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DISCUSSION Although experimental joint inflammation and bone
cancer gave rise to similar pain behaviors in the
The present study shows that chronic pain behaviors absence of spinal neuroinflammation, the underlying
during deep tissue damage occur in the absence of mechanisms differ. Subcutaneous CFA injections
spinal neuroinflammation but in the presence of may result in blood‑brain barrier breakdown and
circulating cytokines. In addition, evidence is provided increase spinal COX‑2 expression, [29,43] that mediates
to indicate that the latter may act on DRG cytokine mechanical pain hypersensitivity. [44] Our work
receptors and that mechanical stimulation increases confirmed increased spinal COX‑2 expression after
spinal cytokine expression. less severe intra‑articular CFA injections suggesting
that it also mediates mechanical allodynia during local
Our work extends a number of previous studies inflammation. However, in contrast to what has been
showing variable spinal GFAP responses across models reported after subcutaneous CFA injection, [27] we did
of inflammatory and cancer pain. [27‑33] It reports the not observe increased spinal cytokine expression in
absence of increased spinal cytokine expression in the absence of mechanical stimulation. Interestingly,
addition to the lack of GFAP up‑regulation in models in addition to IL‑1β and TNF‑α, peripheral IL‑6 also
of moderate deep tissue pain. Indeed, the fact that we increases central COX‑2 expression and pain sensitivity
injected less biologically‑active agents and induced less during inflammation. [45] Since we observed increased
severe pain behavior and tissue damage, compared to circulating IL‑6 concentrations, we propose that
previous reports, [27,29,38] may partly explain discrepancies IL‑6‑induced spinal COX‑2 upregulation underlies
concerning spinal neuroinflammation between studies. mechanical allodynia after intra‑articular CFA injection.
Alternatively, circulating IL‑6 may have acted on
However, studies employing concentrations and routes of IL‑6 receptors in DRG, [23,24] accessible to circulating
administration of biologically‑active agents comparable molecules, [22] to induce COX‑2.
to those used currently have shown increased spinal
GFAP and cytokine expression. [16,30,31] Interestingly, In the absence of increased spinal cytokine and
these studies, like those using higher concentrations of COX‑2 expression during bone cancer pain behavior,
disease biologically‑active agents, imposed mechanical we considered nervous system actions of peripheral
stimulation or movement on animals. Non‑noxious cytokines. We confirmed tumor MCP‑1/CCL2 production
palpation of bone tumor‑containing paws increases and showed for the first time increased circulating
transcription factor expression in the spinal cord. [32,33] MCP‑1/CCL2 and constitutive CCR2 protein expression
We show here that the palpation induced spinal in murine DRG nociceptors. These findings are important
c‑Fos expression, although not to the same extent as given that circulating molecules can access DRGs and
CFA‑provoked articular inflammation. Since sensory that MCP‑1/CCL2 increases nociceptor excitability, [22,46]
nerve stimulation can induce CNS IL‑1β expression, [34,41] and suggest that circulating MCP‑1/CCL2 action on
we tested the effect of paw palpation on spinal cytokine DRGs contributes to bone cancer pain behavior.
expression. Our observation that palpation increased
spinal IL‑1β and IL‑1R1 expression in mice with joint In conclusion, our present work shows that in two
inflammation indicates that afferent nerve stimulation different types of deep tissue lesions, inflammatory
during deep tissue injury can indeed induce hallmarks and neoplastic, signs of spontaneous, chronic pain
of neuroinflammation. Results obtained in models are not correlated to spinal neuroinflammation, but
employing imposed mechanical stimulation to assess rather to peripheral cytokines. In addition, we present
pain sensitivity should, therefore, be interpreted with evidence indicating that mechanical stimulation of
caution. the body segment containing the lesion, similar to
what may occur during pain sensitivity testing, can
As we hypothesized that imposed paw stimulation induce spinal cytokine expression as a hallmark of
during deep tissue injury influences spinal gene neuroinflammation. As such, our work provides
expression, we assessed spontaneous behaviors important new insights into the occurrence and role
indicating pain, such as decreased food intake and of spinal neuroinflammation in chronic pain. Finally,
exploration as well as paw guarding, [36,42] and we studied our findings suggest that circulating cytokine action
allodynia only minutes before sacrifice. Although in dorsal root ganglia may contribute to experimental
joint inflammation affected exploration more than joint inflammation and bone cancer pain.
bone cancer, both conditions reduced food intake and
provoked hind paw guarding. The latter behavior is in ACKNOWLEDGMENTS
accordance with earlier studies and has been shown
to be reversed by morphine, [38,42] thus suggesting that We thank Pascale Roux and Alexandra Serre for skilful
paw guarding reflects spontaneous pain. assistance in PCR and immunohistochemical experiments,

158 Neuroimmunol Neuroinflammation | Volume 1 | Issue 3 | December 2014