Age-induced alterations of granulopoiesis generate atypical neutrophils that aggravate stroke pathology.

Aging accounts for increased risk and dismal outcome of ischemic stroke. Here, we investigated the impact of age-related changes in the immune system on stroke. Upon experimental stroke, compared with young mice, aged mice had increased neutrophil clogging of the ischemic brain microcirculation, leading to worse no-reflow and outcomes. Aged mice showed an enhanced granulopoietic response to stroke that led to the accumulation of CD101+CD62Llo mature and CD177hiCD101loCD62Llo and CD177loCD101loCD62Lhi immature atypical neutrophils in the blood, endowed with increased oxidative stress, phagocytosis and procoagulant features. Production of CXCL3 by CD62Llo neutrophils of the aged had a key role in the development and pathogenicity of aging-associated neutrophils. Hematopoietic stem cell rejuvenation reverted aging-associated neutropoiesis and improved stroke outcome. In elderly patients with ischemic stroke, single-cell proteome profile of blood leukocytes identified CD62Llo neutrophil subsets associated with worse reperfusion and outcome. Our results unveil how stroke in aging leads to a dysregulated emergency granulopoiesis impacting neurological outcome.

Exploring thrombus composition in cerebral venous thrombosis: the first case report with initial insights and implications for treatment advancements.

BACKGROUND: Endovascular thrombectomy (EVT) is a treatment option in patients with a cerebral venous thrombosis (CVT) who deteriorate despite anticoagulant treatment. Assessment of thrombus composition in CVT may provide insights into the pathophysiology of the disease and suggest new therapeutic strategies. CASE REPORT: A 47-year-old woman (smoking habit and estradiol/progesterone-releasing intra-uterine device) diagnosed with massive CVT underwent EVT (complete recanalization via aspiration catheter and stentriever) due to acute-onset left-sided weakness and dysarthria despite 72 h of full-dose subcutaneous low-molecular heparin. Two main reddish clots (maximum diameter 15 mm) were retrieved. Microscopic assessment showed an erythrocyte-rich thrombus (83.9% of entire thrombus surface) with layers of platelets/fibrin (lines of Zahn: 13.9% fibrin and 38.5% platelet [CD61+]). The immunological profile was dominated by neutrophils (30% MPO+), with neutrophil extracellular traps (NETs) in 1.9% of thrombus surface. T- (CD3+), B-lymphocytes (CD20+), and monocytes/macrophages (CD68+) were rather rare (2.2%, 0.7%, and 2.0% respectively). We found no evidence (0.0%) of hemosiderin and endothelial cells (CD34+). Full clinical recovery occurred prior to discharge. CONCLUSION: This is the first case report of a CVT with histologic assessment of the thrombus retrieved via EVT. Evaluating thrombi in CVT can provide key insights into disease pathophysiology and guide treatment advancements.

A systems-level analysis highlights microglial activation as a modifying factor in common epilepsies.

AIMS: The causes of distinct patterns of reduced cortical thickness in the common human epilepsies, detectable on neuroimaging and with important clinical consequences, are unknown. We investigated the underlying mechanisms of cortical thinning using a systems-level analysis. METHODS: Imaging-based cortical structural maps from a large-scale epilepsy neuroimaging study were overlaid with highly spatially resolved human brain gene expression data from the Allen Human Brain Atlas. Cell-type deconvolution, differential expression analysis and cell-type enrichment analyses were used to identify differences in cell-type distribution. These differences were followed up in post-mortem brain tissue from humans with epilepsy using Iba1 immunolabelling. Furthermore, to investigate a causal effect in cortical thinning, cell-type-specific depletion was used in a murine model of acquired epilepsy. RESULTS: We identified elevated fractions of microglia and endothelial cells in regions of reduced cortical thickness. Differentially expressed genes showed enrichment for microglial markers and, in particular, activated microglial states. Analysis of post-mortem brain tissue from humans with epilepsy confirmed excess activated microglia. In the murine model, transient depletion of activated microglia during the early phase of the disease development prevented cortical thinning and neuronal cell loss in the temporal cortex. Although the development of chronic seizures was unaffected, the epileptic mice with early depletion of activated microglia did not develop deficits in a non-spatial memory test seen in epileptic mice not depleted of microglia. CONCLUSIONS: These convergent data strongly implicate activated microglia in cortical thinning, representing a new dimension for concern and disease modification in the epilepsies, potentially distinct from seizure control.

