The role of 5-HT7R in the memory impairment of mice induced by long-term isoflurane anesthesia.

General anesthesia is widely utilized in the clinic for surgical and diagnostic procedures. However, growing evidence suggests that anesthetic exposure may affect cognitive function negatively. Unfortunately, little is known about the underlying mechanisms and efficient prevention and therapeutic strategies for the anesthesia-induced cognitive dysfunction. 5-HT7R, a serotonin receptor family member, is functionally associated with learning and memory. It has recently become a potential therapeutic target in various neurological diseases as its ligands have a wide range of neuropharmacological effects. However, it remains unknown the role of 5-HT7R in the long-term isoflurane anesthesia-induced memory impairment and whether prior activation or blockade of 5-HT7R before anesthesia has modulating effects on this memory impairment. In this study, 5-HT7R selective agonist LP-211 and 5-HT7R selective antagonist SB-269970 were pretreated intraperitoneally to mice before anesthesia; their effects on the cognitive performance of mice were assessed using fear conditioning test and novel object recognition test. Furthermore, the transcriptional level of 5-HT7R in the hippocampus was detected using qRT-PCR, and proteomics was conducted to probe the underlying mechanisms. As a result, long-term exposure to isoflurane anesthesia caused memory impairment and an increase in hippocampal 5-HT7R mRNA expression, which could be attenuated by SB-269970 pretreatment but not LP-211pretreatment. According to the proteomics results, the antiamnestic effect of SB-269970 pretreatment was probably attributed to its action on the gene expression of Slc6a11, Itpka, Arf3, Srcin1, and Epb41l2, and synapse organization in the hippocampus. In conclusion, 5-HT7R is involved in the memory impairment induced by long-term isoflurane anesthesia, and the prior blockade of 5-HT7R with SB-269970 protects the memory impairment. This finding may help to improve the understanding of the long-term isoflurane anesthesia-induced memory impairment and to construct potential preventive and therapeutic strategies for the adverse effects after long-term isoflurane exposure.

Effects of 5-HT7 receptors on circadian rhythm of mice anesthetized with isoflurane.

The role of the serotonin 7 receptor (5-HT7 receptor) subtype in a number of domains has been widely recognized, but its role in the regulation of changes of the circadian rhythm after anesthesia is still unclear. We used intraperitoneal injection of 5-HT7 receptor agonist LP-211 or antagonist SB-269970 in mice to influence the level of 5-HT7 receptor protein in the SCN and to observe the role of this receptor on circadian rhythm changes after isoflurane anesthesia. Our results show the appropriate dose of SB-269970 significantly alleviated the circadian rhythm disorder induced by isoflurane anesthesia, while LP-211 significantly aggravated it after anesthesia, which is different from the phase shift that can be caused by the administration of LP-211 before anesthesia. These findings may indicate the 5-HT7 receptor plays a complex role in the regulation of circadian rhythm after anesthesia. Our findings may provide some positive significance for alleviating circadian rhythm disorder in patients after anesthesia and ultimately promoting rapid postoperative recovery.

Time-restricted feeding alters isoflurane-induced memory deficits.

Food consumption during the rest phase promotes circadian desynchrony, which is corrected with harmful physiological and mental disorders. Previously, we found that circadian desynchrony was involved in isoflurane-induced cognitive impairment. Here, we scheduled food access to modulate daily rhythm to examine its impact on isoflurane-induced cognitive impairments. Mice were randomly transferred to restricted feeding (RF) time groups: Control group (Zeitgeber time (ZT) 0-ZT24, ad libitum feeding), Day-Feeding group (ZT0-ZT12, misaligned feeding), and Night-Feeding group (ZT12-ZT24, aligned feeding). Then, some of them were subjected to 5 h of 1.3% isoflurane anaesthesia from ZT14 to ZT19 and were divided into the Control + Anes group, the Day-Feeding + Anes group, and the Night-Feeding + Anes group. Mini-Mitter was used to monitor the daily rhythm. Fear conditioning system was conducted to assess cognition of mice. We observed that the Night-Feeding group adapted to RF gradually, whereas the Day-Feeding group exhibited a disturbed daily rhythm. The Night-Feeding + Anes group exhibited a partially enhanced daily rhythm, whereas the Day-Feeding + Anes group exhibited sustained phase advances and diurnality score increase 7 days after isoflurane anaesthesia. Notably, in tests of hippocampus-dependent contextual memory, the Night-Feeding + Anes group demonstrated decreased deficits; the Day-Feeding + Anes group showed prolonged post-anaesthetic deficits 14 days after isoflurane anaesthesia. However, amygdala-dependent cued-fear conditioning post-anaesthesia was not altered by the RF schedule. In conclusion, we demonstrated that misaligned feeding disturbed the daily rhythm and led to persistent post-anaesthetic cognitive dysfunction. Aligned feeding enhanced the daily rhythm partially and improved post-anaesthetic cognitive dysfunction.

