The experimental study on multimodal ultrasound evaluation of changes in the rabbit gastrocnemius muscle after acute cold exposure and peritoneal lavage treatment

To quantitatively evaluate changes in the gastrocnemius muscle of rabbits following acute cold exposure and subsequent peritoneal lavage rewarming therapy using multimodal ultrasound. Thirty healthy adult male New Zealand white rabbits, each weighing approximately (4.0 ± 0.2) kg, were selected and randomly divided into three groups( n = 10 per group): the Peritoneal Lavage + Rewarming Platform group, the Rewarming Platform group, and Blank Control group. Models for cold exposure and rewarming therapy were established across four distinct time points: T1 (normothermia), T2 (hypothermia), T3 (rewarming stage 1), and T4 (rewarming stage 2). B-mode ultrasound, shear wave elastography (SWE), and contrast-enhanced ultrasound (CEUS) were employed to measure the following parameters in the rabbit gastrocnemius muscle at each time point and in each group: muscle belly thickness, tendon thickness, pennation angle, mean Young’s modulus (Emean), maximum Young’s modulus (Emax), and CEUS parameters. This was performed to evaluate changes in peripheral microcirculation following acute cold exposure and peritoneal lavage rewarming therapy. Parameters were compared within each group across different time points and among the three groups at the same time point. For pathological examination, three rabbits from each group were selected for tissue sampling at the T4 time point. Additionally, a separate cohort of 15 rabbits underwent tissue sampling as follows: 3 rabbits at T1, 3 rabbits at T2, and 3 rabbits from each group at T3. The collected gastrocnemius muscle samples were processed for transmission electron microscopy (TEM) and immunohistochemical (IHC) staining to observe morphological and cellular changes. (1) Body Temperature Comparison: Body temperature in all three groups decreased significantly at T2 compared to T1 ( P < 0.05). By T4, body temperature had returned to normal levels in all groups, with no significant difference compared to T1 ( P > 0.05). The Peritoneal Lavage + Rewarming Platform Group showed a significantly faster body temperature recovery rate during the T2–T3 period compared to the Rewarming Platform Group and the Blank Control Group ( P < 0.05). No significant difference in recovery rate was observed between the Rewarming Platform Group and the Blank Control Group during T2–T3 ( P > 0.05). There was no significant difference in rewarming rate among the three groups during the T3–T4 period ( P > 0.05). (2) Conventional Ultrasound Parameters: At T2, the muscle belly thickness, tendon thickness, and pennation angle of the gastrocnemius muscle showed statistically significant differences compared to T1 and T4 within each group ( P < 0.05). No significant difference was found between T1 and T4 ( P > 0.05). There were no significant differences in these parameters among the three groups at any corresponding time point ( P > 0.05). (3) SWE Parameters: In all three groups, both Emax and Emean at T2 were significantly different compared to T1 and T4 ( P < 0.05). No significant difference was observed between T4 and T1 ( P > 0.05). No significant differences were found among the three groups at any corresponding time point ( P > 0.05). (4) CEUS Parameters: At T2, parameters including Rising Time (RT), Time to Peak (TTP), Descent Slope (DS), Falling Time (FT), and mean Transition Time (mTT) showed statistically significant differences compared to T1 and T3 ( P < 0.05). No significant difference was observed between T3 and T1 ( P > 0.05). There were no significant differences among the three groups at any corresponding time point ( P > 0.05). (5) Transmission Electron Microscopy and Immunohistochemistry: TEM revealed swelling of some organelles in gastrocnemius muscle cells after acute cold exposure, which returned to normal after rewarming therapy. Immunohistochemical results showed no statistically significant differences in the Average Optical Density (AOD) of BNIP-3 in the gastrocnemius muscle across the different time points ( P > 0.05). Under acute cold exposure conditions, the gastrocnemius muscle entered a contracted state, characterized by an increase in Young’s modulus values, a decrease in the pennation angle, and a slowdown in peripheral microcirculation. The rewarming rate achieved with peritoneal lavage therapy was faster than that in the other two groups, and the resulting prognosis showed no significant difference from the control group. Multimodal ultrasound can rapidly and effectively assess morphological changes and alterations in microcirculatory perfusion in the rabbit gastrocnemius muscle following acute cold exposure and rewarming therapy. Specifically, contrast-enhanced ultrasound enables the quantitative evaluation of the improvement in microcirculation and the effectiveness of the rewarming treatment.