LABORATORY SCIENCES. Confocal scanning laser Doppler flowmetry

Transcription

1 LABORATORY SCIENCES Confocal Scanning Laser Doppler Flowmetry in the Rat Retina Origin of Flow Signals and Dependence on Scan Depth Balwantray C. Chauhan, PhD; Paula K. Yu, PhD; Stephen J. Cringle, PhD; Dao-Yi Yu, MD, PhD Objectie: To inestigate the origin of signals from scanning laser Doppler flowmetry (SLDF) and the influence of axial scan depth on the measurement of blood flow in the rat retina. Methods: We performed SLDF in 5 adult Sprague- Dawley rats using a specially modified Heidelberg retina flowmeter. Axial scans were obtained from 2 diopters (D) to 3 D (in steps of.25 D) or from 1 Dto 2D (in steps of.125 D) relatie to the retinal surface. Fluorescein isothiocyanate dextran angiograms were obtained in whole-mounted retinas to isualize the angioarchitecture and identify measurement locations in the SLDF flow maps. Axial SLDF flow profiles were obtained in an artery, ein, arteriole, enule, and capillary bed using the mean blood flow alues in 2 2, 4 4, and 1 1 pixel measurement windows. Results: The SLDF images showed good correspondence with the angiograms and resolution to thirdorder arterioles and enules; howeer, neither the superficial nor deep capillary circulations were isualized. Flow was imaged from large choroidal essels. Measured flow from capillaries was independent of depth and indistinguishable from background leels. Conclusion: The technique of SLDF images blood flow in larger retinal essels but not in capillaries. Clinical Releance: Scanning laser Doppler flowmetry may not reliably measure capillary blood flow. Arch Ophthalmol. 26;124: Author Affiliations: Retina and Optic Nere Research Laboratory, Department of Ophthalmology and Visual Sciences, and Department of Physiology and Biophysics, Dalhousie Uniersity, Halifax, Noa Scotia (Dr Chauhan); and Centre for Ophthalmology and Visual Science, Lions Eye Institute, Uniersity of Western Australia, Nedlands (Drs Cringle, P. K. Yu, and D.-Y. Yu). MEASUREMENT OF RETInal blood flow can proide aluable clinical information in the management of patients with diabetic retinopathy, agerelated macular degeneration, and other retinopathies. Angiographic techniques using conentional optical 1-3 or scanning laser image 4 acquisition with conersion of angiographic data to flow alues hae been described 5 ; howeer, these methods hae not been fully alidated using experimental or clinical models. Laser Doppler techniques 6-8 are noninasie and therefore offer an adantage oer angiographic techniques, which require an intraenous injection of fluorescent dye. Considerable controersy exists about the origin of the laser Doppler signal, 9,1 specifically regarding the caliber of contributing essels and whether they are situated superficially or deeper in the sampled tissue. Confocal scanning laser Doppler flowmetry (SLDF) has the potential adantage of proiding a spatially rich blood flow image. 11,12 Bench experiments hae shown that measured flow in arbitrary units (AU) changes linearly with actual flow up to a peak elocity of approximately 1 mm/s parallel to the detector axis. 13 Howeer, with this technique it is not clear whether signals arise from mainly superficial essels or whether deeper essels also contribute. One study 14 suggests that SLDF measures predominantly superficial blood flow in the optic nere head, although to date no reports hae been published on important issues such as the effect of focus of the incident laser on SLDF signals or whether the technique can reliably measure flow signals from the deeper retina or capillaries. In this study, we used SLDF for noninasie retinal blood flow measurement in rats. The effect of changing the focus depth of the incident laser light was inestigated on flow measurements, whereas the origin of the signals in indiidual retinas was inestigated by mapping the measured flow alues with the retinal asculature labeled with fluorescein isothiocyanate conjugated (FITC) dextran. (REPRINTED) ARCH OPHTHALMOL / VOL 124, MAR Downloaded From: on 8/16/ American Medical Association. All rights resered.

