Impaired sodium levels in the suprachiasmatic nucleus are associated with the formation of cardiovascular deficiency in sleep-deprived rats

Abstract

Biological rhythms are a ubiquitous feature of all higher organisms. The rhythmic center of mammals is located in the suprachiasmatic nucleus (SCN), which projects to a number of brainstem centers to exert diurnal control over many physiological processes, including cardiovascular regulation. Total sleep deprivation (TSD) is a harmful condition known to impair cardiovascular activity, but the molecular mechanisms are unknown. As the inward sodium current has long been suggested as playing an important role in driving the spontaneous firing of the SCN, the present study aimed to determine if changes in sodium expression, together with its molecular machinery (Na-K ATPase) and rhythmic activity within the SCN, would occur during TSD. Adult rats subjected to different periods of TSD were processed for time-of-flight secondary ion mass spectrometry, Na-K ATPase assay, and cytochrome oxidase (COX) (an endogenous bioenergetic marker for neuronal activity) histochemistry. Cardiovascular dysfunction was determined through analysis of heart rate and changes in mean arterial pressure. Results indicated that, in normal rats, strong sodium signals were expressed throughout the entire SCN. Enzymatic data corresponded well with spectrometric findings in which high levels of Na-K ATPase and COX were observed in this nucleus. However, following TSD, all parameters including sodium imaging, sodium intensity as well as COX activities were drastically decreased. Na-K ATPase showed an increase in responsiveness following TSD. Both heart rate and mean arterial pressure measurements indicated an exaggerated pressor effect following TSD treatment. As proper sodium levels are essential for SCN activation, reduced SCN sodium levels may interrupt the oscillatory control, which could serve as the underlying mechanism for the initiation or development of TSD-related cardiovascular deficiency.

Fig. 1

Time-of-flight secondary ion mass spectrometry positive ion spectrum (A,B) and histogram (C) showing quantitative intensity (total ion counts per 500 μm²) and normalized spectral intensity of Na⁺ (expressed as percentage above the baseline) in the suprachiasmatic nucleus of normal untreated, control for total sleep-deprived (TSC) and total sleep-deprived (TSD) rats. In normal untreated rats (A), the quantitative intensity of Na⁺ was 9.88 × 10⁴ (total ion counts per 500 μm²). However, following 5 days of TSD (B), the Na⁺ intensity was much lower (4.35 × 10⁴; total ion counts per 500 μm²). Similar findings were observed in the normalized spectral intensity (C), in which data obtained from untreated rats (7.011 ± 0.18% for the day group and 4.003 ± 0.16% for the night group) were significantly higher than from TSD rats (2.734 ± 0.15% for the day group and 2.112 ± 0.22% for the night group following 5 days of TSD). It is noteworthy that, no matter at which time-point the sample was performed (day or night group), the normalized spectral intensity showed a decrease in the expression after TSD treatment. *P<0.05 compared with the day group of normal untreated rats; ††P<0.05 compared with the night group of normal untreated rats.

Fig. 2

Time-of-flight secondary ion mass spectrometry positive ion image showing Na⁺ expression in the suprachiasmatic nucleus (SCN) of normal untreated (A) and total sleep-deprived rats (TSD) with different duration and sample time-points (B–F). The molecular imaging of Na⁺ signals is expressed by a color scale in which bright colors represent high levels of Na⁺. In normal untreated rats (A), many neurons in the SCN exhibited strong Na⁺ signals. However, following various periods of TSD (B–F), the ionic image of Na⁺ was decreased in intensity, with the maximal change observed in animals subjected to 5 days of TSD (D,F). It is noteworthy that the reduction of the Na⁺ signal in the SCN following TSD was similar in both the day (D) and night (N) sample groups (C vs. E and D vs. F). V, third ventricle. Scale bar = 150 μm.

Fig. 3

Lower (A) and higher (B–D) magnification of light photomicrographs showing cytochrome oxidase (COX) reactivity in the suprachiasmatic nucleus (SCN) of normal untreated (A,B) and total sleep-deprived (C,D) rats. In normal untreated rats (A,B), numerous moderate to strong COX-reactive neurons were identified in the SCN. However, following 5 days of total sleep deprivation (TSD) (C,D), only a few neurons with weak COX staining intensity were detected (C,D). Also note that the reduction of COX expression was evident in both groups at different sample time-points (C,D). D, day; N, night; V, third ventricle; op, optic chiasma; Scale bar = 400 μm in (A) and 200 μm in (B–D).

Fig. 4

Histogram showing quantitative cytochrome oxidase (COX) staining intensity [expressed as true optical density (OD)] in the suprachiasmatic nucleus (SCN) of normal untreated control for total sleep-deprived (TSC) and total sleep-deprived rats (TSD) with different sample time-points. In normal untreated and TSC rats, numerous COX-reactive neurons with high true OD were detected in the SCN. In contrast, the staining intensity of COX in the SCN decreased progressively over the duration of TSD. Decreasing COX reactivity was apparent in both the day and night sample groups. *P<0.05 as compared with the day group of normal untreated rats; ††P<0.05 as compared with the night group of normal untreated rats.

Fig. 5

Histogram showing the Na-K ATPase activity in the suprachiasmatic nucleus (SCN) of normal untreated control for total sleep-deprived (TSC) and total sleep-deprived (TSD) rats with different sample time-points. Note that the Na-K ATPase activity of the SCN had significantly increased with extension of TSD. Also note that, even among samples at different time-points (day or night group), up-regulation of Na-K ATPase activity was clearly observable in both groups following TSD treatment. *P<0.05 compared with the day group of normal untreated rats; ††P<0.05 compared with the night group of normal untreated rats.

Fig. 6

Histograms showing the mean arterial pressure (MAP) (A) and heart rate (HR) (B) of normal untreated, control for total sleep-deprived (TSC) and total sleep-deprived (TSD) rats. Data are expressed as the averaged value detected from both the day and night groups. In normal untreated rats, MAP and HR were 120 ± 4.7 mmHg and 402 ± 6 beats min^–1. In the TSC group, MAP and HR were 118 ± 3.9 mmHg and 407 ± 2 beats min^–1. In rats treated with TSD for 5 days, however, both MAP and HR were considerably elevated to 157 ± 7.8 mmHg and 463 ± 3 beats min^–1, respectively, indicating a deficiency in cardiovascular regulation. *P<0.05 as compared with normal untreated values.