Abstract
Background
Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting adverse effect of anticancer therapy and is frequently accompanied by anxiety-related symptoms. Effective preventive treatments are lacking, and available pharmacological options provide limited relief, highlighting the need for adjunctive non-pharmacological strategies in cancer care. Emerging evidence suggests that astrocyte-neuron crosstalk within the anterior cingulate cortex (ACC) contributes to chronic pain and pain-associated emotional disorders. Electroacupuncture (EA) has shown beneficial effects in CIPN; however, the central mechanisms underlying its therapeutic actions remain unclear. This study investigated whether astrocyte-mediated neuronal activation in the ACC is associated with the analgesic and anxiolytic effects of EA in CIPN.
Methods
A mouse model of CIPN was established by repeated intraperitoneal administration of paclitaxel. Mice received EA or sham EA once daily for seven consecutive days. Mechanical allodynia, thermal hyperalgesia, and anxiety-like behaviors were assessed using the von Frey test, Hargreaves test, and open field test, respectively. Astrocytic and neuronal activation, GAT-1 expression, and GABA levels in the ACC were evaluated by immunofluorescence staining, Western blotting, and ELISA. Chemogenetic approaches were used to selectively activate or inhibit ACC astrocytes.
Results
CIPN mice exhibited significant mechanical and thermal hypersensitivity and anxiety-like behaviors. EA significantly alleviated pain hypersensitivity and anxiety-like behaviors while suppressing astrocytic activation, downregulating GAT-1 expression, restoring GABA levels, and reducing neuronal activation. Chemogenetic inhibition of ACC astrocytes alleviated pain- and anxiety-like behaviors in CIPN mice, whereas astrocytic activation markedly attenuated the beneficial effects of EA.
Conclusion
EA alleviates CIPN-induced pain hypersensitivity and anxiety-like behaviors by suppressing astrocytic activation, normalizing GAT-1 expression and GABA levels, and attenuating neuronal activation in the ACC. These findings identify astrocyte-mediated neuronal activation as a novel mechanism involved in EA-induced analgesia and a potential therapeutic target for CIPN and its associated affective comorbidities. Overall, this study provides preclinical mechanistic evidence for further evaluating EA as an adjunctive non-pharmacological approach for the integrated management of neuropathic pain and affective symptoms in patients with CIPN.
Keywords: electroacupuncture, chemotherapy-induced peripheral neuropathy, anterior cingulate cortex, astrocyte
Introduction
Chemotherapy-induced peripheral neuropathy (CIPN) represents a major neurological complication of anticancer treatment and a leading cause of dose reduction or treatment discontinuation.1 A recent global meta-analysis involving 33667 patients estimated an overall CIPN prevalence of 51.9%.2 Characterized by persistent nociceptive hypersensitivity, including mechanical allodynia and thermal hyperalgesia, CIPN is frequently accompanied by affective disturbances such as anxiety and depression, imposing a substantial burden on patients’ overall well-being.3,4 According to the American Society of Clinical Oncology guidelines, no pharmacological agent is recommended for CIPN prevention, and duloxetine is the only drug supported by appropriate evidence, although its clinical benefit is limited.5 Thus, identifying novel therapeutic strategies and elucidating their underlying mechanisms remain important clinical challenges.
