Fascin-1 Is Highly Expressed Specifically in Microglia After Spinal Cord Injury and Regulates Microglial Migration

doi:10.21203/rs.3.rs-258359/v1

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Fascin-1 Is Highly Expressed Specifically in Microglia After Spinal Cord Injury and Regulates Microglial Migration

https://doi.org/10.21203/rs.3.rs-258359/v1

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Background

Recent research indicates that after spinal cord injury (SCI), microglia accumulate at the borders of lesions between astrocytic and fibrotic scars and perform inflammation-limiting and neuroprotective functions; however, the mechanism of microglial migration remains unclear. Fascin-1 is a key actin-bundling protein that regulates cell migration, invasion and adhesion, but its role during SCI has not been reported.

Methods

A mouse model of thoracic (T10) spinal cord compression injury was used. We employed Western blotting, and immunohistochemistry to assess expression levels of Fascin-1 protein and analyze cell localization of Fascin-1 after SCI. We employed Scratch assay and Transwell assay to evaluate the effect of Fascin-1 on the function of microglia in vitro.

Results

We found that at 7-14 days after SCI in mice, Fascin-1 is significantly upregulated, mainly distributed around the lesion, and specifically expressed in CX3CR1-positive microglia. However, Fascin-1 is not expressed in GFAP-positive astrocytes, NeuN-positive neurons, NG2-positive cells, PDGFRβ-positive cells, or blood-derived Mac2-positive macrophages infiltrating into the lesion core. The expression of Fascin-1 is correspondingly decreased after microglia are specifically depleted in the injured spinal cord by the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. The upregulation of Fascin-1 expression is observed when microglia are activated by myelin debris in vitro, and microglial migration is prominently increased. The inhibition of Fascin-1 expression using small interfering RNA (siRNA) markedly suppresses the migration of microglia, but this effect can be reversed by treatment with myelin. The M1/M2 polarization of microglia does not affect the expression of Fascin-1.

Conclusions

Our results suggest that Fascin-1 is highly expressed specifically in microglia after SCI and can play an important role in the migration of microglia and the formation of microglial scars. Hence, the elucidation of this mechanism will provide novel therapeutic targets for the treatment of SCI.

Neurobiology of Disease

Fascin-1

microglia

migration

spinal cord injury

Spinal cord injury (SCI) induces a complex heterogeneous inflammatory response largely mediated by resident microglia and infiltrating monocyte-derived macrophages [1, 2]. It has been difficult to distinguish between activated microglia and infiltrating macrophages in the injured spinal cord, since they share many similar markers and phenotypes [3]. Recent advances in conditional gene targeting and chimeric analysis have allowed the identities and functions of these cell populations to become increasingly clear [2, 4, 5]. It has been shown that after SCI, infiltrating macrophages expressing high Mac2 levels but low CX3CR1 levels accumulate in the lesion core, which are overloaded with myelin debris, and trigger a sustained inflammatory reaction [6, 7]. In contrast, resident microglia expressing high CX3CR1 levels accumulate around the lesion core and form a border, which is named the “microglial scar”, to seal the lesion and block the spread of damage [8]. However, the mechanism of microglial scar formation is far from clear.

Fascin-1, an actin-bundling protein, plays a key role in the assembly and stability of cell protrusions and other actin-based structures that aid in cell motility, migration and invasion [9]. A multitude of studies have shown that Fascin-1 supports the migratory and metastatic capacities of carcinomas [10–12]. In addition, it has recently been reported that Fascin-1 is upregulated predominantly in the microglia in the posterior horn of the spinal cord in a rat model of neuropathic pain and that Fascin-1 contributes to neuropathic pain by promoting inflammation [13]. However, the cellular localization and function of Fascin-1 after SCI have not been reported.

In the present study, we found that Fascin-1 was highly expressed specifically in microglia that accumulated at the lesion border after SCI. Fascin-1 expression decreased accordingly after the specific depletion of microglia in the injured spinal cord, and this effect was accompanied by disorganized astrocytic and fibrotic scars and scattered macrophages in the injured spinal cord. Fascin-1 knockdown markedly suppressed microglial migration, but this effect could be reversed by myelin treatment. Our results suggest that Fascin-1 may play a key role in microglial migration and microglial scar formation in SCI.

