Lithium prevents the neurotoxic effects of paclitaxel mediated through TRPA1 channels

Abstract

Paclitaxel (PTX) is a drug commonly used in cancer chemotherapy despite its neurotoxicity. TRPA1 channels are essential mediators of sensory transduction and nociception. These cation channels are linked to PTX-induced neurotoxicity, which Li⁺ prevents. This study aimed to examine the effects of Li+ on PTX-induced neurotoxicity and on TRPA1 channels. We utilized the SH-SY5Y cell line to assess cell viability via the MTT assay. Intracellular Ca²⁺ concentration in Fura-2-loaded cells was measured using spectrofluorometry. TRPA1 channel activity was evaluated with whole-cell patch-clamp recordings. The effects of PTX, Li⁺, and TRPA1 agonists and antagonists were tested. Motor function, thermal response, and cognitive performance were assessed in adult Wistar rats with neuropathy induced by PTX. PTX (100 nM) significantly reduced cell viability, and Li⁺ (10 mM) alleviated this effect. AITC (300 µM), a TRPA1-selective agonist, decreased cell viability, with a more pronounced impact when PTX was present. A967079 (10 µM), a selective TRPA1 antagonist, significantly lessened the cytotoxicity caused by PTX. Li⁺ reduced the cytotoxic effects of TRPA1 activation both with and without PTX. PTX increased TRPA1 currents and amplified TRPA1-mediated intracellular Ca²⁺ increase, while Li⁺ neutralized both effects. Additionally, PTX causes sensorimotor and cognitive neuropathy, which was reversed by Li⁺ treatment. These findings suggest that Li⁺ may act as a neuroprotective agent, preventing neuronal damage caused by PTX via TRPA1 channel pathways.

Introduction

Paclitaxel (PTX) is a microtubule-stabilizing drug that is often used as the first-line chemotherapy for the most common cancer types. ¹ However, PTX is not without risks, as it can cause numerous side effects, including peripheral and central neuropathy. ² Peripheral neuropathy (PTX-induced peripheral neuropathy, PIPN) is characterized by pain, tingling, numbness, and sensory disturbances in the hands and feet, significantly impacting patients’ quality of life and potentially leading to dose reductions or treatment discontinuation. Central neuropathy (PTX-induced cognitive impairment, PICI) involves symptoms such as cognitive impairment, memory deficits, and mood changes, ³ which can affect patient quality of life and may necessitate modifications in treatment regimens. These conditions are caused by severe and irreversible neurotoxicity through various mechanisms, including the disruption of calcium (Ca²⁺) homeostasis, axonal degeneration, impaired neurogenesis, neuroinflammation, and apoptosis. ⁴

Transient receptor potential (TRP) channels are widely expressed calcium transporters, and they are essential actors in Ca²⁺ homeostasis because of their ability to respond to various stimuli, such as mechanical distension, pH variations, arachidonic acid, and cannabinoids. ⁵ One type of TRP channel, TRP ankyrin 1 (TRPA1) protein channels, are cation channels with high selectivity for Ca²⁺ ions and are responsible for various processes, such as sensory transduction and cognitive function.^6,7 TRPA1 is extensively expressed in neurons and nonneuronal cells, such as epithelial cells. However, it is expressed mainly in small-diameter C or A delta fibers of sensory neurons, such as dorsal root ganglia (DRGs) and trigeminal neurons, and is involved in nociception and neurogenic inflammation.^8–10 TRPA1 expression has also been demonstrated in SHSY5Y cells, the cellular model employed in this study. ¹¹

TRPA1 primarily detects an extremely wide variety of exogenous stimuli that may produce cellular lesions. This includes many electrophilic compounds with toxic potential. In addition, TRPA1 has been reported to be activated by cold, heat, and mechanical stimuli, and multiple factors, including Ca²⁺, trace metals, pH, and reactive oxygen, modulate its function. Although TRPA1 has been mainly associated with cold nociception,^12,13 this channel is also involved in heat nociception, as demonstrated in mice,^14,15 rats,^16,17 and Drosophila. ¹⁸ TRPA1 is also involved in acute and chronic pain and inflammation and plays key roles in the pathophysiology of nearly all organ systems. ¹⁹