Although extensive research on CIPN has been conducted at peripheral and spinal levels, increasing evidence has highlighted an essential contribution of supraspinal circuits to the pathogenesis of CIPN and its associated affective disorders.6,7 Within the pain matrix and limbic system, the anterior cingulate cortex (ACC) is a key region involved in the integration of nociceptive sensory processing with affective appraisal.8–10 Enhanced neuronal excitability and aberrant synaptic plasticity within the ACC have been widely reported in neuropathic pain models, contributing to pain hypersensitivity and anxiety-like behaviors.11–14 In addition, recent research has shown that interventions targeting ACC neuronal signaling attenuated mechanical and thermal hypersensitivity in CIPN mice.15 Notably, inhibition of ACC neuronal activity alleviates both nociceptive and affective symptoms, highlighting the ACC as a potential therapeutic target for CIPN.16,17
In addition to neurons, astrocytes have emerged as active regulators of pain-related neural circuits.18 Astrocytic activation is highly associated with chronic pain states and has been observed in multiple brain regions, including the ACC.19–21 Activated astrocytes can profoundly influence neuronal excitability by regulating neurotransmitter homeostasis and excitatory-inhibitory balance.22 γ-aminobutyric acid (GABA)-mediated inhibition, specifically, is linked to nociceptive modulation.23 Previous research has established that reductions in GABAergic signaling contribute to cortical hyperexcitability and central sensitization during neuropathic pain.24,25 Restoration of GABA levels or enhancement of GABA receptor activity effectively attenuates pain-related behaviors.26–28
As a predominant transporter for GABA reuptake in the central nervous system, GABA transporter-1 (GAT-1) is essential for controlling extracellular GABA availability.29,30 Excessive GAT-1 activity can enhance GABA clearance from the synaptic environment, thereby weakening inhibitory neurotransmission and promoting neuronal hyperexcitability.31,32 Notably, increased GAT-1 expression has been reported in rodents with paclitaxel (PTX)-induced neuropathic pain, whereas GAT-1 inhibitor NO-711 can inhibit thermal hyperalgesia and allodynia in CIPN mice, indicating that dysregulation of GAT-1/GABA signaling may contribute to CIPN pathogenesis.33,34
Acupuncture has been increasingly recognized as a promising non-pharmacological therapy for CIPN. Clinical and experimental studies have demonstrated its efficacy in reducing neuropathic pain and pain-associated emotional disturbances.35–37 Besides, it has been reported that EA intervention can modulate astrocyte activation and the GABAergic system within the ACC region, thus alleviating pain-anxiety comorbidity.38,39 Our previous work demonstrated that electroacupuncture (EA) attenuates PTX-induced pain hypersensitivity through spinal mechanisms.40 However, whether EA exerts its anti-nociceptive and anxiolytic effects via modulation of supraspinal astrocyte-neuron interactions remains unexplored. Therefore, this study aimed to investigate the role of ACC astrocytes in the therapeutic effects of EA on CIPN. We hypothesized that EA ameliorates CIPN-induced hyperalgesia and anxiety-like behaviors by suppressing astrocytic activation, normalizing GAT-1/GABA-related changes, and reducing neuronal activation within the ACC.
Materials and Methods
Experimental Animals
Eight-week-old male C57BL/6J mice, purchased from Shanghai SLAC Laboratory Animal Co., Ltd., were housed under SPF conditions in groups of 4–6 per cage and maintained on a 12-h light/dark cycle with food and water ad libitum. Environmental conditions were controlled at a temperature of 22–26°C and a relative humidity of 40–50%. All experimental procedures were performed in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (No. FJTCM-IACUC-2024191).
CIPN Model Induction
A mouse model of CIPN was established by repeated administration of PTX, following the procedure described by Kyte et al,41 Briefly, mice were administered PTX (2 mg/kg; MedChemExpress, USA) via intraperitoneal injection on day 1, day 3, day 5, and day 7, yielding a total dose of 8 mg/kg. Age-matched controls were given sterile saline in equal volumes according to the same treatment schedule.
EA Intervention
Following CIPN model establishment, mice received EA intervention beginning on day 8, following the final PTX injection. EA was administered once daily for 30 min over 7 consecutive days. During treatment, animals were lightly anesthetized with 0.5–1.0% isoflurane (RWD, China) and maintained on a thermostatically controlled heating pad. Pairs of acupuncture needles were bilaterally inserted subcutaneously at the hindlimb wrist-ankle acupuncture sites corresponding to the lower one-fifth region of the leg. Specifically, one needle was inserted at the midpoint between the anterior borders of the tibia and fibula (“Lower 4”), and a second needle was inserted at the midpoint of the posterior fibula border (“Lower 5”). Needles were advanced superficially along the longitudinal axis of the hindlimb with the needle tips oriented toward the sciatic nerve. The needles were connected to an electroacupuncture stimulator (HANS-200A, Nanjing Jisheng Medical Technology Co., China), and biphasic alternating dense-disperse stimulation (2/100 Hz) was delivered at an intensity of 1 mA. For sham EA, acupuncture needles were inserted at the same Lower 4 and Lower 5 sites and retained for 30 min with no electrical stimulation. To control for the potential effects of repeated light anesthesia, mice in all experimental groups were exposed to 0.5–1.0% isoflurane for 30 min once daily during the EA intervention period and maintained on a thermostatically controlled heating pad.
