Effects of VEGF blockade on the dynamics of the inflammatory landscape in glioblastoma-bearing mice.

BACKGROUND: Targeting angiogenesis has been and continues to be an attractive therapeutic modality in glioblastoma (GBM) patients. However, GBM rapidly becomes refractory to anti-VEGF therapies. Myeloid cell infiltration is an important determinant of tumor progression. Given that VEGF is a modulator of the innate immune response we sought to analyze the dynamics of this response in a mouse model of GBM undergoing anti-VEGF therapy. METHODS: We grafted GL261-DsRed cells in transgenic Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined recurrent spectral two-photon imaging with multiparametric cytometry, immunostaining, and brain clearing to characterize at two critical stages of tumor development (day 21 and day 28 after tumor grafting) the nature and spatial distribution of the innate response in control and bevacizumab-treated mice. RESULTS: We report that at an early stage (21 day), VEGF blockade has a detectable effect on the number of microglial cells but only a mild effect on the number of infiltrating myeloid cells. At a later stage (day 28), the treatment resulted in a specific adjustment of dendritic cell subsets. In treated mice, the number of monocytes and their monocyte-derived dendritic cells (moDC) progeny was increased by approximately twofold compared to untreated mice. In agreement, by in vivo quantitative imaging, we observed that treatment increased the number of LysM-EGFP cells traveling in tumor blood vessels and doubled the densities of both infiltrated LysM-EGFP monocytes and double-labeled EGFP/EYFP moDC. The treatment also led to an increased density of conventional cDCs2 subset together with a decrease of cDCs1 subset, necessary for the development of anti-tumor immunity. Finally, we describe differential spatial cell distributions and two immune cell-traveling routes into the brain. LysM-EGFP cells distributed as a gradient from the meninges towards the tumor whereas CD11c-EYFP/MHCII+ cells were located in the basal area of the tumor. Brain clearing also revealed a flow of CD11c-EYFP cells following the corpus callosum. CONCLUSION: We uncovered new features in the dynamics of innate immune cells in GBM-bearing mice and deciphered precisely the key populations, i.e., DC subsets controlling immune responses, that are affected by VEGF blockade. Since despite differences, human pathogenesis presents similarities with our mouse model, the data provide new insights into the effect of bevacizumab at the cellular level.

Inhibitor of Apoptosis Proteins Determines Glioblastoma Stem-Like Cell Fate in an Oxygen-Dependent Manner.

In glioblastomas, apoptosis inhibitor proteins (IAPs) are involved in apoptotic and nonapoptotic processes. We previously showed that IAP inhibition induced a loss of stemness and glioblastoma stem cells differentiation by activating nuclear factor-κB under normoxic conditions. Hypoxia has been shown to modulate drug efficacy. Here, we investigated how IAPs participate in glioblastoma stem-like cell maintenance and fate under hypoxia. We showed that in a hypoxic environment, IAPs inhibition by GDC-0152, a small-molecule IAPs inhibitor, triggered stem-like cell apoptosis and decreased proliferation in four human glioblastoma cell lines. We set up a three-dimensional glioblastoma spheroid model in which time-of-flight secondary ion mass spectrometry analyses revealed a decrease in oxygen levels between the periphery and core. We observed low proliferative and apoptotic cells located close to the hypoxic core of the spheres and glial fibrillary acidic protein+ cells at their periphery. These oxygen-dependent GDC-0152 antitumoral effects have been confirmed on human glioblastoma explants. Notably, serine-threonine kinase activation analysis revealed that under hypoxic conditions, IAP inhibition activated ataxia telangiectasia and Rad3-related protein signaling. Our findings provide new insights into the dual mechanism of action of IAP inhibitors that depends on oxygen level and are relevant to their therapeutic application in tumors. Stem Cells 2019;37:731-742.

Diversity of innate immune cell subsets across spatial and temporal scales in an EAE mouse model.

