The urotensin II receptor triggers an early meningeal response and a delayed macrophage-dependent vasospasm after subarachnoid hemorrhage in male mice.

Subarachnoid hemorrhage (SAH) can be associated with neurological deficits and has profound consequences for mortality and morbidity. Cerebral vasospasm (CVS) and delayed cerebral ischemia affect neurological outcomes in SAH patients, but their mechanisms are not fully understood, and effective treatments are limited. Here, we report that urotensin II receptor UT plays a pivotal role in both early events and delayed mechanisms following SAH in male mice. Few days post-SAH, UT expression is triggered by blood or hemoglobin in the leptomeningeal compartment. UT contributes to perimeningeal glia limitans astrocyte reactivity, microvascular alterations and neuroinflammation independent of CNS-associated macrophages (CAMs). Later, CAM-dependent vascular inflammation and subsequent CVS develop, leading to cognitive dysfunction. In an SAH model using humanized UTh+/h+ male mice, we show that post-SAH CVS and behavioral deficits, mediated by UT through Gq/PLC/Ca2+ signaling, are prevented by UT antagonists. These results highlight the potential of targeting UT pathways to reduce early meningeal response and delayed cerebral ischemia in SAH patients.

Single-Cell RNA Sequencing Identifies Response of Renal Lymphatic Endothelial Cells to Acute Kidney Injury.

SIGNIFICANCE STATEMENT: The renal lymphatic vasculature and the lymphatic endothelial cells that make up this network play important immunomodulatory roles during inflammation. How lymphatics respond to AKI may affect AKI outcomes. The authors used single-cell RNA sequencing to characterize mouse renal lymphatic endothelial cells in quiescent and cisplatin-injured kidneys. Lymphatic endothelial cell gene expression changes were confirmed in ischemia-reperfusion injury and in cultured lymphatic endothelial cells, validating renal lymphatic endothelial cells single-cell RNA sequencing data. This study is the first to describe renal lymphatic endothelial cell heterogeneity and uncovers molecular pathways demonstrating lymphatic endothelial cells regulate the local immune response to AKI. These findings provide insights into previously unidentified molecular pathways for lymphatic endothelial cells and roles that may serve as potential therapeutic targets in limiting the progression of AKI. BACKGROUND: The inflammatory response to AKI likely dictates future kidney health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Owing to the relative sparsity of lymphatic endothelial cells in the kidney, past sequencing efforts have not characterized these cells and their response to AKI. METHODS: Here, we characterized murine renal lymphatic endothelial cell subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI 72 hours postinjury. Data were processed using the Seurat package. We validated our findings by quantitative PCR in lymphatic endothelial cells isolated from both cisplatin-injured and ischemia-reperfusion injury, by immunofluorescence, and confirmation in in vitro human lymphatic endothelial cells. RESULTS: We have identified renal lymphatic endothelial cells and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin-injured conditions. After AKI, renal lymphatic endothelial cells alter genes involved in endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models were also identified with renal lymphatic endothelial cells further demonstrating changed gene expression between cisplatin and ischemia-reperfusion injury models, indicating the renal lymphatic endothelial cell response is both specific to where they lie in the lymphatic vasculature and the kidney injury type. CONCLUSIONS: In this study, we uncover lymphatic vessel structural features of captured populations and injury-induced genetic changes. We further determine that lymphatic endothelial cell gene expression is altered between injury models. How lymphatic endothelial cells respond to AKI may therefore be key in regulating future kidney disease progression.

In Utero Alcohol Exposure Impairs Retinal Angiogenesis and the Microvessel-Associated Positioning of Calretinin Interneurons.

