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. 2014 Sep 3;35(1):137–146. doi: 10.1007/s10571-014-0105-2

Possible Involvement of Nitric Oxide Modulatory Mechanism in the Protective Effect of Retigabine Against Spinal Nerve Ligation-Induced Neuropathic Pain

Raghavender Pottabathini 1, Anil Kumar 1,✉, Archana Bhatnagar 2, Sukant Garg 3
PMCID: PMC11486261  PMID: 25182225

Abstract

Decreasing the hyperexcitability of neurons through opening of voltage-gated potassium (Kv7) channels has been suggested as one of the protective mechanisms in the effective management of neuropathic pain. Reactive oxygen/nitrogen species are well implicated in the pathophysiology of neuropathic pain. Further, M current generated by opening of voltage-gated potassium channels (Kv7) has been modulated by reactive oxygen/nitrogen species. The present study has been designed to elucidate the nitric oxide modulatory mechanism in the protective effect of retigabine against spinal nerve ligation-induced neuropathic pain in rats. Ligation of L5/L6 spinal nerves resulted in alterations in various behavioral (as evident from marked increase in thermal and mechanical hyperalgesia, and allodynia) and biochemical (raised lipid peroxidation, nitrite, and depletion of GSH, SOD, and catalase) cascades as compared to sham treatment. Administration of retigabine (10 mg/kg) for 28 days attenuated these behavioral and biochemical cascades as compared to control rats. Further, l-arginine (100 mg/kg) pretreatment with retigabine (5 mg/kg) significantly reversed the protective effect of retigabine in spinal nerve-ligated rats. However, l-NAME (10 mg/kg) pretreatment with retigabine (5 mg/kg) significantly potentiated their protective effects which were significant as compared to their effect per se, respectively. The present study highlights the possible involvement of nitric oxide modulatory mechanism in the protective effect of retigabine against L5/L6 spinal nerve ligation-induced behavioral and biochemical alterations in rats.

Keywords: Spinal nerve ligation, Retigabine, KCNQ channels, Nitric oxide

Introduction

Neuropathic pain is defined as “pain caused by a lesion or disease of the somatosensory system” (Jensen et al. 2011). Neuropathic pain associated with peripheral nerve injury is characterized by spontaneous pain, hyperalgesia (an increased response to a stimulus which is normally painful), and allodynia (pain as a result of a stimulus which does not normally provoke pain). The molecular mechanisms underlying neuropathic pain are not yet clearly understood. However, various mechanisms such as damage to the sensory neurons resulting in adaptive changes in the density, distribution and functional activities of several voltage, and ligand-gated ion channels, receptors and enzymes in the damaged DRG and spinal neurons (Rasband et al. 2001; Waxman and Zamponi 2014) have been proposed. These mechanistic changes in part have been demonstrated to be responsible for the state of hyperexcitability (Abdulla and Smith 2001, 2002).

Opening of potassium channels plays an essential role in regulating resting membrane potential through hyper polarization of cell membrane resulting in decreased excitability. There are five different types of voltage-gated K+ channel subunits (Kv7.1–7.5) which are the members of KCNQ/M channels family (Brown and Passmore 2009). A slowly activating (tens of milliseconds), low threshold (activate at about −60 mV), non inactivating, steady voltage-dependent outward current (M current) generated by these channels exerts a suppressing effect on repetitive or burst-firing as well as on general excitability of the neurons (Brown and Passmore 2009; Delmas and Brown 2005). Drug therapies which are aimed at these Kv7 channels have been recently shown to be effective in different types of inflammatory (Xu et al. 2010; Nielsen et al. 2004), visceral (Hirano et al. 2007), and neuropathic painful conditions in experimental models (Blackburn-Munro and Jensen 2003; Zheng et al. 2013; Nodera et al. 2011).

Besides, role of reactive oxygen species (ROS) has also been well documented in persistent chronic pain (Kim et al. 2004; Naik et al. 2006). Further, free radical scavengers have been shown to be effective in ameliorating neuropathic pain. Indeed, retigabine, a potent Kv7 channel opener, has been shown to produce antioxidant as well as neuroprotective effect in different in vitro studies (Boscia et al. 2006; Seyfried et al. 2000). Ori and his group proposed the potential role of M current and its modification by nitric oxide modulator in trigeminal ganglion neurons regulation (Ooi et al. 2013). However, the relationship between KCNQ/M channels and NO pathway in neuropathic painful condition and their influence on various behavioral manifestations have not been studied so far.

Taken together, therefore, the present study has been designed to explore the relationship between KCNQ/M channels and NO modulators in L5/L6 spinal nerve ligation-induced neuropathic pain.

Materials and Methods

Animals

Male SD rats (180–220 g) bred in Central Animal House, Panjab University, Chandigarh were used in this study. Animals were acclimatized to laboratory conditions prior to experimentation. They were kept under standard laboratory conditions, maintained on 12-h light/dark cycle and had free access to food and water. All the experiments were performed between 9:00 and 17:00 h. The experimental protocol was approved by Institutional Animal Ethics Committee (IAEC) of Panjab University (Protocol no- 282/30/8/12/UIPS-42) and carried out in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA), Government of India and Indian National Science Academy Guidelines for the use and care of experimental animals. All the behavioral studies were carried out by a person who is blinded to the treatment groups.

Spinal Nerve Ligation

The procedure of ligation of the spinal nerves was performed as per modified method of Chung (Chung et al. 2004). Briefly, under chloral hydrate (350 mg/kg) anesthesia, individual animal was placed in a prone position and left paraspinal muscles were separated from the spinous processes at the L4-S2 level. Part of the L6 transverse process was carefully separated, and L4–L6 spinal nerves were identified. The L5–L6 spinal nerves were carefully exposed and tightly ligated distal to the dorsal root ganglion and proximal to the formation of the sciatic nerve with 6–0 silk thread. Following complete haemostasis, the wound was sutured. The procedure of the sham-treated animals was identical to that of the experimental group except spinal nerves ligation. All surgical procedures were carried out under normal sterile conditions.