Behavioral Assessments
Von Frey Test
Mechanical allodynia was quantified using calibrated von Frey monofilaments (Stoelting, Wood Dale, Illinois, USA). Prior to assessments, mice were individually habituated in transparent acrylic chambers positioned on a wire-mesh platform for a minimum of 30 min. Filaments were vertically applied to the hind paw plantar surface until bending and maintained for 2–3 s. Responses such as paw withdrawal, flinching, licking, or shaking immediately following stimulation were scored as positive. The paw withdrawal threshold (PWT, g) was determined based on the up-down method.
Hargreaves Test
Thermal hyperalgesia was evaluated using the Hargreaves test. Animals were habituated individually in transparent chambers on a glass platform for a minimum of 30 min before testing. A radiant heat source generated by a Hargreaves apparatus (PL-200, Taimeng, China) was directed at the mid-plantar hind paw region. The latency to paw withdrawal (PWL, s) was automatically recorded. A 30-s cutoff time was implemented to avoid potential tissue injury, and stimulus intensity was maintained at a fixed setting of 30% for all measurements.
Open Field Test
Anxiety-like behaviors were evaluated using the open field test (OFT). Mice were placed individually in the central zone of an open-field arena and allowed to roam freely for 5–10 min under controlled lighting conditions. The chamber was wiped with 75% ethanol after each session to minimize the influence of residual olfactory cues. Animal trajectories were recorded via an overhead camera and processed with automated tracking software. Total distance traveled and time spent in the center region were quantified as measures of locomotor function and anxiety-like behavior, respectively.
Stereotaxic Viral Injection
For stereotaxic procedures, mice were anesthetized with 1.5–4% isoflurane and positioned in a stereotaxic frame with a thermostatically controlled heating pad, and the corneas were protected from drying with erythromycin ophthalmic ointment. After exposing the skull, small craniotomies were made above the target coordinates of the ACC based on the Paxinos and Franklin mouse brain atlas (anteroposterior (AP), +1.0 mm; mediolateral (ML), ±0.3 mm; and dorsoventral (DV), −1.8 mm relative to bregma).
For chemogenetic manipulation of astrocytes in the ACC, rAAV-GfaABC1D-mCherry-SV40 polyA (titer: 5.10 × 1012 vg/mL, BrainVTA) or rAAV-GfaABC1D-hM3D-mCherry-SV40 polyA (titer: 5.00 × 1012 vg/mL, BrainVTA) or rAAV-GfaABC1D-hM4D-mCherry-SV40 polyA (titer: 5.50 × 1012 vg/mL, BrainVTA) was bilaterally injected into the ACC through a Hamilton microsyringe connected to a microinjection pump. Viral vectors were delivered at 100 nL/min, with a total volume of 150 nL per hemisphere.
Following each infusion, the needle was left in situ for 10 min and then withdrawn slowly to minimize reflux. Mice were allowed to recover fully and were housed for at least 3 weeks to ensure stable viral expression. Chemogenetic receptors were activated or inhibited by intraperitoneal administration of clozapine-N-oxide (CNO; 2 mg/kg, BrainVTA) 30 min prior to behavioral assessments. Vehicle-treated mice received the corresponding volume of solvent according to the same schedule.
