Korean J Pain.  2023 Oct;36(4):441-449. 10.3344/kjp.23162.

Involvement of the spinal γγ-aminobutyric acid receptor in the analgesic effects of intrathecally injected hypertonic saline in spinal nerve-ligated rats

• Karm MH
• Kwon HJ
• Shin E
• Bae H
• Kim YK
• Choi SS

• KMID: 2546706
• DOI: http://doi.org/10.3344/kjp.23162

Abstract

Background
Hypertonic saline is used for treating chronic pain; however, clinical studies that aid in optimizing therapeutic protocols are lacking. We aimed to determine the concentration of intrathecally injected hypertonic saline at which the effect reaches its peak as well as the underlying γ-aminobutyric acid (GABA) receptor-related antinociceptive mechanism.
Methods
Spinal nerve ligation (SNL; left L5 and L6) was performed to induce neuropathic pain in rats weighing 250–300 g. Experiment 1: one week after implanting the intrathecal catheter, 60 rats were assigned randomly to intrathecal injection with 0.45%, 0.9%, 2.5%, 5%, 10%, and 20% NaCl, followed by behavioral testing at baseline and after 30 minutes, 2 hours, 1 day, and 1 week to determine the minimal concentration which produced maximal analgesia. Experiment 2: after determining the optimal intrathecal hypertonic saline concentration, 60 rats were randomly divided into four groups: Sham, hypertonic saline without pretreatment, and hypertonic saline after pretreatment with one of two GABA receptor antagonists (GABA_A [bicuculline], or GABA_B [phaclofen]). Behavioral tests were performed at weeks 1 and 3 following each treatment.
Results
Hypertonic saline at concentrations greater than 5% alleviated SNL-induced mechanical allodynia and had a significant therapeutic effect, while showing a partial time- and dose-dependent antinociceptive effect on thermal and cold hyperalgesia. However, pretreatment with GABA receptor antagonists inhibited the antinociceptive effect of 5% NaCl.
Conclusions
This study indicates that the optimal concentration of hypertonic saline for controlling mechanical allodynia in neuropathic pain is 5%, and that its analgesic effect is related to GABA_A and GABA_B receptors.

Keyword

Analgesia; Bicuculline; GABA Antagonists; Hyperalgesia; Hypertonic Saline; Neuralgia; Phaclofen; Receptors, GABA; Injections, Spinal

Figure

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  Fig. 1 Flowchart of the NaCl concentration experiment. Behavioral tests include the von Frey filaments, hot-plate, and cold-plate tests. NaCl: sodium chloride, SNL: spinal nerve ligation, IT: intrathecal.

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  Fig. 2 Flowchart of the GABA antagonist experiment. Behavioral tests include the von Frey filaments, hot-plate, and cold-plate tests. GABA: γ-aminobutyric acid, SNL: spinal nerve ligation, IT: intrathecal, PBS: phosphate-buffered saline, S: sham, H: hypertonic, B: bicuculline, P: phaclofen, NaCl: sodium chloride.

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  Fig. 3 Time course of the anti-allodynic and hyperalgesic effects of NaCl administered intrathecally in the rats with mechanical allodynia and thermal and cold hyperalgesia after L5 and L6 spinal nerve ligation. All the behavioral tests were performed at baseline and after 30 minutes, 2 hours, 1 day, and 1 week of administering 0.45%, 0.9%, 2.5%, 5%, 10%, and 20% NaCl intrathecally. (A) VFF test. The withdrawal threshold (grams) to mechanical stimuli is presented on the y-axis, and time on the x-axis. (B) Hot-plate test. The withdrawal latency (seconds) to hot (45°C) stimuli are presented on the y-axis, and time on the x-axis. (C) Cold-plate test. The withdrawal latency (seconds) to cold (4°C) stimuli is presented on the y-axis and time on the x-axis. Vertical bars indicate the standard deviation of the mean. Ten animals were used in each group. Data represent the mean ± standard deviation. VFF: von Frey filaments. aP < 0.05 compared to the baseline. bP < 0.05 compared to the 0.9% NaCl group (0.45% NaCl = open circle; 0.9% NaCl = filled square; 2.5% NaCl = filled up-pointing triangle; 5% NaCl = filled down-pointing triangle; 10% NaCl = open square; 20% NaCl = filled circle).

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  Fig. 4 Effect of 5% hypertonic saline on SNL-induced mechanical allodynia and thermal and cold hyperalgesia in rats pretreated with a GABAA or GABAB antagonist. (A) Effect of the GABA antagonists on mechanical allodynia. (B) Effect of the GABA antagonists on thermal hyperalgesia. (C) Effect of the GABA antagonists on cold hyperalgesia. The rats in the bicuculline and phaclofen groups were pretreated intrathecally with bicuculline or phaclofen. All the behavioral tests were performed at weeks 1 and 3 following the intrathecal administration of 5% NaCl. Each box represents the following: white (first box) = sham group; blue (second box) = hypertonic group; purple (third box) = bicuculline group; green (fourth box) = phaclofen group. Vertical bars indicate the standard deviation of the mean. Fifteen animals were used in each group. Data represent the mean ± standard deviation. SNL: spinal nerve ligation, GABA: γ-aminobutyric acid receptor. aP < 0.05 compared to the sham group. bP < 0.05 compared to the hypertonic group.

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