
==== Front
Anesth Analg
Anesth Analg
ANE
Anesthesia and Analgesia
0003-2999
1526-7598
Lippincott Williams & Wilkins Hagerstown, MD

38294950
AA-D-23-01074
00020
10.1213/ANE.0000000000006884
3
38
Original Research Articles
Original Laboratory Research Report
Analgesic Effect of Exercise on Neuropathic Pain via Regulating the Complement Component 3 of Reactive Astrocytes
Wang Chenghao PhD *†
He Hui PhD †‡
Gao Tianchi BS *
Sun Xinzheng MS †
Du Lixia PhD §
Yang Yayue MS *
Zhu Jianyu PhD *
Yang Yachen BS *
Wang Yanqing PhD *
Mi Wenli MD, PhD *
From the * Department of Integrative Medicine and Neurobiology, School of Basic Medical Science, Institutes of Integrative Medicine, Shanghai Key Laboratory of Acupuncture Mechanism and Acupoint Function, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Shanghai Medical College, Fudan University, Shanghai, China
† China Institute of Sport and Health Science, Beijing Sport University, Beijing, China
‡ Key Laboratory of Physical Fitness and Exercise, Ministry of Education, Beijing Sport University, Beijing, China
§ Department of Biochemistry, School of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Address correspondence to Wenli Mi, MD, PhD, Department of Integrative Medicine and Neurobiology, Shanghai Medical College, Fudan University, Shanghai 200032, China. Address e-mail to wenlimi@fudan.edu.cn.
31 1 2024
10 2024
139 4 840850
21 11 2023
Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Anesthesia Research Society.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

BACKGROUND:

Exercise has been proven to be an efficient intervention in attenuating neuropathic pain. However, the underlying mechanisms that drive exercise analgesia remain unknown. In this study, we aimed to examine the role of complement component 3 (C3) in neuropathic pain and whether antinociceptive effects are produced by exercise via regulating C3 in mice.

METHODS:

In this study, using a spared nerve injury (SNI)-induced neuropathic pain mice model, C57BL/6J mice were divided into 3 groups: Sham mice, SNI mice, and SNI + Exercise (Ex) mice with 30-minute low-intensity aerobic treadmill running (10 m/min, no inclination). Paw withdrawal threshold; thermal withdrawal latency; and glial fibrillary acidic protein, C3, tumor necrosis factor-α, and interlukin-1β expression in the spinal cord were monitored. C3 knockout (KO) mice were further used to verify the role of C3 in neuropathic pain.

RESULTS:

von Frey test, acetone test, and CatWalk gait analysis revealed that treadmill exercise for 4 weeks reversed pain behaviors. In addition, exercise reduced astrocyte reactivity (SNI mean = 14.5, 95% confidence interval [CI], 12.7–16.3; SNI + Ex mean = 10.3, 95% CI, 8.77–11.9, P = .0003 SNI + Ex versus SNI) and inflammatory responses in the spinal cord after SNI. Moreover, it suppressed the SNI-induced upregulation of C3 expression in the spinal cord (SNI mean = 5.46, 95% CI, 3.39–7.53; SNI + Ex mean = 2.41, 95% CI, 1.42–3.41, P = .0054 SNI + Ex versus SNI in Western blot). C3 deficiency reduced SNI-induced pain and spinal astrocyte reactivity (wild type mean = 7.96, 95% CI, 6.80–9.13; C3 KO mean = 5.98, 95% CI, 5.14–6.82, P = .0052 C3 KO versus wild type). Intrathecal injection of recombinant C3 (rC3) was sufficient to produce mechanical (rC3-Ex mean = 0.77, 95% CI, 0.15–1.39; rC3 mean = 0.18, 95% CI, −0.04 to 0.41, P = .0168 rC3-Ex versus rC3) and cold (rC3-Ex mean = 1.08, 95% CI, 0.40–1.77; rC3 mean = 3.46, 95% CI, 1.45–5.47, P = .0025 rC3-Ex versus rC3) allodynia in mice. Importantly, exercise training relieved C3-induced mechanical and cold allodynia, and the analgesic effect of exercise was attenuated by a subeffective dose of intrathecal injection of C3.

CONCLUSIONS:

Overall, these results suggest that exercise suppresses neuropathic pain by regulating astroglial C3 expression and function, thereby providing a rationale for the analgesic effect of exercise as an acceptable alternative approach for treating neuropathic pain.

STI2030 - Major Projects 2022ZD0204700Wenli MiNational Natural Science Foundation of China 10.13039/501100001809 82271248 Wenli MiNational Natural Science Foundation of China 10.13039/501100001809 82271258 Yanqing WangInnovative research team of high-level local universities in ShanghaiWenli MiZJ LabYanqing WangShanghai Center for Brain Science and Brain-Inspired Technology 10.13039/100020441 Yanqing WangOPEN-ACCESSTRUE
SDCT
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pmcKEY POINTS

Question: What are the underlying mechanisms that drive exercise analgesia in neuropathic pain?

Findings: Exercise suppresses neuropathic pain by regulating astroglial component 3 (C3) expression and function.

