Optometry and Vision Sciences - Research Publications
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ItemNo Preview AvailableReversibility of retinal ganglion cell dysfunction due to chronic IOP elevation.(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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ItemNo Preview AvailableResponse of the Rat Optic Nerve to Acute Intraocular and Intracranial Pressure Changes(Springer Singapore, 2019)Glaucoma is a neurodegenerative disease, characterized by the progressive death of retinal ganglion cells. Elevated intraocular pressure (IOP) is known to be an important risk factor for glaucoma; however, it is not the only force acting on the optic nerve. Intracranial pressure (ICP) also exerts an effect on the optic nerve head, effectively opposing the force applied by IOP. Indeed, this balance of forces creates a pressure gradient (or the translaminar pressure gradient) across the optic nerve head [1]. Increasingly it is thought that the pressure difference between IOP and ICP, the translaminar pressure (TLP), may be critical for the integrity of the retina and optic nerve [2], and thus ICP may be an important risk factor for glaucoma [2–6].
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ItemA Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice(Journal of Visualized Experiments, 2018)The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.
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ItemUtility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina(MARY ANN LIEBERT, INC, 2019-11-01)Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing "kamikaze" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were initially designed to target Streptococcus pyogenes Cas9 (SpCas9) and after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein [YFP]), in the presence of mCherry. Both constructs were packaged into AAV2 vectors and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks, the expression of SpCas9 and the efficacy of YFP gene disruption were quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated YFP/SpCas9 targeting CRISPR/Cas compared with those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (∼85.5% reduction compared with LacZ/SpCas9 targeting CRISPR/Cas) in the transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive "kamikaze" CRISPR/Cas system can be used as a robust tool for genome editing in the retina, without compromising on-target efficiency.
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ItemNo Preview AvailableEvaluating retinal biomarkers in a mouse model of Parkinson's disease(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : The retina, an accessible outpouching of the central nervous system, may manifest cortical changes that occur with Parkinson’s disease (PD), lending itself as a potential biomarker. PD is characterised by reduced dopamine levels, a neurotransmitter found in amacrine cells. Human PD patients have also shown structural changes in the outer retina. This work aims to determine if retinal function and structure are altered in a murine model of PD and whether deficits can be ameliorated with L-DOPA treatment. Methods : A PD model was induced in adult C57BL6/J mice using MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 4x i.p. injections, 20mg/kg) and vehicle control and examined at day 21 and 45. Another MPTP group was administered L-DOPA (L-3,4-dihydroxyphenylalanine 0.2 mg/ml) or control in their drinking water and assessed at day 45 (n=12–15/group). In ketamine:xylazine anaesthetised (80:10mg/kg) mice full-field dark- and light-adapted electroretinography (ERG) was assessed to target dopamine-related responses. Optical coherence tomography (OCT) was used to quantify thickness of retinal layers. Retinal and cortical tissue were collected for immunohistochemical assessment of changes in tyrosine hydroxylase (TH)and imaged using confocal microscopy. Data (mean±SEM) were compared using unpaired ANOVA and t-tests as appropriate. Results : At day 21 no retinal changes were found. At day 45 dark and light adapted ERGs showed slower amacrine cell responses (oscillatory potential, p<0.05), a finding which reversed with L-DOPA treatment (p<0.05). Other components of the ERG were unchanged. TH staining showed a trend towards decreased retinal levels in MPTP mice but this did not reach significance (p=0.10). Reduced levels of TH were found in the ventral hippocampus of MPTP mice compared with control (p<0.05). OCT revealed thinning of the outer plexiform layer at day 45, and the L-DOPA group exhibited a thinning of the outer nuclear layer (p<0.05). Conclusions : This study shows for the first time that the MPTP model recapitulates key dopaminergic changes previously reported in humans. In particular, electroretinographic changes that correspond with dopaminergic retinal cells occur in the Parkinson’s model and reverse with therapeutic treatment. Structural thinning of the outer retinal layers also occur, which parallels some human findings. This work paves the way for retinal measures as preclinical screening tools in drug development.
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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.
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ItemNo Preview AvailableThe effect of ageing on the recovery of retinal function and structure following intraocular pressure elevation in mice(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : To investigate the effect of ageing on the capacity of the eye to cope with acute intraocular pressure (IOP) elevation in mice Methods : IOP was elevated to 50 mmHg for 30 minutes in anaesthetised (ketamine/xylazine) 3- and 12-month old (3mo and 12mo) C57Bl/6 mice by infusing Hanks’ Balance Salt Solution through a glass micropipette (~50μm tip) inserted into the anterior chamber of one randomly selected eye. The contralateral eye served as an untreated control. Retinal function was assessed using electroretinogram to provide an index of the health of the major cell classes in the eye. Retinal structure was assessed using optical coherence tomography (OCT) which returns thickness for a range of retinal layers. Responses were collected one week prior to and at 3 (n=13 3mo, n=11 12mo), 7 (n=13 3mo, n=10 12mo), 14 (n=10 3mo, n=11 12mo) or 28 (n=11 3mo, n=11 12mo) days after IOP elevation. Responses in the high IOP eye were expressed relative (%) to their contralateral control eye (mean±SEM). As retinal ganglion cell (RGC) responses are influenced by input from the outer retina, we expressed the functional recovery of RGC as the % difference between relative RGC (output cells) and photoreceptor (input cells) function. The effect of age on RGC functional recovery and retinal structural changes at the various recovery time points was analysed using two-way ANOVA. Results : In 3-month old eyes, 3 days after IOP elevation, RGC function was -37.3±7.0% worse than expected from photoreceptoral input. By 7 days after IOP elevation, RGC responses were similar to photoreceptor responses (-5.7±7.2%) and remained so at 14 (-9.7±6.0%) and 28 (15.6±16.4%) days of recovery. In contrast, 12-month old eyes showed slower recovery. RGC responses were worse than expected from photoreceptoral responses at 3 (-58.1±6.1%) and 7 (-34.8±10.5%) days. Only at 14 (-9.4±10.0%) and 28 (1.9±13.1%) days had RGC responses returned to levels comparable with photoreceptoral responses in 12-month old eyes. Two-way ANOVA confirmed a significant age effect in the functional recovery (p<0.05). There was, however, no significant differences in retinal layers measured using OCT with age. Conclusions : RGC function was more affected by acute IOP elevation than photoreceptoral responses. Ageing slowed down the functional recovery of RGC following an acute IOP stressor but appears to have little effect on retinal structure.
