Establishing standardized transthoracic echocardiography reference ranges for mouse models: insights into the impact of anesthesia, sex, and age

Figures

• 

  Figure 1. COV analysis of data, stratified by sex (female and male), anesthetic regime, and age (EA and LA), identified one parameter, respiration rate, with excess variability (COV > 30%) in LA males and females under anesthesia that was excluded from further analysis (white bar). Parameters in blue were below the COV threshold of 30% and were retained for further analysis.

• 

  Figure 2. Histograms presenting the distribution of each selected TTE parameter for male (blue) and female (red) mice separately. On visual inspection, no sexual dimorphism is apparent. (a) Recorded in the conscious state in EA [subpanels (A–I)], and LA mice [subpanels (J–R)]. (b) Recorded under isoflurane anesthesia in EA [subpanels (A–I)], and LA mice [subpanels (J–R)]. (c) Recorded under tribromoethanol anesthesia in EA [subpanels (A–E)], with five (FS, HR, LVIDd, LVIDs, and LVPWd) out of nine TTE parameters. No late adult data are available for tribromoethanol anesthesia.

• 

  Figure 3. Comparison of the anesthetic regimes with the conscious state recordings. Distribution of the nine selected TTE parameters presented by histograms, stratified for female and male mice in EA [(A–I)], and LA populations [(J–R)]. No late adult data are available, and only FS, HR, LVIDd, LVIDs, and LVPWd parameters in EA are available for tribromoethanol anesthesia. Color code: conscious, isoflurane, and tribromoethanol anesthesia.

• 

  Figure 4. Reference ranges split by an anesthetic regimen showing median and 95% reference ranges (2.5th percentile and 97.5th percentile). Female data are directly above male data for EA [(A–I)] and LA populations [(J–R)]. No late adult data are available, and only FS, HR, LVIDd, LVIDs, and LVPWd parameters in EA are available for tribromoethanol anesthesia. Color code: conscious, isoflurane, and tribromoethanol anesthesia.

• 

  Figure 5. Violin plot representing full distribution of the numeric data, including the density of each TTE variable. Female data are directly above the male data for LA and EA populations for easy comparison. (a) Recorded in the conscious state in EA and LA. (b) Recorded under isoflurane anesthesia. No late adult data are available for tribromoethanol anesthesia.

• 

  Figure 6. A two-tailed t-test is applied to test the difference between the means of EA and LA BW, stratified for female and male mice in (EA and LA populations split by an anesthetic regimen. (a) Recorded in the conscious state in EA and LA. (b) Recorded under isoflurane anesthesia. No BW data are available for tribromoethanol anesthesia. p-values < 0.001 (***) are reached for all comparisons, indicating high statistical significance between age groups.

• 

  Figure 7. Pearson correlation with a linear regression line presenting negligible to weak correlations for all nine TTE parameters with BW (g), stratified for female and male mice in EA and LA populations split by an anesthetic regimen. (a) Recorded in the conscious state in EA and LA. (b) Recorded under isoflurane anesthesia. No late adult data are available for tribromoethanol anesthesia. LVIDd and LVIDs, left ventricular internal diameter in diastole and systole; LVPWd and LVPWs, posterior wall thicknesses in diastole and systole.

• 

  Figure 8. Independent, non-IMPC study on six of the founder strain mice reported in “the Collaborative Cross: A Recombinant Inbred Mouse Population for the Systems Genetic Era” (20) study, including 129S1/SvlmJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/HlLtJ, and PWK/PhJ inbred strains show a close alignment with the reference ranges reported herein for CO, EF, LVIDd, LVIDs, FS, HR, LVPWd, LVPWs, and SV based on multiple C57BL/6N substrains indicating good utility for those reference ranges. Mice were conscious, split by sex, and ∼12 weeks of age, equivalent to the IMPC EA time point. The red dotted lines depict the boundaries of the sex-specific reference ranges calculated from the C57BL/6N IMPC animals described earlier, for each parameter. LVIDd and LVIDs, left ventricular internal diameter in diastole and systole; LVPWd and LVPWs, posterior wall thicknesses in diastole and systole.

• 

  Figure 9. Independent, non-IMPC reference ranges of the ESC multicenter study including a total of 300 TTE examinations, stratified by HR <450 or >450 bpm, performed on control/healthy C57BL/6 adult mice across 10 different laboratories reported in “Towards standardization of echocardiography for the evaluation of left ventricular function in adult rodents: a position paper of the ESC Working Group on Myocardial Function” (9). IMPC mice were under isoflurane anesthesia, whereas ESC mice were under isoflurane or sevoflurane anesthesia. ESC mice were pooled for sex and ∼12 weeks of age, equivalent to the IMPC EA time point. In both sets, <450 or >450 bpm, CO, HR, LVPWd, LVPWs, and SV show a close alignment with the reference ranges generated herein. There are minor shifts that fall beyond the reference ranges generated herein for EF and FS and concurrently LVIDd and LVIDs. Color code: Reference ranges generated herein, and reference ranges of the ESC study, pooled for sex and stratified by <450 (upper panel) or >450 bpm (lower panel). LVIDd and LVIDs, left ventricular internal diameter in diastole and systole; LVPWd and LVPWs, posterior wall thicknesses in diastole and systole.