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Inhibition of KCC2 in Mouse Spinal Cord Neurons Leads to Hypersensitivity to Thermal Stimulation

Austin, Thomas M. MD*; Delpire, Eric PhD*,†

doi: 10.1213/ANE.0b013e31822e0a5d
Analgesia: Research Reports
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BACKGROUND: KCC2, a neuronal-specific K-Cl cotransporter, is involved in pain perception physiology through its effects on postsynaptic inhibition in spinal cord neurons. We injected a newly identified, highly potent and selective inhibitor of KCC2 (D4), an inactive structural variant (D4.14), and the Na-K-2Cl cotransporter (NKCC1) inhibitor, bumetanide, into the intrathecal space of mice to measure their effect on heat-evoked nociceptive responses.

METHODS: Commercially available intrathecal catheters were modified and surgically placed into 2 cohorts of 10 mice. After recovery from the procedure, the mice were injected with D4, D4.14, and bumetanide through this catheter. Nociceptive measurements (hotplate assay, tail flick assay) were performed after injection of each of the test drugs and compared with vehicle controls.

RESULTS: Two mice in each cohort were omitted because of postprocedure complications. There was a statistically significant decrease (P < 0.01) in withdrawal latency after injection of the active KCC2 inhibitor but not after injection of the inactive compound (P = 0.78), as measured by hotplate assay at 55°C. Injection of bumetanide significantly increased withdrawal latency (P = 0.02) at the same temperature. These results were confirmed using tail flick assays performed at 49°C.

CONCLUSIONS: Inhibition of KCC2 by D4 led to decreased heat-evoked withdrawal latency in mice, as measured by hotplate and tail flick assays, whereas inhibition of NKCC1 by bumetanide resulted in increased response latencies to heat stimuli as measured by both of these nociceptive tests.

Published ahead of print September 29, 2011 Supplemental Digital Content is available in the text.

From the Departments of *Anesthesiology and Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee.

Supported by National Institutes of Health grant GM74771 (to Eric Delpire).

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Eric Delpire, PhD, Department of Anesthesiology, Vanderbilt University Medical School, T-4202 MCN, 1161 21st Ave. South, Nashville, TN 37232-2520. Address e-mail to eric.delpire@vanderbilt.edu.

Accepted July 6, 2011

Published ahead of print September 29, 2011

Neurons located in the dorsal horn of the spinal cord process sensory information coming from the periphery and relay this information to the brain through ascending fibers. This information is modulated in part by γ-aminobutyric acid A receptors located in postsynaptic relay neurons as well as in the presynaptic terminals of sensory afferent fibers (presynaptic). A neuronal-specific K-Cl cotransporter (KCC2) expressed in spinal cord neurons, drives the intracellular Cl concentration below its electrochemical equilibrium potential, thereby strengthening γ-aminobutyric acid A hyperpolarizing responses and postsynaptic inhibition.13 Similarly, a Na-K-2Cl cotransporter (NKCC1) expressed in afferent neurons drives the intracellular Cl concentration above its equilibrium potential, thereby facilitating primary afferent depolarization and presynaptic inhibition.46 These 2 cotransporters are critical in gating sensory information from the peripheral to the central nervous system.

Several studies have examined the role of KCC2 in nociception. Whereas one study showed hyposensitivity of KCC2 hypomorphic mice to tactile and thermal stimulation,7 several other studies demonstrated that decreased KCC2 expression in the spinal cord is associated with increased nociception.1,812 However, because of unanticipated effects from possible compensatory mechanisms, a complementary pharmacological approach would be useful. This is now possible because we recently identified, using a high-throughput screening strategy, novel compounds that inhibit KCC2 with high potency and selectivity.13

In this study, we made use of one of these compounds (called D4) and one of its inactive structural variants (D4.14), and developed an intrathecal catheter delivery method to assess the effect of pharmacological inhibition of KCC2 on heat-evoked nociceptive responses in mice. We also used bumetanide, a highly selective inhibitor of NKCC1, to test the effect of pharmacological inhibition of this transporter on nociception.

