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Inflammation and macrophage infiltration exacerbate adult incision response by early life injury

BMC Anesthesiology volume 25, Article number: 165 (2025) Cite this article

Abstract

Background

Neonatal hindpaw incision can evoke long-lasting changes in nociceptive processing following repeat injury in adulthood. Studies have focused on the effects and mechanisms in the spinal cord and brain, however changes in inflammation and macrophages in the periphery, especially at the site of early life injury, remain poorly defined. In this paper, we investigated the role of macrophages in the injured tissue in pain hypersensitivity caused by repeat hindpaw incisions and primed by neonatal injury.

Methods

Hindpaw incision was performed in anesthetized adult rats. Among them, some had neonatal hindpaw incisions on postnatal day 3. To assess the role of inflammatory response in the priming of adult incision pain, the rats were treated with clodronate liposome, a macrophage depletion agent, and ketorolac tromethamine, the commonly used anti-inflammatory drug following surgery. Their mechanical pain sensitivity was measured via von Frey filaments. Inflammation induced by hindpaw incision was evaluated via Enzyme-linked Immunosorbent Assay, H&E, and immunofluorescence staining. The phenotypes of macrophages were examined by analyzing their surface markers by flow cytometry.

Results

Mechanical pain hypersensitivity caused by the hindpaw incision in the adult rats was enhanced by previous neonatal injury, which also significantly increased microglial activation in the spinal dorsal horn, aggravation of inflammation, and infiltration of both M1 and M2 macrophages in damaged hindpaw tissue after the repeat incision in the adult rats on POD 5. Intraperitoneal injection of clodronate liposome alleviates nociceptive and inflammatory responses in neonatal injured rats. Intramuscular injection of ketorolac tromethamine decreased mechanical hyperalgesia and inflammatory responses primed by prior neonatal injury.

Conclusions

Neonatal tissue injury exacerbated mechanical hypersensitivity, infiltration, and activation of macrophages evoked by repeat hindpaw incision in adulthood.

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Introduction

Early life is when the developing nervous system is sensitive to changes in sensory experience. Increasing evidence has suggested associations between early life painful stimuli, which neonates and infants in a neonatal intensive care unit are exposed to, and adverse neurodevelopment outcomes, altered response to future pain, and even persistent changes in sensory processing throughout life, indicating a localized “priming” of spinal nociceptive circuits following early trauma [1,2,3]. It has suggested neonatal tissue damage or inflammation can significantly change the pattern of gene expression across the adult dorsal horn, including genes that are known to be involved in synaptic transmission [4], and alter synaptic signaling within the mature spinal cord such as dampening both GABAergic and glycinergic feedforward inhibition onto adult spinal projection neurons and enhancing the strength of the direct primary afferent input to adult projection neurons [5]. Although convincing evidence accumulates and contributes to understanding the persistent modifications in nociceptive processing led by neonatal injury, the underlying cellular and molecular mechanisms remain unclear.

Macrophages are one of critical immune cells recruited to the wound site following skin injury to regulate the wound healing process. It is well-established that the phenotype of macrophages is associated with stages of tissue injury and wound healing [6, 7]. M1 macrophages, also referred to as pro-inflammatory macrophages, are initially recruited to the wound to clean bacteria, foreign debris, and dead cells. Following initial cleaning, the overall macrophage population transitions to M2 macrophages, also referred to as anti-inflammatory macrophages, which help the injured tissue begins to repair [8]. Accumulating evidence also suggests that inflammatory cells, neutrophils, and macrophages infiltrating the wound site link to inflammatory pain. M1 macrophages produce proinflammatory cytokines such as tumor necrosis factor-alpha (TNFα) and interleukins 6 (IL-6), and interleukins one beta (IL-1β), which promote nociceptor sensitization resulting in the generation of inflammatory pain, whereas M2 macrophages produce anti-inflammatory cytokines such as chemokine (C-C motif) ligand 2 (CCL2), interleukins 10 (IL-10) and transforming growth factor-β (TGFβ), and promote wound healing resulting in pain relief. Because of different phenotypes, macrophages can exacerbate or alleviate pain sensitivity under various conditions [9]. Thus, the transition from M1 to M2 phenotypes and the balance of M1/M2 are crucial for the resolution of inflammation and inflammatory pain relief [10]. The role of macrophages in regulating the enhanced pain sensitivity led by neonatal injury after repeat injury in adulthood has not been intensively studied and remains unclear.