TNF-α antagonism rescues the effect of ageing on stroke: Perspectives for targeting inflamm-ageing.

AIMS: Epidemiologic evidence links ischemic stroke to age, yet the mechanisms that underlie the specific and independent effects of age on stroke remain elusive, impeding the development of targeted treatments. This study tested the hypothesis that age directly aggravates stroke outcomes and proposes inflamm-aging as a mediator and potential therapeutic target. METHODS: 3 months- (young) and 18-20 months-old (old) mice underwent transient middle cerebral artery occlusion (tMCAO) for 30 minutes followed by 48 hours of reperfusion. Old animals received weekly treatment with the TNF-α neutralizing antibody adalimumab over 4 weeks before tMCAO in a separate set of experiments. Plasma levels of TNF- α were assessed in patients with ischemic stroke and correlated with age and outcome. RESULTS: Old mice displayed larger stroke size than young ones with increased neuromotor deficit. Immunohistochemical analysis revealed impairment of the blood-brain barrier in old mice, i.e. increased post-stroke degradation of endothelial tight junctions and expression of tight junctions-digesting and neurotoxic matrix metalloproteinases. At baseline, old animals showed a broad modulation of several circulating inflammatory mediators. TNF-α displayed the highest increase in old animals and its inhibition restored the volume of stroke, neuromotor performance, and survival rates of old mice to the levels observed in young ones. Patients with ischemic stroke showed increased TNF-α plasma levels which correlated with worsened short-term neurological outcome as well as with age. CONCLUSIONS: This study identifies TNF-α as a causative contributor to the deleterious effect of aging on stroke and points to inflamm-aging as a mechanism of age-related worsening of stroke outcomes and potential therapeutic target in this context. Thus, this work provides a basis for tailoring novel stroke therapies for the particularly vulnerable elderly population.

Microglia proliferation plays distinct roles in acquired epilepsy depending on disease stages.

OBJECTIVE: Microgliosis occurs in animal models of acquired epilepsy and in patients. It includes cell proliferation that is associated with seizure frequency and decreased neuronal cells in human epilepsy. The role of microglia proliferation in the development of acquired epilepsy is unknown; thus, we examined its contribution to spontaneous seizure, neurodegeneration, and cognitive deficits in different disease phases. METHODS: We used a model of acquired epilepsy triggered by intra-amygdala kainic acid in C57BL6N adult male mice. Mice were electroencephalographically (EEG) monitored (24/7) during status epilepticus and in early and chronic disease. Microglia proliferation was blocked by GW2580, a selective CSF1 receptor inhibitor, supplemented in the diet for 21 days from status epilepticus onset. Then, mice were returned to placebo diet until experiment completion. Control mice were exposed to status epilepticus and fed with placebo diet. Experimental mice were tested in the novel object recognition test (NORT) and in Barnes maze, and compared to control and sham mice. At the end of the behavioral test, mice were killed for brain histopathological analysis. Additionally, seizure baseline was monitored in chronic epileptic mice, then mice were fed for 14 days with GW2580 or placebo diet under 24/7 EEG recording. RESULTS: GW2580 prevented microglia proliferation in mice undergoing epilepsy, whereas it did not affect microglia or basal excitatory neurotransmission in the hippocampus of naive mice. Mice with occluded microglia proliferation during early disease development underwent status epilepticus and subsequent epilepsy similar to placebo diet mice, and were similarly impaired in NORT, with improvement in Barnes maze. GW2580-treated mice displayed neuroprotection in the hippocampus. In contrast, blockade of microglia proliferation in chronic epileptic mice resulted in spontaneous seizure reduction versus placebo mice. SIGNIFICANCE: Microglia proliferation during early disease contributes to neurodegeneration, whereas in late chronic disease it contributes to seizures. Timely pharmacological interference with microglia proliferation may offer a potential target for improving disease outcomes.

