Appearance and demographic data for the animals in the study are presented in . At baseline, the appearance was identical in the three groups while the heart rate, LVEF, and mean SI were similar amongst the groups. Also shown are the number of animals scanned, sacrificed, and experiencing an unexpected death at each time point in the study. Animals with inadequate cardiac gating (and consequent imaging artifacts) at the time of CMR were not included in the analysis. By week 2, animals receiving low or high doses of DOX showed a significantly worse appearance relative to the animals in the NS group (p<0.0034 from the Kruskal Wallis test for three group comparison, p=0.0023 for the 1.5mg/kg/wk, and p=0.028 for the 2.5 mg/kg/wk DOX, respectively)). The worsening appearance scores persisted at 4 weeks and beyond (p<0.001 at week 4 (for overall and both 2-way comparisons), p<0.002 at week 7 (for overall and both 2-way comparisons), and p<0.021 at week 10 (for overall and both 2-way comparisons).
Two weeks after receipt of DOX, the LVEF remained relatively unchanged (). At 7 weeks however, the LVEF averaged 78%±2%, 72%±3%, and 55%±6%, in the animals receiving NS, 1.5 mg/kg DOX, and 2.5 mg/kg DOX, respectively (). As shown in , of the animals receiving 1.5 mg/kg of DOX, 6 of 19 experienced a primary event (3 developed a LVEF drop <65% and 3 died unexpectedly overnight). Of the 14 animals receiving 2.5 mg/kg of DOX, 10 experienced a primary event: all with a substantial drop in LVEF.
As shown in , the LVEF was similar as baseline and at 2 weeks in animals receiving NS, DOX without a primary event, and DOX with a primary event. Relative to the baseline values, the LVEF remained relatively constant throughout the study in NS animals and those receiving DOX without an event. In the animals receiving DOX with an event, an initial small change (not meeting the criteria for a cardiac event) in LVEF occurred relative to baseline at 2 weeks (74±2% at baseline and 70±1% at 2 weeks, p=0.05).
LVEF and signal intensity in animals with and without events (mean±standard error)
The mean SI averaged 5.3±1.8, 3.0±1.0, and 6.0±1.4 at baseline in animals receiving NS, 1.5 mg/kg DOX, and 2.5 mg/kg DOX, respectively. Throughout the experiment, the average SI did not exceed 1 standard deviation of the baseline value at the 2, 4, 7, or 10-week interval in the animals receiving NS (). The average SI in the animals receiving 1.5 mg/kg of DOX also did not increase by more than 1 standard deviation in value from the baseline measure until the 7th week of the experiment. In the animals receiving 2.5 mg/kg of DOX, the SI increased to 12.8±5.1, at the 2-week sample point and further thereafter to a value of 28.3±8.1. In animals receiving the 2.5 mg/kg of DOX, the SI was significantly higher than the SI observed in the other 2 groups, as well as the SI relative to baseline (p<0.05 for all comparisons).
For those animals that experienced a primary event (), the mean SI at 7 weeks was higher compared to either the animals receiving DOX that did not experience a primary event (p= 0.02), or the animals receiving NS (p=0.03). At the time of the drop in LVEF, the mean Gd-SI was 33.5±5.2 versus 6.6±3.2 in animals that did not experience a primary event (p=0.0001). The difference in SI in those experiencing a primary event versus the combined NS and animals receiving DOX without an event was high (p=0.004). Relative to the NS controls and the animals receiving DOX that did not experience a primary event, the average SI at 2 weeks significantly increased in the animals that subsequently experienced a primary event later in the study (p=0.03). Examples of histograms from animals demonstrating receipt of NS versus those animals receiving DOX with and without a cardiac event are shown in .
Serial histograms and histopathology
To determine if measures of Gd enhanced SI could be used to predict future primary events (a measure of potential clinical utility), measures of Gd-SI from exams prior to events were assessed relative to baseline measures, as well as measures obtained from the preceding intermediate period (). In the animals with an event, the mean signal intensities at the measurement point prior to the event averaged 22.3±3.5 vs. 3.0±2.9 in animals without an event (p<0.0001). In these same animals with and without events, when the difference between the measurement point Gd-SI prior to the event was compared to the measurement at a point in time two intervals before the event, the mean difference in SI was 14.8±5.4 in the animals that experienced an event versus 2.3±4.3 in animals without an event (p=0.098).
The change from baseline in Gd-SI to the measurement point prior to a primary event was 17.0±3.8 versus 0±3.2 in animals without a primary event (p=0.0009). Similarly, the change in SI over time was equally different between the sample point in measurements prior to the primary event versus baseline relative to animals without a primary event (p=0.0007). Results were similar after accounting for the time intervals associated with these changes occurring relative to baseline (p=0.001 for both intervals 6 and 7 in ).
We did not perform a formal test/retest assessment of the SI measurements, however we examined the change from baseline for all NS animals and found that the average difference was 2.58 with a standard deviation of 4.5, indicating that there is some variability in this measure with “healthy” animals over time. When we examined the magnitude of the changes seen in the DOX treated animals, the observed change in SI was several fold larger than the standard deviation of SIs found in NS animals. In fact, the effect is nearly 5 times as large as the standard deviation for the change in SI across all NS measures.
A receiver operating curve was generated to determine the optimal times to assess characteristics in SI at time points prior to an event that would forecast a future event. As shown in , a value in SI >8.2 in a prior sample period was predictive of a future cardiac event. This remained true even if one excluded the 3 unexpected deaths and only assessed LVEF drop. Similarly, an increase in SI of >1.0 between 2 exams prior to an event, or an increase in SI >2.9 from baseline were also predictive of an event.
Prediction of future primary events
To investigate potential etiologies associated with increased SI, histopathologic examination of the heart was accomplished in sections from which the imaging planes were selected to obtain the data for the study. As shown in , there was no significant increase in fibrosis, myocellular death, or apoptosis among the hearts of animals experiencing a drop in LVEF compared to those animals that did not. Importantly however, clear intracytoplasmic myocellular vacuoles and variation in myofiber size and tinctorial quality was present regularly in animals with a drop in LVEF and an increase in CMR SI ( and ). These findings are consistent with vacuolar degeneration resulting from intracellular edema.12
None of the animals without a change in Gd-SI developed myocellular degeneration.