Anatomy and Neuroscience - Research Publications
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ItemThe Role of Histamine in the Retina: Studies on the Hdc Knockout Mouse(PUBLIC LIBRARY SCIENCE, 2014-12-29)The role of histamine in the retina is not well understood, despite it regulating a number of functions within the brain, including sleep, feeding, energy balance, and anxiety. In this study we characterized the structure and function of the retina in mice that lacked expression of the rate limiting enzyme in the formation of histamine, histidine decarboxylase (Hdc-/- mouse). Using laser capture microdissection, Hdc mRNA expression was assessed in the inner and outer nuclear layers of adult C57Bl6J wildtype (WT) and Hdc(-/-)-retinae. In adult WT and Hdc(-/-)-mice, retinal fundi were imaged, retinal structure was assessed using immunocytochemistry and function was probed by electroretinography. Blood flow velocity was assessed by quantifying temporal changes in the dynamic fluorescein angiography in arterioles and venules. In WT retinae, Hdc gene expression was detected in the outer nuclear layer, but not the inner nuclear layer, while the lack of Hdc expression was confirmed in the Hdc-/- retina. Preliminary examination of the fundus and retinal structure of the widely used Hdc-/- mouse strain revealed discrete lesions across the retina that corresponded to areas of photoreceptor abnormality reminiscent of the rd8 (Crb1) mutation. This was confirmed after genotyping and the strain designated Hdcrd8/rd8. In order to determine the effect of the lack of Hdc-alone on the retina, Hdc-/- mice free of the Crb1 mutation were bred. Retinal fundi appeared normal in these animals and there was no difference in retinal structure, macrogliosis, nor any change in microglial characteristics in Hdc-/- compared to wildtype retinae. In addition, retinal function and retinal blood flow dynamics showed no alterations in the Hdc-/- retina. Overall, these results suggest that histamine plays little role in modulating retinal structure and function.
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ItemStudying Age-Related Macular Degeneration Using Animal Models(LIPPINCOTT WILLIAMS & WILKINS, 2014-08)Over the recent years, there have been tremendous advances in our understanding of the genetic and environmental factors associated with the development of age-related macular degeneration (AMD). Examination of retinal changes in various animals has aided our understanding of the pathogenesis of the disease. Notably, mouse strains, carrying genetic anomalies similar to those affecting humans, have provided a foundation for understanding how various genetic risk factors affect retinal integrity. However, to date, no single mouse strain that develops all the features of AMD in a progressive age-related manner has been identified. In addition, a mutation present in some background strains has clouded the interpretation of retinal phenotypes in many mouse strains. The aim of this perspective was to describe how animals can be used to understand the significance of each sign of AMD, as well as key genetic risk factors.
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ItemATP-Induced Photoreceptor Death in a Feline Model of Retinal Degeneration(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2014-12)PURPOSE: To develop and characterize a feline model of retinal degeneration induced by intravitreal injection of adenosine triphosphate (ATP). METHODS: Nineteen normally sighted adult cats received 100 Ī¼L intravitreal injections of ATP with a final concentration of 11, 22, or 55 mM at the retina. Four animals were euthanized 30 hours after injection and retinal sections examined for apoptosis using a TUNEL cell death assay. In the remaining animals, structural and functional changes were characterized over a 3-month period using a combination of electroretinography (ERG) and optical coherence tomography (OCT). RESULTS: Using a TUNEL cell death assay, we detected widespread photoreceptor death 30 hours after injection with 55 mM intravitreal ATP. All concentrations of ATP caused loss of retinal function and gross changes in retinal structure within 2 weeks of injection. Intravitreal injection of ATP led to a rapid loss of rod photoreceptor function and a gradual loss of cone photoreceptor function within 3 months. Outer nuclear layer thickness was globally reduced by 3 months, with the inner nuclear layer including the retinal nerve fiber layer remaining intact. Structural abnormalities were observed, including focal retinal detachment with evidence of both intravitreal and intraretinal inflammation in some eyes. CONCLUSIONS: Development of an ATP-induced feline model of retinal degeneration provides a rapid and effective large-eyed animal model for research into vision restoration.
