Introduction

Skeletal muscle, a highly vascularized tissue, relies on a dynamic interaction with blood vessels to maintain homeostasis under normal physiological conditions1,2. Microcapillaries play a critical role in supplying oxygen and nutrients to skeletal muscle, while myocytes contribute to vasodilation, regulating capillary surface area1,3. In response to increased metabolic demands, skeletal muscle activates angiogenesis—the process through which new blood vessels form from pre-existing ones—thereby enhancing nutrient and oxygen delivery4. This process involves the degradation of the basement membrane surrounding blood vessels, followed by the proliferation and migration of endothelial cells (ECs) to form new capillaries4. Angiogenesis is tightly regulated by a network of pro-angiogenic and anti-angiogenic factors, including growth factors, cytokines, and extracellular matrix proteins.

Peripheral nerve injury leads to nerve degeneration and subsequent skeletal muscle denervation5. Neural activity is essential for maintaining normal muscle structure and function, and its loss disrupts this balance. Following denervation, myocytes undergo molecular changes within hours, activating signaling pathways that lead to muscle fiber degeneration and prolonged atrophy6,7. Macrophages play a crucial role in these processes by mediating tissue repair through pro-angiogenic and anti-angiogenic mechanisms. Additionally, denervation results in hypoxic conditions within the muscle, driven by factors such as impaired blood flow regulation, increased metabolic demands, reduced capillary density, and insufficient angiogenesis.

Hypoxia-inducible factors (HIFs), the master regulators of angiogenesis, play a central role in modulating macrophage behavior under these hypoxic conditions8,9. HIFs regulate genes involved in oxygen homeostasis, cell survival, inflammation, and angiogenesis. These HIF1-regulated genes mediate the cellular response to low oxygen levels and, when activated, upregulate pro-angiogenic genes that promote blood vessel development. Angiogenesis is not only vital for physiological processes such as wound healing10 but also plays a central role in pathological conditions such as cancer8, diabetic retinopathy11, and rheumatoid arthritis12.

Despite extensive studies on skeletal muscle degradation following denervation, the response of blood vessels to denervation is poorly understood, particularly in the early stages. Much of the existing literature focuses on the long-term effects of denervation on vascular remodeling13,14, with fewer studies addressing the acute phase of denervation. This study aims to investigate the vascular response in skeletal muscle during both the acute and chronic phases of denervation, shedding light on the underlying mechanisms of angiogenesis and the role of macrophages in this process.

Results

This study investigates the vascular responses to muscle denervation, utilizing a tightly ligated sciatic nerve model in mice. The lumbrical and tibialis anterior (TA) muscles, located in the hind leg and paw, respectively, were analyzed for changes in muscle capillaries following nerve injury.

Early angiogenesis in denervated muscles

Upon nerve injury, no immediate disruption in blood flow was observed in the lumbrical muscle, which does not share its blood supply with the sciatic nerve (Fig. 1A). To assess circulation, 150 kDa FITC-dextran was injected into the tail vein of wild-type mice 30 min after nerve ligation. The distribution of FITC was comparable between the contralateral and ipsilateral sides, suggesting that sciatic nerve ligation did not disrupt blood flow to the lumbrical muscle (Fig. 1B).

Fig. 1
figure 1

Angiogenesis Caused by Neurodegeneration in Denervated Muscle. (A) Schematic representation of the experimental design. (B) Representative images showing blood vessels labeled with FITC-dextran 70 kDa in the lumbrical muscle 1 h after sciatic nerve ligation. The left image shows blood vessel distribution on the contralateral side, and the right image shows the distribution on the ipsilateral side. The graph quantifies the FITC-labeled blood vessel area for both sides (n=3, 8 images per individual). Unpaired t-test. (C) Representative GFP expression and bungarotoxin (BTX) staining in the lumbrical muscle of Thy1-GFP mice at post-operation (PO) day 3. The left image shows intact nerves on the contralateral side, and the right image shows ipsilateral nerves at PO day 3. (D) Representative td-Tomato expression z-stack (30 slices) images in the lumbrical muscles of Tie2-Tomato mice at PO day 3. The graph quantifies the percentage of td-Tomato-expressing blood vessel area for both contralateral and ipsilateral sides (n=3, 8 images per individual). ****P<0.001, by unpaired t-test. (E) Representative image showing td-Tomato-expressing blood vessels with EdU staining in the ipsilateral lumbrical muscle at PO day 3. White arrows indicate EdU-positive blood vessels. (F) High magnification image of sprouting td-Tomato-positive blood vessels in the ipsilateral lumbrical muscle at 3 days after sciatic nerve ligation. White arrowheads indicate branching blood vessels.

