Edited by: John D. Imig, Medical College of Wisconsin, United States
Reviewed by: Brenda Lilly, The Research Institute at Nationwide Children's Hospital, United States; Michael J. Ryan, University of Mississippi Medical Center School of Dentistry, United States
*Correspondence: Xiaolin Huang
This article was submitted to Vascular Physiology, a section of the journal Frontiers in Physiology
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Cognitive impairment is a serious mental deficit caused by stroke that can severely affect the quality of a survivor's life. Repetitive transcranial magnetic stimulation (rTMS) is a well-known rehabilitation modality that has been reported to exert neuroprotective effects after cerebral ischemic injury. In the present study, we evaluated the therapeutic efficacy of rTMS against post-stroke cognitive impairment (PSCI) and investigated the mechanisms underlying its effects in a middle cerebral artery occlusion (MCAO) rat model. The results showed that rTMS ameliorated cognitive deficits and tended to reduce the sizes of cerebral lesions. In addition, rTMS significantly improved cognitive function via a mechanism involving increased neurogenesis and decreased apoptosis in the ipsilateral hippocampus. Moreover, brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), were clearly upregulated in ischemic hippocampi after treatment with rTMS. Additionally, further studies demonstrated that rTMS markedly enhanced the expression of the apoptosis-related B cell lymphoma/leukemia gene 2 (Bcl-2) and decreased the expression of the Bcl-2-associated protein X (Bax) and the number of TUNEL-positive cells in the ischemic hippocampus. Both protein levels and mRNA levels were investigated. Our findings suggest that after ischemic stroke, treatment with rTMS promoted the functional recovery of cognitive impairments by inhibiting apoptosis and enhancing neurogenesis in the hippocampus and that this mechanism might be mediated by the BDNF signaling pathway.
Stroke is now the third leading cause of death worldwide and the leading cause of death and disability in China (Yang et al.,
The hippocampus is a functional region of the brain that is involved in human cognition. The hippocampus is especially important for spatial learning and memory. Hippocampal lesions caused by a stroke or secondary damage caused by a focal ischemic stroke contribute to the pathogenesis of PSCI (Grysiewicz and Gorelick,
rTMS is a noninvasive brain-stimulating technique with potential neuroprotective activity that has recently received an increasing amount of interest (Nierat et al.,
Several signaling molecules have been demonstrated to be essential for neurogenesis (Faigle and Song,
The present study was designed to investigate the effect of 10 Hz rTMS on cognitive dysfunction following focal cerebral ischemia. Our results confirm that the rTMS-induced neuroprotective effect is associated with changes in apoptosis and the expression of BDNF signaling pathway components, which cooperatively regulate hippocampal neurogenesis. These findings reveal a potential mechanism by which rTMS may improve cognitive impairment, indicating that rTMS is a promising candidate for the development of clinical strategies to treat ischemic stroke.
Adult male
The rats were randomly divided into 7- and 14-day groups, each of which was further divided into Sham, MCAO, and rTMS groups. Apart from the Sham group, the remaining groups underwent MCAO surgery, and only the rTMS group received rTMS treatment.
The rats were anesthetized using 10% chloral hydrate (400 mg/kg, i.p.). The right middle cerebral artery was occluded for 90 min and subsequently reperfused (Longa et al.,
All rats were examined using a Discovery MR750 3.0 T magnetic resonance imaging (MRI) scanner (GE, USA) equipped with a radio frequency (RF) coil for animals. MRI was performed at 24 h, 7 and 14 days after surgery. Each rat was deeply anesthetized and then placed in a prone position with its head in the middle of the coil. Diffusion-weighted images (DWI) were obtained using a two-dimensional spin-echo echo-planar imaging sequence (SE-EPI) using the following parameters: 2,000/minimum; field of view, 50 × 5 mm; thickness, 2 mm and image acquisition matrix, 96 × 64. The infarct volume was expressed as a percentage (%) of the whole brain volume. Apparent diffusion coefficient (ADC) maps were calculated from the DWI data. The values of the ADCs were then measured for the ischemic slice and the mirrored contralateral region. Lesion volumes (LV) were evaluated from ADC images as the ratio of the ischemic region to the corresponding region in the contralateral hemisphere (mean ±
rTMS was delivered using a customized magnetic stimulator (YRD-CCI, Wuhan, China). In conscious rats, a round prototype coil (6 cm in diameter with 3.5T peak magnetic welds) was positioned perpendicular to the cortex ~5 mm to the right of bregma (Linden et al.,
Bromodeoxyuridine (BrdU) (50 mg/kg in saline, Sigma-Aldrich, USA) was intraperitoneally injected once daily for 7 consecutive days starting 24 h after surgery in all groups (
Learning and memory performance was assessed using the Morris water maze test beginning on the 12th day after surgery. The principles and technical details of the task have been described previously (Han et al.,
To avoid any potential impact on the final results, we excluded rats that exhibited abnormal behavioral effects, such as seizures, during treatment. All rats were deeply anesthetized using 10% chloral hydrate (400 mg/kg, i.p.) at different time points. A subset of the rats was transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The brains of these rats were removed and post-fixed in the same fixative at 4°C overnight and then immersed consecutively in 20 and 30% sucrose at 4°C until they sank. The brains of rats injected with BrdU (
Frozen 30-μm-thick sections were incubated in blocking solution (10% normal donkey serum or 10% normal goat serum and 0.3% Triton X-100 in PBS, pH 7.5) for 2 h at room temperature (RT) and then incubated with primary antibodies in 5% serum and PBS for 24 h at 4°C. The sections were subsequently incubated for 3 h at RT with fluorophore-conjugated secondary antibodies. To prepare the sections for BrdU staining, they were incubated in 2 N HCl for 0.5 h at 37°C.