Meaning: The study provides a rationale for the analgesic effect of exercise as an acceptable alternative approach for treating neuropathic pain.

Neuropathic pain, a common type of chronic pain, is typically caused by a lesion or disease of the somatosensory nervous system and has a prevalence of approximately 5% worldwide. It often leads to disability, loss of function, and reduced quality of life1 and remains difficult to treat despite extensive research efforts. The currently used pharmacologic regimens for neuropathic pain exhibit limited efficacy and cause potentially dangerous side effects. Exercise, a nonpharmacological therapeutic approach, has been demonstrated to alleviate various types of chronic pain, including inflammatory pain, musculoskeletal pain, and neuropathic pain.2–5 In addition to being cost-effective, exercise improves locomotor function and exerts positive effects on multiple organs and systems. Thus, exercise has been recommended as a first-line treatment option for patients with fibromyalgia and lower back pain.6 However, the underlying mechanisms of how exercise relieves neuropathic pain remain to be elucidated.

There is emerging evidence that astrocytes in the spinal dorsal horn (SDH) play a vital role in modulating the pathogenesis of pain, especially neuropathic pain.7 After noxious stimulation or nerve injury, astrocytes undergo various morphological and functional alterations; this process is known as reactive astrogliosis. For instance, astrocyte hypertrophy, with process extension and a high expression of astrocyte marker proteins (such as glial fibrillary acidic protein [GFAP]) in the SDH, has been reported after peripheral nerve injury in mice.7,8 However, in addition to morphological changes, reactive astrocytes undergo molecular and functional changes. There are several distinct states of astrocyte reactivity, including a proinflammatory “A1” state, a proregenerative “A2” state, and a proresolving phenotype known as “A3-like astrocyte.”7 Liddelow et al9 reported that neuroinflammation and ischemia induce a toxic astrocyte, which is characterized by the upregulation of complement component 3 (C3) genes. C3 expression has also been detected in several central nervous system (CNS) diseases, such as neuromyelitis optica10 and status epilepticus.11

Furthermore, spinal astrocyte hyperactivity has been reported to be involved in the analgesic effect of exercise.12 Exercise training is purported to suppress astrocyte reactivity and normalize GFAP expression in neuropathic pain.12 However, whether astroglial C3 plays a key role in the analgesic effect of exercise remains unclear. In this study, we demonstrated that exercise training eliminated spared nerve injury (SNI) neuropathic pain and astrocyte reactivity in the spinal cord. C3 knockout (KO) reduced SNI-induced pain and spinal astrocyte reactivity. Furthermore, intrathecal injection of recombinant C3 (rC3) was sufficient to produce mechanical and cold allodynia in mice, which could be attenuated by exercise training. Moreover, a subeffective dose of C3 significantly suppressed the analgesic effect of exercise on SNI mice. Our results demonstrated that exercise alleviated neuropathic pain by regulating astrocyte reactivity via C3 in the spinal cord.

METHODS

Animals

Adult C57BL/6J mice (6–8 weeks old; 20–25 g) were purchased from the Shanghai Experimental Animal Center of Chinese Academy of Sciences. C3 KO (B6.129S4-C3tm1Crr/J) mice in the background of C57BL/6J were provided by Prof Jianhua Li, School of Basic Medical Sciences, Shanghai Medical College of Fudan University. Before the experimental manipulations, the mice were habituated at least 1 week in groups of 4 mice per cage under controlled conditions (23° ± 1°C, 7 am to 7 pm alternating light–dark cycle) with food and water available ad libitum. All experiments were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the International Association for the Study of Pain, as well as the Animal Research Welfare Council of School of Basic Medical Science of Fudan University (Protocol number 20200306-083). All efforts were made to minimize the number of animals used and their suffering.

Surgical Procedure

C57BL/6J mice were subjected to peripheral neuropathy induced by SNI.13 Surgery operation was performed under isoflurane anesthesia using 5% for induction and 1.5% for maintenance. Under sterile conditions, the common peroneal and tibial nerves were tightly ligated with 5.0 silk and sectioned distal to the ligation, removing 2 to 4 mm of the distal nerve stump. Care was taken to avoid touching or stretching the spared sural nerve. The muscles and skin were closed layer by layer. For the sham surgeries, the sciatic nerve was exposed without any manipulation of the nerves.

Intrathecal Injection

Intrathecal injections were performed under isoflurane anesthesia as described above.14 10 μL recombinant mouse C3 (rC3, R&D Systems Inc, 8085) or phosphate-buffered saline (PBS) solution was injected into intervertebral space by using a 10-μL microinjection syringe as previously described. The syringe was inserted into the intervertebral space of a mouse between the lumbar 5 (L5) and lumbar 6 (L6) regions of the spinal cord. A reflexive flick of the tail was considered to be an indicator of the accuracy of each injection.

