Optometry and Vision Sciences - Research Publications

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Hypercapnia Impairs Vascular Responses to Changes in Ocular Perfusion Pressure in Rat Retina

Cull, G ; Wang, L ; Bui, BV (Association for Research in Vision and Ophthalmology, 2018-07-01)

Purpose : Retinal vascular resistance is constantly regulated by both myogenic and metabolic mechanisms. While most studies have investigated these mechanisms separately, how they interact to impact the vascular resistance is unclear. We considered whether hypercapnia (HC) modified the effect of ocular perfusion pressure (OPP) lowering, induced by lowering blood pressure (BP) or increasing intraocular pressure (IOP) on retinal vessel diameter (Ø). Methods : In pentobarbital anesthetized Brown Norway rats, breathing ~30% O2 air, normocapnia (NC) and hypercapnia (HC) were achieved by controlled ventilation. A gradual decrease in OPP, at the same rate for BP lowering or IOP elevation, was induced by drawing blood (1ml/min) from a femoral artery or by increasing the IOP manometrically from 10 to 70 mmHg (9.78 mmHg/min) in two subgroups for each NC (BP=7, IOP=9) and HC (BP=9, IOP=5). Arterial CO2 partial pressure (pCO2) was measured. In all groups, image sequences centered on the optic nerve were acquired with a confocal scanning laser ophthalmoscope (cSLO) every 1.5 minutes until OPP was < 20 mmHg. Arteries and veins at 1-disc diameter from optic nerve were analyzed. Change in Ø (%) was normalized to its own baseline (before pressure manipulation) and differences between NC and HC groups were compared. Results : Average pCO2levelwas 35 ±5 mmHg (n=12) in NC and 77 ±18 mmHg (n=10) in HC (p<0.001), and pO2 was >80 mmHg for all animals. In the NC group, lowering BP induced progressive arterial and venous dilation (P<0.0001 and 0.005, respectively. Fig 1); increasing IOP caused vasodilatation in arteries (P<0.0001), but not in veins (P>0.05, Fig 2). Vasodilation was evident when OPP dropped to 50-60 mmHg for both BP and IOP modification. During HC, IOP and BP induced arterial vasodilation was significantly attenuated and venous diameter showed greater compression (P<0.0001 both, 2-way ANOVA) compared to the NC groups. Conclusions : Carbon dioxide levels significantly modifies the capacity for retinal blood vessel to cope with reduction in OPP. This data show that the metabolic status of the retina profoundly impact vascular autoregulation, which has implication for metabolic diseases. This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Response of the trilaminar retinal vessel network to intraocular pressure elevation in rat eyes

Bui, BV ; Zhao, D ; Wang, L ; Fortune, B ; He, Z (Association for Research in Vision and Ophthalmology, 2018-07-01)

Purpose : It is known that inner retinal blood flow is autoregulated to compensate for changes in ocular perfusion pressure (OPP). However, studies have focused on the superficial vessels. Here we test the hypothesis that the superficial, intermediate and deep retinal vascular plexus show different responses to intraocular pressure (IOP) elevation. Methods : Anesthetized (60:5mg/kg ketamine:xylazine) adult Long Evans rats (n=14) were imaged using optical coherence tomography angiography (OCTA) at baseline (IOP 10mmHg) and in follow up mode to examine the vasculature during IOP elevation (10mmHg steps to 110mmHg, each lasting 3 min). We imaged a 20 x 10-degree field starting at one disc diameter from the optic disc margin. Capillary area (i.e. diameter) within a 2D projection image was determined (% region of interest) for three layers based on segmentation of the structural OCT: superficial vascular complex (SVC), intermediate capillary plexus (ICP) and deep capillary plexus (DVP). Increases and decreases in this parameter can be interpreted as functional “vasodilation” and “vasoconstriction”, respectively, of the column of blood flowing above threshold. Comparisons were made between layers (2-way repeated measures ANOVA, layer vs IOP) following normalisation to baseline (% relative to 10mmHg). Results : Group mean arterial blood pressure at baseline was 125±5 mmHg, thus for the IOPs examined OPP spanned 115±5 to -9±4 mmHg. For OPPs from 115±5 to 77±4 mmHg capillaries within the DCP (9±8%, p<0.05) and ICP (11±10%, p<0.05) showed significant “vasodilation”, whereas those in the SVC showed constriction (-14±6%, p<0.05). For OPPs between 63±4 and 38±4 mmHg, capillary diameter was maintained, by for OPPs below 38mmHg, all layers showed linear attenuation. Significant compression in tissue thickness (retinal nerve fibre, ganglion cell and inner plexiform layers and total retinal thickness) for the same regions were not found until OPP fell below 38mmHg. Conclusions : These data show that the intermediate and deep vascular plexus in the rat retina have a greater capacity for autoregulation against IOP elevation. This might reflect a redistribution of blood flow to the deeper layers during stress. This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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.
Evaluating retinal biomarkers in a mouse model of Parkinson's disease

