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Study of Choroidal Blood Flow by Comparison of SLO Fluorescein Angiography and Microspheres
Exp. Eye Res. (1996), 63, 693–704
Study of Choroidal Blood Flow by Comparison of SLO Fluorescein Angiography and Microspheres H. F E R D I N A N D A. D U I J M*†‡¶, A L E X A N D E R H. F. R U LO * , M A R I A A S T I N§, OLAV MA> EPEAs, T H O M A S J. T. P. V A N D E N B E R G†‡ E R I K L. G R E V E* * Glaucoma Center (Department of Ophthalmology) and † Department of Medical Physics, University of Amsterdam ; ‡ The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands ; § Prostaglandin research, Pharmacia Uppsala AB and s Department of Ophthalmology, University Hospital, University of Uppsala, Sweden. (Received Lund 22 November 1995 and accepted in revised form 6 June 1996) Choroidal hemodynamics estimated with parameters describing the dye build-up curves obtained with video fluorescein angiography, were compared with a classical regional blood flow measurement : radioactively labelled microspheres. Video fluorescein angiograms (Rodenstock’s SLO 101) and microspheres blood flow measurements were made in 13 anaesthetized pigmented rabbits. Ocular perfusion pressures were varied from 60 to 15 mmHg by changing the intraocular pressure. The angiographically derived dye build-up curves were described by means of an exponential model. One of the model parameters is the time constant τ theoretically reflecting local blood refreshment time. Labelled microspheres act as a non-recirculating blood flow indicator, enabling the estimation of regional blood flows. The relation between choroidal blood flow and perfusion pressure is nearly linear, suggesting the passive nature of choroidal vasculature. There is a significant correlation between τ and microspheres flow (R ¯ 0±67, P ! 0±01). According to the rheological model the product of blood flow and τ corresponds to the relevant blood volume. Hence, a function for the volume of the choriocapillaris as a function of perfusion pressure was established. The model parameter τ can be interpreted as the local blood refreshment time. Since the parameter τ, unlike microspheres, can be used clinically, τ may be used to retrieve information on choroidal hemodynamics in clinical practice. Information on the spatial distribution of choroidal hemodynamics is also obtained. # 1996 Academic Press Limited Key words : ocular blood flow ; microspheres ; fluorescein angiography ; computer-assisted image analysis ; choroid ; rabbit.
1. Introduction The choroidal circulation plays an important role in the blood supply of the eye, it nourishes large parts of the optic disk and the outer part of the retina. In the rabbit the choroid nourishes the retina almost completely, since in this animal the retinal circulation is virtually nonexistent. The role of a disturbed blood flow in the posterior pole has been suggested and made plausible in several ocular diseases, e.g.—normal pressure—glaucoma. However, in clinical practice assessment of the choroidal circulation proves to be difficult. The intricate three dimensional structure and the partially blocking effect of the pigments restrict interpretation of choroidal angiographic studies. Several methods have been developed to quantify ocular blood flow, e.g. duplex doppler, laser doppler velocimetry, scanning laser doppler flowmetry. Other methods involve angiography in the determination of blood flow. Ever since the introduction of fluorescein angiography the retinal circulation has been studied extensively, mostly via qualitative means. Various methods have been developed to quantify retinal circulation using photographic methods (Riva, Feke ¶ Corresponding author.
0014–4835}96}12069312 $25.00}0
and Ben-Sira, 1978 ; Bulpitt and Dollery, 1971 ; Hickham and Frayser, 1965) and in recent times using video techniques and computerized image analysis (Wolf et al., 1989, 1992, 1993). In general these methods aim to quantify retinal circulation by data derived from fluorescence intensity curves. In other words, from the shape of the dye curves, information on rheology is inferred, e.g. passage or circulation times and arterial dye velocities. Attempts have been made to quantify choroidal circulation using indocyanine green angiography and image processing (Flower and Klein, 1990 ; Klein, Baumgartner and Flower, 1990 ; Flower, 1993). In these methods the pulsatile character of the choroidal blood flow is described. As in the retinal fluorescein angiographical methods, with the aid of indocyanine green, arterial, venous and capillary filling times in the choroid can also be estimated (Pru$ nte and Niesel, 1988). Also, with the help of laser doppler velocimetry, choroidal hemodynamics in the macular area in humans were measured (Riva et al., 1994b). Also, fluorescein has been used to study choroidal hemodynamics. A vast amount of qualitative knowledge has been obtained on physiologic, normal choroidal blood flow (Hyvarinen et al., 1969 ; Oosterhuis and Boen-Tan, 1971 ; Shimizu, Yokochi # 1996 Academic Press Limited
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F. 1. (A) The graphical representation of the model curve, used to analyse the individual dye build-up curves. The time course is described by four parameters, initial fluorescence (Fmin), maximum fluorescence (Fmax), the time of first dye appearance (t ) and tau, the time-constant of the model, representing the refreshment time. (B) A few examples of choroidal dye build-up ! curves obtained in a rabbit, with the approximations using the exponential model.
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and Okano, 1974 ; Hayreh, 1974, 1990), as well as on pathological conditions such as glaucoma (Raitta and Sarmela, 1970 ; Laatikainen, 1971 ; Geijssen, 1991). In an attempt to quantify choroidal capillary hemodynamics a physical, rheological model, describing the filling process of the choriocapillaris during fluorescein angiography, was used to analyse fluorescence intensity time curves (Lambrou, Van den Berg and Greve, 1989 ; Lambrou, 1993 ; VanStokkum, Lambrou and Van den Berg, 1995). For each location of the posterior pole the dye build-up curve is analysed using the following model : Fmin 1
F(t) ¯ 2
Fmax\(1®e 3
; −(t−t!) τ
)Fmin ;
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0!t!t t&t
! (1)
!
where ; Fmin, initial fluorescence, dependent on pseudofluorescence and possible residual fluorescence of previous angiograms ; Fmax, maximum fluorescence, dependent on the amount of blood with fluorescein, blocking of pigments etc. ; t , the onset of the filling ! process, reflecting the delay time between injection site and the eye ; τ, tau, the time-constant of the model. The model curve is shown in Fig. 1(A). In Fig. 1(B) a few actual dye curves with the fitted model approximations are presented. The fit using the afore-mentioned four parameters of the dye curves was found to be accurate. This simple model describes approximately 90 % of dye curve information. There is a small, though statistically significant improvement in fit by the introduction of an extra time-constant in the model (VanStokkum et al., 1995). The advantage of analysis using this mathematical model is not only the parametrization and description of the dye curves, but also the possibility of interpretation in terms of a rheological model. At first, a simple one compartmental model was proposed for the filling process of the choriocapillaris, resulting in the above mentioned function (Eqn 1). In this context the time-constant τ of the simple model (Eqn 1) can be regarded as the local blood refreshment time : flow dV}dt 1 ¯ ¯ ¯ τ−" volume V τ
(2)
In Eqn 2 dV}dt represents the volume replaced per unit of time in a local compartment with a volume V. The term 1}τ represents the fraction replaced per unit of time, therefore equivalent to the local blood refreshment rate. This interpretation does not take into account potential effects of extravasation of fluorescein. A refinement could be to define dV}dt and V in such way that they include the relevant extravasal space. However, this problem is beyond the scope of the present paper. A basic assumption in the application of the one compartmental model is that the dye arrives in a stepwise manner ; as if the dye would have been
injected into the choroidal arteries. In a clinical setting, as well as in this study, fluorescein is injected into a peripheral vein. The dye does not arrive in a stepwise manner. To take into account influences of the circulation between injection, or in other words to take into account the form of the dye front as it enters the choriocapillaris, a second order model to analyse the choroidal dye curves would make a better choice. This refinement, the introduction of a second timeconstant into the model, corresponds with a rheological two-compartmental model : a systemic compartment and a choroidal compartment with a local choroidal blood refreshment time. Although this twocompartmental model is rheologically more realistic, its mathematical function only marginally improved the description of the dye curves (Van Stokkum et al., 1995). The build-up curves of the retinal arteries can be regarded as an indication for the form of the dye bolus entering the eye. In rabbits this approaches the step function much better. Moreover, since computing time for the two-compartmental model increases manyfold, in the present paper the one-compartment model (Eqn 1) is used. Special interest concerning the interpretation of the model parameter τ arises from clinical findings. Choroidal hemodynamics, determined by scanning laser angiography and parametrization of the peripapillary choroid (SLAPPC) in (especially normal pressure) glaucoma patients as compared to normal, healthy subjects were slowed down. The average of the parameter tau in the peripapillary area of these patients was significantly larger (Greve et al., 1994). By comparing choroidal hemodynamics in glaucoma patients before and after trabeculectomy, it appeared that choroidal hemodynamics improved significantly after surgery (Greve et al., 1995). Although from a theoretical point of view the above-mentioned analysis may seem reliable, and clinical results are promising, its rheological meaning, such as the interpretation of τ as refreshment time, has not been proven. The objective of the present study was to investigate the relationship between the choroidal capillary dye build-up curve and its description using Eqn 1 with a reference method for measuring blood flow. As a reference method, regional blood flow determination using the radioactively labelled microsphere technique was chosen (Alm and Bill, 1972). Numerous methods exist to quantify ocular blood flow in an experimental setting, e.g. auto radiography using tritiated iodoantiprine (Quigley et al., 1985), calorimetry (Armaly and Araki, 1975), direct measurements from choroidal veins (Bill, 1962 ; Sperber and Bill 1985, 1989), radioactive krypton desaturation (Friedman, Kopald and Smith, 1964), colored microspheres (Geijer and Bill, 1979), hydrogen clearance (Yu et al., 1988), laser Doppler velocimetry (Riva, Grunwald and Sinclair, 1982, Riva et al., 1994a, Riva et al., 1994b) and methods using the laser speckle
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phenomenon (Tamaki et al., 1994). In animal research however, partly because of its simplicity and straightforwardness, the labelled microspheres method is more attractive. The use of radioactively labelled microspheres in ocular blood flow research enables determination of blood flow in separate ocular tissues, e.g. retina and choroid (Alm et al., 1972). The technique has been used in numerous studies and on several laboratory animals such as cats, rabbits and monkeys (Alm et al., 1972 ; Alm and Bill, 1973 ; Bill, 1974). The microspheres are injected in the left ventricle of the animal and act as a nonrecirculating blood flow indicator. The microspheres become evenly mixed with blood, follow the blood flow to the various body tissues and get stuck in the capillary bed proportionally to the regional blood flow. Consequently, sample radioactivity is proportional to regional blood flow. From the results of a reference blow flow from e.g. the femoral artery, it is possible to calculate tissue blood flow in an absolute sense. Results obtained from blood flow determinations using different diameters of microspheres have shown that 15 µm spheres provide a reliable estimation of blood flow measurements in the rabbit choroid (Alm, To$ rnquist and Stjernschantz, 1977). Although the labelled microspheres technique seems straightforward, it has its own problems and intricacies ; these will be discussed later on. In the present study two different kinds of blood flow techniques were used. The use of these two methods to measure choroidal blood flow not only enables a mere comparison of two methods, but may also help to gain more insight into the relation between the angiographically derived hemodynamic parameters, i.c. τ, and more classical blood flow measurements. Although the model parameter τ−" theoretically reflects refreshment rate, its relation with basic processes and parameters, such as perfusion pressure and regional blood flow, may become more lucid : is it legitimate to interpret τ−" as the local choroidal blood refreshment rate ?
