The role of dark adaptation in understanding early AMD.

The main aim of the paper is to discuss current knowledge on how Age Related Macular Degeneration (AMD) affects Dark Adaptation (DA). The paper is divided into three parts. Firstly, we outline some of the molecular mechanisms that control DA. Secondly, we review the psychophysical issues and the corresponding analytical techniques. Finally, we characterise the link between slowed DA and the morphological abnormalities in early AMD. Historically, DA has been regarded as too cumbersome for widespread clinical application. Yet the technique is extremely useful; it is widely accepted that the psychophysically obtained slope of the second rod-mediated phase of the dark adaptation function is an accurate assay of photoreceptor pigment regeneration kinetics. Technological developments have prompted new ways of generating the DA curve, but analytical problems remain. A simple potential solution to these, based on the application of a novel fast mathematical algorithm, is presented. This allows the calculation of the parameters of the DA curve in real time. Improving current management of AMD will depend on identifying a satisfactory endpoint for evaluating future therapeutic strategies. This must be implemented before the onset of severe disease. Morphological changes progress too slowly to act as a satisfactory endpoint for new therapies whereas functional changes, such as those seen in DA, may have more potential in this regard. It is important to recognise, however, that the functional changes are not confined to rods and that building a mathematical model of the DA curve enables the separation of rod and cone dysfunction and allows more versatility in terms of the range of disease severity that can be monitored. Examples are presented that show how analysing the DA curve into its constituent components can improve our understanding of the morphological changes in early AMD.

Slowed Dark Adaptation in Early AMD: Dual Stimulus Reveals Scotopic and Photopic Abnormalities.

Purpose: The recovery of visual sensitivity after a photobleach in early AMD is slowed in rods but cones also may be abnormal. The purpose of this article was to test different stimulus locations to investigate cone function and its relation to rod abnormalities. Methods: Stimuli were presented at two locations, 3.0° and 5.5°, in the inferior visual field. Post photobleach dark adaptation (DA) curves from 50 early-AMD patients were compared with those from 15 healthy controls of similar age. Curves were characterized in terms of four parameters: ct, cone threshold; α, the transition point from cone to rod function; S2, the slope of the second rod-mediated component; and ß, the transition from the second to the third rod-mediated component. Results: There were strong location effects for the healthy group and the AMD group. Cone threshold was higher for the outer compared with the inner stimulus (P = 0.001), S2 was steeper for outer compared with inner (P < 0.001), α was shorter for outer (P = 0.004), and ß was shorter for outer than inner (P = 0.002). The high variance in the patient data, particularly for α and ß, explained the absence of a group*location interaction in the statistics. Conclusions: The data provide a novel perspective on abnormal cone- and rod-sensitivity recovery in early dry AMD. The comparison of pairs of DA curves from different locations highlights the involvement of cones in the underlying pathology of AMD. Dynamic measures of visual function are particularly sensitive to early AMD.

Slowed dark adaptation in older eyes; effect of location.

PURPOSE: The rate of rod sensitivity recovery following a photobleach is a basic measure of the integrity of the outer retina. Rods are selectively impaired in aging and many disorders of the retina, notably Age-Related Macular Degeneration (AMD). It is not known for certain whether the age-related deficit is a pan-retinal effect or if there are localised regions of impaired rod function. To address this important issue a dual arc stimulus was developed that samples sensitivity recovery in two retinal locations. METHODS: Arc-shaped stimuli were presented on a black CRT screen at two locations, in the inferior visual field. Following a bleach, which was localised to the stimuli, recovery of sensitivity was measured using a modified method of adjustment technique. Neutral density filters were used to extend the luminance range of the CRT. Sensitivity recovery functions were fitted by non-linear regression to a seven-parameter model. RESULTS: Pairs of sensitivity recovery functions were generated from the stimuli. The cone phases of these functions were identical. The slopes of the S2 sections of the curves were steeper for the outer stimuli for both young (p < 0.001) and older (p = 0.003) observers. The difference between the two was the same for the two groups. The α point was reached slightly earlier for the young observers and with the outer stimulus but neither of these effects reached statistical significance. The ß point occurred earlier for the outer stimuli and this effect was statistically significant only for the older group. CONCLUSIONS: The method places minimal demands on observers. The fact that rod sensitivity recovery is slowed in the older normal eye to the same extent in the two locations suggests that this deficit may be uniform across the retina. As there are localised losses in scotopic function in AMD, the technique is ideally suited to distinguishing impaired recovery dynamics due to normal ageing from those caused by disease.

Macular pigment in ophthalmic practice; a survey.

