Optometry and Vision Sciences - Research Publications
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ItemImplantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats(JOURNAL OF VISUALIZED EXPERIMENTS, 2016-06-01)The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.
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ItemAAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2016-06)PURPOSE: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS: Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS: Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS: Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.
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ItemReversal of functional loss in a rat model of chronic intraocular pressure elevation(WILEY, 2017-01)PURPOSE: This pilot study considered whether intraocular pressure (IOP) lowering could reverse ganglion cell dysfunction in a rat model of chronic ocular hypertension. METHODS: A circumlimbal suture was applied in one eye to induce ocular hypertension (n = 7) in Long-Evans rats. The contralateral eye served as an untreated control. After 8 weeks of IOP elevation the suture was removed to lower IOP for the remaining 7 weeks. Electroretinogram (ERG) and optical coherence tomography (OCT) were measured at baseline, 2, 4, 8, 12 and 15 weeks. Retinae were collected for histology at week 15. RESULTS: In sutured eyes, IOP was elevated by 7-11 mmHg above control eyes (12 ± 0.2 mmHg [standard error of the mean]). Eight weeks of chronic IOP elevation resulted in a reduction of the ganglion cell mediated positive Scotopic Threshold Response (pSTR, -25 ± 7% of baseline), as well as smaller photoreceptor (-7 ± 4%) and bipolar cell mediated responses (-6 ± 5%). After suture removal, IOP recovered to normal. By 15 weeks the a-wave (0 ± 6%), b-wave (-2 ± 6%) and pSTR had recovered back to baseline (from -25 ± 7% to -4 ± 6%). The retinal nerve fiber layer was thinned by -9 ± 3% at week 8 and showed no further decline at week 15 (-10 ± 2%). Cell numbers in the ganglion cell layer were similar between suture removal and control eyes at week 15 (3543 ± 478 vs 4057 ± 476 cells mm-2 ). CONCLUSIONS: The circumlimbal suture model might be a useful platform to study the reversibility of neuronal dysfunction from chronic IOP challenge.
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ItemConscious Wireless Electroretinogram and Visual Evoked Potentials in Rats(PUBLIC LIBRARY SCIENCE, 2013-09-12)The electroretinogram (ERG, retina) and visual evoked potential (VEP, brain) are widely used in vivo tools assaying the integrity of the visual pathway. Current recordings in preclinical models are conducted under anesthesia, which alters neural physiology and contaminates responses. We describe a conscious wireless ERG and VEP recording platform in rats. Using a novel surgical technique to chronically implant electrodes subconjunctivally on the eye and epidurally over the visual cortex, we are able to record stable and repeatable conscious ERG and VEP signals over at least 1 month. We show that the use of anaesthetics, necessary for conventional ERG and VEP measurements, alters electrophysiology recordings. Conscious visual electrophysiology improves the viability of longitudinal studies by eliminating complications associated with repeated anaesthesia. It will also enable uncontaminated assessment of drug effects, allowing the eye to be used as an effective biomarker of the central nervous system.
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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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ItemReactivity in the human retinal microvasculature measured during acute gas breathing provocations(NATURE PORTFOLIO, 2017-05-18)Although changes in vessel diameter following gas perturbation have been documented in retinal arterioles and venules, these responses have yet to be quantified in the smallest vessels of the human retina. Here, using in vivo adaptive optics, we imaged 3-25 µm diameter vessels of the human inner retinal circulation and monitored the effects of altered gas-breathing conditions. During isocapnic hyperoxia, definite constrictions were seen in 51% of vessel segments (mean ± SD for pre-capillary arterioles -9.5 ± 3.0%; capillaries -11.8 ± 3.3%; post-capillary venules -6.3 ± 2.8%); these are comparable with responses previously reported in larger vessels. During isoxic hypercapnia, definite dilations were seen in 47% of vessel segments (mean ± SD for pre-capillary arterioles +9.8 ± 1.5%; capillaries +13.7 ± 3.8%; post-capillary venules +7.5 ± 4.2%); these are proportionally greater than responses previously reported in larger vessels. The magnitude of these proportional changes implies that the capillary beds themselves play an important role in the retinal response to changes in carbon dioxide levels. Interestingly, the distribution of microvascular responses shown here differs from our previously reported responses to flicker stimulation, suggesting differences in the way blood supply is coordinated following gas perturbation and altered neural activity.