Rat monoclonal anti-BrdU (1:100; Abcam, UK) primary antibodies were used to mark proliferating cells, mouse monoclonal anti-Nestin (1:100; BD Pharmingen, USA) primary antibodies were used as a marker for NSCs, and mouse monoclonal anti-NeuN (1:100; Chemicon, USA) primary antibodies were used to label mature neurons. The following secondary antibodies were used: Alexa Fluor 594-labeled donkey anti-rat IgG and Alexa Fluor 488-labeled donkey anti-mouse IgG (1:200 for both; Invitrogen, USA).
The stained slides were dehydrated, cover-slipped in anti-quenching agent (p-phenylenediamine, PPD) and analyzed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan). The number of positive cells was counted in a blinded manner in the ipsilateral SGZ using 20X and 40X objectives in OLYMPUS FV10-ASW Viewer.
Frozen 10-μm-thick sections were prepared for TUNEL staining.
Anti-rabbit BDNF (1:1,000; Santa Cruz, Inc., CA, USA), TrkB (1:1,000; Santa Cruz, Inc., CA, USA), p-AKT (1:1,000; Santa Cruz, Inc., CA, USA), Bcl-2 (1:1,000, Cell Signaling Technology, Inc., MA, USA), and Bax (1:1,000, Cell Signaling Technology, Inc., MA, USA) antibodies were used. Images were acquired using an X-ray film processor. Normalization was performed using mouse monoclonal GAPDH antibodies (1:500; Santa Cruz, Inc., CA, USA). The bands were quantitated using Gel-Pro Analyzer 4.0 software (Media Cybernetics, USA).
Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to analyze the expression levels of the BDNF and TrkB mRNAs in ipsilateral hippocampal tissues. RT-PCR was performed in duplicate for each RNA sample. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The fold-changes in BDNF and TrkB expression were calculated relative to GAPDH expression using the comparative Ct method (2−ΔΔCT). mRNA levels were then expressed as fold-changes after normalization to GAPDH levels.
All data are presented as the mean ±
Lesion volumes (LV) were evaluated from ADC images as the ratio of the ischemic region to the corresponding region in the contralateral hemisphere of the rats. We calculated LV for all groups on the first day, 7th day, and 14th day post-occlusion (Figure
Effect of rTMS on lesion volume ratios in ischemic rats at 1, 7, and 14 d after surgery.
First, we compared the results in the three groups at different time points (Figure
We performed an additional analysis to determine whether there were differences among the groups at different time points (Figure
Cognitive performance was assessed by averaging the values for latency to reach the platform and counting the frequency of swimming across the platform in the spatial probe trial. An analysis of escape latencies showed that there were significant differences among the Sham, MCAO, and rTMS groups. Escape latency was longer (
Effects of rTMS on cognitive impairment in the Morris water maze task.
In addition, an analysis of the frequency of swimming across the platform during the 60 s observation period revealed that there were significant differences among the Sham, MCAO, and rTMS groups. The frequency of swimming across the platform was lower (
To evaluate the proliferation of NSCs in response to rTMS, double immunofluorescence staining was performed for BrdU and Nestin at 7 days after treatment (Figure
rTMS increased the number of BrdU (red) and Nestin (green) co-immunofluorescence-labeled cells in the ipsilateral SGZ at 7 days after surgery. Panels
To further explore the differentiation of newborn cells, which incorporate BrdU, in response to rTMS, we performed double immunofluorescence staining using antibodies against BrdU and a marker of mature neuronal nuclei (NeuN) in the SGZ at 14 days after treatment (Figure
rTMS increased the number of BrdU (red) and NeuN (green) co-immunofluorescence-labeled cells in the ipsilateral SGZ at 14 days after surgery. Panels
To investigate the effect of rTMS on the BDNF pathway, western blot analysis, and RT-PCR were performed to examine the expression of crucial signaling molecules in the ipsilateral hippocampus. As shown in Figure
Effects of rTMS on the expression of BDNF pathway members in the hippocampus at 14 days after surgery.
