Exp. Eye Res. (1992) 55, 499-506
Laser
Doppler
C. E. RIVA”, University
of Pennsylvania,
(Received
Chicago
Flowmetry
S. HARINO, Department
7 October
in the
B.L. PETRIG
AND
Optic
Nerve
R. D.SHONAT
of Ophthalmology PA, U.S.A.
and Scheie Eye Institute,
1991 and accepted
in revised form 29 January
Philadelphia,
7992)
Laser Doppler flowmetry (LDF) is a technique that measuresrelative averagevelocity, number and flux (number times velocity) of red blood cells in a tissue. In this paper, we demonstrate its application in the optic nerve headtissue,describethe laserdelivery and light scatteringdetectionschemes and investigate
the effect of the distancebetweenthe sitesof illumination and detection.We alsoprovide evidencethat the flow measuredby LDF varies linearly with actual blood flow in the optic nerve and examine the question of the depth of the sampledvolume. Experimentsin anesthetizedcats illustrate potential applicationswhich makeuseof the high temporalresolutionof LDF. These include the responseof blood flow to changesin the compositionof the breathing gasesand changesinducedby neuronalstimulation with multiple and single flashes. Key words: ocular circulation ; optic nerve blood flow ; laser Doppler flowmetry ; hyperoxia : hypoxia : hypercapnia : luminance flicker: neuronal stimulation.
1. Introduction The study of hemodynamics in the optic nerve head (ONH) tissue requires a technique capable of measuring blood flow in this region. Ideally, this technique should be non-invasive, in view of its application in humans, sensitive, so that pathological changes can be detected as early as possible, reproducible and accurate. Furthermore, it should have a short response time to allow the temporal characterization of blood flow regulatory processes in response to various induced physiological stimuli and, consequently, help identify the mechanisms involved in these processes. Quantitative investigations of ONH hemodynamics have been performed using unlabeled microspheres (Geijer and Bill, 1979), iodoantipyrine (Sossi and Anderson, 1983), hydrogen clearance (Kimura et al., 198 7) and the non-invasive laser Doppler velocimetry (LDV) and laser Doppler flowmetry (LDF). Early studies with LDV have dealt only with measurements of relative blood velocity in the microcirculation of the ONH (Riva, Grunwald and Sinclair, 1982). More recent ones have measured relative blood flow in the ONH either by combining LDV with three wavelength reflectometry (Sebag et al., 1985, 1986) or by using LDF (Riva et al., 1990, 1991). In LDF, a laser beam illuminates a small volume of a tissue and some of the light scattered by the tissue and red blood ceils (RBCs) is detected by a photodetector. Relative tissue blood flow is obtained by electronic processingof the photocurrent, a technique which has now been validated for various organs, including skin, nasal and intestinal mucosa, kidney * For correspondence at: Scheie Eye Institute. 51 North 39th Street. Philadelphia. PA 19104, U.S.A. 0014-4835/92/090499+08
$08.00/O
and peripheral nerve tissue (Shepherd and ijberg, 1990) and in the choroid of anesthetized cats (Gherezghiher et al., 1991). This paper demonstrates that also ONH blood flow can be monitored by LDF, examines new aspects of this particular extension of LDF and presentsexamples of applications in anesthetized cats that illustrate its potential. 2. Materials and Methods Laser Doppler Flowmetry LDF is based on the Doppler effect: laser light scattered by a moving particle is shifted in frequency by an amount Af = (2n/h) (IS-K,). V. Ki and K, are the wave vectors of the incident and scattered light, respectively. V is the velocity vector of the particle and h is the wavelength of the incident light. When the laser beam impinges on RBCsmoving in a tissue with different velocity vectors, the spectrum of the scattered light, the so-called Doppler shift power spectrum (DSPS), has a width that is due not only to the multiplicity of Vs, but also to the effect of scattering of the laser light by the tissueitself. As a result, the RBCs do not receive light only in the direction of the incident beam, but rather from numerous random directions. Furthermore, light scattered by a RBC can reach the detector along various directions due to additional scattering by the tissue or other RBCs (Fig. 1). These processesgive rise to a multiplicity of K, and &-vectors and, consequently, to a broader DSPS than would otherwise result solely from the various Vs. In the application of LDF to the ONH, an optical system mounted to a Topcon TRC FE fundus camara allows delivery of one or two laser beams to any site of the posterior pole of the eye (Riva, Petrig and Grunwald, 198 7 ; Petrig and Riva, 1991). Different 0 1992 Academic
Press Limited
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ET AL.
independent of the intensity of the scattered light. The mode of operation of these instruments has been described in detail elsewhere (Nilsson, 1990: Borgos, 1990).
/
Animal Preparation
FIG. 1. Schematic diagram of laser light impinging on a tissue and leaving it in the direction of the detector after having been scattered by the tissue (static scatterers) and red blood cells, RBCs. Adapted from Bonner and Nossal. 1990, Fig. 2-2.
lasers can be interchanged to allow for measurements at different wavelengths, such as the 543 and 632.8 nm lines of the helium-neon laser and various diode lasers lines between 670-8 12 nm. In this work, lasers at 785 and 812 nm were used. In addition to the probing laser light, an area of the fundus (30”) is illuminated by a tungsten source for observation or a flash source for diffuse flicker illumination of the fundus. The spectrum of this illumination can be varied using interference or color glass filters placed in the fundus illumination beam. Each incident beam is focused at the disk, at sites away from visible vessels. The scattered laser light emerging from the pupil (approximately 5” cone) is collected in the retinal image plane of the fundus camera by an optical fiber (output fiber) having a nominal aperture of approximately 400 ,um. The projected image of this aperture at the fundus is approximately 160 ,um. The image of the aperture of the output fiber is usually placed slightly (approximately 100 pm) off the site of the incident laser beam. The collected light is guided by the fiber to a photodetector (Riva, Petrig and Grunwald, 198 7 ; Petrig and Riva, 199 1) and the resulting photocurrent is amplified before being fed into an electronic interface whose function is to separately amplify the AC and DC components. These two components are then processed by the electronic system of a PeriFlux PF3 (Perimed, Inc., Stockholm, Sweden) or a TSI LaserFlo (Vasamedics, Minneapolis, U.S.A.) laser Doppler flowmeter, after filtering of the low frequency artifacts caused by tissue motion. The output signals are the relative number, Vol (cc volume of moving blood) and flux (F) of the RBCs in the sampled volume (Borgos, 1990). The TSI LaserFlow also gives the average velocity (V) of the RBCs. All three flow parameters are
Twenty adults cats, weighing 2.0-3.5 kg, were used. Each cat was premeditated with atropine (0.04 mg kg-‘, subcutaneously) and anesthetized with intramuscular ketamine hydrochloride (22 mg kg-‘) and acepromazine maleate (2 mg kg-‘). Catheters were placed in a femoral artery and vein and a tracheostomy performed. A loading dose of pancuronium bromide (0.2 mg kg-‘) was given intravenously and the animal was ventilated with 2 1 Y00,, 50% N,O and 29% N, using a variable volume respirator. Arterial blood pressure, tidal CO, and heart rate were monitored continuously. Arterial pH. PaCO,, and PaO, were monitored intermittently using a blood gas analyser and adjustments of the inspired gas mixture, tidal volume and respiration rate were made to keep approximately pH 7.4, PaCO, 31 mmHg and PaO, 3 90 mmHg and mean blood pressure between 85-l 10 mmHg. Rectal temperature was maintained at approximately 38°C. Halothane (0.7-1.5 %) and pancuronium bromide was administered (0.15 mg kg-’ hr-‘) infused continuously. The pupils were dilated with 1% tropicamide and 10% phenylephrine and the cat was placed prone on a table with the head secured in a special clamp. A ring was sutured to the eye with three stitches at the limbus and held in a iixed position to prevent eye motion. A zero diopter contact lens was placed on the cornea protected with Healon TM. All experimental procedures conformed to the ARVO Resolution on the Use of Animals in Research and The Presbyterian Medical Center of Philadelphia Guidelines on Animal Research. In three cats, the flow parameters V, Vol and F were measured as a function of the distance (r) between the center of the directly illuminated site and the center of the aperture of the output fiber. The incident beam had a wavelength of 812 nm. r was varied by moving the output fiber at a known speed using a motorized mechanical system. Calibration of r was achieved by comparing the width of the optic disk measured in vivo with that measured from a flat preparation of the same retina. In three cats, we tested the relationship between perfusion pressure, P and F by simultaneously recording femoral artery blood pressure, intraocular pressure (IOP) and F following a lethal injection of pentobarbital. P was calculated as the difference between femoral artery blood pressure and IOP. The IOP was measured using a needle inserted into the anterior chamber and connected to a Harvard Apparatus pressure transducer. To demonstrate potential applications of the technique, various physiological maneuvers were carried
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out and their effect on F documented. These included hyperoxia, hypoxia, multiple and single flash stimulation of the retina. Hyperoxia and hypoxia. Hyperoxia was produced by having each of 16 cats breathe 100% oxygen. V, Vol and F were measured at two sites of the disk, one at the rim, the other at the center. both away from vessels that were visible when the fundus was observed in white light. Pure oxygen was given for approximately 5 min. Hypoxia was induced in each of five cats by having them breathe mixtures of 10% 0, and 90% N,. In all these experiments, the output fiber aperture was placed at a distance r approximately 100 /lrn from the incident beam. Multiple
and single flash stimulation
of the retina.