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ItemCharacterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels.(Frontiers Media SA, 2017)To consider whether a circumlimbal suture can be used to chronically elevate intraocular pressure (IOP) in mice and to assess its effect on retinal structure, function and gene expression of stretch sensitive channels. Anesthetized adult C57BL6/J mice had a circumlimbal suture (10/0) applied around the equator of one eye. In treated eyes (n = 23) the suture was left in place for 12 weeks whilst in sham control eyes the suture was removed at day two (n = 17). Contralateral eyes served as untreated controls. IOP was measured after surgery and once a week thereafter. After 12 weeks, electroretinography (ERG) was performed to assess photoreceptor, bipolar cell and retinal ganglion cell (RGC) function. Retinal structure was evaluated using optical coherence tomography. Retinae were processed for counts of ganglion cell density or for quantitative RT-PCR to quantify purinergic (P2x7, Adora3, Entpd1) or stretch sensitive channel (Panx1, Trpv4) gene expression. Immediately after suture application, IOP spiked to 33 ± 3 mmHg. After 1 day, IOP had recovered to 27 ± 3 mmHg. Between weeks 2 and 12, IOP remained elevated above baseline (control 14 ± 1 mmHg, ocular hypertensive 19 ± 1 mmHg). Suture removal at day 2 (Sham) restored IOP to baseline levels, where it remained through to week 12. ERG analysis showed that 12 weeks of IOP elevation reduced photoreceptor (-15 ± 4%), bipolar cell (-15 ± 4%) and ganglion cell responses (-19 ± 6%) compared to sham controls and respective contralateral eyes (untreated). The retinal nerve fiber layer was thinned in the presence of normal total retinal thickness. Ganglion cell density was reduced across all quadrants (superior -12 ± 5%; temporal, -7% ± 2%; inferior -9 ± 4%; nasal -8 ± 5%). Quantitative RT-PCR revealed a significant increase in Entpd1 gene expression (+11 ± 4%), whilst other genes were not significantly altered (P2x7, Adora3, Trpv4, Panx1). Our results show that circumlimbal ligation produces mild chronic ocular hypertension and retinal dysfunction in mice. Consistent with a sustained change to purinergic signaling we found an up-regulation of Entpd1.
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ItemGlial Cell Contribution to Basal Vessel Diameter and Pressure-Initiated Vascular Responses in Rat Retina(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2017-01)PURPOSE: The purpose of this study was to test the hypothesis that retinal glial cells modify basal vessel diameter and pressure-initiated vascular regulation in rat retina. METHODS: In rats, L-2-aminoadipic acid (LAA, 10 nM) was intravitreally injected to inhibit glial cell activity. Twenty-four hours following injection, retinal glial intracellular calcium (Ca2+) was labeled with the fluorescent calcium indicator Fluo-4/AM (F4, 1 mM). At 110 minutes after injection, intraocular pressure (IOP) was elevated from 20 to 50 mm Hg. Prior to and during IOP elevation, Ca2+ and retinal vessel diameter were assessed using a spectral-domain optical coherence tomography/confocal scanning laser ophthalmoscope. Dynamic changes in Ca2+ and diameter from IOP elevation were quantified. The response in LAA-treated eyes was compared with vehicle treated control eyes. RESULTS: L-2-Aminoadipic acid treatment significantly reduced F4-positive cells in the retina (LAA, 16 ± 20 vs. control, 55 ± 37 cells/mm2; P = 0.02). Twenty-four hours following LAA treatment, basal venous diameter was increased from 38.9 ± 3.9 to 51.8 ± 6.4 μm (P < 0.0001, n = 20), whereas arterial diameter was unchanged (from 30.3 ± 3.5 to 30.7 ± 2.8 μm; P = 0.64). In response to IOP elevation, LAA-treated eyes showed a smaller increase in glial cell Ca2+ around both arteries and veins in comparison with control (P < 0.001 for both). There was also significantly greater IOP-induced vasoconstriction in both vessel types (P = 0.05 and P = 0.02, respectively; n = 6 each). CONCLUSIONS: The results suggest that glial cells can modulate basal retinal venous diameter and contribute to pressure-initiated vascular responses.