To determine whether rTMS affects cell survival, we counted apoptotic nuclei and detected the levels of the apoptosis-related proteins Bcl-2 and Bax in the ipsilateral hippocampus after focal cerebral ischemia. Apoptotic neuronal death was evaluated using TUNEL staining (Figure
Effect of rTMS on the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells in the hippocampus.
Effects of rTMS on Bcl-2 and Bax expression levels in the hippocampus.
Our analysis of the whole hippocampus revealed that there were clearly significant signs of neuronal loss in the experimental rats. As shown in Figure
Because the balance between Bcl-2/Bax is involved in the regulation of apoptotic cell death, we next investigated whether rTMS influenced Bcl-2/Bax expression. As shown in Figure
Cognitive impairment and memory dysfunction are common symptoms observed in stroke survivors that significantly affect their quality of life. In the present study, we show that 10 Hz rTMS improves cognitive impairment, stimulated neurogenesis and inhibited apoptosis when focal cerebral ischemia was induced in the hippocampus. In addition, we found that the BDNF signaling pathway is a crucial contributor to the effects of rTMS.
rTMS is a promising approach for treating PSCI stroke-induce cognitive impairment. Several previous investigations showed that chronic high-frequency rTMS ameliorated cognitive impairment in normally aging individuals (Guse et al.,
Although, previous studies have shown that rTMS exerts positive effects on cognition, its clinical effects have remained the subject of dispute (Kim et al.,
Despite the potential of rTMS, the specific mechanisms by which it improves PSCI have not been investigated. Neurogenesis may restore cognitive functions that are impaired by ischemic stroke, and an increasing amount of evidence indicates that a sufficient number of neurons are required for normal hippocampal functions. Hence, we sought to determine the mechanism by which 10 Hz rTMS affects cognitive impairment in MCAO rats. Our results incorporate several lines of evidence demonstrating that treatment with rTMS increases neurogenesis and decreases apoptosis in the hippocampus. First, the results of BrdU+/Nestin+ immunofluorescence staining show that applying 10 Hz rTMS to MCAO rats induced proliferation in NSCs in the SGZ. Second, the results of double immunostaining for BrdU and NeuN show that applying 10 Hz rTMS to MCAO rats enhanced differentiation in newborn cells in the SGZ, and these new neurons were integrated into the existing hippocampal circuitry. Finally, TUNEL labeling revealed that treatment with 10 Hz rTMS down-regulated neuronal apoptosis. All of the above data support the notion that rTMS is an efficient therapeutic treatment for patients with cognitive impairment and that the effects of rTMS on these symptoms may involve increasing neurogenesis and suppressing apoptosis in the hippocampus. To our knowledge, this is the first study to explore the effects of and mechanisms underlying rTMS in PSCI. Nevertheless, our results are in agreement with the results of previous studies that have indicated that rTMS plays an important role in both neurogenesis and apoptosis. Ueyama et al. reported that chronic rTMS increased hippocampal neurogenesis in healthy rats (Ueyama et al.,
In some studies, it has been proposed that rTMS supports improved memory formation and motor learning by enhancing activity at the level of neurotransmitters or neurotrophic factors. Wang et al. reported that rTMS improved the restoration of cognitive ability and exerted a neuroprotective effect in vascular dementia rats and that this effect may have been the result of increases in the levels of BDNF, TrkB, and SYN in the CA1 region (Wang et al.,
The major aim of the present study was to investigate the mechanisms underlying the neuroprotective efficacy of rTMS in PSCI. Increasing our understanding of the environment in the neurogenic niche and the mechanisms involved in maintaining it are paramount to efforts aimed at enhancing endogenous recovery processes. We show that 10 Hz rTMS promotes neurogenesis and inhibits neuronal apoptosis in the ipsilateral hippocampus after focal cerebral ischemia. We also demonstrate that the expression levels of BDNF signaling components are increased and that the expression levels of apoptosis-related proteins are altered in the ischemic hippocampus after rTMS. These results suggest that the cognitive recovery induced by rTMS could be enhanced by manipulating extracellular factors in the hippocampus that are associated with these neurotrophic factors. Additional factors known to be important during these processes should also be explored to further clarify the neuroprotective role of rTMS in PSCI.
In conclusion, the results presented in this study indicate that rTMS improves PSCI, promotes neurogenesis and suppresses neuronal apoptosis in the hippocampus of adult rats with focal cerebral ischemia. Moreover, the mechanisms underlying the neuroprotective effects of rTMS might be associated, at least in part, with the activation of the BDNF signaling pathway. These results collectively suggest that up-regulating BDNF signaling using rTMS affects neurogenesis and apoptosis in ischemic hippocampus and is essential for improving PSCI.
XLH and XHH contributed to the research design and data analysis. FG carried out the experiment, analyzed the data and wrote the manuscript. JL and YD participated in the experiment of the behavior test, rTMS treatment and immunostaining. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to thank Professor Wenzhen Zhu, Yihao Yao, Jingjing Shi for their help with the MRI scan of the study. We also want to thank Fengxia Zhang and Qun Xu for their assistance in rTMS treatment.