Nguyen, CTO ; Tran, K ; Lim, JKH ; Wong, VHY ; Shahandeh, A ; Vingrys, AJ ; Bui, BV ; Finkelstein, D (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.
How ganglion cell responses to IOP elevation are impacted by blood pressure and intracranial pressure

Bui, BV ; van Koeverden, A ; He, Z ; Vingrys, AJ ; Nguyen, CTO ; Zhao, D (Association for Research in Vision and Ophthalmology, 2019-07-01)

Purpose : The extent to which blood pressure or intracranial pressure modifies ganglion cell responses to acute intraocular pressure (IOP) elevation incompletely understood. Using the electroretinogram (ERG) we measure ganglion cell mediated responses in rat retina, whilst acutely modifying IOP, BP and ICP in a systematic manner. We quantify the relationship between ganglion cell function and ocular perfusion pressure (BP - IOP) at low, normal and high ICP. Methods : Six groups of adult Long-Evans rats (n=7-11 eyes/group, total animals = 25) were anaesthetised (60:5mg/kg ketamine:xylazine) and underwent acute pressure modification. A femoral artery and vein were cannulated for blood pressure measurement and manipulation (sodium nitroprusside to lower and angiotensin II to elevate pressure). ICP was set to -5, 5 or 25 mmHg via a dual cannula (30G infusion needle inside a 23G measurement needle) placed into the lateral ventricle (-1.5mm from bregma, ±2mm from midline) on the ipsilateral side to the cannulated eye (30G, vitreal chamber). At each ICP (-5, 5 or 25 mmHg) and BP setting (normal or high), IOP was raised from 10 to 90 mmHg in 10 mmHg steps (3 min each). At each IOP level ganglion cell function was assessed using the scotopic threshold response (-5 log cd.s/m2, 20 repeats). Data were compared using one- and two-way ANOVA. Results : Average blood pressure at baseline was similar for the normal blood pressure groups (ICP-5 93±3; ICP5 99±5; ICP25 105±3mmHg, p=0.8). There was significant BP elevation in all the high blood pressure groups (ICP-5 160±3; ICP5 157±3; ICP25 157±5mmHg p<0.001). Compared with normal blood pressure groups (32.0±2.0μV), animals with high blood pressure (24.5±1.8μV) had significantly smaller baseline STR amplitudes (p<0.01). There was also a significant ICP effect (p<0.01), with larger baseline amplitudes in the 25mmHg ICP group (34.8±1.6μV) compared with normal (26.4±2.5μV) and low ICP groups (23.9±2.5μV). The ocular perfusion pressure (BP-IOP) relationship fully could not account for difference in ganglion cell function between ICP levels. Conclusions : Ganglion cell function is dependent on ocular perfusion pressure, excessive low or high perfusion attenuates function. Higher intracranial pressure appears to protect against acute ocular perfusion stress.
Normative retrobulbar measurements of the optic nerve using ultra high field magnetic resonance imaging

Nguyen, BN ; Cleary, JO ; Glarin, R ; Kolbe, SC ; Moffat, BA ; Ordidge, RJ ; Bui, BV ; McKendrick, AM (Association for Research in Vision and Ophthalmology, 2019-07-01)

