The treatment of peripheral nerve injury is a major clinical challenge due to the limited nerve regenerative potential and nerve graft sources. There have been great advances in tissue engineering and implantable artificial biomaterials for treatment of branchial plexus or sciatic nerve injuries. Treatment of sacral nerve injury that is due to spinal bifida, tethered cord syndrome and sacral fracture, which leads to urinary dysfunctions, is very challenging.¹ Compared with other nerve injuries that usually cause motor dysfunctions, the sacral nerve is a mixed nerve comprising motor, sensory and autonomic nerves (parasympathetic nerves) that mainly innervate the bladder and bowel. Damage to autonomic nerve fibres in the sacral nerve can lead to detrusor underactivity and cause neurogenic bladder.^2⇓-4

Injury to peripheral nerves with regenerative potential can result in incomplete functional recovery.⁵ The prevailing theory focuses on microenvironment, which is initially pro-inflammatory but evolves into the pro-regeneration state.⁶ The window phase inhibits the nerve fibre from reaching the far-end, resulting in neural degeneration of the distal stump. The microenvironment encloses the nerve fibres that coordinate axon elongation and myelination,^7,8 where immune cells such as macrophages, neutrophils and lymph cells of diverse phenotypes are highly involved. Macrophages act as a switch between pro-inflammatory and pro-regeneration states.^{9⇓⇓⇓-13} Inflammatory monocytes are recruited from the blood by multiple chemokines and differentiate into macrophages, which would intermingle with resident macrophages to be activated and clustered into certain groups with symbolic marker and function. The complicated macrophage polarisation was conventionally summarised as M1/M2 phenotypes. Classically activated macrophages (M1) express Tnfa, Il1b, Il6, as well as Il12 and help clear debris and eliminate apoptotic cells, that is to say, aggravate inflammation. Alternatively activated macrophages, which are marked with Retnla, Mrc1, Ym1 and Arg1 facilitate the extension of axons and secretion of growth factors.¹⁴ Manipulation of macrophage polarisation can impact the regeneration process, as the previous studies prevailed.

Electrical stimulation (ES) has promising therapeutic effects on peripheral nerve injury.^8,15 Clinical trials involving median, digital, ulnar, brachial plexus and facial nerves have reported on the significance of ES in promoting peripheral nerve injury regeneration,¹⁶ however, the involved mechanisms have yet to be reported. It has been postulated that ES helps restore the balance of neuronal potentials under certain electrical fields. Zhao et al.¹⁷ found that direct current ES promoted Schwann cell proliferation, migration as well as remyelination via the activation of mitogen-activated protein kinase (MAPK) signalling pathway. In our previous study, ES enhanced Wallerian degeneration.¹⁸ However, these studies did not report on the significance of macrophages, which regulate microenvironmental regeneration Macrophage polarisation can be manipulated via physical or chemical stimulation, including ES. Keane et al.¹⁹ found that intraoperative ES induced M2 polarisation and promoted axon elongation. Similar findings were noted in wound healing,^20,21 limb amputation²² and amyotrophy.²³ However, the mechanisms via which ES coordinates macrophage polarisation have yet to be fully established.

The ES of nerves is limited by electric burns and mechanical irritation by electrodes.²⁴ The dielectric material dispersed the quantity of electric charge and reduced the stress, making it a main research direction. The nerve guide conduits made of natural or synthetic biopolymers such as chitosan, gelatin, collagen, polylactide, poly(lactic-co-glycolic acid) or polycaprolactone (PCL) offer supportive mechanical and biochemical cues for nerve regeneration, which can be chaperoned by conductive materials such as polypyrrole (PPy),¹⁷ polyaniline,²⁵ carbon nanotube^26⇓-28 or reduced-graphene oxide.^29,30

We electrospun the PCL and silk fibroin (SF) membrane and coated it with PPy via in situ polymerisation. The compound scaffold, which exhibited electrical conductivity, was used for ES on crushed sacral nerve. We observed accelerated nerve regeneration and alleviated the neurogenic bladder through regulation of macrophage polarisation in the early regenerative microenvironment. High-throughput sequencing presented that signal transducer and activator of transcription (STAT) expression was adjusted in variation which determined the differential responses of macrophages to pro-inflammatory or pro-regenerative signals.

PCL/SF membranes were electrospun as described in our previous study³¹ with some modification. The PCL pellets (average M_n 80,000, 440744, Sigma-Aldrich, St. Louis, MO, USA) and SF powder (particle size 5 μm, MB4792, Meilunbio, Cambridge, MA, USA) were separately diluted to 8% (w/v) in hexafluoroisopropanol (H1-7501, Aladdin, Shanghai, China), and stirred overnight. The electrospun solutions of PCL and SF were mixed in different ratios and loaded to the mechanical arm connecting positive high voltage. The mixed PCL/SF solutions with positive charges were poured on the negatively charged roller at a rotating speed of 2800 r/min to produce the PCL/SF fibre. The air-dried membranes (2 cm × 2 cm) were immersed in pyrrole (2.5 mM, 8.07492, Sigma-Aldrich) and supplemented with sodium p-toluenesulfonate (2.5 mM, 152536, Sigma-Aldrich). After 2 hours of shaking on ice, they were supplemented with iron chloride (3 mM, dissolved in 1% HCl, I112064, Aladdin) to induce polymerisation under shaking for 6 hours to fully deposit the PPy.^32⇓-34 The membrane was rinsed in distillation-distillation H₂O (ddH₂O) until impurities became invisible, and sterilised with ⁶⁰Co exposure.