femoral arteries for registration of arterial blood pressure and collection of reference blood samples, and into one femoral vein for i.v. injections. Another catheter, for injection of microspheres, was inserted into the left heart ventricle via the right brachial artery. Tropicamide (Mydriacyl 0±5 %, Alcon) was topically administered in the experimental eye to induce mydriasis. In the experimental eye two anterior chamber needles (30 G) were inserted. The needles were connected by a polyethylene tubing (PE 20) to a reservoir in order to raise or lower the intraocular pressure (IOP) and to a pressure transducer for registration of IOP. Before cannulation of the eye, indomethacin (20 mg kg−" i.v.) was given. pO , pCO and pH of the arterial blood were checked # # with a blood gas analyser (ABL 300, Radiometer, Copenhagen) during the experiment and if necessary, adjusted by changing the ventilation or by i.v. administration of sodium bicarbonate (50 mg ml−", ACO, Sweden). A servo controlled heating pad was used to maintain normal body temperature of the animals. The arterial pressure and IOP of the experimental eye were continuously registered during the course of the experiment.
2. Materials and Methods
The perfusion pressure was controlled by adjusting IOP. The first blood flow determinations were made under a ‘ high ’, normal perfusion pressure, approximately 50 mmHg (MAP³.. 70±0³5±6 mmHg ; PP³.. 51±5³4±8 mmHg). The second injection under moderately lowered perfusion pressures (MAP³.. 67±6³8±1 mmHg ; PP³.. 38±1³4±2 mmHg), and the third under low perfusion pressures (MAP³. 68±3³10 mmHg ; PP³.. 23±5³6±4 mmHg). After stabilization of the perfusion pressure, a bolus dose of approximately 1±5¬10' labelled microspheres was injected into the left heart ventricle. This amount of microspheres was chosen to ensure that at least 400 microspheres were caught in the peripapillary choroid. On statistical grounds this will give a measurement
This study was performed in accordance with the ‘ARVO Resolution on the Use of Animals in Research ’.
Preparation Young pigmented rabbits (Swedish Loop) (n ¯ 13), weighing 1±8 to 2±3 kg, were used in the experiments. To induce general anaesthesia, pentobarbital (Mebumal, ACO, Sweden ; 30 mg kg−", i.v.) was used except for two animals which received urethane. Anaesthesia was maintained by continuous i.v. infusion of pentobarbital. The animals were tracheostomized and artificially ventilated. Heparinized polyethylene tubings (PE50) were inserted into both
Experimental Protocol Regional blood flow was measured using radioactively labelled microspheres as described by Alm et al. (1972). Three different labeled microspheres, diameter 15³2 µm, (Co-57, Sn-113 or Ru-103, Medical Dupont, New England Nuclear) were used, enabling three blood flow measurements under three different perfusion pressure conditions in each experiment. Perfusion pressure (PP) was defined as the difference between the mean arterial pressure (MAP) and intraocular pressure : pperfusion ¯ map®iop ¯ part syst"( part syst®part diast)®iop $ (3)
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error of 5 % (Buckberg et al., 1971 ; Hillerdal, Sperber and Bill, 1987). A reference blood sample was collected under free flow condition from one femoral artery (ref blood) during the first minute from the start of each injection of the microspheres. In addition, arterial blood samples were taken just before each injection to determine blood gasses. After the experiment the animal was killed by an overdose of a mixture of pentobarbital and ethanol. Both eyes, experimental and control, were enucleated, and several ocular tissues were dissected : the iris, ciliary body, peripheral and peripapillary choroid, retina and optic nerve. The peripapillary choroid was taken from the eye globe by punching an area centered around the disk with the aid of a trephane (φ9 mm). Radioactivity was measured in the tissues and blood samples with a gamma counter (Compugamma 1282 LKB Wallace) and expressed in counts per minute (cpm). Blood flow in a tissue (mg min−") could be calculated as : blood flowref blood cpmref blood
blood flowtissue ¯ cpmtissue\
(4)
Fluorescein Angiography and Model Analysis After each injection of microspheres, two to three angiograms were recorded under equal flow conditions. Because of the image analysis residual fluorescence of the previous angiograms does not affect the analysis of subsequent angiograms. Therefore angiograms could be repeated within three minutes. Moreover, due to the small amount of fluorescein (15 µl 25 %) that was injected, and also due to the relatively rapid decline of residual fluorescence, the residual fluorescence, Fmin, was low. After proper focussing of the Scanning Laser Ophthalmoscope (Rodenstock, fixed gain, 50 µW blue argon laser) 15 µl fluorescein 25 % (Alcon) was injected in the femoral vein. The fluorescein angiograms were recorded on super-VHS video tape (Panasonic AG-7350). The composition of the picture was such that the center of the picture was located on the temporal part of the retina with the medullary rays just adjacent to the optic disk. The angiogram was digitized into 50 pictures. The spatial resolution is 380¬228 pixels ; the temporal resolution is 5 pictures second−". The pictures were aligned by a specially made automatic procedure using cross correlation of pictures, analogous to methods used in radiology for image alignment (Appledorn, Oppenheim and Wellman, 1980). For the central 228¬228 pixels the model analysis was performed. In Fig. 2(A), nine examples of a series of 50 digitized angiogram frames are shown. For each of the 228¬228 pixels a dye-curve is constructed. Fluorescence intensity is plotted on the ordinate, time is plotted on the abscissa. Every dye curve is analysed according to the previously
mentioned model, Eqn 1 (Lambrou, 1993). Hence, for each location of the posterior pole hemodynamics are parametrized, described by the afore-mentioned parameters. In Fig. 1(B) some examples of dye curves with the fitted model function are given. In order to study the spatial distribution the values of the parameters can be translated into shades of grey. In this way a distribution map of t , Fmax or blood ! refreshment rate (τ−") is obtained. In Fig. 2(B) the three distribution maps for the model parameters are given. The left-hand picture shows Fmax, dark shades indicating low maximum fluorescence and vice versa. The centre picture depicts t , light shades indicating ! early onset of filling. The right-hand picture shows the blood refreshment, light shades indicating high blood refreshment rate. To determine an average blood refreshment rate for the choroid, the disc area and pixels overlying retinal vessels were excluded by an automated image analysis algorithm (roughly 10–15 % of all the pixels in the frame). The parametrization of the dye curves with low fluorescence intensities renders unreliable estimations of the parameters. Therefore, pixels with very low fluorescence were excluded as well (approximately 5 % of all the pixels). The shaded area in Fig 2(C) represents the area that is not included in the average choroidal blood refreshment rate. Statistical analysis involved analysis of variance using the F statistics. Statistical significance was defined as P ! 0±01 (except when stated otherwise).
3. Results In Table I the average blood flow values determined with the aid of microspheres are given for the baseline measurements of the experimental and control eyes. Values for e.g. the peripapillary choroid are in the order of 70 000 counts with a counting period of 10 min, corresponding to several hundreds of caught microspheres. Typical values for retina and optic nerve are 400 counts. As this corresponds to only a few caught microspheres, the results of retina and optic nerve are not included. In Fig. 3 the relation between blood flow of the iris, ciliary body, peripheral and peripapillary choroid and the perfusion pressure is given. Two animals, indicated by arrows, did not follow the general trend. The condition of one animal deteriorated during the experiment (very small flow values in Fig. 3), and was hemodynamically very unstable during the microspheres injections. The second one, unlike all other animals, showed hardly any influence of perfusion pressure. It is assumed that the perfusion pressure registration was at fault. These animals were excluded from further analysis. In Fig. 4 the relation between the model parameter τ−" (calculated from the fluorescein angiograms) and perfusion pressure is given.
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(A)
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F. 2. (A–B) For legend see facing page.
