Sphingosine kinase 2-deficiency mediated changes in spinal pain processing

Abstract

Chronic pain is one of the most burdensome health issues facing the planet (as costly as diabetes and cancer combined), and in desperate need for new diagnostic targets leading to better therapies. The bioactive lipid sphingosine 1-phosphate (S1P) and its receptors have recently been shown to modulate nociceptive signaling at the level of peripheral nociceptors and central neurons. However, the exact role of S1P generating enzymes, in particular sphingosine kinase 2 (Sphk2), in nociception remains unknown. We found that both sphingosine kinases, Sphk1 and Sphk2, were expressed in spinal cord (SC) with higher levels of Sphk2 mRNA compared to Sphk1. All three Sphk2 mRNA-isoforms were present with the Sphk2.1 mRNA showing the highest relative expression. Mice deficient in Sphk2 (Sphk2(-/-)) showed in contrast to mice deficient in Sphk1 (Sphk1(-/-)) substantially lower spinal S1P levels compared to wild-type C57BL/6 mice. In the formalin model of acute peripheral inflammatory pain, Sphk2(-/-) mice showed facilitation of nociceptive transmission during the late response, whereas responses to early acute pain, and the number of c-Fos immunoreactive dorsal horn neurons were not different between Sphk2(-/-) and wild-type mice. Chronic peripheral inflammation (CPI) caused a bilateral increase in mechanical sensitivity in Sphk2(-/-) mice. Additionally, CPI increased the relative mRNA expression of P2X4 receptor, brain-derived neurotrophic factor and inducible nitric oxide synthase in the ipsilateral SC of wild-type but not Sphk2(-/-) mice. Similarly, Sphk2(-/-) mice showed in contrast to wild-type no CPI-dependent increase in areas of the dorsal horn immunoreactive for the microglia marker Iba-1 and the astrocyte marker Glial fibrillary acidic protein (GFAP). Our results suggest that the tightly regulated cell signaling enzyme Sphk2 may be a key component for facilitation of nociceptive circuits in the CNS leading to central sensitization and pain memory formation.

Keywords: CFA; RT-PCR; dorsal horn; formalin; knock-out mouse; sphingosine 1-phosphate.

Figure 1

Expression of sphingosine kinases in mouse spinal cord (SC). Relative mRNA-expression levels as mean normalized expression (MNE) for (A) the two sphingosine kinase isoform, Sphk1 and Sphk2, at different levels of the mouse SC. Sphk2 is expressed significantly higher compared to the Sphk1 isoform in thoracic and lumbosacral SC levels (n = 5, one-way ANOVA, *p < 0.05); (B) the three Sphk2 mRNA-isoforms at different levels of the mouse SC. The isoforms were significantly different expressed at all SC levels with Sphk2.1 isoform showing the highest relative mRNA-expression (n = 5, one-way ANOVA, Bonferroni’s post hoc test, **p < 0.01); (C) the Sphk1isoform in SC of C57BL/6 and Sphk2^−/− mice. The relative expression levels were not different between strains (n = 5, t-test). Values are expressed as mean ± SEM. (D) Western Blot confirmed the absence of the Sphk2 protein in Sphk2-deficient mice. Sphk2 protein was present in SC and DRG and could be detected at a molecular weight of about 50 kDa. βIII tubulin used as reference.

Figure 2

Sphingosine kinase activity and sphingosine 1-phosphate (S1P) levels in mouse SC. (A) Assessment of the amount of spinal S1P in C57BL/6 and Sphk-deficient mice. Whereas the amount of S1P was substantially reduced in SCs from Sphk2^−/− mice in comparison to C57BL/6 it was virtually unchanged in Sphk1^−/− mice (n = 5, one-way ANOVA, Bonferroni’s post hoc test, ***p < 0.001). (B) The Sphk1 activity was not different between SCs of C57BL/6 and Sphk2^−/− mice (n = 5, paired t-test).

Figure 3

Licking response to acute peripheral inflammation induced by formalin. (A) Time course of the response of C57BL/6 mice (black squares) and Sphk2^−/− mice (black circles) to injection of 2% formalin. Time points represent mean ± SEM response of eight animals during 5 min intervals. Asterisks indicate significantly greater time licking (*p < 0.05, two-way ANOVA, Bonferroni’s test). (B) Time course of the response of C57BL/6 mice (black squares) and Sphk2^−/− mice (black circles) to injection of 0.5% formalin. The response to injection of 0.5% formalin was not different between strains. (C–F) Cumulative paw licking time in intervals. Values represent mean ± SEM. (C) Overall time spent licking in response to 2% formalin (unpaired t-test, *p < 0.05). (D) Overall response to 0.5% formalin. (E) Time spent licking between 10–25 min (unpaired t-test, *p < 0.05). (F) Time spent licking between 25–45 min (unpaired t-test, *p < 0.05).