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ItemEvidence of Flicker-Induced Functional Hyperaemia in the Smallest Vessels of the Human Retinal Blood Supply(PUBLIC LIBRARY SCIENCE, 2016-09-12)Regional changes in blood flow are initiated within neural tissue to help fuel local differences in neural activity. Classically, this response was thought to arise only in larger arterioles and venules. However, recently, it has been proposed that a) the smallest vessels of the circulation make a comparable contribution, and b) the response should be localised intermittently along such vessels, due to the known distribution of contractile mural cells. To assess these hypotheses in human neural tissue in vivo, we imaged the retinal microvasculature (diameters 3-28 μm) non-invasively, using adaptive optics, before and after delivery of focal (360 μm) patches of flickering visible light. Our results demonstrated a definite average response in 35% of all vessel segments analysed. In these responding vessels, the magnitude of proportional dilation (mean ± SEM for pre-capillary arterioles 13 ± 5%, capillaries 31 ± 8%, and post-capillary venules 10 ± 3%) is generally far greater than the magnitudes we and others have measured in the larger retinal vessels, supporting proposition a) above. The dilations observed in venules were unexpected based on previous animal work, and may be attributed either to differences in stimulus or species. Response heterogeneity across the network was high; responses were also heterogeneous along individual vessels (45% of vessel segments showed demonstrable locality in their response). These observations support proposition b) above. We also observed a definite average constriction across 7% of vessel segments (mean ± SEM constriction for capillaries -16 ± 3.2%, and post-capillary venules -18 ± 12%), which paints a picture of dynamic redistribution of flow throughout the smallest vessel networks in the retina in response to local, stimulus-driven metabolic demand.
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ItemRetinal and Cortical Blood Flow Dynamics Following Systemic Blood-Neural Barrier Disruption(FRONTIERS MEDIA SA, 2017-10-12)To consider whether imaging retinal vasculature may be used as a marker for cortical vessels, we compared fluorescein angiography flow dynamics before and after pharmacological disruption of blood-neural barriers. Sodium fluorescein (1%, 200 μl/kg) was intravenously delivered in anesthetized adult Long Evans rats (n = 44, brain = 18, retina = 26). In the brain cohort, a cranial window was created to allow direct visualization of surface cortical vessels. Video fluorescein angiography was captured using a rodent retinal camera at 30 frames/second and fluorescence intensity profiles were evaluated for the time to reach 50% brightness (half-rise), 50% decay (half-fall), and the plateau level of remnant fluorescence (offset, %). Cortical vessels fluoresced earlier (artery half-rise: 5.6 ± 0.2 s) and decayed faster (half-fall: 10.3 ± 0.2 s) compared to retinal vasculature. Cortical vessels also had a considerably higher offset, particularly in the capillaries/extravascular space (41.4 ± 2.7%) whereas pigment in the retina reduces such residual fluorescence. In a sub-cohort of animals, sodium deoxycholate (DOC, 0.06 M dissolved in sterile saline, 1 mL) was delivered intravenously to cause simultaneous disruption of the blood-brain and blood-retinal barriers. A separate group received saline as vehicle control. Fluorescein angiography was re-measured at 6 and 24 h after drug infusion and evaluated by comparing flow dynamics to the upper quartile (75%) of the control group. Retinal vasculature was more sensitive to DOC-induced disruption with a higher fluorescence offset at 6 h (47.3 ± 10.6%). A delayed effect was seen in cortical vessels with a higher offset evident only at 24 h (65.6 ± 10.1%). Here we have developed a method to quantitatively compare fluorescein angiography dynamics in the retina and superficial cortical vessels. Our results show that systemic disruption of blood-neural barriers causes vascular leakage in both tissues but earlier in the retina suggesting that pharmacological blood-neural barrier disruption may be detected earlier in the eye than in cortical vasculature.