Introduction
Surgical patients often report pre- and post-surgical sleep disorders, and this is mainly due to anxiety, depression, stress, and the use of opioids [
12]. Perioperative sleep disturbance is also a risk factor for persistent pain after surgery [
51]. Wright et al. examined presurgical sleep efficiency and found that patients with poor sleep on the night before surgery reported greater pain 1 week after surgery [
50]. Another study of postsurgical sleep examined 75 orthopedic patients who underwent major surgery and reported similar results. Most patients in this study (89.3%) experienced pain at the surgical site, reported a Visual Analogue Scale (VAS) pain score of “4” or “5” (range: 0 to 10), said the pain persisted at least 3 days, and declared that this pain are usually accompanied by extremely poor sleep quality [
5]. These and other studies thus indicate that pre- and post-surgical sleep disturbance affects postsurgical pain.
Persistent postsurgical pain after healing of a surgical incision, which has an incidence of about 10%, is a significant clinical problem. More than 320 million people worldwide undergo surgery every year, and persistent postsurgical pain is a significant public health issue [
17]. This pain can be severe enough to cause serious functional impairment or even disability resulting in decreased quality of life [
33]. As the population ages and the number of surgeries continue to increase, persistent postsurgical pain will become an increasingly serious problem. Long-term pain after surgery can increase use of health resources, and thereby greater disability and suffering [
26]. Therefore, there is an urgent need to understand the mechanism of persistent postsurgical pain and to find new predictors and therapeutic targets to prevent and control persistent postsurgical pain.
Previous studies have examined the mechanisms underlying the transition from acute pain to chronic pain in an effort to prevent the development of persistent postsurgical pain, but there has been little clinical progress. Previously, clinicians believed that peripheral nerve injury during surgery was the major cause of persistent postsurgical pain. However, many surgical patients have symptoms of nerve damage but report no pain. For example, after osteotomy of the mandible, only about 10% of patients with severe neurological injury (partial axonal trigeminal nerve lesion) during surgery have clinically significant neuropathic pain, and the others manifests as numbness and paresthesia. Therefore, nerve injury alone cannot explain the extended duration of acute pain after surgery [
22].
The dorsal root ganglion (DRG) is the key to neurotransmission between the peripheral and central nervous systems. Previous studies confirmed that neuropeptides and ion channels, such as calcium channels, in DRG neurons control sensory responses and pain [
25,
44,
52]. Recent large-scale and high-quality trials have demonstrated that gabapentin and pregabalin can reduce postsurgical pain and improve sleep quality [
16]. These agents inhibit the α
2δ subunit of voltage-gated calcium channels (VGCCs), thus suggesting that these channels play an important role in the development of persistent postsurgical pain. VGCCs are essential for the physiological activities of excitable cells, including neurons, and analysis of their biophysical properties has led to their classification as low-voltage-activated (LVA) channels and high-voltage-activated (HVA) channels. Depending on the Ca
vα
1 subunit, HVA channels can be classified as N-, L-, R-, or P/Q-type channels. Changes in the expression and function of these channels can affect the development and persistence of several pain states [
28]. However, little is known about the role of HVA channels in the development and persistence of postsurgical pain, or about the clinical effects of changes in the expression and function of these channel proteins.
To mimic the effect of reduced sleep time in experimental animals, previous researchers have used different types of mild stimuli to keep these animals awake for a long time, such as rapid eye movement sleep deprivation (REM-SD) [
11,
43] and sleep restriction (SR) [
45]. There is evidence that long-term continuous or intermittent REM-SD in naive experimental animals significantly increases their hyperalgesia to heat and pressure stimulation [
39]. In contrast, short-term sleep deprivation does not affect basal pain perception, but it does increase the sensitivity to postsurgical painful stimuli [
48]. However, the mechanism of short-term sleep deprivation on postsurgical pain hypersensitivity is not fully understood. We therefore examined the effect of a short-term sleep deprivation on postsurgical pain by using a previously described sleep deprivation procedure [
19,
37].
