Role of anoctamin-1 and bestrophin-1 in spinal nerve ligation-induced neuropathic pain in rats

Adult female Wistar rats (140–160 g, 6–7 weeks) from our own breeding facilities were used in this study. Female rats were used based on the fact that previous studies from our laboratory have found no differences in tactile allodynia between female and male rats [29]. These animals were housed in a controlled environment, on a 12-h light/dark cycle, with free access to food and water. All experiments were conducted according to the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985), Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals [30] and were approved by our local Ethics Committee (Protocol 0042-13, Cinvestav, Mexico City, Mexico). In addition, every effort was made to minimize pain and suffering, and the number of rats used was the least required to obtain significant statistical power.

Neuropathic pain was induced by spinal nerve ligation [31]. Briefly, rats were anesthetized with a mixture of ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). After surgical preparation and exposure of the dorsal vertebral column, the left L5 and L6 spinal nerves were exposed and tightly ligated with 6-0 silk suture distal to the DRG. For sham-operated rats, the nerves were exposed but not ligated. Rats exhibiting motor deficiency such as paw dragging were discarded from the study.

Tactile allodynia was determined as previously described [32]. Fourteen days after surgery, animals were placed in cages with a mesh grid floor and allowed to acclimate for a minimum of 30 min before performing the experiment. Von Frey filaments (Stoelting, Wood Dale, IL, USA) were used to determine the 50% paw withdrawal threshold using the up-down method of Dixon [33]. A series of filaments, starting with one that had a buckling weight of 2 g, were consecutively applied in consecutive sequence to the plantar surface of the right hind paw with a pressure causing the filament to buckle. Lifting of the paw indicated a positive response and prompted the use of the next weaker filament, whereas absence of paw withdrawal after 5 s indicated a negative response and prompted the use of the next heavier filament in a series. This paradigm continued until four more measurements were made after the initial change of the behavioral response or until five consecutive negative (assigned a score of 15 g) or four consecutive positive (assigned a score of 0.25 g) responses occurred. The resulting scores were used to compute the 50% withdrawal threshold by using the formula: 50% g threshold = 10^{(Xf +κδ)}/10,000, where Xf = value (in log units) of the final von Frey filament used, κ = the value from table published by Dixon [33] for the pattern of positive and/or negative responses, and δ = the mean difference (in log units) between stimulus strengths. Allodynia was considered to be present when paw withdrawal thresholds were less than 4 g.

The Hargreaves test measured the latency to radiant heat using a paw thermal stimulator [34]. Briefly, rats were placed individually in Plexiglass cubicles and allowed to acclimate for 20–30 min before the experiment. A radiant heat stimulus was applied to the base of the paw as it rested on a glass plate maintained at 30 ± 0.1°C at 0, 1, 2, 4, 6 and 8 h after drug administration. A timer was automatically actuated when the light source was turned on. The response latency was defined as the time required for abrupt withdrawal of the paw. In all cases, a cut-off of 20 s was employed to avoid tissue injury. Each test was repeated three times and the average paw withdrawal latency was calculated based on three measurements.

Motor coordination was assessed in the Rotarod system [35]. Animals were acclimated 2 days before evaluation by walking them each day on an accelerating Rotarod apparatus (Panlab Harvard Apparatus, Barcelona, Spain). On the third day, animals were tested starting 1.5 h before and immediately after drug administration as well as 1.5 and 3 h later (time of peak drug effect). Evaluation was carried out with an acceleration from 4 to 40 rpm in 10 min. Motor coordination was considered as the time latency to fall off the Rotarod apparatus. It was determined from the mean time in three trials for each rat at each time.

Seven days after spinal nerve ligation surgery, rats were again anesthetized with a ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) combination and placed in a stereotaxic head holder in order to expose the atlanto-occipital membrane [36]. After piercing the membrane, a PE-10 catheter (7.5 cm) was passed intrathecally to the level of the thoracolumbar junction and the wound was sutured. Rats were allowed to recover from surgery for 7 days in individualized cages before use. Animals showing any signs of motor impairment were eliminated from the study and euthanized with a CO₂ chamber.

