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Thalidomide alleviates postoperative pain and spatial memory deficit in aged rats
Biomedicine & Pharmacotherapy 95 (2017) 583–588
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Original article
Thalidomide alleviates postoperative pain and spatial memory deficit in aged rats Peng Guoa, Si-Ping Hub, a b
MARK
⁎
Department of Anesthesiology, The Central Hospital of Shaoxing City, Shaoxing 312030, Zhejiang, China Department of Anesthesiology, Huzhou Central Hospital, Huzhou 313000, Zhejiang, China
A R T I C L E I N F O
A B S T R A C T
Keywords: postoperative cognitive dysfunction (POCD) thalidomide pain NMDA receptors inflammation
Pain is a major risk factor of post-operative cognitive dysfunction (POCD) in aged population. We investigated the effects of thalidomide, an anti-inflammatory and analgesic drug, on POCD in aged rats, and also explored the underlying mechanisms. Laparotomy was performed under anesthesia in aged rats (24–25 months) to establish POCD models. Thalidomide (5–50 mg/kg) was intraperitoneally administered immediately after laparotomy. Within 12 h after the operation, pain symptoms were assessed by rat grimace scale (RGS). Within postoperative day (POD) 3–14, spatial memory was evaluated using performance errors in a radial arm maze. Protein levels of inflammatory cytokines and N-methyl-D-aspartate (NMDA) receptors were measured on POD 14. POCD rats treated with thalidomide showed decreased RGS and performance errors, compared with saline-treated POCD rats. Single administration of thalidomide significantly reduced production of cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-1β) in serum but not in the brain, and attenuated upregulation of NMDA receptor (NR) 2A/B subunits in the hippocampus at POD 14. MK-801 abolished thalidomide-induced attenuation of spatial memory deficits. Our results support that thalidomide could disrupt the development of post-operative memory deficit in aged rats through its long-term regulation of NMDA receptors (NRs) in the hippocampus. Therefore, thalidomide might provide a new means to prevent the development of POCD.
1. Introduction Patients subjected to anesthesia and surgery show attention deficits, mild cognitive impairment, and spatial memory dysfunction. These syndromes can be categorized as postoperative cognitive dysfunction (POCD) [1]. POCD has higher prevalence in elderly patient, 14% of which experience cognitive decline and confusion up to 4 months after surgery [2,3]. The risk factors include ageing, preoperative stress, drugs, trauma injury, and postoperative pain [1]. Although pathophysiology of POCD is uncertain, management of postoperative pain is one of options proved effective to prevent POCD [4]. One clinical study revealed that elderly patients with non-cardiac surgery who received postoperative anesthesia is less likely to experience POCD [5]. Similarly, postoperative acute pain is found to exacerbate memory deficits after laparotomy in aged rats [6]. The goal of post-operative pain management is to relieve pain while minimizing side-effects. Although opioids are on the front line of effective analgesics, they also carry many undesirable side-effects, including sedation, respiratory depression, nausea and vomiting, overdose, and addiction [7]. Non-steroidal anti-inflammatory drugs
(NSAIDs) are useful for postoperative pain management, especially for mild and moderate, but not severe pain [8]. Therefore, new and more effective treatment modalities are needed for postoperative pain management. Thalidomide was first introduced in 1957 as a non-barbiturate sedative drug for the treatment of sleep disorders and morning sickness. Because thalidomide inhibits the synthesis and expression of multiple cytokines including interleukine-1β (IL-1β), IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α) [9], it has been used in inflammatory disorders and cancers [10,11]. Consistent with the notion that inflammation plays key roles in pain sensitization [12], thalidomide effectively relieves complex regional pain syndrome [10] and neuropathic pain, such as chronic sciatic nerve injury, lumbar 5 ventral root transection neuropathy, and chemotherapy-related pain [11,13,14]. Recent studies indicate that inhibition of systemic inflammatory process may interrupt pathogenesis of POCD [15,16]. In the present study, we revealed that thalidomide could disrupt the development of post-operative memory deficit in aged rats through its long-term regulation of NMDA receptors (NRs) in the hippocampus. Therefore,
Abbreviations: POCD, postoperative cognitive dysfunction; RGS, grimace scale; POD, postoperative day; NMDA, N-methyl-D-aspartate; TNF-α, tumor necrosis factor-α; IL, interleukin ⁎ Corresponding author: Department of Anesthesiology, Huzhou Central Hospital, No.198 Hongqi Rd, Huzhou 313000, Zhejiang, China. E-mail address: [email protected] (S.-P. Hu). http://dx.doi.org/10.1016/j.biopha.2017.08.114 Received 23 May 2017; Received in revised form 18 August 2017; Accepted 24 August 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 95 (2017) 583–588
P. Guo, S.-P. Hu
thalidomide might provide a new means to prevent the development of POCD.
correct arm choices (the first visit to reward arms); number of incorrect arm choices (the first visit to non-reward arms).
2. Methods
2.6. Western blot
2.1. Animal All animal care and experimental protocols were approved by Animal Care and Research Committee in The Central Hospital of Shaoxing City. Aged male Wistar rats (24–25 months old, 550 and 640 g) were used in this study. All rats were housed at 22 ± 2 °C, 45–75% relative humidity with 12 h light–dark cycle, and water and food were provided ad libitum.
