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Laboratory Sciences |

Measurement of Regional Choroidal Blood Flow in Rabbits and Monkeys Using Fluorescent Microspheres FREE

T. Michael Nork, MD; Charlene B. Y. Kim, PhD; Dhanansayan Shanmuganayagam, BS; Michael S. Van Lysel, PhD; James N. Ver Hoeve, PhD; John D. Folts, PhD
[+] Author Affiliations

Author Affiliation: Departments of Ophthalmology and Visual Sciences (Drs Nork, Kim, and Ver Hoeve) and Medicine (Drs Van Lysel and Folts and Mr Shanmuganayagam), University of Wisconsin School of Medicine and Public Health, Madison.


Arch Ophthalmol. 2006;124(6):860-868. doi:10.1001/archopht.124.6.860.
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Published online

Objective  To develop a quantitative measure of regional variation in choroidal blood flow (ChBF).

Methods  Five million 15-μm fluorescent microspheres were injected into the left ventricles of 4 rabbits and 3 monkeys. The fixed globes were bleached, flat mounted, and photomicrographed. After image analysis to locate each microsphere, regional densities and blood flow were determined.

Results  Regional variation in ChBF was clearly evident. In the rabbit, a high density of spheres was seen in the visual streak. This was surrounded by a middle peripheral area of low sphere density and a far peripheral region of moderately high density. In the monkeys, sphere density was markedly greater in the macula compared with the periphery. Contour plots produced lines of constant flow that were oval and extended farther nasally than temporally in the monkeys. The ratio of central to peripheral ChBF was much greater in the monkeys than in the rabbits.

Conclusion  Quantitative assessment of regional ChBF can be performed using a modification of the fluorescent microsphere impaction method.

Clinical Relevance  This method of determining regional ChBF will be useful for studying the vascular effects of pharmacologic agents and for characterizing animal models of human disease involving the outer retina.

Figures in this Article

Choroidal blood flow (ChBF) accounts for 85% of the circulation to the eye.13 Knowing how it is affected by disease and pharmacologic intervention is critically important. However, measurement of ChBF is problematic because its vasculature is partially hidden from view by the overlying retinal pigment epithelium (RPE). Numerous methods have been developed, none of which is completely satisfactory for all experimental purposes. A brief review of some of these methods follows.

Total ChBF can be determined invasively by cutting the vortex veins and measuring output.4 A noninvasive method is color Doppler ultrasonic flowmetry of the ophthalmic artery,59 which serves as an estimate of ChBF given that 85% of its flow serves the choroid. Laser interferometry1012 and laser Doppler flowmetry13 are also noninvasive techniques, but ChBF measurement is limited to the fovea. Radioactive krypton desaturation,14 hydrogen clearance,15 and calorimetric methods16 have also been used. Choroidal blood flow in localized areas has been estimated using fluorescein dye–filling curves17 and indocyanine green dye.18 A recently described novel means of studying ChBF in small portions of the choroid uses laser rupture of dye-laden liposomes.19,20

Except for direct measurement at the vortex veins, all of these methods are limited in that they are approximations of ChBF, show only relative blood flow, or look at selected small regions of the choroid. One additional, highly accurate means of determining blood flow is the use of arterially injected nonrecirculating labeled microspheres. The density of spheres in any given tissue is proportional to the blood flow through that tissue at the time of injection. An absolute blood flow determination can be made by simultaneously withdrawing a reference sample of arterial blood. Multiple injections using differentially labeled spheres is also possible, permitting the technique to be used to determine acute changes in blood flow in response to changes in various physiologic variables, such as blood pressure, oxygenation, and pharmacologic intervention.

The use of radiolabeled microspheres to measure blood flow in nonsurvival experiments was introduced by Rudolph and Heymann in 1967.21 Since then, it has become the accepted standard for determining arterial flow to capillary beds in various organs, including the choroids of the cynomolgus (Macaca fascicularis) monkey1,22,23 and several other species.22,24 More recently, fluorescent microspheres have been used.2528 In 1993, Austin et al29 and Glenny et al30 described quantitative comparisons of the 2 types of microspheres and found that they give similar results in terms of blood flow. The technique using fluorescent microspheres has since been shown to be at least as reliable as that using radiolabeled microspheres,3034 and without the health and environmental risks or costs of tissue disposal and storage.

In the present study, we investigated the use of fluorescent microspheres to determine regional differences in blood flow of the choriocapillaris. Buckberg et al35 analyzed the statistical variability of the distribution of microspheres and concluded that at least 384 spheres are needed in a given volume of tissue to have a precision of 10% at the 95% confidence level. Although the choroid is a high-flow system, the total volume of tissue is small compared with other organs, thus limiting the total number of microspheres that reach the choroid. Dividing the choroid into small segments might be problematic in terms of getting enough spheres for adequate statistical analysis. It is possible to compensate for this somewhat by injecting a larger number of spheres systemically. Almegard and Andersson36 administered 2 injections of 4 to 6 × 106 radiolabeled microspheres (15 μm in diameter) each into cynomolgus monkeys weighing 2.2 to 3.6 kg without a marked reduction in blood pressure. Orgul et al injected as many as 100 × 106 colored microspheres (10.2 μm in diameter) into rabbits37 and rhesus monkeys.38 An alternative approach to increasing the precision of the analyses is to use a larger number of animals.35 Finally, if the differences in flow between regions are greater than 10%, a lower density of spheres might suffice.

Although most studies of ChBF using the microsphere impaction method have looked at total blood flow, a few investigators have attempted to identify regional variation in flow. Alm and Bill24 examined 7 areas of the choroid in the cat and found no regional differences in flow. However, these same investigators found significant differences in the monkey (Macaca irus) comparing peripapillary, foveal, intermediary, and peripheral locations.1 In both studies, the choroid was divided into pieces that were analyzed separately. Autoradiographs of flat mounts of whole choroids have been performed in monkeys1 and rabbits.39 Although no attempt was made to quantitate the results, the researchers concluded that the microspheres had greater density in the macula of the monkey and in the visual streak of the rabbit compared with the periphery in both species. Stern and Ernest3 visualized colored microspheres in flat mounts of rhesus monkey choroids. They observed that the concentration of spheres at the region of the fovea was 5 to 6 times greater than the concentration at the temporal equator.