Immunofluorescence
After deep anesthesia was induced by intraperitoneal injection of Avertin (20 μL/g, MMouse, China), mice received transcardiac perfusion with sterile saline and subsequently with 4% PFA. Brain tissues were harvested, post-fixed overnight at 4°C, cryopreserved sequentially in 15% and 30% sucrose solutions, and cut into 14-μm coronal sections on a cryostat microtome (RWD, China). Tyramide signal amplification assay kit (AFIHC023, AiFang) was used for fluorescent staining. Briefly, tissue sections were incubated in 3% H2O2 to block endogenous peroxidase activity and subsequently blocked with goat serum at ambient temperature. Sections were incubated overnight at 4°C with anti-GAT-1 (1:2000, Proteintech) or anti-c-Fos (1:2000, Abcam) primary antibodies, followed by HRP-conjugated secondary antibodies and TSA-mediated fluorescent signal amplification using TYR-570 or TYR-480 reagents. After removal of unbound antibodies at 37°C, sections were incubated with anti-GFAP (1:2000, Proteintech) primary antibody, paired with TYR-480 fluorescent dyes. Nuclear counterstaining was performed with DAPI, and fluorescence images were collected using a Leica LSM980 and quantitative analyses were performed using ImageJ software.
Western Blot
Mice were euthanized by cervical dislocation, and brain tissues were rapidly extracted after deep anesthesia with intraperitoneal injection of Avertin. The ACC was rapidly dissected and lysed in RIPA buffer (Beyotime, China). Protein concentrations were quantified using a BCA assay kit (Beyotime, China). Samples were resolved by SDS-PAGE and subsequently transferred onto PVDF membranes. After 1-h blocking at room temperature, membranes were incubated overnight at 4°C with anti-GAT-1 (1:10000, Proteintech) and anti-GAPDH (1:5000, Proteintech) primary antibodies. Following TBST washes (5 min each), membranes were exposed to HRP-conjugated goat anti-rabbit secondary antibodies (1:10000, Proteintech) for 1 h at ambient temperature. Immunoreactive bands were detected using ECL (Bioscience, China), visualized with a chemiluminescence detection system (Bio-Rad), and analyzed using ImageJ software.
Elisa
Following deep anesthesia with intraperitoneal injection of Avertin, mice were euthanized by cervical dislocation. ACC tissues were rapidly collected and homogenized in PBS supplemented with protease inhibitors. Tissue homogenates were centrifuged for 15 min at 3000 rpm and 4°C, and the supernatants were collected for further analysis. GABA concentrations were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (CEA900Ge, Cloud-Clone) following the manufacturer’s instructions. Samples and standards were run in duplicate, and the absorbance value was determined at 450 nm with a microplate reader.
Statistical Analysis
Statistical analyses were carried out using SPSS (version 23.0). Data were presented as mean ± SEM. Pairwise comparisons were conducted using two-tailed Student’s t-tests, while one- or two-way repeated-measures analysis of variance (ANOVA) followed by Tukey’s post hoc correction was applied for multiple group comparisons. All comparisons adopted a significance threshold of P < 0.05.
Results
EA Alleviated Mechanical and Thermal Hypersensitivity and Anxiety-Like Behaviors Induced by CIPN by Inhibiting Astrocytic Activation in the ACC
To investigate the therapeutic actions of EA in CIPN, mice received EA treatment for seven consecutive days following PTX administration (Figure 1A). Behavioral assessments revealed that PTX-induced CIPN mice developed pronounced mechanical and thermal pain hypersensitivity, as reflected by a significant reduction in PWT and PWL relative to saline-treated controls. Notably, EA treatment markedly alleviated both mechanical allodynia and thermal hyperalgesia in CIPN mice. In contrast, sham EA did not significantly alter either PWT or PWL relative to the CIPN group, and both measures remained significantly lower in the sham EA group than in the EA group (Figure 1B and C). Given the high prevalence of affective disturbances associated with chronic neuropathic pain, anxiety-like behaviors were further assessed using the OFT. Compared with control mice, CIPN mice exhibited reduced exploration of the central area, indicating increased anxiety-like behaviors. Sham EA did not significantly increase center-zone exploration relative to the CIPN group, and the time spent in the center remained significantly lower than that observed in the EA group. In contrast, EA treatment restored center-zone exploration without affecting overall locomotor activity, suggesting an anxiolytic effect independent of motor function (Figure 1D–F).
