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Purpose: To evaluate the recovery of retinal function after acute IOP elevation., Methods: The electroretinogram (ERG) was measured before, during, and after IOP increased to 50 and 70 mm Hg at different durations in anesthetized, dark-adapted rats (n = 5-7). Signals were collected for dim and bright flashes (-4.95 and 1.0 log cd . s/m(2)) and analyzed in terms of the photoreceptoral (P3), postreceptoral (P2), and inner retinal (negative scotopic threshold response [nSTR]) responses. Parameters (treatment/baseline, %) were compared across time by using repeated-measures ANOVA and t-tests. The rate of recovery was quantified with a logistic function and compared by bootstrap., Results: IOP spikes induce greater loss (P < 0.01) and slower recovery (P < 0.001) in the nSTR compared with the P2 and P3 responses. IOP spikes having common integral (pressure x duration, 2100 mm Hg x minutes) for insult gave significantly greater P2 and nSTR dysfunction at the higher pressure (70 vs. 50 mm Hg, nSTR reduced to -2.5% +/- 0.5% vs. 20.3% +/- 6.5%, P < 0.05). The higher pressure also produced significantly slower nSTR recovery (50% recovery time [t(0.5)] 70 vs. 50 mm Hg: 33.1 vs. 21.7 minutes; P < 0.05). At a given IOP (70 mm Hg), t(0.5) showed a linear relationship with duration (15 vs. 30 vs. 60 minutes' exposure: t(0.5) 16.7 vs. 33.1 vs. 63.2 minutes; P < 0.05) and integral., Conclusions: Ganglion cell function recovers slower than the outer retina after IOP insult, with peak IOP being the principle determinant of functional loss and recovery. For a fixed pressure, functional recovery is linearly related to exposure.

We assessed the growth of the rat photopic electroretinogram (ERG) during light adaptation and the mechanisms underlying this process. Full field ERG responses were recorded from anesthetized adult Brown-Norway rats at each minute for 20 min of light adaptation (backgrounds: 1.8, 2.1, 2.4 log scotopic cd m(-2)). The rat photopic b-wave amplitude increased with duration of light adaptation and its width at 33% maximal amplitude narrowed (by approximately 40 ms). These effects peaked 12-15 min after background onset. The narrowing of the b-wave reflected steepening of the b-wave recovery phase, with little change in the rising phase. OP amplitudes grew in proportion to the b-wave. Inhibition of inner retinal responses using TTX resulted in a greater relative growth of b-wave and OP amplitude compared with fellow control eyes, and delayed the change in recovery phase by approximately 5 min. Inhibition of all ionotropic glutamate receptors with CNQX/D-AP7 delayed both rising and recovery phases equally (approximately 12 ms) without altering b-wave width or the time course of adaptation changes. These outcomes suggest that inner retinal light responses are not directly responsible for b-wave amplitude growth, but may contribute to the change in its recovery phase during adaptation. A TTX-sensitive mechanism may help to hasten this process. The cone a-wave was isolated using PDA/L-AP4 or CNQX/L-AP4. A-wave amplitude (35 ms after stimulus onset) also increased with time during light adaptation and reached a maximum (130 +/- 29% above baseline) 12-15 min after background onset. B-wave amplitude growth in fellow control eyes closely followed the course and relative magnitude of cone a-wave amplitude growth. Hence, the increase of the cone response during light adaptation is sufficient to explain b-wave amplitude growth.

