Effects of inflammation on cutaneous nociceptor response properties
In the first series of experiments, we used an ex vivo
skin/saphenous nerve/DRG/spinal cord preparation to characterize the peripheral response properties of different functional types of cutaneous sensory neurons in naïve and CFA injected C3H/Bl6 mice. In this set of experiments, a total of 270 characterized cutaneous A- and C-fiber nociceptors were recorded in naïve (14 male and 12 female; n = 142) and inflamed (7 male and 5 female; n = 128) C3H/Bl6 mice. Nociceptors were identified based on their peripheral response properties and the presence of an inflected somal action potential [22
]. Afferents conducting faster than 1.2 m/s were classified as A-fibers while all others were classified as C-fibers. A-fiber nociceptors were divided into two groups; those that responded to both mechanical and thermal stimuli (APM) and those that only responded to mechanical (AM). C-fibers were further divided based on their peripheral response properties to mechanical and thermal stimuli [10
]. These groups included: fibers that responded to mechanical and heat and sometimes cold stimuli (CPM); C-mechanocold (CMC) fibers responded to mechanical and cold stimuli on the cutaneous receptive field (RF); C-mechano (CM) fibers responded to mechanical but not to thermal stimulation of the skin (hot or cold); C-cold (CC) fibers responded only to cold/cooling and not to mechanical or heat stimuli; C-heat (CH) fibers responded to heat, but not to mechanical or cold stimuli. There were no differences found between the response properties of cutaneous sensory neurons recorded in naïve male and female mice (e.g. CPM heat thresholds male = 42.1 ± 0.6°C; female 40.8 ± 1.0°C; p = 0.25). There were also no differences between fibers recorded in male and female mice following CFA injection (e.g. CPM heat thresholds; male 38.8 ± 1.1°C; female 38.6 ± 1.0°C; p = 0.9). Therefore the results of male and female mice were combined for analysis.
In naïve C3H/Bl6 mice the average heat threshold of CPM-fibers was 41.7 ± 0.5°C, n = 73; (Fig. ). One and three days after subcutaneous injection of CFA into the dorsum of the hindpaw, the average heat threshold of CPM-fibers was significantly reduced to 38.1 ± 0.8°C, at day 1 (p < 0.001) and to 37.6 ± 1.3°C at day 3 (p < 0.001). By day 5, the heat threshold of CPM-fibers had returned to near naïve levels (40.2 ± 0.7°C). When combining CPM-fibers from all 3 time points after inflammation, the resulting average heat threshold was still significantly lower than those in naive animals (CPM = 38.7 ± 0.8°C, n = 46; p < 0.001). The magnitude of the responses of CPM-fibers in naïve and inflamed animals also differed. One day after inflammation, CPM-fibers responded to the heat ramp with significantly higher mean firing rates at 38°C and higher temperatures (2-way ANOVA, Bonferoni p < 0.001; Fig. ). This persisted at 3 days post CFA injection when mean firing rates were significantly higher at temperatures above 42°C (2-way ANOVA, Bonferoni p < 0.05; Fig. ) and at 5 days post CFA injection at temperatures higher than 44°C (2-way ANOVA, Boneroni p < 0.05; Fig. ). As with heat thresholds, the combined responses from all three time points was also significantly higher to noxious temperatures above 44°C (2-way ANOVA, Bonferoni p < 0.001; Fig. ). However, there was no change in the response of CPMs to cold, or to mechanical stimulation (e.g. naïve CPM mechanical threshold 14.8 ± 2.0 mN vs inflamed 18.2 ± 2.9 mN). This was also true for the other C-fiber types (CMC, CM, CC). Whereas there was a trend toward a decrease in the mean heat threshold of CH fibers, this change was not statistically significant (naïve = 41.8 ± 1.6°C, n = 11; combined inflamed = 39.1 ± 1.7°C, n = 11; p = 0.3). The mean firing rate of the CH neurons during application of the heat ramp after inflammation was also unchanged (Fig. ). Following inflammation we also recorded from a total 14 mechanically sensitive A-fibers (AM); none of these AM nociceptors responded to heat. As we found for CPM fibers (Fig. ), the response to mechanical stimulation was unchanged in AM nociceptors following inflammation (Fig. ).