TRPA1 channels are directly implicated in the neurotoxicity induced by PTX. PTX increases TRPA1 expression in DRG neurons by releasing tumor necrosis factor-α (TNFα) from satellite glial cells in the DRG. ²⁰ This proinflammatory cytokine is an essential mediator of PTX-induced learning and memory impairment. ²¹ Additionally, activation of TRPA1 channels induces calcitonin gene-related peptide release in DRG neurons, a process inhibited by PTX. ²² In preclinical models of PTX-induced peripheral neuropathy, TRPA1 channels are sensitized by proteinase-activated receptor 2 activation, ²³ and the inhibition of TRPA1 channels reduces cold and mechanical allodynia induced by PTX. ²⁴

Lithium (Li⁺) is a free cation that exerts a neuroprotective effect via numerous mechanisms of action. ²⁵ Li⁺ decreases GSK3β activity, promoting its phosphorylation; attenuates phosphatidylinositide signaling; increases the gene expression of neurotrophic factors, including BDNF and VEGF; and regulates inflammatory homeostasis, promoting neurogenesis and preventing apoptosis.^26,27 In preclinical models, Li⁺ pretreatment reversed PTX-induced peripheral neuropathy by restoring calcium signaling and preventing neuronal calcium sensor 1 (NCS-1) degradation.^28,29 Recent studies have shown that Li⁺ treatment before and after PTX administration rescues cognitive decline via protein kinase C (PKC) inhibition. ³⁰ Interestingly, the involvement of PKC signaling in PTX-induced peripheral neuropathy has also been reported. ³¹ TRPA1 channels can be regulated by PKC phosphorylation in this context.^11,23

Although there is no published evidence regarding the effect of Li⁺ on neuronal viability or the regulation of TRPA1 channels in the context of PTX-induced neurotoxicity (PIN), studies have previously shown the regulatory effects of Li⁺ on other members of the TRP family, such as TRPP2 ³² and TRPV4, ³³ along with other neuroprotective effects. Considering the activation of the TRPA1 channel by PTX and its association with neurotoxicity, this study aimed to determine the potential neuroprotective effect of Li⁺ on the neurotoxicity induced by PTX and TRPA1 in both neuronal cell culture and preclinical models.

Methods

Cellular model

Cell cultures

The human neuroblastoma SH-SY5Y cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mixture Ham’s F-12 (1:1) supplemented with 10% fetal bovine serum (FBS; Gibco, CA, USA), 1% penicillin–streptomycin-neomycin (PSN) antibiotic mixture (Gibco, CA, USA), and 1% MEM nonessential amino acid solution (Sigma) at 37 °C in humidified 95% air with 5% CO ² . The medium was replaced every 2 or 3 days. When the cells reached 80% confluence, they were detached with TripLE Express (1X; Gibco) and seeded in 6-well and 96-well plates on the basis of the experimental requirements.

Cell viability assays

Cell viability was determined via the 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma–Aldrich). Briefly, SH-SY5Y cells were seeded at a density of 2 × 10⁴ cells/well in 96-well plates and incubated at 37°C in a 5% CO₂ incubator. After overnight incubation and subsequent reagent exposure, MTT solution (0.5 mg/ml) was added to each well, and the mixture was incubated with the cells for 4 h. After incubation, the MTT solution was removed, and 100 µl of DMSO was added to dissolve the formazan crystals successfully. The absorbance was measured at 570 and 630 nm with a microplate reader (ELX800; BioTek Instruments, Inc., Winooski, VT, USA).

