The tricyclic antidepressant (TCA) amitriptyline is a first-line drug for treating neuropathic pain.1,2 The number needed to treat for 50% pain relief by TCA is lower than that of other first-line drugs for neuropathic pain.1 Studies have suggested that the analgesic effect of amitriptyline against neuropathic pain results from enhancement of the serotonergic and noradrenergic descending inhibitory systems,2 which constitute an important mechanism for inhibition of neuropathic pain. However, amitriptyline utilizes a variety of mechanisms for analgesia in neuropathic pain,3,4 and it is unclear which mechanism is most important.
The serotonin (5-HT) and noradrenaline reuptake inhibitor (SNRI) duloxetine and gabapentinoids that block the α2δ subunit of voltage-gated calcium channels, including pregabalin and gabapentin, are other first-line drugs used to treat neuropathic pain.1 Recent studies have suggested that analgesic mechanism of SNRIs5,6 and gabapentinoids7–9 for neuropathic pain strongly depends on the brainstem-spinal descending noradrenergic system. The descending noradrenergic system inhibits nociceptive signaling from primary afferent neurons to the spinal dorsal horn.10 In the present study, we investigated the requirement of descending noradrenergic systems for the analgesic effect of these first-line drugs for treatment of neuropathic pain. We used a rat model of neuropathic pain caused by spinal nerve ligation (SNL), and assessed the antihyperalgesic effects of 5 daily injections of amitriptyline, duloxetine, pregabalin, or gabapentin. To determine whether these drugs require noradrenergic systems for their effects, we pretreated SNL rats with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), a toxin that selectively degenerates noradrenergic fibers.11 We also examined whether amitriptyline modified the descending noradrenergic systems.
The time course of the experiments is shown in Figure 1. This manuscript adheres to the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines.
The Animal Care and Use Committee of the Gunma University Graduate School of Medicine approved the study protocol. Male Sprague-Dawley rats (200 g, SLC, Shizuoka, Japan) were used in all experiments. The animals were housed (n = 6/cage) on soft bedding under a 12-hour light-dark cycle, with free access to food and water. SNL was performed as described previously.12 Briefly, after anesthetizing the rats with isoflurane (2%) in oxygen, the right L6 transverse process was removed, and the right L5 spinal nerve was tightly ligated with a 5-0 silk suture and cut just distal to the ligature. The wound was then closed, and the animals were allowed to recover for 3 days, and the rats were started to habituate to the person doing behavioral test. We used 144 of 151 rats. The reason of exclusion was a paralysis of right lower limb due to SNL surgery.
The primary aim of our study was to investigate the requirement of descending noradrenergic systems for the analgesic effects of the first-line drugs used to treat neuropathic pain. We used SNL rats pretreated with DSP-4 and assessed the antihyperalgesic effects of 5 daily injections of amitriptyline, duloxetine, pregabalin, or gabapentin. The antihyperalgesic effects were determined based on behavioral testing. The person conducting the behavioral testing was blinded to the drug administered to the animals. The 37215 Analgesy-meter (37215; Ugo Basile, Comerio, Italy) was used to measure the nociceptive threshold to increasing pressure applied to the hind paw, expressed in grams, as described previously.13 Immediately after withdrawal of the paw, the applied pressure was released, and a cutoff of 250 g was used to avoid tissue injury. Animal training for this test was performed with the device for 3 days before baseline values were recorded. During the recovery period, the animals were handled to allow them to habituate to the person doing the behavioral test.