Plantar hindpaw incision produces robust hyperalgesia is an established model of postoperative pain [11]. Initial incision during the neonatal period exacerbates and prolongs hyperalgesia and the spinal microglial response following subsequent incision [12, 13]. In this study, by examining the inflammatory responses and phenotypes of macrophages after hindpaw incision, we demonstrated that neonatal injury leads to increased inflammatory responses in injured tissue and the associated level of the spinal cord and altered macrophage phenotype transitions within the wounded area following repeat injury in adulthood.

Materials and methods

Animals

Sprague-Dawley rats (Hunan SLAC Laboratory Animal) were housed in a temperature-controlled (25–28 °C) and specific pathogen-free room on a 12‐hr light/dark cycle with free access to autoclaved food and water. All experiments adhered to animal welfare guidelines established by the Institutional Ethics Committee of Xiangya hospital of Central South University and the Committee for Research and Ethical Issues of the IASP. The ethics approval document number was (201806892).

Planter hindpaw incision

Neonatal Sprague-Dawley rats were randomly divided into neonatal incision group and sham operation group using the random number table method. Neonatal Sprague-Dawley rats (postnatal days 3) were anesthetized with 2–3% isoflurane (Sigma-Aldrich, Co.St.Louis, MO, USA). As previously described, A midline incision was performed on to the plantar, and the underlying plantaris muscle was elevated and incised longitudinally [11]. The skin was immediately closed with a 5 − 0 silk suture. Sham operation littermates were subjected to the same manipulation, anesthetized, but no incision was made; this group was to exclude effects of maternal deprivation on pain perception in rats. On the post-operation day (POD) 56 (postnatal day 59–60), all rats with or without previous neonatal surgery were anesthetized with 2–3% isoflurane and received hindpaw incisions the same way as described above, and the incision extended from the distal midpoint of the heel to the level of the first footpad to approximate the same relative length of incision in the hindpaw of pups (postnatal day 3, P3). The skin was closed with 4 − 0 silk suture [13].

Drug treatment

Adult rats with neonatal surgery were injected intraperitoneally either with clodronate liposomes (5 mg/ml/kg, Cat: 40337ES10, Yeasen, Shanghai, China) or control liposomes (Cat: 40338ES10, Yeasen, Shanghai, China) immediately and three days after the hindpaw incision.

Immediately after the hindpaw incision, adult rats received either 5 mg/kg ketorolac tromethamine (KT) (Cat: HY-B0580, MCE, Shanghai, China) diluted in 100 µl saline or an equal volume of saline. The solution was administered via intramuscular injection into the right biceps femoris muscle every 6 h for 48 h using 34G needles.

Behavioral assessment

Spontaneous pain

Spontaneous pain, the ongoing pain present without applying a stimulus, was measured according to the method described initially by Brennan et al. [14]. The paws were closely observed six times every 5 min. The guarding behavior was scored as 0 (no guarding, paw flat on the floor), 1 (the paw touched the mesh gently without any blanching or distortion), or 2 (the paw was completely off the mesh). These scores were recorded just before each application of the von Frey filament, which would be used to measure static allodynia behaviors. The sum is presented as the spontaneous pain score.

Static allodynia

Static mechanical sensitivity was tested by applying a series of von Frey filaments (Stoelting, Wood Dale, IL, USA) ranging from 0.41 g to 15.0 g to the heel region of the paws. A cutoff value of 15 g was assigned to animals that did not respond to the highest filament strength used. Rats were placed in an individual chamber (22 × 12 × 12 cm) with a plastic mesh floor and allowed to acclimate 20 min before testing. The lowest force to evoke three consistent withdrawal responses was the paw withdrawal threshold (PWT). For all behavioral experiments, the tester was blinded to the experimental groups.

Histology

Rats were perfused with 0.1 M phosphate buffer until the clear fluid was seen, followed by perfusion with 4% Paraformaldehyde for 20 min. Standard H&E staining was used to verify histological features of paw tissue inflammation. The lumbar spinal cord (5 mm sections) was retrieved and preserved. Paw tissue (skin and the underlying muscle) taken from the area within 1 mm in either direction from the central wound site (4 μm, paraffin-embedded) was cut and processed for H&E staining. For immunohistochemistry, paw tissue was cut at 10 μm on a cryostat after post-fixation in 4% PFA, 0.1 M phosphate buffer for 2 h, and cryoprotected in 30% sucrose in PBS at 4 °C overnight. The sections were incubated with the following primary antibodies: mouse anti-Iba1(Abcam Cambridge, MA, ab283319, diluted 1:200), a general marker for macrophages and microglia; Rabbit anti-CD11b (Abcam Cambridge, MA, ab8878 diluted 1:200); Rabbit anti-CD163 (1:200; ab182422; Abcam); or Rabbit anti-CD86 (1:100; MA5-32078; Thermo Fisher) overnight at 4 °C, followed by incubation with the secondary antibody goat anti-mouse Alexa Fluor 488 (1:200, Abcam) and goat anti-rabbit Alexa Fluor 488 (1:200, Servicebio, Wuhan, Hu Bei, China) for two hours at room temperature. Images were obtained from multiple skin sections randomly selected without regard to the signal level and were captured using a Leica DM5000B microscope (Leica Biosystems, Wetzlar, Germany). Histopathological analysis was scored according to the previously described standards. For inflammation score: 0, no inflammatory cells; 1, a few scattered inflammatory cells; 2, organization of inflammatory infiltrates around blood vessels; and 3, extensive perivascular cuffing with extension into adjacent parenchyma or parenchyma [15].

