Impairment of tactile responses and Piezo2 channel mechanotransduction in mice following chronic vincristine treatment

Abstract

Loss of the sense of touch or numbness in fingertips and toes is one of the earliest sensory dysfunctions in patients receiving chemotherapy with anti-cancer drugs such as vincristine. However, mechanisms underlying this chemotherapy-induced sensory dysfunction is poorly understood. Whisker hair follicles are tactile organs in non-primate mammals which are functionally equivalent to human fingertips. Here we used mouse whisker hair follicles as a model system to explore how vincristine treatment induces the loss of the sense of touch. We show that chronic treatment of mice with vincristine impaired in vivo whisker tactile behavioral responses. In vitro electrophysiological recordings made from whisker hair follicle afferent nerves showed that mechanically evoked whisker afferent impulses were significantly reduced following vincristine treatment. Furthermore, patch-clamp recordings from Merkel cells of whisker hair follicles revealed a significant reduction of mechanically activated currents via Piezo2 channels in Merkel cells. Collectively, our results suggest that Piezo2 channel dysfunction in Merkel cells contribute to the loss of the sense of touch following the chemotherapy treatment regimen with vincristine.

Introduction

The sense of touch is essential in tasks such as environmental explorations, social interactions, and tactile discrimination (1). Loss of the sense of touch occurs under many pathological conditions such as chemotherapy-induced neuropathy (2, 3). Vincristine and its analogs are important chemotherapy drugs for treating a number of cancers but these drugs often induce sensory abnormalities including numbness, tingling sensation, and pain (2, 3). These sensory dysfunctions occur soon after the start of a chemotherapy regimen and persist from months to years beyond the completion of chemotherapy (4, 5), which negatively impacts function and quality of life and is a significant clinical problem. These sensory dysfunctions are dose-limiting factors that prevent patients to continue effective treatment regimens with chemotherapy drugs. Chemotherapy-induced sensory dysfunctions are typically present in patients with a “stocking and glove” distribution in the feet and hands, particularly at toes and fingertips (6). Previous studies have mainly focused on the pain aspect of chemotherapy-induced peripheral neuropathy. The numbness aspect of chemotherapy-induced peripheral neuropathy is poorly understood. This is largely because of our lesser understanding of molecular mechanisms underlying the sense of touch.

The sense of touch is mainly initiated at tactile end organs, including Merkel discs, Pacinian corpuscles, Meissner’s corpuscles, and other highly specialized structures in the periphery of mammals (1, 7). Merkel disc, also known as Merkel cell-neurite complex, is a main type of tactile end organs highly abundant in human fingertips, whisker hair follicles, touch domes and other tactile-sensitive spots throughout mammalian bodies (8, 9). Structurally, Merkel discs are composed of Merkel cells and their associated Aβ-afferent nerve endings to form a structure of disk-shaped expansion (8, 10). Merkel discs have high tactile acuity and are very sensitive to skin indentation, pressure, hair movement and other tactile stimuli. Tactile stimuli to Merkel discs in the touch domes of the skin and whisker hair follicles result in slowly adapting type 1 (SA1) responses, the characteristic Aβ-afferent impulses for tactile encoding (1, 10). Functionally, SA1 responses in fingertips and whisker hair follicles are essential for tactile discrimination of an object’s texture, shape and other physical properties. Recent studies have demonstrated that Merkel cells are mechanotransducing cells using Piezo2 channels to convey tactile stimuli, which is essential for tactile behaviors in mammals (11–13). Piezo2 channels have also been found to be expressed at lanceolate endings which are rapidly adapting mechanoreceptors of hair follicles (14). More recently, studies have shown that Merkel cells in whisker hair follicles release 5-HT in response to mechanical stimulation, which in turn transmit tactile signals by exciting whisker afferent nerves (15).

