Bang V. Bui

Ganglion cell contributions to the rat full-field electroretinogram

The purpose of this study was to determine what contributions are made to the rat full‐field electroretinogram (ERG) by ganglion cells (GCs). To that end, the ERG was assessed longitudinally following optic nerve transection (ONTx). Additional studies were conducted using intravitreal injections of pharmacologically active substances. The ERG was recorded simultaneously from both eyes of anaesthetized adult Brown‐Norway rats (ketamine: xylazine: acepromazine, 55: 5: 1 mg kg−1) using custom silver chloride electrodes. Stimuli were brief, white xenon discharges delivered via a Ganzfeld under dark‐adapted and light‐adapted conditions (150 cd m−2). ERGs were obtained 1, 2, 3, 4 and 9 weeks after ONTx (n= 8) or sham (n= 8) operations. ONTx reduced both positive and negative components of the scotopic threshold response (pSTR and nSTR). Scotopic ERG responses to brighter flashes, including a‐waves, b‐waves and oscillatory potentials (OPs) were unaffected by ONTx. ONTx reduced the photopic b‐wave and OPs. TTX (6 μm) reduced the pSTR and nSTR, but not the scotopic a‐wave, b‐wave or OPs. TTX had dramatic effects on the photopic ERG, surpassing the effects of ONTx. TTX application 9 weeks post‐ONTx had little additional effect on the STR. Inhibition of inner retinal responses using GABA (10 mm) or NMDA (0.8 mm) reduced the nSTR substantially. Similar results were obtained with antagonists of AMPA/KA ionotropic glutamate receptors 6‐cyano‐7‐nitroquinoxaline‐2,3(1H,4H)‐dione (CNQX, 0.2 mm) or cis‐2,3‐piperidinedicarboxylic acid (PDA, 5 mm); however, both also reduced the scotopic b‐wave by ∼40 %. By contrast, the NMDA receptor antagonist D(‐)‐2‐amino‐7‐phosphonoheptanoic acid (D‐AP7, 0.2 mm) had no effect alone, but the combination of D‐AP7 and CNQX completely abolished the STR. The results of this study indicate that: (1) both pSTR and nSTR components in the rat depend directly upon intact GC responses, and that amacrine cell contributions to these components are relatively small; (2) scotopic ERG response components to brighter flashes receive little influence from GCs; (3) the rat photopic ERG also reflects GC signals and may serve as an additional useful test of GC function; (4) TTX had dramatic effects on the rat photopic ERG that were not attributable to GC currents, but rather to voltage‐gated sodium currents in amacrine or interplexiform cells; (5) a small residual negative STR persisted after ONTx that was likely to be generated by graded responses of third‐order retinal cells, most likely amacrine cells.

Early Inner Retinal Dysfunction in Streptozotocin-Induced Diabetic Rats

PURPOSE Diabetes is known to alter retinal function, as measured with the electroretinogram (ERG), which shows a propensity toward inner retinal oscillatory potential (OPs) abnormalities. However, the effect that diabetes has on other ganglion cell-related responses is not known. This study was a systematic evaluation of streptozotocin (STZ) diabetes-related ERG changes in rats for the first 11 weeks after diabetogenesis. METHODS Thirty Sprague-Dawley rats were randomly assigned to treated (50 mg/kg STZ (n = 16) and control groups (1 mL/kg citrate buffer, n = 14) at 6 weeks of age. Two control animals and four STZ animals were excluded because of blood glucose criteria or systemic complications. Diabetic animals were given daily SC injections of 1 to 2 units of long-acting insulin. ERGs were measured at 4, 8, and 11 weeks after treatment. The a-wave was used as an index of outer retinal function, whereas the b-wave, OPs, and the scotopic threshold response (STR) were used as indices of inner retinal function. RESULTS Photoreceptoral (a-wave) and bipolar cell (b-wave) responses were not significantly reduced by STZ treatment. OPs were significantly reduced by 8 weeks (-25% +/- 7%, P < 0.05). The most severely affected component was the ganglion cell-dominated positive STR, which was significantly decreased from the first time point (-51% +/- 11% at 4 weeks, P < 0.05), but the negative component was unaffected over the 11-week period. CONCLUSIONS The ganglion cell dominated pSTR showed large losses in STZ treated rats.

The role of blood pressure in glaucoma

Although intraocular pressure (IOP) remains an important risk factor for glaucoma, it is clear that other factors can also influence disease development and progression. More recently, the role that blood pressure (BP) has in the genesis of glaucoma has attracted attention, as it represents a clinically modifiable risk factor and thus provides the potential for new treatment strategies beyond IOP reduction. The interplay between blood pressure and IOP determines the ocular perfusion pressure (OPP), which regulates blood flow to the optic nerve. If OPP is a more important determinant of ganglion cell injury than IOP, then hypotension should exacerbate the detrimental effects of IOP elevation, whereas hypertension should provide protection against IOP elevation. Epidemiological evidence provides some conflicting outcomes of the role of systemic hypertension in the development and progression of glaucoma. The most recent study showed that patients at both extremes of the blood pressure spectrum show an increased prevalence of glaucoma. Those with low blood pressure would have low OPP and thus reduced blood flow; however, that people with hypertension also show increased risk is more difficult to reconcile. This finding may reflect an inherent blood flow dysregulation secondary to chronic hypertension that would render retinal blood flow less able to resist changes in ocular perfusion pressure. Here we review both clinical and experimental studies that have attempted to clarify the relationships among blood pressure, OPP and blood flow autoregulation in the pathogenesis of glaucoma.

