systemic hypoxia because of the large volume of oxygenated blood in the choroid. However, it remains possible that mild systemic hypoxia could influence the nonpulsatile component of ChBF. This latter possibility awaits confirmation by measurements of the ChBF with LDF after inhalation of 12% O2 in nitrogen. Overall, the present state of knowledge suggests that the retinal circulation is likely more sensitive than the choroid to transient mild systemic hypoxia.

10.6.4Hyperoxia, Hypercapnia, and Retinal Function

In addition to studies on the effects of altered blood gases on ocular blood flow, the effects of hyperoxia and hypercapnia on the neural retinal function have also been assessed. Systemic hyperoxia had minimal effects on the function of the photoreceptors and bipolar cells as indexed by photopic fERG a- and b-waves [64]. However, when the retina was dark adapted, hypercapnia significantly reduced the amplitude of OP5, one index of amacrine cell function [65]. These results provide additional support to the growing body of evidence indicating that neural function in the retina is more vulnerable to changes in blood gases and vascular perfusion levels when the retina is dark adapted.

10.6.5Hypoxia, Hyperoxia, and Retinal Function

While transient hypoxia attenuates the function of the neural generators of the photopic fERG b-wave and specific OPs, it does not affect the amplitude or the implicit time of the photopic ERG a-wave [66]. This is consistent with the fact that the choroidal blood oxygen tension is highest at the RPE-photoreceptor junction and rapidly decreases toward zero by the middle of the retina [45, 67] (Fig. 10.1b). Consequently, bipolar cells that populate the retinal zone where the choroidal and retinal vasculatures provide the lowest oxygen tension would be particularly vulnerable to the effects of transient systemic hypoxia as was

indicated by the reduction in the photopic fERG b-wave. While transient systemic hypoxia does not alter the function of photoreceptors in the outer retinal layer, it does reduce the amplitude and delays the N95 component of the pERG that is principally generated by the ganglion cells in the innermost retinal layer [68]. This greater vulnerability of the ganglion cells to transient hypoxia may be explained by the much smaller oxygen tension in the retinal blood perfusing the inner two-thirds of the retina. The consequence of this reduced level of oxygen tension in retinal blood is that a mild transient systemic hypoxia is likely enough physiological stress to compromise ganglion cell function and thus results in compromised pERGs. The additional observation that transient systemic hyperoxia did not affect the function of ganglion cells supported the interpretation that the much smaller degree of oxygen tension in retinal blood flow may be just adequate to support normal neural function in the innermost retinal layer. Thus, transient systemic hyperoxia providing more oxygen to the retinal vasculature would not be expected to compromise the function of ganglion cells found in the inner retinal layer, and this is what was found experimentally [68].

10.7Regional Choroidal Perfusion

While vascular autoregulation in the retinal vasculature has been demonstrated in many studies over the last 20 years or so, evidence for vascular regulation in the human choroid remains limited but the notion for choroidal regulation is gaining broader acceptance in the ocular blood flow section of the vision science community. For many years, it was thought that there was no need for regulation of ChBF because of the large choroidal volume and flow rate relative to the retinal vasculature. However, seminal work by Linsenmeier et al. [45] revealed a partial pressure of O2 (PO2) that decreased rapidly from the choroid and approached a zero value near the middle of the retina (Fig. 10.1b). This implied that the retina consumed virtually all of the O2 that came from the choroid, and any disruption of blood

flow to the RPE-photoreceptor complex could have serious consequences on neural retinal function and vision.

Fortunately, a global reduction in ChBF is not a common clinical occurrence. However, there is increasing evidence that a regional reduction in ChBF occurs in patients with age-related macular degeneration (ARMD) even before there is any clinical evidence of a significant disruption in the foveomacular anatomy [69]. Thus, a subnormal choroidal perfusion of the macula may be a major risk factor for this age-related macular disease [70]. It may be that a reduction in subfoveal ChBF prior to the typical clinical signs and symptoms of ARMD is the result of defective regulation. If this hypothesis is correct, traditional fluorescein and choroidal angiographies may not be adequate for detecting blood flow that is not capable of sustaining normal neural function during transient vascular stress. Consequently, a more productive method for detecting weakened ChBF would be to quantify choroidal hemodynamic responses during an increased metabolic demand as induced by flicker or safe levels of altered OPP to determine the integrity of choroidal regulation. Absence of regulatory responses could identify individuals at high risk for macular disease and the need to initiate procedures to preserve macular structure and function.

