Transcranial Intermittent Theta-Burst Stimulation Reverses Neurodegeneration in the Somatosensory Motor Cortex after Spinal Cord Transection in Rats

Abstract

Background:

Complete spinal cord injury (SCI) leads to a disconnection between the brain and the body below the injury level, resulting in the functional silencing, degeneration, and apoptosis of sensorimotor cortex (SMC) neurons, which is of crucial importance to the pathological process.

Methods:

In this study, a rat model of spinal cord transection was employed to explore the activation of neurons in the SMC and the reversal of neurodegeneration after the rats were treated with transcranial intermittent theta-burst stimulation (T-iTBS).

Results:

The results demonstrated that the expression of the immediate early gene c-Fos and the synaptic plasticity-associated activity-regulated cytoskeleton (Arc) gene in the neurons of the SMC was increased in the T-iTBS group 4 weeks after SCI. Transcriptome sequencing revealed that neuronal activation-, neuronal metabolism-, synaptic activity-, and neural regeneration-related genes were significantly upregulated in the T-iTBS group compared with those of the sham-iTBS group, but the expression was similar to that in the normal group. Western blot analysis indicated that the expression of Cle-caspase-3 (CC3) in the SMC was significantly reduced in the T-iTBS group, and the number of CD68-positive cells in the SMC was close to that of normal rats but significantly less than that in the sham-iTBS and SCI groups. These results are in line with those of the transcriptome sequencing. Correlation analysis of the expression rate between c-Fos and Arc, CC3, and CD68 further suggested that T-iTBS improved the immune microenvironment and prevented neurodegeneration by regulating the activation and synaptic plasticity of SMC neurons in the early stages of injury.

Conclusions:

Collectively, our findings offer support for the utilization of T-iTBS, a non-invasive neural stimulation treatment, to prevent SMC degeneration following severe SCI.

Graphical abstract

Keywords

spinal cord transection / transcranial intermittent theta-burst stimulation / sensorimotor cortex / neurodegeneration / neuroplasticity

1. Introduction

Complete spinal cord injury (SCI) gives rise to a loss of neural connections between the sensory motor cortex (SMC) and the spinal cord below the level of injury [1, 2, 3]. This brings about neuronal functional silencing, apoptosis, and degeneration of the SMC following the progression of secondary injury and further gray matter atrophy that is observable on imaging [3, 4, 5]. Currently, SCI treatment pays more attention to the injury site rather than brain pathology. Nevertheless, it is of crucial significance for nerve regeneration and functional reconstruction to protect neurons in the SMC and prevent the advancement of neurodegeneration [3, 6, 7, 8].

Several studies have demonstrated that rehabilitation training and physical therapy based on neural stimulation after incomplete SCI have a positive effect on the recovery of sensory and motor function in paralyzed limbs [9, 10, 11]. However, transverse SCI induces a complete loss of neural connection between the SMC and the body below the level of injury. Pathological changes occur in SMC neurons in the early stages of injury, including histological, gene expression, and functional changes, and it is urgent to find therapeutic strategies that can reverse these adverse processes in time [3, 12]. The aim of this study was to explore the efficacy of a neural stimulation therapy, which is easy to apply clinically, in saving SMC neurons.

Repetitive transcranial magnetic stimulation (rTMS) can regulate the plasticity of neural networks by modifying the excitability of neurons at the stimulation site [13, 14, 15]. rTMS is extensively utilized as a clinical therapy for numerous central nervous system diseases, such as pain, depression, and multiple-system atrophy [16, 17, 18, 19]. In recent years, transcranial intermittent theta-burst stimulation (T-iTBS), which is a novel rTMS paradigm with advantage like noninvasiveness and a short treatment duration [20], has been increasingly used for neural rehabilitation [21, 22, 23, 24, 25]. T-iTBS has been certified to promote axonal regeneration and motor function repair at the site of SCI [3, 18, 26, 27, 28]. Nevertheless, the biological effects on the SMC in the early stages of SCI following the application of T-iTBS need to be further investigated.

The SMC initiates a series of nociceptive cascade reactions when it receives damage signals from a SCI [3, 29, 30]. The timely rescue of SMC degeneration is crucial for preserving the regeneration ability of corticospinal tracts and other brain-spinal tracts and restoring voluntary motor function [31, 32, 33]. Therefore, we applied T-iTBS to rats in the early stages of injury after transected SCI to rescue neurodegeneration by regulating the excitability of SMC neurons.

2. Materials and Methods

2.1 Animals

The experimental animals were adult female SD rats (weighing 180–220 g) procured from the Experimental Animal Center of Sun Yat-sen University.