Purpose: To document the existence of idiopathic bilateral optic atrophy (BOA) in rhesus macaque monkeys and to characterize the structural and functional consequences of this condition., Methods: In vivo assessment of retinal and optic nerve structure included fundus biomicroscopy and stereophotography. Functional analyses included transient pattern-reversal electroretinography (PERG) and full-field flash ERG, with both white flashes while dark adapted and red flashes on a blue background used to assess the photopic negative response (PhNR). Also measured were visual evoked cortical potentials (VEPs) and multifocal (mf)ERGs, with both a standard fast and slowed (7F) stimulation sequence. Post mortem histologic evaluation was performed on a subset of five animals with BOA and compared with data from 22 healthy normal animals. Blood tests, including vitamin E, B(12), folate, lead, and complete blood cell count with differential were obtained on the four animals that remained alive., Results: Animals with BOA showed temporal pallor of the optic nerve head and thinning of the retinal nerve fiber layer (RNFL) between the temporal vascular arcades (i.e., of the papillomacular bundle). Severity of optic atrophy and RNFL loss varied between animals from mild to severe, but was similar in the two eyes of each animal. Functional changes included greater loss of the PERG N95, compared with the P50 component and substantial reduction of mfERG high-frequency components. The mfERG low-frequency components were slightly larger than normal. None of the full-field flash ERG amplitudes (a-wave, b-wave, oscillatory potentials, or PhNR) was significantly different from normal. There were no consistent abnormalities found in the results of any blood test. Histologic findings included axonal loss and gliosis limited to the temporal optic nerve, reduction of nuclei within the retinal ganglion cell layer, and thinning of the temporal retinal RNFL., Conclusions: The existence of BOA in nonhuman primates warrants caution on the part of investigators who use these animals in experimental models of ophthalmic disease.

Purpose: To characterize retinal function during a period of acutely elevated intraocular pressure (IOP) across a wide range of IOPs, including those typically observed in animals with experimental glaucoma., Methods: Unilateral elevation of IOP was achieved manometrically in adult Brown Norway rats (nine experimental groups; n=4-7 in each; 10-100 mmHg and sham control). Full-field ERGs were recorded simultaneously from treated and control eyes, beginning 75 minutes after IOP elevation. Scotopic ERG stimuli were brief white flashes (-6.1 to 2.7 log cd-s/m2). Photopic ERGs were recorded (1.2-2.7 log cd-s/m2) after 15 minutes of light adaptation (150 cd/m2). Relative amplitude (treated/control, %) of ERG components versus IOP was described with a cumulative normal function., Results: Resting IOP was 12.1 +/- 2.8 mmHg and mean femoral artery pressure was 97.6 +/- 10.7 mmHg. ERG components showed a graded effect dependent on IOP. Systematic delays in the timing of the scotopic threshold response (STR) and photopic b-wave were observed between IOPs of 30 and 40 mmHg. Analysis of amplitudes revealed that the negative STR component (nSTR) and the photopic OPs were the most sensitive to acute IOP elevation. These components were first significantly affected at 50 mmHg, whereas all parameters of middle and outer retinal function (scotopic P2 and P3) remained normal. The nSTR and photopic OPs declined by 50% at IOP <61 mmHg. The scotopic P2, OPs, and positive STR (pSTR) had intermediate sensitivity, such that they were reduced by 50% at IOPs between 61 and 66 mmHg. Scotopic P2 amplitude, but not sensitivity, was significantly reduced by 60 mmHg. At 60 and 70 mm Hg, the decline in P2 amplitude was not attributable to changes in photoreceptor response (P3) amplitude or sensitivity. The least sensitive component was the scotopic a-wave (RmP3) showing a 50% reduction at an IOP of 71 mmHg., Conclusions: During acute IOP elevation, functional changes progress from the proximal to the distal retina. Alterations in ganglion-cell-related ERG potentials occurred at IOPs (30-50 mmHg) commonly observed in rat experimental glaucoma models. Nonspecific functional changes were observed at acute IOP above 50 mmHg, suggesting that IOP should be maintained below this level in experimental glaucoma models if selective ganglion cell injury is to be sought. Repeated IOP spikes above this level may cause permanent, nonspecific damage, perhaps via ischemic mechanisms. Thus, IOP should be monitored frequently in these models.

