Gene expression timecourse in zebrafish whole eye in response to optic nerve crush
ABSTRACT: It is well-established that neurons in the adult mammalian central nervous system are terminally differentiated and, if injured, will be unable to regenerate their connections. In contrast to mammals, zebrafish and other teleosts display a robust neuroregenerative response. Following optic nerve crush (ONX), retinal ganglion cells (RGC) regrow their axons to synapse with topographically correct targets in the optic tectum, such that vision is restored in ~21 days. What accounts for these differences between teleostean and mammalian responses to neural injury is not fully understood. A time course analysis of global gene expression patterns in the zebrafish eye after optic nerve crush can help to elucidate cellular and molecular mechanisms that contribute to a successful neuroregeneration. We used microarrays to detail the global gene expression patterns underlying a successful regeneration or the optic nerve following injury. Experiment Overall Design: Microarray analysis was performed on total RNA extracted from whole eye following optic nerve crush (ONX) or sham surgery at defined intervals (4, 12, & 21 days).
Project description:Retinal ganglion cell (RGC) death is the final consequence of many blinding diseases, where there is considerable variation in the time course and severity of RGC loss. Indeed, this process appears to be influenced by a wide variety of genetic and environmental factors. In this study we explored the genetic basis for differences in ganglion cell death in two inbred strains of mice. We found that RGCs are more susceptible to death following optic nerve crush in C57BL/6J mice (54% survival) than in DBA2/J mice (62% survival). Using the Illumina Mouse-6 microarray, we identified 1,580 genes with significant change in expression following optic nerve crush in these two strains of mice. Our analysis of the changes occurring after optic nerve crush demonstrated that the greatest amount of change (44% of the variance) was due to the injury itself. This included changes associated with ganglion cell death, reactive gliosis, and abortive regeneration. The second pattern of gene changes (23% of the variance) was primarily related to differences in gene expressions observed between the C57BL/6J and DBA/2J mouse strains. The remaining changes in gene expression represent interactions between the effects of optic nerve crush and the genetic background of the mouse. We extracted one genetic network from this dataset that appears to be related to tissue remodeling. One of the most intriguing sets of changes included members of the crystallin family of genes, which may represent a signature of pathways modulating the susceptibility of cells to death. Differential responses to optic nerve crush between two widely used strains of mice were used to define molecular networks associated with ganglion cell death and reactive gliosis. These results form the basis for our continuing interest in the modifiers of retinal injury. 18 Samples: 9 per strain (C57BL/6J & DBA/2J); 3 conditions per strain
Project description:Reactive gliosis is a complex process that involves profound changes in gene expression. We used microarray to indentify differentially expressed genes and to investigate the molecular mechanisms of reactive gliosis in optic nerve head in response to optic nerve crush injury. C57Bl/6 female mice were 6-8 weeks old at the time of optic nerve crush surgery. The optic nerve in the left eye was crush 1 mm behind the globe for 10 seconds and the right eye served as contralateral control. The animals were allowed to recover for 1 day, 3 day, 1 week, 3 weeks and 3 months before the optic nerve heads were collected. The naive control mice did not receive any surgery in either eye. Due to the small tissue size of the mouse optic nerve head, two optic nerve heads were pooled together for each microarray chip. The left eyes and the right eyes of two mice were combined respectively to form one pair of experiment and control samples. There were five biological replicates (10 mice) for each condition.
Project description:The optic nerve is a white matter tract that conveys visual information to the brain. A detailed investigation of the proteome of the normal human retrobulbar optic nerve may help facilitate studies of the biology and pathophysiology of the optic nerve. We conducted an in-depth proteomic analysis of optic nerve from five adults. Proteins were fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. We identified 2,711 non-redundant proteins in the human retrobulbar optic nerve, including the astrocytic marker glial fibrillary acidic protein, several proteins expressed by oligodendrocytes (laminin, proteolipid protein, and fibronectin), myelin proteins (myelin basic protein, myelin-associated glycoprotein), paranodal structural proteins (neurofascin, contactin, α, β, and γ adducins, septin 2, endophilin, ankyrin β, spectrin), proteins involved in neuronal protection and regeneration (α crytallins A and B, dedicator of cytokinesis proteins, ciliary neurotrophic factor), proteins associated with open-angle glaucoma (thioredoxin, heat shock protein-70), and proteins associated with optic neuritis (aquaporin-4). Twenty-one unambiguous protein isoforms were identified in the optic nerve.
