An Enriched Environment Promotes Motor Function through Neuroprotection after Cerebral Ischemia.

Stroke seriously affects human health. Many studies have shown that enriched environment (EE) can promote functional recovery after stroke, but the intrinsic mechanisms remain unclear. In order to study the internal mechanisms of EE involved in functional recovery after ischemic stroke and which mechanism plays a leading role in the recovery of limb function after cerebral infarction, key proteins potentially involved in neuronal protection and synaptic remodeling in the ischemic penumbra have been investigated. In this study, adult C57BL/6 mice after permanent middle cerebral artery occlusion (pMCAO) were assigned to the EE and standard housing (SH) groups 3 days after operation. The EE house was spacious that contained a large variety of small toys; the SH was a normal sized cage. Sham-operated mice without artery occlusion were housed under standard conditions and were fed a normal diet. On days 3, 7, 14, and 21, postoperative motor functional recovery was tested using the modified neurological severity score (mNSS) and the Rotarod test. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), growth-associated protein-43 (GAP-43), and synaptophysin (SYN) was examined by western blotting and immunofluorescence staining. The motor functional recovery (based on the mNSS and Rotarod test 3, 7, 14, and 21 days post operation) of mice in the EE group improved significantly compared to the SH group. The expression of GAP-43 and SYN and the ratio of Bcl-2/Bax were all upregulated in the EE group compared to the SH group. In addition, we also explored the relationship between neuronal protection and synaptic remodeling in the EE-mediated recovery of limb function after cerebral infarction by correlation analysis. Correlation analysis showed that compared with the increase of Bcl-2/Bax ratio, the increased expression of GAP-43 and SYN was more closely related to the recovery of limb function in ischemic mice. These data support the hypothesis that EE can promote the process of improvement of limb dysfunction induced by ischemic stroke, and this behavior restoration may, via promoting neuroprotection in the ischemic penumbra, be dependent on the regulation of the expression of GAP-43, SYN, Bcl-2, and Bax. A limitation of the study was that we only observed several representative key indicators of synaptic remodeling and neuronal apoptosis, without an in-depth study of the potential mechanisms involved.

The Muscle Activation Differences in Post-Stroke Upper Limb Flexion Synergy Based on Spinal Cord Segments: A Preliminary Proof-of-Concept Study.

Objective: This study examined the activation difference of muscles innervated by cervical cord 5-6 (C5-C6) and cervical cord 8- thoracic cord 1 (C8-T1) in upper limb flexion synergy after stroke. Methods: Surface electromyography (sEMG) signals were collected during elbow flexion in stroke patients and healthy controls. The study compared normalized activation of two pairs of muscles that could cause similar joint movement but which dominated different spinal cord segments (clavicular part of the pectoralis major, PC vs. Sternocostal part of the pectoralis major, PS; Flexor carpi radialis, FCR vs. Flexor carpi ulnaris, FCU). In each muscle pair, one muscle was innervated by the same spinal cord segment (C5-C6), dominating the elbow flexion and the other was not. The comparison of the activation of the same muscle between patients and healthy controls was undertaken after standardization based on the activation of the biceps brachii in elbow flexion. Results: There was no difference between the PC and PS's normalized activation in healthy controls while the PC's normalized activation was higher than PS in stroke patients during elbow flexion. Similarly, there was no significant difference in normalized activation between FCR and FCU in healthy controls, and the same is true for stroke patients. However, the standardized activation of both FCR and FCU in stroke patients was significantly lower than that in healthy controls. Conclusion: After stroke, the activation of the distal muscles of the upper limb decreased significantly regardless of the difference of spinal cord segments; while the activation of the proximal muscles innervated by the same spinal cord segment (C5-C6) dominating the elbow flexion showed higher activation during flexion synergy. The difference in muscle activation based on spinal cord segments may be the reason for the stereotyped joint movement of upper limb flexion synergy.

Cortical excitability signatures for the degree of sleepiness in human.

