Figure 1
Baseline melatonin profiles for rats prior to MCAo. Melatonin profiles were obtained for all animals prior to MCAo to determine baseline timing. Measurements were made for up to six days, and, in all cases, the daily melatonin timing was stable. Therefore, only a single day's tracing of each individual is shown in this composite illustration. Animals exhibit a wide range of timing profiles. There is a broader range of MT-on times compared to MT-off times. The melatonin duration is defined as the time between the MT-on and MT-off. The MT-on is defined as the time when melatonin rises to 20% of the daily maximum (set to 100%). The MT-off is defined as the time when the melatonin level falls to 20% of the daily maximum. The gray shaded area represents time of lights off.
Figure 2
Effect of MCAo on rhythmic secretion of melatonin. Melatonin secretion was measured in animals before and after surgery. Representative animals undergoing MCAo (A) and sham surgery (B) are shown. Tracings represent daily melatonin profiles superimposed upon the time of the day, with the gray shaded area representing lights off. Each day is demonstrated in a different color. D-2 and D-1 are two and one day before surgery. D0 is the day of the surgery, which was performed in the daytime. Days one to four (D1 to D4) are post-operative days. MCAo caused an advance of MT-on that persisted through four days of monitoring (A). MCAo slightly delayed the MT-off. Due to technical reasons, the data for the third postoperative day was lost (D3*). TTC stained brain sections are displayed to demonstrate that MCAo induces cortical and subcortical infarction. Sham surgery (B) did not affect MT-on or MT-off timing and did not cause brain infarction.
Figure 3
Heterogeneous responses of melatonin timing after MCAo. MT-on and MT-off times were obtained for each day of the experiment and are displayed in a composite timeline. Individual rats' melatonin profiles are depicted by unique colors. The left side shows MT-on tracings with black-filled circles, whereas the right side shows MT-off tracings with white-filled circles. As before, the day of MCAo was defined as Day 0. Due to technical difficulty, data was not available for some days for specific animals (dotted lines were used when days were skipped).
Figure 4
Effect of MCAo on the duration of nightly melatonin secretion. Melatonin duration was calculated by computing the time between MT-on and MT-off for each day of the experiment. Since melatonin duration differs among individuals in the cohort, the melatonin duration was therefore normalized to the stable pre-stroke melatonin duration (set to 100%). Melatonin duration plots are displayed for each individual in a different color. As before, the day of MCAo was defined as Day 0. Dotted lines are used to signify days when data were not available for technical reasons.
Figure 5
Effect of MCAo on the shift of nightly melatonin onset. The impact of stroke on the melatonin rhythm for each animal was calculated by averaging the absolute daily melatonin onset shift (relative to pre-stroke baseline); see methods for a discussion of this calculation. The averages of these values determined for all animals with MCAo or sham surgery are displayed in bar graph format. The difference between average melatonin onset shift in the MCAo group was statistically significant relative to the control group (p < 0.02).
Figure 6
Lack of relationship between the phase angle of MT-on and the circadian response to MCAo. As seen in Figure 1, rats demonstrate a wide range of baseline phase angles of MT-on. We explored the relationship between the phase angle (on the day of the stroke; shown on the X-axis) of MT-on, MT-off, and melatonin duration, relative to the baseline values before MCAo. The r2 values are shown; no relationship was found.
Figure 7
Change in melatonin duration is related to the change in MT-on after MCAo. Results from the day of the MCAo were examined here. Data displayed include the change in MT-on, MT-off, and melatonin duration relative to respective pre-MCAo values. (A) There was no relationship between shifts in MT-on and MT-off after stroke. Animals exhibited all four possible combinations of directions of shift. (B) There was no relationship between the shifts in MT-off and melatonin duration. (C) Change in the melatonin duration after stroke was strongly linked to the shift in MT-on. The r2 values are shown.
Figure 8
Day to day changes in MT parameters after stroke. The day-to-day changes in MT-on and melatonin duration were defined as the change in melatonin timing compared to the time on the previous day. As expected, on Day -1, there is no shift since the melatonin rhythm is stable. However, on Day 0 (the day of the stroke) there is a change in the melatonin timing compared to before stroke. Only five animals, represented by different colors, were used for this analysis. Other animals in the study could not be used because of gaps in data. (A) MT-on changes markedly in most animals from day-to-day. In all animals, there is a remarkable oscillation of the daily changes in melatonin timing. In general, the amplitude of the oscillation decreases over time. (B) A similar trend is seen with the daily change in melatonin duration. (C) There is a strong relationship between the daily shift in MT-on and melatonin duration among most animals. The r2 coefficients (for each animal) were: 0.85 (ID2658), 0.56 (ID2833), 0.15 (ID2839), 0.92 (ID2892), and 0.99 (ID2900).
Figure 9
Infarct volume and its relationship to shift in MT timing. Infarct volumes for all animals undergoing MCAo were computed. Each rat's infarct volume was plotted against the Day 0 shift in MT-on, melatonin duration, or MT-off relative to the baseline time before stroke. There was no linear relationship between infarct volume and any of the quantitative measures of circadian changes. The r2 values are shown; no relationship was found.