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METHODS

Catheter Modification

The intrathecal catheter (catalog no. 0007743; Alzet Osmotic Pumps, Cupertino, CA) was modified by trimming the thinnest portion of the catheter to a length of 1.0 cm and trimming the thickest portion the catheter to a length of 0.3 cm. The length of the thin portion was selected because the distance from the superficial muscle to the intrathecal space was measured to be approximately 0.5 cm. Because the catheter is inserted at the L5-6 intervertebral space, this length places the tip of the catheter at the L3-4 level. The thick end of the catheter was shortened as much as possible to decrease the chance of removal while still being functional. A 0.2-cm portion of the thick tubing was fastened to the distal end of the middle portion of the catheter using epoxy (Fig. 1). This extra tubing acted as a tether for the fascial suture and was placed to decrease the chance of catheter withdrawal. After modification, the guiding wire was reinserted into the catheter. The nature of the polyurethane material in the catheter permitted sterilization by autoclaving. The volume within the modified catheter was determined to be 1.3 μL by calculating the difference in weight between a catheter filled with distilled water and the same dry catheter.

Figure 1

Figure 1

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Animal Studies

All animal procedures and experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee and were in compliance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. Two cohorts of 10 mice (65-day-old C57BL/6J males) were used for our experiments.

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Catheter Insertion

The intrathecal catheter procedure was adapted from Wu et al.14 Anesthesia was induced with 5% isoflurane, using a Vapomatic vaporizer (A.M. Bickford, Inc., Wales Center, NY). The anesthesia was maintained with 3% to 5% isoflurane by titrating the concentration to both the respiratory rate and response to surgical stimuli. The lower backs of the mice were shaved using electric clippers, and prepped successively with Betadine and 70% ethanol (Fig. 2A). Postoperative analgesia was provided by injecting ketoprofen (10 mg/kg) subcutaneously before incision. The pelvic girdle was then palpated and a 1-cm midline incision was made directly above the cephalad border of the girdle. The skin was then retracted to expose the lower lumbar vertebrae. The mouse was held firmly by the pelvic girdle in one hand while gently flexing its spine, and the intervertebral space was accessed through an opening in the overlying muscle created by a 22-gauge needle (Fig. 2B). The catheter was then placed in a paravertebral manner by pushing into one side of the L5-6 process at an angle of approximately 20 to 30 degrees above the vertebral column and 20 to 30 degrees to the midline of the vertebra. When the sign of dural penetration (sudden movement of the tail or hindlimb) was observed, the guidewire was withdrawn to avoid nerve damage by the metal wire. The thinnest portion of the catheter was then inserted entirely into the intrathecal space and the catheter was secured by 1 fascial suture (4.0 polypropylene; Ethicon, San Angelo, TX) using the fabricated tether (Fig. 2C). The skin incision was then closed with the catheter exiting the most caudal aspect of the incision (Fig. 2D). The end of the catheter was then sealed with a small piece of transparent film dressing (Tegaderm™; 3M, St. Paul, MN).

Figure 2

Figure 2

One day after implantation, ketoprofen (10 mg/kg) was given subcutaneously, the catheter dressing was removed, and 2.5 μL of 2% lidocaine (Hospira, Inc., Lake Forest, IL) followed by 5 μL normal saline was injected through the catheter. Immediate motor paralysis of the hindlimbs was taken as correct placement of the intrathecal catheter.

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Timeline

After surgery, the mice were allowed to recover for 2 days before testing. Before each test, the animals were transferred to a behavioral unit and allowed to habituate for a minimum of 1 hour. First, mice were injected with vehicle and a baseline measurement was performed at the appropriate time point. Second, the same mice were injected with a test drug and the behavioral measurement was repeated at the same time point. The mice were then allowed to recover for 24 hours to ensure redistribution and metabolism of the test drug. The following baseline measurement was used to confirm complete elimination of prior drug effect. For the time course experiment, D4 was injected and repeated behavioral tests were performed at specific time points.