Protein from the hippocampus, the PFC, and the amygdala was extracted for standard western blot assay. Rabbit anti-NR2A/B (1:500, Millipore, Billerica, MA, USA) and mouse anti-GAPDH (1:10000, Abcam, Cambridge, MA, USA) were used as primary antibodies. Antibody–protein complexes were visualized by chemiluminescent reagents. Band density was quantified by imaging quantification. Ratios of the band density for the protein of interest to that for GAPDH were calculated.
2.2. Anesthesia, laparotomy and drug treatment
2.7. ELISA assay of TNF-α and IL-1β
Rats were randomly divided into 5 experimental groups (n = 10 rats/group): A sham group received anesthesia, but without laparotomy; other 4 surgery groups all received anesthesia and laparotomy, and by the end of these treatments, individual surgery groups were respectively subjected to single intraperitoneal injection of saline (vehicle), 5, 20, or 50 mg/kg thalidomide. Simple laparotomy was performed according to previously reported methods [6]. Briefly, anesthesia was induced with 3% isoflurane, and later maintained with 1.2% isoflurane for 2 h. A 1.0-cm midline longitudinal incision was made through the skin, the abdominal muscle, and the peritoneum. The muscle layers were sutured with 5-0 Vicryl sutures, and the skin was closed with tissue adhesive glue. Thalidomide and MK-801 were purchased from Sigma Aldrich (USA), and were dissolved in saline. MK-801 (0.1 mg/kg) was given intraperitoneally (i.p.) before surgery; saline or thalidomide was given i.p. after laparotomy.
TNF-α and IL-1β in serum and brain were measured using ELISA ready-SET-Go!® assay kits (eBioscience, San Diego, USA), according to the instruction provided by the manufacturer. Protein level was expressed as pg/μg of total proteins determined over an albumin standard curve. 2.8. Statistical analysis Data are presented as mean ± SD. Statistical significance was determined by one- or two-way ANOVA with Dunnett’s or Tukey’s posthoc tests using the GraphPad Prism® 5 software, depending on whether the data passes the normality test. Difference was considered statistically significant when P values were less than 0.05. 3. Results 3.1. Effect of thalidomide on postoperative acute pain in aged rats
2.3. Pain measurement Rat grimace score (RGS) was used to evaluate the intensity of pain. Thalidomide (5–50 mg/kg) was administrated by i.p. injection after laparotomy. Compared with sham group, all surgical groups developed significant acute pain starting 2 h postoperatively, evidenced by higher RGS (p < 0.05, One-way ANOVA), and recovered to baseline level 12 h after surgery (p > 0.05, One-way ANOVA) (Fig. 1a). The concentration-response relationship showed (Fig. 1a) that single injection of thalidomide attenuated pain intensity in a dose-dependent manner, with statistical significance at doses of 20 and 50 mg/kg (p < 0.01). Additionally, thalidomide at 20 and 50 mg/kg induced a faster recovery of postoperative pain, compared with saline treatment (p < 0.05) (Fig. 1a). These data suggest that single administration of thalidomide has anti-analgesic effect on acute pain following laparotomy. Open-field test on post-operative day 3 (POD 3) showed that there was no difference in locomotor activity among experimental groups (p > 0.05) (Fig. 1b). Time lapsed before arm choice in the maze was analyzed for evaluation of task motivation. As shown in Fig. 1c, latency for arm choice did not differ between groups (p > 0.05). These data indicate that neither surgery nor thalidomide treatment changed locomotion and task motivation in aged rats.
Acute postoperative pain intensity was determined using a rat grimace scale (RGS) as previously reported [17]. Briefly, rats were acclimated in a clear plastic cage with a camera immediately outside of one wall for 10 mins. The activity of the rats was digitally videotaped during acclimation phase. Frame of front view of the head was grabbed randomly. Each frame should include the head with most of action units visible (i.e., nose, cheek, whiskers, eyes, and ears). Five facial features as indices of pain were defined: orbital tightening, nose/cheek fluttering, ear position, and whisker changes. Pain intensity ratings were coded using a three point scale from 0 (not present) to 2 (severe) to facial features presented. 2.4. Open field test for measurement of locomotion Rats were subjected to open field test on postoperative day 3. They were placed in a rectangle chamber, which was equipped with photobeams. The activity of the rats was quantified as total count of beam breaks in 60 mins. 2.5. Radial arm maze for measurement of spatial memory
3.2. Effect of thalidomide on spatial memory performance A standard radial arm maze was used to measure spatial memory, as previously reported [18]. Briefly, the maze contains a central platform with a diameter of 34, which was surrounded with a Plexiglass wall and 12 radial arms (50 cm long and 11 cm wide, with 20 cm transparent wall). A reward pellet (45 mg) was placed randomly at the end of 6 out of 12 arms in a plastic cup. Rats were first acclimated in maze for 2 days. During the trial, rats were placed in the center of the maze and allowed to explore all arms. The following data were record: the latency for per arm choice; the arm (reward or non-reward) the rat entered each time; the total time spent for obtaining all 6 food rewards; number of
Spatial memory performance was analyzed using a radial arm maze from POD 3–14. Compared to non-surgical sham group, the working memory errors in all surgical groups were significantly higher than those in sham group from POD 5–14 (p < 0.05) (Fig. 2a, b), suggesting that laparotomy resulted in spatial memory deficit. Single injection of thalidomide by the end of surgery significantly reduced memory errors in a dose-dependent manner at POD 8, 10, 12, 14, with significant difference at 20 and 50 mg/kg (p < 0.05) (Fig. 2a, b). The results suggest that thalidomide not only effectively relieved postoperative 584
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Fig. 1. Effects of thalidomide on postoperative RGS and locomotion in aged rats. Aged rats were subjected to laparotomy, and doses of thalidomide (5–50 mg/kg) were intraperitoneally administered by the end of laparotomy. (a) Acute pain was assessed with rat grimace scores (RGS) before and 2–12 h after operation. (b) Locomotor activity in an open field was measured at post-operative day (POD) 3. (c) The average of latency for the choices of individual arms was determined. Data are presented as mean ± SEM (n = 10 in each group). One-way ANOVA with post hoc Tukey test was used for statistical analyses. * p < 0.05 and ** p < 0.01 vs. Sham group; # p < 0.05 and ## p < 0.01 vs. Vehicle group.
