Laser Doppler Velocimefry Study of the Effect of Pure Oxygen Breathing on Retinal Blood Flow

Transcription

1 Laser Doppler Velocimefry Study of the Effect of Pure Oxygen Breathing on Retinal Blood Flow C. E. Riva, J. E. Grunwald,* and S. H. Sinclair The noninvasive Laser Doppler Velocimetry (LDV) technique was used in normal volunteers to determine retinal blood flow, F, during pure oxygen breathing at atmospheric pressure. Changes in blood flow were calculated based on Poiseuille relation, F = (TTD 2 /4) V^, where V^ is the red blood cell maximum or center-line velocity and D, the diameter of the vessel at the site of the LDV recordings. After five minutes of pure oxygen breathing, V max decreased by about 53%, vessel diameter by 12%, and retinal blood flow by about 60%. In arteries, pulsatility of red blood cell velocity (V max, Systoie/V max, diastole) during the cardiac cycle increased by about 10%. Decrease in red blood cell velocity was detectable within 1-1'/z min. From these LDV flow measurements and the changes in retinal mean circulation time (MCT) reported by other investigators, based on the relation F = v/mct, a 45% decrease in retinal blood volume (v) during 100% O 2 breathing was obtained. Invest Ophthalmol Vis Sci 24:47-51, 1983 Retinal arteries and veins normally dilate when a subject breathes gas with a low oxygen tension and constrict with the breathing of pure oxygen. 1 " 3 This change in caliber suggests that shifts in retinal oxygen tension are opposed by alterations in blood flow. Hickam and Frayser 4 first attempted to quantitate the changes in human retinal blood flow in response to alterations of blood oxygen tension. In their technique, arterial and venous fluorescein dilution curves were constructed from serial fundus angiograms to determine the mean circulation time of fluorescein, t r, through the whole retina. Retinal blood flow, F, was calculated from the relation F = v/f r, where v is the volume of blood in the whole retinal vasculature. Since v could not be determined directly, its change from one physiologic state to another was estimated from changes in the square of the mean diameter of the large retinal veins. It is difficult to estimate the accuracy of the blood From the Department of Ophthalmology, University of Pennsylvania, School of Medicine and the Scheie Eye Institute, Philadelphia, Pennsylvania. Supported by NIH Grant EY and Research Career Development Award EY (C. E. Riva) from the National Eye Institute, Research to Prevent Blindness, and the Edwin M. Lavino Trust. Submitted for publication July 2, * On leave from the Department of Ophthalmology, Beilinson Medical Center, University of Tel Aviv, Israel. Reprint requests: Dr. Charles Riva, Scheie Eye Institute, 51 North 39th Street, Philadelphia, PA flow measurements obtained with this technique because of (1) the uncertainty in the computation of the retinal vascular volume (v), and (2) the errors in l T resulting from measuring the vessel cross-sectional dye fluorescence intensity instead of the mean flow dye concentration. 56 Laser Doppler Velocimetry (LDV) provides direct means to determine retinal blood flow and its change in response to alterations of physiologic conditions. It enables noninvasive measurements of the maximum or centerline speed (V max ) of red blood cells (RBCs) at discrete locations along retinal arteries and veins. 7 Blood flow is calculated from this speed and the cross-section of the vessel at the site of measurement. Using this technique, we have investigated changes in retinal blood flow in response to the breathing of pure oxygen (100% O 2 ) at normal atmospheric pressure in normal subjects. Materials and Methods In the bidirectional LDV technique, 7 ' 8 Doppler shift frequency spectra (DSFS) of laser light scattered from RBCs in retinal vessels are recorded simultaneously for two directions of the scattered light. V max is determined according to the relation V max = KAf, where K is a constant related to the scattering geometry and the wavelength of the laser light. The cutoff frequencies of the DSFS recorded in the two directions are Af = f 2ma x - fimax', the cutoff frequencies of the DSFS recorded in the two directions are f, max /83/0100/047/$ 1.05 Association for Research in Vision and Ophthalmology 47

2 48 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / January 1983 Vol. 24 Fig. 1. Typical pair of Doppler shift frequency spectra (DSFS) recorded during pure oxygen breathing. Recording time: 0.05 seconds during diastole. Cutoff frequencies (arrows) were determined by visual inspection; Af = f 2max - f, max is equal to 2289 Hz Frequency (Hz) and f 2ma x. Relative changes in V max during 100% O 2 are thus obtained from the relation, equation 1: * max, O2/ * max, air = Alo 2 /Al a j r. Doppler shift frequency spectra were recorded with a fundus camera laser Doppler system 8 from 15 major retinal arteries and five veins of five normal volunteers, ages The photocurrent signals produced by the light scattered by the RBCs in two directions were recorded on a Honeywell 5600C magnetic tape system for approximately 20 seconds at 30-second intervals during 5 min of room air breathing, 6 to 8 min of oxygen breathing, and then room air breathing again. One of the photocurrent signals could be heard through a loudspeaker. Between each recording, the fundus camera was recentered; the position of the incident laser beam and light collecting optical fibers remained unchanged. On playback, only the portions of tape containing a distinct pulsatile arterial or a more uniform high frequency venous pitch were analyzed by a two-channel spectrum analyzer (Unigon, model 4520). For 10,000 12,000 most of the arteries, measurements were performed only during diastole, with an analysis time shorter than 0.32 seconds. For veins, the DSFS were recorded for 0.32 seconds, randomly during the heart cycle since no significant pulsatility of speed was detected in these vessels. For measurements of pulsatility in retinal arteries, the spectrum analyzer was activated for 0.05 sec at various phases of the cardiac cycle using an electronic gating module fed by the signal from a finger plethysmograph. The DSFS at each phase represented an average over cycles. A typical pair of DSFS recorded in diastole from an artery during 100% O 2 breathing is shown in Figure 1. At least five such pairs of DSFS were recorded to provide an average Af during the breathing of room air, Af air, and during 100% O 2, Af^. In order to obtain DSFS with distinct cutoff frequencies, the laser beam and light-collecting optical fibers must be sharply focussed and precisely centered on the vessel. This was achieved easily with our instrument in our subjects who had clear media and