2.1 Animals

All the animal procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Anhui Medical University (No. LLSC20160052). C57BL/6J female mice at 6–8 weeks of age were purchased from the Experimental Animal Center of Anhui Medical University. The mice were housed in a temperature- and humidity-controlled room with a 12:12-light/dark cycle and allowed free access to food and water.

2.2 SCI model

All the surgical procedures were performed under pentobarbital anesthesia. The skin around the injury site was shaved and disinfected using iodophor. Subsequently, the T10 spinal cord was exposed via a dorsal laminectomy, and moderately severe crush SCIs were made using No. 5 Dumont forceps (Fine Science Tools, 11252-20, Heidelberg, Germany) ground down to a tip with a width of 0.5 mm by compressing the cord laterally from both sides for 5 s [14]. Then, twitching of the hind limbs and movement of the tail were observed, which indicated that the SCI model was successfully established. Finally, the wound was sutured with 3 − 0 silk threads. The mice with SCI were examined daily to monitor their recovery, and their bladders were expressed manually three times a day until the return of reflexive bladder control. The sham group were subjected to laminectomy alone. The mice were sacrificed at 3, 7 and 14 days after SCI.

2.3 Microglia depletion.

To eliminate microglia, mice were administered PLX5622 (MedChemExpress, HY-114153) at 130 mg/kg by oral gavage once a day for 17 consecutive days. PLX5622 was diluted in 5% DMSO, 40% polyethylene glycol 300, 5% polysorbate 80, and 50% saline, according to the manufacturer’s instructions. An equal volume of vehicle was used as the control. SCI was established on the third day after gavage.

2.4 Preparation of myelin debris

Myelin debris was isolated as previously described [15]. Briefly, 6- to 8-week-old mice were euthanized, and their brain tissues were harvested and homogenized in ice-cold 0.32 M sucrose. Myelin debris was isolated from the brain tissues by sucrose density gradient centrifugation. The endotoxin concentration of the myelin debris was below the limit of detection of the Limulus Amebocyte Lysate assay (Lonza, Switzerland). Myelin debris was added to cells at a final concentration of 1 mg/mL in all the experiments.

2.5 Cell culture and transfection.

The BV-2 microglial cell line was obtained from the American Type Culture Collection (CRL-3265, ATCC, Manassas, VA, USA) and cultured and maintained in Dulbecco's modified Eagle's medium (DMEM, HyClone, SH30021) supplemented with 10% fetal bovine serum (FBS, Gibco, 10270106), 100 U/ml penicillin and 100 g/ml streptomycin (Gibco, Grand Island, NY, USA). The cells were incubated in a humidified chamber at 37°C in a 95% O₂ and 5% CO₂ atmosphere. Small interfering RNA (siRNA) was transfected into these cells using jetPRIME (Polyplus Transfection, 114 − 15), according to the manufacturer's instructions. siRNA targeting mouse Fascin-1 (siRNA: 5’- GAUGCCAACCGUUCCAGUUTT − 3’) and nonspecific control siRNA (NC) were purchased from GenePharma (Shanghai, China).

2.6 Microglial polarization

BV-2 cells were plated in poly-d-lysine (PDL, Sigma, P7280)-coated 6-well plates at a density of 1×10⁶ cells/ml and cultured overnight. After serum deprivation for 24 h, the BV-2 cells were polarized toward the M1 phenotype by treatment with lipopolysaccharide (LPS; 100 ng/ml, Beyotime Biotechnology, ST1470, Shanghai, China) and IFNγ (20 ng/ml, Beyotime Biotechnology, P6137) or toward the M2 phenotype by treatment with IL-4 (20 ng/ml, Beyotime Biotechnology, P5916), and the cells were cultured for 24 h [16].