Multiple flashes (flicker) diffusely illuminating an area of the fundus centered at the optic nerve head and having a diameter of approximately 30” were delivered by means of a fiberoptic cable whose input collected the light from a Xenon arc lamp placed at the focal point of a parabolic reflector (Grass visual stimulator, model PS22). The output was placed against the light bulb used for fundus illumination in the previously described LDV system (Riva, Petrig and Grunwald, 198 7). We used flashes of 20 ,usec duration, delivered at a frequency of 10 Hz. In these experiments, we also compared the results obtained with the Periflux and the TSI flowmeters by simultaneously supplying the input of each instrument with the same photocurrent during flicker stimulation. Furthermore, the presence of spatial variations in the time course and magnitude of the change in F during luminance flicker was explored in nine cats. simultaneously recording F from two sites of the disk, using two near-infrared lasers. Reproducibility of the flicker-induced change in F was determined in one cat by recording 15 successive F responses under identical experimental conditions, i.e. 45 set of diffuse luminance flicker at 10 Hz and the coefficient of variation (standard deviation/mean) was calculated. In one cat, we measured F during 10 Hz flicker stimulation simultaneously in two different ways. Using two output fibers, but one incident laser beam, we placed one of the fibers on top of the laser beam at the optic disk and the other at a distance r of approximately 150 pm. The photocurrent signals were simultaneously analysed with the TSI LaserFlow and PeriFlux. In three cats, while F was measured continuously, single 20 psec flashes (approximately 40 PJ cm-” per flash) were delivered in succession after dark adapting the animal for more than 1 hr. A period of at least 2 min separated the flashes.
two cats tested. In particular, F and Vol always decreased rapidly with r. A typical plot of F as a function of perfusion pressure (P) after a lethal injection of pentobarbital is shown in Fig. 3. A linear fit shows a highly significant correlation between F and P (r = 0.98, P < 0.01)). Identical results were obtained in the other two cats tested, with correlation coefficients of 0.98 and 0.99 (P < 0.01). The effect of hyperoxia on V, Vol and F is demonstrated by the typical recordings shown in Fig 4(A). In these recordings, arterial blood PaO, increased from 100 to 450 mmHg. This measurement was performed with the incident laser beam focused at the rim of the optic disk. In all 16 cats measured, F decreased markedly during approximately 5 min of pure oxygen breathing. The decrease in F was significantly (P < 0.01, paired t-test) greater at the rim than at the center of the nerve (49 &- 15 Y0versus 39 ? 12 %, mean &-95 % confidence interval of the mean). It occurred entirely through a decrease in Vol, since the group average change in V was not significant (P > 0.05). The effect of hypoxia resulted in the changes in V, Vol and F typified by the recordings in Fig. 4(B). Similar results were obtained in the other four cats. When F reached a plateau (between 3-4 min of hypoxia), its value relative to baseline (air breath201
Ot0
I 100
I 200
/ 300
/ 400
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I (pm)
FIG. 2. Relative V (H), Vol (0) and F (0) of r for the same position of the laser beam.
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3. Results Figure 2 represents V(r), Vol(r) and F(r) obtained when the incident laser beam was placed in the nasal part of the disk. Similar curves were consistently found at other peripheral locations of the disk in the other
I 500
i 8( PerfusIon
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FIG. 3. Relationship between ONH F and perfusion pressure (femoral artery mean blood pressure-IOP) after injection of a lethal dose of pentobarbital.
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(A)
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FIG. 4. Typical time coursesof V, Vol and F during the breathing of (A) 100% 0, and (B) a mixture of 10% 0, and 90% N,, which decreasedthe arterial bloodPaO,from 93 to 33 mmHg.The incident laserbeamwasfocusedat the disk, away from visible vessels.Time constant of the flow monitor was 5 sec.The shadedrectangle representsthe time during which gas mixtures other than air were administered.
ing) was increased by 67, 75, 80, 81 and 89% in the five cats, respectively. The increases in F were mainly produced by increases in V since the maximum change in Vol among the five animals was an increase of 13%. The measurements in Fig. 5(A) illustrate the flicker evoked change in F and also allow a comparison between the PeriFlux and the TSI flowmeters. Both instruments provided quantitatively similar results. A clear increase in F was consistently observed within seconds after the initiation of the flicker stimulation. Although not shown here, the increase in F was predominantly the result of an increase in Vol. Based on 15 measurements,the coefficient of variation of the flicker-induced F response,expressedas 100 % x standard deviation/mean, was found to be 12%. Recordings obtained simultaneously from two sitesof the disk in nine cats revealed spatial variations in the time course and magnitude of the F response.For example, in the recordings shown in Fig. 5(B), the increase in F was faster and larger at the center of the nerve than at the periphery. However, based on the results in seven cats, we found no significant difference in the F responsebetween center and periphery, since in four animals the F increase was larger in the center and in three cats larger at the periphery. Simultaneous recordings of F obtained with two output fibers placed at r = 0 and approximately 150 pm during 10 Hz flicker (20 ,usecflashes) are shown in Fig. S(C). The
recording with r = 0 is noisier and less stable. Fig. 6(A) shows six F responsesto a single 20 ksec flash and Fig. 6(B) the mean and the 95% confidence interval of the mean of these responses. Similar responsesto single flashes in terms of time course and magnitude of the F change were obtained in the two other cats studied [Fig. 6(C)]. 4. Discussion In conventional LDF, where the tissue is directly exposed,the laserbeam is delivered through an optical fiber (input fiber) and the scattered light is collected and guided to a photodetector by a second optical fiber [output fiber: Fig. 7(A)]. These two fibers, which are in direct contact with the tissue, are separated by a distance r of 250 pm (Perimed) or 500 ,um (Medpacific andTS1).Apart from technical convenience, separating the illumination and collection areas by 250-500 pm appears to provide an optimal compromise in terms of signal-to-noise ratio and depth of the sampledvolume. Smaller r values yield too shallow depth and greater values decrease the signal-to-noise ratio by reducing the total scattered light impinging on the photodetector (Nilsson, 1990). In ONH LDF. such a dual fiber system in contact with the tissue cannot be implemented without surgical intervention and, therefore, for the technique to be non-invasive, a measuring scheme of the type
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(A) I
I ml”
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Time (mm)
FIG. 5. Change in F induced by diifuse flicker illumination at 10 Hz (a) of a % 30” area centered at the disk of a dark adapted cat retina. A, The photocurrent was simultaneously fed into the TSI LaserFlo and Periflux flow monitors for comparison of the results obtained with these two instruments. Time constants of the flow monitors were 5 and 3 sec. respectively. B, F changes obtained simultaneously at two different areas of the optic disk, rim and center, using two systems of near-infrared laser, output fiber and flowmeter. C, In another cat, we recorded flicker induced F changes obtained when the output fiber aperture was placed on top of the incident laser beam at the disk and off the incident beam by approximately 150 pm. The three F responses were obtained at different flicker wavelengths.
