Cardiovascular function and autonomic regulation in urethane-anesthetized and conscious mice

Abstract

Urethane is widely used for its ability to induce prolonged anesthesia. Variability in previously reported cardiovascular parameters in murine models makes it challenging to definitively evaluate the cardiovascular effects of urethane anesthesia. We aimed to address these challenges, thereby advancing our understanding of urethane's effects on cardiovascular function in mice. In this study, we investigated how urethane anesthesia, with and without supplemental oxygen, impacts heart rate, arterial oxygen saturation ([Formula: see text]), blood pressure, and heart rate variability in mice. First, we conducted a literature review and found that data in mice were both limited and lacking in reproduction. Next, we conducted a series of physiological measurements to address gaps in the literature and subjected C57BL/6J mice to three conditions: 1) conscious, 2) urethane-anesthetized, and 3) urethane-anesthetized with supplemental oxygen. Blood pressure, heart rate, [Formula: see text], and heart rate variability (via time, frequency, and M-curve analyses) were assessed. We observed an increase in heart rate with urethane anesthesia (P = 0.012) compared with the conscious state. Urethane caused a decrease in heart rate variability, which was independent of oxygen supplementation. Urethane anesthesia caused a significant reduction in arterial blood pressure (P < 0.001) with oxygen-supplemented mice remained hypotensive. Urethane decreased [Formula: see text] (P = 0.001), which was restored by oxygen supplementation (P < 0.001). We did not observe sex effects of urethane anesthesia on blood pressure, heart rate, heart rate variability, or [Formula: see text]. Taken together, these results underscore the importance of a cautious approach when using urethane in mice, as urethane significantly impacts arterial blood pressure, heart rate, oxygen saturation, and heart rate variability.NEW & NOTEWORTHY This investigation documents cardiovascular outcomes in mice receiving urethane anesthesia, emphasizing sex as a biological variable, and considering oxygen supplementation during anesthesia. This is the first report of M-curve analysis in rodents as a heart rate-independent variability analysis.

Keywords: blood pressure; heart rate; heart rate variability; mice; urethane.

Figure 1.. Experimental design.

Each mouse underwent a series of randomized measurements of heart rate HR and blood pressure (BP) under three different conditions (Conscious, Urethane, Urethane + Oxygen Supplementation). Blood pressure was measured non-invasively (CODA) and beat-by-beat heart rate, pulse peak intervals, and arterial oxygen saturation were measured by arterial pulse oximetry (MouseOx). Measurements were initially conducted on conscious animals on Day 1 and Day 2, and then repeated on Day 3 with urethane, with and without supplemental oxygen. The sequence of these two latter conditions was assigned randomly. Additionally, measurements were randomized at each time point (CODA vs MouseOx). A darkened nose cone was used at the front of the chamber for the conscious experiments, although not shown in the image. Mice were kept on a heated platform and tail base surface temperature was maintained at 34–35°C. The figure was created using biorender.com.

Figure 2.

Impact of urethane on mean, systolic, and diastolic pressures, compared to conscious measurements (A-C). Data reported as (mean ± SD). A mixed-effect model was used to assess the immediate impact of urethane on blood pressure (Effect of urethane p<0.001 and order of oxygen supplementation p=0.049). Supplemental Table 1.) with Tukey’s adjustment for multiple comparisons. ^{a b} Superscripted letters that differ indicate differences between groups (p<0.05). Urethane decreased mean, systolic, and diastolic arterial pressure (^bp<0.001) and the effect remained after supplemental oxygen was provided(^bp<0.001). Open circles – male mice, closed circles – female mice. Conscious n=40; urethane n=10; urethane O₂=38.

Figure 3.

(A) Arterial oxygen saturation (SpO₂) in conscious mice, with urethane, and with urethane + supplemental oxygen. A mixed-effect model was used to assess the impact of urethane on arterial saturation with Tukey’s adjustment for multiple comparisons (effect of urethane p=0.001; effect of oxygen supplementation p<0.001) ^{a b c} Superscripted letters that differ indicate differences between groups (p < 0.05) Urethane depressed arterial oxygen saturation (^bp<0.001) but was restored by supplemental oxygen (^cp=0.002). Conscious n=31; urethane n=29; urethane O_2; n=31. (B) Changes in SpO₂ were not correlated with changes in heart rate (conscious:urethane n=23, R²=0.06; conscious:urethane O₂ n=23, R²=0.09) (C) or mean arterial pressure (conscious:urethane n=6, R²=0.08; conscious:urethane O₂ n=7, R²=0.03). (A-C) Open circles – male mice, closed circles – female mice.

Figure 4.

(A) Heart rate in three different conditions (conscious breathing room air, urethane–anesthetized, and urethane-anesthetized group with 100% supplemental oxygen). A mixed-effect model was used to assess the immediate impact of urethane on heart rate (effect of urethane p=0.012) with Tukey’s adjustment for multiple comparisons. Conscious vs urethane anesthetized (^ap<0.001) ^{a b} Superscripted letters that differ indicate differences between groups (p < 0.05). Conscious n=31; urethane n=29; urethane O₂=31. (B) A semi-logarithmic M-curve plot from a single representative mouse demonstrates the correlation between the logarithm of root mean square of successive heartbeats (ln (RMSSD)) and mean heart rate (beats/min). (C) Combined correlations of each group. The mixed effect model analysis is outlined in Supplemental Table 3. Tukey’s comparison p<0.001 conscious vs urethane and conscious vs urethane O₂. (A-C) Open circles – male mice, closed circles – female mice.