2.7 Tissue processing

For Western blot analysis, the mice were anesthetized and transcardially perfused with 0.1 M phosphate-buffered saline (PBS) to remove the blood. Spinal cord tissues of 5 mm centered at the injury site were harvested. For histological analysis, once the blood was removed, the mice were transcardially perfused with 4% paraformaldehyde (PFA), and a 5-mm portion of the spinal cord encompassing the injury epicenter was extracted and embedded in paraffin. Then, the tissues were sagittally sectioned at thicknesses of 6 µm on a microtome (Leica RM2235). Every tenth section was collected and mounted onto a series of slides.

2.8 Immunofluorescence analysis

Immunohistochemistry. Six-micrometer, paraffin-embedded spinal cord sections were dried, dewaxed, hydrated and subjected to antigen repair. Next, 10% donkey serum albumin (DSA, Solarbio, SL050) containing 0.3% Triton X-100 (Solarbio, T8200) was added and incubated at room temperature for 1 h. Then, primary antibodies were added and incubated at 4°C overnight. The primary antibodies used were as follows: mouse anti-Fascin-1 (1:50, Santa Cruz, sc-21743), rabbit anti-Fascin-1 (1:100, Abcam, ab126772), goat anti-Iba1 (1:200, Novus Biologicals, NB100-1028), rabbit anti-CX3CR1 (1:500, Abcam, ab8021), rabbit anti-GFAP (1:100, Proteintech, 16825-1-AP), mouse anti-GFAP (1:100, Proteintech, 60190-1-Ig), goat anti-PDGFRβ (1:40, R&D Systems, AF1042-SP), rabbit anti-PDGFRβ (1:200, Abcam, ab32570), mouse anti-Mac2 (1:100, GB12246, Servicebio), rabbit anti-NG2 (1:100, Proteintech, 55027-1-AP), rabbit anti-iNOS (1:100, Affinity, AF0199), rabbit anti-Arg1 (1:100, Affinity, DF6657), and rabbit anti-NeuN (1:500, Abcam, ab177487). The secondary antibodies were diluted in 1% donkey serum in PBS and incubated for 1 h at room temperature. The following secondary antibodies were used: Alexa Fluor 488 and Alexa Fluor 594 (1:500, Thermo Fisher Scientific, A-21206, A-21202, A-21203, A-21207, A-11058). The nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (1 µg/ml, Thermo Fisher Scientific). The fluorescence signals were obtained using an Axio Scope A1 microscope (Zeiss, Germany). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for quantitative analysis.

Immunocytochemistry. Cells were fixed with 4% PFA for 10–15 min and blocked with 5% donkey serum in PBS for 30 min at 20–25°C. The primary antibodies (as listed above) were diluted in 1% donkey serum in PBS and incubated overnight at 4°C. The secondary antibodies (as listed above) were diluted in 1% donkey serum in PBS and incubated for 1 h at room temperature. Images were acquired as described above.

2.9 Imaging analysis and quantification

Every tenth 6-µm-thick, paraffin-embedded section was quantified, resulting in 5 analyzed slides per animal that included the entire injured spinal cord. The total number of Fascin-1⁺, CX3CR1⁺, and Fascin-1⁺CX3CR1⁺ double-positive cells in each of the sagittal sections of the spinal cord was counted under a 20× objective lens with Zeiss ZEN imaging software (Zeiss, Germany).

2.10 Western blot analysis

Tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma, R0278) supplemented with protease inhibitors (Roche, 04693124001) and phosphatase inhibitors (Roche, 04906845001). The cells were washed with cold PBS, homogenized in RIPA buffer on ice for 30 min, and then centrifuged at 12,000 rpm at 4°C for 30 min. The protein extracts were quantified by using a Pierce BCA protein assay kit (Beyotime Biotechnology, P0010S). Aliquots of the protein samples containing equal protein concentrations were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and subsequently transferred to polyvinylidene (PVDF) membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.5% Tween-20 (TBST) for 1 h at room temperature and then incubated with primary antibodies, including mouse anti-GAPDH (1:2000, Proteintech, 60004-1-Ig), rabbit anti-Fascin-1 (1:5000, Abcam, ab126772), rabbit anti-iNOS (1:3000, AF0199, Affinity) and rabbit anti-CD206 (1:1000, ab64693, Abcam), at 4°C overnight. After three washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies (1:10000, Sigma, A4416) and HRP-conjugated goat anti-rabbit secondary antibodies (1:10000, Sigma, A0545) for 1 h at room temperature. The protein band signals were obtained using an ECL detection kit (ECL, Thermol Biotech Inc., USA) and Tanon 5200 system (Tanon, Shanghai, China). ImageJ was used for quantification analysis. The intensity of the GAPDH bands was used for normalization.

