Intermittent theta-burst stimulation improves motor function by inhibiting neuronal pyroptosis and regulating microglial polarization via TLR4/NFκB/NLRP3 signaling pathway in cerebral ischemic mice.

BACKGROUND: Neuronal pyroptosis and neuroinflammation with excess microglial activation are widely involved in the early pathological process of ischemic stroke. Repetitive transcranial magnetic stimulation (rTMS), as a non-invasive neuromodulatory technique, has recently been reported to be anti-inflammatory and regulate microglial function. However, few studies have elucidated the role and mechanism of rTMS underlying regulating neuronal pyroptosis and microglial polarization. METHODS: We evaluated the motor function in middle cerebral artery occlusion/reperfusion (MCAO/r) injury mice after 1-week intermittent theta-burst rTMS (iTBS) treatment in the early phase with or without depletion of microglia by colony-stimulating factor 1 receptor (CSF1R) inhibitor treatment, respectively. We further explored the morphological and molecular biological alterations associated with neuronal pyroptosis and microglial polarization via Nissl, EdU, TTC, TUNEL staining, electron microscopy, multiplex cytokine bioassays, western blot assays, immunofluorescence staining and RNA sequencing. RESULTS: ITBS significantly protected against cerebral ischemia/reperfusion (I/R) injury-induced locomotor deficits and neuronal damage, which probably relied on the regulation of innate immune and inflammatory responses, as evidenced by RNA sequencing analysis. The peak of pyroptosis was confirmed to be later than that of apoptosis during the early phase of stroke, and pyroptosis was mainly located and more severe in the peri-infarcted area compared with apoptosis. Multiplex cytokine bioassays showed that iTBS significantly ameliorated the high levels of IL-1ß, IL-17A, TNF-α, IFN-γ in MCAO/r group and elevated the level of IL-10. ITBS inhibited the expression of neuronal pyroptosis-associated proteins (i.e., Caspase1, IL-1ß, IL-18, ASC, GSDMD, NLRP1) in the peri-infarcted area rather than at the border of infarcted core. KEGG enrichment analysis and further studies demonstrated that iTBS significantly shifted the microglial M1/M2 phenotype balance by curbing proinflammatory M1 activation (Iba1+/CD86+) and enhancing the anti-inflammatory M2 activation (Iba1+/CD206+) in peri-infarcted area via inhibiting TLR4/NFκB/NLRP3 signaling pathway. Depletion of microglia using CSF1R inhibitor (PLX3397) eliminated the motor functional improvements after iTBS treatment. CONCLUSIONS: rTMS could alleviate cerebral I/R injury induced locomotor deficits and neuronal pyroptosis by modulating the microglial polarization. It is expected that these data will provide novel insights into the mechanisms of rTMS protecting against cerebral I/R injury and potential targets underlying neuronal pyroptosis in the early phase of stroke.

High-Intensity Interval Training Improves Physical Function, Prevents Muscle Loss, and Modulates Macrophage-Mediated Inflammation in Skeletal Muscle of Cerebral Ischemic Mice.

Although skeletal muscle is the main effector organ largely accounting for disability after stroke, considerably less attention is paid to the secondary abnormalities of stroke-related skeletal muscle loss. It is necessary to explore the mechanism of muscle atrophy after stroke and further develop effective rehabilitation strategy. Here, we evaluated the effects of high-intensity interval (HIIT) versus moderate-intensity aerobic training (MOD) on physical function, muscle mass, and stroke-related gene expression profile of skeletal muscle. After the model of middle cerebral artery occlusion (MCAO) was successfully made, the blood lactate threshold corresponding speed (S LT) and maximum speed (S max) were measured. Different intensity training protocols (MOD < S LT; S LT < HIIT < S max) were carried out for 3 weeks beginning at 7 days after MCAO in the MOD and HIIT groups, respectively. We found that both HIIT and MOD prevented stroke-related gastrocnemius muscle mass loss in MCAO mice. HIIT was more beneficial than MOD for improvements in muscle strength, motor coordination, walking competency, and cardiorespiratory fitness. Furthermore, HIIT was superior to MOD in terms of reducing lipid accumulation, levels of IL-1ß and IL-6 in paretic gastrocnemius, and improving peripheral blood CD4+/CD8+ T cell ratio, level of IL-10. Additionally, RNA-seq analysis revealed that the differentially expressed genes among HIIT, MOD, and MCAO groups were highly associated with signaling pathways involved in inflammatory response, more specifically the I-kappaB kinase/NF-kappaB signaling. Following the outcome, we further investigated the infiltrating immune cells abundant in paretic muscles. The results showed that HIIT modulated macrophage activation by downregulating CD86+ (M1 type) macrophages and upregulating CD163+ (M2 type) macrophages via inhibiting the TLR4/MyD88/NFκB signaling pathway and exerting an anti-inflammatory effect in paretic skeletal muscle. It is expected that these data will provide novel insights into the mechanisms and potential targets underlying muscle wasting in stroke.

