Rat model of cancer-induced bone pain: changes in nonnociceptive sensory neurons in vivo

Abstract

Introduction: Clinical data on cancer-induced bone pain (CIBP) suggest extensive changes in sensory function. In a previous investigation of an animal model of CIBP, we have observed that changes in intrinsic membrane properties and excitability of dorsal root ganglion (DRG) nociceptive neurons correspond to mechanical allodynia and hyperalgesia.

Objectives: To investigate the mechanisms underlying changes in nonnociceptive sensory neurons in this model, we have compared the electrophysiological properties of primary nonnociceptive sensory neurons at <1 and >2 weeks after CIBP model induction with properties in sham control animals.

Methods: Copenhagen rats were injected with 10⁶ MAT-LyLu rat prostate cancer cells into the distal femur epiphysis to generate a model of CIBP. After von Frey tactile measurement of mechanical withdrawal thresholds, the animals were prepared for acute electrophysiological recordings of mechanically sensitive neurons in the DRG in vivo.

Results: The mechanical withdrawal threshold progressively decreased in CIBP model rats. At <1 week after model induction, there were no changes observed in nonnociceptive Aβ-fiber DRG neurons between CIBP model rats and sham rats. However, at >2 weeks, the Aβ-fiber low-threshold mechanoreceptors (LTMs) in CIBP model rats exhibited a slowing of the dynamics of action potential (AP) genesis, including wider AP duration and lower AP amplitude compared with sham rats. Furthermore, enhanced excitability of Aβ-fiber LTM neurons was observed as an excitatory discharge in response to intracellular injection of depolarizing current into the soma.

Conclusion: After induction of the CIBP model, Aβ-fiber LTMs at >2 weeks but not <1 week had undergone changes in electrophysiological properties. Importantly, changes observed are consistent with observations in models of peripheral neuropathy. Thus, Aβ-fiber nonnociceptive primary sensory neurons might be involved in the peripheral sensitization and tumor-induced tactile hypersensitivity in CIBP.

Keywords: Cancer-induced bone pain; Electrophysiology; Rat model; Sensory neurons.

Figure 1.

Bone tumors induce structural changes and nociception. (A) Comparison of 50% withdrawal threshold between control and cancer rats. Withdrawal threshold in response to mechanical stimulation of the plantar surface of the ipsilateral hind paw with von Frey filaments was recorded on the same day immediately before the acute electrophysiological experiment in control (<1 week, n = 6; >2 weeks, n = 6) and cancer (<1 week, n = 6; >2 weeks, n = 6) animals. The significant differences between each group animals are shown in Table 1. (B) Representative radiographs of rat ipsilateral hind legs. Radiographs of the ipsilateral hind leg from control (a, 6 days; c, 18 days) and MLL cell–injected (b, 6 days; d, 18 days) rats displaying structural changes following model induction. *P < 0.05. The absence of an asterisk indicates the lack of a statistically significant difference. Mann–Whitney U test.

Figure 2.

Changes of action potential configuration of Aβ-fiber low-threshold mechanoreceptor neurons in control and cancer rats. Scatter plots show the distribution of the variables with the median (horizontal line) superimposed in nonnociceptive Aβ-fiber low-threshold mechanoreceptor neurons. Panels are as follows: (A) conduction velocity (CV); (B) resting membrane potential (Vm); (C) action potential (AP) amplitude (APA); (D) AP duration at base (APdB); (E) AP rise time (APRT); (F) AP fall time (APFT); (G) afterhyperpolarization amplitude below Vm (AHPA); and (H) afterhyperpolarization duration to 50% recovery (AHP50). The significant differences between each group animals are shown in Table 1. An asterisk in the figure indicates the significant differences between cancer (<1 week) and cancer (>2 weeks). *P < 0.05, ***P < 0.001. The absence of an asterisk indicates the lack of a statistically significant difference. Mann–Whitney U test.

Figure 3.

Examples of action potentials recorded from subtypes of Aβ-fiber low-threshold mechanoreceptor neurons. Somatic evoked and recorded intracellularly selected to represent the action potential duration values for each of the different groups of neuron in control (left) and cancer-induced bone pain (right) animals. (A) Muscle spindle neurons; (B) rapidly adapting neurons; (C) slowly adapting neurons; (D) GF neurons.

Figure 4.

Decreased excitability threshold in nonnociceptive Aβ-fiber low-threshold mechanoreceptor neurons. (A) The current threshold was defined as the minimum current required to evoke an action potential (AP) by intracellular current injection (20 ms). Excitability of the dorsal root ganglion somata was significantly increased at >2 weeks cancer-induced bone pain rats, as indicated by the decreased activation threshold in nonnociceptive Aβ-fiber neurons (A). (B and C) A comparison of the repetitive discharge characteristics of dorsal root ganglion cells produced by intracellular current injection. Columns indicate the number of APs evoked by different magnitudes of intracellular depolarizing current injection in nonnociceptive Aβ-fiber low-threshold mechanoreceptor neurons. (B) 1 nA, 100 milliseconds; (C) 2 nA, 100 milliseconds; (D) Representative examples of raw recordings to demonstrate the greater number of APs evoked by intracellular current injection in muscle spindle neurons at >2 weeks cancer-induced bone pain rat (E) vs at >2 weeks control rats (D). The significant differences between each group animals are shown in Table 1. An asterisk in the figure indicates the significant differences between cancer (<1 week) and cancer (>2 weeks). *P < 0.05, **P < 0.01. The absence of an asterisk indicates the lack of a statistically significant difference. Mann–Whitney U test.

Figure 5.

The mechanical threshold in nonnociceptive Aβ-fiber low-threshold mechanoreceptor (LTM) neurons. The mechanical response threshold in nonnociceptive Aβ-fiber LTM neurons was measured by application of von Frey filaments to the peripheral receptive fields. Scatter plots show the threshold distribution of the variables with the median (horizontal line) superimposed in nonnociceptive Aβ-fiber LTM neurons including slowly adapting and rapidly adapting neurons. There is no significant difference in all groups. The differences between each group animals are shown in Table 1. An asterisk in the figure indicates the significant differences between cancer (<1 week) and cancer (>2 weeks). *P < 0.05, **P < 0.01. The absence of an asterisk indicates the lack of a statistically significant difference. Mann–Whitney U test.