2.11 Scratch assay

Cell migration was assessed by performing a scratch assay. Briefly, BV-2 cells were seeded into PDL-coated 6-well plates at a density of 2 × 10⁵ cells/well and incubated for 24 h. The cell layers were scratched using a 200-µl pipette tip to form a wound-like gap. The cells were then maintained in DMEM with 2% FBS, and images were captured at 0 h, 24 h and 48 h after cell scratching. ImageJ was used to analyze the wound width.

2.12 Transwell assay

A 24-well plate containing a 3-µm chamber (Costar, 3415) was used to assess the migration abilities of the cells. BV-2 cells were transfected and activated in a PDL-coated 6-well plate before inoculation into the chamber. Then, the cells were suspended in serum-free media and seeded into the upper chamber (5 × 10⁴ cells per chamber). The lower chamber contained complete media. After incubating for 12 h, the nonmigrating cells on the inside of the membrane were carefully removed with a cotton swab, and the migrating cells on the outside of the membrane were fixed with 4% PFA at room temperature for 20 min and stained with 0.1% crystal violet for 15 min. The cells were observed under a microscope and counted. At least 5 random fields were photographed, and the cells in each field were counted.

2.13 Statistical analysis

All the experiments were independently performed with at least three replicates and quantified in a blinded manner. The data are presented as the mean ± standard error of the mean (SEM). The statistical analysis was carried out with SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) software. Student’s t test to compare the difference between 2 groups and One-way analysis of variance followed by Tukey’s post hoc test was used to compare differences among multiple groups. Differences were considered statistically significant if P < 0.05.

3.1 Fascin-1 is significantly upregulated and distributed at the edge of the lesion core after SCI

To determine the changes in Fascin-1 expression during SCI, Western blot was used to detect the relative expression levels of Fascin-1 before and 3–14 days after SCI. As shown in Figs. 1a and 1b, Fascin-1 expression was significantly increased at 7 and 14 days after SCI compared with that before injury (^*P < 0.05 and ^***P < 0.001, respectively). To further analyze the distribution of Fascin-1, we carried out immunofluorescence detection of Fascin-1 and GFAP, which is used to label the astrocytic scar formed around the injury site. The fluorescence signal of Fascin-1 was also obviously increased at 7 and 14 days after SCI and gradually accumulated at the edge of the lesion core, intermingling but not colocalizing with GFAP⁺ astrocytic scars (Fig. 1c). These results indicate that the expression of Fascin-1 is prominently upregulated and mainly distributed at the lesion border; Fascin-1 expression has a spatiotemporal distribution pattern similar to that of microglia after SCI [8].

3.2 Fascin-1 is specifically expressed in microglia after SCI

The cellular localization of Fascin-1 was then detected by immunofluorescence staining. We found that Fascin-1 was highly colocalized with CX3CR1-labeled microglia. The percentage of Fascin-1⁺CX3CR1⁺ costained cells relative to the total number of Fascin-1⁺ or CX3CR1⁺ cells in the injured spinal cord reached 94.06 ± 0.82% or 87.65 ± 1.08%, respectively (Figs. 2a and 2d). These costained cells were mainly located around the SCI site (Fig. 2a), which was consistent with previous studies [8]. Fascin-1 was not expressed in the GFAP⁺ astrocytes surrounding the lesion core or in the PDGFRβ⁺ pericytes accumulated in the epicenter (Figs. 2b and 2c).

