Transcranial direct current stimulation-mediated miR-298-5p downregulation enhances autophagy by targeting LC3 to promote motor recovery after spinal cord injury

doi:10.21203/rs.3.rs-4355457/v1

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Transcranial direct current stimulation-mediated miR-298-5p downregulation enhances autophagy by targeting LC3 to promote motor recovery after spinal cord injury

https://doi.org/10.21203/rs.3.rs-4355457/v1

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While transcranial direct current stimulation (tDCS) has been shown to contribute to motor recovery after spinal cord injury (SCI), the underlying mechanisms behind this process remain unclear. In the present study, we sought to explore whether tDCS can inhibit apoptosis, activate autophagy, and promote functional recovery. To achieve this aim, SCI was induced in rats using a modified Allen’s method and managed with tDCS. MicroRNAs responding to tDCS administration were detected using microRNA sequencing and validated using a quantitative real-time polymerase chain reaction. Dual-luciferase reporter analysis and miRNA overexpression were applied to verify the possible mechanisms of tDCS regulation. Stimulation of PC12 cells with hydrogen peroxide (H2O2) to simulate SCI models in vitro allowed for the detection of the effect of miR-298-5p on neuronal apoptosis and autophagy induced by SCI. The findings revealed that miR-298-5p was upregulated after SCI and decreased after tDCS. In vitro, miR-298-5p silencing was found to promote autophagy and reduce apoptosis in SCI, whereas miR-298-5p overexpression was associated with enhanced SCI-induced neuronal injury. LC3 was demonstrated to be the functional target of miR-298-5p, and tDCS was found to enhance autophagy flux, reduce neuronal apoptosis, improve nerve fiber regeneration, and minimize motor deficits after SCI in vivo. However, all tDCS-induced effects were counteracted after overexpression of miR-298-5p by agomir. In conclusion, this study shows that while miR-298-5p could be detrimental to SCI, tDCS can increase autophagy flux and inhibit neuronal apoptosis by negatively regulating miR-98-5p, thereby improving the recovery of motor function in SCI.

Spinal cord injury

transcranial direct current stimulation

motor cortex

autophagy

apoptosis

neuroprotection

motor function recovery

Spinal cord injury (SCI) is a serious and complicated neurological disorder that can result in the dysfunction or loss of multiple functions below the injured spinal cord segments[1, 2]. Generally, people with SCI consider motor function to be the highest priority in terms of recovery[3, 4]. Indeed, such loss of motor control is largely attributable to the interruption of descending control signals from the motor cortex (M1) of the brain past the lesion site[5]. Restoring this control requires reconstruction of the fibrous connections between the spinal motor neurons below the injury and the specific neurons of M1, which remains a major surgical challenge[6, 7].

Most SCIs are incomplete, thereby allowing for the opportunity to foster the reconnection of the brain with the spinal cord below the lesion by sprouting residual nerve fibers from spared and/or inured axon populations[5, 8]. However, to date, only a few satisfactory therapies for neural repair have been established[9, 10]. This is likely due to the multidimensional pathophysiological changes that develop in injured regions[10–12]. The pathophysiology of SCI involves primary and secondary injury mechanisms[13]. Specifically, mechanical events can lead to compression and tearing of the spinal cord, which would be referred to as the primary injury. The primary injury is followed by a secondary injury cascade, which consists of the activation of neuronal apoptosis and the inhibition of autophagy flux, which ultimately lead to massive tissue destruction[14, 15]. Autophagy is a cellular response that maintains homeostasis in the cell structure and function and exhibits close interactions with apoptosis[16]. However, autophagy is often inhibited after SCI,[17] which can further aggravate the damage and lead to continued functional deficits[6]. Generally, secondary injury leads to cystic cavitation in the lesion, which makes the local microenvironment unfavorable for the regeneration of nerve fibers[6, 18]. Because secondary injury involves a relatively long and reversible process, it is often an available target for therapeutic mediation[17, 19].

MicroRNA (miRNA) is a type of endogenous, noncoding, small-molecule RNA that can participate in the post-transcriptional regulation of the gene expressions of various biological functions[20, 21]. Extensive work has been done to study changes in gene expression by screening miRNAs, of which a small proportion may play a vital role in the modulation of the secondary injury cascade after SCI[22]. In one of our previous studies, we discovered that blocking the expression of miR-106-5p in a rat SCI model can promote motor recovery by modulating neuronal apoptosis and autophagic flux[15]. In a related vein, He et al. concluded that overexpression of miR-92a-3p could promote functional recovery by inhibiting apoptosis in SCI mice. Therefore, certain miRNAs may serve as promising therapeutic targets for SCI rehabilitation[23, 24].

