Publications by authors named "Marco Bacigaluppi"

39 Publications

Cerebral thrombi of cardioembolic etiology have an increased content of neutrophil extracellular traps.

J Neurol Sci 2021 04 21;423:117355. Epub 2021 Feb 21.

Neuroimmunology Unit, Institute of Experimental Neurology (INSPE), IRCCS San Raffaele Institute and University Vita- Salute San Raffaele, Via Olgettina 60, 20132 Milan, Italy; Neurology Department, IRCCS San Raffaele Institute and University Vita- Salute San Raffaele, Via Olgettina 60, 20132 Milan, Italy. Electronic address:

Background: Inflammation is emerging as an essential trigger for thrombosis. In the interplay between innate immunity and coagulation cascade, neutrophils and neutrophil extracellular traps (NETs) can promote thrombus formation and stabilization. In ischemic stroke, it is uncertain whether the involvement of the inflammatory component may differ in thrombi of diverse etiology. We here aimed to evaluate the presence of neutrophils and NETs in cerebral thrombi of diverse etiology retrieved by endovascular thrombectomy (EVT).

Methods: We performed a systematic histological analysis on 80 human cerebral thrombi retrieved through EVT in acute ischemic stroke patients. Thrombus composition was investigated in terms of neutrophils (MPO cells) and NET content (citH3 area), employing specific immunostainings. NET plasma content was determined and compared to NET density in the thrombus.

Results: Neutrophils and NETs were heterogeneously represented within all cerebral thrombi. Thrombi of diverse etiology did not display a statistically significant difference in the number of neutrophils (p = 0.51). However, NET content was significantly increased in cardioembolic compared to large artery atherosclerosis thrombi (p = 0.04), and the association between NET content and stroke etiology remained significant after adjusted analysis (beta coefficient = -6.19, 95%CI = -11.69 to -1.34, p = 0.01). Moreover, NET content in the thrombus was found to correlate with NET content in the plasma (p ≤ 0.001, r = 0.62).

Conclusion: Our study highlights how the analysis of the immune component within the cerebral thrombus, and specifically the NET burden, might provide additional insight for differentiating stroke from diverse etiologies.
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http://dx.doi.org/10.1016/j.jns.2021.117355DOI Listing
April 2021

Fishing an anemone in the brain: embolized cardiac fibroelastoma revealed after stroke thrombectomy.

Eur Heart J 2021 Jan 31. Epub 2021 Jan 31.

Neurology Department, San Raffaele Scientific Institute and Vita-Salute University San Raffaele, Milan, Italy.

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http://dx.doi.org/10.1093/eurheartj/ehab019DOI Listing
January 2021

FABP4 a novel therapeutic target in ischaemic stroke.

Eur Heart J 2020 09;41(33):3181-3183

Neuroimmunology Unit, Institute of Experimental Neurology, San Raffaele Hospital and Vita Salute San Raffaele University, Milan, Italy.

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http://dx.doi.org/10.1093/eurheartj/ehaa230DOI Listing
September 2020

Leukocyte Counts and Ratios Are Predictive of Stroke Outcome and Hemorrhagic Complications Independently of Infections.

Front Neurol 2020 3;11:201. Epub 2020 Apr 3.

Neuroimmunology Unit, Division of Neuroscience, Institute of Experimental Neurology, San Raffaele Hospital, Milan, Italy.