In addition to brain disorders, which constitute a devastating consequence of prenatal alcohol exposure (PAE), eye development is also significantly affected. Given that the retina is a readily accessible part of the central nervous system, a better understanding of the impact of ethanol on retinal development might provide ophthalmological landmarks helpful for early diagnosis of fetal alcohol syndrome. This study aimed to provide a fine morphometric and cellular characterization of the development of retinal microvasculature and neurovascular interactions in a mouse model of fetal alcohol spectrum disorder (FASD). The data revealed that PAE impaired superficial vascular plexus development. In particular, progression of the vascular migration front was significantly decreased in PAE retinas, supporting a delay in plexus progression. Moreover, a significant decrease in the vessel density and number of perforating vessels was quantified in PAE mice, supporting less angiogenesis. The present study provides also the first evidence of a close interaction between migrating calretinin-positive interneurons and perforating microvessels in the inner nuclear layer of the developing retina. This neurovascular association was significantly impaired by PAE. Moreover, projections of amacrine cells were abnormally distributed and densified in stratum S1 and S2. In humans, comparison of a five-month-old control infant with a three-month-old alcohol-exposed case revealed a similar mispositioning of calretinin-positive interneurons. This opens new research avenues regarding a neurovascular contribution in the deleterious effects of alcohol in the developing retina and support that ophthalmological examination could become a promising approach for early detection of alcohol-exposed infants presenting with neurovascular brain defects.

Regulation and impact of cardiac lymphangiogenesis in pressure-overload-induced heart failure.

AIMS: Lymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphangiogenesis in non-ischaemic cardiomyopathy following pressure-overload remains to be determined. Here, we investigated cardiac lymphangiogenesis following transversal aortic constriction (TAC) in C57Bl/6 and Balb/c mice, and in end-stage HF patients. METHODS AND RESULTS: Cardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, oedema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. We found that VEGFR3-signalling was essential to prevent cardiac lymphatic rarefaction after TAC in C57Bl/6 mice. While anti-VEGFR3-induced lymphatic rarefaction did not significantly aggravate myocardial oedema post-TAC, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, whereas inhibition of lymphangiogenesis did not aggravate interstitial fibrosis, it increased perivascular fibrosis and accelerated development of left ventricular (LV) dilation and dysfunction. In clinical HF samples, cardiac lymphatic density tended to increase, although lymphatic sizes decreased, notably in patients with dilated cardiomyopathy. Similarly, comparing C57Bl/6 and Balb/c mice, lymphatic remodelling post-TAC was linked to LV dilation rather than to hypertrophy. The striking lymphangiogenesis in Balb/c was associated with reduced cardiac levels of macrophages, B cells, and perivascular fibrosis at 8 weeks post-TAC, as compared with C57Bl/6 mice that displayed weak lymphangiogenesis. Surprisingly, however, it did not suffice to resolve myocardial oedema, nor prevent HF development. CONCLUSIONS: We demonstrate for the first time that endogenous lymphangiogenesis limits TAC-induced cardiac inflammation and perivascular fibrosis, delaying HF development in C57Bl/6 but not in Balb/c mice. While the functional impact of lymphatic remodelling remains to be determined in HF patients, our findings suggest that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.

SELENOT Deficiency in the Mouse Brain Impacts Catecholaminergic Neuron Density: An Immunohistochemical, in situ Hybridization and 3D Light-Sheet Imaging Study.

BACKGROUND: Selenoprotein T (SELENOT), a PACAP-regulated thioredoxin-like protein, plays a role in catecholamine secretion and protects dopaminergic neurons. However, the role of SELENOT in the establishment of the catecholaminergic (CA) neuronal system is not known yet. METHODS: We analyzed by immunohistochemistry and RNAscope in situ hybridization the distribution of SELENOT and the expression of its mRNA, respectively. In addition, 3D imaging involving immunostaining in toto, clearing through the iDISCO+ method, acquisitions by light-sheet microscopy, and processing of 3D images was performed to map the CA neuronal system. A semi-automatic quantification of 3D images was carried out. RESULTS: SELENOT protein and mRNA are widely distributed in the mouse brain, with important local variations. Three-dimensional mapping, through tyrosine hydroxylase (TH) labeling, and semi-automated quantification of CA neurons in brain-specific SELENOT knockout mice showed a significant decrease in the number of TH-positive neurons in the area postrema (AP-A2), the A11 cell group (A11), and the zona incerta (ZI-A13) of SELENOT-deficient females, and in the hypothalamus (Hyp-A12-A14-A15) of SELENOT-deficient females and males. CONCLUSION: These results showed that SELENOT is diffusely expressed in the mouse brain and that its deficiency impacts CA neuron distribution in different brain areas including Hyp-A12-A14-A15, in both male and female mice.