Figure 1 Inflammation-induced heat sensitization of TRPV1-negative CPM-fibers. A) Heat thresholds of CPM-fibers were significantly reduced 1 (dark grey bar) and 3 days (medium grey bar) after the injection of CFA and indistinguishable from those in naïve (more ...)
Figure 2 CH-fibers lack heat sensitization and AM and CPM-fibers lack mechanical sensitization after inflammation. A) After inflammation, CPM-fibers encode heating of the cutaneous receptive field with significantly higher firing rates while B) CH-fibers are not (more ...)
The role of TRPV1 in inflammation
The results of these initial experiments suggested that the CPM fibers (that do not normally express TRPV1) are sensitized to heat following inflammation. In order to determine whether this inflammation-induced sensitization was truly a TRPV1-independent process, we next examined the effects of inflammation on the response properties of cutaneous CPM fibers in TRPV1-/- and wildtype mice. In this series of experiments we focused on a single time point (3 days) following CFA injection.
Inflammation was induced as before by a subcutaneous injection of CFA into the dorsum of the foot of adult male TRPV1-/- (n = 10) and wildtype (WT) C57BL6 (n = 10) mice. Three days later, all mice exhibited inflamed hindpaws. Behavioral testing revealed that WT C57BL6 mice had significantly shorter paw withdrawal latencies in response to noxious radiant heat delivered to the plantar surface of the inflamed hindpaw (naïve C57BL6 = 9.2 ± 1.4 s, inflamed C57BL6 = 6.7 ± 1.4 s, p < 0.05; Fig. ), while TRPV1-/- mice did not develop thermal hyperalgesia (naïve TRPV1-/- = 10.3 ± 0.9 s, inflamed TRV1-/- = 11.4 ± 1.0 s).
Figure 3 CPM-fibers exhibit decreased heat thresholds in inflamed TRPV-/- mice. A) Only C57/BL6 mice have reduced latencies when responding to a radiant heat stimulus to the plantar surface of the hindpaw after inflammation. As previously described, in TRPV1-/- (more ...)
Following behavioral testing we used the ex vivo preparation to determine if CPM neurons in these same TRPV1-/- and C57/Bl6 mice exhibited sensitization as seen in the C3H/Bl6 mouse strain. We intracellularly recorded from a total of 307 cutaneous C-fibers in naïve and inflamed C57BL6 and TRPV1-/- animals. Similar to that observed for CPM-fibers in C3H/Bl6 mice, the average heat threshold of CPMs in C57BL6 wildtype mice dropped significantly from 40.8 ± 0.6°C (n = 51) in naïve mice to 38.0 ± 0.5°C (n = 54; p < 0.01) 3 days after CFA injection (Fig. ). In addition, these fibers exhibited an increased sensitivity to heat evidenced by an increased firing rate in response to noxious heat temperatures (2-way ANOVA, Bonferoni p < 0.001) (Fig. ). In TRPV1-/- mice, the average heat threshold of CPM-fibers was also significantly reduced 3 days after inflammation (naïve TRPV1-/- = 40.9 ± 0.6°C, n = 62; inflamed TRPV1-/- = 38.7 ± 0.6°C, n = 45; p < 0.01). However, unlike wildtype mice, CPM-fibers from inflamed TRPV1-/- animals did not exhibited any change in the mean firing rates during the heat ramp (Fig. ).
The response of CPM-fibers to an ascending series of constant force stimuli was also not different after inflammation in WT and TRPV1-/- mice (Fig. ). Interestingly, the lack of increased mechanical and thermal sensitivity of CPM fibers in inflamed TRPV1-/- mice was correlated with a trend towards reduced paw edema following CFA injection in these animals. The average area of the inflamed foot 16.2 ± 1.2 mm2 in C57BL6 and 12.9 ± 1.1 mm2 in TRPV1-/- mice (p = 0.06). Additionally, in agreement with the previous set of experiments, CH fibers in wildtype mice were unaffected by inflammation as their heat thresholds (naïve, 40.5 ± 2.4°C, n = 7; inflamed, 39.9 ± 1.9°C, n = 8) and firing rates were also unchanged (data not shown).
