AJA Asian Journal of Anesthesiology

Advancing, Capability, Improving lives

Research Paper
Volume 54, Issue 3, Pages 81-87
Sheng-JieShiue 1.┼ , Chi-Hsu Wang 1.2.┼ , Tao-Yeuan Wang 2.3 , Yi-Chun Chen 1 , Jen-Kun Cheng 1.2
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Abstract

Objective

T-type channel (TCC) CaV3.2 plays a pivotal role in pain transmission. In this study, we examined the effects of intrathecal TCC blockers on CaV3.2 expression in a L5/6 spinal nerve ligation (SNL) pain model. The neurotoxicity of TCC blockers were also evaluated.

Methods

Male Sprague-Dawley rats (200–250 g) were used for right L5/6 SNL to induce neuropathic pain. Intrathecal infusion of saline or TCC blockers [mibefradil (0.7 μg/h) or ethosuximide (60 μg/h)] was started after surgery for 7 days. Fluorescent immunohistochemistry and Western blotting were used to determine the expression pattern and protein level of CaV3.2. Hematoxylin–eosin and toluidine blue staining were used to evaluate the neurotoxicity of tested agents.

Results

Seven days after SNL, CaV3.2 protein levels were upregulated in ipsi-lateral L5/6 spinal cord and dorsal root ganglia (DRG) in immunofluorescence and Western blotting studies. Compared with the saline-treated group, rats receiving mibefradil or ethosuximide showed significant lower CaV3.2 expression in the spinal cord and DRG. No obvious histopathologic change in hematoxylin–eosin and toluidine blue staining were observed in all tested groups.

Conclusion

In this study, we demonstrate that SNL-induced CaV3.2 upregulation in the spinal cord and DRG was attenuated by intrathecal infusion of mibefradil or ethosuximide. No obvious neurotoxicity effects were observed in all the tested groups. Our data suggest that continuous intrathecal infusion of TCC blockers may be considered as a promising alternative for the treatment of nerve injury-induced pain.

Keywords

CaV3.2; intrathecal; neuropathic pain; neurotoxicity; T-type calcium channel;


1. Introduction

Neuropathic pain is difficult to treat clinically, in part due to an incomplete understanding of the mechanisms involved. Neuropathic pain can be caused by damage or disease affecting the nervous system. Many studies have investigated the mechanisms for acute activation of nociceptors, but there is a large gap in knowledge regarding the mechanisms underlying the induction of nerve injury-induced neuropathic pain.12 Symptoms of neuropathic pain such as allodynia, dysesthesia, and paresthesia can be very difficult to manage. Drugs used to treat neuropathic pain such as opioids, antidepressants, or antiepileptics have limited efficacy and can be accompanied by various side effects.34 Therefore, there is an urgent need for new treatments with a better efficacy and fewer side effects. One potential molecular target for neuropathic pain is CaV3.2, one subtype of the T-type Ca2+ channels (TCCs; CaV3 family).567

Activation of Ca2+ channels is critical for neurotransmitter release, neuronal excitability, and postsynaptic Ca2+ signaling. In the spinal lamina I neurons, the N-, L-, and TCCs are involved in depolarization-induced increase of [Ca2+]i.8 CaV3.2 are also expressed in nociceptive neurons in the dorsal root ganglia (DRG) and contribute to the initiation and maintenance of neuropathic pain.9 Specifically, CaV3.2 expression is upregulated in DRG after L5/6 spinal nerve ligation (SNL),9 and the channel current density in DRG neurons is upregulated in models of neuropathic pain.1011Furthermore, CaV3.2 knockout mice have reduced pain responses12 and various blockers of CaV3.2 channels have been shown to induce antinociception in models of inflammatory and neuropathic pain.913 Targeting CaV3.2 channels may be a useful strategy for the development of neuropathic pain therapeutics.