In both multiple sclerosis and its model experimental autoimmune encephalomyelitis (EAE), the extent of resident microglia activation and infiltration of monocyte-derived cells to the CNS is positively correlated to tissue damage. To address the phenotype characterization of different cell subsets, their spatio-temporal distributions and contributions to disease development we induced EAE in Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined high content flow cytometry, immunofluorescence and two-photon imaging in live mice and identified a stepwise program of inflammatory cells accumulation. First on day 10 after induction, EGFP+ neutrophils and monocytes invade the spinal cord parenchyma through the meninges rather than by extravasion. This event occurs just before axonal losses in the white matter. Once in the parenchyma, monocytes mature into EGFP+/EYFP+ monocyte-derived dendritic cells (moDCs) whose density is maximal on day 17 when the axonal degradation and clinical signs stabilize. Meanwhile, microglia is progressively activated in the grey matter and subsequently recruited to plaques to phagocyte axon debris. LysM-EGFP//CD11c-EYFP mice appear as a powerful tool to differentiate moDCs from macrophages and to study the dynamics of immune cell maturation and phenotypic evolution in EAE.

A Delay between Motor Cortex Lesions and Neuronal Transplantation Enhances Graft Integration and Improves Repair and Recovery.

We previously reported that embryonic motor cortical neurons transplanted immediately after lesions in the adult mouse motor cortex restored damaged motor cortical pathways. A critical barrier hindering the application of transplantation strategies for a wide range of traumatic injuries is the determination of a suitable time window for therapeutic intervention. Here, we report that a 1 week delay between the lesion and transplantation significantly enhances graft vascularization, survival, and proliferation of grafted cells. More importantly, the delay dramatically increases the density of projections developed by grafted neurons and improves functional repair and recovery as assessed by intravital dynamic imaging and behavioral tests. These findings open new avenues in cell transplantation strategies as they indicate successful brain repair may occur following delayed transplantation.SIGNIFICANCE STATEMENT Cell transplantation represents a promising therapy for cortical trauma. We previously reported that embryonic motor cortical neurons transplanted immediately after lesions in the adult mouse motor cortex restored damaged cortical pathways. A critical barrier hindering the application of transplantation strategies for a wide range of traumatic injuries is the determination of a suitable time window for therapeutic intervention. We demonstrate that a 1 week delay between the lesion and transplantation significantly enhances graft vascularization, survival, proliferation, and the density of the projections developed by grafted neurons. More importantly, the delay has a beneficial impact on functional repair and recovery. These results impact the effectiveness of transplantation strategies in a wide range of traumatic injuries for which therapeutic intervention is not immediately feasible.

HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix.

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.

Combination of an optical parametric oscillator and quantum-dots 655 to improve imaging depth of vasculature by intravital multicolor two-photon microscopy.

Simultaneous imaging of different cell types and structures in the mouse central nervous system (CNS) by intravital two-photon microscopy requires the characterization of fluorophores and advances in approaches to visualize them. We describe the use of a two-photon infrared illumination generated by an optical parametric oscillator (OPO) on quantum-dots 655 (QD655) nanocrystals to improve resolution of the vasculature deeper in the mouse brain both in healthy and pathological conditions. Moreover, QD655 signal can be unmixed from the DsRed2, CFP, EGFP and EYFP fluorescent proteins, which enhances the panel of multi-parametric correlative investigations both in the cortex and the spinal cord.

Overview of Innovative Mouse Models for Imaging Neuroinflammation.

Neuroinflammation demands a comprehensive appraisal in situ to gain in-depth knowledge on the roles of particular cells and molecules and their potential roles in therapy. Because of the lack of appropriate tools, direct visualization of cells has been poorly investigated up to the present. In this context, reporter mice expressing cell-specific fluorescent proteins, combined with multiphoton microscopy, provide a window into cellular processes in living animals. In addition, the ability to collect multiple fluorescent colors from the same sample makes in vivo microscopy uniquely useful for characterizing many parameters from the same area, supporting powerful correlative analyses. Here, we present an overview of the advantages and limitations of this approach, with the purpose of providing insight into the neuroinflammation field. We also provide a review of existing fluorescent mouse models and describe how these models have been used in studies of neuroinflammation. Finally, the potential for developing advanced genetic tools and imaging resources is discussed. © 2016 by John Wiley & Sons, Inc.

Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice.

Inflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP(+) cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP(+) cells. Fluorescent invading cells were conventional XCR1(+) and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP(+)/EYFP(+) monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response.

Advances in Intravital Non-Linear Optical Imaging of the Central Nervous System in Rodents.

