Free
Pain Medicine  |   June 2014
Enhanced Excitability of Primary Sensory Neurons and Altered Gene Expression of Neuronal Ion Channels in Dorsal Root Ganglion in Paclitaxel-induced Peripheral Neuropathy
Author Affiliations & Notes
  • Haijun Zhang, M.D.
    From the Department of Pain Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas. Current address: Department of Anesthesiology, The University of Texas Medical School at Houston, Houston, Texas (H.Z.).
  • Patrick M. Dougherty, Ph.D.
    From the Department of Pain Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas. Current address: Department of Anesthesiology, The University of Texas Medical School at Houston, Houston, Texas (H.Z.).
  • Submitted for publication April 1, 2013. Accepted for publication January 8, 2014.
    Submitted for publication April 1, 2013. Accepted for publication January 8, 2014.×
  • Correspondence: Address correspondence to Dr. Zhang or Dr. Dougherty: Department of Pain Medicine, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Y6.5710, U110, Houston, Texas 77030. haijun.hj@gmail.com or pdougherty@mdanderson.org. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
Article Information
Pain Medicine / Basic Science / Central and Peripheral Nervous Systems / Pain Medicine / Practice Management
Pain Medicine   |   June 2014
Enhanced Excitability of Primary Sensory Neurons and Altered Gene Expression of Neuronal Ion Channels in Dorsal Root Ganglion in Paclitaxel-induced Peripheral Neuropathy
Anesthesiology 06 2014, Vol.120, 1463-1475. doi:10.1097/ALN.0000000000000176
Anesthesiology 06 2014, Vol.120, 1463-1475. doi:10.1097/ALN.0000000000000176
Abstract

Background:: The mechanism of chemotherapy-induced peripheral neuropathy after paclitaxel treatment is not well understood. Given the poor penetration of paclitaxel into central nervous system, peripheral nervous system is most at risk.

Methods:: Intrinsic membrane properties of dorsal root ganglion neurons were studied by intracellular recordings. Multiple-gene real-time polymerase chain reaction array was used to investigate gene expression of dorsal root ganglion neuronal ion channels.

Results:: Paclitaxel increased the incidence of spontaneous activity from 4.8 to 27.1% in large-sized and from 0 to 33.3% in medium-sized neurons. Paclitaxel decreased the rheobase (nA) from 1.6 ± 0.1 to 0.8 ± 0.1 in large-sized, from 1.5 ± 0.2 to 0.6 ± 0.1 in medium-sized, and from 1.6 ± 0.2 to 1.0 ± 0.1 in small-sized neurons. After paclitaxel treatment, other characteristics of membrane properties in each group remained the same except that Aδ neurons showed shorter action potential fall time (ms) (1.0 ± 0.2, n = 10 vs. 1.8 ± 0.3, n = 9, paclitaxel vs. vehicle). Meanwhile, real-time polymerase chain reaction array revealed an alteration in expression of some neuronal ion channel genes including up-regulation of hyperpolarization-activated cyclic nucleotide-gated channel 1 (fold change 1.76 ± 0.06) and Nav1.7 (1.26 ± 0.02) and down-regulation of Kir channels (Kir1.1, 0.73 ± 0.05, Kir3.4, 0.66 ± 0.06) in paclitaxel-treated animals.

Conclusion:: The increased neuronal excitability and the changes in gene expression of some neuronal ion channels in dorsal root ganglion may provide insight into the molecular and cellular basis of paclitaxel-induced neuropathy, which may lead to novel therapeutic strategies.

In rats, the spontaneous activity of medium and large neurons was increased after paclitaxel treatment. Gene array studies demonstrated that the expression of several ion channels was altered by paclitaxel treatment potentially providing an explanation for the electrophysiological changes.

What We Already Know about This Topic
  • Chemotherapy-induced peripheral neuropathy is a major clinical problem caused by agents such as paclitaxel

What This Article Tells Us That Is New
  • In rats, the spontaneous activity of medium and large neurons was increased after paclitaxel treatment

  • Gene array studies demonstrated that the expression of several ion channels was altered by paclitaxel treatment potentially providing an explanation for the electrophysiological changes

CHEMOTHERAPY-INDUCED peripheral neuropathy (CIPN) is a very common side effect of paclitaxel (Taxol®; Bristol-Myers Squibb, New York, NY), a widely used chemotherapeutic agent, which severely limits its anticancer application.1  Although the mechanisms are not well understood, the symptoms of paclitaxel CIPN include prominent numbness, tingling, and occasionally shooting/burning pain,2–4  suggesting a polyneuropathy affecting both myelinated (Aβ and Aδ) and, less frequently, unmyelinated (C) fibers.2  Only a very low level of paclitaxel was detected in spinal cord and other parts of central nervous system after repeated administration, but a high concentration of paclitaxel was found in dorsal root ganglion (DRG),5  possibly due to the dense vascularization in DRG by highly permeable capillaries.6  The accumulation of paclitaxel in the peripheral nervous system imposes a greater risk for damage to these tissues.
Primary sensory neurons in DRG receive signals generated from peripheral nerve endings, integrate, and transfer these signals to spinal cord. Because DRG neurons play a critical role in transducing sensory signals including pain, numerous studies have focused on its involvement in the development of acute and chronic pain in various pathological conditions (for review, see the study by Gold and Gebhart7 ). Extensive studies have revealed that the intrinsic membrane properties of DRG neuronal soma, including both myelinated and unmyelinated cells, are significantly altered after peripheral nerve injury or inflammation and play important roles in the development of spontaneous pain, hyperalgesia, and allodynia. For example, an increased incidence of ectopic discharges originating from the soma of different populations of DRG neurons are commonly found after peripheral nerve injury,8–11  direct compression of the ganglion,12–20  and inflammation.21,22  A substantial number of DRG neurons showed decreased rheobase and increased responses to either electrical or chemical stimulation applied to the soma.14,15,17,18,20,22–24  More importantly, blocking such enhanced activities of DRG neurons attenuate chronic pain behavior including spontaneous pain and tactile allodynia.22,25–30 
It is unknown whether the intrinsic membrane properties of primary sensory neurons in DRG are altered after exposure to paclitaxel chemotherapy. This gap in knowledge was addressed in the current study by using in vitro intracellular recordings from the somata of both myelinated and unmyelinated neurons in an intact DRG preparation using rats that had received paclitaxel or vehicle treatment. In addition, multiple-gene real-time polymerase chain reaction (rtPCR) array for neuronal ion channels was conducted to examine the changes in gene expression of neuronal ion channels in DRG from animals with paclitaxel CIPN.
Materials and Methods
Animals
Adult male Sprague–Dawley rats (8 to 12 weeks; Harlan, Houston, TX) housed in a 12-h light/dark cycle with free access to food and water were used in all experiments. The studies were approved by the Institutional Animal Care and Use Committee at The University of Texas MD Anderson Cancer Center and were performed in accordance with the National Institutes of Health Guidelines for Use and Care of Laboratory Animals.
Paclitaxel CIPN Model
Animals were treated with paclitaxel as previously described.31–35  Paclitaxel (TEVA Pharmaceuticals, Inc., North Wales, PA) solution was given intraperitoneally at a dosage of 2 mg/kg in 0.9% saline every other day for a total of four injections (day 1, 3, 5, and 7). Control animals received an equivalent volume of vehicle (cremophor EL:ethanol, 1:1). Mechanical hypersensitivity is induced with this treatment protocol by day 7 and lasts at least several weeks (data not shown).32,33,35,36 
In Vitro Intracellular Recording of Intact DRG Neurons
Intracellular recordings were performed after mechanical hypersensitivity has established (usually on days 8 to 14 after chemotherapy).14  The L4 and L5 ganglia with attached sciatic nerve were removed and then incubated in 1 mg/ml Collagenase P (Roche, South San Francisco, CA) and 0.4 mg/ml Protease Type XIV (Sigma, St. Louis, MO) for 15 min at 37°C in oxygenated artificial cerebrospinal fluid. Artificial cerebrospinal fluid contains: 130 mM of NaCl, 24 mM of NaHCO3, 3.5 mM of KCl, 1.25 mM of NaH2PO4, 1.2 mM of MgCl2, 1.2 mM of CaCl2, and 10 mM of dextrose (pH = 7.3). After incubation, the ganglia were transferred to a recording chamber and superfused continuously with artificial cerebrospinal fluid bubbled with 95% O2 plus 5% CO2 at 37°C. The peripheral nerve was stimulated by a suction electrode. All chemicals were purchased from Sigma except where noted.