The L5/6 SNL pain model can induce neuropathic pain, which is characterized by mechanical allodynia (pain due to a stimulus which does not normally provoke pain) and thermal hyperalgesia (an increased response to a stimulus which is normally painful), and refractory to conventional treatment approaches.14 Using the SNL model, we have reported that continuous intrathecal infusion of mibefradil and ethosuximide, all of which have been reported to block TCC or CaV3.2 currents,15161718 could attenuate nerve injury-induced mechanical allodynia and thermal hyperalgesia.9 In this study, we further examined the effects of intrathecal infusion of these two agents on SNL-induced CaV3.2 overexpression. In addition, the possible neurotoxicity of these two agents were examined.

2. Methods

This study was conducted in accordance with the guidelines of the International Association for the Study of Pain19 and appropriate aspects of the Animal Research: Reporting of In Vivo Experiments guidelines. The study protocol was approved by the Institutional Animal Care and Use Committee of MacKay Memorial Hospital, Taipei, Taiwan. All experiments were performed using male Sprague-Dawley rats (BioLASCO Taiwan Co., Ltd.), each weighing 200–250 g on the day of surgery. Rats were housed individually in plastic cages with soft bedding at room temperature and maintained on a 12-hour light/12-hour dark cycle with free access to food and water.

2.1. Surgical procedures for L5/6 SNL

All the surgical procedures were performed under inhalation anesthesia with isoflurane in 100% oxygen, induced at 5% and maintained at 2%. Adequate anesthesia was ascertained by the lack of the ocular reflex and by the absence of a withdrawal response to a pinch of the hind limb. During surgery, the percentage of isoflurane was increased if inadequate anesthesia was noted. Neuropathic pain was induced following the methods of Kim and Chung.14 Rats were anesthetized and placed under a microsurgical apparatus in a prone position. A midline incision was made on the back, and the right paraspinal muscles were separated from the spinous processes at the L4–S2 levels. The L5 transverse process was carefully removed, and the L4–L5 spinal nerves were identified. The nerves were gently separated, and the L5 nerve was tightly ligated with a 6-0 silk thread. Then the right L6 spinal nerve was located just caudal and medial to the sacroiliac junction and tightly ligated with a silk thread. For sham surgery, the right L5 and L6 spinal nerves were exposed but not ligated. Immediately after the surgery, the following procedures of intrathecal catheterization and infusion pump implantation were performed.

2.2. Intrathecal catheterization and implantation of infusion pump

For drug infusion, intrathecal catheters were inserted during isoflurane anesthesia by passing a PE-5 catheter (filled with test drugs or normal saline) through an incision in the atlanta-occipital membrane to a position 8-cm caudal to the cisterna at the level of lumbar enlargement, as previously described.920 An infusion pump (Model 2001, ALZET, Cupertino, CA, USA) with a flow rate of 1 μL/h was filled with normal saline, mibefradil (0.7 μg/μL), or ethosuximide (60 μg/μL) and connected to the intrathecal catheter (n = 6 for each group). The pump was implanted subcutaneously and the wound was closed with 3-0 silk sutures. Rats showing neurological deficits after surgery were euthanized with deep isoflurane anesthesia and intraperitoneal pentobarbital (120 mg/kg).

2.3. Drugs

Mibefradil and ethosuximide were purchased from Sigma-Aldrich (St Louis, MO, USA). All drugs were dissolved in normal saline. The infusion doses of tested drugs were determined according to our previous study.9

