## 2 Materials and Methods
### Measurement of Blood Pressure and Area under the Curve (AUC) Analysis
This method does not allow for continuous monitoring of arterial pressure as compared to the direct indwelling catheter method but has the main advantages of not requiring invasive surgery (especially difficult for small, young mice) and allowing for repeated measurements over a long time period. The CODA system directly measures systolic and diastolic blood pressures and calculates mean arterial pressure for individual readings. An illustration of the experimental setup is shown in Figure 1. Mice were placed into a polycarbonate cylindrical holding tube consisting of holes for air, a cone where the nose was placed, and a rear gate. The tail extended outside of the rear gate of the tube to allow the occlusion cuff and volume pressure recording (VPR) cuff to be placed around the tail base. The mice were secured into the tube at both ends to minimize movement and stress during the collection period. The holding tube was placed over a heating pad to maintain the mouse's core body temperature and increase blood flow to the caudal artery [25]. Tail base temperature was maintained in the range of 32-35 \({}^{\circ}\)C and monitored frequently using an infrared thermometer (General Tools & Instruments LLC, Secaucus, NJ, USA) aimed at the tail base. Measurement collection began when the mouse's tail base temperature reached at least 32 \({}^{\circ}\)C, indicating that the caudal artery was dilated and well perfused with blood. The mice were allowed 5 min to acclimate after placement in the holding tube before caudal artery blood pressure measurement collection began. After the 5 min rest period and attainment of an adequate tail base temperature, 20 cycles of blood pressure measurements were recorded (5 initial acclimation measurements and 15 experimental measurements). When the experiment was ended, mice were removed from the holder and immediately returned to their home cage. The data were exported from CODA to an Excel file for processing. A minimum of 15 valid cycles were deemed acceptable, and these cycles were used to calculate the average of replicate measurements for each blood pressure parameter (systolic, diastolic, and mean) for each mouse. For each diet group, a grand average and SEM (\(n=8\)) was then calculated using the individual mouse average values. Figure 1: Blood pressure measurement using the CODA Noninvasive Blood Pressure System (Kent Scientific Corporation). A mouse was placed into a cylinder fitted with a rear gate and front gate to minimize movement, placed on a heating pad. An occlusion cuff and volume pressure recording (VRP) cuff were secured around the tail base and lines were connected to the CODA Monitor. Area under the curve (AUC) analysis is useful in capturing the overall level of blood pressure across time and has been used previously to summarize interval measurements of blood pressure in longitudinal studies in humans [27] and acute blood pressure measurements in mice over several minutes of continuous recording using implanted blood pressure radio transmitters [28]. Here, we used AUC analysis to summarize overall levels of systolic, diastolic, and mean arterial blood pressure in single mice using the data obtained from biweekly measurements between the ages of 5 and 29 weeks. Specific intervals of interest for AUC analysis included (1) age of 5-15 weeks (AUC1), (2) age of 15-25 weeks (AUC2), and (3) age of 5-29 weeks for the entire study duration (AUC3). Additionally, the difference in AUC value was calculated for AUC2 (15-25 weeks) and AUC1 (5-15 weeks) to compare overall blood pressure between \"adulthood\" versus \"adolescence\", respectively. AUC values were calculated using IgorPro, version 9.0 (WaveMetrics). Figure 2 illustrates the basic steps involved in processing data for the age intervals of interest. The basic steps are as follows: STEP 1 (Figure 2A): Copy data for a single mouse into IgorPro and make an XY-waveform pair. In this step, the raw data are interpolated, i.e., additional points are inserted between raw data points.