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To confirm axonal degeneration in neuromuscular junction (NMJ), motor endplates were labeled with α-bungarotoxin (BTX) conjugates in Thy1 GFP mice. At postoperative day 3 (PO day 3), all NMJ on the contralateral side displayed overlap of the pre-synapse and post-synapse, while a loss of pre-synaptic overlap at the motor endplates on the ipsilateral side was observed, showing axonal degeneration in the lumbrical muscle (Fig. 1C). Next, the vascular response to muscle denervation was investigated. Endothelial cells (ECs) tagged with Td-Tomato fluorescence from Tie2Cre; R26RTd-Tomato mice was visualized, and their sciatic nerve was ligated as shown in Fig. 1A. On the ipsilateral side of the denervated muscle at PO day 3, a more complex microvascular network with increased capillary branching compared to the contralateral side was noted, along with a significant increase in vessel density (Fig. 1D).

To assess endothelial cell (EC) proliferation, EdU, a thymidine analogue incorporated into the DNA of proliferating cells, was injected. EdU-positive ECs were predominantly located at the base of the vessel branches (Fig. 1E), in line with their role in vascular sprouting. These ECs exhibited extended cytoplasmic projections (Fig. 1F) characteristic of tip cells involved in angiogenesis. These findings suggest that denervation induces angiogenesis in the acute phase.

Endothelial cell activation in denervated muscle

Under normal conditions, ECs are quiescent and maintain the integrity of the vascular barrier15. After activation, they undergo structural changes that contribute to neovascularization16,17,18,19. To assess EC activation, the expression of leukocyte adhesion molecules (Vcam1, Icam1, and Selectin), proteins commonly upregulated in activated ECs, was measured. These molecules were significantly elevated in the denervated lumbrical muscle compared to the contralateral side at PO day 3 (Fig. 2A). Immunohistochemistry for CD105, a marker of activated ECs, revealed extensive CD105-positive ECs in the denervated muscle (Fig. 2B), further confirming EC activation.

Fig. 2
figure 2

Neurodegeneration Activates Endothelial Cells in Denervated Muscle. (A) mRNA expression levels of Vcam, Icam, and Selectin in the contralateral or ipsilateral lumbrical muscle at PO Day 3. ****P<0.001, ***P<0.005, by unpaired t-test. (B) Representative images showing td-Tomato expression (red) and immunostaining of CD105 or collagen IV (green) in contralateral and ipsilateral TA muscles of Tie2-Tomato mice at PO day 3. (C) mRNA expression levels of Agp2, Pgf, Pdgfβ, Mmp2, and Mmp3 in the contralateral or ipsilateral lumbrical muscle at PO day 3. ****P<0.001, ***P<0.005, **P<0.01, by unpaired t-test.

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Next, vessel integrity was assessed by examining Type IV collagen, a major component of the vascular basal membrane20,21. In the contralateral muscle, blood vessels were overlapped with Type IV collagen (Fig. 2B), while many vessels in the denervated muscle lacked this immunoreactivity, suggesting immature and destabilized vessels22,23. Furthermore, mRNA expression of pro-angiogenic factors, including angiopoietin 2 (Agp2), platelet-derived growth factor β (Pdgfβ), placental growth factor (Pgf), matrix metalloproteases 2 (Mmp2) and 3 (Mmp3), was elevated in the denervated lumbrical muscle at PO day 3 (Fig. 2C), supporting the occurrence of angiogenesis.

Angiogenesis becomes inactive in Long-Term denervated muscle

At the chronic stage (PO day 28), the microvascular network remained complex, and vessel density was still higher in the denervated muscle compared to the contralateral side (Fig. 3A and B). However, no further increase in vessel density was observed after PO day 3, suggesting angiogenesis had plateaued.