2.2 SCI Surgery

A total of 55 SD rats were divided into four groups: SCI group (n = 15), sham-iTBS group (n = 15), T-iTBS group (n = 15), and normal group (n = 10). The animals were anesthetized with 1% sodium pentobarbital (40 mg/kg, intraperitoneally, No.P3761, Sigma-Aldrich Millipore, Burlington, MA, USA) and the T9-T10 vertebral level of laminae were exposed by cutting through the skin and muscles. The dura matter was cut in a longitudinal incision with microscopic scissors and the segment of spinal cord (2.0 mm) at T9 level was completely removed, including the associated spinal roots. The rats received extensive post-operation care including intramuscular injection of penicillin (80,000 units, No.1224, Shandong Shengwang Pharmaceutical Co., Ltd., Shandong, China) and analgesics (Meloxicam, 0.1 mg/kg, No.T0826, TargetMol Chemicals Inc., Boston, MA, USA) daily for one week, and they were artificially urinated until their autonomous urination function was restored. The SCI group, the sham-iTBS group and the T-iTBS group underwent complete transection of the spinal cord at the T9 level. Rats in the T-iTBS group were treated for four weeks after surgery, the sham-iTBS group received spurious stimulation after SCI for four weeks. The normal group received neither surgery nor stimulation. (Table 1 and Fig. 1E).

2.3 T-iTBS Therapy

The rats in the T-iTBS group received T-iTBS treatment 72 hours subsequent to surgery using a YRD CCY-I Magnetic Stimulator (Wuhan Yiruide Medical Equipment New Technology, Wuhan, Hubei, China), which was equipped with a special circular coil (r = 32 mm) and a pulsed magnetic field with a peak intensity of 3 Tesla [3]. During the procedure, the focal point of the magnetic field generated by the coil was positioned adjacent to the midsection of the skull. T-iTBS therapy was administered repeatedly from 3:00 to 5:00 PM five days a week, the output intensity was 0.675 Tesla, with a total of 600 pulses. It encompassed three-pulse bursts delivered at a frequency of 50 Hz, and the entire burst was reiterated at a rate of 5 Hz. In light of relevant literature, a 90% movement threshold (0.75 Tesla) was chosen as the output intensity of the stimulus [3, 15]. During the concurrent time period, the rats within the sham-iTBS group were exposed to the spurious stimuli. Since T-iTBS does not engender pain or any other discomfort, rats can undergo T-iTBS treatment while remaining awake, and there is no requisite for restraint.

2.4 Cortical Motor-Evoked Potential Detection

Cortical motor-evoked potentials (CMEPs) were assessed in five rats from each group using the TMS instrument (YRD CCY-I, YIRUIDE Medical Equipment, Wuhan, Hubei, China), four weeks post-injury. For the procedure, the rats were anesthetized with 1% sodium pentobarbital (30 mg/kg, intraperitoneally, No.P3761, Sigma-Aldrich Millipore, Burlington, MA, USA) and then positioned on a wooden board for stability. The electrophysiological setup involved placing the recording electrode in the anterior tibialis muscle of the rat’s paralyzed hind limb. Concurrently, the reference electrode was placed adjacent to the calcaneal process, while the ground electrode was inserted into the dorsal skin for effective signal grounding. The instrument stimulation output was set to 100% intensity. The TMS coil was aligned at the center of the rat’s skull to ensure precise stimulation. CMEPs were recorded once the rat had regained consciousness. For each rat, 10 separate CMEP records were acquired and expressed as average amplitude.

2.5 RNA Extraction and Sequencing

Three rats from each group were chosen for sampling and detection through RNA sequencing (RNA-seq) 4 weeks after SCI, and the targeted tissue was the SMC. For each rat, 200 mg of SMC tissue was employed for RNA extraction. Total RNA of different samples was lysed and extracted with TRIzol reagent (15596026, Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The qualities of the RNA samples were assessed through a NanoDrop spectrophotometer (ND-1000, Bio-Rad, Hercules, CA, USA), and the concentrations of RNA samples were examined with a Qubit fluorometer (QX200, Bio-Rad, Hercules, CA, USA) before RNA-seq analysis. RNA-seq was run at the laboratory of Genergy Bio-Technology Co., Ltd. (Shanghai, China). The sequencing data were assessed through Consensus Assessment of Sequence And Variation (CASAVA) base calling after data were generated using a high-throughput sequencer. The gene expression levels were calculated with the help of StringTie (StringTie 2.2.1, Johns Hopkins University, Baltimore, MD, USA), and DESeq2 (DESeq2 1.40.0, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA) was applied to analyze differential genes in pair-wise comparisons. A p value of 0.05 and fold change of >1.5 or 0.667 were set as the criteria for differentially expressed genes (DEGs).

