Anatomy and Neuroscience - Research Publications
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ItemNo Preview AvailableFailure of Autophagy-Lysosomal Pathways in Rod Photoreceptors Causes the Early Retinal Degeneration Phenotype Observed in Cln6nclf Mice(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2018-10)PURPOSE: Vision loss caused by photoreceptor death represents one of the first symptoms in neuronal ceroid lipofuscinosis, a condition characterized by accumulation of intracellular waste. Cln6nclf mice have a naturally occurring mutation in ceroid-lipofuscinosis neuronal (CLN) protein 6 and are a model of this disorder. In order to identify the effect intracellular waste (lipofuscin) accumulation plays in driving retinal degeneration, the time course of degeneration was carefully characterized functionally using the electroretinogram and structurally using histology. METHODS: Cln6nclf and C57BL/6J, wild-type, mice were studied at postnatal day 18 (P18), P30, P60, P120, and P240, and retinal degeneration was correlated with changes in the retinal pigment epithelial (RPE) and neuronal autophagy-lysosomal pathways using super-resolution microscopy. RESULTS: In Cln6nclf mice there was significant loss of rod photoreceptor function at P18, prior to photoreceptor nuclei loss at P60. In contrast, cone pathway function was not affected until P240. The loss of rod photoreceptor function correlated with significant disruption of the autophagy-lysosomal degradation pathways within photoreceptors, but not in the RPE or other retinal neurons. Additionally, there was cytosolic accumulation of P62 and undigested mitochondrial-derived, ATP synthase subunit C in the photoreceptor layers of Cln6nclf mice at P30. CONCLUSIONS: These results suggest that rod photoreceptors have an increased sensitivity to disturbances in the autophagy-lysosomal pathway and the subsequent failure of mitochondrial turnover, relative to other retinal cells. It is likely that primary failure of the rod photoreceptors rather than the RPE or other retinal neurons underlies the early visual dysfunction that occurs in the Cln6nclf mouse model.
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ItemCorrelation of Histologic Features with In Vivo Imaging of Reticular Pseudodrusen(ELSEVIER SCIENCE INC, 2016-06)PURPOSE: To determine the histologic and cellular correlates in the retina and retinal pigment epithelium (RPE) with the presence of optical coherence tomography-defined reticular pseudodrusen (RPD). DESIGN: Observation case using immunocytochemistry of an exenterated eye with immediate fixation after removal. PARTICIPANTS: Two patients, one with confirmed RPD and the other with mid-peripheral drusen, underwent multimethod imaging before exenteration and immediate fixation of the posterior eyecup for high-resolution immunocytochemical analysis. METHODS: Optical coherence tomography (OCT) was compared with high-resolution immunocytochemistry using a range of cellular markers to determine changes in the RPE, photoreceptors, and gliosis. MAIN OUTCOME MEASURES: Correlations of the appearance of reticular pseudodrusen on OCT and immunocytochemical analysis. RESULTS: Reticular pseudodrusen were deposits juxtaposed to photoreceptor outer segments extending through the outer nuclear layer and even beyond the outer limiting membrane. Deposits were rich in vitronectin, photoreceptor-associated proteins, and Iba1-immunoreactive immune cells. In contrast to conventional drusen the lipid stain Oil Red O failed to stain RPD. Cellular analysis revealed that RPD were associated with photoreceptor disruption and loss and localized gliosis. In addition, anomalies in the RPE were observed. CONCLUSIONS: Reticular pseudodrusen represent subretinal deposits that extend through the outer nuclear layer, affect photoreceptor integrity, and are associated with retinal gliosis and RPE damage.
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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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ItemInner retinal change in a novel rd1-FTL mouse model of retinal degeneration(FRONTIERS MEDIA SA, 2015-07-29)While photoreceptor loss is the most devastating result of inherited retinal degenerations such as retinitis pigmentosa, inner retinal neurons also undergo significant alteration. Detailing these changes has become important as many vision restorative therapies target the remaining neurons. In this study, the rd1-Fos-Tau-LacZ (rd1-FTL) mouse model was used to explore inner retinal change at a late stage of retinal degeneration, after the loss of photoreceptor nuclei. The rd1-FTL model carries a mutation in the phosphodiesterase gene, Pde6b, and an axonally targeted transgenic beta galactosidase reporter system under the control of the c-fos promoter. Retinae of transgenic rd1-FTL mice and control FTL animals aged 2-12 months were processed for indirect fluorescence immunocytochemistry. At 2 months of age, a time when the majority of photoreceptor nuclei are lost, there was negligible c-fos reporter (FTL) expression, however, from 4 months, reporter expression was observed to increase within subpopulations of amacrine and ganglion cells within the central retina. These areas of inner retinal FTL expression coincided with regions that contained aberrant Müller cells. Specifically, these cells exhibited reduced glutamine synthetase and Kir4.1 immunolabelling, whilst showing evidence of proliferative gliosis (increased cyclinD1 and glial fibrillary acidic protein expression). These changes were limited to distinct regions where cone photoreceptor terminals were absent. Overall, these results highlight that distinct areas of the rd1-FTL central retina undergo significant glial alterations after cone photoreceptor loss. These areas coincide with up-regulation of the c-fos reporter in the inner retina, which may represent a change in neuronal function/plasticity. The rd1-FTL mouse is a useful model system to probe changes that occur in the inner retina at later stages of retinal degeneration.
