Improvements in impaired GABA and GAD65/67 production in the spinal dorsal horn contribute to exercise-induced hypoalgesia in a mouse model of neuropathic pain

Abstract

Background: Physical exercise effectively attenuates neuropathic pain, and multiple events including the inhibition of activated glial cells in the spinal dorsal horn, activation of the descending pain inhibitory system, and reductions in pro-inflammatory cytokines in injured peripheral nerves may contribute to exercise-induced hypoalgesia. Since fewer GABAergic hypoalgesic interneurons exist in the dorsal horn in neuropathic pain model animals, the recovery of impaired GABAergic inhibition in the dorsal horn may improve pain behavior. We herein determined whether the production of gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) in the dorsal horn is restored by treadmill running and contributes to exercise-induced hypoalgesia in neuropathic pain model mice. C57BL/6 J mice underwent partial sciatic nerve ligation (PSL). PSL-Runner mice ran on a treadmill at 7 m/min for 60 min/day, 5 days/week, from two days after PSL.

Results: Mechanical allodynia and heat hyperalgesia developed in PSL-Sedentary mice but were significantly attenuated in PSL-Runner mice. PSL markedly decreased GABA and GAD65/67 levels in neuropils in the ipsilateral dorsal horn, while treadmill running inhibited these reductions. GABA+ neuronal nuclei+ interneuron numbers in the ipsilateral dorsal horn were significantly decreased in PSL-Sedentary mice but not in PSL-Runner mice. Pain behavior thresholds positively correlated with GABA and GAD65/67 levels and GABAergic interneuron numbers in the ipsilateral dorsal horns of PSL-Sedentary and -Runner mice.

Conclusions: Treadmill running prevented PSL-induced reductions in GAD65/67 production, and, thus, GABA levels may be retained in interneurons and neuropils in the superficial dorsal horn. Therefore, improvements in impaired GABAergic inhibition may be involved in exercise-induced hypoalgesia.

Keywords: Gamma-aminobutyric acid; exercise-induced hypoalgesia; glutamic acid decarboxylase; neuropathic pain; treadmill running.

Figure 1.

Treadmill running protocol and changes in pain behaviors in mice in the five experimental groups. (a) Adult male C57BL/6 J mice were divided into five experimental groups (n = 6). All groups, except for Naive mice, were subjected to Running-1 and Running-2 prior to PSL or Sham surgery. In the Running-1 program, mice ran at a speed of 7 m/min for 10 min/day for 5 days/week, and in the Running-2 program, ran at a speed of 7 m/min for 20 to 60 min/day (an increment of 10 min per day), 5 days/week. PSL- and Sham-Runner mice ran at a speed of 7 m/min for 60 min/day, 5 days/week, from 2 days after surgery (Running-3). Mice were sacrificed for the immunohistochemical analysis on the morning after the final treadmill running session. (b) von Frey and (c) plantar tests were performed in Naive (n = 6), Sham-Sedentary (n = 6), Sham-Runner (n = 6), PSL-Sedentary (n = 6), and PSL-Runner (n = 6) mice two days before and two, three, five, and six days after surgery. (b) Mechanical withdrawal thresholds were significantly higher in PSL-Runner mice than in PSL-Sedentary mice five and six days after PSL (*p < 0.01). (c) Thermal withdrawal latencies were significantly higher in PSL-Runner mice than in PSL-Sedentary mice five and six days after PSL (#p < 0.05, *p < 0.01). The significance of differences among groups was determined by a repeated-measures ANOVA followed by Tukey’s post hoc test.

Figure 2.

Changes in GABA immunoreactivities in the superficial dorsal horn following treadmill running. Lumbar spinal cord (L4-5) sections in (a) Naive, (b) Sham-Sedentary, (c) Sham-Runner, (d) PSL-Sedentary, and (e) PSL-Runner mice were immunostained with GABA (green) and NeuN (red) antibodies. The right and left sides of the dorsal horns indicate the contralateral and ipsilateral sides, respectively. The dotted lines drawn in each photomicrograph indicate the boundary between the dorsal horn and white matter. Bars: (a–e) = 200 µm. (f–h) Lumbar spinal cord sections in Naive mice were immunostained with GABA and NeuN antibodies, and nuclei were stained with DAPI. (f) and (g) indicate the immunoreactivities of GABA (green) and NeuN (red) in the superficial dorsal horn, respectively, and (h) three images including DAPI were merged. A square located in (a) indicates the typical position acquired in enlarged photomicrographs of the superficial dorsal horn. Arrowheads and asterisks indicate GABA+ NeuN+ and GABA− NeuN+ neurons, respectively. Bars: (f-h) = 20µm. (i) As shown in (a), a square of 10⁴µm² in size was placed on the lateral, central, and medial parts of the superficial dorsal horns in microscope images, and the immunofluorescence intensity of GABA within it was quantified. GABA immunoreactivity was significantly weaker in PSL-Sedentary mice than in the other groups (n = 6, *p < 0.01). The significance of differences among groups was determined by a one-way ANOVA and Tukey’s post hoc test.