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ItemMapping kainate activation of inner neurons in the rat retina(WILEY-BLACKWELL, 2013-08-01)Kainate receptors mediate fast, excitatory synaptic transmission for a range of inner neurons in the mammalian retina. However, allocation of functional kainate receptors to known cell types and their sensitivity remains unresolved. Using the cation channel probe 1-amino-4-guanidobutane agmatine (AGB), we investigated kainate sensitivity of neurochemically identified cell populations within the structurally intact rat retina. Most inner retinal neuron populations responded to kainate in a concentration-dependent manner. OFF cone bipolar cells demonstrated the highest sensitivity of all inner neurons to kainate. Immunocytochemical localization of AGB and macromolecular markers confirmed that type 2 bipolar cells were part of this kainate-sensitive population. The majority of amacrine (ACs) and ganglion cells (GCs) showed kainate responses with different sensitivities between major neurochemical classes (Ī³-aminobutyric acid [GABA]/glycine ACs > glycine ACs > GABA ACs; glutamate [Glu]/weakly GABA GCs > Glu GCs). Conventional and displaced cholinergic ACs were highly responsive to kainate, whereas dopaminergic ACs do not appear to express functional kainate receptors. These findings further contribute to our understanding of neuronal networks in complex multicellular tissues.
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ItemEarly remodeling of muller cells in the rd/rd mouse model of retinal dystrophy(WILEY, 2013-08-01)We studied the anatomical remodeling and gliosis of retinal MĆ¼ller cells in the rd/rd mouse model of photoreceptor degeneration. A computational calculation of glutamine synthetase immunoreactivity was developed so we could specifically quantify changes in MĆ¼ller cell anatomy between control mice (C57Bl/6) and the dystrophic strain. We found no change in the number of MĆ¼ller cell somata between mice strains, indicating no cell proliferation as a function of development and degeneration. The retinal area occupied by the total MĆ¼ller cell body (soma and processes) was significantly less in the rd/rd mouse retina compared with control mice. When only the outer retina was considered, we found rd/rd MĆ¼ller cell processes were dramatically reduced during the cone phase of photoreceptor degeneration. However, at older ages an increase in MĆ¼ller cell processes was seen. Conversely, glial fibrillary acidic protein (GFAP) expression showed a significant increase during cone degeneration followed by a reduction in older ages. MĆ¼ller cell electrophysiology, particularly K(+) currents and membrane potential, was similar between rd/rd and control MĆ¼ller cells during cone degeneration. Together, these results show that glial remodeling in the rd/rd retina follows separate phases-an initial conservative glial response involving the loss of MĆ¼ller cells processes, hyperexpression of GFAP, and preservation of normal electrophysiology followed by an active growth of MĆ¼ller cell processes, glial seal formation, and attenuation of GFAP expression after complete photoreceptor loss.
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ItemAdenosine Triphosphate-Induced Photoreceptor Death and Retinal Remodeling In Rats(WILEY, 2014-09-01)Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; MĆ¼ller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision.
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ItemMapping cation entry in photoreceptors and inner retinal neurons during early degeneration in the P23H-3 rat retina(CAMBRIDGE UNIV PRESS, 2013-05)The proline-23-histidine line 3 (P23H-3) transgenic rat carries a human opsin gene mutation leading to progressive photoreceptor loss characteristic of human autosomal dominant retinitis pigmentosa. The aim of the present study was to evaluate neurochemical modifications in the P23H-3 retina as a function of development and degeneration. Specifically, we investigated the ion channel permeability of photoreceptors by tracking an organic cation, agmatine (1-amino-4-guanidobutane, AGB), which permeates through nonspecific cation channels. We also investigated the activity of ionotropic glutamate receptors in distinct populations of bipolar, amacrine, and ganglion cells using AGB tracking in combination with macromolecular markers. We found elevated cation channel permeation in photoreceptors as early as postnatal day 12 (P12) suggesting that AGB labeling is an early indicator of impending photoreceptor degeneration. However, bipolar, amacrine, or ganglion cells displayed normal responses secondary to ionotropic glutamate receptor activation even at P138 when about one half of the photoreceptor layer was lost and apoptosis and gliosis were observed. These results suggest that possible therapeutic windows as downstream neurons in inner retina appear to retain normal function with regard to AGB permeation when photoreceptors are significantly reduced but not lost.