Enzyme-linked immunosorbent assay (ELISA)

Total protein was isolated from the tissue surrounding the wounds on POD5. The following inflammatory cytokines: IL-6, IL-1β, TNFα, CCL2, IL-10, and TGFβ were detected with commercially rat-specific ELISA kits. (E-EL-R0015c for IL-6, E-EL-R0012c for IL-1β, E-EL-R2856c for TNFα, E-EL-R0633c for CCL2, E-EL-R0016c for IL-10, and E-EL-R1015c for TGFβ, Elabscience Biotechnology Co., Ltd., Wuhan, China) following the manufacturer’s instructions as described previously [16].

Western blot

Injured paw tissue taken from the area within 1 mm in either direction from the central wound site was collected and homogenized in RIPA lysis buffer (10 µL/mg tissue) with protease and phosphatase inhibitors cocktail (NCM Biotech Newport, RI) at 4 °C. After homogenization, 50 µg of the sample was loaded per lane, and proteins were separated by 10% polyacrylamide gels (Thermo Fisher Scientific, #NW04125BOX). Proteins were then transferred to Immun-Blot PVDF membranes (Millipore, MA) and incubated with rabbit anti-CD11b (1:1000; ab8878; Abcam), rabbit anti-CD163 (1:1000; ab182422; Abcam), or rabbit anti-CD86 (1:2000; MA5-32078; Thermo Fisher) and rabbit anti-β-tubulin (1:3000, Cell signaling technology Danvers, MA) used as a loading control for 2 h at room temperature. The membranes were then washed and probed with anti-mouse or rabbit secondary antibody conjugated with horseradish peroxidase (HRP; 1:5000, Jackson ImmunoResearch Laboratories West Grove, PA). Protein bands were visualized by enhanced chemiluminescence (Millipore) and quantified using Image Lab software (Universal Hood III; Bio-Rad, Hercules). The expression of each detected protein was normalized by β-tubulin.

Surface antibody staining for flow cytometry

Injured paw tissue was collected and cut into 2–4 mm small pieces. Single-cell suspensions of the skin tissue were generated using the Skeletal Muscle Dissociation Kit and gentleMACS Dissociator (Miltenyi Biotec Technology Shanghai, China) according to the manufacturer protocol. The dissociated 5 × 106 cells were resuspended in 100 µL of 0.1 M PBS and blocked with FcBlock (mouse Anti-Rat CD32,1:100) on ice for 10 min, followed by incubation with APC-CYTM7 mouse anti-rat-CD45 (1:100, BD Biosciences San Jose, CA), FITC mouse anti-rat-CD11b (1:100, BD Biosciences), CD68/SR-D1 antibody(ED1) [PE] (1:100, Novus Biologicals Centennial, CO), BV421 mouse Anti-Rat CD86 (1:100, BD Biosciences), and CD163 antibody (GHI/61) Alexa Fluor® 647 (1:100 Novus Biologicals) on ice for 30 min. After the unbound antibodies were removed by two washing, the cells were resuspended and analyzed using a FACScan system and FlowJo X 10.0 software (BD Biosciences).

Data analysis

GraphPad Prism version 7 software (La Jolla, CA, USA) was used for statistical analysis. Behavioral time course data were analyzed using two-way repeated measures ANOVA with Holm-Sidak multiple comparisons posttest to determine on which days experimental groups differed. Comparisons between groups in other experiments were performed with a two-tailed unpaired Student’s t-test or Mann-Whitney nonparametric test. Significance was ascribed for p < 0.05. Data are presented as Mean ± SEM.

