Effect of electroconvulsive stimulation on messenger RNA expression in the prefrontal cortex in a rat pain model

  • Authors:
    • Yusuke Kimura
    • Masashi Ishikawa
    • Yoko Hori
    • Tadashi Okabe
    • Atsuhiro Sakamoto
  • View Affiliations

  • Published online on: September 30, 2015     https://doi.org/10.3892/br.2015.525
  • Pages: 802-806
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Abstract

Previous reports have shown that electroconvulsive therapy (ECT) is efficacious in the treatment of neuropathic pain; however, its mechanism of action remains unclear. The present study aimed to understand these mechanisms by investigating the alterations in the expression of neuropeptide Y (NPY) and interleukin‑1β (IL‑1β) in the prefrontal cortex. A rat model of neuropathic pain produced by chronic constrictive injury of the sciatic nerve was used, and mechanical and thermal hyperalgesia were evaluated starting 2 days after the injury. Using a pulse generator, ECT was administered to the rodents for 6 days from days 7‑12 after the injury. Thermal and mechanical stimulation were administered to assess pain thresholds. Quantitative polymerase chain reaction, used to measure gene expression levels in the prefrontal cortex, showed that NPY and IL‑1β gene expression levels in the prefrontal cortex increased following the injury. The present results indicate that these gene expression level variations may be associated with the mechanisms underlying the effect of ECT in treating neuropathic pain.

Introduction

Electroconvulsive therapy (ECT) has been widely used as an effective treatment for schizophrenia and refractory depression; however, the underlying mechanism remains unclear. Numerous studies in the field of neuropsychiatry have indicated that the expression levels of genes encoding various neurotransmitters are altered following ECT, suggesting that these changes may be a possible mechanism of action of ECT (14). Certain studies have reported the efficacy of ECT in treating neuropathic pain (59). However, ECT is not widely used for treating neuropathic pain as the underlying mechanism of action has not been sufficiently elucidated.

Chronic pain causes changes in the expression of messenger RNAs (mRNAs) or proteins that regulate cytokines, neurotransmitters and receptors in the brain and dorsal root ganglia (10,11). The differential expression of multiple pain-associated genes, such as interleukin- (IL-1β), has an important role in the development and maintenance of chronic pain (1215). Electroconvulsive stimulation (ECS) improves neuropathic pain symptoms in rats (16). In our previous report, altered expression of neuropeptide Y (NPY) in the brain was identified as a possible mechanism of action of ECT for treating neuropathic pain (16).

The prefrontal cortex is known to participate in executive function, emotions and pain. Certain studies have shown prefrontal cortex dysfunction in chronic pain patients (17,18), suggesting it has an important role in pain. However, there are only a few studies examining the participation of the prefrontal cortex in neuropathic pain and response to ECT. We hypothesized that changes in NPY and IL-1β gene expression levels in the prefrontal cortex may have an important role in the mechanisms underlying neuropathic pain and the effect of ECS in its treatment.

Materials and methods

Experimental animals

All the experimental procedures were approved by the Institutional Committee on Laboratory Animals of Nippon Medical School, Tokyo, Japan (approval no. 27-042) and were performed under the guidelines of the International Association for the Study of Pain (19).

Male Sprague-Dawley rats [6–7 weeks of age, ~250 g (Tokyo Laboratory Animals Science Co., Tokyo, Japan)] were housed in clear, plastic cages with sawdust bedding at standard room temperature under a 12-h light/dark cycle. All the rats received food and water ad libitum. The rats were divided into three experimental groups: i) Rats subjected to the chronic constrictive injury (CCI) protocol and administered ECS (ECS group, n=7); ii) those subjected to the CCI protocol with no ECS (CCI group, n=7); and iii) those subjected to a sham-CCI surgery (control group, n=7) (16).

