Baseline characteristics of the transient pattern electroretinogram in non-human primates: inter-ocular and inter-session variability.

This study assessed the inter-ocular and inter-session variability of the transient pattern electroretinogram (PERG) in a group of non-human primates. The transient PERG was measured both eyes of 29 non-human primates, and again after three months in 23 eyes of 23 of these animals. Signals were elicited using a contrast (90%, 75 cdm(-2)) reversing (5 reversals sec(-1)) checkerboard pattern (0.56 cpd). PERGs were also measured for stimuli of varied spatial frequency (n=8, 0.07-2.22 cpd), contrast (n=4, 20-100%), mean luminance (n=4, 4.7-75 cdm(-2)) and defocus (n=5, +1, +2, +3 diopters). The inter-eye and inter-session limits-of-agreement (LOA; 95%) were determined for each PERG parameter. Variability was also compared with previous studies using the coefficient-of-variability (COV). Pharmacological blockade of the inner retinal contributions to the PERG measured under these conditions was conducted in one animal using intravitreal injection of tetrodotoxin (approximately 6 microM) and N-methyl-D-aspartic acid (approximately 6 microM). The N95 component of the primate transient PERG showed spatial tuning, with a peak between 0.14 and 0.28cpd. This spatial tuning was not as apparent for the P50 component. A linear relationship between P50 and N95 amplitude was found with contrast and mean luminance. Both components were attenuated with the introduction of +2 diopters or more of defocus. The inter-session COV for the P50 and N95 components were 23.8 and 19.2%, respectively, while the LOA were 58 and 46%, respectively. The N95:P50 ratio had smaller inter-session variability, was robust to changes in contrast, mean luminance and defocus, and was effective for characterization of inner-retinal dysfunction after pharmacologic block.

Correlating retinal function and amino acid immunocytochemistry following post-mortem ischemia.

We wanted to determine the characteristics associated with electrophysiological and neurochemical changes secondary to ischemic insult as well as correlate these electrophysiological and neurochemical changes. A Ganzfeld source was used to elicit electroretinograms in anesthetized adult Sprague-Dawley rats. Following baseline recordings, one eye was removed for control quantitative amino acid immunocytochemistry, and ischemic insult was induced by cervical dislocation. Following the induction of ischemia, a single electroretinogram signal was collected at 1, 2, 4, 6, 8, 16, 32 or 64 min, after which the eye was removed for immunocytochemistry. The post-receptoral b-wave was undetectable after 1 min post-ischemia, whereas phototransduction declined more gradually and persisted for up to 16 min post-mortem. Both phototransduction saturated amplitude and sensitivity decayed with a similar time course (tc=3.06 (2.73, 3.48) versus 3.29 (2.61, 4.62)min). Significant elevation of amino acid neurotransmitter levels was not observed until 6 min post-mortem. Between 8 and 16 min post-ischemia, glutamate and GABA were significantly accumulated in neurons and Müller cells (p<0.05). Beyond 16 min, the neurotransmitter elevation in neurons and Müller cells was relatively attenuated. Aspartate immunoreactivity was significantly elevated at 4 and 6 min post-ischemia in neurons, prior to a change in any other amino acid. Moreover, of the amino acids assessed the post-ischemic change in aspartate immunoreactivity showed the best correlation with phototransduction decay (r2=0.68). Our findings show that complete impairment of phototransduction coincides with the accumulation of amino acid neurotransmitter. The correlation of aspartate immunoreactivity and phototransduction provides evidence of heightened glutamate oxidation during ischemic insult.

ACE inhibition salvages the visual loss caused by diabetes.

AIMS: We 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. METHODS: Sprague-Dawley rats (n=44) were randomly assigned to control (C=12, C(p)=4, C(a)=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, C(p)=4, D(p)=8) or an inhibitor of advanced glycation end product formation (aminoguanidine, C(a)=4, D(a)=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. RESULTS: Streptozotocin 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. CONCLUSIONS: Our 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.

Retinal anatomy and function of the transthyretin null mouse.