2.4. Immunofluorescence study

Fluorescent immunohistochemistry was prepared to examine the expression of CaV3.2 in rat spinal cord and DRG 7 days after sham surgery or nerve ligation. After deep anesthesia by intraperitoneal injection of sodium pentobarbital (120 mg/kg), rats were perfused transcardially with normal saline followed by 4% paraformaldehyde in 0.1M phosphate buffer (0.08M K2HPO4, 0.02 MNaH2PO4, pH 7.4) for 20 minutes. L5/6 spinal cord segments and DRGs were removed and postfixed in the same fixative overnight at 4°C. All specimens were cryoprotected in 30% (weight/volume) sucrose in 0.1M phosphate buffer and cut with a cryostat into 15-μm sections and mounted directly onto gelatin-coated slides. Nonspecific binding was blocked by 3% normal goat serum plus 2% bovine serum albumin in Tris-buffered saline containing 0.3% Triton X-100 for 1.5 hours. The following rabbit anti-CaV3.2 primary antibody was used (1:400, Cayman, Los Angeles, CA, USA). Secondary antibodies included Alexa fluor 594-conjugated donkey anti-rabbit immunoglobulin-G (1:500; Invitrogen, Eugene, OR, USA). Sections on slides were mounted with the antifading medium Fluoromount-G (Southern Biotech, Birmingham, AL, USA) under cover slips. Images were collected using an Olympus FV300 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) and processed with Adobe Photoshop 8.0 software (Adobe Systems, Mountain View, CA, USA).

2.5. Quantitative measurements

To obtain the cell profiles of CaV3.2, the naïve and nerve-ligated rats were killed and only L5 DRG was used from each rat. In each L5 DRG, four sections were sampled approximately in the middle part of DRG with a distance of every 80 μm. After immunostaining, CaV3.2+ neurons with obvious nuclear localization at or near the center of cells were measured for diameter and counted. For somatic size distribution of CaV3.2, the quantify immunoreactivity procedures of Chien et al21 were followed.

2.6. Western blotting

To examine the protein level of CaV3.2 in spinal cord and DRG, animals were euthanized with sodium pentobarbital (120 mg/kg, intraperitoneal) at Day 7 after surgery. The L5/6 spinal segments and DRG were quickly removed and homogenized in a sodium dodecyl sulfate sample buffer containing a mixture of proteinase and phosphatase inhibitors (Sigma). Protein samples (40 μg) were separated on NuPAGE Bis-Tris gradient gel (4–12%; Life Technologies, Thermo Fisher Scientific Inc, Waltham, MA, USA) and transferred to polyvinylidene difluoride blots. The blots were blocked with 5% milk and incubated overnight at 4°C with diluted rabbit anti-CaV3.2 primary antibody (1:1000, Cayman) and actin (internal control; 1:5000, Millipore, Temecula, CA, USA). These blots were further incubated with horseradish peroxidase-conjugated secondary antibody, developed in enhanced chemiluminescence solution, and images were taken and analyzed using a cooled charge coupled device (CCD) system (LAS4000, Multi Gauge version 3.0 software, FujiFilm, Tokyo, Japan).

2.7. Hematoxylin and eosin staining

To examine the neurotoxicity of tested drugs, naïve rats received intrathecal infusion of saline (1 μL/h), mibefradil (0.7 μg/μL), or ethosuximide (60 μg/μL) for 7 days (n = 6 in each group). After the infusion, rats were anesthetized and intracardially perfused with 4% paraformaldehyde. The hematoxylin and eosin (HE) staining procedures of Chu et al20 were followed. Histopathological changes were evaluated in a blinded fashion by the senior pathologist (T.-Y.W.) according to a scoring system described previously:22 0 = absence of abnormal cells; 1 = presence of hemorrhage and glial cell reaction in several areas; 2 = presence of prominent necrosis in the gray matter, widespread hemorrhage or demyelination, fibrosis, and inflammatory cells.

2.8. Toluidine blue staining

Toluidine blue staining was performed to evaluate the neurotoxic effect of tested drugs on cauda equina. After receiving intrathecal infusion of saline (1 μL/h), mibefradil (0.7 μg/μL), or ethosuximide (60 μg/μL) for 7 days (n = 6 in each group), rats were anesthetized and perfused intracardially with a phosphate-buffered–2% paraformaldehyde–2.5% glutaraldehyde fixative. For evaluating the neurotoxic effect of these two drugs, the toluidine blue staining procedures of Chu et al20 were followed. Neuropathological examination was conducted using light microscopy by the senior pathologist (T.-Y.W.) who was masked to the group assignment. Quantitative analysis of nerve injury was performed using the sections obtained from the specimen. Each fascicle present in the cross section was assigned an injury score of 0–3 [0: normal (no edema; no injured nerve fibers), 1: mild (mild edema; little or no nerve fiber degeneration or demyelination), 2: moderate (< 50% of nerve fibers with degeneration and demyelination), and 3: severe (> 50% of nerve fibers with degeneration and demyelination)], as published previously.23 The injury score for each cross section was then calculated as the average score of all the fascicles present in the cross section.

