Propentofylline Relieves Incisional Hyperalgesia by Regulating ERK1/2 Phosphorylation in Spinal Astrocytes and Microglia

Summary

This study investigates the effect of intrathecally administered propentofylline on attenuating incisional hyperalgesia and whether this effect is associated with changes in spinal glial markers and in the phosphorylation of extracellular signal-regulated kinase 1 and 2 in rats.

Abstract

Spinal astrocytes, microglia and extracellular signal-regulated kinase 1 and 2 (ERK1/2) have been implicated in pain processing. This study investigated whether preoperative intrathecal propentofylline (PPF) attenuates acute incisional hyperalgesia in rats and whether the effect is associated with spinal glial markers and ERK1/2 phosphorylation. Sixty-six male Sprague–Dawley rats were randomly assigned to blank, incisional pain, normal saline, dimethyl sulphoxide, PPF and U0126 groups. Mechanical and thermal hyperalgesia were assessed at baseline and 2, 4, 8, 24 and 72 h after incision. Spinal neuronal and glial markers, p-ERK1/2 and inflammatory mediators were evaluated by Western blotting and immunofluorescence. Primary spinal astrocyte experiments were additionally used to examine ERK1/2-dependent inflammatory mediator release. Preoperative intrathecal PPF attenuated mechanical and thermal hyperalgesia throughout the 72-h observation period, whereas U0126 attenuated hyperalgesia mainly during the early post-incision period. At 4 h after incision, both treatments were associated with reduced spinal GFAP, Iba-1 and p-ERK1/2 expression and reduced TNF-α and COX-2 immunoreactivity. In cultured astrocytes, PPF reduced LPS-induced inflammatory mediator release, whereas constitutively active ERK1/2 attenuated this effect. These findings support an association between the antihyperalgesic effect of PPF and modulation of spinal glial, ERK1/2-related and inflammatory responses after incision.

Introduction

Incisional pain, a common type of acute pain following surgical procedures, generally does not persist longer than 7 days but has complex pathological mechanisms that differ from those of inflammatory and neuropathic pain^1,2. If incisional pain is poorly controlled during its development, it may further progress to chronic pain syndrome, and its nature may shift from acute tissue injury pain to neuropathic or mixed pain³. Generally, the administration of analgesics, such as opioids and non-steroidal anti-inflammatory drugs, is the most common form of analgesia following incision. However, many side effects may occur with the continuous or high-dose use of these, a circumstance that highlights the need for safer and more effective analgesic strategies.

Reliable evidence indicates that propentofylline (PPF) relieves hyperalgesia across different pain processes^4,5. Furthermore, PPF’s analgesic effect can be partly achieved by inhibiting the spinal expression of mitogen-activated protein kinases (MAPKs), such as p38 and c-Jun N-terminal kinases (JNKs)⁶. Although PPF’s effects on p38 and JNK pathways have been established, its potential modulation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway, another critical MAPK subfamily implicated in pain signaling, remains unexplored in acute incisional pain. This knowledge gap is particularly important given that ERK1/2 activation shows distinct temporal and cellular patterns compared with p38 and JNK, suggesting potentially unique therapeutic opportunities.

Glial cells, including astrocytes, microglia, and oligodendrocytes, were initially thought to be the connecting and supporting structure of nervous Tissue. However, recent studies have shown that astrocytes and microglia play an important role in regulating synaptic plasticity and transmitting pain information⁷. As the resident macrophages of the brain and spinal cord, microglia can be activated by several extracellular stimulus signals in the very early stage of the pain process and then proliferate and release large amounts of inflammatory cytokines, which increase the conduction of pain signals from the spinal cord to the brain^8,9. Concurrently, activated astrocytes can upregulate the release of the excitatory neurotransmitter glutamate and send information directly to sensory neurons by enhancing intercellular communication¹⁰.

Studies have shown that astrocyte activation occurs in the early stage (within the first 24 h) of incision-induced pain, whereas microglial activation appears much later^11,12. Additionally, one study indicated that activated microglia have a limited effect on mechanical hyperalgesia in incision-induced pain¹³. These findings highlight ongoing questions about the distinct roles of astrocytes and microglia in acute pain. Propentofylline, a glial modulating agent, has been reported to relieve hyperalgesia by regulating the activation of astrocytes and microglia following chronic nerve-constrictive injury, acute nerve injury, and acute Tissue injury^4,5. However, to the best of current knowledge, no study has examined PPF’s effects on astrocyte or microglial activation in acute incision-induced pain.

Extracellular signal-regulated kinase 1 and 2, a major MAPK subfamily activated downstream of mitogen-activated protein kinase 1 and 2 (MEK1/2), have been widely implicated in nociceptive processing and neuronal plasticity^14,15. Previous studies have shown that phospho-ERK1/2 (p-ERK1/2) can be induced in spinal neurons by nociceptive stimulation and can also be detected in astrocytes and microglia in inflammatory or neuropathic pain states^16,17,18,19. In addition, the pharmacological inhibition of MEK1/2–ERK1/2 signaling has been reported to attenuate mechanical and thermal hyperalgesia in several pain models^{20,21,22,23,24,25}.

However, the cellular distribution of spinal ERK1/2, particularly in astrocytes and microglia, activated by an acute incision and its role in incision-induced pain remains insufficiently defined. Previous studies by the research group indicate that PPF relieves acute incisional hyperalgesia in rats by reducing spinal JNK and p38 signaling, but whether PPF also modulates ERK1/2-related signaling in spinal glial cells during incision-induced pain remains unclear. Thus, the present study should be regarded as an extension of the existing glial and MAPK frameworks in pain research rather than a completely independent mechanistic paradigm. Its main distinction from prior work lies in its focus specifically on acute incisional pain, its examination of ERK1/2 rather than p38/JNK as the MAPK component of interest, and its evaluation of the early spinal cellular distribution of p-ERK1/2 in relation to PPF treatment.

This study was designed to investigate whether preoperative intrathecally administered PPF attenuates incisional hyperalgesia in a rat plantar incision model and whether this effect is associated with ERK1/2 phosphorylation or changes in the expression of spinal neuronal and glial markers and inflammatory mediators, tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). More specifically, this study differs from prior reports in its combination of behavioral assessment with temporal Western blot analysis; its pharmacological comparison with the MEK1/2 inhibitor U0126; and its qualitative immunofluorescence localization of p-ERK1/2 in spinal neurons, astrocytes, and microglia within an acute incisional pain model. Although its mechanistic framework builds on previous literature linking glial modulation and MAPK signaling to pain, the present work is intended to provide additional evidence for the possible involvement of spinal ERK1/2-related glial responses in the early phase of incisional hyperalgesia.

Compared with conventional postoperative analgesics, pathway-targeting approaches can test a defined signaling component but may provide only time-limited benefit and can require invasive delivery; MEK1/2–ERK1/2 inhibition has shown antihyperalgesic effects in several experimental pain models^{20,21,22,23,24,25}. PPF differs from a single-pathway inhibitor because it modulates glial responses and has been linked to more than one MAPK-associated process^4,5,6. Practical translation remains limited by the preclinical design, preoperative intrathecal administration, use of male rats only, and the absence of dose-ranging and safety studies in larger surgical models.

