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Descending Serotonergic Controls Regulate Inflammation-Induced Mechanical Sensitivity and methyl-CpG-binding Protein 2 Phosphorylation in the Rat Superficial Dorsal Horn

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Descending Serotonergic Controls Regulate Inflammation-Induced Mechanical Sensitivity and methyl-CpG-binding Protein 2 Phosphorylation in the Rat Superficial Dorsal Horn

Sandrine M Géranton et al. Mol Pain.

Abstract

Background: Regulation of pain states is, in part, dependent upon plastic changes in neurones within the superficial dorsal horn. There is also compelling evidence that pain states are under the control of descending projections from the brainstem. While a number of transcription factors including Methyl-CpG-binding protein 2 (MeCP2), Zif268 and Fos have been implicated in the regulation of dorsal horn neurone sensitization following injury, modulation of their activity by descending controls has not been investigated.

Results: Here, we describe how descending controls regulate MeCP2 phosphorylation (P-MeCP2), known to relieve transcriptional repression by MeCP2, and Zif268 and Fos expression in the rat superficial dorsal horn, after CFA injection into the hind paw. First, we report that CFA significantly increased P-MeCP2 in Lamina I and II, from 30 min post injection, with a maximum reached after 1 h. The increase in P-MeCP2 paralleled that of Zif268 and Fos, and P-MeCP2 was expressed in large sub-populations of Zif268 and Fos expressing neurones. Serotonergic depletion of the lumbar spinal cord with 5,7 di-hydroxytryptamine creatinine sulphate (5,7-DHT) reduced the inflammation evoked P-MeCP2 in the superficial dorsal horn by 57%, and that of Zif268 and Fos by 37.5% and 30% respectively. Although 5,7-DHT did not change primary thermal hyperalgesia, it significantly attenuated mechanical sensitivity seen in the first 24 h after CFA.

Conclusion: We conclude that descending serotonergic pathways play a crucial role in regulating gene expression in the dorsal horn and mechanical sensitivity associated with an inflammatory pain state.

Figures

Figure 1
Figure 1
Time course of P-MeCP2 expression in the superficial dorsal horn after CFA injection. CFA injection in the hind paw significantly increased MeCP2 phosphorylation in the superficial dorsal horn, ipsilateral to the injection. Changes in P-MeCP2 expression were seen exclusively in Lamina I and II, with the exception of expression in Lamina 3 to 4 at 6 h. Data show mean ± standard error of the mean. *P < 0.05, **P < 0.001, ***P < 0.0001, always vs naïve within the same dorsal horn area.
Figure 2
Figure 2
Expression of P-MeCP2 in the superficial dorsal horn after CFA injection. Pictures show typical P-MeCP2 immunoreactivity in the dorsal horn, ipsilateral to the injection, 30 min, 1 h and 2 h after CFA injection in the hind paw. Scale bars, top: 100 μm; bottom: 50 μm.
Figure 3
Figure 3
Expression of P-MeCP2, Zif268 and Fos in the superficial dorsal horn after CFA injection. There was a significant effect of CFA on MeCP2 phosphorylation and Zif268 and Fos expression, with maximum changes at 1 h and 2 h. Data show mean ± standard error of the mean. *P < 0.05, **P < 0.001, ***P < 0.0001, always vs naïve for each of the transcription regulator.
Figure 4
Figure 4
Time course of Zif268 expression in the superficial dorsal horn after CFA injection. Pictures show typical Zif268 immunoreactivity in the dorsal horn, ipsilateral to the injection, before (naïve) and 30 min, 1 h, 2 h, 6 h and 24 h after CFA injection in the hind paw. Scale bars, 50 μm.
Figure 5
Figure 5
P-MeCP2 is expressed in sub-populations of Zif268 and Fos expressing neurones after CFA injection. Tissue was taken 1 h after CFA injection. Spinal cord sections were double-labelled with antibodies against P-MeCP2 (green) and A/ Zif268 (red) or B/ Fos (red). Double labelling resulted in yellow staining (arrows). Images were taken using confocal microscopy. Scale bars, 20 μm.
Figure 6
Figure 6
Intrathecal 5,7-DHT causes serotonin depletion but no glial proliferation or cell death. Tissue was taken 7 days after 5,7-DHT treatment and dorsal horn sections were stained for 5-HT, GFAP and NeuN. 5-HT staining confirmed a complete depletion of 5-HT in the superficial dorsal horn 7 days after 5,7-DHT, and GFAP and NeuN staining indicated that, at the concentration used in this study, 5,7-DHT did not cause glial proliferation or hypertrophy or abnormal cell death. Scale bar, 50 μm.
Figure 7
Figure 7
5,7-DHT treatment reduces CFA-induced P-MeCP2, Zif268 and Fos expression. Seven days after 5,7-DHT or saline treatment, animals received CFA and were sacrificed after 1 h. A/ P-MeCP2, Zif268 and Fos expression 1 h after CFA injection in the hind paw were significantly reduced by 5,7-DHT. N = 4/6 in each group. Data show mean ± standard error of the mean; *P < 0.05, ***P < 0.0001. B/ Pictures show typical P-MeCP2 immunoreactivity in the dorsal horn, ipsilateral to the injection, 1 h after CFA injection in the hind paw. 5,7-DHT reduced P-MeCP2 immunoreactivity compared to saline, especially in the medial area of the superficial dorsal horn. Scale bars, 40 μm.
Figure 8
Figure 8
The correlation between P-MeCP2 and Zif268 regulation is stronger than that between P-MeCP2 and Fos. The graphs represent the mean number of Zif268 (A) or Fos (B) expressing neurones per dorsal horn section, ipsilateral to the CFA injection, per individual animal, against the number of P-MeCP2 immuno-positive neurones. The correlation is considerably stronger between P-MeCP2 and Zif268 (correlation coefficient: R2 = 0.8) than that between P-MeCP2 and Fos (R2 = 0.4), suggesting a parallel regulation for P-MeCP2 and Zif268.
Figure 9
Figure 9
5,7-DHT attenuates mechanical sensitivity but not thermal sensitivity. Seven days after 5,7-DHT or saline treatment, animals received CFA in the hind paw. A/ 5,7-DHT did not change the increased thermal sensitivity seen after CFA injection in the hind paw as measured by the Hargreaves test. N = 5/6.B/ 5,7-DHT significantly attenuated the inflammation-induced decrease in mechanical threshold measured by the automatic Von Frey from 1 h after CFA injection. The response was still attenuated 24 h later. N = 7/8. For A, and B, data show mean ± standard error of the mean; *P < 0.05, **P < 0.001.

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