Validation of the Chinese version of the Amsterdam Preoperative Anxiety and Information Scale (APAIS).

BACKGROUND: Preoperative anxiety is an unpleasant state of tension that may impact patients' post-operative pain and satisfaction. The level of preoperative anxiety should be routinely identified. The Amsterdam Preoperative Anxiety and Information Scale (APAIS) is a self-reported questionnaire that is used to quickly assess preoperative anxiety and information needs with good psychometric properties. OBJECTIVES: To validate the Chinese version of the Amsterdam Preoperative Anxiety and Information Scale (APAIS) and to explore coping strategies used by patients in dealing with surgery and anesthetic. METHODS: The cross-cultural validation of APAIS involved the translation of a Chinese version of APAIS and an investigation of its psychometric properties and clinical applicability. Forward-back translation and a pilot study were performed to produce a Chinese adaptation of APAIS. The inpatients of the orthopedic, otolaryngology, and general surgery department scheduled for general anesthesia surgery were enrolled to complete psychometric testing. The reliability was assessed using Cronbach's alpha. Exploratory factor analysis and confirmatory factor analysis were calculated to assess construct validity. The criteria validity was analyzed using the correlation between APAIS and State-trait anxiety inventory-state (STAI-S) and Visual analogue scale-anxiety (VAS-A). Coping styles were evaluated using the Medical Coping Modes Questionnaire (MCMQ) score that covered three domains: confrontation, avoidance, and resignation. The impact of different coping styles on patients' anxiety was explored. RESULTS: A total of 204 valid questionnaires were collected the day before surgery. Cronbach's alpha coefficients were 0.862 for the anxiety scale and 0.830 for the information scale. Exploratory factor analysis with oblique rotation revealed two factors that explained 76.45% of the total variances. A confirmatory factor analysis showed a two-factor model with an adequate model fit (root mean square error of approximation: 0.073, goodness-of-fit: 0.966). The APAIS anxiety score significantly correlated with STAI-S (r = 0.717, P < 0.01) and VAS-A (r = 0.720, P < 0.01). For the three coping strategies, preoperative anxiety had a low correlation with confrontation (r = 0.33, P < 0.01) and resignation (r = 0.22, P < 0.05). CONCLUSIONS: The Chinese version of APAIS is a valid and reliable instrument for assessing preoperative anxiety. Use of this measurement tool for Chinese patients is feasible and shows promising results.

The role of CaMKII in neuropathic pain and fear memory in chronic constriction injury in rats.

Purpose/Aim of the study In this study, we sought to observe the effects of Ca2+/calmodulin-dependent protein kinase II (CaMKII) on neuropathic pain and fear memory in a rat model of chronic constriction injury (CCI). Materials and methods Rats were randomly divided into the Sham, Control, CCI and m-AIP groups. In the m-AIP group, an intrathecal injection of m-AIP, the specific antagonist of CaMKII, was given either pretreatment or posttreatment in rats. Mechanical allodynia and thermal hyperalgesia tests were used to test pain behavior, and the passive avoidance test was used to measure fear memory in rats. Results The right side of hippocampus tissues were taken at varying time points. The expression levels of CaMKII-α, pCaMKII-α, CaMKII-ß, pCaMKII-ß, NR2A, pNR2A, NR2B and pNR2B were detected by Western blot analysis. Significant pain behaviors and impaired cognitive function were shown after CCI surgery, accompanied by the upregulation of proteins in the hippocampus. Pretreatment with m-AIP appeared to provide a temporary improvement in pain and fear memory and decreased the expression of the above proteins in the hippocampus seven days after surgery. Furthermore, postoperative treatment with m-AIP provided relief for pain behavior and protein expression but did not affect fear memory. Conclusions These data suggested that CaMKII played an important role in the crosstalk between neuropathic pain and fear memory, indicating that CaMKII may be a potential therapeutic target for neuropathic pain treatment.