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ItemChanges in morphology of retinal ganglion cells with eccentricity in retinal degeneration(SPRINGER, 2016-05)Ganglion cells are the output neurons of the retina and are known to remodel during the subtle plasticity changes that occur following the death of photoreceptors in inherited retinal degeneration. We examine the influence of retinal eccentricity on anatomical remodelling and ganglion cell morphology well after photoreceptor loss. Rd1 mice that have a mutation in the β subunit of phosphodiesterase 6 were used as a model of retinal degeneration and gross remodelling events were examined by processing serial sections for immunocytochemistry. Retinal wholemounts from rd1-Thy1 and control Thy1 mice that contained a fluorescent protein labelling a subset of ganglion cells were processed for immunohistochemistry at 11 months of age. Ganglion cells were classified based on their soma size, dendritic field size and dendritic branching pattern and their dendritic fields were analysed for their length, area and quantity of branching points. Overall, more remodelling was found in the central compared with the peripheral retina. In addition, the size and complexity of A2, B1, C1 and D type ganglion cells located in the central region of the retina decreased. We propose that the changes in ganglion cell morphology are correlated with remodelling events in these regions and impact the function of retinal circuitry in the degenerated retina.
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ItemNo Preview AvailableNanosecond laser therapy reverses pathologic and molecular changes in age-related macular degeneration without retinal damage(FEDERATION AMER SOC EXP BIOL, 2015-02)Age-related macular degeneration (AMD) is a leading cause of vision loss, characterized by drusen deposits and thickened Bruch's membrane (BM). This study details the capacity of nanosecond laser treatment to reduce drusen and thin BM while maintaining retinal structure. Fifty patients with AMD had a single nanosecond laser treatment session and after 2 yr, change in drusen area was compared with an untreated cohort of patients. The retinal effect of the laser was determined in human and mouse eyes using immunohistochemistry and compared with untreated eyes. In a mouse with thickened BM (ApoEnull), the effect of laser treatment was quantified using electron microscopy and quantitative PCR. In patients with AMD, nanosecond laser treatment reduced drusen load at 2 yr. Retinal structure was not compromised in human and mouse retina after laser treatment, with only a discrete retinal pigment epithelium (RPE) injury, and limited mononuclear cell response observed. BM was thinned in the ApoEnull mouse 3 mo after treatment (ApoEnull treated 683 ± 38 nm, ApoEnull untreated 890 ± 60 nm, C57Bl6J 606 ± 43 nm), with the expression of matrix metalloproteinase-2 and -3 increased (>260%). Nanosecond laser resolved drusen independent of retinal damage and improved BM structure, suggesting this treatment has the potential to reduce AMD progression.
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ItemVesicular expression and release of ATP from dopaminergic neurons of the mouse retina and midbrain(FRONTIERS MEDIA SA, 2015-10-06)Vesicular nucleotide transporter (VNUT) is required for active accumulation of adenosine tri-phosphate (ATP) into vesicles for purinergic neurotransmission, however, the cell types that express VNUT in the central nervous system remain unknown. This study characterized VNUT expression within the mammalian retina and brain and assessed a possible functional role in purinergic signaling. Two native isoforms of VNUT were detected in mouse retina and brain based on RNA transcript and protein analysis. Using immunohistochemistry, VNUT was found to co-localize with tyrosine hydroxylase (TH) positive, dopaminergic (DA) neurons of the substantia nigra and ventral tegmental area, however, VNUT expression in extranigral non-DA neurons was also observed. In the retina, VNUT labeling was found to co-localize solely with TH-positive DA-cells. In the outer retina, VNUT-positive interplexiform cell processes were in close contact with horizontal cells and cone photoreceptor terminals, which are known to express P2 purinergic-receptors. In order to assess function, dissociated retinal neurons were loaded with fluorescent ATP markers (Quinacrine or Mant-ATP) and the DA marker FFN102, co-labeled with a VNUT antibody and imaged in real time. Fluorescent ATP markers and FFN102 puncta were found to co-localize in VNUT positive neurons and upon stimulation with high potassium, ATP marker fluorescence at the cell membrane was reduced. This response was blocked in the presence of cadmium. These data suggest DA neurons co-release ATP via calcium dependent exocytosis and in the retina this may modulate the visual response by activating purine receptors on closely associated neurons.
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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; Müller 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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ItemNo Preview AvailableElectronic restoration of vision in those with photoreceptor degenerations(WILEY, 2012-09)Complete loss of vision is one of the most feared sequelae of retinal disease. Currently, there are few if any treatment options available to patients that may slow or prevent blindness in diseases caused by photoreceptor loss, such as retinitis pigmentosa and age-related macular degeneration. Electronic restoration of vision has emerged over recent years as a safe and viable option for those who have lost substantial numbers of photoreceptors and who are severely vision impaired. Indeed, there has been a dramatic increase in our understanding of what is required to restore vision using an electronic retinal prosthesis. Recent reports show that for some patients, restoration of vision to the point of reading large letters is possible. In this review, we examine the types of implants currently under investigation and the results these devices have achieved clinically. We then consider a range of engineering and biological factors that may need to be considered to improve the visual performance of newer-generation devices. With added research, it is hoped that the level of vision achieved with newer generation devices will steadily improve, resulting in enhanced quality of life for those with severe vision impairment.