Iba1 is a common marker of microglia, but during SCI, both resident microglia and infiltrating macrophages can be identified by Iba1 [6]. Our results showed that costaining of Fascin-1 and Iba1 was mainly confined to the edge of the injury site, which was consistent with the localization of microglia (Fig. 3a). Moreover, Fascin-1 was not colocalized with Mac2⁺ macrophages located at the lesion core (Fig. 3b). In addition, Fascin-1 was not expressed in NeuN⁺ neurons or NG2⁺ cells in the injured area (Figs. 3c and 3d). Taken together, these results show that Fascin-1 is highly expressed specifically in microglia after SCI.

3.3 Depletion of microglia correspondingly reduces Fascin-1 expression after SCI

To confirm that Fascin-1 is derived from microglia, PLX5622, a selective inhibitor of colony-stimulating factor 1 receptor (CSF1R) that crosses the blood–brain barrier (BBB) and eradicates nearly all the microglia in the central nervous system (CNS), was administered to mice [17]. A solution containing PLX5622 was administered to mice by gavage starting 3 days prior to SCI and continuing until sacrifice. As shown in Figs. 4a and 4c, the number of Fascin-1⁺CX3CR1⁺ microglia in the PLX5622 group was significantly decreased compared to that in the control group (^****P < 0.0001). The expression of Fascin-1 decreased accordingly with the depletion of CX3CR1⁺ microglia. The percentages of Fascin-1⁺ cells and CX3CR1⁺ cells in the PLX5622 groups relative to those in the untreated control groups were 26.14 ± 0.61% and 27.38 ± 1.07%, respectively (Fig. 4d). After the depletion of microglia, we observed that both GFAP⁺ astrocytic scars and PDGFRβ⁺ fibrotic scars became less compact and disorganized, and infiltrating Mac2⁺ macrophages were diffusely scattered outside of the lesion core (Fig. 4b). These results indicate that Fascin-1 is mainly derived from microglia after SCI. The significant upregulation of Fascin-1 may play an important role in the formation of microglial scars.

3.4 High expression of Fascin-1 can promote microglial migration

To assess the effect of Fascin-1 on the function of microglia, we cultured the BV-2 microglial cell line in vitro. Western blot (Figs. 5a and 5b) and immunocytochemistry (Fig. 5c) analyses revealed that compared to the negative controls, Fascin-1 siRNA (siFascin-1) could knockdown the expression of Fascin-1, while myelin treatment could upregulate the expression of Fascin-1. The migration ability of the microglia was reduced after Fascin-1 knockdown but enhanced by myelin addition, as observed by scratch and Transwell assays (Fig. 6). Furthermore, the inhibition of Fascin-1 expression and microglial migration using siFascin-1 could be reversed by treatment with myelin (Figs. 5 and 6). These results suggest that Fascin-1 can regulate microglial migration.

3.5 Polarization of microglia does not affect the expression of Fascin-1

Previous studies have shown that microglia can be polarized into either a pro- inflammatory (M1) or anti-inflammatory (M2) phenotype and involved in the regulation of the SCI microenvironment [18]. Next, we detected the effect of microglial polarization on Fascin-1 expression. Western blot analysis showed that M1 microglia significantly expressed iNOS and M2 microglia significantly expressed CD206 (Figs. 7a and 7b). In addition, immunocytochemistry was used to further confirm the reliability of microglial polarization (Figs. 7c and 7d). However, the expression of Fascin-1 in the polarized microglial populations was not significantly different (Fig. 7). The results show that microglial polarization has no effect on the expression of Fascin-1.

Microglia, the resident immune cells of the CNS, are cells that rapidly respond to CNS injury [19, 20]. Following SCI, these cells migrate or project processes toward sites of injury, where they release neurotrophic agents and confer neuroprotection [8, 21]. In this study, we demonstrate that the actin-bundling protein Fascin-1 is highly expressed specifically in microglia after SCI and is indispensable for microglial migration. The expression of Fascin-1 is correspondingly reduced after the specific elimination of microglia by the CSF1R inhibitor PLX5622, and this effect is associated with disordered astrocytic and fibrotic scars and widespread macrophages in the injured spinal cord. Thus, we highlight that Fascin-1 can play a vital role in regulating microglial migration and microglial scar formation after SCI.

