Video Camera Method for Simultaneous ... - SAGE Journals

Journal of Cerebral Blood Flow an d Metabolism

2:421-428 © 1982 Raven Press, New York

Video Camera Method for Simultaneous Measurement of Blood Flow Velocity and Pial Vessel Diameter

F. Gotoh, F. Muramatsu, Y. Fukuuchi, H. Okayasu, K. Tanaka, N. Suzuki, and M. Kobari Department of Neurology, School of Medicine, Keio University, Tokyo, Japan

Summary: A new method for the simultaneous measurement of blood flow velocity and pial vessel diameter is described. The system consists basically of a high-sensitivity vidicon camera, camera control, width analyzer, video den sitometer, TV monitor, desktop computer, and multi-pen recorder. The pial vessels are visualized through a cranial window at 25 - 200 x magnification on the TV monitor. The diameter of three target vessels can be recorded simulta neously on the recorder by adjustment of controllable video signal gates using the width analyzer. At the same time, the optical densities of two targets at points upstream and downstream of the pial vessel are measured continuously with video densitometers, and their outputs are recorded on the polygraph and analyzed by the computer. The time difference in the two peaks of time-con centration curves, produced every 2- 3 s at the highest frequency by the injec tion of a small amount of saline through the lingual artery, is measured on-line using the computer. The flow velocity in the vessel is calculated from the time difference and the distance between the two targets. The system was shown to be stable, reliable, and rapid in response. This method may provide a useful tool for research in the field of blood circulation in the brain or any other organ.

Key Words: Cerebral circulation-Cranial window-Flow velocity-Pial ves sel diameter-Video camera.

on various aspects of cerebrovascular responses (Purves, 1972) and several improvements of the classical technique have been made (Forbes, 1928; Gurdjian et aI., 1958; Wahl et aI., 1973; Harper and MacKenzie, 1977; Auer and Haydn, 1979; Okayasu et aI., 1979). This method, however, has yielded only indirect information about the level of the blood flow (Purves, 1972). Recently, Busija et ai. (1981) developed a method for the virtually con tinuous measurement of changes in cerebral blood flow through a cranial window by means of a Dop pler velocity meter and electronic micrometer or image-splitter. Their new technique, however, has some limitations regarding calibration of the flow velocity and application to small vessels. We have devised another new method for the si multaneous measurement of blood flow velocity

Recent advances in the technology of cerebral blood flow measurement have provided quantitative information on the changes in local cerebral blood flow produced under various physiological and pathological conditions (Purves, 1972; Heiss, 1981; Lassen, 1981). However, the methods used hitherto (indicator clearance methods, microsphere meth ods, and autoradiographic methods) cannot eluci date hemodynamic changes in terms of basic hemodynamic parameters, i.e., vascular diameter and the blood flow velocity in the vessels. Direct observation of the pial vessels (the cranial window method) has provided valuable information

Address correspondence and reprint requests to Dr. Gotoh at Department of Neurology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.

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and pial vessel diameter. Our method can provide absolute values of the flow velocity on-line every 2-3 s, even in small vessels with diameters less than 50 JLm, together with continuous measurement (sampling rate 60/s) of three target vessels, under conditions in which the arachnoid-pial membranes and pial vessels are not disturbed. The purpose of the present paper is to introduce this new method and to describe its characteristics and application in the field of cerebral circulation research. SYSTEM DESCRIPTION Total system

The system consists of an inverted 24-mm or 50-mm lens (Nikkor), extension tube (Nikon), vidi con camera (C 1000-01, Hamamatsu TV), camera control (Hamamatsu TV), width analyzer, video timer (VTG-33, FORA), video densitometer, TV monitor (PM-121, Ikegami), multi-pen recorder (R-16M, Rikadenki), AID converter (R 488-AD, Adtek), and desktop computer (SEIKO 8500) (Fig. 1). The light target of the vidicon camera was made of cadmium-selenium derivatives to give high reso lution properties and to eliminate image persis tence. The standard scanning rates of the horizontal and vertical directions were 16.53 kHz and 60 Hz, respectively. The standard number of raster lines is 512 at 2: 1 interlace, but it was made variable de pending on the speed required for the measure ments. The illumination was provided by a glass fiber tube (Narishige) equipped with a 15 V-150 W halogen tungsten lamp (Philips). The images were visualized at 25-200 x magnification on the TV monitor. The video signals from the vidicon camera were transferred to two separate circuits. One was con nected to the width analyzer and its analog output was continuously recorded on the multi-pen re-

