The influence of rapid eye movement sleep deprivation on nociceptive transmission and the duration of facial allodynia in rats: a behavioral and Fos immunohistochemical study

Experiments were conducted following an ethical review by the Institutional Animal Care and Use Committee of The Catholic University of Korea (approval no. UJA 2014-08A, UJA 2014-12A). Male Sprague-Dawley rats (190–220 g) were placed in standardized plastic cages with sawdust bedding in a temperature-controlled room (22 °C ± 2 °C) with a 12-h light–dark cycle. All procedures took place in a quiet room during the light phase, between 10:00 and 13:00.

We used the single-platform technique to induce REM sleep deprivation. The animals were individually maintained for 96 h in water chambers measuring 22.0 cm long, 22.0 cm wide, and 35.0 cm high. Within the chambers, the rats were placed onto a 6.5-cm-diameter platform atop a 15-cm-high pedestal that was immersed in water up to 1.0 cm below the platform’s upper surface. Whenever rats on the platforms lapsed into REM sleep, the loss of muscle tone caused them to make facial contact with the water and they would subsequently awaken [24]. Control rats were kept in a similar chamber filled with sawdust bedding instead of water.

All rats were habituated to the experimental environment for one hour per day for two consecutive days preceding the onset of the study. The purpose of this routine was to train each animal to balance on the platform to avoid excessive falling into the water during the sleep deprivation period. Body weights and the amount of food eaten during the previous day were estimated daily. Stainless steel clamps held the food container in place within easy access of the platform; drinking water was provided with an overhead bottle.

A surgical procedure involving placement of a supradural indwelling catheter was performed according to a protocol previously published by Wieseler et al. [22]. Briefly, the rats were anesthetized using isoflurane and placed in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA, USA). All surgical tools were sterilized before use. After shaving the surgical site, a 1–2-cm skin incision was made to expose the bregma while trying to minimize bleeding. A dental drill was used with a Dremel #107 engraving cutter bit to bore two 2-mm-wide, 10-mm-long troughs into the skull. These two troughs were positioned 3–4 mm parallel to the midsagittal suture. Troughs were drilled such that the dura was exposed 1 mm caudal to bregma, while trying not to penetrate the dura. After the bilateral troughs were drilled, polyethylene catheters (PE-10; BD, Franklin Lakes, NJ, USA) were carefully inserted horizontally along the troughs. The catheters were immobilized with Superglue (3 M, Maplewood, MN, USA) and allowed to dry. The external seal of each catheter was then clipped, and the catheters were flushed with 5 μl of sterile saline, with care being taken not to introduce any air or occlusion into the catheter. The scalp wound was sealed with 9-mm stainless steel wound clips. Subsequently, the rats were individually housed once ambulatory. As an attempt to prevent clogging, 2 μm of heparin was flushed through the catheter at 72 h after surgery.

Four days after supradural catheter insertion, capsaicin 100 nM (1% ethanol in saline) was injected at a total amount of 16 μl (capsaicin: 10 μl, sterile saline in catheter: 6 μl) serially over four minutes using an infusion pump (Harvard Apparatus, Holliston, MA, USA) while the rat was freely moving. The injector remained connected to the catheter for one-minute following infusion to allow diffusion of the solution. The same procedure was repeated after two hours. Catheter placement was verified at the completion of all experiments.

The sensory threshold to mechanical stimuli at the facial area was verified by the von Frey monofilament (VFMF) threshold test. All testing was performed with blinding with respect to group assignment. For the VFMF test, each rat was placed in an atraumatic plastic tube restraint, into which the animal entered uncoaxed. Animals were placed in the testing apparatus for acclimation through a training period. The sensory thresholds were determined by applying the VFMF apparatus (North Coast Medical, Inc., Gilroy, CA, USA) to the face over the periorbital region on both sides. The monofilaments were presented in either a sequential ascending or descending order for determining the threshold of response, as published previously [25, 26]. A positive response to the VFMF test was noted when the rat moved its head to avoid stimulus or struck its face with its ipsilateral forepaw. The 66% probability threshold of a positive response, defined as a positive response to two of three trials during the VFMF test, was determined.

All male Sprague-Dawley rats were randomly assigned to four groups using the random function (=RAND) in Microsoft Excel (Microsoft Corp., Redmond, WA, USA), as follows:

In the first part of the study, the effects of REM sleep deprivation on sensory threshold to mechanical stimuli and neuronal activation were assessed in the NonSD group and the REMSD group. In these experiments, the VFMF thresholds were assessed every day during sleep deprivation. After sleep deprivation, the brains of each group were analyzed by immunohistochemical staining.

In the second part of the study, the effects of REM sleep deprivation on facial allodynia induced by supradural capsaicin infusion were assessed in the NonSD-Capsaicin group and the REMSD-Capsaicin group. Animals involved in the first part of the study did not participate in any sessions of the second part of the study. In this part, the VFMF threshold test was performed for 28 days after REMSD.