Scanning electron microscopy images were obtained by field-emission scanning electron microscopy (Quanta FEG 250, ThermoFisher Scientific, Waltham, MA, USA) at an acceleration voltage of 20 kV.³⁵ Fibre diameters were determined using ImageJ (v1.53t, modded with DiameterJ as previously described).¹⁷

Fourier transform infrared spectroscopy spectra were recorded on a Nicolet iS50 series (ThermoFisher Scientific) in the region of 4000-400 cm^-1 using KBr pellets (P116274, Aladdin).

Electrical conductivity of PPy-coated PCL/SF or uncoated PCL/SF was measured by four-probe (RTS-8, 4 Probes Tech, Guangzhou, China) at room temperature.³⁶ Conductivity was determined using the Van der Pauw equation.

Tensile mechanical properties of PPy-coated PCL/SF were tested on the low-load tensor (C610M, Labthink, Jinan, China) at room temperature.¹⁷ Equal lengths of the prepared membranes were clamped at both ends. The constant crosshead speed was set to 10 mm/min to determine the stress-strain curve under tensile test.¹⁷

For the biodegradability, samples were soaked in DMEM (11965092, Gibco, Waltham, MA, USA) and incubated at 37°C in a 5% CO₂ atmosphere. The dry weight of samples was tested every day. The biosafety properties of PPy-coated PCL/SF on RAW264.7 macrophages (American Type Culture Collection, Manassas, VA, USA, RRID: CVCL_0493), RSC96 Schwann cells (Bioresource Collection and Research Center, Hsinchu, China, Cat# 60507, RRID: CVCL_4694) and ND27 neurons (European Collection of Authenticated Cell Cultures, Salisbury, UK, Cat# 92090912, RRID: CVCL_4257) were determined via the cell counting kit-8 (CCK-8) assay (CA1210, Solarbio, Beijing, China), PrestoBlue^TM Cell Viability Reagent (A13262, Invitrogen, Waltham, MA, USA) and lived/dead cytometry assay (L3224, Invitrogen), as instructed by the manufacturers. We implanted the PPy-coated PCL/SF at lower back subcutaneously and examined the muscles next to the films 7 days later with haematoxylin & eosin staining.

All animal experiments were in accordance with the Guide for the Care and Use of Laboratory Animals.³⁷ The Administration Committee of Experimental Animals, the Chinese PLA General Hospital, China approved the use of animals in this study (approval No. 2016-x9-07).

The urethra of females is straighter and shorter, making it easier for urodynamic testing. Therefore, 8-week-old female Sprague-Dawley rats were acquired from Spfbiotech (Beijing, China; license No. SCXK (Jing) 2024-0001) and anaesthetised by intraperitoneal injection of pentobarbital sodium (0.9%, 4 g/kg, P3761, Sigma-Aldrich). The rats were then randomised into sham, crush, scaffold and ES (with scaffold) groups, with six rats in each group. Sham rats received sacral laminectomies and laparotomy of sacral nerves without further crush, while the rats of crush groups received the same surgery and crush as follows: bilateral 2^nd sacral laminectomies were performed under the posterior incision to expose the trunk and the root of the bilateral sacral nerves. Crush injuries of the bilateral sacral nerves were performed twice by a microneedle hold maximally clamping (10 seconds each time with 10-second interval). The rats in scaffold group had crushed nerves wrapped and temporarily implanted with PPy-coated PCL/SF scaffold. The scaffold was longer enough to cover the contact of electrode, which is approximately 6 mm. Moreover, the platinum-iridium electrode (Beijing PINS Medical Co., Ltd., Beijing, China) was fixed using an 8-0 suture material onto scaffold with rats of ES groups. Bilateral ES was conducted as single proximal ES protocol guided (impulse wave of 20 Hz, 100 ns per pulse and 3 V for 1 hour).^16,38,39 The membrane was then removed and the skin was sutured. The surgical procedures were depicted in Figure 1A and B.

Rats were housed in the Experimental Animal Center of Chinese PLA General Hospital with free access to water and food. Their bladders were emptied once a day using the crede procedure for 4 weeks until euthanasia using pentobarbital sodium. The investigator was individually blinded to the housed rats as they were unmarked.

Sacral nerves were harvested, washed three times with cold phosphate buffer saline (PBS) and transferred into the Hank’s balanced salt solution supplemented with collagenase II (0.5 mg/mL, C8490, Solarbio), collagenase II (0.5 mg/mL, C8150, Solarbio) and DNase I (25 μg/mL, D8070, Solarbio) for dissociation. Tissues were cut into pieces and incubated at 37°C for 35 minutes with vortexing every 5 minutes. After filtration, the cell suspensions were centrifuged and incubated with the Zombie Aqua Fixable Viability kit (423102, BioLegend, San Diego, CA, USA) for 15 minutes in the dark. They were washed with PBS, incubated with APC-Cy^TM7 Anti-RAT CD45 (202216, BioLegend), APC anti-RAT CD86 (200314, BioLegend) as well as FITC anti-RAT CD163 (MCA342F, Bio-Rad, Hercules, CA, USA) and treated with the permeabilization medium (GAS002S100, Invitrogen) for 30 minutes. The cell suspensions were incubated with PE anti-RAT CD68 (MCA341PE, Bio-Rad) for 1 hour in the dark. After repeated washes with PBS, the samples were subjected to flow cytometry (canto II, BD, Franklin Lakes, NJ, USA). All results were analysed using DIVA 8.0 (BD).

