Rab7a activation promotes degradation of select tight junction proteins at the blood-brain barrier after ischemic stroke.

The stability of tight junctions (TJs) between endothelial cells (ECs) is essential to maintain blood-brain barrier (BBB) function in the healthy brain. Following ischemic stroke, TJ strand dismantlement due to protein degradation leads to BBB dysfunction, yet the mechanisms driving this process are poorly understood. Here, we show that endothelial-specific ablation of Rab7a, a small GTPase that regulates endolysosomal protein degradation, reduces stroke-induced TJ strand disassembly resulting in decreased paracellular BBB permeability and improved neuronal outcomes. Two pro-inflammatory cytokines, TNFα and IL1ß, but not glucose and oxygen deprivation, induce Rab7a activation via Ccz1 in brain ECs in vitro, leading to increased TJ protein degradation and impaired paracellular barrier function. Silencing Rab7a in brain ECs in vitro reduces cytokine-driven endothelial barrier dysfunction by suppressing degradation of a key BBB TJ protein, Claudin-5. Thus, Rab7a activation by inflammatory cytokines promotes degradation of select TJ proteins leading to BBB dysfunction after ischemic stroke.

Vim-Thalamic Deep Brain Stimulation for Cervical Dystonia and Upper-Limb Tremor: Quantification by Markerless-3D Kinematics and Accelerometry.

Background: Deep Brain Stimulation (DBS) for dystonia is usually targeted to the globus pallidus internus (GPi), though stimulation of the ventral-intermediate nucleus of the thalamus (Vim) can be an effective treatment for phasic components of dystonia including tremor. We report on a patient who developed a syndrome of bilateral upper limb postural and action tremor and progressive cervical dystonia with both phasic and tonic components which were responsive to Vim DBS. We characterize and quantify this effect using markerless-3D-kinematics combined with accelerometry. Methods: Stereo videography was used to record our subject in 3D. The DeepBehavior toolbox was applied to obtain timeseries of joint position for kinematic analysis [1]. Accelerometry was performed simultaneously for comparison with prior literature. Results: Bilateral Vim DBS improved both dystonic tremor magnitude and tonic posturing. DBS of the hemisphere contralateral to the direction of dystonic head rotation (left Vim) had greater efficacy. Assessment of tremor magnitude by 3D-kinematics was concordant with accelerometry and was able to quantify tonic dystonic posturing. Discussion: In this case, Vim DBS treated both cervical dystonic tremor and dystonic posturing. Markerless-3D-kinematics should be further studied as a method of quantifying and characterizing tremor and dystonia.

Machine Learning for 3D Kinematic Analysis of Movements in Neurorehabilitation.

PURPOSE OF REVIEW: Recent advances in the machine learning field, especially in deep learning, provide the opportunity for automated, detailed, and unbiased analysis of motor behavior. Although there has not yet been wide use of these techniques in the motor rehabilitation field, they have great potential. In this review, I describe how the current state of machine learning can be applied to 3D kinematic analysis, and how this will have an impact on neurorehabilitation. RECENT FINDINGS: Applications of deep learning methods, in the form of convolutional neural networks, have been revolutionary for image analysis such as face recognition and object detection in images, exceeding human level performance. Recent studies have shown applicability of these deep learning approaches to human posture and movement classification. It is to be expected that portable stereo-camera systems will bring 3D pose estimation into the clinical setting and allow the assessment of movement quality in response to interventions. Advances in machine learning can help automate the process of obtaining 3D kinematics of human movements and to identify/classify patterns of movement.

A Step-by-Step Implementation of DeepBehavior, Deep Learning Toolbox for Automated Behavior Analysis.

Understanding behavior is the first step to truly understanding neural mechanisms in the brain that drive it. Traditional behavioral analysis methods often do not capture the richness inherent to the natural behavior. Here, we provide detailed step-by-step instructions with visualizations of our recent methodology, DeepBehavior. The DeepBehavior toolbox uses deep learning frameworks built with convolutional neural networks to rapidly process and analyze behavioral videos. This protocol demonstrates three different frameworks for single object detection, multiple object detection, and three-dimensional (3D) human joint pose tracking. These frameworks return cartesian coordinates of the object of interest for each frame of the behavior video. Data collected from the DeepBehavior toolbox contain much more detail than traditional behavior analysis methods and provides detailed insights to the behavior dynamics. DeepBehavior quantifies behavior tasks in a robust, automated, and precise way. Following the identification of behavior, post-processing code is provided to extract information and visualizations from the behavioral videos.

DeepBehavior: A Deep Learning Toolbox for Automated Analysis of Animal and Human Behavior Imaging Data.

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.

Meningeal Mast Cells as Key Effectors of Stroke Pathology.

Stroke is the leading cause of adult disability in the United States. Because post-stroke inflammation is a critical determinant of damage and recovery after stroke, understanding the interplay between the immune system and the brain after stroke holds much promise for therapeutic intervention. An understudied, but important aspect of this interplay is the role of meninges that surround the brain. All blood vessels travel through the meningeal space before entering the brain parenchyma, making the meninges ideally located to act as an immune gatekeeper for the underlying parenchyma. Emerging evidence suggests that the actions of immune cells resident in the meninges are essential for executing this gatekeeper function. Mast cells (MCs), best known as proinflammatory effector cells, are one of the long-term resident immune cells in the meninges. Here, we discuss recent findings in the literature regarding the role of MCs located in the meningeal space and stroke pathology. We review the latest advances in mouse models to investigate the roles of MCs and MC-derived products in vivo, and the importance of using these mouse models. We examine the concept of the meninges playing a critical role in brain and immune interactions, reevaluate the perspectives on the key effectors of stroke pathology, and discuss the opportunities and challenges for therapeutic development.

