Blockade of Trks and skeletal pain
In the present study we show that early/sustained administration of a Trk inhibitor significantly inhibited sprouting and neuroma formation by sensory nerve fibers and reduced bone cancer pain-related behaviors by 50-60%. As the Trk inhibitor has a 50:1 plasma to CSF ratio, the anti-hyperalgesic actions of the inhibitor would appear to occur primarily outside the blood brain barrier. Previous reports have demonstrated that following peripheral inflammation and tissue injury, a variety of inflammatory, immune and stromal cells upregulate the expression of NGF, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) whose cognate receptors are TrkA, TrkB and TrkC respectively. Many studies have shown that peripheral NGF can drive pain and have suggested that NGF and perhaps peripherally released BDNF and NT-3 may play a role in modulating pain [
14,
15]. As the present results show that blockade of all three Trks reduces bone cancer pain, a key question is which neurotrophins and Trks are most likely the major contributors to the generation and maintenance of bone cancer pain.
Previous results have demonstrated that in the adult NGF can directly activate and sensitize sensory neurons involved in the conduction of pain originating from the skin [
16,
17], viscera and skeleton. NGF is thought to excite and sensitize sensory neurons by binding to its cognate receptor TrkA which is expressed by a subpopulation of mostly unmyelinated and thinly myelinated sensory neurons [
18]. NGF binding to TrkA has been shown to directly activate TrkA-expressing nociceptors
in vivo and
in vitro and that binding of NGF to TrkA directly lowers the threshold for depolarization in these neurons [
19,
20]. Additionally, NGF has been shown to modulate and/or sensitize a variety of neurotransmitters, receptors, ion channels and structural molecules expressed by nociceptors [
17]. It has also been shown that NGF lowers the threshold and enhances the response of nociceptors to mechanical stimuli [
21], suggesting that NGF activation of TrkA may play a role in activating/sensitizing mechanotransducers expressed by sensory nerve fibers. The NGF produced by target tissues or tumor cells activates TrkA receptors expressed on the terminals of C-fibers [
18,
22] presumably including those innervating the skeleton. Whether the Trk inhibitor used here is exerting its effect by interfering with the retrograde signal (the internalized NGF/TrkA complex) that exerts transcriptional control in the neuronal cell body or by local modulation at the nociceptor terminal is not clear. However, in a model of bone fracture pain, with Trk inhibition at 2 days post fracture, where nerve sprouting has not yet occurred, the full analgesic effect is achieved 6-8 hours following acute administration [
23]. These data suggest that local modulation of nerve fibers must be involved, as transport of the NGF/TrkA from the nerve terminals in the femoral fracture site to the cell bodies of sensory neurons that innervate the femur (which are located in the L1-L3 ganglia) would be expected to take significantly longer than 8 hours [
24].
While there is strong evidence that TrkB receptors expressed by post-synaptic spinal cord neurons play a significant role in pain transmission [
17] there is significantly less agreement about the role of TrkB expressed by sensory neurons in driving pain. Previous reports have suggested that TrkB receptors are expressed by a subpopulation of the DRG, nodose and trigeminal neurons and their terminals in the spinal dorsal horn and trigeminal nucleus [
25‐
27] and that peripheral inflammation in some tissues results in an increase in BDNF levels [
16]. Interestingly, in pancreatitis BDNF content was reported to be correlated with pain intensity [
28] and exogenous application of BDNF has been shown to excite and sensitize some cutaneous nociceptive terminals [
29] (apparently via TrkB). However, the effects of BDNF on TrkB expressing sensory neurons in driving any type of chronic pain remain poorly understood [
17]. Similarly, there is relatively little evidence to suggest that peripheral TrkC receptors expressed by myelinated nerve fibers play a significant role in the generation and maintenance of pain in the adult. Thus, while local injection of NT-3 has been reported to induce mild pain at the injection site [
30], other reports suggest that NT-3 does not sensitize nociceptive primary afferent fibers [
20] and appears to be anti-nociceptive in some pain models such as complete Freund's adjuvant-induced skin inflammation [
31].