Ischemic stroke patients show alterations in peripheral leukocyte counts that may result from the sterile inflammation response as well as the occurrence of early infections. We here aimed to determine whether alterations of circulating leukocytes in acute ischemic stroke are associated with long-term functional outcome and hemorrhagic complications, independently of the occurrence of infections. Blood laboratory values of patients with acute ischemic stroke, presenting within 4.5 h from symptom onset, were collected. Leukocyte subsets were analyzed in relation to 3-month functional outcome, mortality, and parenchymal hemorrhagic transformation (PH). A multivariable logistic regression analysis, considering the occurrence of early post-stroke infections, was performed for each outcome measure. Five-hundred-ten patients were included in the study. Independently of infections, good functional outcome was associated with a lower neutrophil to lymphocyte ratio (NL-R, OR 0.906 [95% CI 0.822-0.998]), a higher lymphocyte count (OR 1.547 [95% CI 1.051-2.277]), a higher eosinophil count (OR 1.027 [95% CI 1.007-1.048]), and a higher eosinophil to leukocyte ratio (EoLeu-R, OR 1.240 [95% CI 1.071-1.436]) at admission. Death within 3 months was associated with higher NL-R (OR 1.103 [95% CI 1.032-1.179]) as well as with lower eosinophil counts (OR 0.909 [95% CI 0.827-0.999]). Patients developing parenchymal hemorrhagic transformation had higher neutrophil counts (OR 1.420 [95% CI 1.197-1.684]) as well as a higher NL-R (OR 1.192 [95% IC 1.088-1.305]). Leukocyte subtype profiles in the acute phase of ischemic stroke represent a predictor of outcome independently of infections. Stroke-evoked sterile inflammation is a pathophysiological relevant mechanism that deserves further investigation.
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http://dx.doi.org/10.3389/fneur.2020.00201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145963PMC
April 2020

Endogenous neural precursor cells in health and disease.

Brain Res 2020 03 24;1730:146619. Epub 2019 Dec 24.

Neuroimmunology Unit and Department of Neurology, Institute of Experimental Neurology, San Raffaele Hospital and Università Vita- Salute San Raffaele, Via Olgettina 60, 20132 Milano, Italy.

Neurogenesis persists in the adult brain of mammals in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone (SGZ) of the dentate gyrus (DG). The complex interactions between intrinsic and extrinsic signals provided by cells in the niche but also from distant sources regulate the fate of neural stem/progenitor cells (NPCs) in these sites. This fine regulation is perturbed in aging and in pathological conditions leading to a different NPC behavior, tailored to the specific physio-pathological features. Indeed, NPCs exert in physiological and pathological conditions important neurogenic and non-neurogenic regulatory functions and participate in maintaining and protecting brain tissue homeostasis. In this review, we discuss intrinsic and extrinsic signals that regulate NPC activation and NPC functional role in various homeostatic and non-homeostatic conditions.
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http://dx.doi.org/10.1016/j.brainres.2019.146619DOI Listing
March 2020

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

Neurobiol Dis 2020 02 10;134:104705. Epub 2019 Dec 10.

Neural Stem Cell Biology Unit, Division of Neuroscience, San Raffaele Scientific Institute, Via Olgettina 58, Milan 20132, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.nbd.2019.104705DOI Listing
February 2020

Letter by Semerano et al Regarding Article, "Platelet-Rich Emboli in Cerebral Large Vessel Occlusion Are Associated With a Large Artery Atherosclerosis Source".

Stroke 2019 10 13;50(10):e297. Epub 2019 Sep 13.

Neurology and Neuroimmunology Unit, Institute of Experimental Neurology, IRCCS San Raffaele Hospital and Vita Salute San Raffaele University, Milano, Italy.

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http://dx.doi.org/10.1161/STROKEAHA.119.026662DOI Listing
October 2019

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

Glia 2020 01 3;68(1):95-110. Epub 2019 Sep 3.

Division of Neuroscience, INSPE, IRCCS San Raffaele Scientific Institute, Milan, Italy.

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.
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http://dx.doi.org/10.1002/glia.23705DOI Listing
January 2020

Insights from thrombi retrieved in stroke due to large vessel occlusion.

J Cereb Blood Flow Metab 2019 08 18;39(8):1433-1451. Epub 2019 Jun 18.

1 Neurology and Neuroimmunology Unit, Insititute of Experimental Neurology, San Raffaele Scientific Institute, Milano, Italy.