Results

Repeat incision enhanced hyperalgesia and microglial activation in neonatal injury rats

To investigate the effect of prior neonatal injury on mechanical hypersensitivity induced by tissue damage in adulthood, we compared mechanical pain behaviors between two groups of adult rats: animals with a neonatal incision at P3 and repeat incision eight weeks later in adulthood (neonatal incision plus adult incision; nIN-IN); and animals with neonatal sham surgery at P3 and undergoing incision at eight weeks of age (adult incision; n sham-IN). Prior neonatal incisions did not change the baseline mechanical sensitivity of adult rats. Paw incisions induced robust mechanical hypersensitivity and spontaneous pain in both groups, characterized as very fast onset, reaching a peak within one day after surgery and then starting recovery (Fig. 1A). However, mechanical hypersensitivity was enhanced and prolonged in the nIN-IN group with the threshold for paw withdrawal remaining significantly lower than rats undergoing single incision on POD 5 and 7 (nIN-IN versus nsham-IN, P < 0.001, Fig. 1A). Higher spontaneous pain scores were also recorded in the nIN-IN group from POD 3 to POD7 (nIN-IN versus nsham-IN, P < 0.05, Fig. 1B). As previous studies, we found that there was no sex difference in pain response between male and female rats following subsequent adult re-incision (results did not show), so we used male rats for follow-up experiments.

Besides comparing the mechanical hypersensitivity between the nIN-IN and the nsham-IN groups, we also compared microglia activation in the spinal cord after paw incision in the adult on POD 5, when mechanical hypersensitivity showed a significant difference. The intensity of Iba1 immunofluorescence in the ipsilateral spinal cord increased significantly in neonatally primed animals compared with the contralateral sides or the nsham-IN groups (Fig. 1C-D). On POD5, microglia in the ipsilateral spinal cords of the nIN-IN rats were changed to hypertrophied morphology with enlarged cell bodies, thicker, shorter, and less branched processes (Fig. 1D). These results demonstrated that the activation of microglia was higher in the affected dorsal spinal cord in the nIN-IN groups, suggesting neonatal injury enhanced inflammatory response to repeat paw incision in the spinal cord.

Fig. 1
figure 1

Neonatal injury enhances mechanical hypersensitivity, spontaneous pain, and microglia activation in adult rats’ spinal cord dorsal horns after a repeat hindpaw incision. (A) Mechanical withdrawal threshold to von Frey test. (B) Cumulative spontaneous pain score. **P < 0.01, ***P < 0.001; the significant difference between the nIN-IN and the nsham-IN groups at the indicated time points (N = 8, two-way repeated measures ANOVA with Holm-Sidak posttest). (C) Example sections of spinal dorsal horn immunolabeled with microglia marker Iba-1 (green) five days after paw incision. Scale bar = 200 μm. (D) Quantitative summary indicated IBA1 expressions were upregulated after repeat hindpaw incision in the ipsilateral spinal dorsal horn. **P < 0.01, ***P < 0.001 significant difference between the labeled groups. (N = 4, unpaired Student t-test). (E) The magnification of the white squares showed the hypertrophy of microglia with an enlarged, darkened soma and shorter, thicker, less branched processes after repeating the hindpaw incision. Scale bar = 50 μm

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Neonatal injury enhances inflammatory response to repeat paw incision in the injured paw tissue

Inflammatory responses associated with tissue damage directly contribute to the generation of pain hypersensitivity through inflammatory mediators. We compared inflammatory responses in injured skin tissues using H&E staining. On POD 5, increased infiltration of inflammatory cells into the epidermis, dermis, and hypodermis was observed in both the nsham-IN and the nIN-IN groups but to a greater level in animals with prior neonatal incision (Fig. 2A-B). The impacts of neonatal injury on inflammatory response were also assessed by measuring the specific inflammatory cytokines with ELISA. IL-6, IL-1β, and TNFα were increased in inIN-IN rats, suggesting that neonatal injury trigged proinflammatory reactions. IL-10 acted as a suppressor of the inflammatory response and was not changed after a repeat paw incision. Chemokines CCL2 and growth factor TGFβ which may affect wound healing were also not different on POD 5 between the two groups (Fig. 2C).