Transcranial direct current stimulation (tDCS) is considered a promising non-invasive neuromodulation approach, and its effects on improving motor function in persons with SCI conditions have been universally supported[25, 26]. In particular, the latest research emphasizes that tDCS can induce plasticity among the residual neural circuits at the lesion site to facilitate motor output after an SCI; however, knowledge of the inner molecular mechanisms at play here has yet to be obtained[5, 27]. Nevertheless, despite these limitations, Sharif et al. found that neuromodulation delivered over M1 can drive corticospinal tract sprouting by reactivating the axon growth-promoting molecular pathways [6]. In another study, Zhang et al. demonstrated how the tDCS-induced effects of neuroplasticity were associated with the anti-apoptosis mechanism in stroke[28], and Guo et al. showed that tDCS may exert neuroprotective effects in cases of vascular dementia via the modulation of autophagy[29]. Against this background, this study advances the hypothesis that certain miRNAs might be involved in the tDCS-mediated repair of motor function after SCI by regulating apoptosis and autophagy. Specifically, we suggest that the associated in-depth molecular mechanisms investigated in this study may be involved in the tDCS-mediated repair of motor function after SCI by regulating apoptosis and autophagy.

Animals

All female adult Sprague-Dawley rats (eight weeks old, weighing 180–220 g) were purchased from the Animal Experimental Center (Guangxi Medical University, Nanning, China). All experimental procedures were approved by the Guangxi Medical University Ethics Committee (approval No. 202105013) in May 2021 and were performed in strict accordance with the National Institutes of Health guidelines for laboratory animal care and safety. Following SCI, many animals suffer from urination dysfunction. The urethra of female rats is wide and short, which is convenient for squeezing urine out of the bladder. Thus, female rats were chosen for this experiment. All rats were housed in an Specefic Pathogen Free (SPF) environment at 22–24℃ under a 12-hour light-dark cycle, with food and water consistently available. The rats were randomized into the following five groups: the sham group, the SCI group, the SCI + tDCS group, the SCI + tDCS + miR-negative control (NC) group, and the SCI + tDCS + miR-agomir group.

Establish SCI animal model

The SCI rat model was implemented on the basis of a modified version of Allen’s method. Briefly, pentobarbital (1%, 50 mg/kg) was used to anesthetize each rat, which was then immobilized in the prone position. Then, the rat underwent a laminectomy at the thoracic vertebra level to expose the T10 spinal segment. Afterward, a stereotaxic apparatus (Ruiwode Life Science Co., Ltd., Shenzhen, China) was used to secure the rat, and the dorsal surface of the spine was submitted to weight-drop injury using a 10-g impact rod with a diameter of 3 mm that was released from a 5-cm vertical height through a glass tube[15, 30, 31].The striking force on the dura mater and spinal cord measured 50 g (Fig. 1a and Fig. 1b). The rats with signs of hyperaemia and edema on the surface of the spinal cord surrounding T10, spasmodic tail-wagging reflection, retraction flutter of the body and lower limbs, and flaccid paralysis of both hind limbs represented a successful application of the model[10, 17, 32]. In the sham group, the rats underwent laminectomy without further spinal cord contusion. Finally, for all of the rats, the muscles and skin were disinfected and sutured separately. After the operation, each rat received an intraperitoneal injection of penicillin daily for three consecutive days. Their bladders were checked and manually massaged twice a day to help in urinating.

Motor function evaluation

According to the Basso–Beattie–Bresnahan (BBB) scale, the hind limb function of the rats was evaluated blindly before and one, three, five, and seven days after each operation in an open field by two trained independent observers. The evaluation time for each rat was 5 min, and the scores ranged from 0 to 21 points (0 points indicated complete paralysis, and 21 points meant normal locomotion)[33]. Rats with BBB scores higher than 3 on the first postoperative day were excluded due to modeling failure, and backup rats were then supplemented in the model.

The swim test is another scoring system for assessing functional recovery. One week before surgery, all the rats were trained to swim from one end of the glass-filled tank to the other for five consecutive days. The rats in each group were then scored by the Louisville Swimming Scale (LSS)[34], which evaluates forelimb dependence, hindlimb movement and alternation, trunk instability, and body angle. Mean scores were calculated when tested more than twice for each ra

tDCS-protocol

Twenty-four hours after the SCI, the rats underwent a tDCS procedure (1 m A, 20 min, 10 min per side) each day for seven days[35-37]. First, the fur around the bregma was shaved to ensure the tight attachment of the electrodes. Then, the anode and cathode electrodes—both pediatric electrocardiogram electrodes with conductive hydrogel—were trimmed to a contact area of 1.5 cm² for a better fit [38].The anode was then fixed to the skin with an adhesive tape over the M1 area (3 mm to the left of the bregma, and 2 mm in front of the interaural line) and then connected to a battery-driven stimulator. (ActivaTek lnc., Gilroy, CA, USA). The cathode electrode was placed over the supraorbital area (midpoint between the lateral angles of both eyes)[39]. The rats were restrained by a soft cloth during stimulation[40, 41](Fig. 2).

High-throughput miRNA sequencing

To assess possible changes of miRNA in response to tDCS after SCI, rats were anesthetized at seven days post-tDCS, and a 10-mm long spinal cord segment (at a distance of approximately 0.5 cm from the injury epicenter) was harvested. The total RNA was extracted using Trizol (Invitrogen, CA), and the concentration was quantified. Regarding microarray detection, hybridization and analysis were performed using mirDeep2 software based on the miRBase database. The miRNA sequencing was performed by Sangon Biotech (Shanghai, China). The threshold set for upregulated and downregulated genes was a p-value < 0.05 and a |log2 fold change (FC)| > 1.

