RNAi‐mediated ephrin‐B2 silencing attenuates astroglial‐fibrotic scar formation and improves spinal cord axon growth

Summary

Aims

Astroglial‐fibrotic scar formation following central nervous system injury can help repair blood‐brain barrier and seal the lesion, whereas it also represents a strong barrier for axonal regeneration. Intensive preclinical efforts have been made to eliminate/reduce the inhibitory part and, in the meantime, preserve the beneficial role of astroglial‐fibrotic scar.

Methods

In this study, we established an in vitro system, in which coculture of astrocytes and meningeal fibroblasts was treated with exogenous transforming growth factor‐β1 (TGF‐β1) to form astroglial‐fibrotic scar‐like cell clusters, and thereby evaluated the efficacy of RNAi targeting ephrin‐B2 in preventing scar formation from the very beginning. We further tested the effect of RNAi‐based mitigation of astroglial‐fibrotic scar on spinal axon outgrowth on a custom‐made microfluidic platform.

Results

We found that siRNA targeting ephrin‐B2 significantly reduced both the number and the diameter of cell clusters induced by TGF‐β1 and diminished the expression of aggrecan and versican in the coculture, and allowed for significantly longer extension of outgrowing spinal cord axons into astroglial‐fibrotic scar as assessed on the microfluidic platform.

Conclusions

These results suggest that astroglial‐fibrotic scar formation and particularly the expression of aggrecan and versican could be mitigated by ephrin‐B2 specific siRNA, thus improving the microenvironment for spinal axon regeneration.

1. INTRODUCTION

Currently available therapies for central nervous system (CNS) injury, including medication, surgery, hyperbaric oxygen, and cell transplantation, often yield unsatisfactory outcomes. One cardinal inhibition of CNS axon regeneration is the unfavorable microenvironment of astroglial‐fibrotic scar which is rapidly formed after injury.1 Multiple cell types, mainly including astrocytes and meningeal fibroblasts (MFb),2, 3, 4 are involved in the formation of astroglial‐fibrotic scar, a physical‐chemical barrier that hinders the outgrowth of axons across the injury site and thus inhibits CNS regeneration and repair.5, 6 Despite a lot of attempts and endeavors made preclinically to eliminate astroglial‐fibrotic scar, the therapeutic benefits have been limited so far, probably because of a relatively late intervention as majority of the treatment was focused on reducing the astroglial‐fibrotic scar that has been formed.3, 7, 8 In this regard, it is possible to achieve better therapeutic effect if the scar formation is ameliorated from the very beginning.

Recent studies have demonstrated that the contact‐dependent bidirectional ephrin B2‐EphB2 signaling of astrocyte‐MFb interaction is an early event in the cellular cascades that lead to the formation of astroglial‐fibrotic scar.4 Early after spinal cord injury (SCI), the expression of the ligand ephrin‐B2 was up‐regulated in astrocytes, and the receptor EphB2 was also up‐regulated on the cytoplasmic membrane in MFb. The contact of migrating astrocytes and MFb enables the binding of ephrin‐B2 to EphB2, which in turn triggers bidirectional signaling cascade in both astrocytes and MFb, resulting in the formation of the scar.3, 4 In addition, depletion of ephrin‐B2 ligand in reactive astrocytes via conditional knockout significantly improves axonal regeneration after SCI in mice.9 Therefore, the up‐regulated expression of ephrin‐B2 and EphB2 seems to be one of the detrimental biological events early after SCI.3 Based on this, we hypothesized that astroglial‐fibrotic scar formation and related inhibitory barrier can be mitigated by down‐regulation of ephrin‐B2 or EphB2 in the early phase after SCI.

Previously, RNA interference (RNAi) can exert an anticancer effect by inhibiting the expression of EphB2 or ephrin‐B2 in cancer cells,10 and injection of specific siRNA can reduce the expression of ephrin‐B2 and relieve neuropathic pain.11 Therefore, the formation of astroglial‐fibrotic scar and the expression of inhibitory substances, such as chondroitin sulfate proteoglycans (CSPGs), can be inhibited, at least in part, by knocking down the expression of ephrin‐B2 or EphB2 via RNAi; on the other hand, the axonal growth‐supporting role12 and injury‐restricting and blood‐brain barrier repairing action13 of astrocytes can be maintained.

