Preoperative anxiety induces chronic postoperative pain by activating astrocytes in the anterior cingulate cortex region

SUMMARY

OBJECTIVE

The study aims to explore the relationship between preoperative anxiety and chronic postoperative pain.

METHODS

A total of forty rats were divided into four groups, control, single-prolonged stress alone, Hysterectomy alone, and SPS+ Hysterectomy. The paw withdrawal mechanical thresholds (PWMT) were examined. qRT-PCR and western blotting assay were performed to detect the GFAP expression in astrocytes isolated from the anterior cingulate cortex (ACC) region. In addition, the long-term potentiation (LTP) in ACC was examined.

RESULTS

Rats in the SPS group or the Hysterectomy alone group had no significant effect on chronic pain formation, but SPS can significantly induce chronic pain after surgery. Astrocytes were still active, and the LTP was significantly increased three days after modeling in the SPS+Hysterectomy group.

CONCLUSIONS

anxiety can induce chronic pain by activating astrocytes in the ACC region.

Ansiedade; Pain, postoperative; Astrocytes

RESUMO

OBJETIVO

O objetivo deste estudo é explorar a relação entre a ansiedade no pré-operatório e a dor crônica no pós-operatório.

MÉTODOS

Um total de 40 ratos foram divididos em quatro grupos: controle, estresse prolongado (SPS), histerectomia e SPS + histerectomia. Os limiares de retirada da pata em resposta a estímulo mecânico (PWMT) foram examinados. Ensaios qRT-PCR e imunoenzimáticos (western blotting) foram realizados para detectar a expressão de GFAP em astrócitos isolados da região do córtex cingulado anterior (CCA). Além disso, a potenciação de longa duração (LTP) no CCA também foi examinada.

RESULTADOS

Os ratos no grupo de estresse prolongado e no grupo de histerectomia não apresentaram nenhum efeito significativo na formação de dor crônica. Porém, o estresse prolongado foi capaz de induzir dor crônica significativamente após a cirurgia. Três dias após o modelo, o grupo de SPS + histerectomia ainda apresentava astrócitos ativos e LTP significativamente maior.

CONCLUSÃO

A ansiedade pode provocar dor crônica através da ativação de astrócitos na região do CCA.

Ansiedade; Pós-operatória, dor; Astrócitos

INTRODUCTION

Clinical investigations have demonstrated that patients with serious anxiety have lower pain threshold and higher sensitivity to pain. In particular, patients who experience serious preoperative anxiety may suffer from postoperative chronic pain ¹,². The mechanism proposed is that anxiety could increase the central sensitivity of pain by regulating corticotropin-releasing and inflammatory factors such as IL-1, IL-6, IL-10, TNF-α, and noradrenaline, which would increase the pain feeling. ³-⁵ Anxiety can also have a negative impact on the treatment of painful diseases by enhancing the pain feeling. It is important to use psychological intervention to relief patients’ pain and improve their quality of life. However, those treatments sometimes may not be effective. Therefore, it is urgent that we find how to mediate pain enhanced by anxiety.

Neuroactive substances and receptors are abundant on the neurons in the posterior horn of the spinal cord ⁶,⁷. Previous studies have generally believed that only neurons and their associated neurotransmitters played important roles in pain development, while the role of glial cells was ignored. Recently, studies have found that glial cells in the nervous system play various roles. Glial cells are essential in the propagation of pain signaling, especially in the course of neuropathic pain ⁸. It was found that the expression of glial fibrillary acidic protein (GFAP) was significantly increased in a rat model of hyperalgesia, suggesting that glial cells may be activated and participate in the process of pain sensitization. Some studies have also found that the activation of glial cells (including astrocytes and microglia) were usually observed during many chronic pain model⁹-¹².