In all cases the blood flow is strongly influenced by the perfusion pressure. Regression analysis showed that there was no statistically significant difference between on the one hand single regression using only perfusion pressure, and multiple regression using intraocular and mean arterial pressure separately (analysis of variance with F statistics) on the other. Hence, it appears that only the difference between mean arterial pressure and intraocular pressure is of influence on blood flow when measured with the aid of microspheres. On analysing the results of the microspheres, the blood flow in the various tissues of the experimental eye seemed systematically higher (Table I). In a
separate paper an analysis is presented showing that blood flow not only depends on perfusion pressure, but also, to a lesser extent, on the individual animal and on the hyperaemic state of the eye (Duijm et al., submitted). Figure 5 shows the correspondence between the model parameter, τ−" and the microspheres measurement having a correlation coefficient of 0±67. For each microspheres measurement the tau values calculated from the matching angiograms were averaged. The average standard deviation of the belonging τ−" values was 0±061, corresponding to a coefficient of variation of 6±1 %. In general blood flow in a tissue describes the
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perfusion pressure. The data show variability of the vascular bed for which there may be two reasons. On the one hand, hyperaemia influences vascular volume in the experimental eye (Duijm et al., submitted) ; this hyperaemia causes increased circulatory volume. On the other hand, the size of the vascular bed as it is, may vary from one animal to another (Duijm et al., submitted). These two factors may affect the volume estimation using Eqn. 6. In Fig. 6 (lower graph) blood volumes corrected for these two effects are presented. These volumes represent the values for an ‘ average ’ animal in normal (non-hyperaemic) condition.
(C)
Discussion
F. 2. (A) Nine examples from a digitized video angiogram sequence. Left-right, top-bottom : 1st t ¯ 0 sec, 2nd t ¯ 3 sec, subsequent time lapse 0.5 sec. (B) The three distribution maps of the model parameters. Left : maximum fluorescence (Fmax) ; middle : the time of first appearance of dye (t ) ; right : blood refreshment rate. Light shades are ! indicating high Fmax, early t and high blood refreshment ! rate. Dark shades are indicating low Fmax, late t and low ! blood refreshment rate. (C) The shaded area is the area that excluded from the average choroidal blood refreshment rate.
T I Average baseline blood flow measurements (mean³S.D.), determined with microspheres, for several ocular tissues Blood flow (mg min−")
Experimental eye
Control eye
Iris Ciliary body Peripheral choroid Peripapillary choroid
184³113 227³144 536³227 129³44
126³64 185³76 495³189 120³41
amount of blood that is being refreshed in a certain time lapse : blood flow ¯
volume time
(5)
The model parameter τ corresponds to the time needed to replace a volume equal to the total volume of the vascular bed of the considered tissue. This leads to the following equation : blood volumetissue ¯ blood flowmicrospheres\τ (6) With the help of Eqn 6 it is theoretically possible to estimate the blood volumes in the tissues of this study. In Fig. 6 (upper graph) the calculated volumes for the choroid are plotted against the corresponding
This study was intended to study the parameter τ−", estimated from fluorescein angiograms, as blood refreshment rate in the choroid and to delineate its relation to blood flow. The reproducibility of the parameter τ−" proved to be good, with a coefficient of variation of 6±1 %, and was linearly related to blood flow (Fig. 5, R ¯ 0±67). Furthermore a function was found for the volume change of the choriocapillaris with perfusion pressure (Fig. 6). In order to evaluate τ−" in this study, the reference technique should provide a reliable blood flow measurement of the choroid. Although the radioactively labelled microspheres method is straightforward, some authors have raised questions (Von Ritter et al., 1988 ; Prinzen and Glenny, 1994 ; Kiel, 1995). The choice of the microspheres for example is of some concern. An important prerequisite of the microspheres method is that no recirculation of the spheres occurs. If the microspheres are too small they will pass through the vascular bed resulting in underestimation of the regional blood flow. On the other hand, spheres that are too large will be trapped proximal to the tissue resulting in an underestimation of the blood flow. Another problem related to the size of the microspheres is axial streaming. Large, heavy spheres tend to migrate to the center of the vessel, thus causing systematic errors. From the literature, it appears that 15 µm spheres present the best choice for examination of regional ocular blood flow in rabbits (Alm et al., 1977). When the results of 9 and 15 µm microspheres in the rabbit are compared, it shows that even 9 µm microspheres are trapped in the choroidal circulation, presumably due to the flattened capillaries of the choriocapillaris. In the cat it was found that 95 % of the 15 µm spheres stayed in the uvea for more than 1 min (Alm et al., 1972). However, around 50 % of the 9 µm spheres passed through the vascular bed of the anterior uvea in the rabbit. There was no indication that axial streaming disturbed the blood flow measurements using the 15 or 9 µm spheres in the rabbit. A problem regarding the premiss of nonrecirculation is that
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F. 4. The inverse of the model parameter tau, the local refreshment rate (seconds−"), measured with the fluorescein angiograms as a function of perfusion pressure (mmHg).
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trapped microspheres may come gradually loose, thus resulting in artificially lower blood flow readings. It has been mentioned that results of microsphere studies ‘ might not be valid ’ if the experiment lasts considerably longer than 1 min (Alm et al., 1977). On the other hand, if spheres larger than 15 µm would be used, they may occlude larger vessels, thus interfering with the circulation. Furthermore, to increase spatial resolution— especially useful in small tissues such as the optic nerve—smaller microspheres, e.g. 9 µm, can be used. Also, if the total mass of injected spheres is limited to reasonable quantities, accuracy is improved when smaller microspheres are used, due to the fact that a larger number of smaller spheres can be injected. In this study, however, the microspheres method should give reliable blood flow values for tissue samples that were not small and with a relatively high blood flow, such as the peripapillary choroid. Therefore, 15 µm spheres were used.
The accuracy of the microspheres method depends, amongst other things, on the number of caught microspheres in the vascular bed studied. As special care was taken to catch at least 400 spheres in a specific tissue, the statistical fluctuation in this number (5 % for 400 caught microspheres) is much better than the physiological variation (Figs 3 and 6) (Buckberg et al., 1971 ; Hillerdal et al., 1987). Another problem using the microspheres technique, is that multiple microspheres injections might change local flow distributions. It has been speculated that, after repeated microsphere injections in cats after a period of artificially induced ischemia, blood is shunted away from the choroid towards the retina, due to obstruction of the choroid and subsequent release of vaso-dilatating agents (Roth and Pietrzyk, 1994). However, in dogs a study did not show any hemodynamic alterations that could be attributed to the number of microspheres used (Von Ritter et al., 1988). Also, the data of the present study (not shown) did not reveal blood flows in the control eye to decrease with the number of microsphere injections. Summarizing, although the blood flow measurement technique, used in this study as a ‘ standard ’, seems a relatively good choice, it still contains some uncertainties. Therefore, some care has to be taken with regard to its use. Figure 3 (blood flow versus perfusion pressure) shows considerable variation in flow amongst the animals. This variation is comparable with findings in literature : in cats Alm et al. (1972) find similar flow pressure relations. In order to understand and explain the variation it was attempted to describe the blood flow data using a model (Duijm et al., submitted). Apart from the evident influence of the perfusion pressure, the blood flow measurements using the microspheres also appeared to be influenced by other factors. In the experimental eye a vasodilatory effect or hyperaemia could be quantitatively estimated. This hyperaemia was influenced by two factors : the individual animal and the tissue type studied. Towards the posterior pole hyperaemia decreased. Several possible causes of the quantified, experimental hyperaemia could be given. The two anterior chamber needles may have touched the iris thus stimulating the release of vasoactive substances, like prostaglandins, notwithstanding the fact that indomethacin was given. Another possibility may be the topically administered tropicamide, a parasympathicolytic, cholinergic blocking substance. Also the combination of trauma (anterior chamber needles) and factors like general anaesthesia with pentobarbital may increase ocular blood flow (Bill and Stjernschantz, 1980 ; Bill, 1991). Furthermore, from the analysis of the experimental hyperaemia it appears that hyperaemia is mainly present in the anterior segment. The peripapillary choroid, the tissue of special interest in this study, showed practically no hyperaemia. Consequently, the
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experimental hyperaemia will have influenced peripapillary choroidal blood flow determinations only to a limited degree. As has already been mentioned, choroidal blood flow measured with radioactively labelled microspheres shows an evident influence of the perfusion pressure. There seems to exist a more or less proportional, linear relation between choroidal blood flow and perfusion pressure. Autoregulation would tend to maintain a relatively constant blood flow despite changes in variables such as perfusion pressure. Hence, this finding implies that there is no autoregulation of the choroidal circulation, at least not in the experimental setup used in this study. This corresponds with numerous studies suggesting that the choroid behaves like a passive vascular bed without autoregulation. Using several methods, a linear relation between blood flow and perfusion pressure has been found (Bill, 1962 ; Alm and Bill, 1970 ; Alm et al., 1972, 1973 ; Yu et al., 1988). In contrast, the retinal vasculature shows evident autoregulatory behavior in rabbits (Bill, 1974). However, in some recent studies using laser doppler flowmetry, evidence was found of myogenic autoregulation of choroidal circulation. This may be partly due to the procedure of alteration of the perfusion pressure. In the studies, which did find indications for myogenic autoregulation, the mean arterial pressure was altered and showed that the autoregulation was most effective if the arterial pressure was altered and the IOP was not controlled (Kiel and Van Heuven, 1992 ; Kiel and Shepherd, 1992 ; Kiel, 1994). In the present study as well as in most other studies, the intraocular pressure was manipulated in order to change perfusion pressure. Apart from this, peripapillary choroidal tissue as defined in the present study is approximately 60 times larger than the sample area used in the studies of Kiel. There may exist considerable local difference in choroidal blood flow (Flower, Fryczkowski and McLeod, 1995). The latter and perhaps the different techniques may explain the differences in findings. Another consideration in investigating autoregulation is the condition of the vascular bed during the experiments. Hyperaemia has been found in the experimental eye. Consequently, experimental findings regarding e.g. vasotonus or autoregulatory behavior may not reflect physiologic, in vivo vasculatory behavior. However, hyperaemia had limited effect in the choroid as opposed to the anterior segment. Hence, this may not have greatly affected the τ comparisons. The primary aim of the present study was to investigate the angiographically derived parameter τ−". The parameter τ represents the time needed to replace the total volume of the vascular bed of the tissue studied, in this case the choriocapillaris. As has already been discussed, a premiss regarding this interpretation of the parameter τ as the blood refreshment time of the choriocapillaris, is that the dye
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arrives at the choriocapillaris in a stepwise manner. Although it seems likely that the front approximates a step, this prerequisite is not fully fulfilled. Since the parameter τ represents the time needed to replace the total volume of the vascular bed of the choriocapillaris, possible changes in this volume have to be taken into account when the two methods are compared. There exists an evident dependence of the estimated volume on the perfusion pressure (Fig. 6). At physiologic perfusion pressures (60 mmHg) the estimated volume is approximately 10 µl. The fluorescein angiography analysis basically gives a blood refreshment rate of the choriocapillaris. Consequently, the 10 µl would correspond to the volume of the choriocapillaris. This value is in accordance with values which can be calculated for the rabbit. If one assumes that the average diameter of the eye globe is 17 mm while the thickness of the choriocapillaris is 20 µm (80 % blood) over two-thirds of the globe’s surface, the volume of the choriocapillaris would be 9 µl. Figure 6 indicates the volume-perfusion pressure relation of the choriocapillaris. To our knowledge no reference material for the choriocapillaris is available and therefore the relation needs verification by means of other techniques. According to literature, most volume-pressure relations of beds of small vessels are more or less logarithmical (VanBavel and Mulvany, 1994). However, one has to be aware that the choriocapillaris is a vascular bed with peculiar properties. Because of its geometric network, its large flattened capillaries and its encapsulation by a rather inelastic sclera, it cannot be easily compared with other vascular beds. It is tempting to relate this study to humans. However, there is a large difference regarding ocular vasculature between rabbits and humans. In the rabbit the retinal vascular layer is practically nonexistent, and fluorescence will be almost entirely choroidal of origin. In the normal human fundus the retinal vasculature cannot be ignored. In fluorescein angiography the large, superficial, retinal vessels, with high contrast and delicate detail, are prominent. The retinal microcirculatory network on the other hand contributes only very little to the fluorescence due to its small volume and absence of dye leakage. Additionally, in areas of delayed or nonexistent filling or in case of targeted dye delivery, small retinal vessels can be discerned, but not the retinal capillary network, or a capillary fluorescent haze. Therefore, fluorescence from fundus locations not overlying distinct retinal vessels very likely originates from the choroid. Because of absorption by e.g. hemoglobin and intra and extravasal fluorescein, the penetration depth of the exciter light in fluorescein angiography is about 30 µm. Therefore, the large choroidal vessels contribute only very little to the fluorescence intensity. Large choroidal vessels are only seen in case of very thin or atrophic choriocapillaris.
