A new approach to assess the gastrocnemius muscle volume in rodents using ultrasound; comparison with the gastrocnemius muscle index

Abstract

Introduction: The purpose of this study was to determine the reliability and validity of a new non-invasive ultrasound technique to measure gastrocnemius muscle atrophy after nerve denervation in an animal model.

Methods: In sixteen rodents an eight mm sciatic nerve gap was created. In the following 8 weeks, each week, two rodents were euthanized and the gastrocnemius muscle was examined using two different ultrasound systems and two investigators. The standardized ultrasound measurement protocol consisted of identifying pre-defined anatomical landmarks: 1) the fibula, 2) the fibular nerve, and 3) the junction between the most distal point of the semitendinosus muscle and gastrocnemius muscle. Consequently, we measured the muscle thickness as the length of the line between the fibula and the junction between the two muscles, perpendicular to the fibular nerve. After the ultrasound recording, the muscle mass was determined.

Results: A steep decline of muscle weight of 24% was observed after one week. In the following weeks, the weight further decreased and then remained stable from 6 weeks onwards, resulting in a maximal muscle weight decrease of 82%. The correlation coefficient was >0.96 between muscle diameter and weight using both ultrasound systems. The inter-rater reliability was excellent for both devices on the operated side (ICC of 0.99 for both ultrasound systems) and good for the non-operated site (ICC's: 0.84 & 0.89). The difference between the muscle mass ratio and the muscle thickness ratio was not more than 5% with two outliers of approximately 13%.

Discussion: We have developed an innovative, highly reliable technique for quantifying muscle atrophy after nerve injury. This technique allows serial measurements in the same animal over time. This is a significant advantage compared to the conventional technique for quantifying muscle atrophy, which requires sacrificing the animal.

Figure 1. Experimental set-up.

The experimental set-up of the ultrasound recording using the Philips L15-7io probe. (A) Position of the ultrasound probe in relation to the rodent, (B) illustration of the position of the tibial bone relative to the probe, (C) angulation of the probe (20 degrees in both the Z-Y axis) and (D) the position of the probe from above.

Figure 2. Anatomy.

An overview of the anatomy of the rat hind limb. (A) Depicts the anatomy of the muscles, (B) shows the skeletal system and (C) illustrates the major nerves.

Figure 3. Ultrasound recordings.

Serial ultrasound recordings with both the Philips iU22 and the Titan SonoSite of the gastrocnemius muscle after denervation. The thick interrupted line depicts the gastrocnemius muscle. The thin interrupted line depicts the peroneal nerve in the image of the healthy muscle recorded with the Philips iU22. The thick interrupted line illustrates the circumference of the gastrocnemius muscle. The non-interrupted line indicates the standardized muscle diameter measurement.

Figure 4. Decrease of muscle mass after denervation.

The weight-based GMI and ultrasound-based muscle diameter in the operated leg expressed as a percentage of the non-operated side. To show the pattern without averaging, only the weekly data of the first animal are displayed.

Figure 5. A scatter plot of the gastrocnemius muscle and the measured muscle diameter with ultrasound.

Scatter plot of the gastrocnemius mass of the affected limb and the diameter of the muscle measured with ultrasound. Correlation coefficients ranged between 0,957 and 0.971 (p<0.001 for all) for both raters and both ultrasound machines.

Figure 6. The difference between muscle diameter and the gastrocnemius muscle weight.

A Bland Altman plot to visualize the difference between the muscle diameter measured and the gastrocnemius muscle weight (GMI) in percentage of atrophy. The GMI is plotted on the x-axis and the difference between the GMI and the muscle thickness ratio on the y-axis.