Based on previous studies from our lab and others, we hypothesized that mechanically insensitive TRPV1-positive CH fibers would be sensitized to heat, while CPM fibers, that normally do not express TRPV1, would not be sensitized in inflamed mice. However, following inflammation we found no change in the heat sensitivity of CH fibers while CPM fibers exhibited reduced heat thresholds and increased firing rates during heating of the skin in both C3H/Bl6 and C57Bl6 wildtype, but only exhibited a decease in heat threshold in TRPV1-/- mice. We also found, contrary to expectations, that mechanical sensitivity was unchanged in this population of fibers in all mouse strains (wildtype and TRPV1-/-) following inflammation.
Lack of change in mechanical sensitivity following inflammation
We did not observe any change in the mechanical sensitivity of either A- or C-fibers following CFA injection into the dorsum of the hindpaw in these preparations, consistent with previous studies in rodents showing a lack of mechanical sensitization of C-fibers following inflammation [14, 15, 23]. However, this differs from other studies showing increased mechanical sensitivity after peripheral inflammation [16, 24]. One possible reason for this discrepancy is the nature of the ex vivo preparation. Previous studies using intact preparations have shown that during inflammation, edema produces increased tension of the skin and that increased tension of the skin is correlated with a reduction in mechanical thresholds of afferent fibers [25, 26]. In the ex vivo preparation, the skin is freed from the underlying tissue, removing any stretching caused by the edema. Moreover, those reporting mechanical sensitization used mostly in vivo preparations , while those that reported a lack of sensitization generally have used some type of ex vivo preparation [15, 23]. Thus, the results observed here suggest that increased mechanical sensitivity of nociceptors following inflammation requires changes in tensile loading of the skin and is not solely due to changes in receptor/channel function by inflammatory mediators. However, it should be noted that some in vivo studies have demonstrated mechanical sensitization within short time frames following injection of inflammatory mediators before edema formation suggesting that changes in receptor/channel function may also play a significant role in mechanical sensitization [e.g. , but also see, [27, 28]].
Changes in CH-fibers following inflammation
Mechanically insensitive C-fibers (MIA) have been described in several species, including mouse [10, 12, 29], rat  non-human primates [24, 31], and humans [32, 33]. Similar to our recent findings in mice , many MIA afferents in humans and other species can be activated by heat stimuli and capsaicin .
The results from previous studies [6, 7, 10, 12, 34] suggest that the population of TRPV1 positive, mechanically insensitive CH fibers play an important role in the development of inflammation induced heat hyperalgesia. Here we found an apparent lack of sensitization of these fibers following inflammation. Although the total number of CH fibers examined in each case is relatively small (11) when compared to the number of CPM fibers (46), this observation is not consistent with the idea that CH fibers play an important role. There are a couple of possible explanations for these findings. It is possible that the process of establishing the ex vivo preparation normally sensitizes these specific afferents thus masking any inflammation induced sensitization. While we do not know of any evidence for this possibility, it is something that we cannot completely rule out. Another possible explanation is recruitment of previously silent CH fibers. Previously, we found that there was an apparent increase in the numbers of functional CH fibers following saphenous nerve regeneration and that some CH fibers gained mechanical sensitivity after nerve injury . While we did not observe the same significant increase in functional CHs here, we have found that some CH fibers are apparently gaining mechanical sensitivity as indicated by the number of the CPM fibers (11%) stained positively for TRPV1 (in naïve animals this number is 0 [10, 35]). We and others have reported increases in expression and/or protein levels of TRPV1 following peripheral injury [35–40]. While there was no change in the numbers of TRPV1 positive cells in the DRG observed in the current study, two of the previous studies report an increase in the numbers of TRPV1 positive cells following inflammation. A major difference between those studies and the current one is that they were carried out in rat, where the distribution of TRPV1 is quite different than in mouse. For example, there is a large degree of overlap between TRPV1 and IB4 binding in rat and very little overlap seen in mouse . The current finding is also in agreement with our previous observations in mice following nerve injury  where we found an increase in TRPV1 expression but no increase in the number of TRPV1 positive cells. In addition, in a separate study in our laboratories no change in the number of TRPV1-positive cells following inflammation was observed (B.M. Davis, unpublished observation). Therefore the difference observed in the current studies and the previous ones could be due to species differences. However, it should also be noted that since we are counting the total numbers of cells, small changes in the numbers of fibers innervating the inflamed skin maybe masked.
We have also previously shown that CH fibers have significantly slower CVs than CPM fibers  and here, as in the previous study following regeneration , we found a significant decrease in the average CV of CPMs after inflammation. Together these findings suggest that some of the mechanically insensitive CHs have changed phenotypes by acquiring mechanical sensitivity. This change in phenotype should result in a decrease in percentage of C-fibers innervating inflamed skin characterized as CH fibers. However, this was not observed suggesting the possibility that some previously "silent" (mechanical and heat insensitive) fibers gained heat sensitivity following inflammation. However we cannot completely rule out other possible explanations for these findings. For example, there maybe a population of silent TRPV1-positive CPM fibers that have slower CV than naïve CPM fibers, and that these fibers gain mechanical and heat sensitivity following inflammation.