COMPARISON OF CHOROIDAL BLOOD FLOW
There are some disadvantages using the microspheres method in that the measurements are discontinuous in time, they cannot be applied in humans, and only a limited number of determinations can be made. Fluorescein angiography on the other hand enables many measurements (over 10 angiograms per rabbit in approximately 1 hr) and is applicable in humans. An attractive characteristic of the model parameter τ is its independence of the tissue size : whereas blood flow varies with the size of tissue, τ is normalized for tissue size. Blood flow as well as blood volume of a tissue may be assumed to be proportionally related to tissue sample size. Consequently, the model parameter τ does not depend on the size of the vascular bed. In conclusion this study has given a relation between flow and the inverse of tau, τ−", the local blood refreshment rate, and a function for the relation between volume, presumably of the choriocapillaris, and perfusion pressure. For clinical applications the model parameter tau can be considered as an interesting measure in determining hemodynamics of the choriocapillaris. Acknowledgements The authors would like to thank professor A. Alm for the use of the scanning laser ophthalmoscope and thank professors A. Alm, A. Bill and S. S. Hayreh for their valuable and critical comments on the manuscript. This work was supported by the National Society for the Blind and Visually Handicapped, Rotterdamse Vereniging Society for the Blind and Visually Handicapped, Rotterdamse Vereniging Blindenbelangen, Stichting Blindenpenning and Gelderse Blinden Vereniging.
References Alm, A. and Bill, A. (1970). Blood flow and oxygen extraction in the cat uvea at normal and high intraocular pressures. Acta Physiol. Scand. 80, 19–28. Alm, A. and Bill, A. (1972). The oxygen supply to the retina. II. Effects of high intraocular pressure and of increased arterial carbon dioxide tension on uveal and retinal blood flow in cats. A study with radioactively labelled microspheres including flow determinations in brain and some other tissues. Acta Physiol. Scand. 84, 306–19. Alm, A. and Bill, A. (1973). Ocular and optic nerve blood flow at normal and increased intraocular pressures in monkeys (Macaca irus) : a study with radioactively labelled microspheres including flow determinations in brain and some other tissues. Exp. Eye Res. 15, 15–29. Alm, A., To$ rnquist, P. and Stjernschantz, J. (1977). Radioactively labelled microspheres in regional ocular blood flow determinations. (9th European Conference Microcirculation, Antwerp 1976). Bibl. Anat. 16, 24–9 (Karger, Basel, 1977). Appledorn, C. R., Oppenheim, B. E. and Wellman, H. N. (1980). An automated method for the alignment of image pairs. J. Nucl. Med. 21, 165–7. Armaly, M. F. and Araki, M. (1975). Effect of ocular pressure on choroidal circulation in the cat and Rhesus monkey. Invest. Ophthalmol. 14, 584–91. Bill, A. (1962). Intraocular pressure and blood flow through the uvea. Arch. Ophthalmol. 67, 336–48.
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Bill, A. (1974). Effects of acetazolamide and carotid occlusion on the ocular blood flow in unanesthetized rabbits. Invest. Ophthalmol. 13, 954–8. Bill, A. (1991). (The 1990 Endre Balazs Lecture.) Effects of some neuropeptides on the uvea. Exp. Eye Res. 53, 3–11. Bill, A. and Stjernschantz, J. (1980). Cholinergic vasoconstrictor effects in the rabbit eye : vasomotor effects of pentobarbital anesthesia. Acta Physiol. Scand. 108, 419–24. Buckberg, G. D., Luck, J. C., Payne, D. B., Hoffman, J. I. E., Archie, J. P. and Fixler, D. E. (1971). Some sources of error in measuring regional blood flow with radioactive microspheres. J Appl. Physiol. 31, 598–604. Bulpitt, C. J. and Dollery, C. T. (1971). Estimation of retinal blood flow by measurement of the mean circulation time. Cardiovasc. Res. 5, 406–12. Duijm, H. F. A., Rulo, A. H. F., Asrin, M., Ma$ epea, O., Van den Berg, T. J. T. P. and Greve, E. L. Analysis of variance of microspheres blood flow measurements in rabbits. Accepted for publication in Exp. Eye Res. Flower, R. W. (1993). Extraction of choriocapillaris hemodynamic data from ICG fluorescence angiograms. Invest. Ophthalmol. Vis. Sci. 34, 2720–9. Flower, R. W. and Klein, G. J. (1990) Pulsatile flow in the choroidal circulation : a preliminary investigation. Eye 4, 310–8. Flower, R. W., Fryczkowski, A. W. and McLeod, D. S. (1995). Variability in choriocapillaris blood flow distribution. Invest. Ophthalmol. Vis. Sci. 36, 1247–58. Friedman, E., Kopald, H. H. and Smith, T. R. (1964). Retinal and choroidal blood flow determined with krypton-85 in anesthetized animals. Invest. Ophthalmol. 3, 539–47. Geijer, C. and Bill, A. (1979). Effects of raised intraocular pressure on retinal, prelaminar, laminar, and retrolaminar optic nerve blood flow in monkeys. Invest. Ophthalmol. Vis. Sci. 18, 1030–42. Geijssen, H. C. (1991). Studies on Normal Pressure Glaucoma. Kugler Publications : Amstelveen. Greve, E. L., Buimer, R., Duijm, H. F. A. and Geijssen, H. C. (1995). SLO fluorescein angiography of the peripapillary choroid before and after trabeculectomy. Invest. Ophthalmol Vis. Sci. 36, S436 (abstract). Greve, E. L., Duijm, H. F. A., Geijssen, H. C., Van den Berg, T. J. T. P. (1994). Scanning laser angiography and parametrization of the peripapillary choroid in glaucoma patients. In : Glaucoma Update 1995. (Ed. Krieglstein, G. K.). Pp. 75–80. Dr Kaden : Heidelberg. Hayreh, S. S. (1974). The choriocapillaris. Albrecht Van Graefes Arch. Klin. Exp. Ophthalmol. 192, 165–79. Hayreh, S. S. (1990). In vivo choroidal circulation and its watershed zones. Eye 4, 273–89. Hickham, J. B. and Frayser, R. (1965). A photographic method for measuring the mean retinal circulation time using fluorescein. Invest. Ophthalmol. 4, 876–84. Hillerdal, M., Sperber, G. O. and Bill, A. (1987). The microsphere method for measuring low blood flows : theory and computer simulations applied to findings in the rat cochlea. Acta Physiol. Scand. 130, 229–35. Hyvarinen, L., Maumenee, A. E., George, T. and Weinstein, G. W. (1969). Fluorescein angiography of the choriocapillaris. Am. J. Ophthalmol. 67, 653–66. Kiel, J. W. (1994). Choroidal myogenic autoregulation and intraocular pressure. Exp. Eye Res. 58, 529–43. Kiel, J. W. and Van Heuven, W. A. J. (1995). Ocular perfusion pressure and choroidal blood flow in the rabbit. Invest. Ophthalmol. Vis. Sci. 36, 579–85. Kiel, J. W. and Shepherd, A. P. (1992). Autoregulation of choroidal blood flow in the rabbit. Invest. Ophthalmol. Vis. Sci. 33, 2399–410.