These methods of determining regional ChBF all have their limitations. Physically dividing the choroid into small pieces and counting the spheres in each piece, apart from being time-consuming, introduces errors because of the inaccuracy introduced in trying to cut the pieces into equal sizes. Furthermore, the sampling bin size is fixed, which limits the analyses that are possible. Autoradiography of the entire choroid, while giving a good subjective sense of regional differences in flow, is difficult to quantitate because an overlying film emulsion is used and what is recorded is not the exact location of the individual spheres but the paths of the particles emitted by the radionuclide as they pass through the emulsion at various angles. Depending on the exposure time, variability relating to decay rate is introduced as well. Directly visualizing individual colored or fluorescent spheres is problematic in pigmented animals because the RPE (whose pigment itself is variable across the eye)40 blocks the view of an undetermined portion of the spheres.

In this article, we describe a new method for preparing flat whole mounts of choroids from rabbits and monkeys that have been injected with fluorescent microspheres. Potassium permanganate is used to bleach the RPE, and the cartesian coordinates of each individual sphere are determined in a semi-automated manner. From these data, quantitative analyses are performed and density plots are generated that show contour lines of equal blood flow. Note that the term ChBF in the context of the fluorescent microsphere impaction method means the blood flow in the choriocapillaris because that is where the microspheres are impacted. No microspheres remain in the large choroidal vessels.

MICROSPHERE INJECTION

Institutional guidelines regarding animal experimentation were followed. Five million fluorescent polystyrene microspheres (mean ± SD diameter, 15.5 ± 0.42 μm) (Molecular Probes, Eugene, Ore) were injected in 25 seconds into the left ventricle via a catheter inserted into the right femoral artery of 4 New Zealand Black rabbits, a rhesus (Macaca mulatta) monkey, and 2 cynomolgus (M fascicularis) monkeys. Each animal's left ventricular and aortic pressure was monitored during the injection procedure using a physiologic recorder (WindoGraf 930; Gould Instrument Systems, Valley View, Ohio). The stock concentration of microspheres was 1 million per milliliter, with excitation and emission peaks of either 505 and 515 nm, respectively (yellow-green fluorescent), or 580 and 650 nm (red fluorescent). The left femoral artery was also catheterized and used for obtaining reference blood samples. For the reference samples, a syringe pump (Harvard Apparatus Inc, Holliston, Mass) was fitted with a 10-mL syringe containing 0.6 mL of heparin sodium (1000 USP U/mL) and was used to aspirate the arterial blood at a rate of 1 mL/min for 2 minutes. The reference sample was withdrawn simultaneously with the fluorescent microsphere injection. Immediately after the fluorescent microsphere injection, a 3-mL volume of heparinized saline (20 U/mL in 0.9% sodium chloride solution) was injected to flush the fluorescent microspheres through the catheter. After aspiration, the syringe and the connecting catheter were rinsed twice with 3 mL of 2% Tween 80. The blood plus the rinse solution were deposited in a 50-mL plastic collection tube that was weighed before and after to determine the weight of the blood plus rinse solution. The arterial blood in the catheter was drawn into the reference sample syringe, and the contents were deposited in a 50-mL conical polypropylene centrifuge tube. The syringe was then rinsed twice as described previously herein.

GLOBE PREPARATION

The animals were humanely killed and their eyes were enucleated, placed in 4% paraformaldehyde overnight at 4°C, and stored in 0.1M phosphate buffer (pH, 7.6) at 4°C. After the anterior segments of the eyes were removed, the retinas were carefully dissected away from the underlying RPE and choroid. A 1.5-mm trephine was then used to free the retina at the optic nerve. The anterior cut edge of the RPE/choroid was then tacked to the underlying sclera with cyanoacrylate glue. This last step was done to help prevent shifting of the RPE/choroid when it was being flattened (see later in this article). The posterior poles were incubated in 0.25% potassium permanganate at 37°C for 30 to 60 minutes. They were then placed in 5% oxalic acid for 5 minutes, flattened with 8 radial cuts, laid out on a layer of nonhardening modeling clay (sclera against the clay), covered with 2.5% hydroxypropylmethylcellulose, and pressed between 2 glass slides. Contact tape was wrapped around the 2 ends of the slides to hold them together.

ChBF DETERMINATION

The total number of microspheres in the samples was calculated by manually counting the spheres in 40-μL aliquot samples from the well-agitated (by means of sonication and vortexing) collection tube. The aliquots were placed on large microscope slides and coverslipped. An epifluorescence microscope (Olympus BH2; Olympus America Inc, Melville, NY) was used to visualize the spheres. A sufficient number of aliquots were used so that at least 400 microspheres were counted per sample. Assuming that the liquid in the collection tube was 1 mL/g, the total number of microspheres in the reference sample (Sr) was calculated as

Sr = Sa(Vt/Va),

where Sa is the total number of microspheres in the aliquots, Vt is the volume of liquid in the collection tube, and Va is the total volume of all of the aliquots used for counting.

The microspheres in the flattened and bleached globes were photographed using a digital camera attached to an epifluorescence microscope. Approximately 50 micrographs were needed to image the entire specimen using a ×2 objective lens. A single high-resolution image composed of the 50 micrographs was produced using Adobe Photoshop 7.0 (Adobe Systems Inc, San Jose, Calif). The cartesian coordinates of each microsphere were then determined using NIH ImageJ v1.31 (National Institutes of Health, Bethesda, Md). Approximately half of the total number of spheres could be detected automatically using ImageJ, and the remaining half had to be identified manually (because they were touching other spheres) using the ImageJ crosshair tool. The data were processed using a customized program in MATLAB v6.5.1 (The MathWorks, Natick, Mass), written by one of us (J.N.V.H.), which tabulates and plots the number of microspheres in bins of any size (0.25-mm2 bins worked well for this study). Blood flow was calculated using the following formula:

ChBF = R(Scc/Sr),

where R is the rate at which blood was withdrawn for the reference sample, in microliters per minute; Scc is the number of spheres per square millimeter of tissue; and Sr is the total number of spheres in the reference sample. These values were then imported into SigmaPlot 2001 (SPSS Inc, Chicago, Ill) along with the cartesian coordinates of the bins. A 3-dimensional smoothing function was used (locally weighted scatterplot smooth [loess (from the SigmaPlot 2001 program)], fourth-order polynomial) to produce a contour plot of blood flow in microliters per minute per square millimeter.