2 METHODS GENERAL PREPARATION Adult Sprague-Dawley rats were anesthetized with an intraperitoneal injection of 1 mg/kg of 5-ethyl-5-(1 -methylpropyl)-2-thiobarbituate (Inactin; Sigma Chemical Co, St Louis, Mo). Atropine sulfate (2 µg) was administered intramuscularly to minimize saliation. Body temperature was monitored and maintained at 37.5 C using a rectal thermometer and a homeostatic blanket (Harard Apparatus, Holliston, Mass). Animals were killed with an anesthetic oerdose. All procedures complied with guidelines from the institutional committee on the care of laboratory animals. SCANNING LASER DOPPLER FLOWMETRY Retinal blood flow was measured using the Heidelberg Retina Flowmeter (HRF; Heidelberg Engineering GmbH, Dossenheim, Germany), a confocal scanning laser Doppler flowmeter. The technique relies on measuring time-related intensity ariations of backscattered light from an illuminated spot on the fundus. These intensity ariations are due to interference between backscattered light from stationary structures such as tissue and essel walls and from moing blood particles. The intensity ariation measurements are subjected to a fast Fourier transform to obtain the power spectrum of the multiple frequency shift components. Thereafter, 3 hemodynamic ariables, elocity, olume, and flow, are computed from the power spectrum in AU. 15 The instrument and its operation hae been detailed elsewhere. 11 Briefly, a diode laser (waelength, 78 nm) is used to scan an area of (in the human retina) after it has been focused at the desired axial plane (for example, the retina-itreous interface). The image resolution is picture elements (pixels). Each of the 64 horizontal lines is scanned 128 times with a line repetition rate of 4 khz. The total image acquisition time is 2.5 seconds. The 128 intensity measurements at each location are made by a photodiode behind a confocal pinhole. The result of each processed scan is a 2-dimensional perfusion map of the imaged area. The measurements deried with SLDF hae been shown to hae a linear relationship to actual flow rates in experimental bench model systems. 13,16 For the current experiment, we used a specially modified optical system for the HRF identical to that used for obtaining topographic images of the rat optic nere head. 17 Briefly, the modifications inoled changes in the laser output and scanning angles and the use of a microscope objectie and a glass planoconcae contact lens. The minimum increment in focus was.125 D as opposed to the.25 D in the standard instrument. An additional stereotaxic framework was specifically manufactured for mounting the HRF camera. This deice allowed centralization of the HRF laser through the pupil and adjustment of the angle and distance of the HRF objectie from the eye. The rat was placed prone in a modified stereotaxic frame (model 514, Stoelting Co, Wood Dale, Ill). An eye ring was sutured to the conjunctia at the limbus and fixed to a custombuilt stereotaxic frame, which could be displaced laterally and ertically with respect to the HRF. In each animal, 3 scan locations (approximately 2 disc diameters inferior, inferior nasal, and inferior temporal) were chosen for imaging. These locations typically contained at least one artery and ein, in addition to arterioles and enules. After the camera was focused on the retinal surface with the stereotaxic apparatus and the focus set to D, sequential images were acquired from 2 D to 3 D (in increments of.25 D) relatie to the -D setting or 1 D to 2 D (in increments of.125 D) relatie to the -D setting. This procedure was repeated for the other 2 locations. Hence, 3 sets of images, with each containing a depth or axial series at each location, were obtained. After animal preparation, all the images were obtained in approximately 45 to 6 minutes. FLUORESCENCE ANGIOGRAPHIC TECHNIQUE After the imaging session with the animal still under deep anesthesia, both carotid arteries were cannulated within 5 to 1 minutes for perfusion fixation. Both jugular eins were seered to allow outflow of blood and perfusate. Red blood cells were flushed out of the ocular circulation using carbogen bubbled Krebs solution that contained heparin (1 U/mL). A total of 5 ml of 3% paraformaldehyde in.1m phosphatebuffered solution was used as a fixatie. To stain the asculature, 1 ml of a mixture of 5% FITC-conjugated dextran and 3% gelatin dissoled in warmed.1m Krebs solution was infused into the carotid arteries until outflow was isible from the seered jugular eins. The eyes were enucleated and further fixed oernight in ice-cold 3% paraformaldehyde to aid the setting of the gelatin-fitc-dextran mixture. After 24 hours, the retina was carefully dissected and whole mounted. The portions of the retina that were imaged with the HRF were digitally imaged under epifluorescence with the same location imaged at 3 axial distances, capturing the superficial and deep retinal (capillary) asculature. DATA ANALYSIS The raw SLDF data from each image were processed using the HRF software (ersion 1.4W) to obtain the flow images. The image quality of the flow maps in each of the 3 sets of images was carefully examined for exposure and detail of the perfusion pattern. One set of images was then selected for analysis based on image quality. Using the pattern of essels from the FITC-dextran angiograms, 5 locations in the SLDF images were carefully chosen for obtaining flow measurements in an artery, ein, arteriole, enule, and capillary bed (identified in the angiograms). The cartesian coordinates of the location were noted and sequential depth measurements were always made at this location, since there was no eye or HRF moement during the axial scans for a gien location. Using the software, aerage measurements in a 2 2, 4 4, and 1 1 pixel window centered on the gien location were made. These flow alues were digitally recorded using the software. We carefully compared the pattern of the asculature from the axial series of SLDF flow images to the pattern of the retinal asculature with the axial angiograms. To supplement this qualitatie isual analysis, we determined whether the measured flow alues for different locations obtained as described aboe changed as a function of retinal depth to address the possibility of extracting flow-related information in SLDF images that do not show obious flow patterns that resemble the asculature. For each animal and location, flow profiles (in axial depth) obtained for each of the 3 measurement windows were plotted. RESULTS A total of 5 animals were used in this study. In 2 animals, the axial SLDF images were obtained in increments of.25 D, whereas in the remaining 3 they were obtained in increments of.125 D. The pattern of the retinal asculature isualized with the SLDF images showed good spatial correspondence (REPRINTED) ARCH OPHTHALMOL / VOL 124, MAR Downloaded From: on 8/16/ American Medical Association. All rights resered.