Fig. 3
figure 3

Inactive Angiogenesis in Long-Term Denervated Muscle. (A) Representative high magnification z-stack (30 slices) images of td-Tomato expression in lumbrical muscles of Tie2-Tomato mice at 28 days post-sciatic nerve ligation. (B) Graph showing the percentage of td-Tomato-expressing blood vessel area for contralateral and ipsilateral sides at PO days 3, 7, and 28 (n=3, 8 images per individual). ****P<0.001, by one-way ANOVA with Dunnett’s multiple-comparison test. (C) Representative images showing td-Tomato expression (red) and EdU labeling (green) in contralateral and ipsilateral TA muscles of Tie2-Tomato mice at PO days 3 and 28. (D) Graph quantifying the number of EdU-positive cells per 1000 × 1000 μm in contralateral and ipsilateral muscles at PO day 28. ***P<0.001, ***P<0.005, by one-way ANOVA with Dunnett’s multiple-comparison test. (E) mRNA expression levels of Vcam, Icam, and Selectin in contralateral or ipsilateral lumbrical muscle at PO days 3 and 28. ****P<0.001, ***P<0.005, *P<0.05, by one-way ANOVA with Dunnett’s multiple-comparison test. (F) Representative images showing td-Tomato expression (red) and immunostaining for CD105 (green) in contralateral and ipsilateral TA muscles of Tie2-Tomato mice at PO day 28. (G) Representative images showing td-Tomato expression (red) and TUNEL staining (green) in contralateral and ipsilateral TA muscles of Tie2-Tomato mice at PO day 28.

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The EdU assay revealed a significant decrease in EC proliferation at PO day 28 compared to PO day 3 (Fig. 3C and D). Moreover, the expression of leukocyte adhesion molecules (Vcam1, Icam1, Selectin) was reduced in the denervated muscle at PO day 28 (Fig. 3E), indicating decreased EC activation. Immunohistochemistry for CD105 confirmed the reduction of activated ECs in the long-term denervated muscle (Fig. 3F). Additionally, TUNEL assays detected apoptosis in ECs, suggesting that angiogenesis was inactive and that vascular pruning and regression were occurring (Fig. 3G)24,25,26.

Hypoxia and macrophage involvement in angiogenesis

Tissue hypoxia is a key driver of angiogenesis, regulated by HIFs. To evaluate hypoxia in denervated muscle, mice were injected with hypoxyprobe-1 (pimonidazole hydrochloride) that forms immunofluorescent detectable protein adducts in hypoxic cells (pO2 < 10 mm Hg). The hypoxic cells were observed in the ipsilateral TA muscle at PO day 3, suggesting that the denervated muscle is starved of oxygen. (Fig. 4A). Hypoxia decreased by PO day 28, confirming the acute phase of denervation was hypoxic. qPCR analysis showed that HIF-1α expression, along with its target genes (Vegf and Glut1), was upregulated at PO day 3 and downregulated at PO day 28 (Fig. 4B), indicating activation of the hypoxia signaling pathway in the acute phase.

Fig. 4
figure 4

Macrophages Sensing Hypoxia in Denervated Muscle Activates HIF Signaling. (A) Representative images showing Hypoxiprobe-1 distribution in ipsilateral lumbrical muscle at PO days 3 and 28. The graph quantifies the number of Hypoxiprobe-1-positive cells per 1000 × 1000 μm. ****P<0.001, ***P<0.005, *P<0.05, by one-way ANOVA with Dunnett’s multiple-comparison test. (B) mRNA expression levels of Hif1-α, Vegf, and Glut1 in contralateral and ipsilateral lumbrical muscles at PO days 3 and 28. ****P<0.001, ***P<0.005, **P<0.01, by one-way ANOVA with Dunnett’s multiple-comparison test. (C) Representative images of immunostaining for CD68 in contralateral and ipsilateral TA muscles at PO day 3. (D) Representative images showing double immunostaining for CD68 (red) and Hypoxiprobe-1 (green) in ipsilateral TA muscles at PO day 3. The pie chart shows the cell composition of Hypoxiprobe-1-positive cells in ipsilateral TA muscle. (E) Representative immunostaining for CD68 in PLX3397-treated TA muscle. The left image shows the contralateral nerve treated with PLX 3397, the middle image shows the ipsilateral nerve at PO day 3, and the right image shows the ipsilateral nerve treated with PLX 3397 at PO day 3. The graph quantifies the number of CD68-positive cells per 1000 × 1000 μm. ***P<0.005, **P<0.01, by one-way ANOVA with Dunnett’s multiple-comparison test. (F) mRNA expression levels of Hif1-α, Vegf, and Glut1 in PLX3397-treated contralateral and ipsilateral lumbrical muscles at PO day 3. ****P<0.001, ***P<0.005, **P<0.01, by one-way ANOVA with Dunnett’s multiple-comparison test.