In addition to examining the peripheral response properties of the cutaneous nociceptors, we also compared the distribution of the different types of unmyelinated cutaneous nociceptors before and after inflammation in wildtype C57/Bl6, C3H/Bl6 and TRPV1 KO mice (Fig. ). There were no differences in distribution of C-fibers between wildtype C3H/Bl6 and C57/Bl6 mice before or after inflammation; therefore, the data was combined for presentation. The largest group of identified C-fibers in both naïve and inflamed animals was the CPM-fibers (naïve = 68.9%; combined inflamed = 70.8%). In naïve mice, we observed similar numbers of CH- (13.2%), CMC (13.2%), CMs (3%) and CC (1.8%) fibers. After inflammation there was not a significant change in the distribution of fiber types (χ2
, p < .17). We have previously reported a lack of CH-fibers in naïve TRPV1-/- animals [10
]. Here we also found that inflamed TRPV1-/- mice lack CH fibers (not shown). In naïve TRPV1-/- 75.3% of all identified C-fibers were CPMs, 9.4% were CMs, 4.7% were CCs and the remaining 10.6% were CMCs. The distribution of identified C-fibers in TRPV1-/- mice was similar after inflammation; 77.8% were CPMs, 7.9% were CMs, 3.2% were CC, and 11.1% were CMC-fibers.
Figure 4 Distribution of C-fiber types is unchanged following inflammation. The prevalence of CH-fibers does not increase after inflammation compared to naïve mice. (p value < 0.17; χ2 test.) Neither naïve (not shown) nor inflamed (more ...)
After the characterization of the cutaneous RFs, selected neurons were filled with neurobiotin and the DRGs later stained for IB4 binding and TRPV1 immunoreactivity. In Table , 81% (31/38) of characterized CPM-fibers in naïve mice labeled for IB4 and none (0/34) were immunoreactive for TRPV1. After inflammation, a similar number of CPM-fibers 83% (30/36) bound IB4. However, following inflammation 11% of CPMs (4/36) were found to stain positively for TRPV1 (Fig. ) and these cells did not bind IB4 suggesting that some of the CH cells may have acquired mechanical sensitivity following inflammation. In addition, all 10 of the CH fibers from both naïve and inflamed mice were found to be TRPV1-positive and IB4-negative. Cell counts in the same ganglia also revealed that the numbers of TRPV1 positive sensory neurons in the L3 ganglia C57/Bl6 mice were unchanged following CFA injections (naive 66.6 ± 4.0, vs inflamed 72.9 ± 4.4, p = 0.29; t-test).
TRPV1 and IB4 staining of identified CPM-fibers in naïve and inflamed mice
Figure 5 Example of TRPV1-positive CPM-fiber following inflammation. Light micrographs of a L3 DRG from the ex vivo recording preparation containing a neurobiotin-filled polymodal C-fiber (CPM) immunoreactive for TRPV1. A) A Neurobiotin labeled (green) CPM neuron (more ...)
Another difference between naïve and inflamed WT CPMs was a significant decrease in mean conduction velocity following inflammation (naïve = 0.53 +/- 0.01 m/s vs inflamed = 0.5 +/- 0.01 m/s; p value < 0.05; student's t-test). Interestingly, this change in CPM conduction velocity was not observed in the TRPV-/- mice (naïve = 0.53 +/- 0.01 m/s vs inflamed = 0.51 +/- 0.01 m/s; p value >0.05; student's t-test) mice after inflammation. This drop in conduction velocity was also not observed for any other fiber type. For example, the mean conduction velocity of CH fibers was unchanged following inflammation (naïve = 0.36 +/-0.02 m/s vs inflamed = 0.36 +/- 0.01 m/s).