Endoplasmic reticulum stress contributes to high-dose radiation-induced chronic mechanical and thermal neuropathic pain

Abstract

Introduction

Exposure to high doses of radiation can cause serious harm to human health and lead to long-term chronic pain. However, the mechanisms underlying pain caused by radiation overexposure remain largely unknown.

Methods

In this study, the double hind paws and tail of mice were exposed to 40Gy electron beams to establish a radiation-induced pain model.

Results

Even after the inflammation of the irradiated skin subsided, the mice continued to exhibit prolonged mechanical and thermal pain. The results indicated that the neuron injury marker activating transcription factor 3 (ATF3) was markedly increased and neuronal excitability was significantly increased 1 month post-radiation in the lumbar dorsal root ganglion (DRG). Yet, radiation-induced chronic pain could not be mitigated by common analgesics. We discovered that endoplasmic reticulum (ER) stress was activated in the DRG, and intraperitoneal injections of the ER stress antagonists 4-phenylbutyric acid (4-PBA) and TUDCA significantly alleviated mechanical and thermal allodynia. Additionally, 4-PBA and TUDCA reduced the neuronal hyperexcitability caused by radiation. Moreover, 4-PBA also ameliorated the motor dysfunction induced by high-dose radiation.

Interpretation

Our findings highlight the pivotal role of ER stress activation in the development of chronic neuropathic pain induced by high doses of radiation. Furthermore, our findings suggest that targeting ER stress may offer a promising approach for preventing chronic abnormal pain resulting from high doses of radiation.

Introduction

Radiation is increasingly significant in various sectors, including industrial, medical, and military applications. ¹ Radiation therapy (RT) serves as a fundamental treatment for many malignant tumors. However, it can also harm the human body. ² Previous research has shown that RT induces peripheral neuropathic pain, characterized by mechanical and heat allodynia. ³ Apart from RT-related injuries, radiation overexposure accidents occur sporadically, resulting in more severe long-term health consequences. ⁴ Reports have indicated that high-dose accidental radiation exposure causes significant local skin and soft tissue damage, often accompanied by pain. ⁵ In 2014, a patient exposed to a very high dose of Iridium-192 (192Ir) was treated; despite the healing of the radiation-induced skin lesion and limb preservation, the patient suffered from persistent local pain and experienced poor quality of life over nearly 5 years of follow-up. ⁶ According to the International Association for the Study of Pain (IASP), pain that persists for more than three months is classified as chronic. ⁷ Chronic pain causes significant distress to patients and necessitates comprehensive treatment. ⁸ Thus, in the 11^th revision of the International Classification of Diseases, chronic painful radiation-induced neuropathy and chronic post-radiotherapy pain were recognized. ⁹ However, the mechanisms underlying chronic pain from radiation overexposure remain unclear, and effective treatment methods for allodynia are lacking.

ER stress results from structural and functional disturbances within the ER. ¹⁰ Following stimulation, cells experience an accumulation of misfolded and unfolded proteins in the ER lumen and dysregulation of Ca²⁺ balance, activating signaling pathways such as the unfolded protein response (UPR), ER overload response, and caspase-12-mediated apoptosis pathway. ¹¹ ER stress is primarily mediated by three sensory pathways: PKR-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and ATF6. The primary role of the UPR is to restore ER homeostasis and enable cellular adaptation to ER stress. However, if homeostasis cannot be reestablished, the UPR initiates apoptosis to eliminate stressed cells. ¹² GRP78, a heat shock chaperone protein, is a master of the UPR during ER stress. ¹³ After tissue injury, the accumulation of misfolded proteins in the ER lumen leads to a series of ER dysfunction, resulting in the dissociation of GRP78 from PERK, IRE1 and ATF6, which in turn leads to ER stress. ¹⁴ Endoplasmic reticulum stress can increase the activity of PERK protein kinase, which phosphorylates eIF2, thereby inhibiting protein transcription. ¹⁵ Finally, selective translation of ATF4 and CHOP induced proadaptive signaling. ¹⁶ Previous studies have identified ER stress as a significant factor in generating pain hypersensitivity in conditions such as diabetic neuropathic pain, ¹⁷ multiple sclerosis, ¹⁸ and vasculitic peripheral neuropathy. ¹⁹ Yet, few studies have explored the role of ER stress in chronic pain resulting from radiation accidents.

