Oxygen Measurements in Animals

Core Messages

¥Retinal oxygen measurements in animals and humans, made with several techniques, provide information about the effectiveness of the circulation in meeting the metabolic demands of the retina.

¥Under normal conditions, PO2 in the outer retina of cats and monkeys varies

from a high of about 50 mmHg at the choriocapillaris to a low of 5 mmHg or less around the photoreceptor inner segments.

¥PO2 in the inner retina averages about 20 mmHg, but is heterogeneous.

¥Hyperoxia dramatically increases PO2 in the outer retina. Increases in the inner

retina are much smaller. Hypercapnia makes all these changes larger.

¥Light decreases oxygen utilization in the outer retina by as much as a factor of

two, and increases PO2. Steady light does not change the metabolism of the

inner retina, but ßickering light can increase it relative to darkness.

R.A. Linsenmeier, Ph.D.

Departments of Biomedical Engineering, Neurobiology and Ophthalmology, Northwestern University,

2145 Sheridan Road, Evanston, IL 60208-3107, USA e-mail: r-linsenmeier@northwestern.edu

¥Hypoxemia, retinal vascular occlusions, retinal detachment, and later stages of diabetic retinopathy all lead to decreased

retinal PO2. Changes glaucoma and early diabetic retinopathy are uncertain.

4.1 Introduction

Measurements of oxygen partial pressure (PO2) and blood oxygen saturation (SO2) are not equivalent to measurements of blood ßow, but, on a moment-to-moment basis, the most critical job of the circulation in most organs is the delivery of oxygen. One of the important reasons for studying blood ßow in the Þrst place is the hope that it will be a surrogate for tissue metabolism and the condition of the tissue, but the ability to understand normal physiology or pathophysiology on the basis of blood ßow measurements alone is limited. Coupling blood ßow measurements with blood oxygen saturation measurements provides more information about retinal metabolism, as discussed further below. Tissue oxygen measurements on their own, without simultaneous blood ßow measurements, show the result of what the circulation provides to the cells. Information about oxygen is therefore critical to an understanding of the performance of the retinal and choroidal circulations under physiological and pathological conditions.

For the purposes of oxygen supply and metabolism, the retina is best thought of as two separate

organs, the inner retina and the outer retina, which are about equal in thickness. The oxygen supply of the inner retina shares many properties with that of the brain, but has unusual aspects because of the proximity of the vitreous, the leakiness of the arterioles to oxygen, and the interaction between the retinal and choroidal circulations in providing oxygen under certain conditions. The oxygen supply to the outer retina (RPE and photoreceptors) is unlike that of any other organ, because its main supply, the choroid, is quite unusual and because the metabolically demanding photoreceptors are in an avascular layer.

Work on the role of altered oxygenation in retinal diseases began in the 1950s by Ashton [24, 26] and Patz [144, 145] for retinopathy of prematurity and arterial occlusion, and the Þrst important measurements of oxygen in the eye of animals were made in the early 1970s by Alm and Bill [14, 16]. Since these publications, there has been a steady stream of work so that good descriptions of many phenomena, both physiological and pathophysiological, can be given, and several reviews have appeared focusing on different aspects of oxygen measurement, oxygen supply, and oxidative metabolism [23, 92, 113, 188, 189, 205, 229]. This section will discuss the several ways in which oxygen measurements are made in animals, explain basic physiological properties of retinal and optic nerve head oxygenation, and relate these to pathophysiology.

4.2Measurements of PO2 and Saturation

The importance of oxygen in the eye has stimulated the development of several methods of measurement, each of which has both advantages and disadvantages [92, 205, 229]. The methods discussed here are (1) oxygen electrodes, (2) histology with Hypoxyprobe, (3) magnetic resonance imaging, (4) phosphorescent dyes, and (5) reßection oximetry or spectrophotometry. These are all used in vivo, sometimes in conjunction with measurements of blood ßow. For understanding the use of oxygen in the retina, in vitro measurements have also proven to be important

and usually involve the use of oxygen electrodes, although not always microelectrodes.

4.2.1Oxygen Electrodes

Oxygen-sensitive electrodes work on the same principle as those in blood gas machines, that is, polarography. When a noble metal such as gold or platinum is polarized at about −0.7 V with respect to a reference electrode, it becomes a cathode at which oxygen is chemically reduced [45, 209]. Nothing besides oxygen is reduced in tissue, and the electrode is selective for oxygen, unlike the situation when an electrode is polarized at a positive potential and can generate various oxidation products. The reduction of oxygen generates a current proportional to the oxygen tension (PO2) in the medium, which could be as little as a few picoamps or as much as a few nanoamps, depending on the size of the cathode. While these electrodes consume oxygen, for microelectrodes, it is a small amount that does not substantially change the oxygenation of the tissue. If the stoichiometry at the cathode is O2 +4e− +2H2O → 4OH− [45] and the microelectrode produces 6 pA of current, a typical value in the inner retina, then making simple unit conversions allows one to show that the electrode uses the same amount of oxygen as a region 5 mm in radius that has an oxygen consumption of 4 ml O2-100 g−1- min−1, also a typical value for inner retina. That is, the electrode simply acts like one more cell in the retina. Oxygen electrodes of different types have been used in the vitreous humor (e.g., [12, 16, 64, 158, 224]), the retina (e.g., [6, 37, 116, 156, 227]), and the optic nerve head (e.g., [3, 63, 104, 107, 136, 148, 188]). For intraretinal measurements, there is an advantage in using double-barreled microelectrodes, in which one barrel is an oxygen cathode and the other measures voltage (Fig. 4.1). The voltage barrel records the intraretinal electroretinogram, which indicates the physiological state of the retina and the quality of the electrode penetration. However, to the extent that it can be determined, single-barreled microelectrodes (i.e., without a voltage barrel) give similar intraretinal measurements.