ACE inhibition salvages the visual loss caused by diabetes

AimsWe consider the nature of retinal dysfunction in streptozotocin rats and assess the functional benefits of administering an angiotensin enzyme inhibitor or an inhibitor of advanced glycation end product formation.MethodsSprague-Dawley rats (n=44) were randomly assigned to control (C=12, Cp=4, Ca=4) and diabetic groups (Streptozotocin, D=24). Diabetes was diagnosed based on a range of physiological and biochemical parameters at 4, 8 and 12 weeks. Streptozotocin animals were administered insulin daily (4 units protophane). Animals were treated with either an Angiotensin Converting Enzyme inhibitor (perindopril, Cp=4, Dp=8) or an inhibitor of advanced glycation end product formation (aminoguanidine, Ca=4, Da=8). Dark-adapted electroretinograms were measured on anaesthetized animals at 12 weeks following streptozotocin treatment. Photoreceptoral and inner retinal responses were extracted, modelled and compared using ANOVA.ResultsStreptozotocin injection increased blood glucose, glycosylated haemoglobin, fluid intake and urine volume, whereas body weight was decreased. Perindopril treatment produced improvements (p<0.05) in all indices, whereas aminoguanidine therapy produced some improvement in blood glucose and water intake. Streptozotocin rats showed losses of photoreceptoral-P3 (−27%), postreceptoral-P2 (−15%) and oscillatory potential (−19%) amplitudes of a similar magnitude. Perindopril therapy returned photoreceptoral and inner retinal function to within control limits. However, aminoguanidine treatment gave no significant functional improvement.ConclusionsOur findings provide evidence for a selective neuropathy in diabetes with a primary photoreceptoral lesion. Treatment with perindopril, an angiotensin converting enzyme inhibitor, ameliorates the neuropathy.

The contribution of cone responses to rat electroretinograms

The contribution of rods and cones to the scotopic electroretinogram (ERG) of small animals is unclear, with a recent report suggesting that the mouse has no cone a‐wave. The present study considered the contribution of cones to the ERG of the rat. Dark‐adapted Long Evans rats (n = 4) had ERG signals collected following a single flash, which stimulated rods and cones (mixed response), or a twin‐flash paradigm (short interstimulus interval, 1 s), which isolated cone responses. Rod signals were derived by digital subtraction of the cone signal from the mixed rod/cone ERG. The rat a‐wave was found to be dominated by rod responses but cone responses contributed substantially (45%) to post‐receptoral waveforms (b‐wave) at higher light levels.

Functional Changes in the Retina during and after Acute Intraocular Pressure Elevation in Mice

PURPOSE To examine retinal function using the full-field electroretinogram (ERG) during and after acute intraocular pressure (IOP) elevation in wild-type mice. METHODS IOP was elevated by anterior chamber cannulation in wild-type C57/BL6 mice. The pressure-function relationship was determined by IOP elevation in steps from baseline to 80 mm Hg. The rate of functional recovery was assessed for 60 minutes after an IOP spike of 50 mm Hg for 30 minutes. During and immediately after IOP elevation, scotopic ERG signals were recorded in response to dim and bright flashes (-4.54, -2.23, and 0.34 log cd x s x m(-2)) and analyzed for photoreceptoral (a-wave), ON-bipolar (b-wave), oscillatory potentials (OPs), and scotopic threshold responses (positive [p]STR/negative [n] STR). A full ERG protocol was collected 2 days before and 7 days after the single 50-mm Hg IOP spike. RESULTS The pSTR was most sensitive to IOP elevation with 50% amplitude loss (mu) at 41 mm Hg (mu, 95% confidence limits (CL): 37.7, 45.6) followed by nSTR at 45 mm Hg (95% CL: 41.0, 49.1). pSTR was significantly more sensitive than the b-wave (95% CL: 41.4, 49.1), a-wave (95% CL: 47.6, 55.3), and OPs (95% CL: 49.6, 59.2). pSTR showed slower recovery immediately after the 50 mm Hg spike compared with the b-wave (P = 0.02). One week after the 50-mm Hg spike, pSTR (-30% +/- 6%, P < 0.001) and OP (-27% +/- 2%, P < 0.001) amplitudes were reduced, whereas other components were unaffected. CONCLUSIONS The STR in mice is more sensitive to acute IOP elevation and recovers slower than other ERG components. Reduction in pSTR and OP amplitude at 1 week suggests persistent impairment of inner retinal function can occur after a single IOP spike.

Extraction and modelling of oscillatory potentials.