These recent studies on tactile transduction and transmission have led us to hypothesize that reduction of tactile transduction and/or transmission is an underlying mechanism of chemotherapy-induced numbness. In the present study we set out to determine whether Merkel cells are targeted by the chemotherapy drug vincristine to account to chemotherapy-induced loss of tactile sensitivity.

Materials and Methods

Animals:

C57BL/6 mice were obtained from Harlan Laboratories. At the age of 4–8 weeks mice were used for in vivo tactile behavioral assessments, in vitro whisker hair follicle afferent fiber recordings, and patch-clamp recordings from Merkel cells of whisker hair follicles. Animal care and use conformed to NIH guidelines for the care and use of experimental animals. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alabama at Birmingham.

Vincristine treatment and tactile behavioral assessment:

Vehicle (sterile phosphate-buffered saline, PBS) or vincristine dissolved in vehicle was i.p. administered for 5 consecutive days, then 2 day breaks, and 5 consecutive days again. The dose of vincristine was at 1 mg/kg each injection. Whisker tactile behavioral tests were performed daily for up to 19 days. The whisker tactile test was performed in a blinded manner in that one examiner conducted vincristine injections and animal grouping, and another examiner who did not know the grouping performed in vivo whisker tactile behavioral tests on these animals. To begin the tests, mice were placed in a cage and habituated for 10 min. During habituation and subsequent experiments, the testing room only had a red light on so that animals could not see the examiner and the tactile stimulation filament. After the habituation, a single whisker hair (D1 whisker) was displaced up to 2 mm in caudal-rostral direction by the tactile stimulation filament, and the whisker tactile test was performed 20 times with an interval of 1 min between trials. A positive whisker tactile behavioral response was considered when the testing animal exhibited an avoidance reaction to the tactile stimulation.

Whisker hair follicle afferent fiber recordings.

Whisker hair follicles were harvested from vehicle-injected or vincristine-treated groups 7 to 17 days following the last injections of vehicle or vincristine, respectively. Whisker hair follicle preparations and whisker hair follicle afferent fiber recordings were performed using our previously described method (13). In brief, mice were anesthetized with isoflurane and sacrificed by decapitation. Whisker hair follicles with attached afferent fiber bundles were dissected out and anchored in a recording chamber. The whisker hair follicles were submerged and perfused in oxygenated Krebs solution that contained (in mM): 117 NaCl, 3.5 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃, and 11 glucose bubbled with 95% O₂ and 5% CO₂, had pH of 7.3 and osmolarity of 325 mOsm, and was maintained at 24°C. Unless otherwise indicated, the end of the follicle capsule was cut open to facilitate bath diffusion into whisker hair follicles. To record whisker hair follicle afferent nerve impulses elicited by whisker deflections, compound action potentials conducted on whisker hair follicle afferent fibers were recorded using a suction electrode. Signals of nerve impulses were amplified using a Multiclamp 700A amplifier and sampled 10 KHz with low pass filter set at 1 KHz.

Patch-clamp recordings from Merkel cells in situ in whisker hair follicles:

Whisker hair follicles were harvested from vehicle-injected and vincristine-treated groups 7 to 17 days following the last injections of vehicle or vincristine. In brief, mice were anesthetized with isoflurane and sacrificed by decapitation. Whisker hair follicles were dissected out from whisker pad and capsule of each hair follicle removed under a dissection microscope. Whisker hair follicles (without capsules) were then affixed in a recording chamber with a tissue anchor and perfused with oxygenated Krebs solutions at 24°C. The recording chamber was mounted on the stage of an Olympus IX50 microscope that was equipped with IR-DIC and fluorescent imaging systems. The whisker hair follicles were exposed to 0.05% dispase II and 0.01% collagenase in Krebs solution for 8~11 min, then the enzymes were washed off with Krebs solution. The ring sinus and glassy membranes were removed using a glass electrode controlled by a micromanipulator. The whisker hair follicles were then incubated with 0.3 µM quinacrine in Krebs solution for 15 min to vital-stain Merkel cells. The whisker hair follicles were continuously perfused with oxygenated Krebs solution at a flow rate of 1.5 ml/min. Quinacrine-labeled Merkel cells were identified using the fluorescent imaging system.

