Algis J. Vingrys

Rodent electroretinography: Methods for extraction and interpretation of rod and cone responses

The flash electroretinogram (ERG) represents a serial ensemble of neural responses that can be used to objectively evaluate retinal function on a layer-by-layer basis. In this review, the seminal concepts of Granit are developed within the modern context to demonstrate how the ERG waveform can be decomposed to isolate the activity of individual neural populations and their circuitry. The contribution of rods and cones to the ERG waveform can be precisely defined with simple methods that yield the veridical cone response, which allows identification of rod-isolated components. This knowledge will afford an enhanced capacity to understand retinal development and ageing as well as to interpret the effects of insult, genetic manipulation and disease processes on photoreceptor and neuron-specific components. This review integrates conclusions drawn from a large body of past work and presents new data that enables the provision of detailed methodology for ERG assessment in rodents. Emphasis is placed on protocols that allow efficient acquisition of useful information for the major ERG components with minimal complexity. In particular, specific guidelines for the isolation of rod and cone contributions from the full-field ERG in rodents are provided. This is complemented with detailed and novel methodology for determining parameters that describe individual neuronal generators of rod and cone responses. The effect of stimulus energy on the kinetics of ERG response recovery and photopigment bleaching and regeneration are also discussed. The guidelines presented here are applicable to a wide range of investigations of retinal disease in rodent models.

The Effects of Dietary α-Linolenic Acid Compared with Docosahexaenoic Acid on Brain, Retina, Liver, and Heart in the Guinea Pig

The aim of this study was to compare two different strategies to elevate brain, retina, liver, and heart docosahexaenoic acid (DHA) levels in guinea pigs. Fist, we used an increasing dose of α-linolenic acid (AIA) relative to a constant linoleic acid (LA) intake, and second, we used two levels of dietary DHA provided in conjunction with dietary arachidonic acid (AA). The percentage DHA and AA of total phospholipids in retina, liver, and heart, and in the brain phosphotidylethanolamine and phosphatidylcholine was studied in female pigmented guinea pigs (3 wk old) fed one of five semisynthetic diets containing 10% (w/w) lipid for 12 wk. The LA content in the diets was constant (17% of total fatty acids), with the ALA content varying from 0.05% (diet SFO), to 1% (diet Mix), and to 7% (diet CNO). Two other diets LCP) and LCP3) had a constant LA/ALA ratio (17.5∶1) but varied in the levels of dietary AA and DHA supplementation. Diet LCP1 was structured to closely replicate the principal long chain polyunsaturated fatty acids (PUFA) found in human breast milk and contained 0.9% AA and 0.6% DHA (% of total fatty acids) whereas diet LCP3 contained 2.7% AA and 1.8% DHA. At the end of the study, animals were sacrificed and tissues taken for fatty acid analyses. We found no significant effects of diets on the growth of guinea pigs. Diets containing ALA has profoundly different effects on tissue fatty acid compositions compared with diets which contained the long chain PUFA (DHA and AA). In the retina and brain phospholipids, high-ALA diets or dietary DHA supplementation produced moderate relative increases in DHA levels. There was no change in retinal or brain AA proportions following dietary AA supplementation, even at the highest level. This was in contrast to liver and heart where tissue DHA proportions were low and AA predominated. In these latter tissues, dietary ALA had little effect on tissue DHA proportions although the proportion of AA was slightly depressed at the highest dietary ALA intake, but dietary DHA and AA supplements led to large increases (up to 10-fold) in the proportions of these PUFA. Tissue uptake of dietary AA and DHA appeared maximal for the LCP1 diet (replicate of breast milk) in the heart. There were no significant changes in the plasma levels of 11-dehydrothromboxane B2 (a thromboxane A2 metabolite), for any diet. The data confirm that dietary ALA is less effective than dietary DHA supplementation (on a gram/gram basis) in increasing tissue DHA levels and that tissues vary greatly in their response to exogenous AA and DHA, with the levels of these long chain metabolites being most resistant to change in the retina and brain compared with liver and heart. Dietary DHA markedly increased tissue DHA proportions in both liver and heart, whereas the major effect of dietary AA was in the liver. Future studies of the effects of dietary DHA and AA supplementation should examine a variety of tissues rather than focusing only on neural tissue.

The effect of docosahexaenoic acid on the electroretinogram of the guinea pig.