Treadmill Exercise

To test the effect of physical exercise on neuropathic pain, animals were performed low-intensity aerobic treadmill running (SA101C, SANS) for 30 minutes (10 m/min, no inclination, 5 days per week for 4 weeks) as described previously.12 Before surgery, animals were familiarized with the treadmill for 3 to 5 days (10 m/min, no inclination, 10 min/session). Throughout the treadmill running period, a black cloth was placed over the end of the treadmill to prompt the mice to run and no any mechanical or electrical stimulation was used to guarantee training compliance. Exercise training program was applied at 19:00 to 21:00 pm and began on the third day after induction of nerve injury. Animals of the untrained groups (Sham, SNI) were exposed to the same environment by placing them on the treadmill without motion for the same amount of time as the SNI + Exercise (Ex) groups.

Immunohistochemistry and Sholl Analysis

Mice were deeply anesthetized and perfused intracardially with saline followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). L4 to L6 spinal tissues were removed, fixed in 4% paraformaldehyde overnight at 4°C, then transferred to 30% sucrose in 0.1 M phosphate buffer at 4°C. Subsequently, the spinal cord segments were cut in a freezing microtome (Leica 2000) at 30 μm thickness. Sections were blocked for 60 mins with 10% donkey serum in Tris-buffered saline (TBS) containing 0.3% Triton X-100 (Sigma-Aldrich), then incubated for 16 hours at 4° with primary antibodies, including mouse antiGFAP (1:200, Cell Signaling Technology, 3670), goat antiComplement C3 (1:200, R&D Systems, AF2655), rabbit anti-Iba1 (1:400, Wako, 019-19741). After washing, free-floating sections were incubated for 1 hour at room temperature with the corresponding Alexa Fluor 488- and 594-conjugated secondary antibodies (1:1000; Invitrogen). Fluorescent images were visualized by confocal scanning laser microscope (FV1000, Olympus), and mean fluorescence intensity (MFI) was quantified with Image J (http://rsbweb.nih.gov/ij/).

Sholl analysis was performed to evaluate astrocyte cell morphology as previously described.15 Confocal images immunostained with GFAP antibody was adopted for analysis. Six to eight astrocytes from superficial laminae of the SDH of each animal were chosen to draw serial concentric circles around the cell starting from the center of the DAPI signal and radiating outward at increasing radial increments of 2 μm. The plugin of Sholl analysis applied in ImageJ automatically measured the number of intercepts of GFAP processes in each circle and end radius (length of the longest astroglial process).

Figure 1. Effect of exercise on mechanical allodynia, cold allodynia, and gait behavior in mice after SNI. A, Experimental protocol for mice in the SNI + Ex group. Left, schematic illustration of pain-like behavior testing, and right, protocol for the training program. B and C, Effect of exercise on PWT in both male and female mice after SNI. Line graphs (B) showing time course of group differences for PWT (2-way repeated-measures ANOVA followed by Bonferroni post hoc comparisons indicated that the groups were statistically different at indicated time points. ***P < .001 SNI versus Sham group on week 4, ###P = .001 SNI + Ex versus SNI group on week 4). C, AUC of the durations of PWT in von Frey test from B showed that SNI + Ex group was significantly different from SNI group (1-way ANOVA followed by Bonferroni post hoc comparisons. ***P = .0008 SNI + Ex versus SNI group). D and E, Effect of exercise on cold pain threshold in SNI mice. Line graphs (D) showing time course of group differences for cold sensitivity by 2-way repeated-measures ANOVA test. Post hoc Bonferroni test indicated that the groups were statistically different at indicated time points. ***P < .0001 SNI versus Sham group on week 4, ###P = .0001 SNI + Ex versus SNI group on week 4. E, AUC of the durations of licking/lifting in acetone test from D. n = 15–18 per group. F, Representative print view and timing view of the right hind paw in Catwalk gait testing. G–I, Effect of exercise training on the SNI-induced changes of the body speed (***P = .0051 SNI versus Sham group; * P < .0258 SNI + Ex versus SNI group) (G), print length (H), and swing speed (***P < .0001 SNI versus Sham group) (I) of the ipsilateral hind paw. G–I, 1-way ANOVA followed by Bonferroni post hoc test was used. ANOVA indicates analysis of variance; AUC, area under the curve; PWT, paw withdrawal threshold; SNI, spared nerve injury.

Figure 2. Effect of exercise on astrocyte reactivity and inflammatory factors. One-way ANOVA was used to test the statistical difference (n = 5–8 per group). A, Top, illustrations of immunostaining astrocytes (GFAP) and bottom, representative image for Sholl analysis of an astrocyte in the stratum radiatum from the GFAP-stained image in top image. The interval of the concentric circles is 2 μm. B, MFI of GFAP displayed a significant difference between SNI + Ex and SNI group (*** P = .0042). Scale bars: 100 μm. n = 10 spinal slices from 5 mice for each group. C–E, The morphological features of astrocytes in groups. The changes of the numbers of process intersections (C and D) and ending radius (E) in GFAP positive astrocytes (* P = .0299). F and G, Bar graph showing mRNA expression of inflammatory factors, including IL-1β (* P = .0333) (F) and TNF-α (*P = .0470) (G) after exercise training. Post hoc Bonferroni test indicated that inflammatory factors were decreased in SNI + Ex group. ANOVA indicates analysis of variance; GFAP, glial fibrillary acidic protein; IL-1β, interlukin-1β; MFI, mean fluorescence intensity; mRNA, messenger RNA; SNI, spared nerve injury; TNF-α, tumor necrosis factor-α.