Drug and Treatment Schedule

Study protocol includes ten treatment groups, consisting of six animals in each. Retigabine was obtained from Sigma-Aldrich, suspended in 10 % v/v Tween 80 and administered orally (p.o.) as per body weight (5 ml/kg). l-arginine and l-NAME [N(G)-nitro-l-arginine methyl ester] (Sigma Chemicals, St. Louis, MO, USA) were diluted with saline (pH 7.4) and administered intraperitoneally (i.p.) 30 min prior to retigabine treatment.

The study was conducted in two phases; the effect of retigabine was explored in the first phase and its interaction with NO modulators in the second. Group-1: Naive (without treatment), Group-2: Sham (surgery performed, vehicle administered), Group-3: SNL (L5/L6 spinal nerves were ligated), Group-4: SNL + Retigabine (5 mg/kg, p.o.), Group-5: SNL + Retigabine (10 mg/kg, p.o.), Group-6: SNL + l-NAME (10 mg/kg, i.p.), Group-7: SNL + l-NAME (10 mg/kg, i.p.) + Retigabine (5 mg/kg, p.o.), Group-8: SNL + l-arginine (100 mg/kg, i.p.), Group-9: SNL + l-arginine (100 mg/kg, i.p.) + Retigabine (5 mg/kg, p.o.), and Group-10: Sham + Retigabine (10 mg/kg, p.o.).

Each group received drug treatment daily in the morning 10:00 am, for 28 days starting from the day after SNL. Drugs and their dose selection were made as per the reported literature (Dost et al. 2004; Patil et al. 2006). All the behavioral tests were performed after 1 h of drug administration.

Behavioral Assessments

Mechanical Allodynia

Rats were placed individually in a clear plastic cage containing mesh (1 cm2 perforations). Animals were adapted to the testing environment for at least 30 min before the assessment of mechanical allodynia. Mechanical allodynic response was quantified by using vonFrey anesthesiometer and rigid vonFrey filaments. A polypropylene rigid tip of 0.5 mm diameter was used to apply the force to the plantar region of the hind paw. The force that leads to paw withdrawal was recorded by the anesthesiometer. The test was repeated for five times at the interval of 5 min, and the mean value was recorded (Negi et al. 2010).

Chemical Allodynia

The cold allodynia was assessed by spraying a 200 μL of acetone onto the plantar surface of rat paw (placed over a wire mesh), without touching the skin. The total time duration that the animal spent on lifting, shaking, or licking against acetone treatment was recorded for 2 min immediately after acetone application (Choi et al. 1994).

Mechanical Hyperalgesia

Paw pressure threshold was assessed with Randall–Selitto paw pressure analgesia meter (IITC Life Science, Woodland Hills, CA). Increasing pressure at a linear rate of 10 g/s was applied to the center of the hind paw. The pressure at which the animal withdraws its paw was recorded by an analgesia meter and expressed in mass units (grams), with a cut-off of 250 g to avoid potential tissue injury. Five tests were recorded at the interval of at least 10 min for each animal and expressed as a mean value (Santos-Nogueira et al. 2012).

Heat Hyperalgesia

Hyperalgesic response through hot-plate is an involvement of both central and peripheral mechanisms. In this test, animals were individually placed on a hot-plate (Eddy’s Hot-Plate) with the temperature adjusted to 52 ± 1 °C. The latency to the first sign of paw licking or jumping response was taken as an index of pain threshold; the cut-off time was 15 s in order to avoid paw damage (Kanaan et al. 1996).

Dissection and Homogenization

Immediately after behavioral assessments on day 28, animals were sacrificed by decapitation, and sciatic nerve (starting from the point of emergence from the spinal cord to its trifurcation, so that it covers the ligated portion along with proximal and distal ends) was dissected out and stored at −20 °C. On the next day, biochemical estimations were performed. For the biochemical estimations, 10 % (w/v) tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 × g at 4 °C for 15 min. Aliquots of supernatants were separated and used for biochemical estimations.

Biochemical Estimations

Estimation of Lipid Peroxidation

The quantitative measurement of lipid peroxidation in sciatic nerve was performed as per the method of Wills (Wills 1966). The amount of malondialdehyde (MDA), a measure of lipid peroxidation, was assessed by the reaction with thiobarbituric acid at 532 nm by using Perkin Elmer Lambda 20 Spectrophotometer (Norwalk, CT, USA). The values were calculated using molar extinction coefficient of chromophore (1.56 × 105 M−1 cm−1) and expressed as nanomoles of malondialdehyde per milligram of protein.

Estimation of Nitrite

The accumulation of nitrite concentration in the supernatant, an indicator of the production of NO, was determined with a colorimetric assay with Greiss reagent (0.1 % N-(1-naphthyl) ethylenediamine dihydrochloride, 1 % sulfanilamide, and 2.5 % phosphoric acid) as described by (Green et al. 1982). Equal volumes of supernatant and Greiss reagent were mixed, which were then incubated for 10 min at room temperature in the dark. Absorbance was determined at 540 nm using Perkin Elmer Lambda 20 Spectrophotometer (Norwalk, CT, USA). The concentration of nitrite in the supernatant was determined from a sodium nitrite standard curve and expressed as micromole per milligram of protein.