TRPA1 electrophysiological recordings

The whole-cell patch-clamp technique was used to record the membrane current (voltage clamp) and membrane potential (current clamp) in cultured SH-SY5Y cells. The cells were placed in a recording chamber attached to an inverted microscope (Nikon, Tokyo, Japan). Patch pipettes (Clark PG150T glass, from Harvard Apparatus Ltd., Edenbridge, Kent, UK) were pulled and polished (P-97, Sutter Instrument, Novato, CA, USA) to resistances of 5–10 MΩ. After a seal with a resistance greater than 5 GΩ was obtained, the membrane currents were recorded via an Axopatch 200B amplifier with a CV203BU headstage (Axon Instruments Inc., Union City, CA, USA). Voltage clamp signals were generated by a Digidata 1440A interface (Axon Instruments, Inc.). The membrane currents were filtered at 2 kHz and digitized at a sampling rate of 10 kHz. The signals were acquired and analyzed via pCLAMP 10.0 (Axon Instruments Inc.). At the beginning of each experiment, the junction potential between the pipette solution and the bath solution was electronically adjusted to zero. The macroscopic current values were normalized as pA/pF.

The standard external solution contained (in mM) 30 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose, and 180 mannitol, and the pH was adjusted to 7.4 at 25°C with 1 M NaOH. The standard pipette solution contained (in mM) 25 KCl, 5 K-gluconate, 5 NaCl, 4 CaCl₂, 4 MgCl₂, 10 HEPES, 5 glucose, 20 BAPTA, and 180 mannitol, and the pH was adjusted to 7.1 at 25°C with 1 M NaOH. The osmolarity of these solutions was adjusted to 290 mOsm/l using 5 M mannitol. The free Ca²⁺ concentration was estimated via Maxchelator software (Dr. C. Patton, Stanford University); with this software, a value of 102 nM was derived for the intrapipette solution. In the Na⁺-free solutions, Na⁺ was replaced with NMDG⁺ at equimolar concentrations. EGTA (1 mM) was added to the Ca²⁺-free solutions to chelate trace contaminants of this ion. For Cl^-free intrapipette solutions, NaCl and KCl were replaced with Na-gluconate and K-gluconate, respectively. The osmolarities of all the solutions were measured via an Advanced Model 3320 Micro-Osmometer (Advanced Instruments, Norwood MA, USA). The solutions were switched using a cFlow V2.x flow controller (Cell Microcontrols, Norfolk, VA, USA).

Measurements of the intracellular calcium concentration ([Ca²⁺]_i)

SH-SY5Y cells were loaded with Fura-2 (5 µmol/l) by incubation in HBS for 30 min at 20°C, followed by 15 min at 37°C. The cell suspension was then centrifuged, and the cells were resuspended in the appropriate experimental medium before being transferred to a cuvette. Fluorometer measurements were performed for 300 s at 37°C with magnetic stirring (FP-6500 spectrophotometer, Jasco, Tokyo, Japan) during the measurements. The dye was alternately excited at 380 and 340 nm, and the fluorescence emission was measured at 510 nm. Using a previous method, the 380 nm/340 nm signal ratio was calibrated before every experiment. ³⁴ Briefly, the fluorescence ratio was measured in HBS without CaCl₂ and supplemented with EGTA (1 mmol/l) and in 2 mmol/l Ca²⁺ HBS supplemented with ionomycin (300 nmol/l), a concentration of Ca²⁺ at which Fura-2 is saturated. The maximal and minimal ratios (R_max and R_min) were obtained under these two conditions, and the [Ca²⁺]_i values were derived via the following equation:

$${[{\mathrm{Ca}}^{2+}]}_{\mathrm{i}}={\mathrm{K}}_{\mathrm{d}}([{\mathrm{R}-\mathrm{R}}_{\min }]/[{\mathrm{R}}_{\max }-\mathrm{R}])({\mathrm{Sf}}_2{/\mathrm{Sb}}_2),$$

where K_d is the dissociation constant for Fura-2 (224 nmol/l), R is the experimentally measured ratio, Sf₂ is the fluorescence measured at 380 nm under Ca²⁺-free conditions, and Sb₂ is the fluorescence measured at 380 nm with saturating Ca²⁺ (2 mmol/l).

