Nociception and inflammatory hyperalgesia evaluated in rodents using infrared laser stimulation after Trpv1 gene knockout or resiniferatoxin lesion

Abstract

TRPV1 is expressed in a subpopulation of myelinated Aδ and unmyelinated C-fibers. TRPV1+ fibers are essential for the transmission of nociceptive thermal stimuli and for the establishment and maintenance of inflammatory hyperalgesia. We have previously shown that high-power, short-duration pulses from an infrared diode laser are capable of predominantly activating cutaneous TRPV1+ Aδ-fibers. Here we show that stimulating either subtype of TRPV1+ fiber in the paw during carrageenan-induced inflammation or following hind-paw incision elicits pronounced hyperalgesic responses, including prolonged paw guarding. The ultrapotent TRPV1 agonist resiniferatoxin (RTX) dose-dependently deactivates TRPV1+ fibers and blocks thermal nociceptive responses in baseline or inflamed conditions. Injecting sufficient doses of RTX peripherally renders animals unresponsive to laser stimulation even at the point of acute thermal skin damage. In contrast, Trpv1-/- mice, which are generally unresponsive to noxious thermal stimuli at lower power settings, exhibit withdrawal responses and inflammation-induced sensitization using high-power, short duration Aδ stimuli. In rats, systemic morphine suppresses paw withdrawal, inflammatory guarding, and hyperalgesia in a dose-dependent fashion using the same Aδ stimuli. The qualitative intensity of Aδ responses, the leftward shift of the stimulus-response curve, the increased guarding behaviors during carrageenan inflammation or after incision, and the reduction of Aδ responses with morphine suggest multiple roles for TRPV1+ Aδ fibers in nociceptive processes and their modulation of pathological pain conditions.

Keywords: Analgesia; Capsaicin; Chemoreceptors; Chronic Pain; Nociception.

Conflict of interest statement

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Conflict of Interest

The authors declare that they have no conflicts of interest.

Copyright © 2014. Published by Elsevier B.V.

Figures

Fig. 1

Characterization of laser-evoked withdrawal responses. (A) Linear relationship between laser power per area and heating rate measured as change in skin surface temperature per second. (B) Stimulus-response curve showing the frequency of paw withdrawal following activation of Aδ fibers at increasing laser intensity (n = 16 rats). (C) Response severity based on behavioral ratings increases with increasing laser intensity. (D, E) Effect of unilateral hind-paw inflammation on Aδ responses. Probability of paw withdrawal was determined for the ipsilateral (D) carrageenan-injected doses of 2 and 6 mg in 150 μL and for the contralateral (E), saline-injected paws (n = 5 rats). (D) Note the leftward shift in the stimulus response curve in the inflamed paw after carrageenan, consistent with sensitization, P < 0.05. (E) Withdrawal frequency was decreased in the uninflamed contralateral paw, suggesting the animals inhibited the abrupt transfer of weight to the inflamed paw, P < 0.05. (F) The Aδ stimulus provoked a brisk and vigorous paw withdrawal even if the hind paw was inflamed and the graph of the first observable movement following Aδ stimulus was identical for the inflamed hind paw and the withdrawal latency from a noninflamed rat (ie, neither paw was inflamed). (G) Licking and guarding nocifensive behaviors are significantly increased following Aδ laser stimulation under inflamed conditions compared to noninflamed rats, P < 0.001. (H) C-fiber stimulation of the hind paw. The scatterplot of withdrawal latencies for uninflamed animals (n = 16 rats) shows the distribution about the mean (8.66 ± 0.25 s). (I) Effect of unilateral hind-paw inflammation on C-fiber responses (n = 5 rats). Both 2 mg and 6 mg carrageenan produced significant decreases in withdrawal latency consistent with hyperalgesia (P < 0.001), but no further sensitization occurred with the 6 mg dose of carrageenan.

Fig. 2

Effect of intrathecal RTX on withdrawal responses to noxious thermal stimulation. RTX at 20, 200 and 2000 ng was administered intrathecally to ablate the TRPV1+ fibers in the cauda equina and cell bodies in the lumbar ganglia. (A) Paw withdrawal frequency following 2 intensities of Aδ-fiber stimulation in rats 10 days after intrathecal administration of vehicle or RTX (n = 6 rats). Complete suppression of Aδ responses occured with 200 or 2000 ng RTX, depending on stimulus intensity. (B) C-fiber laser stimulation shows a dose-dependent increase in paw-withdrawal latency in RTX-treated rats compared to vehicle (n = 6 rats). (C, D) RTX-induced TRPV1+ fiber ablation significantly attenuates behavioral responses for either (C) Aδ- or (D) C-fiber stimulation of inflamed hind paw compared to the saline-injected contralateral paw (n = 6 rats). The animals were tested within 24 hours after carrageenan injection for (C) Aδ- or (D) C-fiber response.