Fine-Tuning of Sox17 and Canonical Wnt Coordinates the Permeability Properties of the Blood-Brain Barrier.

RATIONALE: The microvasculature of the central nervous system includes the blood-brain barrier (BBB), which regulates the permeability to nutrients and restricts the passage of toxic agents and inflammatory cells. Canonical Wnt/ß-catenin signaling is responsible for the early phases of brain vascularization and BBB differentiation. However, this signal declines after birth, and other signaling pathways able to maintain barrier integrity at postnatal stage are still unknown. OBJECTIVE: Sox17 (SRY [sex-determining region Y]-box 17) constitutes a major downstream target of Wnt/ß-catenin in endothelial cells and regulates arterial differentiation. In the present article, we asked whether Sox17 may act downstream of Wnt/ß-catenin in inducing BBB differentiation and maintenance. METHODS AND RESULTS: Using reporter mice and nuclear staining of Sox17 and ß-catenin, we report that although ß-catenin signaling declines after birth, Sox17 activation increases and remains high in the adult. Endothelial-specific inactivation of Sox17 leads to increase of permeability of the brain microcirculation. The severity of this effect depends on the degree of BBB maturation: it is strong in the embryo and progressively declines after birth. In search of Sox17 mechanism of action, RNA sequencing analysis of gene expression of brain endothelial cells has identified members of the Wnt/ß-catenin signaling pathway as downstream targets of Sox17. Consistently, we found that Sox17 is a positive inducer of Wnt/ß-catenin signaling, and it acts in concert with this pathway to induce and maintain BBB properties. In vivo, inhibition of the ß-catenin destruction complex or expression of a degradation-resistant ß-catenin mutant, prevent the increase in permeability and retina vascular malformations observed in the absence of Sox17. CONCLUSIONS: Our data highlight a novel role for Sox17 in the induction and maintenance of the BBB, and they underline the strict reciprocal tuning of this transcription factor and Wnt/ß-catenin pathway. Modulation of Sox17 activity may be relevant to control BBB permeability in pathological conditions.

Neural Stem Cells of the Subventricular Zone Contribute to Neuroprotection of the Corpus Callosum after Cuprizone-Induced Demyelination.

Myelin loss occurring in demyelinating diseases, including multiple sclerosis, is the leading cause of long-lasting neurological disability in adults. While endogenous remyelination, driven by resident oligodendrocyte precursor cells (OPCs), might partially compensate myelin loss in the early phases of demyelinating disorders, this spontaneous reparative potential fails at later stages. To investigate the cellular mechanisms sustaining endogenous remyelination in demyelinating disorders, we focused our attention on endogenous neural precursor cells (eNPCs) located within the subventricular zone (SVZ) since this latter area is considered one of the primary sources of new OPCs in the adult forebrain. First, we fate mapped SVZ-eNPCs in cuprizone-induced demyelination and found that SVZ endogenous neural stem/precursor cells are recruited during the remyelination phase to the corpus callosum (CC) and are capable of forming new oligodendrocytes. When we ablated SVZ-derived eNPCs during cuprizone-induced demyelination in female mice, the animals displayed reduced numbers of oligodendrocytes within the lesioned CC. Although this reduction in oligodendrocytes did not impact the ensuing remyelination, eNPC-ablated mice experienced increased axonal loss. Our results indicate that, in toxic models of demyelination, SVZ-derived eNPCs contribute to support axonal survival.SIGNIFICANCE STATEMENT One of the significant challenges in MS research is to understand the detrimental mechanisms leading to the failure of CNS tissue regeneration during disease progression. One possible explanation is the inability of recruited oligodendrocyte precursor cells (OPCs) to complete remyelination and to sustain axonal survival. The contribution of endogenous neural precursor cells (eNPCs) located in the subventricular zone (SVZ) to generate new OPCs in the lesion site has been debated. Using transgenic mice to fate map and to selectively kill SVZ-derived eNPCs in the cuprizone demyelination model, we observed migration of SVZ-eNPCs after injury and their contribution to oligodendrogenesis and axonal survival. We found that eNPCs are dispensable for remyelination but protect partially from increased axonal loss.