The retrograde urodynamic test was performed at the end of the observation period as previously described,² with some modifications. Briefly, rats were anaesthetised by intraperitoneal injection of urethane (10%, 1 g/kg, 94300, Sigma-Aldrich), the dose of which was adjusted based on toe pinching reflex. A sterile PE-50 catheter was retro-inserted into the bladders through the urethras, after which bladder emptying of was induced by pushing on the abdomen. Then, the bladders were perfused with 0.9% normal saline using an infusion pump (Harvard Apparatus, Holliston, MA, USA) at a rate of 5 mL/h until the first drop of urine could be seen. An electrophysiology recorder (Millar, Houston, TX, USA) with a pressure transducer was connected to PE-50 for cystometrogram with the initial bladder pressure rectified to zero. Urodynamic parameters for each rat (leakage point pressure, and maximum bladder volume) were directly recorded from respective urination cycles. Bladder compliance was calculated as:

Postvoid residual was measured by a portable digital ultrasound imaging system (Lanmage, Shenzhen, China) to assess urination function. The four groups of rats were anesthetised with inhalational isoflurane (5%, RWD Life Science Co., Shenzhen, China) delivered by a gas anaesthesia machine (Medical Supplies & Services Inc. Ltd., West Yorkshire, UK). Their lower abdomens were shaved with a depilatory paste, covered with the coupling agent (Jinya Co., Tianjin, China) and scanned with a high frequency linear probe (65 channels, 10 MHz, 38 mm). Axial and sagittal section images with the largest areas were acquired. First, the probe was placed in an axial position to capture the oval-shaped bladder and simultaneously measure their widths (D1) and depths (D3). With the probe rotated 90°, wedgy-shaped bladders in the sagittal position were captured and their lengths (D3) measured. Bladder volumes were estimated using the formula:

After experiments, rats were humanely euthanised using overdosed pentobarbital sodium after which the sacral nerves and bladders were harvested. The gross appearance of bladders was imaged. The nerves and bladders were fixed in 4% paraformaldehyde and dehydrated with 30% sucrose solution (powder, S112224, Aladdin). For immunofluorescence, all tissues were embedded in O.C.T Compound (Sakura Tissue Tek, Nagano, Japan) and sectioned into 10 μm using a freezing microtome (CM1950, Leica, Hesse, Germany). For haematoxylin & eosin and Masson’s trichrome staining, the tissues were processed by sequential dehydration and waxing using a dehydrator (ASP200S, Leica). The tissues were embedded with an embedding machine (HistoCore Arcadia C, Leica) and sectioned with a pathological microtome (RM2016, Leica). The bladders were stained with a Hematoxylin & Eosin staining kit (G1120, Solarbio) and Masson’s trichrome staining kit (G1340, Solarbio). Nerves were stained with Toluidine blue (1%, G3668, Solarbio) for 15 mintes and washed twice. The morphology and distribution of myelin can be observed with confocal scanner. Bladder wall thicknesses were measured after which the ratios of muscles and collagen were calculated using ImageProPlus v6.0 (Media Cybernetics, Rockville, MD, USA). Immunofluorescence staining was conducted as previously described.³¹ The slides were incubated in the presence of primary antibodies, including rabbit anti-S100β monoclonal antibody (unconjugated, Clone EP1576Y, 1:200, Abcam, Cat# ab52642, RRID: AB_882426), mouse monoclonal anti-neurofilament 200 (Phos. and Non-Phos.) antibody (1:300, Sigma-Aldrich, Cat# N0142, RRID: AB_477257), rabbit recombinant anti-collagen I antibody [EPR24331-53] (1:400, Abcam, Cat# ab270993, RRID: AB_2927551), mouse anti-collagen III monoclonal antibody (unconjugated, Clone FH-7A, 1:200, Abcam, Cat# ab6310, RRID: AB_305413), mouse CD68 antibody [ED1] (1:400) and CD206/MRC1(E6T5J)XP@rabbit mAb (1:800). Slides was washed thrice using PBS, incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) (1:200, Abcam, Cambridge, UK, Cat# ab150080) and Goat Anti-Mouse IgG H&L (Alexa Fluor 488) preadsorbed Antibody (1:200, Abcam, Cat# ab150117) for 1 hour in the dark, washed three times with PBS, then stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:5000) for 15 minutes, washed three times with PBS and covered with a coverslip. The immunofluorescence images were captured using confocal scanner. All samples were analysed with ImageJ 1.52a.

The isolation and induction of bone marrow-derived macrophages (BMDMs) was performed as previously reported.^{40⇓⇓⇓-44} Briefly, cells were rinsed from the femurs of C57BL/6 mice (Spfbiotech) and treated with cold red blood cell lysis buffer (C3702, Beyotime, Shanghai, China) at 4°C for 10 minutes. Resuspended cells were cultured in Dulbecco’s modified eagle medium (DMEM; 11965092, Gibco, Waltham, MA, USA), penicillin-streptomycin (5000 U/mL, 15070063, Gibco) and murine macrophage colony-stimulating factor (M-CSF; 10 ng/mL, AF-315-02, Peprotech, Cranbury, NJ, USA). After 6 hours of stationary culture at 37°C in a 5% CO₂ atmosphere, the suspended cells were collected, reseeded into new petri dishes and incubated. The culture medium was refreshed every 3 days. Mature BMDMs were harvested on the 7^th day for subsequent assays.

The BMDMs were seeded in a 12-well plate with PPy-coated PCL/SF membrane and their polarisation towards M1 phenotype induced by lipopolysaccharides (LPS; 100 ng/mL, L8880, Solarbio) and interferon-γ (IFN-γ; 20 ng/mL, P00106, Solarbio) for 24 hours.⁴⁵ The home-made platinum electrodes fitting the 12-well plate were sterilised and placed over the plate to make sure the tips were submerged for at least 2 mm. The direct current impulse waves (2.0 V, 20 Hz, 100 μs) for stimulation were generated by connecting the signal generator (DG811, RIGOL, Beijing, China) to the electrode for 1 hour. After 12 hours, the cells were harvested for immunofluorescence staining and quantitative polymerase chain reaction (qPCR) assay.