The above results, together with the present data demonstrating that the analgesic efficacy of the Trk inhibitor in blocking bone cancer pain is similar to that of anti-NGF sequestering therapy, suggest that TrkA plays the prominent role in driving bone cancer pain [
32]. One unique aspect of the sensory innervation of bone, which may partially explain why Trk inhibition is effective in relieving skeletal pain, is that the majority of C-fibers that innervate the bone are CGRP-expressing fibers, and nearly all CGRP
+ fibers co-express TrkA [
18,
22]. Thus, most C-fibers that innervate both human [
33] and rodent [
34] vertebral discs and bone [
35] appear to be CGRP/TrkA expressing fibers and few unmyelinated non-peptidergic IB4/RET
+ nerve fibers are present in these tissues [
10,
35]. Thus, since bone appears to lack the redundancy of the C-fiber non-peptidergic IB4/RET
+ nerve fibers that are present in skin [
10], blocking TrkA activation may be particularly efficacious in relieving bone pain vs. skin pain.
Trks and their involvement in the development of nerve sprouting and neuroma formation
Although Trks clearly play an essential role in the growth and survival of sensory neurons in the developing animal [
36,
37], much less is known about the role the Trks play in the maintenance and survival of adult sensory neurons. In the adult, neurotrophins appear to be expressed in most tissues at very low levels, whereas the levels of NGF (and in some tissues BDNF and NT-3) are dramatically up-regulated by inflammation and/or injury [
14,
16]. However, it has been shown that chronic NGF deprivation (by exogenous administration of an anti-NGF polyclonal antibody or autoimmunization to NGF) results in a modest hypoalgesia, where animals are less sensitive to some thermal and algogenic stimuli [
38]. Whether this hypoalgesic effect observed in rats with polyclonal antibodies or autoimmunization will also be observed in humans treated with Trk inhibitors is unclear as is how much endogenous NGF, BDNF or NT-3 is required to maintain normal sensory nerve function in the adult.
The present studies suggest that similar to the tumor, sensory nerve fibers undergo a highly pathological and active reorganization as tumor cells invade the bone. Thus, as the tumor and associated stromal cells invade the bone, there is significant sprouting by CGRP
+ and NF200
+ sensory nerve fibers and these sensory nerve fibers are intermingled among the tumor/stromal cells that have invaded and remodeled the bone. These newly sprouted CGRP
+ and NF200
+ nerve fibers have a very dense and highly disorganized morphology that is never observed in the normal bone. In addition to the sprouting of nerve fibers, in approximately 1 out of 2 tumor-bearing bones we observe the appearance of neuroma-like structures that looked very similar to neuromas that have been described in both animals and humans following traumatic nerve injury. These structures appear as a disordered mass of CGRP
+ and NF200
+ blind ending axons that generally run parallel to each other and have an interlacing or whirling morphology [
39,
40]. It should be emphasized that we have never observed these neuroma-like structures in the sham vehicle-treated or naïve bones. However, these data would agree with previous findings suggesting that NGF is involved in neuroma formation and when provided with the appropriate trophic factor, sensory nerve fibers can grow at a remarkable pace, sprouting several millimeters a day [
41].
Previous studies have shown that injury to peripheral nerves associated with trauma, amputation, compression, or surgery can lead to painful neuromas [
13,
42,
43], which have a morphology similar to the neuroma-like structures observed in the tumor-bearing mouse bones. In humans, these non-malignant neuromas frequently cause chronic and severe pain [
13,
43] and can produce spontaneous ectopic discharges [
44,
45] in part by up-regulation of sodium channels [
43,
46]. Problematically, painful neuromas can be largely refractory to current medical treatment [
43]. It is not currently known whether there is up-regulation of sodium channels and spontaneous discharge by these neuroma-like structures in the tumor bearing mouse bone. However, movement may not be required for these ectopic discharges to occur suggesting that this mechanism is a possible explanation for spontaneous breakthrough pain.