Cell-penetrating, antioxidant SELENOT mimetic protects dopaminergic neurons and ameliorates motor dysfunction in Parkinson's disease animal models.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor dysfunction for which there is an unmet need for better treatment options. Although oxidative stress is a common feature of neurodegenerative diseases, notably PD, there is currently no efficient therapeutic strategy able to tackle this multi-target pathophysiological process. Based on our previous observations of the potent antioxidant and neuroprotective activity of SELENOT, a vital thioredoxin-like selenoprotein, we designed the small peptide PSELT from its redox active site to evaluate its antioxidant properties in vivo, and its potential polyfunctional activity in PD models. PSELT protects neurotoxin-treated dopaminergic neurons against oxidative stress and cell death, and their fibers against neurotoxic degeneration. PSELT is cell-permeable and acts in multiple subcellular compartments of dopaminergic neurons that are vulnerable to oxidative stress. In rodent models of PD, this protective activity prevented neurodegeneration, restored phosphorylated tyrosine hydroxylase levels, and led to improved motor skills. Transcriptomic analysis revealed that gene regulation by PSELT after MPP+ treatment negatively correlates with that occurring in PD, and positively correlates with that occurring after resveratrol treatment. Mechanistically, a major impact of PSELT is via nuclear stimulation of the transcription factor EZH2, leading to neuroprotection. Overall, these findings demonstrate the potential of PSELT as a therapeutic candidate for treatment of PD, targeting oxidative stress at multiple intracellular levels.

Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction.

OBJECTIVE: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-CC156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4+ and CD8+ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. CONCLUSIONS: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-CC156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Glucose homeostasis is impaired in mice deficient in the neuropeptide 26RFa (QRFP).

INTRODUCTION: 26RFa (pyroglutamyl RFamide peptide (QRFP)) is a biologically active peptide that has been found to control feeding behavior by stimulating food intake, and to regulate glucose homeostasis by acting as an incretin. The aim of the present study was thus to investigate the impact of 26RFa gene knockout on the regulation of energy and glucose metabolism. RESEARCH DESIGN AND METHODS: 26RFa mutant mice were generated by homologous recombination, in which the entire coding region of prepro26RFa was replaced by the iCre sequence. Energy and glucose metabolism was evaluated through measurement of complementary parameters. Morphological and physiological alterations of the pancreatic islets were also investigated. RESULTS: Our data do not reveal significant alteration of energy metabolism in the 26RFa-deficient mice except the occurrence of an increased basal metabolic rate. By contrast, 26RFa mutant mice exhibited an altered glycemic phenotype with an increased hyperglycemia after a glucose challenge associated with an impaired insulin production, and an elevated hepatic glucose production. Two-dimensional and three-dimensional immunohistochemical experiments indicate that the insulin content of pancreatic ß cells is much lower in the 26RFa-/- mice as compared with the wild-type littermates. CONCLUSION: Disruption of the 26RFa gene induces substantial alteration in the regulation of glucose homeostasis, with in particular a deficit in insulin production by the pancreatic islets. These findings further support the notion that 26RFa is an important regulator of glucose homeostasis.

Three-dimensional mapping of tyrosine hydroxylase in the transparent brain and adrenal of prenatal and pre-weaning mice: Comprehensive methodological flowchart and quantitative aspects of 3D mapping.