3 No. 1 LASER DOPPLER VELOCIMETRY STUDY / Rivo er ol Fig. 2. Relative changes in diastolic V max in a retinal artery during 100% oxygen breathing at atmospheric pressure. Each data point was calculated based on at least five values of Af. < 0.8 o <j Hilt M 100 O # 21 0 J I I Minutes minimal astigmatism and who were able to fixate steadily for the duration of a few heart beats. Changes in vessel diameter, D, were measured at the site of the LDV recordings from photographic negatives that were taken in monochromatic light at 570 nm to maximize the contrast between vessel and background. 9 Since fundus photography could not be performed during the LDV recordings, an identical O 2 -breathing experiment was performed in each subject for photography. Percent changes in blood flow, F, between air and 100% O2 breathing were calculated from the relation, equation 2: F = 7r(D 2 /4)V max, which is valid for Poiseuille flow. For retinal arteries, blood flow changes were determined from the DSFS obtained during diastole only because, as reported later, neglecting the 10% increase in speed pulsatility during 100% O 2 results in an overestimation of flow changes of only 3-4%. During the LDV recordings, the cardiac frequency was continuously monitored as well as the O 2 inspiratory and CO 2 expiratory concentration. The arterial blood pressure was monitored by sphygmomanometry. In two subjects, the intraocular pressure was measured by pneumatonometry during air and after 5 min of O 2 breathing. Results Figure 2 shows a typical time course of the change in Af oc V max obtained from an artery during diastole as the subject switched from breathing room air to breathing pure oxygen at normal atmospheric pressure. In this recording, a relative change in V max was statistically detectable (P < 0.001) after about 1.5 min. In a few cases, such changes were detected within 1 min. The return of V max to normal again after room air breathing followed a similar time course. Changes in V max from the veins were similar in their magnitude and time course to changes recorded from arteries. After 5 min of 100% O 2 breathing, the diameter of the vessels decreased by an average of 12.05% (SD = 3.85%). The maximum RBC velocity decreased by an average of 53.1% (SD = 10.7%) and bloodflowby an average of 64.3% (SD = 9.9%). In 6 of the 20 experiments, either f lmax or f 2max could not be measured, and therefore the average blood flow decrease was calculated from the changes in f lmax or f 2max alone and was found to be 67% (SD = 7.7%), not significantly different (P > 0.05) from the decrease based on changes in V max obtained in the other 14 experiments. Figure 3 displays the pulsatile variation of V max in an artery during the breathing of room air (a) and after 5 min of breathing 100% O 2. The pulsatility of speed, defined as V maxsystole /V maxjdiastole5 was obtained from measurements on nine arteries of five subjects, and ranged between 2.12 and 3.25 (mean: 2.69 ±0.38) during room air breathing; it ranged between 2.31 and 3.66 (mean: 2.96 ± 0.44) and after 5 min of 100% O 2, ie, a significant increase of 10% (0.002 < P < 0.05 for paired-t statistics). There was no significant change in arterial pressure, intraocular pres-