3 A B C a a a DC +1 D D 2 D DC DC Flw Flw Flw a a a Vol Vol Vol Vel Vel Vel Figure 1. Representatie fluorescein isothiocyanate (FITC) dextran angiograms and in io confocal scanning laser Doppler flowmetry (SLDF) in 1 animal. Angiograms obtained in whole-mounted retinas at 3 planes focused on the superficial circulation (A), circulation at an intermediate location (B), and circulation at the deep capillary layer (C). Reflectiity image (DC) and SLDF flow map (Flw) were obtained at 3 ( 1 D, D, and 2 D) of the 2 axial planes imaged in this animal. Corresponding olume (Vol) and elocity (Vel) maps are also shown. The SLDF flow maps that contain only Doppler-shifted information depict blood flow in essels not shown in the reflectiity image. Note that the depth of A, B, and C does not correspond to the illustrated axial SLDF images. The corresponding artery (a), ein (), arteriole (white arrow), and enule (unfilled white arrows) in the angiograms and SLDF flow maps are also shown; howeer, capillaries (yellow arrows) are not imaged by SLDF. At the 2-D focal leel, the enule draining the deep capillary circulation is clearly isible (unfilled white arrows); howeer, another essel (blue arrow) that is not apparent in the angiograms is also isible at this leel and faintly at the other 2 anterior focal planes and is choroidal in origin. Scale bar in angiogram=1 µm. with the FITC-dextran angiograms for the larger essels. In many cases, the SLDF images showed resolution to third-order arterioles (Figure 1); howeer, some of the arterioles, often of first or second order, shown in the angiograms were not apparent in the flow images. Venules draining the deep capillary circulation were also clearly resoled by the SLDF images. Howeer, neither the superficial nor deep capillary circulations could be isualized (Figure 1) in any of the axial SLDF images of any animal. At deeper locations, flow information from apparently large essels that did not correspond to essels in the angiograms was also isualized. Since only the retina was dissected and whole mounted for obtaining the angiograms, it was concluded that this information likely originated from choroidal essels. Careful examination of superficial SLDF images also showed blood flow information from relatiely large essels in the choroid (Figure 1). Representatie flow profiles of one animal for all locations and 3 measurement windows are shown in Figure 2. The data for measurements in artery, ein, arteriole, and enule showed that the flow alues depended on axial depth, indicating that the highest flow alues were obtained when the focal plane corresponded to the axial location of the essel. The flow profiles obtained from the capillary bed did not seem dependent on axial depth, confirming the isual obseration that the SLDF images do not contain information from the capillaries. The fact that the flow alues at these locations were low and did not change with axial depth suggests that flow information from the capillaries cannot be distinguished from the background noise leel. The mean axial blood flow data obtained from the 1 1 pixel measurement window for all 5 measurement locations in all animals are shown in Figure 3. The data for the 2 2 and 4 4 pixel measurement window were similar and are not shown. The peak flow alues in the arteries aried from 2 to 7 AU and in eins from 35 to 55 AU. In all cases, flow measurement alues in the artery and ein showed dependence on scan depth with a single peak. Howeer, in one animal (R-387), measurements in the ein yielded 2 peaks because of interference from a choroidal essel. Although measurements from the arteriole and enule locations also depended on scan depth, the flow alues were lower and peaks not as distinct, especially for the enules. Measurements from the capillary locations were generally ery low and not systematically dependent on scan depth. In 2 cases (R-348 and R-386), flow alues decreased slightly with scan depth, whereas in 1 case the opposite (R-387) was noted. COMMENT Techniques for measuring blood flow in the retina and optic nere head hae the potential to proide important clues about the pathophysiology of many retinal and optic nere disorders. They can also play an important role in the diagnosis and management of these diseases. (REPRINTED) ARCH OPHTHALMOL / VOL 124, MAR Downloaded From: on 8/16/ American Medical Association. All rights resered.