All rats were intraperitoneally injected with DSP-4 (50 mg/kg) or saline from 7 days after SNL. DSP-4 was dissolved in 1 mL of saline. The animals received 5 daily intraperitoneal injections of amitriptyline (10 mg·kg−1·day−1), duloxetine (10 mg·kg−1·day−1), pregabalin (10 mg·kg−1·day−1), or gabapentin (50 mg·kg−1·day−1) from 21 days after SNL surgery. The paw withdrawal threshold was determined before SNL surgery, 15 minutes before drug injection (days 0–4 in Figure 1A), and at predetermined time points after drug injection (days 5, 6, 8, 10, and 12 in Figure 1A) to assess the chronic effect of the drugs. Duloxetine was dissolved by mixing with 50% dimethylsulfoxide and saline (vehicle). Amitriptyline, pregabalin, or gabapentin was dissolved in saline. The drugs were administered in a volume of 1 mL. Amitriptyline was purchased from LKT Laboratories, Inc (St Paul, MN). Duloxetine was purchased from Wako Pure Chemical Industries (Osaka, Japan). Pregabalin was kindly donated by Pfizer, Inc (Groton, CT). Gabapentin was purchased from Toronto Research Chemicals, Inc (North York, ON, Canada).
To examine the role of noradrenaline in the spinal cord in amitriptyline-induced analgesia in detail, we used the α2-adrenoceptor antagonist idazoxan. Idazoxan (30 µg/10 µL, dissolved in saline) was administered intrathecally to SNL rats. The rats were intraperitoneally injected with DSP-4 (50 mg/kg) or vehicle from 7 days after SNL surgery and were given daily intraperitoneal injections of amitriptyline (10 mg·kg−1·day−1) for 5 days from 21 days after SNL surgery. At 24 hours after the last injection of amitriptyline, intrathecal administration from the L5–L6 intervertebral space was performed under anesthesia with 2% isoflurane in oxygen, using a 30-gauge stainless-steel needle. The withdrawal thresholds were determined before SNL surgery, 15 minutes before amitriptyline injection, and 15 minutes before and 60 minutes after idazoxan injection. Idazoxan was purchased from Sigma-Aldrich (St Louis, MO).
To determine whether the 5 daily injections of amitriptyline activate noradrenergic neurons in the locus coeruleus (LC) and spinal cord, and to investigate the effect of DSP-4 on the noradrenergic system, we performed immunohistochemistry using antibodies for c-Fos (a marker for neuronal activation) and dopamine beta-hydroxylase (DβH, an enzyme that labels noradrenergic fibers). The SNL animals were injected with DSP-4 and amitriptyline at the same dose, route, and time points used for the behavioral studies. At 24 hours after the last amitriptyline injection, the animals were anesthetized by an intraperitoneal injection of pentobarbital (100 mg/kg) and intracardially perfused with 0.01 M phosphate-buffered saline (PBS) containing 1% sodium nitrite at 4°C, followed by 4% paraformaldehyde in 0.1 M PBS. The brainstems and lumbar (L4–5) spinal cord segments were dissected out, postfixed in the same fixative, and cryoprotected with 30% sucrose in 0.1 M PBS. Four days later, a cryostat was used to cut the brain containing the LC and spinal cord segments into 20-µm transverse sections, which were then mounted on glass slides.
After pretreatment with 3% normal donkey serum (Jackson Immuno Research, West Grove, PA) containing 0.3% Triton X-100 (Sigma-Aldrich) for 2 hours to prevent nonspecific labeling, the sections were incubated with a rabbit monoclonal anti–c-Fos antibody (1:500, 4; Santa Cruz Biotechnology, Dallas, TX) and a mouse monoclonal anti-DβH antibody (1:500, MAB 308; Millipore, Billerica, MA) overnight in 1.5% normal donkey serum. The sections were then washed in PBS and incubated in the appropriate secondary antibody including CY3-conjugated donkey anti-rabbit IgG (1:600; Millipore) and CY2-conjugated donkey anti-mouse IgG (1:200; Millipore) for 3 hours at room temperature. The sections were rinsed thoroughly in PBS again, and incubated in 4′,6-diamidino-2-phenylindole (1:40,000; Life Technologies, Eugene, OR) for 5 minutes.