The recent advances of endovascular procedures to treat stroke due to large cerebral vessel occlusion have made it possible to analyze the retrieved thrombus material. Analysis of cerebral thrombi is emerging as a relevant opportunity to complement the diagnostic workup of etiology, to develop new lytic approaches and to optimize the acute treatment of stroke due to large vessel occlusion. Nonetheless, retrieved thrombi are frequently discarded since their informative potential is often neglected and standards are missing. This review provides an overview of the current knowledge and expanding research relating to thrombus composition analysis in large vessel occlusions. We first discuss the heterogeneity of thrombogenic factors that underlie the thrombotic formation in stroke and its implications to identify stroke etiology and thrombus age. Further, we show that understanding structural characteristics of thrombus is pivotal for the development of new-targeted lytic therapies as well as to improve, through thrombus modeling, the development of thrombectomy devices. Finally, we discuss the on-going attempts to identify a signature of thrombus composition indirectly through imaging and peripheral blood biomarkers, which might in future assist treatment decision-making as well as secondary prevention. Thrombus analysis might contribute to the advancement and optimization of personalized stroke treatments.
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http://dx.doi.org/10.1177/0271678X19856131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681524PMC
August 2019

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

J Neurosci 2019 07 28;39(28):5481-5492. Epub 2019 May 28.

Neuroimmunology Unit, Institute of Experimental Neurology, Division of Neuroscience, IRCCS San Raffaele Hospital, 20132 Milan, Italy,

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. 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.
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http://dx.doi.org/10.1523/JNEUROSCI.0227-18.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616285PMC
July 2019

Thrombus hallmarks reveal atherothrombotic stroke aetiology.

J Neurol 2019 06 19;266(6):1533-1535. Epub 2019 Mar 19.

Neuroimmunology Unit, Institute of Experimental Neurology, San Raffaele Scientific Institute, Via Olgettina 60, 20132, Milan, Italy.

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http://dx.doi.org/10.1007/s00415-019-09279-zDOI Listing
June 2019

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

Circ Res 2019 02;124(4):511-525

From the FIRC Institute of Molecular Oncology Foundation (IFOM), Milan, Italy (M.C., F.O., F.B., G.V.B., A.A.M., E.D.).

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/β-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/β-catenin in endothelial cells and regulates arterial differentiation. In the present article, we asked whether Sox17 may act downstream of Wnt/β-catenin in inducing BBB differentiation and maintenance.

Methods And Results: Using reporter mice and nuclear staining of Sox17 and β-catenin, we report that although β-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/β-catenin signaling pathway as downstream targets of Sox17. Consistently, we found that Sox17 is a positive inducer of Wnt/β-catenin signaling, and it acts in concert with this pathway to induce and maintain BBB properties. In vivo, inhibition of the β-catenin destruction complex or expression of a degradation-resistant β-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/β-catenin pathway. Modulation of Sox17 activity may be relevant to control BBB permeability in pathological conditions.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407809PMC
February 2019

Neural precursor cell-secreted TGF-β2 redirects inflammatory monocyte-derived cells in CNS autoimmunity.

J Clin Invest 2017 Nov 25;127(11):3937-3953. Epub 2017 Sep 25.

Neuroimmunology Unit.

In multiple sclerosis, the pathological interaction between autoreactive Th cells and mononuclear phagocytes in the CNS drives initiation and maintenance of chronic neuroinflammation. Here, we found that intrathecal transplantation of neural stem/precursor cells (NPCs) in mice with experimental autoimmune encephalomyelitis (EAE) impairs the accumulation of inflammatory monocyte-derived cells (MCs) in the CNS, leading to improved clinical outcome. Secretion of IL-23, IL-1, and TNF-α, the cytokines required for terminal differentiation of Th cells, decreased in the CNS of NPC-treated mice, consequently inhibiting the induction of GM-CSF-producing pathogenic Th cells. In vivo and in vitro transcriptome analyses showed that NPC-secreted factors inhibit MC differentiation and activation, favoring the switch toward an antiinflammatory phenotype. Tgfb2-/- NPCs transplanted into EAE mice were ineffective in impairing MC accumulation within the CNS and failed to drive clinical improvement. Moreover, intrathecal delivery of TGF-β2 during the effector phase of EAE ameliorated disease severity. Taken together, these observations identify TGF-β2 as the crucial mediator of NPC immunomodulation. This study provides evidence that intrathecally transplanted NPCs interfere with the CNS-restricted inflammation of EAE by reprogramming infiltrating MCs into antiinflammatory myeloid cells via secretion of TGF-β2.
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http://dx.doi.org/10.1172/JCI92387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663358PMC
November 2017

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

Mol Ther 2017 Dec 10;25(12):2727-2742. Epub 2017 Aug 10.