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FIG. 6. Flash evoked increase in F after more than one hour of dark adaptation. A, Measurements performed at the center of the ONH. One flash of 20 psec duration was administered (at time = 0) every 2 min, for a total of six flashes. Time constant of the flow monitor was 5 sec. B, Time course of mean F obtained from the six recordings shown in the top part of the figure. C, F response obtained in two other animals tested. For the upper curve, the intensity of the flash was three times that of the flash used to record the lower curve.
504
C. E. RIVA (A)
Laser
To detector
hght
-0ptml
fibers
-
Lam
FIG. 7. Optical arrangement for non-invasively measuring
tissue blood flow. A, In an exposed tissue such as the skin, a dual optical fiber system is used to deliver the incident laser light and detect the scattered light. B, In the tissue of the optic nerve head, the laser beam is focused at the optic disk and the scattered light is detected in an image plane of the retina by an optical fiber. The site of laser beam and site of detection can be controlled independently.
shown in Fig. 7(B) must be used. In an emmetropic eye, the laser beam is delivered as a parallel beam to the cornea and focused by the optical system of the eye on the disk tissue. The diameter of the area directly illuminated by the beam at the disk depends upon the width of the beam at the cornea, the wavelength of the laser light and the refractive power of the eye. The scattered light is collected by an optical fiber in the retinal image plane of the fundus camera. When deciding upon the placement of the aperture of this fiber relative to the incident beam at the disk, two main factors must be considered, namely the maximum laser irradiance (W cm-2) that can be safely delivered to the fundus for a given wavelength and the vascular anatomy of the disk. Using irradiances below the maximum permissible level for humans, a detectable signal is obtained only for small rs, as the intensity of the scattered light reaching the detector decreases rapidly with increasing r [Fig. 2(A)]. Furthermore the presence of numerous retinal arterioles or venules close to each other in the disk severely limits r, in particular if measurements are done in eyes possessing a central retinal vasculature, such as those of monkeys or humans. Figure 2 shows that F depends strongly upon r. The main reason for the rapid drop in F is that the incident
ET AL.
beam was placed at the periphery and that, in all cats measured, F was always smaller at the center of the disk than in the periphery, as we observed when the disk was scanned, keeping r constant (unpubl. res.). In previous work with LDV in the ONH (Riva, Grunwald and Sinclair, 1982; Sebag et al., 1985, 1986) r was always chosen equal to zero. There are advantages in using r > 0, namely increased signal-to-noise ratio of the F recordings [Nilsson, 1990: Fig. 5(C)] and increased simple volume (see below). We believe that with r = 0, specular reflection of the incident laser beam and its variation with small motion of the eye is responsible for the increase in noise fluctuations. The strong dependence of F upon r (Fig. 2) makes it important to keep r constant if one wants to compare results obtained from different sites of the disk or from the same place at different times. This does not present any difficulty in experiments involving changes in the breathing conditions or flicker stimulation, if the optic disk does not move relative to the incident laser beam during measurements. In order to obtain valid relative measurements of ONH blood flow, the measured F must vary linearly with actual flow. If changes in P occur faster than any
regulatory change in vascular resistance, then the change in F through this system will follow linearly that of P, as described by the relationship F = P/R, where R is the vascular resistance. Previous measurements have shown that, following a step drop in P, it takes about 1.5 min for the autoregulatory process to complete its response (Riva, Grunwald and Sinclair, 1982). This suggests that during the 15 set it took for the blood pressure to drop from 80 to 5 mmHg (Fig. 3), regulatory mechanism had little effect. Therefore, the simultaneous measurements of F and P following a lethal injection of pentobarbital provide a valid method of testing the linearity between ONH blood flow and the measured F. The measurements in three cats show that a linear function provided an excellent fit to the F
versus P data (Fig. 3). Linearity between actual flow and F has been documented in other tissues, such as the skin, skeletal muscle, cerebral cortex, nerves and others (Shepherd and ijberg, 1990). This is also expected from theoretical arguments in so far as the value of F depends upon the calibration factor 2m/f( fi), where @Iis the number of times a photon has been scattered by a moving RBC before being detected and f(m) is a function of EI (Bonner and Nossal, 1990). This factor has a value of 1 for small m which is the case if the fraction of light that is Doppler shifted by RBCs is small compared to the total scattered light that is detected. Laser Doppler signals recorded from the ONH, a tissue where capillaries occupy only approximately 2.5% of the volume (Quigley, Hohmann and Addicks, 1982). show that this condition is fulfilled, since the root-mean-square value of the fluctuating photocurrent, the one due primarily to shifted light, was consistently found to represent not more than 10% of the total photocurrent. This condition insures
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that the heterodyne detection mode prevails and in this case both blood flow instruments respond linearly to flux changes. The high temporal resolution of LDF makes this technique ideal for studying rapid blood flow changes induced by breathing various types of gases, neuronal stimulation and pharmacological agents. With hyperoxia [Fig. 4(A)], F decreased with a time course and magnitude similar to blood flow in the retinal circulation (Riva, Grunwald and Sinclair, 1983). The decrease in F was significantly (P < 0.0 1, paired t-test) greater at the rim than at the center of the nerve, most probably due to oxygen leaking into the tissue from the large arterioles of the cat eye leaving the disk at the rim (Harino et al., 1990; Riva, Pournaras and Tsacopoulos, 1986). With hypoxia, the magnitude of the increases in F (between 67-89x) is within the range observed in the brain circulation (McDowall. 1966). The finding that F is strongly affected by flicker and single flashes of diffuse luminance [Figs S(A)-(C)] is important. It suggests that ONH blood flow is modulated by the ganglion cell firing rate. The possibility that the stimulus itself produces the observed change in F can be discarded for reasons which have been discussed elsewhere (Riva et al., 1991). Combining LDF and local partial pressure of oxygen (PaO,) measurements (Riva et al., 1991; Shonat et al.. 1991) during flicker stimulation may provide new insights into the coupling between blood flow, metabolism and visual function. Moreover, in contrast to experiments involving changes in the breathing conditions, which affect the supply of oxygen to the ocular tissues and the removal of waste products, the flicker stimulation appears to produce local changes in oxygen consumption, as evidenced by the results of Sperber and Bill (1989) and the drop of PaO, following the onset of the stimulus (Shonat et al., 1991). The simultaneous recordings of the flicker induced F response at two sites of the disc demonstrated local variations in the magnitude of this response that cannot be attributed to differences in the flowmeters. The nature of these variations is not known and needs to be systematically investigated to identify the factors that may play a role in this effect. Interestingly, the time course of the F increase induced by a single flash (Fig. S(A)] is very similar in the three animals tested. The reason for the variability between the F response in the same animal needs to be explored. It may result from variations in the time course of ganglion cells discharges following the flash. Measurements combining simultaneous recordings of mass activity and blood flow in the ONH are necessary to answer this question. The recordings obtained during hyperoxia and hypoxia (Fig. 4) suggest that there must be several mechanisms involved in ONH blood flow regulation, since the changes in F occur in one case predominantly through changes in blood volume (hyperoxia) and in