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Macrophages, known to sense hypoxia, are crucial for angiogenesis in response to tissue damage. Immunohistochemistry for CD68, a pan-macrophage marker, revealed extensive macrophage infiltration in the denervated muscle at PO day 3 (Fig. 4C). Co-localization of hypoxyprobe-1 and CD68 confirmed that macrophages were the primary cells experiencing hypoxia, comprising 79.67% of hypoxic cells (Fig. 4D). To evaluate macrophage activation of HIF1 signaling in denervated muscle, macrophage depletion using PLX3397, a CSF1 receptor inhibitor, was performed. The number of macrophages was significantly reduced in denervated muscle of PLX3397-treated mice (Fig. 4E). Macrophage depletion significantly reduced HIF signaling, with no increase in Hif-1α, Vegf, or Glut1 expression (Fig. 4F).

Macrophages are essential for angiogenesis in denervated muscle

To assess the role of macrophages in angiogenesis, blood vessel formation in macrophage-depleted mice was examined. In PLX3397-treated Tie2-Tomato mice, no significant difference in blood vessel density was observed between the ipsilateral and contralateral sides (Fig. 5A). Similarly, the number of proliferating ECs, as assessed by EdU incorporation, was significantly reduced in the denervated muscle (Fig. 5B). Additionally, the expression level of Vcam, Icam, and Selectin, markers of activated endothelial cells, was significantly lower (Fig. 5C), suggesting that macrophage depletion inhibited angiogenesis in denervated muscle. qPCR analysis revealed a significant downregulation of pro-angiogenic genes, such as Pgf, Mmp2, and Mmp3, in macrophage-depleted mice (Fig. 5D), supporting the critical role of macrophages in angiogenesis. These findings highlight that macrophages are essential for activating ECs and driving angiogenesis in denervated muscle via HIF signaling.

Fig. 5
figure 5

Contribution of Macrophages to Angiogenesis in Denervated Muscle. (A) Representative td-Tomato expression in lumbrical muscles of Tie2-Tomato mice treated with PLX3397 at PO day 3. The graph quantifies the percentage of td-Tomato-expressing blood vessel area for contralateral and ipsilateral sides with or without PLX3397 treatment at PO day 3 (n=3, 8 images per individual). ****P<0.001, by one-way ANOVA with Dunnett’s multiple-comparison test. (B) Representative images showing td-Tomato expression (red) and EdU labeling (green) in contralateral and ipsilateral TA muscles of Tie2-Tomato mice with or without PLX3397 treatment at PO day 3. (C) mRNA expression levels of Vcam, Icam, and Selectin in contralateral and ipsilateral lumbrical muscles at PO Day 3. ****P<0.001, ***P<0.005, **P<0.01, by one-way ANOVA with Dunnett’s multiple-comparison test. (D) mRNA expression levels of Agp2, Pgf, Pdgfβ, Mmp2, and Mmp3 in contralateral and ipsilateral lumbrical muscles at PO day 3. ****P<0.001, ***P<0.005, **P<0.01, by one-way ANOVA with Dunnett’s multiple-comparison test.

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Discussion

This study demonstrates that muscle denervation induces a robust angiogenic response in the acute phase, marked by significant endothelial cell (EC) activation, vessel sprouting, and the formation of an immature vasculature. However, as time progresses, angiogenesis becomes inactive, and vascular remodeling leads to vessel pruning.

Peripheral nerve injury frequently results in skeletal muscle denervation, which significantly contributes to morbidity and disability, especially among young, otherwise healthy individuals27,28. While numerous studies have explored the biological mechanisms behind denervated muscle, particularly focusing on the pathological processes leading to muscle weakness, the impact of denervation on skeletal muscle blood vessels remains less understood. The present study provides new insights into how denervation influences the vasculature, highlighting the role of hypoxia and angiogenesis in the acute phase following nerve injury.

Hypoxia in denervated muscle has been previously reported29,30 consistent with the findings of this study. Under normal conditions, muscle contractions generate mechanical stimuli that enhance blood flow31. However, denervated muscle loses its ability to contract and relax, leading to a reduction in blood flow and, consequently, hypoxia32,33. Additionally, denervated muscle experiences increased oxygen consumption due to the upregulation of proteolytic and glycolytic activity associated with muscle fiber degeneration34,35. These factors likely contribute to the hypoxic conditions observed in this model of denervation.