Prostaglandin D2 synthase modulates macrophage activity and accumulation in injured peripheral nerves.

We have previously reported that prostaglandin D2 Synthase (L-PGDS) participates in peripheral nervous system (PNS) myelination during development. We now describe the role of L-PGDS in the resolution of PNS injury, similarly to other members of the prostaglandin synthase family, which are important for Wallerian degeneration (WD) and axonal regeneration. Our analyses show that L-PGDS expression is modulated after injury in both sciatic nerves and dorsal root ganglia neurons, indicating that it might play a role in the WD process. Accordingly, our data reveals that L-PGDS regulates macrophages phagocytic activity through a non-cell autonomous mechanism, allowing myelin debris clearance and favoring axonal regeneration and remyelination. In addition, L-PGDS also appear to control macrophages accumulation in injured nerves, possibly by regulating the blood-nerve barrier permeability and SOX2 expression levels in Schwann cells. Collectively, our results suggest that L-PGDS has multiple functions during nerve regeneration and remyelination. Based on the results of this study, we posit that L-PGDS acts as an anti-inflammatory agent in the late phases of WD, and cooperates in the resolution of the inflammatory response. Thus, pharmacological activation of the L-PGDS pathway might prove beneficial in resolving peripheral nerve injury.

Enhanced SPARCL1 expression in cancer stem cells improves preclinical modeling of glioblastoma by promoting both tumor infiltration and angiogenesis.

Glioblastoma (GBM) is the most malignant brain tumor of adults and is characterized by extensive cell dissemination within the brain parenchyma and enhanced angiogenesis. Effective preclinical modeling of these key features suffers from several shortcomings. Aim of this study was to determine whether modulating the expression of extracellular matrix (ECM) modifiers in proneural (PN) and mesenchymal (MES) cancer stem cells (CSCs) and in conventional glioma cell lines (GCLs) might improve tumor invasion and vascularization. To this end, we selected secreted, acidic and rich in cysteine-like 1 (SPARCL1) as a potential mediator of ECM remodeling in GBM. SPARCL1 transcript and protein expression was assessed in PN and MES CSCs as well as GCLs, in their xenografts and in patient-derived specimens by qPCR, WB and IHC. SPARCL1 expression was then enforced in both CSCs and GCLs by lentiviral-based transduction. The effect of SPARCL1 gain-of-function on microvascular proliferation, microglia activation and advanced imaging features was tested in intracranial xenografts by IHC and MRI and validated by chorioallantoic membrane (CAM) assays. SPARCL1 expression significantly enhanced the infiltrative and neoangiogenic features of PN and MES CSC/GCL-induced tumors, with the concomitant activation of inflammatory responses associated with the tumor microenvironment, thus resulting in experimental GBMs that reproduced both the parenchymal infiltration and the increased microvascular density, typical of GBM. Overall, these results indicate that SPARCL1 overexpression might be instrumental for the generation of CSC-derived preclinical models of GBM in which the main pathognomonic hallmarks of GBMs are retrievable, making them suitable for effective preclinical testing of therapeutics.

AAV-PHP.B-Mediated Global-Scale Expression in the Mouse Nervous System Enables GBA1 Gene Therapy for Wide Protection from Synucleinopathy.

The lack of technology for direct global-scale targeting of the adult mouse nervous system has hindered research on brain processing and dysfunctions. Currently, gene transfer is normally achieved by intraparenchymal viral injections, but these injections target a restricted brain area. Herein, we demonstrated that intravenous delivery of adeno-associated virus (AAV)-PHP.B viral particles permeated and diffused throughout the neural parenchyma, targeting both the central and the peripheral nervous system in a global pattern. We then established multiple procedures of viral transduction to control gene expression or inactivate gene function exclusively in the adult nervous system and assessed the underlying behavioral effects. Building on these results, we established an effective gene therapy strategy to counteract the widespread accumulation of α-synuclein deposits throughout the forebrain in a mouse model of synucleinopathy. Transduction of A53T-SCNA transgenic mice with AAV-PHP.B-GBA1 restored physiological levels of the enzyme, reduced α-synuclein pathology, and produced significant behavioral recovery. Finally, we provided evidence that AAV-PHP.B brain penetration does not lead to evident dysfunctions in blood-brain barrier integrity or permeability. Altogether, the AAV-PHP.B viral platform enables non-invasive, widespread, and long-lasting global neural expression of therapeutic genes, such as GBA1, providing an invaluable approach to treat neurodegenerative diseases with diffuse brain pathology such as synucleinopathies.

