Anatomy and Neuroscience - Research Publications

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    Reversibility of retinal ganglion cell dysfunction due to chronic IOP elevation.
    Zhao, D ; Wong, VHY ; He, Z ; Nguyen, CTO ; Jobling, AI ; Fletcher, E ; Chinnery, H ; Jusuf, P ; Lim, JKH ; Vingrys, AJ ; Bui, BV (Association for Research in Vision and Ophthalmology, 2018-07-01)
    Purpose : To determine the duration of chronic IOP elevation beyond which ganglion cell function can no longer recover using the mouse circumlimbal suture model. Methods : IOP elevation was induced in anaesthetized (isoflurane) adult male C57BL6/J mice by attaching a circumlimbal suture (nylon, 10/0) around the equator of one eye, with the contralateral eye serving as a control. The suture was left in place for 8, 12 and 16 weeks (n=27, 23 and 27), respectively, and animals underwent electroretinography and optical coherence tomography at these time points. In two other groups, the suture was removed after 8 and 12 weeks (n=26 and 28), and the capacity for recovery assessed 4 weeks later. IOP was measured weekly (Tonolab). Retinal ganglion cell (RGC) function (or integrity) was assessed with the positive scotopic threshold response (pSTR) and retinal nerve fibre layer (RNFL) thickness. Data (mean ± SEM) were compared using t-test (control vs. treatment) and one-way ANOVA (within groups). Results : IOP in sutured eyes was higher than control eyes (8wk: 17.1 ± 0.3 vs. 26.8 ± 0.6 mmHg, 12wk: 13.8 ± 0.3 vs. 19.5 ± 0.5 mmHg, 16wk: 17.1 ± 0.2 vs. 27.4 ± 0.6 mmHg; all P<0.001). After suture removal, IOP returned to levels comparable to control eyes (8+4wk: 16.9 ± 0.3 vs. 16.1 ± 0.3 mmHg; P=0.08, 12+4wk: 17.3 ± 0.2 vs. 17.1 ± 0.3 mmHg; P=0.5). With IOP elevation, RGC function declined to 75% ± 8% (8wk), 78% ± 7% (12wk) and 59% ± 4% (16wk, all P<0.001) of control eyes. RNFL thinning was also evident (8wk: 84% ± 4%, 12wk: 83% ± 5%; 16wk: 83% ± 3%; P<0.001) but no change in total retinal thickness was noted (P=0.33). Suture removal at week 8 facilitated full recovery of RGC function (97% ± 7%, P=0.9 vs. baseline) 4 weeks later. However, there was no recovery in RNFL thickness (87% ± 3%, P<0.001 vs. baseline). When the suture was removed at week 12, neither function (79% ± 9%, P<0.05) nor RNFL thickness recovered (89% ± 3%, P<0.01) 4 weeks later. Conclusions : RGC dysfunction can be recovered 4 weeks after an 8-week period of mild IOP elevation, but not after a 12-week period. Beyond 12 weeks, IOP reversal only served to prevent further functional decline. This identifies a critical chronic IOP duration that results in irreversible ganglion cell dysfunction. This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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    Treatments targeting autophagy ameliorate the age-related macular degeneration phenotype in mice lacking APOE (apolipoprotein E)
    Vessey, KA ; Jobling, A ; Tran, MX ; Wang, AY ; Greferath, U ; Fletcher, EL (TAYLOR & FRANCIS INC, 2022-02-24)
    Age-related macular degeneration (AMD) is a leading cause of vision loss with recent evidence indicating an important role for macroautophagy/autophagy in disease progression. In this study we investigate the efficacy of targeting autophagy for slowing dysfunction in a mouse model with features of early AMD. Mice lacking APOE (apolipoprotein E; B6.129P2-Apoetm1UncJ/Arc) and C57BL/6 J- (wild-type, WT) mice were treated with metformin or trehalose in the drinking water from 5 months of age and the ocular phenotype investigated at 13 months. Control mice received normal drinking water. APOE-control mice had reduced retinal function and thickening of Bruch's membrane consistent with an early AMD phenotype. Immunohistochemical labeling showed reductions in MAP1LC3B/LC3 (microtubule-associated protein 1 light chain 3 beta) and LAMP1 (lysosomal-associated membrane protein 1) labeling in the photoreceptors and retinal pigment epithelium (RPE). This correlated with increased LC3-II:LC3-I ratio and alterations in protein expression in multiple autophagy pathways measured by reverse phase protein array, suggesting autophagy was slowed. Treatment of APOE-mice with metformin or trehalose ameliorated the loss of retinal function and reduced Bruch's membrane thickening, enhancing LC3 and LAMP1 labeling in the ocular tissues and restoring LC3-II:LC3-I ratio to WT levels. Protein analysis indicated that both treatments boost ATM-AMPK driven autophagy. Additionally, trehalose increased p-MAPK14/p38 to enhance autophagy. Our study shows that treatments targeting pathways to enhance autophagy have the potential for treating early AMD and provide support for the use of metformin, which has been found to reduce the risk of AMD development in human patients.Abbreviations:AMD: age-related macular degeneration; AMPK: 5' adenosine monophosphate-activated protein kinase APOE: apolipoprotein E; ATM: ataxia telangiectasia mutated; BCL2L1/Bcl-xL: BCL2-like 1; DAPI: 4'-6-diamidino-2-phenylindole; ERG: electroretinogram; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; IS/OS: inner and outer photoreceptor segments; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; OCT: optical coherence tomography; ONL: outer nuclear layer; OPs: oscillatory potentials; p-EIF4EBP1: phosphorylated eukaryotic translation initiation factor 4E binding protein 1; p-MAPK14/p38: phosphorylated mitogen-activated protein kinase 14; RPE: retinal pigment epithelium; RPS6KB/p70 S6 kinase: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; TP53/TRP53/p53: tumor related protein 53; TSC2: TSC complex subunit 2; WT: wild type.