Stem Cell and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy; National Research Council (CNR), Institute of Neuroscience, 20129 Milan, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ymthe.2017.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768559PMC
December 2017

Treatment Challenges of a Primary Vertebral Artery Aneurysm Causing Recurrent Ischemic Strokes.

Case Rep Neurol Med 2017 10;2017:2571630. Epub 2017 Jan 10.

Stroke Unit, Department of Neurology and Neurophysiology, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy.

. Extracranial vertebral artery aneurysms are a rare cause of embolic stroke; surgical and endovascular therapy options are debated and long-term complication may occur. . A 53-year-old man affected by neurofibromatosis type 1 (NF1) came to our attention for recurrent vertebrobasilar embolic strokes, caused by a primary giant, partially thrombosed, fusiform aneurysm of the left extracranial vertebral artery. The aneurysm was treated by endovascular approach through deposition of Guglielmi Detachable Coils in the proximal segment of the left vertebral artery. Six years later the patient presented stroke recurrence. Cerebral angiography and Color Doppler Ultrasound well characterized the unique hemodynamic condition developed over the years responsible for the new embolic event: the aneurysm had been revascularized from its distal portion by reverse blood flow coming from the patent vertebrobasilar axis. A biphasic Doppler signal in the left vertebral artery revealed a peculiar behavior of the blood flow, alternately directed to the aneurysm and backwards to the basilar artery. Surgical ligation of the distal left vertebral artery and excision of the aneurysm were thus performed. . This is the first described case of NF1-associated extracranial vertebral artery aneurysm presenting with recurrent embolic stroke. Complete exclusion of the aneurysm from the blood circulation is advisable to achieve full resolution of the embolic source.
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http://dx.doi.org/10.1155/2017/2571630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5259648PMC
January 2017

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

J Neurosci 2016 10;36(41):10529-10544

Neuroimmunology Unit, Institute of Experimental Neurology and

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.
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http://dx.doi.org/10.1523/JNEUROSCI.1643-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059427PMC
October 2016

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

J Neurosci 2016 10;36(41):10529-10544

Neuroimmunology Unit, Institute of Experimental Neurology and

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.
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http://dx.doi.org/10.1523/JNEUROSCI.1643-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059427PMC
October 2016

Neurogenic and non-neurogenic functions of endogenous neural stem cells.

Front Neurosci 2014 29;8:92. Epub 2014 Apr 29.

Neuroimmunology Unit, Division of Neuroscience, Institute of Experimental Neurology, San Raffaele Scientific Institute Milan, Italy.

Adult neurogenesis is a lifelong process that occurs in two main neurogenic niches of the brain, namely in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus. In the 1960s, studies on adult neurogenesis have been hampered by the lack of established phenotypic markers. The precise tracing of neural stem/progenitor cells (NPCs) was therefore, not properly feasible. After the (partial) identification of those markers, it was the lack of specific tools that hindered a proper experimental elimination and tracing of those cells to demonstrate their terminal fate and commitment. Nowadays, irradiation, cytotoxic drugs as well as genetic tracing/ablation procedures have moved the field forward and increased our understanding of neurogenesis processes in both physiological and pathological conditions. Newly formed NPC progeny from the SVZ can replace granule cells in the olfactory bulbs of rodents, thus contributing to orchestrate sophisticated odor behavior. SGZ-derived new granule cells, instead, integrate within the DG where they play an essential role in memory functions. Furthermore, converging evidence claim that endogenous NPCs not only exert neurogenic functions, but might also have non-neurogenic homeostatic functions by the release of different types of neuroprotective molecules. Remarkably, these non-neurogenic homeostatic functions seem to be necessary, both in healthy and diseased conditions, for example for preventing or limiting tissue damage. In this review, we will discuss the neurogenic and the non-neurogenic functions of adult NPCs both in physiological and pathological conditions.
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http://dx.doi.org/10.3389/fnins.2014.00092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010760PMC
June 2014

Neurogenesis or non-neurogenesis: that is the question.

