Comprehensive assessment of HF-rTMS treatment mechanism for post-stroke dysphagia in rats by integration of fecal metabolomics and 16S rRNA sequencing.

Background: The mechanism by which high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) improves swallowing function by regulating intestinal flora remains unexplored. We aimed to evaluate this using fecal metabolomics and 16S rRNA sequencing. Methods: A Post-stroke dysphagia (PSD) rat model was established by middle cerebral artery occlusion. The magnetic stimulation group received HF-rTMS from the 7th day post-operation up to 14th day post-surgery. Swallowing function was assessed using a videofluoroscopic swallowing study (VFSS). Hematoxylin-eosin (H&E) staining was used to assess histopathological changes in the intestinal tissue. Intestinal flora levels were evaluated by sequencing the 16S rRNA V3-V4 region. Metabolite changes within the intestinal flora were evaluated by fecal metabolomics using liquid chromatography-tandem mass spectrometry. Results: VFSS showed that the bolus area and pharyngeal bolus speed were significantly decreased in PSD rats, while the bolus area increased and pharyngeal transit time decreased after HF-rTMS administration (p < 0.05). In the PSD groups, H&E staining revealed damaged surface epithelial cells and disrupted cryptal glands, whereas HF-rTMS reinforced the integrity of the intestinal epithelial cells. 16S rRNA sequencing indicated that PSD can disturb the intestinal flora and its associated metabolites, whereas HF-rTMS can significantly regulate the composition of the intestinal microflora. Firmicutes and Lactobacillus abundances were lower in the PSD group than in the baseline group at the phylum and genus levels, respectively; however, both increased after HF-rTMS administration. Levels of ceramides (Cer), free fatty acids (FA), phosphatidylethanolamine (PE), triacylglycerol (TAG), and sulfoquinovosyl diacylglycerol were increased in the PSD group. The Cer, FA, and DG levels decreased after HF-rTMS treatment, whereas the TAG levels increased. Peptococcaceae was negatively correlated with Cer, Streptococcus was negatively correlated with DG, and Acutalibacter was positively correlated with FA and Cer. However, these changes were effectively restored by HF-rTMS, resulting in recovery from dysphagia. Conclusion: These findings suggest a synergistic role for the gut microbiota and fecal metabolites in the development of PSD and the therapeutic mechanisms underlying HF-rTMS.

High-frequency rTMS alleviates cognitive impairment and regulates synaptic plasticity in the hippocampus of rats with cerebral ischemia.

Poststroke cognitive impairment (PSCI) is a common complication of stroke, but effective treatments are currently lacking. Repetitive transcranial magnetic stimulation (rTMS) is gradually being applied to treat PSCI, but there is limited evidence of its efficacy. To determine rTMS effects on PSCI, we constructed a transient middle cerebral artery occlusion (tMCAO) rat model. Rats were then grouped by random digital table method: the sham group (n = 10), tMCAO group (n = 10) and rTMS group (n = 10). The shuttle box and Morris water maze (MWM) tests were conducted to detect the cognitive functions of the rats. In addition, synaptic density and synaptic ultrastructural parameters, including the active zone length, synaptic cleft width, and postsynaptic density (PSD) thickness, were quantified and analyzed using an electron microscope. What's more, synaptic associated proteins, including PSD95, SYN, and BDNF were detected by western blot. According to the shuttle box and MWM tests, rTMS improved tMCAO rats' cognitive functions, including spatial learning and memory and decision-making abilities. Electron microscopy revealed that rTMS significantly increased the synaptic density, synaptic active zone length and PSD thickness and decreased the synaptic cleft width. The western blot results showed that the expression of PSD95, SYN, and BDNF was markedly increased after rTMS stimulation. Based on these results, we propose that 20 Hz rTMS can significantly alleviate cognitive impairment after stroke. The underlying mechanism might be modulating the synaptic plasticity and up-regulating the expression PSD95, SYN, and BDNF in the hippocampus.

Effect of high-frequency repetitive transcranial magnetic stimulation on swallowing function and pneumonia in poststroke dysphagia in rats.