Figure 1.
EA ameliorated pain hypersensitivity and anxiety-like behaviors in CIPN. (A) Representative schematic diagram for the time course of CIPN model induction, EA intervention and behavioral tests. (B and C) Comparison of paw withdrawal thresholds and thermal withdrawal latency. Two-way ANOVA with Tukey post hoc tests; ***P<0.001, CIPN group versus saline group; ###P<0.001, EA group versus CIPN group; %%%P<0.001, Sham EA group versus EA group, n = 8/group. (D) Representative movement traces and quantitative analyses of (E) total distance traveled and (F) time spent in the center zone in the OFT. n = 8/group. (G) Representative immunofluorescence images showing GFAP (green) in the ACC and (H) quantitative analyses. Scale bar = 20 µm. n = 4/group. One-way ANOVA with Tukey post hoc tests, ***P<0.001.
Accumulating evidence implicates astrocytic activation within the ACC in the development of chronic pain and pain-associated emotional dysregulation. Therefore, we examined GFAP expression in the ACC by immunofluorescence staining. As shown in Figure 1G and H, PTX administration induced a robust increase in GFAP expression within the ACC, while EA treatment significantly reduced GFAP levels relative to the CIPN group, indicating that EA suppresses astrocytic activation in the ACC. Together, these results suggest that EA effectively ameliorates both sensory and emotional manifestations of CIPN, which are accompanied by inhibition of ACC astrocyte activation.
EA Attenuated Neuronal Activation in the ACC by Suppressing Astrocytic GAT-1 Overexpression and Increasing GABA Levels
Given that EA suppressed astrocytic activation in the ACC, we next investigated whether astrocyte-mediated GABAergic signaling correlates with the inhibitory effects of EA on ACC neuronal activity. Immunofluorescence staining demonstrated that a subset of GAT-1 immunoreactivity overlapped with GFAP-positive astrocytes in the ACC. Compared with saline-treated controls, CIPN mice exhibited increased colocalization of GAT-1 and astrocytes, whereas this increase was attenuated following EA treatment (Figure 2A and B). Consistent with the development of CIPN, Western blot analysis demonstrated a significant elevation of GAT-1 protein expression in the ACC following PTX administration compared with saline-treated controls. Notably, EA treatment markedly reduced GAT-1 expression (Figure 2C and D). Consistent with the changes in GAT-1, ELISA analysis showed that PTX administration resulted in a marked reduction in GABA levels, whereas EA treatment increased GABA concentrations toward control levels (Figure 2E).
Figure 2.
EA suppressed aberrant astrocytic GAT-1 upregulation and restored GABA levels in the ACC. (A) Representative confocal images showing immunofluorescent labeling of GFAP (green) and GAT-1 (red) in the ACC and (B) quantitative analyses. Scale bar = 20 µm. n = 4/group. (C) Representative immunoblots and (D) quantification of GAT-1 expression in the ACC. n = 4/group. (E) Quantification of GABA levels in the ACC. n = 4/group. (F) Representative immunofluorescence images of c-Fos (green) in the ACC and (G) quantitative analyses. Scale bar = 20 µm. n = 4/group. One-way ANOVA with Tukey post hoc tests, ***P<0.001.
As GABAergic signaling is a major regulator of cortical excitability, we subsequently examined neuronal activation in the ACC by c-Fos immunofluorescence staining. PTX-treated mice exhibited a marked elevation in c-Fos+ cell counts within the ACC, indicative of neuronal activation following CIPN. Importantly, EA markedly reduced c-Fos expression compared with the CIPN group, indicating suppression of ACC neuronal activation (Figure 2F and G). Collectively, these findings suggest that EA treatment is associated with reduced GAT-1 expression, increased GABA levels, and attenuated neuronal activation in the ACC of CIPN mice.