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Nociceptive Measurements

Hotplate assay was performed by placing the mice individually on a platform maintained at 52°C to 55°C (Hot-Plate Analgesia Meter; Columbus Instruments, Columbus, OH). A plastic cylinder 15 cm in diameter and 20 cm high confined the mouse to the surface of the hotplate. The time necessary for the mouse to respond to the thermal stimulus (hindpaw fluttering, licking, or withdrawal) was measured with a stopwatch. After the initial response or the maximal cutoff time of 15 seconds, the mouse was removed from the hotplate and returned to the home cage. A minimal recovery period of 1 hour was implemented between hotplate assay sessions.

For the tail flick assay, the mouse was gently restrained using a 50-mL polypropylene conical tube and the distal 2 cm of its tail was inserted into a thermostatically controlled water bath. The latency to withdraw or flick the tail was recorded using a stopwatch. The tests were performed at a temperature of 49°C, with a minimal recovery period of 1 hour between trials. A maximal cutoff time of 20 seconds was implemented to prevent permanent injury.

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Activity of Novel Compounds and Bumetanide on KCC2

KCC2-overexpressing HEK293 cells13 were grown in 10-cm plastic Petri dishes in a 37°C incubator, 95% air and 5% CO2. The dishes contained 10 mL DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 50 U/mL penicillin, 50 pg/mL streptomycin (Invitrogen), and 2 μg/mL puromycin (Sigma, St. Louis, MO). Confluent dishes were trypsinized and resuspended in identical culture medium. A homogeneous cell suspension was plated on polylysine-coated 35-mm plastic dishes (2 mL/dish) and placed in the 37°C incubator. Two hours later, the medium from 3 dishes was aspirated and replaced with 1 mL of an HEPES-buffered isosmotic saline containing 100 μM ouabain (Na/K pump inhibitor) and 500 μM N-ethylmaleimide (K-Cl cotransporter activator) at room temperature for 15-minute preincubation. The medium was then aspirated and replaced with 1 mL saline containing 100 μM ouabain, 1 μCi/mL 86Rb in the presence or absence of drugs (0.1–30 μM) for a 15-minute uptake period. Next, the radioactive medium was aspirated and the cells were washed 3 times with ice-cold saline. Eight groups of 3 dishes (triplicate measurements) were staggered every 2 minutes to allow precise uptake measurements. At the end of the experiment, the cells were lysed with 500 μL 0.25 N NaOH for 1 hour and neutralized with 250 μL glacial acetic acid. A 300-μL aliquot per dish was used for β-scintillation counting and a 30-μL aliquot was used for Bradford protein assay (BioRad, Hercules, CA). KCC2-mediated K+ influx was expressed as pmol K+ mg protein−1 min−1. Data were fitted, by nonlinear regression sigmoidal dose response, using Prism 3.0 (GraphPad, San Diego, CA).

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Drugs

The synthesis of D4 (Pubchem CID7211972: VU0240551-2-D4) was previously described in the article by Delpire et al.13 Active and inactive variants of D4 were synthesized by the Vanderbilt Institute of Chemical Biology, Chemical Synthesis Core, Vanderbilt University (Nashville, TN). Bumetanide and DIOA (2-[[(2S)-2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-3H-inden-5-yl]oxy]) were obtained from Sigma. All drugs were resuspended in dimethyl sulfoxide as 50 mM stock solutions, diluted in saline to the appropriate concentration, and filter sterilized using Millipore Millex 0.22-mm syringe filters (Millipore, Bedford, MA).