In the current study, we examined the role of ER stress in a mouse model of chronic pain induced by 40 Gy electron beams, simulating radiation overexposure accidents. Findings indicate that the activation of transcription factor ATF3, a marker of neuron injury, is upregulated and neuronal excitability increases in mouse DRG post-radiation. The results suggest that ER stress is crucial for the development of radiation-induced chronic mechanical and heat allodynia in mice. Furthermore, inhibitors of ER stress have been shown to alleviate chronic pain caused by high-dose radiation. These findings may contribute to the treatment of chronic pain in cases of radiation overexposure.

Methods and materials

Animals

Adult ICR mice (22–25 g) were sourced from the Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (Hangzhou, China). Four mice were housed in each cage, with access to food and water ad libitum. All mice were maintained at a controlled room temperature (22°C) and humidity (60%–80%), with a 12 h/12 h light/dark cycle (lights on from 6:00 a.m. to 6:00 p.m.). The animal study received approval from the University Committee on Animal Care at Soochow University (No. 2012–03) and was conducted according to the guidelines for Animal Use and Care of Jiangsu Province (2008). The Ethics Committee of the Laboratory Animal Center at Soochow University approved the study prior to commencement (Batch number: SUDA20230323A02).

Reagents

Detailed information is provided in Supplementary Data Table 1.

Radiation-induced allodynia model

Mice were anesthetized with an intraperitoneal (i.p.) injection of pentobarbital sodium (70 mg/kg) and immobilized on a plastic plate using adhesive tape to minimize movement during irradiation. A 3-cm thick lead shield was used to localize the radiation field and protect adjacent areas. A single 40 Gy dose of electron beam was administered to the double hind paws and tail at a rate of 600 cGy/min using an Elektra Synergy linear accelerator (Sweden). Control group mice received a 0 Gy electron beam dose. Following irradiation, mice were returned to normal conditions. Pain behavior tests were conducted in the subsequent days.

Behavioral tests

Behavioral tests were performed in a quiet, temperature-controlled room between 9 a.m. and 5 p.m., by an operator unaware of the treatment groups. Mechanical allodynia was evaluated using the “up-and-down” method with Von Frey filaments. Heat allodynia was measured by the tail-flick test, and cold hyperalgesia was assessed using the acetone stimulation test. ³ Thresholds were recorded before (baseline), after IR, and following drug treatments. Testing ceased when thresholds returned to normal or showed significant increase.

Drug administration

4-PBA was dissolved in two doses of 40 mg/kg and 100 mg/kg, and TUDCA was configured in two doses of 100 mg/kg and 300 mg/kg, which were administered by intraperitoneal injection once daily starting the day before radiation. Morphine was diluted to a concentration of 0.5 mg/ml with normal saline and administered at a dose of 5 mg/kg by intraperitoneal injection. Gabapentin was administered at a dose of 10 mg/kg by intraperitoneal injection. Indomethacin was dissolved in corn oil and administered at a final dose of 10 mg/kg by gavage.

RNA isolation and quantitative real-time polymerase chain reaction

Sciatic nerve (SN) and dorsal root ganglia (DRGs) from L4 to L6 were collected from mice treated with radiation at various time points. Total RNA was extracted using Trizol Reagent. cDNA was synthesized with the Revert Aid First Strand cDNA Synthesis Kit. Quantitative real-time polymerase chain reaction (qPCR) was performed using SYBR Green PCR Master Mix and a real-time PCR Detection System. Relative expression levels were calculated using the 2^^-ΔΔCt method after normalization to GAPDH expression. Primers are listed in Supplementary Data Table 2.

Protein extraction and western blot analysis

SN and DRGs from L4 to L6 were collected from mice treated with radiation. Tissue samples were homogenized in radioimmunoprecipitation assay lysis buffer that included protease and phosphatase inhibitors. Protein concentration was determined by the bicinchoninic acid assay. Proteins (30 μg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene fluoride membrane. Blots were blocked for 30 minutes at room temperature and incubated with primary antibodies overnight at 4°C. After 60 minutes of incubation at room temperature with HRP-conjugated secondary antibody, reactive bands were visualized using ECL luminescence reagent.

Histology and immunofluorescence

Mice were anesthetized with entobarbital sodium (70 mg/kg, i.p.) at various time points after radiation treatment and perfused transcardially with physiological saline, followed by 4% paraformaldehyde. Paraffin sections of paw skin (10 μm) were stained with hematoxylin and eosin (H&E) for histological examination. Cryosections (15 μm) of DRGs (L4–L6) were incubated with the primary antibody overnight at 4 °C. Sections were then incubated with fluorescent secondary antibodies and images captured using a Leica confocal fluorescence microscope. Finally, the proportion of positively labeled cells in all cells was calculated.