Estimation of Superoxide Dismutase (SOD) Activity

Superoxide dismutase (SOD) activity was accessed as per the method of Kono (Kono 1978), wherein the reduction of nitrobluetetrazolium (NBT) was inhibited by superoxide dismutase and measured at 560 nm using spectrophotometer. Briefly, the reaction was initiated by the addition of the hydroxylamine hydrochloride to the mixture containing NBT and sample. The results were expressed as unit/mg protein, where one unit of enzyme is defined as the amount of enzyme inhibiting the rate of reaction by 100 %.

Estimation of Reduced Glutathione (GSH)

Reduced glutathione in sciatic nerve was estimated as per the method of Ellman (Ellman 1959). 1 ml supernatant was precipitated with 1 ml of 4 % sulfosalicylic acid and cold digested at 4 °C for 1 h. The sample was centrifuged at 1,200 rpm for 15 min at 4 °C. To 1 ml of this supernatant, 2.7 ml of 0.1 M phosphate buffer (pH 8) and 0.2 ml of 5,5-dithiobis 2-nitrobenzoic acid (DTNB) were added. The absorbance was read immediately at 412 nm, and the results were calculated using molar extinction coefficient of chromophore (1.36 × l04 M−1 cm−1) and expressed as micromole GSH per milligram protein.

Catalase Estimation

Catalase activity was assayed by the method of Luck (Luck 1963), wherein breakdown of hydrogen peroxides (H2O2) is measured at 240 nm. Briefly, assay mixture consisted of 3 ml of H2O2 phosphate buffer and 0.05 ml of supernatant of tissue homogenate (10 %), and the change in absorbance was recorded at 240 nm. The results were then expressed as micromole H2O2 decomposed per milligram of protein/min.

Protein Estimation

The protein was measured by biuret method using bovine serum albumin as standard (Gornall et al. 1949).

Statistical Analysis

A group of six animals (n = 6) were assigned to a specific drug treatment. Results were expressed as mean ± S.E.M. The data were analyzed by two analysis of variance (ANOVA) followed by Bonferroni post tests for behavioral parameters and one way analysis of variance (ANOVA) followed by Tukey’s test for biochemcial estimations. p < 0.05 was considered to be statistically significant. Graph Pad Prism (Graph Pad Software, San Diego, CA) was used for all statistical analyses.

Results

Effect of Retigabine on Mechanical Allodynia and its Interactions with Nitric Oxide Modulators in Spinal Nerve Ligation-Induced Neuropathic Pain

Sham treatment did not show any significant effect on paw withdrawal threshold as compared to naive animals (data not shown). Ligation of L5/L6 spinal nerves in SNL control group significantly (p < 0.001) decreased paw withdrawal threshold as compared to sham group. Retigabine (10 mg/kg, p.o.) treatment significantly (p < 0.01) improved paw withdrawal threshold from day 14 as compared to SNL control group (Fig. 1a). However, lower dose of retigabine (5 mg/kg, p.o.) significantly improved paw withdrawal threshold from day 21 as compared to SNL control group. Further, retigabine (10 mg/kg) per se treatment did not show any significant effect on paw withdrawal threshold as compared to sham treatment (Fig. 1a).

Fig. 1.

Fig. 1

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a Effect of retigabine on mechanical allodynia in spinal nerve ligation-induced neuropathic pain. Data were expressed as mean ± S.E.M. a p < 0.05 compared to sham group; b p < 0.05 compared to SNL group; c p < 0.05 compared to Ret (5); (Two way ANOVA followed by Bonferroni posttests). SNL Spinal nerve ligation, Ret Retigabine. b Effect of retigabine on mechanical hyperalgesia in spinal nerve ligation-induced neuropathic pain. Data were expressed as mean ± S.E.M. a p < 0.05 compared to sham group; b p < 0.05 compared to SNL group; c p < 0.05 compared to Ret (5); (Two way ANOVA followed by Bonferroni posttests). SNL Spinal nerve ligation, Ret Retigabine

Further, l-NAME (10 mg/kg, i.p.) pretreatment with retigabine (5 mg/kg, p.o.) significantly potentiated their protective effect (increased paw withdrawal threshold) on 21st and 28th days as compared to their effect per se in SNL-treated group (p < 0.05). Conversely, l-arginine (100 mg/kg, i.p.) pretreatment with retigabine (5 mg/kg, p.o.) significantly (p < 0.05) reversed the protective effect (shortened paw withdrawal threshold) of retigabine on 3rd and 4th weeks in SNL-treated group (Fig. 2a).

Fig. 2.

Fig. 2

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a Effect of retigabine along with the nitric oxide modulators on mechanical allodynia in spinal nerve ligation-induced neuropathic pain. Data were expressed as mean ± S.E.M. a p < 0.05 compared to sham group; b p < 0.05 compared to SNL group; c p < 0.05 compared to Ret (5); d p < 0.05 compared to l-NAME (10); (Two way ANOVA followed by Bonferroni posttests). SNL Spinal nerve ligation, Ret Retigabine, l -ARG l-arginine. b Effect of retigabine along with the nitric oxide modulators on mechanical hyperalgesia in spinal nerve ligation-induced neuropathic pain. Data were expressed as mean ± S.E.M. a p < 0.05 compared to sham group; b p < 0.05 compared to SNL group; c p < 0.05 compared to Ret (5); d p < 0.05 compared to l-NAME (10); (Two way ANOVA followed by Bonferroni posttests). SNL Spinal nerve ligation, Ret Retigabine, l -ARG l-arginine

Effect of Retigabine on Mechanical Hyperalgesia and its Interactions with Nitric Oxide Modulators in Spinal Nerve Ligation-Induced Neuropathic Pain

Sham treatment did not show any significant effect on mechanical hyperalgesia (change in paw withdrawal threshold) as compared to naive animals, whereas SNL significantly (p < 0.001) decreased paw withdrawal threshold as compared to sham group. Retigabine (10 mg/kg, p.o.) treatment significantly (p < 0.05) improved paw withdrawal threshold as compared to SNL control group. However, lower dose of retigabine (5 mg/kg, p.o.) significantly improved paw withdrawal threshold from day 14 onwards as compared to SNL control group. Further, retigabine (10 mg/kg) per se treatment did not produce any significant effect on mechanical hyperalgesia as compared to sham treatment (Fig. 1b).