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Statistical Analysis

All statistics were performed using Instat 3.01 (GraphPad). Because all data passed the normality test with P > 0.05 using the method of Kolmogorov and Smirnov, t tests instead of nonparametric tests were used. Because the behavior was recorded for each mouse before and after drug injection, our data are first presented as paired values and analyzed using paired t tests. To account for repeated-measure differences that go in opposite directions, our data are also presented as averages and analyzed using unpaired t tests. The 2 statistical analyses reach similar conclusions. Finally, to ensure consistency between sessions, we analyzed our baseline data using repeated-measure analysis of variance and found no significant differences between sessions (P > 0.5). Repeated-measure analysis of variance was also used to analyze the time course data.

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RESULTS

The purpose of this study was to develop a method to deliver small molecules to the subarachnoid space of the spinal cord of a mouse and measure their effect on heat-evoked nociceptive responses. The biological targets were 2 cation-chloride cotransporters, KCC2 and NKCC1. In a previous study, we identified a highly potent and selective inhibitor of KCC2 (D4), and identified several putative structural variants with little to no effect on the cotransporter.13 To demonstrate the potency of D4 on KCC2, and the absence of inhibitory effect of D4.14 (a structurally related variant of D4) and of bumetanide on KCC2, we provide concentration response curves for all 3 compounds on KCC2-mediated K+ influx, as measured in HEK293 cells (Fig. 3). Note the half-maximal inhibition of KCC2 by D4 in the submicromolar range and the complete absence of inhibition of D4.14 and bumetanide at concentrations up to 30 μM.

Figure 3

Figure 3

Intrathecal catheters were successfully placed in 10 mice using the procedure described in the Methods section. Gross motor function was examined 1 day after catheter implantation by observing for any gait dysfunction, weakened hindlimb withdrawal reflex, or limited toe spread. One mouse was noted to have significant left hindlimb paresis and was euthanized. In addition, the catheter of another mouse had been displaced overnight and was nonfunctional. This mouse was also euthanized.

For the initial hotplate assay, a temperature of 52°C was selected based on the statistically significant response observed between wild-type and SPAK knockout mice.15 For this experiment, the mice were slowly injected with 8 μL saline containing dimethyl sulfoxide (as vehicle for drugs) and tested at 2 time points: 20 minutes and 80 minutes after intrathecal injection. These 2 time points were chosen based on the time effect of furosemide and bumetanide on capsaicin-induced nociceptive response16 and the 1-hour recovery period between hotplate assay sessions. After baseline measurements, the same mice were injected with 8 μL of the active KCC2 inhibitor (D4), resulting in a final drug concentration of 20 μM, and the assays were performed again at the 2 time points. As seen in Figure 4A, there was no statistical difference in the pain response times between the vehicle and D4, 20 minutes after injection. However, there was a small (13%) but statistically significant decrease in withdrawal latency in mice injected with D4 after 80 minutes. Based on these results, we increased the hotplate temperature to 55°C and omitted the 20-minute measurement. Increasing the hotplate temperature had been shown in previous work to lead to more significant differences in pain latencies.5,15

Figure 4

Figure 4

When measured at 55°C, a larger decrease in withdrawal latency (28%) after D4 injection compared with vehicle was observed (Fig. 4B). Importantly, there was no appreciable difference in pain withdrawal after injection of the inactive KCC2 compound (Fig. 4B). To determine the duration of the D4 effect, we performed a time-course experiment using a separate cohort of animals. After injection of the KCC2 inhibitor, the hotplate assay was performed after 50, 110, 170, and 290 minutes. Figure 5A shows these time points as well as the 20- and 80-minute time points of the previous experiment. As seen in the figure, the D4 effect is no longer observed after 3 and 6 hours. Next, to assess the potency of D4 in vivo, we diluted the compound to reach final concentrations of 10, 5, 2.5, and 1 μM. As seen in Figure 5B, there was no statistical difference at 1 μM, whereas the effect became statistically significant at 2.5 μM and higher. We also tested DIOA, a known KCC2 inhibitor, at a concentration of 10 μM and observed no decrease in withdrawal latency (9.93 ± 2.07 seconds vs 10.35 ± 2.21 seconds, P = 0.46). To address the participation of the Na-K-2Cl cotransporter in nociception, we tested the effect of 20 μM bumetanide. At this concentration, the loop diuretic inhibits NKCC1 with high selectivity without affecting the activity of KCC2 (Fig. 3). Bumetanide significantly increased withdrawal latency compared with vehicle (Fig. 6). To allow ample time for drug redistribution and metabolism, we waited approximately 24 hours between injections of the test drugs. Individual control times were compared with one another to ensure consistent baseline pain response before injection of test drug. Statistical analysis of the baseline measurements showed no significant difference between them.