The proposal that choroidal regulation may also involve a differential perfusion across the ocular fundus is based on several previous observations in blood flow. In subhuman primates, flicker stimulation elicited differential perfusion by the retinal vasculature according to regional metabolic demands [71]. As such, only differential ChBF across the retina may be able to satisfy large differences in local metabolic demand. At the systemic level, it is well established that blood flow is shunted to regions with increased metabolic demand such as preferential blood flow to the large leg muscles while running, even to the detriment of blood flow to internal organs [72]. Finally, data in Sects. 10.7.3 and 10.7.4 present findings that support the hypothesis of differential choroidal perfusion in favor of retinal areas with increased metabolic requirements.

10.7.1Cones Versus Rods: Structure and Function

The degree to which a flash elicits an electrophysiological response from the retina depends on two variables: (1) the physical properties of the flash and (2) the anatomical location and density distribution of the cone and rod photoreceptors across the retina. Variables in the flash stimulus include wavelength composition, luminance level, and presentation frequency (Hz). With respect to retinal location, the short, medium, and long wavelength sensitive cones (“Blue,” “Green,” and “Red” cones) totaling ~6 million [73], are distributed within the central ~20° around the foveola. The G and R cones have the highest density in the central 5° (~200 K cones/mm2) while the blue cones for the same central area have a density of ~2 K cones/mm2 [74]. Rhodopsin-containing rods estimated at ~120 million [73] are found in all regions of the retina except the foveola and occur in greatest density within a concentric annular zone that extends between ~5° and ~50° from the foveola, and thereafter decrease in density toward the periphery.

This differential distribution of cones and rods across the retina is the basis for the rationale presented in a later section of this chapter that repeated stimulation of extra foveal blue-sensi- tive cones and especially the blue-sensitive rods greatly increased the metabolic need of more eccentric retinal sites, and consequently, through some unknown mechanism, the ChBF was directed away from the fovea in favor of the midperiphery of the retina. As such, it was concluded that the choroid that nourishes the RPEphotoreceptor complex can preferentially divert blood flow in the macula from one site to another to support the metabolic demands of retinal sites with heightened neuronal activity, as was reported to be the case for the retinal vasculature [71].

10.7.2 Choroidal Angioarchitecture

The angioarchitecture of the choroid at the posterior pole is described as a honeycomb, nonlobular structure that gradually changes into a mosaic of

clearly defined lobules with anastomosing capillaries from the peripapillary zone to the retinal periphery. While the distinct lobular pattern of the choriocapillaris in the periphery determines how blood will flow from one point to another, the direction of blood flow in the foveal zone is less obvious. The choriocapillaris in the foveomacular zone has a homogenous structure and blood flow in that area does not match what is seen by fluorescein and indocyanine angiographies [2]. It is likely that foveomacular blood flow is determined by pressure gradients and differences in metabolic requirements within the macular retina [2]. Thus, the choroidal lobuli comprising the choriocapillaris layer of the choroid determine the flow of blood by both structure (lobuli) and function (neuroretinal activity).

The technological challenge for this new level of clinical diagnostics based on blood flow measurements requires the development of a system that can penetrate the RPE (IR probe) and make measurements of blood flow in the retina and/or choroid in arbitrary sites of the retina, as well as a series of sites that correspond to visual field thresholds for vision. Perhaps most importantly, such a system should provide variable luminance focal flicker to determine threshold changes in blood flow, as well as stimulus–response profiles for suspect areas of the fundus.

10.7.3 Dark Adaptation

Photopic vision is mediated by the cone photoreceptors that provide high spatial resolution and color perception. Scotopic vision is a function of rod photoreceptors and is characterized by achromatic vision and low spatial resolution. The inverse distribution of cone vs. rod density across the retina, and the drop in choriocapillaris vessel density from the fovea to the periphery, may be an evolutionary adaptation that attempts to balance metabolic needs with energy supplies. Inasmuch as the retina consumes most of the O2 that the choroid can provide [75], topographic change in choriocapillaris vessel density may reflect the metabolic needs of the photoreceptors in the overlying retina. To date, a correlation