We have employed fos-tau-LacZ (FTL) transgenic mice to examine functional activation in the visual areas of the nervous system. The FTL mice express the marker gene lacZ in neurons and their processes following many different stimuli, and allow the imaging of activation from the level of the entire brain surface through individual neurons and their projections. Analysis of FTL expression in the retinas of mice following diurnal exposure to light shows that bipolar cells, specific classes of amacrine cells, ganglion cells, and a dense network of processes in the inner plexiform layer are functionally activated. In animals deprived of light, there is almost no activity in the retina. In the lateral geniculate nucleus (LGN), light exposure appears responsible for FTL expression in dorsal nuclei, but not for expression in the ventral nuclei or the intergeniculate leaflet. In the superficial layers of the superior colliculus, FTL expression is highly dependent on light exposure. Similarly, light exposure is required for FTL expression in primary visual cortex (area 17), but some expression remains in area 18 of dark-adapted animals. Finally, using mice with one or both eyes missing, we have determined which parts of the visual system are dependent on the presence of a functional connectivity from the eye. These data demonstrate the usefulness of the FTL mice to map functional activation within the entire visual system. Furthermore, we can capture visual activation in a conscious animal. Our findings give an insight into the architecture of activity within the retina and throughout the visual system.

We assessed the effect of the in vivo application of monocarboxylate transport inhibitors on retinal function and amino acid immunocytochemistry. We wanted to determine the impact that altered aerobic metabolite availability has on retinal function and the characteristics of amino acid shunting into metabolic pools. Electroretinograms were collected from anaesthetized rats at various times after intravitreal injection of the monocarboxylate transport inhibitors alpha-cyano-4-hydroxycinnamate (4-CIN; 2 micro L, 0.1-10 mm) or p-(dipropylsulphamoyl)benzoic acid (probenecid; 1-10 mm). Changes in retinal function were compared with quantitative amino acid immunocytochemical changes in retinas harvested 20 and 40 min after either 4-CIN or vehicle treatment. The injection of 4-CIN resulted in a dose-dependent reduction of the ON-bipolar cell P2 wave amplitude (20-80%) and delay in its implicit time. The phototransduction sensitivity was mildly reduced whereas the ON-bipolar cell P2 sensitivity was unaffected. Probenecid induced functional changes similar to those observed with 4-CIN. We also mapped the amino acid alterations within specific cell classes induced by 4-CIN application. All neurones displayed a reduced glutamate content averaging 48%; reduced GABA (31%) and glycine (28%) were found within amacrine cells and glutamine was reduced in all cell classes except photoreceptor and Müller cells. All cell classes in the retina demonstrated increases in aspartate (57%), whereas leucine (24%) and ornithine (21%) were only significantly increased in photoreceptor and bipolar cells. The reduction in glutamate immunolabelling in specific retinal cell classes was mirrored by an increase in aspartate levels at these locations. In addition, attenuated glutamine immunolabelling also closely matched the spatial pattern observed for glutamate. Our immunocytochemical analysis provides evidence that monocarboxylate transport inhibition induces a shift in the equilibrium of glutamate transamination reactions involving aspartate throughout the retina whereas photoreceptor and bipolar cells also use glutamate transamination reactions involving ornithine and leucine. The distribution pattern of glutamine secondary to monocarboxylate inhibition suggests that this amino acid is a major precursor for glutamate throughout the retina.

Purpose: To characterize retinal functional consequences of elevated intraocular pressure (IOP) in a rat model of experimental glaucoma., Methods: Unilateral elevation of IOP was produced by hypertonic saline injection into an episcleral vein in 20 adult male Brown-Norway rats. IOP was measured in both eyes of awake animals four to five times per week. After 5 weeks, animals were dark adapted overnight (>12 hours) and full-field electroretinograms (ERGs) were obtained simultaneously from both eyes. Scotopic ERG stimuli were brief white flashes (-6.64-2.72 log cd-s/m(2)). Photopic responses were also obtained (0.97-2.72 log cd-s/m(2)) after 15 minutes of light adaptation (150 cd/m(2)). Eyes were processed the following day for histologic evaluation by light microscopy, including masked determination of optic nerve injury grade (ONIG; 1, normal; 5, severe, diffuse damage)., Results: Among experimental eyes, the group average IOP (+/-SD) was 34.5 +/- 4.1 mm Hg, whereas the average for control eyes was 28.1 +/- 0.5 mm Hg (t = 7.1, P < 0.0001). The average ONIG for experimental and control eye groups, respectively, was 3.4 +/- 1.7 and 1.0 +/- 0.02 (t = 6.3, P < 0.0001). The ONIG increased with mean IOP in experimental eyes (r(2) = 0.78, P < 0.0001) and was unrelated to mean IOP in control eyes (r(2) = 0.09, P = 0.18). In experimental eyes with relatively mild IOP elevation (mean IOP < 31 mm Hg) and no structural (histologic) damage to the optic nerve evident by light microscopy (ONIG = 1.1 +/- 0.2, n = 5), there was a selective reduction of the positive scotopic threshold response (pSTR; P < 0.001), whereas other ERG components remained unaltered. In four of the five eyes, pSTR amplitude was reduced by more than 50%, whereas all five had normal scotopic a-wave, b-wave, and OP amplitudes. Eyes with mean IOP of more than 35 mm Hg had reduced a-wave, b-wave, and oscillatory potential (OP) amplitudes., Conclusions: As demonstrated by prior studies, selective loss of the pSTR is indicative of selective retinal ganglion cell (RGC) injury. In this rat model of experimental glaucoma, selective RGC functional injury occurred before the onset of structural damage, as assessed by light microscopy of optic nerve tissue. The highest IOP levels resulted in nonselective functional loss. Thus, in rodent models of experimental glaucoma, lower levels of chronically elevated IOP may be more relevant to human primary chronic glaucoma.