Luciferase Reporter Assay

A bioinformatics analysis was then performed using TargetScan (http://www.targetscan.org/vert_72/) to identify the favorable binding site between miR-298-5p and LC3. A wild-type (WT) LC3 3’UTR fragment containing the putative miR-298-5p binding sequence, along with the mutant version (MU), was ordered from Sangon Biotech. (Shanghai, China). The above sequences were cloned into a psiCHECK-2 dual-luciferase vector (Promega, USA) to generate LC3-WT and LC3-MU recombinant vectors. PC-12 cells were plated in 96-well plates for co-transfection with LC3-WT/LC3-MU plasmids and rno-miR-298-5p mimic or NC. Quantification of luciferase activity after 48 hours of transfection was determined using a luciferase detection system (Madison, USA, USA) according to the manufacturer’s protocol.

Lentivirus (LV) Construction and Intrathecal Injections

To further explore the function of miR-298-5p, miR-298-5p lentiviral vectors were constructed using GeneChem Co. Ltd. (Shanghai, China). In the SCI + tDCS + miR-agomir group, the rats were intrathecally injected with LV-rno-miR-298-5p-agomir; in contrast, the rats in the SCI + tDCS + miR-NC group were intrathecally injected with LV-rno-miR-298-5p-NC. The original titers of the lentiviral vector were 6×10⁸ transduction units (TU) / mL. Before injection, lentiviral vectors (6×10⁸ TU/mL) were mixed with 0.01 M phosphate buffer solution (PBS; Servicebio, Wuhan, China) on ice to obtain a final lentiviral vector concentration of 4×10⁸ TU/mL. Subsequently, intrathecal injections of agomir or NC were performed daily for three consecutive days in these rats[42-44]. Experiments were performed one week later.

Haematoxylin & Eosin (H&E) and Nissl Staining

The 10-mm spinal cord segments centered at the injury epicenter were carefully harvested, fixed with 4% paraformaldehyde immersion for 24 hours, and then embedded with paraffin.A cross-section (5μm thick) was then placed on a glass slide coated with poly-L-lysine for analysis of the diseased tissue after deaffinity and rehydration. H&E staining was employed to examine the tissue histopathology. After being stained with toluidine blue (1%) for Nissl bodies, the spinal tissue sections were visualized under an optical microscope. (BX53, Olympus, Tokyo, Japan).

Immunofluorescence analysis

The paraffin-embedded sections were added and incubated with the primary antibodies for an entire night at 4°C. Then, dewaxing was performed to ensure transparency, after which hydrogen peroxide (H₂O₂) was applied to repair the antigen. Finally, the sections were incubated with a secondary antibody at 37°C for 30 min, incubated with diaminobenzidine (DAB) for 10 min, counterstained with hematoxylin for 3 min, and attached to coverslips. The results were visualized and photographed under an optical microscope. (BX53, Olympus, Tokyo, Japan).

Transmission electron microscope (TEM)

The tissue segments were then dissected and fixed in glutaraldehyde (2.5%) in sodium phosphate buffer (0.1 M) for 24 hours. After rinsing with PBS, the tissues were post-fixed with osmium tetroxide (1%) in a sodium phosphate buffer. The specimens of spinal tissues were then dehydrated with gradient ethyl alcohol solutions and embedded in epoxy resin. Ultrathin sections were prepared and stained with 2% uranyl acetate and 2.6% lead citrate, which were later visualized using a transmission electron microscope (7800; Hitachi, Tokyo, Japan).

Cell culture and cell model establishment

PC-12 cell lines are often selected as research tools for in vitro SCI studies.[93,59] In line with this approach, PC-12 cells purchased from the Chinese Academy of Sciences (Shanghai, China) were cultured at 37°C with 5% CO₂ in Dulbecco’s modified Eagle’s medium (Gibco, CA, USA) supplemented with 8% fetal bovine serum (Gibco) and 1% penicillin-streptomycin solution mixture (Gibco) .The PC-12 cells were randomly divided into the following six groups: a control group, H₂O₂ group, H₂O₂ + miR-mimics group,H₂O₂ + miR-mimics-NC group, H₂O₂ + miR-inhibitors group, and H₂O₂ + miR-inhibitors-NC group. An application of H₂O₂ (100 µM for six hours) was used to mimic neuronal injury, according to the results of the half-maximal inhibitory concentration (IC₅₀) values (additional files 1). The PC-12 cells were then transiently transfected with Lipofectamine™ 6000 (Invitrogen, USA), as directed by the manufacturer. The miR-298-5p mimics and inhibitors, along with their corresponding NC mimics and NC inhibitors, were purchased from Jikai Co. (Shanghai, China).