Production of a neuropathic pain model

Experimental neuropathy was produced according to previously described methods (20). All the surgical procedures were performed on rats that were anesthetized with sodium pentobarbital [50 mg/kg intraperitoneally (i.p.)]. CCI was induced on the left (ipsilateral) side by exposing the common sciatic nerve in the left mid-thigh and loosely ligating the nerve using 4-0 silk thread in 4 regions spaced at ~1-mm intervals. As a control, the right (contralateral) sciatic nerve was similarly exposed, but not ligated. Rats in the control group were subjected to exposure of both sciatic nerves without nerve ligation.

ECS

ECS was administered transauricularly using metal forceps, as previously described (1,2,21,22). ECS was administered to the ECS group once daily for 6 days, starting on day 7 postoperation and continuing to day 12, using a pulse generator [frequency, 100 pulses; pulse width, 0.5 msec; shock duration, 0.8 sec; current, 50 mA (57800 ECT Unit; Ugo Basile, Comero, Italy)]. The shock elicited a full tonic-clonic seizure lasting ~30 sec in all rats. Rats in the CCI and control groups were exposed to the forceps once daily from days 7 to 12 postoperation; these animals, however, did not receive a current (16).

Behavioral tests

Two behavioral tests (thermal and mechanical stimulation) were performed 8 times to evaluate pain thresholds, as previously described (22,23). They were performed the day before surgery (day 0) and on days 2, 4, 6, 8, 10, 12 and 14 after the surgery. During the period of ECS, the behavioral tests were performed subsequent to ECS (days 8, 10 and 12) and 48 h after the last ECS (day 14). The plantar test (Ugo Basile) was used to analyze thermal allodynia. Rats were placed on a glass plate with radiant heat (a 50-W halogen reflector bulb) positioned underneath. Following an acclimation period, radiation heat was independently applied to either the ipsilateral or contralateral hind paw pad. The latency of paw withdrawal from thermal stimulus was measured 3 times at 5-min intervals, and the average value was used as the latency response. Mechanical hyperalgesia was measured using an electronic von Frey instrument (IITC Life Science, Woodland Hills, CA, USA). The instrument consisted of a disposable tip, a hand-held probe, a display unit and a connector cable. A polypropylene pipette tip was mounted on the probe and used for mechanical stimulation. Each rat was set on a metallic mesh floor and covered with a plastic box. Subsequently, the tip was applied from under the mesh floor to the plantar surface of either the ipsilateral or contralateral hind paw. When the rat felt pain and raised its leg, the mechanical stimuli was immediately stopped and the force shown on the digital display was recorded. Each paw was stimulated with the tip 3 times at 10-sec intervals in individual trials, and the average value was used as the latency response (16).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

On day 14 postoperation, rats were anesthetized with pentobarbital at a dose of 300 mg/100 g i.p. before being sacrificed. The brains were immediately removed, and the prefrontal cortex, containing a coronal slice of the most anterior 3 mm of the brain (without the olfactory bulbs) was dissected as previously described (24). The prefrontal cortex was separated into right and left hemispheric regions. Each sample was placed in RNAlater® solution (Applied Biosystems, Foster City, CA, USA) and stored at −80°C until use. Prior to use, the samples were defrosted and the RNAlater® solution was immediately separated from the samples. Total RNA was extracted from the samples using an mirVana™ miRNA isolation kit® (Applied Biosystems), according to the manufacturer's instructions. RNA quality and quantity were evaluated by absorbance using a NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). Samples with an A260/280 ratio between 1.8 and 2.0 were of sufficient quality for use in subsequent analysis. Total RNA (10 µg) was used as a template for RT reactions with 10 µl of 10X RT buffer, 4 µl of 25X deoxyribonucleotide triphosphate mix, 10 µl of 10X RT random primers, 5 µl of MultiScribe™ RT, 5 µl of RNase inhibitor and nuclease-free H2O (Applied Biosystems). The RT reaction was carried out using a PCR Express (Thermo Fisher Scientific, Yokohama, Japan) at 42°C for 60 min, 99°C for 10 min and 4°C for 5 min.

The amplification of NPY and IL-1β was performed in a fast 96-well reaction plate (Applied Biosystems, Foster City, CA, USA), as previously described (25). Reactions were carried out in a 20-µl volume containing 10 µl of TaqMan Universal PCR master mix, 1 µl of TaqMan gene expression assays (Applied Biosystems), 8 µl of RNase-free water (Wako, Tokyo, Japan) and 20 ng of cDNA. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to assess DNA integrity.