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 microM) 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 approximately 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.

Purpose: To test the proposal that inhibiting monocarboxylate transport in the rat retina results in altered retinal function measured using the electroretinogram (ERG) and to evaluate the efficacy of exogenous metabolic substrates to restore any functional deficit., Methods: Full-field white-flash ERGs were measured after monocarboxylate transport inhibition with intravitreal injection of alpha-cyano-4-hydroxycinnamic acid (4-CIN, 10 mM), and functional recovery was assessed after the introduction of various exogenous metabolic substrates (10 mM): lactate, pyruvate, alpha-ketoglutarate, alanine, succinate, and glutamine. The efficacy of glutamine as a metabolic substrate was also considered in the presence of phosphate-activated glutaminase inhibition (6-diazo-5-oxo-norleucin, 10 mM) or aminotransferase inhibition (aminooxyacetic acid, 10 mM). Pyruvate and alanine recovery was also assessed after aminooxyacetic acid application., Results: 4-CIN application resulted in an increased phototransduction amplitude but a mild reduction of gain. A greater reduction of postreceptoral b-wave and oscillatory potential amplitudes (80%) was observed, along with delayed implicit times (35 ms). Partial recovery of b-wave amplitudes was achieved with exogenous lactate (24%), pyruvate (27%), alpha-ketoglutarate (27%), alanine (25%), and succinate (26%), whereas glutamine provided 62% recovery. However, none of the substrates improved phototransduction gain. Both 6-diazo-5-oxo-norleucin and aminooxyacetic acid completely suppressed the glutamine-induced b-wave recovery. Aminooxyacetic acid also abolished the b-wave recovery from 4-CIN afforded by pyruvate and alanine., Conclusions: The greater loss of the b-wave and oscillatory potentials may reflect preferential routing of amino acid carbon skeletons to oxidative metabolic pathways, which in turn reduces glutamate availability for neurotransmission between photoreceptors and ON-bipolar cells. The reduction in log S provides evidence that inhibition of monocarboxylate transport produced some metabolic dysfunction in the rat.

Purpose: To assess the inter-ocular and inter-session reliability for a range of parameters derived from the photopic electroretinogram (ERG) in a group of normal non-human primates., Methods: Inter-ocular differences for photopic ERGs were assessed in a group of normal anesthetized adult rhesus monkeys (Macaca mulatta, n=29); inter-session reliability was assessed for 23 eyes of 23 animals tested 3 months later. Signals were acquired using Burian-Allen contact lens electrodes, whereby the contralateral cornea served as a reference. Photopic ERGs were elicited using red Ganzfeld flashes (-0.5-0.67 log photopic cd.sm(-2)) on a rod suppressing blue-background (30 scotopic cdm(-2)). Measurement reliability was established for a-wave, b-wave, photopic negative response (PhNR) and oscillatory potential (OP) amplitudes, as well as for their implicit times, by calculation of the 95% limits-of-agreement (LOA) and the coefficient-of-variation (COV) for each parameter., Results: OP and a-wave amplitudes increased with intensity up to 0.67 log photopic cd.sm(-2), following a typical saturating function, whereas b-wave and PhNR amplitudes both declined above 0.42 log photopic cd.sm(-2). Inter-session variability was greater than inter-ocular variability. The inter-session COVs for PhNR amplitude (10-20%) were similar to the other photopic ERG components (a-wave: 12-17%, b-wave: 12-17%, OPs: 13-19%). Inter-session LOAs were also similar across components, but on average, were smallest for responses to moderate intensities (0.0-0.42 log photopic cd.sm(-2))., Conclusion: In non-human primates, the 95% LOA for inter-session measurements of the photopic ERG a-wave, b-wave, OPs and PhNR are all similar. Inner-retinal damage may best be measured using the PhNR amplitude for moderately bright stimulus intensities. B-wave and PhNR amplitudes for brighter flashes are smaller and more variable. The ratio of PhNR:b-wave amplitudes manifests smaller variability and may therefore be useful for detection of selective PhNR loss.