UNITS OF MEASURE

In the literature, ChBF is often expressed in milligrams per minute, which is a shorthand notation for milligrams per minute per tissue. In other words, the total blood flow through the choroid would be measured in milligrams per minute per choroid. In this article, we assume that the specific gravity of blood is approximately 1.0 and thus substitute microliters for milligrams. Also, we are interested in regional variations in blood flow in what is essentially a 2-dimensional capillary bed (the choriocapillaris). Dividing by area, the units then become microliters per minute per square millimeter. Flow measured in this way is sometimes referred to as flux density, or volume of a fluid per time through a given area.

The Table gives the total ChBF of each eye tested. Although variable among animals, these flows were similar to those reported by other researchers in rabbits39,4145 and monkeys1,22,42,46 using nonrecirculating microspheres (see the “Comment” section).

Table Graphic Jump LocationTable. Characteristics of the 7 Study Animals

Figure 1 illustrates the appearance of the posterior pole of a rabbit eye (rabbit 4) after it had been bleached and flattened between 2 glass slides. The retina had been removed before bleaching. Some choroidal pigmentation remained after bleaching, especially in the peripapillary and far peripheral regions. However, the RPE was fully bleached everywhere so that it did not interfere with visualization of the fluorescent microspheres (Figure 2).

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Figure 1.

Posterior pole of the left eye of a rabbit (rabbit 4) after it had been bleached, placed on modeling clay, and flattened between 2 glass slides. The retina had been removed before bleaching. The reddish hue corresponds to residual blood in the choroid. Some choroidal pigment remains in the periphery. Just below (ventral) to the optic nerve (white oval) is the visual streak, in which the pigment around the short posterior ciliary arteries can be seen (small dots). The background shading was digitally created to improve contrast with tissue.

Graphic Jump Location
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Figure 2.

Epifluorescence image from the visual streak of a rabbit that had undergone processing similar to that shown in Figure 1. Because the microspheres are all impacted in the choriocapillaris and the retinal pigment epithelium is fully bleached, none of the spheres are hidden by either blood or melanin despite the fact that both remain in the deeper choroid. This figure is reduced in size. The original digital image was enlarged to permit manual localization of the individual microspheres in the clusters.

Graphic Jump Location

Once photographed using an epifluorescence microscope, the cartesian coordinates of each individual sphere were determined in a semi-automated manner (see the “Methods” section) and plotted (Figure 3). By simple inspection of these plots, it was evident that there was a much greater density of microspheres in the portion of the eye corresponding to the visual streak in all of the rabbit eyes. Further quantitative analysis involved sorting the data into either 0.25- or 1-mm2 bins (Figure 4). Blood flow corresponding to each bin was calculated according to the equation ChBF = R(Scc/Sr) (see the “Methods” section). These data were then smoothed and replotted as described previously. No attempt was made to compensate for the missing areas produced by the radial cuts, which gave the smoothed graphs a lumpy appearance in the periphery (Figure 5 and Figure 6). From the smoothed plots, it was apparent that in addition to the higher blood flow in the visual streak, there were 2 areas of greatly lower blood flow corresponding to the choroid just dorsal to and just ventral to the visual streak. Blood flow was also found to be greater toward the far periphery everywhere except directly dorsal to the optic nerve.

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Figure 3.

Plot of all 22 305 microspheres in the left eye of a rabbit (rabbit 4). A composite image consisting of approximately 50 photomicrographs was assembled, and individual sphere locations were determined. The highest density of spheres is seen along a horizontal line corresponding to the visual streak. The position of the optic nerve is denoted by a black oval.

Graphic Jump Location
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Figure 4.

Quantitative density analysis for the rabbit eye shown in Figure 3. In this example, a bin size of 0.5 mm (0.25 mm2) was used. The position of the optic nerve is denoted by a white oval.

Graphic Jump Location
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Figure 5.

Plot of data shown in Figure 4 after the data had been smoothed using a locally weighted scatterplot smooth (loess) algorithm (see the “Methods” section). A horizontal ridge of greater density is present at the level of the visual streak. Elevations are also evident in the periphery. Origin is arbitrary. ChBF indicates choroidal blood flow.

Graphic Jump Location
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Figure 6.

Color contour plot of the same data used for Figure 5. The visual streak and peripheral elevations are evident. The position of the optic nerve is denoted by a white oval.

Graphic Jump Location

Contour plots using lines of equal ChBF showed that the high- and low-flow areas differed by a factor of approximately 3. The features of these plots were relatively insensitive to the size of the bins chosen (Figure 7). The monkeys (rhesus and cynomolgus) showed a much different pattern of blood flow than was the case for the rabbits. In these animals, there was a marked concentration of microspheres in the central macula and a much reduced density in the periphery (cynomolgus 2) (Figure 8). This was especially evident in the smoothed plots (Figure 9). Examination of the contour plots revealed that the lines representing equal ChBF were not simply concentric circles but rather fell off more slowly in the nasal retina (corresponding to the temporal visual field) than they did in the temporal retina (nasal visual field) (Figure 10). In contrast to the rabbit, the high- and low-flow regions differed by a factor of as much as 13.

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Figure 7.

Contour plots for rabbit 4 showing lines of equal choroidal blood flow using data sorted into either 0.5-mm bins (A) or 1.0-mm bins (B). There is little difference between the plots, illustrating that the shape of the smoothed plots is not critically dependent on bin size. The position of the optic nerve is denoted by a black oval. The numbers indicate blood flow in microliters per minute per square millimeter.

Graphic Jump Location
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Figure 8.

Plot of all 10 474 microspheres in the left eye of a cynomolgus monkey (cynomolgus 2). A composite image consisting of approximately 50 photomicrographs was assembled, and individual sphere locations were determined. The highest density of spheres is seen in the macula. The position of the optic nerve is denoted by a black oval.

Graphic Jump Location
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Figure 9.

Plot of data shown in Figure 8 after the data had been smoothed using a locally weighted scatterplot smooth (loess) algorithm (see the “Methods” section). A prominent central peak in flow is present in the macula. Note the depression corresponding to the optic nerve. Elevations are also evident in the periphery. Origin is arbitrary. ChBF indicates choroidal blood flow.

Graphic Jump Location
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Figure 10.