The REMSD and NonSD groups were sacrificed by injection with 0.9% saline (300 mL), followed by 4% paraformaldehyde (300 mL) in 0.1 M phosphate-buffered saline (PBS; pH: 7.4) via the ascending aorta. The brain and cervical spinal cord were stored overnight in the same fixative and then placed in a cryoprotectant solution (30% sucrose in 0.1 M PBS) for 48 h. After serial cutting using a freezing cryostat, sections (30 μm) that spanned the hypothalamus, periaqueductal gray (PAG), trigeminocervical complex (composed of the trigeminal nucleus caudalis and cervical spinal cord levels C1, C2, and C3; TCC) were collected in PBS. Tissue sections were processed as free-floating sections. Sections were placed in and rinsed with 0.1 M PBS in 24-well plates and then incubated in 3% H₂O₂ in 50% ethanol (Merck, Darmstadt, Germany) for 15 min. Each section was incubated in a blocking solution (10% normal goat serum in 0.1 M PBS), followed by incubation overnight with a rabbit primary anti-Fos antibody (1,5000; ab99515; Abcam, Cambridge, UK) diluted in 4% BSA, 2% normal goat serum, and 0.2% Triton X-100 in PBS. The next morning, sections were incubated in a secondary antibody (a biotinylated goat anti-rabbit antibody; Vector, Burlingame, CA, USA) diluted at 1:500 in 4% BSA, 2% normal goat serum, and 0.2% Triton X-100 in PBS for 20 min, followed by further washes in PBS. Sections were then incubated with ExtrAvidin–peroxidase (Sigma-Aldrich, St Louis, MO, USA) for two hours. Finally, the sections were incubated with 3,3′-diaminobenzidine tetrahydrochloride dihydrate containing nickel (Vector, Burlingame, CA, USA) before being washed and mounted on slides, air-dried, dehydrated, and mounted in a distyrene plasticizer xylene medium under a coverslip (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany). The omission of either the primary or the secondary antibody abolished the immunohistochemical staining completely, indicating specificity. Fos immunoreactivity was distinguishable by cellular location. That is, the nucleus was always identified by an intense dark brown-to-black color, indicating the presence of the Fos protein. All sections were examined under an Olympus Universal microscope equipped with a digital camera (Olympus, Shinjuku, Tokyo, Japan) by an investigator blinded to the animal group being assessed.

Cytoarchitecturally identified regions of the hypothalamus, PAG, and TCC of hemisections were used for confirmation of Fos-positive cells. Localization of the hypothalamus, PAG, and TCC was done with reference to the obex according to coordinates provided by Paxinos and Franklin [27]. For the hypothalamus, four brain regions were selected for quantitative analysis: the posterior hypothalamus (P-HT), dorsomedial hypothalamus (DM-HT), ventromedial hypothalamus, and lateral hypothalamus. Three brain regions were selected for quantitative analysis for the PAG; these were the dorsomedial periaqueductal gray, dorsolateral periaqueductal gray, and ventrolateral periaqueductal gray (VL-PAG). Lastly, three brain regions were selected for quantitative analysis for the TCC: the superficial layer (spinal trigeminal tract or lamina I/II; Supf C), spinal trigeminal nucleus, and the area around the central commissure.

At each level, four randomly selected sections were assessed, and the average number of Fos-positive cells per hemisection were calculated for each level to investigate the distribution pattern in each nucleus without prior knowledge of the treatment of each animal. All counts were performed by the same experimenters to maintain consistency in application of the criteria used to select Fos-positive cells and to reduce the likelihood of individual variability.

The VFMF threshold of the REMSD group significantly decreased during the sleep deprivation period versus the baseline. In comparison with the NonSD group, the REMSD group had a significantly lower VFMF threshold after one day of sleep deprivation (p = 0.003). This difference lasted for the rest of the sleep deprivation period (Fig. 1).

An integrated count of Fos-positive cells for each group is presented in Fig. 2. In the hypothalamus, the number of Fos-positive cells in the P-HT (p = 0.010) and DM-HT (p = 0.024) in the REMSD group were significantly higher compared to the NonSD group (Table 1, Fig. 3). In the PAG, the number of Fos-positive cells in the VL-PAG was significantly higher in the REMSD group compared to the NonSD group (p = 0.016). In the TCC, the number of Fos-positive cells in the Supf C was significantly higher in the REMSD group compared to the NonSD group (p = 0.019).

Following supradural capsaicin infusion, the decreased VFMF threshold of the NonSD-Capsaicin group was maintained until Day 10 PSD (p = 0.000) and returned to the baseline at Day 14 PSD (p = 0.300). In the REMSD-Capsaicin group, the reduced VFMF threshold was maintained until Day 25 PSD (p = 0.003) and returned to the baseline at Day 28 PSD (p = 0.172). Based on a comparison between the two groups, there were significant differences in VFMF threshold from Day 7 PSD (p = 0.014) to Day 25 PSD (p = 0.013) (Fig. 4).