Further, l-NAME (10 mg/kg, i.p.) pretreatment with retigabine (5 mg/kg, p.o.) significantly potentiated their protective effect (increased paw withdrawal threshold) from day 21 onwards as compared to their effects per se in SNL-treated animals (p < 0.05). Conversely, l-arginine (100 mg/kg) pretreatment with lower dose of retigabine (5 mg/kg, p.o.) significantly reversed the protective effect of retigabine on day 28 in SNL-treated group (Fig. 2b).

Effect of Retigabine on Cold Allodynia and its Interactions with Nitric Oxide Modulators in Spinal Nerve Ligation-Induced Neuropathic Pain

Sham treatment did not produce any significant effect on cold allodynia [paw withdrawal duration (lifting and licking)] as compared to naive animals. SNL treatment significantly (p < 0.001) increased cold allodynia (increased paw withdrawal duration, caused more licking and lifting) in response to acetone treatment that persisted throughout the study period as compared to sham group. Retigabine (10 mg/kg, p.o.) treatment significantly attenuated cold allodynia (decreased paw withdrawal duration, reduced licking and lifting) from day 7 onwards (p < 0.05) as compared to SNL control group. However, treatment with lower dose of retigabine (5 mg/kg, p.o.) significantly reversed cold allodynia from day 21 onwards as compared to SNL control group. Retigabine (10 mg/kg) per se treatment did not show any significant effect on cold allodynia as compared to sham group (Fig. 3a).

Fig. 3.

Fig. 3

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a Effect of retigabine on cold allodynia and b on heat hyperalgesia in spinal nerve ligation-induced neuropathic pain. Data were expressed as mean ± S.E.M. a p < 0.05 compared to sham group; b p < 0.05 compared to SNL group; c p < 0.05 compared to Ret (5); (Two way ANOVA followed by Bonferroni posttests). SNL Spinal nerve ligation, Ret Retigabine. c Effect of retigabine along with the nitric oxide modulators on cold allodynia and d on heat hyperalgesia in spinal nerve ligation-induced neuropathic pain. Data were expressed as mean ± S.E.M. a p < 0.05 compared to sham group; b p < 0.05 compared to SNL group; c p < 0.05 compared to Ret (5); d p < 0.05 compared to l-NAME (10); (Two way ANOVA followed by Bonferroni posttests). SNL Spinal nerve ligation, Ret Retigabine, l -ARG l-arginine

Further, l-NAME (10 mg/kg, i.p.) pretreatment with retigabine (5 mg/kg, p.o.) significantly potentiated their protective effect (decreased paw withdrawal duration, reduced licking and lifting) from day 14 onwards as compared to their effects per se (p < 0.05) in SNL-treated animals. Conversely, l-arginine (100 mg/kg, i.p.) pretreatment with lower dose of retigabine (5 mg/kg, p.o.) reversed the protective effect of retigabine from day 14 onwards which is significant as compared to retigabine (5 mg/kg, p.o.) treatment alone in SNL-treated group (Fig. 3c).

Effect of Retigabine on Heat Hyperalgesia and its Interactions with Nitric Oxide Modulators in Spinal Nerve Ligation-Induced Neuropathic Pain

Sham treatment did not show any significant effect on paw withdrawal latency as compared to naive animals, whereas SNL treatment significantly (p < 0.001) decreased paw withdrawal latency in response to heat stimulation and remained stable for 4 weeks as compared to sham group. Administration of retigabine (10 mg/kg, p.o.) significantly (p < 0.05) delayed paw withdrawal latency from day 21 onwards, whereas lower dose of retigabine (5 mg/kg, p.o.) shown significant effect on paw withdrawal latency on day 28 (p < 0.05) as compared to SNL control group. Further, retigabine (10 mg/kg) per se treatment did not show any significant effect on heat hyperalgesia as compared to sham animals (Fig. 3b).

Further, l-NAME (10 mg/kg, i.p.) pretreatment with retigabine (5 mg/kg, p.o.) significantly (p < 0.05) potentiated their protective effect (delayed paw withdrawal latency) from day 14 onwards as compared to their effects per se in SNL-treated animals. Further, l-arginine (100 mg/kg) pretreatment with lower dose of retigabine (5 mg/kg, p.o.) significantly reversed the protective effect (shortened paw withdrawal latency) of retigabine in SNL-treated group (Fig. 3d).

Effect of Retigabine on Oxidative Damage and its Interaction with Nitric Oxide Modulators in Spinal Nerve-Ligated Rats

Sham treatment did not produce any significant effect on oxidative damage parameters as compared to naive group. SNL treatment significantly (p < 0.001) increased lipid peroxidation, nitrite concentration, depleted GSH, SOD, and catalase enzyme activity in sciatic nerves as compared to sham group. Treatment with retigabine (10 mg/kg) for 28 days significantly (p < 0.05) attenuated lipid peroxidation (p < 0.001), nitrite concentration (p < 0.001), restored GSH (p < 0.001), SOD (p < 0.001), and catalase enzyme (p < 0.001) activity as compared to SNL control group.