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ItemNo Preview AvailableRetinal amino acid neurochemistry in health and disease(TAYLOR & FRANCIS LTD, 2013-05)Advances in basic retinal anatomy, genetics, biochemical pathways and neurochemistry have not only provided a better understanding of retinal function but have also allowed us to link basic science to retinal disease. The link with disease allowed measures to be developed that now provide an opportunity to intervene and slow down or even restore sight in previously 'untreatable' retinal diseases. One of the critical advances has been the understanding of the retinal amino acid neurotransmitters, related amino acids, their metabolites and functional receptors. This review provides an overview of amino acid localisation in the retina and examples of how retinal anatomy and amino acid neurochemistry directly links to understanding retinal disease. Also, the implications of retinal remodelling involving amino acid (glutamate) receptors are outlined in this review and insights are presented on how understanding of detrimental and beneficial retinal remodelling will provide better outcomes for patients using strategies for the preservation or restoration of vision. An internet-based database of retinal images of amino acid labelling patterns and other amino acid-related images in health and disease is located at http://www.aminoacidimmunoreactivity.com.
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ItemNo Preview AvailableA Naturally Occurring Mouse Model of Achromatopsia: Characterization of the Mutation in Cone Transducin and Subsequent Retinal Phenotype(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2013-05)PURPOSE: This work investigates a novel, naturally occurring mouse model of achromatopsia. The specific missense mutation within the Gnat2 gene was identified and the subsequent retinal phenotype characterized. METHODS: The Gnat2 sequence was amplified using PCR from BALB/c and Gnat2(c.518A>G) retinae and the product sequenced. Retinal function was assessed at 3, 6, 9, and 12 months using the electroretinogram. Transducin and opsin expression were assessed at 3 and 12 months using immunohistochemistry and quantitative PCR. Retinal remodeling and MĆ¼ller cell gliosis were investigated using immunocytochemistry. RESULTS: An A to G missense mutation at position 518 of the Gnat2 gene was identified that resulted in an aspartic acid to glycine substitution. Gnat2(c.518A>G) animals showed no cone response, while the rod response was normal except for a decrease in the photoreceptor response at 12 months (a-wave, -14%). Gnat2(c.518A>G) retinal sections showed no transducin immunolabeling; however, protein was detected via Western blot. Gnat2 gene expression was only decreased at 12 months of age (-27%). There was reduced cone number at 12 months (-27%) and M-opsin showed evidence of mislocalization. Displaced photoreceptor terminals and altered horizontal cell, cone/rod bipolar cell morphology were evident at 3 months, becoming more extensive at 12 months with the emergence of MĆ¼ller cell gliosis. CONCLUSIONS: The Gnat2(c.518A>G) mouse contains a missense mutation that results in no cone function due to a misfolding of transducin. Cone photoreceptors also show signs of opsin mislocalization, retinal remodeling and degeneration. This naturally occurring model shows all the hallmark signs of achromatopsia.
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ItemRelationship between the Magnitude of Intraocular Pressure during an Episode of Acute Elevation and Retinal Damage Four Weeks later in Rats(PUBLIC LIBRARY SCIENCE, 2013-07-29)PURPOSE: To determine relationship between the magnitude of intraocular pressure (IOP) during a fixed-duration episode of acute elevation and the loss of retinal function and structure 4 weeks later in rats. METHODS: Unilateral elevation of IOP (105 minutes) was achieved manometrically in adult Brown Norway rats (9 groups; nā=ā4 to 8 each, 10-100 mm Hg and sham control). Full-field ERGs were recorded simultaneously from treated and control eyes 4 weeks after IOP elevation. Scotopic ERG stimuli were white flashes (-6.04 to 2.72 log cd.s.m(-2)). Photopic ERGs were recorded (1.22 to 2.72 log cd.s.m(-2)) after 15 min of light adaptation (150 cd/m(2)). Relative amplitude (treated/control, %) of ERG components versus IOP was described with a cummulative normal function. Retinal ganglion cell (RGC) layer density was determined post mortem by histology. RESULTS: All ERG components failed to recover completely normal amplitudes by 4 weeks after the insult if IOP was 70 mmHg or greater during the episode. There was no ERG recovery at all if IOP was 100 mmHg. Outer retinal (photoreceptor) function demonstrated the least sensitivity to prior acute IOP elevation. ERG components reflecting inner retinal function were correlated with post mortem RGC layer density. CONCLUSIONS: Retinal function recovers after IOP normalization, such that it requires a level of acute IOP elevation approximately 10 mmHg higher to cause a pattern of permanent dysfunction similar to that observed during the acute event. There is a 'threshold' for permanent retinal functional loss in the rat at an IOP between 60 and 70 mmHg if sustained for 105 minutes or more.