BACKGROUND: Tyrosine hydroxylase (TH) catalyzes the rate-limiting step for the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline. Although its distribution in different organs, species and stages of development has been the subject of numerous studies, the recent emergence of 3D imaging techniques has created the potential to shed new light on the dynamics of TH expression during the development of the mammalian central and peripheral nervous systems. NEW METHOD: Here, we describe a flowchart summarizing different protocols adapted to developmental stage-specific tissues to generate a 3D atlas of the catecholaminergic system in the brain and peripheral nervous system in mice from embryonic to pre-weaning stages. The procedures described allowed a quantitative assessment of developing TH-positive neuronal populations and pathways, previously understudied due to dimensional limitations. RESULTS: Our approach allowed us to reveal in 3D the dynamics of the onset and the establishment of the catecholaminergic system in embryonic and developing central and peripheral nervous system. Quantitative analyses applied to 3D images yielded accurate measurements of neuron population volumes and numbers, and tract pathway dimensions for selected TH-positive brain structures. COMPARISON WITH EXISTING METHODS: We applied a set of different protocols to yield a comprehensive flowchart for 3D imaging and a precise quantitative assessment of specific neuronal populations during the course of their development up to adulthood in mice. CONCLUSION: The procedures described and the extensive 3D mapping of TH immunoreactivity at early embryonic and postnatal stages provide a comprehensive view of the onset and development of the catecholaminergic system in the mouse brain and sympathoadrenal nervous system.

Correction to: Tyrosine-hydroxylase immunoreactivity in the mouse transparent brain and adrenal glands.

Unfortunately, the given name and family name of the fourth author was incorrectly tagged in the xml data, therefore it is abbreviated wrongly as ''Goazigo AR'' in Pubmed. The correct given name is Annabelle and family name is Reaux­Le Goazigo.

Tyrosine-hydroxylase immunoreactivity in the mouse transparent brain and adrenal glands.

Working on catecholamine systems for years, the neuropharmacologist Arvid Carlsson has made a number of important and pioneering discoveries, which have highlighted the key role of these neuronal and peripheral neurotransmitters in brain functions and adrenal regulations. Since then, major advances have been made concerning the distribution of the catecholaminergic systems in particular by studying their rate-limiting enzyme, tyrosine hydroxylase (TH). Recently new methods of tissue transparency coupled with in toto immununostaining and three-dimensional (3D) imaging technologies allow to precisely map TH immunoreactive pathways in the mouse brain and adrenal glands. High magnification images and movies obtained with combined technologies (iDISCO+ and light-sheet microscopy) are presented in this review dedicated to the pioneer work of Arvid Carlsson and his collaborators.

Cone degeneration is triggered by the absence of USH1 proteins but prevented by antioxidant treatments.

Usher syndrome type 1 (USH1) is a major cause of inherited deafness and blindness in humans. The eye disorder is often referred to as retinitis pigmentosa, which is characterized by a secondary cone degeneration following the rod loss. The development of treatments to prevent retinal degeneration has been hampered by the lack of clear evidence for retinal degeneration in mutant mice deficient for the Ush1 genes, which instead faithfully mimic the hearing deficit. We show that, under normal housing conditions, Ush1g-/- and Ush1c-/- albino mice have dysfunctional cone photoreceptors whereas pigmented knockout animals have normal photoreceptors. The key involvement of oxidative stress in photoreceptor apoptosis and the ensued retinal gliosis were further confirmed by their prevention when the mutant mice are reared under darkness and/or supplemented with antioxidants. The primary degeneration of cone photoreceptors contrasts with the typical forms of retinitis pigmentosa. Altogether, we propose that oxidative stress probably accounts for the high clinical heterogeneity among USH1 siblings, which also unveils potential targets for blindness prevention.

Tridimensional Visualization and Analysis of Early Human Development.