Figure 3. Effect of exercise on C3 expression after SNI. One-way ANOVA was used to test the statistical difference. Data are represented as mean ± SEM. A, Immunofluorescence staining of C3 in spinal and quantitative analysis of mean immunofluorescence intensity in groups (B). Post hoc Bonferroni test indicated C3 was downregulated in SNI + Ex group compared with SNI group (* P = .0338); scale bars: 100 μm, n = 10 spinal slices from 5 mice for each group. C, Bar graph showing normalized C3 mRNA expression after training; n = 6–8 per group. D, Western blot analysis of C3 in spinal cord. Representative bands are shown on the top, and a data summary is shown on the bottom. There was a significant difference between SNI + Ex and SNI group (** P = .0054); n = 5 mice for each group. E, Double immunostaining of C3 (green) with GFAP in the spinal dorsal horn of naïve mice. Scale bars =50 μm. ANOVA indicates analysis of variance; C3, component 3; GFAP, glial fibrillary acidic protein; mRNA, messenger RNA; SEM, standard error of the mean; SNI, spared nerve injury.

Details on behavioral tests, real time-quantitative polymerase chain reaction, and Western blot are described in Supplemental Digital Content 1, Supplemental Material, https://links.lww.com/AA/E705.

Statistical Analysis

Antinociceptive effect of 4-week exercise (Figure 1B, D) and changes of chronic pain after C3 KO (Figure 4B, D) were analyzed using 2-way repeated-measures analysis of variance (ANOVA) (for multiple time points comparisons) followed by Bonferroni’s post hoc tests. Gait analysis was tested by 1-way ANOVA after the exercise program (Figure 1G–I). For the comparison of molecules and behavior differences between 2 groups (WT and C3 KO) in Figure 4, Student t tests were used. Changes in astrocyte reactivity and C3 level in 3 groups were tested by 1-way ANOVA followed by Bonferroni’s post hoc tests. The sample sizes are based on results from our preliminary studies and pilot studies as well as calculations using G Power. In all cases, the sample calculations were based on an assumption of 0.80 power and an α < .05. Supplemental Digital Content 2, Supplemental Table 1, http://links.lww.com/AA/E706, provides the details of animal allocation. All data are presented as the mean ± SEM in the figures. Differences were considered statistically significant if P < .05. All statistical analyses were performed using GraphPad Prism 8.0 software.

RESULTS

Exercise Training Reverses SNI-induced Neuropathic Pain-Like Behavior

In this study, the effect of exercise training on SNI-induced neuropathic pain was observed (Figure 1A). Paw withdrawal threshold (PWT) was significantly decreased from week 1 to week 4 after nerve injury (SNI mean = 0.03, 95% confidence interval [CI], 0.02–0.04) compared with that in the Sham group (Sham mean = 0.59, 95% CI, 0.44–0.74, P < .0001 SNI versus Sham on week 4). Running on a treadmill since the third day after SNI for 4 weeks remarkably increased the PWT starting from week 2 and maintained to week 4 compared with the SNI group (SNI + Ex mean = 0.20, 95% CI, 0.12–0.28, P = .001 SNI + Ex versus SNI on week 4, Figure 1B). Area under the curve (AUC) quantification also revealed that exercise training (SNI + Ex mean = 10.1, 95% CI, 8.07–12.1) markedly enhanced the SNI-induced decrease of the AUC value (SNI mean = 5.10, 95% CI, 3.84–6.38, P = .0008 SNI + Ex versus SNI, Figure 1C). Similarly, treadmill exercise markedly attenuated SNI-induced cold allodynia in the acetone test (SNI + Ex mean = 3.75, 95% CI, 3.04–4.47; SNI mean = 8.06, 95% CI, 6.47–9.64, P = .0001 SNI + Ex versus SNI on week 4, Figure 1D; SNI + Ex mean = 89.9, 95% CI, 80.7–99.1; SNI mean = 175, 95% CI, 151–200, P = .0001 SNI + Ex versus SNI, Figure 1E).

CatWalk gait analysis is used for the objective assessment of chronic pain behavior in neuropathic pain models.16 Accordingly, we used CatWalk gait analysis to determine spontaneous pain behavior in SNI mice. SNI induced a significant decrease in body speed (SNI mean = 19.6, 95% CI, 17.2–22.0; Sham mean = 26.1, 95% CI, 22.4–39.7, P = .0051 SNI versus Sham), ipsilateral hind paw print length (SNI mean = 0.45, 95% CI, 0.12–0.78; Sham mean = 0.96, 95% CI, 0.85–1.07, P = .0032 SNI versus Sham), and average ipsilateral swing speed (SNI mean = 32.9, 95% CI, 22.3–43.5; Sham mean = 62.4, 95% CI, 53.9–70.8, P < .0001 SNI versus Sham) on week 4 (Figure 1G–I). In addition, running on the treadmill improved body speed (SNI + Ex mean = 24.7, 95% CI, 21.3–28.1, P = .0258 SNI + Ex versus SNI); however, the hind paw print length and average ipsilateral swing speed remained unaffected.