Images of the LC and spinal cord were captured on an Olympus FSX100 microscope (Olympus Co, Tokyo, Japan) using fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate filters and a 20× objective with a resolution of 1360 × 1024 pixels. The number of c-Fos–positive noradrenergic cells within a defined threshold in the LC was counted using image analysis software (Image J; NIH Image, National Institutes of Health, Bethesda, MD). 4′,6-diamidino-2-phenylindole staining was used to identify the nuclei of LC neurons. Image J was used to quantify changes in immunofluorescence (DβH). The optical density threshold was set to include and match the immunoreactivity, providing an image with immunoreactive (IR) material appearing as red pixels. For quantification of DβH immunofluorescence, a fixed area (250 × 250 pixels) was placed in the center of the LC cells and the middle one-third of the mediolateral extent of the spinal cord dorsal horn, and the number of pixels of DβH-IR cells within the threshold value was quantified. Data are shown as the number of pixels in the area. All rats were killed and processed on the same day, and the same threshold value was applied to all images for a given antibody. The person quantifying the sections was blind to the treatment.
All of the data are presented as the mean ± SD. We assessed behavior as the primary outcome based on our hypothesis that pretreatment with DSP-4 decreases the antihyperalgesic effects of amitriptyline, duloxetine, pregabalin, and gabapentin. We divided SNL rats into 4 groups: saline + vehicle, saline + drug, DSP-4 + saline, and DSP-4 + drug. The time points analyzed were before SNL surgery (pre in Figure 1A), 15 minutes before drug injection (days 0–4 in Figure 1A), and predetermined time points after drug injection (days 5, 6, 8, 10, and 12 in Figure 1A). For idazoxan testing, we divided SNL rats that received 5 daily injections of amitriptyline into the following groups: saline + saline, saline + idazoxan, DSP-4 + saline, and DSP-4 + idazoxan. The time points analyzed were before SNL surgery (pre-SNL in Figure 1B), 15 minutes before amitriptyline injection (control in Figure 1B), and 15 minutes before and 60 minutes after idazoxan injection (15 minutes before intrathecal treatment and 60 minutes after intrathecal treatment in Figure 1B). Behavioral data were analyzed by 2-way repeated-measures analyses of variance (ANOVA) where the group and time were considered as independent variables, and Student t test with Bonferroni correction was used for multiple comparisons. Immunohistochemistry data were analyzed by 1-way ANOVA followed by Student t test with Bonferroni correction. P < .01 was considered statistically significant. The estimated treatment effect (difference between groups) and 99% confidence interval for main outcomes (mechanical withdrawal threshold in SNL rats) are reported. The statistical analysis was conducted using SigmaPlot 12 (Systat Software Inc, San Jose, CA). We analyzed 144 rats. We performed a power analysis for the primary outcome (mechanical withdrawal threshold in SNL rats) to determine the appropriate sample size, with the assumptions of a mean difference of 50 g in the withdrawal threshold and SD of 30 g in each group according to a previous study.5 We found that 6 rats in each group would result in detection of significant differences with 80% power at a significance level of α = .05.
We examined the antihyperalgesic effects of 5 daily injections of amitriptyline (10 mg·kg−1·day−1), duloxetine (10 mg·kg−1·day−1), pregabalin (10 mg·kg−1·day−1), or gabapentin (50 mg·kg−1·day−1) in DSP-4- or saline-pretreated SNL rats. Table 1 shows the results of the 2-way repeated-measures ANOVA (group, time, and group × time interaction). Table 2 shows the estimated treatment effect (difference between groups) with the 99% confidence interval. Ipsilateral paw withdrawal thresholds were decreased 3 weeks after SNL surgery (day 0) in all groups. In the saline-pretreated groups, paw withdrawal thresholds were increased after 5 daily injections of amitriptyline (Figure 2A), duloxetine (Figure 2B), pregabalin (Figure 3A), and gabapentin (Figure 3B) compared with the vehicle-treated group (P < .001, respectively).
