Here we show that peripheral nerve injury in mice differentially regulates the heat response properties of directly-injured small neurons during the same time course of behavioral hypersensitivity to noxious heat. Furthermore, TRPV1 inhibition completely blocks the heat hypersensitivity at both the sensory neuron membrane and the behavioral levels, indicating that in mice, TRPV1 mediates the heat hyperalgesia induced by spinal nerve ligation.
Whereas IB4 positive neurons from non-injured mice rarely respond to noxious heat, spinal nerve ligation increased their heat responsiveness 4-fold, such that after injury over 50% of the injured IB4 positive neurons responded to noxious heat. In contrast, IB4 negative neurons which are normally very responsive to noxious heat, were decreased in heat sensitivity by spinal nerve ligation. These differential effects of injury on functionally distinct subpopulations of small neurons were not likely due to underlying changes in IB4 binding because the percentage of neurons binding IB4 after injury was unchanged in mice. Other studies in rats21, 32
have shown a marked decrease in IB4 staining in DRG neurons after injury. Glial cell line-derived neurotrophic factor (GDNF) is critical for the survival and function of IB4 positive neurons.2, 43, 67
The differential effect of injury on IB4 binding in mice verses rats may be because mice and rats express different profiles for GDNF production by Schwann cells and satellite cells after nerve injury. 20, 45
Three pieces of evidence indicate that the heat-evoked current in the somata of all neurons from either naive mice or injured mice is mediated entirely by TRPV1. First, a selective TRPV1 antagonist, A-425619, completely and reversibly inhibited all heat-evoked inward currents in naive, sham, SNL injured and SNL adjacent neurons. Second, none of the neurons from TRPV1-mutant mice responded to noxious heat. Third, the heat threshold for all neurons ranged between 42 and 47 °C, which is consistent with the documented threshold for TRPV1 in an expression system.9
Thus, in isolated somata, heat-evoked inward currents in neurons from naive, nerve injured and inflamed mice were entirely due to activation of TRPV1.6
In contrast to the soma, heat responses at the peripheral terminal are less dependent on TRPV1. Recordings from skin-nerve preparations, where heat is applied at the peripheral terminal and responsiveness is recorded in the peripheral axon, indicate that heat-sensitive C fibers with thresholds ~ 42 °C remain partly intact in TRPV1 deficient mice8, 30, 63, 66
(and Stucky C.L., unpublished data), albeit to different degrees in different laboratories. The residual heat responses in peripheral axons may be mediated by heat-sensitive ion channels expressed only at the terminal region of the neuron or by non-neuronal cells in the skin. For example, keratinocytes express functional heat-sensitive channels including TRPV3 and TRPV419, 47
and recent evidence suggest that keratinocytes confer heat responsiveness to nearby sensory terminals via release of ATP.31, 40
Together, these findings indicate that future studies searching for heat transduction molecules should include a component where the sensory terminal remains in its native milieu such that interactions between non-neuronal cells in the target tissue and the sensory neuron membrane can be appropriately modeled and investigated.
When using isolated DRG somata as a model for the sensory terminal, the dissociation and culture procedure may intrinsically injure the neuron, thereby altering the composition and density of ion channels normally expressed by the soma membrane in vivo
Nonetheless, using DRG somata as a model provides an important measure of the overall function of TRPV1 channels in the plasma membrane. Indeed, TRPV1 is expressed by many membrane regions of the sensory neuron, including central terminals in the spinal cord,44
peripheral axons of passage,4
the cell body and the peripheral terminals. In our study, DRG somata from all groups were processed in an identical manner, and therefore, the differences observed were likely due to the injury in vivo
The somata of directly injured neurons no longer have axons that reach peripheral targets. Injured neurons frequently form neuromas at the cut end of an injured nerve. Increased expression of ion channels at the neuroma in the setting of an inflammatory milieu may contribute to spontaneous pain via spontaneous input to the spinal cord and/or sensitization of spinal cord neurons. It has been hypothesized that increased TRPV1 function may contribute to spontaneous activity via a reduction in the thermal threshold, such that in the setting of nerve or tissue injury, TRPV1 becomes activated at body temperature.52, 56
Our data add to a growing body of evidence that IB4 positive C fiber neurons play important roles in injury. The majority of cutaneous C fiber neurons that innervate the epidermis bind IB4.35
They are known to be nociceptors and respond to mechanical stimuli in situ
.1, 17, 60
A variety of animal models of persistent pain indicate that IB4 positive neurons are highly sensitive and malleable to injury and upregulate their functional response properties in response to injury. For example, IB4 positive neurons increase TRPV1 function after peripheral inflammation.6
Interestingly, evidence now indicates that IB4 positive neurons provide input to a special pathway to the brain that is involved in modulating the emotional processing of pain.5, 69
Together, these data suggest that IB4 positive nociceptors may be involved in providing enhanced input to the emotional aspects of neuropathic pain.
Nerve injury pain is due to a mixture of effects from axon injury and inflammation. Inflammation is well-documented to increase TRPV1 function.8, 12
In our study, neurons from animals with sham surgery were very likely exposed to some degree of inflammation. This is suggested by a trend for increased TRPV1-mediated heat responsiveness in medium-large neurons from sham animals compared to naive controls (). In animals with SNL, the L3 adjacent neurons were exposed to minor injury and inflammation, whereas L4 neurons were axotomized and exposed to inflammation. We speculate that these differences in inflammation and axon injury between groups underlies our finding that TRPV1 function was significantly decreased in injured L4 medium-large neurons compared to sham surgery controls, and that the net effect of direct injury on myelinated neurons is decreased TRPV1 function. Thus, the overall change in TRPV1 function in myelinated neurons is due to the combination of inflammation and nerve injury. In different animal models of nerve injury, if inflammation is predominant, TRPV1 function increases; if injury is predominant, TRPV1 function decreases.
In behavioral experiments, L4 SNL induced heat hypersensitivity in the hind paw that was completely reversed by acute intraperitoneal injection with the TRPV1 inhibitor A-425619. In the days following L4 SNL, the plantar paw presumably loses innervation by the L4 afferents but retains innervation by L3 afferent terminals. The heat hypersensitivity is not likely due to sensitization of L3 adjacent neurons because we found no change in TRPV1 function in neurons from the L3 ganglia. Instead, we observed increased TRPV1-mediated heat responses in IB4 positive neurons from the injured L4 ganglia. We propose that increased TRPV1 activity in injured IB4 positive neurons, possibly due to reduced TRPV1 heat threshold and activation by body temperature, contributes to enhanced spontaneous activity in these neurons that subsequently sensitizes spinal cord neurons to respond more to input from intact L3 afferents activated by peripheral heat stimuli.
Knowledge about nerve injury-induced changes in TRPV1 function has important clinical implications. First, injury-induced upregulation of TRPV1 function provides rationale for treatment strategies that inhibit or desensitize TRPV1. Second, identifying specific neuronal subtypes that exhibit increased TRPV1 function may lead to treatments that specifically target these neuronal subtypes. For example, selective inhibition or elimination of IB4 positive C fiber-type neurons during neuropathic pain may help alleviate negative emotional aspects of chronic pain, but leave intact the acute, protective nociceptive responses necessary for safe interaction with the environment.