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    Transcriptomic analysis of choroidal neovascularization reveals dysregulation of immune and fibrosis pathways that are attenuated by a novel anti-fibrotic treatment
    Brandli, A ; Khong, FL ; Kong, RCK ; Kelly, DJ ; Fletcher, EL (NATURE PORTFOLIO, 2022-01-17)
    Neovascular AMD (nAMD) leads to vision loss and is a leading cause of visual impairment in the industrialised world. Current treatments that target blood vessel growth have not been able to treat subretinal fibrosis and nAMD patients continue to lose vision. The molecular mechanisms involved in the development of fibrotic lesions in nAMD are not well understood. The aim of this study was to further understand subretinal fibrosis in the laser photocoagulation model of choroidal neovascularization (CNV) by studying the whole transcriptome of the RPE/choroid following CNV and the application of an anti-fibrotic following CNV. Seven days after laser induced CNV, RPE and choroid tissue was separated and underwent RNAseq. Differential expression analysis and pathway analysis revealed an over representation of immune signalling and fibrotic associated pathways in CNV compared to control RPE/choroid tissue. Comparisons between the mouse CNV model to human CNV revealed an overlap in upregulated expression for immune genes (Ccl2, Ccl8 and Cxcl9) and extracellular matrix remodeling genes (Comp, Lrcc15, Fndc1 and Thbs2). Comparisons between the CNV model and other fibrosis models showed an overlap of over 60% of genes upregulated in either lung or kidney mouse models of fibrosis. Treatment of CNV using a novel cinnamoyl anthranilate anti-fibrotic (OCX063) in the laser induced CNV model was selected as this class of drugs have previously been shown to target fibrosis. CNV lesion leakage and fibrosis was found to be reduced using OCX063 and gene expression of genes within the TGF-beta signalling pathway. Our findings show the presence of fibrosis gene expression pathways present in the laser induced CNV mouse model and that anti-fibrotic treatments offer the potential to reduce subretinal fibrosis in AMD.
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    Selective retinal ganglion cell vulnerability in older mice exposed to acute intraocular pressure elevation and the potential involvement of the P2X7-receptor
    Wang, AY ; Vessey, KA ; Bui, BV ; Wong, VHY ; Lee, PY ; Fletcher, EL (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.
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    Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy
    Mills, S ; Jobling, A ; Dixon, M ; Bui, B ; Vessey, K ; Phipps, J ; Greferath, U ; Venables, G ; Wong, VHY ; Wong, CHY ; He, Z ; Hui, F ; Young, J ; Tonc, J ; Ivanova, E ; Sagdullaev, B ; Fletcher, E ( 2020-06-16)
    Local blood flow control within the CNS is critical to proper function and is dependent on coordination between neurons, glia and blood vessels. Macroglia such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial involvement, since mice lacking Cx3cr1, exhibited no fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan, abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 weeks due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.

    Significance Statement

    This work identifies a novel role for microglia, the innate immune cells of the CNS, in the local control of the retinal vasculature and identifies deficits early in diabetes. Microglia contact neurons and vasculature and express several vasoactive agents. Activation of microglial fractalkine-Cx3cr1 signalling leads to capillary constriction and blocking the renin-angiotensin system (RAS) with candesartan abolishes microglial-mediated vasoconstriction in the retina. In early diabetes, reduced retinal blood flow is coincident with capillary constriction, increased microglial-vessel association, loss of microglial-capillary regulation and altered microglial expression of the RAS pathway. While candesartan restores retinal capillary diameter early in diabetes, targeting of microglial-vascular regulation is required to prevent coincident dilation of large retinal vessels and reduced retinal blood flow.
