Exploring the transcriptome of resident spinal microglia after collagen antibody–induced arthritis (1)
Arthritis and autoimmunity research has been fairly balanced in the consideration of both sexes, most preclinical pain research has been performed in male animals. The consideration of studying pain in arthritis because RA, like other autoimmune diseases, is more likely to occur in women.
For induction of arthritis, adult male and female Balb/cAnNRj were injected intravenously (1.25-1.5 mg) with an anti-collagen type-II cocktail containing 5 different monoclonal autoantibodies at day 0 followed by an intraperitoneal injection of lipopolysaccharide (25 μg in 100 μL) at day 5. The scoring is 1 point was given for every inflamed toe or knuckle. Each paw, ankle, or wrist was awarded 2.5 or 5 points if moderately or severely inflamed, respectively, resulting in a maximum arthritis score of 15 points per leg and 60 points per mouse. Animals that did not reach a minimum of 12 points in the hind paws at the peak of inflammation were excluded from the study. Significant differences in CAIA incidence were shown in between males and females, whereas 90% (27/30) of females developed CAIA, only 53.5% (23/43) of males did. Male and female mice that developed CAIA presented visually apparent joint swelling and redness from day 6, reaching a peak in arthritis score around day 12 and gradually returning to baseline levels around day 30. Mechanical hypersensitivity, which was present from day 6, persisted up until the last testing point at day 52, although joint inflammation was transient in all mice.
No change in expression levels for neuropeptides associated with pain (CGRP, SP, and galanin) in male mice at the time points examined after CAIA induction. Galanin expression levels were modestly but statistically higher in male compared with female CAIA mice in the late phase. Significant increase of the astrocyte marker GFAP was observed only in the late phase of CAIA in both males and females, whereas increase in the signal intensity of the microglia marker IBA-1 was detected in both CAIA phases in both male and female mice.
Pentoxifylline or minocycline, is frequently used to inhibit spinal astrocytic or microglial actions, respectively. 6 hours after the drug administration, both pentoxifylline and minocycline significantly reversed arthritis-induced withdrawal thresholds to control levels in males, but not in females.
No dimorphic differences in the relative number of CD45+, CD11b+ spinal microglial cells, their mean fluorescence intensity, or Cd11b and Iba-1 mRNA expression when comparing naive mice of both sexes. The relative levels of the XY-linked genes Ddx3y, Eif2s3y, and Xist. showed significant differences between males and females. No difference in Cd11b and Iba-1 gene expression between C57BL/6 and Balb/c males or C57BL/6 and Balb/c females was observed. CD45+, CD11b+, and Fcrls+ microglial cells amongst CD45+ immune cells were analyzed, resulting in macrophage infiltration in the lumbar dorsal horns is negligible. The proportion of CD45+, CD11b+ microglia, CD45+ immune cells, and live cells did not significantly differ between saline and CAIA conditions. In addition, a significantly lower percentage of female microglia amongst live cells and amongst CD45+ immune cells.
Joint swelling and mechanical hypersensitivity can be characterized as the “inflammatory phase,” and the period where joint inflammation has resolved but mechanical hypersensitivity persists as the “postinflammatory phase” or “late phase.”
Microglia do not contribute to postinflammatory pain-like behaviour in CAIA model. Instead, astrocytes may contribute to spinal sensitization in the CAIA model in male mice, whereas direct changes in neuronal factors such as galanin are the main drivers in females.