505
the other through changes in blood velocity (hypoxia). ’ Since changes in Vol represents changes in the capacitance vessels (Riva et al., 1990) one or both of the following two processes must take place during hyperoxia : either a decrease in the number of capillaries being perfused locally or a change in the diameter of the venules could account for the change in Vol. Our technique does not permit us to determine which of these processes takes place. In contrast, with hypoxia, the increase in F [Fig. 4(B)] occurs through an increase in V, suggesting a different process, presumably dilatation of the precapillary arteriole(s) feeding the region of measurement. Further investigations are needed to clarify these mechanisms. A central and difficult question in the application of LDF to tissue blood flow is the depth of the sampled volume. In the ONH, depending upon this depth, different vascular beds may contribute to the signal. The theoretical analysis of Bonner and Nossal (1990) shows that the average depth (z) probed by emerging photons before they exit a tissue and are detected is (Bonner and Nossal, 1990; equation 2.34): +> z 0.4
$‘”
p4
where p is the dimensionless absorption coefficient per unit scattering length within the tissue and p = r/L. L is the mean free path of a photon within the tissue, i.e. the average path a photon travels before being scattered by a RBC. This analysis is not valid as r approaches zero. As shown by the equation above, increasing r results in an increase in the sampled depth. For tissues such as the skin and using a laser at 800 nm and probe separation of 500 pm, the analysis predicts a depth of measurement of 0.5-l mm. For brain tissue, a depth greater than 1 mm is suggested by the experiments of Skarphedinsson, Harding and Thoreu (1988). With a probe separation of 150 pm, application of the above equation gives a depth of approximately 260 ,um in the skin and around 520,~m in the brain (grey matter). In the ONH, the penetration depth should be greater than in grey matter because of the greater penetration of nearinfrared light in white matter (Sterenborg et al., 1989). Therefore, with such a separation and based on Hayreh’s scheme of the ONH (Hayreh, 1969), some contribution from the lamina cribrosa to the Doppler signal, in addition to that from the anterior ONH vasculature and lamina choroidalis, is expected. Our results show that the measured ONH circulation has a regulation that is very similar to that of the retina with regard to hyperoxia. This suggests that the LDF technique, as applied in this work, either samples predominantly the surface layer of the nerve which is fed by the retinal circulation, or the deeper layers of the ONH have a regulation that is similar to that of the retina with regard to hyperoxia. The investigation of Harino et al. (1991), demonstrating that the calcium channel blocker nicardipine increases F but not retinal blood flow in cats, provides additional evidence that FliK5i
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the volume sampled in near-infrared LDF includes layers of the ONH deeper than those supplied by retinal vessels. In conclusion, LDF is a powerful technique to investigate changes of blood flow in the ONH of anesthetized animals. Such changes can be induced by physiologic maneuvers involving the breathing of various gases or neuronal stimulation, as demonstrated in this paper, or by the administration of pharmacologic agents (Harino et al., 1991). The technique is highly sensitive, reproducible and accurate. Its fast response time has enabled us to demonstrate changes in blood flow occurring in seconds.This capacity may open new avenues in the elucidation of the mechanismsof blood flow regulation in this complex ocular tissue.
McDowall, D. G. ( 1966). Interrelationships between blood oxygen tension and cerebral blood flow. In Oxygen Measurements in Blood and Tissue (Ed. Hill, J. P. P. D. W.). Pp. 205-14. Churchill, London. Nilsson, G. E. (1990). Perimed’s LDV flowmeter. In LuserDoppler Blood Flowwletry (Vol. 107 of Developments in Cardiovascular Medicine, ch. 4) (Eds Shepherd. A. P. and ijberg, P. A). Pp. 5 7-72. Kluwer Academic Publishers : Boston. Petrig, B. I,. and Riva, C. E. (1991). Near-infrared retinal laser Doppler velocimetry and flowmetry : new delivery and detection techniques. Appl. Opt. 30, 2073-8. Quigley. H. A.. Hohmann, R. M. and Addicks, E. M. (1982). Quantitative study of optic nerve head capillaries in experimental optic disk pallor. Am. 1. Ophthalmol. 93, 689-99. Riva, C. E.. Grunwald, J. E. and Sinclair, S. H. (1982). Laser Doppler measurement of relative blood velocity in the human optic nerve head. Invest. OphthaJmoJ. Vis. Sci 22,
Acknowledgements
Riva, C. E., Harino, Flicker evoked in anesthetized Riva, C. E.. Petrig, infrared retinal
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The authors wish to thank T. McLaughlin and S. Cranstoun for their expert technical help and Drs J. E. Grunwald and Vo Van Toi for their critical reading of the manuscript. Supported by NIH Grants EY03242, EY08413, The Vivian Simkins Lasko Research Fund, the Pennsylvania Lions Sight Conservation & Eye Research Foundation and Alcon Research Institute Award (CER).
References Bonner, R. F. and Nossal, R. (1990). Principles of laserDoppler flowmetry. In Laser-Doppler Blood FJowmetry (Vol. 107 of Developments in Cardiovascular Medicine, ch. 2) (Eds Shepherd, A. P. and Oberg, P. A). Pp. 1745. Kluwer Academic Publishers : Boston. Borgos,J. A. (1990). TSI’s LDV blood flowmeter. In LaserDoppler Blood Flowmetry (Vol. 107 of Developments in Cardiovascular Medicine, ch. 5) (Eds Shepherd, A. P. and iiberg. P. A). Pp. 73-92. Kluwer Academic Publishers: Boston. Geijer, C. and Bill, A. (1979). Effects of raised intraocular pressure on retinal, prelaminar, laminar and retrolaminar optic nerve blood flow in monkeys. Invest. OphthaJmoJ. Vis. Sci. 18, 103042. Gherezghiher, T., Okubo, H. and Koss, M. C. (1991). Choroidal and ciliary blood flow analysis: application of laser Doppler flowmetry in experimental animals. Exp. Eye Res. 53(2), 151-6.
Harino, S..Riva, C. E.,Petrig, B. L. andShonat,R. D. (1991). Effect of nicardipine on retinal and optic nerve blood flow in anesthetized cats. Invest OphthaJmoJ. Vis. Sci. 32(4) (Suppl.), 1031. Harino, S., Riva, C. E., Shonat, R. D., Petrig B. L. and McLaughlin, T. J, (1990). Reactivity of retinal and optic nerve head blood flow to 100% 0, breathing. Invest. Ophthalmol. Iris. Sci 31(4) (Suppl.), 380. Hayreh, S. S. (1969). Blood supply of the optic nerve head and its role in optic atrophy, glaucoma, and oedema of the optic disc. Br. J. OphthaJmoJ. 53, 721-748. Kimura, Y., Nitta, A., Takayama, H. and Shimizu, R. (1987). The effect of raised intraocular pressures on blood flow in the optic nerve head in monkeys. Chibret Int. J. OphthaJmoJ. 5, 24-3 1.
thulmoI
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S., Shonat, R. D. and Petrig, B. L. (1991). increase in optic nerve head blood flow cats. Neurosci. Lett. 128. 291-6. B. L. and Grunwald, J. E. (1987). Near laser Doppler velocimetry. Lasers Oph-
211-15.