Sleep is essential in maintaining physiological homeostasis in the brain. While the underlying mechanism is not fully understood, a 'synaptic homeostasis' theory has been proposed that synapses continue to strengthen during awake and undergo downscaling during sleep. This theory predicts that brain excitability increases with sleepiness. Here, we collected transcranial magnetic stimulation measurements in 38 subjects in a 34 hr program and decoded the relationship between cortical excitability and self-report sleepiness using advanced statistical methods. By utilizing a combination of partial least squares regression and mixed-effect models, we identified a robust pattern of excitability changes, which can quantitatively predict the degree of sleepiness. Moreover, we found that synaptic strengthen occurred in both excitatory and inhibitory connections after sleep deprivation. In sum, our study provides supportive evidence for the synaptic homeostasis theory in human sleep and clarifies the process of synaptic strength modulation during sleepiness.

Differential Changed Excitability of Spinal Motor Neurons Innervating Tibialis Anterior and Peroneus Muscles Cause Foot Inversion After Stroke.

Objective: To study differential post-stroke changes of excitability of spinal motor neurons innervating a group of antagonist muscles of ankle and their effects on foot inversion. Methods: F waves in tibialis anterior (TA) and peroneus muscles (PN) were recorded. The condition of spasticity and foot inversion in stroke patients were also evaluated. The differences of F wave parameters between patients and healthy controls (HC), as well as TA and PN, were investigated. Results: There were natural differences in the persistence of the F waves (Fp) and F/M amplitude ratio (F/M) between TA and PN in HC. Stroke patients showed significantly higher F/M in TA and PN, while there was no difference in Fp comparing to HC. The natural differences in F wave parameters between TA and PN were differentially retained after stroke. The natural difference of the two muscles in Fp remained unchanged and the F/M difference disappeared in those without spasticity or foot inversion, while the Fp difference disappeared and the F/M difference remained in those with spasticity or foot inversion. Conclusion: Based on the natural difference of the number and size of spinal motor neurons innervating TA and PN, their excitability may change differently according to the severity of the stroke, which may be the reason of foot inversion.

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.

The effect of magnetic stimulation on differentiation of human induced pluripotent stem cells into neuron.

The effect of stem cell transplantation in the treatment of neural lesions is so far not satisfactory. Magnetic stimulation is a feasible exogenous interference to improve transplantation outcome. However, the effect of magnetic stimulation on the differentiation of induced pluripotent stem cells (iPSCs) into neuron has not been studied. In this experiment, an in vitro neuron differentiation system from human iPSCs were established and confirmed. Three magnetic stimuli (high frequency [HF], low frequency [LF], intermittent theta-burst stimulation [iTBS]) were applied twice a day during the differentiation process. Immunofluorescence and quantitative polymerase chain reaction (Q-PCR) were performed to analyze the effect of magnetic stimulation. Neural stem cells were obtained on day 12, manifested as floating neurospheres expressing neural precursor markers. All groups can differentiate into neurons while glial cell markers were not detected. Both Immunofluorescence and PCR results showed LF and iTBS increased the transcription and expression of neuronal nuclei (NeuN). HF significantly increased vesicular glutamate transporters2 transcription while iTBS promoted transcription of both synaptophysin and postsynaptic density protein 95. These results indicate that LF and iTBS can promote the generation of mature neurons from human iPSCs; HF may promote differentiate into glutamatergic neurons while iTBS may promote synapse formation during the differentiation.

The Neuronal Activation of Deep Cerebellar Nuclei Is Essential for Environmental Enrichment-Induced Post-Stroke Motor Recovery.

The level of cerebellar activity in stroke patients has been shown to correlate with the extent of functional recovery. We reasoned that the cerebellum may be an important player in post-stroke rehabilitation. Because the neurons in the deep cerebellar nuclei (DCN) represent virtually all of the output from the cerebellum, in this study, using environmental enrichment (EE) to promote rehabilitation, we investigated the influence of the optogenetic neuronal modulation of DCN on EE-induced rehabilitation. We found that neuronal inhibition of the DCN almost completely blocked motor recovery in EE treated mice, but the stroke mice with neuronal activation of the DCN achieved a similar recovery level as those in the EE treated group. No difference was observed in anxiety-like behavior. Moreover, Htr2a in the DCN, the gene encoding 5-HT2A receptor, was shown to be a hub gene in the protein-protein interaction network identified using RNA-seq. This indicated that 5-HT2A receptor-mediated signaling may be responsible for DCN-dependent functional improvement in EE. We further verified this using the 5-HT2A receptor antagonist, MDL100907, to inhibit the function of 5-HT2A receptor in the DCN. This treatment resulted in impaired recovery in EE treated mice, who performed at a level as poor as the stroke-only group. Thus, this work contributes to an understanding of the importance of the DCN activation in EE-induced post-stroke rehabilitation. Attempts to clarify the mechanism of 5-HT2A receptor-mediated signaling in the DCN may also lead to the creation of a pharmacological mimetic of the benefits of EE-induced rehabilitation.