Circadian rhythm resynchronization improved isoflurane-induced cognitive dysfunction in aged mice.

Postoperative cognitive dysfunction (POCD) is a common clinical phenomenon characterized by cognitive deficits in patients after anesthesia and surgery. Advanced age is a significant independent risk factor for POCD. We previously reported that in young mice, sleep-wake rhythm is involved in the isoflurane-induced memory impairment. In present study, we sought to determine whether advanced age increased the risk of POCD through aggravated and prolonged post-anesthetic circadian disruption in the elderly. We constructed POCD model by submitting the mice to 5-h 1.3% isoflurane anesthesia from Zeitgeber Time (ZT) 14 to ZT19. Under novel object recognition assay (NOR) and Morris water maze (MWM) test, We found 5-h isoflurane anesthesia impaired the cognition of young mice for early 3 days after anesthesia but damaged the aged for at least 1 week. With Mini-Mitter continuously monitoring, a 3.22 ±â€¯0.75 h gross motor activity acrophase delay was manifested in young mice on D1, while in the aged mice, the gross motor activity phase shift lasted for 3 days, consistent with the body temperature rhythm trends of change. Melatonin has been considered as an effective remedy for circadian rhythm shift. In aged mice, melatonin was pretreated intragastrically at the dose of 10 mg/kg daily for 7 consecutive days before anesthesia. We found that melatonin prevented isoflurane-induced cognitive impairments by restoring the locomotor activity and temperature circadian rhythm via clock gene resynchronization. Overall, these results indicated that Long-term isoflurane anesthesia induced more aggravated and prolonged memory deficits and circadian rhythms disruption in aged mice. Melatonin could prevent isoflurane-induced cognitive impairments by circadian rhythm resynchronization.

Misaligned Feeding May Aggravate Pain by Disruption of Sleep-Awake Rhythm.

BACKGROUND: Increasing evidence suggests that patients with eating disorders are more likely to develop chronic pain. A misaligned diet has been reported to disrupt the sleep-awake rhythms. Combined with our previous investigation on circadian pain, we aimed to investigate the role of misaligned diet in the pain sensitivity and the underlying mechanisms. METHODS: Two-month-old C57BL/6J male mice were administered chronic constriction injury (CCI) surgery to establish neuropathic pain models. CCI mice were randomized to scheduled food access throughout the whole day (CCI-free), during the daytime (CCI-misaligned), and at night (CCI-aligned), respectively. The paw withdrawal mechanical threshold, indicating pain behavior, was measured by Von Frey. The gross motor activity pattern indicating the sleep-awake rhythm was monitored by Mini-Mitter. Melatonin (Mel) was administered to ameliorate the sleep-awake rhythm (CCI-free + Mel and CCI-misaligned + Mel). The expressions of circadian pain-related proteins were detected by quantitative polymerase chain reaction and western blot. The primary outcome is the pain threshold and the secondary outcome is the sleep-awake rhythm. RESULTS: Misaligned diet during the peri-CCI surgery period significantly decreased the paw withdrawal mechanical threshold compared with the CCI-free mice (day 14: 0.40 ± 0.09 vs 0.64 ± 0.15; P = .03;) and altered the sleep-awake rhythm. Mel pretreatment alleviated the increased pain (day 14, CCI-misaligned + Mel versus CCI-misaligned: day 14: 0.60 ± 0.13 vs 0.35 ± 0.12; P = .022) and the disrupted sleep-awake rhythm caused by misaligned feeding. The mRNA levels of N-methyl-D-aspartate receptor subtype 2B (NR2B), Ca/calmodulin-dependent protein kinase II (CaMKII), and cyclic adenosine monophosphate-response element binding protein (CREB) in the spinal dorsal horn increased in CCI-misaligned mice compared with the CCI-free mice. The phosphor-NR2B, phosphor-CaMKII, and phosphor-CREB also increased in CCI-misaligned mice compared with the CCI-free mice. However, the expressions of NR2B, CaMKII, and CREB were decreased in CCI-misaligned + Mel mice compared to CCI-misaligned mice at both transcriptional and translational levels. CONCLUSIONS: Misaligned diet might aggravate pain sensitivity through the disruption of the sleep-awake cycle, which could be recovered by Mel. NR2B-CaMKII-CREB may participate in the disruption of sleep-awake rhythm-mediated pain aggravation.