Evidence that meningeal mast cells can worsen stroke pathology in mice.

Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine (C-C motif) ligand 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke.

Optogenetic neuronal stimulation promotes functional recovery after 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.

Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.

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 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 hr after stroke, TJs display profound structural defects only after 2 days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 hr after stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.

Defective sphingosine 1-phosphate receptor 1 (S1P1) phosphorylation exacerbates TH17-mediated autoimmune neuroinflammation.

Sphingosine 1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. The 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 an S1pr1 gene encoding phosphorylation-deficient receptors (S1P1(S5A)) developed severe experimental autoimmune encephalomyelitis (EAE) due to autoimmunity mediated by interleukin 17 (IL-17)-producing helper T cells (TH17 cells) in the peripheral immune and nervous system. S1P1 directly activated the Jak-STAT3 signal-transduction pathway via IL-6. Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.

Systemic augmentation of alphaB-crystallin provides therapeutic benefit twelve hours post-stroke onset via immune modulation.

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.

Human neural stem cell grafts modify microglial response and enhance axonal sprouting in neonatal hypoxic-ischemic brain injury.

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 derived from human embryonic stem cells in a rat model of neonatal HI and the mechanisms enhancing brain repair. METHODS: The human neural stem cells 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 human neural stem cells 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 polymerase chain reaction. RESULTS: Bioluminescence imaging permitted real time longitudinal tracking of grafted human neural stem cells. HI transplanted animals significantly improved in their use of the contralateral impeded forelimb and in the Rotorod test. The grafts showed good survival, dispersion, and differentiation. We observed an increase of uniformly distributed microglia cells in the grafted side. Anterograde neuroanatomical tracing demonstrated significant contralesional sprouting. Microarray analysis revealed upregulation of genes involved in neurogenesis, gliogenesis, and neurotrophic support. CONCLUSIONS: These results suggest that human neural stem cell transplants enhance endogenous brain repair through multiple modalities in response to HI.

Efficacy of endovascular stenting in dural venous sinus stenosis for the treatment of idiopathic intracranial hypertension.

Multiple pathophysiological mechanisms have been proposed for the increased intracranial pressure observed in idiopathic intracranial hypertension (IIH). The condition is well characterized, with intractable headaches, visual obscurations, and papilledema as dominant features, mainly affecting obese women. With the advent of MR venography and increased use of cerebral angiography, there has been recent emphasis on the significant number of patients with IIH found to have associated nonthrombotic dural venous sinus stenosis. This has led to a renewed interest in endovascular stenting as a treatment for IIH. However, the assumption that venous stenosis leads to a high pressure gradient that decreases CSF resorption through arachnoid villi requires further evidence. In this paper, the authors analyze the published results to date of dural venous sinus stenting in patients with IIH. They also present a case from their institution for illustration. The pathophysiological mechanism in IIH requires further elucidation, but venous sinus stenosis with subsequent intracranial hypertension appears to be an important mechanism in at least a subgroup of patients with IIH. Among these patients, 78% had complete relief or improvement of their main presenting symptoms after endovascular stenting. Resolution or improvement in papilledema was seen in 85.1% of patients. Endovascular stenting should be considered whenever venous sinus stenosis is diagnosed in patients with IIH.

Functional engraftment of the medial ganglionic eminence cells in experimental stroke model.

Currently there are no effective treatments targeting residual anatomical and behavioral deficits resulting from stroke. Evidence suggests that cell transplantation therapy may enhance functional recovery after stroke through multiple mechanisms. We used a syngeneic model of neural transplantation to explore graft-host communications that enhance cellular engraftment.The medial ganglionic eminence (MGE) cells were derived from 15-day-old transgenic rat embryos carrying green fluorescent protein (GFP), a marker, to easily track the transplanted cells. Adult rats were subjected to transient intraluminal occlusion of the medial cerebral artery. Two weeks after stroke, the grafts were deposited into four sites, along the rostro-caudal axis and medially to the stroke in the penumbra zone. Control groups included vehicle and fibroblast transplants. Animals were subjected to motor behavioral tests at 4 week posttransplant survival time. Morphological analysis demonstrated that the grafted MGE cells differentiated into multiple neuronal subtypes, established synaptic contact with host cells, increased the expression of synaptic markers, and enhanced axonal reorganization in the injured area. Initial patch-clamp recording demonstrated that the MGE cells received postsynaptic currents from host cells. Behavioral analysis showed reduced motor deficits in the rotarod and elevated body swing tests. These findings suggest that graft-host interactions influence the fate of grafted neural precursors and that functional recovery could be mediated by neurotrophic support, new synaptic circuit elaboration, and enhancement of the stroke-induced neuroplasticity.

Assessment of outcome following decompressive craniectomy for malignant middle cerebral artery infarction in patients older than 60 years of age.

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 < or = 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 < 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 < or = 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 < or = 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 < or = 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.

Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat brain.

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.

Lymphangiomatosis in a child: eight years' follow-up without treatment.

Lymphangiomatosis of the spleen with diffuse lymphangiomatosis of the bone is extremely rare and there is no consensus about management at present. Here, the authors report a child presenting initially with back pain and diagnosed as lymphangiomatosis on several sites throughout the bony structures and the spleen. The patient is in a stable state at the end of 8 years without any treatment. The authors also discuss the follow-up option that may be considered as a strategy for management.