Figure 3.

Changes in GABA+ NeuN+ neurons in the superficial dorsal horn following treadmill running. (a,b) Immunoreactivities of GABA (green) and NeuN (red) in the ipsilateral superficial dorsal horn of PSL-Sedentary mice, and (c) three images including DAPI were merged. A typical position acquired in enlarged photomicrographs (a–c) in the ipsilateral dorsal horn was marked by the square located in Figure 3(d). (d,e) Immunoreactivities of GABA (green) and NeuN (red) in the ipsilateral superficial dorsal horn of PSL-Runner mice, and (f) three images including DAPI were merged. A typical position acquired in enlarged photomicrographs (d–f) in the ipsilateral dorsal horn was marked by the square located in Figure 3(e). Arrowheads and asterisks indicate GABA+ NeuN+ and GABA− NeuN+ neurons, respectively. Bars: (a–f) = 20 µm. (g) As shown in Figure 3(a), (d), and (e), squares of 10⁴µm² in size were placed on the lateral, central, and medial parts, respectively, of the superficial dorsal horn in microscope images, and the number of GABA+ NeuN+ neurons within it was counted. The number of GABA+ NeuN+ neurons in the ipsilateral dorsal horn was significantly lower in PSL-Sedentary mice than in the other groups (n = 6, *p < 0.01). The significance of differences among groups was determined by a one-way ANOVA and Tukey’s post hoc test.

Figure 4.

Changes in GAD65/67 immunoreactivities in the superficial dorsal horn following treadmill running. Lumbar spinal cord (L4-5) sections in (a) Naive, (b) Sham-Sedentary, (c) Sham-Runner, (d) PSL-Sedentary, and (e) PSL-Runner mice were immunostained with GAD65/67 (green) and NeuN (red) antibodies. The right and left sides of the dorsal horns correspond to the contralateral and ipsilateral sides, respectively. The square located in (a) was enlarged in (f): GAD65/67 (green) and (g): NeuN (red). The square located in (d) was enlarged in (i): GAD65/67 (green) and (j): NeuN (red), and the square located in (e) was also enlarged in (l): GAD65/67 (green) and (m): NeuN (red). NeuN+ neurons (arrowheads) in (g), (j), and (m) were enlarged in (h), (k), and (n), respectively. Bars: (a–e) = 200 µm, (f, g, i, j, l, and m) = 30 µm, (h, k, and n) = 10 µm. Intense GAD65/67 immunoreactivities were detected in neuropils. (o) The immunofluorescence intensity of GAD65/67 in the superficial dorsal horn was quantified as described in the Methods section. GAD65/67 immunoreactivity levels were significantly lower in PSL-Sedentary mice than in the other groups (n = 6, *p < 0.01). The significance of differences among groups was determined by a one-way ANOVA and Tukey’s post hoc test.

Figure 5.

Relationships between pain behavior thresholds and GABA and GAD65/67 levels or GABAergic interneuron numbers in superficial dorsal horns of PSL-Sedentary and -Runner mice. (a) A positive correlation was observed between GABA levels and the thresholds of von Frey (a, r = 0.874, n = 12, p < 0.001) or plantar tests (b, r = 0.929, n = 12, p < 0.001). A positive correlation was observed between GAD65/67 levels and the thresholds of von Frey (c, r = 0.753, n = 12, p < 0.01) or plantar tests (d, r = 0.817, n = 12, p < 0.01). A positive correlation was observed between the number of GABA + NeuN + neurons and thresholds of von Frey (e, r = 0.645, n = 12, p < 0.05) or plantar tests (f, r = 0.813, n = 12, p < 0.01). Open and closed circles indicate the values of PSL-Sedentary and -Runner mice, respectively.