Enriched Environment Enhances Poststroke Neurological Function Recovery on Rat: Involvement of p-ERK1/2.

BACKGROUND: Increasing evidence shows that exposure to an enriched environment (EE) after cerebral ischemia or reperfusion injury is neuroprotective in animal models, including that EE enhances functional recovery after ischemic stroke. However, the mechanism underlying this effect remains unclear. To clarify this critical issue, the current study investigated the effects of EE on the role of extracellular signal-regulated kinase (ERK) after cerebral ischemia or reperfusion injury of rat. METHODS: Adult rats were subjected to ischemia induced by middle cerebral artery occlusion (MCAO) followed by reperfusion. Ladder walking task and limb-use asymmetry task were used to test the recovery of rat behavior on postoperative days 1, 3, 5, 7, 14 and days 3, 7, 14, respectively. On the eighth day after MCAO, infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride staining. Expressions of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 were examined by western blot, and electron microscopy was used to evaluate the astrocytes morphology surround in the perivascular 14 days after MCAO. RESULTS: EE improves the recovery of coordination and integration of motor movements on rats after cerebral ischemia or reperfusion injury. EE downregulates the level of p-ERK1/2 in the rat cortex after cerebral ischemia or reperfusion injury. Furthermore, EE reduces astrocytic swelling and injury. CONCLUSIONS: These findings suggest that EE could promote rehabilitation after ischemia via regulation of p-ERK1/2 expression, which may provide a therapeutic approach for cerebral ischemia or reperfusion injury. The suppression of postischemic astrocytic swelling in the brain of the ischemic rats through the intervention of EE would be one of the underlying mechanisms in the protective effect of cerebral ischemia.

Long-term radial extracorporeal shock wave therapy for neurogenic heterotopic ossification after spinal cord injury: A case report.

Context: Heterotopic ossification is characterized by abnormal growth of bone in soft tissues. Neurogenic heterotopic ossification is also closely related to central nervous system injuries and has been reported to respond to radial extracorporeal shock wave therapy.Findings: In this case, a radial extracorporeal shock wave therapy (five times per week, lasted for almost one year) was applied to a patient with neurogenic heterotopic ossification on the left hip as a result of spinal cord injury. Throughout the treatment session, the heterotopic ossification lesion was gradually diminished, associated with the increase in joint range of motion, pain mitigation and decrease in serum alkaline phosphatase level.Conclusion/clinical relevance: Long-term radial extracorporeal shock wave therapy offers a promising therapeutic alternative for neurogenic heterotopic ossification.

Environmental enrichment enhances post-ischemic cerebral blood flow and functional hyperemia in the ipsilesional somatosensory cortex.

Environmental enrichment has been reported to promote functional recovery in an ischemic stroke. However, the underlying mechanism remains unclear. This study aimed to investigate the effect of environmental enrichment treatment on post-ischemic cerebral blood flow and functional hyperemia in the ipsilesional primary somatosensory cortex of rats. With laser speckle imaging, we were able to monitor the resting cerebral blood flow alteration in the middle cerebral artery occlusion model. Both 3- and 28-day post-ischemic infarct volumes were then examined with triphenyltetrazolium chloride and cresyl violet staining, respectively. We found that an exposure to environmental enrichment was associated with higher post-ischemic cerebral blood flow and less brain tissue loss in the ipsilesional primary somatosensory cortex compared with the standard cage environment. Furthermore, environmental enrichment also enhanced the cerebral blood flow response to whisker stimulation in the ipsilesional barrel cortex when measured 28 days after the middle cerebral artery occlusion. Together, the data suggested that an exposure to environmental enrichment promoted the restoration of cerebral blood flow in the ipsilesional cortex and contributed to a better coupling between functional activation and cerebral blood flow change, which might be the possible mechanisms underlying the neuroprotective effects of EE after ischemia.