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, is found in consistently high concentrations in the retinae of mammals, yet its role in vision remains unclear. In this study, a mammalian model of variable retinal DHA concentration has been developed, such that the retinal phospholipids of guinea pigs contained between 2.5 and 30.8% DHA. Visual function was assessed using full-field flash electroretinography, over a range of exposure levels spanning six log units. Trend analysis indicated that retinal function was altered by the tissue DHA level, and was described by a second-order polynomial “inverted U-shaped” function. The results suggested that although some amount of DHA is essential for normal retinal function increases in the DHA level past an optimal amount, found to be 19%, provided diminishing returns. In this study, manipulation of the retinal DHA level accounted for 21–35% of the electroretinographic variability.

Increased blood pressure later in life may be associated with perinatal n-3 fatty acid deficiency

Hypertension is a major risk factor for cardiovascular and cerebrovascular disease. Previous work in both animals and humans with high blood pressure has demonstrated the antihypertensive effects of n−3 polyunsaturated fatty acids (PUFA), although it is not known whether these nutrients are effective in preventing hypertension. The predominant n−3 PUFA in the mammalian nervous system, docosahexaenoic acid (DHA), is deposited into synaptic membranes at a high rate during the perinatal period, and recent observations indicate that the perinatal environment is important for the normal development of blood pressure control. This study investigated the importance of perinatal n−3 PUFA supply in the control of blood pressure in adult Sprague-Dawley rats. Pregnant rat dams were fed semisynthetic diets that were either deficient in (DEF) or supplemented with (CON) n−3 PUFA. Offspring were fed the same diets as their mothers until 9 wk; then, half of the rats from each group were crossed over to the opposite diet, creating four groups, i.e., CON-CON; CON-DEF; DEF-DEF, DEF-CON. Mean arterial blood pressures (MAP) were measured directly, at 33 wk of age, by cannulation of the femoral artery. The phospholipid fatty acid profile of the hypothalamic region was determined by capillary gas-liquid chromatography. The tissue phospholipid fatty acid profile reflected the diet that the rats were consuming at the time of testing. Both groups receiving DEF after 9 wk of age (i.e., DEF-DEF and CON-DEF) had similar profiles with a reduction in DHA levels of 30%, compared with rats receiving CON (i.e., CON-CON and DEF-CON). DEF-DEF rats had significantly raised MAP compared with all other groups, with differences as great as 17 mm Hg. DEF-CON rats had raised MAP compared with CON-CON rats, and DEF-DEF rats had higher MAP than CON-DEF rats, despite the fact that their respective fatty acid profiles were not different. These findings indicate that inadequate levels of DHA in the perinatal period are associated with altered blood pressure control in later life. The way in which these long-term effects are produced remains to be elucidated.

The duration of normal visual exposure necessary to prevent form deprivation myopia in chicks

The aim of this study was to determine the minimum daily period of exposure to normal visual stimulation required to prevent occlusion induced myopia in chicks. Chicks were treated with monocular translucent occlusion in a 12 hr light/12 hr dark cycle. Occluders were removed for 0 (constant occlusion), 15, 20, 30, 40, 60, 75, 90, 120, 150, 240 or 720 (no occlusion) minutes each day for either 2 or 3 weeks. Fellow eyes and the eyes of normal chicks (bilaterally unoccluded) were used as controls. Occlusion-induced myopia and axial elongation were found to decrease significantly (P < 0.01) with increasing daily exposure to normal visual stimulation. Application of a time series equation to the data estimates that 30 and 130 min of normal visual exposure per day reduces myopia by 50 and 95% respectively. This study demonstrated that the regulation of ocular growth is affected strongly by short periods of normal visual stimulation in the presence of long periods of abnormal stimulation.

Effect of Dietary n-3 Deficiency on the Electroretinogram in the Guinea Pig

Despite the abundance of n-3 polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA), in the retina of all mammals, their role in vision remains unclear. However, the widely accepted notion that DHA is universally important in the retinal function was questioned in an earlier study (see text) involving guinea pigs, completely depleted of retinal DHA. The purpose of this study was to replicate this mammalian model of such deficiency and to determine any abnormalities in the electroretinogram. Guinea pigs were raised through three generations on 1 of 2 semipurified diets, containing different amounts of n-3 fatty acids, as determined by the supplementary oil which was the sole source of lipid in each diet. The added oils were safflower oil (n-6/n-3 = 72.0) or canola oil (n-6/n-3 = 2.5). Comprehensive electroretinographic assessment was conducted on guinea pigs, aged 6-9 weeks (safflower n = 13, canola n = 12), and retinae were extracted and the phospholipid fatty acid profile analyzed. Phospholipid fatty acid analysis revealed significant DHA deficiency (p < 0.001) in guinea pigs reared on the safflower oil diet (retinal DHA 2.5%) as compared with the canola oil group (retinal DHA 21.0%). Analysis of the electroretinogram showed significant reductions (p < 0.003) in both peak-to-peak and a-waves of safflower oil fed animals.