Generating a precise cellular and molecular cartography of the human embryo is essential to our understanding of the mechanisms of organogenesis in normal and pathological conditions. Here, we have combined whole-mount immunostaining, 3DISCO clearing, and light-sheet imaging to start building a 3D cellular map of the human development during the first trimester of gestation. We provide high-resolution 3D images of the developing peripheral nervous, muscular, vascular, cardiopulmonary, and urogenital systems. We found that the adult-like pattern of skin innervation is established before the end of the first trimester, showing important intra- and inter-individual variations in nerve branches. We also present evidence for a differential vascularization of the male and female genital tracts concomitant with sex determination. This work paves the way for a cellular and molecular reference atlas of human cells, which will be of paramount importance to understanding human development in health and disease. PAPERCLIP.

Hyperosmolarity and Benzalkonium Chloride Differently Stimulate Inflammatory Markers in Conjunctiva-Derived Epithelial Cells in vitro.

Tear hyperosmolarity is known to cause ocular surface inflammation in dry eye syndrome. Benzalkonium chloride (BAK), an eyedrop preservative, is known to induce dry eye in long-term-treated patients. Analyzing the modulation of the proinflammatory potential of hyperosmolarity in the presence of BAK on the conjunctiva could give new insights into the effect of this preservative on the disease. In a hyperosmolar model on a conjunctiva-derived cell line, and in the presence of BAK, we evaluated key inflammatory markers [CCL2, IL-8, IL-6, macrophage migration inhibitory factor (MIF) and intercellular adhesion molecule (ICAM)-1] as well as the osmoprotectant element nuclear factor of activated T cells (NFAT)5 using ELISA, RT-qPCR or immunofluorescence staining. Hyperosmolarity highly stimulated CCL2 and NFAT5 in these cells. BAK alone only increased IL-6 expression. The stress-combined condition stimulated CCL2, NFAT5, MIF and IL-8 secretion. ICAM-1 was not modulated by any of the conditions tested. In this model, hyperosmolarity and BAK induced the release of different proinflammatory mediators, and, when combined, they lead to the release of additional inflammatory cytokines. This in vitro study highlights the importance of avoiding long-term ophthalmic treatments containing BAK, as tear film hyperosmolarity can be a result of its detergent action.

Bilateral neuroinflammatory processes in visual pathways induced by unilateral ocular hypertension in the rat.

BACKGROUND: Glaucoma is one of the leading causes of irreversible blindness in the world. The major risk factor is elevated intraocular pressure (IOP) leading to progressive retinal ganglion cell (RGC) death from the optic nerve (ON) to visual pathways in the brain. Glaucoma has been reported to share mechanisms with neurodegenerative disorders. We therefore hypothesize that neuroinflammatory mechanisms in central visual pathways may contribute to the spread of glaucoma disease. The aim of the present study was to analyze the neuroinflammation processes that occur from the pathological retina to the superior colliculi (SCs) in a rat model of unilateral ocular hypertension induced by episcleral vein cauterization (EVC). RESULTS: Six weeks after unilateral (right eye) EVC in male Long-Evans rats, we evaluated both the neurodegenerative process and the neuroinflammatory state in visual pathway tissues. RGCs immunolabeled (Brn3a(+)) in ipsilateral whole flat-mounted retina demonstrated peripheral RGC loss associated with tissue macrophage/microglia activation (CD68(+)). Gene expression analysis of hypertensive and normotensive retinas revealed a significant increase of pro-inflammatory genes such as CCL2, IL-1ß, and Nox2 mRNA expression compared to naïve eyes. Importantly, we found an upregulation of pro-inflammatory markers such as IL-1ß and TNFα and astrocyte and tissue macrophage/microglia activation in hypertensive and normotensive RGC projection sites in the SCs compared to a naïve SC. To understand how neuroinflammation in the hypertensive retina is sufficient to damage both right and left SCs and the normotensive retina, we used an inflammatory model consisting in an unilateral stereotaxic injection of TNFα (25 ng/µl) in the right SC of naïve rats. Two weeks after TNFα injection, using an optomotor test, we observed that rats had visual deficiency in both eyes. Furthermore, both SCs showed an upregulation of genes and proteins for astrocytes, microglia, and pro-inflammatory cytokines, notably IL-1ß. In addition, both retinas exhibited a significant increase of inflammatory markers compared to a naïve retina. CONCLUSIONS: All these data evidence the complex role played by the SCs in the propagation of neuroinflammatory events induced by unilateral ocular hypertension and provide a new insight into the spread of neurodegenerative diseases such as glaucoma.