Whole cell patch clamp technique

Isolated DRGs were transferred into 5 ml of dissecting fluid and equilibrated for 30 minutes. DRGs were digested in an oxygen-saturated dissection solution containing 1.2 mg/ml trypsin and 1.6 mg/ml collagenase for 90 minutes. DRGs were washed and resuspended in dissection solution with 0.5 mg/ml DNase. Neurons were plated on slides and covered with extracellular solution for 30 minutes. Solutions: The external electrode solution contained: 0.76 g NaCl, 0.037 g KCl, 0.027 g KH2PO4, 25 mL CaCl2•2H2O, 0.01 mL MgCl2, 0.238 g Hepes, 0.18 g Glucose and was adjusted to pH 7.3 with NaOH. The electrode internal solution contained: 0.261 g KCl, 0.015 g NaCl, 0.06 g Hepes, 0.045 g Glucose, 0.048 g EGTA, 25 μL CaCl2•2H2O and was adjusted to pH 7.25 with KOH. The coreless hard glass tube was pulled in two steps using the P97 microelectrode puller. After pulling, the electrode tip was polished with the MF-900 electrode polisher. Finally, the polished electrode tip was heated to make it smooth, free of burrs and with low resistance. Under the microscope, a neuron cell with a smooth surface, clear contour and good activity was located and placed at the center of the field of view for sealing. After stabilization, the cell membrane was ruptured by suction to perform whole-cell current recording. Whole-cell perforated patch-clamp recordings were acquired and analyzed using a Digidata 1440 digitizer, an Axopatch 200B amplifier, and a Clampex 10 software.

The elevated plus maze (EPM)

Mice were placed in the central area of the elevated cross maze facing the open arm, with their backs toward the experimenter. SuperMaze animal behavior analysis software tracked the movement of mice in the maze, and indices were automatically calculated. The experiment lasted for 5 minutes. Afterward, mice were removed and the maze was cleaned with alcohol and paper towels.

Rotarod test

The impact of radiation on motor coordination was assessed using the rotarod test. Prior to testing, mice underwent training on the rotarod apparatus for three consecutive days. The rotarod accelerated to 40 rpm in two minutes and then remained at this speed for three minutes. The duration before mice fell was recorded as the rotarod latency.

Statistical analysis

Data were analyzed using Graph Prism 9 (Graph Pad, CA). Results were presented as mean ± S.E.M. Student’s t-test was utilized for two-group comparisons. One-way or two-way analysis of variance, followed by post hoc Bonferroni’s test, was conducted for multiple comparisons. A p-value of <0.05 was considered statistically significant.

Results

High-dose radiation induces chronic mechanical and thermal pain in mice

In the initial experiments, it was investigated whether exposure to high-dose radiation could induce chronic pain-like behavior following skin recovery. The hind paws and tail of mice received a single 40 Gy radiation dose. As illustrated in Figure 1(a), the hind paws appeared swelling at 1 week post-irradiation, purulent discharge at 2 weeks, scab at 3 weeks, and basically healed at 4 weeks. And H&E-staining was performed in order to verity whether the skin response was consistent with the histological changes. Inflammatory infiltration in the stratum corneum was noted at 2 week but had resolved at 5 week (Figure 1(b)). Subsequently, changes in radiation-induced mechanical, heat, and cold allodynia over time were recorded. The results indicated that radiation induced significant mechanical hypersensitivity following skin recovery, with pain persisting for approximately five months (Figure 1(c), P < 0.001). Thermal allodynia commenced three days post-radiation and resolved by the 103rd day (Figure 1(d), P < 0.001). However, cold allodynia remained unchanged throughout the study period (Figure 1(e)). Both mechanical and thermal pain thresholds were significantly reduced in female mice after radiation (Supplementary Figure 1A–B). And the following experimental studies were all performed in male mice. Collectively, these findings suggest that high-dose radiation leads to sustained mechanical and heat allodynia post-recovery from paw skin injury.

Figure 1.

High-dose radiation-induced pain-like behaviors in mice. (a) Inflammatory responses in the hind limb skin were observed post-radiation and resolved after one month. (b) Morphological recovery of hind paw skin was evident by day 35 post-radiation, as demonstrated by H&E staining. (c-e) The hind limbs and tail of mice were subjected to high-dose radiation, inducing time-dependent mechanical, heat, and cold allodynia. Data are presented as mean ± S.E.M. n = 6-8. ^*p<0.05, ^**p<0.01, ^***p<0.001, compared to control mice at corresponding time points. Analysis was conducted using an unpaired Student's t-test.