In DSP-4–pretreated SNL animals, the antihyperalgesic effects induced by the injections of duloxetine, pregabalin, and gabapentin were reversed compared with the saline-pretreated group (P < .001, respectively). In contrast, the antihyperalgesic effects induced by the injections of amitriptyline were not reversed in DSP-4–pretreated SNL animals (P= .081; Figure 2A). Antihyperalgesic effects were observed on days 4 and 5 in the DSP-4-injected rats compared with the saline + vehicle-treated group (P < .001 on day 4, P= .001 on day 5 by Student t test with Bonferroni correction after 2-way repeated-measures ANOVA; Figure 2A).
Intrathecal treatment with idazoxan, an α2-adrenoceptor antagonist (30 µg), reversed the antihyperalgesic effect of 5 daily injections of amitriptyline in saline-pretreated SNL rats (P < .001 by 2-way repeated-measures ANOVA followed by the Student t test with Bonferroni correction; Figure 4). In DSP-4–pretreated SNL rats, idazoxan did not reverse the antihyperalgesic effect of 5 daily injections of amitriptyline (P = 1.0).
The photomicrographs in Figure 5A depict c-Fos-IR in noradrenergic LC neurons, identified by DβH-IR in SNL rats pretreated with injection of DSP-4 or saline and then given 5 daily injections of amitriptyline or vehicle at the same dose used for the behavioral studies. In the saline-pretreated SNL rats, 5 daily injections of amitriptyline (10 mg·kg−1·day−1) increased the number of DβH-IR pixels in the LC (F3,15 = 155.240; P < .001 by Student t test with Bonferroni correction after 1-way repeated-measures ANOVA; Figure 5B). In DSP-4–pretreated SNL rats, the number of DβH-IR pixels was decreased, and this effect was not changed by 5 daily injections of amitriptyline (P < .001, respectively). Amitriptyline increased c-Fos expression in the LC neurons in SNL rats pretreated with both saline and DSP-4 compared with those pretreated with vehicle injection (F3,15 = 77.956, P < .001 by Student t test with Bonferroni correction after 1-way repeated-measures ANOVA, respectively; Figure 5C).
The photomicrographs in Figure 6A depict DβH-IR fibers in the lumbar spinal dorsal horn in SNL rats, pretreated with an injection of DSP-4 or saline and then treated with 5 daily injections of amitriptyline or vehicle at the same dose used for the behavioral studies. Similarly to the LC, 5 daily injections of amitriptyline (10 mg·kg−1·day−1) increased the number of DβH-IR pixels in the spinal dorsal horn in saline-pretreated SNL rats (F3, 15 = 178.086, P < .001 by Student t test with Bonferroni correction after 1-way repeated-measures ANOVA; Figure 6B). In DSP-4–pretreated SNL rats, the number of DβH-IR pixels was decreased, and this effect was not changed by the 5 amitriptyline injections (P< .001).
In the current study, 5 daily injections of amitriptyline, duloxetine, pregabalin, and gabapentin resulted in an antihyperalgesic effect in SNL rats. When the treatment stopped, however, the withdrawal thresholds gradually decreased to the preinjection level. Pretreatment with DSP-4 followed by 5 daily injections of duloxetine, pregabalin, and gabapentin did not result in antihyperalgesic effects in SNL rats. We found that the antihyperalgesic effect was still present after amitriptyline treatment in SNL rats with DSP-4 pretreatment, and this effect was maintained after intrathecal injection of the α2-adrenoceptor antagonist idazoxan. We also showed that 5 daily injections of amitriptyline increased the number of DβH-IR pixels in the LC and the spinal dorsal horn in SNL rats. With DSP-4 pretreatment, the number of DβH-IR pixels was dramatically decreased regardless of whether or not amitriptyline was administered. The amitriptyline treatment increased the ratio of c-Fos-IR cells in noradrenergic LC neurons in SNL rats regardless of DSP-4 pretreatment. These results suggest that 5 daily injections of amitriptyline produce antihyperalgesic effects in SNL rats, in which the noradrenergic descending inhibitory systems are suppressed.