Figure 5

Figure 5

Figure 6

Figure 6

To provide an independent measure of heat-evoked nociception, we next used tail flick assays. These assays were performed at a temperature of 49°C. This temperature was selected based on previous studies showing a baseline latency of 3 to 4 seconds at a temperature of 52°C.15 Because we were anticipating shorter latencies upon KCC2 inhibition, we slightly decreased the bath temperature to obtain more appropriate baseline times. While performing these assays, we noted that one of the intrathecal catheters leaked during injection. This mouse was subsequently euthanized, decreasing the total number of animals to 7. Similar to the hotplate assay results, we observed a statistically significant decrease in the pain response time after injection of D4 and no statistical difference in withdrawal latency after injection of the inactive compound (Fig. 7A). Finally, we observed a small but statistically significant increase in withdrawal latency after injection of the NKCC1 inhibitor, bumetanide (Fig. 7B). As for the hotplate assays, a 24-hour recovery period was used between drug injections to allow for drug clearance, and there was no statistical difference between the different baseline measurements.

Figure 7

Figure 7

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DISCUSSION

This report describes a simple surgical procedure that allows repeated delivery of small molecules in the intrathecal space of mice. This study also uses this methodology to address the role of the neuronal-specific K-Cl cotransporter (KCC2) on nociception, through the injection of a novel KCC2 inhibitory compound. We chose to place a catheter instead of repeated needle injection to minimize traumatic injury and guarantee that all injections were placed in the same location within the intrathecal space. The intrathecal catheter procedure was simplified from the method of Wu et al.14 The authors had demonstrated that the placement of a catheter in the L5-6 interspace did not produce morphological changes under light microscopy for up to 12 days. First, we used a commercially available catheter that allows easy modification, resulting in reproducible results. Second, we performed only a small incision, reducing postoperative pain and possibility of complications. Third, by not tunneling the catheter, we were able to shorten it and decrease the dead-space volume. Fourth, by using Tegaderm to sterilely cap the catheter instead of repeatedly melting and cutting the tip, we were able to keep the outside portion small. Only 1 animal of 10 displaced the catheter immediately after the procedure. Therefore, this minimally invasive procedure shortened the recovery time and allowed for testing 2 days postimplantation.