Perturbing NR2B-PSD-95 interaction relieves neuropathic pain by inactivating CaMKII-CREB signaling.

Neuropathic pain is characterized by central sensitization. The interaction between N-methyl-D-aspartate receptors (NMDARs) and postsynaptic density protein-95 (PSD-95) plays a major role in central sensitization. Here, we aimed to investigate the analgesic effect of disruption of the interaction between NMDAR and PSD-95. Chronic dorsal root ganglia compression model rats were used to mimic sciatica. Thermal hyperalgesia and mechanical allodynia were evaluated. The expression of spinal phospho-NR2B, PSD-95, calcium/calmodulin-dependent protein kinase II (CaMKII), and cAMP response element binding protein (CREB) was measured using western blotting. A mimetic peptide Myr-NR2B9c was injected intrathecally to disrupt the interaction between PSD-95 and NR2B and detected by coimmunoprecipitation. Chronic dorsal root ganglia compression surgery induced thermal hyperalgesia and mechanical allodynia, and upregulated pain-related proteins such as phospho-NR2B, PSD-95, CaMKII, and CREB expressions in the spinal cord. Myr-NR2B9c disrupted the interaction between NR2B-containing NMDARs and PSD-95 in the spinal cord. Intrathecal administration of Myr-NR2B9c attenuated neuropathic pain behaviors and downregulated the expressions of phospho-NR2B, PSD-95, CaMKII, and CREB in the spinal cord. The present study indicates that dissociation of NR2B-containing NMDARs from PSD-95 inactivates CaMKII and CREB signaling and relieves pain.

Activation of spinal alpha-7 nicotinic acetylcholine receptor shortens the duration of remifentanil-induced postoperative hyperalgesia by upregulating KCC2 in the spinal dorsal horn in rats.

Background Accumulating evidence has shown that the signal from spinal brain-derived neurotrophic factor/tyrosine receptor kinase B-K+-Cl- cotransporter-2 plays a critical role in the process of pain hypersensitivity. The activation of alpha-7 nicotinic acetylcholine receptors could have an analgesic effect on remifentanil-induced postoperative hyperalgesia. Nevertheless, whether intrathecal administration of PNU-120596, an alpha-7 nicotinic acetylcholine receptors selective type II positive allosteric modulator, before surgery could affect the duration of remifentanil-induced postoperative hyperalgesia remains unknown, and the effects of alpha-7 nicotinic acetylcholine receptors activation on the brain-derived neurotrophic factor/tyrosine receptor kinase B-K+-Cl- cotransporter-2 signal in the spinal dorsal horn of rats with remifentanil-induced postoperative hyperalgesia is still enigmatic. Results We demonstrated that the brain-derived neurotrophic factor/tyrosine receptor kinase B-K+-Cl- cotransporter-2 signal played a critical role in the development of remifentanil-induced postoperative hyperalgesia. Intrathecal administration of PNU-120596 (8 µg/kg, 15 min before surgery) was associated with earlier signs of recovery from remifentanil-induced postoperative hyperalgesia. Simultaneously, remifentanil-induced postoperative hyperalgesia-induced K+-Cl- cotransporter-2 downregulation was partly reversed and coincided with a decreased expression of brain-derived neurotrophic factor/tyrosine receptor kinase B in the spinal dorsal horn, approximately correlating with the time course of the nociceptive behavior. Moreover, intrathecal administration of the K+-Cl- cotransporter-2 inhibitor VU0240551 significantly reduced the analgesic effect of PNU-120596 on remifentanil-induced postoperative hyperalgesia. Conclusions The activation of alpha-7 nicotinic acetylcholine receptors induced a shorter duration of remifentanil-induced postoperative hyperalgesia by restoring the brain-derived neurotrophic factor/tyrosine receptor kinase B-K+-Cl- cotransporter-2 signal in the spinal dorsal horn of rats, which provides new insight into treatment in clinical postoperative pain management.