Intracellular recordings were obtained under current clamp (MultiClamp 700A; Axon Instruments, Sunnyvale, CA) by a glass sharp electrode (Sutter Instruments, Novato, CA) with impedances of 60 to 80 MΩ when filled with 3 M KCl. Electrophysiological signals were filtered at 5 kHz, digitized at 20 kHz via a Digidata 1320A interface, and analyzed offline with pClamp 10.0 software (Axon Instruments). All neuron accepted have resting membrane potentials more negative than −45 mV. Baseline activity was first monitored for 2 min without the delivery of any external stimuli to detect spontaneous activity (SA). Cells showing high levels of SA that would confound interpretation of responses to external stimuli were not further characterized. If there was either no or only low levels of SA, a series of electric stimuli (1 to 5 mA, duration 0.05 ms for A-fiber and 0.5 ms for C-fiber) was delivered to the nerve through the suction electrode to induce action potentials (APs) from the soma. The axonal conduction velocity (CV) was calculated by dividing the latency of the evoked AP into the distance between the stimulating electrode and the center of the ganglion (usually 12 to 15 mm). After the completion of peripheral nerve stimulation, the recorded cell was depolarized by a series of 40-ms injecting currents from −0.05 to 3 nA in increments of 0.025 nA to induce one or more APs. The current threshold (rheobase) was defined as the minimal current that evoked an AP through intracellular sharp electrode. The input resistance of each cell was measured based on the steady-state voltage changes at the end of the application of a series of hyperpolarizing/depolarizing currents (−50 pA to 50 pA, ΔI = 25 pA, duration 40 ms). Repetitive discharges of each neuron were measured by counting the spikes evoked by injecting a 1-s depolarizing current at two times the threshold.
Neurons with CV were divided into three groups as previously reported14,15,37,38 : C-neurons with CV less than 1.3 m/s, Aδ neurons with CV 1.3 to 12 m/s, and Aα/β neurons with CV greater than 12 m/s. Additional characteristics of the AP including amplitude, duration, rise time, and fall time, amplitude and 50% duration (measured at an amplitude half-way between) of afterhyperpolarization were measured.
Multiple-gene rtPCR Neuronal Ion Channels Array
Dorsal root ganglion tissues used for rtPCR array were collected on day 7 after paclitaxel or vehicle treatment (12 rats were treated with paclitaxel and 4 rats were treated with vehicle) as previously described.35  Animals were perfused transcardially with cold phosphate-buffered saline before tissue collection. L4 and L5 DRGs from both sides were collected (four ganglia per rat). DRG tissues from animals with the same treatment were pooled together (four rats per group) during tissue processing. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and further purified using the RNeasey Mini Kit (Qiagen, Valencia, CA). The concentration and purity of RNA were detected by measuring the absorbance at 260 and 280 nm using a spectrophotometer (NanoDrop 2000; Thermo Scientific, Odessa, TX). Reverse transcription of complementary DNA from 2 μg of total RNA was performed in a thermal cycler (GeneAmp Polymerase Chain Reaction System 9700; Life Technologies, Carlsbad, CA) by RT2 First Strand Kit (Qiagen) according to the manufacturer’s instruction (RT2 Profiler PCR Array Handbook; Qiagen). The proprietary procedure effectively eliminated possible contaminating genomic DNA from RNA samples before reverse transcription. The amplification of complementary DNA was performed by using RT2 SYBR Green ROX qPCR Mastermix (Cat. no. 330521; Qiagen). The neuronal ion channels array was performed using rat 384-well RT2 Profiler PCR Array Kit (Cat. no. 330231, PARN-036ZA; Qiagen) on a 7900 Sequence Detection system (Life Technologies). Amplification steps consisted of one cycle of 50°C for 2 min plus 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 1 min, and one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s as a dissociation stage. The gene expression data were uploaded and analyzed using the online polymerase chain reaction array data analysis tool (version 3.0; Qiagen).*01  The threshold cycle (Ct; the number of cycles to reach the threshold of detection) was determined for each gene, and the relative expression level of each gene was calculated using the following formula39 : relative expression of messenger RNA = 2−Δ[(Ct sample − Ct control)paclitaxel−(Ct sample − Ct control)vehicle], where Ctsample is the Ct for the target gene and Ctcontrol is the average Ct for several housekeeping genes including β-actin, β-2 microglobulin, hypoxanthine phosphoribosyltransferase 1, lactate dehydrogenase A, and ribosomal protein P1. Positive and negative control wells were used to access reverse transcription efficiency, presence of genomic DNA, and positive rtPCR reactions according to the manufacturer’s instruction. To minimize the variance caused by sample handling and processing, all samples were processed by the same experimenter with the same experiment protocol, all complementary DNA were synthesized and amplified on the same plate at the same time.
Statistical Analysis
All results are presented as means ± SEM (which only reaches a 68% CI) and analyzed with Prism GraphPad 6.0 (GraphPad Software, Inc., La Jolla, CA) or Sigma Plot 11.0 (Systat Software, Inc., San Jose, CA). For electrophysiological results, differences between means were tested for significance using one-way ANOVA with Turkey multiple comparison or Mann–Whitney U test (comparison was made across all six groups within the same family). The difference in incidence was analyzed with chi-square test. For rtPCR results, the difference between paclitaxel and vehicle treatment was expressed by fold changes (comparing with vehicle group) and analyzed using one-sample t test with the hypothesis that the actual fold change of each ion channel after paclitaxel treatment was not different from the hypothesized value of 1.0. The Bonferroni correction was later applied in the multiple testing. The difference is considered as statistically significant with an α value of P less than 0.05, and the term “significant” means statistically significant unless otherwise noted. The investigators were not blinded with the groups.
Results
One hundred eighty DRG neurons, including 111 from paclitaxel-treated rats (23 rats) and 69 from vehicle-treated rats (11 rats), recorded with sharp electrode were included for further analysis. The neurons were first grouped according to their size as small-sized (≤30 um), medium-sized (31 to 44 um), and large-sized (≥45 um) cells. It has been shown by different studies that the classification of DRG neurons by the diameter of the soma yielded virtually the same groups of neurons as a classification by their axonal CV.14,18,40  Seventy large-sized, 21 medium-sized, and 20 small-sized neurons were from paclitaxel-treated animals. Forty-two large-sized, 14 medium-sized, and 13 small-sized neurons were from vehicle-treated animals.
Increased Excitability of DRG Neurons after Paclitaxel Chemotherapy
Spontaneous Activity.
Spontaneous activity was observed in 26 neurons from paclitaxel-treated animals (26 of 111, 23.4%) but in only two neurons from vehicle-treated animals (2 of 69, 2.9%). SA was recorded in 19 of 70 (27.1%) large-sized neurons and in 7 of 21 (33.3%) medium-sized neurons in paclitaxel-treated animals. Two of 42 (4.8%) large neurons from vehicle-treated animals showed SA. No SA was observed in small neurons from either group. The incidence of SA was significantly increased after paclitaxel treatment (fig. 1A).
Fig. 1.