BACKGROUND: Post-stroke dysphagia (PSD) is a common symptom of stroke. Clinical complications of PSD include malnutrition and pneumonia. Clinical studies have shown that high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) can improve the swallowing function in stroke patients. However, few studies have elucidated the underlying molecular mechanisms. METHODS: A PSD rat model was established using transient middle cerebral artery occlusion (tMCAO). Rats were randomly divided into sham-operated groups, PSD groups, PSD + sham-rTMS groups, PSD + 5 Hz-rTMS groups, PSD + 10 Hz-rTMS groups and PSD + 20 Hz-rTMS groups. Rats were weighed and videofluoroscopic swallowing studies were conducted. Pulmonary inflammation, levels of substance P (SP) and calcitonin gene-related peptide (CGRP) in the serum, lung, and nucleus tractus solitarius (NTS), brain-derived neurotrophic factor (BDNF) and 5-hydroxytryptamine (5HT) in NTS were evaluated. RESULTS: Rats in the PSD group experienced weight loss, reduced bolus area and pharyngeal bolus speed, and increased pharyngeal transit time (PTT) and inter-swallow interval (ISI) on day 7 and day 14 after operation. Moreover, PSD rats showed pulmonary inflammation, reduced levels of SP in the lung and serum, increased levels of CGRP in the lung and NTS, reduced levels of BDNF and 5HT in the NTS. There was no significant difference between the PSD group and the PSD + sham-rTMS group in the results of weight and VFSS. Comparing with the PSD group, there significant increases in the bolus area, decreases in PTT of rats following 5 Hz rTMS intervention. HF-rTMS at 10 Hz significantly increased the weight, bolus area, pharyngeal bolus speed and decreased the PTT and ISI of rats. There were also significant increases in the bolus area (p < 0.01) and pharyngeal bolus speed, decreases in PTT and ISI of rats following 20 Hz rTMS intervention. Furthermore, compared with the PSD + 5 Hz-rTMS group, there were significant increases in the bolus area and pharyngeal bolus speed, decreases in ISI in the swallowing function of rats in the PSD + 10 Hz-rTMS group. Besides, compared with the PSD + 5 Hz-rTMS group, there were significant decreases in ISI in the swallowing function of rats in the PSD + 20 Hz-rTMS group. HF-rTMS at 10 Hz alleviated pulmonary inflammation, increased the levels of SP in the lung, serum, and NTS, CGRP in the serum and NTS, 5HT in the NTS of PSD rats. CONCLUSION: Compared with 5 Hz and 20 Hz rTMS, 10 Hz rTMS more effectively improved the swallowing function of rats with PSD. HF-rTMS at 10 Hz improved the swallowing function and alleviated pneumonia in PSD rats. The mechanism may be related to increased levels of SP in the lung, serum and NTS, levels of CGRP in the serum and NTS, 5HT in the NTS after HF-rTMS treatment.

Changes in gene expression and neuroinflammation in the hippocampus of rats with poststroke cognitive impairment.

Poststroke cognitive impairment (PSCI) often occurs during the stroke recovery period and greatly increases the difficulty of rehabilitation. Activation of neuroinflammation and long-term changes in gene expression patterns in the hippocampus could be essential in the development of PSCI. Therefore, this study aimed to identify neuroinflammation and changes in gene expression patterns in the hippocampus in rats with PSCI. Rats underwent transient middle cerebral artery occlusion (tMCAO) or sham surgery. The infarct volume was measured on day 3 after surgery. The Morris water maze (MWM) test was used to assess cognitive function. Microglial activation and white matter (WM) lesions in the hippocampus were evaluated on day 28 after surgery. In addition, we compared differentially expressed genes (DEGs) in the hippocampus between tMCAO group rats and sham group rats by RNA sequencing. Then, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) network analyses were conducted to investigate these DEGs. The results showed that the tMCAO group rats showed extensive infarction and cognitive dysfunction compared with the sham group rats. Microglial activation and WM damage were obvious in the hippocampus of tMCAO group rats. We found 43 DEGs by RNA sequencing: 29 genes with upregulated expression and 14 genes with downregulated expression. The GO analysis indicated that DEGs were mainly involved in cell proliferation and differentiation, cholesterol synthesis, and metabolism. The KEGG pathway analysis suggested that the DEGs were significantly enriched in intestinal immune network for IgA production and steroid biosynthesis. Acta2, Calb2, and Cxcl12 were notable in the PPI analysis. Our results suggest that microglial activation and WM damage are maintained in rats with PSCI. The mechanism may be related to the regulation of steroid biosynthesis, intestinal immunity, and potential key genes such as Acta2, Calb2, and Cxcl12 in the hippocampus.