The placement of the catheter facilitated the delivery of compounds to the subarachnoid space of the spinal cord and the testing of a novel small molecule that inactivates KCC2 but not NKCC1. Pharmacological separation of KCC2 and NKCC1 activities was difficult in the past because high concentrations of furosemide inhibited both transport mechanisms. In this study, we tested the effect of D4 (PubChem, CID7211972), at a final concentration of 20 μM, as well as an inactive variant of the compound to serve as control, on the response of the mouse to nociceptive stimuli. The final drug concentration was estimated based on a cerebrospinal fluid volume of 35 to 40 μL in an adult mouse.1719 Using ion fluxes in KCC2-overexpressing HEK293 cells, we show complete inhibition of KCC2 with 20 μM D4, but no effect of D4.14 or bumetanide at that concentration. Because D4.14 is structurally related to D4, but inactive, this compound constitutes a perfect negative control for this experiment. Furthermore, because bumetanide does not affect KCC2 at 20 μM, but completely inhibits NKCC1 at that concentration (for review see Ref. 20), the use of D4 and bumetanide can nicely separate the functional activities of KCC2 and NKCC1. We therefore tested the effect of D4, D4.14, and bumetanide on nociception using hotplate and tail flick assays. Both tests yielded similar data with D4 and bumetanide resulting in opposite effects on the latency to respond to the heat stimuli, while D4.14 showed no effect. The potency of D4 was demonstrated by diluting the compound to a concentration of 2.5 μM and still observing the effect. The decreased response latency to the noxious stimuli upon KCC2 inhibition is consistent with previous data showing increased nociception associated with reduction in KCC2 expression,1,812 with the exception of 1 study using KCC2 hypomorphic mice, which showed increased latency to heat stimulus or a hyposensitivity.7 In that particular case, however, KCC2 was genetically manipulated from conception, and compensatory mechanisms at play during development could have affected the nociceptive response of these animals. We also tested the effect of DIOA at 10 μM. Although this compound in vitro inhibits K-Cl cotransport in the low micromolar range, we did not observe any significant effect. This lack of effect could be attributable to a reduced potency of the alkanoic acid or to the nonselectivity of this compound. Indeed, DIOA inhibits not only K-Cl cotransport, but also organic solute transport,21 K+22 and Cl23 conductances, as well as a K+(Na+)/H+ exchanger.24 Therefore, the different effect of DIOA and D4 found in this study highlights the research benefits of using an agent with improved potency and specificity. The lack of a DIOA effect could also have been attributable to the possible inhibition of NKCC1,13 which could negate the effect of KCC2 inhibition. In fact, opposite to KCC2 inhibition, we observed increased response latency to the heat stimuli upon NKCC1 inhibition with low dose of bumetanide. These bumetanide data are consistent with previous data showing decreased nociception associated with the knockout of NKCC1,5,25 and the antinociceptive effect of NKCC1 inhibitors in a formalin model of tissue injury–induced pain.16 The data are also consistent with a previous study showing that spinal administration of bumetanide reduces dorsal root reflex activity, mechanical allodynia, and hyperalgesia produced by intradermal injection of capsaicin.26

The tail flick and hotplate assays are 2 methods of assessing pain response in mice that have been well validated and standardized.2731 Both of these tests are based on spinal reflexes that require segmental connections and a propriospinal connection from dorsal horn neurons to ventral motor neurons.28,30 Latencies in these reflexes are dependent on the activation time of cutaneous thermal nociceptors, the conduction time of this impulse through dorsal horn neurons and the central nervous system, and the time for impulse transmission through ventral horn neurons with activation of effector muscles. In addition, supraspinal centers modulate these reflexes through excitatory or inhibitory effects on dorsal horn interneurons. Because KCC2 is expressed in postsynaptic relay neurons, which are intrinsic to these reflex arcs,2,3 these 2 pain sensitivity tests are appropriate for measuring KCC2 activity. It should be noted that because the mice are restrained during the tail flick assay, and restraint is a stressful event,32,33 additional factors (e.g., hormonal, psychological) might modulate the nociceptive response. In this case, because the genetic background of all mice is identical, this variable should similarly affect all mice in this cohort. Because the hotplate assay does not have this complication, it should be preferred when selecting only 1 paradigm for screening of drug effects on nociception.

In summary, inhibition of the K-Cl cotransporter, KCC2, led to decreased heat-evoked response latency in mice, indicating nociceptive hypersensitivity, whereas inhibition of the Na-K-2Cl cotransporter, NKCC1, resulted in increased response latencies to heat stimuli, indicating hyposensitivity to pain.

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DISCLOSURES

Name: Thomas M. Austin, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Thomas M. Austin has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Eric Delpire, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Eric Delpire has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

This manuscript was handled by: Quinn H. Hogan, MD.

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