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Nociceptor-expressed ephrin-B2 Regulates Inflammatory and Neuropathic Pain

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Nociceptor-expressed ephrin-B2 Regulates Inflammatory and Neuropathic Pain

Jing Zhao et al. Mol Pain.

Abstract

Background: EphB receptors and their ephrin-B ligands play an important role in nervous system development, as well as synapse formation and plasticity in the adult brain. Recent studies show that intrathecal treatment with EphB-receptor activator ephrinB2-Fc induced thermal hyperalgesia and mechanical allodynia in rat, indicating that ephrin-B2 in small dorsal root ganglia (DRG) neurons and EphB receptors in the spinal cord modulate pain processing. To examine the role of ephrin-B2 in peripheral pain pathways, we deleted ephrin-B2 in Nav1.8+ nociceptive sensory neurons with the Cre-loxP system. Sensory neuron numbers and terminals were examined using neuronal makers. Pain behavior in acute, inflammatory and neuropathic pain models was assessed in the ephrin-B2 conditional knockout (CKO) mice. We also investigated the c-Fos expression and NMDA receptor NR2B phosphorylation in ephrin-B2 CKO mice and littermate controls.

Results: The ephrin-B2 CKO mice were healthy with no sensory neuron loss. However, pain-related behavior was substantially altered. Although acute pain behavior and motor co-ordination were normal, inflammatory pain was attenuated in ephrin-B2 mutant mice. Complete Freund's adjuvant (CFA)-induced mechanical hyperalgesia was halved. Formalin-induced pain behavior was attenuated in the second phase, and this correlated with diminished tyrosine phosphorylation of N-methyl-D-aspartic acid (NMDA) receptor subunit NR2B in the dorsal horn. Thermal hyperalgesia and mechanical allodynia were significantly reduced in the Seltzer model of neuropathic pain.

Conclusions: Presynaptic ephrin-B2 expression thus plays an important role in regulating inflammatory pain through the regulation of synaptic plasticity in the dorsal horn and is also involved in the pathogenesis of some types of neuropathic pain.