Fig. 2
figure 2

Neonatal injury enhances inflammatory response to repeat paw incision in the injured paw tissue. (A) Samples of cross sections of the paw tissue taken near the incision site were processed with the H&E method five days after the paw incision from the indicated groups. There is greater edema, swelling, and immune cell infiltration in nIN-IN rats than in nsham-IN rats. Scale bar = 100 μm. (B) Inflammation score was significantly increased in nIN-IN group compared with the nsham-IN groups in both skin and muscle tissue surrounding the incision. Data are shown as mean ± SEM. *P < 0.05 vs. nsham-IN group. (N = 4, Mann-Whitney nonparametric test). (C) Neonatal injury increased pro-inflammatory cytokines IL-6, IL-1β, and TNFα, while there was no difference in IL-10, CCL2, and TGFβ between the two groups. *P < 0.05, **P < 0.01 vs. nsham-IN group. (N = 5, unpaired Student t-test)

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Neonatal injury enhances activation of macrophage after repeat incision in adult rats

Inflammatory mediators produced by activated macrophages can sensitize nociceptors and prolong inflammatory pain. To investigate if the activation of macrophages contributes to the enhanced mechanical hypersensitivity in the nIN-IN group, we compared the expression of CD11b, a marker for myeloid cells in the injured tissues on POD 5. The expression of CD11b was increased in the nIN-IN groups (Fig. 3A-B). Furthermore, we investigated the macrophage-specific markers CD86 and CD163 in the injured paw tissue on POD 5. The expressions of CD86 and CD163 were increased to a greater level in the nIN-IN group compared with the nsham-IN groups (Fig. 3C-F).

Fig. 3
figure 3

Neonatal injury enhances macrophage infiltration in the injured skin after repeat hindpaw incision in the adult. A. Fluorescence images of myeloid cells immunostained for CD11b (green) in the hindpaw skin of nsham-IN and nIN-IN groups (B) Band of western blot and quantitative analysis showed the up-regulated expression of CD11b induced in the nIN-IN group. ***P < 0.001, vs. nsham-IN group (N = 6, unpaired Student t test). C. Examples of M1 phenotype macrophage marker CD 86 staining in hindpaw skin of nsham-IN and nIN-IN groups. D. Band of western blot and quantitative analysis showed the expression of CD86 increased in the nIN-IN group. **P < 0.01, vs. nsham-IN group (N = 6, unpaired Student t test). E. M2 phenotype macrophage marker CD 163 staining in the hindpaw skin of nsham-IN and nIN-IN groups (Scale bar = 200 μm). F. Band of western blot and quantitative analysis showed that the expression of CD163 increased in the nIN-IN group. *P < 0.05, vs. nsham-IN group (N = 6, unpaired Student t test)

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Prior neonatal injury alters macrophage polarization responding to subsequent injuries in adult

The transitions of macrophage phenotypes play an essential role in inflammatory pain and wound healing. We next compared the macrophage phenotypes in the injured tissue by examining the expressions of CD86 (M1 marker) and CD163 (M2 marker) on POD 5. The populations of different macrophage phenotypes were analyzed via surface antibody labeling and flow cytometry. The number of CD45+/CD11b + macrophages was higher in the nIN-IN group than in the nsham-IN groups on POD 5 (Fig. 4B). After further distinguishing the CD45+/CD11b + macrophages into the M1 phenotype (CD45+/CD11b+/CD68+/CD86+) and M2 phenotype (CD45+/CD11b+/CD68+/CD163+), we found that the populations of macrophages in both phenotypes were higher in the nIN-IN group than in the nsham-IN group (Fig. 4C-D), suggesting that neonatal injured enhanced macrophage infiltration and polarization in the damaged tissue of adult rats.

Fig. 4
figure 4

The M1 and M2 phenotype macrophages were increased in the adult rats with prior neonatal injury after repeat hindpaw incision. A. Cells collected from the injured hind paws from the nsham-IN and the nIN-IN groups five days after surgery were subjected to flow cytometric analysis. Dot plots circled by red boxes represent rat macrophages stained with fluorochrome-labeled specific antibodies as indicated in the nsham-IN and the nIN-IN groups. Total macrophages are represented as anti-CD45 /anti-CD11b double-labeled dot plots. Derived from total macrophages, the M1 phenotype is represented as anti-CD86 /anti-CD68 double-labeled dot plots, and the M2 phenotype is represented as anti-CD163 /anti-CD68 double-labeled dot plots. Quantifications of total (B), M1 (C), and M2 (D) phenotype macrophages showed that the population of each type of macrophage is significantly increased in the nIN-IN group. *P < 0.05, **P < 0.01, significant difference between the groups (N = 5,unpaired Student t test)

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Ketorolac tromethamine alleviates incision hypersensitivity and inflammatory response in neonatal injured rats

Nonsteroidal anti-inflammatory drug (NSAID) ketorolac tromethamine has been widely used to treat moderately severe pain and inflammation after surgery. We applied ketorolac tromethamine to the nIN-IN group every 6 h for 48 h, starting immediately after a repeat hindpaw incision. The enhanced mechanical hypersensitivity in this group was significantly reduced by ketorolac injection compared to saline injection from POD1 to 5 (nIN-ktIN versus nIN-sIN, Fig. 5A) Ketorolac intramuscular injection also reduced the infiltration of inflammatory cells in the injured tissue, as indicated by H&E staining (Fig. 5B) and the expression of CD11b in the injured tissue (Fig. 5C-D). The expressions of inflammatory cytokines IL-6, IL-1β, and TNFα were inhibited by ketorolac tromethamine. (Fig. 5E).