Patch-clamp recordings were made at room temperature of 23°C from quinacrine-stained cells. Recording electrodes were filled with an internal solution containing (in mM): 135 K-gluconate, 5 KCl, 0.5 CaCl₂, 2 MgCl₂, 5 EGTA, 5 HEPES, 5 Na₂ATP and 0.5 GTP-TRIS salt; the pH of the solution was adjusted to 7.3 with KOH. Signals were amplified and filtered at 2 kHz using the Multiclamp 700A amplifier and sampled at 4 kHz using pCLAMP 10 software (Molecular Devices, San Jose). Membrane and action potential properties of Merkel cells were determined under the whole-cell current clamp mode. Step current pulses were injected into cells through patch-clamp recording electrodes from −60 pA to 220 pA in increments of 20 pA with a pulse duration of 200 ms. Currents evoked by voltages and mechanical stimulation were recorded with Merkel cells voltage-clamped at −75 mV. For voltage-evoked currents, voltage steps were applied from −135 mV to 65 mV in increments of 20 mV and duration of 100 ms.

Mechanical Stimulation.

Hair deflection was used as a tactile stimulus to elicit whisker afferent SA1 impulses. In the present study, we anchored the whisker hair follicles in a recording chamber by affixing the whisker hair shaft onto the bottom of the recording chamber, and perfused them with Krebs solution. A fire-polished blunted glass probe was used for delivering mechanical stimuli. The probe was attached onto the capsule surface at the whisker hair follicle center and controlled by a piezo device. When the mechanical probe displaced the whisker hair follicle, it generated a whisker hair shaft deflection. This modified tactile stimulation method improved consistency of tactile responses. Unless otherwise indicated, hair deflection was induced by a 38-µm forward step to push the hair follicle for a duration of 2.62 s; the step had a 56-ms ramp at the speed of 0.68 µm/ms (dynamic phase) before reaching the 38-µm step (static phase).

Merkel cell mechanical sensitivity was tested using a method described in our previous study (13). In brief, a fire-polished blunted glass probe was used for the mechanical stimulation. It was connected to a computer-programmable piezo device (E-625 LVPZT; Physik Instrumente). The tip of the glass probe was ~3 μm in diameter. It was positioned at an angle of 30 degrees to the surface (the outer root sheath layer) of the hair follicle. The distance from the probe tip to the surface of the hair follicle tissue was set in such a way that the tip would contact the surface if the probe had one step (0.5 μm) forward movement. The stepwise forward movement of the probe was delivered by the piezo device. Merkel cells were displaced indirectly by the probe (indirect displacement stimulation). This was achieved by displacing non-recorded cells so that mechanical force was transmitted across two adjacent cells (~15 µm) to the recorded Merkel cells.

Data Analysis:

Electrophysiological data were analyzed using Clampfit software. Data are presented as mean ± SEM. Statistical significance was evaluated using Student’s t-test or two-way ANOVA with Bonferroni post-hoc tests for multiple groups, * p<0.05, ** p<0.01, and *** p<0.001.

Results

We adopted a vincristine treatment regimen to induce chemotherapy-induced peripheral neuropathy in rats from a previous study (16). The vincristine treatment regimen consisted of a consecutive 5 days of i.p. administration of 1 mg/kg vincristine, a two-day break, and then another consecutive 5 days of i.p. administration of 1 mg/kg vincristine (Figure 1A). Control animals received i.p. vehicle (PBS) injections in the same manner. Tactile behavioral assessments were performed daily in both groups, and a portion of animals in each group was used for in vitro electrophysiological recording experiments (Figure 1A&B). All in vivo behavioral experiments were performed in a blinded manner. As shown in Figure 1C, the vehicle-injected group showed high numbers of responses to tactile stimuli, approximately 18 out of total 20 stimuli, during the course of ~3 weeks of whisker tactile behavioral tests. Vincristine-treated animals also displayed high numbers of whisker tactile behavioral responses during the initial 11 days of the tests, which was not significantly different from those of vehicle-injected group (Figure 1C). However, a significant reduction in the numbers of whisker tactile behavioral responses were observed after 11 days, i.e., 2 days before the end of the vincristine treatment regimen. The significant reduction of whisker tactile behavioral responses continued for approximately a week after the end of the vincristine treatment regimen, and animals did not undergo further whisker tactile behavioral tests after day 19 (Figure 1C).