BMDMs were fixed in 4% paraformaldehyde (P1110, Solarbio) for 15 minutes, washed three times with PBS, blocked in 10% normal goat serum (SL038, Solarbio) for 1 hour, incubated overnight at 4°C with mouse CD68 antibody [ED1] (1:500, Abcam, Cat# ab31630, RRID: AB_1141557), CD206/MRC1(E6T5J)XP@rabbit mAb (1:800, CST, Danvers, MA, USA, Cat# 24595, RRID: AB_2892682), washed three times with PBS, incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) (1:200, Abcam, Cat# ab150080, RRID: AB_2650602) and Goat Anti-Mouse IgG H&L (Alexa Fluor 488) preadsorbed Antibody (1:200, Abcam, Cat# ab150117, RRID: AB_2688012) for 1 hour away from light, washed thrice using PBS, then stained with DAPI (1:5000, ab285390, Abcam) for 15 minutes, washed three times with PBS and covered with a coverslip. The immunofluorescence images were captured using confocal scanner (3DHISTECH, Panoramic Confocal, Budapest, Hungary) and the intensity was measured using ImageJ 1.52a.

BMDMs were plated in a petri dish at a density of 5 × 10⁶ cells/mL, induced and electrically stimulated. The total RNA was extracted using the FastPure Total RNA Isolation kit (RC112-01, Vazyme, Nanjing, China). The nucleic acid analyser (NanoDrop2000, ThermoFisher Scientific) was used to assess the RNA concentration, which was normalised to the control group. Reverse transcription was performed using the ReverTra Ace® qPCR RT Kit (FSQ-201, Toyobo, Osaka, Japan) at 37°C for 15 minutes and at 85°C for 5 seconds. The qPCR was performed using the StepOne^TM Real-Time PCR System (4376357, Applied Biosystems, Waltham, MA, USA): 95°C for 5 minutes, followed by 40 cycles of 15 seconds at 95°C, and 15 seconds at 65°C. Primer was synthesised by Sangon Biotech (Shanghai, China) and sequences for Il1b, Tnfa, Il6, Il12, Arg1, Ym1, Retnla, Mrc1, Stat1, Stat3, Stat6, Jak1, and Jak2 are shown in Table 1. Relative mRNA expressions were determined by normalising to β-actin.

The BMDMs were lysed with Trizol (15596018, Life Technologies, Waltham, MA, USA) for 10 minutes and snap frozen in liquid nitrogen. Library construction, high-throughput sequencing, as well as data collection and analyses were performed at CapitalBio Technology., Inc. (Beijing, China). Briefly, total RNA was extracted and assessed using the Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit Fluorometer (Invitrogen). The RNA-seq libraries of triplicate samples were generated and sequenced by CapitalBio Technology using the NEB Next Ultra RNA library Prep Kit for Illumina. The products were purified and enriched by qPCR to amplify the library DNA, followed by paired-end sequencing with pair end 150-base pair ending length on an Illumina NovaSeq sequencer (Illumina, San Diego, CA, USA). Sequencing qualities were assessed with FastQC (v0.11.5), with reference to the hg38 human genome.⁴⁶ Processed reads from each sample were aligned against the reference genome using HISAT2 (v1.3.3b, http://daehwankimlab.github.io/hisat-genotype/). Gene expression analyses were performed using StringTie (v1.3.3b, https://github.com/gpertea/stringtie) while differential expression genes (DEGs) analysis between samples were analysed using DESeq (v1.28.0, https://bioconductor.org/packages/release/bioc/html/DESeq.html). Independent statistical hypothesis testing was separately conducted on DEGs. Then, a P-value was obtained, which was corrected by the false discovery rate (FDR) method. The corrected P-value (q-value) was obtained using the BH method. The P- or q-values were set as thresholds for significance. The parameters for classifying significantly DEGs were ≥ 2-fold differences (|log2 fold change | ≥ 1) in the transcript abundance and P ≤ 0.05. Subsequently, Gene Ontology (GO) analysis was based on the DEGs results referring to the GO annotation database (https://www.geneontology.org/).^47⇓⇓-50 With R packages “clusterProfiler (https://github.com/YuLab-SMU/clusterProfiler)”, “enrich (https://github.com/wjawaid/enrichR)”, “GOplot”, “ggplot2”and “org.Hs.e.g.db” used, we explored their enrichment in molecular function, biological processes, and cellular components. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to detect enrichment of pathways between groups with the R “clusterProfiler” package. The Sankey analysis (https://github.com/davidsjoberg/ggsankey) was based on the KEGG analysis and were used to attribute the influence of genes to enrichment of pathway. The STRING database (https://cn.string-db.org/)⁵¹ was used to construct a protein-protein interaction network and revisualised using Cytoscape (v3.7.1). The confidence score of protein interactions was set as > 0.9. A set of hub genes was selected based on the maximal clique centrality algorithm and degree according to CytoHubba (v0.1, https://apps.cytoscape.org/apps/cytohubba).⁵¹

Data are expressed as mean standard error of the mean (SEM), and were analysed using IBM SPSS Statistics for Windows (version 27.0, IBM Corp., Armonk, NY, USA) and GraphPad Prism (version 9.4.0 for Windows, GraphPad Software, Boston, MA, USA, www.graphpad.com). Comparisons of means between two groups were performed using the Student’s t-test with P < 0.05 as the threshold for statistical significance. While some comparisons of means between more than three groups were performed using the one-way analysis of variance followed by Bonferroni’s post hoc test with P < 0.0332 as the threshold for statistical significance.