Contour plot of data shown in Figure 8 after the data had been smoothed as in Figure 9. Note that lines of equal choroidal blood flow fall off more gradually in the nasal retina (corresponding to the temporal visual field). The positions of the optic nerve (black oval) and fovea (black plus sign) are indicated. The numbers indicate blood flow in microliters per minute per square millimeter.

Graphic Jump Location

Regional variations in ChBF can be identified and analyzed quantitatively using this new method of detecting the distribution of nonrecirculating fluorescent microspheres. As noted early in this article, it is theoretically possible to accomplish such an analysis by dividing the choroid into numerous small pieces and determining the number of microspheres in each piece (using a scintillation counter for radiolabeled microspheres or a spectrofluorophotometer/particle sorter for fluorescent microspheres). However, this has yet to be done for more than 7 pieces, presumably because it would be difficult to cut the tissue into equal-sized pieces on the order of 0.25 or 1 mm2. Also, such a method would be unwieldy. For example, the MATLAB analysis performed for rabbit 4 mathematically divided the choroid into 1950 bins (Figure 4). Having to physically process each piece separately, even with some sort of automation, would be untenable. Furthermore, reanalysis, such as that shown in Figure 7 using different bin sizes, is not possible once the tissue is physically sectioned.

Dividing the choroid into so many bins results in fewer than the traditional 400 microspheres per sample.35 Nevertheless, use of the loess curve-smoothing function compensates for this because each smoothed value is determined by neighboring bins within a defined span. In other words, several bins are combined in a weighted manner to produce each point on the curve. Of course, 400 microspheres per sample is not an absolute limit. It is only the level at which the statistical variability from random distribution is approximately 10%. Lower numbers are acceptable if the differences to be detected are greater than 10%,35 which is the case with ChBF because the regional variations are approximately several hundred percent. Finally, even if 400 were used as a strict limit, it would still be possible to divide a choroid such as that shown in Figure 2 into 55 segments (albeit of different sizes), each with 400 spheres.

The 15-μm microspheres used in this study represent a fairly standard size for investigating ChBF. There is probably a range of sizes that could be used that would give a similar result in terms of the calculated blood flow. The choice has to do with finding the optimal combination of size and total number of spheres injected for any given capillary bed. Large spheres are almost all entrapped but block the larger precapillary arterioles, which causes systemic effects if injected in large numbers. Because they block larger vessels, the initial microspheres to reach a capillary bed block subsequent spheres, thus giving an artifactitiously low value for blood flow. Small spheres have the advantage that they can be injected in larger numbers (increasing precision), presumably because they do not affect certain capillary beds needed for maintaining cardiovascular stability. If they are too small, the spheres will escape entrapment from the capillary bed of interest, thus giving a value that is below the actual blood flow. Buckberg et al35 found little difference in calculated blood flow when they compared microspheres in the 8- to 50-μm range. Specifically addressing the issue of sphere size and calculated blood flow in the eye, Alm et al22 performed simultaneous injections of 2 different-sized microspheres (each size with distinctive radiolabels). They found that for the choroid in rabbits, the mean ± SD ratio of 15-μm microspheres to 9-μm microspheres was 1.06 ± 0.02. Similarly, the mean ± SD ratio of 35-μm microspheres to 15-μm microspheres in cynomolgus monkey choroid was 1.02 ± 0.07. In other words, the diameter of the microspheres in the studied range made little difference with respect to calculated ChBF, but size was important for other capillary beds in the eye. The 15:9-μm ratios were more than 2 for the iris and ciliary body in rabbits. The 35:15-μm ratio was less than 1 for the retina and iris in the monkey. They concluded that for the choroid at least, 15 μm provided a reasonable balance between precision and accuracy. The choroid is a high-flow system. In the present study, we injected enough of the 15-μm microspheres to achieve a good statistical sampling but not so many as to affect the cardiovascular stability of the animals.

Total blood flow through the choroid varied considerably among animals (Table). However, such variability has been well described. Alm and Bill,24 using the microsphere method for whole choroids, noted that blood flows differed by as much as a factor of 3.5 among cats with similar perfusion pressures. (As in the present study, they carefully monitored the animals' physiologic variables.) Similarly, Stjernschantz et al41 found ChBF that differed by a factor of 4 in rabbits (excluding an outlier animal that would raise the factor to nearly 7). Despite such marked differences in flow among animals, measurement of ChBF has proved useful in part because the fluctuation in flow between eyes and between multiple injections in the same eye is considerably less dramatic (eg, see cynomolgus 1, right and left eyes, in Table).

Although increased ChBF in the visual streak of rabbits and in the macula of monkeys has been observed (at least subjectively) by other researchers, this new method has revealed at least 2 previously undescribed features of ChBF. In the rabbit, there is a region of low flow just ventral to the visual streak centrally and also dorsal to the visual streak above the optic nerve. However, in the other areas of the periphery, including along the axis of the visual steak itself, there is a relative increase in flow. The highest flows are at the nasal and temporal periphery, not at the center of the visual streak. Just why this is so is not obvious. One might speculate that because the rabbit is a prey animal, peripheral vision is more important than central vision. Of course, high ChBF may or may not correlate with good vision. The concentrations of short-wavelength–sensitive cones (S-cones or blue cones) are greatest in the inferior periphery of the rabbit eye.47,48

The other previously undescribed feature of regional ChBF is in the monkey. The contour lines of equal ChBF reach a peak in the central macula, as has previously been shown either subjectively by microsphere impaction or by limited sampling. However, these lines are not radially symmetrical. Instead, they have an increasingly oval shape toward the periphery, extending more nasally than temporally, which would correspond to the temporal and nasal visual fields, respectively. Unlike in rabbits, the peripheral visual acuity in monkeys is known from behavioral testing and is remarkably similar to that seen in humans,49 at least in the central 30°. In humans, the nasal peripheral lines of equal light sensitivity (isopters) of standard Goldmann perimetry extend farther for the nasal field than for the temporal field. Morphologically, similar radial asymmetry of the cone photoreceptors has been found in monkey50,51 and human51,52 retinas. Another interesting feature of the ChBF in monkeys is the much greater range (by a factor of as much as 13) between the central macula and the periphery compared with the rabbit (by a factor of 3) even though total circulation may be similar (see previously in this article).