Changes in Mitochondria-Associated Protein Expression and Mitochondrial Function in Response to 2 Weeks of Enriched Environment Training After Cerebral Ischaemia-Reperfusion Injury.

An enriched environment (EE) can stimulate the recovery of neurological function following a cerebral ischaemia-reperfusion injury; however, the impact of EE's on mitochondrial function has been insufficiently studied. Our research aimed to assess whether EE's therapeutic impact involved the enhancement of mitochondrial dysfunction. Following 2 weeks of EE training, we tested both mitochondrial function and mitochondria-associated protein expression within the cerebral cortex following cerebral ischaemia-reperfusion injury. We subjected Sprague-Dawley rats to transient focal cerebral ischaemia and categorized the rats into three separate groups, i.e. an enriched environment (EE) group, a standard condition (SC) group and a sham control group (no middle cerebral artery embolization). The rats within the EE group were raised in enriched conditions for 2 weeks, while the rats within the SC group, in comparison, were reared in standard conditions for 2 weeks. After 2 weeks, the cerebral cortices of the rats were removed. We then measured a series of indices, i.e. the protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α), nuclear respiratory factor-1 (NRF-1), mitochondrial transcription factor A (TFAM) and mitochondrial protein cytochrome C oxidase subunit IV (COX IV). Furthermore, the number of mitochondria was evaluated through electron microscopy.EE upregulated the protein expression of PGC-1α, NRF-1 as well as TFAM, which function as the master regulators of mitochondrial biogenesis, in comparison with the SC group. The EE group's COX IV protein expression also exhibited an increase. Moreover, the amount of mitochondria in the peri-infarct region of the cortex increased as result of EE training. Over 2 weeks, EE training significantly increased mitochondrial biogenesis-associated protein expression and mitochondrial function. A possible mechanism of the EE leading to the improvement of neurological function is that it increases brain mitochondrial biogenesis after the rats' cerebral ischaemia-reperfusion injury. Mitochondrial biogenesis stimulation or enhancement could become an innovative strategy for neuroprotection in future treatment.

An enriched environment increases the expression of fibronectin type III domain-containing protein 5 and brain-derived neurotrophic factor in the cerebral cortex of the ischemic mouse brain.

Many studies have shown that fibronectin type III domain-containing protein 5 (FDNC5) and brain-derived neurotrophic factor (BDNF) play vital roles in plasticity after brain injury. An enriched environment refers to an environment that provides animals with multi-sensory stimulation and movement opportunities. An enriched environment has been shown to promote the regeneration of nerve cells, synapses, and blood vessels in the animal brain after cerebral ischemia; however, the exact mechanisms have not been clarified. This study aimed to determine whether an enriched environment could improve neurobehavioral functions after the experimental inducement of cerebral ischemia and whether neurobehavioral outcomes were associated with the expression of FDNC5 and BDNF. This study established ischemic mouse models using permanent middle cerebral artery occlusion (pMCAO) on the left side. On postoperative day 1, the mice were randomly assigned to either enriched environment or standard housing condition groups. Mice in the standard housing condition group were housed and fed under standard conditions. Mice in the enriched environment group were housed in a large cage, containing various toys, and fed with a standard diet. Sham-operated mice received the same procedure, but without artery occlusion, and were housed and fed under standard conditions. On postoperative days 7 and 14, a beam-walking test was used to assess coordination, balance, and spatial learning. On postoperative days 16-20, a Morris water maze test was used to assess spatial learning and memory. On postoperative day 15, the expression levels of FDNC5 and BDNF proteins in the ipsilateral cerebral cortex were analyzed by western blot assay. The results showed that compared with the standard housing condition group, the motor balance and coordination functions (based on beam-walking test scores 7 and 14 days after operation), spatial learning abilities (based on the spatial learning scores from the Morris water maze test 16-19 days after operation), and memory abilities (based on the memory scores of the Morris water maze test 20 days after operation) of the enriched environment group improved significantly. In addition, the expression levels of FDNC5 and BDNF proteins in the ipsilateral cerebral cortex increased in the enriched environment group compared with those in the standard housing condition group. Furthermore, the Pearson correlation coefficient showed that neurobehavioral functions were positively associated with the expression levels of FDNC5 and BDNF (r = 0.587 and r = 0.840, respectively). These findings suggest that an enriched environment upregulates FDNC5 protein expression in the ipsilateral cerebral cortex after cerebral ischemia, which then activates BDNF protein expression, improving neurological function. BDNF protein expression was positively correlated with improved neurological function. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Fudan University, China (approval Nos. 20160858A232, 20160860A234) on February 24, 2016.

