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Supplemental material, sj-pdf-1-jcb-10.1177_0271678X231159958 for Harmonization of sensorimotor deficit assessment in a registered multicentre pre-clinical randomized controlled trial using two models of ischemic stroke by Alessia Valente, Jacopo Mariani, Serena Seminara, Mauro Tettamanti, Giuseppe Pignataro, Carlo Perego, Luigi Sironi, Felicita Pedata, Diana Amantea, Marco Bacigaluppi, Antonio Vinciguerra, Susanna Diamanti, Martina Viganò, Francesco Santangelo, Chiara Paola Zoia, Virginia Rodriguez-Menendez, Laura Castiglioni, Joanna Rzemieniec, Ilaria Dettori, Irene Bulli, Elisabetta Coppi, Chiara Di Santo, Ornella Cuomo, Giorgia Serena Gullotta, Erica Butti, Giacinto Bagetta, Gianvito Martino, Maria-Grazia De Simoni, Carlo Ferrarese, Stefano Fumagalli, Simone Beretta and for the TRICS study group in Journal of Cerebral Blood Flow & Metabolism

AM251 treatment has no effect on synaptic activity, cell viability and CB1 receptor expression. A Representative electrophysiological traces and group data of average mEPSC amplitude and frequency of control (n = 10) and AM 251 treated (n = 9) cells (scale bar: 20 pA, 200 ms). B Fluorescence images of Hoechst and propidium iodide double staining in cortical neurons and summary bar graph showing lack of cytotoxic effects after AM251 treatment (scale bar: 40 μm). C, D Confocal microscopy images and summary bar graph showing expression of CB1 receptors in control and AM251 treated cultured neurons (scale bar: 20 μm). All data are expressed as mean ± standard error of the mean. CB1 type 1 cannabinoid receptor, mEPSC miniature excitatory post-synaptic current. (PPT 1143 kb)

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.

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 STATEMENTTissue 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.