TCA, SNRI, and gabapentinoids are strongly recommended for pharmacotherapy of neuropathic pain.1 In clinical doses, antidepressants do not provide acute analgesic effects for neuropathic pain and require chronic administration to treat neuropathic pain.14 Our behavioral results revealed that 5 daily injections of amitriptyline, duloxetine, pregabalin, and gabapentin gradually increased the withdrawal thresholds in rats with SNL. The analgesic effect also gradually decreased after finishing 5 daily injections of the drugs, suggesting that chronic administration is necessary for management of neuropathic pain.
The analgesic mechanisms of TCA, SNRI, and gabapentinoids for neuropathic pain depend on brainstem-spinal descending noradrenergic systems. Previous studies suggested that the main effect of the TCA amitriptyline in neuropathic pain is to enhance the serotonergic and noradrenergic descending inhibitory systems.4 Our previous studies showed that the noradrenergic descending inhibitory system was more important than the serotonergic system for the efficacy of SNRIs in neuropathic pain.6 Gabapentin not only acts on the α2δ subunit of voltage-gated calcium channels in the spinal cord but also activates the noradrenergic descending inhibitory system, resulting in increased levels of noradrenaline in the spinal cord.7,9,15 The α2-adrenoceptors in the spinal dorsal horn play an important role in the mechanism by which noradrenaline inhibits neuropathic pain.10 In the spinal dorsal horn, activation of the α2-adrenoceptor by noradrenaline directly reduces nociceptive transmission by decreasing release of excitatory neurotransmitters such as substance P and glutamate from primary afferent terminals10 and by hyperpolarizing spinal interneurons via G-protein–mediated activation of potassium channels.16 Although α2-adrenoceptor agonists suppress inhibitory spinal cholinergic interneuron activity in normal animals, they excite this system in neuropathic pain states, resulting in acetylcholine release. This mechanism is thought to be important for the analgesic effects of spinal α2-adrenoceptor activation in neuropathic pain.17–19
In the present study, we used DSP-4 to attenuate the effects of the noradrenergic descending inhibitory system. Injection of DSP-4 (50 mg/kg) reduces the tissue level of noradrenaline to 10%–30% and 20% of that in normal rats in the LC11 and spinal cord,20 respectively. In the present study, DSP-4 decreased the ratio of DβH-IR in the LC (38%) and the spinal dorsal horn (14%). However, the DSP-4 experiment revealed that the efficacy of amitriptyline for neuropathic pain was less dependent on the brainstem-spinal descending noradrenergic system than that of SNRI and gabapentinoids.
Noradrenaline in the LC and the spinal cord is not completely eliminated by DSP-4.11 Previous studies showed that DSP-4 does not affect the content of the α2-adrenoceptor in the spinal cord and that the α2-adrenoceptor shows increased affinity of the receptor for noradrenaline.20,21 It is thus possible that amitriptyline uses the residual noradrenergic signaling to produce analgesia, even after DSP-4 treatment. However, in the current study, intrathecal administration of idazoxan did not change the efficacy of amitriptyline in SNL rats pretreated with DSP-4. This result suggests that the α2-adrenoceptor in the spinal cord does not relate to the analgesic efficacy of amitriptyline in SNL rats with DSP-4 pretreatment. Furthermore, amitriptyline did not increase the DβH-IR ratio in the LC and spinal dorsal horn of SNL rats with DSP-4 pretreatment. These results suggest that 5 daily injections of amitriptyline do not activate residual noradrenergic signaling and the other mechanisms of amitriptyline may contribute to achieve analgesia in SNL rats with DSP-4 pretreatment. On the other hand, idazoxan decreased the antihyperalgesic effect of amitriptyline in saline-pretreated rats. We think that in rats without DSP-4 pretreatment, the function of the noradrenergic descending inhibitory system is normally preserved, and thus amitriptyline produced antihyperalgesic effects mainly through inhibiting noradrenaline reuptake. Thus, in the normal condition, the antihyperalgesic effect of amitriptyline might heavily depend on noradrenergic descending inhibitory systems, including α2-adrenoceptor in the spinal dorsal horn.