Astrocytes are particularly sensitive to the changes in the microenvironment around the neurons, and once receiving the signal, activated astrocytes can release massive proinflammatory and neuroactive substances ¹³. The anterior cingulate cortex (ACC) region is a cortical area in the brain that contributes to the regulations of pain and emotions and contains abundant astrocytes. ¹⁴,¹⁵ Some studies using neuroimaging techniques demonstrated increased activity in the ACC during chronic pain formation ¹⁶,¹⁷. In addition, it has been reported that astrocytes in the ACC can be activated in the inflammatory pain model ¹⁸. As such, we wondered if preoperative anxiety can activate astrocytes in the ACC, subsequently leading to the formation of postoperative chronic pain.

This study investigated the effects of preoperative anxiety on postoperative chronic pain by stablishing a preoperative anxiety model. By using techniques such as ACC slice, western blot, RT-PCR, immunohistochemistry, immunofluorescence, and electrophysiology, the alterations of astrocyte activation and LTP in the mice model were evaluated. As a result, we found that preoperative anxiety can induce postoperative chronic pain by activating astrocytes in the ACC region.

METHODS

Animals

Adult female Sprague-Dawley rats (210–260 g) were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. Rats were housed six per cage in a controlled environment, fed a standard rodent food, and allowed distilled water ad libitum. Rats were given at least one week to adapt to the new environment before any manipulation. Forty rats were randomly assigned to four groups (n=10): control group; SPS group (Single-prolonged stress model); Hysterectomy only group; SPS + Hysterectomy group.

Isolation of astrocytes in the rat ACC region.

Five rats in each group were euthanized around three weeks after modeling for astrocytes isolation by placing rats in a CO2 enriched tank. The ACC region in the brain was dissected, and the meninges were removed. The rodent brain was kept in Hank’s Balanced Salt Solution (HBSS) containing 0.05% trypsin and 0.005% DNase at 4 ^OC during the dissection process. The tissue was then triturated for around 5 min by pipetting up and down using a Pasteur pipette. Triturated tissue was then centrifuged at 400 g for 5 min at 22^OC. After removing the supernatant, the pellet was resuspended into the HBSS containing 34 U papain/ml, 0.02% cysteine and 0.005% DNase and triturated for further 5 min. Followed by another centrifugation at 400 g for 5 min at 22^OC, the cells were resuspended in HBSS containing 0.005% DNase and put on ice for 30 min. The supernatant was collected and centrifuged again for another 10 min at 400 g.

Single-prolonged stress (SPS) modeling

When establishing the SPS model, rats were first restrained for 2 hr by placing them in plastic bags individually and immobilizing tightly. Several holes were made on the bag to make sure rats could breathe freely. After restraint, the rats were placed in a swimming pool (24 cm in diameter, 50 cm in height) at 24°C and forced to swim for 20 min. After 15-min rest, the rats in all groups were anesthetized with isoflurane. Thereafter, the rats were placed in a ventilated place until they waked up naturally and returned to the cage.

Hysterectomy

24 hr after the SPS modeling, the rats in the Hysterectomy and SPS+Hysterectomy groups underwent a hysterectomy. The rats were anesthetized with isoflurane and then fixed onto a warm pad. The fur over the surgery area was shaved, and the skin was sterilized with ethanol. The skin was cut with approximately 1.5cm length at 0.5 cm above the midline of the pubic symphysis. The abdominal muscles and peritoneum were bluntly separated. Thereafter, the uterus was exposed by pushing intestine upwards. The lower edge of the cervix was severed, and the cut was sealed with wire. Then, cellulite was removed, and pink or yellow-red ovary was exposed. The fallopian tube, peri-uterine, and fascia were ligatured, and the uterus was removed. At last, the surgical wound was double sutured. A sham operation was performed in the control and SPS groups, and the wounds were sutured only at 0.5 cm above the midline of the pubic symphysis