VIDEO SIGNAL

corder. The other was delivered to a couple of den sitometers. Their analog outputs were registered on the recorder, and were simultaneously converted into digital data and processed by the desktop com puter. Width analyzer

Figure 2 is a block diagram of the width analyzer for continuous measurement of vessel diameter. Video signals from the camera control were con verted to sliced video signals under a threshold level that was determined according to the optical den sities of the target on the TV screen. At the same time, the horizontal drive pulse (16.53 kHz) and vertical drive pulse (60 Hz) were separated from the video signals through the synchronization separa tor, and then supplied to the gate generator. The location and horizontal size of three regions of interest on the monitor screen could be simulta neously defined by adjusting the gate generator, and the signals of the gates were superimposed on the TV picture as line markers. When both sliced video signals and those of the target gate were "on," clock pulses (12.195 MHz) from the generator passed through the" AND" gate and were counted for digitization. The digitized data were then reconverted to analog signals, whose voltage was proportional to the width of the target. The signals were recorded continuously on a poly graph. Video densitometer

Figure 3 is a block diagram of the video den sitometer, which measures the optical densities in a region of interest on the TV monitor image. The position and size of the target were optionally de termined with the character generator utilizing the synchronization pulses, which were separated by the synchronization separator. The voltage of the

ANALOG SIGNAL

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FIG. 1. Block diagram of the video camera system for simultaneous mea surement of flow velocity and pial ves

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sel diameter.

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J Cereb Blood Flow Metabol, Vol. 2, No.4, 1982

PIAL VESSEL DIAMETER AND FLOW VELOCITY

CAMERA CONTROL

COMPARATOR VIDEO SIGNAL

tween the two targets can be optionally determined over the range of 100 JLm to 7 mm, depending on the degree of magnification. These mathematical pro cedures were performed on-line by means of the desktop computer, and the results were displayed on the cathode ray tube ( CRT) display.

CHARACTERISTICS

ANALOG SIGNAL to-IOVI

Width analyzer

RECORDER FIG. 2. Block diagram of the width analyzer for continuous measurement of vessel diameter.

video signal from the target was sampled at a rate of 60 Hz. It was held until the next scanning beam returned to the same target by means of the sam pling hold circuit triggered by the pulse from the character generator. The analog output of the sam pling hold circuit was connected to the recorder and to the computer by the AID converter. In order to demonstrate the position and size of the target, a marker was superimposed on the TV image through the character mixer. By the use of two video den sitometers, changes in the optical densities of two regions of interest could be measured simulta neously. The transit time of an indicator moving through two targets corresponds to the time difference be tween the two deflections of the output signals of both densitometers. The velocity of the indicator was calculated from the transit time and the dis tance between the two targets. The distance be-

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423

The measured values obtained from the width analyzer showed a linear relationship to the values directly measured with a microscale (Nikon) under in vitro conditions (Fig. 4). The stability of the re cording was extremely good and drift was negligible for 4 to 5 h. Duplicate measurements of the widths of the in vitro targets were made over the range of 20 to 300 JLm using a microprocessor voltmeter (Solartron 7065, Schlumberger) connected to the width analyzer. The reproducibility was calculated at each width level from the following formula: 100 x [1 (mean value of first measurement - mean value of second measurement)/mean value of first mea surement]. The resultant reproducibility value at each width level exceeded 99%. The resolution of the system was theoretically 1.98 JLm at 200 x magnification in vitro, while under in vivo conditions, such as the observation of pial vessels, the resolution was de creased to 2.5 JLm at the same magnification due to complex diffraction and refraction signals, which originated mainly from the arachnoid membranes and cranial glass window. -

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FIG. 5. Relationship between the calculated velocity (abscissa) and the velocity obtained with the video camera system (ordinate).

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FIG. 4. Relationship between the calibrated width (abscissa) and the output of the width analyzer (ordinate).

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The reproducibility of the response of the system to a certain intensity level of light was over 99%. The two densitometers in this system exhibited the same characteristics. The velocities of the object on the rotating disk of the cone-plate type viscometer (Tokyo Keiki) were measured with this system using a vertical scanning rate of 60 Hz and a separation distance between the two targets of 2.5 mm. In the range below 40 mm/s, there was close agreement between the measured values of velocity and those calculated from the rotatory rates (Fig. 5). APPLICATION OF THE METHOD TO THE PIAL CIRCULATION

The method was used for simultaneous measure ment of the flow velocity and diameter of feline pial vessels in vivo. The velocity was measured every 2 3 s at the highest frequency, while the diameter was measured continuously. Under anesthesia with a-chloralose and urethane, a burr hole of 1 cm in diameter was made in the parietal region of the skull. The dura was carefully incised so that the brain surface was exposed, and a cranial window -

