Differences in electrophysiological properties of functionally identified nociceptive sensory neurons in an animal model of cancer-induced bone pain

Abstract

Background: Bone cancer pain is often severe, yet little is known about mechanisms generating this type of chronic pain. While previous studies have identified functional alterations in peripheral sensory neurons that correlate with bone tumours, none has provided direct evidence correlating behavioural nociceptive responses with properties of sensory neurons in an intact bone cancer model.

Results: In a rat model of prostate cancer-induced bone pain, we confirmed tactile hypersensitivity using the von Frey test. Subsequently, we recorded intracellularly from dorsal root ganglion neurons in vivo in anesthetized animals. Neurons remained connected to their peripheral receptive terminals and were classified on the basis of action potential properties, responses to dorsal root stimulation, and to mechanical stimulation of the respective peripheral receptive fields. Neurons included C-, Aδ-, and Aβ-fibre nociceptors, identified by their expression of substance P. We suggest that bone tumour may induce phenotypic changes in peripheral nociceptors and that these could contribute to bone cancer pain.

Conclusions: This work represents a significant technical and conceptual advance in the study of peripheral nociceptor functions in the development of cancer-induced bone pain. This is the first study to report that changes in sensitivity and excitability of dorsal root ganglion primary afferents directly correspond to mechanical allodynia and hyperalgesia behaviours following prostate cancer cell injection into the femur of rats. Furthermore, our unique combination of techniques has allowed us to follow, in a single neuron, mechanical pain-related behaviours, electrophysiological changes in action potential properties, and dorsal root substance P expression. These data provide a more complete understanding of this unique pain state at the cellular level that may allow for future development of mechanism-based treatments for cancer-induced bone pain.

Keywords: Bone cancer; behaviour; dorsal root ganglion; electrophysiology; pain; primary afferent; prostate cancer.

Figure 1.

Model timeline, recording sites, and action potential parameters. (a) Illustrative timeline of procedures involving animals. Model induction of MLL cancer cell injected animals and control animals (PBS) occurred on experimental Day 0. Behavioural and electrophysiological procedures were performed between experimental Days 7 and 14. (b) Illustration of stimulation and recording sites for electrophysiological procedures. Recording occurred intracellularly for all neurons. Stimulation was performed at the receptive field by mechanical stimulation with von Frey filaments and also electrically at the dorsal root and the DRG soma. (c) A representative intracellular somatic action potential of a C-fibre neuron evoked by electrical stimulation of the dorsal root demonstrating the electrophysiological parameters measured, including: (1) conduction velocity; (2) resting membrane potential; (3) action potential amplitude; (4) action potential duration at base; (5) action potential rise time; (6) action potential fall time; (7) after hyperpolarization amplitude below Vm; and (8) after hyperpolarization duration to 50% recovery. MLL: MAT-LyLu rat prostate cancer cell line; PBS: phosphate buffer saline; DRG: dorsal root ganglia.

Figure 2.

Model confirmation: structural and histological differences. (a) Representative H&E stained 4 µm thick sections of the ipsilateral distal epiphysis of femurs from control (left) and cancer rats (right). Control bone appears healthy and free of indications of pathology induced by sham injections. In contrast, cancer bone features extensive invasion of cancer cells into areas of the bone marrow and mineralized bone; surfaces of trabecular bone appear ragged and eroded (indicated by arrow). B: mineralized bone; M: marrow; T: tumour cells; G: growth plate. Scale bar represents 300 µm. (b) Representative radiographs of ipsilateral hind limbs of control (left) and cancer rats (right). Control bone appears pathology-free, while cancer bone displays structural modifications and lytic lesions at the injection site in the distal femur epiphysis. All images were taken following fixation of samples from animals 7 to 14 days after model induction. (c) Comparison of 50% withdrawal thresholds between control and cancer groups. Withdrawal threshold to mechanical stimulation of the plantar surface of the ipsilateral hind paw with von Frey filaments was recorded immediately prior to the acute electrophysiological experiment in control (n = 15) and cancer (n = 15) animals. Data are shown as mean ± SEM. ***p < 0.001. H&E: hematoxylin and eosin;

Figure 3.