A calibration (i.e., current vs. PO2) must be obtained for each electrode, and investigators must

0.9% Saline

Fig. 4.1 Double-barreled oxygen microelectrode. The top barrel has a gold cathode, recessed from the tip. This helps to conÞne the diffusion Þeld to the recess and allows measurements with high spatial precision. The LMA is a low-melting-point alloy that serves as a substrate onto which gold is plated. The lower barrel is Þlled with saline and is used to record intraretinal voltages, such as the electroretinogram

assume that the calibration done in vitro applies in vivo. The microenvironment may be slightly different in the eye than in the calibration system in terms of temperature and diffusion properties, however, so PO2s obtained in this way should be considered good approximations rather than exact values. One can have more conÞdence in differences between two situations (e.g., light and dark) in the same animal than in those that rely on comparing measurements across animals. However, sometimes, there is no alternative to drawing conclusions from interanimal comparisons. Absolute measurements can be made with Clark-type oxygen electrodes, which have both the anode and cathode in the same environment under a gas-per- meable membrane, but these are larger and can only be used in the vitreous humor.

Outweighing the disadvantages of microelectrodes for measurements in animals are their great advantages. These include (1) their spatial resolution in giving intraretinal PO2, which is unequaled by any other technique; (2) their temporal resolution, with the time required for 90% of the complete response to a PO2 change of 25Ð50 ms [169], much faster than any biological changes in PO2 in the retina [41, 55]; and (3) their ability to measure tissue PO2 rather than intravascular PO2. Oxygen electrodes have been used primarily in animals, although a few measurements have been made in the vitreous of humans [94, 126, 168].

4.2.2Hypoxyprobe

Pimonidazole (Hypoxyprobe) is a chemical that irreversibly forms adducts in tissue when the PO2

is below some critical level, usually given as 10 mmHg [161]. These pimonidazole-protein adducts can be detected histologically using a monoclonal antibody [52]. Pimonidazole has been used primarily to investigate hypoxia in animal and human tumors, but it has also been used in some normal tissues, including the retina [57, 58, 73, 93].

Pimonidazole histology could be an important compliment to measurements with microelectrodes because there is the possibility of sampling more tissue, of leaving the eye completely undisturbed during measurements, and of making measurements in eyes that are too small or in animals that are too delicate for physiological measurements (e.g., neonatal animals or potentially long-term diabetic rats or mice). The disadvantages are that this technique (1) can give only one set of measurements per animal, (2) only gives a binary readout of PO2 (i.e., either higher or lower than the cutoff PO2), and (3) does not have a wellvalidated cutoff PO2.

4.2.3Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) consumes no oxygen and can even be used in very small eyes of neonatal rodents [30]. MRI has been used in three ways. One method employs the property that the T1-weighted image of hydrogen in the water in the eye is modulated by local PO2 [29, 96]. The water T1 is sensitive to factors other than oxygen, so it does not provide an absolute measurement, but changes in this signal when an animal or person breathes oxygen or carbogen (95% O2 and 5% CO2) do reßect oxygen changes in the retina. The MRI responses have been called DPO2. Because the retina is thinner than a standard voxel, the voxel containing the retina may include choroid as well as inner and outer retina, so detail cannot be resolved. Consequently, the approach has primarily been to make measurements of the row of voxels in the vitreous in front of the retina [96], on the good assumption that the oxygen there has diffused from the retinal circulation (Fig. 4.2). With longer acquisition times, higher Þeld strengths, and good eye

Fig. 4.2 (a) Average DPO2 band derived from two mice during a 2-min carbogen challenge was overlaid on a representative FITC-dextran infused ßat mount. The median panretinal DPO2 was 102 mmHg. Pixels from the superior portion of the retina are at the top of the Þgure. The longer white tick mark in the center of each band represents the posterior pole near the optic nerve; the shorter tick marks represent 0.5-mm increments along the retinal surface [96]. Published with kind permission of © Elsevier 2001. (b) Differential layer-speciÞc BOLD fMRI of the retina. Lamina-speciÞc BOLD fMRI responses to (A) hyperoxia (100% O2) and (B) hypercapnia (5% CO2 in air) from a normal rat. BOLD percent change maps are overlaid on echoplanar images. The color bar indicates 1Ð20% BOLD changes [50]. Reprinted with the kind permission of the National Academy of Sciences, U.S.A. © 2006

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stabilization, it is possible to detect some structure in the retina itself and even do the second type of measurement, BOLD (Blood-oxygen- level-dependent) imaging [50, 61]. BOLD images are dependent on both blood ßow and hemoglobin saturation and again are useful for investigating changes in oxygenation rather than absolute

values. In contrast, the third magnetic resonance method can provide absolute values. This technique relies on placing a small droplet of perßuorocarbon, such as perßuorotributylamine (PFTBA), in the vitreous against the retina [96]. One then measures the inverse T1 of ßuorine (19FNMR), which depends only on oxygen and