Dynamic Changes in Heat Transducing Channel TRPV1 Expression Regulate Mechanically Insensitive, Heat Sensitive C-Fiber Recruitment after Axotomy and Regeneration

Abstract

Peripheral injury leads to a significant increase in the prevalence of mechanically insensitive, heat sensitive C-fibers (CH) that contain the heat transducing TRPV1 channel in mice. We have recently shown that this recruitment of CH fibers is associated with increased expression of the receptor for GDNF family neurotrophic factor artemin (GFRα3), and that in vivo inhibition of GFRα3 prevented the increase in TRPV1 expression normally observed following axotomy. Here we have directly tested the hypothesis that the recruitment of functional CH-fibers following nerve regeneration requires enhanced TRPV1 levels. We utilized in vivo siRNA-mediated knockdown to inhibit the injury-induced expression of TRPV1 coupled with ex-vivo recording to examine response characteristics and neurochemical phenotypes of different functionally defined cutaneous sensory neurons after regeneration. We confirmed that inhibition of TRPV1 did not affect the axotomy induced decrease in CPM heat threshold, but transiently prevented the recruitment of CH neurons. Moreover, a recovery of TRPV1 protein was observed following resolution of siRNA-mediated inhibition that was correlated with a concomitant rebound in CH neuron recruitment. Thus dynamic changes in TRPV1 expression, not absolute levels, may underlie the functional alterations observed in CH neurons and may contribute to the development of heat hyperalgesia after nerve injury.

Dual Modulation of Nociception and Cardiovascular Reflexes during Peripheral Ischemia through P2Y1 Receptor-Dependent Sensitization of Muscle Afferents

“…This particular duplex was previously determined to have the highest targeting efficiency to P2Y1 based on the knockdown efficacy of four different P2Y1-targeting siRNAs in vitro (Jankowski et al, 2012a, data not shown). The one duplex used in the current study and in the experiments of the previous report is as follows: sense: 5Ј-S-S-GAA GUUAUUUCAUCUACAGUU; antisense: 5Ј-P-CUGUAGAUGAAA UAACUUCUU (Jankowski et al, 2012a). Our nontargeting control siRNA used in this report and in others (Jankowski et al, , 2010(Jankowski et al, , 2012a has been determined to not target any gene in the mouse genome (Thermo Fisher Scientific, catalog #D-001206-14-05).…”

“…Specific targeting siRNAs were used to selectively knock down the expression of the ADP receptor P2Y1 (Thermo Fisher Scientific) and conjugated to penetratin-1 (MP Biomedicals) as described by Davidson et al (2004) and Jankowski et al ( , 2010Jankowski et al ( , 2012a. This particular duplex was previously determined to have the highest targeting efficiency to P2Y1 based on the knockdown efficacy of four different P2Y1-targeting siRNAs in vitro (Jankowski et al, 2012a, data not shown). The one duplex used in the current study and in the experiments of the previous report is as follows: sense: 5Ј-S-S-GAA GUUAUUUCAUCUACAGUU; antisense: 5Ј-P-CUGUAGAUGAAA UAACUUCUU (Jankowski et al, 2012a).…”

“…Muscle tissue generates heat (González-Alonso et al, 2000) and releases metabolites, such as protons, ATP, and lactic acid, during contraction (Bockman, 1983;Bockman and McKenzie, 1983;MacLean et al, 1998). The group III/IV muscle sensory neurons are known to respond to thermal stimuli Jankowski et al, 2013;Queme et al, 2013;Ross et al, 2014) and different combinations of these same metabolites (Light et al, 2008). Specifically, there is one population of muscle afferents that responds to a combination of ATP, lactic acid, and protons that resembles the environment in the muscle during moderate exercise, and a second population that responds exclusively to the same metabolites but in a higher concentration, similar to what is produced in muscle tissue during ischemic contractions (Alam and Smirk, 1937;Kniffki et al, 1978;Kaufman and Rybicki, 1987;Light et al, 2008;Jankowski et al, 2013;Pollak et al, 2014;Ross et al, 2014).…”

“…The group III/IV muscle sensory neurons are known to respond to thermal stimuli Jankowski et al, 2013;Queme et al, 2013;Ross et al, 2014) and different combinations of these same metabolites (Light et al, 2008). Specifically, there is one population of muscle afferents that responds to a combination of ATP, lactic acid, and protons that resembles the environment in the muscle during moderate exercise, and a second population that responds exclusively to the same metabolites but in a higher concentration, similar to what is produced in muscle tissue during ischemic contractions (Alam and Smirk, 1937;Kniffki et al, 1978;Kaufman and Rybicki, 1987;Light et al, 2008;Jankowski et al, 2013;Pollak et al, 2014;Ross et al, 2014). Human subjects that received intramuscular injections of these metabolite mixtures reported that the solution associated with moderate exercise produced a sensation of fatigue while injection of the more concentrated mixture caused a feeling of muscle pain (Pollak et al, 2014).…”

“…Evidence shows that, in addition to afferents that respond to these aforementioned metabolites, there are thermally sensitive afferents that also play a role in EPR alterations after injury (Gao et al, 2007;Sluka et al, 2007). One type of purinergic receptor, P2Y1, has been shown to modulate heat sensitivity and phenotypic changes in the expression of transient receptor potential vanilloid 1 in cutaneous C-polymodal fibers during inflammation (Molliver et al, 2011;Jankowski et al, 2012a). We have further shown that P2Y1 is also upregulated in the dorsal root ganglia (DRGs) after muscle ischemia (Ross et al, 2014), and mice with 24 h ischemic injury experience spontaneous pain-like behaviors, decreased mechanical thresholds to von Frey hair stimulation of the plantar surface of the affected forepaw, and decreased muscle strength (Ross et al, 2014).…”