Remote limb ischemic postconditioning promotes motor function recovery in a rat model of ischemic stroke via the up-regulation of endogenous tissue kallikrein.

AIMS: Remote ischemic conditionings, such as pre- and per-conditioning, are known to provide cardioprotection in animal models of ischemia. However, little is known about the neuroprotection effect of postconditioning after cerebral ischemia. In this study, we aim to evaluate the motor function rescuing effect of remote limb ischemic postconditioning (RIPostC) in a rat model of acute cerebral stroke. METHODS: Left middle cerebral artery occlusion (MCAO) was performed to generate the rat model of ischemic stroke, followed by daily RIPostC treatment for maximum 21 days. The motor function after RIPostC was assessed with foot fault test and balance beam test. Local infarct volume was measured through MRI scanning. Neuronal status was evaluated with Nissl's, HE, and MAP2 immunostaining. Lectin immunostaining was performed to evaluate the microvessel density and area. RESULTS: Daily RIPostC for more than 21 days promoted motor function recovery and provided long-lasting neuroprotection after MCAO. Reduced infarct volume, rescued neuronal loss, and enhanced microvessel density and size in the injured areas were observed. In addition, the RIPostC effect was associated with the up-regulation of endogenous tissue kallikrein (TK) level in circulating blood and local ischemic brain regions. A TK receptor antagonist HOE-140 partially reversed RIPostC-induced improvements, indicating the specificity of endogenous TK mediating the neuroprotection effect of RIPostC. CONCLUSION: Our study demonstrates RIPostC treatment as an effective rehabilitation therapy to provide motor function recovery and alleviate brain impairment in a rat model of acute cerebral ischemia. We also for the first time provide evidence showing that the up-regulation of endogenous TK from remote conditioning regions underlies the observed effects of RIPostC.

Efficacy of transcranial alternating current stimulation over bilateral mastoids (tACSbm) on enhancing recovery of subacute post-stroke patients.

BACKGROUND AND AIMS: Transcranial alternating current stimulation (tACS) offers another method of non-invasive brain stimulation in post-stroke rehabilitation. Because it is not known if tACS over bilateral mastoids (tACSbm) can promote the functional recovery in subacute post-stroke patients, we wish to learn the effect of tACSbm on improving neurological function and intracranial hemodynamics of subacute post-stroke patients. METHODS: Sixty subacute post-stroke patients (mean age: 65.4 ± 9.8 years), 15 to 60 days after the onset, were randomly assigned to receiving 15 sessions of usual rehabilitation program without (n = 30) or with tACSbm (20 Hz and < 400 µA for 30-min; n = 30). The outcome measures included the NIH Stroke Scale (NIHSS) and measures of intracranial hemodynamics before and after treatment. RESULTS: At the fifteenth session, when compared with the baseline, the mean NIHSS scores of the patients in the tACSbm group had significantly a larger decrease [18.3 ± 2.6 vs. 10.8 ± 2.7; p < 0.001] than that of the control group [19.1 ± 2.7 vs. 13.0 ± 2.4] [F(1,54) = 4.29, p = 0.043]. After both the first and fifteenth sessions, compared with the control group, the mean blood flow velocity (MFVs) of the tACSbm group had significantly larger increase in the MCA, ACA, and PCA (p < 0.001), the Gosling pulsatility index (PI) of the tACSbm group had also significantly larger decline in the MCA, ACA, and PCA than that of the control group (p < 0.001). The best predictor of the changes in the NIHSS scores was the decline in the pulsatility index in the vascular territory of both lesional and non-lesional MCA measured by the end of the last treatment session. CONCLUSIONS: tACSbm appeared to be effective for enhancing patients' functional recovery and cerebral hemodynamics in the subacute phase. The extent of recovery seems to be associated with the decline of the resistance in vascular bed of the main cerebral arteries. The mechanisms behind this effect should be explored further through research.