High-dose radiation upregulates the expressions of neuronal injury markers

To determine whether the pain induced by high-dose radiation is of neuropathic origin, the expression of neuronal injury markers in mouse DRG was examined 35 days post-radiation. Immunofluorescence staining revealed a marked increase in ATF3 signaling in mouse DRG after 35 days (Figure 2(a), P = 0.0265). Furthermore, qRT-PCR analysis showed significant upregulation in the mRNA levels of Atf3 (Figure 2(b), P = 0.008), Cgrp (Figure 2(c), P < 0.001), Gal (Figure 2(d), P = 0.013), and Vgf (Figure 2(e), P = 0.007) in the DRG from mice 35 days post-radiation. These results demonstrate that peripheral sensory neurons in the DRG are damaged 35 days following radiation.

Figure 2.

Expression of neuron injury markers post-radiation. (a) Representative immunofluorescence staining images showing ATF3 and DAPI in DRG of mice 35 days after radiation, and ATF3 levels were significantly increased in DRG after radiation in the histogram. (b-e) qRT‐PCR results for Atf3, Cgrp, Gal, and Vgf from DRG of mice 35 days post-radiation. Mean ± S.E.M. n ≥ 3; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, analyzed using an unpaired Student’s t‐test. White arrowheads marked DRGs expressing ATF3. The scale of the fluorescence image is 25 μm.

High-dose radiation increases neuronal excitability

To assess the impact of radiation on DRG neuron currents, DRG neurons were isolated for electrophysiological recording. A representative trace from small DRG neurons (< 25 μm) demonstrates that radiation induces action potentials in mice (Figure 3(a)). The results revealed an increase in the resting membrane potential (RMP) (Figure 3(b)B, P < 0.001) and a decrease in the rheobase (RB) compared to the control group (Figure 3(c), P = 0.009). Additionally, the number of action potentials triggered by a 100pA current was elevated post-radiation (Figure 3(d), P = 0.037). These findings indicate that radiation enhances neuronal excitability.

Figure 3.

Electrophysiological properties comparison of small DRG neurons in CON and RAD mice. (a) Representative traces from small DRG neurons (<25 μm) in current-clamp mode. (b) Resting membrane potentials (RMP) were compared between neurons from control and radiation-exposed (RAD) mice. The rheobase (c) and the number of action potentials evoked by different currents (d) are also compared between these groups. Mean ± S.E.M. n = 4; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Statistical analysis was performed using an unpaired Student’s t‐test.

High-dose radiation-induced pain is resistant to common analgesics

Pharmacotherapy, commonly used to alleviate pain, was employed using morphine, gabapentin (GBPT), and indomethacin (IND) on the 35th day post-radiation. ²⁰ According to updated guidelines from the IASP’s Neuropathic Pain Special Interest Group (NeuPSIG), morphine and GBPT are recommended for treating neuropathic pain. ²¹ Despite intraperitoneal injections of morphine (5 mg/kg) and GBPT (10 mg/kg), a single dose failed to alleviate the mechanical (Figure 4(a) and (b)) and thermal pain (Figure 4(d) and (e)) induced by radiation. IND, a nonsteroidal anti-inflammatory drug noted for its potent analgesic properties, was administered orally at a dosage of 10 mg/kg. ²² The results showed no relief from chronic mechanical (Figure 4(c)) and thermal pain (Figure 4(f)) following radiation. It is presumed that radiation-induced chronic pain, specific to radiation injury, is not mitigated by common analgesics.

Figure 4.

Effects of morphine, GBPT, and IND on radiation-induced pain. (a-c) Impact of morphine, GBPT, and IND on radiation-induced mechanical pain. (d-f) Impact of morphine, GBPT, and IND on radiation-induced thermal pain. Data are presented as mean ± S.E.M. n = 6. Analysis conducted using an unpaired Student's t-test at each time point.