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Klein, G. J., Baumgartner, R. H. and Flower, R. W. (1990). An image processing approach to characterizing choroidal blood flow. Invest. Ophthalmol. Vis. Sci. 31, 629–37. Laatikainen, L. (1971). Fluorescein angiographic studies of the peripapillary and perilimbal regions in simple, capsular and low tension glaucoma. Acta Ophthalmol (Copenh.) 111S, 3–83. Lambrou, G. N. (1993). Choroidal Plerometry : A Method for Evaluating the Choroidal Blood Supply in Clinical Practice. Kugler Publications : Amsterdam. Lambrou, G. N., Van den Berg, T. J. T. P. and Greve, E. L. (1989). Vascular plerometry of the choroid. An approach to the quantification of choroidal blood flow using computer-assisted processing of fluorescein angiograms. In Ocular Blood Flow in Glaucoma. (Eds Lambrou, G. N. and Greve, E. L.). Pp. 287–94. Kugler & Ghedeni : Amstelveen. Oosterhuis, J. A. and Boen-Tan, T. N. (1971). Choroidal fluorescence in the normal human eye. Ophthalmologica 162, 246–60. Prinzen, F. W. and Glenny, R. W. (1994). Developments in non-radioactive microsphere techniques for blood flow measurement. Cardiovasc. Res. 28, 1467–75. Pru$ nte, C. and Niesel, P. (1988). Quantification of choroidal blood-flow parameters using indocyanine green videofluorescence angiography and statistical picture analysis. Graefes Arch. Clin. Exp. Ophthalmol. 226, 55–8. Quigley, H. A., Hohman, R. M., Sanchez, R. and Addicks, E. M. (1985). Optic nerve head blood flow in chronic experimental glaucoma. Arch. Ophthalmol. 103, 956–62. Raitta, C. and Sarmela, T. (1970). Fluorescein angiography of the optic disc and the peripapillary area in chronic glaucoma. Acta Ophthalmol. (Copenh.) 48, 303–8. Von Ritter, C., Hinder, R. A., Womack, W., Bauerfeind, P., Fimmel, C. J., Kvietys, P. R., Granger, D. N. and Blum, A. L. (1988). Microsphere estimates of blood flow : methodological considerations. Am. J. Physiol. 254, G275–9. Riva, C. E., Cranstoun, S. D., Mann, R. M. and Barnes, G. E. (1994a). Local choroidal blood flow in the cat by laser Doppler flowmetry. Invest. Ophthalmol. Vis. Sci. 35, 608–18. Riva, C. E., Cranstoun, S. D. Grunwald, J. E. and Petrig, B. L. (1994b). Choroidal blood flow in the foveal region of the human ocular fundus. Invest. Ophthalmol. Vis. Sci. 35, 4273–81. Riva, C. E., Feke, G. T. and Ben-Sira, I. (1978). Fluorescein
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dye-dilution technique and retinal circulation. Am. J. Physiol. 234, H315–22. 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. Ophthalmol. Vis. Sci. 22, 241–8. Roth, S. and Pietrzyk, Z. (1994). Blood flow after retinal ischemia in cats. Invest. Ophthalmol. Vis. Sci. 35, 3209–17. Shimizu, K., Yokochi, K. and Okano, T. (1974). Fluorescein angiography of the choroid. Jap. J. Ophthalmol. 18, 97–108. Sperber, G. O. and Bill, A. (1985). Blood flow and glucose consumption in the optic nerve, retina and brain : effects of high intraocular pressure. Exp. Eye Res. 41, 639–53. Sperber, G. O. and Bill, A. (1989). The 2-deooxyglucose method and ocular blood flow. In : Ocular Blood Flow in Glaucoma. (Eds Lambrou, G. N. and Greve, E. L.). Pp. 73–80. Kugler & Ghedeni : Amstelveen. Tamaki, Y., Araie, M., Kawamoto, E., Eguchi, S. and Fujii, H. (1994). Noncontact, two-dimensional measurement of retinal microcirculation using laser speckle phenomenon. Invest. Ophthalmol. Vis. Sci. 35, 3825–34. VanBavel, E. and Mulvany, M. J. (1994). Role of wall tension in the vasoconstrictor response of cannulated rat mesenteric small arteries. J. Physiol. 477, 103–15. VanStokkum, I. H. M., Lambrou, G. N. and Van den Berg, T. J. T. P. (1995). Hemodynamic parameter estimation from ocular fluorescein angiograms. Graefes Arch. Clin. Exp. Ophthalmol. 233, 123–30. Wolf, S., Arend, O., Sponsel, W. E., Schulte, K., Cantor, L. B. and Reim, M. (1993). Retinal hemodynamics using scanning laser ophthalmoscopy and hemorheology in chronic open-angle glaucoma. Ophthalmology 100, 1561–6. Wolf, S., Arend, O., Toonen, H., Bertram, B. and Reim, M. (1992). Measurement of retinal micro- and macrocirculation in patients with diabetes mellitus with scanning laser ophthalmoscopy. Clin. Vis. Sci. 7, 461–9. Wolf, S., Jung, F., Kiesewetter, H., Ko$ rber, N. and Reim, M. (1989). Video fluorescein angiography : method and clinical application. Graefes Arch. Clin. Exp. Ophthalmol. 227, 145–51. Yu, D. Y., Alder, V. A., Cringle, S. J. and Brown, M. J. (1988). Choroidal blood flow measured in the dog eye in vivo and in vitro by local hydrogen clearance polarography : validation of a technique and response to raised intraocular pressure. Exp. Eye Res. 46, 289–303.
Study of Choroidal Blood Flow by Comparison of SLO Fluorescein Angiography and Microspheres H. F E R D I N A N D A. D U I J M*†‡¶, A L E X A N D E R H. F. R U LO * , M A R I A A S T I N§, OLAV MA> EPEAs, T H O M A S J. T. P. V A N D E N B E R G†‡ E R I K L. G R E V E* * Glaucoma Center (Department of Ophthalmology) and † Department of Medical Physics, University of Amsterdam ; ‡ The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands ; § Prostaglandin research, Pharmacia Uppsala AB and s Department of Ophthalmology, University Hospital, University of Uppsala, Sweden. (Received Lund 22 November 1995 and accepted in revised form 6 June 1996) Choroidal hemodynamics estimated with parameters describing the dye build-up curves obtained with video fluorescein angiography, were compared with a classical regional blood flow measurement : radioactively labelled microspheres. Video fluorescein angiograms (Rodenstock’s SLO 101) and microspheres blood flow measurements were made in 13 anaesthetized pigmented rabbits. Ocular perfusion pressures were varied from 60 to 15 mmHg by changing the intraocular pressure. The angiographically derived dye build-up curves were described by means of an exponential model. One of the model parameters is the time constant τ theoretically reflecting local blood refreshment time. Labelled microspheres act as a non-recirculating blood flow indicator, enabling the estimation of regional blood flows. The relation between choroidal blood flow and perfusion pressure is nearly linear, suggesting the passive nature of choroidal vasculature. There is a significant correlation between τ and microspheres flow (R ¯ 0±67, P ! 0±01). According to the rheological model the product of blood flow and τ corresponds to the relevant blood volume. Hence, a function for the volume of the choriocapillaris as a function of perfusion pressure was established. The model parameter τ can be interpreted as the local blood refreshment time. Since the parameter τ, unlike microspheres, can be used clinically, τ may be used to retrieve information on choroidal hemodynamics in clinical practice. Information on the spatial distribution of choroidal hemodynamics is also obtained. # 1996 Academic Press Limited Key words : ocular blood flow ; microspheres ; fluorescein angiography ; computer-assisted image analysis ; choroid ; rabbit.
1. Introduction The choroidal circulation plays an important role in the blood supply of the eye, it nourishes large parts of the optic disk and the outer part of the retina. In the rabbit the choroid nourishes the retina almost completely, since in this animal the retinal circulation is virtually nonexistent. The role of a disturbed blood flow in the posterior pole has been suggested and made plausible in several ocular diseases, e.g.—normal pressure—glaucoma. However, in clinical practice assessment of the choroidal circulation proves to be difficult. The intricate three dimensional structure and the partially blocking effect of the pigments restrict interpretation of choroidal angiographic studies. Several methods have been developed to quantify ocular blood flow, e.g. duplex doppler, laser doppler velocimetry, scanning laser doppler flowmetry. Other methods involve angiography in the determination of blood flow. Ever since the introduction of fluorescein angiography the retinal circulation has been studied extensively, mostly via qualitative means. Various methods have been developed to quantify retinal circulation using photographic methods (Riva, Feke ¶ Corresponding author.
0014–4835}96}12069312 $25.00}0
and Ben-Sira, 1978 ; Bulpitt and Dollery, 1971 ; Hickham and Frayser, 1965) and in recent times using video techniques and computerized image analysis (Wolf et al., 1989, 1992, 1993). In general these methods aim to quantify retinal circulation by data derived from fluorescence intensity curves. In other words, from the shape of the dye curves, information on rheology is inferred, e.g. passage or circulation times and arterial dye velocities. Attempts have been made to quantify choroidal circulation using indocyanine green angiography and image processing (Flower and Klein, 1990 ; Klein, Baumgartner and Flower, 1990 ; Flower, 1993). In these methods the pulsatile character of the choroidal blood flow is described. As in the retinal fluorescein angiographical methods, with the aid of indocyanine green, arterial, venous and capillary filling times in the choroid can also be estimated (Pru$ nte and Niesel, 1988). Also, with the help of laser doppler velocimetry, choroidal hemodynamics in the macular area in humans were measured (Riva et al., 1994b). Also, fluorescein has been used to study choroidal hemodynamics. A vast amount of qualitative knowledge has been obtained on physiologic, normal choroidal blood flow (Hyvarinen et al., 1969 ; Oosterhuis and Boen-Tan, 1971 ; Shimizu, Yokochi # 1996 Academic Press Limited
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F. 1. (A) The graphical representation of the model curve, used to analyse the individual dye build-up curves. The time course is described by four parameters, initial fluorescence (Fmin), maximum fluorescence (Fmax), the time of first dye appearance (t ) and tau, the time-constant of the model, representing the refreshment time. (B) A few examples of choroidal dye build-up ! curves obtained in a rabbit, with the approximations using the exponential model.