Increased incidence of spontaneous activity (SA) in dorsal root ganglion neurons from animals with paclitaxel neuropathy. (A) Paclitaxel chemotherapy induced a significant increase of SA from dorsal root ganglion myelinated neurons including large- (P = 0.003) and medium-sized (P = 0.027) neurons. *P < 0.05, **P < 0.01, chi-square test. (B) An example of irregular SA recorded from a medium-sized neuron, noticing the subthreshold membrane potential oscillations between each discharge. (C) Continuous discharges were recorded from a spontaneously active large neuron. (D) Spontaneous bursting discharges were recorded from a large neuron after paclitaxel chemotherapy. Subthreshold oscillations (arrows) were obvious between each episode of bursting discharges.
Increased incidence of spontaneous activity (SA) in dorsal root ganglion neurons from animals with paclitaxel neuropathy. (A) Paclitaxel chemotherapy induced a significant increase of SA from dorsal root ganglion myelinated neurons including large- (P = 0.003) and medium-sized (P = 0.027) neurons. *P < 0.05, **P < 0.01, chi-square test. (B) An example of irregular SA recorded from a medium-sized neuron, noticing the subthreshold membrane potential oscillations between each discharge. (C) Continuous discharges were recorded from a spontaneously active large neuron. (D) Spontaneous bursting discharges were recorded from a large neuron after paclitaxel chemotherapy. Subthreshold oscillations (arrows) were obvious between each episode of bursting discharges.
Fig. 1.
Increased incidence of spontaneous activity (SA) in dorsal root ganglion neurons from animals with paclitaxel neuropathy. (A) Paclitaxel chemotherapy induced a significant increase of SA from dorsal root ganglion myelinated neurons including large- (P = 0.003) and medium-sized (P = 0.027) neurons. *P < 0.05, **P < 0.01, chi-square test. (B) An example of irregular SA recorded from a medium-sized neuron, noticing the subthreshold membrane potential oscillations between each discharge. (C) Continuous discharges were recorded from a spontaneously active large neuron. (D) Spontaneous bursting discharges were recorded from a large neuron after paclitaxel chemotherapy. Subthreshold oscillations (arrows) were obvious between each episode of bursting discharges.
×
The patterns of SA were classified as irregular, regular-continuous, and bursting as previously described.8,15,17  Of the 28 spontaneously discharging neurons (26 from paclitaxel- and 2 from vehicle-treated groups), 15 ells displayed irregular discharges, including 10 large- and 5 medium-sized neurons from paclitaxel-treated animals. Consistent with the previous reports on the study of peripheral nerve injury8  and compressed ganglion,15,17  irregular SA was the most common pattern observed (53.6%) in paclitaxel CIPN. In this category, one cell displayed a mixture of doublet and single spikes (data not shown), whereas all others showed irregular interspike intervals (fig. 1B). Nine cells, including six large- and one medium-sized neurons from paclitaxel- and two large neurons from vehicle-treated animals, showed regular and continuous discharges which was the second most common discharge pattern in the study (32.1%) (fig. 1C). Bursting discharge, the least common discharge pattern (14.3%), was observed in four cells including three large- and one medium-sized neurons from paclitaxel-treated animals (fig. 1D).
Resting Membrane Potential.
Both large- and medium-sized neurons with SA were depolarized compared with the quiescent cells (neurons that did not show SA) in the same size group in paclitaxel-treated animals (−51.6 ± 1.7, n = 19 vs. −57.3 ± 0.9, n = 51, P = 0.009 for large cells; −46.3 ± 0.9, n = 7 vs. −57.4 ± 1.6, n = 14, P = 0.003 for medium-sized cells, one-way ANOVA). For quiescent cells, AP was first induced by intracellular current injection to verify the ability of the recorded neuron to fire (fig. 2A). There were no statistically significant differences observed in resting membrane potential for large-sized, medium-sized, or small-sized neurons after paclitaxel treatment (fig. 2B and table 1).
Table 1.
Electrophysiological Properties of DRG Neurons with Different Sizes
Electrophysiological Properties of DRG Neurons with Different Sizes×
Electrophysiological Properties of DRG Neurons with Different Sizes
Table 1.
Electrophysiological Properties of DRG Neurons with Different Sizes
Electrophysiological Properties of DRG Neurons with Different Sizes×
×
Fig. 2.
Dorsal root ganglion neurons showed increased excitability after paclitaxel chemotherapy. (A) Sample recordings of large neurons from paclitaxel- and vehicle-treated animals. The input resistance of each cell was measured based on the steady-state voltage changes at the end of the application of a series of hyperpolarizing/depolarizing currents as indicated by arrowhead. Comparison of electrophysiological properties of dorsal root ganglion neurons with different size, including resting membrane potential (RMP) (B), current threshold (C), number of action potentials (APs) induced by a 1-s two times current threshold intracellular depolarizing current (D) and input resistance (E). *P < 0.001, **P < 0.0001, one-way ANOVA.
Dorsal root ganglion neurons showed increased excitability after paclitaxel chemotherapy. (A) Sample recordings of large neurons from paclitaxel- and vehicle-treated animals. The input resistance of each cell was measured based on the steady-state voltage changes at the end of the application of a series of hyperpolarizing/depolarizing currents as indicated by arrowhead. Comparison of electrophysiological properties of dorsal root ganglion neurons with different size, including resting membrane potential (RMP) (B), current threshold (C), number of action potentials (APs) induced by a 1-s two times current threshold intracellular depolarizing current (D) and input resistance (E). *P < 0.001, **P < 0.0001, one-way ANOVA.
Fig. 2.
Dorsal root ganglion neurons showed increased excitability after paclitaxel chemotherapy. (A) Sample recordings of large neurons from paclitaxel- and vehicle-treated animals. The input resistance of each cell was measured based on the steady-state voltage changes at the end of the application of a series of hyperpolarizing/depolarizing currents as indicated by arrowhead. Comparison of electrophysiological properties of dorsal root ganglion neurons with different size, including resting membrane potential (RMP) (B), current threshold (C), number of action potentials (APs) induced by a 1-s two times current threshold intracellular depolarizing current (D) and input resistance (E). *P < 0.001, **P < 0.0001, one-way ANOVA.
×
Increased Excitability of DRG Neurons after Paclitaxel Chemotherapy.
The excitability of each quiescent cell was measured by current threshold (fig. 2A). The current threshold in each category, including large- (P < 0.0001, one-way ANOVA) and medium-sized (P < 0.001) neurons, was significantly lower in paclitaxel-treated animals compared with that in vehicle-treated animals (fig. 2C and table 1). No difference was observed in small cells (P = 0.067). DRG neurons often showed increased number of APs evoked by either peripheral nerve stimulation15  or a long depolarizing current injected intracellularly14,23  after injury or inflammation. In the current study, the number of APs evoked by a 1-s current injection of two times current threshold through intracellular electrode was compared. Only cells that showed more than one AP to the stimulation were included in the analysis. Although there was no statistically significant difference in the number of evoked AP for any of the groups after paclitaxel treatment, medium-sized neurons tended to have more APs in response to the depolarizing current in paclitaxel-treated group (fig. 2D and table 1). This lack of difference is possibly due to the small number of samples collected. No significant difference was found in input resistance for any of the groups after paclitaxel treatment (fig. 2E).
Characteristics of DRG Neuron APs Evoked by Peripheral Nerve Stimulation.
An AP was evoked by delivering electric stimulation through peripheral nerve in 55 (including seven SA cells) of 111 cells (49.5%) from paclitaxel-treated animals and in 35 (including one SA cell) of 69 cells (50.7%) from vehicle-treated animals (fig. 3A). The proportion of neurons showing nerve stimulation-evoked AP was similar in paclitaxel- and vehicle-treated groups, which was consistent with the report that roughly half of the L4/L5 DRG neurons have axons in the sciatic nerve in rats.41  CV (fig. 3B) and some characteristics of evoked AP including amplitude (fig. 3C), duration (fig. 3D), rise time (fig. 3E), fall time (fig. 3F), afterhyperpolarization amplitude (fig. 3G) and 50% duration (fig. 3H) were measured from these neurons and listed in table 2. No significant difference was observed in CV or any of the characteristics of evoked AP between paclitaxel- and vehicle-treated groups for each size category, except that Aδ neurons showed shorter AP fall time after paclitaxel treatment (fig. 3F; P = 0.035, one-way ANOVA).