Fig. 5
figure 5

Effect of ketorolac tromethamine on incision hypersensitive and inflammatory responses in neonatal injured rats. A. Intramuscular ketorolac tromethamine (nIN-ktIN) reduced incision mechanical hypersensitivity to the von Frey test in neonatal injured rats. *P < 0.05, **P < 0.01, ***P < 0.001; significant difference between the nIN-IN and the nIN-ktIN groups; (N = 8, two-way repeated measures ANOVA with Holm-Sidak posttest). B, Samples of cross sections of the paw tissue taken near the incision site were processed with the H&E method five days after paw incision from the indicated groups. Edema, swelling, and immune cell infiltrations enhanced by prior neonatal injury in the subsequence injured tissue are decreased by ketorolac tromethamine (nIN-ktIN). Scale bar = 100 μm. C, D Examples of CD11b staining (green) in the skin or the muscle as indicated at five days after paw incision with prior neonatal injury from rats received intramuscular ketorolac tromethamine (nIN-ktIN) or saline (nIN-sIN). The morphology of microphage staining for CD11b was presented as 3x magnification in the lower right corner of each picture. Scale bar = 200 μm. E. Histograms showing the expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNFα were decreased after ketorolac tromethamine injection. *P < 0.05, significant difference between the groups (N = 5,unpaired Student t test)

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Macrophage depletion alleviates nociceptive and inflammatory responses in neonatal injured rats

To further investigate the effect of macrophages on neonatal injury-enhanced mechanical hypersensitivity and inflammatory response in adulthood, clodronate liposomes were used to deplete macrophage. We intraperitoneally injected clodronate liposomes to the nIN-IN group on the first and third day after repeated hindpaw incision. Immunofluorescence showed that macrophages labeled by CD 11b were significant depleted in the tissues surrounding the injury (Fig. 6A). The adult incision induced hyperalgesia decreased from day 4 after clodronate liposomes injection (Fig. 6B), while the spontaneous pain measured by cumulative pain score decreased significantly on POD 5 (Fig. 6C). Macrophage depletion also reduced the infiltration of inflammatory cells in the injured tissue, as indicated by H&E staining (Fig. 6D-E).

Fig. 6
figure 6

Macrophage depletion alleviates nociceptive and inflammatory responses in neonatal injured rats. A. Fluorescence images of macrophage immune-stained for CD11b (green) in the incision site of the neonatal injured rats received intraperitoneal injection of clodronate liposomes (nIN-IN-Cld) or control liposome (nIN-IN-Veh). Scale bar = 100 μm. B. Mechanical withdrawal threshold to von Frey test. C. Cumulative spontaneous pain score. **P < 0.01, ***P < 0.001; the significant difference between the nIN-IN-Cld and the nIN-IN-Cld groups at the indicated time points (N = 8, two-way repeated measures ANOVA with Holm-Sidak posttest). D. H&E stain of the cross sections of skin or muscle taken at the incision site showed that edema, swelling, and immune cell infiltrations enhanced by prior neonatal injury in the subsequence injured tissue are decreased after clodronate liposomes injection. E. Inflammation score was significantly reduced in nIN-IN-Cld group compared with the nIN-IN-Veh groups in both skin and muscle tissue surrounding the incision. Data are shown as mean ± SEM. ***P < 0.001 vs. nIN-IN-Veh group. (N = 5, Mann-Whitney nonparametric test)