2.9. Data analysis

All the data were expressed as mean ± standard error of the mean. The nonparametric Mann–Whitney U test was used to compare the injury scores of different groups in HE and toluidine blue staining. The data of Western blot was analyzed using Student t test. A p value < 0.05 was considered statistically significant.

3. Results

3.1. Spinal nerve ligation induced CaV3.2 upregulation in spinal cord and DRG

Firstly, we examined the CaV3.2 expression pattern and level in spinal cord and DRG. The immunofluorescent staining was performed to reveal the expression pattern of CaV3.2 in spinal cord and L5 DRG. Figure 1A shows that CaV3.2 expression is enhanced in the ipsilateral spinal dorsal horn of SNL rats, compared with sham rats. Furthermore, the Western blotting also revealed that SNL induced CaV3.2 upregulation in the ipsilateral spinal cord (p < 0.05, n = 3 in each group; Figure 2A). In L5 DRG, CaV3.2 is expressed in neuron-like cells (Figure 1B), as suggested by our previous report.9 In the Sham group, 39% of DRG neurons (385 of 1000 of total cell counts) showed CaV3.2 in their cell bodies (Figures 1B and 1C). The somatic sizes of CaV3.2+ neurons were measured and divided into three groups: 46% small diameter (< 30 μm), 43% medium diameter (30–40 μm), and 11% large diameter (> 40 μm; Figure 1C). In the SNL group, up to 73% of DRG neurons (728 of 1000 of total cell counts) showed CaV3.2-IR in their cell bodies, and was also divided into three groups: 39% small diameter (< 30 μm), 39% medium diameter (30–40 μm), and 22% large diameter (> 40 μm). Therefore, the percentage of CaV3.2+ neurons was increased from 39% to 73% after SNL, especially in the somatic sizes 20–60 μm groups [from sham 41% (382/936) increased to SNL 75% (710/946); Figure 1C]. The Western blotting data also revealed that the protein level of CaV3.2 in ipsilateral L5/6 DRG was significantly increased after SNL (Figure 2B). These data suggest that SNL induces CaV3.2 upregulation in the ispilateral spinal cord and DRG.

Fig. 1
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Figure 1. Expression pattern and differential distribution of CaV3.2 in rat dorsal spinal cord and L5 dorsal root ganglion (DRG) by immunofluorescent study. (A) The expression of CaV3.2 in ipsilateral spinal dorsal horn was increased 7 days after spinal nerve ligation (SNL). (B) The expression of CaV3.2 in ipsilateral L5 DRG was increased 7 days after SNL. (C) The differential distribution of CaV3.2 in different sized DRG neurons. Scale bar: 200 μM (A) and 50 μM (B). DAPI = 4′,6-diamidino-2-phenylindole.
Fig. 2
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Figure 2. T-type calcium channel blockers mibefradil and ethosuximide attenuated nerve ligation-induced CaV3.2 upregulation in ipsilateral spinal cord and dorsal root ganglion (DRG). Representative Western blots and summarized bar graphs depicting the expression level of CaV3.2 protein in (A) ipsilateral spinal cord and (B) L5/6 DRG sampled from sham rats receiving saline and nerve-ligated rats receiving saline, mibefradil or ethosuximide for 7 days. The top blots are the immunoreactive bands for the CaV3.2 protein and the bottom blots are the bands for the control protein actin. The ordinate of the base graph is the expression level of CaV3.2 protein, taking the Sham/Saline group as 100%. * p < 0.05 compared with the Sham/Saline group. **p < 0.05 compared with the Ligation/Saline group by Student t test.