Preclinical model

Animals

Adult (5-week-old) male Wistar rats were used in this study. The Ethics Committee of Universidad Tecnológica de Pereira reviewed and approved the study (CBE-SYR-162016). Upon arrival, the rats were randomized, assigned to cages, and allowed to habituate for 5 days. The animals were maintained on a 12-h light/dark cycle with food and water ad libitum. All experiments were performed during the light cycle in rats weighing approximately 250–300 g.

To establish chemotherapy-induced peripheral neuropathy, the animals received one intraperitoneal injection of PTX (4 mg/kg) per day or its corresponding vehicle on four subsequent days along 7 days (days 1, 3, 5, 7) to reach a final cumulative dose of 16 mg/kg. PTX was dissolved in Cremophor EL:ethanol (1:1) and then diluted in sterile 0.9% saline solution to a final concentration of 2 mg/ml. For vehicle treatment, Cremophor EL:ethanol (1:1) was diluted in 0.9% saline solution to a final concentration of 3.2%. In addition, the rats were either injected with Li⁺ (12.8 mg/kg, subcutaneously) or the TRPA1 antagonist A967079 (A96, 10 mg/kg, intraperitoneally) before every PTX or vehicle application. Li⁺ was dissolved in 0.9% saline solution to a final concentration of 5 mg/ml, and A96 was dissolved in DMSO to 10 mg/ml. The final volumes administered to the animal were 600–750 µl and 250–300 µl, respectively. All reagents were prepared immediately before use. All the investigators involved with the experiments were blinded throughout the entire process. The well-being of the animals was checked daily.

Thermal algesia assessment

Thermal algesia was assessed using a hot plate apparatus (LE7406, Harvard Apparatus, Massachusetts, USA). The rats were placed in a container with the surface temperature maintained at 52°C. The thermal latency was measured, which was defined as the time (seconds) elapsed from the time the rat was placed on the surface until the time the rat either licked, shook, lifted its hind paws, or jumped out of the container. A cutoff time of 15 s was established to prevent tissue injury. Testing was performed at the end of the PTX protocol (day 8 after the first PTX application) and again 7 days later (day 15 after the first PTX application). These evaluations were conducted after the rotarod performance test (Figure 4(f)).

Cognitive impairment assessment

The Morris water maze (MWM) test was used to assess the spatial learning and memory abilities of the rats. The experiments took place in a tank measuring 110 cm in diameter and 55 cm in height, filled with water to a depth of 40 cm, and maintained at 26°C until testing began. A transparent, round platform (10 cm in diameter) was placed 1 cm below the water surface during the spatial learning sessions (Days 2–5) and positioned just above the water surface during the visible platform session (Day 1). Obvious cues, such as geometric shapes, were attached to the tank walls. Each day, four tests were conducted, with each rat placed in the tank from one of four different directions over a 5-day period. The time taken to reach the platform was recorded. If a rat did not get the platform within 60 s, it was gently guided onto the platform and allowed to stay there for 20 s. For testing on Day 6, the platform was removed for the spatial reference memory probe trial. The variables recorded included the latency to reach the platform during the learning trials (in seconds) and the number of platform crossings on Day 6. An automated system recorded the behaviors of the animals.

Motor coordination assessment

Motor coordination was assessed via the rotarod test. The rats were placed on a rotating rod (LE8305, Harvard Apparatus, Massachusetts, USA) in individual compartments with increasing speed starting at 10 rpm and reaching a maximum speed of 40 rpm in 300 s. To allow the animals to learn the task, training sessions were carried out over 5 days, consisting of three trials per day, with an increase of 75 s daily in the time spent on the rod. The rats were placed back on the rod when they fell off within the designated time. The baseline latency was recorded by measuring the initial latency to fall off the rod on the last day. Subsequently, testing was performed 8 and 15 days after the first PTX application (Figure 4(f)). For each time point, three trials were averaged.