Our study provides novel evidence that 5 daily injections of amitriptyline activate noradrenergic neural cells of the LC in SNL rats. It is known that TCA drugs facilitate glutamate release in the rat LC.22 Glutamate is an important excitatory neurotransmitter for noradrenergic neurons, acting through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.23 In particular, amitriptyline elevates brain-derived neurotrophic factor levels in astrocyte cultures24 and brain-derived neurotrophic factor induces phosphorylation of the AMPA receptor GluA1 and modulates trafficking of the AMPA receptor.25 Amitriptyline might thus increase glutamate release and AMPA receptor levels on the surface of neurons of the LC to provide therapeutic effects in neuropathic pain.
Although amitriptyline may activate the noradrenergic neurons in the LC by enhancing glutamatergic transmission, this mechanism did not contribute to the analgesic effect of amitriptyline in the DSP-4–pretreated group in the present study. Amitriptyline acts as a blocker of voltage-gated sodium and potassium channels in lamina 1–3 of the spinal cord.26 It is also a N-methyl-d-aspartate receptor antagonist,27 an activator of adenosine release and of the adenosine A1 receptor,28 and an enhancer of the γ-aminobutyric acid B receptor29 and opioid receptor,30,31 and it inhibits inflammation by decreasing levels of prostaglandin E and tumor necrosis factor-α.4 A recent study also indicated that amitriptyline activates tyrosine kinase receptors to enhance axonal growth.32,33 Although various mechanisms of action of amitriptyline may contribute to its inhibition for neuropathic pain, we did not determine which mechanism is most important in this study.
Noxious stimulus–induced analgesia, which represents endogenous analgesia, is decreased from 5 weeks after SNL surgery because of attenuation of the descending noradrenergic system.34,35 Our present finding that the antihyperalgesic effect duloxetine, pregabalin, and gabapentin depended on brainstem-spinal descending noradrenergic systems suggests that the analgesic potential of these drugs might be reduced in neuropathic pain. Our finding also indicated that amitriptyline activated noradrenergic neurons in the LC and enhanced noradrenergic signaling in SNL rats. A previous clinical study demonstrated that combined treatment with the TCAs nortriptyline and gabapentin was more effective than either drug alone for neuropathic pain.15 These findings may explain why the combined use of these drugs for the treatment of neuropathic pain is more effective than a single drug alone.
There are some limitations to the present study. DSP-4 not only affects noradrenergic transmission but it also slightly affects serotonergic transmission (20%–40% reduction in rat brain cerebellum and spinal cord).11 Although we did not evaluate serotonergic transmission, there are no reports that the output of noradrenaline-containing LC neurons involves serotonergic or other nonadrenergic neurotransmitters. In the present study, we focused on noradrenergic systems and used DSP-4 because it is commonly used to destroy noradrenaline-containing LC neuron terminals.
In summary, 5 daily injections of amitriptyline produced antihyperalgesic effects in neuropathic pain despite suppression of the noradrenergic descending inhibitory system. Additionally, amitriptyline activated neurons in the LC and increased noradrenergic fiber signaling in SNL rats. These results suggest that amitriptyline may produce analgesia in pathological conditions involving dysfunction of the descending noradrenergic inhibitory system, as observed in long-term neuropathic pain.
We thank Sachiko Ito, MD, for technical support, and Devang Thakor, PhD, for English editing.
Name: Tadanao Hiroki, MD.
Contribution: This author helped conduct the study, collect data, analyze the data, and prepare the manuscript.
Name: Takashi Suto, MD, PhD.
Contribution: This author helped collect data and prepare the manuscript.
Name: Shigeru Saito, MD, PhD.
Contribution: This author helped design the study and prepare the manuscript.
Name: Hideaki Obata, MD, PhD.
Contribution: This author helped design and conduct the study, analyze the data, and prepare the manuscript.
This manuscript was handled by: Gregory J. Crosby, MD.
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