3.2. Electrophysiology measurement
All neurons included in this study were Aβ nonnociceptive neurons judged by sensory testing and by AP features. Electrophysiological properties of Aβ-fiber LTMs in control rats were comparable with those of the CIBP rats at <1 week and >2 weeks. A total of 18 neurons from 6 animals for each group met the acceptance criteria. In terms of the breakdown of different types of Aβ-fiber LTMs, both groups of animals yielded comparable numbers of each neuronal subtype following the criteria of Lawson et al (1997). Aβ-fiber LTMs were included based on the 4 subsets: 4 guard/field hair, 4 RA, 4 SA, and 6 MS in each group.
3.3. Action potential conduction velocity and configuration
Intracellular somatic APs evoked by electrical stimulation of the dorsal root showing the electrophysiological parameters were measured, including: (1) CV; (2) Vm; (3) APdB; (4) APRT; (5) APFT; (6) APA; (7) AHP50%; and (8) AHPA. All data are shown in the scatter plots of Figure 2, illustrating the distributions of various parameters for individual neurons in each neuron type in control and CIBP rats. Table 1 shows the statistical comparison between all groups (control and CIBP rats at <1 and >2 weeks, each).
3.3.1. Conduction velocity
Comparison of the neuronal CV between control and CIBP rats at >2 weeks did not show a significant difference. At >2 weeks, the CV was 12.60 ± 0.68 mm/ms in control vs 11.80 ± 0.85 mm/ms in CIBP rats (P = 0.924). At <1 week, the CV was 12.70 ± 5.56 mm/ms in control vs 11.91 ± 0.83 mm/ms in CIBP neurons (P = 0.962).
3.3.2. Resting membrane potential
Vm of Aβ-fiber LTMs of CIBP rats was not significantly different from Vm of control rats at >2 weeks (control, −62.82 ± 1.70 mV vs CIBP, −63.50 ± 1.86 mV, P = 0.862), while also not significantly different at <1 week group (control, −61.95 ± 1.68 mV vs CIBP, −63.52 ± 2.25 mV, P = 0.729).
3.3.3. Action potential amplitude
There were significant differences in APA between control and CIBP rats at >2 weeks. There was reduced APA in CIBP rats (control, 66.17 ± 2.07 mV vs CIBP, 55.18 ± 2.16 mV, P < 0.001); however, there were no significant differences between control and CIBP rats at <1 week (control, 65.94 ± 2.44 mV vs CIBP, 67.56 ± 2.42 mV, P = 0.987).
3.3.4. Action potential duration at base
In marked contrast to neurons in control rats, neurons in CIBP rats exhibited a wider APdB (control, 1.33 ± 0.17 ms vs CIBP, 1.75 ± 0.37 ms; P = 0.001) at >2 weeks. No significant differences were observed between groups at <1 week (control, 1.31 ± 0.04 ms vs CIBP, 1.31 ± 0.04 ms; P = 0.693).
3.3.5. Action potential rise time
A longer APRT was observed in CIBP rats relative to control at >2 weeks. APRT was 0.46 ± 0.02 milliseconds in control and 0.82 ± 0.27 milliseconds in CIBP rats (P < 0.001). At <1 week, there were no differences between groups; APRT was 0.44 ± 0.02 milliseconds in the control rats and 0.47 ± 0.02 milliseconds in the CIBP rats (P = 0.601).
3.3.6. Action potential fall time
There were no differences in neuronal APFT between groups at either time point. At >2 weeks, APFT in control animals was 0.89 ± 0.17 milliseconds and 0.97 ± 0.38 milliseconds in CIBP rats (P = 0.195). At <1 week, APFT in control animals was 0.87 ± 0.03 milliseconds and 0.88 ± 0.04 milliseconds in the CIBP rats (P = 0.275).
3.3.7. AHP amplitude
No differences in AHPA were observed between any groups. At >2 weeks, AHPA in control animals was 5.30 ± 0.64 mv and 6.41 ± 0.73 mv in CIBP rats (P = 0.658). At <1 week, there were no differences between groups (control, 5.89 ± 0.54 mv vs CIBP 6.62 ± 0.48 mv, P = 0.342).
3.3.8. Afterhyperpolarization duration to 50% recovery
A shorter AHP50 was observed in CIBP rats relative to control at >2 weeks. AHP50 in control animals was 4.37 ± 0.66 ms and 3.62 ± 0.62 ms in CIBP rats, (P = 0.016). No differences in AHP50 were observed between groups at <1 week. AHP50 in control animals was 5.46 ± 0.56 ms and 5.32 ± 0.59 ms in CIBP rats (P = 0.069).
3.4. Changes in action potential configuration in subgroups of Aβ-fiber low-threshold mechanoreceptors
The Aβ-fiber LTM neurons showed significantly slower dynamics of APA, APdB, and APRT. These parameters were also compared for each subset of Aβ-fiber LTMs based on the 4 subsets described above: GF, RA, SA, and MS neurons. Figure 3 shows representative intracellular somatic APs for these subsets. Muscle spindle neurons were the most affected, followed by RA, SA, and GF neurons.