3.2. Intrathecal infusion of mibefradil or ethosuximide attenuated SNL-induced CaV3.2 upregulation

Since SNL could induce CaV3.2 over-expression (Figure 1Figure 2), and intrathecal infusion of mibefradil or ethosuximide could attenuate the development of SNL-induced mechanical allodynia and thermal hyperalgesia.9 We further tested the effect of mibefradil and ethosuximide on CaV3.2 expression in the pain model. Compared with the Ligation/Saline group, the Western blotting showed that CaV3.2 expression was significantly decreased in the tested groups, in either the spinal cord or DRG (Figure 2). These results indicate that intrathecal infusion of TCC blockers could attenuate SNL-induced CaV3.2 upregulation.

3.3. No prominent histopathological change is noted after intrathecal infusion of mibefradil or ethosuximide

To further investigate the neurotoxic effect of the tested agents, we performed the HE staining of spinal cord and toluidine blue staining of cauda equina of rats receiving chronic intrathecal infusion. Figure 3 shows the HE stain of the lumbar spinal cord sections from naïve rats receiving intrathecal saline (1 μL/h), mibefradil (0.7 μg/h), or ethosuximide (60 μg/h) for 7 days. Spinal sections of all these rats demonstrate normal histological appearances. Under microscopic examinations, no gliosis, demyelination, fibrosis, inflammation, hemorrhage, or necrosis was found at the lumbar level of spinal cords of all these animals (Figure 3). According to the scoring system described in the Methods section,22 all these groups were scored at 0 (n = 6 in each group).

Fig. 3
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Figure 3. Representative photomicrograph showing the hematoxylin and eosin stain of rat lumbar spinal cord sections. After receiving intrathecal infusion of saline (A; 1 μL/h), mibefradil (B; 0.7 μg/h), or ethosuximide (C; 60 μg/h) for 7 days, no obvious histopathological change was noted in the white or gray matter of all these rats (n = 6 in each group). 1–9: Higher magnification images of the dorsal, ventral, and central insets in A–C, respectively. Scale bar: 500 μm (A–C), 50 μm (1–9).

For toluidine blue staining, sections obtained from the cauda equina of naïve rats receiving intrathecal infusion of saline or tested agents for 7 days showed no obvious damage in the fascicles. Representative fascicles from animals in each group are shown in Figure 4. The injury scores of rats receiving mibefradil (0.7 μg/h) or ethosuximide (60 μg/h) infusion were 0.24 ± 0.15 and 0.13 ± 0.12, respectively, not significantly different from the score of rats receiving saline infusion (0.22 ± 0.14, p > 0.05, n = 6 in each group). Combined with the data of HE staining, it is suggested that the tested agents, when given intrathecally at the infusion concentrations, do not induce obvious histopathological change.

Fig. 4
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Figure 4. Representative photomicrograph showing the toluidine blue stain of cauda equina fascicle sections from rats after receiving intrathecal infusion of saline (A; 1 μL/h), mibefradil (B; 0.7 μg/h), or ethosuximide (C; 60 μg/h) for 7 days. A1–C1: Higher magnification images of the insets in A–C, respectively. According to the scoring system described in the Methods section, the injury scores of all three groups were not significantly different from each other (n = 6 in each group). Scale bar: 100 μm (A–C), 20 μm (A1–C1).

4. Discussion

The present study shows that the expression pattern of CaV3.2 in the spinal cord and DRG is upregulated in nerve-ligated rats. In the DRG, the expression of CaV3.2 is majorly colocalized with medium- and small-sized DRG neurons, and is up to about a 2-fold increase in SNL rats, especially in the somatic sizes 20–60 μm groups. The Western blotting showed that SNL-induced CaV3.2 upregulation was significantly attenuated by TCC blockers mibefradil and ethosuximide. At the dose tested, none of the tested agents induced obvious neuropathological changes in the spinal cord and cauda equina. Our findings suggest that continuous intrathecal infusion of these two agents may be considered for the treatment of nerve injury-induced pain.