2.6 Western Blotting

Three rats from each group were chosen for sampling and detection through Western blotting 4 weeks after SCI, and the targeted tissue was the SMC. For each rat, 500 mg of SMC tissue was utilized for protein extraction using RIPA lysis buffer (P0013, Beyotime, Shanghai, China). Equal amounts of proteins from each group were electrophoresed through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto 0.2-µm Polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and incubated with primary antibodies at 4 °C overnight. After that, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. The bands were detected by a chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA) with ECL solution (NO. BIO9934, Bioman Technology Co., Ltd, Beijing, China) and quantified by ImageJ (ImageJ For Mac 1.52, LOCI, University of Wisconsin, Madison, WI, USA). A summary of the antibodies used is provided in Supplementary Table 1.

2.7 Immunofluorescence Analyses

All rats intended for immunofluorescence analyses were deeply anaesthetized using 1% sodium pentobarbital (50 mg/kg, intraperitoneally, No.P3761, Sigma-Aldrich Millipore, Burlington, MA, USA), and then they were perfused systemically with 0.9% NaCl containing 0.002% NaNO₂ and 0.002% heparin (PHR8927, Merck, Rahway, NJ, USA), followed with 4% paraformaldehyde (PFA, C10814814; Macklin, Shanghai, China). After perfusion, samples of tissues were placed in 30% sucrose solution to dehydrate. Brain slices were cut at a 25-µm thickness with a cryotome, blocked in 10% goat serum (Biosharp, BL210A, Wuhan, Hubei, China) with 0.3% Triton X-100 (EZ7890C254, BioFroxx, Beijing, China) at room temperature for 2 h, and incubated in primary antibody at 4 °C overnight. Then, the samples were washed three times with PBS and incubated with secondary antibodies at 37 °C for 1 h in the dark. The images were captured by fluorescence microscope, and the number of positive cells was calculated by Image J ( ImageJ 1.53t, National Institutes of Health, Bethesda, MD, USA). A summary of the primary and secondary antibodies used is provided in Supplementary Table 1. Each group consists of five rats, with one rat having four slices chosen (with one taken from every five consecutive slices as a sample) for immunofluorescence analyses.

2.8 Statistical Analyses

All experimental data are presented as the mean \pm standard error of the mean from at least three samples. Pearson or Spearman rank correlation test was utilized to determine the association between two variables according to the distribution. Data were analyzed by GraphPad Prism 9.0 software (Dotmatics, Boston, MA, USA) and differences determined by one-way analysis of variance. If p 0.05, the value was considered statistically significant.

3. Results

3.1 Neurons Were Activated in the SMC after T-iTBS Treatment

The results of normal rat showed that c-Fos was activated in the layer V pyramidal neurons of SMC and the action potentials of the hind limb skeletal muscles were recorded (Fig. 1A–D). After T-iTBS treatment for 4 weeks post-SCI, we assessed the expression levels of the immediate early gene c-Fos and NeuN by immunofluorescence. The results showed that the numbers of c-Fos and NeuN double-positive neurons were significantly higher in the T-iTBS group than the SCI and sham-iTBS groups (Fig. 2A–F). The above evidence indicates that neurons of SMC are functionally silenced after SCI but can be re-activated by T-iTBS treatment.

3.2 T-iTBS Regulated Neural Regeneration and Synaptic Plasticity in the SMC

To further explore the biological effects of 4 weeks of excitatory stimulation with T-iTBS on SMC neurons, we observed the expression of Arc, an activity-regulated cytoskeleton-associated gene, which reflects the occurrence of neuronal regeneration and synaptic plasticity after excitation. The immunofluorescence showed that the T-iTBS treatment group had significantly increased expression levels of Arc on microtubule-associated protein 2-positive (Map2⁺) neurons in SMC after SCI compared with the control group (Fig. 3A–F).

3.3 SCI-Induced Abnormal Gene Expression was Reversed in the T-iTBS Treatment Group

To detect the biological changes attributed to T-iTBS, we utilized RNA-seq to analyze DEGs. The results showed that the expression levels of mRNA related to neurogenesis, neuronal metabolism, synaptic plasticity, and nerve regeneration were upregulated in T-iTBS treatment group compared with the sham-iTBS group (Fig. 4A and Supplementary Table 2). We further investigated the DEGs relating to inflammation and apoptosis. In a heatmap showing considerably upregulated (red) and downregulated (green) genes, the expression levels of proinflammatory and pro-apoptosis genes of the T-iTBS group in the SMC were significantly lower than those in the sham-iTBS group (Fig. 4B and Supplementary Table 3). In addition, we found anti-inflammatory and anti-apoptosis genes were upregulated in the T-iTBS group (Fig. 4B). Gene ontology (GO)-interaction analysis further showed that, compared with the T-iTBS group, the sham-iTBS group showed the significant upregulation of genes related to inflammation and microglia, whereas genes related to nerve regeneration, neuronal metabolism, and synaptic plasticity were significantly downregulated (Fig. 4C).

