamber for anesthesia was assembled for initial anesthesia. Maintenance anesthesia was performed using a nose cone |
Isothesia (Isoflurane, USP) | Butler Schein | 29405 250ML PVL | Or equivalent |
Enviro-pure anesthesia absorbing canister | Surgivet Smiths Medical PM, Inc. | Part #: 32373B10 | Or equivalent |
Ultrasound transmission gel | HM Sonic | N/A | Or equivalent |
Puralube vet ointment | Puralube | NDC 17033-211-38 | Or equivalent |
Deltaphase isothermal pad | Braintree Scientific Inc. | 39DP | Or equivalent |
Hair remover | Nair | N/A | Or equivalent |
Electric razor | Remington | HC-5015 | Or equivalent |
Surgical tape | 3M Micropore | 1530-1 | Or equivalent |
Gauze sponges | Dynarex | 3262 | Or equivalent |