4 50 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / January 1983 Vol ,000 Hz g- 20,000 c 9> 15,000 m 10,000 Q. Q. ^ 5000 Fig. 3. Variation of f max c V max during the cardiac cycle in an artery when breathing room air (a) and after 5 min of 100% O 2 breathing. The error bars represent ±1 standard deviation based on at least five measurements. i i i L Delay (sec) 0.8 sure, cardiac frequency, or CO 2 expiratory concentration when breathing pure O 2 for 5 min. Discussion In this investigation, changes in retinal blood flow were calculated using equation 2. This is valid only if the velocity distribution of the RBCs can be described by a parabolic profile as a function of the radial distance from the axis, R, in a vessel of radius Ro (Poiseuille flow), equation 3: V(R) = V max (l - R 2 / Ro). Studies by Baker and Wayland 10 indicate that at normal hematocrit, the velocity profiles are nearly parabolic for all values of the parameter Vav/2Ro above 6 sec" 1. Vav is the average velocity of the RBCs. In retinal veins with a radius between 50 and 100 fxm, Vav, calculated from Af by equation 5 from the paper by Riva et al 8 and using the relation Vav = V max /2, is between 5 and 8.5 mm/sec, ie, clearly larger than 6 sec" 1. Therefore, assuming that the veins have a circular cross section, equation 2 is applicable. A normal RBC speed of mm/sec in retinal arteries with a radius between /*m would suggest that flow obeys the Poiseuille relation. Blood flow, however, is known to be pulsatile in these vessels, and the question arises as to whether the pulsatility affects the shape of the velocity profile to such an extent that the application of Poiseuilleflowwould be incorrect. The equal magnitude and time course of blood flow change between arteries and veins during oxygen breathing indirectly suggests that Poiseuille relation is valid for the pulsatile flow of blood in retinal arteries. Furthermore, LDV experiments performed in this laboratory 1 ' with whole blood flowing in a pulsatile fashion through glass tubes 70 to 200 /urn internal diameter support this conclusion. With a speed pulsatility as high as 3.3, close agreement was observed between the average velocity calculated by integrating instantaneous V max measurements over the pulse cycle and the average velocity calculated from the measured pump flow rate. The magnitude of the changes in the diameter of the arteries and veins obtained in this study are in good agreement with those measured by Hickam and Frayser. 4 These measurements actually reflect changes in the diameter of the RBC column in vessel internal diameter since both vary proportionally under different conditions of oxygenation. 12 The decrease in flow following 5 min of 100% O 2 breathing obtained in our experiments is significantly larger than the 40% decrease obtained through the application of the dye dilution technique. This most probably occurs because calculations of changes in retinal vascular volume based on the diameter of the large veins tend to underestimate the actual volume changes. 4 A direct estimation of volume changes can be obtained from the relation between flow, volume, and mean circulation time (equation 4): (F O2 /F air ) = (vo 2 /tr,o 2 ) # (fr,air/vair). From our LDV measurements, F O2 /Fai r = 0.64 and from Hickam and Frayser, 4 I r,air/t r,o 2 = Therefore, Vc^/v^ is approxi-

5 No. 1 LASER DOPPLER VELOCIMETRY STUDY / Riva er ol. 51 mately 0.55, corresponding to a decrease in volume of 45%, a value significantly larger than the 29% calculated from the changes in the mean diameter of the large veins. In six experiments, the changes in blood flow induced by 100% O 2 breathing were determined from changes in either fi max or f2 max, which theoretically should be identical to the changes calculated from Af if the scattering geometry (direction of the incident and of the two scattered beams relative to the flow direction) remained constant. Constant scattering geometry was maintained during the experiment by carefully realigning the fundus camera before each recording, as demonstrated by the insignificant difference between percent changes in Af (53 ± 11), fi max (46 ± 7.8), and f 2max (48 ± 7.7), obtained from the 14 experiments. Furthermore, in vitro measurements performed with polysterene spheresflowingin a glass tube (diameter 200 ^m) placed in the "retinal" plane of a Topcon model eye showed that by centering the fundus camera before each measurement, errors in fimax and f2 max could be kept as low as 8%. Therefore, changes in speed, which are measured using only one scattering direction, are valid provided that the fundus camera remains well aligned during the experiment. Key words: laser Doppler velocimetry, red blood cell velocity, retinal bloodflow,oxygen, vessel reactivity, vascular volume References 1. Cusick PL, Benson OO Jr, and Boothby WM: Effect of anoxia and of high concentrations of oxygen on the retinal vessels: Preliminary report. Proc Stf Mtg Mayo Clin 15:500, Duguet J, Dumont P, and Bailliart JP: The effect of anoxia on retinal vessels and retinal arterial pressure. J Aviat Med 18:516, Sieker HO and Hickam JB: Normal and impaired retinal vascular reactivity. Circulation 7:79, Hickam JB and Frayser R: Studies of the retinal circulation in man. Observations on vessel diameter, arterio-venous oxygen difference, and mean circulation time. Circulation 33:302, Riva CE, Feke GT, and Ben-Sira I: Fluorescein dye-dilution technique and retinal circulation. Am J Physiol 234:H315, Patel IC and Sirs JA: Indicator dilution measurements of flow parameters in curved tubes and branching networks. Phys Med Biol 22:714, Riva CE and Feke GT: Laser Doppler velocimetry in the measurement of retinal blood flow. In The Biomedical Laser: Technology and Clinical Applications, Goldman L, editor. New York, Springer-Verlag, 1981, p Riva CE, Grunwald JE, Sinclair SH, and O'Keefe K: Fundus camera based retinal LDV. Appl Optics 20:117, Delori FC and Gragoudas ES: Examination of the ocular fundus with monochromatic light. Ann Ophthalmol 8:703, Baker M and Wayland H: On-line volume flow rate and velocity profile measurement for blood in microvessels. Microvase Res 7:131, Brein K and Riva CE: Laser Doppler velocimetry measurements of pulsatile bloodflowin capillary tubes. Micro vase Res 24:114, Bulpitt CJ, Dollery CT, and Kohner EM: The marginal plasma zone in the retinal microcirculation. Cardiovasc Res 4:207, 1970.