The mechanisms of astroglial‐fibrotic scar formation are complex and yet to be completely understood; however, astrocytes and MFb are the two main cellular components of the scar and are the major source of extracellular molecules that buildup the chemical barrier of the scar.14 Studies have demonstrated that coculture of astrocytes and MFb with addition of transforming growth factor‐β1 (TGF‐β1) could mimic astroglial‐fibrotic scar formation15 and release multiple inhibitory substances into the culture medium which inhibit neuronal growth.15, 16 However, most neuronal perikarya of damaged axons are away from the lesion epicenter in SCI and it is therefore the severed axonal compartment that directly contacts with the inhibitory microenvironment but not the somatic one. This unique pattern of exposure to inhibitory cues makes it difficult to mimic in a conventional cell culture environment until the introduction of microfluidic platform‐Campenot chambers.17 In this system, neuronal somata are cultured in a chamber connected to microchannels, through which axons grow and enter an adjacent chamber, and somata and axons are spatially partitioned as a result. In addition, the microfluidic force between adjacent chambers with different fluid levels allows for only unidirectional flow of the liquid within microchannels which connect the chambers, thus the chemicals within the downstream chamber will not contaminate the upstream one,18, 19 making it possible to exclusively expose the axonal compartment to chemicals of interest. As this microfluidic platform can better mimic the living microenvironment of cells in the body, it has become an effective tool for neurocytobiological research.20, 21

In this study, we established an in vitro astroglial‐fibrotic scar model, and partially mitigated the scar formation and secretion of inhibitory CSPGs using RNAi to specifically knock down the expression of ephrin‐B2. We further utilized a custom‐made microfluidic platform to evaluate the effect of the RNAi‐induced mitigation of astroglial‐fibrotic scar on the outgrowth of spinal motor neuron axons.

2. MATERIALS AND METHODS

2.1. Animals

One‐day‐old Sprague‐Dawley (SD) rats, of special pathogen‐free grade, were from Experimental Animal Center at Nantong University (Nantong, China). All surgical interventions and postoperative animal care were performed in accordance with the Institutional Animal Care guidelines and approved ethically by the Administration Committee of Jiangsu Province, China.

2.2. In vitro astroglial‐fibrotic scar model and RNAi

The ephrin‐B2 specific siRNA was synthesized by Nantong Biomics Biotechnologies Co., Ltd (Nantong, China), using the following sequences: positive‐sense strand GCUAGAAGCUGGUACGAAUdtdt and antisense strand AUUCGUACCAGCUUCUAGCdtdt. RNAi screening of the interference sequences for the most specific and effective one for ephrin‐B2 silencing (Table S1, Fig. S1).

The meninges and spinal cord of newborn rats were dissected routinely for cell culture, from which MFb and astrocytes of about 95% purity were obtained, respectively.

Purified passage 2‐3 astrocytes (1 mL) and MFb (1 mL) were seeded on plates precoated with poly‐l‐lysine (PLL, Sigma, St. Louis, MO, USA) at a density of 0.82 × 10⁵/mL and 0.63 × 10⁵/mL, respectively, or 300 μL from each to a cover glass precoated with PLL, and cultured at 37°C and 5% CO₂. Cells were then cocultured for 2 days, added with TGF‐β1, at a final concentration of 10 ng/mL (R&D, St. Minnesota, USA), to establish an in vitro astroglial‐fibrotic scar model, and finally observed at 4 h and 2, 4, 7, and 14 days after seeding.

The experiment was conducted in four groups: (i) coculture of astrocytes and MFb; (ii) astrocytes/MFb coculture in the presence of exogenous TGF‐β1; (iii) astrocytes/MFb coculture with siRNA treatment; and (iv) astrocytes/MFb coculture with treatment of both TGF‐β1 and siRNA. For TGF‐β1 and/or siRNA treatment, astrocytes and MFb were mixed and cocultured in proportion. When cells grew to 50% confluence, 10 ng/mL TGF‐β1 and/or 30 nmol L⁻¹ siRNA and SuperFectinTMII reagent (in vitro siRNA Transfection Reagent, Pufei Co., Ltd., Shanghai, China) were added respectively to corresponding groups. Four days after TGF‐β1 treatment with or without siRNA, cells were counted and the diameter of cell clusters larger than 40 μm was measured under the low‐power microscopic field. In addition, cell samples that had been treated for 48 h were collected from all groups, from which mRNA and protein were extracted routinely and the cell clusters were subjected to immunofluorescence staining.

2.3. Ventral spinal cord 4.1 (VSC4.1) motoneuron culture

VSC4.1 motoneurons line, generated by fusion of embryonic rat ventral spinal cord neuron with the mouse N18TG2 neuroblastoma cell,22, 23 was a gift from Dr. Weidong Le from Baylor College of Medicine (Houston, USA). VSC4.1 cells in DMEM/F12 containing 5% fetal bovine serum (FBS) were seeded to PLL‐precoated culture flasks for amplification. Before cell differentiation, the medium was replaced with DMEM/F12 containing 0.5% FBS only (Fig. S2).

