Here we set out to determine the contribution of TRPA1 to mechanical hypersensitivity following skin-only incision injury in mice. Our results demonstrate that skin incision induces mechanical hypersensitivity in TRPA1-deficient “knockout” (KO) mice similar to levels measured in wild-type mice. Additionally, we observed no effect of the TRPA1 antagonist HC-030031 on mechanical hypersensitivity following skin injury in wild-type mice. Further, we found no functional up-regulation of TRPA1 in lumbar DRGs from wild-type mice after skin incision injury using calcium imaging. When we recorded from neurons that specifically projected to the plantar region of the ipsilateral hind paw where the skin incision was performed, there was also no functional up-regulation of TRPA1 at the DRG level. Together, these findings indicate that TRPA1 does not mediate mechanical hypersensitivity following cutaneous surgical incision injury in mice.
Several studies have shown that TRPV1 mediates heat hypersensitivity following skin plus deep incision [13
]. Therefore, we investigated the role of TRPV1 in heat hypersensitivity with cutaneous-only injury. We found that TRPV1 KO mice exhibit a modest increase in heat sensitivity following skin incision injury; however, incised TRPV1 knockout mice exhibit significantly less heat hypersensitivity compared to wild-type mice. Since we observed a small, yet significant, increase in heat sensitivity in TRPV1 KO mice following skin incision, other receptors likely play a role in the heat sensitivity. Other contributors to heat hypersensitivity may include TRPV3 and TRPV4 [39
] as well as the recently identified sensory neuron heat channels TRPM3 [44
] and Anoctamin 1 (ANO1) [45
]. Nonetheless, our data indicate that TRPV1 is the major contributor to behavioral heat hypersensitivity following cutaneous incision injury.
Beyond behavioral assays, we show for the first time that TRPV1 is functionally up-regulated in isolated DRG sensory neurons following skin incision injury. The behavioral heat hypersensitivity present after skin incision is likely mediated, at least in part, by this functional up-regulation of TRPV1 which may be attributed to increased TRPV1 protein or mRNA expression, increased receptor translocation to the plasma membrane, or modulation of existing receptors on sensory neurons. Further experimentation is necessary to determine the exact mechanism of TRPV1 functional up-regulation. Interestingly, our data demonstrate that functional expression of TRPV1 increases specifically in IB4-positive neurons, which are a subset of small-diameter neurons that have been shown to terminate more superficially in skin, centrally project to the inner lamina II of the spinal cord, and transmit pain signals into central processing centers that influence the affective components of pain [32
]. TRPV1 has been shown to mediate heat hypersensitivity in other pain models such as inflammation and nerve injury, and studies have shown that these models induce increased TRPV1 expression in IB4-positive sensory neurons [38
]. Accumulating evidence suggests that IB4-positive C fiber-type neurons are the most malleable subset of sensory neurons to sensitization after tissue injury [38
], and evidence from Levine and colleagues strongly suggests that hyperalgesic priming occurs selectively in the IB4-binding population following injury [48
]. Therefore, our data suggest that the mechanism behind heat hypersensitivity in postoperative pain may be largely mediated by cutaneous incision injury-driven TRPV1 functional up-regulation in IB4-positive C fiber-type neurons.
Although we found that skin incision induced functional up-regulation of TRPV1 among non-labeled L3-5 DRG neurons that project to mixed peripheral target tissues, we failed to detect functional up-regulation of TRPV1 among neurons that specifically innervated the injured glabrous skin. Spofford and Brennan [50
] reported tissue-specific expression of growth factors following skin plus deep incision injury, which may explain the disparity in our results. Nerve growth factor (NGF) was found to be over-expressed in skin from 4hr to 10 days following incision [50
], and this growth factor has been previously shown to potentiate functional TRPV1 preferentially in muscle afferent neurons but far less so in cutaneous neurons [51
]. Up-regulation of NGF in incised skin may drive the functional up-regulation of TRPV1 among neurons with mixed peripheral target tissues, which include neurons that terminate in muscle. Down-regulation of other growth factors, including artemin, was previously reported in skin at 24hr post incision [50
]. Artemin has been previously shown to induce TRPV1 sensitization primarily among cutaneous afferents [51
]. The down-regulation of artemin and other growth factors that primarily sensitize cutaneous neurons could explain why we did not observe functional up-regulation of TRPV1 among glabrous skin-specific neurons following skin-only incision. However, a number of studies have shown that cutaneous afferent nerves are
sensitized to heat following skin plus deep incision injury and that the sensitization to heat in cutaneous afferents is mediated by TRPV1 [10
]. When growth factor expression was evaluated in incised muscle
tissue, the expression of artemin was up-regulated at 24hr post skin plus deep incision [50
]. Up-regulation of artemin in injured muscle tissue could possibly sensitize TRPV1 in cutaneous neurons. Taken together, we have found that cutaneous incision injury drives increased functional expression of TRPV1 among neurons with mixed peripheral targets which includes muscle afferents but not among neurons that terminate within the superficial region of glabrous skin injury. These findings along with previous studies may suggest that deep incision injury is required to sensitize TRPV1 among glabrous skin afferents.