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and Okano, 1974 ; Hayreh, 1974, 1990), as well as on pathological conditions such as glaucoma (Raitta and Sarmela, 1970 ; Laatikainen, 1971 ; Geijssen, 1991). In an attempt to quantify choroidal capillary hemodynamics a physical, rheological model, describing the filling process of the choriocapillaris during fluorescein angiography, was used to analyse fluorescence intensity time curves (Lambrou, Van den Berg and Greve, 1989 ; Lambrou, 1993 ; VanStokkum, Lambrou and Van den Berg, 1995). For each location of the posterior pole the dye build-up curve is analysed using the following model : Fmin 1
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0!t!t t&t
! (1)
!
where ; Fmin, initial fluorescence, dependent on pseudofluorescence and possible residual fluorescence of previous angiograms ; Fmax, maximum fluorescence, dependent on the amount of blood with fluorescein, blocking of pigments etc. ; t , the onset of the filling ! process, reflecting the delay time between injection site and the eye ; τ, tau, the time-constant of the model. The model curve is shown in Fig. 1(A). In Fig. 1(B) a few actual dye curves with the fitted model approximations are presented. The fit using the afore-mentioned four parameters of the dye curves was found to be accurate. This simple model describes approximately 90 % of dye curve information. There is a small, though statistically significant improvement in fit by the introduction of an extra time-constant in the model (VanStokkum et al., 1995). The advantage of analysis using this mathematical model is not only the parametrization and description of the dye curves, but also the possibility of interpretation in terms of a rheological model. At first, a simple one compartmental model was proposed for the filling process of the choriocapillaris, resulting in the above mentioned function (Eqn 1). In this context the time-constant τ of the simple model (Eqn 1) can be regarded as the local blood refreshment time : flow dV}dt 1 ¯ ¯ ¯ τ−" volume V τ
(2)
In Eqn 2 dV}dt represents the volume replaced per unit of time in a local compartment with a volume V. The term 1}τ represents the fraction replaced per unit of time, therefore equivalent to the local blood refreshment rate. This interpretation does not take into account potential effects of extravasation of fluorescein. A refinement could be to define dV}dt and V in such way that they include the relevant extravasal space. However, this problem is beyond the scope of the present paper. A basic assumption in the application of the one compartmental model is that the dye arrives in a stepwise manner ; as if the dye would have been
injected into the choroidal arteries. In a clinical setting, as well as in this study, fluorescein is injected into a peripheral vein. The dye does not arrive in a stepwise manner. To take into account influences of the circulation between injection, or in other words to take into account the form of the dye front as it enters the choriocapillaris, a second order model to analyse the choroidal dye curves would make a better choice. This refinement, the introduction of a second timeconstant into the model, corresponds with a rheological two-compartmental model : a systemic compartment and a choroidal compartment with a local choroidal blood refreshment time. Although this twocompartmental model is rheologically more realistic, its mathematical function only marginally improved the description of the dye curves (Van Stokkum et al., 1995). The build-up curves of the retinal arteries can be regarded as an indication for the form of the dye bolus entering the eye. In rabbits this approaches the step function much better. Moreover, since computing time for the two-compartmental model increases manyfold, in the present paper the one-compartment model (Eqn 1) is used. Special interest concerning the interpretation of the model parameter τ arises from clinical findings. Choroidal hemodynamics, determined by scanning laser angiography and parametrization of the peripapillary choroid (SLAPPC) in (especially normal pressure) glaucoma patients as compared to normal, healthy subjects were slowed down. The average of the parameter tau in the peripapillary area of these patients was significantly larger (Greve et al., 1994). By comparing choroidal hemodynamics in glaucoma patients before and after trabeculectomy, it appeared that choroidal hemodynamics improved significantly after surgery (Greve et al., 1995). Although from a theoretical point of view the above-mentioned analysis may seem reliable, and clinical results are promising, its rheological meaning, such as the interpretation of τ as refreshment time, has not been proven. The objective of the present study was to investigate the relationship between the choroidal capillary dye build-up curve and its description using Eqn 1 with a reference method for measuring blood flow. As a reference method, regional blood flow determination using the radioactively labelled microsphere technique was chosen (Alm and Bill, 1972). Numerous methods exist to quantify ocular blood flow in an experimental setting, e.g. auto radiography using tritiated iodoantiprine (Quigley et al., 1985), calorimetry (Armaly and Araki, 1975), direct measurements from choroidal veins (Bill, 1962 ; Sperber and Bill 1985, 1989), radioactive krypton desaturation (Friedman, Kopald and Smith, 1964), colored microspheres (Geijer and Bill, 1979), hydrogen clearance (Yu et al., 1988), laser Doppler velocimetry (Riva, Grunwald and Sinclair, 1982, Riva et al., 1994a, Riva et al., 1994b) and methods using the laser speckle
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phenomenon (Tamaki et al., 1994). In animal research however, partly because of its simplicity and straightforwardness, the labelled microspheres method is more attractive. The use of radioactively labelled microspheres in ocular blood flow research enables determination of blood flow in separate ocular tissues, e.g. retina and choroid (Alm et al., 1972). The technique has been used in numerous studies and on several laboratory animals such as cats, rabbits and monkeys (Alm et al., 1972 ; Alm and Bill, 1973 ; Bill, 1974). The microspheres are injected in the left ventricle of the animal and act as a nonrecirculating blood flow indicator. The microspheres become evenly mixed with blood, follow the blood flow to the various body tissues and get stuck in the capillary bed proportionally to the regional blood flow. Consequently, sample radioactivity is proportional to regional blood flow. From the results of a reference blow flow from e.g. the femoral artery, it is possible to calculate tissue blood flow in an absolute sense. Results obtained from blood flow determinations using different diameters of microspheres have shown that 15 µm spheres provide a reliable estimation of blood flow measurements in the rabbit choroid (Alm, To$ rnquist and Stjernschantz, 1977). Although the labelled microspheres technique seems straightforward, it has its own problems and intricacies ; these will be discussed later on. In the present study two different kinds of blood flow techniques were used. The use of these two methods to measure choroidal blood flow not only enables a mere comparison of two methods, but may also help to gain more insight into the relation between the angiographically derived hemodynamic parameters, i.c. τ, and more classical blood flow measurements. Although the model parameter τ−" theoretically reflects refreshment rate, its relation with basic processes and parameters, such as perfusion pressure and regional blood flow, may become more lucid : is it legitimate to interpret τ−" as the local choroidal blood refreshment rate ?
femoral arteries for registration of arterial blood pressure and collection of reference blood samples, and into one femoral vein for i.v. injections. Another catheter, for injection of microspheres, was inserted into the left heart ventricle via the right brachial artery. Tropicamide (Mydriacyl 0±5 %, Alcon) was topically administered in the experimental eye to induce mydriasis. In the experimental eye two anterior chamber needles (30 G) were inserted. The needles were connected by a polyethylene tubing (PE 20) to a reservoir in order to raise or lower the intraocular pressure (IOP) and to a pressure transducer for registration of IOP. Before cannulation of the eye, indomethacin (20 mg kg−" i.v.) was given. pO , pCO and pH of the arterial blood were checked # # with a blood gas analyser (ABL 300, Radiometer, Copenhagen) during the experiment and if necessary, adjusted by changing the ventilation or by i.v. administration of sodium bicarbonate (50 mg ml−", ACO, Sweden). A servo controlled heating pad was used to maintain normal body temperature of the animals. The arterial pressure and IOP of the experimental eye were continuously registered during the course of the experiment.
2. Materials and Methods
The perfusion pressure was controlled by adjusting IOP. The first blood flow determinations were made under a ‘ high ’, normal perfusion pressure, approximately 50 mmHg (MAP³.. 70±0³5±6 mmHg ; PP³.. 51±5³4±8 mmHg). The second injection under moderately lowered perfusion pressures (MAP³.. 67±6³8±1 mmHg ; PP³.. 38±1³4±2 mmHg), and the third under low perfusion pressures (MAP³. 68±3³10 mmHg ; PP³.. 23±5³6±4 mmHg). After stabilization of the perfusion pressure, a bolus dose of approximately 1±5¬10' labelled microspheres was injected into the left heart ventricle. This amount of microspheres was chosen to ensure that at least 400 microspheres were caught in the peripapillary choroid. On statistical grounds this will give a measurement
This study was performed in accordance with the ‘ARVO Resolution on the Use of Animals in Research ’.