Riva. C. E.. Pournaras, C. J., Poitry-Yamate. C. L. and Petrig. B. L. (1990). Rhythmic changes in velocity, volume and flow of blood in the optic nerve head tissue. Microvusc. Res. 40(l). 3645. Riva, C. E.. Pournaras, C. J. and Tsacopoulos, M. (1986). Regulation of local oxygen tension and blood flow in the inner retina during hyperoxia. 1. Appl. Physiol. 61(2), 592-8. Sebag, J., Delori, F. C., Feke, G. T., et al. (1986). Anterior optic nerve blood flow decreases in clinical neurogenic optic atrophy. Ophthalmology 93, 858-65. Sebag, J.. Feke, G. T., Delori, F. C.. et al. (1985). Anterior optic nerve blood flow in experimental optic atrophy. Invest. Ophthalmol. Vis. Sci. 26, 1415-22. Shepherd, A. P. and ijberg, P. A. (1990). Laser-Doppler Blood Flowmetry. Kluwer Academic Publishers: Boston. Shonat, R. D., Riva, C. E.. Buerk, D. G. and Petrig, B. L. ( 199 1). Simultaneous measurement of local oxygen tension and blood tlow in the optic nerve of the anesthetized cat during diffuse luminance flicker. In Proceedings, Fi,fth World Congress for Microcirculation. Pp. 101. Microcirculatory Society. Skarphedinsson. J. O., Harding, H. S. and Thoreu, P. (1988). Repeated measurements of cerebral blood flow in rats. Comparisons between the hydrogen clearance method and laser Doppler flowmetry. Acta PhysioJ. Stand. 134, 13 3-42. Sossi. N. and Anderson, D. R. (1983). Effect of elevated intraocular pressure on blood flow. Arch. OphthaJmoJ. 101,98-101. Sperber. G. 0. and Bill, A. (1989). The 2-deoxyglucose method and ocular blood flow. In Ocular Blood Flow in Glaucoma: Means, Methods and Measurements. (Eds Lambrou, G. N. and Greve, E. L.). Pp. 73-80. Kugler & Ghedini Publications : Berkley. Sterenborg. H. J., Gemert, M. J. V., Kamphorst. W., Wolbers, J. G. and Hogervorst, W. (1989). The spectral dependence of the optical properties of human brain. Lasers Med. Sci. 4, 221-7. Tsacopoulos. M. and David, N. J. (1973). The effect of arterial P,, on relative retinal blood flow in monkeys. Invest. OphthaJmoJ. 12(5), 33547.
Laser
Doppler
C. E. RIVA”, University
of Pennsylvania,
(Received
Chicago
Flowmetry
S. HARINO, Department
7 October
in the
B.L. PETRIG
AND
Optic
Nerve
R. D.SHONAT
of Ophthalmology PA, U.S.A.
and Scheie Eye Institute,
1991 and accepted
in revised form 29 January
Philadelphia,
7992)
Laser Doppler flowmetry (LDF) is a technique that measuresrelative averagevelocity, number and flux (number times velocity) of red blood cells in a tissue. In this paper, we demonstrate its application in the optic nerve headtissue,describethe laserdelivery and light scatteringdetectionschemes and investigate
the effect of the distancebetweenthe sitesof illumination and detection.We alsoprovide evidencethat the flow measuredby LDF varies linearly with actual blood flow in the optic nerve and examine the question of the depth of the sampledvolume. Experimentsin anesthetizedcats illustrate potential applicationswhich makeuseof the high temporalresolutionof LDF. These include the responseof blood flow to changesin the compositionof the breathing gasesand changesinducedby neuronalstimulation with multiple and single flashes. Key words: ocular circulation ; optic nerve blood flow ; laser Doppler flowmetry ; hyperoxia : hypoxia : hypercapnia : luminance flicker: neuronal stimulation.
1. Introduction The study of hemodynamics in the optic nerve head (ONH) tissue requires a technique capable of measuring blood flow in this region. Ideally, this technique should be non-invasive, in view of its application in humans, sensitive, so that pathological changes can be detected as early as possible, reproducible and accurate. Furthermore, it should have a short response time to allow the temporal characterization of blood flow regulatory processes in response to various induced physiological stimuli and, consequently, help identify the mechanisms involved in these processes. Quantitative investigations of ONH hemodynamics have been performed using unlabeled microspheres (Geijer and Bill, 1979), iodoantipyrine (Sossi and Anderson, 1983), hydrogen clearance (Kimura et al., 198 7) and the non-invasive laser Doppler velocimetry (LDV) and laser Doppler flowmetry (LDF). Early studies with LDV have dealt only with measurements of relative blood velocity in the microcirculation of the ONH (Riva, Grunwald and Sinclair, 1982). More recent ones have measured relative blood flow in the ONH either by combining LDV with three wavelength reflectometry (Sebag et al., 1985, 1986) or by using LDF (Riva et al., 1990, 1991). In LDF, a laser beam illuminates a small volume of a tissue and some of the light scattered by the tissue and red blood ceils (RBCs) is detected by a photodetector. Relative tissue blood flow is obtained by electronic processingof the photocurrent, a technique which has now been validated for various organs, including skin, nasal and intestinal mucosa, kidney * For correspondence at: Scheie Eye Institute. 51 North 39th Street. Philadelphia. PA 19104, U.S.A. 0014-4835/92/090499+08
$08.00/O
and peripheral nerve tissue (Shepherd and ijberg, 1990) and in the choroid of anesthetized cats (Gherezghiher et al., 1991). This paper demonstrates that also ONH blood flow can be monitored by LDF, examines new aspects of this particular extension of LDF and presentsexamples of applications in anesthetized cats that illustrate its potential. 2. Materials and Methods Laser Doppler Flowmetry LDF is based on the Doppler effect: laser light scattered by a moving particle is shifted in frequency by an amount Af = (2n/h) (IS-K,). V. Ki and K, are the wave vectors of the incident and scattered light, respectively. V is the velocity vector of the particle and h is the wavelength of the incident light. When the laser beam impinges on RBCsmoving in a tissue with different velocity vectors, the spectrum of the scattered light, the so-called Doppler shift power spectrum (DSPS), has a width that is due not only to the multiplicity of Vs, but also to the effect of scattering of the laser light by the tissueitself. As a result, the RBCs do not receive light only in the direction of the incident beam, but rather from numerous random directions. Furthermore, light scattered by a RBC can reach the detector along various directions due to additional scattering by the tissue or other RBCs (Fig. 1). These processesgive rise to a multiplicity of K, and &-vectors and, consequently, to a broader DSPS than would otherwise result solely from the various Vs. In the application of LDF to the ONH, an optical system mounted to a Topcon TRC FE fundus camara allows delivery of one or two laser beams to any site of the posterior pole of the eye (Riva, Petrig and Grunwald, 198 7 ; Petrig and Riva, 1991). Different 0 1992 Academic
Press Limited
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C. E. RIVA
Detected )/ light /
,,I
ET AL.
independent of the intensity of the scattered light. The mode of operation of these instruments has been described in detail elsewhere (Nilsson, 1990: Borgos, 1990).