Purpose of review: Highly coordinated cellular interactions occur in the healthy or pathologic adult rodent central nervous system (CNS). Until recently, technical challenges have restricted the analysis of these events to largely static modes of study such as immuno-fluorescence and electron microscopy on fixed tissues. The development of intravital imaging with subcellular resolution is required to probe the dynamics of these events in their natural context, the living brain. Recent findings: This review focuses on the recently developed live non-linear optical imaging modalities, the core principles involved, the identified technical challenges that limit their use and the scope of their applications. We highlight some practical applications for these modalities with a specific attention given to Experimental Autoimmune Encephalomyelitis (EAE), a rodent model of a chronic inflammatory disease of the CNS characterized by the formation of disseminated demyelinating lesions accompanied by axonal degeneration. Summary: We conclude that label-free nonlinear optical imaging combined to two photon imaging will continue to contribute richly to comprehend brain function and pathogenesis and to develop effective therapeutic strategies.

An orthotopic glioblastoma mouse model maintaining brain parenchymal physical constraints and suitable for intravital two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date. Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma. Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development. Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease. The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

Dynamic quantitative intravital imaging of glioblastoma progression reveals a lack of correlation between tumor growth and blood vessel density.

The spatiotemporal and longitudinal monitoring of cellular processes occurring in tumors is critical for oncological research. We focused on glioblastoma multiforme (GBM), an untreatable highly vascularized brain tumor whose progression is thought to critically depend on the oxygen and metabolites supplied by blood vessels. We optimized protocols for orthotopic GBM grafting in mice that were able to recapitulate the biophysical constraints normally governing tumor progression and were suitable for intravital multiphoton microscopy. We repeatedly imaged tumor cells and blood vessels during GBM development. We established methods for quantitative correlative analyses of dynamic imaging data over wide fields in order to cover the entire tumor. We searched whether correlations existed between blood vessel density, tumor cell density and proliferation in control tumors. Extensive vascular remodeling and the formation of new vessels accompanied U87 tumor cell growth, but no strong correlation was found between local cell density and the extent of local blood vessel density irrespective of the tumor area or time points. The technique moreover proves useful for comparative analysis of mice subjected either to Bevacizumab anti-angiogenic treatment that targets VEGF or to AMD3100, an antagonist of CXCR4 receptor. Bevacizumab treatment massively reduced tumoral vessel densities but only transiently reduced U87 tumor growth rate. Again, there was no correlation between local blood vessel density and local cell density. Moreover, Bev applied only prior to tumor implantation inhibited tumor growth to the same extent as post-grafting treatment. AMD3100 achieved a potent inhibition of tumor growth without significant reduction in blood vessel density. These results indicate that in the brain, in this model, tumor growth can be sustained without an increase in blood vessel density and suggest that GBM growth is rather governed by stromal properties.

Long- and short-term intravital imaging reveals differential spatiotemporal recruitment and function of myelomonocytic cells after spinal cord injury.

After spinal cord injury (SCI), resident and peripheral myelomonocytic cells are recruited to the injury site and play a role in injury progression. These cells are important for clearing cellular debris, and can modulate the retraction and growth of axons in vitro. However, their precise spatiotemporal recruitment dynamics is unknown, and their respective roles after SCI remain heavily debated. Using chronic, quantitative intravital two-photon microscopy of adult mice with SCI, here we show that infiltrating lysozyme M (LysM(+)) and resident CD11c(+) myelomonocytic cells have distinct spatiotemporal recruitment profiles, and exhibit changes in morphology, motility, phagocytic activity and axon interaction patterns over time. This study provides the first in vivo description of the influx of inflammatory and resident myelomonocytic cells into the injured spinal cord and their interactions with cut axons, and underscores the importance of precise timing and targeting of specific cell populations in developing therapies for SCI.

Implanting glass spinal cord windows in adult mice with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment. Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows.

Repeated in vivo two-photon imaging of adult mammalian spinal cords, with subcellular resolution, would be crucial for understanding cellular mechanisms under normal and pathological conditions. Current methods are limited because they require surgery for each imaging session. Here we report a simple glass window methodology avoiding repeated surgical procedures and subsequent inflammation. We applied this strategy to follow axon integrity and the inflammatory response over months by multicolour imaging of adult transgenic mice. We found that glass windows have no significant effect on axon number or structure, cause a transient inflammatory response, and dramatically increase the throughput of in vivo spinal imaging. Moreover, we used this technique to track retraction/degeneration and regeneration of cut axons after a 'pin-prick' spinal cord injury with high temporal fidelity. We showed that regenerating axons can cross an injury site within 4 days and that their terminals undergo dramatic morphological changes for weeks after injury. Overall the technique can potentially be adapted to evaluate cellular functions and therapeutic strategies in the normal and diseased spinal cord.