Cell Viability Assay

Cell viability was detected using a Cell Counting Kit-8 assay (CCK-8; Meilun, China). Cells were seeded at a density of 1.5×10⁴ cells/well on the 96-well plate, and then 10 µL of CCK-8 reagent in 90 µl of the medium was added to each well. After 30 min, cell viability was evaluated under a microplate reader (Synergy H1, BioTek, USA) at a wavelength of 450 nm.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Staining

A TUNEL kit (Meilun, China) was used to determine the apoptosis of the neurons. The cells were first immobilized using 4% paraformaldehyde. The previously generated tissue sections and cells were then dipped into a TUNEL reaction mixture (50 μL) at 37°C for one hour; after this, the nuclei were stained for 3 min with 4',6-diamidino-2-phenylindole (DAPI) solution. The fluorescence density was observed under a fluorescence microscope (EVOS™ FL Auto 2, Thermo Fisher).

Immunohistochemical

The cell slides and tissue sections were permeabilized with 0.2% Triton X-100 (Solarbio, China) and blocked with 5% goat serum (Gibco) for 30 min at room temperature. Afterward, these samples were incubated at 4°C overnight with primary antibodies against Bcl2-associated X protein (Bax; 1:100, mouse), p62 (1:100, rabbit), and anti-light chain 3 (LC3; 1:100, rabbit). Tissue sections were also stained with neurofilament protein-200 (NF-200; 1:200, rabbit) and cluster of differentiation 31 (CD31;1:200, mouse). These primary antibodies were all obtained from Proteintech (Wuhan, China) . After washing with PBS, the samples were subsequently stained for two hours with the appropriate secondary antibody (1:400, goat anti-rabbit conjugated to Alexa Fluor 488; 1:200 rat anti-rabbit conjugated to Alexa Fluor 549; UElandy Suzhou, China) at room temperature. Finally, the samples were counterstained using DAPI (1 μg/mL) and covered with coverslips. Images were captured via fluorescence microscopy (BX51, Olympus, Tokyo, Japan).

Western blot analysis (WB)

The total protein in each of the samples was extracted using RIPA buffer (89900, Thermo Fisher, USA). Proteins per sample were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After that, the separated samples were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The membranes were subsequently blocked by 5% skimmed milk in Tris-buffered saline and 1% Tween 20 (TBST, Thermo Fisher, USA) at room temperature for one hour and incubated at 4°C overnight with the primary antibodies, which included rabbit β-Actin, rabbit LC3, rabbit beclin1, rabbit p62, mouse Bax, and rabbit Bcl2 (all diluted 1:2000, Proteintech, China); with rabbit β-Actin serving as an internal control. After washing with TBST, the membranes were incubated in the secondary antibody (goat anti-rabbit IgG, 1:15000, Proteintech, China) for another hour at room temperature. The LiCor Odyssey Scanner was used for blot scanning, and the Odyssey 3.0 software package (LiCor, USA) was used for the analysis.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA in the samples was extracted with the NucleoZol RNA reagent (Macherey–Nagel, Düren, Germany) and then reversely transcribed into cDNAs through a PrimeScript RT reagent kit with a gDNA eraser (code no. RR047A, Takara, Japan) or PrimeScript RT Master Mix (code no. RR036Q/A/B, Takara, Japan). TB GreenprexExTaqII (code number. RR820Q/A/B, Takara, Japan) and the Applied Biosystems 7500 real-time PCR instrument (Applied Biosystems, USA) were used to evaluate the PCR reactions. U6/β-actin was employed as the internal control for miRNA and mRNA, respectively. The relative gene expression levels were calculated by the 2^-ΔΔCt methods. The primer sequences are listed in Table1.

Table 1. Primer sequence for quantitative real-time polymerase chain reaction

	Forward (5’-3’)	Reverse (3’-5’)
rno-miR-298-5p	GGAGGGCTGTTCTTCCCAA	mRQ 3’primer (Takara, Japan)
Bcl-2	GACTGAGTACCTGAACCGGCATC	CTGAGCAGCGTCTTCAGAGACA
Bax	TGGCGATGAACTGGACAACAA	GGGAGTCTGTATCCACATCAGCA
LC3	AGCTCTGAAGGCAACAGCAACA	GCTCCATGCAGGTAGCAGGAA
p62	TGAAGGCTATTACAGCCAGAGTCAA	CCTTCAGTGATGGCCTGGTC
U6	GGAACGATACAGAGAAGATTAGC	TGGAACGCTTCACGAATTTGCG
β-actin	TGTCACCAACTGGGACGATA	GGGGTGTTGAAGGTCTCAAA

