Behavioral and anatomical correlates of chronic episodic hypoxia during sleep in the rat

Abstract

The role played by chronic episodic hypoxia (EHYP) in the neurocognitive morbidity of obstructive sleep apnea (OSA) is unknown. Sleep recordings, Morris water maze experiments, and immunohistochemistry for NMDA NR1 glutamate receptor, c-fos protein, and apoptosis [nuclear immunoreactivity for single-stranded DNA and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assay] were conducted in EHYP-exposed Sprague Dawley male rats. Exposures consisted of up to14 d in an environmental chamber in which O(2) concentrations were cycled between 10 and 21% every 90 sec or 30 min during 12 hr of daylight. For the remaining 12 hr, EHYP rats breathed room air, while controls spent 14 d in room air. Although EHYP induced significant disruption of sleep architecture during the initial day of exposure, sleep patterns normalized thereafter. Marked increases in apoptosis occurred in the CA1 hippocampal region (sevenfold) and cortex (Cx; eightfold) after 1-2 d of EHYP but not in CA3 and were followed by decreases toward normoxic levels by 14 d. Double labeling for NMDA NR1 and c-fos revealed marked architectural disorganization in CA1 and Cx with increases in c-fos over time. Rats exposed to EHYP displayed significantly longer escape latencies and swim path lengths to escape a hidden platform during 12 training trials given over 2 d. Differences in the performances of EHYP and control rats, although reduced, persisted after 14 d of recovery. We conclude that EHYP is associated with marked cellular changes over time within neural regions associated with cognitive functions. Furthermore, EHYP impaired performance during acquisition of a cognitive spatial task without affecting sensorimotor function. Such changes may underlie components of the learning and memory impairments found in OSA.

Figures

Fig. 1.

Mean duration of REMS and NREMS expressed in minutes per 2 hr blocks and spanning over the circadian cycle in six rats during baseline conditions (■) and during day 1 (●), day 2 (Δ), day 7 (▿), and day 14 (⋄) of EHYP exposure. Significant decreases in REMS and NREMS (indicated as *) occurred during day 1 (p < 0.001, ANOVA vs baseline), whereas significant increases in REMS and NREMS emerged during day 2 (p < 0.002, ANOVA vs day 1;p < 0.03, ANOVA vs baseline). The filled horizontal bar indicates the 12 hr lights-off period. Thevertical dashed line indicates the separation between lights-off and lights-on period.

Fig. 2.

A, Mean escape latencies in a water maze during place-training trials in 18 rats exposed to EHYP for 14 d (○) and 19 control rats (▪) on days 1 and 2 of the trials (*, EHYP vs control, p < 0.01, ANOVA).B, Mean swim path lengths in a water maze during place-training trials in 18 rats exposed to EHYP for 14 d (○) and 19 control rats (▪) on days 1 and 2 of the trials (*, EHYP vs control, p < 0.01, ANOVA). C, Mean escape latencies in a water maze during cued trials (right) and mean target quadrant times during probed trials (left) in 18 rats exposed to EHYP for 14 d (○, right; open column,left) and 19 control rats (▪, right;filled column, left) (EHYP vs control, NS). D, Mean escape latencies (left) and swim path lengths (right) in a water maze during place-training trials in 13 rats exposed to EHYP for 14 d (○) and 13 control rats (▪) tested after 14 d of normoxic recovery on days 1 and 2 of the trials (*, EHYP vs control,p < 0.01, ANOVA).

Fig. 3.

A, B, Photomicrographs of coronal sections through the hippocampal formation illustrating NR1 and c-fos immunoreactivity in room air (A) and after EHYP for 14 d (B). C, D, Higher magnification of photomicrographs of the CA1 hippocampal region (lower boxed area in A, B, respectively) illustrating the dense NR1 labeling in this region and the relative paucity of c-fos-labeled neurons during control conditions (C). In contrast, marked architectural disorganization is apparent in the CA1 region after a 14 d exposure to EHYP, and c-fos labeling is also more prominent and colocalizes with NR1-labeled pyramidal neurons (D). E, F, Higher magnification of photomicrographs of the cortex (upper boxed areain A, B, respectively) illustrating abundant NR1 cellular labeling in this region (E). After 14 d of EHYP, NR1-immunoreactive cells are scarce, and instead small cells (possibly glia) emerge and display enhanced c-fos nuclear expression (F). The scale bar is shown on every image in the right-hand bottom corner.

Fig. 4.

Mean (±SD) number of c-fos-positive cells per 100 cells in the CA1 region (filled columns) and CA3 region (open columns) of the hippocampus and the adjacent cortex (cross-hatched columns). Significant increases in c-fos (shown as *) occurred with EHYP in all regions over time (vs control: CA1, p < 0.0001, ANOVA; CA3, p < 0.03, ANOVA; Cx,p < 0.0001, ANOVA) and were more prominent in CA1 and Cx (p < 0.001 vs CA3) (n = 8 rats for each time point; normoxia indicates control rats).

Fig. 5.

Photomicrographs of coronal cortical sections illustrating GFAP immunoreactivity in two control rats (A, C), and in two rats after exposure to EHYP for 14 d (B, D).

Fig. 6.

a–c, Photomicrographs of coronal sections through the hippocampal formation illustrate SS-DNA immunoreactivity in room air (a) and after EHYP for 2 d (b) and 7 d (c). d–f, Higher magnification of photomicrographs of the CA1 hippocampal region illustrates the scarce SS-DNA labeling in this region during normoxic conditions (d) and the marked enhancements in SS-DNA-positive cells at 2 d EHYP (e), followed by some reduction at 7 d EHYP (f).g–i, Similarly, neocortical regions exhibited only occasional SS-DNA nuclear staining in control (g) but marked enhancements after EHYP at 2 d (h) and reductions in SS-DNA labeling at 7 d (i). The scale bar is shown on every image in theleft-hand bottom corner.

Fig. 7.

Mean (±SD) number of SS-DNA-positive cells per 1000 cells in the CA1 region (filled columns) and CA3 region (open columns) of the hippocampus and the adjacent cortex (hatched columns). Significant increases in SS-DNA labeling occurred with EHYP in CA1 (p < 0.001 vs time 0) and Cx (p < 0.001 vs time 0) but not in CA3 (NS vs time 0). A biphasic pattern emerged, by which SS-DNA labeling peaked at 24–48 hr and returned to baseline levels after 14 d of EHYP (n = 4 rats for each time point; *p < 0.05 vs time 0).

Fig. 8.

Photomicrographs of coronal sections of neocortical regions illustrating TUNEL labeling counterstained with methyl green, in a rat exposed to room air (A) and after EHYP for 2 d (B). Similarly, the CA1 region of the hippocampal formation exhibited only occasional labeling in a control animal (C) but marked enhancements after 2 d of EHYP (D). The scale bar is shown on every image in the left-hand bottom corner. Examples of positive TUNEL-labeled cells are indicated by the arrows.

Fig. 9.

Mean (±SD) number of TUNEL-positive cells per 1000 cells in the CA1 region (filled columns) and CA3 region (open columns) of the hippocampus and the adjacent cortex (hatched columns). Significant increases in TUNEL labeling occurred with EHYP in CA1 (p < 0.001 vs time 0) and Cx (p < 0.001 vs time 0), and smaller increases occurred at 2 d of EHYP in CA3 (p < 0.05 vs time 0). A biphasic pattern emerged, such that TUNEL labeling peaked at 48 hr and decreased thereafter, but without returning to baseline values at 14 d (n = 4 rats for each time point; *p < 0.05 vs time 0).