SUMMARY Brain endothelial cells form a paracellular and transcellular barrier to many blood-borne solutes via tight junctions (TJs) and scarce endocytotic vesicles. The blood-brain barrier (BBB) plays a pivotal role in the healthy and diseased CNS. BBB damage after ischemic stroke contributes to increased mortality, yet the contributions of paracellular and transcellular mechanisms to this process in vivo are unknown. We have created a novel transgenic mouse strain whose endothelial TJs are labeled with eGFP and have imaged dynamic TJ changes and fluorescent tracer leakage across the BBB in vivo, using two-photon microscopy in the t-MCAO stroke model. Although barrier function is impaired as early as 6 h post-stroke, TJs display profound structural defects only after two days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 h post-stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.

Clinical and research efforts have focused on promoting functional recovery after stroke. Brain stimulation strategies are particularly promising because they allow direct manipulation of the target area's excitability. However, elucidating the cell type and mechanisms mediating recovery has been difficult because existing stimulation techniques nonspecifically target all cell types near the stimulated site. To circumvent these barriers, we used optogenetics to selectively activate neurons that express channelrhodopsin 2 and demonstrated that selective neuronal stimulations in the ipsilesional primary motor cortex (iM1) can promote functional recovery. Stroke mice that received repeated neuronal stimulations exhibited significant improvement in cerebral blood flow and the neurovascular coupling response, as well as increased expression of activity-dependent neurotrophins in the contralesional cortex, including brain-derived neurotrophic factor, nerve growth factor, and neurotrophin 3. Western analysis also indicated that stimulated mice exhibited a significant increase in the expression of a plasticity marker growth-associated protein 43. Moreover, iM1 neuronal stimulations promoted functional recovery, as stimulated stroke mice showed faster weight gain and performed significantly better in sensory-motor behavior tests. Interestingly, stimulations in normal nonstroke mice did not alter motor behavior or neurotrophin expression, suggesting that the prorecovery effect of selective neuronal stimulations is dependent on the poststroke environment. These results demonstrate that stimulation of neurons in the stroke hemisphere is sufficient to promote recovery. stroke recovery | channelrhodopsin

Real-time imaging of transplanted stem cells is essential for understanding their interactions in vivo with host environments, for tracking cell fate and function and for successful delivery and safety monitoring in the clinical setting. In this study, we used bioluminescence (BLI) and magnetic resonance imaging (MRI) to visualize the fate of grafted human embryonic stem cell (hESC)-derived human neural stem cells (hNSCs) in stroke-damaged rat brain. The hNSCs were genetically engineered with a lentiviral vector carrying a double fusion (DF) reporter gene that stably expressed enhanced green fluorescence protein (eGFP) and firefly luciferase (fLuc) reporter genes. The hNSCs were self-renewable, multipotent, and expressed markers for neural stem cells. Cell survival was tracked noninvasively by MRI and BLI for 2 months after transplantation and confirmed histologically. Electrophysiological recording from grafted GFP(+) cells and immuno-electronmicroscopy demonstrated connectivity. Grafted hNSCs differentiated into neurons, into oligodendrocytes in stroke regions undergoing remyelination and into astrocytes extending processes toward stroke-damaged vasculatures. Our data suggest that the combination of BLI and MRI modalities provides reliable real-time monitoring of cell fate.

Background and Purpose Hypoxic-Ischemic (HI) brain injury in newborn infants represents a major cause of cerebral palsy, development delay and epilepsy. Stem cell-based therapy has the potential to rescue and replace the ischemic tissue caused by HI and to restore function. However, the mechanisms by which stem cell transplants induce functional recovery are yet to be elucidated. In the present study, we sought to investigate the efficacy of human neural stem cells (hNSCs) derived from human embryonic stem cells (hESCs), in the rat model of neonatal HI and the mechanisms enhancing brain repair. Methods The hNSCs were genetically engineered for in vivo molecular imaging and for postmortem histological tracking. Twenty-four hours after the induction of HI, animals were grafted with hNSCs into the forebrain. Motor behavioral tests were performed the fourth week after transplantation. We used immunocytochemistry and neuroanatomical tracing to analyze neural differentiation, axonal sprouting and microglia response. Treatment-induced changes in gene expression were investigated by microarray and quantitative PCR. Results Bioluminescence imaging (BLI) permitted longitudinal tracking of grafted hNSCs in real time. HI transplanted animals significantly improved in their use of the contralateral impeded forelimb and in the rotarod test. The grafts showed good survival, dispersion and differentiation. We observed an increase of uniformly distributed microglia cells in the grafted side. Anterograde neuronanatomical tracing demonstrated significant contralesional sprouting. Microarray analysis revealed upregulation of genes involved in neurogenesis, gliogenesis and neurotrophic support. Conclusions These results suggest that hNSC transplants enhance endogenous brain repair through multiple modalities in response to HI.