impairment [6]. The levels of NR2 subunits in the hippocampus, the PFC, and the amygdala were determined using western blot at POD 14. Rats in surgical group displayed significant increase in NR2 protein levels in the hippocampus (p < 0.01) (Fig. 4a) that were reduced by single injection of thalidomide in a dose-dependent manner, with a statistical significance at doses of 20 mg/kg and 50 mg/kg (p < 0.05) (Fig. 4a). In contrast, there was no significant difference in NR2 expression in the PFC and the amygdala among all groups (p > 0.05) (Fig. 4b and c). The data suggest that NR2 were selectively up-regulated in the hippocampus after surgery in aged rats, and thalidomide could counteract the increase of NR2. Next, we further examined the involvement of NRs in improvement of postoperative memory by thalidomide. Consistent with previous report [20,6], pretreatment with MK-801, a non-competitive NMDAR antagonist, significantly increased working memory errors in surgical group, compared with saline treated group (p < 0.05) (Fig. 5a). Furthermore, we observed that pretreatment of MK-801 abolished thalidomide improvement of postoperative working memory (p < 0.01)
pain, but also attenuated postoperative memory deficits in aged rats. 3.3. Effect of thalidomide on pro-inflammatory cytokines Inflammatory responses have been associated with various pain states and POCD [16,19]. As shown in Fig. 3a, b, both TNF-α and IL-1β in serum were up-regulated after laparotomy (p < 0.01), detected by ELISA on POD 14. Thalidomide reduced TNF-α and IL-1β in serum in a dose-dependent manner, with significant difference at 20 mg/kg and 50 mg/kg, compared with saline treatment (p < 0.05) (Fig. 3a, b). However, the level of TNF-α and IL-1β in the brain did not differ among all groups (p > 0.05) (Fig. 3c, d). The results suggest that thalidomide may attenuate postoperative inflammatory responses in peripheral but not in the central nervous system. 3.4. Effect of thalidomide on NR2 expression NRs have been proposed to participate in pain-related cognitive
Fig. 2. Effect of thalidomide on postoperative spatial memory in aged rats. Postoperative spatial memory at post-operative day (POD) 3–14 was evaluated with memory errors in radial arm maze assay. Both memory errors at each time point (a) and total errors throughout the entire testing period (b) were analyzed with one-way ANOVA with post hoc Tukey test. Data are presented as mean ± SEM (n = 10 in each group). * p < 0.05 and ** p < 0.01 vs. Sham group; # p < 0.05 and ## p < 0.01 vs. Vehicle group.
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Fig. 3. Long-term effects of thalidomide on postoperative cytokine levels in aged rats. TNF-α and IL-1β levels in serum (a) and brain (b) were quantified with ELISA assay. Serum and brain tissues were harvested after completion of behavioral testing at post-operative day (POD) 14. Data are presented as mean ± SEM. One-way ANOVA with post hoc Tukey test was used for statistical analyses. ** p < 0.01 vs. Sham group. # p < 0.05 and ##p < 0.01 vs. Vehicle group.
2b). In these tests, thalidomide relieved acute postoperative pain within 2 h, while improved postoperative spatial memory 5–6 days after surgery. The drastically different time courses of these two effects suggest that diverse underlying mechanisms could exist. As half-life of thalidomide in the body is roughly 5–9 h [9], anti-nociceptive effects of thalidomide could be associated with its immediate action. However, thalidomide enhanced cognitive function in postoperative condition probably through its secondary action following its analgesic effect. Our data suggest the involvement of postoperative pain in POCD and effective management of postoperative pain with thalidomide may be beneficial for the prevention of POCD. Thalidomide can easily cross blood brain barrier after acute systemic administration, and dramatically reduces the expression and synthesis of multiple cytokines in peripheral and central nervous system, including IL-1β, IL-6, and TNF-α [11,9]. Immunomodulatory and anti-inflammatory properties of thalidomide are critical for its antinociceptive effects. We observed that laparotomy resulted in a long lasting peripheral inflammatory response: increased expression in serum TNF-α and IL-1β, at POD 14 (Fig. 3a, b), consistent with other preclinical studies [27,28]. Although at POD 14, thalidomide was no longer present in the body, we still observed reduced levels of the production of TNF-α and IL-1β in the serum (Fig. 3a, b). The result suggests that thalidomide could have long-term anti-inflammatory effects. Considering the important roles of inflammation in neuropathic pain, further investigations are warranted to elucidate whether aged rats received laparotomy show spontaneous pain behaviors co-existing with spatial memory deficit. Previous studies have identified a possible causal relationship between surgery, inflammation, and memory impairment. Evidence suggests that peripheral cytokines can pass signals to the brain through blood and neural routes, and then stimulating production of cytokines in the hippocampus [29,30]. Likewise, i.p. injection of lipopolysaccharide triggers activation of microglia in the brain and upregulation of cytokines (including IL-1β) in the hippocampus, meanwhile, results in memory impairment [31]. This is also supported by an interventional study showing that surgery induced peripheral and hippocampal cytokine expression (TNF-α and IL-1β) is associated with
(Fig. 5b). These results indicate that NRs might be involved in thalidomide-induced improvement of postoperative spatial memory deficits in aged rats.