Chemogenetic Inhibition of Astrocytes in the ACC Ameliorated CIPN-Induced Hyperalgesia and Anxiety
To determine whether ACC astrocytes contribute to CIPN-associated hyperalgesia and anxiety, astrocytes were selectively manipulated using chemogenetic approaches (Figure 3A–C). Immunofluorescence analysis confirmed robust viral expression and extensive colocalization of mCherry with astrocytes, indicating selective targeting of ACC astrocytes (Figure 3D and E). Behavioral assessments demonstrated that chemogenetic inhibition of ACC astrocytes caused significant increases in PWT and PWL of CIPN mice, indicating attenuation of pain hypersensitivity. Conversely, chemogenetic activation of ACC astrocytes in saline-treated mice was sufficient to induce pain hypersensitivity in the absence of chemotherapy (Figure 3F and G). Similarly, the OFT results showed that activation of ACC astrocytes significantly reduced center-zone exploration in control mice, whereas inhibition of astrocytes increased center-zone time in CIPN mice (Figure 3H–J). These findings indicate that aberrant activation of ACC astrocytes is associated with the induction and maintenance of pain- and anxiety-like behaviors in CIPN.
Figure 3.
Chemogenetic modulation of astrocytes in the ACC altered CIPN-induced pain hypersensitivity and anxiety-like behaviors. (A) Representative schematic diagram for the time course of virus injection, CIPN model induction, EA intervention and behavioral tests. (B) Schematic representation of viral injection sites in the ACC. (C) Representative images of viral expression within the ACC. Scale bars = 100 and 20 µm. (D) Representative confocal images showing immunofluorescent labeling of mCherry (red) and GFAP (green) and (E) quantitative analyses of mCherry and GFAP costaining in the ACC. Scale bar = 20 µm. n = 4/group. (F and G) Comparison of paw withdrawal thresholds and thermal withdrawal latency. Two-way ANOVA with Tukey post hoc tests; ***P<0.001, CIPN+mCherry group versus Saline+mCherry group; ###P<0.001, Saline+hM3Dq group versus Saline+mCherry group; and P<0.001, CIPN+hM4Di group versus CIPN+mCherry group, n = 8/group. (H) Representative movement traces and quantitative analyses of (I) total distance traveled and (J) time spent in the center zone in the OFT. One-way ANOVA with Tukey post hoc tests, ***P<0.001, n = 8/group.
Given the important role of neuronal hyperactivity within the ACC in chronic pain processing, we next examined whether astrocytic manipulation influences neuronal activation within the ACC. Immunofluorescence analysis confirmed effective chemogenetic modulation of astrocyte activity, as evidenced by altered GFAP immunoreactivity following hM3Dq- or hM4Di-mediated manipulation (Figure 4A and B). To assess neuronal activation, c-Fos expression was quantified within the ACC. Chemogenetic activation of astrocytes produced a substantial increase in the number of c-Fos+ cells in saline-treated mice. In contrast, astrocytic suppression significantly reduced c-Fos expression in CIPN mice (Figure 4C and D). Taken together, these findings establish a link between astrocytic and neuronal activation in the ACC and suggest that EA-induced improvements in pain hypersensitivity and anxiety-like behaviors were accompanied by reduced astrocytic activation, normalization of GAT-1 expression and GABA levels, and decreased neuronal activation in the ACC.
Figure 4.
Inhibition of astrocytes in the ACC reduced neuronal activation. (A) Representative confocal images of mCherry (red) and GFAP (green) immunofluorescence and (B) quantitative analyses in the ACC. Scale bar = 20 µm. n = 4/group. (C) Representative immunofluorescence images of c-Fos (green) in the ACC and (D) quantitative analyses. Scale bar = 20 µm. n = 4/group. One-way ANOVA with Tukey post hoc tests, ***P<0.001.
Activation of ACC Astrocytes Reversed the Therapeutic Actions of EA in CIPN
To determine whether suppression of astrocytic activity is required for the beneficial effects of EA, ACC astrocytes were chemogenetically manipulated during EA treatment. Chemogenetic activation of ACC astrocytes largely abolished the beneficial effects of EA, indicating that suppression of astrocytic activity is necessary for EA-mediated pain and anxiety relief (Figure 5A–E). To verify the effectiveness of chemogenetic manipulation, GFAP immunofluorescence was performed in the ACC. hM3Dq-mediated astrocytic activation restored GFAP expression despite EA treatment (Figure 5F and G).