A priori, it is not necessarily the case that regional variations in ChBF should reflect visual sensitivity as measured in photopic conditions. Mammalian retinas are duplex, containing rods and cones that each have different physiologic characteristics and functions. Furthermore, these characteristics change with light adaptation, the rods being more metabolically active in dark adaptation.53,54 Therefore, is ChBF a reflection more of the cone retina or of the rod retina? One might guess that the ChBF variation should follow the variations in oxygen consumption of the outer retina. Although extensive regional studies of comparative outer retinal oxygen consumption have not been performed, Yu and Cringle55 did a 2-point comparison using oxygen-sensitive electrodes in rabbits. They found that the oxygen demands of the outer retina were greater in the visual streak than in the vascularized portion of the retina just dorsal to the visual streak (where we found a nadir in ChBF). However, this simple relationship may not hold in the monkey. Ahmed et al56 did a similar 2-point analysis in a pig-tailed macaque (Macaca nemestrina) in which they looked at outer retinal oxygen consumption in the fovea compared with the parafoveal region (about halfway between the fovea and the optic disc). They found that oxygen consumption was actually lower in the fovea. The reason for this is not clear, but if the same holds true for cynomolgus and rhesus monkeys, it would suggest that ChBF does not rigidly follow oxygen demand. Such a parafoveal zone of borderline ChBF might account for some of the bull’s-eye maculopathies seen in humans. Without further studies, such comments are speculative. Other factors, such as temperature regulation and (as yet unmeasured) metabolic needs supplied by the choroid, could be important in addition to oxygen supply.

In summary, using the modified fluorescent microsphere impaction method described in this article, we quantitatively examined the entire choroid in rabbits and monkeys and showed a consistent pattern of regional variations in ChBF among the animals studied. Using this method, it may be possible to study regional variations in ChBF in response to the light-dark cycle. The effects of drugs and animal models of retinal disease on regional ChBF may, for the first time, be measurable as well.

Correspondence: T. Michael Nork, MD, Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, 600 Highland Ave, F4/336, Madison, WI 53792-3220 (tmnork@wisc.edu).

Submitted for Publication: May 15, 2005; final revision received September 7, 2005; accepted October 23, 2005.

Financial Disclosure: None.

Funding/Support: This study was supported by grants R01 EY014041 (Dr Nork) and P30 EY016665 from the National Eye Institute, National Institutes of Health; the Walter H. Helmerich Chair (Dr Nork); Research to Prevent Blindness; the Retina Research Foundation; and the Wisconsin Lions.

Acknowledgment: We thank Carrie Bunger and Derrick Woodward for providing technical assistance.