2.4. Observation of the effect of ephrin‐B2 silencing on the formation of astroglial‐fibrotic scar and axonal growth on the microfluidic platform

We designed a novel three‐chamber microfluidic platform, which includes one axon/scar chamber (15 mm × 10 mm × 5 mm) in the middle and two soma chambers (15 mm × 5 mm × 5 mm) on the two wings. Between the soma and axon/scar chambers were about two hundred microchannels (250 μm × 15 μm × 5 μm) to guide axonal growth (Figure 6A,B). The microfluidic platform was manufactured by Suzhou Wenhao Chip Technology Co., Ltd (Suzhou, China) using the photolithography method.24 A schematic representation of axonal growth on the microfluidic platform was shown in Figure 6C.

Figure 6.

Motor axon outgrown from VSC4.1 culture toward the axon/scar chamber on the microfluidic platform. (A) Diagram showing the design of the microfluidic platform. (B) Photograph of a real fabrication. (C) Schemes showing motor axon growth toward the axon/scar chamber. In the absence of TGF‐β1, astrocytes/MFb coculture allows ingrowth of motor axons into the axon/scar chamber; in the presence of TGF‐β1, the coculture forms cell clusters, which resemble astrocyte/fibrotic scar, and inhibits the ingrowth of motor axons. (D‐I) Photomicrographs showing axonal growth in the microchannels. The axons of VSC4.1 motor neurons in the soma chamber can grow into the microchannels, via which they enter the axon/scar chamber when there was no cell seeding, nor addition of growth‐inhibiting chemicals (D, D′). The growth of motoneuron axons was slowed down as they approached the axon/scar chamber, and retraction bulb structures appeared in axonal terminals, when CSPGs was present in the axon/scar chamber (E, E′), or when the astrocytes/MFb coculture in the axon/scar chamber was treated with TGF‐β1 (H, H′). When the axon/scar chamber was added with astrocytes/MFb coculture (F), or astrocytes/MFb coculture with siRNA (G), VSC4.1 motoneurons extended fine and long axons from the soma chamber and entered the microchannels; after 4‐5 days, they passed through the whole length of the microchannels and entered the axon/scar chamber. When the astrocytes/MFb coculture in the axon/scar chamber with both TGF‐β1 and siRNA (I, I′), the length of VSC4.1 axons was overtly increased as compared to coculture treated with TGF‐β1 alone; however, smaller retraction bulbs were still observed in axonal terminals. (J) Bar chart showing the difference in axonal length 7 days after culturing on the microfluidic platform. **P < 0.01 compared to no cell seeding nor growth‐inhibiting chemicals within the axon/scar chamber; ^▲▲ P < 0.01 compared to CSPGs treatment only; ^## P < 0.01 compared to astrocytes/MFb coculture only; ^★ P < 0.05 compared to TGF‐β1‐treated astrocytes/MFb coculture. Arrows indicate axonal terminals or retraction bulbs of axons. Bar = 50 μm (D‐I), 25 μm (D′, E′, H′, I′)

The microfluidic platform chambers were coated with PLL overnight, rinsed with sterile ultrapure water, and irradiated with ultraviolet rays for 0.5 h. VSC4.1 cells were then added to the soma chamber on both wings of the microfluidic platform at a density of 800 cells/mm², and 200 μL DMEM/F12 solution containing 5% FBS, which was replaced with DMEM/F12 containing 0.5% FBS on the second day of cell culture. On the second day following seeding of VSC4.1 cells, 5 μg/mL CSPGs (Merck‐Millipore, Schwalbach, Germany), or the coculture of astrocytes and MFb were added to the axon/scar chamber in the middle of the platform. On the third day, the coculture of astrocytes and MFb in the axon/scar chamber was treated with 10 ng/mL TGF‐β1 and/or 30 nmol L⁻¹ siRNA, depending on the designated treatment for the specific group. After 1‐week culture, the axon length was measured using DP2‐BSW software (Olympus Corp., Hachioji, Tokyo, Japan).

2.5. Immunocytochemistry

For immunofluorescence staining, cell specimens were fixed in paraformaldehyde, blocked with normal goat serum and incubated with primary antibodies overnight at 4°C. Then, secondary antibodies were added at room temperature for 2 h. The primary antibodies and secondary antibodies are shown in Table 1. Finally, the slides were mounted with mounting medium containing DAPI (Vector laboratories, Burlingame, CA, USA) and observed under a laser scanning confocal microscope (SP8, Leica, Wetzlar and Mannheim, Germany).

Table 1.