Treadmill Exercise Reduces Astrocyte Reactivity and Inflammatory Responses in the Spinal Cord After SNI

Reactive astrocytes in the spinal cord have been reported to play a pivotal role in the pathogenesis of pain.7 Accordingly, we examined the effects of treadmill exercise on astrocyte reactivity after SNI. Immunostaining for GFAP in the ipsilateral dorsal horn of the spinal cord was performed (Figure 2A). The MFI of GFAP was significantly increased in SNI mice (SNI mean = 14.5, 95% CI, 12.7–16.3) compared with control mice (Sham mean = 8.52, 95% CI, 7.66–9.38, P < .0001 SNI versus Sham), but this increase was suppressed in SNI + Ex mice (SNI + Ex mean = 10.3, 95% CI, 8.77–11.9, P = .0003 SNI + Ex versus SNI, Figure 2B).

Despite the highly upregulated GFAP in reactive astrocytes, astrocytes may undergo typical changes such as process extension and hypertrophy, which are considered the phenotypic characteristics of reactive astrocytes.8 To examine the morphological changes that occur on activation, we conducted a Sholl analysis to evaluate the complexity and extension of astroglial processes (Figure 2A). Sholl analysis consists of placing concentric rings at fixed intervals from the soma to count branch intersections at each ring. Sholl analysis of individual reactive astrocytes revealed that the sum of intersections and the number of intersections at each ring significantly increased in SNI astrocytes (SNI mean = 35.4, 95% CI, 29.4–41.3) compared with control astrocytes (Sham mean = 24.6, 95% CI, 19.0–30.3, P = .0041 SNI versus Sham), and treadmill exercise markedly inhibited this upregulation (SNI + Ex mean = 24.7, 95% CI, 20.3–29.1, P = .0042 SNI + Ex versus SNI, Figure 2C, D). Similarly, the ending radius was measured as an indicator of astrocytic territory. Although there was no significant increase in the ending radius in SNI astrocytes, the SNI + Ex astrocytes (SNI + Ex mean = 8.80, 95% CI, 6.41–11.2) showed remarkably reduced ending radius compared with the SNI astrocytes (SNI mean = 11.6, 95% CI, 10.2–13.0, P = .0299 SNI + Ex versus SNI, Figure 2E).

In addition, we investigated changes in the expression of the inflammatory factors interleukin-1β (IL-1β) (Figure 2F) and tumor necrosis factor-α (TNF-α) (Figure 2G) in the spinal cord. We found that SNI significantly increased IL-1β (SNI mean = 1.59, 95% CI, 1.03–2.16; Sham mean = 1.01, 95% CI, 0.90–1.12; P = .0167 SNI versus Sham) and TNF-α (SNI mean = 2.20, 95% CI, 1.52–2.88; Sham mean = 1.11, 95% CI, 0.65–1.57, P = .0021 SNI versus Sham), whereas exercise pronouncedly reduced this upregulation (IL-1β: SNI + Ex mean = 1.07, 95% CI, 0.82–1.32, P = .0333 SNI + Ex versus SNI; TNF-α: SNI + Ex mean = 1.50, 95% CI, 1.24–1.76, P = .0470 SNI + Ex versus SNI).

Treadmill Exercise Suppresses SNI-induced C3 Expression Upregulation in Astrocytes

Neurotoxic astrocytes (A1) are present in most neuroinflammatory and neurodegenerative diseases and are often characterized by highly upregulated C3 expression.9–11 Accordingly, we next investigated whether the expression of spinal C3 was upregulated after SNI. Statistical analysis of the MFI revealed that exercise (SNI + Ex mean = 12.3, 95% CI, 9.21–15.4) significantly suppressed the SNI-induced increase of C3 expression (SNI mean = 18.8, 95% CI, 13.7–24.0, P = .0338 SNI + Ex versus SNI, Figure 3A, B). C3 mRNA in the spinal cord showed an increase after SNI operation (SNI mean = 1.25, 95% CI, 0.90–1.59), but this was greatly inhibited after exercise training (SNI + Ex mean = 0.80, 95% CI, 0.56–1.04, P = .0202 SNI + Ex versus SNI, Figure 3C). Western blot analysis revealed that the protein level of C3 in the spinal cord was significantly increased after SNI (SNI mean = 5.46, 95% CI, 3.39–7.53; Sham mean = 2.83, 95% CI, 1.47–4.20, P = .0147 SNI versus Sham) and decreased after treadmill exercise (SNI + Ex mean = 2.41, 95% CI, 1.42–3.41, P = .0054 SNI + Ex versus SNI, Figure 3D). We also investigated the cellular localization of C3 in the spinal cord and confirmed that C3 immunoreactivity is colocalized with GFAP (Figure 3E).