In the present study of male Sprague Dawley rats, we sought to understand the role of HVA channels in the delayed recovery from postsurgical pain and to find a new therapeutic target for reducing prolonged postsurgical pain. Our basic approach was to implement a perioperative SD procedure and to study its effect on postsurgical pain. We also studied the expression and function of various subtypes of HVA channels in the dorsal root ganglia (DRG) during the development of postsurgical pain.
Discussion
This study of male Sprague Dawley rats demonstrated that short-term SD before and after surgery delayed recovery from postsurgical pain. In addition, our electrophysiological and molecular biology experiments indicated that prolonged postsurgical pain duration was related to the increased expression and activity of L-type calcium channels in the lumbar DRG, and that blocking these channels accelerated postsurgical recovery from pain. These results suggest that the prolonged duration of postsurgical pain that is mediated by perioperative SD depends on the expression and activity of L-type calcium channels.
L-type channels, one of the four subtypes of HVA channels, are encoded by the
Cav1.1–1.4 genes. Mammals have almost no expression of Ca
v1.1 and Ca
v1.4 in their nervous systems, but Ca
v1.2 and Ca
v1.3 are expressed in most excitable cells, including neurons [
28]. These proteins are present in the cell body and also in axons [
9,
36], consistent with our staining results. Also, mammals with
Cav1.3 knockout have a normal pain phenotype [
10]. These previous results led us to focus on the L-type channels encoded by
Cav1.2. As L-type channels are widely distributed in cells that participate in the pain pathway, previous studies have also examined the role of these channels [
1,
38,
41]. Increased L-type channels in spinal cord lamina II mediate hyperalgesia in rats in a chronic constriction injury (CCI) model [
1]. Lack expression of L-type channels in the anterior cingulate cortex of mice correlates with pain relief [
23]. The L-type channel mRNA in the cerebral cortex and thalamus is up-regulated in the presence of a migraine aura [
8]. Moreover, administration of the L-type channel blocker nifedipine can enhance anti-nociceptive effect of opioids, which were microinjected into the midbrain ventrolateral periaqueductal gray (vPAG). This suggests that the activity of the L-type channels of PAG may play an important role in the development of pain [
21]. In addition, L-type channels are also associated with psychological factors, such as stress, anxiety, and depression [
27]. These previous studies thus indicate that L-type channels have major roles in pain occurrence and development.
Our current work demonstrated that L-type channels in the DRG contributed to the SD-induced prolongation of postsurgical pain. Our western blotting and electrophysiology data indicated increased expression and activity of L-type channels of rats that received incision+SD, and our immunofluorescent staining indicated that increased L-type channels were mainly located in medium and large neurons, but they had a lower level in small neurons. Our electrophysiological data also confirm these results. It should be noticed that although the altered L-type channels mostly located in medium and large neurons, which mainly contributed to the mechanical pain, the CGRP+ small neurons still have a proportion of co-labeling with L-type channels. According to previous reports, CGRP+ neurons mainly control thermal pain [
6,
35,
54]. This is consistent with our behavioral data that the thermal pain could be partially reversed. Moreover, to rule out the effects of L-type channels at other anatomical sites in the pain pathway, we performed DRG microinjections with a recombinant AAV to specifically knockdown the L-type channels to confirm our interpretation. These findings extend the limited literature regarding the effect and mechanism of SD before or/and after surgery on recovery from postsurgical pain.
With the continuous improvement of people’s living standards, the quality of sleep has become a topic of increasing concern. Especially for surgical patients, sleep disorders often occur before and after surgery. Many studies have shown that sleep disorders and pain are closely related. In particular, sleep disorders can cause many changes in endogenous regulatory factors, which in turn can cause hyperalgesia. For example, insufficient sleep leads to increased migration of B cells into the brain compartment [
24], activation of complement [
47], and increased levels of IL-1 [
55], thus leading to the onset and aggravation of neuroinflammation, a key factors underlying pain. In addition, stress caused by sleep disorders can cause dysfunction of the HPA axis, causing cortisol dysfunction to trigger, exacerbate, or prolong pain, impair healing, and contribute to chronic disability [
20]. However, a common problem with these regulators is that specific regulation is difficult; for example, it is difficult to inhibit inflammation and regulate plasma cortisol levels to achieve pain relief. Moreover, most previous studies have focused on the relationship between sleep and pain, and few studies have examined postsurgical pain.