Figure 4

Microglia and astrocytes in the dorsal horn in C57BL/6 and Sphk2^−/− mice. (A) Areas with immunoreactivity (IR) for the astrocyte marker GFAP and the microglia marker Iba-1 in the dorsal horn of untreated C57BL/6 and Sphk2^−/− mouse SC normalized to 100,000 μm² (n = 5, unpaired t-test, n.s.). (B) Area with IR for Iba-1 in the dorsal horn of C57BL/6 and Sphk2^−/− mouse SC 60 min post injection of two percent formalin, normalized to 100,000 μm² (n = 10, unpaired t-test, n.s.). (C) Area with IR for GFAP in C57BL/6 and Sphk2^−/− mouse SC dorsal horn 60 min post injection of two percent formalin, normalized to 100,000 μm² (n = 10, unpaired t-test, *p < 0.05). (D) Number of nuclei immunoreactive for NeuN in C57BL/6 and Sphk2^−/− mouse SC dorsal horn 60 min post injection of two percent formalin, normalized to 100,000 μm² (n = 10, unpaired t-test).

Figure 5

IR for c-Fos in the dorsal horn of C57BL/6 and Sphk2^−/− mice in response to acute peripheral inflammation. (A,B) Double- labeling immunohistochemistry for NeuN and c-Fos. Nuclei with immuno-reactivity for NeuN (A) and c-Fos (B) in the ipsilateral SC dorsal horn after injection of formalin into the hindpaw. c-Fos positive nuclei also showed IR for NeuN. The arrows indicate some double-labeled nuclei. The asterisk indicates background labeling of capillaries. Bar = 50 μm. (C) Number of nuclei with IR for c-Fos in the SC dorsal horn of C57BL/6 and Sphk2^−/− mice 60 min after injection of two percent formalin. (D) Number of nuclei with IR for c-Fos in the SC dorsal horn of C57BL/6 and Sphk2^−/− mice 60 min after injection of 0.5% formalin. (E) Number of nuclei with IR for c-Fos in the SC dorsal horn of C57BL/6 and Sphk2^−/− mice 60 min after injection of saline. (F) Number of nuclei with IR for c-Fos in the SC dorsal horn of C57BL/6 and Sphk2^−/− mice in untreated SC. Values represent mean ± SEM, n = 5, one-way ANOVA, Bonferroni’s post hoc test, *p < 0.05.

Figure 6

Paw withdrawal thresholds in response to chronic peripheral inflammation (CPI) induced by CFA in C57BL/6 and Sphk2^−/− mice. (A) Time course of paw withdrawal thresholds in response to intraplantar injection of CFA in C57BL/6 (n = 8, ipsilateral, upwards pointing triangles solid line; contralateral downward pointing triangles solid line) and Sphk2^−/− (n = 8, ipsilateral, circles dashed line; contralateral squares dashed line) hindpaws (two-way ANOVA, Bonferroni’s post hoc test, *p < 0.05). (B) Difference in mid-hindpaw thickness of the between ipsi- and contralateral side in response to CFA injection. Values represent mean ± SEM.

Figure 7

S1P content of ipsi- and contralateral SC in response to CPI induced by CFA in C57BL/6 and Sphk2^−/− mice. Analysis of the amount of S1P in C57BL/6 and Sphk-deficient mice at 7 days post-CFA injection into the hindpaw. The amount of S1P was substantially reduced in ispi- and contralateral SCs from C57BL/6 and Sphk2^−/− mice in comparison to untreated controls (control, n = 5, ispi- and contralateral SC n = 4, one-way ANOVA, Bonferroni’s post hoc test, ***p < 0.001).

Figure 8

Relative mRNA expression in ipsi- and contralateral SC in response to CPI. Relative mRNA expression levels in the SC of C57BL/6 mice and Sphk2-deficient mice (Sphk2^−/−). The data are presented as MNE. (A) Relative mRNA-expression levels for sphingosine kinase 1 (Sphk1) in ipsi- and contralateral lumbosacral SC of C57BL/6 and Sphk2^−/− mice after 7 days CFA. (B) Relative mRNA-expression levels for the P₂X₄ receptor (P2X4) in lumbosacral SC of untreated control mice (control) and in ipsi- (ipsi) and contralateral (contra) lumbosacral SC of C57BL/6 and Sphk2^−/− mice after 7 days CFA. (C) Relative mRNA-expression levels for the Bdnf in lumbosacral SC of untreated control mice (control) and in ipsi- (ipsi) and contralateral (contra) lumbosacral SC of C57BL/6 and Sphk2^−/− mice after 7 days CFA. (D) Relative mRNA-expression levels for the Nos2 (iNos) in lumbosacral SC of untreated control mice (control) and in ipsi- (ipsi) and contralateral (contra) lumbosacral SC of C57BL/6 and Sphk2^−/− mice after 7 days CFA. (E,F) Relative mRNA-expression levels for the Il-1β and Il-6 in lumbosacral SC of untreated control mice (control) and in ipsi- (ipsi) and contralateral (contra) lumbosacral SC of C57BL/6 and Sphk2^−/− mice after 7 days CFA. Values represent mean ± SEM, n = 5–8, one-way ANOVA, Bonferroni post hoc test, *p < 0.05, **p < 0.01.

Figure 9

Microglia and astrocytes in the dorsal horn in C57BL/6 and Sphk2^−/− mice in response to chronic inflammation. Areas of the ipsi- and contralateral (contra) SC dorsal horn with IR for GFAP (A,C), Iba-1 (B,D) and positive staining for Dapi (E) in C57BL/6 (A,B,E) and Sphk2^−/− (C, D) lumbosacral SC 7 days post injection of CFA (CFA) or saline (saline) into the left hindpaw. Values represent mean ± SEM, n = 5, one-way ANOVA, Bonferroni’s post hoc test, *p < 0.05, ***p < 0.001.