temperature, and can therefore be interpreted directly in terms of PO2. The ßuorine technique yields a measurement of the average vitreal PO2 close to the retina, which is taken to be an estimate of the average inner retinal PO2 near the droplet. Measurements made in this way over the human macula give values of 6Ð9 mmHg [211], and measurements in the vitreous of normal rabbits give values of 22 ± 9 mmHg [71] and 39.4 ± 9.2 mmHg [210] over the vascularized retina near the optic disc, in reasonably good agreement with electrode measurements [168].

4.2.4Phosphorescence Decay

Oxygen quenches the phosphorescence of certain palladium-porphyrin compounds, so for these molecules, the phosphorescence lifetime following excitation is inversely proportional to PO2 [121, 196]. Alternatively, in response to time-vary- ing excitation of the dye, one can measure a phase shift in the phosphorescence signal. Unlike the techniques reviewed so far, phosphorescence has been used to give information about intravascular PO2 rather than tissue PO2 because the dyes bind to albumin. Compounds with excitation maxima near 500 nm and photon emission in the deep red or near infrared have been bound to albumin and injected intravenously in rodents to image the larger retinal arteries and veins as well as the optic nerve head [175, 176, 212, 213] (Fig. 4.3a). Unfortunately, because of their toxicity, these dyes are not available for use in humans. As expected, results with these dyes show that the PO2 is higher in retinal arteries than in veins, and it is sometimes possible to see gradients along vessels. This method might also be expected to give PO2s in retinal capillaries as well, but it is uncertain whether signals in areas between arteries and veins are purely from retinal capillaries or contain a contribution from the choroid vessels. A way around this is with retinal slice imaging combined with a phosphorescent dye [170Ð172]. In this technique, the excitation beam and viewing angle are obliquely oriented with respect to the retina rather than being along the optic axis. This clearly reveals retinal vessels and choroidal circulation separately (Fig. 4.3b). In one study, a

different phosphorescent dye, sodium pyrenebutyrate, was added to the Ringer solution bathing a retina in vitro so that the PO2 in extravascular tissue could be imaged [237]. Intravitreal injection of the dye may eventually be developed to allow mapping of tissue PO2 in vivo.

4.2.5Oximetry

In contrast to all the other techniques, oximetry gives values for hemoglobin saturation, or concentration of oxygen, rather than PO2. Saturation is related to PO2 in a nonlinear way that depends on the shape of the hemoglobin saturation curve, but, except in hyperoxia where arterial PO2 increases without an increase in saturation, PO2 and saturation can be interconverted if the parameters characterizing the hemoglobin saturation curve are known. Like phosphorescence, oximetry gives an intravascular PO2 rather than tissue PO2, but, unlike phosphorescence, it can be used in humans. The principles and results were recently reviewed [83], and this method is considered further elsewhere in this book. A number of investigators have tackled the job of determining oxygen saturation in retinal vessels, which is difÞcult for a variety of reasons [83], including possible inßuences of the choroid on the reßected light and differences in background absorption and fundus reßection among subjects. However, the basic information from the various kinds of oximetry is largely consistent with the earliest measurements by Hickam and Frayser [69, 88, 89] in the important result that retinal arterial blood is almost fully saturated and that retinal venous blood has a saturation of about 60%, which is lower than in most organs. While oximetry is sometimes used in animal studies [103], these are often done to validate techniques that are ultimately designed for humans.