Statistical Analysis

The statistical tests of all the data were completed by SPSS software (version 25.0, USA) and GraphPad Prism software (version 8.0, USA), Data are presented as mean ± standard deviation from at least three independent experiments in this study. The measurement data were first analysed for normal distribution. One-way ANOVA was performed, followed by Tukey's post-hoc test for Comparisons among multiple groups. Comparisons between the two groups were conducted using Student’s t-tests. For comparisons of motor scores, repeated-measures analysis of variance (ANOVA) was performed. Statistical significance was denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

tDCS altered miR-298-5p expression flowing injured area of SCI rats

A total of 175 miRNAs were found with |log2 fold change (FC)| > 1 and p < 0.05 between SCI group and SCI + tDCS group (Fig. 3a). Among these miRNAs, miR-298-5p was the most significantly suppressed in the tDCS group compared to the SCI group. To validate the outcome of the sequencing, qRT-PCR was applied. The relative expression of miR-298-5p was significantly upregulated at one, five, seven, and 14 days post-operation in the SCI group compared to the sham group (p <0.05, Fig. 3b). However, the expression was downregulated after tDCS in the SCI + tDCS group compared with the SCI group (p <0.001; Fig. 3c) In conclusion, the expression of miR-298-5p was upregulated after SCI and decreased after tDCS treatment in vivo.

LC3 is a direct target of miR-298-5p

In the present study, we predicted the target gene of miR-298-5p via bioinformatics. Two potential binding sites were identified between LC3 and miR-298-5p by TargetScan (Fig. 4a ). Based on the binding sequences, LC3-WT and LC3-MUT were constructed. The luciferase activity markedly decreased in cells co-transfected with miR-298-5p-mimics and LC3-WT (p < 0.001), , whereas those co-transfected with miR-298-5p-mimics and LC3-MUT did not show a significant change (p > 0.05, Fig. 4b ). Both of these results strongly support the idea that miR-298-5p interacts with LC3 directly. Meanwhile, the WB results showed that LC3 expression was negatively regulated by miR-298-5p, which is consistent with the qRT-PCR results (p <0.05, Fig. 4c and Fig. 4 d and Fig. 4 e).

Mir-298-5p affects the SCI-induced autophagy and apoptosis in vitro

Transfect efficiency was assessed by qRT-PCR. As displayed in Fig. 5a, miR-298-5p expression was significantly increased after injury in the H₂O₂ group compared with the control group (p < 0.05). At that point, miR-298-5p mimics/NC mimics and miR-298-5p inhibitors/NC inhibitors were transfected into the H₂O₂-induced PC-12 cells.

The findings indicated that the viability of the PC-12 cells was reduced after injury (p < 0.001). Following the transfection of miR-298-5p mimics, cell viability had further declined (p < 0.001). However, cell viability was significantly enhanced following transfection with miR-298-5p inhibitors ( (p < 0.001, Fig. 5b. The TUNEL assay revealed that cell apoptosis was enhanced in the H₂O₂ group compared with the control group. The ratio of apoptotic cells was further increased after transfection with the miR-298-5p mimics (p < 0.001), while it was significantly inhibited by miR-298-5p inhibitors (p < 0.001, Fig. 5 c and Fig. 5 d).

The results of the immunofluorescence staining showed that H₂O₂ can activate autophagy and induce apoptosis, which can be manifested as the increased fluorescence intensity of Bax, p62, and LC3. The positive signals of Bax and p62 were markedly enhanced, while LC3 was decreased in the H₂O₂ + miR-mimics group compared with theH₂O₂ group; the opposite results were observed in the H₂O₂+ miR-inhibitors group compared to the H₂O₂ group (Fig. 6a and Fig. 6b). Subsequently, the WB assay showed that the expression of the Bcl-2 protein decreased after H₂O₂ injury. While the expression was further attenuated after miR-298-5p mimic-transfection (p < 0.05), it increased by miR-298-5p inhibitor-transfection(p < 0.01, Fig. 4c and Fig. 6c).

tDCS attenuates tissue damage, ameliorates neural functional status and motor function of SCI rats via downregulating miR-298-5p

To further explore the function of miR-298-5p in the neuroprotection of tDCS for SCI, miR-298-5p-agomir or miR-298-5p-NC was administered. The expression level of miR-298-5p in the injured spinal segment was significantly decreased after tDCS in the SCI + tDCS group compared to the SCI group (p < 0.001). However, miR-298-5p-agomir actually reversed the change in the SCI + tDCS + miR-agomir group (p < 0.001; Fig. 3c).

The functional recovery of the rats after the different treatments was comprehensively evaluated based on the BBB and LSS scales. The preoperative BBB scores were similar among the groups(p>0.05). At one day post-operation, the scores of the sham group were higher than those of the other groups (P<0.01). Starting at five days and ranging up to seven days post-operation, the BBB scores in the SCI + tDCS group were significantly higher than those in the SCI group. However, the effects of tDCS treatment were reversed by miR-298-5p overexpression (all p < 0.01, Fig. 7a and Table 2). The LSS could thus intuitively reflect the differences in motor function among the various groups, and the scores displayed a similar trend as the BBB (Fig. 7b and Fig. 7c,and Table 3 ).

The results also indicated that the structure of the gray-white matter was intact and clear in the sham group. The images show a lesion cavity in the center of the injury, with clear signs of tissue swelling in the SCI group. After the tDCS procedure, a clearer structure and less tissue loss could be observed in the SCI + tDCS group. These results emphasize the role of tDCS in the repair of SCI in rats. However, miR-298-5p overexpression counteracted the tDCS-induced effects, leading to significant necrotic cavities and spinal cord edema in the SCI + tDCS + miR-agomir group and a significant reduction in the number of neuronal cells compared with the +miR-NC group (Fig.7d).