Figure 5.
Chemogenetic activation of astrocytes in the ACC reversed the therapeutic actions of EA. (A and B) Comparison of paw withdrawal thresholds and thermal withdrawal latency. Two-way ANOVA with Tukey post hoc tests; ***P<0.001, EA+mCherry group versus CIPN+mCherry group; ###P<0.001, CIPN+hM4Di group versus CIPN+mCherry group; and P<0.001, EA+hM3Dq group versus EA+mCherry group, n = 8/group. (C) Representative movement traces and quantitative analyses of (D) total distance traveled and (E) time spent in the center zone in the OFT. n = 8/group. (F) Representative confocal images showing immunofluorescent labeling of mCherry (red) and GFAP (green) and (G) quantitative analyses in the ACC. Scale bar = 20 µm. n = 4/group. (H) Representative immunoblots and (I) quantification of GAT-1 expression in the ACC. n = 4/group. (J) Quantification of GABA levels in the ACC. n = 4/group. (K) Representative immunofluorescence images of c-Fos (green) in the ACC and (L) quantitative analyses. Scale bar = 20 µm. n = 4/group. One-way ANOVA with Tukey post hoc tests, *P<0.05, ***P<0.001.
Given our previous observations implicating astrocytic GAT-1/GABA-related changes in ACC neuronal activation, we next examined changes in GAT-1 expression and GABA levels. Chemogenetic activation of astrocytes reversed the effects of EA on suppressing the CIPN-induced upregulation of GAT-1 and restoring GABA concentrations in the ACC (Figure 5H–J). Consistent with the alterations in GABAergic signaling, astrocytic activation restored neuronal c-Fos expression despite EA treatment (Figure 5K and L). Taken together, these results demonstrate that inhibition of ACC astrocytes is required for the therapeutic actions of EA in CIPN.
Discussion
Our study investigated a central mechanism associated with the therapeutic actions of EA in CIPN. We demonstrated that EA effectively attenuated mechanical and thermal pain hypersensitivity and anxiety-like behaviors in PTX-induced CIPN mice via reduced astrocytic activation, normalization of GAT-1 expression and GABA levels, and attenuation of neuronal activation within the ACC. Importantly, chemogenetic activation of ACC astrocytes markedly attenuated the therapeutic actions of EA. Together, these findings suggest that EA alleviates CIPN through modulation of astrocyte-mediated neuronal activation in the ACC.
Accumulating evidence demonstrates that the ACC plays a key role in both the sensory and affective dimensions of chronic pain. Functional imaging studies have consistently demonstrated increased ACC activation in patients with chronic pain. Meanwhile, experimental studies have shown that enhanced ACC neuronal excitability contributes to nociceptive hypersensitivity and anxiety-related behaviors.42–44 In addition to neuronal plasticity, recent studies have highlighted astrocytes as critical regulators of cortical pain circuits.19,45 Astrocytic activation has been reported in CIPN models and is increasingly recognized as an important contributor to pain-related emotional disturbances.22,46,47 Activation of astrocytes in the ACC enhances local neural activity, inducing anxiety-like behaviors; however, this activation can be reversed by duloxetine.48,49 Consistent with these findings, we observed robust astrocytic and neuronal activation in the ACC of CIPN mice. More importantly, chemogenetic activation of ACC astrocytes in healthy mice induced hyperalgesia and anxiety-like behaviors, whereas astrocytic inhibition produced the opposite effects in CIPN mice. These results further support the evidence that ACC astrocytes actively contribute to both nociceptive and affective abnormalities associated with CIPN. Notably, EA markedly suppressed astrocytic and neuronal activation in the ACC, whereas chemogenetic activation of astrocytes attenuated these effects. These findings suggest that regulation of astrocytic activity is an important mechanism underlying the therapeutic actions of EA, and prompt us to further investigate the downstream molecular mechanisms linking astrocytes to neuronal activation.