Total RNA from ipsilateral (injured side) dorsal spinal cord segments L1–S1 as well as ipsilateral DRG (L4–L6) were isolated by the guanidinium-thiocyanate method with TRIzol-reagent according to the manufacturer’s instructions (Invitrogen Corporation, Carlsbad, CA, USA). The concentration and purity of the RNA were determined by measuring the absorbance at 260 and 280 nm in a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Equal amounts (5 μg) of RNA from samples were reversed-transcribed into first-strand complementary DNA (cDNA) using oligo dT and M-MLV-reverse transcriptase (Invitrogen Corporation, Carlsbad, CA, USA). The reaction was performed at 37°C for 60 min, followed by a heat denaturation step at 95°C for 5 min. Amplification of cDNA by polymerase chain reaction (PCR) was performed with specific primers previously reported [23] for bestrophin-1 (forward, 5′-TGGCAGAACAGCTCATCAAC-3′ and reverse, 5′-GCTGCCTCGTTCCAGTACAT-3′, 100 bp product) and anoctamin-1 (forward, 5′-TTCGTCAATCACACGCTCTC-3′ and reverse, 5′-GGGGTTCCCGGTAATCTTTA-3′, 100 bp product). The reaction mixture contained 2.5 mM MgCl₂, 200 μM dNTP, 1 U Taq DNA polymerase (Invitrogen Corporation, Carlsbad, CA, USA), 100 pmol of specific sense and antisense primers for each gene, and 10 ng of first-strand cDNA in a total volume of 50 μL. The optimal number of cycles within the linear range of amplification was selected. The PCR conditions for amplification were 94°C for 5 min. Thirty-five cycles of 94°C for 45 s, 58.5°C for 45 s, 72°C for 90 s, and 72°C for 7 min using a Mastercycler DNA Engine Thermal Cycler (Eppendorf, Hamburg, Germany). To ensure that equal amounts of reverse-transcribed RNA were added to the PCR, the β-actin housekeeping gene was amplified using oligonucleotides previously reported [37]. The PCR products were analyzed by 10% SDS–polyacrylamide gel electrophoresis and ethidium bromide staining and captured by a Chemidoc XRS + imaging system (BioRad, Hercules, CA, USA). Bands were quantified by scanning densitometry using Image Lab 5.0 software (BioRad, Hercules, CA, USA).

Ipsilateral dorsal spinal cord segments L1–S1 and DRG (L4–L6) were homogenized in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1% NP40, 0.1% SDS, 2 mM phenyl-methyl-sulfonyl fluoride, 6.8 µg/mL aprotinin, 4 µg/mL leupeptin, 4 µg/mL pepstatin A, 4 µg/mL soybean trypsin inhibitor and 2 mM NaVO₄) [35]. Homogenates were centrifuged at 4°C for 10 min at 14,000 rpm, and the supernatant fraction was used to measure protein concentration [38]. Total protein (50 or 100 µg, for spinal cord or DRG, respectively) was resolved by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk in phosphate-buffered saline at pH 7.4 containing (in mM) (137 NaCl, 2.7 KCl, 10 Na₂HPO₄ and 2 KH₂PO₄) with Tween 0.05% and they were incubated at 4°C overnight with rabbit anti-bestrophin-1 (ABC-001, 1:200; Alomone Labs, Jerusalem, Israel) or rabbit anti-anoctamin-1 (ACL-011, 1:100; Alomone Labs, Jerusalem, Israel). Horseradish peroxidase-conjugated secondary antibody (anti-rabbit, 711-035-152, 1:6,000; Jackson ImmunoResearch Laboratories Inc, West Grove, PA, USA) was applied for detecting the primary antibody signal using an enhanced chemiluminescence detection system according to the manufacturer´s instructions (Western Lightning Ultra, NEL112001EA, PerkinElmer, Waltham, MA, USA). After bestrophin-1 or anoctamin-1 detection, the membranes were stripped, blocked and incubated with a mouse monoclonal antibody directed against β-actin (MAB1501, 1:10,000; Millipore, Billerica, MA, USA) and its respective horseradish peroxidase-conjugated secondary antibody (anti-mouse, 115-035-003, 1:10,000; Jackson ImmunoResearch Laboratories Inc, West Grove, PA, USA). β-actin expression was used as a loading control to normalize protein expression levels. In addition, antibodies were pre-adsorbed with control bestrophin-1 and anoctamin-1 peptides to validate antibody specificity. Scanning of the immunoblots was performed and the bands were quantified by densitometry using an image analysis program (LabWorks; UVP Inc., Upland, CA, USA).

Rats were anesthetized with a mixture of ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) 14 days after surgery. A laminectomy was performed while the surgical field was bathed with oxygenated (5% CO₂ and 95% O₂) artificial cerebrospinal fluid (CSF) at pH of 7.6 containing (in mM): 117 NaCl, 3.6 KCl, 1.2 MgCl₂, 2.3 CaCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃ and 11 glucose at room temperature. The L5 ganglia attached to dorsal root and spinal nerve were removed. The tissue was transferred to a recording chamber and bathed with artificial CSF solution at room temperature.