/
Animal Preparation
FIG. 1. Schematic diagram of laser light impinging on a tissue and leaving it in the direction of the detector after having been scattered by the tissue (static scatterers) and red blood cells, RBCs. Adapted from Bonner and Nossal. 1990, Fig. 2-2.
lasers can be interchanged to allow for measurements at different wavelengths, such as the 543 and 632.8 nm lines of the helium-neon laser and various diode lasers lines between 670-8 12 nm. In this work, lasers at 785 and 812 nm were used. In addition to the probing laser light, an area of the fundus (30”) is illuminated by a tungsten source for observation or a flash source for diffuse flicker illumination of the fundus. The spectrum of this illumination can be varied using interference or color glass filters placed in the fundus illumination beam. Each incident beam is focused at the disk, at sites away from visible vessels. The scattered laser light emerging from the pupil (approximately 5” cone) is collected in the retinal image plane of the fundus camera by an optical fiber (output fiber) having a nominal aperture of approximately 400 ,um. The projected image of this aperture at the fundus is approximately 160 ,um. The image of the aperture of the output fiber is usually placed slightly (approximately 100 pm) off the site of the incident laser beam. The collected light is guided by the fiber to a photodetector (Riva, Petrig and Grunwald, 198 7 ; Petrig and Riva, 199 1) and the resulting photocurrent is amplified before being fed into an electronic interface whose function is to separately amplify the AC and DC components. These two components are then processed by the electronic system of a PeriFlux PF3 (Perimed, Inc., Stockholm, Sweden) or a TSI LaserFlo (Vasamedics, Minneapolis, U.S.A.) laser Doppler flowmeter, after filtering of the low frequency artifacts caused by tissue motion. The output signals are the relative number, Vol (cc volume of moving blood) and flux (F) of the RBCs in the sampled volume (Borgos, 1990). The TSI LaserFlow also gives the average velocity (V) of the RBCs. All three flow parameters are
Twenty adults cats, weighing 2.0-3.5 kg, were used. Each cat was premeditated with atropine (0.04 mg kg-‘, subcutaneously) and anesthetized with intramuscular ketamine hydrochloride (22 mg kg-‘) and acepromazine maleate (2 mg kg-‘). Catheters were placed in a femoral artery and vein and a tracheostomy performed. A loading dose of pancuronium bromide (0.2 mg kg-‘) was given intravenously and the animal was ventilated with 2 1 Y00,, 50% N,O and 29% N, using a variable volume respirator. Arterial blood pressure, tidal CO, and heart rate were monitored continuously. Arterial pH. PaCO,, and PaO, were monitored intermittently using a blood gas analyser and adjustments of the inspired gas mixture, tidal volume and respiration rate were made to keep approximately pH 7.4, PaCO, 31 mmHg and PaO, 3 90 mmHg and mean blood pressure between 85-l 10 mmHg. Rectal temperature was maintained at approximately 38°C. Halothane (0.7-1.5 %) and pancuronium bromide was administered (0.15 mg kg-’ hr-‘) infused continuously. The pupils were dilated with 1% tropicamide and 10% phenylephrine and the cat was placed prone on a table with the head secured in a special clamp. A ring was sutured to the eye with three stitches at the limbus and held in a iixed position to prevent eye motion. A zero diopter contact lens was placed on the cornea protected with Healon TM. All experimental procedures conformed to the ARVO Resolution on the Use of Animals in Research and The Presbyterian Medical Center of Philadelphia Guidelines on Animal Research. In three cats, the flow parameters V, Vol and F were measured as a function of the distance (r) between the center of the directly illuminated site and the center of the aperture of the output fiber. The incident beam had a wavelength of 812 nm. r was varied by moving the output fiber at a known speed using a motorized mechanical system. Calibration of r was achieved by comparing the width of the optic disk measured in vivo with that measured from a flat preparation of the same retina. In three cats, we tested the relationship between perfusion pressure, P and F by simultaneously recording femoral artery blood pressure, intraocular pressure (IOP) and F following a lethal injection of pentobarbital. P was calculated as the difference between femoral artery blood pressure and IOP. The IOP was measured using a needle inserted into the anterior chamber and connected to a Harvard Apparatus pressure transducer. To demonstrate potential applications of the technique, various physiological maneuvers were carried
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out and their effect on F documented. These included hyperoxia, hypoxia, multiple and single flash stimulation of the retina. Hyperoxia and hypoxia. Hyperoxia was produced by having each of 16 cats breathe 100% oxygen. V, Vol and F were measured at two sites of the disk, one at the rim, the other at the center. both away from vessels that were visible when the fundus was observed in white light. Pure oxygen was given for approximately 5 min. Hypoxia was induced in each of five cats by having them breathe mixtures of 10% 0, and 90% N,. In all these experiments, the output fiber aperture was placed at a distance r approximately 100 /lrn from the incident beam. Multiple
and single flash stimulation
of the retina.
Multiple flashes (flicker) diffusely illuminating an area of the fundus centered at the optic nerve head and having a diameter of approximately 30” were delivered by means of a fiberoptic cable whose input collected the light from a Xenon arc lamp placed at the focal point of a parabolic reflector (Grass visual stimulator, model PS22). The output was placed against the light bulb used for fundus illumination in the previously described LDV system (Riva, Petrig and Grunwald, 198 7). We used flashes of 20 ,usec duration, delivered at a frequency of 10 Hz. In these experiments, we also compared the results obtained with the Periflux and the TSI flowmeters by simultaneously supplying the input of each instrument with the same photocurrent during flicker stimulation. Furthermore, the presence of spatial variations in the time course and magnitude of the change in F during luminance flicker was explored in nine cats. simultaneously recording F from two sites of the disk, using two near-infrared lasers. Reproducibility of the flicker-induced change in F was determined in one cat by recording 15 successive F responses under identical experimental conditions, i.e. 45 set of diffuse luminance flicker at 10 Hz and the coefficient of variation (standard deviation/mean) was calculated. In one cat, we measured F during 10 Hz flicker stimulation simultaneously in two different ways. Using two output fibers, but one incident laser beam, we placed one of the fibers on top of the laser beam at the optic disk and the other at a distance r of approximately 150 pm. The photocurrent signals were simultaneously analysed with the TSI LaserFlow and PeriFlux. In three cats, while F was measured continuously, single 20 psec flashes (approximately 40 PJ cm-” per flash) were delivered in succession after dark adapting the animal for more than 1 hr. A period of at least 2 min separated the flashes.
two cats tested. In particular, F and Vol always decreased rapidly with r. A typical plot of F as a function of perfusion pressure (P) after a lethal injection of pentobarbital is shown in Fig. 3. A linear fit shows a highly significant correlation between F and P (r = 0.98, P < 0.01)). Identical results were obtained in the other two cats tested, with correlation coefficients of 0.98 and 0.99 (P < 0.01). The effect of hyperoxia on V, Vol and F is demonstrated by the typical recordings shown in Fig 4(A). In these recordings, arterial blood PaO, increased from 100 to 450 mmHg. This measurement was performed with the incident laser beam focused at the rim of the optic disk. In all 16 cats measured, F decreased markedly during approximately 5 min of pure oxygen breathing. The decrease in F was significantly (P < 0.01, paired t-test) greater at the rim than at the center of the nerve (49 &- 15 Y0versus 39 ? 12 %, mean &-95 % confidence interval of the mean). It occurred entirely through a decrease in Vol, since the group average change in V was not significant (P > 0.05). The effect of hypoxia resulted in the changes in V, Vol and F typified by the recordings in Fig. 4(B). Similar results were obtained in the other four cats. When F reached a plateau (between 3-4 min of hypoxia), its value relative to baseline (air breath201
Ot0
I 100
I 200
/ 300
/ 400
6 0
I (pm)
FIG. 2. Relative V (H), Vol (0) and F (0) of r for the same position of the laser beam.
as a function
60
3. Results Figure 2 represents V(r), Vol(r) and F(r) obtained when the incident laser beam was placed in the nasal part of the disk. Similar curves were consistently found at other peripheral locations of the disk in the other
I 500
i 8( PerfusIon
pressure
(mmHg)
FIG. 3. Relationship between ONH F and perfusion pressure (femoral artery mean blood pressure-IOP) after injection of a lethal dose of pentobarbital.
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ET AL.
(A)
I.2 t
L
0.6
0.6
c.
,.,I
,
.
,
/
.