In the sham group, the image of Nissl staining showed that the neuronal structures were complete and clear, the nucleus was obvious, and a large number of Nissl bodies were evenly distributed in the cytoplasm. In the SCI group, neurons were swollen and morphologically unclear, and Nissl bodies were reduced. The cell morphology improved, and edema was reduced in response to treatment with tDCS, while the number of Nissl bodies significantly increased in the SCI + tDCS group. However, the image displayed shrunken neuronal cell bodies and Nissl granule dissolution after the overexpression of miR-298-5p in the SCI + tDCS + miR-agomir group compared with the SCI + tDCS + miR-NC group (Fig. 7e). Overall, these results reveal that tDCS promotes autophagy and suppresses SCI-induced apoptosis by downregulating miR-298-5p.

Table 2： BBB scores at various times pre- and postsurgery

BBB score Group	Before surgery	After surgery
BBB score Group	Before surgery	0 Day	Third day	Fifth day	Fifth day
sham	21.00±0.00	21.00±0.00	21.00±0.00	21.00±0.00	21.00±0.00
SCI	21.00±0.00	0.00±0.00†**	1.06±0.40	1.72±0.45†**	2.39±0.49†**
SCI+tDCS	21.00±0.00	0.00±0.00†**	2.78±0.63	3.39±0.49‡**	5.17±0.50‡**
SCI+miR298-5p NC+tDCS	21.00±0.00	0.00±0.00†**	2.67±0.47	3.22±0.42§**	5.11±0.57§**
SCI+ago-miR298-5p+tDCS	21.00±0.00	0.00±0.00†**	1.50±0.50	1.56±0.50	2.11±0.46

Values are presented as mean± standard deviation of 20 rats per group per time point, where lower BBB scores indicate poorer locomotor function. BBB, Basso-Beattie-Bresnahan locomotor scale; SCI, spinal cord injury; tDCS:Transcranial direct current stimulation; NC, negative control. A one-way analysis of variance followed by Tukey’s post-hoc test was performed. †, as

Table 3： LSS scores at various times pre- and postsurgery

LSS score Group	Before surgery	After surgery
LSS score Group	Before surgery	0 Day	Third day	Fifth day	Fifth day
sham	17.00±0.00	17.00±0.00	17.00±0.00	17.00±0.00	17.00±0.00
SCI	17.00±0.00	0.45±0.51†**	1.95±0.69	2.65±0.49†**	3.15±0.67†**
SCI+tDCS	17.00±0.00	0.75±0.44†**	2.45±0.60	4.45±0.51‡*	6.70±0.73‡*
SCI+miR298-5p NC+tDCS	17.00±0.00	0.65±0.59†**	2.45±0.51	4.20±0.70§*	6.35±0.93§*
SCI+ago-miR298-5p+tDCS	17.00±0.00	0.50±0.51†**	1.9±0.40	2.40±0.50	3.00±0.65

Values are presented as mean± standard deviation of 20 rats per group per time point, LSS:Louisville Swim Scale. SCI, spinal cord injury; tDCS:Transcranial direct current stimulation; NC, negative control. A one-way analysis of variance followed by Tukey’s post-hoc test was performed. †, as compared with sham group; ‡, as compared with SCI group;§, as compared with SCI+tDCS+miR-298-5p agomir group.* P < 0.05, ** P < 0.01, *** P < 0.001.

tDCS promotes autophagy and suppresses SCI-induced apoptosis via downregulating miR-298-5p

TEM was used to examine the ultrastructural characteristics (×3,000 magnification) of the injured segment. In the sham group, abnormal structures were not identified in either the nucleus or the organelles. Moreover, no autolysosomes or autophagosomes were observed (Fig. 8a). In contrast, intracellular edema, an irregular nucleus, and an increased number of lysosomes (yellow arrow) and autolysosomes (green arrow) were found in the cells in the SCI group (Fig. 8 b). The SCI + tDCS group showed an improved state and a creased number of autolysosomes (green arrow) and autophagosomes (red arrow) compared with the SCI group (Fig. 8c). In addition, more autophagosomes (red arrows) and autolysosomes (green arrows) were observed in the SCI + tDCS + miR-NC group compared to the SCI group (Fig. 8 d). Finally, in the SCI + tDCS + miR-agomir group, the cells were swollen, and some lysosomes (yellow arrows) could be detected (Fig. 8e). These results clarify that tDCS can heighten the autophagy flux after SCI, while miR-298-5p overexpression can neutralize these effects.

In comparison with the sham group, the number of TUNEL-positive cells markedly increased in the SCI group. In the SCI + tDCS group, the proportion of TUNEL-positive cells was obviously decreased in response to the tDCS procedure (p < 0.001). However, miR-298-5p-agomir was able to reverse the change in tDCS, such that the proportion of TUNEL-positive cells in the SCI + tDCS + miR-agomir group was higher than those in the SCI + tDCS + miR-NC group (p < 0.001，Fig. 9a and Fig. 9b).