C3 KO Reduces SNI-Induced Neuropathic Pain-Like Behavior

To further verify the role of C3 in neuropathic pain, we used C3 KO mice (Figure 4A). Consistent with a previous study,17 the PWT and AUC of C3 KO mice (C3 KO mean = 6.53, 95% CI, 4.24–8.81) significantly increased after SNI during the 2-week measurement period compared with those of the WT control mice (WT mean = 1.91, 95% CI, 0.93–2.89, P = .0013 C3 KO versus WT, Figure 4B, C). Similarly, acetone test revealed that the duration of lifting/licking behaviors and AUC quantification (C3 KO mean = 44.9, 95% CI, 31.8–58.0; WT mean = 91.9, 95% CI, 54.3–129, P = .0126 C3 KO versus WT) decreased significantly in C3 KO mice (Figure 4D, E). However, the C3 KO mice showed no significant difference from the WT mice in terms of cold pain threshold, as measured via the cold plate test (Figure 4F), locomotor activity, as indicated by the center zone time, total distance traveled, and percentage of distance in the center area in an open field test (Figure 4G–J).

Microglial C3 receptor signaling mediates astrocyte–microglia interaction and is a critical driver of microglia activation. Therefore, we investigated the activation level of microglia after SNI. No significant differences in activation and morphological changes of microglia were observed between WT and C3 KO mice (Figure 4K–M). However, GFAP expression in C3 KO mice (C3 KO mean = 5.98, 95% CI, 5.14–6.82) decreased compared with that in WT mice (WT mean = 7.96, 95% CI, 6.80–9.13, P = .0052 C3 KO versus WT), and the ending radius was also significantly decreased in C3 KO mice (C3 KO mean = 10.9, 95% CI, 8.63–13.1; WT mean = 15.2, 95% CI, 10.6–19.7, P = .0440 C3 KO versus WT, Figure 4K N–P). These results suggested that C3 KO suppressed the development of neuropathic pain as well as astrocyte reactivity after SNI.

Exercise Training Relieves C3-Induced Pain-Like Behaviors

To further determine the role of C3 regulation in the analgesic effect of exercise, we first tested whether exogenous administration of rC3 triggered pain behaviors. Our results showed that intrathecal administration of 50 ng rC3 in naive mice decreased the PWT 30 minutes after injection (rC3 mean = 0.24, 95% CI, 0.00–0.47; PBS mean 0.77, 95% CI, 0.49–1.05, P = .0062 rC3-50 ng versus PBS), indicating that C3 could induce mechanical allodynia (Figure 5A). Similarly, cold allodynia was detected after intrathecal injection of 50 ng rC3 in the acetone test (rC3 mean = 2.96, 95% CI, 1.62–4.30; PBS mean = 0.94, 95% CI, 0.46–1.41, P = .0028 rC3-50 ng versus PBS, Figure 5B).

Next, we investigated whether exercise training could suppress C3-induced allodynia. For this, the mice were subjected to 30 minutes of treadmill training after intrathecal administration of rC3 (50 ng) (Figure 5C). Both mechanical (rC3-Ex mean = 0.77, 95% CI, 0.15–1.39; rC3 mean = 0.18, 95% CI, −0.04-0.41, P = .0168 rC3-Ex versus rC3) and cold (rC3-Ex mean = 1.08, 95% CI, 0.40–1.77; rC3 mean = 3.46, 95% CI, 1.45–5.47, P = .0025 rC3-Ex versus rC3) allodynia were significantly improved in the rC3-Ex group compared with the rC3 only group (Figure 5D, E).

Considering that an exogenous administration of 10 ng rC3 (i.t.) did not trigger mechanical allodynia behavior in mice, we then tested whether a subeffective dose of rC3 (10 ng) could reverse the exercise-induced analgesic effect in SNI mice. All animals underwent 2 weeks of treadmill training 3 days after SNI surgery, after which rC3 or PBS was intrathecally injected (Figure 5F). Our results showed that a subeffective dose of rC3 significantly suppressed the analgesic effect of exercise on SNI mice (rC3 mean = 0.06, 95% CI, 0.01–0.11; PBS mean = 0.36, 95% CI, 0.22–0.49, P = .0348 rC3 versus PBS in von Frey test; rC3 mean = 4.18, 95% CI, 2.65–5.72; PBS mean = 2.32, 95% CI, 1.22–3.43, P = .0191 rC3 versus PBS in acetone test; Figure 5G, H).

DISCUSSION

Although the positive effects of exercise training on pain relief and functional recovery after peripheral nerve injury have been reported in previous studies,4,6,12,18–20 the underlying mechanisms remain unclear. The results of our study showed that (1) exercise training reversed mechanical and cold allodynia and improved pain-related gait behaviors in an SNI-induced neuropathic pain mice model; (2) exercise training suppressed astrocyte reactivity in the SDH after SNI; (3) exercise training reversed C3 expression in astrocytes, and C3 KO significantly relieved SNI-induced mechanical and cold allodynia; and (4) exercise training attenuated rC3 i.t. injection-induced mechanical allodynia, and subeffective rC3 suppressed the analgesic effect of exercise on SNI-induced mechanical and cold allodynia. Overall, these data provide evidence that exercise training suppresses neuropathic pain by regulating astrocyte reactivity and normalizing C3 expression in the spinal cord (Figure 6).