J Cereb Blood Flow Metabol, Vol. 2, No.4, 1982

was screwed into the hole. The ipsilateral lingual artery was cannulated for injection of an indicator into the carotid artery as described below. The image of the pial vessels observed through the cra nial window was displayed on the TV monitor. The vidicon camera was rotated to orient the axis of the vessels vertically on the TV screen, so far as was possible. However, in practice it was impossible to have all three target vessels exactly parallel to the vertical direction. After setting up the system, a calibration was made in vessels that were not par allel to the vertical direction to obtain the true changes in diameter from the deflection on the re corder, using the cosine of the angle between the axis of the vessel and the vertical line on the screen. Depending on the background, appropriate green gelatin filters were placed in the extension tube to give a clear image of the vessels with optimum con trast. Data for continuous measurement of vessel diam eter obtained with this system have already been presented elsewhere (Okayasu et aI., 1979; Gotoh et aI., 1980, 1981; Tanaka et aI., 1980, 1981). There was a good linear correlation between the width of the vessels recorded with this system and that mea sured simultaneously by serial photographic tech niques over the range of 10-250 /-tm (r 0.998). In addition to the width measurement, the bright ness of the two sampling points on a given pial ves=


(FUJII UELOC ITY & D IMTER (F PIAL ARTERY> XllAX1

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FIG.6. Example of the CRT display of the computer showing the output of the densitometers and results of subsequent calculation. CH 1 and CH 2 are the recordings at the up stream and downstream points of the pial artery. respec tively.

sel was continuously recorded so that any changes in optical density of the blood in the vessel could be detected immediately. A small amount of saline (0.1 ml) was injected into the lingual artery to obtain an indicator time-concentration curve at each sam pling point on the pial vessel. The time interval be tween the two peaks of the curves (peak-to-peak transit time of the indic3;tor) was measured auto matically by the computer. The flow velocity in the

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vessel was then calculated from the distance be tween the two sampling points and the time interval. An example of the CRT display of the computer is shown in Fig. 6. The time-concentration curves at two separate points (distance 3.312 mm) in a pial artery (diameter 48 /Lm) are displayed. The com puted results for the time difference in the two peaks (175 ms) and the flow velocity (18.926 mm/s) are also included on this display. The transit time of blood from a pial artery to a pial vein could also be measured by recording curves in the pial artery and vein. In this case, the dif ference in the time for each curve that was required for half the area under each curve to elapse offered a more physiological approximation to the transit time than did the difference in the peak of each curve. An example of the resultant polygraphic recordings is shown in Fig. 7, in which the transit time from a pial artery 82 /Lm in diameter to a pial vein 48 /Lm in diameter was 2.5 s, using the above method of calculation. Figure 8 gives an actual recording for simulta neous measurement of the diameter and flow velocity of a pial artery. Gradual vasodilatation and increase of flow velocity were evident during CO2 inhalation. The blood flow in the target vessel, calculated by multiplying the cross-sectional area of the vessel 2 [71' x (diameter/2) ] by the flow velocity, was 0.018 /LUS and 0.041 /LUS before and during CO2 inhala tion, respectively.

FIG. 7. Example of the recording of time-concentration curves in a pial ar

tery (A) and a pial vein (V), together with the systemic arterial blood pres sure (BP).

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426

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inhalation. Td is the time difference between the two peaks of the time-concentration curves and V is flow velocity in the pial artery. The downward deflections in the recording of diameter after the saline injections are artifacts caused by the passage of saline.

DISCUSSION

Measurement of pial vessel caliber through a cra nial window represents one of the classical methods for investigating the cerebral circulation (Purves, 1972), and various microphotographic ( Forbes, 1928; Gotoh et aI., 1975) and high - speed mi crocinephotographic methods (Gurdjian et aI., 1958; Rosenblum, 1969) have long been employed. How ever, such methods are time-consuming and involve difficulties in the detection of transient or rapid changes in the circulation. To observe such changes, television microscopic techniques have been developed since 1963 (Wiederhielm, 1963). The television image-splitting method (Baez, 1966; Wahl et aI., 1973; Harper and MacKenzie, 1977) has increased the accuracy of measurements of caliber change, but gives only intermittent, not continuous, data. Multichannel videoangiometry ( Auer and Haydn, 1979) represents a similar method to our previous system (Okayasu et aI., 1979) for the con tinuous measurement of pial vessel diameter, and both techniques were reported at the same sym posium in 1979. Changes in the diameter of pial vessels, however, provide no reliable index of the changes in pial blood flow except at a constant arterial pressure, since without simultaneous measurement of the local intravascular pressure there is no guarantee that these changes are linearly related (Purves, 1978). To evaluate the pial blood flow, fluorescein angiography (Russel and Simcock, 1966; Rosen-