Pyrrole was polymerised on surfaces of PCL/SF membranes without significantly changing their flexibility (Figure 2A and B), morphologies of which were observed and confirmed by scanning electron microscopy. The granular process above the PCL/SF fibres indicated the appropriate extent of the reaction. There was no excess polymer that may have dissociated and damaged the resident microenvironment. Fourier transform infrared spectroscopy was also performed (Figure 2C). Addition of PPy resulted in higher stress levels than the PCL/SF membrane, which could reach the 11.151 MPa, improving the mechanical property of the membrane (Figure 2D). Conductivity, which was measured by the four-probes revealed significant increases after coating of the PPy (2.4127 ± 0.4989 S/cm; Figure 2E), compared to the uncoated PCL/SF of conductivity lower than the limit of detection. The scanning electron microscopy images showed that the radius of fibre was in the range of 0.1 to 0.15 μm (Figure 2F). The CCK-8 assay, PrestoBlue assay and live/dead cytometry with three major cell types (ND27, RSC96, and RAW264.7 cells) did not reveal significant differences in cell toxicity among all groups (Figure 2G-J). And we found no evidence of necrosis when attached the membrane on rat muscles over 7 days (Figure 2K). In summary, we have constructed a PPy-coated PCL/SF membrane with favourable mechanical, electrical and biological properties for further applications in nerve injury.

To investigate the effects of PPy-PCL/SF scaffold and ES, we crushed the sacral nerves at the S2 level and encircled the injured nerves with the scaffold. The rats with crushed sacral nerves were subjected to 1 hour of localised ES after wrapping, parameters of which were determined as previously described^2, 31 (Figure 1A). At 28 days after surgery, nerves and bladders were harvested and assessed. The neurofilament 200-positive nerve fibres were degenerated and reduced after injury, which was indicated by the intensity (t₍₂₀₎ = 2.771, P = 0.0035 < 0.05) and number of neurofilament 200-positive fibres (t₍₂₀₎ = 5.479, P < 0.0001; Figure 3A–D and F). The PPy-PCL/SF did not exhibit the regenerative effects (intensity: t₍₂₀₎ = 0.7191, P = 0.8597 > 0.05; number: t₍₂₀₎ = 0.3713, P = 0.9767 > 0.05). ES promoted axon regeneration (intensity: t₍₂₀₎ = 4.836, P = 0.0003 < 0.001; number: t₍₂₀₎ = 2.940, P = 0.0241 < 0.05). Axon myelination by Schwann cells is crucial for peripheral nerve regeneration. The intensity and number of S100β revealed the Schwann cells were severelydamaged and their abundance was significantly decreased after acute injury (intensity: t₍₂₀₎ = 2.665, P = 0.0440 < 0.05; number: t₍₂₀₎ = 4.155, P = 0.0015 < 0.01). The situation of Schwann cells was not improved by application of PPy-coated PCL/SF membrane (intensity: t₍₂₀₎ = 0.4982, P = 0.9467 > 0.05; number: t₍₂₀₎ = 0.08785, P = 0.9997 > 0.05), but did not rescued by ES (intensity: t₍₂₀₎ = 2.877, P = 0.0277 < 0.05; number: t₍₂₀₎ = 3.258, P = 0.0118 < 0.05; Figure 3A, E and G).

Toluidine blue staining was performed to assess the microcosmic changes of the axon and myelin. There was myelin regression and loss to varying degrees in the crush and PPy-PCL/SF groups, compared with the ES group (Figure 3B). Transmission electron microscopy revealed significant differences in myelin thickness (Crush vs. Sham: t₍₂₀₎ = 3.811, P = 0.0033 < 0.01; Scaffold vs. Crush: t₍₂₀₎ = 0.9993, P > 0.05; ES vs. Scaffold: t₍₂₀₎ = 2.988, P = 0.0217 < 0.05; Figure 3C and H). Those results demonstrated the neuroprotective effect of single immediate ES after crush injuries as presented results of the improved demyelination and promoted axon regeneration. According to Maeng et al.’s review,⁵² we believed with affirmation that the ES played a role of pro-regeneration through shaping the post-injured microenvironment once for all. It awaited us to determine whether macrophages are engaged in the ES-induced regeneration promotion.

As one of the major important target organs of the sacral nerve, the bladder is susceptible to all types of sacral nerve injury and exhibits various symptoms referred to neurogenic bladder. In Figure 4A, there was swelling and expanding of the bladder, which was alleviated by ES treatment. To investigate the bladder protective effects of ES, haematoxylin & eosin staining was performed to measure bladder thickness (Crush vs. Sham: t₍₂₀₎ = 10.54, P < 0.0001; Scaffold vs. Crush: t₍₂₀₎ = 3.335, P = 0.0099 < 0.01; ES vs. Scaffold: t₍₂₀₎ = 3.553, P = 0.0030 < 0.01; Figure 4A, B and G). The significant changes in thickness were attributed to enlargement of interstitial spaces and abnormal collagen hyperplasia that superseded the atrophic muscles, especially the detrusor, which was confirmed by Masson’s trichrome staining. The muscle and collagen areas were measured (Crush vs. Sham: t₍₂₀₎ = 10.59, P < 0.0001; Scaffold vs. Sham: t₍₂₀₎ = 1.373, P = 0.4589 > 0.05; ES vs. Scaffold: t₍₂₀₎ = 6.881, P < 0.0001; Figure 4C and H). Higher magnification revealed bladder wall vasodilation and inflammation in the crush and scaffold groups. Accompanied by increased collagen levels, there was a switch from collagen 3 to collagen 1 using immunofluorescence as showed in Figure 4D and I.