The expression of the autophagy-related proteins LC3, p62, and Beclin-1, as well as the apoptotic-related proteins Bax and Bcl-2, in the spinal tissues was detected via WB (Fig. 10a and Fig. 10b） and immunohistochemical staining (Fig. 10c and Fig. 10d）. The contents of LC3, p62, and Bax increased, while those of Bcl-2 decreased following SCI. However, tDCS decreased the level of p62 and Bax, as well as increased the expression of Bcl-2 and LC3, compared to the SCI group (all p <0.05). It was also found that miR-298-5p-agomir can reverse the tDCS-induced changes. Additionally, qRT-PCR of LC3, Beclin-1, p62, Bax, and Bcl-2 mRNA further confirmed the above findings (Fig. 10e）. These observations suggest that tDCS can promote autophagy and inhibit apoptosis after SCI by downregulating miR-298-5p.

tDCS facilitates neurogenesis post-SCI via downregulating miR-298-5p

Double immunofluorescent staining for NF-200 (axon marker) and CD31 (vascular marker) was used to investigate nerve fiber regeneration in each group (Fig. 11). The results confirmed that the fluorescence intensity of NF-200 and CD31 in the SCI group significantly decreased compared with the corresponding values in the sham group. In contrast, the NF-200- and CD31-positive signals were significantly inducted after tDCS therapy. Subsequently, these tDCS-induced effects were reversed by miR-298-5p overexpression.

Observation of microstructure and autophagosome changes of each group by transmission electron microscopy.a In the sham group, the nucleus was intact with obvious nucleoli. The myelin sheath was arranged around the axon in concentric circles. (B) In the SCI group, the irregular myelin sheath, pyknotic nuclei, and swelling mitochondrial with several autolysosomes are shown. (C&D) In the SCI+tDCS group and SCI+tDCS+miR-NC group, the morphology of the nuclei was nearly normal, the mitochondria exhibited swelling to a lesser extent, and the number of autophagosomes obviously increased. (E) In the SCI+tDCS+miR-agomir group, neurons showed swelling mitochondrial with vacuolization, karyopyknotic, and the number of autophagosomes has obviously decreased. The microstructure of each group was observed with scale bar = 5 μM, and the autophagosomes were observed with scale bar = 2 μM. The blue asterisk identifies the nuclei; the green points to autophagosomes.

SCI is a serious neurological disease that can cause extensive damage to the structure of the spinal cord, resulting in motor dysfunction[2, 45]. Massive neuronal death and atrophy after SCI are notable pathophysiologies and mechanisms of motor function defects[46]. Increased apoptosis and decreased autophagy can cause secondary injury and negatively impact neuronal survival following SCI[47]. Therefore, the improvement of the neuronal microenvironment in the injured section by regulation of both autophagy and apoptosis is crucial for encouraging the regeneration of nerve fibers[46]. Our data demonstrated that tDCS therapy can promote neuronal survival, neural regeneration, and functional recovery via the molecular mechanism of activating autophagy flux and attenuating cell apoptosis after SCI.

Previously, some studies had suggested that SCI may perturb miRNA homeostasis[11]. Depending on their downstream target genes, the dysregulation of these miRNAs might result in either alleviated or aggravated post-SCI conditions[23, 48]. In the present study, we detected high expression levels of miR-298-5p after SCI. The function of miR-298-5p in apoptosis has been extensively investigated elsewhere. For example, Wallach et al. demonstrated that miR-298-5p can be released from apoptotic cortical neurons, thereby triggering further neuronal apoptosis in the central nervous system[49]. In another study, Wei et al. found that miR-298-5p targets Srpk1 to change cell viability and apoptosis as mediated by sevoflurane[50]. However, little is known about the regulatory role of miR-298-5p in SCI repair. Here, we validated that miR-298-5p acts as a harmful factor after SCI, wherein neural and autophagy flux was enhanced and apoptosis was inhibited after downregulating miR-298-5p through the tDCS procedure, which indicates that tDCS has a regulatory effect by inhibiting the expression of miR-298-5p. An in vitro model of H₂O₂-induced neurotoxicity in PC12 cells was used to further confirm the involvement of miR-298-5p in autophagy and apoptosis.