The Role of NR2B-CREB-miR212/132-CRTC1-CREB Signal Network in Pain Regulation In Vitro and In Vivo.

BACKGROUND: Chronic pain is a debilitating threat to human health, and its molecular mechanism remains undefined. Previous studies have illustrated a key role of cAMP response element-binding protein (CREB) in pain regulation; CREB-regulated transcription coactivator 1 (CRTC1) and microRNA212/132 (miR212/132) are also vital in synaptic plasticity. However, little is known about the interaction among these factors in pain condition. We conducted this experiment mainly to determine the crosstalk between CREB, CRTC1, and miR212/132 in vitro. Moreover, we explored the changes in hyperalgesia on chronic constrictive injury (CCI) mouse in vivo when given CREB-related adenovirus vectors, CRTC1-related adenovirus vectors, and miR212/132-locked nucleic acid (LNA). METHODS: We cultured primary neurons in the spinal cord of mouse embryos. Exogenous glutamate was added to cultured neurons to simulate in vivo pain process. Real-time quantitative polymerase chain reaction was used to determine changes of NR2B, CRTC1, CREB, and miR212/132 at the mRNA level; Western blot was used to detect p-NR2B, p-CREB, and CRTC1 at protein level. Von Frey cilia were used to study mechanical hyperalgesia in a murine model of CCI. CREB-miR (adenovirus vector interfering CREB gene), CREB-AD (adenovirus vector overexpressing CREB gene); CRTC1-miR (adenovirus vector interfering CRTC1 gene), CRTC1-AD (adenovirus vector overexpressing CRTC1 gene), and miR212/132-LNA were injected intrathecally. RESULTS: In vitro, 100 µmol/L glutamate induced p-CREB and miR212/132-LNA. CRTC1 protein was downregulated by CREB-miR and miR212/132-LNA. CRTC1 mRNA was upregulated by CREB-AD and downregulated by CREB-miR and miR212-LNA. P-CREB was upregulated by CRTC1-AD and downregulated by miR212/132. CREB mRNA was upregulated by CRTC1-AD and downregulated by CRTC1-miR. MiR212/132 was upregulated by CRTC1-AD and CREB-AD; downregulated by CREB-miR. In vivo, CRTC1-miR, CREB-miR, and miR212/132-LNA increased paw withdrawal mechanical threshold in various degrees. CONCLUSIONS: The NR2B-CREB-miR212/132-CRTC1-CREB signal network plays an important role in the regulation of pain. Intervening with any molecule in this signal network would reduce pain perception.

Antinociceptive effects of dehydrocorydaline in mouse models of inflammatory pain involve the opioid receptor and inflammatory cytokines.

Dehydrocorydaline (DHC) is an alkaloidal component isolated from Rhizoma corydalis. Previous studies have shown that DHC has anti-inflammatory and anti-tumor effects and that it can protect the cardiovascular system. However, there are few studies of the antinociceptive effects of DHC in vivo. This study explored the antinociceptive effects and possible mechanisms of DHC in mice using two inflammatory pain models: the acetic acid-induced writhing test and the formalin paw test. The intraperitoneal administration of DHC (3.6, 6 or 10 mg/kg) showed a dose-dependent antinociceptive effect in the acetic acid-induced writhing test and significantly attenuated the formalin-induced pain responses in mice. The antinociceptive effects of DHC were not associated with changes in the locomotor activity or motor responses of animals, and no obvious acute or chronic toxic effects were observed in the mice. Furthermore, the use of naloxone confirmed the involvement of the opioid receptor in the central antinociceptive effects of DHC. DHC reduced formalin-induced paw edema, which indicated that DHC may produce an anti-inflammatory effect in the periphery. In the formalin test, DHC decreased the expression of caspase 6 (CASP6), TNF-α, IL-1ß and IL-6 proteins in the spinal cord. These findings confirm that DHC has antinociceptive effects in mice.