Figures

Figure 1
Figure 1
Exon 2 of ephrin-B2 was deleted in DRG in Efnb2 CKO mice. (A) Diagram showing the ephrin-B2 wild-type locus, the targeted locus before and after Cre excision, Nav1.8 wild-type and Nav1.8-Cre locus. Exons are represented as numbered boxes. The loxP site (blank triangles) and the PCR primers (black arrows) are indicated. (B) Genotyping analysis with PCR. The DRGs genomic DNA from Efnb2 CKO mice, Efnb2fl/fl controls, heterozygous floxed exon 2 ephrin-B2 (Efnb2fl/+) mice and C57BL/6 wild-type (WT) mice were examined with PCR. The wild-type band (wt), floxed band (floxed) and Cre band (cre) were amplified with primer sets a/b, a/c and d/e respectively. The knockout band (ko) was found only from Efnb2 CKO mutant mice.
Figure 2
Figure 2
Neural marker expression was normal in DRG neurons of ephrin-B2 CKO mice. (A) Whole DRG sections of Efnb2 CKO mutant mice and Efnb2fl/fl littermate controls were labeled with anti-peripherin (green) and anti-neurofilament (red) antibodies. The right panels are merged images of the left and middle panels. (B) DRG sections were also labeled with anti-CGRP (red) and anti-IB4 (red) antibodies, co-labeled with anti-βIII-tubulin (green). The right panels are erged images of the left and middle panels. (C) The proportions of CGRP, IB4 and neurofilament (N52) expressing neurons were normal in Efnb2 CKO animals compared to Efnb2fl/fl littermate controls. (D) Cross section of lumbar spinal cord (L3 - L5) stained with anti-CGRP (green) antibody and anti-IB4 (red) antibody. In laminae I-II, both CGRP positive terminals and IB4 positive terminals were identified in the Efnb2 CKO mutant mice and Efnb2fl/fl littermate controls. Scale bar = 50 μm.
Figure 3
Figure 3
Acute pain behavior was not affected in ephrin-B2 mutant mice. (A) Rotarod studies showed no motor deficits in Efnb2 CKO animals. (B) Responses to low-threshold mechanical stimulation by von Frey filaments are normal in Efnb2 CKO mutant mice. (C) Acute mechanical pressure applied with the Randall Selitto apparatus demonstrated identical behavior in Efnb2 CKO and Efnb2fl/fl mice. (D) Hargreaves' apparatus also demonstrates identical latencies of response to thermal stimulation in Efnb2 CKO and Efnb2fl/fl mice. (E) Supra-spinal reflexes to heat (50 and 55°C) using the hot plate apparatus demonstrates identical latencies in Efnb2 CKO and Efnb2fl/fl mice. (F) Cold behavior (number of liftings) is the same in Efnb2fl/fl and Efnb2 CKO mice (0°C) using the cold plate apparatus.
Figure 4
Figure 4
Both inflammatory and neuropathic pain behavior are attenuated in ephrin-B2 null mutant mice. (A) Two phases (0 - 10 min, 10 - 60 min) of licking and biting behavior after intraplantar injection of formalin were analyzed, the second phase of which is attenuated in Efnb2 CKO mutant mice. (B) Intraplantar injection of carrageenan caused identical thermal hyperalgesia in Efnb2fl/fl controls and Efnb2 CKO mice. (C) Intraplantar injection of carrageenan caused a long-term mechanical allodynia in both Efnb2 CKO mutants and controls. (D) Intraplantar injection of CFA caused identical thermal hyperalgesia in Efnb2fl/fl controls and Efnb2 CKO mutant mice. (E) However, intraplantar injection of CFA caused a long-term mechanical allodynia that is attenuated after 4 hours in Efnb2 CKO mutants (two-way ANOVA, * p < 0.05). (F) Both Efnb2 CKO mutant mice and Efnb2fl/fl controls showed thermal hyperalgesia after sciatic nerve ligation (Seltzer model). However, thermal hyperalgesia was attenuated in Efnb2 CKO mutants (two-way ANOVA, * p < 0.05). (G) Sciatic nerve ligation following the Seltzer procedure caused a long-lasting mechanical allodynia that was attenuated after 5 days of surgery in Efnb2 CKO mutant mice (two-way ANOVA, * p < 0.05).
Figure 5
Figure 5
c-Fos expression was attenuated in the spinal cord in ephrin-B2 conditional mutant mice after formalin injection in the hindpaw. (A) Representative immunostaining for c-Fos in superficial laminae I, II after formalin injection. The right hemicord is showing the cord ipsilateral to the injection of formalin or saline. (B) Cell counting for c-Fos positive nuclei in the dorsal horn. Compared to Efnb2fl/fl control mice, the number of positive c-Fos nuclei in the ipsilateral side in Efnb2 CKO mutants is significantly reduced after formalin injection. Scale bar = 100 μm. All data presented as means ± SEM; using students t-test, * p < 0.05.
Figure 6
Figure 6
Increase of immunoreactivity for Iba1 was attenuated in the spinal cord in ephrin-B2 conditional mutant mice in a Seltzer neuropathic pain model. (A) Representative immunostaining for Iba1 in superficial laminae I, II after sciatic nerve partial ligation (Seltzer model). The right hemicord is showing the cord ipsilateral to the sciatic nerve partial ligation. (B) Cell counting for Iba1 positive cells in the dorsal horn. The microglia were activated after sciatic nerve partial ligation in both Efnb2fl/fl controls and Efnb2 CKO mice. However, the increase of immunoreactivity for Iba1 was attenuated in Efnb2 CKO mice. Data shown as mean ± SEM, Scale bar = 100 μm. Using students t-test, * p < 0.05; ** p < 0.01.

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