3.4 T-iTBS Treatment Suppressed Inflammation and Apoptosis in the SMC after SCI

To further investigate the anti-apoptosis effect of T-iTBS in the SMC after SCI, we examined the protein levels of pro-caspase-3, Cleaved-caspase-3 (CC3), and \beta-actin. Western blotting showed that the expression levels of CC3 and the ratio of activated caspase-3 were downregulated in the T-iTBS group compared to the SCI and sham-iTBS groups (Fig. 5A,B). For all original western blot figures of Fig. 5A see Supplementary Figures. To further evaluate the level of inflammation after T-iTBS in the SMC, a immunofluorescence assay was performed, which showed that the number of CD68-positive (CD68⁺) cells was significantly reduced after T-iTBS treatment (Fig. 6A–E). These results demonstrated that T-iTBS treatment alleviates the inflammation and apoptosis in the SMC caused by SCI.

3.5 Correlation Analysis of Neuron Activation, Synaptic Plasticity, and Inflammation in the SMC

To determine the relationship between neuron activation, synaptic plasticity, and inflammation in the SMC, we analyzed the correlation between the expression levels of c-Fos and Arc, CD68, and activated caspase-3 from each group. The number of c-Fos⁺/NeuN⁺ cells was positively correlated with the number of Arc⁺/Map2⁺ cells (Fig. 7A). When we analyzed the correlation between neuron activation and inflammation, we found that the number of c-Fos⁺/NeuN⁺ cells was negatively correlated with the number of CD68⁺ cells (Fig. 7B). Moreover, in the correlation between neuron activation and apoptosis, the number of c-Fos⁺/NeuN⁺ cells was negatively correlated with the ratio of activated caspase-3 (Fig. 7C). Four weeks after SCI, the CMEPs of each group of rats indicated that the latency of CMEPs in the T-iTBS group was significantly shorter than that of the control group (Fig. 7D,E). There was a tendency for a higher amplitude, but it was not statistically significant (Fig. 7D,F).

4. Discussion

It is of considerable significance that the SMC neurodegeneration resulting from complete SCI is mitigated promptly to facilitate the reconstruction of voluntary motor function [3]. In this study, we employed T-iTBS to treat rats transected at the T9 level. Our results suggested that treatment with T-iTBS for 4 weeks effectively mitigated the damage cascade caused by SCI by stimulating SMC neurons and regulating neuroplasticity. The expression levels of mRNA related to SMC neuronal activation, neuronal metabolism, synaptic activity, and neural regeneration were significantly downregulated in the sham-iTBS group, but upregulated in the T-iTBS group to nearly normal levels. The expression level of CC3, which is related to apoptosis, and the number of CD68⁺ cells, which are related to inflammation, were significantly lower in the T-iTBS group. These findings imply that T-iTBS could serve as a treatment that is facile to apply clinically for the early prevention of cerebral neurodegeneration after severe SCI.

4.1 Effect of T-iTBS on Regulating the Excitability and Neuroplasticity of SMC

The reconstruction of voluntary motor function after complete SCI is still a global medical challenge [34, 35]. SMC is the core cortex of the brain that integrates somatosensory information and emits motor control information. Recent studies suggest that protecting SMC neurons can play a key role in repairing voluntary motor function [3, 25, 36, 37]. Our results showed that the expression of c-Fos in SMC neurons in T9 transverse SCI rats was significantly reduced after 4 weeks compared to that of normal rats. This demonstrated that the functional silencing of SMC neurons begins in the early stages of SCI; it also predicted that further apoptosis and atrophy would occur. It is necessary to perform early stimulation to regulate the excitability of the neurons.