Ocular inflammation induces trigeminal pain, peripheral and central neuroinflammatory mechanisms.

Ocular surface diseases are among the most frequent ocular pathologies, with prevalence ranging from 20% of the general population. In addition, ocular pain following corneal injury is frequently observed in clinic. The aim of the study was to characterize the peripheral and central neuroinflammatory process in the trigeminal pathways in response to cornea alteration induced by chronic topical instillations of 0.2% benzalkonium chloride (BAC) in male C57BL/6J mice. In vitro BAC induced neurotoxicity and increases neuronal (FOS, ATF3) and pro-inflammatory (IL-6) markers in primary mouse trigeminal ganglion culture. BAC-treated mice exhibited 7days after the treatment reduced aqueous tear production and increased inflammatory cell infiltration in the cornea. Hypertonic saline-evoked eye wipe behavior was enhanced in BAC-treated animals that exhibited increased FOS, ATF3 and Iba1 immunoreactivity in the trigeminal ganglion. Ocular inflammation is associated with a significant increase in IL-6 and TNF-α mRNA expression in the trigeminal ganglion. We reported a strong increase in FOS and Iba1 positive cells in particular in the sensory trigeminal complex at the ipsilateral interpolaris/caudalis (Vi/Vc) transition and Vc/upper cervical cord (Vc/C1) regions. In addition, activated microglial cells were tightly wrapped around activated FOS neurons in both regions and phosphorylated p38 mitogen-activated protein kinase was markedly enhanced specifically in microglial cells during ocular inflammation. Similar data were obtained in the facial motor nucleus. These neuroanatomical data correlated with the increase in mRNA expression of pro-inflammatory (TNF-α, IL-6, CCL2) and neuronal (FOS and ATF3) markers. Interestingly, the suppression of corneal inflammation 10days following the end of BAC treatment resulted in a marked attenuation of peripheral and central changes observed in pathological conditions. This study provides the first demonstration that corneal inflammation induces activation of neurons and microglial p38 MAPK pathway within sensory trigeminal complex. These results suggest that this altered activity in intracellular signaling caused by ocular inflammation might play a priming role in the central sensitization of ocular related brainstem circuits, which represents a significant factor in ocular pain development.

Intraocular pressure reduction and neuroprotection conferred by bone marrow-derived mesenchymal stem cells in an animal model of glaucoma.

