Anatomy and Neuroscience - Research Publications
Permanent URI for this collection
Search Results
1 - 10 of 18
-
ItemNo Preview AvailableSelective retinal ganglion cell vulnerability in older mice exposed to acute intraocular pressure elevation and the potential involvement of the P2X7-receptor(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : P2X7-receptors may contribute to retinal ganglion cell (RGC) death in glaucoma. We examined RGC function following acute intraocular pressure (IOP) elevation in older C57BL/6 (WT) mice and P2X7-receptor knockout (P2X7-KO) mice using a multielectrode array (MEA). Methods : In 13-month-old WT (n=15) and P2X7-KO mice (n=9), the anterior chamber of one eye was cannulated (50μm glass micropipette connected to a height-adjustable Hanks balanced salt solution reservoir) to increase IOP to 50 mmHg for 30 minutes. The contralateral eye was cannulated without increasing IOP (sham). Three days following injury, mice were dark-adapted over-night and retinae were mounted onto an MEA to record RGC spontaneous activityand light-evoked responses. Full field stimuli were 1 second flashes modulated between 0 and 1066 photoisomerisations/rod/sec. To test frequency responsiveness, full field light ON and OFF components were modulated from 1 to 30 Hz. Receptive fields were mapped by calculating the spike triggered average in response to a 32x32 checkerboard stimulus (70µm squares) presented at 12 Hz, with mean luminance of 517 photoisomerations/rod/sec. Cells were analyzed and sorted using Spike2 and classified into ON, OFF, ON-OFF and non-responsive types based on peak firing during light on and off full-field stimuli. Results : In WT mice there was a significant reduction in spontaneous activity (p<0.05) and full-field-evoked spike rates (p<0.05) for OFF RGCs after IOP stress compared to OFF cells of sham eyes. These changes appear to be subtype-specific as ON and ON-OFF cells showed no change in response. There were no further effects of IOP at higher temporal frequencies of full field stimulus, nor were there changes in receptive field size. In P2X7-KO mice, OFF RGCs in IOP stressed eyes showed significantly reduced spontaneous rate (p<0.05) compared to OFF RGCs in WT sham eyes, much like the effect of IOP stress on WT OFF cells. Additionally, ON RGCs from P2X7-KO eyes subjected to IOP stress showed a significant decrease in peak spike rate compared to P2X7-KO sham eyes (p<0.05). Conclusions : These results suggest that even a short period of mild IOP stress can have long lasting effect on RGC function, particularly that of OFF-RGCs. In contrast to previous studies, P2X7-KO did not prevent RGC functional deficits associated with acute mild IOP elevation.
-
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.
-
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.
-
ItemNanosecond Laser Treatment for Age-Related Macular Degeneration Does Not Induce Focal Vision Loss or New Vessel Growth in the Retina(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2018-02)PURPOSE: Subthreshold, nanosecond pulsed laser treatment shows promise as a treatment for age-related macular degeneration (AMD); however, the safety profile needs to be robustly examined. The aim of this study was to investigate the effects of laser treatment in humans and mice. METHODS: Patients with AMD were treated with nanosecond pulsed laser at subthreshold (no visible retinal effect) energy doses (0.15-0.45 mJ) and retinal sensitivity was assessed with microperimetry. Adult C57BL6J mice were treated at subthreshold (0.065 mJ) and suprathreshold (photoreceptor loss, 0.5 mJ) energy settings. The retinal and vascular responses were analyzed by fundus imaging, histologic assessment, and quantitative PCR. RESULTS: Microperimetry analysis showed laser treatment had no effect on retinal sensitivity under treated areas in patients 6 months to 7 years after treatment. In mice, subthreshold laser treatment induced RPE loss at 5 hours, and by 7 days the RPE had retiled. Fundus imaging showed reduced RPE pigmentation but no change in retinal thickness up to 3 months. Electron microscopy revealed changes in melanosomes in the RPE, but Bruch's membrane was intact across the laser regions. Histologic analysis showed normal vasculature and no neovascularization. Suprathreshold laser treatment did not induce changes in angiogenic genes associated with neovascularization. Instead pigment epithelium-derived factor, an antiangiogenic factor, was upregulated. CONCLUSIONS: In humans, low-energy, nanosecond pulsed laser treatment is not damaging to local retinal sensitivity. In mice, treatment does not damage Bruch's membrane or induce neovascularization, highlighting a reduced side effect profile of this nanosecond laser when used in a subthreshold manner.