Table 2.
Intrinsic Membrane Properties of DRG Neurons with CV
Intrinsic Membrane Properties of DRG Neurons with CV×
Intrinsic Membrane Properties of DRG Neurons with CV
Table 2.
Intrinsic Membrane Properties of DRG Neurons with CV
Intrinsic Membrane Properties of DRG Neurons with CV×
×
Fig. 3.
Dorsal root ganglion neurons showed similar conduction velocity (CV) and action potential (AP) properties after paclitaxel chemotherapy. (A) AP was recorded intracellularly from soma of a C-neuron evoked by peripheral nerve stimulation. AP properties were measured as illustrated: (1) AP amplitude, (2) AP rise time, (3) AP fall time, (4) AP duration at base, (5) afterhyperpolarization (AHP) amplitude, and (6) AHP 50% duration. Comparisons of CV (B), AP amplitude (C), AP duration (D), AP rise time (E), AP fall time (F), AHP amplitude (G), and AHP 50% duration (H) were made after paclitaxel chemotherapy. *P < 0.05, one-way ANOVA.
Dorsal root ganglion neurons showed similar conduction velocity (CV) and action potential (AP) properties after paclitaxel chemotherapy. (A) AP was recorded intracellularly from soma of a C-neuron evoked by peripheral nerve stimulation. AP properties were measured as illustrated: (1) AP amplitude, (2) AP rise time, (3) AP fall time, (4) AP duration at base, (5) afterhyperpolarization (AHP) amplitude, and (6) AHP 50% duration. Comparisons of CV (B), AP amplitude (C), AP duration (D), AP rise time (E), AP fall time (F), AHP amplitude (G), and AHP 50% duration (H) were made after paclitaxel chemotherapy. *P < 0.05, one-way ANOVA.
Fig. 3.
Dorsal root ganglion neurons showed similar conduction velocity (CV) and action potential (AP) properties after paclitaxel chemotherapy. (A) AP was recorded intracellularly from soma of a C-neuron evoked by peripheral nerve stimulation. AP properties were measured as illustrated: (1) AP amplitude, (2) AP rise time, (3) AP fall time, (4) AP duration at base, (5) afterhyperpolarization (AHP) amplitude, and (6) AHP 50% duration. Comparisons of CV (B), AP amplitude (C), AP duration (D), AP rise time (E), AP fall time (F), AHP amplitude (G), and AHP 50% duration (H) were made after paclitaxel chemotherapy. *P < 0.05, one-way ANOVA.
×
Changes in Gene Expression of Multiple Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy.
Many molecular and cellular factors, including expression and function of neuronal ion channels, may contribute to the excitability of DRG neurons in chronic pain condition.7  Changes in gene expression of neuronal ion channels in DRG (L4/L5) from animals with verified mechanical hypersensitivity after paclitaxel chemotherapy was detected using multiple-gene rtPCR array. The array detects 84 different neuronal ion channel genes including 43 potassium, 12 calcium, 9 sodium, 12 transient receptor potential, 3 chloride and 3 neuronal amiloride-sensitive cation channels, 1 potassium-chloride transporter, and bestrophin 1 (see appendix for the complete list). By using one-sample t test with Bonferroni correction (P = 0.05/84 = 0.0006), it has shown that none of the changes reached statistical significance. When Bonferroni correction was not performed, 10 genes were found to have significant changes after paclitaxel treatment, including seven potassium, one calcium, one sodium, and one transient receptor potential channels. Seven genes were up-regulated including Kv1.2, Kv11.3, Kir3.1, hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1), transient receptor potential vanilloid 3 (TRPV3), Nav1.7, and brain ryanodine receptor–calcium release channel and three genes were down-regulated including Kir1.1, Kir3.4, and K2P1.1 (table 3). We also highlighted the genes with fold change greater than 1.2 or less than 0.8 compared with vehicle-treated animals and the Ct value less than 33 (which is more conservative than the default cutoff value of 35 by manufacturer’s handbook) in both paclitaxel and vehicle groups because these genes were considered to have marginal changes and may have potential biological impacts in paclitaxel CIPN. By this criterion, another 21 genes were modified by paclitaxel treatment (table 3).
Table 3.
Changes in Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Changes in Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy×
Changes in Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Table 3.
Changes in Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Changes in Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy×
×
Discussion
Paclitaxel Chemotherapy Increases the Excitability of DRG Neurons
By using the similar paclitaxel CIPN animal models, recordings from peripheral nerve fibers have found that paclitaxel treatment induces increased SA of both myelinated (A-) and unmyelinated (C-) fibers42  and enhanced responses of C-fibers to peripheral mechanical stimulation.34  Because both studies used teased fiber recordings of peripheral nerve (sural and saphenous nerves) with transected central end but intact skin innervation, the recorded axonal activities originated from periphery. The data shown here provided evidence that increased excitability of soma of DRG neurons is induced as well in paclitaxel CIPN and hyperexcitability measured by the intrinsic membrane properties is observed in both myelinated (Aβ and Aδ) and unmyelinated (C) cells.
Around one third of myelinated neurons displayed SA when recorded from the soma after paclitaxel chemotherapy, which is higher than the incidence of SA recorded from peripheral A-fibers.42  Because patients with paclitaxel CIPN often complain about spontaneous numbness and tingling which indicate myelinated fiber neuropathy,2–4  the prominent hyperexcitability of Aβ and Aδ neurons in DRG may well account for those major sensory dysfunctions in patients with paclitaxel CIPN, as well as tactile allodynia which is consistently observed in paclitaxel CIPN animal models. The increased incidence of SA (ectopic discharges or ectopia) originated from the soma of DRG neurons, especially myelinated cells, has been found across various models of peripheral nerve injury.8–20  Because the degree of allodynia correlates with the degree of ectopia8,25,43,44  and blocking the increased activities of DRG neurons attenuate chronic pain behavior including spontaneous pain and allodynia,22,25–30  it has been thought that the hyperexcitability of DRG neurons, particularly myelinated cells, plays a critical role in the initiation of spontaneous pain and tactile allodynia.45  In addition to the increased incidence of SA, we also found a robust decrease in the rheobase of DRG non-SA neurons, which is similar to the observation after peripheral nerve injury.11,14,15,17,18,20,23,24,46 
The rheobase of DRG small neurons also decreased after paclitaxel treatment, but such a decrease did not reach statistically significant. Although we did not observe SA from soma of nociceptive neurons in our study, the possibility of such activity could not be excluded. Due to the large heterogeneity of DRG neurons, it is possible that the important effects of paclitaxel may be restricted to particular subclasses of small neurons which were not sampled in the current study. It seems that the incidence of SA originated from the soma of C-cells is quite variable and may depend on different animal models. For example, no or only minimal level of SA in C-cells was observed after spinal nerve axotomy.8,47,48  A low level of SA from C-cells was found after chronic compression of DRG.13–16,18,19,24,49  A robust increase in such activity was observed after modified spinal nerve ligation11,21  or paw inflammation induced by intraplantar Complete Freund Adjuvant injection.21 
Similar to previous studies,34,42  we did not find any change in axonal CV of either myelinated or unmyelinated neurons after paclitaxel chemotherapy. Although it has been reported that paclitaxel induces a reduction of CV in peripheral nerve in experimental animals, these changes seem to depend on the cumulative dosage of paclitaxel because such a reduction only occurred in animals receiving 80 mg/kg but not 16 mg/kg paclitaxel50  or in mice receiving more than 200 mg/kg.51  The impact of chemotherapy may also be variable on different types of peripheral nerves because no reduction of CV was found in digital nerve even from mice receiving 200 mg/kg paclitaxel.51  Finally, the changes in CV may be more prominent closer to the peripheral terminals where die-back occurs because a large increase in CV of muscle afferents after paclitaxel was observed in a recent study.52  Nevertheless, the lack of change in axonal CV reported here, along with previous studies, may correlate with the observation that low dose of paclitaxel did not induce any axonal degeneration at the mid-axon level53  but was still sufficient to induce distal terminal degeneration.33,54 
No changes were found in other intrinsic membrane properties including input resistance, AP and afterhyperpolarization amplitude and duration, except that medium-sized neurons had shorter AP fall time in paclitaxel group compared with that in the same-sized cells in vehicle group. Such a difference could be due to sampling variance because the total duration and rise time of AP in the same group were not changed.