In MS neurons, the slower dynamics of the AP was the most obvious of these parameters studied. There was a reduced APA in CIBP rats at >2 weeks (control, 56.50 ± 8.02 mV vs CIBP, 46.62 ± 8.02, P = 0.04), a longer APdB in CIBP rats at 2 weeks (control, 1.21 ± 0.06 ms vs CIBP, 1.76 ± 0.39 ms; P = 0.007), and a longer APRT in CIBP rats at 2 weeks (control, 0.46 ± 0.10 ms vs CIBP, 0.75 ± 0.20 ms, P = 0.002).
In RA neurons, there was a reduced APA in CIBP rats at >2 weeks (control, 71.25 ± 4.99 mV vs CIBP, 57.90 ± 8.53, P = 0.03) and a longer APdB in CIBP rats at 2 weeks (control, 1.42 ± 0.18 ms vs CIBP, 1.62 ± 0.31 ms; P = 0.005) but no significance difference in the ARPT in CIBP rats at 2 weeks (control, 0.51 ± 0.100 ms vs CIBP, 0.87 ± 0.17 ms, P = 0.03).
In SA neurons, there was a reduced APA in CIBP rats at >2 weeks (control, 73.25 ± 7.81 mV vs CIBP, 59.06 ± 4.38, P = 0.03), a longer APdB in CIBP rats at 2 weeks (control, 1.49 ± 0.13 ms vs CIBP, 1.59 ± 0.25 ms; P = 0.11), and a longer APRT in CIBP rats at 2 weeks (control, 0.44 ± 0.13 ms vs CIBP, 0.89 ± 0.37 ms, P = 0.11).
In GF neurons, there was no difference in APA in CIBP rats at >2 weeks (control, 74.45 ± 5.31 mV vs CIBP, 61.43 ± 7.06, P = 0.06), no difference in APdB in CIBP rats at 2 weeks (control, 1.39 ± 0.11 ms vs CIBP, 1.62 ± 0.27 ms; P = 0.11), and no difference in APRT in CIBP rats at 2 weeks (control, 0.45 ± 0.02 ms vs CIBP, 0.81 ± 0.38 ms, P = 0.34).
3.5. Excitability of neurons
3.5.1. Excitability of the soma measured by responses to injection of depolarizing current
The AP responses to intracellular depolarizing current pulse injection were tested to determine whether there is a difference in soma excitability in CIBP model rats. Figure 4A illustrates the threshold currents that elicited APs in different groups of animals. At >2 weeks, the threshold of Aβ-fiber LTM neurons in CIBP rats showed a significant decrease; activation thresholds were 0.61 ± 0.10 nA (n = 18) in CIBP rats and 0.96 ± 0.08 nA (n = 18) in control rats vs (P = 0.008). There was no significant difference in Aβ-fiber LTM neurons at <1 week (0.93 ± 0.09 nA in control rats, n = 18 vs, 0.95 ± 0.09 nA in CIBP rats, n = 18; P = 0.776).
Figures 4B and C show the number of APs elicited with different current strengths; with a 1 nA, 20-millisecond current injection, the number of APs elicited in control rats at >2 weeks were 0.33 ± 0.14 (n = 18), whereas in CIBP rats, it was 1.83 ± 0.57 (n = 18) (P = 0.047). At <1 week, the number of APs in control rats was 0.44 ± 0.20 (n = 18), whereas in CIBP rats, it was 0.44 ± 0.17 (P = 0.461) (Fig. 3B). With a 2 nA, 100-millisecond current injection, the number of APs elicited in control rats at >2 weeks were 1.00 ± 0.38 (n = 18), whereas in CIBP rats, it was 3.11 ± 0.78 (n = 18) (P = 0.166). At <1 week, the number of APs in control rats was 1.05 ± 0.38 (n = 18), whereas in CIBP rats, it was 0.94 ± 0.37 (P = 0.833) (Fig. 3C). Figures 4D and E show typical discharge patterns of APs elicited in MS neurons by 2 nA current pulses with a duration of 100 milliseconds. In this figure, CIBP rats at <1 week showed 6 APs, whereas at >2 weeks, CIBP rats showed 8 APs with the same current pulse injection, which was the maximal number of APs observed using 2 nA current pluses. Table 1 shows all the comparison P values between the 4 groups.
3.6. Excitability of the receptive field measured by responses to application of von Frey filaments
The mechanical thresholds of DRG neurons tested with von Frey filaments during electrophysiology recording are shown in Figure 5. The mechanical thresholds of RA and SA neurons in control rats and CIBP rats were within the range 0.07 to 4 g and 0.02 to 4 g, respectively. At >2 weeks, the threshold of these LTM neurons in CIBP rats showed no significant difference; activation thresholds were 1.05 ± 1.37 g (n = 8) in CIBP rats vs 1.08 ± 1.33 g (n = 8) in control rats (P = 0.875). There was no significant difference in Aβ-fiber LTM neurons at <1 week (1.04 ± 1.38 g, (n = 8) in control rats vs 0.83 ± 1.44 g (n = 8) in CIBP rats (P = 0.798)). Table 1 shows the comparison P values between the 4 groups.
