Fine structure of the choroidal coat of the avian eye: Lymphatic vessels

PURPOSE: To clarify the fine structure of the avian choroid and thus help explain the mechanisms for normal and abnormal eye function and growth. METHODS: Eyes from normal chickens and from experimental chickens subjected to unilateral paracentesis were fixed either by perfusion or in situ, with or without post-fixation by microwave irradiation, and then processed for light and electron microscopic analysis. RESULTS: The avian choroid contains thin-walled lacunae, whose fine structure is identical to that of lymphatic vessels. The lacunae are much smaller toward the anterior chamber and the Schlemm's canal than posteriorly in the eye bulb. Large lacunae are situated primarily in the suprachoroidea, and their blind-ended capillary branches enter the choriocapillaris and the walls of large veins. The walls of the large veins contain villous structures that protrude into their lumina and are penetrated by thin lacunar branches and by side lines of the venous lumen. In normal chickens, the lacunae usually are devoid of blood cells. After paracentesis of the anterior eye chamber, the lacunae become filled with erythrocytes on the side that was operated on, but not on the contralateral side. CONCLUSIONS: The authors propose that the lacunae of the avian choroid represent a system of posterior short lymphatic vessels, which drain intraocular fluids directly into the eye's venous system, and that the villous structures are sites of communication between lacunae and veins. The demonstration of a choroidal lymphatic system opens new insights into the processes of fluid removal, control of intraocular pressure, and regulation of choroidal thickness in the avian eye under normal and experimental conditions

Shortened primary cilium length and dysregulated Sonic hedgehog signaling in Niemann-Pick C1 disease

The Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder due to mutations in the NPC1 gene, encoding a transmembrane protein related to the Sonic hedgehog receptor, Patched, and involved in intracellular trafficking of cholesterol. We have recently found that the proliferation of cerebellar granule neuron precursors is significantly reduced in Npc1-/- mice due to the downregulation of Shh expression. This finding prompted us to analyze the formation of the primary cilium, a non-motile organelle that is specialized for Shh signal transduction and responsible, when defective, for several human genetic disorders. In this study, we show that the expression and subcellular localization of Shh effectors and ciliary proteins are severely disturbed in Npc1-deficient mice. The dysregulation of Shh signaling is associated with a shortening of the primary cilium length and with a reduction of the fraction of ciliated cells in Npc1-deficient mouse brains and the human fibroblasts of NPC1 patients. These defects are prevented by treatment with 2-hydroxypropyl-β-cyclodextrin, a promising therapy currently under clinical investigation. Our findings indicate that defective Shh signaling is responsible for abnormal morphogenesis of the cerebellum of Npc1-deficient mice and show, for the first time, that the formation of the primary cilium is altered in NPC1 disease

Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain

We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDES). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDES activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDES activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC50 = 0.6 muM) and sildenafil (IC50 = 3.5 nM), Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDES mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment. (C) 2001 Elsevier Science B,V, All rights reserved

Comparative anatomy of nitrergic intrinsic choroidal neurons (ICN) in various avian species

Intrinsic choroidal neurons (ICN) represent a peculiar feature of eyes in higher primates and birds. They account for up to 2000 in human and duck eyes but are virtually absent or rare in all other mammalian species investigated so far. It has been suggested that ICN are involved in regulation of ocular blood supply, hence influencing intraocular pressure, and changes in choroidal thickness, thus influencing accommodation. The present study was undertaken in order to compare differences in various avian species with respect to ICN as well as to provide data on some avian species relevant for experimental ophthalmic research, i.e. chicken and quail. Choroids from 12 avian species were processed for NADPH-diaphorase histochemistry or, in some cases, neuronal nitric oxide synthase immunocytochemistry. ICN were quantified and normalized to mean choroidal area. Three choroids of each galliformes (i.e. chicken, quail, turkey) and anseriformes (i.e. Muscovy duck, Mallard duck, goose) were rastered in squares of 1 mm(2) and x/y coordinates were transferred into a 3D-diagram with the amount of ICN represented in the z-axis. ICN were detected in all species investigated. They were predominantly small cells with soma diameters of 20-30 mum. In turkey, and to a lesser amount in chicken, a subpopulation of ICN with somal diameters of up to 70 mum was observed. Highest mean cell counts were found in goose (6195(.)4; turkey 3558(.)4; chicken 1681(.)4; Muscovy duck 785(.)4; Mallard duck 640(.)8; quail 440(.)2). Normalized to choroidal area, highest mean cell counts were (per mm(2)): 12(.)62 in goose, 4(.)42 in both chicken and turkey, 2(.)86 in quail, 2(.)66 in Mallard duck and 1(.)89 in Muscovy duck. In galliformes, ICN were found to be accumulated temporo-cranial, while in anseriformes they were arranged in a more belt-like fashion, passing from cranio-nasal to temporo-caudal. Our results show that besides Muscovy duck, other avian species appear as suitable models for further functional experiments on ICN. The temporo-cranial accumulation of ICN in galliformes and the belt-like arrangement in anseriformes may reflect special functional requirements in regions of high visual acuity. (C) 2003 Elsevier Ltd. All rights reserved