DRG classification and substance P colocalization. Examples of an evoked AP for each nociceptive neuron type (first column) and micrographs showing representative neurobiotin labelled DRG neurons in sections colabelled for SP. Each micrograph row consists of three images: left panel illustrates cells filled with neurobiotin in red (Texas Red); middle panel illustrates cells immunoreactive to SP in green (AlexaFluor 488); and right panel displays the merged images to show signal colocalization (yellow). Dye-labelled cells are indicated by arrowheads. Scale bar represents 50 µm. DRG: dorsal root ganglia; AP: action potential; SP: substance P.

Figure 4.

Action potential properties of DRG neurons in control and cancer rats. Scatter plots of properties of evoked APs in individual neurons. The median is superimposed as a horizontal line. Each column represents a specific neuron type: C-fibre nociceptive neurons (a), Aδ-fibre nociceptive neurons (b), and Aβ-fibre nociceptive neurons (c). The recorded variable panels are in rows, as follows: (a) CV; (b) resting membrane potential (Vm); (c) APA; (d) APdB; (e) APRT; (f) APFT; (g) AHPA; and (h) after hyperpolarization duration to 50% recovery (AHP50). Asterisks indicate differences between control and cancer rats: *p < 0.05, **p < 0.01. DRG: dorsal root ganglia; AP: action potential; APA: action potential amplitude; APdB: action potential duration at base; APRT: action potential rise time; APFT: action potential fall time; AHPA: after hyperpolarization amplitude below Vm; AHP50: after hyperpolarization duration to 50% recovery; CV = conduction velocity.

Figure 5.

Comparison of the mechanical response threshold of DRG nociceptive neurons to application of von Frey filaments to the peripheral receptive fields of control and cancer rats. The upper row shows the distribution of the mechanical activation thresholds of the individual neurons. Columns indicate distribution of the number of neurons activated by each mechanical stimulus (in grams) in C- (a), Aδ- (b) and Aβ-fibre (c) neurons in the cancer animals (filled bars) compared with the control animals (open bars). The lower row shows the mean mechanical response thresholds of neurons of each classification in control and cancer rats. *p < 0.05, **p < 0.01. DRG: dorsal root ganglia.

Figure 6.

Comparison of the activation threshold of DRG nociceptive neurons in response to intracellular current injection, between control and cancer rats. (A) The current threshold is the minimum current required to evoke an AP by intracellular current injection to the soma of DRG neurons. Excitability of the DRG somata was significantly higher in cancer rats relative to control rats, as indicated by lower mean current activation threshold in all types of nociceptive fibres: C-fibre neurons (a), Aδ-fibre neurons (B) and Aβ-fibre neurons (c). (b) A comparison the repetitive discharge characteristics of DRG cells produced by intracellular current injection at the soma. Columns indicate the number of APs evoked by intracellular injection of different magnitudes (Left = 1.5 nA, 100 ms. Middle = 2 nA, 100 ms. Right = 2.5 nA, 100 ms) of depolarizing current in C-fibres (a–c), Aδ-fibres (d–f) and Aβ-fibres (g–i). Most neuron types in cancer rats produced a greater number of APs in response to depolarizing current than control rats. (C) Representative examples of raw recordings to demonstrate the greater number of APs evoked by intracellular current injection in cancer (d–f) than in control rats (a–c) in all neuron fibre types. APs in these examples were evoked by current pulses of 2 nA, 100 ms. *p < 0.05, **p < 0.01. DRG = dorsal root ganglia; AP: action potential;

Figure 7.

Comparison of current activation threshold of DRG nociceptive neurons in response to stimulation of the dorsal roots, between control and cancer rats. (A) Current activation threshold of DRG nociceptive neurons in response to stimulation of the dorsal roots. Current threshold was defined using the rheobase/chronaxie curve (threshold duration), which was determined as the minimum stimulus current to the dorsal root sufficient to evoke an AP with pulses of 0.02 ms, 0.1 ms, 0.2 ms, 0.4 ms, and 0.6 ms duration. C-fibre neurons (a), Aδ-fibre neurons (b), and Aβ-fibre neurons (c) show a lower rheobase in cancer rats compared with controls. *p < 0.05, **p < 0.01; (B) Representative recordings of the repetitive discharge characteristics of DRG neurons evoked by dorsal root stimulation by current pulses of 1 mA, 0.4 m. DRG = dorsal root ganglia; AP: action potential.