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(r ¼ 0.44) correlated (po0.01) with the neuroretinal rim area (r ¼ 0.44). RFonh showed a weak, borderline significant (po0.10) correlation (r ¼ 0.30) with pattern ERG amplitudes and perimetric mean deviation. From this study, it was concluded that RFonh is abnormally reduced in OHT and EOAG patients, indicating an impairment of luminance (M-cellular)-mediated vasoactivity. Flicker-evoked Fonh changes appear to be only weakly correlated with functional indicators of neural damage, suggesting that a loss of neurovascular coupling of the optic nerve head may occur independently of neural activity loss early in the disease process.

Discussion

FLDF and neurovascular coupling in humans

Previous studies in animals have demonstrated a tight correlation between local RFonh and neuronal activity response, the latter obtained from the flicker-induced changes in the electrical signal (Riva and Buerk, 1998) and K+ ion concentration (Buerk et al., 1995), both measured at the surface of the optic disk. In humans, luminance and heterochromatic flicker stimulation generate a RFonh that displays characteristics comparable to those of ganglion cell spiking activity evoked by the same stimuli, when RFonh is plotted as a function of frequency, modulation depth, and relative chromaticity (Riva et al., 2001). Although these findings had suggested the existence of a coupling between vasoand neuronal activity, the exact relationship between both physiological activities remained to be established. With this goal in mind, and since noninvasive methods to measure local activity at the optic nerve rim are not yet available, the 1F and 2F components of the flicker ERG and the simultaneously recorded RFonh were correlated. Although the pattern ERG, which is generated mainly by ganglion cell activity, would appear to be the most appropriate stimulus to correlate activity and RFonh in humans, the components of the F-ERG were chosen because of the substantial amount of data available from humans. In addition, the hypothesis

of an association between RFonh and the 1F and 2F harmonics of the flicker ERG was justified

based on the origin of these components of retinal neural activity.

The results of investigations in the normal human eye (Falsini et al., 2002) indicated that, under specific experimental conditions, the stimu-

lus-evoked changes in RFonh are similar and significantly correlated to the changes in the flicker

ERG harmonic components. In particular, the associations were the most significant (a) when (Fig. 8) the temporal frequency of the stimulus was changed in the condition of heterochromatic red– green equiluminant flicker; (b) when the mean illuminance was varied for a green illuminance flicker; and (c) when the color ratio, r, was varied for the heterochromatic R-G flicker. In case (a), RFonh was correlated with both 1F and 2F amplitudes. In the others, RFonh was correlated only with the 2F amplitude.

The significant correlation found between RFonh and each of 1F and 2F amplitudes for the case of R-G equiluminant flicker responses recorded as a function of temporal frequency, as well as between

RFonh and 2F amplitudes for R-G flicker responses as a function of r, suggests a coupling

arising from the pooled activity of the middleand long-wavelength sensitive cones. Given the retinal origin of the flicker ERG 2F, the present findings provide support to the hypothesis that blood flow changes recorded at the ONH in response to flicker stimulation parallel the corresponding changes in the neural function of the inner retina. Under some experimental conditions (chromatic flicker modulated at different temporal frequencies), however, RFonh changes may also be similar to changes in the 1F amplitude, implying common properties of optic nerve vasoand neural activity changes in the middle retina (i.e. ON and OFF bipolar cells).

In the case of pure luminance flicker experiments, when changing temporal frequency, no significant association was found between RFonh and both components’ amplitudes. A possible physiological basis for this finding is that the generators underlying RFonh and ERG response to luminance flicker may differ depending on flicker frequency. Multiple generators for both responses, whose relative contribution changes according to

stimulus frequency, may obscure the correlation when measurements are obtained over a range of frequencies. On the other hand, it is possible that the blood flow and neural responses to a specific stimulus frequency, i.e. the luminance flicker at 10 Hz, share common generators in the inner retina, thus revealing the association between

RFonh and ERG 2F amplitude. In contrast, the R-G heterochromatic flicker reveals a significant

coupling between RFonh and both 1F and 2F amplitudes, suggesting a contribution from both retinal layers to the coupling between the neural and vascular responses.

Although several previous studies have suggested a neurovascular coupling in the human retina (Scheiner et al., 1994; Riva et al., 1995), direct evidence of it has not been provided in the past. The observed correlations may not directly prove an effect of retinal neural activity on blood flow measured at the optic disk. It is however reasonable to suggest that the pooled response of neural generators underlying the 1F and 2F components may induce a vaso-active mechanism resulting in a corresponding blood flow change. Putative mediators underlying this neurovascular response have been discussed in detail in previous reports (Buerk et al., 1995, 1996; Kondo et al., 1997; Riva and Buerk, 1998), the most prominent being nitric oxide (NO). The present findings support the hypothesis that the strength of neurovascular coupling may be dependent upon the type of flicker stimulus. From the data shown in Fig. 8 it can be concluded that both luminance and chromatic flicker may be appropriate to study the coupling between RFonh and 2F amplitude, whereas the chromatic equiluminant stimulus appears to be more suitable for investigating the association between RFonh and 1F.

Comments on clinical application of FLDF in glaucoma

The data reviewed here demonstrated that RFonh is diminished in both OHT and EOAG patients compared to normal controls. The group-averaged time course of the flicker-induced increase in Fonh in EOAG was not significantly different from that found in the normal subjects. Presumably, in the

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EOAG patients the flicker-induced vasodilatation in the apparently healthy rim tissue of the anterior optic nerve has a normal temporal evolution.