Enriched Environment Elicits Proangiogenic Mechanisms After Focal Cerebral Ischemia.

Brain has limited capacity for spontaneous recovery of lost function after stroke. Exposure to enriched environment (EE) can facilitate functional recovery, but mechanisms underlying this effect are poorly understood. Here, we used a middle cerebral artery occlusion (MCAO) model to investigate the impact of EE on angiogenesis in the post-ischemic brain in adult male Sprague Dawley rats, and examined whether blood-borne factors may contribute. Compared with standard cage (SC), exposure to EE was associated with greater improvement in neurological function, higher peri-infarct vascular density, and higher chronic post-ischemic cerebral blood flow assessed by laser speckle imaging. The effect persisted for at least 28 days. EE also enhanced the expression of hepatocyte growth factor in the peri-ischemic cortex when measured 15 days after MCAO. Interestingly, serum from rats exposed to EE after MCAO showed elevated levels of hepatocyte growth factor, and plasma or serum from rats exposed to EE after MCAO enhanced the survival and proliferation of cultured endothelial cells, in vitro, when compared with control plasma or serum from SC group after MCAO. Together, our data suggest that exposure to EE promotes angiogenesis in the ischemic brain that may in part be mediated by blood-borne factors.

Effects of Transcutaneous Electrical Acupoint Stimulation on Motor Functions and Self-Care Ability in Children with Cerebral Palsy.

OBJECTIVES: To observe the effects of transcutaneous electrical acupoint stimulation (TEAS) in improving motor functions and self-care abilities in children with cerebral palsy in their early childhood. DESIGN: A preliminary, prospective, cohort study. SETTINGS/LOCATION: Multicenter. SUBJECTS: Children aged 2-6 years old. INTERVENTIONS: Twenty-three children were included in the study and randomly assigned to a control group ([CG] N = 11) or a therapeutic group ([TG] N = 12). In the TG, children were treated with TEAS (Shousanli [LI10] and Waiguan [SJ5]) plus the exercise therapy, while in the control group, they were treated with sham TEAS plus exercise therapy. Therapies were performed five days per week for eight weeks. OUTCOME MEASURES: The Gross Motor Function Measure (GMFM) and the Functional Independent Measurement for children (WeeFIM) were used to evaluate motor functions and self-care abilities before and after the therapies. RESULTS: Greater improvements were observed in the TG concerning all the measurements, although without statistical differences. The increments of the GMFM score and the WeeFIM motor, self-care and total scores were 36.08 ± 18.34 (26%), 16.17 ± 8.21 (33%), 7.67 ± 3.42 (40%) and 20.33 ± 10.08 (28%) in the TG, while 22.73 ± 16.54 (17%), 9.09 ± 9.43 (19%), 5.64 ± 6.73 (29%) and 12.82 ± 11.77 (18%) in the CG, respectively. No statistically significant correlations were shown between functional improvements and the demographics in the TG or the CG. The GMFM improvement was not statistically correlated with the improvements of the WeeFIM motor, self-care or total scores. However, the WeeFIM motor, self-care and total score were significantly positively correlated with one another in both groups (P < 0.01). No adverse effect was recorded during the study. CONCLUSION: TEAS may be effective in improving motor functions and self-care abilities in children with cerebral palsy, in addition to conventional exercise therapy. Larger samples are required to confirm the efficacies.

Apoptotic cell characteristics of rat brain microvascular endothelia induced by different degrees of hypoperfusion.

Hypoperfusion is one of the common causes of ischemic stroke. In this study, decreased blood perfusion and neurological damage were confirmed in ischemic rats. Further, the effect of different perfusion was researched in vivo. We found that hypoperfusion promoted the apoptosis of rats brain microvascular endothelial cells, and the more serious of hypoperfusion, the more obvious of apoptosis. At the same time, this process was related to Tie-2 receptor on cell membranes and Caspase-3 apoptotic pathways. Hemodynamics was one factors affecting the cerebral infarction.

[Effects of standardized three-stage rehabilitation on recovery of neurological function in stroke patients with hemiplegia].