Neural Stem Cell Transplantation Induces Stroke Recovery by Upregulating Glutamate Transporter GLT-1 in Astrocytes.

Ischemic stroke is the leading cause of disability, but effective therapies are currently widely lacking. Recovery from stroke is very much dependent on the possibility to develop treatments able to both halt the neurodegenerative process as well as to foster adaptive tissue plasticity. Here we show that ischemic mice treated with neural precursor cell (NPC) transplantation had on neurophysiological analysis, early after treatment, reduced presynaptic release of glutamate within the ipsilesional corticospinal tract (CST), and an enhanced NMDA-mediated excitatory transmission in the contralesional CST. Concurrently, NPC-treated mice displayed a reduced CST degeneration, increased axonal rewiring, and augmented dendritic arborization, resulting in long-term functional amelioration persisting up to 60 d after ischemia. The enhanced functional and structural plasticity relied on the capacity of transplanted NPCs to localize in the peri-ischemic and ischemic area, to promote the upregulation of the glial glutamate transporter 1 (GLT-1) on astrocytes and to reduce peri-ischemic extracellular glutamate. The upregulation of GLT-1 induced by transplanted NPCs was found to rely on the secretion of VEGF by NPCs. Blocking VEGF during the first week after stroke reduced GLT-1 upregulation as well as long-term behavioral recovery in NPC-treated mice. Our results show that NPC transplantation, by modulating the excitatory-inhibitory balance and stroke microenvironment, is a promising therapy to ameliorate disability, to promote tissue recovery and plasticity processes after stroke. SIGNIFICANCE STATEMENT: Tissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster axonal rewiring and dendritic plasticity and to induce long-term functional recovery. The observed therapeutic effect of neural precursor cells seems to underlie their capacity to upregulate the glial glutamate transporter on astrocytes through the vascular endothelial growth factor inducing favorable changes in the electrical and molecular stroke microenvironment. Cell-based approaches able to influence plasticity seem particularly suited to favor poststroke recovery.

Weighing brain activity with the balance: Angelo Mosso's original manuscripts come to light.

Neuroimaging techniques, such as positron emission tomography and functional magnetic resonance imaging are essential tools for the analysis of organized neural systems in working and resting states, both in physiological and pathological conditions. They provide evidence of coupled metabolic and cerebral local blood flow changes that strictly depend upon cellular activity. In 1890, Charles Smart Roy and Charles Scott Sherrington suggested a link between brain circulation and metabolism. In the same year William James, in his introduction of the concept of brain blood flow variations during mental activities, briefly reported the studies of the Italian physiologist Angelo Mosso, a multifaceted researcher interested in the human circulatory system. James focused on Mosso's recordings of brain pulsations in patients with skull breaches, and in the process only briefly referred to another invention of Mosso's, the 'human circulation balance', which could non-invasively measure the redistribution of blood during emotional and intellectual activity. However, the details and precise workings of this instrument and the experiments Mosso performed with it have remained largely unknown. Having found Mosso's original manuscripts in the archives, we remind the scientific community of his experiments with the 'human circulation balance' and of his establishment of the conceptual basis of non-invasive functional neuroimaging techniques. Mosso unearthed and investigated several critical variables that are still relevant in modern neuroimaging such as the 'signal-to-noise ratio', the appropriate choice of the experimental paradigm and the need for the simultaneous recording of differing physiological parameters.

Caudate functional networks influence brain structural changes with aging.