Tissue plasminogen activator is the only treatment option for stroke victims; however, it has to be administered within 4.5 h after symptom onset, making its use very limited. This report describes a unique target for effective treatment of stroke, even 12 h after onset, by the administration of αB-crystallin (Cryab), an endogenous immunomodulatory neuroprotectant. In Cryab −/− mice, there was increased lesion size and diminished neurologic function after stroke compared with wild-type mice. Increased plasma Cryab was detected after experimental stroke in mice and after stroke in human patients. Administration of Cryab even 12 h after experimental stroke reduced both stroke volume and inflammatory cytokines associated with stroke pathology. Cryab is an endogenous anti-inflammatory and neuroprotectant molecule produced after stroke, whose beneficial properties can be augmented when administered therapeutically after stroke.

Sphingosine-1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. Sphingosine phosphate receptor 1 (S1P1) agonist, FTY-720 (Gilenya™) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring a S1pr1 gene encoding phosphorylation-deficient receptors [S1P1(S5A)] developed severe experimental autoimmune encephalomyelitis (EAE) due to T helper (TH) 17-mediated autoimmunity in the peripheral immune and nervous system. S1P1 directly activated Janus-like kinase–signal transducer and activator of transcription 3 (JAK-STAT3) pathway via interleukin 6 (IL-6). Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.

Detailed behavioral analysis is key to understanding the brain-behavior relationship. Here, we present deep learning-based methods for analysis of behavior imaging data in mice and humans. Specifically, we use three different convolutional neural network architectures and five different behavior tasks in mice and humans and provide detailed instructions for rapid implementation of these methods for the neuroscience community. We provide examples of three dimensional (3D) kinematic analysis in the food pellet reaching task in mice, three-chamber test in mice, social interaction test in freely moving mice with simultaneous miniscope calcium imaging, and 3D kinematic analysis of two upper extremity movements in humans (reaching and alternating pronation/supination). We demonstrate that the transfer learning approach accelerates the training of the network when using images from these types of behavior video recordings. We also provide code for post-processing of the data after initial analysis with deep learning. Our methods expand the repertoire of available tools using deep learning for behavior analysis by providing detailed instructions on implementation, applications in several behavior tests, and post-processing methods and annotated code for detailed behavior analysis. Moreover, our methods in human motor behavior can be used in the clinic to assess motor function during recovery after an injury such as stroke.

Object Decompressive surgery can be life saving after malignant cerebral infarction. However, severe residual disability occurs in a significant number of surviving patients. Most discussion about the benefits of surgery is based on studies performed in patients who are ≤ 60 years of age. Less is known about the benefits of the procedure in the elderly population. The authors undertook a review of the literature on decompressive craniectomy for malignant cerebral infarction and compared the mortality and outcome data published in patients older and younger than 60 years of age. The authors discuss their analysis, with specific reference to the limitations of the studies analyzed, the outcome measures used, and the special considerations required when discussing stroke recovery in the elderly. Methods Studies on decompressive craniectomy for malignant middle cerebral artery infarction reported in the English literature were analyzed. A cutoff point for age of > 60 or ≤ 60 years was set, and the study population was segregated. No studies specifically analyzed patients > 60 years old. A total of 19 studies was identified, 10 of which included patients who were > 60 years of age. A comparison between the 2 age groups was made within the 10 studies and also among all the patients in the 19 studies. Mortality rates and outcome scores were assessed for each study, and a Barthel Index (BI) score of < 60 or a modified Rankin Scale (mRS) score of > 3 was considered to represent a poor outcome. Rates were compared using the Fisher exact test, and p values < 0.05 were considered statistically significant. Results Nineteen studies were found, which included 273 patients undergoing decompressive craniectomy for malignant cerebral infarcts. Ten of these studies included 73 patients (26.7%) who were > 60 years of age. The mean follow-up times ranged from 5.75 to 12.3 months in the > 60-years group and 4.2 to 28 months in the ≤ 60-years group. The mortality rate was significantly higher, at 51.3% in the > 60-years group (37 of 72 patients) compared with 20.8% (41 of 197 patients) in the ≤ 60-years group (p < 0.0001). Similarly, patients who survived in the > 60-years group had significantly higher rates of poor outcomes, at 81.8% (27 of 33), compared with 33.1% (47 of 142) in the ≤ 60-year-old group (p < 0.0001). The BI was the most commonly used primary outcome measure (15 out of 19 studies), followed by the mRS score, which was used in 4 studies. Conclusions The mortality rate and functional outcome, as measured by the BI and mRS, were significantly worse in patients > 60 years of age following decompressive craniectomy for malignant infarction. Age is an important factor to consider in patient selection for surgery. However, cautious interpretation of the results is required because the outcome scores that were used only measure physical disability, whereas other factors, including psychosocial, financial, and caregiver burden, should be considered in addition to age alone.