4 SLDF Measured Flow, AU 12 1 Artery 8 7 Vein Scan Focus,.25-D Increments Arteriole 2 Venule 3 Capillary SLDF Measured Flow, AU Scan Focus,.25-D Increments Figure 2. Axial blood flow profiles obtained with scanning laser Doppler flowmetry (SLDF) at the 5 locations selected from the angiograms in 1 animal. Values shown are means in 2 2, 4 4, and 1 1 pixel measurement windows. AU indicates arbitrary units; D, diopters. Validation studies of clinical deices for measuring blood flow can be problematic, since gold standard methods are either unaailable or inasie. Because the retina contains multilayered ascular beds, it is impossible in human studies to determine the contribution of each layer to the final measurements obtained with a technique such as SLDF. Although studies in experimental animals may hae limited direct releance to clinical measurements, experimental studies proide a powerful start to understanding the alidity and limitations of blood flow measurements for translation into clinical practice. Our study demonstrates that SLDF is capable of measuring signals from larger retinal essels, down to second- or third-order arterioles and enules. This was eidenced by both the FITC-dextran angiograms and the quantitatie measurements performed at these locations. It was notable that not all arterioles and enules that were shown in the angiograms produced a detectable flow signal in the SLDF images. There was no obious trend that the ability to detect SLDF flow signals was related to essel size, because similarly sized essels located in close proximity (as shown by the angiograms) were often not all detected with SLDF. It is possible that the flow signals are critically dependent on image quality and that the relatiely high curature of the rat retina may hae caused some optical distortion and made the Doppler signal difficult to detect. Alternatiely, it is possible that these essels were not perfused at a leel to hae a detectable flow measurement at the time of the imaging session. Many authors hae used SLDF with the assumption that flow measurements originating from capillaries can be made. 11,18,19 Howeer, eidence from this and a recent study 2 strongly suggest that SLDF cannot measure reliably in capillaries. First, in the present study we were unable to isualize any capillaries as localized in the angiograms in either the superficial or deep capillary layers in the respectie SLDF images. In the HRF analyses, the Doppler frequency shifts between 125 and 2 Hz, which correspond to a detection range in the elocity ector parallel to the detector of.5 to.78 mm/s. 13 Since the plane of larger retinal essel is nearly perpendicular to the detector, substantially higher elocities can theoretically be detected. 13 Howeer, because the angle between the elocity ector and the detector axis likely aries, een along an indiidual essel, and since the Doppler shift near 9 for a gien elocity changes rapidly with small ariations in this angle, it is probably not meaningful to compare flow alues among different locations in a flow map. Comparing the same location before and after an interention or oer time may be more meaningful, proiding the properties of the static scatterers hae not changed. The possibility exists that the elocity in capillaries is too low and produces Doppler shifts that escape detection with a lower cutoff frequency of 125 Hz. The SLDF flow maps were recomputed in some animals with a lower cutoff of 31 Hz to address this issue (G. Zinser, PhD, oral communication, January 25). In all cases the recalculated flow maps failed to reeal capillary flow; hence, we do not beliee that the frequency bandpass of the HRF is the reason for not detecting capillary flow. Second, we were unable to show any focus depth dependent changes in flow when measurements were (REPRINTED) ARCH OPHTHALMOL / VOL 124, MAR 26 4 Downloaded From: on 8/16/ American Medical Association. All rights resered.