Further, l-NAME (10 mg/kg) pretreatment with retigabine (5 mg/kg, p.o.) significantly (p < 0.05) potentiated their protective effect (reduced lipid peroxidation, nitrite concentration, restored GSH, SOD, and catalase activity) as compared to their effects per se in SNL-treated animals. Conversely, l-arginine (100 mg/kg) pretreatment with retigabine (5 mg/kg p.o.) significantly reversed the protective effect of retigabine (increased lipid peroxidation, nitrite concentration, depleted GSH, SOD, and catalase activity) in SNL-treated animals. Retigabine (10 mg/kg) per se treatment did not produce any significant effect on oxidative damage parameters as compared to sham group (Table 1).

Table 1.

Effect of retigabine on oxidative damage (lipid peroxidation, SOD, nitrite, GSH, and catalase levels) and its interaction with nitric oxide modulators in spinal nerve ligation-induced neuropathic pain in rats

Treatment (mg/kg) MDA nM/mg protein Mean ± S.E.M. (% of sham) Nitrite µM/mg protein
Mean ± S.E.M. (% of sham)		 SOD Units/mg Protein Mean ± S.E.M. (% of sham) Catalase µM of H2O2 decomposed per min/mg protein Mean ± S.E.M. (% of sham) Reduced glutathione
(nM/mg protein)
Mean ± S.E.M (%Sham)
Sham

1.585 ± 0.05

(100)

67.50 ± 2.08

(100)

13.082 ± 0.33

(100)

2.94 ± 0.34

(100)

31.82 ± 0.53

(100)
SNL

4.398 ± 0.20a

(277.33)

300.83 ± 3.74a

(445.68)

3.142 ± 0.36a

(24.04)

0.39 ± 0.05a

(13.42)

5.32 ± 0.63a

(16.73)
SNL + Ret (5)

3.690 ± 0.11b

(232.77)

234.67 ± 5.16b

(347.65)

5.458 ± 0.24b

(41.73)

1.41 ± 0.10b

(48.00)

11.79 ± 1.00b

(37.05)
SNL + Ret (10)

2.928 ± 0.14b,c

(184.69)

186.67 ± 5.51b,c

(276.54)

7.957 ± 0.36b,c

(60.84)

2.03 ± 0.11b,c

(69.13)

18.10 ± 1.20b,c

(56.89)
SNL + l- NAME (10)

4.083 ± 0.18

(257.63)

290.50 ± 9.08

(430.37)

3.737 ± 0.29

(28.56)

0.47 ± 0.04

(15.93)

7.55 ± 0.59

(23.74)
SNL + l-NAME (10) + Ret (5)

2.765 ± 0.16b,c,d

(174.46)

202.67 ± 4.77b,c,d

(300.25)

7.957 ± 0.40b,c,d

(60.82)

2.43 ± 0.12b,c,d

(82.87)

17.82 ± 1.43b,c,d

(56.01)
SNL + l-ARG (100)

4.465 ± 0.13

(281.67)

310.33 ± 9.34

(459.75)

3.088 ± 0.14

(23.61)

0.43 ± 0.04

(14.61)

6.49 ± 0.65

(20.39)
SNL + l-ARG (100) + Ret (5)

4.385 ± 0.13c

(276.51)

306.17 ± 9.32c

(453.58)

3.377 ± 0.16c

(25.82)

0.95 ± 0.09c

(32.40)

5.88 ± 0.76c

(18.49)
Sham + Ret (10) per se

1.560 ± 0.11

(98.43)

71.67 ± 3.45

(106.17)

14.022 ± 0.59

(107.20)

2.97 ± 0.43

(101.17)

30.23 ± 2.25

(95.03)
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Data are expressed as mean ± S.E.M; in parenthesis, percentage of sham was mentioned

SNL spinal nerve ligation, Ret Retigabine

a p < 0.05 compared to sham group

b p < 0.05 compared to SNL group

c p < 0.05 compared to Ret (5)

d p < 0.05 compared to l-NAME (10); (One way ANOVA followed by Tukey’s test)

Discussion

Injury to peripheral nerve and its branches by transection, crush, or ligation resulted in the development of spontaneous pain, allodynia, and hyperalgesia, well-known characteristics of neuropathic pain (Padi and Kulkarni 2008; Sekiguchi et al. 2009). These behavioral alterations vary in duration and magnitude depending on the experiment model or species. These behavioral alterations last up to several weeks. In the present study, L5/L6 spinal nerve ligation significantly caused hyperalgesia and allodynia to both mechanical as well as thermal stimuli indicating neuropathic-like symptoms. Several nerve injury models have been developed to mimic the neuropathic pain-like symptoms analogous to similar clinical conditions in humans. Few of these models involve loose ligation of a part of the sciatic nerve as in the chronic constriction injury model (Kumar et al. 2011) or tight ligation of portion of the sciatic nerve as in the partial sciatic nerve ligation model (Rose et al. 2011). In both these models, there exists a considerable degree of variability in tightness of the ligation and the extent of nerve damaged by ligation. Both of these difficulties can be resolved by tight ligation of lumbar L5 and L6 spinal branches of the sciatic nerve. This L5 and L6 ligation model also has an advantage of causing separate injury on nerve branches to enable further manipulations in the neuropathic pain conditions. In the present study, SNL resulted in significant alterations in behavioral (mechanical allodynia, mechanical hyperalgesia, cold allodynia, and heat hyperalgesia) and biochemical (lipid peroxidation, nitrite concentration, GSH, SOD, and catalase) parameters in sciatic nerves supporting neuropathic-like symptoms. Our observations are in accordance with the previous experimental studies demonstrating similar behavioral and biochemical alterations against SNL (Sekiguchi et al. 2009; Choi et al. 1996; Chung et al. 2004; Xu et al. 2010; Yowtak et al. 2011).