Purpose : We exploit the improved spatial resolution and signal-to-noise gain of ultra high field (7T) magnetic resonance imaging (MRI) with a dedicated eye coil for more accurate morphometric measurements of the optic nerve ~2.5mm behind the globe. Methods : Coronal T2-weighted oblique images (TR=2000ms, TE=64ms, FOV=155mm, matrix=384 x 384, slice thickness=0.7mm, scan time=2’34”) through the optic nerve were obtained in 21 healthy adults (20-41 years, 11 emmetropes: +0.75 to -0.50D, 10 myopes: -4.5 to -12D) using a 7T Siemens Magnetom scanner (Erlangen, Germany) and 6-channel eye coil (MRI.TOOLS GmbH, Berlin, Germany). Horizontal and vertical outer diameter of the optic nerve, subarachnoid space (fluid gap) and optic sheath were measured by hand using biomedical imaging software (OsiriX, Pixmeo, Switzerland) (Figure). Significant motion artefacts were avoided with customised fixation and preparation techniques. Results : Horizontal and vertical measurements were similar so were averaged. Right and left eye diameters did not differ and were highly correlated (optic nerve: Pearson r=0.9, p<0.001; fluid gap: r=0.8, p<0.001; optic sheath: r=0.7, p<0.001); hence we report left eye data only. Optic nerve diameter (average of horizontal and vertical diameters) ranged from 2.8-4.1mm in emmetropes and 1.5-4.2mm in myopes and correlated with refractive error (Spearman r=0.46, p=0.04). Similarly, fluid gap diameter (emmetropes: 3.6-5.5mm, myopes: 2.5-5.6mm), but not optic sheath diameter (emmetropes: 4.5-6.8mm, myopes: 4.2-6.8mm), correlated with refractive error (r=0.47, p=0.03). Conclusions : Ultra high field MRI with thinner slices enables more accurate demarcation of the optic nerve, surrounding fluid/subarachnoid space and optic sheath without overlapping of neighbouring anatomy (minimal partial volume artefact). Our 7T MRI-derived normative measurements of optic nerve, fluid gap and sheath diameter are comparable with published reports in healthy observers obtained at conventional MRI magnetic fields (1.5-3T). Our findings suggest a trend for retrobulbar optic nerve and subarachnoid space, but not optic sheath, to be smaller in high myopes.
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.
The effect of ageing on the recovery of retinal function and structure following intraocular pressure elevation in mice

Lee, PY ; He, Z ; Wong, VHY ; Crowston, JG ; Bui, BV (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.
A retinal imaging biomarker of Alzheimer's disease

van Wijngaarden, P ; Hadoux, X ; Hui, F ; Lim, J ; Nguyen, C ; Bui, B ; Crowston, J (Wiley, 2019-11-01)

Background: Amyloid-beta (Ab) deposition in the brain is a diagnostic marker for Alzheimer's disease (AD), but current tests are costly and not widely available. Evidence from transgenic rodent models and post-mortem human tissues suggest that retinal accumulation of Ab may serve as a surrogate marker of brain Ab levels. As Ab has a wavelength-dependent effect on light scatter, we investigated the potential for in vivo retinal hyperspectral imaging to serve as a biomarker of brain Ab. Purpose: To develop and validate a retinal imaging biomarker of Alzheimer's disease. Methods: We performed human retinal hyperspectral imaging on individuals with high Ab burden on brain PET imaging and mild cognitive impairment (cases; n = 15), and age-matched PET-negative controls (n = 20). Image analysis methods were developed and validated on a second group of participants with and with (n = 4) and without (n = 13) moderate-to-high brain Ab burden and on transgenic mice (5xFAD) known to accumulate retinal Ab. Results: We show significant differences in retinal reflectance spectra between cases and controls in both cohorts (AUC ROC = 0.82, P = 0.001, 95% CI: 0.67-0.97). There was a moderate positive linear correlation between retinal imaging scores and brain Abburden (r = 0.46, 95%CI: 0.13-0.69, P = 0.008).The technique also enabled discrimination of AD-model mice from wild-type controls. Conclusion: We have developed a novel retinal imaging method to distinguish people with moderate-high brain Ab load from those without. This approach may have value for the diagnostic confirmation of AD.
A tractable preclinical model of optic nerve demyelination

van Wijngaarden, P ; Paul, JP ; Wong, VHY ; Bui, BV ; Merson, TD (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.

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