Primary and secondary antibodies used in this study

Antibody	Dilution	Manufacturer
Rabbit anti EphB2	1:200	Thermo Scientific, MA, USA
Rabbit anti ephrin‐B2	1:200	Thermo Scientific
Mouse anti GFAP	1:500	Millipore
Rabbit anti GFAP	1:600	Abcam, Cambridge, UK
Rabbit anti FN	1:200	Abcam
Mouse anti FN	1:200	Abcam
Rabbit anti Tuj1	1:2000	COVANCE, Princeton, NJ, USA
Cy3‐conjugated goat‐anti‐mouse IgG	1:800	Jackson, West Grove, PA, USA
Cy3‐conjugated goat‐anti‐rabbit IgG	1:800	Jackson
FITC‐affinipure goat‐anti‐mouse IgG	1:800	Jackson
FITC‐affinipure goat‐anti‐rabbit IgG	1:800	Jackson

2.6. ELISA

Supernatants were collected from coculture of astrocytes and MFb in the absence or presence of treatment with TGF‐β1, siRNA or the combination of both as stated above at 4 days of coculturing, and subjected to ELISA for quantitate secreted aggrecan and versican, according to the instruction of respective ELISA kit (Suzhou Shengkang Bio‐Technology Co., Ltd., Suzhou, China). Optical density (OD) of each well was measured at 450 nm on a microplate reader (BioTeK, Winooski, VT, USA).

2.7. qPCR

During astroglial‐fibrotic scar formation, the expression of ephrin‐B2 and EphB2 mRNA and the experiment of RNAi with ephrin‐B2 expression were detected by qPCR at different time points after addition of TGF‐β1 to the coculture using primers synthesized (Table S2) by Invitrogen (Invitrogen Corporation, USA). Total RNA was extracted by QIAcube Automated RNA Extractor (QIAGEN, Chatsworth, CA, USA) and reverse transcribed (Thermo, MA, USA) for real‐time PCR. The result was finally analyzed using GAPDH as an internal reference and the well without addition of the template as the negative control.

2.8. Western blot

Cells were harvested in RIPA lysis buffer (50 mmol L⁻¹ Tris, 150 mmol L⁻¹ NaCl, 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS) with 1 mmol L⁻¹ phenylmethylsulfonyl fluoride (PMSF), vortexed, chilled on ice for 30 min, and centrifuged at 10 000×g for 10 min. The supernatant was saved and subjected to bicinchoninic acid (BCA) assay for protein concentration. Proteins were separated by 10% SDS‐PAGE, electrotransferred to poly‐vinylidene fluoride (PVDF) membrane, and probed with antibodies against EphB2 (Thermo Fisher Scientific, 1:800), ephrinB2 (Thermo Fisher Scientific, 1:500), or GAPDH (Sigma, 1:5000). After incubation with the primary antibodies, the membranes were incubated appropriate secondary antibodies and development with enhanced chemiluminescence (ECL) Western detection kit. The blots were subjected to densitometry analysis using Image lab 5.1 software (Bio‐Rad Laboratories Inc., Hercules, CA, USA).

2.9. Statistical analysis

Data were analyzed and plotted using GraphPad Prism 5 software package (Graphpad Software, San Diego, CA, USA). All data are expressed as mean ± standard deviation (SD). Experimental data were first subjected to test of homogeneity of variance. Data of qPCR and the length of the axon were analyzed by one‐way ANOVA. Some of the ELISA data, the diameter, and number of the cell clusters were analyzed by Student's unpaired t test. P < 0.05 was considered statistically significant.

3. RESULTS

3.1. Morphological change in cell clusters after RNAi

Astrocytes and MFb were cocultured for 2‐4 days, and both showed relatively uniform growth and distribution. Some of these cells began interweaving with each other; however, no cell aggregation into clusters was observed in this condition (Fig. S3A,B). Two days after TGF‐β1 was added, cell migration and aggregation, which formed small cell clusters, were observed. At day 4, most cells aggregated and formed even larger cell clusters of 40‐300 μm in diameter (Fig. S3C, D). When screened for efficacy, siRNA‐2 showed the strongest interfering effect, down‐regulating ephrin‐B2 mRNA level by 86% (Fig. S1), and was thus used for subsequent RNAi experiments. The coculture treated with TGF‐β1 and siRNA‐2 showed not only significantly reduced number but also decreased diameter of cell clusters as compared to that treated with TGF‐β1 alone (P < 0.01) (Figure 1, Fig. S4).

Figure 1.