I.0 0.6 0
0
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4
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FIG. 4. Typical time coursesof V, Vol and F during the breathing of (A) 100% 0, and (B) a mixture of 10% 0, and 90% N,, which decreasedthe arterial bloodPaO,from 93 to 33 mmHg.The incident laserbeamwasfocusedat the disk, away from visible vessels.Time constant of the flow monitor was 5 sec.The shadedrectangle representsthe time during which gas mixtures other than air were administered.
ing) was increased by 67, 75, 80, 81 and 89% in the five cats, respectively. The increases in F were mainly produced by increases in V since the maximum change in Vol among the five animals was an increase of 13%. The measurements in Fig. 5(A) illustrate the flicker evoked change in F and also allow a comparison between the PeriFlux and the TSI flowmeters. Both instruments provided quantitatively similar results. A clear increase in F was consistently observed within seconds after the initiation of the flicker stimulation. Although not shown here, the increase in F was predominantly the result of an increase in Vol. Based on 15 measurements,the coefficient of variation of the flicker-induced F response,expressedas 100 % x standard deviation/mean, was found to be 12%. Recordings obtained simultaneously from two sitesof the disk in nine cats revealed spatial variations in the time course and magnitude of the F response.For example, in the recordings shown in Fig. 5(B), the increase in F was faster and larger at the center of the nerve than at the periphery. However, based on the results in seven cats, we found no significant difference in the F responsebetween center and periphery, since in four animals the F increase was larger in the center and in three cats larger at the periphery. Simultaneous recordings of F obtained with two output fibers placed at r = 0 and approximately 150 pm during 10 Hz flicker (20 ,usecflashes) are shown in Fig. S(C). The
recording with r = 0 is noisier and less stable. Fig. 6(A) shows six F responsesto a single 20 ksec flash and Fig. 6(B) the mean and the 95% confidence interval of the mean of these responses. Similar responsesto single flashes in terms of time course and magnitude of the F change were obtained in the two other cats studied [Fig. 6(C)]. 4. Discussion In conventional LDF, where the tissue is directly exposed,the laserbeam is delivered through an optical fiber (input fiber) and the scattered light is collected and guided to a photodetector by a second optical fiber [output fiber: Fig. 7(A)]. These two fibers, which are in direct contact with the tissue, are separated by a distance r of 250 pm (Perimed) or 500 ,um (Medpacific andTS1).Apart from technical convenience, separating the illumination and collection areas by 250-500 pm appears to provide an optimal compromise in terms of signal-to-noise ratio and depth of the sampledvolume. Smaller r values yield too shallow depth and greater values decrease the signal-to-noise ratio by reducing the total scattered light impinging on the photodetector (Nilsson, 1990). In ONH LDF. such a dual fiber system in contact with the tissue cannot be implemented without surgical intervention and, therefore, for the technique to be non-invasive, a measuring scheme of the type
LDF
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NERVE
(A) I
I ml”
I
100 t
0
2
4
6
0
Time (mm)
FIG. 5. Change in F induced by diifuse flicker illumination at 10 Hz (a) of a % 30” area centered at the disk of a dark adapted cat retina. A, The photocurrent was simultaneously fed into the TSI LaserFlo and Periflux flow monitors for comparison of the results obtained with these two instruments. Time constants of the flow monitors were 5 and 3 sec. respectively. B, F changes obtained simultaneously at two different areas of the optic disk, rim and center, using two systems of near-infrared laser, output fiber and flowmeter. C, In another cat, we recorded flicker induced F changes obtained when the output fiber aperture was placed on top of the incident laser beam at the disk and off the incident beam by approximately 150 pm. The three F responses were obtained at different flicker wavelengths.
(Cl Flash
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FIG. 6. Flash evoked increase in F after more than one hour of dark adaptation. A, Measurements performed at the center of the ONH. One flash of 20 psec duration was administered (at time = 0) every 2 min, for a total of six flashes. Time constant of the flow monitor was 5 sec. B, Time course of mean F obtained from the six recordings shown in the top part of the figure. C, F response obtained in two other animals tested. For the upper curve, the intensity of the flash was three times that of the flash used to record the lower curve.
504
C. E. RIVA (A)
Laser
To detector
hght
-0ptml
fibers
-
Lam
FIG. 7. Optical arrangement for non-invasively measuring
tissue blood flow. A, In an exposed tissue such as the skin, a dual optical fiber system is used to deliver the incident laser light and detect the scattered light. B, In the tissue of the optic nerve head, the laser beam is focused at the optic disk and the scattered light is detected in an image plane of the retina by an optical fiber. The site of laser beam and site of detection can be controlled independently.
shown in Fig. 7(B) must be used. In an emmetropic eye, the laser beam is delivered as a parallel beam to the cornea and focused by the optical system of the eye on the disk tissue. The diameter of the area directly illuminated by the beam at the disk depends upon the width of the beam at the cornea, the wavelength of the laser light and the refractive power of the eye. The scattered light is collected by an optical fiber in the retinal image plane of the fundus camera. When deciding upon the placement of the aperture of this fiber relative to the incident beam at the disk, two main factors must be considered, namely the maximum laser irradiance (W cm-2) that can be safely delivered to the fundus for a given wavelength and the vascular anatomy of the disk. Using irradiances below the maximum permissible level for humans, a detectable signal is obtained only for small rs, as the intensity of the scattered light reaching the detector decreases rapidly with increasing r [Fig. 2(A)]. Furthermore the presence of numerous retinal arterioles or venules close to each other in the disk severely limits r, in particular if measurements are done in eyes possessing a central retinal vasculature, such as those of monkeys or humans. Figure 2 shows that F depends strongly upon r. The main reason for the rapid drop in F is that the incident
ET AL.