It has also been shown that mechanically insensitive cutaneous fibers can rapidly gain mechanical sensitivity following injection of inflammatory mediators (e.g. Davis et al., 1993) . Additionally, microneurography studies in humans showing that following capsaicin application, MIA-fibers can quickly become mechanically sensitive  and investigators report that similar changes in this set of fibers are found in patients with chronic pain disorders [41, 42]. Taken together with the results presented here, these findings suggest that it is the normally mechanically insensitive CH-fibers that exhibit pronounced plasticity following peripheral injury and could contribute to both heat and mechanical hyperalgesia observed following inflammation.
TRPV1-independent heat sensitization of CPM fibers
In the present study we found that inflammation induced sensitization of TRPV1-negative CPM-fibers to heat in two different strains of wildtype mice and this change was correlated with heat hyperalgesia in the C57/Bl6 mice. The results in wildtype mice strongly suggested that this was a TRPV1-independent process, and additional experiments carried out in the inflamed TRPV1-/- mice confirmed that at least part of the heat sensitization process of CPM-fibers (the decrease in heat threshold) is independent of TRPV1. One possible mechanism for these changes could be changes in the expression of purinergic receptors following inflammation. It has been shown that this population of IB4-binding fibers also contains both P2X3 or P2Y1 receptors [43, 44] and it has also been shown that they can play a role in changes in neuronal sensitivity [45–47]. In addition, we have recently shown that this population of cutaneous CPM fibers undergo a very similar decrease in heat thresholds following peripheral nerve injury and regeneration, and that this decrease was correlated with changes in the expression of these two purinergic receptors P2X3 and P2Y1 .
In this study, we have also confirmed the earlier reports [6, 7] showing that TRPV1-/- mice do not develop heat hyperalgesia following inflammation. These findings suggest that although CPMs show a significant decrease in heat thresholds, this change, in and of itself, is not sufficient to induce behavioral changes. It is quite possible that the full sensitization of CPM fibers, including increased firing rates, is necessary to drive behavioral changes. However, It is important to note that Cavanaugh et al.,  have reported recently that the population of IB4-positive/TRPV1-negative CPM fibers expressing the G protein coupled receptor Mrgprd, can be ablated without diminishing acute thermal pain in mice. The results presented here are consistent with these findings and further suggests the possibility that these fibers cannot by themselves signal enhanced acute heat pain following inflammation. However, it should also be noted that TRPV1-/- mice have relatively normal acute thermal pain detection [6, 7] indicating that mechanisms exist that allow mice lacking TRPV1-positive CH fibers, or for that matter any fiber containing TRPV1, to detect noxious thermal stimuli.
A possible effecter role for fibers containing TRPV1 in inflammation-induced heat hyperalgesia
Here we have confirmed our earlier findings that TRPV1-/- mice lack CH-fibers  and show that they are still absent following inflammation. However, TRPV1-/- mice have been shown to develop heat hyperalgesia following neuropathic injury , suggesting the possibility that TRPV1 has a significant contribution to the inflammatory response that is not essential for increased heat sensitivity after neuropathic injuries. While it is possible that this difference reflects compensatory mechanisms in the constitutive knock-out mice, support for the former possibility can be found in recent reports showing that TRPV1-/- mice exhibit less edema and swelling following CFA-induced inflammation [48, 49] but also see Davis et al., 2000 . Given the propensity for TRPV1 to be sensitized by endogenous factors and that activation of TRPV1 has been shown to evoke release of vasoactive peptides in the skin (e.g CGRP) [50, 51] these fibers may be playing an important role in the neurogenic inflammatory response. For example, it has been shown that TRPV1 sensitivity can be significantly modulated by many of the components of the inflammatory milieu (e.g. bradykinin, and NGF) present in inflamed skin as well as by local decreases in pH [2, 52–54], that in turn could result in local peptide release in the inflamed tissue contributing to neurogenic inflammation . It is also of interest to note that in an earlier study in the pig, Lynn et al., (1996)  reported that a vasodilatory response was elicited by stimulation of CH-fibers, but not CPM-fibers.
One possible mechanism that could be responsible for the increase in the magnitude of the response to heat in IB4-positive CPM fibers is modulation of the M-current. The M-current has been shown to be present in CPM fibers innervating mouse skin that also contain the mas gene related G-protein coupled receptor D . Peptides can inhibit the M-current either directly  or indirectly by inducing the release of inflammatory mediators such as bradykinin . Thus, the increased responsiveness to heat in the CPM population of fibers could be mediated by peptide release from TRPV1-positive CH fibers.