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.

AT1 receptor inhibition prevents astrocyte degeneration and restores vascular growth in oxygen-induced retinopathy

We investigated the effect of receptor blockade induced by an angiotensin II type‐1 receptor antagonist (AT1‐RB) on glial and vascular changes in oxygen‐induced retinopathy (OIR), a model of retinopathy of prematurity (ROP). OIR was induced in Sprague‐Dawley rats by exposure to 80% oxygen from postnatal (P) days 0–11, followed by 7 days in room air. Control animals were in room air for the entire duration. One cohort of OIR and control pups received the AT1‐RB valsartan (40 mg/kg/day intraperitoneal) from P11 to P18. The vascular response was examined immunocytochemically using retinal wholemounts and vertical sections labeled with endothelial (Isolectin‐B4) and pericyte (NG2, desmin) markers. Glial cell changes were assessed by measuring cell numbers and immunoreactivity (S100β, connexin‐26, and glial fibrillary acidic protein). OIR resulted in extensive intravitreal neovascularization and under‐development of the outer vascular plexus. Pericyte numbers were not significantly affected in OIR, although pericyte‐endothelial (desmin‐IB4) interactions were impaired. Peripheral astrocyte degeneration occurred between P11 and P13 with prominent Müller cell reactivity at P18. Valsartan imparted a protective effect on glia and blood vessels in OIR. At P18, valsartan‐treated OIR retinae showed significantly greater astrocyte survival, improved revascularization of the retina, and reduced preretinal neovascularization and Müller cell reactivity. This study identifies a glio‐vascular protective effect with AT1‐RB in OIR.

Measuring Rod and Cone Dynamics in Age-Related Maculopathy

PURPOSE A cathode-ray-tube (CRT) monitor-based technique was used to isolate clinically significant components of dark adaptation. The utility of the technique in identifying adaptation abnormalities in eyes with age-related maculopathy (ARM) is described. METHODS A CRT dark adaptometer was developed to assess cone and rod recovery after photopigment bleach. The following measures were obtained: cone recovery rate (R(c); in decades per minute) and absolute threshold (Tf(c); log candelas per square meter), rod recovery rate (R(r); decades per minute), and rod-cone transition (rod-cone break [RCB], in minutes). These components were isolated by appropriately selecting stimulus size, stimulus location, pigment bleach, and test duration and by coupling the CRT with judiciously selected neutral-density (ND) filters. The protocol was developed by using 5 young observers and was tested on 27 subjects with ARM in the study eye and 22 age-matched control subjects. RESULTS The parameters necessary for effective isolation of cone and early phase rod dark adaptation were a 2.6 ND filter (for a standard CRT monitor, 0.08-80 cd . m(-2) luminance output); a 4 degrees foveated, 200-ms, achromatic spot; approximately 30% pigment bleaching; and a 30-minute test duration. These settings returned obvious rod and cone recovery curves in control and ARM eyes that were compatible with conventional test methods and identified 93% of participants with ARM as having delayed dynamics in at least one of the parameters. Cone recovery dynamics were significantly slower in the ARM group when compared with age-matched control subjects (R(c), 0.99 +/- 0.35 vs. 2.63 +/- 0.61 decades . min(-1), P < 0.0001). Three of the 27 eyes with ARM did not achieve RCB during the allowed duration (30 minutes). The remaining eyes with ARM (n = 24) exhibited a significant delay in rod recovery (R(r)(,) ARM, 0.16 +/- 0.03 vs. controls, 0.22 +/- 0.02 decades . min(-1), P < 0.0001) and the average time to RCB (+/-SD) in the ARM group was significantly longer than in the control subjects (19.12 +/- 5.17 minutes vs. 10.40 +/- 2.49 minutes, P < 0.0001). CONCLUSIONS The CRT dark-adaptation technique described in this article is an effective test for identifying abnormalities in cone and rod recovery. Slowed cone and rod recovery and a delayed RCB were evident in the eyes with ARM. The test method is potentially useful for clinical intervention trials in which ARM progression is monitored.

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.