Animal behavioral test

The mechanical paw withdrawal threshold (PWMT) was performed by Von Frey cilia at 0d (before modeling) every three days after the operation. It was examined by Chaplan’s “Up-and-Down” method. Briefly, the rats were placed on a metal frame with a clear plexiglass box cover. The bottom of the box was an empty metal grid. The Electronic von Frey monofilament was used to stimulate the pain at the inner and outer sides of the rat’s paw with a series of upward forces. We gradually increased the force until the appearance of a sharp retraction of the hind paw. The number on the electronic display was recorded as a mechanical pain threshold. The threshold was measured three times for each rat with a 5-10 min break interval. The average of three measurements was taken for statistical analysis

qRT-PCR

One-Step SYBR ® PrimeScript ™ (Takara, Japan) qPCR Kit and 7300 real-time fluorescence quantitative PCR instrument were used to detect GFAP, per kit instructions. The reaction conditions were as follows: 95°C for 30 s; 95°C for 5 s; 60°C for 30 s, 40 cycles. The relative mRNA expression level of GFAP was calculated by the 2^- method. β-actin was used as an internal reference, and the primer sequences were:

GFAP forward-5’-GTACCAGGACCTGCTCAAT-3’, reverse-5’-CAACTATCCTGCTTCTGCTC-3’;

Actin forward-5’-AGAGCTACGAGCTGCCTGAC-3’, reverse-5’-AGCACTGTGTTGGCGTACAG-3’.

Western Blot

Isolated astrocytes from the rats were spin down, and 200 μl of protein lysis buffer was added into each tube and mixed well. After 30 min of lysis, the cell lysates were transferred into another EP tube and centrifuged at 12000 r/min for 10 min. The protein concentration was measured by BCA assay. The SDS buffer was added into cell lysates followed by 10 min protein denature. SDS-PAGE gel electrophoresis was performed, and the wet transfer method was applied to transfer the protein gel onto a PVDF membrane. After 5% skimmed milk powder block at room temperature for 2h, samples were incubated with primary antibody anti-GFAP at 4ºC overnight (ab7260, Abcam, USA, dilution ratio of 1: 10000). On the next day, samples were incubated with corresponding secondary antibodies at room temperature for 1h (Abcam, USA) Membranes were washed three times by TBST containing ECL chemiluminescence solution (Promega). Gel imaging equipment was used for band observation. The results were presented as the ratio of the optical density of the target strip to internal GAPDH.

LTP measurement

Experiments were performed in a recording chamber on the stage of an Axioskop 2FS microscope with infrared DIC optics for visualizing whole-cell patch-clamp recordings. Excitatory postsynaptic currents (EPSCs) were recorded from layer II-III neurons using an Axon 200B amplifier (Axon Instruments, CA) and stimulations were delivered using a bipolar tungsten stimulating electrode placed in layer V of the ACC. The EPSCs were induced by repetitive stimulations at 0.02 Hz, and the neurons were voltage-clamped at -70 mV. LTP was induced within 10 min after obtaining stable EPSCs to prevent the washout effect. The protocol involved paired presynaptic 80 pulses at 2 Hz with postsynaptic depolarization at +30 mV (referred to as pairing training). The neurons were then voltage-clamped at -30 mV and EPSCs were evoked at 0.05 Hz. The access resistance was 15-30 MΩ and was monitored throughout the experiment. Data were discarded if access resistance changed by more than 15% during an experiment.

Statistical analysis

All the statistical analysis was performed using SPSS 17.0(SPSS, USA) and Prism 6 software. Statistical analysis for all the raw data was then performed using student t-test.

RESULTS

PWMT test after modeling

The mechanical PWMT in each group were tested before modeling and every three days after it. As shown in figure 1, these two thresholds in the SPS, Hysterectomy, and SPS+Hysterectomy groups were decreased significantly within the first week compared to day0 or the control group. Notably, there was no obvious variation of PWMT between different model groups for the first week, suggesting the modeling did not have an impact on the acute pain. However, around one week later, the thresholds of the SPS and Hysterectomy alone groups started to increase and recovered back to the control level at around day18. However, rats in the SPS+Hysterectomy did not present any improvement on pain thresholds at all after one week and showed a noticeable difference compared to the SPS or Hysterectomy groups after day10. This indicates chronic pain was formed in the SPS+Hysterectomy group but not in the SPS or Hysterectomy groups.

FIGURE 1
POSTOPERATIVE CHRONIC PAIN FORMATION INDUCED BY SPS+HYSTERECTOMY.