TCCs are considered as important contributors to pain signaling and are crucial mediators of neuropathic pain.6 Among TCCs, Cav3.2 is the most highly expressed in DRG,24 making it a target of choice to regulate pain transmission. In this study, the CaV3.2+ neurons are majorly of the somatic sizes 20–50 μm (Figure 1; small and medium size) in L5 DRG. Small and medium diameter DRG neurons predominantly have slow-conducting unmyelinated C-fibers, while larger diameter neurons have faster A-type myelinated fibers.25 On the basis of their peptide content, the C-fiber nociceptors can be further divided into nonpeptidergic IB4+ and peptidergic CGRP+neurons,26 proposed to be majorly responsible for mechanical and thermal sensations, respectively.27 In our previous study, we have noticed that CaV3.2 is expressed in these two types of neurons.9 In this study, the CaV3.2 expression proportion is increased after SNL (from 39% to 73%), especially in the somatic sizes 20–60 μm groups (Figure 1C), which should contain these IB4+ and CGRP+ neurons. Intrathecal mibefradil and ethosuximide may, at least in part, act on CaV3.2 of these IB4+ and CGRP+ neurons to achieve their antiallodynic and antihyperalgesia effects.

Mibefradil and ethosuximide have been reported to block TCC or CaV3.2 currents in vitro.15161718 In our previous study, intrathecal infusion of these two agents could attenuate nerve injury-induced neuropathic pain to a similar extent.9 The IC50s of mibefradil and ethosuximide in inhibiting the T-type Ca2+ current in rat DRG neurons, in which high levels of CaV3.2 messenger RNA transcripts were reported,28are 3 μmol/L and 23.7 mmol/L, respectively.29 The doses of mibefradil and ethosuximide used in this study were determined considering their IC50s and molecular weights (495.6 g/mol and 141.2 g/mol), as our previous report.9 In this study, the Western blotting showed that these two agents could attenuate SNL-induced CaV3.2 upregulation in the spinal cord and DRG (Figure 2), also with similar inhibitory effects. However, the suppression mechanism remains to be clarified by future studies. Given systemically or intrathecally, TCC blockers have been reported to be effective in other animal pain models.3031 Furthermore, TCC blockers have also been used to attenuate inflammatory pain. For example, preceding and periodic intraplantar treatment with TCC blockers mibefradil and NNC 55-0396 markedly reduced and reversed mechanical hyperalgesia during the acute and subacute phases of carrageenan-induced inflammatory hyperalgesia in mice.32 Taken together, it is suggested that targeting CaV3.2 channels may be a useful strategy for pain management.

For drugs to be tested intrathecally in clinical trials, it is imperative to examine its neurotoxic effect first in animals.33 For instance, intrathecal lidocaine has been found to induce neuropathological changes in spinal cord and cauda equina.34 In this study, no obvious histopathological changes in spinal cord and cauda equina were found after chronic intrathecal infusion of tested agents. Larger doses or more detailed neurotoxicity studies may be warranted in the future. Altogether, our results demonstrate that intrathecal infusion of mibefradil or ethosuximide could attenuate SNL-induced CaV3.2 upregulation without causing obvious neuropathological change. Given their analgesic effects for neuropathy,9 intrathecal infusion of CaV3.2 blockers may be considered as a promising therapeutic management for nerve injury-induced pain.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

This work was supported by grants from Ministry of Science and Technology, Taipei, Taiwan (MOST 103-2314-B-195-003-MY2 and 105-2314-B-195-003-MY3 to J.K.C. and 105-2811-B-195-001 to S.J.S) and MacKay Memorial Hospital, Taipei, Taiwan(MMH 103051037410439104100105481057510621 to J.K.C.).


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