Neurogenesis decline with aging may be associated with brain atrophy. Subventricular zone neuron precursor cells possibly modulate striatal neuronal activity via the release of soluble molecules. Neurogenesis decay in the subventricular zone may result in structural alterations of brain regions connected to the caudate, particularly to its medial component. The aim of this study was to investigate how the functional organization of caudate networks relates to structural brain changes with aging. One hundred and fifty-two normal subjects were recruited: 52 young healthy adults (≤35 years old), 42 middle-aged (36 ≤ 60 years old) and 58 elderly subjects (≥60 years old). In young adults, stepwise functional connectivity was used to characterize regions that connect to the medial and lateral caudate at different levels of link-step distances. A statistical comparison between the connectivity of medial and lateral caudate in young subjects was useful to define medial and lateral caudate connected regions. Atrophy of medial and lateral caudate connected regions was estimated in young, middle-aged and elderly subjects using T1-weighted images. Results showed that middle-aged and elderly adults exhibited decreased stepwise functional connectivity at one-link step from the caudate, particularly in the frontal, parietal, temporal and occipital brain regions, compared to young subjects. Elderly individuals showed increased stepwise functional connectivity in frontal, parietal, temporal and occipital lobes compared to both young and middle-aged adults. Additionally, elderly adults displayed decreased stepwise functional connectivity compared to middle-aged subjects in specific parietal and subcortical areas. Moreover, in young adults, the medial caudate showed higher direct connectivity to the basal ganglia (left thalamus), superior, middle and inferior frontal and inferior parietal gyri (medial caudate connected region) relative to the lateral caudate. Considering the opposite contrast, lateral caudate showed stronger connectivity to the basal ganglia (right pallidum), orbitofrontal, rostral anterior cingulate and insula cortices (lateral caudate connected region) compared to medial caudate. In elderly subjects, the medial caudate connected region showed greater atrophy relative to the lateral caudate connected region. Brain regions linked to the medial caudate appear to be more vulnerable to aging than lateral caudate connected areas. The adjacency to the subventricular zone may, at least partially, explain these findings. Stepwise functional connectivity analysis can be useful to evaluate the role of the subventricular zone in network disruptions in age-related neurodegenerative disorders.

Safety and efficacy of transcranial direct current stimulation in acute experimental ischemic stroke.

BACKGROUND AND PURPOSE: Transcranial direct current stimulation is emerging as a promising tool for the treatment of several neurological conditions, including cerebral ischemia. The therapeutic role of this noninvasive treatment is, however, limited to chronic phases of stroke. We thus ought to investigate whether different stimulation protocols could also be beneficial in the acute phase of experimental brain ischemia. METHODS: The influence of both cathodal and anodal transcranial direct current stimulation in modifying brain metabolism of healthy mice was first tested by nuclear magnetic resonance spectroscopy. Then, mice undergoing transient proximal middle cerebral artery occlusion were randomized and treated acutely with anodal, cathodal, or sham transcranial direct current stimulation. Brain metabolism, functional outcomes, and ischemic lesion volume, as well as the inflammatory reaction and blood brain barrier functionality, were analyzed. RESULTS: Cathodal stimulation was able, if applied in the acute phase of stroke, to preserve cortical neurons from the ischemic damage, to reduce inflammation, and to promote a better clinical recovery compared with sham and anodal treatments. This finding was attributable to the significant decrease of cortical glutamate, as indicated by nuclear magnetic resonance spectroscopy. Conversely, anodal stimulation induced an increase in the postischemic lesion volume and augmented blood brain barrier derangement. CONCLUSIONS: Our data indicate that transcranial direct current stimulation exerts a measurable neuroprotective effect in the acute phase of stroke. However, its timing and polarity should be carefully identified on the base of the pathophysiological context to avoid potential harmful side effects.

Subventricular zone neural progenitors protect striatal neurons from glutamatergic excitotoxicity.

