Coherent anti-Stokes Raman scattering through thick biological tissues by single wavefront shaping

Coherent Anti Stokes Raman Scattering (CARS) offers many advantages for nonlinear bio-imaging, thanks to its sub-cellular spatial resolution and unique chemical specificity. Its working principle requires two incident pulsed laser beams with distinct frequencies to be focused in space and time, which focus quality however rapidly deteriorates when propagating at large depths in biological tissues. The depth limits of CARS and the capability of wavefront correction to overcome these limits are currently unknown. In this work we exploit the spectral correlation properties of the transmission matrix of a scattering medium in a pulsed regime, to recover coherent focusing for two distant incident CARS wavelengths which propagation is initially uncorrelated. Using wavefront shaping with a single spatial light modulator, we recover CARS generation through thick mice spinal cord tissues where initially no signal is measurable due to scattering, and demonstrate point scanning over large field of views of tens of micrometers.Comment: 25 pages, 7 figure

Contribution of Intravital Neuroimaging to Study Animal Models of Multiple Sclerosis

Multiple sclerosis (MS) is a complex and long-lasting neurodegenerative disease of the central nervous system (CNS), characterized by the loss of myelin within the white matter and cortical fbers, axonopathy, and infammatory responses leading to consequent sensory-motor and cognitive defcits of patients. While complete resolution of the disease is not yet a reality, partial tissue repair has been observed in patients which ofers hope for therapeutic strategies. To address the molecular and cellular events of the pathomechanisms, a variety of animal models have been developed to investigate distinct aspects of MS disease. Recent advances of multiscale intravital imaging facilitated the direct in vivo analysis of MS in the animal models with perspective of clinical transfer to patients. This review gives an overview of MS animal models, focusing on the current imaging modalities at the microscopic and macroscopic levels and emphasizing the importance of multimodal approaches to improve our understanding of the disease and minimize the use of animals

Mature oligodendrocytes bordering lesions limit demyelination and favor myelin repair via heparan sulphate production

International audienceMyelin destruction is followed by resident glia activation and mobilization of endogenous progenitors (OPC) which participate in myelin repair. Here we show that in response to demyelination, mature oligodendrocytes (OLG) bordering the lesion express Ndst1, a key enzyme for heparan sulfates (HS) synthesis. Ndst1+ OLG form a belt that demarcates lesioned from intact white matter. Mice with selective inactivation of Ndst1 in the OLG lineage display increased lesion size, sustained microglia and OPC reactivity. HS production around the lesion allows Sonic hedgehog (Shh) binding and favors the local enrichment of this morphogen involved in myelin regeneration. In MS patients, Ndst1 is also found overexpressed in oligodendroglia and the number of Ndst1-expressing oligodendroglia is inversely correlated with lesion size and positively correlated with remyelination potential. Our study suggests that mature OLG surrounding demyelinated lesions are not passive witnesses but contribute to protection and regeneration by producing HS

Contribution à l'étude des bases génétiques de la polymicrogyrie

La polymicrogyrie est un type de malformation corticale dans laquelle on retrouve un excès de gyrations et une surface corticale irrégulière. La polymicrogyrie peut être provoquée par des causes environnementales ou génétiques. C'est ces dernières auxquelles nous nous sommes intéressés et que nous avons étudié afin d'approfondir nos connaissances sur les bases génétiques de la polymicrogyrie. Nous traitons trois projets qui se situent à trois niveaux de recherche différents : étude d'un gène dont la pathogénicité est établie pour le premier, étude de gènes candidats pour le deuxième et recherche de nouveaux gènes candidats pour le troisième. Dans le premier projet, nous avons réussi à prouver l'implication du gène NHEJ1 dans le développement du cortex cérébral. Nous avons montré, grâce à l'ARN interférence in utero que la dérégulation de Nhej1 chez le rat perturbe la migration neuronale, déclenche un phénomène de mort neuronale massive et désorganise les couches corticales. Dans le deuxième projet, après une étude par hybridation génomique comparative sur puce d'ADN, nous avons identifié une duplication dans la région 1p36 chez un patient présentant une polymicrogyrie bilatérale. Nous avons montré que cette duplication casse le gène ENO1 et diminue son expression. L'expression spatio-temporelle d'ENO1 est en accord avec un rôle de celui-ci pendant le développement cérébral. Nous avons également montré que la diminution de l'expression du gène Eno1 perturbe la migration neuronale radiale.Polymicrogyria is a cortical malformation characterized by excessive gyration and an irregular cortex surface. Environmental and genetic causes can be responsible for this disorder. Our principal aim was to better understand the genetic basis of polymicrogyria. Three projects were conducted. The first focused on the NHEJ1 gene. Using RNA interference and in utero electroporation, we showed that deregulation of NHEJ1 disrupts neuronal migration, triggers massive neuronal cell death and disorganizes the cortical layers. In the second project, we identified by comparative genomic hybridization microarray, a duplication in the 1p36 region in a patient with bilateral polymicrogyria. We have shown that this duplication breaks the ENO1 gene and reduces its expression. The spatio-temporal expression of ENO1 and the fact that its deregulation disrupts neuronal migration indicates that ENO1 is a good candidate gene for cortical development. Finally, in the third project, we identified by exome sequencing of familial cases of bilateral polymicrogyria, one coding variation in the GABRA3 gene. Our work allowed us to generate new knowledge for several candidate genes for polymicrogyria