Statistical analysis

The data analysis was conducted using the analysis tools available in GraphPad Prism 8.0. The results are presented as the mean ± standard error of the mean (SEM) or as the median with minimum and maximum values for each data set, where n indicates the number of assays performed or the number of cells tested. Each experimental observation was repeated at least three times. When possible, an unpaired Student’s t-test was used; otherwise, the appropriate nonparametric test was applied. All statistical tests were two-tailed, and a p-value less than 0.05 was considered significant.

Results

Cell viability

We first exposed SH-SY5Y cells to PTX (1 µM) to validate the cytotoxic effects of the compounds through the MTT assay. After 6 h of exposure to PTX, the mean viability significantly decreased (Figure 1). When Li⁺ (10 mM) was administered simultaneously with PTX, the cell survival was similar to the control. Li⁺ alone did not affect cell survival.

Figure 1.

Cell viability was measured using the MTT assay in SH-SY5Y cells treated with various agents: PTX (1 µM), Li⁺ (10 mM), AITC (300 µM), A96 (10 µM), as indicated. Significant differences and p-values are provided for each condition.

To assess the cytotoxic role of TRPA1 channel activation, the selective agonist allyl isothiocyanate (AITC) was administered at a concentration of 300 µM for 6 h alone or in combination with PTX (1 µM). We found a significant decrease in cell survival when AITC alone was administered. This neurotoxic effect was significantly enhanced when AITC was combined with PTX (Figure 1).

We reversed these cytotoxic effects of PTX and AITC when the TRPA1 channel was blocked with the selective antagonist A96 (10 µM).

Measurements of [Ca²⁺]_i

PTX (1 µM) and AITC (300 µM) significantly increased the intracellular Ca²⁺ concentration, which intensified when both compounds were used simultaneously. This PTX-induced Ca²⁺ increase was inhibited when the TRPA1 selective antagonist A96 (10 µM) was added, confirming that TRPA1 is involved in the Ca²⁺ entry pathway (Figure 2(a) and (b)). Next, we tested the effect of Li⁺ under these conditions. When lithium was added, there was a significant decrease in the amount of Ca²⁺ entering the cell induced by PTX and AITC, suggesting that this molecule modulates the TRPA1 channel and its Ca²⁺ dynamics. When cells were treated with Li⁺ alone, there was no increase in intracellular Ca²⁺ concentration (Figure 2(a) and (b)).

Figure 2.

(a) Representative recordings of intracellular Ca²⁺ concentration in Fura-2-loaded SH-SY5Y cells under steady-state conditions and following different treatments, as indicated. Fluorescence was recorded for 150 s. The arrow indicates when the pharmacological agents were added to the external solution. (b) Comparison of the mean maximum intracellular Ca²⁺ concentration in Fura-2-loaded SH-SY5Y cells following different treatments, as indicated. n is specified in each case. Significant differences are also shown with the corresponding p-value.

Electrophysiology recordings

Whole-cell currents in SH-SY5Y cells were recorded using the voltage patch-clamp technique. We applied a descending ramp protocol, starting from a holding potential of −40 mV and then ramping to +100 mV, followed by a ramp to −100 mV. To characterize the TRPA1 current, we first tested the specific agonist AITC (300 µM), which elicited a predominantly inward current with a voltage dependence. These currents were abolished in the presence of the TRPA1 antagonist A96 (10 µM), confirming their identity as I_TRPA1.

Next, currents were recorded in cells treated with PTX (1 µM) alone, in the presence of A96 (10 µM), or Li⁺ (10 mM). I_TRPA1 increased in the presence of PTX, and this increase was wholly or partially abolished by the effect of Li⁺ (Figure 3).

Figure 3.

(a) Typical I-V recordings of current from a SH-SY5Y cell, elicited by a ramp protocol from −100 mV to +100 mV and activated by AITC under control conditions and in the presence of PTX, A96, Li⁺, or combinations of these agents, as indicated. (b) Typical I–V relationships of the AITC-induced current in SH-SY5Y cells under control conditions and with PTX, A96, Li⁺, or their combinations, as indicated. n = 12 in all cases. (c) Typical current (I) recordings at −60 mV with AITC, PTX, and PTX + Li⁺, as shown. (d) Comparison of the mean maximum normalized current recorded at −60 mV and +60 mV under these conditions. n = 12 in all cases. *denotes significant differences (p < 0.0001) with respect to the non-marked bars and the bar marked with**.