Recovery of hippocampal functions and modulation of muscarinic response by electroacupuncture in young diabetic rats

The muscarinic receptor response to acetylcholine regulates the hippocampal-related learning, memory, neural plasticity and the production and processing of the pro-nerve growth factor (proNGF) by hippocampal cells. The development and progression of diabetes generate a mild cognitive impairment reducing the functions of the septo-hippocampal cholinergic circuitry, depressing neural plasticity and inducing proNGF accumulation in the brain. Here we demonstrate, in a rat model of early type-1 diabetes, that a physical therapy, the electroacupuncture, counteracts the diabetes-induced deleterious effects on hippocampal physiology by ameliorating hippocampal-related memory functions; recovering the impaired long-term potentiation at the dentate gyrus (DG-LTP) and the lowered expression of the vesicular glutamate transporter 1; normalizing the activity-dependent release of proNGF in diabetic rat hippocampus. Electroacupuncture exerted its therapeutic effects by regulating the expression and activity of M1- and M2-acetylcholine muscarinic receptors subtypes in the dentate gyrus of hippocampus. Our results suggest that a physical therapy based on repetitive sensory stimulation could promote hippocampal neural activity, neuronal metabolism and functions, and conceivably improve the diabetes-induced cognitive impairment. Our data can support the setup of therapeutic protocols based on a better integration between physical therapies and pharmacology for the cure of diabetes-associated neurodegeneration and possibly for Alzheimer's disease

α7 Nicotinic Acetylcholine Receptors May Improve Schwann Cell Regenerating Potential via Metabotropic Signaling Pathways

Background: Schwann cells (SCs) are glial cells involved in peripheral axon myelination. SCs also play a strategic role after peripheral nerve injury, regulating local inflammation and axon regeneration. Our previous studies demonstrated the presence of cholinergic receptors in SCs. In particular, the α7 nicotinic acetylcholine receptors (nAChRs) are expressed in SCs after peripheral axotomy, suggesting their involvement in the regulation of SC-regenerating properties. To clarify the role that α7 nAChRs may play after peripheral axon damage, in this study we investigated the signal transduction pathways triggered by receptor activation and the effects produced by their activation. Methods: Both ionotropic and metabotropic cholinergic signaling were analyzed by calcium imaging and Western blot analysis, respectively, following α7 nAChR activation. In addition, the expression of c-Jun and α7 nAChRs was evaluated by immunocytochemistry and Western blot analysis. Finally, the cell migration was studied by a wound healing assay. Results: Activation of α7 nAChRs, activated by the selective partial agonist ICH3, did not induce calcium mobilization but positively modulated the PI3K/AKT/mTORC1 axis. Activation of the mTORC1 complex was also supported by the up-regulated expression of its specific p-p70 S6KThr389 target. Moreover, up-regulation of p-AMPKThr172, a negative regulator of myelination, was also observed concomitantly to an increased nuclear accumulation of the transcription factor c-Jun. Cell migration and morphology analyses proved that α7 nAChR activation also promotes SC migration. Conclusions: Our data demonstrate that α7 nAChRs, expressed by SCs only after peripheral axon damage and/or in an inflammatory microenvironment, contribute to improve the SCs regenerating properties. Indeed, α7 nAChR stimulation leads to an upregulation of c-Jun expression and promotes Schwann cell migration by non-canonical pathways involving the mTORC1 activity

Detection of stiff nanoparticles within cellular structures by contact resonance atomic force microscopy subsurface nanomechanical imaging