Figure 4. Effect of C3 KO on SNI-induced neuropathic pain-like behavior. Student t test was used to test the statistical difference. Data are represented as mean ± SEM. A, Schematic of experimental approach. B and C, Effect of C3 KO on PWT in SNI mice. Line graph (B) showing time course of PWT. C, AUC of the PWT in von Frey test from B showing difference between C3 KO and WT group (** P = .0013). D and E, Difference of cold sensitivity in groups. D, Line graph showing time course of cold sensitivity. E, AUC of the durations of licking/lifting in acetone test from C showing difference between C3 KO and WT group (* P = .0126). n = 6–7 per group (F). No difference was found in the cold allodynia threshold assessed by cold plate test. G–J, Locomotor activity assessed by open field test. G, Representative moving tracks from an WT and C3 KO mouse. Bar graph showing no difference on center zone time (H), total distance traveled (I), and percentage of distance in the central area (J) compared with WT mice; n = 5 mice per group. K–P, Effect of C3 KO on activation of microglia and astrocyte in the spinal dorsal horn. K, Immunofluorescence staining of astrocytes (GFAP) and microglia (Iba1) in spinal cord. L and N, Quantitative analysis of mean immunofluorescence intensity of Iba1 (L) and GFAP (N) in groups. Significant difference was found only in GFAP expression between WT and C3 KO mouse (** P = .0052); scale bars: 100 μm, WT, n = 6; C3 KO, n = 7; 2 spinal slices from each mouse were used for analysis. M, O, and P, The morphological features of microglia and astrocytes in groups, including soma size of microglia (M), ending radius (O), and intersections analysis (P) of astrocytes. AUC indicates area under the curve; GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adapter protein 1; C3 KO, C3 knockout; PWT, paw withdrawal threshold; SEM, standard error of the mean; SNI, spared nerve injury; WT, wild type.

Figure 5. Effect of C3 on exercise-induced analgesic effect. A and B, The dose-dependent effect of rC3 (i.t.) on mechanical (A) and cold (B) pain behaviors. One-way ANOVA was used to test the statistical difference and the post hoc Bonferroni test indicates that 50 ng rC3 induced both mechanical (** P = .0062) and cold pain behaviors (** P = .0028). C–E, Analgesic effect of exercise on rC3-induced mechanical and cold allodynia. Schematic of experimental approach (C) and bar graph (D to E) showing significantly higher PWT (* P = .0168) and decreased cold pain behavior (** P = .0025) in the rC3 combined exercise group than rC3 alone group. F–H, The effect of subeffective dose of rC3 on pain threshold of SNI mice after 2 weeks training. Schematic of experimental approach (F) and the changes of PWT (* P = .0348) (G) and cold sensitivity (* P = .0191) (H) were significant after 10 ng rC3 administration; n=7–8 per group. D–H, 2-way ANOVA followed by Bonferroni post hoc test was used to analyze the statistical difference. Data are represented as mean ± SEM. ANOVA indicates analysis of variance; PWT, paw withdrawal threshold; rC3, recombinant C3; SEM, standard error of the mean; SNI, spared nerve injury.

Figure 6. Scheme depicting that analgesic effect of exercise on neuropathic pain via the regulating the C3 of reactive astrocytes. In response to SNI-induced neuropathic pain, the following processes might be triggered: (1) the GFAP expression is upregulated in reactive astrocyte together with corresponding morphological changes, such as process extension, hypertrophy; (2) inflammatory factors are released and inflammatory responses are increased in the spinal cord; and (3) C3 is highly expressed in reactive astrocytes and contributed to neuropathic pain in SNI mice. These processes are reversed by continuous low-intensity exercise training, suggesting that astroglia complement C3 mediates the analgesia produced by exercise in mice with neuropathic pain. C3 indicates component 3; GFAP, glial fibrillary acidic protein; SNI, spared nerve injury.

It is well established that aerobic exercise is an effective type of exercise-induced pain relief after peripheral nerve injury, although different training protocols have shown different results.3 High-intensity exercise training (16 m/s vs 10 m/s) was reported to result in a more complete reversal of sensory hypersensitivity than low-intensity exercise.18 However, low-intensity exercise exerts a therapeutic effect in chronic neuropathic pain, especially when considering exercise capacity limitations due to limb nerve injures.12,19,21,22 It is also reported by others in pain models.23–25 Consistent with several reports showing that prolonged exercise can reduce or fully reverse mechanical and cold allodynia after sciatic nerve injuries,12,19 we found that low-intensity training (10 m/s) significantly reduced cold allodynia from the first week onward, whereas mechanical allodynia was attenuated from the second week of training intervention, which is in line with a previous study.20 In terms of the time course, most running exercise paradigms for chronic pain treatment span for 20 to 120 minutes, and approximately 50% of the paradigms cease running by 20 to 25 minutes. In this study, we found 30 minutes of training to be beneficial for chronic neuropathic pain. The findings of our study further confirm that low-intensity and continuous training is a feasible training regimen having an analgesic effect.