4. Discussion Cardiac surgery or non-cardiac surgery with larger and more invasive procedures, such as abdominal, thoracic, and vascular surgery, present a greater risk than outpatient surgery for a relatively higher incidence of POCD [21,3]. Besides the type of surgery, it is proposed that ageing is another major risk factor of POCD [3,22]. POCD is evident in 26% of elderly patients 7 days after major non-cardiac surgery [23], and still presents 3 months after surgery in 7% of patients aged 60–69 and in 14% of patients over 69 years old [4]. In the present study, we performed abdominal laparotomy in 24–25 months old rats to successfully establish POCD models. Spatial memory in these rats was impaired showing more memory errors in radial arm maze, but no effect on locomotor activity and motivation. Although isoflurane anesthesia is toxic to the ageing brain [24], the link between anesthesia or surgery and postoperative cognitive problems has been elusive [6,25,26]. In the present study, rats in laparotomy plus isoflurane anesthesia displayed more severe postoperative pain and stronger spatial memory deficits in comparison with rats receiving isoflurane only (sham group) (Fig. 1a). These results suggest that laparotomy (surgery) may play an important role in postoperative spatial memory deficits. Studies on pathophysiological mechanisms of POCD have identified that postoperative pain may be associated with POCD. For instance, morphine and non-steroidal anti-inflammatory drugs (NSAIDs), at doses relieving acute postoperative pain, attenuated postoperative memory deficits [25,4]. Here, we tested effects of thalidomide on both acute postoperative pain and spatial memory deficit. Acute postoperative pain was evident 2 h after laparotomy and lasted for 8 h (Fig. 1a). Surgical rats subjected to single dose of thalidomide by the end of the surgery showed not only accelerated recovery from acute postoperative pain, but also reversed deterioration in spatial memory function at POD 14, compared with vehicle-treated rats (Fig. 1a, 2a, 586
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Fig. 4. Thalidomide reversed postoperative upregulation of NR2A/B in rat hippocampus. NR2A/B in the hippocampus (a), the prefrontal cortex (b) and the amygdala (c) at postoperative day (POD) 14 was analyzed with western blot. GAPDH was used as a loading control. Data are presented as mean ± SEM. One-way ANOVA with post hoc Tukey test was used for statistical analyses. * p < 0.05 and ** p < 0.01 vs. Sham group. # p < 0.05 vs. Vehicle group.
spatial memory following laparotomy by thalidomide might be NRsdependent, because 1) thalidomide dose-dependently counteracted the upregulation of NR2; 2) thalidomide at the dose reversed NR2 upregulation improved spatial memory; and 3) the beneficial effect of thalidomide no longer existed after pre-treatment with MK-801 (Fig. 5). At POD 14, we also observed that the upregulation of NR2 subunits was selective in the hippocampus, but not in the PFC and the amygdala (Fig. 4a, b, c).
memory impairment, and both antagonizing and knocking-out IL-1β receptors mitigate the neuro-inflammatory reaction and memory dysfunction following surgery [15]. In contrast, we observed upregulation of TNF-α and IL-1β in serum but not in the brain following laparotomy. The data suggest that central inflammation may not mediate spatial memory deficits in aged rats following laparotomy. Synaptic plasticity is a key cellular mechanism underlying learning and memory, and relies on NMDA receptor (NR) function [32–34]. Either downregulation or upregulation of NRs leads to spatial memory deficit [32,35]. That is, NRs should be kept at optimized levels to maintain normal cognitive functions. This notion could explain our observation that laparotomy upregulated NR2 subunits in the hippocampus and impaired spatial memory, and the behavioral outcome was replicated with antagonizing NRs with MK-801. The improvement of
5. Conclusions In summary, single dose of thalidomide by the end of surgery effectively mitigates postoperative reactions in aged rats, including acute pain symptoms, and long-term peripheral inflammation and spatial 587
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Fig. 5. Thalidomide improved postoperative spatial memory deficit probably by regulating NMDA receptors. Spatial memory performance was evaluated by memory errors in a standard radial arm maze at (POD) 3–14 (a) and total errors over the entire testing period (b). MK-801 pre-treatment abolished the effect of thalidomide (50 mg/kg) on spatial memory performance in rats. Data are presented as mean ± SEM (n = 10 in each group). One-way ANOVA with post hoc Dunnett’s test was used for statistical analyses. * p < 0.05 and ** p < 0.01 vs. Vehicle group. ## p < 0.01 vs. thalidomide group. ‘n.s.’ indicates no statistical difference. [15] M. Cibelli, A.R. Fidalgo, N. Terrando, D. Ma, C. Monaco, M. Feldmann, M. Takata, I.J. Lever, J. Nanchahal, M.S. Fanselow, M. Maze, Role of interleukin-1beta in postoperative cognitive dysfunction, Ann. Neurol. 68 (2010) 360–368. [16] L. Peng, L. Xu, W. 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Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Original article
Thalidomide alleviates postoperative pain and spatial memory deficit in aged rats Peng Guoa, Si-Ping Hub, a b
MARK
⁎