Effect of Modified Pharyngeal Electrical Stimulation on Patients with Severe Chronic Neurogenic Dysphagia: A Single-Arm Prospective Study.

Current treatments for severe chronic neurogenic dysphagia (SCND) are limited. Modified pharyngeal electrical stimulation (mPES) was modified from pharyngeal electrical stimulation (PES). This prospective study aimed to explore the efficacy and safety of mPES on SCND. 30 patients with severe chronic neurogenic dysphagia were recruited. mPES was administered to patients once daily until the functional oral intake scale score (FOIS) reach 3. Videofluoroscopic swallow study (VFSS), flexible endoscopic evaluation of swallowing (FEES), and high-resolution manometry (HRM) were utilized for evaluating the swallowing function. After mPES, 24 of 30 patients (80%) reached the endpoint (FOIS = 3) (P < 0.001). 3 of 6 tracheotomized patients (50%) removed the tracheal tube. The median number of mPES sessions for the 24 patients who met the criteria was 28 (17, 38) and the median period was 43 (29, 63) days. Moreover, a significant increase was observed in hypopharyngeal peak pressure (P = 0.015), hypopharyngeal contraction duration (P = 0.023), velopharyngeal peak pressure (P = 0.044), and velopharyngeal contraction duration (P = 0.031). A reduction was observed in PAS (P < 0.001), secretion (P = 0.001), vallecular residue (P < 0.001), left (P = 0.001), and right (P < 0.001) pyriform sinus residue. The median FOIS of 30 patients at 3-month follow-up was 5 (3, 6). No serious side effects were reported. mPES is a promising effective and safe therapeutic approach that is simple to use in patients with SCND.

Effects of HF-rTMS on microglial polarization and white matter integrity in rats with poststroke cognitive impairment.

Poststroke cognitive impairment (PSCI) occurs frequently after stroke, but effective treatments are lacking. Previous studies have revealed that high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) has a beneficial effect on PSCI, but the mechanism is unclear. This study aimed to evaluate the effect of 10 and 20 Hz HF-rTMS on PSCI and the possible mechanisms. An ischemic stroke rat model was established by transient middle cerebral artery occlusion (tMCAO). The modified neurological deficit score (mNSS) and Morris water maze tests were conducted to assess neurological function and cognitive function. Luxol Fast Blue (LFB) staining was performed to evaluate white matter damage. Proinflammatory and anti-inflammatory cytokines were measured using enzyme-linked immunosorbent assays (ELISA). Immunofluorescence was used to assess microglial activation and polarization. Western blotting was performed to measure JAK2-STAT3 pathway-related protein expression. We found that HF-rTMS decreased the neurological deficit score. Compared with 10 Hz HF-rTMS, 20 Hz HF-rTMS more markedly improved the cognitive function of tMCAO rats at day 28 after operation. Furthermore, 20 Hz HF-rTMS attenuates white matter lesion, decreased proinflammatory cytokine levels, and increased anti-inflammatory cytokine levels. It also decreased the number of CD68- and CD16/32-positive microglia and increased the number of CD206-positive microglia. In addition, p-JAK2, JAK2, p-STAT3 and STAT3 expression was increased. These findings suggest that HF-rTMS improves cognitive function and attenuates white matter lesion in tMCAO rats by shifting microglia toward the M2 phenotype. Mechanisms may be related to regulation JAK2-STAT3 pathways.

Modulating swallowing-related functional connectivity and behavior via modified pharyngeal electrical stimulation: A functional near-infrared spectroscopy evidence.