ER stress is induced in DRG and SN following high-dose radiation

Since commonly used analgesic drugs could not relieve chronic neuropathic pain caused by ionizing radiation, we further searched for the mechanism related to radiation induced pain. ER stress is involved in the development of a variety of neuropathic pain, so we hypothesized that high-dose ionizing radiation could induce ER stress. To verify our hypothesis, expression levels of mRNAs and proteins associated with ER stress were quantified in DRGs and SN on days 7, 14, 28, 35, and 56 post-radiation. On day 35, significant increases were observed in GRP78 mRNA (P = 0.002), ATF4 mRNA (P = 0.02), CHOP mRNA (P < 0.001), and sXBP1 mRNA (P < 0.001) in DRGs compared to controls (Figure 5(a)). Similar trends were noted in SN, with increases in GRP78 mRNA (P < 0.001), CHOP mRNA (P < 0.001), and sXBP1 mRNA (P = 0.037) on day 35, and ATF4 mRNA (P = 0.023) on day 56 (Figure 5(b)). Western blot analysis revealed significant increases in p-PERK/PERK (P < 0.001) and p-eIF2α/eIF2α (P = 0.019) on day 35 post-radiation. Additionally, increases in GRP78 (P = 0.033), ATF4 (P < 0.001), CHOP (P < 0.001), and sXBP1 (P < 0.001) were also noted on day 35 (Figure 5(c) and (d)). In SN, while CHOP expression increased, levels of p-PERK, PERK, p-eIF2α, eIF2α, GRP78, ATF4, and sXBP1 decreased (Supplementary Figure 2A–B). Moreover, protein levels of Bax were upregulated and Bcl2 was downregulated post-radiation (Supplementary Figure 2C–D). These results indicated that ionizing radiation induced ER stress in DRG and SN. However, the expression of ER stress-related proteins decreased in SN due to the severe injury and apoptosis.

Figure 5.

Temporal changes in ER stress-related proteins in DRG and SN post-radiation. (a) Levels of GRP78, ATF4, CHOP, and sXBP1 mRNA in DRG were quantified by qPCR post-radiation. (b) Levels of GRP78, ATF4, CHOP, and sXBP1 mRNA in SN were quantified by qPCR post-radiation. (c-d) Protein expressions of p-PERK/PERK, p-eIF2α/eIF2α, GRP78, ATF4, CHOP, and sXBP1 in DRG were assessed by Western blot analysis, with subsequent quantifications. Data are presented as mean ± S.E.M. n = 4. ^*p<0.05, ^**p<0.01, ^***p < 0.001 compared to control mice. Analysis performed using one-way ANOVA.

ER stress predominantly occurs in CGRP neurons after excessive radiation

To identify the localization of GRP78 within DRG neurons, double-labeled immunofluorescence was performed. The co-staining included GRP78 and CGRP, which labels small nociceptive peptidergic neurons (Figure 6(a)), IB4 for a subpopulation of small, nonmyelinated nociceptive neurons (Figure 6(b)), NF200 for large myelinated non-nociceptive neurons (Figure 6(c)), and GS for glial cells (Figure 6(d)). The results demonstrated that GRP78 was primarily co-localized with CGRP, IB4, and NF200, but not with GS. Immunofluorescence analysis indicated that GRP78-positive staining was predominantly found in CGRP-positive DRG neurons and increased significantly post-radiation (P = 0.015), whereas changes in GRP78-positive staining in IB4- and NF200-positive DRG neurons were minimal (Figure 6(e)). This indicates that ER stress primarily affects CGRP neurons following excessive radiation exposure.

Figure 6.

Localization of GRP78 protein in DRG neurons post-radiation. (a-d) IF images demonstrated that GRP78 did not colocalize with the glial-cell marker GS but was co-labeled with CGRP, IB4, and NF200. (e) GRP78/CGRP levels were significantly increased in DRG on day 35 post-radiation. Data are presented as mean ± S.E.M. n = 3. ^*p<0.05 compared to control mice. Analysis performed using an unpaired Student's t-test. White arrowheads marked double-labeled DRGs. The scale of the fluorescence image is 25 μm.

ER stress inhibitors alleviate mechanical and thermal allodynia induced by high-dose radiation

Recently, the ER stress antagonists 4-PBA has been demonstrated to reduce neuropathic pain by diminishing ER stress. ¹⁸ TUDCA, a bile acid with chaperone properties, also serves as an ER stress inhibitor and may mitigate neurodegenerative disorders. ²³ Above results suggested ER stress’s role in chronic neuropathic pain due to high-dose radiation. It was hypothesized that 4-PBA and TUDCA could lessen mechanical and heat hypersensitivity resulting from high-dose radiation. Mice were administered 4-PBA (40 and 100 mg/kg) and TUDCA (100 and 300 mg/kg) starting the day before radiation and continuing for two months (Figure 7(a)). Significant reductions in mechanical pain were observed with 4-PBA (100 mg/kg) from day 58 (Figure 7(b), P = 0.026), and with 4-PBA (40 mg/kg) from day 65 (Figure 7(b), P = 0.010). TUDCA (100 mg/kg and 300 mg/kg) alleviated mechanical allodynia from day 58 following treatment initiation (Figure 7(c), P < 0.001, P = 0.004). One month post-radiation, both 4-PBA and TUDCA significantly relieved heat pain, with effects persisting for a month (Figure 7(d) and (e)). These findings indicate that ER stress inhibition can reduce mechanical and thermal pain induced by high-dose radiation.