Department of Anesthesiology, The Central Hospital of Shaoxing City, Shaoxing 312030, Zhejiang, China Department of Anesthesiology, Huzhou Central Hospital, Huzhou 313000, Zhejiang, China
A R T I C L E I N F O
A B S T R A C T
Keywords: postoperative cognitive dysfunction (POCD) thalidomide pain NMDA receptors inflammation
Pain is a major risk factor of post-operative cognitive dysfunction (POCD) in aged population. We investigated the effects of thalidomide, an anti-inflammatory and analgesic drug, on POCD in aged rats, and also explored the underlying mechanisms. Laparotomy was performed under anesthesia in aged rats (24–25 months) to establish POCD models. Thalidomide (5–50 mg/kg) was intraperitoneally administered immediately after laparotomy. Within 12 h after the operation, pain symptoms were assessed by rat grimace scale (RGS). Within postoperative day (POD) 3–14, spatial memory was evaluated using performance errors in a radial arm maze. Protein levels of inflammatory cytokines and N-methyl-D-aspartate (NMDA) receptors were measured on POD 14. POCD rats treated with thalidomide showed decreased RGS and performance errors, compared with saline-treated POCD rats. Single administration of thalidomide significantly reduced production of cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-1β) in serum but not in the brain, and attenuated upregulation of NMDA receptor (NR) 2A/B subunits in the hippocampus at POD 14. MK-801 abolished thalidomide-induced attenuation of spatial memory deficits. Our results support that thalidomide could disrupt the development of post-operative memory deficit in aged rats through its long-term regulation of NMDA receptors (NRs) in the hippocampus. Therefore, thalidomide might provide a new means to prevent the development of POCD.
1. Introduction Patients subjected to anesthesia and surgery show attention deficits, mild cognitive impairment, and spatial memory dysfunction. These syndromes can be categorized as postoperative cognitive dysfunction (POCD) [1]. POCD has higher prevalence in elderly patient, 14% of which experience cognitive decline and confusion up to 4 months after surgery [2,3]. The risk factors include ageing, preoperative stress, drugs, trauma injury, and postoperative pain [1]. Although pathophysiology of POCD is uncertain, management of postoperative pain is one of options proved effective to prevent POCD [4]. One clinical study revealed that elderly patients with non-cardiac surgery who received postoperative anesthesia is less likely to experience POCD [5]. Similarly, postoperative acute pain is found to exacerbate memory deficits after laparotomy in aged rats [6]. The goal of post-operative pain management is to relieve pain while minimizing side-effects. Although opioids are on the front line of effective analgesics, they also carry many undesirable side-effects, including sedation, respiratory depression, nausea and vomiting, overdose, and addiction [7]. Non-steroidal anti-inflammatory drugs
(NSAIDs) are useful for postoperative pain management, especially for mild and moderate, but not severe pain [8]. Therefore, new and more effective treatment modalities are needed for postoperative pain management. Thalidomide was first introduced in 1957 as a non-barbiturate sedative drug for the treatment of sleep disorders and morning sickness. Because thalidomide inhibits the synthesis and expression of multiple cytokines including interleukine-1β (IL-1β), IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α) [9], it has been used in inflammatory disorders and cancers [10,11]. Consistent with the notion that inflammation plays key roles in pain sensitization [12], thalidomide effectively relieves complex regional pain syndrome [10] and neuropathic pain, such as chronic sciatic nerve injury, lumbar 5 ventral root transection neuropathy, and chemotherapy-related pain [11,13,14]. Recent studies indicate that inhibition of systemic inflammatory process may interrupt pathogenesis of POCD [15,16]. In the present study, we revealed that thalidomide could disrupt the development of post-operative memory deficit in aged rats through its long-term regulation of NMDA receptors (NRs) in the hippocampus. Therefore,
Abbreviations: POCD, postoperative cognitive dysfunction; RGS, grimace scale; POD, postoperative day; NMDA, N-methyl-D-aspartate; TNF-α, tumor necrosis factor-α; IL, interleukin ⁎ Corresponding author: Department of Anesthesiology, Huzhou Central Hospital, No.198 Hongqi Rd, Huzhou 313000, Zhejiang, China. E-mail address: [email protected] (S.-P. Hu). http://dx.doi.org/10.1016/j.biopha.2017.08.114 Received 23 May 2017; Received in revised form 18 August 2017; Accepted 24 August 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 95 (2017) 583–588
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thalidomide might provide a new means to prevent the development of POCD.
correct arm choices (the first visit to reward arms); number of incorrect arm choices (the first visit to non-reward arms).
2. Methods
2.6. Western blot
2.1. Animal All animal care and experimental protocols were approved by Animal Care and Research Committee in The Central Hospital of Shaoxing City. Aged male Wistar rats (24–25 months old, 550 and 640 g) were used in this study. All rats were housed at 22 ± 2 °C, 45–75% relative humidity with 12 h light–dark cycle, and water and food were provided ad libitum.