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Armaly  MFAraki  M Effect of ocular pressure on choroidal circulation in the cat and rhesus monkey. Invest Ophthalmol 1975;14584- 591
PubMed
van Stokkum  IHLambrou  GNvan den Berg  TJ Hemodynamic parameter estimation from ocular fluorescein angiograms. Graefes Arch Clin Exp Ophthalmol 1995;233123- 130
PubMed
Flower  RW Extraction of choriocapillaris hemodynamic data from ICG fluorescence angiograms. Invest Ophthalmol Vis Sci 1993;342720- 2729
PubMed
Kiryu  JShahidi  MMori  MTOgura  YAsrani  SZeimer  R Noninvasive visualization of the choriocapillaris and its dynamic filling. Invest Ophthalmol Vis Sci 1994;353724- 3731
PubMed
Asrani  SZou  SD'Anna  SGoldberg  MFZeimer  R Noninvasive visualization of blood flow in the choriocapillaris of the rat. Invest Ophthalmol Vis Sci 1996;37312- 317
PubMed
Rudolph  AMHeymann  MA The circulation of the fetus in utero: methods for studying distribution of blood flow, cardiac output and organ blood flow. Circ Res 1967;21163- 184
PubMed
Alm  ATornquist  PStjernschantz  J Radioactively labelled microspheres in regional ocular blood flow determinations. Bibl Anat 1977;1624- 29
PubMed
Alm  ALambrou  GNMaepea  ONilsson  SFPercicot  C Ocular blood flow in experimental glaucoma: a study in cynomolgus monkeys. Ophthalmologica 1997;211178- 182
PubMed
Alm  ABill  A The oxygen supply to the retina, II: effects of high intraocular pressure and of increased arterial carbon dioxide tension on uveal and retinal blood flow in cats: a study with radioactively labelled microspheres including flow determinations in brain and some other tissues. Acta Physiol Scand 1972;84306- 319
PubMed
Gottlieb  GJKubo  SHAlonso  DR Ultrastructural characterization of the border zone surrounding early experimental myocardial infarcts in dogs. Am J Pathol 1981;103292- 303
PubMed
Hale  SLVivaldi  MTKloner  RA Fluorescent microspheres: a new tool for visualization of ischemic myocardium in rats. Am J Physiol 1986;251H863- H868
PubMed
Kinoshita  KHearse  DJBraimbridge  MVManning  AS “Early” ischemia and reperfusion-induced arrhythmias: antiarrhythmic effects of diltiazem in the conscious rat. Can J Cardiol 1988;437- 43
PubMed
Jasper  MSMcDermott  PGann  DSEngeland  WC Measurement of blood flow to the adrenal capsule, cortex and medulla in dogs after hemorrhage by fluorescent microspheres. J Auton Nerv Syst 1990;30159- 167
PubMed
Austin  GETuvlin  MBMartino-Salzman  D  et al.  Determination of regional myocardial blood flow using fluorescent microspheres. Am J Cardiovasc Pathol 1993;4352- 357
PubMed
Glenny  RWBernard  SBrinkley  M Validation of fluorescent-labeled microspheres for measurement of regional organ perfusion. J Appl Physiol 1993;742585- 2597
PubMed
Abel  FLCooper  RHBeck  RR Use of fluorescent latex microspheres to measure coronary blood flow distribution. Circ Shock 1993;41156- 161
PubMed
van Oosterhout  MFPrinzen  FWArts  T  et al.  Asynchronous electrical activation induces asymmetrical hypertrophy of the left ventricular wall. Circulation 1998;98588- 595
PubMed
Deveci  DEgginton  S Development of the fluorescent microsphere technique for quantifying regional blood flow in small mammals. Exp Physiol 1999;84615- 630
PubMed
Gervais  MDemolis  PDomergue  VLesage  MRicher  CGiudicelli  JF Systemic and regional hemodynamics assessment in rats with fluorescent microspheres. J Cardiovasc Pharmacol 1999;33425- 432
PubMed
Buckberg  GDLuck  JCPayne  DBHoffman  JIArchie  JPFixler  DE Some sources of error in measuring regional blood flow with radioactive microspheres. J Appl Physiol 1971;31598- 604
PubMed
Almegard  BAndersson  SE Vascular effects of calcitonin gene-related peptide (CGRP) and cholecystokinin (CCK) in the monkey eye. J Ocul Pharmacol 1993;977- 84
PubMed
Orgul  SCioffi  GAWilson  DJBacon  DRVan Buskirk  EM An endothelin-1 induced model of optic nerve ischemia in the rabbit. Invest Ophthalmol Vis Sci 1996;371860- 1869
PubMed
Orgul  SCioffi  GABacon  DRVan Buskirk  EM An endothelin-1-induced model of chronic optic nerve ischemia in rhesus monkeys. J Glaucoma 1996;5135- 138
PubMed
Bill  A Effects of acetazolamide and carotid occlusion on the ocular blood flow in unanesthetized rabbits. Invest Ophthalmol 1974;13954- 958
PubMed
Boyer  MMPoulsen  GLNork  TM Relative contributions of the neurosensory retina and retinal pigment epithelium to macular hypofluorescence. Arch Ophthalmol 2000;11827- 31
PubMed
Stjernschantz  JAlm  ABill  A Effects of intracranial oculomotor nerve stimulation on ocular blood flow in rabbits: modification by indomethacin. Exp Eye Res 1976;23461- 469
PubMed
Stjernschantz  JBill  A Effect of intracranial stimulation of the oculomotor nerve on ocular blood flow in the monkey, cat, and rabbit. Invest Ophthalmol Vis Sci 1979;1899- 103
PubMed
Bill  A Effects of indomethacin on regional blood flow in conscious rabbits: a microsphere study. Acta Physiol Scand 1979;105437- 442
PubMed
Granstam  ENilsson  SF Non-adrenergic sympathetic vasoconstriction in the eye and some other facial tissues in the rabbit. Eur J Pharmacol 1990;175175- 186
PubMed
Duijm  HFRulo  AHAstin  MMaepea  Ovan den Berg  TJGreve  EL Study of choroidal blood flow by comparison of SLO fluorescein angiography and microspheres. Exp Eye Res 1996;63693- 704
PubMed
Alm  A The effect of topical l-epinephrine on regional ocular blood flow in monkeys. Invest Ophthalmol Vis Sci 1980;19487- 491
PubMed
Juliusson  BBergstrom  ARohlich  PEhinger  Bvan Veen  TSzel  A Complementary cone fields of the rabbit retina. Invest Ophthalmol Vis Sci 1994;35811- 818
PubMed
Famiglietti  EVSharpe  SJ Regional topography of rod and immunocytochemically characterized “blue” and “green” cone photoreceptors in rabbit retina. Vis Neurosci 1995;121151- 1175
PubMed
Harwerth  RSSmith  EL  IIIDeSantis  L Behavioral perimetry in monkeys. Invest Ophthalmol Vis Sci 1993;3431- 40
PubMed
Perry  VHCowey  A The ganglion cell and cone distributions in the monkey's retina: implications for central magnification factors. Vision Res 1985;251795- 1810
PubMed
Curcio  CASloan  KR  JrPacker  OHendrickson  AEKalina  RE Distribution of cones in human and monkey retina: individual variability and radial asymmetry. Science 1987;236579- 582
PubMed
Ahnelt  PK The photoreceptor mosaic. Eye 1998;12531- 540
PubMed
Stefansson  EWolbarsht  MLLanders  MB  III In vivo O2 consumption in rhesus monkeys in light and dark. Exp Eye Res 1983;37251- 256
PubMed
Linsenmeier  RA Effects of light and darkness on oxygen distribution and consumption in the cat retina. J Gen Physiol 1986;88521- 542
PubMed
Yu  DYCringle  SJ Low oxygen consumption in the inner retina of the visual streak of the rabbit. Am J Physiol Heart Circ Physiol 2004;286H419- H423
PubMed
Ahmed  JBraun  RDDunn  R  JrLinsenmeier  RA Oxygen distribution in the macaque retina. Invest Ophthalmol Vis Sci 1993;34516- 521
PubMed

Figures

Place holder to copy figure label and caption
Figure 1.

Posterior pole of the left eye of a rabbit (rabbit 4) after it had been bleached, placed on modeling clay, and flattened between 2 glass slides. The retina had been removed before bleaching. The reddish hue corresponds to residual blood in the choroid. Some choroidal pigment remains in the periphery. Just below (ventral) to the optic nerve (white oval) is the visual streak, in which the pigment around the short posterior ciliary arteries can be seen (small dots). The background shading was digitally created to improve contrast with tissue.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Epifluorescence image from the visual streak of a rabbit that had undergone processing similar to that shown in Figure 1. Because the microspheres are all impacted in the choriocapillaris and the retinal pigment epithelium is fully bleached, none of the spheres are hidden by either blood or melanin despite the fact that both remain in the deeper choroid. This figure is reduced in size. The original digital image was enlarged to permit manual localization of the individual microspheres in the clusters.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Plot of all 22 305 microspheres in the left eye of a rabbit (rabbit 4). A composite image consisting of approximately 50 photomicrographs was assembled, and individual sphere locations were determined. The highest density of spheres is seen along a horizontal line corresponding to the visual streak. The position of the optic nerve is denoted by a black oval.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Quantitative density analysis for the rabbit eye shown in Figure 3. In this example, a bin size of 0.5 mm (0.25 mm2) was used. The position of the optic nerve is denoted by a white oval.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.

Plot of data shown in Figure 4 after the data had been smoothed using a locally weighted scatterplot smooth (loess) algorithm (see the “Methods” section). A horizontal ridge of greater density is present at the level of the visual streak. Elevations are also evident in the periphery. Origin is arbitrary. ChBF indicates choroidal blood flow.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 6.

Color contour plot of the same data used for Figure 5. The visual streak and peripheral elevations are evident. The position of the optic nerve is denoted by a white oval.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 7.