5 SLDF Measured Flow, AU 45 4 R R Scan Focus,.25-D Increments Artery Vein Arteriole Venule Capillary SLDF Measured Flow, AU R R Scan Focus,.25-D Increments R Figure 3. Axial blood flow profiles obtained with scanning laser Doppler flowmetry (SLDF) at the 5 locations selected from the angiograms in all 5 animals. Values shown are the means in a 1 1 pixel measurement window. AU indicates arbitrary units; D, diopters. made in locations deoid of retinal essels except capillaries, as indicated in the angiogram. Additionally, flow measurements in capillaries were ery low and close to noise leels. Finally, in a recent study, we showed that there were no changes in SLDF-measured flow in locations that contained capillaries immediately before and after laser occlusion of the retinal circulation, whereas significant decreases, down to background leels, were noted in all other retinal essels. 2 The SLDF images showed signals that originated from essels that were not detected in the angiograms and were therefore highly likely to be choroidal in origin. These signals were detected een at relatiely superficial retinal locations (Figure 1). In clinical studies, SLDF measurements are usually made at one focal plane. Axial flow profiles are not recorded due to acquisition time, eye moements both during and between axial scans, and the current inability to accurately register axial SLDF images. It is therefore difficult to rule out the interference of choroidal signals in the measurement of retinal blood flow on the basis of a single axial flow image, een when the focus is set to the superficial retina. Because the axial resolution of confocal ophthalmoscopy in the human eye is estimated to be approximately 3 µm, 21 it is likely that despite confocal optics, flow signals from at least the anterior choroid can be detected when the imaging focal plane is set at the superficial retina. Indeed, SLDF measured blood flow in areas of retina affected by a branch ein occlusion where absence of blood flow was confirmed by fluorescein angiography. 22 Thus, clinical studies hae also proided eidence that the choroidal circulation influences SLDF-measured flow in the retina. Based on the ocular constants in the rat eye 23 and the optical setup of the HRF, the axial resolution is estimated to be similar to that in the human eye (G. Zinser, PhD, oral communication, January 25). With the modified HRF setup for use in rats, a 1-D change in focus is equialent to approximately 11 µm (G. Zinser, PhD, oral communication, January 25). Examination of the axial SLDF images (Figure 1) shows that the flow imaged in the large essels at deeper locations are choroidal gien that the rat retina is approximately 325 to 35 µm thick. 24 Our study was necessarily descriptie because it is not meaningful to pool data across animals, since measurement locations were chosen according to the respectie angiograms and not location, size, and depth of essels. Therefore, computing means to examine the effect of focus depth on flow measurements across all animals would hae been inaccurate since the respectie essels may hae been located in different axial locations. This was particularly the case with enules, because they emerged superficially at a steep gradient from the deep capillary bed. We belieed it was more appropriate to present the data from each animal separately. Examining the correspondence between the angiograms and SLDF images was qualitatie and subjectie, because an objectie and quantitatie analysis is not readily feasible with this form of data. Although the rat and human retina and its circulation are not completely equialent, an experimental study such as this can offer many adantages oer clinical studies in understanding the applications and limitations of SLDF. Because the eye was stabilized and the (REPRINTED) ARCH OPHTHALMOL / VOL 124, MAR Downloaded From: on 8/16/ American Medical Association. All rights resered.

6 HRF fixed to the stereotaxic frame, no motion occurred during image acquisition, ensuring high-quality images. In clinical studies, motion artifacts are irtually impossible to eliminate. Additionally, we were able to obtain axial scans at the same transerse location, allowing us to generate axial flow profiles that help us understand the contributions of the different sources of blood flow to the measured alues. Such studies would be difficult to replicate clinically gien the current hardware and software limitations. Correlating the angioarchitecture obtained with the FITC-dextran angiograms to the SLDF images is a powerful method to identify the blood essels that contribute to the SLDF measured flow. In summary, based on angiograms and axial SLDF flow images, we showed that SLDF can image blood flow in arteries, eins, and some arterioles and enules. We were unable to demonstrate that capillary flow could be reliably imaged. Finally, some