GFAP expression in astrocytes isolated from the ACC region

To investigate the reason why chronic pain was formed in the SPS+Hysterectomy group but not in the others, the astrocytes from the ACC region were isolated from the rats in each group three weeks after the modeling. Since GFAP is the activation marker of astrocytes, its expression level was detected in these cells. The western blot results presented that GAFP in the SPS+Hysterectomy group had higher expression than in the other groups. The GAFP expressions in the SPS and Hysterectomy alone groups did not show a significant difference compared to the control, suggesting astrocytes were only activated in the SPS+Hysterectomy group three weeks after the modeling. This observation was further confirmed by RT-PCR(Figure 2).

FIGURE 2
ASTROCYTES WERE STILL ACTIVE 3 WEEKS AFTER MODELING IN THE SPS+HYSTERECTOMY GROUP.

LTP change after modeling

To determine the LTP in different rat models, the ACC region were isolated and sliced for electrophysiological test three weeks after the modeling. As a result, the EPSC amplitude in the ACC region of the SPS+Hysterectomy group was significantly enhanced after induction compared to the control (Figure 3), SPS, and Hysterectomy groups. This suggests the LTP in the SPS+Hysterectomy group was enhanced, and chronic pain occurred after the modeling. Though a small enhancement of LTP in the SPS and Hysterectomy rats was recorded, the difference is not significant compared to the control group. We did not observe any noticeable change of LTP in the ACC region between the SPS and Hysterectomy groups. This indicates that SPS+Hysterectomy affects synaptic potentiation in the ACC.

FIGURE 3
LTP WAS ENHANCED IN THE ACC OF SPS+HYSTERECTOMY RATS.

DISCUSSION

Negative emotions, including anxiety, fear, and depression, are common among patients before operation. Preoperative anxiety, in particular, is very common and apparent. This kind of anxiety can reduce postoperative pain tolerance, diminish treatment efficiency, and damage patients’ physical and mental health. It has been reported that patients who undergo operations tend to stimulate pain after the surgery, especially those receiving local anesthesia such as peripheral nerve block anesthesia, epidural anesthesia, and spinal anesthesia. The body damage caused by the surgery and postoperative pain they experience are usually overestimated, resulting in unnecessary anxiety in patients ¹⁹. Clinical approaches, such as non-steroidal anti-inflammatory drugs and non-medical intervention, have been applied to relieve postoperative pain ²⁰ while the treatment outcomes are not ideal in many cases. Despite an increased focus and development of new standards for pain management, many patients still experience intense pain after surgery. Additional efforts are required to improve patients’ postoperative pain relief ²¹.

The ACC region is important to control the nociceptive emotion. Some studies suggest that astrocytes in this region may be activated and participate in the process of pain sensitization. Therefore, we wondered if the activation of astrocytes could be the link between preoperative anxiety and postoperative pain. In this project, we used a SPS model to investigate the relationship between preoperative anxiety and postoperative pain. SPS is a frequently used rat model of posttraumatic stress disorder (PTSD) that involves exposure to several successive stressors. ²² Hereby, we report a novel animal model which combines SPS and hysterectomy to study the formation of postoperative pain induced by preoperative anxiety. In addition, we found that chronic pain was formed in the SPS+Hysterectomy group but not in the SPS or Hysterectomy groups, indicating preoperative anxiety did enhance the postoperative pain. Moreover, astrocytes were found to be still activated, and increased LTP was also observed in the ACC region of rats in the SPS+Hysterectomy group three weeks after modeling, suggesting the formation of chronic pain.

CONCLUSION

Anxiety can induce chronic pain by activating astrocytes in the ACC region.

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• Funding
  This work was supported by Fund of Six Best Talent of Jiangsu, 2016 (WSW-113), and Fund of Science and Technology of Wuxi (CSE31N1522), and Fund of Science and Technology of Yixing, 2016-11).

Publication Dates

• Publication in this collection
  10 Oct 2019
• Date of issue
  Sept 2019

History

• Received
  08 Mar 2019
• Accepted
  13 May 2019