Figure 1. Impairment of in vivo tactile behavioral responses following chronic vincristine treatment.

(A) Illustration of vincristine treatment regimen and schedules of in vivo and in vitro experiments. Vincristine was i.p. administrated at daily dose of 1 mg/kg. In vivo tactile behavioral assessments were performed daily for up to the days of in vitro recording experiments. (B) Diagram illustrates tactile behavioral assessment to measure animals’ avoidance responses to gentile strikes of D1 whisker hairs by a tactile filament. (C) Tactile behavioral responses measured as numbers of avoidance reactions in animals of vehicle-injected and vincristine-injected groups. Animal numbers were 12 in each group. Data represent Mean ± SEM, **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni post-hoc tests.

We determined if mechanically evoked whisker hair follicle afferent impulses were impaired in vincristine-treated group. In this set of experiments, whisker hair follicles were harvested from vehicle-injected and vincristine-treated animals, and whisker hair follicle afferent impulses evoked by a whisker hair deflection (38 µm) were recorded (Figure 2A). As shown in Figure 2B&C, robust impulses were elicited by the mechanical stimulation in recordings made from whisker hair follicles harvested from vehicle-injected group (n = 7). In contrast, there were much fewer impulses elicited in recordings from whisker hair follicles harvested from vincristine-treated group (Figure 2B&C, n =7). Total impulses in the course of 2.5 sec of whisker hair deflection were 736.5±32.4 (n = 7) in vehicle-injected group and reduced to 233.4±36.8 (n = 7, P < 0.001) in vincristine-treated group.

Figure 2. Reduction of tactile-induced impulses in whisker afferent nerves following chronic treatment of mice with vincristine.

(A) Schematic diagram shows basic structures of a whisker hair follicle and experimental setting for recordings of whisker afferent impulses following tactile stimulation. Whisker afferent impulses were recorded using a suction electrode. Tactile stimuli were delivered by deflecting the whisker hair. (B) Sample traces show whisker afferent nerve impulses evoked by a 38-µm whisker hair displacement in a whisker hair follicle of vehicle-injected group (control, top) and a whisker hair follicle of vincristine-injected group (bottom). (C) Summary data show impulse frequencies over the duration of 2.5 s of the hair displacement. At each data point, the frequency was calculated with the events of impulse in every 100 ms. (D) Comparison of the total impulse numbers evoked during whisker hair displacement for the vehicle group (n = 7) and vincristine-treated group (n = 7). Data represent the mean ± SEM. ***P<0.001, student’s T-test.

We examined membrane and action potential properties of Merkel cells in whisker hair follicles to determine if these properties may be affected by the vincristine treatment regimen in animals. Patch-clamp recordings were performed in Merkel cells pre-identified by the fluorescent dye quinacrine in whisker hair follicles freshly harvested from vehicle-injected group and vincristine-treated group (Figure 3A). Under the whole-cell current-clamp configuration, injections of depolarizing currents resulted in membrane depolarization and action potential firing in more than 80% Merkel cells tested in the vehicle-injected group (Figure 3B&C). Depolarizing currents also led to membrane depolarization and action potential firing in ~60% Merkel cells tested in vincristine-treated group. The percentages of action potential firing cells were lower in vincristine-treated group than in vehicle-injected group, but the differences were not statistically significant. For action potential firing Merkel cells in both groups, action potential firing frequencies were not significantly different between the two groups. A plot of voltage-current relationship (Figure 3D) showed that membrane responses to depolarizing currents were similar in vehicle-injected group (n = 10) and vincristine treated group (n = 10). Resting membrane potentials were −54.8 ±7.2 mV (n = 10) in vehicle-injected group and −33.2 ± 10.5 mV (n = 10) in vincristine-treated group, but the differences between the two groups were not statistically significant (Figure 3D).