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Discussion

Although it is clear that neonatal tissue injuries enhance painful responses to repeat tissue damage in adulthood, much work has been focused on the persistent alters of spinal nociceptive signaling [17]. The critical roles of macrophages in regulating inflammatory pain demand the need to understand how such injuries modulate the activation and polarization of macrophages when encountering repeat injury in adulthood. The present study confirmed previous findings that neonatal surgical injury enhanced pain hypersensitivity and microglia activation in the spinal cord evoked by repeat injury in adulthood in a rat hindpaw incision model [12, 13]. For the first time, we found that the impacts of neonatal surgical injuries on nociceptive signal processing in the adult were only limited to the tissue with a prior neonatal injury and did not spread to the contralateral side. Studies on neonatal incisions demonstrated that in adults with prior neonatal incision, enhanced hyperalgesia and spinal microglial reactivity are independent of peripheral reinjury and can be induced by injuries at distant sites, such as a lateral thigh incision or tibial nerve stimulation. While enhanced hyperalgesia was strongest when subsequent injuries occur within the same or overlapping spinal segments [12]. In our study, enhanced spinal microglial reactivity was induced at other ipsilateral but not contralateral sites, supporting a spinal segmental mechanism. Recent work has suggested the long-term effects of neonatal tissue damage on synaptic signaling for nociception transmission within the mature spinal cord, such as weakening both GABAergic and glycinergic feedforward inhibition and enhancing the strength of the direct primary afferent input to adult projection neurons [5]. Our result suggests that these persistent changes in synaptic integration in the adult spinal cord led by neonatal tissue damage were only limited in the spinal cord region, which directly receives nociceptive input from the damaged tissue. Unlike reported in a previous clinical study that preterm or full-term children aged 9–12 years who had undergone neonatal surgery in infancy have shown mechanical and thermal hyposensitivity in areas around prior tissue damage compared with age and gender-matched controls [18, 19], we did not observe that the baseline mechanical thresholds were significantly different between adult rats with or without prior neonatal injury. This is consistent with previous studies in animal models, in which only decreased thermal sensitivity during development [20] and adulthood [21] was observed in rats with prior neonatal repetitive needle prick stimuli. This difference between human and animal models suggests that we should be very cautious about using the results collected from animal models to interpret the conditions observed in human patients. Another limitation of this study is the exclusive use of male rats, despite evidence from numerous research indicating that enhanced hyperalgesia following adult re-incision is independent of sex [12, 22]. To fully understand whether sex influences the effects of macrophages in the context of re-incision, future studies should incorporate female animals.

Furthermore, we investigated early tissue damage’s effect on the activation and polarization of macrophages after repeating tissue damage in adulthood. It has been pointed out that different types or subsets of macrophages might be involved in various pain conditions. However, even with discovering a series of markers, macrophages often resemble each other and are difficult to distinguish precisely. The expression markers of macrophages exhibit species and organ specificity and even differ between normal and injured states [23]. Because macrophages can exhibit different phenotypes [24], the roles of macrophages in pain regulations can also be distinguished. M1- phenotype has been believed to promote pain through releasing pro-inflammatory cytokines and chemokines, such as IL-6, IL-1β, and TNFα, which directly activate or sensitize nociceptors to generate pain [25,26,27]. On the other hand, the M2 phenotype, considered an immune suppressive cell, resolves pain by releasing anti-inflammatory cytokines and growth factors such as IL-10 and TGFβ, which promote tissue repair [28,29,30]. Thus, the distribution of macrophages in each phenotype and their phenotype transitions at different stages of tissue damage would dramatically affect their regulations for nociceptors. Our results indicated that during the first days of tissue damage, there were robust macrophage infiltration and activation, which also led to a higher population of M1 phenotype in the injured tissue and was associated with mechanical hypersensitivity observed during this stage. Moreover, we also noticed that both the activation of macrophages and the population of M1 phenotype exhibited greater levels in the rats with prior neonatal injury, suggesting that neonatal injury not only persistently alters the nociception processing in the mature spinal dorsal horn but also potentiates inflammatory responses to tissue damage in adulthood.

To further clarify the role of macrophage infiltration in neonatal injury-enhanced adult incision pain. We opted to use clodronate liposomes to decrease the number of macrophages infiltrating at the injury site. Clodronate liposomes serve as a popular method for depleting phagocytic macrophages in mammals [31, 32]. It has been confirmed that local macrophage infiltration is one critical regulator of nociception. The infiltration of macrophages was increased the knee tissue of modified osteoarthritis pain rats, Clophosome injection reduced the numbers of synovial macrophages and produced a significant analgesic effect [33]. Reducing macrophage infiltration in the inflamed tissues could effectively diminish pro-inflammatory cytokine secretion and alleviate inflammatory pain [34]. Our results showed that ablation of macrophages infiltrated at the incision site could alleviate the prolonged hypersensitivity duration in the neonatal injury-enhanced adult incision pain and reduce the inflammation of surrounding tissue. It is important to note that the systemic administration of liposomal clodronate in this study may have targeted macrophages not only at the site of tissue injury but also within the dorsal root ganglia (DRG). Previous study has suggested that DRG macrophages may play a significant role in the neuropathic pain [35]. Consequently, our findings may partially reflect the effects of macrophage depletion in the DRG, in addition to the effects at the local injury site. Future studies should employ localized delivery of clodronate or macrophage-specific labeling techniques to further delineate the local macrophage infiltration in inducing the adult incision response by early life injury.