Figure 7.

Efects of ER stress antagonists on pain-like behaviors post-radiation. (a-b) Mechanical allodynia was evaluated post-intraperitoneal treatment with 4-PBA (40 and 100 mg/kg) and TUDCA (100 and 300 mg/kg) using the Von Frey test. (c-d) Thermal allodynia was evaluated post-intraperitoneal treatment with 4-PBA (40 and 100 mg/kg) and TUDCA (100 and 300 mg/kg) using the tail-flick test. Data are presented as mean ± S.E.M. n = 6. ^*p<0.05, ^**p<0.01, ^***p<0.001 compared to control mice; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 when 4-PBA 40mg or TUDCA 100mg compared to RAD mice; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001 when 4-PBA 100mg or TUDCA 300mg compared to RAD mice. Analysis performed using an unpaired Student's t-test at each time point.

4-PBA and TUDCA reduce ER stress in the DRG of high-dose radiation-treated mice

To explore the mechanisms underlying the analgesic effects of 4-PBA and TUDCA, levels of UPR-related proteins in the DRG were evaluated post-treatment. Significant reductions in GRP78 (P = 0.019), ATF4 (P = 0.005), CHOP (P = 0.019), and sXBP1 (P = 0.002) levels were noted in DRG samples from mice treated with 4-PBA compared to vehicle-treated controls (Figure 8(a) and (b)). Similarly, siginificant upregulations were also observed in GRP78 (P < 0.05), ATF4 (P < 0.01) and CHOP (P < 0.05) after TUDCA treatment. However, sXBP1 levels remained unchanged in TUDCA-treated mice compared to those only exposed to radiation (Figure 8(c) and (d)). These results suggest that the antinociceptive effects of 4-PBA and TUDCA are attributable to their ability to reduce ER stress in the DRG, particularly affecting GRP78, ATF4, and CHOP levels.

Figure 8.

Inhibition of ER stress alleviated ER stress caused by high-dose radiation. (a-b) Expressions of GRP78, ATF4, CHOP, and sXBP1 post-4-PBA treatment in DRG were measured by Western blot, and quantifications were analyzed. (c-d) Expressions of GRP78, ATF4, CHOP, and sXBP1 post-TUDCA treatment in DRG were measured by Western blot, and quantifications were analyzed. Data are presented as mean ± S.E.M. n = 4. ^*p<0.05, ^**p<0.01, ^***p < 0.001 compared to CON mice; ^#p<0.05, ^##p < 0.01 when RAD + 4-PBA compared to RAD mice; ^&p < 0.05, ^&&p < 0.01 when RAD + TUDCA compared to RAD mice. Analysis performed using two-way ANOVA.

Inhibition of ER stress alleviates increased neuronal excitability induced by high-dose radiation

The impact of ER stress inhibitors 4-PBA and TUDCA on the electrophysiological characteristics of DRG neurons post-radiation was investigated. Neuronal excitability was assessed following treatment with 4-PBA and TUDCA (Figure 9(a)). Although 4-PBA and TUDCA did not alter RPM (Figure 9(b)), 4-PBA counteracted the reduction in RB caused by radiation (Figure 9(c), P = 0.039). Furthermore, both TUDCA and 4-PBA mitigated the increase in action potential number post-radiation (Figure 9(d)). These results show that ER stress inhibition can alleviate neuronal excitability induced by high-dose radiation.

Figure 9.

Inhibition of ER stress reduced neuronal excitability increase post-radiation. (a) Small DRG neurons (<25 μm) were isolated from RAD mice treated with vehicle, TUDCA, and 4-PBA, and traces were recorded in current-clamp mode. (b) RMP comparisons were made between neurons from vehicle, TUDCA, and 4-BPA treated RAD mice. RB (c) and the number of action potentials evoked by various currents (d) were also compared. Data are presented as mean ± S.E.M. n = 4. ^*p<0.05, ^**p<0.01, ^***p < 0.001 compared to CON mice; ^#p<0.05, ^###p < 0.001 when RAD + 4-PBA compared to RAD mice; ^&p < 0.05, ^&&&p < 0.001 when RAD + TUDCA compared to RAD mice. Analysis using one-way or two-way ANOVA.