The urodynamic test was performed to assess denervation of the detrusor and atrophy of smooth muscles. The urodynamic curve revealed a smoothly ascending slope and prolonged urination latency in the crush group, compared to the Sham groups, indicating damaged contractability of detrusor and diastolic functions of smooth muscles. There were significant differences in leakage point pressure, while the calculated bladder compliance revealed that it was more sensitive to bladder dysfunctions caused by sacral nerve injury (Crush vs. Sham: t₍₂₀₎ = 8.239, P = 0.0095 < 0.01; ES vs. Scaffold: t₍₂₀₎ = 4.360, P = 0.0491 < 0.05; Figure 4E, J, and K). Thus, uroschesis can be detected by ultrasound and the bladder volume calculated (Crush vs. Sham: t₍₂₀₎ = 5.754, P < 0.0001; ES vs. Scaffold: t₍₂₀₎ = 2.790, P = 0.0335 < 0.05; Figure 4F, and L). Taken together with all the above results, we found that ES with crushed sacral nerves could alleviate the neurogenic bladder symptoms and decrease the histological change compared to the crush groups.

As important coordinators of the microenvironment, macrophages have bidirectional effects: pro-inflammation and pro-regeneration.^9,11 The timing of macrophage polarisation shift, if delayed, can cause excess inflammation and suppress nerve regeneration. There is a need to determine whether ES promotes nerve regeneration by regulating macrophages. We isolated the BMDMs and co-cultured them with the PPy-PCL/SF scaffold. Then, the BMDMs were subjected to ES at the same parameters as in vivo. Cells expressing CD68 occupying most view showed Figure 3A, indicating a purity of BMDMs. Next, we found no significant difference of CD206/CD68 ratio between control and PPy groups (t₍₈₎ = 0.3736, P = 0.9777 > 0.05), which showed PPy-coated PCL/SF membrane could not altered the polarisation. While significant differences in the ratio of CD206/CD68 intensity were noted (PPy + LPS/IFN-γ vs. PPy: t₍₈₎ = 3.423, P = 0.0269 < 0.05; PPy + LPS/IFN-γ+ ES vs. PPy + LPS/IFN-γ: t₍₈₎ = 13.35, P < 0.0001; Figure 5A and E). The in vitro result may not reflect the fate of macrophage of neural regeneration microenvironment. Therefore, we examined the in vivo macrophage polarisation with immunofluorescent staining which showed CD68⁺CD206⁺ macrophage concentrated most in ES group as showed in Figure 5C. Quantitative analysis of flow cytometry confirmed the results. The ratio of CD45⁺CD68⁺CD86⁺CD163⁺ cells to CD45⁺CD68⁺CD86^-CD163^- cells was decreased (t₍₆₎ = 7.543, P = 0.0017 < 0.05), but increased after ES (t₍₆₎ = 5.820, P = 0.0023 < 0.05; Figure 5B and D).

On the transcriptional level, the ES suppressed the expressions of M1-related markers (Il1b: t₍₄₎ = 3.197, P = 0.0330 < 0.05; Il6: t₍₄₎ = 4.024, P = 0.0158 < 0.05; Il12: t₍₄₎ = 5.636, P = 0.0049 < 0.01; Tnfa: t₍₄₎ = 4.936, P = 0.0078 < 0.01; Figure 5F–I). Expressions of the M2-assciated genes were significantly upregulated (Arg1: t₍₄₎ = 3.780, P = 0.0194 < 0.05; Mrc1: t₍₄₎ = 28.26, P < 0.0001; Retnla: t₍₄₎ = 2.942, P = 0.0423 < 0.05; Ym1: t₍₄₎ = 3.011, P = 0.0395 < 0.05; Figure 5F–M).

Studies have aimed at assessing the mechanisms of ES in regulating cell fates. The macrophages exhibiting unique responsiveness to multiple stimuli are activated by different mechanisms that have not been fully established. We comprehensively analysed the gene expression profiles by high-throughput RNA sequencing in non-induced BMDMs (M0), LPS/IFN-γ-induced BMDMs (LPS/IFN-γ) and ES + LPS/IFN-γ induced BMDMs (ES) groups. In Figure 6A, a total of 4557 genes were found to be differentially expressed between the ES + LPS/IFN-γ groups, among which 1178 genes were significantly changed after LPS/IFN-γ treatment, compared with the M0 group. The DEG analysis revealed that the most down-regulated genes in the ES groups were chemokine-related (Ccl5, Irf7, Cxcl10, Ifit1 and Il1b),^53⇓-55 indicating that cascade amplification of inflammation by macrophages was blocked after ES (Figure 6B). The potential of Anxa856 for enhancing M2 polarisation has been reported while the neurotrophic factors (Ngf, Nrep and Edil3)^57, 58 facilitate axon regeneration. The Fn1 encode fibronectin1, which makes up the basal structure of nerve outgrowth.⁵⁹ Thus, after ES, gene expressions of macrophages were alternated to M2 profiles. The genes encoding the voltage-gated channels (Cacnb1, Cacng7, Clcn7 and Prap1) were differentially expressed, and are important in ES-induced transcriptional changes.