-
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.
-
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.
-
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.
-
ItemRetinal Changes in an ATP-Induced Model of Retinal Degeneration(FRONTIERS MEDIA SA, 2016-04-29)In rodents and felines, intravitreal administration of adenosine triphosphate (ATP) has been shown to induce photoreceptor death providing a tractable model of retinal degeneration in these species. This study investigated the long term effects of photoreceptor loss in an ATP induced feline model of retinal degeneration. Six normal sighted felines were unilaterally blinded using intravitreal ATP injections and assessed using electroretinography (ERG) and optical coherence tomography (OCT). At 30 h (n = 3) or 12 weeks (n = 3) post-injection, the animals were euthanized and the eyes enucleated. Retinae were sectioned and labeled using immunohistochemistry for markers of cell death, neural remodeling and gliosis. Ongoing cell death and retinal degeneration was observed in the outer retina at both 30 h and 12 weeks following unilateral ATP injection. Markers of mid to late-stage retinal remodeling such as cell displacement and aberrant neurite growth were observed in the inner retina at 12 weeks post-injection. Ganglion cells appeared to remain intact in ATP injected eyes. Müller cell gliosis was observed throughout the inner and outer retina, in some parts completely enveloping and/or displacing the surviving neural tissue. Our data suggests that the ATP injected feline retina continues to undergo progressive retinal degeneration and exhibits abnormalities consistent with a description of retinal remodeling commonly seen in other models of retinal degeneration. These findings validate the use of intravitreal ATP injection in feline as a large animal model of retinal degeneration which may aid in development of therapies aiming to restore visual function after photoreceptor degeneration.
-
ItemRod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse(PUBLIC LIBRARY SCIENCE, 2012-01-10)The P2X7 receptor (P2X7-R) is expressed in the retina and brain and has been implicated in neurodegenerative diseases. However, whether it is expressed by neurons and plays a role as a neurotransmitter receptor has been the subject of controversy. In this study, we first show that the novel vesicular transporter for ATP, VNUT, is expressed in the retina, verifying the presence of the molecular machinery for ATP to act as neurotransmitter at P2X7-Rs. Secondly we show the presence of P2X7-R mRNA and protein in the retina and cortex and absence of the full length variant 1 of the receptor in the P2X7-R knock out (P2X7-KO) mouse. The role of the P2X7-R in neuronal function of the retina was assessed by comparing the electroretinogram response of P2X7-KO with WT mice. The rod photoreceptor response was found to be similar, while both rod and cone pathway post-photoreceptor responses were significantly larger in P2X7-KO mice. This suggests that activation of P2X7-Rs modulates output of second order retinal neurons. In line with this finding, P2X7-Rs were found in the outer plexiform layer and on inner retinal cell classes, including horizontal, amacrine and ganglion cells. The receptor co-localized with conventional synapses in the IPL and was expressed on amacrine cells post-synaptic to rod bipolar ribbon synapses. In view of the changes in visual function in the P2X7-KO mouse and the immunocytochemical location of the receptor in the normal retina, it is likely the P2X7-R provides excitatory input to photoreceptor terminals or to inhibitory cells that shape both the rod and cone pathway response.
-
ItemAmyloid Precursor Protein Is Required for Normal Function of the Rod and Cone Pathways in the Mouse Retina(PUBLIC LIBRARY SCIENCE, 2012-01-18)Amyloid precursor protein (APP) is a transmembrane glycoprotein frequently studied for its role in Alzheimer's disease. Our recent study in APP knockout (KO) mice identified an important role for APP in modulating normal neuronal development in the retina. However the role APP plays in the adult retina and whether it is required for vision is unknown. In this study we evaluated the role of APP in retinal function and morphology comparing adult wildtype (WT) and APP-KO mice. APP was expressed on neuronal cells of the inner retina, including horizontal, cone bipolar, amacrine and ganglion cells in WT mice. The function of the retina was assessed using the electroretinogram and although the rod photoreceptor responses were similar in APP-KO and WT mice, the post-photoreceptor, inner retinal responses of both the rod and cone pathways were reduced in APP-KO mice. These changes in inner retinal function did not translate to a substantial change in visual acuity as assessed using the optokinetic response or to changes in the gross cellular structure of the retina. These findings indicate that APP is not required for basic visual function, but that it is involved in modulating inner retinal circuitry.