We established a site for T-iTBS stimulation that completely activated layer V pyramidal neurons. Action potentials of hindlimb muscles could be detected during T-iTBS treatment [3, 38]. T-iTBS treatment increased the expression of c-Fos in SMC neurons after SCI, achieving their excitatory regulation. To assess the mitigation of neuronal degeneration, we observed the expression of the activity-regulated cytoskeleton-associated protein Arc, an immediate early gene related to neural regeneration and synaptic plasticity regulation [39]. The results showed SCI caused neuronal silencing in the SMC and decreased the expression of Arc. The expression of Arc is often used as a sensitive indicator to evaluate the progression of neurodegeneration [39]. The number of Arc⁺ neurons of the SMC was upregulated in the T-iTBS group to almost normal levels, which indicated that this excitatory neural regulation may prevent the progression from neuronal functional silencing to neurodegeneration in the early stage of injury. Transcriptome sequencing also demonstrated that mRNA related to neuronal activation, synaptic activity, neuronal metabolism, and regeneration were significantly upregulated in the SMC after T-iTBS treatment and were almost at the levels seen in normal rats. According to above results, we speculated that T-iTBS reversed the progression toward neuronal apoptosis and atrophy by promoting the metabolism and regeneration abilities of neurons. It is necessary to treat in the acute or subacute phase after injury in order to prevent neuronal functional silencing progressing into irreversible apoptosis and atrophy.

4.2 Effect and Mechanism of T-iTBS in Reversing SMC Neurodegeneration

After 4 weeks of T-iTBS treatment, RNA-seq analysis revealed that mRNA related to neuronal apoptosis were significantly downregulated compared with those in the SCI and sham-iTBS group, but mRNA related to anti-apoptosis were significantly upregulated to almost normal levels. The detection of apoptosis protein CC3 also confirmed the amelioration of apoptosis by T-iTBS treatment. According to these results, it can be inferred that T-iTBS promotes neuronal metabolism and regeneration abilities by regulating the excitability of SMC neurons while reversing the apoptosis of these neurons.

Many studies have indicated that neuronal functional silencing is an important factor in neuronal apoptosis, with another important factor being the activation of neuro-inflammation [20, 40, 41, 42]. Due to the damage signals caused by SCI, SMC neurons are in a stressed state and release inflammatory factors to induce a cascade of inflammation [43, 44]. After 4 weeks of SCI, the results of transcriptome sequencing indicated that mRNA related to inflammation were significantly upregulated in the SCI group, but anti-inflammatory genes were significantly downregulated, compared to the T-iTBS group. Moreover, the expression levels of mRNA related to anti-inflammatory activity were upregulated to almost normal levels after T-iTBS treatment. These results suggested that T-iTBS treatment maintained the homeostasis of immune microenvironment, avoided neuronal stress, and synthetized inflammatory factors by regulating the excitability of SMC neurons, in which mRNA for anti-inflammation, synaptic activation, and nerve regeneration were upregulated. Our immunohistochemical analysis confirmed that the number of CD68⁺ cells was reduced and was similar to that of the normal rats in the T-iTBS group, whereas the SCI and sham-iTBS group showed the significant activation and aggregation of CD68⁺ cells. CD68⁺ cells may release inflammatory factors, further inducing neuronal apoptosis and finally leading to the gray matter atrophy that is visible on imaging [3, 45]. We speculated that the functional silencing of neurons and the inflammatory microenvironment induced the downregulation of regenerative genes and increased the expression of apoptotic genes. Hence, after SCI, it is necessary to improve the immune microenvironment of neurons, reverse apoptosis, and enhance the intrinsic regeneration ability of brain-derived axons through excitatory regulation with T-iTBS [3, 46, 47]. After four weeks of T-iTBS treatment, the results of CMEPs imply that the capacity of the spinal cord to convey nerve impulses has begun to show an improvement tendency. We hypothesize that this improvement will become more prominent with the prolongation of T-iTBS treatment.

5. Conclusions

In this study, we analyzed the regulation of neuronal excitability and neuroplasticity by T-iTBS in the SMC by establishing a spinal cord-transected model in rats. Based on the findings, we propose that T-iTBS can regulate the expression of genes and the external microenvironment of SMC and has potential application value for the reconstruction of voluntary motor function. However, this study did not explore the cumulative therapeutic effects of T-iTBS over a longer period, particularly regarding immune regulation and brain-derived nerve regeneration at the injury area. Although T-iTBS has demonstrated positive effects in protecting SMC neurons, it still cannot solely reconstruct motor voluntary function. It is necessary to combine T-iTBS with advanced regenerative medicine technologies such as stem cell tissue engineering to further achieve neural pathway reconstruction at the site of SCI, which will be the direction of our research in the future.

Availability of Data and Materials

Data and materials could be obtained upon reasonable request to the corresponding author.

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Funding

National Key R&D Program of China(2022YFA1105900)

Chinese National Natural Science Foundation(82371400)

Natural Science Foundation of Guangdong Province(2021B1515020045)

Science and Technology Planning Project of Guangdong Province(2023B1212060018)