These results reveal that during the acute phase following nerve injury, denervated muscle undergoes a proliferative response in endothelial cells, leading to the formation of new, branching blood vessels (Fig. 1E-G). This angiogenic response is likely an adaptive mechanism to address hypoxia, as angiogenesis is a well-known physiological and pathological response in a variety of contexts, including embryonic development and disease36,37. Hypoxic conditions trigger rapid endothelial activation, which promotes new blood vessel formation, largely through the upregulation of vascular endothelial growth factor (VEGF)8,18. Consistent with this, increased Vegf expression was observed in denervated muscle at PO day 3 (Fig. 4B). Furthermore, the findings of this study suggest that other pro-angiogenic factors, along with hypoxia-sensing macrophages, contribute to this process (Fig. 5D). These data indicate that the hypoxia-induced upregulation of pro-angiogenic factors drives angiogenesis in short-term denervated muscle19.

An important aspect of these findings is the role of macrophages in the angiogenic response. Approximately 80% of macrophages in denervated muscle were immunoreactive to hypoxyprobe-1, indicating their response to hypoxic conditions. Macrophage depletion impaired the upregulation of hypoxia-inducible factor 1-alpha (HIF-1α) and its downstream target genes, highlighting the central role of macrophages in the hypoxic response. Macrophage infiltration in denervated muscle during the acute phase following nerve injury is well-documented38 and similar phenomena have been observed in neurodegenerative diseases, such as amyotrophic lateral sclerosis39 spinal muscular atrophy40 and Guillain-Barré syndrome41. These findings suggest that macrophages play a pivotal role in denervated muscle, particularly by responding to hypoxic conditions and contributing to angiogenesis. Macrophages are known to rapidly alter their gene expression in response to low oxygen levels42,43,44 and in this study, hypoxic conditions were observed, which are associated with macrophages, resulting in the accumulation of transcription factors such as HIF-1α. These factors bind to hypoxia response elements in various genes, thereby enhancing their transcription45,46,47,48. As expected, macrophage activation in response to hypoxia led to the upregulation of pro-angiogenic molecules, including VEGF, fibroblast growth factor (FGF), and matrix metalloproteinases (MMPs). Notably, elevated mRNA expression of Ccl2 and Il-6, two pro-angiogenic factors regulated by HIF-1, were observed, which coincided with macrophage infiltration at PO day 3. Macrophage depletion significantly reduced the expression of these pro-angiogenic factors and inhibited angiogenesis, further supporting the hypothesis that macrophages are key contributors to angiogenesis in denervated skeletal muscle.

The intriguing role of macrophages in angiogenesis raises important questions regarding the mechanisms underlying this phenomenon. These data suggest that macrophages actively promote angiogenesis in denervated muscle; however, it remains unclear why this angiogenic response is limited to the acute phase following nerve injury. In the chronic phase, a downregulation of endothelial activation markers and pro-angiogenic factors (Figs. 3E and F and 4B), as well as the expression of apoptosis markers in some endothelial cells, was observed (Fig. 3G). Similar declines in angiogenesis have been reported in long-term denervated muscle, where the upregulation of pro-angiogenic factors, including VEGF, is associated with capillary regression49,50.

Two possible explanations for the downregulation of angiogenesis in chronic denervation are: (1) a reduction in the number of macrophages that promote angiogenesis via HIF-1 signaling, and (2) a shift in the functional phenotype of macrophages between the acute and chronic phases following injury.

Macrophages exhibit functional heterogeneity, with their activation status and functions influenced by the local microenvironment51,52. Typically, macrophages are classified into two main phenotypes: M1 (pro-inflammatory) and M2 (anti-inflammatory). In the acute phase, M1 macrophages are activated to promote debris clearance, while M2 macrophages are involved in tissue repair during the chronic phase52,53. Recent studies suggest that hypoxia induces M1 macrophage polarization via HIF-1α, which stabilizes and upregulates genes related to glycolysis and inflammation54. In contrast, M2 macrophages exhibit lower sensitivity to hypoxia, as evidenced by reduced expression of hypoxia-related genes in M2 macrophages compared to M1 macrophages in various hypoxic tissues55. Based on these observations, it was hypothesized that macrophages in short-term denervated muscle may exhibit an M1 phenotype with stabilized HIF-1α, which then transitions to an M2 phenotype in the chronic phase. Further investigation is required to validate this hypothesis and explore the dynamics of macrophage polarization in the context of denervated muscle.