Retigabine is a structural analog of flupirtine, a non opiate. It has a broad spectrum of anticonvulsant activity. Its analgesic activity has been demonstrated in various chronic pain models. Retigabine has reported to reduce mechanical hyperalgesia in chronic constriction injury model and spared nerve injury, but failed to show any significant effect on allodynic response against vonFrey stimulation in both the above models (Blackburn-Munro and Jensen 2003). In the present study, retigabine treatment has been shown to attenuate behavioral alterations such as mechanical allodynia, mechanical hyperalgesia, cold allodynia, and heat hyperalgesia, suggesting its ameliorative effect against SNL-induced neuropathic-like symptoms. Exact reason for these discrepancies is not known. Differences in the site (spinal and sciatic level) and extent of injury are produced because of these models, and the duration of drug treatment might be responsible for the differences observed.

Role of reactive oxygen species (ROS) has been well suggested in the pathophysiology of neuropathic pain (Kim et al. 2004; Yowtak et al. 2011). In the present study, SNL significantly caused oxidative stress as evidenced by an increased lipid peroxidation, nitrite concentration, depletion of GSH, SOD, and catalase in sciatic nerves. 28-day treatment with retigabine significantly attenuated oxidative stress in sciatic nerve as evidenced by reversal of these parameters (reduced lipid peroxidation, nitrite levels, restored GSH, SOD, and catalase) in sciatic nerves suggesting its antioxidant-like effect. Different antioxidants, free radical scavenging substances, and peroxynitrite-decomposition catalysts have been shown to reduce the mechanical allodynia in different models of neuropathic pain (Kim et al. 2004). In support, retigabine has also been demonstrated to reduce reactive oxygen species in rat pheochromocytoma PC-12 cells (Seyfried et al. 2000) as well as in organotypic hippocampal slice cultures (Boscia et al. 2006). In rat pheochromocytoma PC-12 cells, retigabine has been shown to restore glutathione levels and decreased reactive oxygen species production caused by l-glutamate toxicity (Seyfried et al. 2000). Supporting to the above findings, in the present study, retigabine has demonstrated to restore GSH, SOD, and catalase level which are the main antioxidant defense present in the body suggesting its role in promoting antioxidant defense. Therefore, it is likely that an antioxidant defense property of retigabine could be partially responsible for its protective effect as one of the mechanisms. Even though the potential antioxidant effect through the reduction in reactive oxygen species has been studied in vitro, to the best of our knowledge, this is the first study to demonstrate antioxidant-like effect of retigabine in vivo. However, the exact mechanism by which reactive oxygen species contribute to the development and maintenance of hypersensitivity in neuropathic pain is not clearly known. Further studies are required to elucidate the exact mechanism to prove the antioxidant-like effect of retigabine.

Peripheral nerve injury resulted in an up regulation of NOS activity in the DRG and lumbar spinal dorsal horn has been observed in different neuropathic pain models (Choi et al. 2012; Kim et al. 2011; Luo et al. 1999). Increased NO reacts with superoxide radicals to form peroxynitrile, which further takes part in the phosphorylation of NMDA receptors in spinal dorsal horn leading to central sensitization in different neuropathic pain conditions (Kwak et al. 2014). In trigeminal ganglion neurons administration of S-nitroso-N-acetyl-dl-penicillamine, a NO donor inhibited the M current and the same is reversed by the NOS inhibitors and scavenger of NO, suggesting that NO-mediated alteration of M current in these neurons (Ooi et al. 2013). However, modulation of neuronal M current through redox modulatory site has been proposed. Consistent with this, H2O2 resulted in an increase in M current, whereas NO reduced M current. Further, these modulations are attributed to exert through triple cysteine catalytic site present in DRG neurons. Strict control of NO environment around this catalytic site has been proposed to be further responsible for the change in M current. In line with these studies, in the present study, SNL resulted in an increased NO production as indicated by the rise in nitrite levels in sciatic nerves. It seems that rise in NO might further affect M current that is responsible for protective effect of retigabine. Further, l-NAME pretreatment with retigabine significantly potentiated their protective effect. However, l-arginine pretreatment with retigabine significantly reversed its protective effect suggesting the involvement of nitric oxide mechanism in the protective effect of retigabine. This implies the role of NO-mediated alterations in the protective effects of retigabine in neuropathic pain-induced oxidative stress parameters. However, earlier studies involving linopiridine (KCNQ channel blocker) in combination with retigabine in neuropathic pain models has been studied to demonstrate the mechanism of action of retigabine (Dost et al. 2004). It has been proposed that an antiallodynic response of retigabine was mediated through the opening of KCNQ channels and was antagonized by linopiridine in neuropathic pain. But the down stream process that affects the retigabine actions through the opening of KCNQ channels has not been clearly studied. The results of the present study demonstrate that the possible involvement of NO-mediated alterations in the protective effect of retigabine in L5/L6 SNL model of neuropathic pain in rats. In summary, the present study highlights the role of retigabine in SNL-induced behavioral and biochemical alterations. Further, the protective effect of retigabine could be at least in part due to its antioxidant-like effects in addition to its effect on KCNQ channel. The interaction of retigabine along with NO modulators suggests the possible involvement of NO-mediated alterations in the protective effect of retigabine in SNL-induced neuropathic pain. However, the complex pathophysiological process does not rule out the involvement of other possible mechanisms of neuropathic pain. However, further studies are required to demonstrate the exact mechanism of retigabine in neuropathic conditions.

Acknowledgments

The research project sanctioned to Professor Anil Kumar by Indian council of medical research (ICMR), New Delhi is gratefully acknowledged. Raghavender Pottabathini is the SRF in this project.

Conflict of interest

The authors have no competing financial interests to declare. There is no conflict of interest among any of the authors.