Detecting stiff nanoparticles buried in soft biological matrices by atomic force microscopy (AFM) based techniques represents a new frontier in the field of scanning probe microscopies, originally developed as surface characterization methods. Here we report the detection of stiff (magnetic) nanoparticles (NPs) internalized in cells by using contact resonance AFM (CR-AFM) employed as a potentially non-destructive subsurface characterization tool. Magnetite (Fe3O4) NPs were internalized in microglial cells from cerebral cortices of mouse embryos of 18 days by phagocytosis. Nanomechanical imaging of cells was performed by detecting the contact resonance frequencies (CRFs) of an AFM cantilever held in contact with the sample. Agglomerates of NPs internalized in cells were visualized on the basis of the local increase in the contact stiffness with respect to the surrounding biological matrix. A second AFM-based technique for nanomechanical imaging, i.e., HarmoniX™, as well as magnetic force microscopy and light microscopy were used to confirm the CR-AFM results. Thus, CR-AFM was emonstrated as a promising technique for subsurface imaging of nanomaterials in biological samples

Metalloproteinase-9 contributes to inflammatory glia activation and nigro-striatal pathway degeneration in both mouse and monkey models of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism

Inflammation is a predominant aspect of neurodegenerative diseases, manifested by glia activation and expression of pro-inflammatory mediators. Studies on animal models of Parkinson’s disease (PD) suggest that sustained neuroinflammation exacerbates degeneration of the dopaminergic (DA) nigro-striatal pathway. Therefore, insights into the inflammatory mechanisms of PD may help the development of novel therapeutic strategies against this disease. As extracellular matrix metalloproteinases (MMPs) could be major players in the progression of Parkinsonism, we investigated, in the substantia nigra and striatum of mice acutely injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), changes in mRNA expression, protein levels, and cell localization of MMP-9. This protease is mainly neuronal, but early after MPTP injection its mRNA and protein levels, as well as the number of MMP-9-expressing microglia and astrocytes, increase concomitantly to a prominent inflammation. Neuroinflammation and MMP-9+ glia begin to decline within 2 weeks, although protein levels remain higher than control, in association with a partial recovery of DA nigro-striatal circuit. Comparable quantitative studies on MMP-9 knock-out mice, show a significant decrease in both glia activation and loss of DA neurons and fibers, with respect to wild-type. Moreover, in a parallel study on chronically MPTP-injected macaques, we observed that perpetuation of inflammation and high levels of MMP-9 are associated to DA neuron loss. Our data suggest that MMP-9 released by injured neurons favors glia activation; glial cells in turn reinforce their reactive state via autocrine MMP-9 release, contributing to nigro-striatal pathway degeneration. Specific modulation of MMP-9 activity may, therefore, be a strategy to ameliorate harmful inflammatory outcomes in Parkinsonism

Evidence of oligodendrogliosis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism

Aims: Mice and nonhuman primates administered with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) represent elective experimental models of Parkinsonism, in which degeneration of the nigrostriatal dopaminergic pathway is associated with prominent neuroinflammation, characterized by activated microglia and astrocytes in both substantia nigra (SN) and striatum. To date, it is unknown whether oligodendrocytes play a role in these events. Methods: We performed a detailed qualitative and quantitative analysis of oligodendrocyte-associated changes induced by acute and chronic MPTP treatment, in the SN and striatum of mice and macaques respectively. Oligodendrocytes were immunolabelled by cell-specific markers and analysed by confocal microscopy. Results: In both experimental models, MPTP treatment induces an increase in oligodendrocyte cell number and average size, as well as in the total area occupied by this cell type per tissue section, accompanied by evident morphological changes. This multifaceted array of changes, herein referred to as oligodendrogliosis, significantly correlates with the reduction in the level of dopaminergic innervation to the striatum. Conclusions: This event, associated with early damage of the dopaminergic neurone axons and of the complex striatal circuits of which they are part, may result in an important, although neglected, aspect in the onset and progression of Parkinsonism

Microglia reactivity entails microtubule remodeling from acentrosomal to centrosomal arrays

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the behavior of the microtubule cytoskeleton during these changes remains unexplored. Here we show that activated microglia provide an example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state, microglia nucleate microtubules at Golgi outposts, and activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation, inhibition of microtubule dynamics may provide a different strategy to reduce microglia reactivity in inflammatory disease