RT-qPCR consisted of an initial denaturation at 95°C for 3 sec, incubation at 60°C for 30 sec and subsequent measurement of the fluorescence signal following each cycle. The TaqMan probe labeled with 6-fluorescein amidite was cleaved during amplification and generated a fluorescent signal. The assay used an instrument capable of measuring fluorescence in real-time (ABI PRISM 7500 Fast Sequence Detector; Applied Biosystems). RT-qPCR data are presented as threshold cycle (Ct) values, where Ct indicates a unit-less value defined as the fractional cycle number at which the sample fluorescence signal passes a fixed threshold above baseline. Triplicate samples with significantly different values due to inaccurate operation were omitted. Relative amounts of all the mRNAs were calculated using the comparative Ct method (Applied Biosystems). ΔCt is the difference in the Ct values derived from the experimental samples and the GAPDH control, and ΔΔCt represents the difference between paired samples, as calculated by the following formula: ΔΔCt = (ΔCt of targets of CCI + ECS group) - (ΔCt of control of CCI + sham-ECS group). The expression ratio indicates the relative quantity of the target gene (Xtarget) to the control gene (Xcontrol). The expression ratio was computed using the formula: Xtarget/Xcontrol = 2−ΔΔCt.

Statistical analysis

All the numerical data are expressed as mean ± standard errors of the mean. A paired t-test was used to compare the latencies or threshold values in behavioural tests between the ipsilateral and contralateral sides (ECS group, n=7; CCI group; n=7). Dunnett's test for multiple comparisons was used to compare latencies, threshold values or different scores obtained in behavioural tests performed prior to the procedures (day 0, n=7) with those obtained in tests performed following the procedures (days 2, 4, 6, 8, 10, 12 and 14; n=7). Analysis of variance followed by Tukey's test was performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Behavioral tests

There was no difference in the latency of paw withdrawal in the plantar test and threshold values in the electronic von Frey test between the ipsilateral and contralateral sides in the control group. The CCI group showed a significant decrease in the latency of paw withdrawal in the plantar test and threshold values in the electronic von Frey test for the ipsilateral side on postoperative days (2, 4, 6, 8, 10, 12 and 14) compared with the corresponding values on day 0 (P<0.01). However, significant decreases were not observed on the contralateral side. Paw withdrawal thresholds in response to mechanical stimulation were not significantly altered following ECS. Compared with the values on day 0, in the ECS group, latencies of paw withdrawal from thermal stimulation on the ipsilateral side were decreased on days 2, 4, 6, 8 and 10 (P<0.01). After ECS, the latencies of paw withdrawal from thermal stimulation on the ipsilateral side were increased on days 12 and 14 (Figs. 1 and 2).

RT-qPCR of mRNAs

In the right prefrontal cortex, expression of NPY was 1.7 times greater in the CCI group compared with the ECS and control groups (P<0.01); expression levels in the ECS and control groups were similar. No significant differences among the 3 groups were observed in the left prefrontal cortex. In the right prefrontal cortex, the expression of IL-1β was 1.4 times greater in the CCI group compared with the ECS and control groups (P<0.01). In addition, expression of IL-1β in the ECS group was lower compared with the control group. In the left prefrontal cortex, there were no significant differences observed in IL-1β gene expression between the CCI and control groups. However, IL-1β gene expression was lower in the ECS group compared with the control group [P<0.01 (Fig. 3].