In our behavior studies, the mechanical withdrawal threshold response decreased with increasing duration of the CIBP animal model. Multiple mechanisms could account for these changes in nociception, including lowered activation threshold of nociceptive small Aδ-fiber neurons and C-fiber neurons. Studies from our laboratory and others have also suggested a possible role of Aβ-fiber LTMs in nociceptive mechanisms, such as allodynia and mechanical hypersensitivity.1,17,20,28,37,39 One possible explanation is that some Aβ-fiber LTM neurons may take up a new functional role in nociception and begin to convey signals along novel pathways leading to nociception during CIBP model development. We found that after 2 weeks, Aβ-fiber LTM neurons in CIBP model animals show differences in AP configurations and excitability, similar to what has been reported in an animal model of peripheral neuropathy.37,39 The correlation of the changes between the function of Aβ-fiber LTM neurons and behavioral nociception suggests the potential participation of Aβ-fiber LTM neurons in bone cancer pain generated in the present model.
Previous studies have indicated that the CIBP state includes aspects of nociceptive, neuropathic, and inflammatory pain.7,8 This prompted us to further question the role that Aβ-fiber LTM neurons fulfil in CIBP. In various animal models of chronic pain, there is evidence that inflammation and neuropathic etiologies affect distinct populations of DRG neurons. In peripheral models of inflammatory pain induced by injecting complete Freund adjuvant subcutaneously, only small Aδ-fiber neurons and C-fiber neurons undergo significant changes in electrophysiological properties.33 In hind leg joint inflammation models, indirect evidence suggests that large, nonnociceptive A-fiber neurons are unaffected.3,15
On the contrary, in peripheral neuropathic pain models, changes in large Aβ-fiber LTM neurons are commonly reported, such as in the complete sciatic nerve transection model,2 the partial sciatic nerve transection model,19 and the sciatic nerve cuff model.37,39 Although in some studies on neuropathic models changes in C-fiber neurons have been reported,1,17,21 such changes are less prominent than those in A-fiber neurons. Therefore, we propose that the electrophysiological changes in Aβ-fiber LTM neurons may be associated with a neuropathic etiology that follows model induction of animal models of CIBP. In fact, the observed changes in AP configuration in Aβ-fiber LTM neurons, including wider AP duration, and lower APA, reflect slowed dynamics of depolarization that are consistent with observations in models of peripheral neuropathy.37,39
It is not clear what is driving the changes in Aβ-fiber LTM neurons or how these neurons are affected. A possible explanation is that tumor growth induces peripheral nerve lesions on sensory neurons. Tumor cells invade the normal tissue, come into contact, compress, and injure the processes of sensory neurons including Aβ-fiber LTM neurons; Cain et al showed degeneration of nerve fibers in the skin in their murine model of cancer pain4 This implies that a component of CIBP is of neuropathic origin. A slowing of the dynamics of AP configuration in these neurons suggests a change in sodium currents in these neurons, either a functional change or a change in expression of channels.10,14 However, the specific ionic mechanisms remain unknown.
We did not find a change in the threshold of activation of the peripheral receptive field of these neurons as measured by responses to application of von Frey filaments in the CIBP rats. This is an important observation in view of earlier suggestion that CIBP is at least partially a neuropathic pain, which is characterized by tactile hypersensitivity. Specifically, we have reported that in a rat model of prostate CIBP, all 3 types of primary sensory neurons undergo increases in excitability corresponding to increases in CIBP behaviors.37
Given that behavioral reflex studies demonstrated a decrease in mechanical withdrawal threshold but the Aβ-fiber LTM neurons did not show a change in activation threshold of the peripheral receptive fields, we interpret these data to suggest that the behavioral change may be due to increased ectopic activity in Aβ-fiber LTM neurons, increased excitability of the soma of Aβ-fiber LTM neurons in a CIBP model as reported in a model of peripheral neuropathic pain37,39
Results from this study support the concept that nonnociceptive Aβ LTM neurons undergo changes in the model of CIBP. Importantly, there is a delayed onset of electrophysiological changes in these neurons, corresponding with changes in nociceptive behavioral scoring. The time course of development of the phenotypic changes in sensory neurons in these models may relate to the transient episodes of intense pain that characterize CIBP and the changes specifically in Aβ low-threshold LTM neurons might account for the relatively refractory nature of this type of pain, especially in more advanced stages.
The authors have no conflicts of interest to declare.
This study was supported by the Canadian Institutes of Health Research, Prostate Cancer Canada, and a postdoctoral fellowship for YFZ from the Michael G. DeGroote Institute for Pain Research and Care.
Author contributions: Y. F. Zhu did the electrophysiological experiments, analyzed the data, and performed statistical analyses. R. Ungard performed cell culture and induced animal models, N. Zacal performed radiography. Y. F. Zhu wrote the initial draft of the manuscript. All authors contributed to modification of the manuscript. J. D. Huizinga and J. L. Henry provided expertise and advice for the conception and design of the project. G. Singh and J. L. Henry worked on refining this draft. All authors have read and approved the final manuscript.
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Keywords:© 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain.
Cancer-induced bone pain; Electrophysiology; Sensory neurons; Rat model