The crosstalk between apoptosis and autophagy has been observed elsewhere[39, 51]. Several studies have indicated that the appropriate activation of autophagy can protect neurons in the spinal cord from undergoing apoptosis, while the dysfunction of autophagy could further lead to neuronal cell injury[16, 47, 52]. In this study, we confirmed that miR-298-5p can directly target LC3 via the dual-luciferase reporter system. LC3 is correlated with the control of autophagosome elongation and is a reliable indicator for monitoring autophagy induction in mammals[39, 53]. Moreover, p62, an autophagy cargo protein, can interact directly with LC3 to bring damaged mitochondria and other autophagosomes into the autolysosomes for degradation through specific autophagy-lysosome pathways. Thus, the accumulation of p62 indicates a blockage in autophagy[54, 55]. Our data indicated that tDCS conditions markedly enhanced LC3 levels and reduced p62 and Beclin-1 expression in SCI rats, which was accompanied by the accumulation of autolysosome and autophagosome observed by TEM, thereby indicating that functional autophagic flux in the SCI + tDCS group was significantly heightened by tDCS. Also, the inhibitory effect of tDCS on neuronal apoptosis, which was accompanied by the increase of autophagy flux, was also observed in the present study as a gradual decrease in the apoptotic protein Bax and Bcl-2 levels and the proportion of TUNEL-positive cells. These findings suggest that tDCS has the therapeutic effect of strengthening autophagic flux and inhibiting SCI-induced apoptosis.

SCI leads to the interruption of neural connectivity, resulting in serious neurological dysfunction. Functional restoration relies on the formation of new fibrous connections and neural circuits[56]. The remodeling of functional neural circuits between the spinal cord and brain may need to be driven by rehabilitation[57]. One study has suggested that tDCS, a non-invasive neuromodulation technique targeting the promotion of neuronal plasticity, seems to be an effective approach[9]. Previous studies have found that suitable conditions for neuronal survival and plasticity can be induced by enhancing autophagy flux and inhibiting neuronal apoptosis after SCI[10, 58]. The intriguing finding related to Nissl staining in our study demonstrated that neuronal function and survival were significantly increased after tDCS treatment. We also observed that the fluorescence intensity of NF-200 in the SCI + tDCS group was significantly higher than that in the SCI group. NF-200 is an axon-specific intermediate filament protein that is used as a “growth” or “plasticity” marker in the regeneration of nerve fibers[18, 46, 59]. The higher expression of NF-200 in the SCI + tDCS group hinted that tDCS treatment could promote the regeneration of nerve fibers in spinal tissue after SCI. The results of TEM and H&E staining further demonstrated that the local microenvironment in the injury segment and the ultrastructure of neurons were markedly modified after tDCS therapy. These effects coincided with the trend of restored motor capacity in the SCI rats evaluated by BBB and LSS scores, which therefore explained the significant functional recovery after tDCS treatment.

After SCI, neural repair and plasticity require the formation of blood vessels to supply sufficient oxygen and nutrients to the injured area[60]. The blood vessel–specific marker CD31 reflects vascular structure and function.[43] In this study, immunofluorescence staining showed that the level of CD31 protein decreased in the spinal tissues after SCI and increased after tDCS. These findings illustrate that tDCS promotes angiogenesis. Moreover, double staining with NF-200 and CD31 in the lesions of the rats suggested that nerve fiber regeneration was associated with the formation of vessels. To better illustrate the potential role of miR-298-5p in the treatment of tDCS after SCI, we next attempted to treat rats with tDCS in combination with intrathecal miR-298-5p agomir. However, the protective effects of tDCS could be counteracted by the overexpression of miR-298-5p. Nevertheless, these observations show that tDCS exerted its effects against SCI by inhibiting miR-298-5p expression.

The present study has several limitations. First, we did not provide evidence of the best time points or treatment parameters for the repair of SCI by tDCS. Second, we did not detect changes in neurotrophic factor content or neurite extension in the in-vivo experiments. Third, the present study was carried out only with adult female rats. Thus, it is not clear whether the use of rats of different ages and sexes may affect the experimental data. Furthermore, we chose PC-12 cells to simulate an SCI model in vitro, which would be more convincing if primary spinal cord neuronal cells were selected to further corroborate our conclusions.

Our results provide new insights for explaining the therapeutic mechanism of tDCS for SCI-related motor dysfunction. Through the regulation of autophagy and apoptosis by the downregulation of miR-298-5p, tDCS might promote neural repair and enhance motor functional recovery after SCI. Therefore, the results of the present study provide experimental basic evidence for the application of tDCS in clinical treatments for SCI.

Acknowledgements

TThe authors would like to express their appreciation to Shu-Hui Guo and Chen-Xi Liang for their support on the experiment.

Funding

This study was supported by the National Natural Science Foundation of China (No. 81960417), Guangxi Key Research and Development Program (No. GuiKeAB20159027), and Guangxi Natural Science Foundation (No. 2018GXNSFAA050033).n of China (Grant number. 82172531).

Author Contribution

JWX, YJS and YL designed the study; JWX provided funding support; QHP, XLL and YT conducted the in vitro experiment; YY, FCL，KWW and YCG performed the in vivo experiment; QHP and WFZ analysed data; JMC drafted and wrote the manuscript. All authors approved the final version of the paper.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics Approval and Consent to Participate All animal care and experimental procedures in this study were approved by the Guangxi Medical University Animal Research Ethics Committee ( No. 202105013)

Consent for Publication All authors agree to the publication of this manuscript

Competing Interests The authors declare no competing interests.

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No competing interests reported.

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Transcranial direct current stimulation-mediated miR-298-5p downregulation enhances autophagy by targeting LC3 to promote motor recovery after spinal cord injury

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