Our current study focused on the L-type calcium channel, and found the endogenous factor Egr-1 might have an important in the regulation of these channels in DRG. This suggests that specific regulation of the activity of Egr-1 and L-type channels has potential for the therapeutic management of peripheral postsurgical pain. Most previous studies in this field have examined the effects of SD on pain in naive animals. These studies demonstrated that long-term consecutive or intermittent SD caused abnormal nociceptive sensitivity at the basal level [
11,
19]. However, it is still unclear whether short-term SD before and after surgery actually affects postsurgical pain. We established an animal model of perioperative SD, in which rats with SD had slightly greater pain sensitivity than control rats (no SD) from 1 to 5 days after surgery, although these differences were not statistically significant. This may be due to factors such as the strain of the rat, feeding conditions, and other details of the experimental model. This model thus simulates a common clinical situation, because most surgical patients have short-term SD before and after surgery, and this short-term SD does not cause changes in pain perception.
The relationship between central nervous system activity and delayed postoperative pain recovery is currently unclear. It is possible, although uncertain, whether pre- and post-surgical SD initially affects the central nervous system (CNS) and then the peripheral nervous system (PNS), eventually leading to a delay of postsurgical recovery. There is much evidence that SD causes a series of changes in the CNS. For example, mice subjected to sleep disturbance produce more Ly-6C
high monocytes and less hypocretin (a neuropeptide that promotes wakefulness) in the lateral hypothalamus [
34]. More importantly, long-term lack of sleep in rats can lead to increased neural activity in the periaqueductal grey (PAG) and the nucleus accumbens (NAc), which are closely related to perception of pain [
42]. Studies of animal models of chronic pain have identified some molecular mechanisms and neurobiological activities that are associated with the transition from acute pain to chronic pain. The most studied descending pain pathway projects from the midbrain periaqueductal grey (PAG) to the rostral ventromedial medulla (RVM). Electrical stimulation of the PAG can block the spinal cord’s response to noxious stimuli, and stimulation of the RVM can inhibit and/or promote pain signaling [
57]. In addition, insufficient sleep can cause release of glucocorticoids from the adrenal gland [
2] while endogenous glucocorticoids can interact with Egr-1 [
7]. Therefore, we speculate that changes in glucocorticoid levels after short-term SD may also be associated with persistent postsurgical pain. Overall, we believe that a regulatory mechanism first affects the CNS and then the PNS, so that short-term SD before and after surgery delays postsurgical recovery from pain. This regulatory mechanism, which may be related to neuroimmunity, neural circuits, and/or endocrine systems, is a topic of our future studies.
A limitation of the present study is that we did not examine the effect of overexpression of L-type channels in DRG to verify that recovery from postsurgical pain is delayed upregulation and activation of these channels. To the best of our knowledge, upregulation of L-type channels leads to hyperalgesia. For example, in one animal model of chronic pain, the chronic constriction injury (CCI) model, up-regulation of L-type channels markedly decreases the pain threshold in rats. Moreover, use of L-type calcium channel blockers reduces the frequency of spontaneous excitatory postsynaptic currents, thereby providing relief from pain [
1]. Thus, if we overexpressed the L-type channels in the DRG, the rats would likely remain in a constant state of hyperalgesia, and this could be difficult to distinguish from postsurgical pain. Another limitation is that we did not perform genetic knock-out of
Cav1.2 to confirm our results. This was because of the technical difficulties in performing knock-out of the
Cacna1c gene in rats. Instead, we performed lumbar DRG microinjection of Cacna1c-shRNA to specifically eliminate Ca
v1.2 in L4/5 DRG; an advantage of our approach is that it was specific to the lumbar region. The results of our shRNA microinjection experiments confirmed that L-type calcium channels function in the prolongation of postsurgical pain.
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