The number (A) and diameter (B) of cell clusters formed in coculture of astrocytes and MFb in the presence of TGF‐β1 and/or siRNA. Four groups of astrocytes/MFb cocultures with varying treatment were included (i) treated with TGF‐β1 alone (TGF‐β1), (ii) treated with both TGF‐β1 and siRNA (TGF‐β1+si), (iii) treated with siRNA alone and (iv) without treatment. No cell cluster larger than 40 μm in diameter was observed in the coculture without treatment, nor in that treated with siRNA alone, and thus no quantitative data were included for these two groups. **P < 0.01 compared to TGF‐β1 group

Immunostaining of cocultures showed that the astrocytes and MFb expressed GFAP and fibronectin (FN), respectively. The two cell types were interwoven with each other at the interface without overt cell aggregation. At this stage, the fluorescence of ephrin‐B2 in astrocytes and that of EphB2 in MFb were very weak, suggesting that the two cell types expressed at very low level ephrin‐B2 and EphB2, respectively (Figure 2A‐C). Four days after addition of TGF‐β1, the cell clusters were observed to comprise both astrocytes and MFb, where the expression of ephrin‐B2 and EphB2 was significantly higher than the cocultures in the absence of TGF‐β1 (Figure 2D‐F). In addition, the expression of ephrin‐B2 was dominantly located on astrocytes and EphB2 mainly on MFb (Figure 2E,F). Although the cell clusters were still composed of interwoven astrocytes and MFb after RNAi treatment, they were significantly smaller than those treated with TGF‐β1 only; in addition, the intensity of ephrin‐B2 and EphB2 immunofluorescence staining was lower in the presence than in the absence of RNAi (Figure 2G‐I).

Figure 2.

Immunofluorescence cytochemistry staining of cocultured astrocytes/MFb or cell clusters. (i) After purified astrocytes (GFAP as a marker, green) and MFb (FN as a marker, red) were seeded separately on a single cover glass in a well, the two types of cells grew interweaving into each other at the interface without forming cell clusters (A). Astrocytes and MFb were seen to weakly express ephrin‐B2 (B, ephrin‐B2 in red, GFAP in green) and EphB2 (C, EphB2 in green, FN in red), respectively. (ii) Dense cell clusters were formed by both GFAP‐positive astrocytes and FN‐positive MFb after supplementation of TGF‐β1 (D). The clusters of cells expressed overtly higher level of ephrin‐B2 (E) and EphB2 (F), in astrocytes and MFb, respectively, compared to the coculture in the absence of exogenous TGF‐β1. (iii) After cotreatment with siRNA and TGF‐β1, the cell clusters were still formed by interwoven aggregation of astrocytes and MFb (G), but the size of these cluster‐like structures became smaller as compared to TGF‐β1 treatment alone; the fluorescence intensity of ephrin‐B2 (H) and EphB2 (I) was apparently reduced compared to TGF‐β1 treatment alone. Bar = 100 μm

3.2. Ephrin‐B2 and EphB2 expression in cell clusters treated with TGF‐β1 and RNAi targeting ephrin‐B2

Changes in the expression of ephrin‐B2 and EphB2 mRNA at different time points after addition of TGF‐β1 to the coculture were detected by qPCR. The results showed that the expression of ephrin‐B2 mRNA after addition of TGF‐β1 increased with time (P < 0.05 or 0.01) except for that at 4 h, when the expression of ephrin‐B2 mRNA showed no significant change as compared with the cocultures in the absence of TGF‐β1(P > 0.05). The expression of ephrin‐B2 mRNA reached the peak at day 14, when it was significantly different from that at day 7 (P < 0.05) (Figure 3A). The expression of EphB2 mRNA was significantly up‐regulated at all time points (P < 0.05 or 0.01) except at 4 h, when the expression of EphB2 mRNA in the presence of TGF‐β1 did not show significant increase as compared to coculture alone (P > 0.05). However, the expression of EphB2 mRNA slightly decreased at day 14, though it was not significantly different from that at day 7 (P > 0.05) (Figure 3B).

Figure 3.

qPCR and Western blots showing expression of ephrin‐B2 and EphB2 across time following coculturing astrocytes and MFb in the presence of TGF‐β1. (A, B) Bar charts showing quantitation ephrin‐B2 and Eph B2 mRNA. (C) Representative blots of ephrin‐B2 and Eph B2 protein. (D, E) Bar charts showing quantitation ephrin‐B2 and Eph B2 protein. From day 2 and onwards, TGF‐β1 treated cocultures showed significant increase in the expression of both ephrin‐B2 and EphB2, and these proteins further increased at 7‐14 days following treatment. *P < 0.05, **P < 0.01 compared coculture without TGF‐β1 treatment; ^▲ P < 0.05, ^▲▲ P < 0.01 compared to day 7; ^ΔΔ P < 0.01 compared to day 2. *P < 0.05, **P < 0.01 compared coculture without TGF‐β1 treatment

Changes in protein expression of ephrin‐B2 and EphB2 at different time points after addition of TGF‐β1 to the coculture were detected by Western blots. The results showed that ephrin‐B2 expression increased from day 2 and onwards after addition of TGF‐β1 (P < 0.01) and further increased at day 14 (Figure 3C,D). The expression of EphB2 was significantly up‐regulated at all time points (P < 0.05 or 0.01). However, the expression of EphB2 slightly decreased at day 14, though it was not significantly different from that at day 7 (P > 0.05) (Figure 3C,E).