between ChBF in the foveal and the perifoveal area and the transition from cone to rod function has not been demonstrated in man. However, a recent study did investigate such a coupling between central photoreceptor function and ChBF in the human retina [76]. A continuously recording NI-LDF was used to record foveomacular ChBF changes throughout a 26-min dark adaptation interval. A 10-s recording of the ChBF was made in the foveal and perifoveal zones every 2 min for 26 min of dark adaptation. The group averaged ChBF decreased by ~17% (Fig. 10.17). This indicated that the gain in light sensitivity by the retina in the dark was accompanied by a decrease in the resting level of ChBF in the cone-rich foveomacular zone and that volumetric change in the flow presumably went to the rod-rich area because rod sensitivity to light was maximized after dark adaptation. Since rods outnumber cones by ~20:1 in the human retina, and the highest rod density is found ~15–18° peripheral to the fovea, it was hypothesized that the reduction in foveal ChBF was a manifestation of ChBF regulation wherein blood was moved toward a retinal site with higher physiological activity and metabolic demand. Since retinal blood flow increases in darkness [77, 78], presumably to support the metabolic needs of rod photoreceptors, and the photoreceptor “dark current,” choroidal blood that nourishes photoreceptors from the scleral side may also move in the same direction, i.e., away from the fovea, toward the peripherally located rods in proportion to their activity. The reduction in the subfoveal ChBF during dark adaptation raised the possibility that choroidal blood was shunted radially outward toward the paramacular annular zone that is densely populated by rod photoreceptors. Measurements of ChBF in perimacular regions during dark adaptation would be needed to confirm empirically the hypothesis of a redistribution of choroidal blood from cone-dominated to roddominated areas in the retina. Figure 10.17 presents the graphical illustration of the changes in subfoveal ChBF as the retina transited from light to dark adaptation over a 26-min recording interval. During dark adaptation, the subfoveal ChBF decreased by about 6% in the first 10 min of dark

ChBF (AU)

5.0

4.8

4.6

4.4

Fig. 10.17 Group-averaged subfoveal ChBF as a function of time into dark adaptation. ChBF was measured by NI-LDF. The dim speckle appearance of the probing laser beam does not affect dark adaptation of the retina. In the first 6 min, the ChBF decreased by about 6%, and then by another ~11% during the remaining time. The dashed biphasic line has been drawn to show the resemblance between the change in the subfoveal ChBF in dark adaptation and the psychophysically measured dark adaptation

curve (see Inset). A linear regression model through the data points confirmed the reduction in ChBF throughout dark adaptation. The reduction in subfoveal choroidal blood suggests a routing of this blood outward toward retinal zones with increasing metabolic needs. Rod photoreceptors populating the perifoveal retinal zones are physiologically logical recipients of the subfoveal blood shunted to eccentric retinal sites because of their increasing metabolic requirements

adaptation and then decreased rapidly by about 11% within the next 8.5 min. Thereafter, the ChBF remained at a constant level of ~17% below the initial ChBF measurement in normal ambient lighting. The biphasic attenuation of the subfoveal ChBF during dark adaptation resembled the psychophysical dark adaptation curve for the human retina shown in the inset of Fig. 10.17.

This likely was the first study that raised the possibility of a correlation between the changes in the subfoveal ChBF during the switch from cone to rod vision in the human retina with the psychophysically measured changes in light detection thresholds across the retina as it changed from a light to dark-adapted state. Because the amplitude and timing of the rod-cone break in dark adaptation is strongly influenced by the level of light adaptation of the retina, and because blood flow changes likely precede perceptual changes in light thresholds by yet unknown amounts, the degree and timing of the subfoveal ChBF changes in dark adaptation likely

do not correlate perfectly with the subjective measurements of retinal dark adaptation.

Nonetheless, the objective quantification of blood flow changes that may precede functional changes in vision could provide significant advantages in the differential diagnoses for photoreceptor dysfunction, neuronal atrophy, and ultimately permanent vision loss. Further research is required to evaluate the clinical utility of this objective procedure for subfoveal changes in the ChBF during dark adaptation.

10.7.4 Protracted Blue Flicker

Recent studies reported that flicker stimulation of the retina increased blood flow in the retinal vasculature and the ONH [79–81], but not in the choroid [82, 83]. It was rationalized that the absence of blood flow change in the choroid meant that it was independent of changes in retinal metabolism.