Protein from the hippocampus, the PFC, and the amygdala was extracted for standard western blot assay. Rabbit anti-NR2A/B (1:500, Millipore, Billerica, MA, USA) and mouse anti-GAPDH (1:10000, Abcam, Cambridge, MA, USA) were used as primary antibodies. Antibody–protein complexes were visualized by chemiluminescent reagents. Band density was quantified by imaging quantification. Ratios of the band density for the protein of interest to that for GAPDH were calculated.
2.2. Anesthesia, laparotomy and drug treatment
2.7. ELISA assay of TNF-α and IL-1β
Rats were randomly divided into 5 experimental groups (n = 10 rats/group): A sham group received anesthesia, but without laparotomy; other 4 surgery groups all received anesthesia and laparotomy, and by the end of these treatments, individual surgery groups were respectively subjected to single intraperitoneal injection of saline (vehicle), 5, 20, or 50 mg/kg thalidomide. Simple laparotomy was performed according to previously reported methods [6]. Briefly, anesthesia was induced with 3% isoflurane, and later maintained with 1.2% isoflurane for 2 h. A 1.0-cm midline longitudinal incision was made through the skin, the abdominal muscle, and the peritoneum. The muscle layers were sutured with 5-0 Vicryl sutures, and the skin was closed with tissue adhesive glue. Thalidomide and MK-801 were purchased from Sigma Aldrich (USA), and were dissolved in saline. MK-801 (0.1 mg/kg) was given intraperitoneally (i.p.) before surgery; saline or thalidomide was given i.p. after laparotomy.
TNF-α and IL-1β in serum and brain were measured using ELISA ready-SET-Go!® assay kits (eBioscience, San Diego, USA), according to the instruction provided by the manufacturer. Protein level was expressed as pg/μg of total proteins determined over an albumin standard curve. 2.8. Statistical analysis Data are presented as mean ± SD. Statistical significance was determined by one- or two-way ANOVA with Dunnett’s or Tukey’s posthoc tests using the GraphPad Prism® 5 software, depending on whether the data passes the normality test. Difference was considered statistically significant when P values were less than 0.05. 3. Results 3.1. Effect of thalidomide on postoperative acute pain in aged rats
2.3. Pain measurement Rat grimace score (RGS) was used to evaluate the intensity of pain. Thalidomide (5–50 mg/kg) was administrated by i.p. injection after laparotomy. Compared with sham group, all surgical groups developed significant acute pain starting 2 h postoperatively, evidenced by higher RGS (p < 0.05, One-way ANOVA), and recovered to baseline level 12 h after surgery (p > 0.05, One-way ANOVA) (Fig. 1a). The concentration-response relationship showed (Fig. 1a) that single injection of thalidomide attenuated pain intensity in a dose-dependent manner, with statistical significance at doses of 20 and 50 mg/kg (p < 0.01). Additionally, thalidomide at 20 and 50 mg/kg induced a faster recovery of postoperative pain, compared with saline treatment (p < 0.05) (Fig. 1a). These data suggest that single administration of thalidomide has anti-analgesic effect on acute pain following laparotomy. Open-field test on post-operative day 3 (POD 3) showed that there was no difference in locomotor activity among experimental groups (p > 0.05) (Fig. 1b). Time lapsed before arm choice in the maze was analyzed for evaluation of task motivation. As shown in Fig. 1c, latency for arm choice did not differ between groups (p > 0.05). These data indicate that neither surgery nor thalidomide treatment changed locomotion and task motivation in aged rats.
Acute postoperative pain intensity was determined using a rat grimace scale (RGS) as previously reported [17]. Briefly, rats were acclimated in a clear plastic cage with a camera immediately outside of one wall for 10 mins. The activity of the rats was digitally videotaped during acclimation phase. Frame of front view of the head was grabbed randomly. Each frame should include the head with most of action units visible (i.e., nose, cheek, whiskers, eyes, and ears). Five facial features as indices of pain were defined: orbital tightening, nose/cheek fluttering, ear position, and whisker changes. Pain intensity ratings were coded using a three point scale from 0 (not present) to 2 (severe) to facial features presented. 2.4. Open field test for measurement of locomotion Rats were subjected to open field test on postoperative day 3. They were placed in a rectangle chamber, which was equipped with photobeams. The activity of the rats was quantified as total count of beam breaks in 60 mins. 2.5. Radial arm maze for measurement of spatial memory
3.2. Effect of thalidomide on spatial memory performance A standard radial arm maze was used to measure spatial memory, as previously reported [18]. Briefly, the maze contains a central platform with a diameter of 34, which was surrounded with a Plexiglass wall and 12 radial arms (50 cm long and 11 cm wide, with 20 cm transparent wall). A reward pellet (45 mg) was placed randomly at the end of 6 out of 12 arms in a plastic cup. Rats were first acclimated in maze for 2 days. During the trial, rats were placed in the center of the maze and allowed to explore all arms. The following data were record: the latency for per arm choice; the arm (reward or non-reward) the rat entered each time; the total time spent for obtaining all 6 food rewards; number of
Spatial memory performance was analyzed using a radial arm maze from POD 3–14. Compared to non-surgical sham group, the working memory errors in all surgical groups were significantly higher than those in sham group from POD 5–14 (p < 0.05) (Fig. 2a, b), suggesting that laparotomy resulted in spatial memory deficit. Single injection of thalidomide by the end of surgery significantly reduced memory errors in a dose-dependent manner at POD 8, 10, 12, 14, with significant difference at 20 and 50 mg/kg (p < 0.05) (Fig. 2a, b). The results suggest that thalidomide not only effectively relieved postoperative 584
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Fig. 1. Effects of thalidomide on postoperative RGS and locomotion in aged rats. Aged rats were subjected to laparotomy, and doses of thalidomide (5–50 mg/kg) were intraperitoneally administered by the end of laparotomy. (a) Acute pain was assessed with rat grimace scores (RGS) before and 2–12 h after operation. (b) Locomotor activity in an open field was measured at post-operative day (POD) 3. (c) The average of latency for the choices of individual arms was determined. Data are presented as mean ± SEM (n = 10 in each group). One-way ANOVA with post hoc Tukey test was used for statistical analyses. * p < 0.05 and ** p < 0.01 vs. Sham group; # p < 0.05 and ## p < 0.01 vs. Vehicle group.