OBJECTIVE: To explore the effects of standardized three stages' rehabilitation on the neurological function in stroke patients with hemiplegia. METHODS: All 52 patients firstly are brought into two blocks: primary cerebral infarction and primary cerebral hemorrhage then are divided into treated group and controlled group randomly. Patients in the treated group are given Standardized Three Stages' Rehabilitation, while those in the controlled group are only given normal internal medicine treatments that are the same as the treated group but without Standardized Three Stages' Rehabilitation. All patients would be assessed with the scale of Clinical Neurological Function Defects (CNFD) at the entering time, the end of 1st month, 3rd month and 6th month respectively after stroke. RESULTS: The scores of the treated group are lower than those of the controlled group (P < 0.001) at every stage, the margins between every stage's scores in the treated group are greater than those in the controlled group (P < 0.001). The scores of the treated group's patients are about 51%, 34%, 18% and 8% of total scores at the entering time, the end of 1st month, 3rd month and 6th month after stroke differently, but that of the controlled group are about 58%, 54%, 42% and 37% of total scores differently. The margins between the scores of entering time and that of the end of 1st month, 3rd month and 6th month in the treated group are 17%, 33% and 43% of total scores respectively, but that of the controlled group are about 5%, 16% and 21% of total scores differently. CONCLUSION: Standardized three stages' rehabilitation could promote stroke patients' motor function of every stage obviously.

Autophagy suppression by exercise pretreatment and p38 inhibition is neuroprotective in cerebral ischemia.

Autophagy is a degradative mechanism for cellular proteins and organelles, but its role in the nervous system is still not clear. In the present study, we found that exercise pretreatment and p38 inhibition had influence on autophagic process after cerebral ischemia, contributing to their neuroprotective effects. We examined the levels of p62 and phosphorylated ERK1/2 as an autophagic marker and cell-survival marker respectively after cerebral ischemic injury. The brain infarction volume after ischemia was measured as well. Both treadmill training pretreatment and p38 inhibition decreased the degradation of p62, promoted the phosphorylation of ERK1/2, and alleviated the brain infarction, indicating that these treatments could provide neuroprotection in cerebral ischemic injury via autophagy suppression.

Long-term three-stage rehabilitation intervention alleviates spasticity of the elbows, fingers, and plantar flexors and improves activities of daily living in ischemic stroke patients: a randomized, controlled trial.

To investigate the effects of rehabilitation interventions on spasticity and activities of daily living (ADL) in ischemic stroke patients. A total of 165 ischemic stroke patients were recruited and assigned randomly to a control group (CG, n=82) or a therapeutic group (TG, n=83). Rehabilitation interventions were performed in the TG. The Modified Ashworth Scale was used to evaluate the severity of spasticity in the fingers, elbows, and plantar flexors, and the Modified Barthel Index (MBI) was used to measure ADL performance. Evaluations were performed at baseline (M0) and at the end of the first, third, and sixth months (M1, M3, M6) after enrollment. At M0, 20.8% (16/77) in the CG and 29.9% (23/77) in the TG developed spasticity, whereas at M6, the incidence of spasticity increased to 36.4% (28/77) in the TG and 42.9% (33/77) of patients in the CG. Fewer patients developed spasticity in the fingers, elbows, and ankles in the TG than CG, respectively. Both groups showed significant improvements in MBI scores (M6 vs. M0, P<0.01). MBI scores correlated negatively with the severity of spasticity in both groups at M6. Long-term standardized rehabilitation interventions alleviate spasticity and promote ADL with the presence of minor spasticity (Supplementary video, Supplemental digital content 1, http://links.lww.com/WNR/A291).

A prospective, randomized, single-blinded trial on the effect of early rehabilitation on daily activities and motor function of patients with hemorrhagic stroke.

To investigate whether early rehabilitation has a positive impact on the recovery of the activities of daily living and motor function after intracerebral hemorrhagic stroke, 364 patients with hemorrhagic stroke were selected and randomly divided into a rehabilitation group and a control group. The rehabilitation group underwent a standardized, three-stage rehabilitation program. The control group was treated with standard hospital ward, internal medical intervention. The simplified Fugl-Myere assessment scale (FMA) and Modified Barthel Index (MBI) were administered at various time points. The magnitude of improvement was significantly higher in the rehabilitation group than in the control group for both the FMA (p<0.05) and MBI scores (p<0.05). The greatest improvement was observed in the first month post-stroke. Thus, our study shows that early rehabilitation can significantly improve the daily activities and motor functions of patients with stroke.