Astrocytes regulate neuronal excitability through multiple pathways, among which maintenance of GABAergic inhibition is particularly important.50 GABA functions as the major inhibitory neurotransmitter in the central nervous system, and aberrations in GABAergic neurotransmission are associated with chronic pain states and their emotional comorbidities.51 Both clinical and experimental studies have demonstrated decreased GABA levels in chronic pain states, and restoration of GABAergic transmission effectively alleviates pain hypersensitivity and negative emotional behaviors.24,52,53 Further, transplantation of GABAergic interneuron precursors into the ACC alleviated pain-related behaviors in CIPN mice.54 GAT-1, a major GABA transporter expressed in the central nervous system, regulates extracellular GABA levels through reuptake and clearance.55 Increased GAT-1 expression has been reported in PTX-induced CIPN rats, and pharmacological inhibition of GAT-1 attenuates pain-related behaviors.56 In the present study, increased GAT-1 expression and reduced GABA levels in the ACC were observed in CIPN mice. Furthermore, GAT-1 was colocalized with astrocytes, suggesting that astrocytic GABA transport correlates with local GABA dysregulation. These alterations were accompanied by increased c-Fos expression, indicating a relationship between astrocytic GAT-1-related GABAergic signaling and neuronal activation. Importantly, EA reversed the CIPN-induced increase in GAT-1 expression, restored GABA levels, and reduced neuronal activity within the ACC. Moreover, chemogenetic activation of astrocytes markedly attenuated these regulatory effects of EA and reinstated hyperalgesia and anxiety-like behaviors. Together, these results indicate that astrocytic GAT-1/GABA signaling represents a critical mechanism of astrocyte-neuron crosstalk in CIPN and may be associated with the analgesic and anxiolytic effects of EA.
Several limitations need to be considered. First, the relatively small sample sizes may limit the precision of the estimated effects, and future studies with prospectively determined sample sizes and independent replication are therefore warranted. Second, the present study only employed a single PTX-induced CIPN model, which does not fully reproduce the clinical heterogeneity of CIPN arising from different anticancer agents. Third, neuronal activation was inferred primarily from c-Fos expression; thus, future studies employing electrophysiological approaches, such as whole-cell patch-clamp recordings, will be required to directly characterize alterations in neuronal excitability and excitatory-inhibitory balance associated with CIPN. Finally, while our findings identify the astrocytic GAT-1/GABA pathway as a potential mediator of EA-induced analgesia, direct manipulation of GAT-1 and GABA using pharmacological inhibitors or genetic approaches would provide stronger evidence for causality. Despite these limitations, the findings provide a preclinical rationale for evaluating EA as an adjunctive non-pharmacological strategy for addressing both neuropathic pain and affective symptoms in patients with CIPN.
Conclusions
In conclusion, the present study demonstrates that astrocyte-dependent disruption of GABA homeostasis is associated with ACC neuronal activation and the development of hyperalgesia and anxiety-like behaviors in PTX-induced CIPN. EA effectively reverses these pathological alterations by suppressing astrocytic activation, normalizing GAT-1-associated GABA regulation, and thus reducing neuronal activation in the ACC. Together, these findings identify the astrocytic GAT-1/GABA-related changes as a novel mechanism involved in EA-induced analgesia in CIPN.
Funding Statement
This study was supported by the National Natural Science Foundation of China (Nos. 82274658, 82575226), the Fujian Provincial Joint Funding Project for Scientific and Technological Innovation (2025Y9581), and the University-Industry Cooperation Project of the Science and Technology Department of Fujian Province (2026Y4013).
Abbreviations
ACC, anterior cingulate cortex; ANOVA, analysis of variance; CIPN, chemotherapy-induced peripheral neuropathy; EA, electroacupuncture; GABA, γ-aminobutyric acid; GAT-1, GABA transporter-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PTX, paclitaxel; PWL, paw withdrawal latency; PWT, paw withdrawal threshold.
Data Sharing Statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author (Z.W.) on reasonable request.
Disclosure
The authors declare no conflicts of interest.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author (Z.W.) on reasonable request.