To evoke action potentials, electrical stimulation (0.3 ms of duration) was applied to the peripheral cut end of the spinal nerve with a suction electrode. The compound action potential (CAP) was recorded in the dorsal root with a suction electrode. The recording electrode was connected to a DC amplifier (World Precision Instruments, Sarasota, FL, USA) with a bandwidth of DC to 10 kHz. Unless otherwise noted, the recordings shown represent the average of 10 stimuli applied every 10 s. The threshold (T) of the faster Aα/β fiber compound action potential (CAP) was determined by increasing the stimulus strength until a visible action potential was evoked 50% of the time. The maximal CAP of the putative C fibers was evoked stimulating at 50xT.

5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), anthracene-9-carboxylic acid (9-AC) and niflumic acid (NFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The specific anoctamin-1 inhibitor (T16A_inh-A01) was purchased from Tocris Bioscience (Avon, Bristol, England). The specific calcium-activated chloride channel inhibitor (CaCC_inh-A01) was purchased from Merck Millipore (Billerica, MA, USA). All drugs were dissolved in 30% dimethyl sulfoxide (DMSO) in all doses tested.

In order to determine the role of CaCCs in neuropathic pain, neuropathic (14 days after spinal nerve ligation) and sham animals received an intrathecal (10 μL) or peripheral (into the dorsal surface of the left hind paw, 50 μL) injection of vehicle (30% DMSO) or increasing concentrations of non-selective (NPPB, 9-AC or NFA) or selective (T16A_inh-A01 or CaCC_inh-A01) CaCCs inhibitors 5 min before evaluation of tactile allodynia, thermal hyperalgesia or motor coordination. The antiallodynic and antihyperalgesic effects were evaluated for the following 8 h. All behavioral tests were scored by a single investigator who was blind to the treatment received by the subject. Furthermore, in order to assess the role of the CaCCs bestrophin-1 and anoctamin-1 in neuropathic rats, we determined the expression of bestrophin-1 and anoctamin-1 mRNA and protein levels in the ipsilateral dorsal section of the spinal cord and ipsilateral DRG at 1, 7 and 14 days after nerve injury.

As spinal nerve ligation increased anoctamin-1 expression, we next determined its anoctamin-1 mRNA and protein expression in the presence and absence of the most effective CaCCs inhibitors. For this, we intrathecally administered CaCC_inh-A01 (10 μg), T16A_inh-A01 (10 μg) or NFA (300 μg) every 6 h for five times starting on day 12 after ligation. Samples of spinal cord and DRG were obtained on day 14 after nerve ligation.

Considering that bestrophin-1 mRNA or protein expression levels were not modified by spinal nerve ligation and the lack of specific inhibitors for this channel, we next determined bestrophin-1 mRNA and protein expression in the presence and absence of its specific antibody. For this study, we intrathecally injected either the anti-bestrophin-1 or anti-anoctamin-1 antibody (2 μg) every 6 h for five times starting on day 12 after ligation, as previously reported for other proteins [39‐41]. Withdrawal threshold was evaluated every 6 h after each administration and rats were sacrificed on day 14 after nerve ligation to obtain spinal cord and DRG.

To investigate whether the antiallodynic effect of CaCCs inhibitors was mediated by a reduction of the peripheral nerve injury-induced hyperexcitability, we recorded the C component of the CAP in L5 dorsal roots of naïve, sham and neuropathic rats in the presence and absence of the selective CaCCs inhibitors T16A_inh-A01 (20 μM) and CaCC_inh-A01 (20 μM).

All behavioral results are reported as the mean ± SEM for six animals/group. Curves were constructed by plotting the paw withdrawal threshold or withdrawal latency as a function of time. An increase of the 50% withdrawal threshold or withdrawal latency was considered as antiallodynic or antihyperalgesic effect, respectively.

In order to determine the dose required to reduce 50% of the possible maximal effect (ED₅₀), we calculated the percent of maximum possible effect (%MPE) according to the following equation:where AUC is the area under the curve of the time-course of the 50% withdrawal threshold per rat of each group. The dose–response curves were constructed by plotting the %MPE versus dose and the experimental points were fitted using least-square linear regression. ED₅₀ ± standard error (SE) was calculated according to Tallarida [42].

For mRNA and protein expression, all results are reported as the mean relative intensity ± SEM for three independent animals/group.

For the electrophysiological recordings, all results are reported as the mean of the normalized area under the curve of the C component of the CAP ± SEM for six animals/group.