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.

Purpose: To assess the structural and functional consequences of local ganglion cell (GC) loss in an experimental model of a retinal nerve fiber layer (NFL) bundle defect. To evaluate and compare three commonly used multifocal electroretinogram (mfERG) stimuli, as well as the standard transient pattern-reversal ERG (pERG) and the photopic full-field ERG, for detection of local GC damage., Methods: Intraretinal axotomy was achieved by multiple treatments with a diode laser adapted to a slit lamp biomicroscope. Retinal laser burns were applied along an arc, subtending approximately 60 degrees, about one disc diameter superotemporal to the optic nerve. Functional measures were acquired before laser application and at numerous time points thereafter. These included mfERGs for three different stimuli: a standard fast m-sequence flicker, a global-field flash paradigm (MOFO), and a slowed m-sequence (with seven dark frames inserted to each m-step [7F]). pERGs were measured for a 24 degrees x 32 degrees checkerboard stimulus (0.56 cyc/deg, 90% contrast, 75 cd/m(2), 5 reversals/s). Photopic full-field ERGs were measured for red flashes (0.42 log photopic cd-s/m(2)) on a blue rod-saturating background (30 scotopic cd/m(2)). Retinal photography, fluorescein angiography and postmortem histologic evaluation of the optic nerve, NFL, and retinal tissues were performed., Results: After six laser sessions, the NFL bundle defect appeared to be complete and contiguous and was visible both proximal to and distal to the site of the photoablation by clinical examination of the fundus and stereoscopic photographs. Histologic evaluation demonstrated localized loss of GC axons, confirmed at the level of the retrobulbar optic nerve. Retinal cross sections in the temporal retina (distal to the axotomy) showed loss of GC soma and NFL degeneration, whereas all other layers appeared intact. mfERGs showed loss of high-frequency components (HFCs) for responses located within the arcuate region corresponding to the NFL defect. Local GC damage was most easily detected using the slowed 7F m-sequence stimulus. This stimulus elicited relatively large HFCs that were significantly reduced from local responses after axotomy and that were tetrodotoxin (TTX)-sensitive in a control experiment. Low-frequency component loss with the 7F stimulus did not reach statistical significance. The photopic full-field ERG was not significantly affected. pERG amplitudes declined significantly from baseline but remained within normal limits., Conclusions: Focal loss of GC function in the macaque retina is most easily detected using the slowed-sequence mfERG. Local 7F HFCs depend on intact GC function.

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.