Introduction: Modified pharyngeal electrical stimulation (mPES) is a novel therapeutic modality for patients with neurogenic dysphagia. However, the underlying neural mechanism remains poorly understood. This study aimed to use functional near-infrared spectroscopy (fNIRS) to explore the influence of mPES on swallowing-related frequency-specific neural networks and ethology. Methods: Twenty-two healthy right-handed volunteers participated in the study. Each participant was randomly assigned to either the sham or the mPES group and provided a 10-min intervention program every day for 5 days. Oxyhemoglobin and deoxyhemoglobin concentration changes verified by fNIRS were recorded on days 1, 3, and 5. Five characteristic frequency signals (0.0095-2 Hz) were identified using the wavelet transform method. To calculate frequency-specific functional connectivity, wavelet phase coherence (WPCO) was adopted. Furthermore, behavioral performance was assessed pre- and post-mPES using a 150 ml-water swallowing stress test. Results: Compared with sham stimulation on day 1, the significantly decreased WPCO values were mainly associated with the dorsolateral prefrontal lobe, Broca's area, and middle temporal lobe. Compared with the sham mPES on day 1, the mPES showed a noticeable effect on the total swallow duration. Compared with the baseline, the WPCO values on days 3 and 5 showed a stepwise decrease in connectivity with the application of mPES. Furthermore, the decreased WPCO was associated with a shortened time per swallow after mPES. Conclusions: The mPES could modulate swallowing-related frequency-specific neural networks and evoke swallowing cortical processing more efficiently. This was associated with improved performance in a water swallowing stress test in healthy participants.

Different training patterns at recovery stage improve cognitive function in ischemic stroke rats through regulation of the axonal growth inhibitor pathway.

Running wheel exercise training (RWE) and skilled reaching training (SRT) are physical training approaches with positive effects on cognitive function. However, few studies have compared the different effects of these exercises on long-term memory, and their mechanism remains unknown. This study investigated the effects of SRT and RWE, at the recovery stage, on the cognitive function of transient middle cerebral artery occlusion (tMCAO) rats and explored their association with NgR1/Rho-A/ROCK/LOTUS/LGI1 signaling. Adult Sprague-Dawley rats (n = 55) were divided into four groups after pretraining: SRT, RWE, tMCAO, and Sham. Rats were subjected to modified neurological severity score (mNSS) measurements and forelimb grip strength and the Morris water maze tests. Using immunofluorescence and western blotting, we evaluated axonal growth inhibitor expression in the peri-infarct cortex on days 28 and 56 after tMCAO. Results showed the mNSS reduced, whereas the grip strengths improved in RWE and SRT groups. The escape latency in the Morris water maze test was shorter, whereas the number of times of crossing the platform was higher in both the SRT and RWE groups than in the tMCAO group on day 56; furthermore, the parameters in the SRT group improved compared to those in the RWE group. Physical exercise training could improve cognitive functions by reducing the expression of the NgR1/RhoA/ROCK axon growth inhibitors and increasing the expression of the endogenous antagonists LOTUS/LGI1. Exercise training beginning at the recovery stage could improve the cognitive function in tMCAO rats through a mechanism probably associated with the axonal growth inhibitor pathway.

Corrigendum: Modulating swallowing-related functional connectivity and behavior via modified pharyngeal electrical stimulation: A functional near-infrared spectroscopy evidence.

[This corrects the article DOI: 10.3389/fneur.2022.1006013.].

NgR1 pathway expression in cerebral ischemic Sprague-Dawley rats with cognitive impairment.

OBJECTIVES: This study aimed to determine the effect of ischemic occlusion duration and recovery time course on motor and cognitive function, identify optimal conditions for assessing cognitive function with minimal interference from motor deficits, and elucidate the underlying mechanism of axonal inhibitors. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were randomly allocated to the transient middle cerebral artery occlusion (tMCAO) 60-min (tMCAO60min), tMCAO90min, tMCAO120min, and sham groups. We conducted forelimb grip strength, two-way shuttle avoidance task, and novel object recognition task (NORT)tests at three time points (14, 21, and 28 days). Expression of Nogo receptor-1 (NgR1), the endogenous antagonist lateral olfactory tract usher substance, ras homolog family member A (Rho-A), and RhoA-activated Rho kinase (ROCK) was examined in the ipsilateral thalamus. RESULTS: There was no difference in grip strength between sham and tMCAO90min rats at 28 days. tMCAO90min and tMCAO120min rats showed lower discrimination indices in the NORT than sham rats on day 28. Compared with that in sham rats, the active avoidance response rate was lower in tMCAO90min rats on days 14, 21, and 28 and in tMCAO120min rats on days 14 and 21. Furthermore, 50-54% of rats in the tMCAO90min group developed significant cognitive impairment on day 28, and thalamic NgR1, RhoA, and ROCK expression were greater in tMCAO90min rats than in sham rats. CONCLUSION: Employing 90-min tMCAO in SD rats and assessing cognitive function 28 days post-stroke could minimize motor dysfunction effects in cognitive function assessments. Axonal inhibitor deregulation could be involved in poststroke cognitive impairment.