Project description:The normal gene expression profiles of the tissues in the eye are a valuable resource for considering genes likely to be involved with disease processes. This is based on the assumption that transcript abundances in healthy tissue are correlated to the continued health of that tissue. Expression values were compared with publically available EST and RNA-sequencing resources. The estimated gene and exon level abundances are available online on the Ocular Tissue Database. Ten different tissues were obtained from 6 different individuals and RNA was pooled. The tissues included: retina, optic nerve head (ONH), optic nerve (ON), ciliary body (CB), trabecular meshwork (TM), sclera, lens, cornea, choroid/RPE and iris.
Project description:retinal ganglion cells die after optic nerve injury, either crush or transection. The molecular causesunderlying this degeneration are largely unkwon; the purpose of this job is to find which (if any) gene regulation triggers RGC death with the final goal of design neuroprotective protocols Experiment Overall Design: 3 groups: naive, IONT (intraorbital nerve transection) IONC (intraorbital nerve crush). IONT and IONC lesioned animals were kept the appropriate times postlesion (12h,. 24h, 48h, 3d, 7d, and 15d). For each time point 8-12 animals were used. RNA from 4 animals was pooled and extracted to hybridaze 1 array replica. All replicas were pooled biological replicas: 5 for naive RNA (each replica 4 retinas, therefore 5 independent RNA extractions were done with a total of 20 retinas), IONT: 12h 3 replicas, 24h 2 replicas, 48h 3replicas, 3d 2 replicas, 7d 3 replicas, 15d 2 replicas. IONC: 3 replicas per time point: 12h, 24h, 48h, 3d and 7d)
Project description:Axon regeneration in the central nervous system (CNS) requires reactivating injured neurons’ intrinsic growth state and enabling growth in an inhibitory environment. Using an inbred mouse neuronal phenotypic screen, we find that CAST/Ei mouse adult dorsal root ganglion neurons extend axons more on CNS myelin than the other eight strains tested, especially when pre-injured. Injury-primed CAST/Ei neurons also regenerate markedly in the spinal cord and optic nerve more than those from C57BL/6 mice and show greater spouting following ischemic stroke. Heritability estimates indicate that extended growth in CAST/Ei neurons on myelin is genetically determined, and two whole-genome expression screens yield the Activin transcript Inhba as most correlated with this ability. These screens are presented here. Biological quadruplicate - Mouse tissue - Naïve Dorsal Root Ganglia (DRG) and 5 day post sciatic nerve crush DRG - x9 strains.
Project description:Reactive astrocytes are typically studied in models that cause irreversible mechanical damage to axons, neuronal cell bodies, and glia. We evaluated the response of astrocytes in the optic nerve head to a subtle injury induced by a brief, mild elevation of the intraocular pressure. Astrocytes demonstrated reactive remodeling showing hypertrophy, process retraction and simplification of their shape. We used microarray to indentify differentially expressed genes and to investigate the molecular mechanisms of astrogliosis in response to this subtle injury. Six- to eight-week old C57Bl/6 male mice were used in this experiment. One eye underwent an elevation in intraocular pressure to 30 mmHg for 1 hour and then allowed to recover for 3 days. The contralateral eye served as a control. Due to the small tissue size of the mouse optic nerve head, two optic nerve heads were pooled together for each microarray chip. We used 10 mice to generate five biological replicates for each condition.
Project description:In this study that was specifically designed to identify early stages of glaucoma in DBA/2J mice, we used genome-wide expression profiling and a series of computational methods. Our methods successfully subdivided eyes with no detectable glaucoma by conventional assays into molecularly defined stages of disease. These stages represent a temporally ordered sequence of glaucoma states. Using an array of tools, we then determined networks and biological processes that are altered at these early stages. Our strategy proved very sensitive, suggesting that similar approaches will be valuable for uncovering early processes in other complex, later-onset diseases. Early changes included upregulation of both the complement cascade and endothelin system, and so we tested the therapeutic value of separately inhibiting them. Mice with a mutation in the complement component 1a gene (C1qa) were robustly protected from glaucoma with the protection being among the greatest reported. Similarly, inhibition of the endothelin system was strongly protective. Since EDN2 is potently vasoconstrictive and was produced by microglial/macrophages, our data provide a novel link between these cell types and vascular dysfunction in glaucoma. Targeting early events such as the upregulation of the complement and endothelin pathways may provide effective new treatments for human glaucoma. Genome-wide assessment of gene expression changes was performed in DBA/2J mice. The optic nerve head and retina from 40 DBA/2J eyes at 10.5 months of age were separately profiled. These eyes were selected as they encompassed a range of glaucoma severity. Two control groups were also included; 10 eyes from 10.5 months old D2-Gpnmb+ mice (age and strain matched, no glaucoma control) and 10 eyes from 4.5 months old DBA/2J mice (young, pre-glaucoma).