The expression level of ephrin‐B2 mRNA in astrocyte/MFb cocultures was significantly reduced with combined treatment of TGF‐β1 and siRNA as compared to TGF‐β1 treatment only or no treatment groups (P < 0.01); the TGF‐β1 treated group showed significantly higher level of ephrin‐B2 mRNA than the cocultures without treatment (P < 0.01) (Figure 4A). Similarly, astrocyte/MFb cocultures showed significantly decreased expression level of EphB2 mRNA when treated with both TGF‐β1 and siRNA as compared to that treated with TGF‐β1 alone (P < 0.01), a group in which significantly increased level of EphB2 mRNA was detected when compared to the cocultures without treatment (P < 0.05) (Figure 4B).

Figure 4.

qPCR showing level of ephrin‐B2 and EphB2 mRNA after coculturing astrocytes and MFb with addition of TGF‐β1 and/or siRNA targeting ephrin‐B2. While TGF‐β1 treatment increased the mRNA level of ephrin‐B2 (A) and EphB2 (B) by over 6‐ and 4‐folds in the coculture, respectively, combined treatment with siRNA targeting ephrin‐B2 prevented the TGF‐β1‐induced increase in ephrin‐B2 and EphB2. However, siRNA targeting ephrin‐B2 reduced the mRNA level of ephrin‐B2 but not EphB2. **P < 0.01 compared to coculture without treatment; ^## P < 0.01 compared to TGF‐β1 treatment alone

3.3. Effect of RNAi on the expression of aggrecan and versican in cell clusters

ELISA results showed that combined treatment of TGF‐β1 and siRNA significantly decreased the protein expression level of both aggrecan and versican in cocultures than TGF‐β1 treatment only (P < 0.05), despite that TGF‐β1 treatment significantly increased the expression of aggrecan as compared to coculture without treatment (P < 0.01). No statistically significant change was observed between any two of other three groups (P > 0.05) (Figure 5).

Figure 5.

ELISA showing excretion of aggrecan (A) and versican (B) by cocultured astrocytes and MFb after simultaneous addition of TGF‐β1 and/or siRNA. **P < 0.01 compared to coculture without treatment; ^# P < 0.05 compared to TGF‐β1 treatment alone

3.4. Axonal growth in astroglial‐fibrotic scar with RNAi on the microfluidic platform

We employed the three‐chamber microfluidic platform to evaluate the effect of RNAi‐based down‐regulation of ephrin‐B2 expression on the permeability for axonal ingrowth into astroglial‐fibrotic scar (Figure 6A,B). In the microfluidic platform, the axons of VSC4.1 spinal motoneurons can grow from the soma chambers on two wings into the central axonal/scar chamber where astroglial‐fibrotic scar was mimicked by coculturing of astrocytes and MFb in the presence of exogenous TGF‐β1 (Figure 6C). Motor axons were observed to grow for 4‐5 days along the microchannels and entered the astrocytes/MFb coculture in the axon/scar chamber without TGF‐β1 treatment (Figure 6F). Although motor axons could also grow into the microchannels toward the axon/scar chamber of astrocytes/MFb coculture in the presence of exogenous TGF‐β1, or as a positive control, CSPGs, axonal growth appeared to be inhibited when they approached the axon/scar chamber, evidenced by retraction bulbs at the axonal terminals (Figure 6E,H). Intriguingly, when treated astrocytes/MFb coculture without both TGF‐β1 and siRNA, or with siRNA alone, motor axons were able to extend axons farther and entered the axon/scar chamber (Figure 6G). Compared to that treated with TGF‐β1 alone, the coculture treated with both TGF‐β1 and siRNA significantly increased the length of axonal growth; however, small bulb‐like structures were also seen at axonal terminals (Figure 6I).