The functional significance of adult neural stem and progenitor cells in hippocampal-dependent learning and memory has been well documented. Although adult neural stem and progenitor cells in the subventricular zone are known to migrate to, maintain and reorganize the olfactory bulb, it is less clear whether they are functionally required for other processes. Using a conditional transgenic mouse model, selective ablation of adult neural stem and progenitor cells in the subventricular zone induced a dramatic increase in morbidity and mortality of central nervous system disorders characterized by excitotoxicity-induced cell death accompanied by reactive inflammation, such as 4-aminopyridine-induced epilepsy and ischaemic stroke. To test the role of subventricular zone adult neural stem and progenitor cells in protecting central nervous system tissue from glutamatergic excitotoxicity, neurophysiological recordings of spontaneous excitatory postsynaptic currents from single medium spiny striatal neurons were measured on acute brain slices. Indeed, lipopolysaccharide-stimulated, but not unstimulated, subventricular zone adult neural stem and progenitor cells reverted the increased frequency and duration of spontaneous excitatory postsynaptic currents by secreting the endocannabinod arachidonoyl ethanolamide, a molecule that regulates glutamatergic tone through type 1 cannabinoid receptor (CB(1)) binding. In vivo restoration of cannabinoid levels, either by administration of the type 1 cannabinoid receptor agonist HU210 or the inhibitor of the principal catabolic enzyme fatty acid amide hydrolase, URB597, completely reverted the increased morbidity and mortality of adult neural stem and progenitor cell-ablated mice suffering from epilepsy and ischaemic stroke. Our results provide the first evidence that adult neural stem and progenitor cells located within the subventricular zone exert an 'innate' homeostatic regulatory role by protecting striatal neurons from glutamate-mediated excitotoxicity.

Harmonization of sensorimotor deficit assessment in a registered multicentre pre-clinical randomized controlled trial using two models of ischemic stroke.

Multicentre preclinical randomized controlled trials (pRCTs) are a valuable tool to improve experimental stroke research, but are challenging and therefore underused. A common challenge regards the standardization of procedures across centres. We here present the harmonization phase for the quantification of sensorimotor deficits by composite neuroscore, which was the primary outcome of two multicentre pRCTs assessing remote ischemic conditioning in rodent models of ischemic stroke. Ischemic stroke was induced by middle cerebral artery occlusion for 30, 45 or 60 min in mice and 50, 75 or 100 min in rats, allowing sufficient variability. Eleven animals per species were video recorded during neurobehavioural tasks and evaluated with neuroscore by eight independent raters, remotely and blindly. We aimed at reaching an intraclass correlation coefficient (ICC) ≥0.60 as satisfactory interrater agreement. After a first remote training we obtained ICC = 0.50 for mice and ICC = 0.49 for rats. Errors were identified in animal handling and test execution. After a second remote training, we reached the target interrater agreement for mice (ICC = 0.64) and rats (ICC = 0.69). In conclusion, a multi-step, online harmonization phase proved to be feasible, easy to implement and highly effective to align each centre's behavioral evaluations before project's interventional phase.

Neural stem cell transplantation in patients with progressive multiple sclerosis: an open-label, phase 1 study.

Innovative pro-regenerative treatment strategies for progressive multiple sclerosis (PMS), combining neuroprotection and immunomodulation, represent an unmet need. Neural precursor cells (NPCs) transplanted in animal models of multiple sclerosis have shown preclinical efficacy by promoting neuroprotection and remyelination by releasing molecules sustaining trophic support and neural plasticity. Here we present the results of STEMS, a prospective, therapeutic exploratory, non-randomized, open-label, single-dose-finding phase 1 clinical trial ( NCT03269071 , EudraCT 2016-002020-86), performed at San Raffaele Hospital in Milan, Italy, evaluating the feasibility, safety and tolerability of intrathecally transplanted human fetal NPCs (hfNPCs) in 12 patients with PMS (with evidence of disease progression, Expanded Disability Status Scale ≥6.5, age 18-55 years, disease duration 2-20 years, without any alternative approved therapy). The safety primary outcome was reached, with no severe adverse reactions related to hfNPCs at 2-year follow-up, clearly demonstrating that hfNPC therapy in PMS is feasible, safe and tolerable. Exploratory secondary analyses showed a lower rate of brain atrophy in patients receiving the highest dosage of hfNPCs and increased cerebrospinal fluid levels of anti-inflammatory and neuroprotective molecules. Although preliminary, these results support the rationale and value of future clinical studies with the highest dose of hfNPCs in a larger cohort of patients.