Protocol

The experiments conducted were approved by the Ethics Committee of the Second Affiliated Hospital, Lanzhou University, Gansu Province, China (ethics approval no. 2017-070). All procedures followed the International Association for the Study of Pain guidelines²⁶. A total of 66 adult male Sprague–Dawley rats obtained from an accredited laboratory animal centre and weighing 200–250 g were used. Only male rats were included in this study to reduce potential variability related to sex-dependent hormonal influences. All protocols were performed in accordance with the International Association for the Study of Pain guidelines²⁶, and the necessary efforts to minimize suffering were taken. Detailed information on the reagents, consumables, instruments, and software used in this study is provided in the Table of Materials.

Animals

The rats were housed in pairs before operation and individually after incision with food and water ad libitum. The room was maintained in a 12-h light–12-h dark cycle and temperature-controlled at 24 °C ± 2 °C.

Intrathecal injection and incisional pain model

For the intrathecal administration of solutions, rats were anesthetized with 1.5%–3% sevoflurane (delivered through a nose cone) and were placed in the prone position with the vertebral column flexed. The fur over the lumbar region was shaved, and the skin over the L5–L6 intervertebral space was disinfected. A microsyringe was inserted percutaneously at the L5–L6 intervertebral space. Successful entry into the subarachnoid space was indicated by a sudden tail flick or tail movement during needle advancement and was further confirmed by the appearance of cerebrospinal fluid upon gentle aspiration. If these signs were not observed, the needle position was adjusted before injection. Injection was performed only after both confirmation criteria had been satisfied, and animals showing abnormal neurological signs after injection were excluded. The respective solution was then injected slowly, and the skin was cleaned again after injection. The surgical procedure lasted approximately 3 min, and the rats generally recovered from anesthesia within 5–10 min.

The rat model of incisional pain was established as previously described²⁷. After anesthesia, the plantar surface of the hind paw was disinfected with 10% povidone–iodine. A 1-cm longitudinal incision was made in the plantar skin, beginning approximately 0.5 cm from the heel and extending towards the toes. The incision penetrated the skin and fascia. The plantar muscle was gently elevated with ophthalmic forceps and incised longitudinally while preserving overall tissue continuity. The muscle was then repositioned, gentle pressure was applied to achieve hemostasis, and the wound was closed with two mattress sutures using 3-0 nylon. The incision site was disinfected again after wound closure. Following surgery, the rats were housed individually and allowed to recover in a quiet, warm environment, protected from strong light and with free access to food and water. The animals were monitored postoperatively, and those not meeting the predefined criteria were excluded promptly. Wound infection was determined based on redness, swelling, and increased local temperature around the wound, abnormal yellow-green purulent exudate with malodour, delayed wound healing with persistent wound opening, necrosis and enlargement, and systemic signs including elevated body temperature, lethargy, and refusal to feed. Additional exclusion criteria included a failure to recover from anesthesia within 30 min, intraoperative muscle transection or neurovascular injury, postoperative self-mutilation, unsuccessful model establishment, and neurological deficits, such as hind limb paralysis or an inability to bear weight that was unrelated to incisional pain. Rats with wound infection or dehiscence were excluded from the study.

Behavioral measurement

Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured by an investigator blinded to group allocation. Testing was performed in a quiet room at a consistent time of day after at least 10 min of acclimatisation; the enclosure, mesh or glass floor, ambient light, and heat-source intensity were kept constant across groups. For PWMT testing, calibrated von Frey filaments (0.4 g, 0.6 g, 1.0 g, 1.4 g, 2.0 g, 4.0 g, 6.0 g, 8.0 g, 10.0 g, and 15.0 g) were applied perpendicular to the plantar surface adjacent to the wound until the filament bent by approximately 1–2 mm. Consecutive stimulations were separated by 2 min, and the 50% PWMT was determined using the up-and-down method²⁸. For PWTL testing, rats were placed in individual transparent enclosures on a glass floor, and the radiant heat source was aligned with the plantar surface beside the wound. A 30 s cut-off prevented tissue injury, consecutive exposures were separated by 2 min, and the mean of three measurements was recorded²⁹. Paw lifting, shaking, or licking was considered a positive withdrawal response; trials affected by grooming, locomotion, or loss of heat-source alignment were repeated after reacclimatization.

Western blot analysis

At the preset times, rats were euthanized by cervical dislocation, and the L4–L6 lumbar spinal cord was rapidly harvested into radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitor at a ratio of 100:1. Tissue was homogenised, kept on ice for 20 min, and centrifuged at 15,000 × g for 20 min at 4 °C. The supernatant was mixed with 4× loading buffer at a ratio of 3:1, heated in boiling water for 5 min, and stored at −80 °C. Protein samples (30 µg per lane) were separated on 10% sodium dodecyl sulfate–polyacrylamide gels and transferred to 0.45 or 0.22 µm polyvinylidene fluoride membranes. Successful transfer was checked by confirming the expected migration of the prestained molecular-weight marker and the absence of obvious uneven or interrupted transfer regions. Membranes were blocked with 5% skim milk and 3% bovine serum albumin for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies against NeuN (1:8,000), GFAP (1:4,000), Iba-1 (1:3,000), p-ERK1/2 (1:1,000), t-ERK1/2 (1:3,000), or GAPDH (1:2,000). After washing in Tris-buffered saline containing 0.05% Tween 20 (pH 7.4), membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (1:5,000) for 1 h at room temperature. Signals were developed with enhanced chemiluminescence and recorded on film using the same exposure range within each target. An investigator blinded to group allocation analyzed band density with image-analysis software. Images were converted to 8-bit grayscale, an identical rectangular region of interest was applied to each band, local background from an adjacent blank region was subtracted, and the integrated density was recorded. The same region size and background procedure were used for all lanes on a blot. NeuN, GFAP, Iba-1, and t-ERK1/2 were normalized to GAPDH; p-ERK1/2 was normalized to t-ERK1/2; and values were then expressed relative to the blank group.