Vitamin A (retinol) is vital for the normal development and function of many tissues in the body including the eye. The purpose of this project was to characterize the retinal anatomy and function of the transthyretin (TTR) null mouse. Mice lacking TTR have been constructed by homologous recombination. Immunocytochemistry was performed to localize short and mid-long wavelength cone opsins as well as morphological examination of the entire retina in wild-type and TTR null mice. Visual function was assessed using the electroretinogram (ERG) and resulting waveforms were analysed in terms of receptoral and postreceptoral components. Retinal morphology of the TTR null mouse was normal. In addition, short and mid-long wavelength cone opsins were localized normally in both TTR null and wild-type retinae. Consistent with these findings, TTR null mice show no anomalies of receptoral (P3) nor post-receptoral (b-wave) ERG components compared with wild-type mice. The results suggest that although circulating plasma levels of retinol and retinol binding protein (RBP) are extremely low, this reduction has little effect on the retinal structure or function of the TTR null mouse. These data are consistent with the existence of mechanisms for the transport of retinol to the retina independent of the classical retinol-RBP-TTR complex.

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-fash 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.

Development of postreceptoral function in pigmented and albino guinea pigs.

Retinal neurons are generated in overlapping growth spurts with ganglion cell and cone populations peaking sooner than rod and bipolar cell numbers. As such the functional development of the inner and outer retinal components and elements within these strata (rods vs. cones) may differ. We considered the postnatal development of the postreceptoral components of the ERG (P2, oscillatory potentials) in the guinea pig. ERGs were also evaluated across albino and pigmented strains in order to consider the role that pigmentation has for functional development. Electroretinograms were collected on postnatal days PDI to PD60 (n = 4-7 per time point). The postreceptoral P2 amplitude and implicit time was extracted (digital subtraction of modelled P3 and filtering, 0.5-49 Hz). Intensity-response relationships were described using Naka-Rushton functions whose parameters were compared using a nonparametric bootstrap. Oscillatory potentials (OPs) were extracted following signal conditioning and filtering to remove the a- and b-waves and were described using a Gabor function. OP response parameters were compared using repeated measures ANOVA. Postreceptoral P2 amplitudes mature soon after birth (PD10-PD12). Oscillatory potentials show a similar postnatal amplitude development (PD10-PD12) but a later maturation in timing (PD20) compared with the postreceptoral waveform. All components (P3, P2, and OPs) declined at the same relative rate with age after PD12. Albino animals gave larger, faster, and more sensitive waveforms at all ages but showed the same age-related trends as did pigmented animals. Early development of inner retinal synapses in guinea pigs may underlie the rapid postnatal maturation of their postreceptoral response. These appear to be constrained by the development of receptoral responses. All components declined at the same rate suggesting either a change in the photoreceptoral response or changes to ocular impedance with age.

Effects of dietary n-3 fatty acid deficiency and repletion in the guinea pig retina.

PURPOSE: To investigate the nature and reversibility of biochemical and functional changes in the retina encountered over a single generation of dietary n-3 polyunsaturated fatty acid deficiency in guinea pigs. METHODS: Dunkin-Hartley guinea pigs were fed for 16 weeks after weaning with diets supplemented with safflower seed oil (n-3 deficient) or canola oil (n-3 sufficient, control). A number of deficient animals were repleted at 6 weeks with canola oil for 5 or 10 weeks, or at 11 weeks for 5 weeks. Electroretinograms (0.8 and 4.3 log scot td x sec) were collected at 6, 11, and 16 weeks after weaning. Conventional waveforms (a- and b-waves), oscillatory potentials, and receptoral and postreceptoral subcomponents (PIII and PII, respectively) were evaluated. Cone pathway function was assessed with 30-Hz flicker at the brighter intensity. Retinal phospholipid fatty acids were measured by capillary gas-liquid chromatography. RESULTS: Electroretinographic amplitudes showed statistically significant losses in b- and a-waves after 6 and 16 weeks of dietary n-3 deficiency, respectively. The response amplitude to 30-Hz flicker was reduced 42% after 16 weeks. Retinal docosahexaenoic acid (DHA) levels of animals maintained on the safflower oil diet for 16 weeks were 42% of levels in age-matched control subjects. There were significant losses in maximum response amplitudes (R(mPIII) and R(mPII)), although the major effect was a reduction in sensitivity of the receptoral response. Complete functional recovery was observed only in animals repleted for 10 weeks. CONCLUSIONS: Functional deficits in PIII and PII of the electroretinogram were apparent in first-generation guinea pigs fed an n-3 deficient diet. These losses showed a correlation with age and retinal DHA level, although varying degrees of dependence on the DHA level were found. All functional deficits were reversed after 10 weeks of dietary n-3 repletion. The results suggest that DHA may serve several functional and structural roles in the retina and further emphasize the requirement for DHA in the normal development of vision.