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ItemProgressive impairments in executive function in the APP/PS1 model of Alzheimer’s disease as measured by translatable touchscreen testing( 2019-08-21)Executive function deficits in Alzheimer’s disease (AD) occur early in disease progression and may be predictive of cognitive decline. However, no preclinical studies have identified deficits in rewarded executive function in the commonly used APP/PS1 mouse model. To address this, we assessed 12-26 month old APP/PS1 mice on rewarded reversal and/or extinction tasks. 16-month-old, but not 13- or 26-month-old, APP/PS1 mice showed an attenuated rate of extinction. Reversal deficits were seen in 22-month-old, but not 13-month-old APP/PS1 animals. We then confirmed that impairments in reversal were unrelated to previously reported visual impairments in both AD mouse models and humans. Age, but not genotype, had a significant effect on markers of retinal health, indicating the deficits seen in APP/PS1 mice were directly related to cognition. This is the first characterisation of rewarded executive function in APP/PS1 mice, and has great potential to facilitate translation from preclinical models to the clinic.
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ItemAge-Specific Retinal and Cerebral Immunodetection of Amyloid-β Plaques and Oligomers in a Rodent Model of Alzheimer's Disease(IOS PRESS, 2020)BACKGROUND: Amyloid-β soluble oligomers (Aβo) are believed to be the cause of the pathophysiology underlying Alzheimer's disease (AD) and are normally detected some two decades before clinical onset of the disease. Retinal pathology associated with AD pathogenesis has previously been reported, including ganglion cell loss, accumulation of Aβ deposits in the retina, and reduction of nerve fiber layer thickness as well as abnormalities of the microvasculature. OBJECTIVE: This study's aim is to better understand the relationship between brain and retinal Aβo deposition and in particular to quantify levels of the toxic Aβo as a function of age in the retina of a rodent model of AD. METHODS: Retinas and brain tissue from 5×FAD mice were stained with Congo red, Thioflavin-T (Th-T), and Aβ plaque-specific and Aβo-specific antibodies. RESULTS: We show that retinas displayed an age-dependent increase of Th-T-specific amyloid fibrils. Staining with anti-Aβ antibody confirmed the presence of the Aβ plaques in all 5×FAD retinas tested. In contrast, staining with anti-Aβo antibody showed an age-dependent decrease of retinal Aβo. Of note, Aβo was observed mainly in the retinal nuclear layers. Finally, we confirmed the localization of Aβo to neurons, typically accumulating in late endosomes, indicating possible impairment of the endocytic pathway. CONCLUSION: Our results demonstrate the presence of intraneuronal Aβo in the retina and its accumulation inversely correlated with retinal Aβ plaque deposition, indicating an age-related conversion in this animal model. These results support the development of an early AD diagnostic test targeting Aβo in the eye.
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ItemNo Preview AvailableA tractable preclinical model of optic nerve demyelination(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : Progress in the development of therapies to enhance remyelination in demyelinating diseases has been hampered by a lack of appropriate preclinical models - functional measures are often lacking or variable. We sought to develop a tractable and reproducible model of optic nerve demyelination with precise structural and functional measures. Methods : Oligodendrocytes of MBP-DTR 100a transgenic mice express diphtheria toxin receptor (DTR) and systemic diphtheria toxin (DT) administration induces diffuse demyelination of the central nervous system. In the present study we used retrobulbar DT injection to induce focal demyelination of the optic nerves of 3-month-old MBP-DTR 100a mice. Dose optimisation: anaesthetised mice underwent unilateral retrobulbar DT injection with 5, 10 or 15ng/kg DT (n=7 per dose, 1 µL per injection). Tissues were harvested three weeks after injection. Time-course study: Following baseline visual evoked potential (VEP) recording, electroretinogram (ERG) and optical coherence tomography (OCT), mice underwent retrobulbar DT injection with 15ng/kg DT or 1µL PBS. Follow-up measurements were taken at 2 (n=5 DT, 5 PBS), 4 (n=6 DT, 6 PBS), 8 (n=9 DT, 9 PBS) or 12-weeks (n=7 DT, 7 PBS). Animals were culled at each timepoint for tissue analysis. Tissue analysis: Optic nerves were resin embedded, sectioned (1µm) and stained with toluidine blue for myelin analysis, or cryosectioned for immunofluorescence, and retinas were flat-mounted for ganglion cell counts. Results : 3 weeks after injection with 15ng/kg DT, optic nerves showed colocalisation of activated caspase 3 & olig2, consistent with the apoptosis of oligodendroglia. Gliosis and axonal degeneration were evident.