Immunofluorescence

At the preset times, rats were anesthetized with 3% pentobarbital sodium (1.5 mL/kg) and perfused through the heart with 300 mL of precooled 0.01 mol/L phosphate-buffered saline (PBS; pH 7.4), followed by 250 mL of precooled 4% paraformaldehyde. The L4–L6 spinal cord was post-fixed in 4% paraformaldehyde for 6 h, cryoprotected overnight in 20%–30% sucrose–PBS at 4 °C and cut into 10 µm sections at −22 °C. Sections were washed in 0.01 mol/L PBS, blocked in 10% goat serum for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against NeuN (1:100), GFAP (1:1,000), Iba-1 (1:100), p-ERK1/2 (1:100), TNF-α (1:200) or COX-2 (1:50). After three PBS washes, sections were incubated with the corresponding green- or red-fluorophore-conjugated secondary antibody (1:400) for 1 h at room temperature, washed three further times in PBS and mounted with antifade medium and a coverslip. Negative-control sections processed without the primary antibody were included in each staining batch. Images were acquired by a blinded investigator with a fluorescence microscope. Low-magnification overview images were acquired using a 10× objective, whereas cellular immunoreactivity and co-localization images were acquired using 20× or 40× objectives according to the corresponding figure panel. For comparisons within the same figure and staining batch, objective magnification, illumination intensity, exposure time, detector gain, camera settings, and image-processing parameters were fixed after optimization below saturation. Separate fluorescence channels were acquired sequentially to minimize channel overlap. Three non-overlapping fields in the ipsilateral dorsal horn were captured per animal. Fluorescence intensity was quantified with image-analysis software after 8-bit conversion, background subtraction using a cell-free region, and application of an identical region of interest and threshold within each staining batch; the field values were averaged to obtain one value per animal. Fields with overexposure, tissue folding, tearing, poor focus, or uneven background were excluded and reacquired under the same non-saturating settings. Staining was accepted when tissue architecture was intact, the target signal exceeded the no-primary-antibody control, and no major fold, tear, or saturated region affected the analysis.

In vitro primary spinal astrocyte experiments

Primary rat spinal astrocytes were cultured under standard sterile conditions in complete culture medium at 37 °C in a humidified 5% CO₂ incubator. Cells were used when they reached approximately 70%–80% confluence and were assigned to control, LPS-stimulated, LPS + PPF, LPS + U0126, and ERK1/2 rescue groups. PPF and U0126 were added at 10 µM. LPS stimulation, drug treatment, and harvest conditions were kept identical across parallel wells within each experiment. For pathway-selectivity analysis, cell lysates were collected for Western blotting of p-ERK1/2, t-ERK1/2, p-p38, total p38, p-JNK, total JNK, and the corresponding loading control. Culture supernatants from parallel wells were collected, clarified by centrifugation, and analyzed for TNF-α, PGE2, and IL-6 using enzyme-linked immunosorbent assay according to the manufacturer’s instructions. Western blot densitometry followed the same image-analysis principles described above. For rescue experiments, endogenous ERK1/2 was suppressed by siRNA transfection, and a constitutively active ERK1/2 (CA-ERK1/2) expression construct was introduced before treatment. Knockdown and overexpression efficiency were confirmed by Western blotting before cytokine data were interpreted. Cells exposed to the transfection reagent without active siRNA or construct served as procedural controls when applicable.

Experimental design

Subgrouping of experimental animals

The rats were assigned to six groups using a random number table method, and group allocation was completed before behavioral testing and tissue collection. The six groups comprised a blank group (n = 6), an incisional pain (IP) group (n = 18), a normal saline (NS) group (0.9% NS, 10 µL; n = 9), a PPF group (10 µg in 10 µL; n = 12), a dimethyl sulfoxide (DMSO) group (10% DMSO, 10 µL; n = 9) and a U0126 group (10 µg in 10 µL; n = 12). Rats in the blank group received neither a plantar incision nor an intrathecal injection and served as untreated controls. Rats in the IP group underwent plantar incision without pretreatment by intrathecal injection. Rats in the NS, PPF, DMSO, and U0126 groups received intrathecal administration of their respective agents 30 min before plantar incision. The NS group served as the vehicle control for PPF, whereas the DMSO group served as the vehicle control for U0126.

Doses of propentofylline and U0126

Propentofylline is a glial modulator, and a dose of 10 µg was selected because it had relieved acute hyperalgesia in earlier studies by the research group. U0126 is a non-adenosine triphosphate-competitive MEK1/2 inhibitor used to block ERK1/2 phosphorylation. Effective intrathecal doses of 1–10 µg have been reported across pain models^30,31,32; the 10 µg dose was used in the present study. U0126 was dissolved in 10% dimethyl sulfoxide, whereas PPF was dissolved in 0.9% normal saline. Final concentrations were 1 µg/µL. Drugs and vehicles were prepared before surgery, maintained at 4 °C, and used within 12 h.

Effect of the preoperative intrathecal administration of propentofylline or U0126 on hyperalgesia

Six rats from each group were randomly drawn for behavioral measurement 24 h before the operation, and the resultant PWMTs and PWTLs were designated as baseline. Intrathecal injections were administered 30 min before the operation. To assess the effects of the preoperative intrathecal administration of PPF and U0126 on mechanical and thermal hyperalgesia, PWMT and PWTL were assessed at 2 h, 4 h, 8 h, 24 h, and 72 h post-incision.

Experimental design for evaluating the effects of preoperative intrathecally administered propentofylline or U0126 on spinal neuronal and glial marker expression following incision

To determine the appropriate post-incision timepoint for treatment–effect assessment, a preliminary time course analysis was first performed in the IP group. Specifically, rats in the IP group were euthanized at 2 h, 4 h, 24 h, and 72 h after incision, and the expression levels of the cell markers NeuN, GFAP, and Iba-1 in the spinal cord were measured by Western blot (n = 3 per timepoint). Based on this preliminary analysis, the first 4 h post-incision were selected as an early post-incision observation window because NeuN expression reached its highest level at the 4 h timepoint and because early molecular changes were considered most relevant to the initial development of incisional hyperalgesia. However, the 4 h timepoint was not intended to represent all temporal molecular changes after incision.

At this selected timepoint, rats that had been administered an intrathecal injection were euthanized for Western blot analysis of different cell markers (n = 3 per group) and immunofluorescence evaluation of the morphology of different cell types (n = 3 per group). In the blank group, rats were randomly terminated to determine the basic expression levels of different cell markers (NeuN, GFAP, and Iba-1) in the spinal cord by Western blot (n = 3) and the morphology of different cells (neurons, astrocytes, and microglia) by immunofluorescence (n = 3). Thus, the blank group served as the non-incised, non-treated baseline control.

Treatment effects on spinal p-ERK1/2 and inflammatory mediators

To determine the appropriate post-incision timepoint for evaluating treatment effects on p-ERK1/2 expression and levels of inflammatory cytokines, a preliminary time course analysis was first performed in the IP group. Specifically, rats in the IP group were euthanized at 2 h, 4 h, 24 h and 72 h after incision, and spinal p-ERK1/2 expression was measured by Western blot (n = 3 per timepoint). Based on this time course experiment, together with the timepoint at which NeuN expression was highest after incision, 4 h after incision was selected as the fixed timepoint for the subsequent evaluation of the effects of preoperative intrathecally administered PPF or U0126 on spinal p-ERK1/2 expression and inflammatory cytokines. This timepoint was selected as an early mechanistic observation window rather than as a comprehensive representation of all temporal changes after incision.

At this selected timepoint, rats that had been administered an intrathecal injection were euthanized for Western blot analysis of p-ERK1/2 expression (n = 3 per group) and immunofluorescence evaluation of inflammatory cytokines (n = 3 per group). In the blank group, rats were randomly terminated to determine the basic expression levels of p-ERK1/2 in the spinal cord by Western blot (n = 3) and of inflammatory cytokines (TNF-α and COX-2) by immunofluorescence (n = 3). Thus, the blank group served as the non-incised, non-treated baseline control.