choroidal essels can exert an influence on blood flow measurements obtained from retinal locations. Submitted for Publication: February 14, 25; final reision receied June 28, 25; accepted June 3, 25. Correspondence: Balwantray C. Chauhan, PhD, Department of Ophthalmology and Visual Sciences, Dalhousie Uniersity, Second Floor, Centennial Building, Queen Elizabeth II Health Sciences Centre, Halifax, Noa Scotia, Canada B3H 2Y9 (bal@dal.ca). Author Contributions: All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Financial Disclosure: None. Funding/Support: This study was supported by grant MOP from the Canadian Institutes of Health Research, Ottawa, Ontario (Dr Chauhan), and the National Health and Medical Research Council of Australia, Canberra (Drs D.-Y. Yu and Cringle). Acknowledgment: We are grateful to Gerhard Zinser, PhD, Heidelberg Engineering, for modifying the HRF for use in rats, computing the axial resolution of the HRF in the rat eye, and recalculating the SLDF flow maps at the lower-frequency cutoff. REFERENCES 1. Nagin P, Schwartz B, Reynolds G. Measurement of fluorescein angiograms of the optic disc and retina using computerized image analysis. Ophthalmology. 1985; 92: Preussner PR, Richard G. Determination of the circulation in the human retina using image analysis of ideo-angiograms. Fortschr Ophthalmol. 1989;86: Wolf S, Jung F, Kiesewetter H, Korber N, Reim M. Video fluorescein angiography: method and clinical application. Graefes Arch Clin Exp Ophthalmol. 1989; 227: Arend O, Harris A, Sponsel WE, et al. Macular capillary particle elocities: a blue field and scanning laser comparison. Graefes Arch Clin Exp Ophthalmol. 1995; 233: Harris A, Chung HS, Ciulla TA, Kagemann L. Progress in measurement of ocular blood flow and releance to our understanding of glaucoma and age-related macular degeneration. Prog Retin Eye Res. 1999;18: Ria C, Ross B, Benedek GB. Laser Doppler measurements of blood flow in capillary tubes and retinal arteries. Inest Ophthalmol. 1972;11: Ria CE, Grunwald JE, Sinclair SH. Laser Doppler Velocimetry study of the effect of pure oxygen breathing on retinal blood flow. Inest Ophthalmol Vis Sci. 1983; 24: Ria CE, Harino S, Petrig BL, Shonat RD. Laser Doppler flowmetry in the optic nere. Exp Eye Res. 1992;55: Hayreh SS. Ealuation of optic nere head circulation: reiew of the methods used. J Glaucoma. 1997;6: Petrig BL, Ria CE, Hayreh SS. Laser Doppler flowmetry and optic nere head blood flow. Am J Ophthalmol. 1999;127: Michelson G, Schmauss B. Two dimensional mapping of the perfusion of the retina and optic nere head. Br J Ophthalmol. 1995;79: Michelson G, Schmauss B, Langhans MJ, Harazny J, Groh MJ. Principle, alidity, and reliability of scanning laser Doppler flowmetry. J Glaucoma. 1996; 5: Chauhan BC, Smith FM. Confocal scanning laser Doppler flowmetry: experiments in a model flow system. J Glaucoma. 1997;6: Wang L, Cull G, Cioffi GA. Depth of penetration of scanning laser Doppler flowmetry in the primate optic nere. Arch Ophthalmol. 21;119: Bonner RF, Nossal R. Principles of laser-doppler flowmetry. In: Shepherd AP, Ödberg PÅ, eds. Laser-Doppler Blood Flowmetry. Boston, Mass: Kluwer Academic Publishers; 199: Tsang AC, Harris A, Kagemann L, et al. Brightness alters Heidelberg retinal flowmeter measurements in an in itro model. Inest Ophthalmol Vis Sci. 1999; 4: Chauhan BC, Pan J, Archibald ML, et al. Effect of intraocular pressure on optic disc topography, electroretinography, and axonal loss in a chronic pressureinduced rat model of optic nere damage. Inest Ophthalmol Vis Sci. 22; 43: Nicolela MT, Hnik P, Schulzer M, Drance SM. Reproducibility of retinal and optic nere head blood flow measurements with scanning laser Doppler flowmetry. J Glaucoma. 1997;6: Carlsson AM, Chauhan BC, Lee AA, LeBlanc RP. The effect of brimonidine tartrate on retinal blood flow in patients with ocular hypertension. Am J Ophthalmol. 2;129: Yu DY, Townsend R, Cringle SJ, Chauhan BC, Morgan WH. Improed interpretation of flow maps obtained by scanning laser Doppler flowmetry using a rat model of retinal artery occlusion. Inest Ophthalmol Vis Sci. 25;46: Woon WH, Fitzke FW, Bird AC, Marshall J. Confocal imaging of the fundus using a scanning laser ophthalmoscope. Br J Ophthalmol. 1992;76: Squirrell DM, Watts A, Eans D, Mody C, Talbot JF. A prospectie ealuation of the Heidelberg retina flowmeter in diagnosing ischaemia following branch retinal ein occlusion: a masked, controlled comparison with fluorescein angiography. Eye. 21;15: Block MT. A note on the refraction and image formation of the rat s eye. Vision Res. 1969;9: Yu DY, Cringle SJ. Oxygen distribution and consumption within the retina in ascularised and aascular retinas and in animal models of retinal disease. Prog Retin Eye Res. 21;2: (REPRINTED) ARCH OPHTHALMOL / VOL 124, MAR Downloaded From: on 8/16/ American Medical Association. All rights resered.