In conclusion, these findings provide compelling evidence for the contribution of macrophages to angiogenesis in denervated skeletal muscle during the acute phase following nerve injury. These results offer new insights into the cellular and molecular mechanisms underlying muscle responses to denervation and may inform future therapeutic strategies aimed at promoting muscle regeneration following peripheral nerve injury.

Materials and methods

Animals

All animal procedures were approved by the Niigata University Institutional Animal Care and Use Committee (approval number SA01198). This study is reported in accordance with ARRIVE guidelines. Male C57BL/6 mice (8–12 weeks old) were used in this experimental study. Additionally, the following genetically modified mouse strains were used: Tie2Cre; R26RTd-tomato (referred to as Tie2 Tomato) and Thy1 GFP mice. Mice were housed under standard laboratory conditions in a temperature-controlled room with a 12-hour light/dark cycle, and they had free access to food and water.

Surgical procedures

Prior to surgery, mice were anesthetized with a mixture of three anesthetic agents—medetomidine (0.75 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg)—administered intraperitoneally at a dosage of 0.1 ml/10 g.

The left sciatic nerve was exposed and tightly ligated with a 6 − 0 silk suture to create the complete axon degeneration model. After the injury, the surgical site was closed using sutures.

Vascular circulation assay

To evaluate vascular circulation in the denervated muscle, mice subjected to sciatic nerve ligation were injected with 2.5 mg of 150 kDa FITC-dextran (TdBLabs, 21059) in 50 µl of normal saline via the lateral tail vein, 30 min after the surgery. The dye was allowed to circulate for 1 h, after which tissues were harvested for further analysis.

Tissue Preparation and immunohistochemistry

Following deep anesthesia, mice were intracardially perfused with 20 ml of 4% paraformaldehyde (PFA) to fix the tissues. The fixed tissues were processed into 16-µm-thick cryosections. These sections were washed in PBS and blocked with 3% normal goat serum for 30 min at room temperature. Subsequently, tissue sections were incubated overnight at 4 °C with primary antibodies (detailed in Table 1). After incubation, sections were incubated with appropriate fluorescent secondary antibodies for 1 h at room temperature.

EdU proliferation detection

To assess cell proliferation, mice were injected intraperitoneally with 5-ethynyl-2´-deoxyuridine (EdU, Baseclick, BCK-EdU488IM100) at 50 mg/kg daily for 3 days, beginning on the day of nerve injury. After 6 h post-injection, mice were intracardially perfused with 4% PFA. The dissected lumbrical muscles were then incubated with a reaction cocktail (6-FAM Azide, reaction buffer, reactor system, and buffer additive; Baseclick, BCK-EdU488IM100) for 1 h at room temperature.

TUNEL apoptosis detection

Apoptotic cells in the tibialis anterior (TA) muscle were detected using a TUNEL assay (Roche, 11684795910). Frozen muscle sections were processed and stained following the manufacturer’s protocol for in situ apoptosis detection.

Quantitative real-time PCR (qPCR) analysis

RNA was isolated from contralateral or ipsilateral lumbrical muscle at 3, 7, and 28 days post-injury using the RNeasy kit (QIAGEN)56. Reverse transcription and quantitative PCR were performed using the qPCR Master Mix kit (Promega, A6001) on a Quant Studio3 real-time PCR machine (ThermoFisher). The Ct values were normalized to Gapdh, and fold changes were determined using the ΔCt method. Primer sequences are provided in Table 2.

Hypoxia analysis

Hypoxia in the denervated muscle was assessed using the Hypoxyprobe-1 kit (Hypoxyprobe, HP3-100). Mice were administered pimonidazole HCl (1.5 mg/mouse, Hypoxyprobe-1) intraperitoneally. After 3 h, mice were perfused with 4% PFA, and tissues were collected for further analysis.

Macrophage depletion

Macrophages were depleted using PLX3397, a CSF1R inhibitor, administered via oral gavage. PLX3397 (Chemgood LLC) was incorporated into AIN-76 A standard chow at a concentration of 290 mg/kg, and mice were provided with this chow for 7 days prior to sciatic nerve ligation. This protocol was employed to specifically deplete macrophages during the experimental period.

Table 1 Antibody list.
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Table 2 qPCR primer list.
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Statistical analysis

Data was analyzed using GraphPad Prism software. One-way ANOVA with Dunnett’s multiple comparison test and t-tests were applied as appropriate. A p-value of less than 0.05 was considered statistically significant.