Modulation of Multiple Neuronal Ion Channels May Contribute to Increased Excitability of DRG Neurons after Paclitaxel Chemotherapy
The excitability of DRG neurons is fundamentally determined by the functional activities of neuronal ion channels which could be modulated by many intracellular and extracellular factors in various conditions of chronic pain.7  In a recent study, multiple-gene rtPCR array has been used to largely confirm the gene changes in both DRG and spinal cord after peripheral nerve injury and inflammation detected by microarray method.55  This array has also been successfully used to detect robust changes in gene expression of neurotrophin and proinflammatory cytokine in various tissues including DRG after paw plantar incision.56 
With multiple-gene rtPCR array, we have found prominent changes in gene expression of some neuronal ion channels in DRG after paclitaxel chemotherapy. Although the changes in expression of these genes did not reach statistical significance after Bonferroni correction, each did show a statistical significance when Bonferroni correction was not performed. Considering the increased probability of false negatives after Bonferroni correction and the high chance of reaching statistical significance when individual rtPCR was performed, we presented these data here.
Although it is impossible to tell which particular channel plays a major role, the robust down-regulation of voltage-gated potassium channels such as inwardly rectifying potassium channels (Kir 1.1 and Kir 3.4) and up-regulation of HCN1 and voltage-gated sodium channel Nav1.7 suggest the possible involvement of these channels in paclitaxel CIPN. The hyperpolarization-activated current (Ih) medicated by HCN1 is expressed on DRG myelinated neurons.57,58  The increase of HCN1 may facilitate the increased SA in these neurons observed in the current study. Another striking observation is the increased expression of Nav1.7 in DRG after paclitaxel. Nav1.7 is expressed in both large- and small-diameter DRG neurons and in most functionally identified nociceptors.59,60  Studies have shown that gain-of-function mutations of Nav1.7 are strongly linked to some inherited pain disorders such as inherited erythromelalgia.61–63  Recent studies have established a link between the gain-of-function mutation of Nav1.7 and painful peripheral neuropathy.64,65  Interestingly, loss-of-function of Nav1.7 has been found being related to insensitivity to pain.66–68  In addition, animal studies have found that the Nav1.7 expression level in DRG neurons is increased in response to various stimulation and knockdown of Nav1.7 attenuates some pathological pain (for review, see the study by Dib-Hajj et al.69 ). The specific role of these neuronal ion channels revealed by the current rtPCR study in paclitaxel CIPN remains to be fully established. The ongoing work in the laboratory is focused on addressing these issues.
There are certainly many limitations to interpret the changes of ion channel gene expression with rtPCR array. For example, the function of these channels depends on multiple factors, such as the rates of protein translation and degradation, post-translational modification, and the number of channels expressed on the plasma membrane. Tying the gene expression of these channels to their ultimate function and their specific role in paclitaxel CIPN will need further investigation. Some discrepancies between our study and previous studies have also been noticed. A recent study has shown that TRPV1 mRNA in DRG was increased after paclitaxel and blocking TRPV1 prevented or attenuated mechanical and thermal hypersensitivity of paclitaxel CIPN.70  A marginal increase in DRG TRPV1 expression was also found in our study, but the difference did not reach statistically significant (appendix). Another example is that Nav1.2 has been found to be expressed predominantly in brain71  and only at a very low level in DRG.72,73  Our data showed that the level of Nav1.2 expression in DRG was comparative with that of Nav1.1, Nav1.9, and Nav1.6 as judged by Ct values (appendix). These discrepancies could be contributed by several factors such as the variance in the efficiency of RNA extraction or complementary DNA synthesis between different laboratories, and the different design of probes.
In conclusion, we show that paclitaxel chemotherapy induces increased excitability of primary sensory neurons in DRG, which is demonstrated by the increased incidence of SA originated from the soma of myelinated cells and decreased rheobase in both myelinated and unmyelinated cells. These alterations in the intrinsic membrane properties of DRG neurons, along with increased activities of peripheral nerve fibers reported by other studies, suggest that the pathology of peripheral nervous system may play critical roles in the development of paclitaxel CIPN. The prominent changes in gene expression of some neuronal ion channels in DRG such as up-regulation of HCN1 and Nav1.7 channels and down-regulation of Kir channels suggest the possible roles of these channels in paclitaxel CIPN. Our data suggest the underlying channelopathy mechanism of paclitaxel CIPN and provide potential therapeutic targets.
Acknowledgments
This work was supported by grants from National Institutes of Health, Bethesda, Maryland (NS 046606), and National Cancer Institute, Bethesda, Maryland (CA 124787).
Competing Interests
The authors declare no competing interests.
References
Cavaletti, G, Marmiroli, P Chemotherapy-induced peripheral neurotoxicity.. Nat Rev Neurol. (2010). 6 657–66 [Article] [PubMed]
Dougherty, PM, Cata, JP, Cordella, JV, Burton, A, Weng, HR Taxol-induced sensory disturbance is characterized by preferential impairment of myelinated fiber function in cancer patients.. Pain. (2004). 109 132–42 [Article] [PubMed]
Loprinzi, CL, Reeves, BN, Dakhil, SR, Sloan, JA, Wolf, SL, Burger, KN, Kamal, A, Le-Lindqwister, NA, Soori, GS, Jaslowski, AJ, Novotny, PJ, Lachance, DH Natural history of paclitaxel-associated acute pain syndrome: Prospective cohort study NCCTG N08C1.. J Clin Oncol. (2011). 29 1472–8 [Article] [PubMed]
Reeves, BN, Dakhil, SR, Sloan, JA, Wolf, SL, Burger, KN, Kamal, A, Le-Lindqwister, NA, Soori, GS, Jaslowski, AJ, Kelaghan, J, Novotny, PJ, Lachance, DH, Loprinzi, CL Further data supporting that paclitaxel-associated acute pain syndrome is associated with development of peripheral neuropathy: North Central Cancer Treatment Group trial N08C1.. Cancer. (2012). 118 5171–8 [Article] [PubMed]
Cavaletti, G, Cavalletti, E, Oggioni, N, Sottani, C, Minoia, C, D’Incalci, M, Zucchetti, M, Marmiroli, P, Tredici, G Distribution of paclitaxel within the nervous system of the rat after repeated intravenous administration.. Neurotoxicology. (2000). 21 389–93 [PubMed]