GO analysis showed that the biological process was enriched in immunological response and inflammation as a cascade of cytokines and chemokines was differentially regulated (Figure 6C). The extracellular organelle and extracellular membrane-bound organelle were significantly changed, indicating the activation of infiltration. The focal adhesion was also altered. The extracellular vesicle and extracellular exosome which were enriched may implied the activation of secretion of EVs. For the molecular function, some metabolite binding was significantly regulated, for example, anion, carbohydrate derivative and nucleoside. It is usually speculated that those terms correlated with transcriptional regulation.

Enrichment analysis was performed to identify the significantly differentially enriched pathways between the ES and LPS/IFN-γ groups using the KEGG database (Figure 6D). The adenosine 5′-monophosphate-activated protein kinase (AMPK) signalling pathway,⁶⁰ peroxisome proliferators-activated receptor (PPAR) signalling pathway,^10,61 JAK-STAT signalling pathway⁶² and PI3K-Akt signalling pathway⁶³ are of intermediate importance in macrophage polarisation. The cytokine-cytokine receptor interaction pathway and chemokine signalling pathway were significantly enriched, however, most of which were downstream proteins regulated by the signal transduction harmonised between the ES and pro-inflammation stimuli. The calcium signalling pathway exhibited unique intermediate effects between the ES effects and transcriptional regulation.^64,65

The traditional KEGG visualised analysis may not have assessed the impact of the most variably expressed genes in KEGG pathways, therefore, Sankey analysis, which focuses on the most significantly expressed genes and pathways, was performed. The nerve growth factor (NGF) signalling was upregulated, with genes such as Mapkapk2,⁶⁶ Fgfr1,⁶⁷ Src⁶⁸ and Csf3r that are favourable for wound healing. The Col1a2,⁶⁹ Mmp14,⁷⁰ Sdc1 and Sdc3 were highly correlated with extracellular matrix organisation by macrophages, which may have an important role in nerve regeneration.⁷¹ Besides, macrophages coordinate foreign body reactions via the pathway to promote “scar-like” tissue formation while also resolving inflammation by accelerating the formation of connective tissues to bridge the minor gaps.⁷² Platelet activation promotes regeneration via release of platelet granules, leading to endothelial activation and growth factor secretion.

The protein-protein interaction network was constructed and the hub genes identified. In Figure 6E and F, Stat1 was modulated by ES. Zhang et al.⁷³ reported that there are four JAKs and seven STATs mediating the transduction of almost 60 cytokines, hormones and growth factors. Homodimers of JAKs can phosphorylate themselves in response to cytokine binding and pass the phosphates to STATs to become dimers and move intranuclearly to activate target gene transcriptions. Canonical JAKs and STATs are highly selective. The STAT1 and STAT3 transduce pro-inflammation signals like IFN-γ, while STAT6 transduce pro-regeneration signals.^62,73,74 The qPCR results indicated that Stat1 (t₍₄₎ = 3.359, P = 0.0283 < 0.05), Stat3 (t₍₄₎ = 4.523, P = 0.0106 < 0.05) were down-regulated while Stat6 (t₍₄₎ = 9.603, P = 0.0007 < 0.001) expressions were up-regulated. However, Jak1 (t₍₄₎ = 0.9585, P = 0.3921 > 0.05) and Jak2 (t₍₄₎ = 0.7565, P = 0.9433 > 0.05) expressions were not affected, as showed in Additional Figure 1A. In the meantime, we found a significant increase of intracellular calcium concentration after ES (t₍₄₎ = 7.990, P = 0.0013 < 0.05), which agreed with the up-regulated genes that encoded calcium channel substrate. However, the mechanism on the calcium regulating STATs transcription was still hidden in the mist.

As the ES may post paradox impacts on various component besides macrophage, we performed ES-manipulated macrophage transplantation experiments after crush injury as described above (Figure 7A) and observed improved nerve regeneration. The intensity of S100β (t₍₈₎ = 3.074, P = 0.0153 < 0.05) and neurofilament 200 (t₍₈₎ = 1.698, P = 0.1279 > 0.05) was mildly changed with replantation of non-treated BMDMs compared to those crushed (Figure 7B, C and E). It was even worse for those transplanted with LPS/IFN-γ induced BMDMs (S100β: t₍₈₎ = 3.556, P = 0.0074 < 0.01; neurofilament 200: t₍₈₎ = 2.378, P = 0.0447 < 0.05), however, better for those with macrophage induced by ES and LPS/IFN-γ (S100β: t₍₈₎ = 3.827, P = 0.0050 < 0.01; neurofilament 200: t₍₈₎ = 3.699, P = 0.0061 < 0.01). Then, we evaluated the histological reconstruction of neurogenic bladder. The Masson’s trichrome staining showed the ratio of collagen to muscle was unchanged when transplanted with BMDMs without induction (t₍₈₎ = 0.6957, P = 0.5063 > 0.05; Figure 7D and F). The significant increase was found in BMDMs (LPS/IFN-γ) group (t₍₈₎ = 2.689, P = 0.0275 < 0.05). However, electrically stimulated BMDMs remodeled the neurogenic bladder accordingly (t₍₈₎ = 3.531, P = 0.0077 < 0.01). Similar tendency was observed in collagen type (Figure 7G and K). The bladder function was measured with urodynamic test as the leaking point pressure decreased with BMDMs (LPS/IFN-γ + ES) so as to bladder compliance (leakage point pressure: t₍₈₎ = 4.790, P = 0.0014 < 0.01; compliance: t₍₈₎ = 2.982, P = 0.0176 < 0.05; Figure 7H and I). The volume of bladder was tested with high frequency microultrasoundgraphy was significantly different in crushed rats transplanted with BMDMs (LPS/IFN-γ) (t₍₈₎ = 8.186, P < 0.0001). Meanwhile, it decreased in BMDMs (LPS/IFN-γ + ES) group (t₍₈₎ = 3.437, P = 0.0089 < 0.01; Figure 7J). Generally, we found that crushed nerve without ES suffered from worse regeneration from the transplantation of LPS/IFN-γ-induced BMDMs. The BMDM polarisation induced by LPS/IFN-γ was reversed by ES, remodelling the microenvironment accordingly.