Statistical analysis showed that compared to the group in which the axon/scar chamber was neither seeded with cells nor challenged with axonal growth‐inhibiting chemicals, the length of motor axon outgrowth in the microchannels was significantly reduced in groups in which the axon/scar chamber was added with CSPGs, or coculture of astrocytes/MFb in the presence of TGF‐β1, or astrocytes/MFb coculture with both TGF‐β1 and siRNA (P < 0.01). The length of motor axon outgrowth was significantly longer when the axon/scar chamber was added with astrocytes/MFb coculture only, or the coculture in the presence of siRNA treatment only, as compared to the group in which axon/scar chamber was supplemented with CSPGs only (P < 0.05 or 0.01). Although TGF‐β1 treatment of the coculture significantly reduced motor axon outgrowth regardless of RNAi when compared to astrocytes/MFb coculture without treatment (P < 0.01), down‐regulation of ephrin‐B2 with siRNA in TGF‐β1‐treated astrocytes/MFb coculture showed significantly longer axonal outgrowth as compared to that treated with TGF‐β1 alone (P < 0.05) (Figure 6J).

4. DISCUSSION

The interaction of Ephs with their ligands Ephrins has recently been proposed to play a pivotal role in initiating the formation of glial‐fibrotic scar after SCI and contribute to the failure of axonal regeneration after SCI.3 Growing evidences showed that ephrins play a number of different roles and influence the CNS injury response at multiple levels; for instance, accumulation of EphA4 in the proximal axon stumps of the lacerated corticospinal tract prevent axonal regeneration through interaction with up‐regulated ephrin B2, one of the ligands for EphA4, in astrocytes in the glial scar.25 After SCI, astrocytes expressing ephrin‐B2 have been observed to encapsulate the lesion cavity filled with EphB2‐bearing fibroblasts and form a characteristic glial scar around the lesion site; these two cell types contact with each other during scar formation, and bidirectional signaling between astrocytes expressing ephrin‐B2 and fibroblasts expressing EphB2 has been proposed to initiate intracellular signaling cascades that are essential for regulating glial‐fibrotic scar formation after SCI.26

After SCI, the blood‐brain barrier is damaged, which induces a series of inflammatory responses and generates multiple inflammatory cytokines that in turn activate astrocytes27 and finally glial scar formation.28, 29 Furthermore, up‐regulation of TGF‐β1 at the injured site would promote MFb to proliferate, invade the injured tissue, and secrete extracellular matrix (ECM) to form fibrous scars.30, 31 Migration, arrangement, and demarcation of the two cell types are distinct, and they work together to form astroglial‐fibrotic scars.32 Consequently, we established the in vitro astroglial‐fibrotic scar model using TGF‐β1 in the present study, despite that other cell types especially inflammation‐related cells may also participate in the process.33, 34 Intercellular spaces in scars are filled with large amount of ECM, especially CSPGs which form a basal layer in the lateral aspect of astrocytes and inhibit axon regeneration.35 This process is caused by a series of pathological changes after up‐regulation of ephrin‐B2 and EphB2 following SCI,1 and thus it is also a potential target to inhibit astroglial‐fibrotic scar formation from the very beginning.

We, therefore, postulated whether it would be possible to prevent astroglial‐fibrotic scar formation by inhibiting the up‐regulation of ephrin‐B2 expression using the siRNA technique. A contemporary study by Ren et al 9 also showed that proliferation of astrocytes and glial scar formation was inhibited in ephrin‐B2 knockout mice, allowing for better regeneration of corticospinal tract axons. These results are in line with the present study, however, in their in vitro experiment, they only used ephrin‐B2^+/+ or ephrin‐B2^−/− astrocyte and ephrin‐B2^siRNA astrocytes to the coculture with neurons, indicated that ephrin‐B2 knockout or interference could promote axonal growth. However, most scars in CNS injuries are composed of both astrocytes and MFb but not astrocytes alone. Of note, a recent study12 showed that reactive astrocytes in glial scar are permissive and beneficial rather than inhibitory for axonal growth after SCI, and CSPGs are of multiple cell origins and ablation of glial scar formation does not reduce CSPGs production. Therefore, it is novel for the study described here to utilize astrocytes and MFb together to gain further insights into the effect of ephrin‐B2 specific RNAi on astroglial‐fibrotic scar formation and axonal growth.

We demonstrated that the addition of TGF‐β1 accelerated mutual aggregation of astrocytes and MFb, forming cell clusters, an in vitro astroglial‐fibrotic scar model, which is consistent with previous studies.15, 30, 36 After TGF‐β1 treatment, ephrin‐B2 and EphB2 were also up‐regulated on astrocytes and MFb, respectively, and significant up‐regulation of aggrecan and versican, the main components of CPSGs, was detected in the cell clusters. The up‐regulations of ephrin‐B2 and EphB2 suggested that their interaction was involved during the cell clusters formation from the very beginning. Thus, we used the siRNA technique to interfere with ephrin‐B2 mRNA expression and inhibit its translation for the sake of preventing astrocytes and MFb from aggregating and forming cell cluster after addition of TGF‐β1. Our data demonstrated that ephrin‐B2 silencing could decrease the number and diameter of the cell clusters as compared with TGF‐β1 group, validating the concept we proposed as stated above.