Of particular importance in evaluating these data is the fact that these studies used flicker composed of wavelengths that included all or large parts of the photopic spectral sensitivity curve. Such flicker would have activated the blue-, green-, and red-sensitive cones (or short-, medium-, and long-wave-sensitive cones) in proportion to their respective spectral response profiles [74]. Anatomically, the “green” and “red” cones are mainly located in the foveal zone, while the “blue” cones are found in highest density outside the fovea in a ~5° perifoveal ring, and in half the density in a second ring that extends a further 10° outward. By population, ~64% of cones are red, ~32% green, and ~2% blue. The spectral property of the flicker used in those studies allows a prediction that the foveal retina would likely have been stimulated most strongly, followed by the perifovea, and then the more peripheral retina to the lowest degree. This differential stimulation across the retina has particularly important implications for predicting the retinal zones where the most significant changes in blood flow could be expected to occur.

In a recent study, blue flicker was presented over a broad luminance range to selectively stimulate rods and short-wave cones [84]. The hypothesis in that study was that narrowband blue flashes were more likely to activate rods populating the greater macular periphery, and therefore the subfoveal ChBF would be redistributed toward the retinal sites with greater metabolic activity. Subfoveal choroidal hemodynamics were quantified by LDF while the retina was stimulated by dim blue flashes delivered at low-to-high frequencies and then step increments in luminance using the same frequency sequence. In the last stage of the experiment, the order of step changes in flicker luminance was reversed, keeping the same order of increasing frequency. Flash ERGs elicited by the same wavelength (= 473 ± 11 nm) were subsequently recorded in response to a series of bright-to-dim and dim-to-bright blue flashes (over a 4.0-log range) to help explain the different ChBF responses.

The resulting data shown in the top graph in Fig. 10.18 revealed that blue flicker with increasing luminance steps caused a ~32% reduction in

75Flicker luminance increasing

Fig. 10.18 The subfoveal ChBF as changed by shortwave narrow-bandpass flicker. Blue flicker with luminance increased from 0.0375 to 375 cd/m2 in equal steps caused a linear reduction in the subfoveal ChBF due to a decrease in blood volume. In contrast, blue flicker with changes in luminance delivered in reverse order stimulus, i.e., from 375 to 0.0375 cd/m2, had no effect on the subfoveal ChBF

the subfoveal ChBF that was caused by a reduction in volume but no change in velocity. Surprisingly, the same flicker frequencies with luminance steps presented in reverse order (high- to-low) had no measurable effect on the ChBF, volume, or velocity. The absence of blood flow

ERG waveform

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in 1 log steps

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375

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Fig. 10.19 Objective recordings of retinal responses to diffuse blue flashes of increasing luminance (Frame a) vs. decreasing luminance (Frame b). Blue traces indicate ERGs recorded before the frequency flicker series, while the red traces show ERGs taken after the flicker series. These ERG waveforms confirmed that the same stimuli

could elicit different retinal responses (waveform, amplitude, and timing) if they were preceded by flashes presented in reverse order of luminance. These findings supported the conclusion that blood flow measurements could also show opposite trends when the flash intensity series was presented in reverse order

changes during the reversal in luminance steps appeared erroneous but was subsequently validated by fERGs. These electrophysiological indices of neural function provided objective measurements of changes in photoreceptor function that also explained the profiles of change in blood flow. Stated succinctly, blue flashes of increasing luminance elicited progressively larger rod-dominated retinal responses from the blue-sensitive rhodopsin containing rods (Fig. 10.19a; blue recordings). Such flashes elicited ERGs with a progressive increase in the b-wave component for each 1.0-log increase in flash luminance. At the highest flash luminance, rod activity was suppressed and only blue-sensi- tive cones contributed to the fERG. As such, these flashes progressively increased metabolic activity in rods that populate the far macular zone and thereby drew blood away from the subfoveal choroid. In contrast, flashes of decreasing luminance first strongly activated foveally

located blue-sensitive cones with the brightest flash and subsequently had smaller effects on rods with decreasing flash luminance as is shown by the rapid attenuation of the fERG b-wave (Fig. 10.19b; blue recordings). As the effect on rods was reduced, the flashes progressively increased their effect on centrally located photoreceptors and thereby required the subfoveal ChBF to remain unchanged. The red recordings in Frames a and b show the ERGs elicited at each flash intensity at the end of each flicker frequency series. A comparison of the red ERG recordings for identical flash intensities in Frame a vs. b reveals that the ERGs differed widely for the 3.75 and 0.375 flash intensities. For example, in Frame a, a 0.375-cd/m2 flash elicited virtually equal amplitude ERGs before and after the flicker frequency series. In contrast, in Frame b, the same flash intensity presented after the flicker frequency series elicited an ERG that was nearly extinguished.