References

  1. Abdulla FA, Smith PA (2001) Axotomy- and autotomy-induced changes in Ca2+ and K+ channel currents of rat dorsal root ganglion neurons. J Neurophysiol 85(2):644–658 [DOI] [PubMed] [Google Scholar]
  2. Abdulla FA, Smith PA (2002) Changes in Na(+) channel currents of rat dorsal root ganglion neurons following axotomy and axotomy-induced autotomy. J Neurophysiol 88(5):2518–2529. doi:10.1152/jn.00913.2001 [DOI] [PubMed] [Google Scholar]
  3. Blackburn-Munro G, Jensen BS (2003) The anticonvulsant retigabine attenuates nociceptive behaviours in rat models of persistent and neuropathic pain. Eur J Pharmacol 460(2–3):109–116 [DOI] [PubMed] [Google Scholar]
  4. Boscia F, Annunziato L, Taglialatela M (2006) Retigabine and flupirtine exert neuroprotective actions in organotypic hippocampal cultures. Neuropharmacology 51(2):283–294. doi:10.1016/j.neuropharm.2006.03.024 [DOI] [PubMed] [Google Scholar]
  5. Brown DA, Passmore GM (2009) Neural KCNQ (Kv7) channels. Br J Pharmacol 156(8):1185–1195. doi:10.1111/j.1476-5381.2009.00111.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Choi Y, Yoon YW, Na HS, Kim SH, Chung JM (1994) Behavioral signs of ongoing pain and cold allodynia in a rat model of neuropathic pain. Pain 59(3):369–376 [DOI] [PubMed] [Google Scholar]
  7. Choi Y, Raja SN, Moore LC, Tobin JR (1996) Neuropathic pain in rats is associated with altered nitric oxide synthase activity in neural tissue. J Neurol Sci 138(1–2):14–20 [DOI] [PubMed] [Google Scholar]
  8. Choi JI, Kim WM, Lee HG, Kim YO, Yoon MH (2012) Role of neuronal nitric oxide synthase in the antiallodynic effects of intrathecal EGCG in a neuropathic pain rat model. Neurosci Lett 510(1):53–57. doi:10.1016/j.neulet.2011.12.070 [DOI] [PubMed] [Google Scholar]
  9. Chung JM, Kim HK, Chung K (2004) Segmental spinal nerve ligation model of neuropathic pain. Methods Mol Med 99:35–45. doi:10.1385/1-59259-770-x:035 [DOI] [PubMed] [Google Scholar]
  10. Delmas P, Brown DA (2005) Pathways modulating neural KCNQ/M (Kv7) potassium channels. Nat Rev Neurosci 6(11):850–862 [DOI] [PubMed] [Google Scholar]
  11. Dost R, Rostock A, Rundfeldt C (2004) The anti-hyperalgesic activity of retigabine is mediated by KCNQ potassium channel activation. Naunyn Schmiedebergs Arch Pharmacol 369(4):382–390. doi:10.1007/s00210-004-0881-1 [DOI] [PubMed] [Google Scholar]
  12. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82(1):70–77 [DOI] [PubMed] [Google Scholar]
  13. Gornall AG, Bardawill CJ, David MM (1949) Determination of serum proteins by means of the biuret reaction. J Biol Chem 177(2):751–766 [PubMed] [Google Scholar]
  14. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126(1):131–138 [DOI] [PubMed] [Google Scholar]
  15. Hirano K, Kuratani K, Fujiyoshi M, Tashiro N, Hayashi E, Kinoshita M (2007) Kv7.2–7.5 voltage-gated potassium channel (KCNQ2-5) opener, retigabine, reduces capsaicin-induced visceral pain in mice. Neurosci Lett 413(2):159–162. doi:10.1016/j.neulet.2006.11.043 [DOI] [PubMed] [Google Scholar]
  16. Jensen TS, Baron R, Haanpää M, Kalso E, Loeser JD, Rice ASC, Treede R-D (2011) A new definition of neuropathic pain. Pain 152(10):2204–2205. doi:10.1016/j.pain.2011.06.017 [DOI] [PubMed] [Google Scholar]
  17. Kanaan SA, Saadé NE, Haddad JJ, Abdelnoor AM, Atweh SF, Jabbur SJ, Safieh-Garabedian B (1996) Endotoxin-induced local inflammation and hyperalgesia in rats and mice: a new model for inflammatory pain. Pain 66(2–3):373–379 [DOI] [PubMed] [Google Scholar]
  18. Kim HK, Park SK, Zhou J-L, Taglialatela G, Chung K, Coggeshall RE, Chung JM (2004) Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain. Pain 111(1–2):116–124. doi:10.1016/j.pain.2004.06.008 [DOI] [PubMed] [Google Scholar]
  19. Kim KH, Kim J-I, Han JA, Choe M-A, Ahn J-H (2011) Upregulation of neuronal nitric oxide synthase in the periphery promotes pain hypersensitivity after peripheral nerve injury. Neuroscience 190:367–378. doi:10.1016/j.neuroscience.2011.05.064 [DOI] [PubMed] [Google Scholar]
  20. Kono Y (1978) Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys 186(1):189–195 [DOI] [PubMed] [Google Scholar]
  21. Kumar A, Meena S, Kalonia H, Gupta A, Kumar P (2011) Effect of nitric oxide in protective effect of melatonin against chronic constriction sciatic nerve injury induced neuropathic pain in rats. Indian J Exp Biol 49(9):664–671 [PubMed] [Google Scholar]
  22. Kwak K-H, Jung H, Park JM, Yeo J-S, Kim H, Lee HC, Byun SH, Kim J-C, Park S-S, Lim DG (2014) A peroxynitrite decomposition catalyst prevents mechanical allodynia and NMDA receptor activation in the hind-paw ischemia reperfusion injury rats. Exp Ther Med 7(2):508–512. doi:10.3892/etm.2013.1440 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Luck H (1963) Catalase spectrophotometric method. In: Methods in Enzymatic Analysis. Academic Press, New York