Figure 3. Effects of chronic vincristine treatment on Merkel cell excitability.

(A) Image shows Merkel cells labeled by quinacrine vital staining in a fresh whisker hair follicle preparation. A patch-clamp recording electrode is indicated in the image. (B) Sample traces show membrane responses and action potentials recorded from Merkel cells pre-identified with quinacrine. Left and right panels are recordings from a Merkel cells of control group and a Merkel cell of vincristine-treated group. Membrane responses were elicited by injections of current steps each at 20 pA. (C) Left panel, graph shows percent of Merkel cells that could (red) or could not (black) fire action potentials. Control group, n = 10; vincristine-treated group, n = 10. Right panel, Summary data of AP frequency in Merkel cells of vehicle control group (n = 10) and vincristine-treated group (n = 10). (D) Voltage-current relationship of membrane responses in Merkel cells of vehicle group (n = 10) and vincristine-treated group (n = 10). Inset, bar graph shows resting membrane potentials of Merkel cells of vehicle group (n = 10) and vincristine-treated group (n = 10). Data represent Mean ± SEM, ns, not significantly different, student’s T-test.

Under the voltage-clamp configuration, we examined voltage-activated currents in Merkel cells harvested from both vehicle-injected and vincristine-treated groups. Similarly to our previous studies (13, 15), whole-cell currents activated by depolarizing voltages were dominated by outward currents (Figure 4A–D). Voltage-activated currents measured in both initial phase (Figure 4C) and later phase (Figure 4D) showed no significant difference between vehicle-injected (n =10) and vincristine-treated groups (n =10).

Figure 4. Effects of chronic vincristine treatment on voltage-gated currents in Merkel cells.

(A) Sample traces of voltage-activated currents recorded from a Merkel cells of vehicle-injected group. (B) Sample traces of voltage-activated currents recorded from a Merkel cells of vincristine-treated group. (C) I-V curves of voltage-activated currents at initial phase recorded from Merkel cells of vehicle-injected group (n = 10) and vincristine-treated group (n = 10). (D) I-V curves of voltage-activated currents at late phase recorded from Merkel cells of vehicle-injected group (n = 10) and vincristine-treated group (n = 10). Data represent Mean ± SEM, ns, not significantly different, two-way ANOVA.

We determined whether chronic vincristine treatment impaired mechanical transduction of Merkel cells in whisker hair follicles. In this set of experiments, whole-cell patch-clamp recordings were performed on Merkel cells harvested from vehicle-injected and vincristine-treated animals and mechanically activated currents (MA) were elicited by displacement of Merkel cell membranes with a mechanical probe (Figure 5A). As shown in Figure 5B&C, mechanically activated (MA) currents mediated by Piezo2 channels in Merkel cells (12, 13, 17) were evoked in a displacement dependent manner. However, with displacements of 1.5 and 2 µm, the amplitudes of MA currents were significantly smaller in Merkel cells of vincristine-treated group than in vehicle-injected group (Figure 5C).

Figure 5. Suppression of mechanically activated currents in Merkel cells following chronic vincristine treatment.