Jimenez-Andrade, JM, Herrera, MB, Ghilardi, JR, Vardanyan, M, Melemedjian, OK, Mantyh, PW Vascularization of the dorsal root ganglia and peripheral nerve of the mouse: Implications for chemical-induced peripheral sensory neuropathies.. Mol Pain. (2008). 4 10 [Article] [PubMed]
Gold, MS, Gebhart, GF Nociceptor sensitization in pain pathogenesis.. Nat Med. (2010). 16 1248–57 [Article] [PubMed]
Liu, CN, Wall, PD, Ben-Dor, E, Michaelis, M, Amir, R, Devor, M Tactile allodynia in the absence of C-fiber activation: Altered firing properties of DRG neurons following spinal nerve injury.. Pain. (2000). 85 503–21 [Article] [PubMed]
Liu, CN, Devor, M, Waxman, SG, Kocsis, JD Subthreshold oscillations induced by spinal nerve injury in dissociated muscle and cutaneous afferents of mouse DRG.. J Neurophysiol. (2002). 87 2009–17 [PubMed]
Wall, PD, Devor, M Sensory afferent impulses originate from dorsal root ganglia as well as from the periphery in normal and nerve injured rats.. Pain. (1983). 17 321–39 [Article] [PubMed]
Djouhri, L, Fang, X, Koutsikou, S, Lawson, SN Partial nerve injury induces electrophysiological changes in conducting (uninjured) nociceptive and nonnociceptive DRG neurons: Possible relationships to aspects of peripheral neuropathic pain and paresthesias.. Pain. (2012). 153 1824–36 [Article] [PubMed]
Zhang, JM, Donnelly, DF, Song, XJ, Lamotte, RH Axotomy increases the excitability of dorsal root ganglion cells with unmyelinated axons.. J Neurophysiol. (1997). 78 2790–4 [PubMed]
Song, XJ, Zhang, JM, Hu, SJ, LaMotte, RH Somata of nerve-injured sensory neurons exhibit enhanced responses to inflammatory mediators.. Pain. (2003). 104 701–9 [Article] [PubMed]
Zhang, H, Mei, X, Zhang, P, Ma, C, White, FA, Donnelly, DF, Lamotte, RH Altered functional properties of satellite glial cells in compressed spinal ganglia.. Glia. (2009). 57 1588–99 [Article] [PubMed]
Zhang, JM, Song, XJ, LaMotte, RH Enhanced excitability of sensory neurons in rats with cutaneous hyperalgesia produced by chronic compression of the dorsal root ganglion.. J Neurophysiol. (1999). 82 3359–66 [PubMed]
Ma, C, LaMotte, RH Multiple sites for generation of ectopic spontaneous activity in neurons of the chronically compressed dorsal root ganglion.. J Neurosci. (2007). 27 14059–68 [Article] [PubMed]
Ma, C, Greenquist, KW, Lamotte, RH Inflammatory mediators enhance the excitability of chronically compressed dorsal root ganglion neurons.. J Neurophysiol. (2006). 95 2098–107 [Article] [PubMed]
Ma, C, LaMotte, RH Enhanced excitability of dissociated primary sensory neurons after chronic compression of the dorsal root ganglion in the rat.. Pain. (2005). 113 106–12 [Article] [PubMed]
Song, XJ, Hu, SJ, Greenquist, KW, Zhang, JM, LaMotte, RH Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia.. J Neurophysiol. (1999). 82 3347–58 [PubMed]
Song, Y, Li, HM, Xie, RG, Yue, ZF, Song, XJ, Hu, SJ, Xing, JL Evoked bursting in injured Aβ dorsal root ganglion neurons: A mechanism underlying tactile allodynia.. Pain. (2012). 153 657–65 [Article] [PubMed]
Djouhri, L, Koutsikou, S, Fang, X, McMullan, S, Lawson, SN Spontaneous pain, both neuropathic and inflammatory, is related to frequency of spontaneous firing in intact C-fiber nociceptors.. J Neurosci. (2006). 26 1281–92 [Article] [PubMed]
Weng, X, Smith, T, Sathish, J, Djouhri, L Chronic inflammatory pain is associated with increased excitability and hyperpolarization-activated current (Ih) in C- but not Aδ-nociceptors.. Pain. (2012). 153 900–14 [Article] [PubMed]
Zheng, JH, Walters, ET, Song, XJ Dissociation of dorsal root ganglion neurons induces hyperexcitability that is maintained by increased responsiveness to cAMP and cGMP.. J Neurophysiol. (2007). 97 15–25 [Article] [PubMed]
Xie, Y, Zhang, J, Petersen, M, LaMotte, RH Functional changes in dorsal root ganglion cells after chronic nerve constriction in the rat.. J Neurophysiol. (1995). 73 1811–20 [PubMed]
Sukhotinsky, I, Ben-Dor, E, Raber, P, Devor, M Key role of the dorsal root ganglion in neuropathic tactile hypersensibility.. Eur J Pain. (2004). 8 135–43 [Article] [PubMed]
Zhang, JM, Li, H, Brull, SJ Perfusion of the mechanically compressed lumbar ganglion with lidocaine reduces mechanical hyperalgesia and allodynia in the rat.. J Neurophysiol. (2000). 84 798–805 [PubMed]
Malan, TP, Ossipov, MH, Gardell, LR, Ibrahim, M, Bian, D, Lai, J, Porreca, F Extraterritorial neuropathic pain correlates with multisegmental elevation of spinal dynorphin in nerve-injured rats.. Pain. (2000). 86 185–94 [Article] [PubMed]
Lyu, YS, Park, SK, Chung, K, Chung, JM Low dose of tetrodotoxin reduces neuropathic pain behaviors in an animal model.. Brain Res. (2000). 871 98–103 [Article] [PubMed]
Blenk, KH, Häbler, HJ, Jänig, W Neomycin and gadolinium applied to an L5 spinal nerve lesion prevent mechanical allodynia-like behaviour in rats.. Pain. (1997). 70 155–65 [Article] [PubMed]
Xie, W, Strong, JA, Meij, JT, Zhang, JM, Yu, L Neuropathic pain: Early spontaneous afferent activity is the trigger.. Pain. (2005). 116 243–56 [Article] [PubMed]
Polomano, RC, Mannes, AJ, Clark, US, Bennett, GJ A painful peripheral neuropathy in the rat produced by the chemotherapeutic drug, paclitaxel.. Pain. (2001). 94 293–304 [Article] [PubMed]
Cata, JP, Weng, HR, Dougherty, PM The effects of thalidomide and minocycline on taxol-induced hyperalgesia in rats.. Brain Res. (2008). 1229 100–10 [Article] [PubMed]
Boyette-Davis, J, Xin, W, Zhang, H, Dougherty, PM Intraepidermal nerve fiber loss corresponds to the development of taxol-induced hyperalgesia and can be prevented by treatment with minocycline.. Pain. (2011). 152 308–13 [Article] [PubMed]
Dina, OA, Chen, X, Reichling, D, Levine, JD Role of protein kinase Cepsilon and protein kinase A in a model of paclitaxel-induced painful peripheral neuropathy in the rat.. Neuroscience. (2001). 108 507–15 [Article] [PubMed]
Zhang, H, Yoon, SY, Zhang, H, Dougherty, PM Evidence that spinal astrocytes but not microglia contribute to the pathogenesis of Paclitaxel-induced painful neuropathy.. J Pain. (2012). 13 293–303 [Article] [PubMed]
Cata, JP, Weng, HR, Dougherty, PM Increased Excitability of WDR Neurons Accompanies Paclitaxel-induced Hyperalgesia in Rats. 2004 Abstract Viewer/Itinerary Planner. (2004). Washington, DC Society for Neuroscience
Waddell, PJ, Lawson, SN Electrophysiological properties of subpopulations of rat dorsal root ganglion neurons in vitro.. Neuroscience. (1990). 36 811–22 [Article] [PubMed]
Waddell, PJ, Lawson, SN, McCarthy, PW Conduction velocity changes along the processes of rat primary sensory neurons.. Neuroscience. (1989). 30 577–84 [Article] [PubMed]
Schmittgen, TD, Livak, KJ Analyzing real-time PCR data by the comparative C(T) method.. Nat Protoc. (2008). 3 1101–8 [Article] [PubMed]
Harper, AA, Lawson, SN Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurones.. J Physiol. (1985). 359 31–46 [PubMed]
Devor, M, Govrin-Lippmann, R Neurogenesis in adult rat dorsal root ganglia.. Neurosci Lett. (1985). 61 189–94 [Article] [PubMed]