Cellular localization of spinal p-ERK1/2 in neurons, astrocytes and microglia

To further assess the cellular distribution of spinal p-ERK1/2 after incision, immunofluorescence staining was performed in the Blank and IP groups at 4 h after incision (n = 3).

Statistical analysis

Data are expressed as mean ± standard deviation. Sample size was determined with reference to earlier studies by the research group and preliminary experiments. Behavioral data were analyzed by two-way repeated-measures analysis of variance (ANOVA), with group as the between-subject factor and time as the within-subject factor. Time-course Western blot data were evaluated by one-way ANOVA followed by Bonferroni-adjusted pairwise comparisons. Fixed-time Western blot and immunofluorescence data were evaluated by one-way ANOVA with Bonferroni-adjusted planned comparisons; two-group comparisons were evaluated by Student’s t-test. Normality and homogeneity of variance were assessed with the Shapiro–Wilk and Levene tests, respectively. A two-sided p-value < 0.05 was considered significant. Statistical analyses were performed with the statistical analysis software listed in the Table of Materials. For the in vitro astrocyte experiments, independent culture experiments were treated as the unit of analysis; Western blot densitometry and cytokine concentrations were compared using one-way ANOVA followed by Bonferroni-adjusted planned comparisons or Student’s t-test for prespecified two-group comparisons, as appropriate.

DATA AVAILABILITY:

Data supporting the findings of this study are provided in Supplementary File 1.

Results

The effects of incision and of the preoperative intrathecal administration of propentofylline or U0126 on hyperalgesia after incision

Mechanical hyperalgesia

There were no significant differences in PWMTs between the six groups at 24 h before operation (p > 0.05; Figure 1A,B). In the IP group, the PWMT decreased after incision and remained significantly lower than baseline from 2 h to 72 h after incision (p < 0.05 vs baseline; Figure 1A). In the NS and DMSO groups, there were no significant differences in PWMTs between timepoints after incision (p > 0.05 vs the IP group; Figure 1B). In the PPF group, the PWMT was significantly higher than that in the IP group from 2 h to 72 h after incision (p < 0.05 vs the IP group; Figure 1A), and at 72 h after incision, it was no longer significantly different from baseline (p > 0.05 vs baseline; Figure 1A). In the U0126 group, PWMTs were significantly higher than those in the IP group during the first 24 h after incision (p < 0.05 vs the IP group; Figure 1A), but this effect was not maintained at 72 h after incision, when the PWMT remained significantly lower than baseline and was no longer significantly different from that in the IP group (p < 0.05 vs baseline; p > 0.05 vs the IP group; Figure 1A).

Thermal hyperalgesia

There were no significant differences in PWTLs between the six groups at 24 h before operation (p > 0.05; Figure 1C,D). In the IP group, PWTL decreased from 24.85 s ± 1.06 s before operation to 4.38 s ± 1.02 s at 4 h after incision and then partially increased to 18.37 s ± 1.38 s at 72 h after incision, but remained significantly lower than baseline throughout the observation period (p < 0.05 vs baseline; Figure 1C). In the NS and DMSO groups, there were no significant differences in PWTLs between timepoints after incision (p > 0.05 vs the IP group; Figure 1D). In the PPF group, PWTL was significantly higher than that in the IP group throughout the 72 h observation period (p < 0.05 vs the IP group; Figure 1C), and at 72 h after incision, it had returned to a level that was not significantly different from baseline (p > 0.05 vs baseline; Figure 1C). In the U0126 group, PWTL was significantly higher than that in the IP group during the first 24 h after incision (p < 0.05 vs the IP group; Figure 1C); this effect was not maintained at 72 h after incision, when the PWTL remained significantly lower than baseline and was no longer significantly different from that in the IP group (p < 0.05 vs baseline; p > 0.05 vs the IP group; Figure 1C).

Western blot analysis of temporal changes in spinal cell marker expression and extracellular signal-regulated kinase 1 and 2 phosphorylation after incision

There were several bands identified at exact molecular weights with a specific antibody on the PVDF membranes, including NeuN (48 and 50 kD), GFAP (50 kD), Iba-1 (17 kD), p-ERK1/2 (p-ERK1 = 44 kD; p-ERK2 = 42 kD), t-ERK1/2 (t-ERK1 = 44 kD; t-ERK2 = 42 kD), and GAPDH (36 kD). The GAPDH band was used as the control for NeuN, GFAP, Iba-1, and t-ERK1/2, and the t-ERK1/2 band was used as the control for p-ERK1/2.

Western blotting data from the spinal cords in the IP group showed increases of varying degrees after incision (Figure 2A). Neuron-specific nuclear protein expression reached its highest value at 4 h after incision (p < 0.001 vs baseline; Figure 2A) and remained significantly elevated 72 h after incision (p < 0.001 vs baseline; Figure 2A). Glial fibrillary acidic protein levels increased gradually over the 72-h observation period (p < 0.001 vs baseline; Figure 2A), with the highest level observed at 72 h among the measured timepoints. Similarly, Iba-1 also reached a significantly increased level at 4 h after incision (p < 0.001 vs baseline; Figure 2A) and fluctuated within a small range. Notably, Western blotting data from the spinal cords in the IP group showed ERK1/2 activation only increased during the first 24 h after incision (p < 0.05 vs baseline; Figure 2B) but was decreased at 72 h after incision (p < 0.05 vs baseline; Figure 2B), whereas the expression of t-ERK/2 showed no significant change at any timepoint.

Western blot analysis of the effects of the preoperative intrathecal administration of propentofylline or U0126 on the expression of cell markers and phospho-extracellular signal-regulated kinase 1 and 2 in the spinal cord

Western blotting data showed that NeuN expression reached its highest level at 4 h after incision, when the behavioral measurement was at its lowest threshold. Therefore, the effects on cell marker expression and ERK1/2 phosphorylation of PPF and U0126 administered intrathecally 30 min before operation were assessed at 4 h after incision.

Western blotting data showed that, compared with the blank group, the spinal expression of all cell markers was significantly increased in the IP group at 4 h after incision (p < 0.05 vs baseline; Figure 3). Compared with the IP group, preoperative intrathecal PPF administration reduced the spinal expression of NeuN (p < 0.05 vs the IP group; Figure 3A), GFAP (p < 0.05 vs the IP group; Figure 3B), and Iba-1 (p < 0.01 vs the IP group; Figure 3C). Compared with the IP group, preoperative intrathecal U0126 administration also reduced the spinal expression of NeuN (p < 0.01 vs the IP group; Figure 3D), GFAP (p < 0.05 vs the IP group; Figure 3E), and Iba-1 (p < 0.01 vs the IP group; Figure 3F). When compared with the blank group, however, NeuN expression in the PPF group remained significantly higher than baseline (p < 0.01 vs baseline; Figure 3A), whereas NeuN expression in the U0126 group was significantly lower than that in the blank group (p < 0.01 vs baseline; Figure 3D). For GFAP and Iba-1, the text now emphasises the comparison with the IP group, as shown in Figure 3. The preoperative intrathecal administration of NS and DMSO had no significant effect on the expression of any cell markers.

