Phagocyte migration and cellular stress induced in liver, lung, and intestine during sleep loss and sleep recovery

Abstract

Sleep is understood to possess recuperative properties and, conversely, sleep loss is associated with disease and shortened life span. Despite these critical attributes, the mechanisms and functions by which sleep and sleep loss impact health still are speculative. One of the most consistent, if largely overlooked, signs of sleep loss in both humans and laboratory rats is a progressive increase in circulating phagocytic cells, mainly neutrophils. The destination, if any, of the increased circulating populations has been unknown and, therefore, its medical significance has been uncertain. The purpose of the present experiment was to determine the content and location of neutrophils in liver and lung tissue of sleep-deprived rats. These are two principal sites affected by neutrophil migration during systemic inflammatory illness. The content of neutrophils in the intestine also was determined. Sleep deprivation in rats was produced for 5 and 10 days by the Bergmann-Rechtschaffen disk method, which has been validated for its high selectivity under freely moving conditions and which was tolerated and accompanied by a deep negative energy balance. Comparison groups included basal conditions and 48 h of sleep recovery after 10 days of sleep loss. Myeloperoxidase (MPO), an enzyme constituent of neutrophils, was extracted from liver, lung, and intestinal tissues, and its activity was determined by spectrophotometry. Leukocytes were located in vasculature and interstitial spaces in the liver and the lung by immunohistochemistry. Heme oxygenase-1, also known as heat shock protein-32 and a marker of cellular stress, and corticosterone also were measured. The results indicate neutrophil migration into extravascular liver and lung tissue concurrent with cell stress and consistent with tissue injury or infection induced by sleep loss. Plasma corticosterone was unchanged. Recovery sleep was marked by increased lung heme oxygenase-1, increased intestinal MPO activity, and abnormally low corticosterone, suggesting ongoing reactive processes as a result of prior sleep deprivation.

Fig. 1.

Myeloperoxidase (MPO) activity during sleep loss and sleep recovery. MPO activities in the lung (left), liver (middle), and intestine (right) from baseline controls (open bars) and from yoked (shaded bars) and sleep-deprived rats (solid bars) during 5 and 10 days of sleep deprivation and during 2 days of recovery sleep subsequent to 10 days of sleep deprivation are shown. Values are means (SE); n = 6 per group at each time point. P < .016 or better for the comparison with *baseline controls (Bsln), or †day 5 or ¶day 10 of the same group. P < .001 for the comparison with **baseline control or ‡day 5 of the same group.

Fig. 2.

Representative sections of lung and liver tissue showing immunohistochemical staining of granulocytes and the effect of sleep deprivation Left: representative section of frozen lung stained with HIS48 antibody (A) illustrative of the distribution of granulocytes (stained brown) among bronchiole, alveoli, and vascular interstitium, compared with negative control staining (a). Right: representative section of liver stained with HIS48 antibody showing positive labeling in vasculature and diffusely throughout the extravascular space (B), compared with negative control staining (b). Magnification ×200.

Fig. 3.

Liver (left) and lung (right) heme oxygenase-1 content during sleep loss and sleep recovery. Data are expressed as the chemiluminescence intensity of the specimen relative to a standard. Data from baseline controls (open bar) included n = 8 for the liver and n = 5 for the lung, the latter of which was combined with data from colony controls (n = 2; stippled bar) to increase the degrees of freedom for statistical tests These data are shown relative to those from yoked (shaded bars) and sleep-deprived animals (solid bars) during 5 and 10 days of sleep deprivation and during 2 days of recovery sleep subsequent to 10 days of sleep deprivation. Values are expressed as means ± SE; n = 5 or 6 per yoked or sleep-deprived group at each time point. P < .016 for the comparison with *baseline+colony, or †day 5 or ¶day 10 of the same group. P < .001 for the comparison with **baseline or baseline+colony.

Fig. 4.

Mean basal plasma corticosteroid concentrations during sleep deprivation and sleep recovery. Results from yoked (shaded bars) or sleep-deprived groups (solid bars) were not statistically different from baseline controls (n = 6, open bar) until the recovery period of 48 h of sleep ad libitum after 10 days of partial or total sleep deprivation. Values are expressed as means ± SE, n = 6 per group except sleep-deprived rats studied on day 10, for which there were 5 specimens. P < .016 for the comparison with †day 5 and ¶day 10 of the same group. P < .001 for the comparison with **baseline control and §day 10 of the same group.