Total sleep deprivation inhibits the neuronal nitric oxide synthase and cytochrome oxidase reactivities in the nodose ganglion of adult rats

Abstract

Sleep disorders are a form of stress associated with increased sympathetic activity, and they are a risk factor for the occurrence of cardiovascular disease. Given that nitric oxide (NO) may play an inhibitory role in the regulation of sympathetic tone, this study set out to determine the NO synthase (NOS) reactivity in the primary cardiovascular afferent neurons (i.e. nodose neurons) following total sleep deprivation (TSD). TSD was performed by the disc-on-water method. Following 5 days of TSD, all experimental animals were investigated for quantitative nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d, a co-factor of NOS) histochemistry, neuronal NOS immunohistochemistry and neuronal NOS activity assay. In order to evaluate the endogenous metabolic activity of nodose neurons, cytochrome oxidase (COX) reactivity was further tested. All the above-mentioned reactivities were objectively assessed by computerized image analysis. The clinical significance of the reported changes was demonstrated by alterations of mean arterial blood pressure (MAP). The results indicated that in normal untreated rats, numerous NADPH-d/NOS- and COX-reactive neurons were found in the nodose ganglion (NG). Following TSD, however, both the labelling and staining intensity of NADPH-d/NOS as well as COX reactivity were drastically reduced in the NG compared with normal untreated ganglions. MAP was significantly higher in TSD rats (136+/-4 mmHg) than in normal untreated rats (123+/-2 mmHg). NO may serve as an important sympathoinhibition messenger released by the NG neurons, and decrease of NOS immunoexpression following TSD may account for the decrease in NOS content. In association with the reduction of NOS activity, a defect in NOS expression in the primary cardiovascular afferent neurons would enhance clinical hypertension, which might serve as a potential risk factor in the development of TSD-relevant cardiovascular disturbances.

Fig. 1

Histograms showing the mean arterial pressure (MAP) of untreated, controls for total sleep-deprived (TSC) and total sleep-deprived (TSD) rats. Note that the MAP of untreated and TSC rats is estimated to be 123 ± 2 and 125 ± 3 mmHg, respectively. However, in rats subjected to TSD, MAP is significantly increased to 136 ± 4 mmHg.

Fig. 2

Light photomicrographs showing NADPH-d histochemistry in the nodose ganglion (NG) of untreated (A,C) and total sleep deprived (TSD) rats (B,D). Numerous NADPH-d(+) neurons with various staining intensities are scattered throughout the NG of untreated rats (A). However, following TSD (B), NADPH-d reactivity was drastically reduced in the NG in terms of labelling frequency and staining intensity. (C) Higher magnification showed that the NADPH-d reaction product was mainly deposited in the cytoplasm of NG neurons. (D) Following TSD, the reduction of NADPH-d reactivity was easily identified by the diminished labelling of the histochemical reaction. Scale bars = 100 µm (A,B); 50 µm (C,D).

Fig. 3

Light (A,B,D) and fluorescence (C) photomicrographs showing neuronal NOS immunohistochemistry (A,B), neuronal NOS immunofluorescence (C) and NADPH-d histochemistry (D) in the nodose ganglion (NG) of untreated (A,C,D) and total sleep deprived (TSD) rats (B). Numerous NOS(+) neurons were scattered throughout the NG in the untreated rats (A). However, following TSD (B), NOS reactivity was drastically reduced in the NG with regard to both labelling frequency and staining intensity. Most NOS immunofluorescent neurons were co-localized with NADPH-d-reactive cells (arrows in C and D). Scale bars = 75 µm (A,B); 100 µm (C,D).

Fig. 4

Histograms showing the percentage (A) and true optical density (true OD) (B) of NADPH-d-positive [NADPH-d(+)] neurons in the nodose ganglion (NG) of untreated, control for total sleep-deprived (TSC) and total sleep-deprived (TSD) rats. Note that in A, about 43 and 41% of the nodose neurons were positively stained for NADPH-d histochemistry in the untreated and TSC groups, respectively. Also note that no significant difference (n.d.) was observed between the untreated and TSC groups. However, following 5 days of TSD, the proportion of NADPH-d(+) neurons was markedly decreased to nearly 20% as compared with that of untreated rats. In B, the true OD of NADPH-d(+) neurons in the NG was not significantly different between the untreated and TSC groups. After 5 days of TSD, the staining intensity of NADPH-d(+) neurons was markedly decreased to 0.51 ± 0.11 as compared with that of untreated values (1.66 ± 0.17).

Fig. 5

Histograms showing neuronal NOS activity in the nodose ganglion (NG) of untreated, control for total sleep-deprived (TSC) and total sleep-deprived (TSD) rats. Note that in the NG of untreated and TSC rats, no obvious change in NOS activity was detected. However, following 5 days of TSD, neuronal NOS activity in the NG was noticeably down-regulated to nearly half the value of the normal untreated rats.

Fig. 6

Light photomicrographs (A,B) and histograms (C) showing cytochrome oxidase (COX) reactivity in the nodose ganglion (NG) of untreated (A), control for total sleep-deprived (TSC) and total sleep-deprived (TSD) (B) rats. Note that in the untreated rats, numerous COX-reactive neurons, with moderate to dark staining, were identified in the NG (A). The true optical density (true OD) of COX-reactive neurons was 1.59 ± 0.22 in the NG of untreated rats (C). However, following TSD, both the labelling intensity and the true OD of COX-reactive neurons were drastically decreased in the NG. In rats subjected to TSD, almost all neurons in the NG were lightly stained for COX histochemistry (B). The true OD of COX-reactive neurons was significantly reduced to nearly 0.39 ± 0.08 after 5 days of TSD (C).