Preparation Young pigmented rabbits (Swedish Loop) (n ¯ 13), weighing 1±8 to 2±3 kg, were used in the experiments. To induce general anaesthesia, pentobarbital (Mebumal, ACO, Sweden ; 30 mg kg−", i.v.) was used except for two animals which received urethane. Anaesthesia was maintained by continuous i.v. infusion of pentobarbital. The animals were tracheostomized and artificially ventilated. Heparinized polyethylene tubings (PE50) were inserted into both
Experimental Protocol Regional blood flow was measured using radioactively labelled microspheres as described by Alm et al. (1972). Three different labeled microspheres, diameter 15³2 µm, (Co-57, Sn-113 or Ru-103, Medical Dupont, New England Nuclear) were used, enabling three blood flow measurements under three different perfusion pressure conditions in each experiment. Perfusion pressure (PP) was defined as the difference between the mean arterial pressure (MAP) and intraocular pressure : pperfusion ¯ map®iop ¯ part syst"( part syst®part diast)®iop $ (3)
COMPARISON OF CHOROIDAL BLOOD FLOW
697
error of 5 % (Buckberg et al., 1971 ; Hillerdal, Sperber and Bill, 1987). A reference blood sample was collected under free flow condition from one femoral artery (ref blood) during the first minute from the start of each injection of the microspheres. In addition, arterial blood samples were taken just before each injection to determine blood gasses. After the experiment the animal was killed by an overdose of a mixture of pentobarbital and ethanol. Both eyes, experimental and control, were enucleated, and several ocular tissues were dissected : the iris, ciliary body, peripheral and peripapillary choroid, retina and optic nerve. The peripapillary choroid was taken from the eye globe by punching an area centered around the disk with the aid of a trephane (φ9 mm). Radioactivity was measured in the tissues and blood samples with a gamma counter (Compugamma 1282 LKB Wallace) and expressed in counts per minute (cpm). Blood flow in a tissue (mg min−") could be calculated as : blood flowref blood cpmref blood
blood flowtissue ¯ cpmtissue\
(4)
Fluorescein Angiography and Model Analysis After each injection of microspheres, two to three angiograms were recorded under equal flow conditions. Because of the image analysis residual fluorescence of the previous angiograms does not affect the analysis of subsequent angiograms. Therefore angiograms could be repeated within three minutes. Moreover, due to the small amount of fluorescein (15 µl 25 %) that was injected, and also due to the relatively rapid decline of residual fluorescence, the residual fluorescence, Fmin, was low. After proper focussing of the Scanning Laser Ophthalmoscope (Rodenstock, fixed gain, 50 µW blue argon laser) 15 µl fluorescein 25 % (Alcon) was injected in the femoral vein. The fluorescein angiograms were recorded on super-VHS video tape (Panasonic AG-7350). The composition of the picture was such that the center of the picture was located on the temporal part of the retina with the medullary rays just adjacent to the optic disk. The angiogram was digitized into 50 pictures. The spatial resolution is 380¬228 pixels ; the temporal resolution is 5 pictures second−". The pictures were aligned by a specially made automatic procedure using cross correlation of pictures, analogous to methods used in radiology for image alignment (Appledorn, Oppenheim and Wellman, 1980). For the central 228¬228 pixels the model analysis was performed. In Fig. 2(A), nine examples of a series of 50 digitized angiogram frames are shown. For each of the 228¬228 pixels a dye-curve is constructed. Fluorescence intensity is plotted on the ordinate, time is plotted on the abscissa. Every dye curve is analysed according to the previously
mentioned model, Eqn 1 (Lambrou, 1993). Hence, for each location of the posterior pole hemodynamics are parametrized, described by the afore-mentioned parameters. In Fig. 1(B) some examples of dye curves with the fitted model function are given. In order to study the spatial distribution the values of the parameters can be translated into shades of grey. In this way a distribution map of t , Fmax or blood ! refreshment rate (τ−") is obtained. In Fig. 2(B) the three distribution maps for the model parameters are given. The left-hand picture shows Fmax, dark shades indicating low maximum fluorescence and vice versa. The centre picture depicts t , light shades indicating ! early onset of filling. The right-hand picture shows the blood refreshment, light shades indicating high blood refreshment rate. To determine an average blood refreshment rate for the choroid, the disc area and pixels overlying retinal vessels were excluded by an automated image analysis algorithm (roughly 10–15 % of all the pixels in the frame). The parametrization of the dye curves with low fluorescence intensities renders unreliable estimations of the parameters. Therefore, pixels with very low fluorescence were excluded as well (approximately 5 % of all the pixels). The shaded area in Fig 2(C) represents the area that is not included in the average choroidal blood refreshment rate. Statistical analysis involved analysis of variance using the F statistics. Statistical significance was defined as P ! 0±01 (except when stated otherwise).
3. Results In Table I the average blood flow values determined with the aid of microspheres are given for the baseline measurements of the experimental and control eyes. Values for e.g. the peripapillary choroid are in the order of 70 000 counts with a counting period of 10 min, corresponding to several hundreds of caught microspheres. Typical values for retina and optic nerve are 400 counts. As this corresponds to only a few caught microspheres, the results of retina and optic nerve are not included. In Fig. 3 the relation between blood flow of the iris, ciliary body, peripheral and peripapillary choroid and the perfusion pressure is given. Two animals, indicated by arrows, did not follow the general trend. The condition of one animal deteriorated during the experiment (very small flow values in Fig. 3), and was hemodynamically very unstable during the microspheres injections. The second one, unlike all other animals, showed hardly any influence of perfusion pressure. It is assumed that the perfusion pressure registration was at fault. These animals were excluded from further analysis. In Fig. 4 the relation between the model parameter τ−" (calculated from the fluorescein angiograms) and perfusion pressure is given.
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(A)
(B)
F. 2. (A–B) For legend see facing page.
In all cases the blood flow is strongly influenced by the perfusion pressure. Regression analysis showed that there was no statistically significant difference between on the one hand single regression using only perfusion pressure, and multiple regression using intraocular and mean arterial pressure separately (analysis of variance with F statistics) on the other. Hence, it appears that only the difference between mean arterial pressure and intraocular pressure is of influence on blood flow when measured with the aid of microspheres. On analysing the results of the microspheres, the blood flow in the various tissues of the experimental eye seemed systematically higher (Table I). In a
separate paper an analysis is presented showing that blood flow not only depends on perfusion pressure, but also, to a lesser extent, on the individual animal and on the hyperaemic state of the eye (Duijm et al., submitted). Figure 5 shows the correspondence between the model parameter, τ−" and the microspheres measurement having a correlation coefficient of 0±67. For each microspheres measurement the tau values calculated from the matching angiograms were averaged. The average standard deviation of the belonging τ−" values was 0±061, corresponding to a coefficient of variation of 6±1 %. In general blood flow in a tissue describes the
COMPARISON OF CHOROIDAL BLOOD FLOW
699
perfusion pressure. The data show variability of the vascular bed for which there may be two reasons. On the one hand, hyperaemia influences vascular volume in the experimental eye (Duijm et al., submitted) ; this hyperaemia causes increased circulatory volume. On the other hand, the size of the vascular bed as it is, may vary from one animal to another (Duijm et al., submitted). These two factors may affect the volume estimation using Eqn. 6. In Fig. 6 (lower graph) blood volumes corrected for these two effects are presented. These volumes represent the values for an ‘ average ’ animal in normal (non-hyperaemic) condition.
(C)
Discussion
F. 2. (A) Nine examples from a digitized video angiogram sequence. Left-right, top-bottom : 1st t ¯ 0 sec, 2nd t ¯ 3 sec, subsequent time lapse 0.5 sec. (B) The three distribution maps of the model parameters. Left : maximum fluorescence (Fmax) ; middle : the time of first appearance of dye (t ) ; right : blood refreshment rate. Light shades are ! indicating high Fmax, early t and high blood refreshment ! rate. Dark shades are indicating low Fmax, late t and low ! blood refreshment rate. (C) The shaded area is the area that excluded from the average choroidal blood refreshment rate.
T I Average baseline blood flow measurements (mean³S.D.), determined with microspheres, for several ocular tissues Blood flow (mg min−")
Experimental eye
Control eye
Iris Ciliary body Peripheral choroid Peripapillary choroid
184³113 227³144 536³227 129³44
126³64 185³76 495³189 120³41
amount of blood that is being refreshed in a certain time lapse : blood flow ¯
volume time
(5)
The model parameter τ corresponds to the time needed to replace a volume equal to the total volume of the vascular bed of the considered tissue. This leads to the following equation : blood volumetissue ¯ blood flowmicrospheres\τ (6) With the help of Eqn 6 it is theoretically possible to estimate the blood volumes in the tissues of this study. In Fig. 6 (upper graph) the calculated volumes for the choroid are plotted against the corresponding
This study was intended to study the parameter τ−", estimated from fluorescein angiograms, as blood refreshment rate in the choroid and to delineate its relation to blood flow. The reproducibility of the parameter τ−" proved to be good, with a coefficient of variation of 6±1 %, and was linearly related to blood flow (Fig. 5, R ¯ 0±67). Furthermore a function was found for the volume change of the choriocapillaris with perfusion pressure (Fig. 6). In order to evaluate τ−" in this study, the reference technique should provide a reliable blood flow measurement of the choroid. Although the radioactively labelled microspheres method is straightforward, some authors have raised questions (Von Ritter et al., 1988 ; Prinzen and Glenny, 1994 ; Kiel, 1995). The choice of the microspheres for example is of some concern. An important prerequisite of the microspheres method is that no recirculation of the spheres occurs. If the microspheres are too small they will pass through the vascular bed resulting in underestimation of the regional blood flow. On the other hand, spheres that are too large will be trapped proximal to the tissue resulting in an underestimation of the blood flow. Another problem related to the size of the microspheres is axial streaming. Large, heavy spheres tend to migrate to the center of the vessel, thus causing systematic errors. From the literature, it appears that 15 µm spheres present the best choice for examination of regional ocular blood flow in rabbits (Alm et al., 1977). When the results of 9 and 15 µm microspheres in the rabbit are compared, it shows that even 9 µm microspheres are trapped in the choroidal circulation, presumably due to the flattened capillaries of the choriocapillaris. In the cat it was found that 95 % of the 15 µm spheres stayed in the uvea for more than 1 min (Alm et al., 1972). However, around 50 % of the 9 µm spheres passed through the vascular bed of the anterior uvea in the rabbit. There was no indication that axial streaming disturbed the blood flow measurements using the 15 or 9 µm spheres in the rabbit. A problem regarding the premiss of nonrecirculation is that
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F. 3. Blood flow measured with microspheres of the iris, ciliary body, peripheral and peripapillary choroid (mg min−"), plotted as a function of perfusion pressure (mmHg). Arrows indicating two animals not following the ‘ general ’ trend.
1 0.8 0.6
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τ –1 = 0.012 pp + 0.58 r = 0.79
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τ–1 = 0.0020 bf + 0.86 r = 0.67
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F. 4. The inverse of the model parameter tau, the local refreshment rate (seconds−"), measured with the fluorescein angiograms as a function of perfusion pressure (mmHg).
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F. 5. Comparison of the inverse of the model parameter tau, the local refreshment rate, τ−" (ordinate) and the blood flow of the peripapillary choroid determined with the help of microspheres (mg min−").