Preclinical model

The hot plate algesiometric test evaluated PTX-induced sensory neuropathy by measuring heat hyperalgesia. The PTX group exhibited significantly lower latency starting from Day 8 after the initial PTX administration, with relatively stable hyperalgesic scores on Day 15 for both the pretreatment and control groups. The concurrent administration of Li⁺ or the TRPA1 antagonist A96 reversed neuropathy, as indicated by a latency time similar to that of the control group. Additionally, when a channel agonist (AITC) was introduced, an increase in PTX-induced neuropathy was observed (Figure 4(a)).

Figure 4.

(a) Reaction times measured by the hot plate (HP) algesimeter test under various conditions; n is indicated for each case. Significant differences are shown with their p-values. (b) Latencies to reach the platform in the MWM test during training trials, under different conditions as noted. *indicates significant differences (p < 0.0001) for rats treated with PTX compared to the control. Note that there are no significant differences between the control and any other conditions. (c) Platform crossing times in the MWM test, under the same conditions as in (b). (d) Representative swimming paths in the MWM test for rats under the specified conditions. (e) Fall latencies measured by rotarod (R) testing. Note that there were no significant differences between the control group and rats treated with PTX. As a result, further experiments under other conditions were not conducted. (f) Timeline of the experiments performed in the animal model. The short arrow indicates the days on which the drugs were administered to the animals, as specified in the protocol. The larger arrow indicates the days on which HP and R testing were performed. *denotes the days when the training tests were done in WMW to record the latency time to the platform. **denotes the day when testing without the platform was performed to record the number of platform crossings. HP: hot plate. MWM: Morris water maze. R: Rotarod.

Spatial learning and memory in the neuropathic pain rat model were assessed via the MWM test. The results of the training days indicated that the average escape latencies and platform crossing times in all the groups gradually decreased after the training sessions. Statistical analysis revealed a significant effect of PTX treatment, increasing both variables. This effect was completely reversed by treatment with Li⁺ and A96, a TRPA1 antagonist (Figure 4(b)–(d)).

PTX did not affect animal motor performance, as shown by the rotarod test. The PTX group exhibited no differences from the vehicle group in motor coordination and balance, and both groups of rats showed similar improvements in motor performance over time compared to pre-PTX scores, indicating they had comparable ability to learn motor skills (Figure 4(e)). When the PTX dose was increased to 6 mg/kg in an effort to produce any effects, all the rats died.

Discussion

Li⁺ is a therapeutic agent with many physiological effects that are not fully understood. Here, we show how this molecule can modulate the TRPA1 channel to prevent the development of PIN through a mechanism involving the rescue of Ca²⁺ dynamics, with no effect on cell viability.

Among the several mechanisms associated with PTX function, PTX exerts its chemotherapeutic effect by stabilizing microtubules, resulting in mitotic arrest in rapidly dividing cancer cells. This antineoplastic mechanism may not be the primary driver in the development of peripheral neuropathy. Evidence suggests that PTX binds to neuronal calcium sensor 1 (NCS-1) and activates TLR4, sensitizes TRPV4 and the inositol trisphosphate (InsP3) receptor, disrupts [Ca²⁺]_i dynamics, triggers Ca²⁺-dependent calpains, induces a proinflammatory signal, and ultimately leads to axonal degeneration.^2,35 New evidence provided by our research group has shown that NCS-1 directly interacts with TRPV4 and TRPA1 channels, modifying their expression and activity.^11,33,36,37 This constitutes an alternative pathway that could explain why PTX induced an increase in TRPA1-induced Ca²⁺ entrance, as shown by our results.