Western blotting data showed that spinal t-ERK1/2 expression did not differ significantly among the Blank, IP, NS, and PPF groups at 4 h after incision (Figure 4A). In contrast, compared with the blank group, p-ERK1/2 expression was significantly increased in the IP and NS groups at 4 h after incision (p < 0.001 vs the blank group; Figure 4B). Compared with the IP group, preoperative intrathecal PPF administration significantly reduced p-ERK1/2 expression at 4 h after incision (p < 0.001 vs the IP group; Figure 4B). Compared with the NS group, p-ERK1/2 expression in the PPF group was also significantly lower (p < 0.01 vs the NS group; Figure 4B). However, compared with the blank group, p-ERK1/2 expression in the PPF group remained significantly elevated at 4 h after incision. Similarly, spinal t-ERK1/2 expression did not differ significantly among the Blank, IP, DMSO, and U0126 groups at 4 h after incision (Figure 4C). Compared with the blank group, p-ERK1/2 expression was significantly increased in the IP and DMSO groups at 4 h after incision (p < 0.001 vs the blank group; Figure 4D). Compared with the IP group, preoperative intrathecal U0126 administration significantly reduced p-ERK1/2 expression (p < 0.001 vs the IP group; Figure 4D). Compared with the DMSO group, p-ERK1/2 expression in the U0126 group was also significantly lower (p < 0.001 vs the DMSO group; Figure 4D). Notably, compared with the blank group, p-ERK1/2 expression in the U0126 group was not significantly different from that in the blank group at 4 h after incision. Pretreatment with NS or DMSO had no significant effect on p-ERK1/2 expression when compared with the IP group.

Immunofluorescence observations of spinal cell markers and inflammatory mediators

The results of immunofluorescence qualitatively showed stronger NeuN immunoreactivity in the ipsilateral dorsal horn at 4 h after incision compared with the contralateral side (Figure 5). In the blank group, representative images showed baseline NeuN, GFAP, and Iba-1 immunoreactivity in the ipsilateral spinal cord dorsal horn (Figure 6A–C). In the IP group, NeuN immunoreactivity was qualitatively stronger than that in the blank group (Figure 6D vs Figure 6A), GFAP immunoreactivity was also stronger (Figure 6E vs Figure 6B), and Iba-1 immunoreactivity was increased as well (Figure 6F vs Figure 6C). Concurrently, astrocytes and microglia in the IP group appeared hypertrophic, with enlarged cell bodies and thicker, more highly ramified processes (Figure 6E,F).

In the PPF group, representative images suggested weaker NeuN immunoreactivity than in the IP group (Figure 6G vs Figure 6D), weaker GFAP immunoreactivity (Figure 6H vs Figure 6E), and weaker Iba-1 immunoreactivity (Figure 6I vs Figure 6F). In the U0126 group, representative images similarly suggested weaker NeuN immunoreactivity than in the IP group (Figure 6J vs Figure 6D), weaker GFAP immunoreactivity (Figure 6K vs Figure 6E), and weaker Iba-1 immunoreactivity (Figure 6L vs Figure 6F). In both the PPF and U0126 groups, astrocytes and microglia appeared less hypertrophic, with smaller cell bodies and fewer, thinner processes (Figure 6H,I,K,L). The corresponding fluorescence-intensity analysis of NeuN, GFAP, and Iba-1 was consistent with the representative immunofluorescence observations (Figure 6M–O, respectively).

At 4 h after incision, TNF-α and COX-2 immunoreactivity and relative fluorescence intensity were increased in the ipsilateral dorsal horn of the IP group compared with the blank group. Both measures were lower after preoperative intrathecal PPF or U0126 treatment (Figure 7A–C).

Cellular localization of spinal p-ERK1/2 by immunofluorescence

The results of immunofluorescence showed that in the blank group, p-ERK1/2 immunoreactivity was weak in the spinal cord and observed only in NeuN-positive neurons (Figure 8A–C). By contrast, at 4 h after incision, p-ERK1/2 immunoreactivity was markedly increased in the ipsilateral spinal cord dorsal horn and co-localised not only with neurons but also with GFAP-positive astrocytes and Iba-1-positive microglia (Figure 8D–I). This finding indicates that incision induced a broader cellular distribution of p-ERK1/2, from neurons alone under baseline conditions to neurons, astrocytes, and microglia after injury.

In vitro validation of the ERK1/2-dependent mechanism in spinal astrocytes

To further evaluate whether PPF could suppress inflammatory mediator release through ERK1/2-related signaling at the cellular level, primary rat spinal astrocytes were stimulated with LPS and treated with PPF or U0126 (10 µM). Western blot analysis showed that PPF reduced LPS-induced p-ERK1/2 expression, whereas p-p38 and p-JNK phosphorylation were not evidently changed (Figure 9A), supporting relative ERK1/2 pathway selectivity under these conditions. In parallel, PPF reduced TNF-α, PGE2 (a downstream product of COX-2 activity), and IL-6 levels in the culture supernatant compared with the Stim group; U0126 produced similar inhibitory effects (Figure 9B).

To test whether persistent ERK1/2 activation could weaken the anti-inflammatory effect of PPF, rescue experiments were performed. Endogenous ERK1/2 was knocked down using siRNA, and a constitutively active ERK1/2 (CA-ERK1/2) construct was subsequently introduced (Figure 9C). In astrocytes expressing CA-ERK1/2, PPF no longer reduced TNF-α, PGE2, or IL-6 levels to the same extent (Figure 9D), indicating that suppression of ERK1/2 phosphorylation contributes to the anti-inflammatory effect of PPF in this in vitro astrocyte system.

Collectively, these in vitro findings provide mechanistic support at the cellular level that PPF attenuates inflammatory mediator release through an ERK1/2-dependent mechanism in spinal astrocytes, complementing the in vivo observational data.

Figure 1: Effects of preoperative intrathecal PPF or U0126 on incision-induced mechanical and thermal hyperalgesia. (A) PWMT in the blank, IP, PPF, and U0126 groups. (B) PWMT in the IP and vehicle-control groups. (C) PWTL in the blank, IP, PPF, and U0126 groups. (D) PWTL in the IP and vehicle-control groups. Data are mean ± SD (n = 6 rats per group) and were analyzed by two-way repeated-measures ANOVA. ***p < 0.001 versus baseline; #p < 0.05, ##p < 0.01 and ###p < 0.001 versus the IP group. Please click here to view a larger version of this figure.

Figure 2: Time course of spinal cell-marker expression and ERK1/2 phosphorylation after incision. (A) Quantification and representative Western blots of NeuN, GFAP, and Iba-1 in the IP group from baseline to 72 h after incision. (B) Quantification and representative Western blots of t-ERK1/2 and p-ERK1/2 over the same period. Values were normalized to the blank group (100%) and are shown as mean ± SD (n = 3 rats per timepoint). Overall differences were analyzed by one-way ANOVA with the indicated pairwise comparisons. *p < 0.05, **p < 0.01 and ***p < 0.001 versus baseline. Please click here to view a larger version of this figure.

