In the present investigation, animals subjected to 96 hours of PSD exhibited an increased pain response after mechanical noxious stimuli, and the results suggest that there is a correlation between PSD-induced hyperalgesia and the increased NO system activity in the dorsolateral periaqueductal gray matter.
A significant reduction of the withdrawal threshold to von Frey filament application was observed after PSD. The control animals were less sensitive to pain as their paw withdrawal thresholds were higher than those of the PSD-treated rats. These data are consistent with previous studies that showed a PSD-induced hyperalgesia effect, as evaluated through mechanical noxious test [7, 13]. In these studies, only one of the hindpaws was challenged with the von Frey filament. In the present study, we demonstrated that a PSD-induced hyperalgesic response to noxious mechanical stimuli is detected independently of the hindpaw selected.
An increase in NOS is associated with sleep deprivation and the development of hypersensitivity to pain in inflammatory and neuropathic pain models [22–24]. In the present study, we observed that PSD-treated animals presented an increased number of NADPH-d-positive cell bodies in the dlPAG. Because NADPH-d is a common marker for NOS and because its activity parallels NO production, this increased expression could indicate that dlPAG- NO plays a role in PSD-induced hyperalgesia. Jang and collaborators also described a NO involvement in dlPAG pain modulation . These authors observed an increased dlPAG NOS activity in a peripheral neuropathy model, which was evoked through streptozotocin-induced diabetes, and a reduction after acupunctural treatment.
The observed hyperalgesia and increased NOS activity in the dlPAG were both reverted after the administration of L-NAME (50 mg/kg), a dose which was previously established as antinociceptive to mechanical, chemical and thermal noxious stimulus [33–35]. Wei and collaborators  also described a reduction of PSD-induced hyperalgesia through L-NAME, confirming that the nitrergic system plays an important role in this process.
In our PSD model, the increase in NADPH-d-positive cell bodies in the dlPAG might be responsible for the increased pain sensitivity because this region plays an important role in the modulation of nociception and the antinociceptive effects of morphine [20, 21]. The neuronal pathways and molecular events involved in NO-supraspinal pain modulation are not completely understood. This effect is likely mediated through multiple neurobiological components. A recent study described a reversed NO-induced nociceptive hypersensitivity through the blockade of a supraspinal signaling pathway involving a PKC-dependent CREB (cAMP response element-binding protein), STAT1 (signal transducer and activator of transcription 1) and NF-κB (nuclear factor kappa B) activation in the PAG and thalamus . Spinal glutamate has also been associated with mechanical hypersensitivity following sleep deprivation in rats, and the intrathecal administration of MPEP (an antagonist of mGluR5) and MK-801 (an NMDA glutamate receptor antagonist) reverts PSD-induced hyperalgesia . In a previous study, we described a reduction in dopaminergic activity in the lateral PAG in paradoxical sleep-deprived rats and a reversion of hyperalgesia through L-DOPA (a dopamine precursor) treatment .
The PAG contains a dense plexus of cholinergic nerve terminals derived from the pontine tegmentum; these nerves mediate analgesia at least partly via the endocannabinoid signaling system . The sleep-wake cycle also controls the activity of cholinergic neurons in the basal forebrain . Thus, further studies are needed to characterize the neuronal pathways that are associated with sleep deprivation-induced hyperalgesia.
The PSD method used in this work induces physiologic signs of stress; however, a few studies have reported that stress produces a reduction in pain-related behavior [39, 40] that is different from the PSD results obtained in this work.
Thus we propose that the hyperalgesia observed in PSD-treated rats, observed after mechanical noxious stimuli, is associated with increased NOS activity in the dlPAG and NO-signaling pathway activation, presumably influencing the descending antinociceptive pathway. Because there is a great prevalence of sleep complaints in individuals suffering from chronic pain, this knowledge will be of great pharmacological interest.