Astrocytes are distinguished from other CNS glial cell types based on the expression of GFAP, which is present in all major branches and processes of astrocytes and dynamically changes during their transition to reactive states in injury-induced allodynia.7 Furthermore, astrocytes exhibit plasticity via changes in their morphology and function in response to stimuli.26 Consistent with previous reports,12,27 our Sholl analysis revealed that exercise inhibited the sum of intersections and the number of intersections at each ring induced by SNI, suggesting that the complexity and extension of astroglial processes can be improved by exercise. Hence, our data suggested that exercise induced the inhibition of astrocyte reactivity and astrocyte morphology recovery in accordance with behavior normalization of mechanical and cold allodynia.

After nerve injury, proinflammatory mediators, such as cytokines of the TNF and IL-1 families, release and augment the development of neuropathic pain. The expression of TNF-α has been shown to increase mechanical allodynia in neuropathy model.28,29 In this study, we found that upregulated IL-1β and TNF-α expression in the spinal cord of SNI mice was reduced after the training intervention, consistent with previous studies.4,12,30,31 These data suggested that exercise training prevented SNI-induced astrocyte reactivity and normalized immune changes in the spinal cord.

Astrocytes undergo phenotypic diversification in the presence of damage- or disease-associated signals, and they can be classified into A1 and A2 phenotypes.9,32 A1 astrocytes are abundant in various human neurodegenerative diseases9,32 and are involved in the maintenance of chronic pain.7,8 The activation of the complement system due to injury increases inflammation by producing complement factors, a key component of the immune system, which recruit and activate immune cells.33 Although C3 has been reported to be present in microglia,34 Schwann cells35 and even satellite cells,36 it is the most characteristic and significantly upregulated gene in A1 astrocytes and is a well-recognized marker of neurotoxic A1 reactive astrocytes, coexpressed with GFAP and S100β immunoreactive astrocytes.9 C3 has been reported to be upregulated in chronic postsurgical pain, paclitaxel-induced neuropathic pain, and spinal cord injury.37–39 Similarly, we observed that astrocytic C3 expression was upregulated in the spinal cord after SNI surgery.

In addition, C3 has been shown to elicit nociception when injected into mouse hind paws.40 Here, we found that the PWT was reduced within 30 minutes and maintained for 3 hours after i.t. injection of rC3, suggesting an algogenic effect of C3 in the spinal cord segment. Importantly, this mechanical allodynia was attenuated after 30 minutes of exercise training. Moreover, a subeffective dose of rC3 reversed the analgesic effect of exercise. Although the timing of the rC3 study does not allow for direct comparison with the SNI study, it illustrated the potential role of spinal C3 in pain as well as exercise analgesia. Furthermore, C3 KO remarkedly attenuated SNI-induced pain, mimicking the analgesic effect of exercise. To overcome the limitations of global KO mice, astrocyte-specific C3 conditional KO mice should be used in future studies. The results of this study demonstrated that exercise exerts its analgesic effect by regulating astroglial C3 in the spinal cord.

CONCLUSIONS

Our study showed that astroglial C3 contributes to the development of SNI-induced allodynia, and exercise training attenuates pain behaviors, suppresses astrocyte reactivity, and decreases neuroimmune responses in the spinal cord of mice. Thus, we can conclude that continuous, low-intensity exercise training is a safe and effective treatment for neuropathic pain.

DISCLOSURES

Name: Chenghao Wang, PhD.

Contribution: This author helped conduct the study, analyze the data, and write the article.

Name: Hui He, PhD.

Contribution: This author helped perform the experiments, analyze the data, and prepare with the article.

Name: Tianchi Gao, BS.

Contribution: This author helped perform the behavioral test.

Name: Xinzheng Sun, MS.

Contribution: This author helped perform the behavioral test.

Name: Lixia Du, PhD.

Contribution: This author participated in Western blotting and immunofluorescence.

Name: Yayue, Yang, MS.

Contribution: This author participated in Western blotting and immunofluorescence.

Name: Jianyu, Zhu, PhD.

Contribution: This author participated in Western blotting and immunofluorescence.

Name: Yachen Yang, BS.

Contribution: This author participated in Western blotting and immunofluorescence.

Name: Yanqing Wang, PhD.

Contribution: This author helped prepare with the article.

Name: Wenli Mi, MD, PhD.

Contribution: This author helped design the study, supervise the project, and write the article.

This manuscript was handled by: Jianren Mao, MD, PhD.

ACKNOWLEDGMENTS

We are grateful to Prof. Jianhua Li (Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, China) for providing us the C3 KO mice.

Supplementary Material

Reprints will not be available from the authors.

Funding: This work was supported by STI2030 – Major Projects 2022ZD0204700, National Natural Science Foundation of China (82271248, 82271258), Innovative research team of high-level local universities in Shanghai, ZJ Lab, and Shanghai Center for Brain Science and Brain-Inspired Technology.

The authors declare no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website.

C. Wang and H. He contributed equally to this work.

All authors agreed for the publication of this article.

Ethical review: The present research was approved by the Animal Research Welfare Council of School of Basic Medical Science of Fudan University (Process No. 20200306-083).

The data that support the findings of this study are available from the corresponding author on request.
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