This paper considers the recommendation that Oscillatory Potentials (OP) be extracted by filtering in the frequency domain. This recommendation presumes that filtering isolates OPs from other ERG waveforms. However, we show that the leading edge of the a-wave has substantial frequency overlap with the OP spectrum at high intensities and that it contaminates these wavelets in the frequency domain. We propose a method of signal conditioning that removes a-waves prior to filtering. When this is done, the OPs show a bimodal distribution in the frequency domain that is well approximated by two Gaussians having means (±std. dev.) of 91.0±14.6 Hz and 153.1±17.1 Hz. This implies that two functions can be used to model the OPs in the time domain. However, we show that as most of the power of the Fourier spectrum (74%) is contained in a single Gaussian, a reasonable OP model can be derived by using a single function in the time domain. We test such a model on humans (n=5) and pigmented (n=14) and albino (n=14) guinea-pigs and show that it provides excellent fits to data across a range of flash exposures. Furthermore, changes in OP amplitude and timing between strains of guinea-pigs are easily detected with this model. We show that there is no statistical justification for making the model more complex by including multiple functions. Such paramatisation of the OP envelope provides a valuable and intuitive description of the OP waveforms in the time domain. The model provides an excellent description of OPs obtained with the current paradigm, however the single gaussian model may be deficient under stimulus conditions which produce highly asymmetric OP envelopes.

Blood Pressure Modifies Retinal Susceptibility to Intraocular Pressure Elevation

Primary open angle glaucoma affects more than 67 million people. Elevated intraocular pressure (IOP) is a risk factor for glaucoma and may reduce nutrient availability by decreasing ocular perfusion pressure (OPP). An interaction between arterial blood pressure and IOP determines OPP; but the exact contribution that these factors have for retinal function is not fully understood. Here we sought to determine how acute modifications of arterial pressure will affect the susceptibility of neuronal function and blood flow to IOP challenge. Anaesthetized (ketamine:xylazine) Long-Evan rats with low (∼60 mmHg, sodium nitroprusside infusion), moderate (∼100 mmHg, saline), or high levels (∼160 mmHg, angiotensin II) of mean arterial pressure (MAP, n = 5–10 per group) were subjected to IOP challenge (10–120 mmHg, 5 mmHg steps every 3 minutes). Electroretinograms were measured at each IOP step to assess bipolar cell (b-wave) and inner retinal function (scotopic threshold response or STR). Ocular blood flow was measured using laser-Doppler flowmetry in groups with similar MAP level and the same IOP challenge protocol. Both b-wave and STR amplitudes decreased with IOP elevation. Retinal function was less susceptible to IOP challenge when MAP was high, whereas the converse was true for low MAP. Consistent with the effects on retinal function, higher IOP was needed to attenuated ocular blood flow in animals with higher MAP. The susceptibility of retinal function to IOP challenge can be ameliorated by acute high BP, and exacerbated by low BP. This is partially mediated by modifications in ocular blood flow.

AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo

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.

Retinal and choroidal TGF-β in the tree shrew model of myopia: Isoform expression, activation and effects on function

A visually evoked signalling cascade, which begins in the retina, transverses the choroid, and mediates scleral remodelling, is considered to control eye growth. The ubiquitous cytokine TGF-beta has been associated with alterations in ocular growth, where alterations in scleral TGF-beta isoforms mediate the scleral remodelling that results in myopia. However, while the TGF-beta isoforms have been implicated in the scleral change during myopia development, it is unclear whether alterations in retinal and choroidal isoforms constitute part of the retinoscleral cascade. This study characterised the retinal and choroidal TGF-beta isoform profiles and TGF-beta2 activation during different stages of myopia development, as induced by form deprivation, in a mammalian model of eye growth. Using quantitative real-time PCR, the mRNA for all three mammalian isoforms of TGF-beta was detected in tree shrew retina and choroid. Distinct tissue-specific isoform profiles were observed for the retina (TGF-beta1:TGF-beta2:TGF-beta3=20:2085:1) and choroid (TGF-beta1:TGF-beta2:TGF-beta3=16:23:1), which remained constant over the development period under investigation. The active and latent pools of retinal TGF-beta2 were quantified using ELISA with the majority (>94%) of total TGF-beta2 found in the latent form. Unlike previous scleral data showing early and continuous decreases in TGF-beta isoform expression during myopia development, the levels of the three isoforms remained within normal ranges for retinal (TGF-beta1, -14 to +14%; TGF-beta2, -2 to +20%; TGF-beta3, -10 to +26%) and choroidal (TGF-beta1, -19 to +21%; TGF-beta2, -26 to +8%; TGF-beta3, -11 to +28%) tissues during myopia development (induction times of 3h, 7h, 11h, 24h, and 5 days). A 40% decrease in retinal TGF-beta2 activation was observed after 5 days of myopia development, however, there was no functional correlate of altered TGF-beta2 activity, as assessed by the retinal ERG response. Overall, these data highlight the specific nature of TGF-beta isoform expression, which reflects the differences in tissue structure and function. While TGF-beta isoforms are involved in scleral regulation during myopia development in mammals, they do not have a primary role in the retinal and choroidal signals. Thus, the regulation of eye growth via the retinoscleral cascade involves more than one factor, which is likely to be tissue-specific in nature.