Inhibition of ER stress alleviates the comorbidity of chronic pain induced by high-dose radiation

Chronic pain is often accompanied by multiple physical and psychological issues, including depression, anxiety, trauma, stress response, cognitive dissonance, and reduced exercise.^24,25 To assess the impact of radiation on pain comorbidities, the elevated plus maze (EPM) and rotarod experiments were conducted. There were no changes in open-arm entry time and counts in mice 8 weeks after radiation. The data indicated that radiation did not induce anxiety behaviors in mice (Figure 10(a) and (b)). However, the rotarod test revealed a decrease in endurance (Figure 10(c)), with mice showing reduced rotarod times post-radiation (Figure 10(d), P = 0.002). Treatment with 4-PBA extended these reduced times (Figure 10(d), P = 0.039). These results suggest that inhibiting ER stress ameliorates the decline in exercise capacity caused by high-dose radiation, although it does not significantly affect anxiety.

Figure 10.

Effects of radiation on anxiety and exercise capacity. (a) Tracking movement of mice in the elevated cross maze. (b) Time spent in open arms and number of entries did not significantly change compared to control mice. (c-d) Schematic and duration analysis of mice on the rotarod. Data are presented as mean ± S.E.M. ^*p<0.05, ^**p<0.01 compared to control mice; ^#p<0.05 when RAD + 4-PBA compared to RAD mice (n = 6). Analysis using two-way ANOVA.

Discussion

Our findings indicate that overexposure to radiation induces mechanical and thermal allodynia, with DRGs exhibiting elevated levels of ER stress markers. Intraperitoneal injections of ER stress antagonists efficiently mitigated radiation-induced neuropathic pain behaviors in mice simulating radiation overexposure incidents. Recent studies have linked ER stress to various pain types, including diabetic neuropathic pain, ¹⁷ multiple sclerosis, ¹⁸ and vasculitic peripheral neuropathy. ¹⁹ However, the role of ER stress in pain resulting from radiation overexposure remains underexplored. This study highlights the significance of ER stress in sensory neurons in mediating pain associated with radiation exposure accidents.

Accidental radiation incidents cause severe injuries, such as those seen in the 1986 Chernobyl nuclear power plant accident. ²⁶ Radiation encompasses ionizing (IR) and non-ionizing forms. IR includes photon radiation (X-rays and γ-rays) and particle radiation (electrons, protons, neutrons, carbon ions, alpha, and beta particles). ²⁷ Our previous research explored pain mechanisms following a single therapeutic dose of X-rays. ³ However, studies on the effects of high-dose radiation on pain are limited. Therefore, we exposed the double hind limbs and tails of mice to 40 Gy electron beam radiation to simulate an accident. Our results demonstrated that both male and female mice developed chronic mechanical allodynia and thermal hyperalgesia similar to that observed in patients exposed to high radiation doses. Immunofluorescence also showed peripheral sensory neuron damage and upregulation of the neuron injury marker ATF3 in the DRG post-irradiation, confirming that our mouse model effectively mimics chronic neuropathic pain behaviors directly induced by radiation overexposure.

In some instances, electrical activity triggered by nociceptive receptors becomes uncoupled from normal physiological and pathological functions, leading to pain production without appropriate stimulation. This phenomenon is known as neuropathic pain. ²⁸ In our study, a significant increase in the excitability of DRG neurons was observed following a single radiation treatment of both hind limbs and the tail of mice. These results suggest that while the DRG is not directly exposed to radiation, radiation from the skin can still induce long-term abnormal activity in DRG neurons.