4.3Vitreal, Intraretinal,

and Intravascular Oxygen in Holangiotic Retinas

While all vertebrates have a choroidal circulation, not all have a retinal circulation. The species that do have a retinal circulation are all mammals

Fig. 4.3 (a) Phosphorescence intensity images (A and E) and colorized two-dimensional maps of PO2 in one mouse retina (BÐD) and one rat retina (FÐH) at different inspiratory oxygen fractions. Images were taken through a 10× microscope objective (mouse) or 4× microscope objective (rat). FiO2 is indicated for each mouse map, while actual arterial blood gas oxygen tensions are indicated for each rat map. The arterial (A), venous (V), and capillary (C) regions are indicated [176]. Reprinted with the kind

permission of the Biomedical Engineering Society © 2003. (b) Optical section phosphorescence image shows the retinal and choroidal vasculatures displaced in depth. The retina and choroid are on the left and right side of the image, respectively, as indicated by arrowheads. Retinal artery, vein, capillaries, and choroid are indicated by the arrows. Phosphorescence is quenched more at higher PO2, so the vein is brighter than the choroid or artery [170]. Reprinted with the kind permission of Informa PLC © 2006

and are said to have holangiotic retinas. These include nonhuman primates, dogs, pigs, cats, rats, mice, cows, and some other species [220]. The following section focuses on results in these species.

4.3.1Vitreal Oxygen

Measurements of vitreal PO2 close to the retina or preretinal vitreous PO2 (PvrO2) date to the 1950s [76]. Vitreal measurements continue to be useful

Fig. 4.4 Preretinal vitreal PO2 proÞles during air breathing measured near an artery (open and closed circles), vein (open and closed squares), and intermediate position (open and closed diamonds). The open symbols were obtained during the withdrawal of the microelectrode from the retina and the closed symbols from a subsequent advance to the retina [7]. Reprinted with the kind permission of Informa PLC © 1985

Oxygen current (nA)

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Distance from retina (μm)

because they are less invasive and sometimes use Clark-type electrodes that have the advantage of an internal reference [8, 12, 16, 42, 158, 181Ð 183]. In cats, which appear to be representative, the average PvrO2 under baseline conditions (i.e., not hyperoxic, hypoxic, or ischemic) is about 20 mmHg (variously reported as having average values of 18.9 [16], 20Ð30 [42], 15Ð20 [114], 20.2 [6], and 19 [182] mmHg). In rat, the average in the midvitreous, which is only about 500 mm from the retina, is 22.6 mmHg [12]. While the oxygen in the vitreous must come largely from the retina [42, 114], the details of the oxygen gradients within a few hundred micrometers of the vitreal-retinal interface are complex [12, 46]. Near arterioles and venules in cat [7] and rat [12], there is a gradient of decreasing PO2 from the retina into the vitreous (Fig. 4.4). In contrast, in regions away from the ophthalmoscopically visible vessels, the PO2 is generally lower at the retinal surface than further out in the vitreous in cat [7, 44] and in some (Lau and Linsenmeier, unpublished observations), but not all [12, 225], measurements in rat. These differences in the gradients have at least two interesting implications. First, measurements of PvrO2 tend to overestimate the PO2 in much of the inner retina, so, when possible, intraretinal measurements are preferable. In a series of measurements

in cat that included both the inner retina and vitreous, the vitreal PO2 was higher than inner retinal PO2 in normal regions, but frequently lower than inner retinal PO2 after photocoagulation of the outer retina [44], so, while photocoagulation had a signiÞcant effect on intraretinal PO2, its effect on vitreal PO2 was not signiÞcant. Second, the vitreous must be supplying much of the retina with some oxygen, and this includes at least part of the fovea in primates [2, 233]. Of course, ultimately, this oxygen is derived from the retinal circulation, but from somewhat remote vessels. Ordinarily, the diffusion of oxygen from the vitreous probably provides little of the demand of the inner retina, but the exact amount has not been determined.

4.3.2Intraretinal Oxygen

Intraretinal microelectrode measurements have been made under many conditions in cat and rat and, to a lesser extent, in primates, pigs, rabbits, and guinea pigs. Examples of oxygen ÒproÞlesÓ recorded in dark adaptation for the central retina of primate, cat, and rat are shown in Fig. 4.5. These proÞles were measured during electrode withdrawal from the choroid to the vitreous. There is a relatively high PO2 at the choroid and