The proliferation of CX3CR1⁺ residual microglia has been observed beginning at 3 days after SCI [6]. Microglia are rapidly recruited around the lesion epicenter, exhibit a round morphology, and upregulate the lysosome-associated protein CD68, which suggests a potential increase in their phagocytic activity. Moreover, microglia continue to proliferate and accumulate around the site of the lesion epicenter, peaking at 14 days after SCI and forming microglial scars [8]. Our results also show that Fascin-1 is prominently expressed in microglia beginning at 3 days and peaks at 14 days after SCI. Olah et al. determined the global gene expression changes in microglia during demyelination and remyelination via microarray analysis, and the results suggested that the primary functions of CNS-resident microglia are repair and maintenance of tissue homeostasis [22]. Using the lysozyme M EGFP-knock-in mouse, in which the expression of EGFP is specifically promoted in hematogenous macrophages but in not microglia, Greenhalgh and David showed that microglia are the predominant cells that contact and phagocytose damaged and degenerating tissue 3 days after SCI; however, EGFP⁺ infiltrating macrophages are less efficient at processing CNS debris, and their death in situ may contribute to secondary damage after SCI [23]. In the latest study, the transplantation of CX3CR1^GFP/+ neonatal microglia or adult microglia treated with peptidase inhibitors into the spinal cord lesions of adult mice improved wound healing and axon regeneration [24]. Consistent with previous studies, our results also confirmed that CX3CR1⁺ microglia surround Mac2⁺ macrophages at 14 days after SCI. Moreover, when microglia are eliminated, the compact form of astrocytic scars is destroyed, and macrophages are scattered, indicating that microglial scarring plays an important role in maintaining astrocytic scarring and limiting inflammation. However, the mechanism by which microglia accumulate to form protective scars remains unclear.

Fascin-1 is a cytoskeleton-organizing protein localized at the core actin bundles within microvillar projections and filopodial extensions of migrating cells [25, 26]. Fascin-1 is expressed in neurons, dendritic cells and myofibroblasts [9, 27]. Fascin-1 can promote structural changes in cell membranes and affect the integrity of intercellular interactions to promote the invasion and metastasis of tumor cells [28, 29]. Compared with the vector control, overexpression of Fascin-1 in colonic epithelial cells increases their motility on two-dimensional laminin surfaces and enhances their migration through extracellular matrix (ECM)-coated filters [30]. However, tumor metastasis of lung adenocarcinoma cells is blocked when Fascin-1 is knocked out with short hairpin RNA (shRNA), indicating that Fascin-1 plays a mechanical role in driving tumor cell migration and invasion [31]. These studies show that Fascin-1 may be essential for cell migration. In the CNS, Fascin-1 plays an indispensable role in the development and polarization of filopodia (early neuritis) and growth cones, which can guide neurite outgrowth and branching [32]. Fascin-1 can also bind to MHC-II and B7-2 to play a role in the antigen presentation of dendritic cells [27]. B Wang et al. studied the role of microglia in neuropathic pain and found that microglia upregulate the expression of Fascin-1 in the posterior horn of the L4-L6 spinal cord, which then participates in the process of antigen presentation and the regulation of the secretion of the inflammatory factors TNF-α and IL-6 [13]. Considering that microglia are professional antigen-presenting cells in the CNS, we hypothesized that Fascin-1 may also regulate the migration and functional activity of microglia after SCI. Our data show that Fascin-1 is highly expressed specifically in microglia after SCI but not in neurons, astrocytes, NG2⁺ cells, pericytes, or blood-derived macrophages. Hence, Fascin-1 is expected to become a specific marker of microglia during CNS injury. Fascin-1 also plays an important role in regulating the migration of microglia, as demonstrated by gain- and loss-of-function studies. Thus, Fascin-1 may be a key regulatory protein for the formation of microglial scars, which can envelop inflammatory macrophages and promote the repair of SCI.