Figure 3: Effects of preoperative intrathecal PPF or U0126 on spinal neuronal and glial markers at 4 h after incision. (A–C) NeuN, GFAP, and Iba-1 in the blank, IP, normal-saline, and PPF groups. (D–F) NeuN, GFAP, and Iba-1 in the blank, IP, dimethyl-sulfoxide, and U0126 groups. Representative Western blots are shown at right. Values were normalized to the blank group (100%) and are shown as mean ± SD (n = 3 rats per group). *p < 0.05, **p < 0.01 and ***p < 0.001 versus blank; #p < 0.05 and ##p < 0.01 versus IP; Δp < 0.05, ΔΔp < 0.01 and ΔΔΔp < 0.001 versus the corresponding vehicle group. Please click here to view a larger version of this figure.

Figure 4: Effects of preoperative intrathecal PPF or U0126 on spinal ERK1/2 phosphorylation at 4 h after incision. (A,B) t-ERK1/2 and p-ERK1/2 in the blank, IP, normal-saline, and PPF groups. (C,D) t-ERK1/2 and p-ERK1/2 in the blank, IP, dimethyl-sulfoxide, and U0126 groups. Representative Western blots are shown at right. Values were normalized to the blank group (100%) and are shown as mean ± SD (n = 3 rats per group). **p < 0.01 and ***p < 0.001 versus blank; ###p < 0.001 versus IP; ΔΔp < 0.01 and ΔΔΔp < 0.001 versus the corresponding vehicle group. Please click here to view a larger version of this figure.

Figure 5: Ipsilateral and contralateral spinal NeuN immunoreactivity after incision. A representative transverse lumbar spinal cord section from the IP group at 4 h after incision shows NeuN staining in the contralateral and ipsilateral dorsal horns. The image is representative of n = 3 rats and is provided for qualitative anatomical comparison. Scale bar = 500 µm. Please click here to view a larger version of this figure.

Figure 6: Effects of PPF and U0126 on spinal neuronal and glial immunoreactivity at 4 h after incision. (A–C) NeuN, GFAP, and Iba-1 in the blank group. (D–F) Corresponding staining in the IP group. (G–I) Corresponding staining after PPF treatment. (J–L) Corresponding staining after U0126 treatment. (M–O) Relative fluorescence intensities of NeuN, GFAP, and Iba-1, respectively. Bars show mean ± SD (n = 3 rats per group); *p < 0.05, **p < 0.01 and ***p < 0.001 for the comparisons indicated. Scale bars = 200 µm. Please click here to view a larger version of this figure.

Figure 7: Spinal TNF-α and COX-2 immunoreactivity after incision and treatment. (A) Representative TNF-α and COX-2 images from the blank, IP, PPF, and U0126 groups at 4 h after incision. (B) Relative TNF-α fluorescence intensity. (C) Relative COX-2 fluorescence intensity. Bars show mean ± SD (n = 3 rats per group); ***p < 0.001 for the comparisons indicated. Scale bars = 200 µm. Please click here to view a larger version of this figure.

Figure 8: Cellular localization of spinal p-ERK1/2 at 4 h after incision. (A–C) p-ERK1/2 co-staining with NeuN, GFAP and Iba-1, respectively, in the blank group. (D–F) Corresponding co-staining in the IP group; arrows identify representative co-localised cells. Red indicates p-ERK1/2, green indicates the cell marker, and yellow indicates co-localization. (G–I) Relative fluorescence intensities associated with NeuN-, GFAP-, and Iba-1-positive regions, respectively. Bars show mean ± SD (n = 3 rats per group); **p < 0.01 and ***p < 0.001 for the comparisons indicated. Scale bars = 100 µm. Please click here to view a larger version of this figure.

Figure 9: ERK1/2-dependent anti-inflammatory effects of PPF in primary rat spinal astrocytes. (A) Representative Western blots showing that PPF reduced LPS-induced p-ERK1/2 without an evident effect on p-p38 or p-JNK. (B) TNF-α, PGE2, and IL-6 concentrations in culture supernatants after LPS, PPF, or U0126 treatment. (C) Western blots confirming siRNA-mediated ERK1/2 knockdown and expression of constitutively active ERK1/2 (CA-ERK1/2). (D) TNF-α, PGE2, and IL-6 concentrations after CA-ERK1/2 expression with or without PPF. Bars show mean ± SD from independent in vitro experiments; ***p < 0.001 for the comparisons indicated; ns, not significant. Please click here to view a larger version of this figure.

Supplementary File 1: Data supporting the findings of this study.Please click here to download this file.

Discussion

Most patients suffer from acute pain after surgery, not only physically but even at times mentally³³, yet much is still unknown about the detailed mechanisms of acute postoperative pain. Along with the powerful effects of analgesic medicines come their inevitable side effects, making the exploration of safer, effective analgesics of critical importance. This study was based on a previously validated incisional rat model for simulating acute postoperative pain that was easy and feasible to prepare²⁷. The present study evaluated changes in spinal neuronal and glial markers, ERK1/2 phosphorylation, and inflammatory mediators after incision and examined whether the antihyperalgesic effect of preoperative intrathecal PPF administration was associated with parallel changes in spinal ERK1/2 phosphorylation and inflammatory cytokine expression.

Following incision, rats in the IP group developed robust mechanical and thermal hyperalgesia, whereas vehicle pretreatment had no detectable effect on either behavioral or molecular outcomes. These findings support the stability of the model and indicate that the observed effects in the PPF and U0126 groups were unlikely to be attributable to the injection procedure or vehicle itself. In addition, although NeuN expression increased after incision, this change should be interpreted cautiously as a change in neuronal marker expression rather than direct evidence of neuronal activation. Together, these observations provide the basis for examining glial and ERK1/2-related responses in the early development of incisional pain.

Glial cells have been shown to play an important role in synaptic formation, synaptic information transmission and integration, and synaptic plasticity regulation. The immunologic and proinflammatory functions of glial cells in pain processing, and especially of activated spinal astrocytes and microglia, may enhance the central sensitisation involved in mediating hyperalgesia and postoperative behavioral changes^7,34. Maladaptive neurological changes in the spinal cord may lead to an increased responsiveness to noxious or non-noxious stimuli. Considerable evidence indicates that activation of spinal astrocytes and microglia is involved in the development and maintenance of hyperalgesia in models of spinal nerve injury³⁵, tissue plasminogen activator-induced pain¹⁶, incisional pain^11,12, and bone cancer pain^19,31. Therefore, spinal glial activation appears to be a common feature of multiple pain states, and a further exploration of its role in incisional pain may extend the current understanding of postoperative pain mechanisms.