Accumulating data have shown that Li⁺ can prevent and diminish the painful symptoms of PIPN.^28,29 This therapeutic molecule is the first-line treatment for bipolar disorder; it can be beneficial for other neurologic conditions, including traumatic brain injury, ²⁵ spinal cord injury, ³⁸ ischemia, ³⁹ excitotoxicity, ⁴⁰ and neurodegenerative diseases. ⁴¹ The underlying mechanisms are still debated but include neurotrophic, neuroprotective, antioxidant, and anti-inflammatory actions. ⁴² The evidence suggests that Li⁺ is a neuroprotective agent against neurodegeneration by preventing inflammation, oxidative stress, apoptosis, and mitochondrial dysfunction via the PI3/Akt/GSK3β and PI3/Akt/CREB/BDNF signaling pathways. ⁴³

Our data did not reveal a protective effect of Li⁺ against PTX-induced death in SH-SY5Y cells. We hypothesize that the neuroprotective role of Li⁺ against PIPN is not related to acute PTX-mediated toxicity. Nevertheless, it prevents chronic Ca²⁺ dyshomeostasis by interfering with the binding of PTX to NCS-1, inhibiting the sensitization of the TRPA1 channel and the downstream activation of Ca²⁺-dependent caspases. Our electrophysiology and Ca²⁺ measurements support this hypothesis by showing how Li⁺ rescues the PTX/AITC-induced increases in I_TRPA1 and [Ca²⁺]_i.

Previous studies have reproduced a model of PTX-induced neuropathy using different doses, some as low as a total dose of 4 mg/kg and others as high as 80 mg/kg.^44,45 In our study, we established PTX-induced sensory peripheral neuropathy and a central neuropathy, with a final cumulative dose of 28 mg/kg; the fatal dose was 42 mg/kg in total.

Li⁺ was shown to prevent the development of PIPN and CICI in a mouse model. ²⁹ We obtained similar results in our preclinical model with Wistar rats, where Li⁺ and TRPA1 antagonists reversed PTX-induced sensory and cognitive neuropathy. Moreover, treatment with a TRPA1 agonist enhanced this effect, which confirms that the mechanism driving PTX neurotoxicity involves TRPA1 activation. Furthermore, in a previous study, our group demonstrated that PTX increased TRPA1 protein expression, ¹¹ indicating that the effect of PTX on TRPA1 may occur at both the molecular and functional levels.

One limitation of this study is that other ion channels, such as TRPV1 and TRPM3, can be activated by heat at the temperatures used, ¹⁵ and TRPA1 may undergo desensitization at temperatures higher than 40°C, ⁴⁶ effects not ruled out by the methodology employed. Further studies considering these limitations should be conducted. Another limitation is that the impact of learning, habituation, or sensitization induced by repeated exposure to thermal or motor tasks were not assessed in this study; therefore, their influence on the results is unpredictable.

Nevertheless, these results demonstrate that the TRPA1 channel plays a role in the development of PIN. ²⁹ Additionally, these data suggest a new mechanism of action for Li⁺ as an indirect modulator of TRP channels, with great potential for treating PIPN, PICI, and other TRP-related conditions. Li⁺ could serve as a therapeutic tool to help oncologists maintain higher doses of PTX, enhancing treatment efficacy without harming patients’ neurological health. This potential is significant in cancers where PTX-based treatments are standard, such as breast, ovarian, and lung cancers.

Although these promising findings are encouraging, further experimental studies and clinical trials are needed to clarify the mechanisms of action, identify potential drug interactions, determine optimal dosing schedules, evaluate long-term safety, and assess efficacy across different cancer types and patient populations. Additionally, interdisciplinary collaboration among pharmacologists, physiologists, oncologists, neurologists, and psychiatrists is essential to safely incorporate Li⁺ into cancer treatment protocols. Future research on this pathway should include TRP channels as part of the mechanism involved in the actions of Li⁺ and PTX.

In conclusion, Li⁺ offers a new strategy to prevent paclitaxel-induced peripheral neuropathy, providing hope for improved cancer treatment outcomes and better patient quality of life. Ongoing research and clinical trials are essential to fully explore Li⁺’s potential in this field and turn these findings into practical solutions for cancer care.