J Clin Invest 2014 Mar 24;124(3):970-3. Epub 2014 Feb 24.

Neural stem/precursor cells (NPCs) that reside within germinal niches of the adult CNS have more complex roles than previously expected. In addition to their well-documented neurogenic functions, emerging evidence indicates that NPCs exert non-neurogenic functions that contribute to the regulation and preservation of tissue homeostasis under both physiological and pathological conditions. In this issue of the JCI, Mohammad et al. found that DCs efficiently patrol the CNS only when the germinal niche of the subventricular zone functions properly. Indeed, DCs traveled from the ventricles along the rostral migratory stream to the olfactory bulb (a cervical lymph node access point) to dampen anti-CNS immune responses. The authors' findings further support a non-neurogenic role for NPCs in maintaining tissue homeostasis and promoting tissue protection in the adult brain.
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http://dx.doi.org/10.1172/JCI74419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938270PMC
March 2014

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

Stroke 2013 Nov 27;44(11):3166-74. Epub 2013 Aug 27.

From the Neuroimmunology Unit (L.P.-J., M.B., M.G., S.S., G.C., G.M.) and Experimental Neurophysiology Unit, Division of Neuroscience, Institute of Experimental Neurology (INSPE), DIBIT-II, San Raffaele Scientific Institute (M.C., M.C., L.T., G.C., L.L.), Vita-Salute San Raffaele University, Milan, Italy; and Dulbecco Telethon Institute, Biomolecular NMR Laboratory c/o Center for Translational Genomics and Bioinformatics, Ospedale San Raffaele, Milan, Italy (E.G., S.M., G.M.).

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.
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http://dx.doi.org/10.1161/STROKEAHA.113.001687DOI Listing
November 2013

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

Brain 2014 Feb 17;137(Pt 2):621-33. Epub 2013 May 17.

1 Vita-Salute San Raffaele University, I-20132 Milan, Italy.

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.
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http://dx.doi.org/10.1093/brain/awt091DOI Listing
February 2014

Emerging subspecialties in Neurology: transcranial stimulation.

Neurology 2013 Jan;80(4):e33-5

Vita-Salute San Raffaele University, Neuroimmunology Unit, Institute of Experimental Neurology (INSPE), Division of Neuroscience, DIBIT-II, San Raffaele Scientific Institute, Milan, Italy.

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http://dx.doi.org/10.1212/WNL.0b013e3182833d74DOI Listing
January 2013

Angelo Mosso (1846-1910).

J Neurol 2012 Nov 23;259(11):2513-4. Epub 2012 Aug 23.

Vita-Salute San Raffaele University, 20132, Milan, Italy.

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http://dx.doi.org/10.1007/s00415-012-6632-1DOI Listing
November 2012

Subventricular zone neural progenitors protect striatal neurons from glutamatergic excitotoxicity.

Brain 2012 Nov 24;135(Pt 11):3320-35. Epub 2012 Sep 24.

Neuroimmunology Unit, INSPE, Division of Neuroscience, San Raffaele Scientific Institute Via Olgettina 58, 20132 Milan, Italy.

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.
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http://dx.doi.org/10.1093/brain/aws194DOI Listing
November 2012

Learning from default mode network: the predictive value of resting state in traumatic brain injury.

J Neurosci 2012 Feb;32(6):1915-7

Graduate Program in Psychology-Cognitive Neuroscience, Vita-Salute San Raffaele University, I-20132 Milan, Italy.

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http://dx.doi.org/10.1523/JNEUROSCI.5637-11.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6621708PMC
February 2012

Intracerebroventricularly delivered VEGF promotes contralesional corticorubral plasticity after focal cerebral ischemia via mechanisms involving anti-inflammatory actions.

Neurobiol Dis 2012 Mar 17;45(3):1077-85. Epub 2011 Dec 17.

Department of Neurology, University Hospital, Essen, Germany.

