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. 2018 Nov 9;19(1):72.
doi: 10.1186/s12868-018-0470-8.

Glycine Receptors Expression in Rat Spinal Cord and Dorsal Root Ganglion in Prostaglandin E2 Intrathecal Injection Models

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Free PMC article

Glycine Receptors Expression in Rat Spinal Cord and Dorsal Root Ganglion in Prostaglandin E2 Intrathecal Injection Models

Hung-Chen Wang et al. BMC Neurosci. .
Free PMC article

Abstract

Background: Glycine receptors (GlyRs) are involved in the development of spinal pain sensitization. The GlyRα3 subunit has recently emerged as a key factor in inflammatory pain pathways in the spinal cord dorsal horn (DH). Our study is to identify the extent of location and cell types expressing different GlyR subunits in spinal cord and dorsal root ganglion (DRGs). To tease out the possible actions of GlyRs on pain transmission, we investigate the effects produced by GlyRs on acute inflammatory pain by behavioral testing using prostaglandin E2 (PGE2) intrathecal injection models. Furthermore, we investigate the changes of GlyR expression in DRGs and spinal cord in rats after the induction of acute inflammatory pain.

Results: Compared to the vehicle administration, the PGE2 intrathecal injection model produced significantly higher hyperalgesia, which started 3 h after PGE2 injection and lasted more than 5 h. PGE2 intrathecal injection significantly decreased GlyRα1 and GlyRα3 protein expressions in the L5 DH at 1 h and lasted to 5 h, and similar results were observed in the L5 DRG at 5 h. Confocal microscopic images showed the co-existence of punctate gephyrin and GlyRα3 immunoreactivity (IR) throughout the gray matter of the spinal cord, mainly in DH laminae I-III neurons and in ventral horn neurons. It also showed the co-existence of punctate gephyrin and GlyRα3 IR in DRG neurons.

Conclusions: In this study, PGE2 intrathecal injection significantly decreased protein expression of gephyrin, GlyRα1 and GlyRα3 in spinal cord DH and DRG. The gephyrin and GlyRα3 were localized on neuron cells both in the DH and DRG.

Keywords: Dorsal root ganglion; Glycine receptors; Inflammatory pain; Prostaglandin E2; Spinal cord dorsal horn.

Figures

Fig. 1
Fig. 1
Intrathecal injection with PGE2 induced hyperalgesic responses in rats. In the PGE2 group, the hyperalgesic response was significantly different at 3 h post PGE2 injection compared with vehicle injection and lasted to 5 h. Significant differences of behavior hypersensitivity were as indicated. Mann–Whitney U-test, Error bars represent SE. **p < 0.01, and ***p < 0.001 (between the PGE2 and vehicle groups); h, hour
Fig. 2
Fig. 2
a Western blot and quantitative analysis of b gephyrin, c GlyRα3 expressions in L5 spinal cord dorsal horn at 1 h and 5 h after PGE2 intrathecal injection. The GlyRα3 protein expressions were significantly decreased in the PGE2 group at 1 h and lasted up to 5 h. The gephyrin expression decreased significantly at 5 h after PGE2 injection. d Confocal microscopic imaging showed expression of gephyrin throughout the gray matter of spinal cord. e Expression of GlyRα3 was mainly in the superficial layer of dorsal horn and in the ventral horn. Each group had n = 3–6 rats. Scale Bars: 200 μm; **p < 0.01, and ***p < 0.001, One-way ANOVA, followed by the LSD test
Fig. 3
Fig. 3
Triple immunofluorescence staining showing GlyRα3, Gephyrin and NeuN co-localization in the L5 spinal cord. Positive for GlyRα3 are shown in green (a), positive for Gephyrin are shown in red (b) and positive for NeuN are shown in blue (c). Double-labelled images of GlyRα3 and Gephyrin (d), GlyRα3 and NeuN (e) are indicated. f The merged image demonstrates co-localization of GlyRα3, Gephyrin in Neurons. Scale bars = 200 µm, 20 µm
Fig. 4
Fig. 4
a Western blot and quantitative analysis of b gephyrin, c GlyRα1, and d GlyRα3 protein expressions in L5 DRG at 1 h and 5 h after PGE2 intrathecal injection. The gephyrin, GlyRα1 and GlyRα3 protein expressions were significantly decreased in the PGE2 group at 5 h. Confocal microscopic imaging showed expression of e gephyrin, f GlyRα1 and g GlyRα3 localized mainly in the neuron cells. Each group had n = 3–6 rats. Scale Bars: 20 μm; **p < 0.01, and ***p < 0.001, One-way ANOVA, followed by the LSD test
Fig. 5
Fig. 5
Triple immunofluorescence staining showing GlyRα3, Gephyrin and NeuN co-localization in the L5 DRG. Positive for GlyRα3 are shown in green (a), positive for Gephyrin are shown in red (b) and positive for NeuN are shown in blue (c). Double-labelled images of GlyRα3 and Gephyrin (d), GlyRα3 and NeuN (e) are indicated. f The merged image demonstrates co-localization of GlyRα3, Gephyrin in Neurons of DRG. IF experiments from three different sham controls were performed. Secondary Abs only were included as negative controls. Scale bars = 20 µm

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