beam was placed at the periphery and that, in all cats measured, F was always smaller at the center of the disk than in the periphery, as we observed when the disk was scanned, keeping r constant (unpubl. res.). In previous work with LDV in the ONH (Riva, Grunwald and Sinclair, 1982; Sebag et al., 1985, 1986) r was always chosen equal to zero. There are advantages in using r > 0, namely increased signal-to-noise ratio of the F recordings [Nilsson, 1990: Fig. 5(C)] and increased simple volume (see below). We believe that with r = 0, specular reflection of the incident laser beam and its variation with small motion of the eye is responsible for the increase in noise fluctuations. The strong dependence of F upon r (Fig. 2) makes it important to keep r constant if one wants to compare results obtained from different sites of the disk or from the same place at different times. This does not present any difficulty in experiments involving changes in the breathing conditions or flicker stimulation, if the optic disk does not move relative to the incident laser beam during measurements. In order to obtain valid relative measurements of ONH blood flow, the measured F must vary linearly with actual flow. If changes in P occur faster than any
regulatory change in vascular resistance, then the change in F through this system will follow linearly that of P, as described by the relationship F = P/R, where R is the vascular resistance. Previous measurements have shown that, following a step drop in P, it takes about 1.5 min for the autoregulatory process to complete its response (Riva, Grunwald and Sinclair, 1982). This suggests that during the 15 set it took for the blood pressure to drop from 80 to 5 mmHg (Fig. 3), regulatory mechanism had little effect. Therefore, the simultaneous measurements of F and P following a lethal injection of pentobarbital provide a valid method of testing the linearity between ONH blood flow and the measured F. The measurements in three cats show that a linear function provided an excellent fit to the F
versus P data (Fig. 3). Linearity between actual flow and F has been documented in other tissues, such as the skin, skeletal muscle, cerebral cortex, nerves and others (Shepherd and ijberg, 1990). This is also expected from theoretical arguments in so far as the value of F depends upon the calibration factor 2m/f( fi), where @Iis the number of times a photon has been scattered by a moving RBC before being detected and f(m) is a function of EI (Bonner and Nossal, 1990). This factor has a value of 1 for small m which is the case if the fraction of light that is Doppler shifted by RBCs is small compared to the total scattered light that is detected. Laser Doppler signals recorded from the ONH, a tissue where capillaries occupy only approximately 2.5% of the volume (Quigley, Hohmann and Addicks, 1982). show that this condition is fulfilled, since the root-mean-square value of the fluctuating photocurrent, the one due primarily to shifted light, was consistently found to represent not more than 10% of the total photocurrent. This condition insures
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that the heterodyne detection mode prevails and in this case both blood flow instruments respond linearly to flux changes. The high temporal resolution of LDF makes this technique ideal for studying rapid blood flow changes induced by breathing various types of gases, neuronal stimulation and pharmacological agents. With hyperoxia [Fig. 4(A)], F decreased with a time course and magnitude similar to blood flow in the retinal circulation (Riva, Grunwald and Sinclair, 1983). The decrease in F was significantly (P < 0.0 1, paired t-test) greater at the rim than at the center of the nerve, most probably due to oxygen leaking into the tissue from the large arterioles of the cat eye leaving the disk at the rim (Harino et al., 1990; Riva, Pournaras and Tsacopoulos, 1986). With hypoxia, the magnitude of the increases in F (between 67-89x) is within the range observed in the brain circulation (McDowall. 1966). The finding that F is strongly affected by flicker and single flashes of diffuse luminance [Figs S(A)-(C)] is important. It suggests that ONH blood flow is modulated by the ganglion cell firing rate. The possibility that the stimulus itself produces the observed change in F can be discarded for reasons which have been discussed elsewhere (Riva et al., 1991). Combining LDF and local partial pressure of oxygen (PaO,) measurements (Riva et al., 1991; Shonat et al.. 1991) during flicker stimulation may provide new insights into the coupling between blood flow, metabolism and visual function. Moreover, in contrast to experiments involving changes in the breathing conditions, which affect the supply of oxygen to the ocular tissues and the removal of waste products, the flicker stimulation appears to produce local changes in oxygen consumption, as evidenced by the results of Sperber and Bill (1989) and the drop of PaO, following the onset of the stimulus (Shonat et al., 1991). The simultaneous recordings of the flicker induced F response at two sites of the disc demonstrated local variations in the magnitude of this response that cannot be attributed to differences in the flowmeters. The nature of these variations is not known and needs to be systematically investigated to identify the factors that may play a role in this effect. Interestingly, the time course of the F increase induced by a single flash (Fig. S(A)] is very similar in the three animals tested. The reason for the variability between the F response in the same animal needs to be explored. It may result from variations in the time course of ganglion cells discharges following the flash. Measurements combining simultaneous recordings of mass activity and blood flow in the ONH are necessary to answer this question. The recordings obtained during hyperoxia and hypoxia (Fig. 4) suggest that there must be several mechanisms involved in ONH blood flow regulation, since the changes in F occur in one case predominantly through changes in blood volume (hyperoxia) and in
505
the other through changes in blood velocity (hypoxia). ’ Since changes in Vol represents changes in the capacitance vessels (Riva et al., 1990) one or both of the following two processes must take place during hyperoxia : either a decrease in the number of capillaries being perfused locally or a change in the diameter of the venules could account for the change in Vol. Our technique does not permit us to determine which of these processes takes place. In contrast, with hypoxia, the increase in F [Fig. 4(B)] occurs through an increase in V, suggesting a different process, presumably dilatation of the precapillary arteriole(s) feeding the region of measurement. Further investigations are needed to clarify these mechanisms. A central and difficult question in the application of LDF to tissue blood flow is the depth of the sampled volume. In the ONH, depending upon this depth, different vascular beds may contribute to the signal. The theoretical analysis of Bonner and Nossal (1990) shows that the average depth (z) probed by emerging photons before they exit a tissue and are detected is (Bonner and Nossal, 1990; equation 2.34): +> z 0.4
$‘”
p4
where p is the dimensionless absorption coefficient per unit scattering length within the tissue and p = r/L. L is the mean free path of a photon within the tissue, i.e. the average path a photon travels before being scattered by a RBC. This analysis is not valid as r approaches zero. As shown by the equation above, increasing r results in an increase in the sampled depth. For tissues such as the skin and using a laser at 800 nm and probe separation of 500 pm, the analysis predicts a depth of measurement of 0.5-l mm. For brain tissue, a depth greater than 1 mm is suggested by the experiments of Skarphedinsson, Harding and Thoreu (1988). With a probe separation of 150 pm, application of the above equation gives a depth of approximately 260 ,um in the skin and around 520,~m in the brain (grey matter). In the ONH, the penetration depth should be greater than in grey matter because of the greater penetration of nearinfrared light in white matter (Sterenborg et al., 1989). Therefore, with such a separation and based on Hayreh’s scheme of the ONH (Hayreh, 1969), some contribution from the lamina cribrosa to the Doppler signal, in addition to that from the anterior ONH vasculature and lamina choroidalis, is expected. Our results show that the measured ONH circulation has a regulation that is very similar to that of the retina with regard to hyperoxia. This suggests that the LDF technique, as applied in this work, either samples predominantly the surface layer of the nerve which is fed by the retinal circulation, or the deeper layers of the ONH have a regulation that is similar to that of the retina with regard to hyperoxia. The investigation of Harino et al. (1991), demonstrating that the calcium channel blocker nicardipine increases F but not retinal blood flow in cats, provides additional evidence that FliK5i
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the volume sampled in near-infrared LDF includes layers of the ONH deeper than those supplied by retinal vessels. In conclusion, LDF is a powerful technique to investigate changes of blood flow in the ONH of anesthetized animals. Such changes can be induced by physiologic maneuvers involving the breathing of various gases or neuronal stimulation, as demonstrated in this paper, or by the administration of pharmacologic agents (Harino et al., 1991). The technique is highly sensitive, reproducible and accurate. Its fast response time has enabled us to demonstrate changes in blood flow occurring in seconds.This capacity may open new avenues in the elucidation of the mechanismsof blood flow regulation in this complex ocular tissue.
McDowall, D. G. ( 1966). Interrelationships between blood oxygen tension and cerebral blood flow. In Oxygen Measurements in Blood and Tissue (Ed. Hill, J. P. P. D. W.). Pp. 205-14. Churchill, London. Nilsson, G. E. (1990). Perimed’s LDV flowmeter. In LuserDoppler Blood Flowwletry (Vol. 107 of Developments in Cardiovascular Medicine, ch. 4) (Eds Shepherd. A. P. and ijberg, P. A). Pp. 5 7-72. Kluwer Academic Publishers : Boston. Petrig, B. I,. and Riva, C. E. (1991). Near-infrared retinal laser Doppler velocimetry and flowmetry : new delivery and detection techniques. Appl. Opt. 30, 2073-8. Quigley. H. A.. Hohmann, R. M. and Addicks, E. M. (1982). Quantitative study of optic nerve head capillaries in experimental optic disk pallor. Am. 1. Ophthalmol. 93, 689-99. Riva, C. E.. Grunwald, J. E. and Sinclair, S. H. (1982). Laser Doppler measurement of relative blood velocity in the human optic nerve head. Invest. OphthaJmoJ. Vis. Sci 22,
Acknowledgements
Riva, C. E., Harino, Flicker evoked in anesthetized Riva, C. E.. Petrig, infrared retinal
241-8.
The authors wish to thank T. McLaughlin and S. Cranstoun for their expert technical help and Drs J. E. Grunwald and Vo Van Toi for their critical reading of the manuscript. Supported by NIH Grants EY03242, EY08413, The Vivian Simkins Lasko Research Fund, the Pennsylvania Lions Sight Conservation & Eye Research Foundation and Alcon Research Institute Award (CER).
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