Vascular endothelial growth factor (VEGF) has potent angiogenic and neuroprotective effects in the ischemic brain. Its effect on axonal plasticity and neurological recovery in the post-acute stroke phase was unknown. Using behavioral tests combined with anterograde tract tracing studies and with immunohistochemical and molecular biological experiments, we examined effects of a delayed i.c.v. delivery of recombinant human VEGF(165), starting 3 days after stroke, on functional neurological recovery, corticorubral plasticity and inflammatory brain responses in mice submitted to 30 min of middle cerebral artery occlusion. We herein show that the slowly progressive functional improvements of motor grip strength and coordination, which are induced by VEGF, are accompanied by enhanced sprouting of contralesional corticorubral fibres that branched off the pyramidal tract in order to cross the midline and innervate the ipsilesional parvocellular red nucleus. Infiltrates of CD45+ leukocytes were noticed in the ischemic striatum of vehicle-treated mice that closely corresponded to areas exhibiting Iba-1+ activated microglia. VEGF attenuated the CD45+ leukocyte infiltrates at 14 but not 30 days post ischemia and diminished the microglial activation. Notably, the VEGF-induced anti-inflammatory effect of VEGF was associated with a downregulation of a broad set of inflammatory cytokines and chemokines in both brain hemispheres. These data suggest a link between VEGF's immunosuppressive and plasticity-promoting actions that may be important for successful brain remodeling. Accordingly, growth factors with anti-inflammatory action may be promising therapeutics in the post-acute stroke phase.
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http://dx.doi.org/10.1016/j.nbd.2011.12.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4578250PMC
March 2012

Therapeutic stem cell plasticity orchestrates tissue plasticity.

Brain 2011 Jun;134(Pt 6):1585-7

Neuroimmunology Unit, Institute of Experimental Neurology (INSpe), Division of Neuroscience, DIBIT-II, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.

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http://dx.doi.org/10.1093/brain/awr115DOI Listing
June 2011

Post-acute delivery of erythropoietin induces stroke recovery by promoting perilesional tissue remodelling and contralesional pyramidal tract plasticity.

Brain 2011 Jan;134(Pt 1):84-99

Dementia and Ageing Disorders, Department of Neurology, University Hospital Essen, Hufelandstr 55, D-45122 Essen, Germany.

The promotion of post-ischaemic motor recovery remains a major challenge in clinical neurology. Recently, plasticity-promoting effects have been described for the growth factor erythropoietin in animal models of neurodegenerative diseases. To elucidate erythropoietin's effects in the post-acute ischaemic brain, we examined how this growth factor influences functional neurological recovery, perilesional tissue remodelling and axonal sprouting of the corticorubral and corticobulbar tracts, when administered intra-cerebroventricularly starting 3 days after 30 min of middle cerebral artery occlusion. Erythropoietin administered at 10 IU/day (but not at 1 IU/day), increased grip strength of the contralesional paretic forelimb and improved motor coordination without influencing spontaneous locomotor activity and exploration behaviour. Neurological recovery by erythropoietin was associated with structural remodelling of ischaemic brain tissue, reflected by enhanced neuronal survival, increased angiogenesis and decreased reactive astrogliosis that resulted in reduced scar formation. Enhanced axonal sprouting from the ipsilesional pyramidal tract into the brainstem was observed in vehicle-treated ischaemic compared with non-ischaemic animals, as shown by injection of dextran amines into both motor cortices. Despite successful remodelling of the perilesional tissue, erythropoietin enhanced axonal sprouting of the contralesional, but not ipsilesional pyramidal tract at the level of the red and facial nuclei. Moreover, molecular biological and histochemical studies revealed broad anti-inflammatory effects of erythropoietin in both hemispheres together with expression changes of plasticity-related molecules that facilitated contralesional axonal growth. Our study establishes a plasticity-promoting effect of erythropoietin after stroke, indicating that erythropoietin acts via recruitment of contralesional rather than of ipsilesional pyramidal tract projections.
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http://dx.doi.org/10.1093/brain/awq344DOI Listing
January 2011