Role of ASIC3 in the primary and secondary hyperalgesia produced by joint inflammation in mice

Abstract

The acid sensing ion channel 3 (ASIC3) is critical for the development of secondary hyperalgesia as measured by mechanical stimulation of the paw following muscle insult. We designed experiments to test whether ASIC3 was necessary for the development of both primary and secondary mechanical hyperalgesia that develops after joint inflammation. We used ASIC3 -/- mice and examined the primary (response to tweezers) and secondary hyperalgesia (von-Frey filaments) that develops after joint inflammation comparing to ASIC3 +/+ mice. We also examined the localization of ASIC3 to the knee joint afferents innervating the synovium using immunohistochemical techniques before and after joint inflammation. We show that secondary mechanical hyperalgesia does not develop in ASIC3 -/- mice. However, the primary mechanical hyperalgesia of the inflamed knee joint still develops in ASIC3 -/- mice and is similar to ASIC3 +/+ mice. In knee joint synovium from ASIC3 +/+ mice without joint inflammation, ASIC3 was not localized to joint afferents that were stained with an antibody to protein gene product (PGP) 9.5 or calcitonin gene-related peptide (CGRP). ASIC3 was found, however, in synoviocytes of the knee joint of uninflamed mice. In ASIC3 +/+ mice with joint inflammation, ASIC3 co-localized with PGP 9.5 or CGRP in joint afferents innervating the synovium. We conclude that the decreased pH that occurs after inflammation would activate ASIC3 on primary afferent fibers innervating the knee joint, increasing the input to the spinal cord resulting in central sensitization manifested behaviorally as secondary hyperalgesia of the paw.

Figure 1

A. The mechanical withdrawal thresholds of the paw are shown before and after joint inflammation in ASIC3 +/+ and ASIC3 -/- mice for the ipsilateral (circles) and the contralateral (squares) hindpaws. The withdrawal thresholds were significantly less in the ASIC3 -/- mice after inflammation when compared to the withdrawal thresholds from ASIC3 +/+ mice for both the ipsilateral and the contralateral hindpaws. *, P<0.05. B. The mechanical withdrawal threshold of the knee joint before and after joint inflammation in ASIC3 +/+ and ASIC3 -/- mice for the ipsilateral (circles) and the contralateral (squares) knees. There was no difference between ASIC 3 +/+ and ASIC3 -/- mice with decreases in withdrawal thresholds occurring 24 h after joint inflammation in both groups.

Figure 2

A. Cross section through the knee joint of a mouse without joint inflammation stained with H & E depicting the location of the synovium. B. High power magnification of the synovium from the knee joint showing synoviocytes lining the joint cavity and fat cells below. C. Double label for PGP 9.5 (green, arrows) showing the nerve fibers innervating the synovium. Red is labeled for ASIC3 and shows localization in the synovium. D. Shows ASIC3 localization in synovioctyes lining the joint cavity in ASIC3 +/+ mice. E. Shows lack of ASIC3 staining in synoviocytes from ASIC3 -/- mice.

Figure 3

Immunohistochemistry staining of the synovium from ASIC3 +/+ mice with joint inflammation stained for ASIC3 (A), PGP 9.5 (B). The merged image shows co-localization of ASIC3 and PGP 9.5 (C) depicted by arrows. Some PGP 9.5 labeled nerve fibers in the synovium do not localize with ASIC3 (arrowhead). Immunohistochemistry staining from ASIC3 -/- mice with joint inflammation stained for ASIC3 (D), PGP 9.5 (E). There is no staining for ASIC3 the ASIC3 -/- mice. The merged image shows no co-localization of ASIC3 and PGP 9.5 (E; arrowheads). Bar = 25 μm

Figure 4

Immunohistochemsitry staining for ASIC3 and CCRP from sections in whole specimens showing the synovium from ASIC3 +/+ and ASIC3 -/- mice without joint inflammation and those 24 h after joint inflammation. A-F shows immunoreactivity in animals without joint inflammation and G-L shows immunoreactivity in animals with joint inflammation. ASIC3 staining is shown in A,D,G and J; CGRP staining is shown in B,E,H and K; and the merged images of ASIC3 and CGRP are shown in C,F,I and L. CGRP staining occurs in all groups, both ASIC3 +/+ and ASIC3 -/-. No immunoreactivity for ASIC3 was observed in ASIC3 -/- mice; and nerve fibers did not stain for ASIC3 in ASIC3 +/+ mice. After joint inflammation, ASIC3 was located in nerve fibers innervating the synovium in ASIC3 +/+ mice. Some of these fibers co-localize with CGRP (arrows). However some fibers stain positive for CGRP that do not localize with ASIC3 (arrowheads), and some fibers stain positive for ASIC3 that do not localize with CGRP (not shown).