Xiao, WH, Bennett, GJ Chemotherapy-evoked neuropathic pain: Abnormal spontaneous discharge in A-fiber and C-fiber primary afferent neurons and its suppression by acetyl-L-carnitine.. Pain. (2008). 135 262–70 [Article] [PubMed]
Sheen, K, Chung, JM Signs of neuropathic pain depend on signals from injured nerve fibers in a rat model.. Brain Res. (1993). 610 62–8 [Article] [PubMed]
Yoon, YW, Na, HS, Chung, JM Contributions of injured and intact afferents to neuropathic pain in an experimental rat model.. Pain. (1996). 64 27–36 [Article] [PubMed]
Devor, M Ectopic discharge in Abeta afferents as a source of neuropathic pain.. Exp Brain Res. (2009). 196 115–28 [Article] [PubMed]
Zhu, YF, Henry, JL Excitability of Aβ sensory neurons is altered in an animal model of peripheral neuropathy.. BMC Neurosci. (2012). 13 15 [Article] [PubMed]
Liu, X, Eschenfelder, S, Blenk, KH, Jänig, W, Häbler, H Spontaneous activity of axotomized afferent neurons after L5 spinal nerve injury in rats.. Pain. (2000). 84 309–18 [Article] [PubMed]
Han, HC, Lee, DH, Chung, JM Characteristics of ectopic discharges in a rat neuropathic pain model.. Pain. (2000). 84 253–61 [Article] [PubMed]
Ma, C, Shu, Y, Zheng, Z, Chen, Y, Yao, H, Greenquist, KW, White, FA, LaMotte, RH Similar electrophysiological changes in axotomized and neighboring intact dorsal root ganglion neurons.. J Neurophysiol. (2003). 89 1588–602 [Article] [PubMed]
Authier, N, Gillet, JP, Fialip, J, Eschalier, A, Coudore, F Description of a short-term Taxol-induced nociceptive neuropathy in rats.. Brain Res. (2000). 887 239–49 [Article] [PubMed]
Carozzi, VA, Canta, A, Oggioni, N, Sala, B, Chiorazzi, A, Meregalli, C, Bossi, M, Marmiroli, P, Cavaletti, G Neurophysiological and neuropathological characterization of new murine models of chemotherapy-induced chronic peripheral neuropathies.. Exp Neurol. (2010). 226 301–9 [Article] [PubMed]
Chen, X, Green, PG, Levine, JD Abnormal muscle afferent function in a model of Taxol chemotherapy-induced painful neuropathy.. J Neurophysiol. (2011). 106 274–9 [Article] [PubMed]
Flatters, SJ, Bennett, GJ Studies of peripheral sensory nerves in paclitaxel-induced painful peripheral neuropathy: Evidence for mitochondrial dysfunction.. Pain. (2006). 122 245–57 [Article] [PubMed]
Flatters, SJ, Bennett, GJ Ethosuximide reverses paclitaxel- and vincristine-induced painful peripheral neuropathy.. Pain. (2004). 109 150–61 [Article] [PubMed]
LaCroix-Fralish, ML, Austin, JS, Zheng, FY, Levitin, DJ, Mogil, JS Patterns of pain: Meta-analysis of microarray studies of pain.. Pain. (2011). 152 1888–98 [Article] [PubMed]
Spofford, CM, Brennan, TJ Gene expression in skin, muscle, and dorsal root ganglion after plantar incision in the rat.. Anesthesiology. (2012). 117 161–72 [Article] [PubMed]
Gao, LL, McMullan, S, Djouhri, L, Acosta, C, Harper, AA, Lawson, SN Expression and properties of hyperpolarization-activated current in rat dorsal root ganglion neurons with known sensory function.. J Physiol. (2012). 590Pt 19 4691–705 [Article] [PubMed]
Acosta, C, McMullan, S, Djouhri, L, Gao, L, Watkins, R, Berry, C, Dempsey, K, Lawson, SN HCN1 and HCN2 in Rat DRG neurons: Levels in nociceptors and non-nociceptors, NT3-dependence and influence of CFA-induced skin inflammation on HCN2 and NT3 expression.. PLoS One. (2012). 7 e50442 [Article] [PubMed]
Dib-Hajj, SD, Cummins, TR, Black, JA, Waxman, SG Sodium channels in normal and pathological pain.. Annu Rev Neurosci. (2010). 33 325–47 [Article] [PubMed]
Djouhri, L, Newton, R, Levinson, SR, Berry, CM, Carruthers, B, Lawson, SN Sensory and electrophysiological properties of guinea-pig sensory neurones expressing Nav 1.7 (PN1) Na+ channel alpha subunit protein.. J Physiol. (2003). 546Pt 2 565–76 [Article] [PubMed]
Yang, Y, Wang, Y, Li, S, Xu, Z, Li, H, Ma, L, Fan, J, Bu, D, Liu, B, Fan, Z, Wu, G, Jin, J, Ding, B, Zhu, X, Shen, Y Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia.. J Med Genet. (2004). 41 171–4 [Article] [PubMed]
Cummins, TR, Dib-Hajj, SD, Waxman, SG Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy.. J Neurosci. (2004). 24 8232–6 [Article] [PubMed]
Dib-Hajj, SD, Rush, AM, Cummins, TR, Hisama, FM, Novella, S, Tyrrell, L, Marshall, L, Waxman, SG Gain-of-function mutation in Nav1.7 in familial erythromelalgia induces bursting of sensory neurons.. Brain. (2005). 128Pt 8 1847–54 [Article] [PubMed]
Hoeijmakers, JG, Faber, CG, Lauria, G, Merkies, IS, Waxman, SG Small-fibre neuropathies—Advances in diagnosis, pathophysiology and management.. Nat Rev Neurol. (2012). 8 369–79 [Article] [PubMed]
Faber, CG, Hoeijmakers, JG, Ahn, HS, Cheng, X, Han, C, Choi, JS, Estacion, M, Lauria, G, Vanhoutte, EK, Gerrits, MM, Dib-Hajj, S, Drenth, JP, Waxman, SG, Merkies, IS Gain of function Naν1.7 mutations in idiopathic small fiber neuropathy.. Ann Neurol. (2012). 71 26–39 [Article] [PubMed]
Ahmad, S, Dahllund, L, Eriksson, AB, Hellgren, D, Karlsson, U, Lund, PE, Meijer, IA, Meury, L, Mills, T, Moody, A, Morinville, A, Morten, J, O’donnell, D, Raynoschek, C, Salter, H, Rouleau, GA, Krupp, JJ A stop codon mutation in SCN9A causes lack of pain sensation.. Hum Mol Genet. (2007). 16 2114–21 [Article] [PubMed]
Cox, JJ, Reimann, F, Nicholas, AK, Thornton, G, Roberts, E, Springell, K, Karbani, G, Jafri, H, Mannan, J, Raashid, Y, Al-Gazali, L, Hamamy, H, Valente, EM, Gorman, S, Williams, R, McHale, DP, Wood, JN, Gribble, FM, Woods, CG An SCN9A channelopathy causes congenital inability to experience pain.. Nature. (2006). 444 894–8 [Article] [PubMed]
Goldberg, YP, MacFarlane, J, MacDonald, ML, Thompson, J, Dube, MP, Mattice, M, Fraser, R, Young, C, Hossain, S, Pape, T, Payne, B, Radomski, C, Donaldson, G, Ives, E, Cox, J, Younghusband, HB, Green, R, Duff, A, Boltshauser, E, Grinspan, GA, Dimon, JH, Sibley, BG, Andria, G, Toscano, E, Kerdraon, J, Bowsher, D, Pimstone, SN, Samuels, ME, Sherrington, R, Hayden, MR Loss-of-function mutations in the Nav1.7 gene underlie congenital indifference to pain in multiple human populations.. Clin Genet. (2007). 71 311–9 [Article] [PubMed]
Dib-Hajj, SD, Yang, Y, Black, JA, Waxman, SG The Na(V)1.7 sodium channel: From molecule to man.. Nat Rev Neurosci. (2013). 14 49–62 [Article] [PubMed]
Hara, T, Chiba, T, Abe, K, Makabe, A, Ikeno, S, Kawakami, K, Utsunomiya, I, Hama, T, Taguchi, K Effect of paclitaxel on transient receptor potential vanilloid 1 in rat dorsal root ganglion.. Pain. (2013). 154 882–9 [Article] [PubMed]
Trimmer, JS, Rhodes, KJ Localization of voltage-gated ion channels in mammalian brain.. Annu Rev Physiol. (2004). 66 477–519 [Article] [PubMed]
Black, JA, Dib-Hajj, S, McNabola, K, Jeste, S, Rizzo, MA, Kocsis, JD, Waxman, SG Spinal sensory neurons express multiple sodium channel alpha-subunit mRNAs.. Brain Res Mol Brain Res. (1996). 43 117–31 [Article] [PubMed]
Ho, C, O’Leary, ME Single-cell analysis of sodium channel expression in dorsal root ganglion neurons.. Mol Cell Neurosci. (2011). 46 159–66 [Article] [PubMed]
Appendix.
Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy×
Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy×
×
Fig. 1.
Increased incidence of spontaneous activity (SA) in dorsal root ganglion neurons from animals with paclitaxel neuropathy. (A) Paclitaxel chemotherapy induced a significant increase of SA from dorsal root ganglion myelinated neurons including large- (P = 0.003) and medium-sized (P = 0.027) neurons. *P < 0.05, **P < 0.01, chi-square test. (B) An example of irregular SA recorded from a medium-sized neuron, noticing the subthreshold membrane potential oscillations between each discharge. (C) Continuous discharges were recorded from a spontaneously active large neuron. (D) Spontaneous bursting discharges were recorded from a large neuron after paclitaxel chemotherapy. Subthreshold oscillations (arrows) were obvious between each episode of bursting discharges.
Increased incidence of spontaneous activity (SA) in dorsal root ganglion neurons from animals with paclitaxel neuropathy. (A) Paclitaxel chemotherapy induced a significant increase of SA from dorsal root ganglion myelinated neurons including large- (P = 0.003) and medium-sized (P = 0.027) neurons. *P < 0.05, **P < 0.01, chi-square test. (B) An example of irregular SA recorded from a medium-sized neuron, noticing the subthreshold membrane potential oscillations between each discharge. (C) Continuous discharges were recorded from a spontaneously active large neuron. (D) Spontaneous bursting discharges were recorded from a large neuron after paclitaxel chemotherapy. Subthreshold oscillations (arrows) were obvious between each episode of bursting discharges.
Fig. 1.
Increased incidence of spontaneous activity (SA) in dorsal root ganglion neurons from animals with paclitaxel neuropathy. (A) Paclitaxel chemotherapy induced a significant increase of SA from dorsal root ganglion myelinated neurons including large- (P = 0.003) and medium-sized (P = 0.027) neurons. *P < 0.05, **P < 0.01, chi-square test. (B) An example of irregular SA recorded from a medium-sized neuron, noticing the subthreshold membrane potential oscillations between each discharge. (C) Continuous discharges were recorded from a spontaneously active large neuron. (D) Spontaneous bursting discharges were recorded from a large neuron after paclitaxel chemotherapy. Subthreshold oscillations (arrows) were obvious between each episode of bursting discharges.
×
Fig. 2.
Dorsal root ganglion neurons showed increased excitability after paclitaxel chemotherapy. (A) Sample recordings of large neurons from paclitaxel- and vehicle-treated animals. The input resistance of each cell was measured based on the steady-state voltage changes at the end of the application of a series of hyperpolarizing/depolarizing currents as indicated by arrowhead. Comparison of electrophysiological properties of dorsal root ganglion neurons with different size, including resting membrane potential (RMP) (B), current threshold (C), number of action potentials (APs) induced by a 1-s two times current threshold intracellular depolarizing current (D) and input resistance (E). *P < 0.001, **P < 0.0001, one-way ANOVA.
Dorsal root ganglion neurons showed increased excitability after paclitaxel chemotherapy. (A) Sample recordings of large neurons from paclitaxel- and vehicle-treated animals. The input resistance of each cell was measured based on the steady-state voltage changes at the end of the application of a series of hyperpolarizing/depolarizing currents as indicated by arrowhead. Comparison of electrophysiological properties of dorsal root ganglion neurons with different size, including resting membrane potential (RMP) (B), current threshold (C), number of action potentials (APs) induced by a 1-s two times current threshold intracellular depolarizing current (D) and input resistance (E). *P < 0.001, **P < 0.0001, one-way ANOVA.
Fig. 2.
Dorsal root ganglion neurons showed increased excitability after paclitaxel chemotherapy. (A) Sample recordings of large neurons from paclitaxel- and vehicle-treated animals. The input resistance of each cell was measured based on the steady-state voltage changes at the end of the application of a series of hyperpolarizing/depolarizing currents as indicated by arrowhead. Comparison of electrophysiological properties of dorsal root ganglion neurons with different size, including resting membrane potential (RMP) (B), current threshold (C), number of action potentials (APs) induced by a 1-s two times current threshold intracellular depolarizing current (D) and input resistance (E). *P < 0.001, **P < 0.0001, one-way ANOVA.
×
Fig. 3.
Dorsal root ganglion neurons showed similar conduction velocity (CV) and action potential (AP) properties after paclitaxel chemotherapy. (A) AP was recorded intracellularly from soma of a C-neuron evoked by peripheral nerve stimulation. AP properties were measured as illustrated: (1) AP amplitude, (2) AP rise time, (3) AP fall time, (4) AP duration at base, (5) afterhyperpolarization (AHP) amplitude, and (6) AHP 50% duration. Comparisons of CV (B), AP amplitude (C), AP duration (D), AP rise time (E), AP fall time (F), AHP amplitude (G), and AHP 50% duration (H) were made after paclitaxel chemotherapy. *P < 0.05, one-way ANOVA.
Dorsal root ganglion neurons showed similar conduction velocity (CV) and action potential (AP) properties after paclitaxel chemotherapy. (A) AP was recorded intracellularly from soma of a C-neuron evoked by peripheral nerve stimulation. AP properties were measured as illustrated: (1) AP amplitude, (2) AP rise time, (3) AP fall time, (4) AP duration at base, (5) afterhyperpolarization (AHP) amplitude, and (6) AHP 50% duration. Comparisons of CV (B), AP amplitude (C), AP duration (D), AP rise time (E), AP fall time (F), AHP amplitude (G), and AHP 50% duration (H) were made after paclitaxel chemotherapy. *P < 0.05, one-way ANOVA.
Fig. 3.
Dorsal root ganglion neurons showed similar conduction velocity (CV) and action potential (AP) properties after paclitaxel chemotherapy. (A) AP was recorded intracellularly from soma of a C-neuron evoked by peripheral nerve stimulation. AP properties were measured as illustrated: (1) AP amplitude, (2) AP rise time, (3) AP fall time, (4) AP duration at base, (5) afterhyperpolarization (AHP) amplitude, and (6) AHP 50% duration. Comparisons of CV (B), AP amplitude (C), AP duration (D), AP rise time (E), AP fall time (F), AHP amplitude (G), and AHP 50% duration (H) were made after paclitaxel chemotherapy. *P < 0.05, one-way ANOVA.
×
Table 1.
Electrophysiological Properties of DRG Neurons with Different Sizes
Electrophysiological Properties of DRG Neurons with Different Sizes×
Electrophysiological Properties of DRG Neurons with Different Sizes
Table 1.
Electrophysiological Properties of DRG Neurons with Different Sizes
Electrophysiological Properties of DRG Neurons with Different Sizes×
×
Table 2.
Intrinsic Membrane Properties of DRG Neurons with CV
Intrinsic Membrane Properties of DRG Neurons with CV×
Intrinsic Membrane Properties of DRG Neurons with CV
Table 2.
Intrinsic Membrane Properties of DRG Neurons with CV
Intrinsic Membrane Properties of DRG Neurons with CV×
×
Table 3.
Changes in Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Changes in Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy×
Changes in Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Table 3.
Changes in Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Changes in Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy×
×
Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy×
Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy
Gene Expression of Neuronal Ion Channels in DRG after Paclitaxel Chemotherapy×
×