Our results strongly suggest that the decrease in RFonh reflects an altered ganglion cell activity response to the flicker. Nevertheless, other possible explanations of this decrease need to be addressed to. One possibility could be that resting blood flow in the rim tissue of the OHT and EOAG patients could be too low in comparison with the amount of tissue it supplies to satisfy the metabolic needs. Consequently, it would be unable to fully respond to the additional stress evoked by the flicker stimulation. However, the lack of correlation between RFonh and Fonh (Riva et al., 2004b) goes against this hypothesis. Another possibility is that in the EOAG group, because the nerve fiber layer is thinner than in the normal control and OHT groups, the choroid would contribute more to the LDF signal. This is unlikely since the markedly higher flow velocities in the choriocapillaris did not result in a significantly greater RVelonh at rest in the EOAG compared to the control group. Furthermore, RFonh was also found to be reduced in the OHT group, although the nerve fiber layer thickness in this group was not significantly different from the control group.

Studies in the human eye using a range of flicker frequencies and modulations suggest that the RFonh loss observed in our patients when using a 15 Hz luminance flicker occurs predominantly at the level of the ganglion cell M-cellular pathway (Riva et al., 2001; Falsini et al., 2002). This hypothesis agrees with the large body of anatomical evidence indicating that, in early glaucoma, large ganglion cells, subserving primarily the M-cellular pathway, are selectively or predominantly damaged (Quigley et al., 1989; Glovinski et al., 1991; Kerrigan-Baumrind et al., 2000).

Patients’ RFonh, in contrast, were poorly correlated with functional indicators of early damage, such as pattern ERG and Humphrey perimetric indices. Thus, the data obtained from the OHT patients show that, in some of these patients, the

pattern ERG was normal whereas RFonh was substantially diminished. In other OHT patients,

the opposite was found. This poor correlation may be related to the inherent variability of the

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techniques employed in this study, or, alternatively, may reflect an altered neurovascular coupling in early stages of diseases. Alteration of the neurovascular coupling could occur at various points of the chain of events coupling the activity to the local vasodilatation (Zonta et al., 2003), such as the flicker-induced release of NO, K+, or other substances (Buerk et al., 1996; Kondo et al., 1997; Zonta et al., 2003), the glutamate release from axonal terminals, the activation of astrocytes and subsequent release of vasodilating products and possibly others. Clearly, future work should be aimed at elucidating this important question.

RFonh was positively correlated with neuroretinal rim area when the two groups of patients were pooled. In EOAG patients, although this correlation did not reach significance, possibly because of the low number of patients, both neuro-

retinal rim area and RFonh were smaller in the average than the corresponding values in OHT

and normal controls. It has been previously shown that a reduction in neuro-retinal rim area, together with an increase in cup/disk area ratio and cup shape measure, may be highly accurate in detecting early glaucomatous damage (Uchida et al., 1996; Blumenthal and Weinreb, 2001). These results indicate that a reduction in ONH vaso-activity assessed by flicker stimulation is associated with early loss of nerve fiber layer in eyes with EOAG.

A number of studies have compared Fonh measurements obtained in normal subjects and in glaucoma patients. Although the results vary considerably between studies (Piltz-Seymour et al.,

2001), Fonh has been found to be reduced in primary open-angle glaucoma patients compared

to normal controls (Michelson et al., 1996; Nicolela et al., 1996; Hafez et al., 2003; Riva et al., 2004b). Furthermore, Fonh also tended to be reduced in the OHT patients (Riva et al., 2004b).

The decrease of Fonh in glaucoma has been interpreted by a number of investigators as evidence of an actual reduction in blood flow, although some caution about the validity of this interpretation has been expressed in view of the limitations of the LDF technique. As discussed elsewhere (Riva and Petrig, 2003), comparisons between Fonh values in terms of actual blood flow are strictly valid only if the scattering properties of

the tissue from which Fonh is measured are identical. This is due to the fact that the DSPS depends not only upon the number and velocity of RBCs in the sampled volume, but also on the optical characteristics (i.e. the absorption and scattering) of the tissue (nonmoving scatterers) sampled. In general, increased tissue scattering broadens the power spectrum, causing an artificial increase in the measured flow. To our present knowledge, it is not clear yet how the scattering of light by the glaucomatous rim tissue differs from that of the healthy eye and therefore how it may

affect Fonh.

In contrast to Fonh, RFonh is not affected by the scattering properties of the tissue. Changes in light

scattering may be expected during increased neural activity, but these changes are too fast and probably too small to have a notable effect on

the LDF spectrum and Fonh (Gratton and Fabiani, 2001). Furthermore, the changes in Fonh are

proportional to the actual flow changes as they are within the range of linearity of the LDF technique (Riva et al., 2000). Both of these aspects make the LDF technique most appropriate to investigate the regulation of Fonh in response to various physiological stimuli.

Using flicker to investigate this regulation offers additional advantages over previously used physiological stimuli such as decreases in ocular perfusion pressure (OPP) achieved by increasing the IOP with a suction cup (Pillunat et al., 1997; Riva et al., 1997), increases in OPP by means of isometric exercises (Movaffaghy et al., 1998), and the breathing of various gas mixtures (Harris et al., 1996). Flicker is not invasive and a more physiological stimulus since modulation of light exposure is the most natural stimulus for the visual system, leaving also the systemic circulation unperturbed.

The LDF measurements were obtained by directing the probing laser beam at the temporal site of the neuro-retinal rim of the optic disk. In humans, as in monkeys (Petrig et al., 1999), LDF is probably predominantly sensitive to blood flow changes occurring only in the most superficial layers (supplied by the retinal circulation) of the optic nerve head. However, regardless of which layer of the optic nerve head circulation was in fact