The present findings support the view that spinal astrocytes and microglia participate in the early neuroinflammatory response following incision¹². Rather than showing identical temporal patterns, GFAP, Iba-1, and p-ERK1/2 exhibited partially distinct time courses, suggesting that different glial-related processes may contribute to different phases of postoperative pain development^31,35. Immunofluorescence findings further supported this interpretation by showing hypertrophic morphological changes in astrocytes and microglia following incision. Together with the increases in TNF-α and COX-2 immunoreactivity, these observations are consistent with the concept that glial-associated inflammatory signaling contributes to acute incisional hyperalgesia^7,36.

Propentofylline, a derivative of methylxanthine, was initially studied as a cerebrovascular active drug because of its antiplatelet activity and support of long-term memory maintenance³⁷. However, the regulatory effect of PPF on glial cells makes it potentially valuable as an analgesic drug as well. Studies indicate that PPF can impede the activation of microglia by inhibiting the decomposition of intracellular cyclic adenosine monophosphate and reducing the reuptake of extracellular adenosine^4,5. In an in vitro study, PPF inhibited the proliferation of astrocytes by hindering the TNF-α-induced mammalian target of rapamycin pathway⁶. Thus, it is the regulatory effect of PPF on glial cells that makes its participation in the modulation of pain processes possible.

The sustained antihyperalgesic effect of PPF in the present study, together with the reductions in GFAP and Iba-1 expression, supports the interpretation that glial modulation is an important component of PPF’s analgesic action in this model. Previous studies have also suggested that PPF may suppress glial-related inflammatory signaling in pain states^5,31. In addition, the parallel reduction in TNF-α and COX-2 suggests that PPF may attenuate incisional pain partly by dampening glial-associated inflammatory responses. However, because the present data are associative rather than mechanistic, these findings should be interpreted as support for glial involvement rather than proof of a direct causal pathway.

The involvement of ERK1/2, a pair of important intracellular signal-regulated kinases, in the development and maintenance of different pain processes (by mediating neuronal excitability and synaptic plasticity in the brain and spinal cord) has been proven²¹. For example, a study showed that the injection of capsaicin or formalin induced a short and rapid increase of p-ERK1/2 in the spinal cord, but spinal ERK1/2 activation persisted much longer following the injection of complete Freund’s adjuvant or bee venom (after a short latency)^38,39. Conversely, spinal ERK1/2 activation induced by nerve injury peaked extremely early in another study and persisted for some time, with hyperalgesia developing after the peak of p-ERK1/2³⁵. Therefore, the phosphorylation of ERK1/2 may occur at different stages in different pain processes, and upregulated p-ERK1/2 in the spinal cord may be important for both immediate induction and long-term gene response.

In the present study, the incision-induced phosphorylation of ERK1/2 significantly increased in the first 24 h after incision compared with the blank group, and the expression of p-ERK1/2 peaked at 4 h after incision (as described previously)²⁴, coinciding with the timepoint at which NeuN expression was highest. Concurrently, as revealed by the results of immunofluorescence, the incision-induced phosphorylation of ERK1/2 was simultaneously distributed in neurons, astrocytes, and microglia in the ipsilateral spinal cord dorsal horn, supporting the view that ERK1/2-related signaling is particularly relevant to the early phase of incisional hyperalgesia. In addition, the preoperative intrathecal administration of U0126 reduced the expression of spinal glial markers, NeuN, and p-ERK1/2 at 4 h after incision and relieved both mechanical and thermal hyperalgesia during the early post-incision period, suggesting that spinal p-ERK1/2 may be involved in the early stage of acute hyperalgesia after incision. However, because GFAP, Iba-1, p-ERK1/2, and behavioral outcomes showed different temporal profiles, the present mechanistic interpretation should be limited to the early post-incision stage represented by the 4 h timepoint rather than generalised to the entire postoperative period.

Furthermore, in the present study, preoperative intrathecal PPF administration also reduced ERK1/2 phosphorylation at 4 h after incision, suggesting that the observed changes in spinal NeuN, GFAP, and Iba-1 expression may be associated, at least in part, with ERK1/2-related signaling. Preoperative intrathecal U0126 administration also decreased TNF-α and COX-2 immunoreactivity, supporting a possible association between ERK1/2 phosphorylation and inflammatory mediator expression following incision. The additional in vitro astrocyte experiments shown in Figure 9 provided cellular-level support for an ERK1/2-dependent anti-inflammatory component, because persistent ERK1/2 activation weakened the ability of PPF to reduce TNF-α, PGE2, and IL-6 release. However, because these rescue experiments were performed in cultured astrocytes rather than in the intact spinal cord, and because in vivo rescue or cell-type-selective manipulation was not performed, the in vivo conclusions should still be interpreted as supportive rather than definitive proof of a causal ERK1/2-dependent mechanism.

To further discuss the possible relationships among glial cell activation, p-ERK1/2 expression and inflammatory cytokine release, the spatial localization patterns observed with immunofluorescence were considered. After incision, p-ERK1/2 was co-expressed with GFAP-positive astrocytes and Iba-1-positive microglia in the spinal dorsal horn at 4 h after incision. Combined with the Western blot and immunofluorescence findings showing that both PPF and U0126 were associated with reduced p-ERK1/2, TNF-α, and COX-2 expression, these data suggest that ERK1/2 phosphorylation in spinal glial cells may be involved in the inflammatory response following incision. The in vitro data in primary astrocytes further support a direct cellular link between PPF treatment, ERK1/2 suppression, and inflammatory mediator release. Nevertheless, the in vivo evidence demonstrates association and treatment-related parallel changes, whereas the cellular experiments complement but do not replace in vivo cell-type-specific causal validation. Because the immunofluorescence analysis was based on a limited number of animals and fields, these observations should be interpreted cautiously and in conjunction with the Western blot and in vitro findings.

Several procedural steps have a disproportionate effect on reproducibility. Intrathecal injection requires both a characteristic tail response and cerebrospinal fluid confirmation; resistance during injection, absent confirmation, or a new neurological deficit should prompt exclusion rather than reinjection at the same site. Behavioral testing is sensitive to acclimatisation, enclosure conditions, stimulus placement, and investigator expectation, so blinding, fixed intervals, and consistent environmental conditions are essential. Tissue collection time, uniform protein transfer, antibody incubation, and identical microscopy settings within each staining batch are equally important. Common problems include variable withdrawal thresholds from inadequate acclimatisation, weak or uneven Western blot bands from incomplete transfer or overexposure, and high fluorescence background from insufficient washing, excessive antibody concentration, or saturated acquisition settings.

The protocol can be modified according to the scientific question. A broader time course can be used when late glial responses are of interest, and dose-ranging or alternative administration routes can be added when translational feasibility is prioritised. Compared with inflammatory or neuropathic pain models, the plantar-incision model is relatively rapid and practical and reproduces a defined postoperative injury²⁷. Combining behavioral testing, Western blotting, and cell-associated immunofluorescence provides complementary functional, molecular, and spatial information; however, it is more resource-intensive than a single-endpoint assay and does not by itself establish cell-specific causality. Pharmacological comparison with U0126 strengthens pathway interpretation but remains less specific than genetic or cell-type-selective manipulation^{20,21,22,23,24,25}.