The histological feature of astroglial‐fibrotic scars is a mechanical barrier that is difficult for growing axons to pass through. More importantly, CSPGs in scar tissues may serve as a chemical barrier to induce collapse of axon growth cones, resulting in failure of axonal regeneration.37, 38 CSPGs, including aggrecan, versican, brevican, neurocan, phosphacan, NG2, and others,3, 39 show two distinct patterns of change in expression, either an initial decrease followed by increase or prolonged increase, after SCI. After SCI, neurocan, brevican, and versican immunolabeling increased within days in injured spinal cord parenchyma surrounding the lesion site and peaked at 2 weeks; neurocan and versican were persistently elevated for 4 weeks postinjury, and brevican expression persisted for at least 2 months.40 On the other hand, phosphacan immunolabeling decreased in the same region immediately following injury but later recovered and then peaked after 2 months40; SCI caused significant decrease in aggrecan, and partial recovery in some aggrecan species was evident by 2 weeks after injury.41 Given that aggrecan and versican, respectively, represent CSPGs of the distinct patterns of change following SCI,42, 43 we examine these two CSPGs in our cell culture model. We found that the expression of aggrecan and versican was increased significantly after addition of TGF‐β1. After reducing the expression of ephrin‐B2 by siRNA, both the number and the size of cell clusters were reduced, and the expression of aggrecan and versican was also decreased significantly, indicating that ephrin‐B2 specific RNAi not only reduced the expression level of the target gene but also reduced the scar‐like tissue formation and inhibited the secretion of CSPGs, such as aggrecan and versican.

To objectively evaluate the effect of ephrin‐B2 silencing on the formation of astroglial‐fibrotic scar and axonal growth on astroglial‐fibrotic scar, we designed a three‐chamber microfluidic platform. In the middle of the platform is an axon/scar chamber for astroglial‐fibrotic scar formation via coculture of astrocytes and MFb in the presence of TGF‐β1, and on both sides are two soma chambers for seeding neurons, with about two hundred microchannels for guiding axonal growth in between adjacent chambers. These microchannels, 5 μm in height, 15 μm in width, and 250 μm in length, can effectively prevent somata from leaving the chamber and differentiate between axons and dendrites. The results showed that fine and long axons were able to enter the axon/scar chamber along these microchannels. The coculture of nonactivated astrocytes and MFb in the axon/scar chamber did not exhibit an inhibitory effect on axonal growth. However, when cell clusters appeared after addition of TGF‐β1 to the cocultured cells, axonal growth was inhibited markedly, and at the same time, the growth cone shrank to form a bulb‐like structure. This phenomenon is similar to the situation after addition of CSPGs alone to the axon/scar chamber. When TGF‐β1 and siRNA were added simultaneously to the axon/scar chamber, axonal growth was improved remarkably, indicating that reducing the expression of ephrin‐B2 by RNAi could partially inhibit astroglial‐fibrotic scar formation and reduce the production of CSPGs, thus allowing for better axonal growth. We postulate that inhibitory cues in the axon/scar chamber can diffuse, to a certain distance, into the microchannels toward the soma chamber. This might contribute to the retraction bulbs observed in the case of TGF‐ß1‐treated cocultures in the axon/scar chamber. Actually, similar phenomenon was also observed by Hur and colleagues, who documented that “some axons that managed to exit the channels grew along the permissive–inhibitory border or turned back and reentered the microchannels to avoid CSPGs”.38

Although astroglial‐fibrotic scar formation has a significantly negative effect on nerve regeneration, it exhibits a positive effect on the repair of the damaged blood‐brain barrier and separation of damaged area from healthy tissue.13 In this regard, it is necessary to suitably control the degree of inhibition on astroglial‐fibrotic scar formation. We found that RNAi was able to reduce the expression of ephrin‐B2 properly and prevent astroglial‐fibrotic scar formation partially, and may thus represent a promising approach to fine‐tune the formation of astroglial‐fibrotic scar for maximizing the benefits and minimizing the harms, which deserves further investigation.

In conclusions, knocking down of ephrin‐B2 by siRNA could mitigate the TGF‐β1‐stimulated formation of astroglial‐fibrotic scar and production of CSPGs, and thereby allow more axons to grow across the scar.

CONFLICT OF INTEREST

We declare that we do not have any conflict of interest.

Supporting information

 

 

 

 

 

Li Y, Chen Y, Tan L, et al. RNAi‐mediated ephrin‐B2 silencing attenuates astroglial‐fibrotic scar formation and improves spinal cord axon growth. CNS Neurosci Ther. 2017;23:779–789. 10.1111/cns.12723