Purpose: To consider how aerobic and anaerobic metabolic processes limit posthypoxemic decay in retinal function, measured by electroretinogram (ERG)., Methods: The hypothesis that lowering metabolic demand would prolong endogenous metabolic stores was tested by comparing the rate of ERG decay in rats in dark- (n = 5) versus light-adapted (15 minutes, 112 cd/m(2), n = 5) conditions and with serial versus single (n = 5 at each of seven time points) light stimulation. Postmortem hypoxemia was induced by cervical dislocation. Glucose (10 and 100 mM) and glutamine or lactate (100 mM) were injected into the vitreous 10 minutes before hypoxemic insult, to consider glycolytic-oxidative versus oxidative metabolism, respectively., Results: Lowering the metabolic drain by light adaptation or serial stimulation significantly improved the photoreceptoral saturated amplitude during the first 5 to 7.5 minutes after postmortem hypoxemia. Increasing substrate availability with exogenous glucose preloading delayed the loss of the photoreceptoral response, thereby extending the delay constant from 4.8 to 10.9 minutes. Postreceptoral amplitudes were not improved by any exogenous substrate. Providing glucose at 5 minutes after hypoxemia provided no benefits. Similar to glucose, glutamine and lactate loading significantly delayed photoreceptoral decay over the first 7.5 minutes, after which time glucose was the more effective substrate., Conclusions: The postmortem decay of photoreceptoral function reflects depletion of both endogenous oxygen and carbon substrate reserves. The findings provide evidence that a transition between aerobic and anaerobic metabolism occurs after approximately 8 minutes of complete hypoxemia.

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.

Purpose: To consider whether growth restriction secondary to chronic placental insufficiency results in postnatal deficits in retinal structure and function., Methods: Chronic placental insufficiency was induced just before midgestation in guinea pigs through unilateral ligation of the uterine artery. Eight weeks after birth, electroretinograms were recorded from prenatally compromised (PC, n = 6) and control (n = 15) animals. Data were collected for b-wave amplitude and implicit time, also the modeled receptoral (P3) response and oscillatory potentials were extracted. After electroretinography, retinas were prepared for structural analysis (PC, n = 6; control, n = 7). A separate cohort of PC (n = 8) and control (n = 9) animals underwent tyrosine hydroxylase immunoreactivity (TH-IR, dopaminergic neurons) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry (neuronal nitric oxide synthase, nNOS)--these being markers of amacrine cell subpopulations., Results: Electroretinography revealed two PC guinea pigs with marked changes to saturated receptoral amplitude (Rm(P3)), sensitivity (log S) and postreceptoral waveforms. Grouped PC data revealed significantly reduced Rm(P3), whereas log S was not affected. The b-wave amplitudes were normal, but b-wave implicit times were delayed (P < 0.05) in PC animals. Amplitudes and peak times of oscillatory potentials were also significantly reduced and delayed (P < 0.05). Morphologic analysis revealed significant reductions in all cellular and plexiform (synaptic) layers in both the central (P < 0.05) and peripheral (P < 0.05) retina in PC animals. The outer retina, which contains the photoreceptors and the outer plexiform layer was particularly affected. The reduced growth of plexiform layers suggests a reduction in the growth of the neuropile in PC animals compared with control animals. The total number (P < 0.03) and density (P < 0.05) of TH-IR neurons was reduced, whereas the total number and density of nNOS-positive amacrine cells was not significantly different between PC and control animals., Conclusions: Chronic placental insufficiency results in morphologic and functional alterations to the retina. Electroretinogram deficits in PC animals indicated both inner and outer retinal anomalies. Such affects could contribute to the visual impairments reported in very-low-birth-weight children, some of whom are growth restricted.

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.

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., ((C) 2001 Academic Press.)

BACKGROUND: The retinal response to flickering stimuli (steady state ERG) recruits many retinal elements and is a sensitive indicator of early retinal dysfunction. This study reports the post-natal maturation of the steady state ERG response in guinea pigs. METHODS: The steady state ERG response to flickering stimuli (0.6 to 20 Hz) was recorded from dark adapted (more than 12 hrs) English Shorthair guinea pigs (n = 7) using flashes that produced rod and cone dominated responses. Temporal sensitivity functions and critical fusion frequencies (CFF) were derived over a range of ages from postnatal day (PND) 1 to 45. RESULTS: Guinea pig rod and cone temporal sensitivity functions show shape characteristics and CFF similar to humans. Furthermore, the post-natal development of the guinea pig temporal characteristics is also similar to that of humans - they are present at birth and mature rapidly post-natally. The time-course of CFF maturation is similar for rod and cone mediated responses. CONCLUSIONS: These data show that the temporal response and its maturation in the guinea pig retina is similar to that in humans. Therefore, we propose that the guinea pig is a particularly useful animal model to study retinal disease in early childhood.

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.

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.

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.

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.

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.

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.