Contour plots for rabbit 4 showing lines of equal choroidal blood flow using data sorted into either 0.5-mm bins (A) or 1.0-mm bins (B). There is little difference between the plots, illustrating that the shape of the smoothed plots is not critically dependent on bin size. The position of the optic nerve is denoted by a black oval. The numbers indicate blood flow in microliters per minute per square millimeter.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 8.

Plot of all 10 474 microspheres in the left eye of a cynomolgus monkey (cynomolgus 2). A composite image consisting of approximately 50 photomicrographs was assembled, and individual sphere locations were determined. The highest density of spheres is seen in the macula. The position of the optic nerve is denoted by a black oval.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 9.

Plot of data shown in Figure 8 after the data had been smoothed using a locally weighted scatterplot smooth (loess) algorithm (see the “Methods” section). A prominent central peak in flow is present in the macula. Note the depression corresponding to the optic nerve. Elevations are also evident in the periphery. Origin is arbitrary. ChBF indicates choroidal blood flow.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 10.

Contour plot of data shown in Figure 8 after the data had been smoothed as in Figure 9. Note that lines of equal choroidal blood flow fall off more gradually in the nasal retina (corresponding to the temporal visual field). The positions of the optic nerve (black oval) and fovea (black plus sign) are indicated. The numbers indicate blood flow in microliters per minute per square millimeter.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable. Characteristics of the 7 Study Animals