CX3CR1 is a receptor of fractalkine and directly mediates the adhesion and migration of leukocytes and microglia [33, 34]. Wang X et al. labeled infiltrating macrophages in chimeric mice and showed that after SCI, infiltrating macrophages expressing higher Mac2 levels accumulated at the epicenter and microglia expressing higher CX3CR1 levels were distributed at the edges of the lesion [6]. In CX3CR1^−/− mice with demyelinating disease, the clearance of myelin debris by microglia was substantially inhibited, affecting the integrity of the axon and myelin sheaths and thus preventing remyelination [35]. Moreover, we found that Fascin-1 is expressed specifically in CX3CR1⁺ microglia after SCI, and the expression of Fascin-1 decreases accordingly with the depletion of CX3CR1⁺ microglia. We hypothesize that CX3CR1 may be the upstream regulator of Fascin-1 and mediate the changes in its expression during SCI.

Microglia play an important role in inflammation and nerve remodeling [36]. It has been shown that microglia can be polarized into M1-neurotoxic or M2-neuroprotective states and produce a variety of cytokines involved in the regulation of the SCI microenvironment [37, 38]. However, in this study, we found that the polarization of microglia does not affect the expression of Fascin-1.

In summary, this study found that Fascin-1 is highly expressed specifically in microglia after SCI and can regulate microglial migration. The migration and accumulation of microglia at the lesion border after SCI may be closely related to the specific upregulation and cellular localization of Fascin-1. Hence, the elucidation of this mechanism will provide new insights into novel therapeutic targets for the treatment of SCI.

SCI: Spinal cord injury; CSF1R: Colony-stimulating factor 1 receptor; siRNA: Small interfering RNA; DMEM: Dulbecco's modified Eagle's medium; FBS: Fetal bovine serum; NC: Nonspecific control; PDL: Poly-d-lysine; LPS: Lipopolysaccharide; PBS: Phosphate-buffered saline; PFA: Paraformaldehyde; DSA: Donkey serum albumin; DAPI: 4′,6-diamidino-2-phenylindole; ANOVA: Analysis of variance; RIPA: Radioimmunoprecipitation; TBST: Tris-buffered saline with 0.5% Tween-20; HRP: Horseradish peroxidase; CNS: Central nervous system ; BBB: Blood–brain barrier; ECM: Extracellular matrix.

Ethics approval and consent to participate

Ethics approval was obtained by the Ethics Committee of Anhui Medical University (No. LLSC20160052).

Consent for publication

Not applicable.

Availability of data and materials

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

Competing interests

The authors declare that there is no conflict of interest regarding the publication of this article.

Authors’ contributions

SY and JJ conceived and designed the project. SY, ZL, FY, and YL performed research. SY, LC, YCL, and ZZ analyzed data, and prepared figures. SY and MZ wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 81801220 and 81671204) and Key Research and Development Projects of Anhui Province (No. 202004j07020042).

Acknowledgments

We would like to thank the Research Center of the Second Affiliated Hospital of Anhui Medical University for providing technical assistance.

Authors' information

Department of Orthopaedics, The Second Hospital of Anhui Medical University, No. 678 Furong Road, Hefei 230601, Anhui, China

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Fascin-1 Is Highly Expressed Specifically in Microglia After Spinal Cord Injury and Regulates Microglial Migration

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Animals

2.2 SCI model

2.3 Microglia depletion.

2.4 Preparation of myelin debris

2.5 Cell culture and transfection.

2.6 Microglial polarization

2.7 Tissue processing

2.8 Immunofluorescence analysis

2.9 Imaging analysis and quantification

2.10 Western blot analysis

3. Results

3.1 Fascin-1 is significantly upregulated and distributed at the edge of the lesion core after SCI

3.2 Fascin-1 is specifically expressed in microglia after SCI

3.3 Depletion of microglia correspondingly reduces Fascin-1 expression after SCI

3.4 High expression of Fascin-1 can promote microglial migration

3.5 Polarization of microglia does not affect the expression of Fascin-1

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

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