Development of receptoral responses in pigmented and albino guinea-pigs (Cavia porcellus).

We describe the postnatal development of the electroretinogram (ERG) receptoral response in the guinea pig. In addition, the time course and nature of maturation was compared between albino and pigmented strains to consider the role that melanogenesis might have in this process. Electroretinograms were collected on groups of albino and pigmented animals from postnatal day (PD) PD1 to PD60. A-wave amplitudes and implicit times were extracted from filtered data (0-75 Hz). Receptoral components were modelled using the delayed gaussian model of Hood and Birch [1] fitted as an ensemble to the raw data. Guinea pigs show saturated amplitudes (RmP3) that are 50% of adult values at birth, these mature by PD12. Receptoral delay (t(d)) also undergoes some postnatal maturation, while phototransduction gain (log S) is adult-like at birth. Albino animals had significantly (p<0.05) larger RmP3 and log S across all ages. Guinea pigs have significant postnatal development in their receptoral response. Maturation of RmP3 implies a postnatal increase in rod outer segment length. Whereas the adult values of log S implies a mature phototransduction process at birth. We argue that the likely cause for the larger log S of albino eyes is compatible with theories of increased levels of internal light. Whereas the larger RmP3, even after allowing for increased light effectiveness, may reflect a lower ocular resistance in albino eyes due to their lower levels of melanin. Furthermore, decreased RmP3 and log S with age is observed in the pigmented group only and is consistent with increased ocular resistance due to melanin development in this strain.

Electroretinograms of albino and pigmented guinea-pigs (Cavia porcellus).

PURPOSE/METHODS: The present study compares the electroretinogram responses of albino and pigmented guinea-pigs (Cavia porcellus) with data published for the rat and mouse. RESULTS: We found that albino guinea-pigs gave significantly larger amplitudes and faster implicit times for a-wave and peak-to-peak responses than did pigmented animals. In contrast, pigmented and albino rats and mice do not show such differences between strains. CONCLUSION: We suggest that the guinea-pig may be a better model of the retinal response in human albinism as their response characteristics are consistent with those published for humans.

Comparison of guinea pig electroretinograms measured with bipolar corneal and unipolar intravitreal electrodes.

This study considers the precision and accuracy of bipolar corneal electrodes compared with unipolar intravitreal methods in collecting electroretinographic (ERG) recordings from a small animal. Flash ERGs were obtained from 9 adult guinea pigs on three occasions. Corneal bipolar (Burian-Allen) electrodes were used to collect data on the first two occasions whereas unipolar intravitreal electrodes were used on the last. We identified the a-wave, b-wave, oscillatory potentials, PIII and PII responses. Intensity-response functions were fit using a Naka-Rushton relationship with a bootstrap estimating the 95% confidence limits. Discrepancy analysis was applied to determine the coefficient of agreement. We found significantly larger amplitudes with unipolar intravitreal electrodes (ANOVA; a-wave, p<0.002; b-wave, p<0.001; Oscillatory potentials (OPs), p<0.005) especially at high intensities. Implicit times showed little differences between electrodes for the a-wave, significantly faster (p<0.03) b-waves at some intensities, and significantly slower (p<0.005) OP implicit times across all intensities. The PIlI amplitude (log microV), sensitivity and timing were not significantly different (p>O.05) if expressed in logarithmic units but PII amplitude (log microV) was significantly smaller with corneal electrodes. We suggest that a conversion factor (x1.35) should be applied to data collected with bipolar corneal electrodes to estimate the amplitudes of the modelled parameters accurately. The corneal electrode gave a precision of +/-39 microV which yields a statistical power of 0.90 for a sample size of 7 subjects. We conclude that bipolar corneal electrodes provide smaller electroretinogram amplitudes due to their location and reduced span of the retinal generators.