INTRODUCTION: Glaucoma is a sight-threatening retinal neuropathy associated with elevated intraocular pressure (IOP) due to degeneration and fibrosis of the trabecular meshwork (TM). Glaucoma medications aim to reduce IOP without targeting the specific TM pathology, Bone-marrow mesenchymal stem cells (MSCs) are used today in various clinical studies. Here, we investigated the potential of MSCs therapy in an glaucoma-like ocular hypertension (OHT) model and decipher in vitro the effects of MSCs on primary human trabecular meshwork cells. METHODS: Ocular hypertension model was performed by cauterization of 3 episcleral veins (EVC) of Long-Evans male rat eyes. MSCs were isolated from rat bone marrow, amplified in vitro and tagged with quantum dot nanocrystals. Animals were distributed as 1) MSCs group receiving 5.10(5)cells/6µl Minimum Essential Medium and 2) MEM group receiving 6µl MEM (n = 10 each). Injections were performed into the anterior chamber of 20 days-hypertensive eyes and IOP was monitored twice a week for 4 weeks. At the end of experiment, cell distribution in the anterior segment was examined in confocal microscopy on flat mounted corneas. Moreover, we tested in vitro effects of MSCs conditioned medium (MSC-CM) on primary human trabecular meshwork cells (hTM cells) using Akt activation, myosin phosphorylation and TGF-ß2-dependent profibrotic phenotype in hTM cells. RESULTS: We demonstrated a rapid and long-lasting in vivo effect of MSCs transplantation that significantly reduced IOP in hypertensive eyes induced by EVC. MSCs were located to the ciliary processes and the TM. Enumeration of RGCs on whole flat-mounted retina highlighted a protective effect of MSCs on RGCs death. In vitro, MSC-CM promotes: (i) hTM cells survival by activating the antiapoptotic pathway, Akt, (ii) hTM cells relaxation as analyzed by the decrease in myosin phosphorylation and (iii) inhibition of TGF-ß2-dependent profibrotic phenotype acquisition in hTM cells. CONCLUSIONS: MSCs injection in the ocular anterior chamber in a rat model of OHT provides neuroprotective effect in the glaucoma pathophysiology via TM protection. These results demonstrate that MSCs constitute promising tool for treating ocular hypertension and retinal cell degeneration.

Combined 3DISCO clearing method, retrograde tracer and ultramicroscopy to map corneal neurons in a whole adult mouse trigeminal ganglion.

Tissue clearing and subsequent imaging of intact transparent tissues have provided an innovative way to analyze anatomical pathways in the nervous system. In this study, we combined a recent 3-dimensional imaging of solvent cleared organ (3DISCO) procedure, light-sheet microscopy, fluorescent retrograde tracer, and Imaris software to 3D map corneal sensory neurons within a whole adult mouse trigeminal ganglion (TG). We first established the optimized steps to easily and rapidly clear a fixed TG. We found that the 3DISCO procedure gave excellent results and took less than 3 h to clear the TG. In a second set of experiments, a retrograde tracer (cholera toxin B Alexa 594-conjugated) was applied to de-epithelialized cornea to retrograde-labeled corneal sensory neurons. Two days later, TGs were cleared by the 3DISCO method and serial imaging was performed using light-sheet ultramicroscopic technology. High-resolution images of labeled neurons can be easily and rapidly obtained from a 3D reconstructed whole mouse TG. We then provided a 3D reconstruction of corneal afferent neurons and analyzed their precise localization in the TG. Thus, we showed that neurons supplying corneal sensory innervation exhibit a highly specific limited dorsomedial localization within the TG. We report that our combined method offers the possibility to perform manual (on 20 µm sections) and automated (on 3D reconstructed TG) counting of labeled cells in a cleared mouse TG. To conclude, we illustrate that the combination of the 3DISCO clearing method with light-sheet microscopy, retrograde tracer, and automatic counting represents a rapid and reliable method to analyze a subpopulation of neurons within the peripheral and central nervous system.

A simple method for 3D analysis of immunolabeled axonal tracts in a transparent nervous system.

Clearing techniques have been developed to transparentize mouse brains, thereby preserving 3D structure, but their complexity has limited their use. Here, we show that immunolabeling of axonal tracts followed by optical clearing with solvents (3DISCO) and light-sheet microscopy reveals brain connectivity in mouse embryos and postnatal brains. We show that the Robo3 receptor is selectively expressed by medial habenula axons forming the fasciculus retroflexus (FR) and analyzed the development of this commissural tract in mutants of the Slit/Robo and DCC/Netrin pathways. Netrin-1 and DCC are required to attract FR axons to the midline, but the two mutants exhibit specific and heterogeneous axon guidance defects. Moreover, floor-plate-specific deletion of Slit ligands with a conditional Slit2 allele perturbs not only midline crossing by FR axons but also their anteroposterior distribution. In conclusion, this method represents a unique and powerful imaging tool to study axonal connectivity in mutant mice.