To investigate the effect of common clinical treatment on neonatal injury-enhanced mechanical hypersensitivity and inflammatory response in adulthood, in the final part of the present study, we used ketorolac tromethamine, an NSAID and having both analgesic and anti-inflammatory activity to reduce activation and polarization of macrophages by blocking the production of prostaglandins [36]. When applied to the nIN-IN group, ketorolac tromethamine inhibits inflammatory responses and reduces mechanical hypersensitivity. It is widely agreed that significant painful stimuli such as repeated interventions, which neonates and infants are exposed to in neonatal intensive care unit, are associated with adverse neurodevelopmental outcomes, persistent changes in sensory processing, and altered responses to future pain [37]. These persistent effects of neonatal injuries on developing nociceptive pathways are non-reversible and increase the difficulties of managing chronic pain generated by subsequent tissue damage in this population group. The long-term changes in sensory response can be modulated by preventive strategies at the time of neonatal injury or more selectively targeted by mechanism-based interventions at the time of subsequent repeat injury [38]. Administration of systemic morphine at the time of neonatal injury or inflammation prevented long-term changes in sensory response [39, 40]. Blocking afferent activity from the injured tissue with peri-operative sciatic nerve local anesthetic prevented long-term changes in RVM signaling [41], and prolonged perioperative blockade throughout the first 6–8 h also prevented the enhanced response to subsequent incision [42]. In the present study, we found that neonatal tissue damage also alters the immune response to subsequence injury in the same site of tissue in adults. Thus, modulating local inflammation evoked by tissue injury, especially the distribution of macrophage phenotype may be a new strategy to relieve enhanced hyperalgesia led by early tissue damage. It is worth noting that repeated intramuscular injections in this study may potentially increase muscular sensitivity due to local tissue trauma or inflammation, which could confound pain-related behavioral outcomes. For instance, intraperitoneal or subcutaneous routes may offer viable alternatives for this therapeutic approach.

Conclusions

In summary, in the present study, we found neonatal injury enhanced painful and local inflammatory responses to subsequence tissue damage in the adult. Ablation of macrophages infiltrated at the incision site alleviated nociceptive and inflammatory responses. Intramuscular ketorolac tromethamine relieved mechanical hyperalgesia and reduced inflammation at the injury site. Since we found that neonatal injury enhanced the domination of M1 phenotype macrophage infiltration after repeat tissue damage, in the future study, we are going to focus on whether neonatal tissue damage changes the transitions between different macrophage phenotypes when responding to future damage and prolongs wound healing in the adult.

Data availability

All data will be accessible at written request after the scientific results are published. All experimental materials are commercially available.

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Acknowledgements

The authors thank all participants involved in this study.

Funding

This work was supported by the National Natural Science Foundation of China (82071248, 82371238 to Z.S. 81901143 to Z.D.); Hunan Provincial Natural Science Foundation of China (2023JJ40934 to Z.D.); Program of National Clinical Research Center for Geriatric Disorders (Xiangya Hospital, grant number: 2021LNJJ14 to Z.S.).

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  1. Zhuofeng Ding and Zongbin Song contributed equally to the work.

Authors and Affiliations

  1. 1Department of Anesthesiology, Xiangya Hospital Central South University, Changsha, 410008, China

    Tong Wan, Anqi Wei, Zhuofeng Ding, Sarel Chavarria Gonzalez, Jian Wang, Xinran Hou, Xiao Luo, Liqiong He & Zongbin Song

  2. Department of Anesthesiology, Guiqian International General Hospital, Guiyang, 550024, China

    Anqi Wei

  3. Clinical Research Center for Geriatric Disorders, Xiangya Hospital Central South University, Changsha, 410008, China

    Zongbin Song

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Contributions

TW, ZD and AW performed experiments, analyzed data, prepared figures, and drafted the manuscript. QH, S.C.G, XL and JW performed experiments, and analyzed data. ZD and ZS designed and supervised the experiments, and edited the manuscript. All authors read and approved the final manuscript.

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Wan, T., Wei, A., Ding, Z. et al. Inflammation and macrophage infiltration exacerbate adult incision response by early life injury. BMC Anesthesiol 25, 165 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12871-025-03029-7

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BMC Anesthesiology

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