Discussion

Functional brain imaging has revealed that the mechanism of pain is not simply an alteration of sensory neural transmission from the periphery to the cerebrum, such as in the spinothalamic tract. With respect to chronic pain, it has been suggested that shifting the focus from ‘an infringed sensory system’ to ‘an emotion system’ is essential for advancing mechanistic understanding (26). In addition, it is thought that pain arising from damage and inflammation is associated with mental and physical stress (27). Mental stress has been shown to activate the cerebral cortex (such as prefrontal cortex) and limbic system prior to spreading to the central amygdaloid nucleus or bed nucleus of stria terminalis (BST), which results in the activation of the hypothalamo-pituitary-adrenocortical axis (28). Although it is known that NPY is associated with appetite, blood pressure and memory, it has also been recently suggested that NPY could be involved in pain (29). Furthermore, BST contains neurons that are associated with the unpleasantness of pain, and it was also reported that NPY controls the function of these neurons (30). On the basis of the above data, the present study was interpreted to find that elevated NPY expression in the prefrontal cortex of the CCI group normalized to control levels in the ECS group according to the following hypothesis: Activity of neurons associated with pain in BST was promoted by the stress associated with CCI, and NPY gene expression increased in the prefrontal cortex as a compensatory response. As pain was decreased by ECS, it is thought that NPY expression correspondingly returned to control values in the prefrontal cortex. Briefly, the alterations in NPY expression between the prefrontal cortex and BST were thought to occur to control the CCI-induced mental stress. NPY expression was altered in the right prefrontal cortex (as the left sciatic nerve was ligated) as it utilizes the spinothalamic tract, which innervates the contralateral side of the spinal cord.

The effects of IL-1β are varied and are recognized as important for controlling immunity and inflammation. Apkarian et al (24) showed that IL-1β mRNA expression in the prefrontal cortex increased in the spinal nerve injury model rat. Although a similar alteration in IL-1β expression following CCI was not reported, the study also failed to obtain effective allodynia in the CCI model rats. In the present study, effective allodynia was achieved in the CCI model rats and provided results that were equivalent to their study in CCI model rats. The neuropathic pain symptoms were improved by performing ECS as expression of IL-1β decreased in the ECS group. Furthermore, the laterality of IL-1β expression was consistent with that of NPY expression. Although it was expected that the alterations in expression would be normalized to control levels following ECS, as with NPY, the expression of IL-1β fell to levels below that of the control group. Notably, this phenomenon was observed in the left and right prefrontal cortex.

The results of the present study indicate that ECS is effective for curing thermal allodynia, but not for treating mechanical hyperalgesia. This result is similar to those of previous studies (16,22). Up to postoperative day 10, the development of thermal allodynia and mechanical hyperalgesia was shown on the affected side in the CCI and ECS groups, and a significant difference was corroborated between the preoperative and postoperative data. As there was not a significant difference in the scores of the thermal and mechanical stimulant tests between the 2 groups, it can be suggested that there was no difference in the levels of CCI between the two groups.

In conclusion, the present study showed that the symptoms of neuropathic pain improved, and the level of expression of the NPY and IL-1β genes in the prefrontal cortex decreased in CCI model rats following ECS. The NPY and IL-1β gene expression levels in the prefrontal cortex were increased in CCI model rats. The possibility exists that changes in the levels of expression of NPY and IL-1β in the prefrontal cortex are associated with the mechanism of action of ECT in treating neuropathic pain.

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Kimura Y, Ishikawa M, Hori Y, Okabe T and Sakamoto A: Effect of electroconvulsive stimulation on messenger RNA expression in the prefrontal cortex in a rat pain model. Biomed Rep 3: 802-806, 2015
APA
Kimura, Y., Ishikawa, M., Hori, Y., Okabe, T., & Sakamoto, A. (2015). Effect of electroconvulsive stimulation on messenger RNA expression in the prefrontal cortex in a rat pain model. Biomedical Reports, 3, 802-806. https://doi.org/10.3892/br.2015.525
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Kimura, Y., Ishikawa, M., Hori, Y., Okabe, T., Sakamoto, A."Effect of electroconvulsive stimulation on messenger RNA expression in the prefrontal cortex in a rat pain model". Biomedical Reports 3.6 (2015): 802-806.
Chicago
Kimura, Y., Ishikawa, M., Hori, Y., Okabe, T., Sakamoto, A."Effect of electroconvulsive stimulation on messenger RNA expression in the prefrontal cortex in a rat pain model". Biomedical Reports 3, no. 6 (2015): 802-806. https://doi.org/10.3892/br.2015.525