Regulation of the NR2B-CREB-CRTC1 Signaling Pathway Contributes to Circadian Pain in Murine Model of Chronic Constriction Injury.

BACKGROUND: Numerous clinical investigations have revealed the circadian rhythm changes in the perception of chronic pain, and most clinical chronic pain types peak in the night. However, it is still undiscovered whether circadian rhythm of pain exists in rodents and the specific mechanism that may underlie it. Our study was conducted to investigate the rhythmic changes of hyperalgesia behavior in a chronic constrictive injury (CCI) model of rodents and to explore the role of the N-methyl-d-aspartate receptor 2B (NR2B)-cAMP response element binding protein (CREB)-CREB-regulated transcription coactivator 1 (CRTC1) signaling pathway in this pain rhythm. METHODS: A CCI operation was performed to mimic clinical chronic pain. Paw mechanical withdrawal threshold and paw withdrawal thermal latency were used to test pain behavior in rats; a von Frey cilia test was used to test mechanical hyperalgesia in mice at Zeitgeber time (ZT) 4, ZT10, ZT16, and ZT22 for 14 contiguous days. The relative mRNA and protein expression of NR2B, CREB and CRTC1 in the suprachiasmatic nuclei and the dorsal horn were measured by real-time polymerase chain reaction and Western blot. CRTC1 and CREB interference adenovirus vectors were injected intrathecally at 2 time points, respectively (ZT12 and ZT0), to further explore the proper time point for pain treatment. RESULTS: During the period of chronic pain state, the pain behavior of CCI rodents showed a circadian rhythm with the peak at ZT4 or ZT10 daily. The pain thresholds were significantly different between the activity period and the rest period. The expressions of NR2B, CRTC1, and CREB at the spinal level were consistent with the pain rhythm. The intrathecal treatment with CRTC1 or CREB interference adenovirus from day 7 to day 9 after CCI surgery markedly improved pain behaviors. Nevertheless, when given at ZT0, they were both more effective at relieving peak pain than drugs given at ZT12. CONCLUSIONS: Pain behavior in the chronic pain of CCI displayed circadian rhythm and was associated with circadian secretion of pain-related receptors. The NR2B-CREB-CRTC1 signaling pathway may play a crucial role in this rhythm. Moreover, our results suggest that measures to relieve pain should be taken before pain reaches its peak.

Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep-wake rhythm in mice.

BACKGROUND: Sleep plays an important role in memory processing. However, its role in anesthesia-induced cognitive dysfunction was not revealed. Our study sought to investigate the connection between the cognition decline and sleep-wake rhythm disorders after long-term isoflurane anesthesia in mice. Also, we examined the effect of exogenous melatonin pretreatment on both cognitive function and circadian rhythm. Furthermore, we discussed whether NR2B (N-methyl-D-aspartate receptor 2B subunit)-CREB (cAMP-response element binding protein) signaling pathway was involved in this course. METHODS: 2-month-old male C57/BL-6J mice were submitted to long-term anesthesia using 1% isoflurane from CT (Circadian Time) 14 to CT20. Melatonin pretreatment were conducted before anesthesia for 7 Days. Intellicage for mice and Mini-Mitter were applied to monitor spatial memory and gross motor activity which can reflect cognition and sleep-wake rhythm. Messenger RNA and protein expression of right hippocampus NR2B and CREB were examined by RT-PCR and Western blot. RESULTS: 6h isoflurane anesthesia led to impaired spatial memory from Day 3 to Day 10 in mice accompanied by the disruption of sleep-wake rhythm. Meanwhile, the hippocampus CREB and NR2B expression declined in step. Melatonin pretreatment ameliorated disturbed sleep-wake cycle, improved isoflurane-induced cognitive dysfunction, and reversed the down-regulation of CREB and NR2B expression. CONCLUSIONS: Our data demonstrate that sleep-wake rhythm is involved in the isoflurane-induced cognition impairment and pretreatment of melatonin has a positive effect on circadian normalization and cognition reversal. Also, NR2B-CREB signaling pathway has a critical role in this process. This study provides us a new strategy for anesthesia-induced cognitive dysfunction therapy.