BACKGROUND: Macular pigment (MP) might provide some protection against age-related eye disease, and it is now being measured in ophthalmic practice. The purpose of the survey described here was to determine the distribution of MP in a random population of patients in a typical UK ophthalmic practice. METHODS: Macular pigment optical density (MPOD) was measured in 56 patients aged 11 to 87 years, mean 52 ± 19, over a 3-month period. Typically, the test requires setting flicker thresholds for a centrally and peripherally viewed blue/green alternating target. Here we describe the results when an age-based estimate of the peripheral value is used, thus avoiding the peripheral setting. In 32 observers, a comparison was made between this and values obtained with the centre and periphery method. Information on smoking habits, iris colour, diabetic status, and ethnicity were recorded. RESULTS: The overall average MPOD for the population obtained with the centre-only approach for 56 individuals was 0.400 ± 0.165. The centre-only technique was an accurate predictor of values based on centre and peripheral measures, with 95% limits of agreement of 0.137 OD units. Pearson's correlation coefficient showed a high correlation between right and left eyes (r = 0.7 (p < 0.001)). There was a small difference between males and females that did not reach statistical significance (r = -0.22). There was a non-statistically significant age-related decline in MPOD in this particular population (r = -0.17). Dark irides were significantly associated with high MPOD (r = 0.28, p < 0.05). MPOD in Type II diabetic patients was 27% lower than that in non-diabetics (r = 0.29, p < 0.05). CONCLUSION: The technique provides similar values of MP optical density to previous reports. As with other HFP-based methods, in a small percentage of older patients, more than one measurement is required before satisfactory results are obtained.

Assessment of age changes and repeatability for computer-based rod dark adaptation.

PURPOSE: To characterize the rate of rod-mediated sensitivity decline with age using a PC-driven cathode ray tube (CRT) monitor. To provide data regarding the repeatability of the technique. METHODS: Dark adaptation was monitored for 30 min following a minimum 30 % pigment bleach, using a white 1° stimulus (modulated at 1 Hz), presented 11° below fixation on a CRT monitor. Thirty-three subjects with no ocular pathology and normal fundus photographs were divided into two groups: older (≥45, n = 16) and younger (<45, n = 17). RESULTS: Rod recovery was assessed using component S2 of dark adaptation. S2 was significantly slower in the older (0.19 ± 0.03 log cd.m(-2).min(-1)) compared with the younger group (0.23 ± 0.03 log cd.m(-2).min(-1), t = -4.05, p < 0.0003), despite no difference in visual acuity and fundus appearance. Faster rates of S2 recovery were correlated with lower threshold at 30 min (T30) (r = -0.49). Correlation coefficients between first and second measurements for S2 and T30 were 0.49 (p < 0.009) and 0.84 (p < 0.0001) respectively. The coefficient of repeatability was 0.07 log cd.m(-2).min(-1) for S2 and 0.35 log cd.m(-2) for T30. The coefficients of variation for S2 and T30 were 15 % and 10 % respectively. CONCLUSIONS: Dark adaptation is slowed in normal ageing. CRT-based dark adaptometry is easily implemented and highly repeatable. The technique described in this article would be useful for documenting visual changes in future clinical trials assessing retinal health in the older eye with and without ocular pathology.

Lutein supplementation over a one-year period in early AMD might have a mild beneficial effect on visual acuity: the CLEAR study.

PURPOSE: We investigated the effect of daily supplementation with lutein (L) capsules on macular pigment optical density (MPOD) and visual acuity (VA) in patients with early age-related macular degeneration (AMD). METHODS: A randomized, double-blind, placebo-controlled, two-center investigation of the effects of L supplementation in early AMD was conducted. The duration of the trial was 12 months. The centers were Manchester, United Kingdom and Maastricht, the Netherlands. L capsules (10 mg Ester) or a placebo (P) were taken daily. There were 72 patients (mean age 70.5 ± 8.7) assigned randomly to either L (n = 36) or P (n = 36) groups. MPOD using a flicker-based technique (MPS9000) and best corrected VA (LogMAR) were measured at the beginning and at 4-month intervals over the duration of the 12-month supplementation period. Blood serum samples were collected to monitor compliance. RESULTS: At the end of the trial, an overall increase in the mean MPOD level was found for the L group from 0.38 ± 0.19 to 0.53 ± 0.22 optical density (OD) units. According to a mixed design ANOVA, this was statistically significant (P < 0.001). No change in MPOD was found for the P group. There was no significant change in VA in the L group (n = 36). The P group (n = 36) showed a statistically significant deterioration from 0.05 ± 0.13 to 0.09 ± 0.13 (P < 0.05). When comparing the change in VA over the supplementation period, there was a significant difference between the two groups (P < 0.05). To avoid ceiling effects, 2 subgroups of patients with VA worse than 0.06 at baseline were reanalyzed. In the L subgroup (n = 19) a mean improvement in VA from 0.23 ± 0.12 at baseline to 0.16 ± 0.10 at visit 4 was observed (P < 0.05). In the P subgroup (n = 14), there was a small deterioration from 0.18 ± 0.13 to 0.19 ± 0.12 (P = 0.70). The improvement in VA in the L subgroup was compared to the deterioration in VA in the P group and this effect reached statistical significance (P < 0.05). CONCLUSIONS: L supplementation increases MPOD levels in early stage AMD patients. According to the VA measurements, the progress of the disease might be slowed in some patients with augmented levels of MP. (ClinicalTrials.gov number NCT01042860.).

