Spinal Cord Toll-Like Receptor 4 Mediates Inflammatory and Neuropathic Hypersensitivity in Male But Not Female Mice

Abstract

The innate immune system is increasingly appreciated to play an important role in the mediation of chronic pain, and one molecule implicated in this process is the Toll-like receptor 4 (TLR4). Here using pharmacological and genetic manipulations we found that activating TLR4 in the spinal cord, with the agonist lipopolysaccharide (LPS), causes robust mechanical allodynia but only in male mice. Spinal LPS had no pain-producing effect in female mice. TLR4 also has a sex-specific role in inflammatory (complete Freund’s adjuvant) and neuropathic (spared nerve injury) pain: pain behaviors were TLR4-dependent in males but TLR4-independent in females. The sex differences appear to be specific to the spinal cord, as LPS administered to the brain or the hindpaw produces equivalent allodynia in both sexes, and specific to pain, as intrathecal LPS produces equivalent hypothermia in both sexes. The involvement of TLR4 in pain behaviors in male mice is dependent on testosterone, as shown by gonadectomy and hormone replacement. We found no sex differences in spinal Tlr4 expression at baseline or after LPS, suggesting the existence of parallel spinal pain processing circuitry in female mice not involving TLR4s.

Sex differences in pain and pain inhibition: multiple explanations of a controversial phenomenon

“…Symbols represent mean ± standard error of the mean von Frey fibre withdrawal thresholds before and after spared nerve injury. Further experiments using a selective agonist (LPS) and antagonist (LPS from Rhodobacter sphaeroides) of TLR4 revealed no apparent involvement of spinal TLR4 in pain processing in female mice 53 . Why was this not already known?…”

“…The distinction between the latter two types can be considered the difference between 'quantitative' and 'qualitative' sex differences. Although attention has mostly been paid to documenting quantitative sex differences in pain, a growing number of examples of qualitative differences in pain have been reported 31,39,43,[53][54][55][56][57][58][59][60][61][62][63][64] , and these promise to be far more important in the long run. As a practical matter, analgesics are routinely titrated according to their effect, which will effectively mitigate any sex differences along with other sources of inter-individual variability.…”

“…104); female C3H/HeJ mutant mice displayed normally robust allodynic responses (see the figure; data are from REF. 53). Symbols represent mean ± standard error of the mean von Frey fibre withdrawal thresholds before and after spared nerve injury.…”

“…Steroids with sex-specific pain-associated effects include androgens 53 , oestrogens 107 and progesterone 81 .…”

Laboratory environmental factors and pain behavior: the relevance of unknown unknowns to reproducibility and translation

“…The human sufferers of chronic pain are overwhelmingly female 14 , middle-aged or elderly 15 , and of heterogeneous genetic background, whereas the animal subjects in pain experiments are overwhelmingly young-adult, male Sprague Dawley rats or C57BL/6 mice 13,16,17 . Both quantitative and robust qualitative differences in pain processing have been documented between strains 18 and the sexes 19,20 , confounding simple conclusions. The most common chronic pain syndromes in humans are low back pain, arthritis of the joints, and headache 21 , whereas the most common chronic pain assays in current use for animal subjects involve experimental ligations of afferent fibers serving, and injection of inflammatory substances into, the hind paw 22 .…”