COMPARISON OF CHOROIDAL BLOOD FLOW
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12
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Raw values v = 0.18 pp + 0.37 r = 0.74
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Corrected values v = 0.18 pp – 0.36 r = 0.93
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F. 6. Calculated volumes of the choriocapillaris plotted as a function of perfusion pressure. Upper graph : raw blood flow values ; lower graph : standardized, for hyperaemia corrected blood flow values.
trapped microspheres may come gradually loose, thus resulting in artificially lower blood flow readings. It has been mentioned that results of microsphere studies ‘ might not be valid ’ if the experiment lasts considerably longer than 1 min (Alm et al., 1977). On the other hand, if spheres larger than 15 µm would be used, they may occlude larger vessels, thus interfering with the circulation. Furthermore, to increase spatial resolution— especially useful in small tissues such as the optic nerve—smaller microspheres, e.g. 9 µm, can be used. Also, if the total mass of injected spheres is limited to reasonable quantities, accuracy is improved when smaller microspheres are used, due to the fact that a larger number of smaller spheres can be injected. In this study, however, the microspheres method should give reliable blood flow values for tissue samples that were not small and with a relatively high blood flow, such as the peripapillary choroid. Therefore, 15 µm spheres were used.
The accuracy of the microspheres method depends, amongst other things, on the number of caught microspheres in the vascular bed studied. As special care was taken to catch at least 400 spheres in a specific tissue, the statistical fluctuation in this number (5 % for 400 caught microspheres) is much better than the physiological variation (Figs 3 and 6) (Buckberg et al., 1971 ; Hillerdal et al., 1987). Another problem using the microspheres technique, is that multiple microspheres injections might change local flow distributions. It has been speculated that, after repeated microsphere injections in cats after a period of artificially induced ischemia, blood is shunted away from the choroid towards the retina, due to obstruction of the choroid and subsequent release of vaso-dilatating agents (Roth and Pietrzyk, 1994). However, in dogs a study did not show any hemodynamic alterations that could be attributed to the number of microspheres used (Von Ritter et al., 1988). Also, the data of the present study (not shown) did not reveal blood flows in the control eye to decrease with the number of microsphere injections. Summarizing, although the blood flow measurement technique, used in this study as a ‘ standard ’, seems a relatively good choice, it still contains some uncertainties. Therefore, some care has to be taken with regard to its use. Figure 3 (blood flow versus perfusion pressure) shows considerable variation in flow amongst the animals. This variation is comparable with findings in literature : in cats Alm et al. (1972) find similar flow pressure relations. In order to understand and explain the variation it was attempted to describe the blood flow data using a model (Duijm et al., submitted). Apart from the evident influence of the perfusion pressure, the blood flow measurements using the microspheres also appeared to be influenced by other factors. In the experimental eye a vasodilatory effect or hyperaemia could be quantitatively estimated. This hyperaemia was influenced by two factors : the individual animal and the tissue type studied. Towards the posterior pole hyperaemia decreased. Several possible causes of the quantified, experimental hyperaemia could be given. The two anterior chamber needles may have touched the iris thus stimulating the release of vasoactive substances, like prostaglandins, notwithstanding the fact that indomethacin was given. Another possibility may be the topically administered tropicamide, a parasympathicolytic, cholinergic blocking substance. Also the combination of trauma (anterior chamber needles) and factors like general anaesthesia with pentobarbital may increase ocular blood flow (Bill and Stjernschantz, 1980 ; Bill, 1991). Furthermore, from the analysis of the experimental hyperaemia it appears that hyperaemia is mainly present in the anterior segment. The peripapillary choroid, the tissue of special interest in this study, showed practically no hyperaemia. Consequently, the
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experimental hyperaemia will have influenced peripapillary choroidal blood flow determinations only to a limited degree. As has already been mentioned, choroidal blood flow measured with radioactively labelled microspheres shows an evident influence of the perfusion pressure. There seems to exist a more or less proportional, linear relation between choroidal blood flow and perfusion pressure. Autoregulation would tend to maintain a relatively constant blood flow despite changes in variables such as perfusion pressure. Hence, this finding implies that there is no autoregulation of the choroidal circulation, at least not in the experimental setup used in this study. This corresponds with numerous studies suggesting that the choroid behaves like a passive vascular bed without autoregulation. Using several methods, a linear relation between blood flow and perfusion pressure has been found (Bill, 1962 ; Alm and Bill, 1970 ; Alm et al., 1972, 1973 ; Yu et al., 1988). In contrast, the retinal vasculature shows evident autoregulatory behavior in rabbits (Bill, 1974). However, in some recent studies using laser doppler flowmetry, evidence was found of myogenic autoregulation of choroidal circulation. This may be partly due to the procedure of alteration of the perfusion pressure. In the studies, which did find indications for myogenic autoregulation, the mean arterial pressure was altered and showed that the autoregulation was most effective if the arterial pressure was altered and the IOP was not controlled (Kiel and Van Heuven, 1992 ; Kiel and Shepherd, 1992 ; Kiel, 1994). In the present study as well as in most other studies, the intraocular pressure was manipulated in order to change perfusion pressure. Apart from this, peripapillary choroidal tissue as defined in the present study is approximately 60 times larger than the sample area used in the studies of Kiel. There may exist considerable local difference in choroidal blood flow (Flower, Fryczkowski and McLeod, 1995). The latter and perhaps the different techniques may explain the differences in findings. Another consideration in investigating autoregulation is the condition of the vascular bed during the experiments. Hyperaemia has been found in the experimental eye. Consequently, experimental findings regarding e.g. vasotonus or autoregulatory behavior may not reflect physiologic, in vivo vasculatory behavior. However, hyperaemia had limited effect in the choroid as opposed to the anterior segment. Hence, this may not have greatly affected the τ comparisons. The primary aim of the present study was to investigate the angiographically derived parameter τ−". The parameter τ represents the time needed to replace the total volume of the vascular bed of the tissue studied, in this case the choriocapillaris. As has already been discussed, a premiss regarding this interpretation of the parameter τ as the blood refreshment time of the choriocapillaris, is that the dye
H. F. A. D U I J M E T A L.
arrives at the choriocapillaris in a stepwise manner. Although it seems likely that the front approximates a step, this prerequisite is not fully fulfilled. Since the parameter τ represents the time needed to replace the total volume of the vascular bed of the choriocapillaris, possible changes in this volume have to be taken into account when the two methods are compared. There exists an evident dependence of the estimated volume on the perfusion pressure (Fig. 6). At physiologic perfusion pressures (60 mmHg) the estimated volume is approximately 10 µl. The fluorescein angiography analysis basically gives a blood refreshment rate of the choriocapillaris. Consequently, the 10 µl would correspond to the volume of the choriocapillaris. This value is in accordance with values which can be calculated for the rabbit. If one assumes that the average diameter of the eye globe is 17 mm while the thickness of the choriocapillaris is 20 µm (80 % blood) over two-thirds of the globe’s surface, the volume of the choriocapillaris would be 9 µl. Figure 6 indicates the volume-perfusion pressure relation of the choriocapillaris. To our knowledge no reference material for the choriocapillaris is available and therefore the relation needs verification by means of other techniques. According to literature, most volume-pressure relations of beds of small vessels are more or less logarithmical (VanBavel and Mulvany, 1994). However, one has to be aware that the choriocapillaris is a vascular bed with peculiar properties. Because of its geometric network, its large flattened capillaries and its encapsulation by a rather inelastic sclera, it cannot be easily compared with other vascular beds. It is tempting to relate this study to humans. However, there is a large difference regarding ocular vasculature between rabbits and humans. In the rabbit the retinal vascular layer is practically nonexistent, and fluorescence will be almost entirely choroidal of origin. In the normal human fundus the retinal vasculature cannot be ignored. In fluorescein angiography the large, superficial, retinal vessels, with high contrast and delicate detail, are prominent. The retinal microcirculatory network on the other hand contributes only very little to the fluorescence due to its small volume and absence of dye leakage. Additionally, in areas of delayed or nonexistent filling or in case of targeted dye delivery, small retinal vessels can be discerned, but not the retinal capillary network, or a capillary fluorescent haze. Therefore, fluorescence from fundus locations not overlying distinct retinal vessels very likely originates from the choroid. Because of absorption by e.g. hemoglobin and intra and extravasal fluorescein, the penetration depth of the exciter light in fluorescein angiography is about 30 µm. Therefore, the large choroidal vessels contribute only very little to the fluorescence intensity. Large choroidal vessels are only seen in case of very thin or atrophic choriocapillaris.
COMPARISON OF CHOROIDAL BLOOD FLOW
There are some disadvantages using the microspheres method in that the measurements are discontinuous in time, they cannot be applied in humans, and only a limited number of determinations can be made. Fluorescein angiography on the other hand enables many measurements (over 10 angiograms per rabbit in approximately 1 hr) and is applicable in humans. An attractive characteristic of the model parameter τ is its independence of the tissue size : whereas blood flow varies with the size of tissue, τ is normalized for tissue size. Blood flow as well as blood volume of a tissue may be assumed to be proportionally related to tissue sample size. Consequently, the model parameter τ does not depend on the size of the vascular bed. In conclusion this study has given a relation between flow and the inverse of tau, τ−", the local blood refreshment rate, and a function for the relation between volume, presumably of the choriocapillaris, and perfusion pressure. For clinical applications the model parameter tau can be considered as an interesting measure in determining hemodynamics of the choriocapillaris. Acknowledgements The authors would like to thank professor A. Alm for the use of the scanning laser ophthalmoscope and thank professors A. Alm, A. Bill and S. S. Hayreh for their valuable and critical comments on the manuscript. This work was supported by the National Society for the Blind and Visually Handicapped, Rotterdamse Vereniging Society for the Blind and Visually Handicapped, Rotterdamse Vereniging Blindenbelangen, Stichting Blindenpenning and Gelderse Blinden Vereniging.
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