A new desktop instrument for measuring macular pigment optical density based on a novel technique for setting flicker thresholds.

A rapid portable technique for estimating macular pigment optical density (MPOD) in large populations is described. The new instrument utilises a novel method for setting flicker thresholds which is undemanding for naïve and elderly observers and easily operated by a non-technical person. The method has good repeatability (r = 0.97) and the data are comparable with an optical method based on retinal reflectometry (r = 0.78). MPOD spatial profiles are presented for seven normal observers and these are well described (r = 0.99) by a decaying exponential function consistent with previous reports. MPOD values are presented from 5581 (2435 females and 3146 males) individuals measured in 48 optometric practices. The mean MPOD of this population was 0.33 (S.D. +/- 0.187) which is similar to previous large scale studies of MP.

Correspondence between retinal reflectometry and a flicker-based technique in the measurement of macular pigment spatial profiles.

A comparison of macular pigment optical density (MPOD) spatial profiles determined by an optical and a psychophysical technique is presented. We measured the right eyes of 19 healthy individuals, using fundus reflectometry at 0, 1, 2, 4, 6, and 8 deg eccentricity; and heterochromatic flicker photometry (HFP) at 0, 0.5, 1, 2, 3, 4, 5, 6, and 7 deg, and a reference point at 8 deg eccentricity. We found a strong correlation between the two techniques. However, the absolute estimates obtained by fundus reflectometry data were higher than by HFP. These differences could partly be explained by the fact that at 8 deg eccentricity the MPOD is not zero, as assumed in HFP. Furthermore, when performing HFP for eccentricities of <1 deg, we had to assume that subjects set flicker thresholds at 0.4 deg horizontal translation when using a 1-deg stimulus. MPOD profiles are very similar for both techniques if, on average, 0.05 DU is added to the HFP data at all eccentricities. An additional correction factor, dependent on the steepness of the MPOD spatial distribution, is required for 0 deg.

The mode of action of cell wall-degrading enzymes and their interference with Nod factor signalling in Medicago sativa root hairs.

Medicago sativa L. (alfalfa) root hairs respond to Nod factors [NodRm-IV(C16:2,S)] in a host-specific manner with depolarization and rapid ion fluxes. Protoplasts prepared from these cells using the cell wall-digesting enzymes pectolyase and cellulase do not, or to a rather small extent, respond to Nod factors. In an effort to understand this activity loss we analyzed the mode of action of both enzymes with respect to their effects on the root hairs as well as their interference with the Nod factor response. (i) In the presence of the enzymes, Nod factor at saturating concentrations neither depolarized the plasma membrane of root hairs nor caused ion fluxes. Even after removal of the enzymes, Nod factor responses were strongly refractory. (ii) After a lag-phase of 12-18 s, pectolyase depolarized the plasma membrane, alkalized the external space, acidified the cytosol and increased the cytosolic Ca(2+) activity. (iii) Cellulase, without a lag-phase, depolarized the plasma membrane, acidified the cytosol, but only marginally increased the cytosolic Ca(2+) activity. Unlike pectolyase, the cellulase response was only weakly refractory to a second addition. (iv) Neither enzyme increased the membrane conductance, but pectolyase inhibited the H(+)-pump. (v) Pectolyase shows all the signs of an elicitor, while cellulase yields a mixed response. (vi) Denatured enzymes yielded strong effects similar to those of untreated enzymes. We conclude that the effects shown do not originate from enzymatic activity, but from interactions of the proteins with cell wall or plasma membrane constituents. It is further concluded that these enzymes (pectolyase more so than cellulase) trigger defense-related signal pathways, which makes protoplasts prepared with such enzymes unsuitable for studies of symbiotic or defense-related signalling.

Single-cell measurements of the contributions of cytosolic Na(+) and K(+) to salt tolerance.

Ion concentrations in the roots of two barley (Hordeum vulgare) varieties that differed in NaCl tolerance were compared after exposure to NaCl. Triple-barreled H(+)-, K(+)-, and Na(+)-selective microelectrodes were used to measure cytosolic activities of the three ions after 5 and 8 d of NaCl stress. In both varieties of barley, it was only possible to record successfully from root cortical cells because the epidermal cells appeared to be damaged. The data show that from the 1st d of full NaCl stress, there were differences in the way in which the two varieties responded. At 5 d, the tolerant variety maintained a 10-fold lower cytosolic Na(+) than the more sensitive variety, although by 8 d the two varieties were not significantly different. At this time, the more tolerant variety was better at maintaining root cytosolic K(+) in the high-NaCl background than was the more sensitive variety. In contrast to earlier work on K(+)-starved barley (Walker et al., 1996), there was no acidification of the cytosol associated with the decreased cytosolic K(+) activity during NaCl stress. These single-cell measurements of cytosolic and vacuolar ion activities allow calculation of thermodynamic gradients that can be used to reveal (or predict) the type of active transporters at both the plasma membrane and tonoplast.