(A) Schematic diagram illustrates the patch-clamp recording of mechanically activated (MA) currents from Merkel cells. MA currents were evoked by a mechanical probe which delivered step-wise forward movements to indirectly displace Merkel cell membranes. (B) Sample traces of whole-cell MA currents recorded from a Merkel cell of vehicle-injected group (black) and a Merkel cell of vincristine-treated group (read). Displacement distance, 2 µm. Cells were voltage-gated at holding potential of −70 mV. (C) Summary data of MA currents recorded from Merkel cells of vehicle-injected group (n = 10) and vincristine-treated group (n = 10). MA currents were evoked at displacement steps up to 2 µm with a 0.5-µm increment each step. Data represent Mean ± SEM, *P < 0.05. **P < 0.01, two-way ANOVA with Bonferroni post-hoc tests.

Discussion

In the present study we have for the first time shown that treatment of animals with vincristine results in the impairment of in vivo and in vitro tactile responses and reduction of mechanically activated currents in Merkel cells. These findings suggest that the loss of function of mechanical transduction in Merkel cells is a mechanism underlying the impairment of the sense of touch in chemotherapy with vincristine.

We have used whisker hair follicles, which functionally resemble human fingertips (18–20), to show that in vivo whisker tactile behavioral responses are impaired following the vincristine treatment regimen. Chemotherapy with vincristine and a number of other anti-cancer drugs in human patients induces sensory dysfunctions including numbness, tingling sensation, and pain in the body, with fingertips and toes are more severely affected (6). These symptoms are collectively termed chemotherapy-induced peripheral neuropathy because it has been thought that damage of afferent nerves account for these sensory dysfunctions (2, 21, 22). However, the present study shows that Merkel cells are sites where vincristine impairs Merkel cell mechanical transduction and tactile responses. This finding suggests that Merkel cells are very vulnerable to the cytotoxicity of chemotherapy drugs.

Whisker hair follicles contain several types of mechanoreceptors including Merkel discs, lanceolate endings and reticular endings (23). Merkel discs are a major type of mechanoreceptors in whisker hair follicles, which largely account for whisker afferent impulses following tactile stimulation of whisker hairs. Therefore, the impairment of mechanical transduction functions of Merkel cells by vincristine would significantly contribute to the reduced tactile responses measured with both impulses of whisker hair follicle afferent nerves and whisker tactile behavioral responses. In the present study, we also tested whether vincristine affects Merkel cell membrane and action potential properties to subsequently affect tactile transmission from Merkel cells to whisker afferent nerves. There was a tendency of a reduction in the number of AP firing Merkel cells, AP firing frequency, and resting membrane potentials following vincristine treatment. However, the differences in the properties were not statistically significant between the vehicle and vincristine-treated groups. We show that mechanically activated currents were reduced following vincristine treatment. Previous studies have shown that MA currents in Merkel cells are mediated by Piezo2 channels (12, 13, 17). Thus, the reduction of MA currents in Merkel cells is likely due to a down-regulation of the expression and/or functions of Piezo2 channels. Since the reduction of MA currents could be observed days after the termination of vincristine regiment, it is more likely that vincristine caused down-regulation of Piezo2 channel expression. Alternatively, vincristine may impair Piezo2 channel functions via its effects on the cytoskeleton in Merkel cells if Pieozo2 channel functions require the cytoskeleton. This is because vincristine and other vinca alkaloids are known to inhibit microtubule polymerization to disrupt cytoskeleton in cells. Piezo2 channels are also expressed on Merkel disc nerve endings and lanceolate endings in hair follicles (14). This raises the possibility that Piezo2 channel-mediated mechanical transduction in these nerve endings may be also impaired following the chronic treatment with vincristine. It would be interesting in future studies to explore this possibility and also to explore whether Piezo2 channels may be targeted by other anti-cancer drugs to account for chemotherapy-induced sensory dysfunctions.

Highlights.

• Chronic treatment of mice with vincristine impaired whisker tactile behavioral responses, reduced mechanically evoked whisker afferent impulses, and decreased Piezo2 currents in Merkel cells. Loss of functions of Piezo2 channels in Merkel cells may contribute to the loss of the sense of touch following the chemotherapy treatment regimen with vincristine.