Different combinations of high-frequency rTMS and cognitive training improve the cognitive function of cerebral ischemic rats.

Poststroke cognitive impairment (PSCI) occurs frequently after stroke, but lacks effective treatments. Previous studies have revealed that high-frequency repetitive transcranial magnetic stimulation (rTMS) has a beneficial effect on PSCI and is often used with other cognitive training methods to improve its effect. This study aimed to evaluate the effect of different combinations of rTMS and cognitive training (rTMS-COG) on PSCI and identify the optimal combination protocol. A cerebral infarction rat model was established by transient middle cerebral artery occlusion (tMCAO). The Morris water maze test was conducted to assess the cognitive function of rats. RNA sequencing and bioinformatics analysis were employed to study the underlying mechanisms. rTMS, COG and rTMS-COG all had beneficial effects on PSCI, while cognitive training immediately after rTMS (rTMS-COG0h) achieved a better effect than cognitive training 1 h and 4 h after rTMS, rTMS and COG. We identified 179 differentially expressed genes (DEGs), including 24 upregulated and 155 downregulated genes, between the rTMS-COG0h and rTMS groups. GO analysis revealed that the major categories associated with the DEGs were antigen procession and presentation, regulation of protein phosphorylation and axoneme assembly. KEGG analysis showed that the DEGs were enriched in processes related to phagosome, circadian entrainment, dopaminergic synapse, apelin signaling pathway, long-term depression, neuroactive ligand-receptor interaction, axon guidance and glucagon signaling pathway. PPI analysis identified Calb2, Rsph1, Ccdc114, Acta2, Ttll9, Dnah1, Dlx2, Dlx1, Ccdc40 and Ccdc113 as related genes. These findings prompt exploration of the potential mechanisms and key genes involved in the effect of rTMS-COG0h on PSCI.

High-Frequency rTMS Improves Cognitive Function by Regulating Synaptic Plasticity in Cerebral Ischemic Rats.

Poststroke cognitive impairment (PSCI) is one of the most severe sequelae of stroke and lacks effective treatment. Previous studies have shown that high-frequency repetitive transcranial magnetic stimulation (rTMS) may be a promising PSCI therapeutic approach, but the underlying mechanism is unclear. To uncover the effect of rTMS on PSCI, a transient middle cerebral artery occlusion (tMCAO) model was established. Modified Neurological Severity Score (mNSS) test and Morris Water Maze (MWM) test were performed to assess the neurological and cognitive function of rats. Furthermore, to explore the underlying mechanism, differentially expressed genes (DEGs) in the hippocampus of rats in the rTMS group and tMCAO group were compared using RNA sequencing. Then, bioinformatics analysis, including gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and protein-protein interaction (PPI) network analysis, was conducted to elaborate these DEGs. Our results indicated that high-frequency rTMS could significantly improve neurological and cognitive function, according to mNSS and MWM tests. We found 85 DEGs, including 71 upregulated genes and 14 downregulated genes, between the rTMS group and tMCAO group. The major functional category was related to chemical synaptic transmission modulation and several DEGs were significantly upregulated in processes related to synaptic plasticity, such as glutamatergic synapses. Calb2, Zic1, Kcnk9, and Grin3a were notable in PPI analysis. These results demonstrate that rTMS has a beneficial effect on PSCI, and its mechanism may be related to the regulation of synaptic plasticity and functional genes such as Calb2, Zic1, Kcnk9, and Grin3a in the hippocampus.