We developed a novel conductive membrane for in vivo sacral nerve ES by combining electrospinning of PCL/SF and in situ polymerisation of pyrrole. Obviously, we’ve seen many studies concerning the ES on injured nerves, most of which paid close attention to axon elongation or Schwann cells activation.^{8,75⇓ - 77} Macrophage, however was less reported. This work demonstrated the important role of macrophage polarisation in peripheral nerve regeneration boosted by ES. Some researcher may argue that the traditional M1/M2 theory overcomplicated the vague and versatile macrophage spectrum therefore diminished the credibility of conclusion of experiments in vitro. In fact, our results from “back-transplantation” confirmed the direct ES on macrophage reversed the LPS/IFN-γ-induced pro-inflammation phenotype and, in the meantime, yielded an exclusive pro-regenerative effect when implanted back to crushed nerves. The idea came from a similar research⁷⁸ and was first applied in peripheral nerve-regeneration studies.

Moreover, our findings demonstrate that ES regulates macrophage activity during the initial phase of nerve regeneration, even when minimal macrophages are recruited to the site of injury, thereby attenuating inflammation. Previous study may insist on periodical stimulation^79⇓-81 based on the theory that macrophage may fall easily into M1 without constant M2-related stimulation. According to our results, brief ES immediately after injury could alter immune microenvironment once for all to polarised recruited circulating macrophages. It is still unclear how it works and demands our further investigation.

Here, we found JAK-STAT signalling pathway and calcium signalling pathway involved in ES on macrophage through high-throughput sequencing analysis for the first time. We will carry out further analysis of the calcium channel blocker to show whether the voltage-gated calcium opening is the initial steps leading to polarisation shift. STAT family has seven members, expression of which can be distinguishedly regulated according to our results. In a word, macrophage response to same stimuli in varied extent due different STAT expression. However, the precise mechanisms by which calcium uptake influences the expression of STATs are currently under investigation.

The utilization of ES on the sacral nerve has gained significant attention due to promising clinical application of sacral nerve modulation in the treatment of neurogenic bladder, which has shown favourable rates of restoring bladder function. We took a step forward and found macrophage as a crucial regulator of sacral nerve regeneration. Next, Wang et al.⁸² reported that the sacral spinal-vagal pathway may participate in the regulation of the GI disorder by sacral ES. The mechanisms of sacral nerve modulation have been clarified in similar studies conducted in other regions. By initiating stimulation at the onset of injury, we have provided evidence that early-stage ES facilitates sacral nerve regeneration after crush injury, thereby expanding the applicability of nerve modulation as a therapeutic modality.

Dielectric materials have long been investigated due to the susceptibility of nerves to mechanical damage, electric burns, and electrolytic effects. PPy possessed with excellent conductivity, was not suitable for biomaterial application for hardness. Most researcher take it as dopant in biocompatible material.^17,83 In our study, we developed a membrane composed of PCL/SF with a PPy coating, which exhibited adequate biocompatibility and conductivity through in situ polymerization for in vivo ES, which was seldom used before. The method did not impair the excellent mechanical nature of electrospun PCL/SF fibre evidently for that the polymer was just coated onto surface fibre. Additionally, we used less pyrrole this way which attenuated possible remanent toxic monomer. We also over-dosed the oxidising agent compared to previous study⁸⁴ just in that case. However, we removed the membrane after 1 hour of ES, as its retention did not contribute to the regeneration of the crushed sacral nerve. This approach limited any potential damage.

In conclusion, the clinical management of sacral nerve injuries, which often involve complex damage, poses significant challenges. In our study, we employed ES as a therapeutic approach for sacral nerve crush injuries using a PPy-coated PCL/SF membrane. As we hypothesised, the ES improved neural regenerative potential and rescued the neurogenic bladder. Cell cytometry showed that macrophage polarisation shifted to M2 at 7^th day post-injury in contrast to the enrichment of M1 in the damaged sacral nerve. Combining in vitro studies with transcriptome analysis, we have demonstrated that ES promotes the transition of macrophages from an M1 to an M2 phenotype by regulating the expression of Stat1/Stat3/Stat6. These findings highlight the distinctive contribution of macrophages to ES-induced nerve regeneration, unveiling a novel role in this process.

Author contributions

HC: Conceptualization, methodology, investigation and writing - original draft; JB: software, resources, data curation, writing - original draft and visualisation; XZ: validation, investigation, formal analysis and writing - original draft; NC: resources, investigation and data curation; QJ: methodology and formal analysis; ZR: visualisation and writing - review & editing; XL: validation and writing - review & editing; TS: formal analysis and visualisation; LL: methodology and software; WJ: supervision; YW: supervision, writing - review & editing and guarantor; JP: supervision and project administration; AS: supervision and funding acquisition. All authors approved the final version of the manuscript.

Financial support

This study was supported by Capital’s Funds for Health Improvement and Research (No. CFH2022-2-5022).

Acknowledgement

We are grateful to Beijing PINS Medical Co., Ltd. for their support in providing the electrodes and pulse generator and technical data. And authors would like to thank all the reviewers who participated in the review and MJEditor (www.mjeditor.com) for its linguistic assistance during the preparation of this manuscript.

Conflicts of interest statement

The authors declare no conflict of the interest.

Open access statement

This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.