impairment [6]. The levels of NR2 subunits in the hippocampus, the PFC, and the amygdala were determined using western blot at POD 14. Rats in surgical group displayed significant increase in NR2 protein levels in the hippocampus (p < 0.01) (Fig. 4a) that were reduced by single injection of thalidomide in a dose-dependent manner, with a statistical significance at doses of 20 mg/kg and 50 mg/kg (p < 0.05) (Fig. 4a). In contrast, there was no significant difference in NR2 expression in the PFC and the amygdala among all groups (p > 0.05) (Fig. 4b and c). The data suggest that NR2 were selectively up-regulated in the hippocampus after surgery in aged rats, and thalidomide could counteract the increase of NR2. Next, we further examined the involvement of NRs in improvement of postoperative memory by thalidomide. Consistent with previous report [20,6], pretreatment with MK-801, a non-competitive NMDAR antagonist, significantly increased working memory errors in surgical group, compared with saline treated group (p < 0.05) (Fig. 5a). Furthermore, we observed that pretreatment of MK-801 abolished thalidomide improvement of postoperative working memory (p < 0.01)
pain, but also attenuated postoperative memory deficits in aged rats. 3.3. Effect of thalidomide on pro-inflammatory cytokines Inflammatory responses have been associated with various pain states and POCD [16,19]. As shown in Fig. 3a, b, both TNF-α and IL-1β in serum were up-regulated after laparotomy (p < 0.01), detected by ELISA on POD 14. Thalidomide reduced TNF-α and IL-1β in serum in a dose-dependent manner, with significant difference at 20 mg/kg and 50 mg/kg, compared with saline treatment (p < 0.05) (Fig. 3a, b). However, the level of TNF-α and IL-1β in the brain did not differ among all groups (p > 0.05) (Fig. 3c, d). The results suggest that thalidomide may attenuate postoperative inflammatory responses in peripheral but not in the central nervous system. 3.4. Effect of thalidomide on NR2 expression NRs have been proposed to participate in pain-related cognitive
Fig. 2. Effect of thalidomide on postoperative spatial memory in aged rats. Postoperative spatial memory at post-operative day (POD) 3–14 was evaluated with memory errors in radial arm maze assay. Both memory errors at each time point (a) and total errors throughout the entire testing period (b) were analyzed with one-way ANOVA with post hoc Tukey test. Data are presented as mean ± SEM (n = 10 in each group). * p < 0.05 and ** p < 0.01 vs. Sham group; # p < 0.05 and ## p < 0.01 vs. Vehicle group.
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Fig. 3. Long-term effects of thalidomide on postoperative cytokine levels in aged rats. TNF-α and IL-1β levels in serum (a) and brain (b) were quantified with ELISA assay. Serum and brain tissues were harvested after completion of behavioral testing at post-operative day (POD) 14. Data are presented as mean ± SEM. One-way ANOVA with post hoc Tukey test was used for statistical analyses. ** p < 0.01 vs. Sham group. # p < 0.05 and ##p < 0.01 vs. Vehicle group.
2b). In these tests, thalidomide relieved acute postoperative pain within 2 h, while improved postoperative spatial memory 5–6 days after surgery. The drastically different time courses of these two effects suggest that diverse underlying mechanisms could exist. As half-life of thalidomide in the body is roughly 5–9 h [9], anti-nociceptive effects of thalidomide could be associated with its immediate action. However, thalidomide enhanced cognitive function in postoperative condition probably through its secondary action following its analgesic effect. Our data suggest the involvement of postoperative pain in POCD and effective management of postoperative pain with thalidomide may be beneficial for the prevention of POCD. Thalidomide can easily cross blood brain barrier after acute systemic administration, and dramatically reduces the expression and synthesis of multiple cytokines in peripheral and central nervous system, including IL-1β, IL-6, and TNF-α [11,9]. Immunomodulatory and anti-inflammatory properties of thalidomide are critical for its antinociceptive effects. We observed that laparotomy resulted in a long lasting peripheral inflammatory response: increased expression in serum TNF-α and IL-1β, at POD 14 (Fig. 3a, b), consistent with other preclinical studies [27,28]. Although at POD 14, thalidomide was no longer present in the body, we still observed reduced levels of the production of TNF-α and IL-1β in the serum (Fig. 3a, b). The result suggests that thalidomide could have long-term anti-inflammatory effects. Considering the important roles of inflammation in neuropathic pain, further investigations are warranted to elucidate whether aged rats received laparotomy show spontaneous pain behaviors co-existing with spatial memory deficit. Previous studies have identified a possible causal relationship between surgery, inflammation, and memory impairment. Evidence suggests that peripheral cytokines can pass signals to the brain through blood and neural routes, and then stimulating production of cytokines in the hippocampus [29,30]. Likewise, i.p. injection of lipopolysaccharide triggers activation of microglia in the brain and upregulation of cytokines (including IL-1β) in the hippocampus, meanwhile, results in memory impairment [31]. This is also supported by an interventional study showing that surgery induced peripheral and hippocampal cytokine expression (TNF-α and IL-1β) is associated with
(Fig. 5b). These results indicate that NRs might be involved in thalidomide-induced improvement of postoperative spatial memory deficits in aged rats.