NCAM180 regulates Ric8A membrane localization and potentiates ß-adrenergic response.

Cooperation between receptors allows integrated intracellular signaling leading to appropriate physiological responses. The Neural Cell Adhesion Molecule (NCAM) has three main isoforms of 120, 140 and 180 kDa, with adhesive and signaling properties, but their respective functions remains to be fully identified. Here we show that the human NCAM180 intracellular domain is a novel interactor of the human guanosine exchange factor (GEF) Ric8A using the yeast two hybrid system and immunoprecipitation. Furthermore, NCAM, Ric8A and G(αs) form a tripartite complex. Colocalization experiments by confocal microscopy revealed that human NCAM180 specifically induces the recruitment of Ric8A to the membrane. In addition, using an in vitro recombinant system, and in vivo by comparing NCAM knock-out mouse brain to NCAM heterozygous and wild type brains, we show that NCAM expression dose dependently regulates Ric8A redistribution in detergent resistent membrane microdomains (DRM). Previous studies have demonstrated essential roles for Ric8 in G(α) protein activity at G protein coupled receptors (GPCR), during neurotransmitter release and for asymmetric cell division. We observed that inhibition of Ric8A by siRNA or its overexpression, decreases or increases respectively, cAMP production following ß-adrenergic receptor stimulation. Furthermore, in human HEK293T recombinant cells, NCAM180 potentiates the G(αs) coupled ß-adrenergic receptor response, in a Ric8A dependent manner, whereas NCAM120 or NCAM140 do not. Finally, in mouse hippocampal neurons expressing endogenously NCAM, NCAM is required for the agonist isoproterenol to induce cAMP production, and this requirement depends on Ric8A. These data illustrate a functional crosstalk between a GPCR and an IgCAM in the nervous system.

Chemical optimization of new ligands of the low-density lipoprotein receptor as potential vectors for central nervous system targeting.

Drug delivery to the central nervous system is hindered by the presence of physiological barriers such as the blood-brain barrier. To accomplish the task of nutrient transport, the brain endothelium is endowed with various transport systems, including receptor-mediated transcytosis (RMT). This system can be used to shuttle therapeutics into the central nervous system (CNS) in a noninvasive manner. Therefore, the low-density lipoprotein receptor (LDLR) is a relevant target for delivering drugs. From an initial phage display biopanning, a series of peptide ligands for the LDLR was optimized leading to size reduction and improved receptor binding affinity with the identification of peptide 22 and its analogues. Further real-time biphoton microscopy experiments on living mice demonstrated the ability of peptide 22 to efficiently and quickly cross CNS physiological barriers. This validation of peptide 22 led us to explore its binding on the extracellular LDLR domain from an NMR-oriented structural study and docking experiments.

Cortical and subventricular zone glioblastoma-derived stem-like cells display different molecular profiles and differential in vitro and in vivo properties.

BACKGROUND: Cellular self-renewal capacity in glioblastomas is heterogeneous, with only stem-like cells having this property. These cells generate a specific tumor phenotype, but no link with tumor location or molecular characteristics has ever been made. METHODS: Two cells lines, established from cell-dissociated glioblastomas and A2B5+ magnetic cell sorting, were used to decipher the mechanisms of cell migration in glioblastomas. GBM6 was derived from a glioblastoma close to the subventricular zone, whereas GBM9 was derived from a cortical glioblastoma and contained a high number of CD133(+) cells. RESULTS: Orthotopic injections in both the subventricular zone and the cortex of nude mice showed that GBM6 and GBM9 cells had a differential pattern of migration that mirrored that of adult and fetal normal neural stem cells, respectively. GBM6 demonstrated higher tumorigenicity than GBM9, and whichever cell line was injected, subventricular zone-implanted tumors were larger than cortical ones. In vitro, GBM6 and GBM9 displayed high autorenewal and proliferation rates, and their expression profiles and genomic status showed that they had distinctive molecular signatures: GBM6 was classified as a mesenchymal glioblastoma and GBM9 as a proneural glioblastoma. CONCLUSIONS: Altogether, our findings suggest that tumor location in addition to molecular signature influence tumor growth and migration pattern.