Promoting Myelin Repair through In Vivo Neuroblast Reprogramming

International audienceDemyelination is frequently observed in a variety of CNS insults and neurodegenerative diseases. In rodents, adult neural stem cells can generate oligodendrocytes and participate to myelin repair. However, these cells mainly produce migratory neuroblasts that differentiate in the olfactory bulb. Here, we show that, in the demyelination context, a small subset of these neuroblasts can spontaneously convert into myelinating oligodendrocytes. Furthermore, we demonstrate that the contribution of neuroblasts to myelin repair can be improved by in vivo forced expression of two transcription factors: OLIG2 and SOX10. These factors promote directed fate conversion of endoge-nous subventricular zone neuroblasts into mature functional oligodendrocytes, leading to enhanced remyelination in a cuprizone-induced mouse model of demyelination. These findings highlight the unexpected plasticity of committed neuroblasts and provide proof of concept that they could be targeted for the treatment of demyelinated lesions in the adult brain

Commentary: "Promoting Myelin Repair through In vivo Neuroblast Reprogramming"

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Oligodendrogenesis in the normal and pathological central nervous system

Oligodendrocytes (OLGs) are generated late in development and myelination is thus a tardive event in the brain developmental process. It is however maintained whole life long at lower rate, and myelin sheath is crucial for proper signal transmission and neuronal survival. Unfortunately, OLGs present a high susceptibility to oxidative stress, thus demyelination often takes place secondary to diverse brain lesions or pathologies. OLGs can also be the target of immune attacks, leading to primary demyelination lesions. Following oligodendrocytic death, spontaneous remyelination may occur to a certain extent. In this review, we will mainly focus on the adult brain and on the two main sources of progenitor cells that contribute to oligodendrogenesis: parenchymal oligodendrocyte precursor cells (OPCs) and subventricular zone (SVZ)-derived progenitors. We will shortly come back on the main steps of oligodendrogenesis in the postnatal and adult brain, and summarize the key factors involved in the determination of oligodendrocytic fate. We will then shed light on the main causes of demyelination in the adult brain and present the animal models that have been developed to get insight on the demyelination / remyelinations process. Finally, we will synthetize the results of studies searching for factors able to modulate spontaneous myelin repair

Contribution of Intravital Neuroimaging to Study Animal Models of Multiple Sclerosis

International audienceMultiple sclerosis (MS) is a complex and long-lasting neurodegenerative disease of the central nervous system (CNS), characterized by the loss of myelin within the white matter and cortical fibers, axonopathy, and inflammatory responses leading to consequent sensory-motor and cognitive deficits of patients. While complete resolution of the disease is not yet a reality, partial tissue repair has been observed in patients which offers hope for therapeutic strategies. To address the molecular and cellular events of the pathomechanisms, a variety of animal models have been developed to investigate distinct aspects of MS disease. Recent advances of multiscale intravital imaging facilitated the direct in vivo analysis of MS in the animal models with perspective of clinical transfer to patients. This review gives an overview of MS animal models, focusing on the current imaging modalities at the microscopic and macroscopic levels and emphasizing the importance of multimodal approaches to improve our understanding of the disease and minimize the use of animals

Longitudinal Intravital Microscopy Reveals Axon Degeneration Concomitant With Inflammatory Cell Infiltration in an LPC Model of Demyelination

International audienceDemyelination and axon degeneration are major events in all neurodegenerative diseases, including multiple sclerosis. Intoxication of oligodendrocytes with lysophosphatidylcholine (LPC) is often used as a selective model of focal and reversible demyelination thought to have no incidence for neurons. To characterize the cascade of cellular events involved in LPC-induced demyelination, we have combined intravital coherent antistoke Raman scattering microscopy with intravital two-photon fluorescence microscopy in multicolor transgenic reporter mice. Moreover, taking advantage of a unique technique of spinal glass window implantation, we here provide the first longitudinal description of cell dynamics in the same volume of interest over weeks after insults. We have detected several patterns of axon–myelin interactions and classified them in early and advanced events. Unexpectedly, we have found that oligodendrocyte damages are followed by axon degeneration within 2 days after LPC incubation, and this degeneration is amplified after the recruitment of the peripheral proinflammatory cells at day 4. Beyond day 7, the recovery of axon number and myelin takes 3 more weeks postlesion and involves a new wave of anti-inflammatory innate immune cells at day 14. Therefore, recurrent imaging over several weeks suggests an important role of peripheral immune cells in regulating both the axonal and oligodendroglial fates and thereby the remyelination status. Better understanding the recruitment of peripheral immune cells during demyelinating events should help to improve diagnosis and therapy

Alpha-Synuclein: The Spark That Flames Dopaminergic Neurons, In Vitro and In Vivo Evidence

Mitochondria, α-syn fibrils and the endo-lysosomal system are key players in the pathophysiology of Parkinson’s disease. The toxicity of α-syn is amplified by cell-to-cell transmission and aggregation of endogenous species in newly invaded neurons. Toxicity of α-syn PFF was investigated using primary cultures of dopaminergic neurons or on aged mice after infusion in the SNpc and combined with mild inhibition of GBA. In primary dopaminergic neurons, application of α-syn PFF induced a progressive cytotoxicity associated with mitochondrial dysfunction, oxidative stress, and accumulation of lysosomes suggesting that exogenous α-syn reached the lysosome (from the endosome). Counteracting the α-syn endocytosis with a clathrin inhibitor, dopaminergic neuron degeneration was prevented. In vivo, α-syn PFF induced progressive neurodegeneration of dopaminergic neurons associated with motor deficits. Histology revealed progressive aggregation of α-syn and microglial activation and accounted for the seeding role of α-syn, injection of which acted as a spark suggesting a triggering of cell-to-cell toxicity. We showed for the first time that a localized SNpc α-syn administration combined with a slight lysosomal deficiency and aging triggered a progressive lesion. The cellular and animal models described could help in the understanding of the human disease and might contribute to the development of new therapies