4. Discussion Cardiac surgery or non-cardiac surgery with larger and more invasive procedures, such as abdominal, thoracic, and vascular surgery, present a greater risk than outpatient surgery for a relatively higher incidence of POCD [21,3]. Besides the type of surgery, it is proposed that ageing is another major risk factor of POCD [3,22]. POCD is evident in 26% of elderly patients 7 days after major non-cardiac surgery [23], and still presents 3 months after surgery in 7% of patients aged 60–69 and in 14% of patients over 69 years old [4]. In the present study, we performed abdominal laparotomy in 24–25 months old rats to successfully establish POCD models. Spatial memory in these rats was impaired showing more memory errors in radial arm maze, but no effect on locomotor activity and motivation. Although isoflurane anesthesia is toxic to the ageing brain [24], the link between anesthesia or surgery and postoperative cognitive problems has been elusive [6,25,26]. In the present study, rats in laparotomy plus isoflurane anesthesia displayed more severe postoperative pain and stronger spatial memory deficits in comparison with rats receiving isoflurane only (sham group) (Fig. 1a). These results suggest that laparotomy (surgery) may play an important role in postoperative spatial memory deficits. Studies on pathophysiological mechanisms of POCD have identified that postoperative pain may be associated with POCD. For instance, morphine and non-steroidal anti-inflammatory drugs (NSAIDs), at doses relieving acute postoperative pain, attenuated postoperative memory deficits [25,4]. Here, we tested effects of thalidomide on both acute postoperative pain and spatial memory deficit. Acute postoperative pain was evident 2 h after laparotomy and lasted for 8 h (Fig. 1a). Surgical rats subjected to single dose of thalidomide by the end of the surgery showed not only accelerated recovery from acute postoperative pain, but also reversed deterioration in spatial memory function at POD 14, compared with vehicle-treated rats (Fig. 1a, 2a, 586
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Fig. 4. Thalidomide reversed postoperative upregulation of NR2A/B in rat hippocampus. NR2A/B in the hippocampus (a), the prefrontal cortex (b) and the amygdala (c) at postoperative day (POD) 14 was analyzed with western blot. GAPDH was used as a loading control. Data are presented as mean ± SEM. One-way ANOVA with post hoc Tukey test was used for statistical analyses. * p < 0.05 and ** p < 0.01 vs. Sham group. # p < 0.05 vs. Vehicle group.
spatial memory following laparotomy by thalidomide might be NRsdependent, because 1) thalidomide dose-dependently counteracted the upregulation of NR2; 2) thalidomide at the dose reversed NR2 upregulation improved spatial memory; and 3) the beneficial effect of thalidomide no longer existed after pre-treatment with MK-801 (Fig. 5). At POD 14, we also observed that the upregulation of NR2 subunits was selective in the hippocampus, but not in the PFC and the amygdala (Fig. 4a, b, c).
memory impairment, and both antagonizing and knocking-out IL-1β receptors mitigate the neuro-inflammatory reaction and memory dysfunction following surgery [15]. In contrast, we observed upregulation of TNF-α and IL-1β in serum but not in the brain following laparotomy. The data suggest that central inflammation may not mediate spatial memory deficits in aged rats following laparotomy. Synaptic plasticity is a key cellular mechanism underlying learning and memory, and relies on NMDA receptor (NR) function [32–34]. Either downregulation or upregulation of NRs leads to spatial memory deficit [32,35]. That is, NRs should be kept at optimized levels to maintain normal cognitive functions. This notion could explain our observation that laparotomy upregulated NR2 subunits in the hippocampus and impaired spatial memory, and the behavioral outcome was replicated with antagonizing NRs with MK-801. The improvement of
5. Conclusions In summary, single dose of thalidomide by the end of surgery effectively mitigates postoperative reactions in aged rats, including acute pain symptoms, and long-term peripheral inflammation and spatial 587
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Fig. 5. Thalidomide improved postoperative spatial memory deficit probably by regulating NMDA receptors. Spatial memory performance was evaluated by memory errors in a standard radial arm maze at (POD) 3–14 (a) and total errors over the entire testing period (b). MK-801 pre-treatment abolished the effect of thalidomide (50 mg/kg) on spatial memory performance in rats. Data are presented as mean ± SEM (n = 10 in each group). One-way ANOVA with post hoc Dunnett’s test was used for statistical analyses. * p < 0.05 and ** p < 0.01 vs. Vehicle group. ## p < 0.01 vs. thalidomide group. ‘n.s.’ indicates no statistical difference. [15] M. Cibelli, A.R. Fidalgo, N. Terrando, D. Ma, C. Monaco, M. Feldmann, M. Takata, I.J. Lever, J. Nanchahal, M.S. Fanselow, M. Maze, Role of interleukin-1beta in postoperative cognitive dysfunction, Ann. Neurol. 68 (2010) 360–368. [16] L. Peng, L. Xu, W. 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