VEGFR2 (KDR/Flk1) signaling mediates axon growth in response to semaphorin 3E in the developing brain.

Common factors are thought to control vascular and neuronal patterning. Here we report an in vivo requirement for the vascular endothelial growth factor receptor type 2 (VEGFR2) in axon tract formation in the mouse brain. We show that VEGFR2 is expressed by neurons of the subiculum and mediates axonal elongation in response to the semaphorin (Sema) family molecule, Sema3E. We further show that VEGFR2 associates with the PlexinD1/Neuropilin-1 (Nrp1) receptor complex for Sema3E and becomes tyrosine-phosphorylated upon Sema3E stimulation. In subicular neurons, Sema3E triggers VEGFR2-dependent activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway that is required for the increase in axonal growth. These results implicate VEGFR2 in axonal wiring through a mechanism dependent on Sema3E and independent of vascular endothelial growth factor (VEGF) ligands. This mechanism provides an explanation as to how a semaphorin can activate an axon growth promoting response in developing neurons.

Polysialic acid neural cell adhesion molecule (PSA-NCAM) is an adverse prognosis factor in glioblastoma, and regulates olig2 expression in glioma cell lines.

BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive and frequent brain tumor, albeit without cure. Although patient survival is limited to one year on average, significant variability in outcome is observed. The assessment of biomarkers is needed to gain better knowledge of this type of tumor, help prognosis, design and evaluate therapies. The neurodevelopmental polysialic acid neural cell adhesion molecule (PSA-NCAM) protein is overexpressed in various cancers. Here, we studied its expression in GBM and evaluated its prognosis value for overall survival (OS) and disease free survival (DFS). METHODS: We set up a specific and sensitive enzyme linked immunosorbent assay (ELISA) test for PSA-NCAM quantification, which correlated well with PSA-NCAM semi quantitative analysis by immunohistochemistry, and thus provides an accurate quantitative measurement of PSA-NCAM content for the 56 GBM biopsies analyzed. For statistics, the Spearman correlation coefficient was used to evaluate the consistency between the immunohistochemistry and ELISA data. Patients' survival was estimated by using the Kaplan-Meier method, and curves were compared using the log-rank test. On multivariate analysis, the effect of potential risk factors on the DFS and OS were evaluated using the cox regression proportional hazard models. The threshold for statistical significance was p = 0.05. RESULTS: We showed that PSA-NCAM was expressed by approximately two thirds of the GBM at variable levels. On univariate analysis, PSA-NCAM content was an adverse prognosis factor for both OS (p = 0.04) and DFS (p = 0.0017). On multivariate analysis, PSA-NCAM expression was an independent negative predictor of OS (p = 0.046) and DFS (p = 0.007). Furthermore, in glioma cell lines, PSA-NCAM level expression was correlated to the one of olig2, a transcription factor required for gliomagenesis. CONCLUSION: PSA-NCAM represents a valuable biomarker for the prognosis of GBM patients.

Polysialic acid glycomimetic promotes functional recovery and plasticity after spinal cord injury in mice.

Regeneration after injury of the central nervous system is poor due to the abundance of molecules inhibiting axonal growth. Here we pursued to promote regeneration after thoracic spinal cord injury in young adult C57BL/6J mice using peptides which functionally mimic polysialic acid (PSA) and human natural killer cell-1 (HNK-1) glycan, carbohydrate epitopes known to promote neurite outgrowth in vitro. Subdural infusions were performed with an osmotic pump, over 2 weeks. When applied immediately after injury, the PSA mimetic and the combination of PSA and HNK-1 mimetics, but not the HNK-1 mimetic alone, improved functional recovery as assessed by locomotor rating and video-based motion analysis over a 6-week observation period. Better outcome in PSA mimetic-treated mice was associated with higher, as compared with control mice, numbers of cholinergic and glutamatergic terminals and monaminergic axons in the lumbar spinal cord, and better axonal myelination proximal to the injury site. In contrast to immediate post-traumatic application, the PSA mimetic treatment was ineffective when initiated 3 weeks after spinal cord injury. Our data suggest that PSA mimetic peptides can be efficient therapeutic tools improving, by augmenting plasticity, functional recovery when applied during the acute phase of spinal cord injury.