References

Alm  ABill  A Ocular and optic nerve blood flow at normal and increased intraocular pressures in monkeys (Macaca irus): a study with radioactively labelled microspheres including flow determinations in brain and some other tissues. Exp Eye Res 1973;1515- 29
PubMed
Bill  A Blood circulation and fluid dynamics in the eye. Physiol Rev 1975;55383- 417
PubMed
Stern  WHErnest  JT Microsphere occlusion of the choriocapillaris in rhesus monkeys. Am J Ophthalmol 1974;78438- 448
PubMed
Alm  ABill  A Blood flow and oxygen extraction in the cat uvea at normal and high intraocular pressures. Acta Physiol Scand 1970;8019- 28
PubMed
Rojanapongpun  PDrance  SMMorrison  BJ Ophthalmic artery flow velocity in glaucomatous and normal subjects. Br J Ophthalmol 1993;7725- 29
PubMed
Galassi  FNuzzaci  GSodi  ACasi  PVielmo  A Color Doppler imaging in evaluation of optic nerve blood supply in normal and glaucomatous subjects. Int Ophthalmol 1992;16273- 276
PubMed
Harris  ASergott  RCSpaeth  GLKatz  JLShoemaker  JAMartin  BJ Color Doppler analysis of ocular vessel blood velocity in normal-tension glaucoma. Am J Ophthalmol 1994;118642- 649
PubMed
Butt  ZO'Brien  CMcKillop  GAspinall  PAllan  P Color Doppler imaging in untreated high- and normal-pressure open-angle glaucoma. Invest Ophthalmol Vis Sci 1997;38690- 696
PubMed
Kaiser  HJSchoetzau  AStumpfig  DFlammer  J Blood-flow velocities of the extraocular vessels in patients with high-tension and normal-tension primary open-angle glaucoma. Am J Ophthalmol 1997;123320- 327
PubMed
Schmetterer  LFLexer  FUnfried  CJSattmann  HFercher  AF Topical measurement of fundus pulsations. Opt Eng 1995;34711- 716
Schmetterer  LDallinger  SFindl  OEichler  HGWolzt  M A comparison between laser interferometric measurement of fundus pulsation and pneumotonometric measurement of pulsatile ocular blood flow, 1: baseline considerations. Eye 2000;1439- 45
PubMed
Fuchsjager-Mayrl  GWally  BGeorgopoulos  M  et al.  Ocular blood flow and systemic blood pressure in patients with primary open-angle glaucoma and ocular hypertension. Invest Ophthalmol Vis Sci 2004;45834- 839
PubMed
Riva  CECranstoun  SDGrunwald  JEPetrig  BL Choroidal blood flow in the foveal region of the human ocular fundus. Invest Ophthalmol Vis Sci 1994;354273- 4281
PubMed
Friedman  EKopald  HHSmith  TR Retinal and choroidal blood flow determined with krypton-85 anesthetized animals. Invest Ophthalmol 1964;3539- 547
PubMed
Yu  DYAlder  VACringle  SJBrown  MJ Choroidal blood flow measured in the dog eye in vivo and in vitro by local hydrogen clearance polarography: validation of a technique and response to raised intraocular pressure. Exp Eye Res 1988;46289- 303
PubMed
Armaly  MFAraki  M Effect of ocular pressure on choroidal circulation in the cat and rhesus monkey. Invest Ophthalmol 1975;14584- 591
PubMed
van Stokkum  IHLambrou  GNvan den Berg  TJ Hemodynamic parameter estimation from ocular fluorescein angiograms. Graefes Arch Clin Exp Ophthalmol 1995;233123- 130
PubMed
Flower  RW Extraction of choriocapillaris hemodynamic data from ICG fluorescence angiograms. Invest Ophthalmol Vis Sci 1993;342720- 2729
PubMed
Kiryu  JShahidi  MMori  MTOgura  YAsrani  SZeimer  R Noninvasive visualization of the choriocapillaris and its dynamic filling. Invest Ophthalmol Vis Sci 1994;353724- 3731
PubMed
Asrani  SZou  SD'Anna  SGoldberg  MFZeimer  R Noninvasive visualization of blood flow in the choriocapillaris of the rat. Invest Ophthalmol Vis Sci 1996;37312- 317
PubMed
Rudolph  AMHeymann  MA The circulation of the fetus in utero: methods for studying distribution of blood flow, cardiac output and organ blood flow. Circ Res 1967;21163- 184
PubMed
Alm  ATornquist  PStjernschantz  J Radioactively labelled microspheres in regional ocular blood flow determinations. Bibl Anat 1977;1624- 29
PubMed
Alm  ALambrou  GNMaepea  ONilsson  SFPercicot  C Ocular blood flow in experimental glaucoma: a study in cynomolgus monkeys. Ophthalmologica 1997;211178- 182
PubMed
Alm  ABill  A The oxygen supply to the retina, II: effects of high intraocular pressure and of increased arterial carbon dioxide tension on uveal and retinal blood flow in cats: a study with radioactively labelled microspheres including flow determinations in brain and some other tissues. Acta Physiol Scand 1972;84306- 319
PubMed
Gottlieb  GJKubo  SHAlonso  DR Ultrastructural characterization of the border zone surrounding early experimental myocardial infarcts in dogs. Am J Pathol 1981;103292- 303
PubMed
Hale  SLVivaldi  MTKloner  RA Fluorescent microspheres: a new tool for visualization of ischemic myocardium in rats. Am J Physiol 1986;251H863- H868
PubMed
Kinoshita  KHearse  DJBraimbridge  MVManning  AS “Early” ischemia and reperfusion-induced arrhythmias: antiarrhythmic effects of diltiazem in the conscious rat. Can J Cardiol 1988;437- 43
PubMed
Jasper  MSMcDermott  PGann  DSEngeland  WC Measurement of blood flow to the adrenal capsule, cortex and medulla in dogs after hemorrhage by fluorescent microspheres. J Auton Nerv Syst 1990;30159- 167
PubMed
Austin  GETuvlin  MBMartino-Salzman  D  et al.  Determination of regional myocardial blood flow using fluorescent microspheres. Am J Cardiovasc Pathol 1993;4352- 357
PubMed
Glenny  RWBernard  SBrinkley  M Validation of fluorescent-labeled microspheres for measurement of regional organ perfusion. J Appl Physiol 1993;742585- 2597
PubMed
Abel  FLCooper  RHBeck  RR Use of fluorescent latex microspheres to measure coronary blood flow distribution. Circ Shock 1993;41156- 161
PubMed
van Oosterhout  MFPrinzen  FWArts  T  et al.  Asynchronous electrical activation induces asymmetrical hypertrophy of the left ventricular wall. Circulation 1998;98588- 595
PubMed
Deveci  DEgginton  S Development of the fluorescent microsphere technique for quantifying regional blood flow in small mammals. Exp Physiol 1999;84615- 630
PubMed
Gervais  MDemolis  PDomergue  VLesage  MRicher  CGiudicelli  JF Systemic and regional hemodynamics assessment in rats with fluorescent microspheres. J Cardiovasc Pharmacol 1999;33425- 432
PubMed
Buckberg  GDLuck  JCPayne  DBHoffman  JIArchie  JPFixler  DE Some sources of error in measuring regional blood flow with radioactive microspheres. J Appl Physiol 1971;31598- 604
PubMed
Almegard  BAndersson  SE Vascular effects of calcitonin gene-related peptide (CGRP) and cholecystokinin (CCK) in the monkey eye. J Ocul Pharmacol 1993;977- 84
PubMed
Orgul  SCioffi  GAWilson  DJBacon  DRVan Buskirk  EM An endothelin-1 induced model of optic nerve ischemia in the rabbit. Invest Ophthalmol Vis Sci 1996;371860- 1869
PubMed
Orgul  SCioffi  GABacon  DRVan Buskirk  EM An endothelin-1-induced model of chronic optic nerve ischemia in rhesus monkeys. J Glaucoma 1996;5135- 138
PubMed
Bill  A Effects of acetazolamide and carotid occlusion on the ocular blood flow in unanesthetized rabbits. Invest Ophthalmol 1974;13954- 958
PubMed
Boyer  MMPoulsen  GLNork  TM Relative contributions of the neurosensory retina and retinal pigment epithelium to macular hypofluorescence. Arch Ophthalmol 2000;11827- 31
PubMed
Stjernschantz  JAlm  ABill  A Effects of intracranial oculomotor nerve stimulation on ocular blood flow in rabbits: modification by indomethacin. Exp Eye Res 1976;23461- 469
PubMed
Stjernschantz  JBill  A Effect of intracranial stimulation of the oculomotor nerve on ocular blood flow in the monkey, cat, and rabbit. Invest Ophthalmol Vis Sci 1979;1899- 103
PubMed
Bill  A Effects of indomethacin on regional blood flow in conscious rabbits: a microsphere study. Acta Physiol Scand 1979;105437- 442
PubMed
Granstam  ENilsson  SF Non-adrenergic sympathetic vasoconstriction in the eye and some other facial tissues in the rabbit. Eur J Pharmacol 1990;175175- 186
PubMed
Duijm  HFRulo  AHAstin  MMaepea  Ovan den Berg  TJGreve  EL Study of choroidal blood flow by comparison of SLO fluorescein angiography and microspheres. Exp Eye Res 1996;63693- 704
PubMed
Alm  A The effect of topical l-epinephrine on regional ocular blood flow in monkeys. Invest Ophthalmol Vis Sci 1980;19487- 491
PubMed
Juliusson  BBergstrom  ARohlich  PEhinger  Bvan Veen  TSzel  A Complementary cone fields of the rabbit retina. Invest Ophthalmol Vis Sci 1994;35811- 818
PubMed
Famiglietti  EVSharpe  SJ Regional topography of rod and immunocytochemically characterized “blue” and “green” cone photoreceptors in rabbit retina. Vis Neurosci 1995;121151- 1175
PubMed
Harwerth  RSSmith  EL  IIIDeSantis  L Behavioral perimetry in monkeys. Invest Ophthalmol Vis Sci 1993;3431- 40
PubMed
Perry  VHCowey  A The ganglion cell and cone distributions in the monkey's retina: implications for central magnification factors. Vision Res 1985;251795- 1810
PubMed
Curcio  CASloan  KR  JrPacker  OHendrickson  AEKalina  RE Distribution of cones in human and monkey retina: individual variability and radial asymmetry. Science 1987;236579- 582
PubMed
Ahnelt  PK The photoreceptor mosaic. Eye 1998;12531- 540
PubMed
Stefansson  EWolbarsht  MLLanders  MB  III In vivo O2 consumption in rhesus monkeys in light and dark. Exp Eye Res 1983;37251- 256
PubMed
Linsenmeier  RA Effects of light and darkness on oxygen distribution and consumption in the cat retina. J Gen Physiol 1986;88521- 542
PubMed
Yu  DYCringle  SJ Low oxygen consumption in the inner retina of the visual streak of the rabbit. Am J Physiol Heart Circ Physiol 2004;286H419- H423
PubMed
Ahmed  JBraun  RDDunn  R  JrLinsenmeier  RA Oxygen distribution in the macaque retina. Invest Ophthalmol Vis Sci 1993;34516- 521
PubMed

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