  24. Luo ZD, Chaplan SR, Scott BP, Cizkova D, Calcutt NA, Yaksh TL (1999) Neuronal nitric oxide synthase mRNA upregulation in rat sensory neurons after spinal nerve ligation: lack of a role in allodynia development. J Neurosci 19(21):9201–9208 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Naik AK, Tandan SK, Dudhgaonkar SP, Jadhav SH, Kataria M, Prakash VR, Kumar D (2006) Role of oxidative stress in pathophysiology of peripheral neuropathy and modulation by N-acetyl-l-cysteine in rats. Eur J Pain 10(7):573–579. doi:10.1016/j.ejpain.2005.08.006 [DOI] [PubMed] [Google Scholar]
  26. Negi G, Kumar A, Sharma SS (2010) Concurrent targeting of nitrosative stress-PARP pathway corrects functional, behavioral and biochemical deficits in experimental diabetic neuropathy. Biochem Biophys Res Commun 391(1):102–106. doi:10.1016/j.bbrc.2009.11.010 [DOI] [PubMed] [Google Scholar]
  27. Nielsen AN, Mathiesen C, Blackburn-Munro G (2004) Pharmacological characterisation of acid-induced muscle allodynia in rats. Eur J Pharmacol 487(1–3):93–103. doi:10.1016/j.ejphar.2004.01.017 [DOI] [PubMed] [Google Scholar]
  28. Nodera H, Spieker A, Sung M, Rutkove S (2011) Neuroprotective effects of Kv7 channel agonist, retigabine, for cisplatin-induced peripheral neuropathy. Neurosci Lett 505(3):223–227. doi:10.1016/j.neulet.2011.09.013 [DOI] [PubMed] [Google Scholar]
  29. Ooi L, Gigout S, Pettinger L, Gamper N (2013) Triple cysteine module within M-type K+ channels mediates reciprocal channel modulation by nitric oxide and reactive oxygen species. J Neurosci 33(14):6041–6046. doi:10.1523/jneurosci.4275-12.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Padi SSV, Kulkarni SK (2008) Minocycline prevents the development of neuropathic pain, but not acute pain: possible anti-inflammatory and antioxidant mechanisms. Eur J Pharmacol 601(1–3):79–87. doi:10.1016/j.ejphar.2008.10.018 [DOI] [PubMed] [Google Scholar]
  31. Patil CS, Padi SV, Singh VP, Kulkarni SK (2006) Sildenafil induces hyperalgesia via activation of the NO-cGMP pathway in the rat neuropathic pain model. Inflammopharmacology 14(1–2):22–27. doi:10.1007/s10787-006-1511-y [DOI] [PubMed] [Google Scholar]
  32. Rasband MN, Park EW, Vanderah TW, Lai J, Porreca F, Trimmer JS (2001) Distinct potassium channels on pain-sensing neurons. Proc Natl Acad Sci U S A 98(23):13373–13378. doi:10.1073/pnas.231376298 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rose K, Ooi L, Dalle C, Robertson B, Wood IC, Gamper N (2011) Transcriptional repression of the M channel subunit Kv7.2 in chronic nerve injury. Pain 152(4):742–754. doi:10.1016/j.pain.2010.12.028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Santos-Nogueira E, Redondo Castro E, Mancuso R, Navarro X (2012) Randall-Selitto test: a new approach for the detection of neuropathic pain after spinal cord injury. J Neurotrauma 29(5):898–904. doi:10.1089/neu.2010.1700 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sekiguchi M, Sekiguchi Y, Konno S-I, Kobayashi H, Homma Y, Kikuchi S-I (2009) Comparison of neuropathic pain and neuronal apoptosis following nerve root or spinal nerve compression. Eur Spine J 18(12):1978–1985. doi:10.1007/s00586-009-1064-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Seyfried J, Evert BO, Rundfeldt C, Schulz JB, Kovar KA, Klockgether T, Wüllner U (2000) Flupirtine and retigabine prevent L-glutamate toxicity in rat pheochromocytoma PC 12 cells. Eur J Pharmacol 400(2–3):155–166 [DOI] [PubMed] [Google Scholar]
  37. Waxman SG, Zamponi GW (2014) Regulating excitability of peripheral afferents: emerging ion channel targets. Nat Neurosci 17(2):153–163. doi:10.1038/nn.3602 [DOI] [PubMed] [Google Scholar]
  38. Wills ED (1966) Mechanisms of lipid peroxide formation in animal tissues. Biochem J 99(3):667–676 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Xu W, Wu Y, Bi Y, Tan L, Gan Y, Wang K (2010) Activation of voltage-gated KCNQ/Kv7 channels by anticonvulsant retigabine attenuates mechanical allodynia of inflammatory temporomandibular joint in rats. Mol Pain 6:49. doi:10.1186/1744-8069-6-49 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yowtak J, Lee KY, Kim HY, Wang J, Kim HK, Chung K, Chung JM (2011) Reactive oxygen species contribute to neuropathic pain by reducing spinal GABA release. Pain 152(4):844–852. doi:10.1016/j.pain.2010.12.034 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zheng Q, Fang D, Liu M, Cai J, Wan Y, Han J-S, Xing G-G (2013) Suppression of KCNQ/M (Kv7) potassium channels in dorsal root ganglion neurons contributes to the development of bone cancer pain in a rat model. Pain 154(3):434–448. doi:10.1016/j.pain.2012.12.005 [DOI] [PubMed] [Google Scholar]

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