Future applications include testing both sexes, larger surgical injuries, clinically feasible dosing routes, additional postoperative timepoints, and interventions targeting specific glial populations. The workflow is practical for screening candidate analgesic or glial-modulating treatments because the incision model is brief, behavioral endpoints are repeatable, and the molecular assays use standard laboratory methods. Reproducibility nevertheless depends on prespecified exclusion criteria, blinded outcome assessment, consistent image acquisition, and complete reporting of raw data and analysis settings.

Limitations

This study has several limitations. First, the plantar paw incision model represents a relatively minor surgical injury and may not fully reflect larger surgical procedures, such as thoracotomy or laparotomy. Therefore, caution is required when extrapolating the present findings to other forms of postoperative pain. In addition, only male Sprague–Dawley rats were included, limiting the generalisability of these findings across sexes. Given the known sex-based differences in pain responses, analgesic effects, and glial activation patterns, further studies including female animals are warranted.

Principal molecular and histological observations were focused on the 4 h post-incision timepoint. Because different endpoints, including GFAP, Iba-1, p-ERK1/2, and behavioral outcomes, showed distinct temporal profiles following incision, the mechanistic interpretation of the present findings should be limited mainly to the early post-incision stage. Moreover, the molecular experiments herein were performed with a relatively limited sample size, potentially reducing the statistical robustness of these observations.

Extracellular signal-regulated kinase 1 and 2 were evaluated together as p-ERK1/2; their individual contributions were not examined separately. Therefore, the respective roles of ERK1 and ERK2 in incision-induced pain processing could not be determined in the present study. In addition, fluorescence-intensity quantification was based on a limited number of animals and fields and did not include stereological cell counts or formal co-localization coefficients. These observations should therefore be interpreted cautiously, together with the Western blot findings.

Finally, although the in vitro astrocyte rescue experiments provided mechanistic support for ERK1/2-dependent inflammatory mediator regulation, the upstream regulatory events after incision and the downstream events in intact spinal Tissue were not investigated in depth. The in vitro culture system also cannot fully reproduce interactions among neurons, astrocytes, microglia, and infiltrating immune cells in the spinal dorsal horn. Accordingly, the present study supports an ERK1/2-related mechanism but does not establish complete in vivo cell-specific causality.

Conclusion

The present study demonstrates that incision-induced increases in spinal NeuN, GFAP, and Iba-1 expression were attenuated by the preoperative intrathecal administration of PPF. This effect was accompanied by the relief of acute mechanical and thermal hyperalgesia and by reductions in ERK1/2 phosphorylation and TNF-α and COX-2 expression following incision. However, these findings support (rather than provide definitive mechanistic proof of) an association between the antihyperalgesic effect of PPF and reduced ERK1/2 phosphorylation in spinal glial cells (in association with reduced inflammatory mediator expression). The antihyperalgesic effect of PPF persisted throughout the observational period following incision, whereas the effect of the MEK1/2 inhibitor U0126 was mainly limited to the early post-incision period. These findings suggest that ERK1/2 signaling may be particularly relevant during the early development of incisional pain. The longer-lasting effect of PPF indicates that its mechanism of action may involve additional pathways beyond ERK1/2 inhibition alone. Thus, PPF may represent a potential therapeutic intervention, rather than a biological target, for improving the management of acute postoperative pain.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.01 M PBS buffer	Solarbio	P1020
1.0 M Tris-HCl buffer (pH 6.8)	Solarbio	T1020
1.5 M Tris-HCl buffer (pH 8.8)	Solarbio	T1010
10% sodium dodecyl sulfate (SDS)	Solarbio	S1010
10× TBST stock solution	Solarbio	T1081
30% acrylamide	Solarbio	A1010
4× protein loading buffer	Solarbio	P1041
Adobe Photoshop	Adobe	—
Ammonium persulfate	Solarbio	A1030
Automatic flake ice machine	Changshu Xueke	IMS-40
Bovine serum albumin (BSA)	Solarbio	A8020
Cryostat CM150	Leica	CM150
Digital pH meter	—	PHS-3C
Dimethyl sulfoxide (DMSO)	Solarbio	D8371
Electronic analytical balance	Cany	—
Enhanced chemiluminescence (ECL) reagent	Solarbio	PE0010
Film	Kodak	—
Fluorescence microscope DP71	Olympus	DP71
Frozen section embedding medium	Sakura	4583
Gel electrophoresis apparatus	BIO-RAD	—
Glycerol	Solarbio	IG0910
Glycine	Solarbio	G8200
Goat anti-mouse Alexa Fluor 594 secondary antibody	Proteintech	SA00013-3
Goat anti-mouse horseradish peroxidase-conjugated secondary antibody	ZSGB-BIO	ZB2305
Goat anti-rabbit Alexa Fluor 488 secondary antibody	Proteintech	SA00013-2
Goat anti-rabbit horseradish peroxidase-conjugated secondary antibody	ZSGB-BIO	ZB2301
Goat serum	Solarbio	SL038
GraphPad Prism	GraphPad Software	5
High-speed refrigerated centrifuge 3K15	Sigma	3K15
Horizontal orbital shaker TS-1000	Haimen Qilinbeier	TS-1000
ImageJ	National Institutes of Health	1.51j8
Methanol	Tianjin Guangfu	—
Mouse anti-rat GAPDH primary antibody	Proteintech	60004-1-Ig
Mouse anti-rat p-ERK1/2 primary antibody	CST	9101S
Na2HPO3	Tianjin Beichen Fangzheng	—
NaH2PO3	Tianjin Damao	—
Paraformaldehyde	Biosharp	BL539A
Pathology-grade microscope slides	Shitai	—
Phenylmethanesulfonyl fluoride (PMSF)	Beyotime	ST506
Plantar Test 37370	UGO BASILE	37370
Polyvinylidene fluoride membrane	Solarbio	—
Propentofylline (PPF)	Taosu	T19812
Rabbit anti-rat COX-2 primary antibody	Abcam	Ab179800
Rabbit anti-rat GFAP primary antibody	Proteintech	16825-1-AP
Rabbit anti-rat Iba-1 primary antibody	Proteintech	10904-1-AP
Rabbit anti-rat NeuN primary antibody	Abcam	Ab279297
Rabbit anti-rat t-ERK1/2 primary antibody	Proteintech	11257-1-AP
Rabbit anti-rat TNF-α primary antibody	Proteintech	29652-1-AP
RIPA lysis buffer	Solarbio	R0010
SDS	Solarbio	S8010
Skim milk powder	Solarbio	D8340
SPSS	IBM	22
Tetramethylethylenediamine (TEMED)	Solarbio	T8090
Tris	Solarbio	T8060
Triton X-100	Solarbio	T8200
U0126-EtOH	Selleck	S1102
Ultra-low temperature freezer	Haier	—
Ultra-low temperature freezer	Sanyo	—
Ultrapure water purification system	Heal Force	—
von Frey filaments	North Coast Medical	—