Fig. 3

Trpv1 gene knockout and RTX-mediated TRPV1+ fiber lesion attenuate Aδ nocifensive behavioral responses. (A) Ascending Aδ stimuli (2–6 W/mm2) were applied to plantar surface of uninflamed wt mouse hind paws. Withdrawal frequency plateaued at 100% with a power of 4 W/mm2 (n = 6 mice). Trpv1−/− produced nearly complete reduction at 2, 3, and 4 W/mm2. Attenuated yet significant withdrawal occurred at 5 and 6 W/mm2, suggesting the presence of TRPV1 ion channel independent mechanism(s) for withdrawal at high-intensity stimulations. Removal of the TRPV1 fibers with RTX produced a more complete suppression of withdrawal, even with the 5 and 6 W/mm2 high-intensity stimulations. (B) Aδ-mediated withdrawal frequency in RTX-treated wt and Trpv1−/− mice was determined at baseline and 24 hours postinflammation (n = 6). Inflammation caused a leftward shift in the stimulus response (black lines) compared to noninflamed wt and Trpv1−/− mice, but not in mice treated with RTX. For Trpv1−/− mice, significant sensitization occurred with the high-intensity stimuli (red line). Sensitization to low-intensity stimuli (2–4 W/mm2) was TRPV1 ion channel-dependent. Loss of the TRPV1+ peripheral ending blocked sensitization; however, some residual behavioral responsiveness was detected. (C) Comparison of withdrawal latency in wt, Trpv1−/−, and RTX-treated mice showed that C-fiber behavioral responses were sensitized to inflammation only in wt mice. An elevated withdrawal latency occurred in both Trpv1−/− and RTX-treated wt mice compared to wt mice; both manipulations blocked thermal sensitization (n = 6).

Fig. 4

Withdrawal responses and skin damage to high-intensity and supramaximal infrared diode laser stimuli. (A) Paw withdrawal frequency was determined in intraplantar high-dose (250 ng) RTX- and vehicle-treated rats to Aδ stimulation at supramaximal laser intensities (n = 6). Rats were stimulated only once per energy setting to minimize skin damage. (B) Rats showed significantly elevated withdrawal latencies following intraplantar RTX treatment. In 4 of the tests, responses were blocked up to 30 seconds of stimulation, an arbitrarily chosen late cut-off (n = 6 rats). Filled circles indicate the presence of skin damage or edema post stimulation. High-dose, local RTX blockage of response to damaging stimuli is consistent with the presence of a low amount of TRPV1 on these damage-responsive afferent fibers. (C) In mice, withdrawal frequencies were tested to Aδ thermal pulses at 4.46 W/mm2. Stimulation with noxious (100 ms; 0.45 J/mm2) and damaging (200 ms; 0.90 J/mm2) durations resulted in similar withdrawal frequencies between Trpv1−/− and wt mice, whereas wt mice treated with 10 ng intraplantar RTX exhibited significantly reduced withdrawal frequencies (n = 6). (D, E) Epidermal neutrophil infiltration in paw skin of RTX-treated rats 24 hours after C-fiber stimulation with (D) sub-damaging and (E) damaging stimuli. Arrows show neutrophils immunostained for S100A8, a highly expressed neutrophil marker. C = cornified layer; S = spinous layer; D = dermis. (F) S100A8 immunostaining following progressively increasing Aδ thermal stimulation in an anesthetized rat. Behavioral withdrawal occurs at laser powers well below those that cause tissue damage. Increasing the duration to 200 ms produced a subclinical effect because no damage was seen with hematoxylin-eosin staining (not shown), but did provoke neutrophil diapedesis just under the zone of thermal stimulation. Increasing the duration to 300 ms produced a small burn in the skin, much greater neutrophil infiltration, and visible damage with hematoxylin-eosin staining (not shown).

Fig. 5

Intraplantar RTX eliminates guarding and Aδ-mediated withdrawal behaviors following experimental incision. (A) Incisional injury produces prolonged guarding activity, which is eliminated following intraplantar RTX (250 ng) treatment. (B) Experimental incision (Inc) sensitizes rats to Aδ stimulation (3.5 W/mm2) compared to baseline (n = 6). The hyperalgesic response is reversed by intraplantar RTX.

Fig. 6

Assessment of TRPV1 localization in myelinated primary afferents. Immunocytochemical staining was performed for (A) neurofilament 200 protein, a myelination marker in primary afferent neurons, and (B) TRPV1. The proportions of single- and double-labeled neuronal perikarya were counted in L4-L5 DRG sections from 3 rats (C). A total of 1086 cells were counted. Of the 479 TRPV1+ neurons, the majority were singly labeled (430), with 11.4% coexpressing NF200 to varying extents, as indicated (D). This proportion is within the parameters of other published studies, which report a range of colocalization between 5% and 30% (see text).

Fig. 7

Effect of systemic morphine on Aδ- and C-fiber responses at baseline and during inflammation. (A) Morphine dose-dependently blocked paw withdrawal to the Aδ stimulus at 5.6 W/mm2. Withdrawal frequency was almost completely inhibited in rats treated with 5 mg/kg morphine (n = 10 rats). (B) The withdrawal latency to C-fiber stimulation at 0.0815 W/mm2 was significantly increased compared to vehicle at the highest dose only, 25 mg/kg (n = 10 rats). The dashed line at 14 seconds represents the trial cut-off to prevent tissue injury. (C) During carrageenan-induced unilateral inflammation, 5 mg/kg morphine reversed thermal hyperalgesia to the C-fiber stimulus without increasing baseline withdrawal latency (ie, producing analgesia) in the contralateral paw (n = 15 rats). With the Aδ stimulus, 5 mg/kg morphine (D) attenuated the withdrawal frequency in inflamed rats, (E) reduced the response intensity, and (F) inhibited Aδ-evoked guarding (n = 15 rats).