After extensive research, ER stress has been identified as a pivotal factor in the pathophysiology of various widespread diseases. Conditions such as diabetes, neurodegeneration, stroke, pulmonary fibrosis, viral infections, inflammatory diseases, cancer, and heart disease have been associated with both ER stress and the unfolded protein response (UPR).^13,29 Specifically, our investigations into diabetic peripheral neuropathic pain revealed that ER stress plays a crucial role in the underlying mechanisms of neuropathic pain, offering new insights into its mechanisms and treatments. ¹⁷ GRP78, a chaperone heat shock protein, acts as a master regulator of the UPR in ER stress. ¹⁴ The accumulation of misfolded proteins in the ER lumen leads to a series of dysfunctions, resulting in the dissociation of GRP78 from PERK, IRE1, and ATF6, thereby initiating ER stress. ³⁰ This stress enhances PERK protein kinase activity, which then phosphorylates eIF2α, inhibiting protein synthesis. ¹⁵ Consequently, a pro-adaptive signaling pathway is activated through the selective translation of ATF4 and CHOP. ¹⁶ In this study, we demonstrated that the primary chaperone GRP78 was activated, followed by activation of the PERK-eIF2 and IRE-XBP1 pathways in the DRG of a radiation-induced neuropathic pain model. In the SN, although ER stress-related protein expression decreased, apoptosis was observed. Thus, it is hypothesized that while ER stress occurs in the SN, severe damage to the SN leads to decreased protein expression. Furthermore, we anticipate that the ATF6 pathway may function differently in a radiation-induced neuropathic pain model compared to the IRE-XBP1 and PERK-elF2 pathways.

The impact of ER stress on neuronal excitability required further investigation. Existing research has indicated that ER stress can influence calcium homeostasis. ER stress impaired the function of calcium regulatory proteins, leading to abnormal intracellular Ca²⁺ signals and neuronal dysfunction. ³¹ Additionally, CHOP has been proven to interact with GABA_B receptors, resulting in the down - regulation of GABA_B receptors on the cell surface. ³² This also affected neuronal excitability and activity.

Current treatments for pain typically include NSAIDs, opioids, and antidepressants. However, these therapies are minimally effective against radiation-induced neuropathic pain. This resulted in a clinical scenario where there are currently no effective therapeutic approaches for chronic pain induced by high-dose radiation injury. Additionally, during the follow-up period, we identified that the patient had been experiencing persistent pain for an extended duration. Therefore, we need to explore the relevant mechanisms to discover drugs. As antagonists of ER stress, 4-PBA and TUDCA reduce radiation-induced ER stress and alleviate chronic neuropathic pain caused by radiation. Additionally, inhibition of ER stress can reduce DRG neuronal excitability induced by radiation. These results suggest that ER stress antagonists could serve as therapeutic targets for managing radiation-induced chronic neuropathic pain in humans and companion animals.

Epidemiological studies have shown that individuals with chronic pain are more prone to psychiatric disorders. ³³ Emotional factors significantly influence pain perception, and negative emotions can entirely negate the analgesic effects of therapeutic doses of remifentanil. ³⁴ Therefore, the EPM test was conducted to assess whether high-dose radiation induces anxiety. The findings indicated that the mice did not exhibit anxiety behaviors post-radiation, suggesting that chronic neuralgia induced by high-dose radiation is not strongly associated with anxiety.

Moreover, pain is closely linked to exercise. On one hand, pain acts as a protective response to injury, mitigated by avoiding exercise; on the other hand, prolonged or dysfunctional neuromuscular adaptations in response to pain can lead to the chronicity of various pain conditions.^35–37 The rotarod test results revealed a significant decrease in motor ability, while 4-PBA extended the latency period. Thus, it is hypothesized that inhibition of ER stress can alleviate the comorbidities associated with chronic pain induced by high-dose radiation.

There are some limitations in our current study. Firstly, the mechanism of ER stress in primary DRG neuronal cells post-radiation was not elucidated. Secondly, although changes in electrical signals were recorded using patch clamp, changes in ion channel function were not explored. Furthermore, we have only investigated the mechanism in male mice, and have not yet explored whether ER stress leaded to neuropathic pain in female mice. Despite these shortcomings, our study demonstrated that high-dose radiation induces chronic neuropathic pain and identified ER stress antagonists as potential targets for pain treatment. In summary, our research confirmed that ER stress contributes to radiation-induced neuropathic allodynia. The experimental data indicated that blocking ER stress is beneficial for relieving allodynia and reducing abnormal neuronal excitability induced by radiation. Therefore, ER stress antagonists appear to be suitable prevention strategies for high-dose radiation-induced allodynia in clinical practice.

ORCID iDs

Liya Dai https://orcid.org/0009-0002-0654-8694

Cunjin Su https://orcid.org/0000-0003-3728-3377

Yusong Zhang https://orcid.org/0000-0003-0474-5870

Ethical considerations

The Ethics Committee of Laboratory Animal Center of Soochow University has been approved before conducting animal experiments (Batch number: SUDA20230323A02).

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.*