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    Potential mechanisms of retinal ganglion cell type-specific vulnerability in glaucoma
    Wang, AYM ; Lee, PY ; Bui, B ; Jobling, A ; Greferath, U ; Brandli, A ; Dixon, MA ; Findlay, Q ; Fletcher, EL ; Vessey, KA (WILEY, 2020-09-01)
    Glaucoma is a neurodegenerative disease characterised by progressive damage to the retinal ganglion cells (RGCs), the output neurons of the retina. RGCs are a heterogenous class of retinal neurons which can be classified into multiple types based on morphological, functional and genetic characteristics. This review examines the body of evidence supporting type-specific vulnerability of RGCs in glaucoma and explores potential mechanisms by which this might come about. Studies of donor tissue from glaucoma patients have generally noted greater vulnerability of larger RGC types. Models of glaucoma induced in primates, cats and mice also show selective effects on RGC types - particularly OFF RGCs. Several mechanisms may contribute to type-specific vulnerability, including differences in the expression of calcium-permeable receptors (for example pannexin-1, P2X7, AMPA and transient receptor potential vanilloid receptors), the relative proximity of RGCs and their dendrites to blood supply in the inner plexiform layer, as well as differing metabolic requirements of RGC types. Such differences may make certain RGCs more sensitive to intraocular pressure elevation and its associated biomechanical and vascular stress. A greater understanding of selective RGC vulnerability and its underlying causes will likely reveal a rich area of investigation for potential treatment targets.
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    Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy
    Mills, SA ; Jobling, A ; Dixon, MA ; Bui, B ; Vessey, KA ; Phipps, JA ; Greferath, U ; Venables, G ; Wong, VHY ; Wong, CHY ; He, Z ; Hui, F ; Young, JC ; Tonc, J ; Ivanova, E ; Sagdullaev, BT ; Fletcher, EL (NATL ACAD SCIENCES, 2021-12-21)
    Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.
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    Reversibility of Retinal Ganglion Cell Dysfunction From Chronic IOP Elevation
    Zhao, D ; Wong, VHY ; Nguyen, CTO ; Jobling, AI ; Fletcher, EL ; Vingrys, AJ ; Bui, BV (ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2019-09-01)
    Purpose: To test the hypothesis that the capacity for retinal ganglion cells to functionally recover from chronic IOP elevation is dependent on the duration of IOP elevation. Methods: IOP elevation was induced in one eye in anesthetized (isoflurane) adult C57BL6/J mice using a circumlimbal suture. Sutures were left in place for 8 and 16 weeks (n = 30 and 28). In two other groups the suture was cut after 8 and 12 weeks (n = 30 and 28), and ganglion cell function (electroretinography) and retinal structure (optical coherence tomography) were assessed 4 weeks later. Ganglion cell density was quantified by counting RBPMS (RNA-binding protein with multiple splicing)-stained cells. Results: With IOP elevation (∼10 mm Hg above baseline), ganglion cell function declined to 75% ± 8% at 8 weeks and 59% ± 4% at 16 weeks relative to contralateral control eyes. The retinal nerve fiber layer was thinner at 8 (84% ± 4%) and 16 weeks (83% ± 3%), without a significant difference in total retinal thickness. Ganglion cell function recovered with IOP normalization (suture removal) at week 8 (97% ± 7%), but not at week 12 (73% ± 6%). Ganglion cell loss was found in all groups (-8% to -13%). Conclusions: In the mouse circumlimbal suture model, 12 weeks of IOP elevation resulted in irreversible ganglion cell dysfunction, whereas retinal dysfunction was fully reversible after 8 weeks of IOP elevation.
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    Failure of Autophagy-Lysosomal Pathways in Rod Photoreceptors Causes the Early Retinal Degeneration Phenotype Observed in Cln6(nclf) Mice
    von Eisenhart-Rothe, P ; Grubman, A ; Greferath, U ; Fothergill, LJ ; Jobling, A ; Phipps, JA ; White, AR ; Fletcher, EL ; Vessey, KA (ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2018-10-01)
    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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    Correlation of Histologic Features with In Vivo Imaging of Reticular Pseudodrusen
    Greferath, U ; Guymer, RH ; Vessey, KA ; Brassington, K ; Fletcher, EL (ELSEVIER SCIENCE INC, 2016-06-01)
    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.