J Cereb Blood Flow Metabol. Vol. 2. No.4. 1982

blum, 1970) has been used, but this is not very accurate and is rather time-consuming in calculating the fluorescein transit time. Furthermore, repeated measurements are difficult because of the increasing background fluorescence. Later, a dual-slit, cross correlation method (Ma et aI., 1974; Intaglietta et aI., 1975) was developed, which can measure the red cell velocity in the vessel. However, this method cannot be applied to vessels where the red cell flow is continuous and homogeneous. Application of this method is thus limited to studies of the capillary or very small vessel circulation. The video camera system presented in this paper was designed to explore an unknown domain in ce rebral hemodynamics, which cannot be approached by the conventional techniques described above. By employing our method, the local cerebral circu lation could be quantified in terms of two basic hemodynamic parameters: the vascular dimensions and the blood flow velocity in the vessels. Vascular reactions along cerebrovascular trees can thus be analyzed in relation to simultaneous blood flow changes. In addition, the temporal profile of changes in cerebral hemodynamics can be followed by the rapid repetitive injection of saline into the lingual artery, so that application of this method may be of particular value for assessing the control mechanisms of the cerebral circulaton. This video camera system, however, does pre sent a few problems, which must be given careful consideration. In the strict sense, our method mea sures neither the actual diameter of the vessel nor


the size of the actual vascular lumen, but rather the width of the red cell column in the vessel. In prac tice, however, the red cell column can be regarded as representing the lumen of the vessel because the blood flow in pial vessels with a diameter above 20 JLm appears to be turbulent, and the thin plasma layer along the endothelium produced by axial drift of red cells is said not to extend further than 2-5 JLm from the wall, if it exists at all (Bayliss, 1959; Charm and Kurland, 1972). Second, the placement of the targets of the den sitometers on the vessel image must be discussed. Since the flow velocity is relatively low near the wall and highest in the center of the vessel (Rosenblum, 1972), particularly when the flow is laminar (Charm and Kurland, 1972), it is necessary for both the upstream and downstream targets to be place d in the same location relative to the vessel wall. In other words, the flow velocity can be mea sured either in the marginal area or in the central area of the vascular lumen, depending on the pur pose of the investigation. Third, there is an upper limit of flow velocity that can be measured with the video camera system. When the targets of the densitometers are separated by a given distance on the television image, they are correspondingly separated in time during the pro cess of deriving video signals. Thus, if an event oc curs simultaneously at both targets, the output of one target would appear delayed in time. This in herent characteristic leads to some error in the mea surement of a high blood flow velocity. As shown in Fig. 5, however, velocities of up to 40 mmls can be measured accurately, and this was sufficient for the study on pial vessels with diameters below 100-150 JLm in the preliminary experiments. A faster scan ning system would resolve this problem. Finally, concerning the problems with the sys tem, while the rapid repetitive injection of small amounts of saline does not affect the responsive ness of the pial vessels, the moment-to-moment changes in flow velocity cannot be continuously monitored by this method in the strict sense. In practice, however, the flow velocity can be mea sured every 2 3 s at the highest frequency. In addition to our system, a method for the si multaneous measurement of pial artery diameter and velocity has recently been reported by Busija et al. (1981). Their method combines two techniques: diameter is measured by a television image-splitting method or with an electronic micrometer, and ve-

427

locity is measured with a pulsed Doppler meter and a single piezoelectric crystal placed under the pial vessel. Their new method, however, has a number of limitations. (1) Absolute values of flow velocity cannot be obtained, unless the exact angle between the crystal and the blood velocity vector is known; only relative changes in flow velocity were pre sented in their paper. (2) It cannot be applied to the small vessels. (3) Positioning of the crystal with re spect to the target vessel (insertion of the crystal beneath the pial vessel) is technically difficult and requires considerable expertise on the part of the operator. (4) Measurement of vessel diameter can be performed only every 2-4 s. On the other hand, our method has the following advantages. (1) Absolute values of flow velocity can be obtained easily on-line even for a small vessel with a diameter less than 50 p,m. (2) The method involves neither dissection of the arachnoid-pial membranes nor placement of any device under the pial vessel. (3) The diameters of three target vessels can be measured continuously (sampling rate 60/s for each vessel), which makes it possible to follow rapid or transient changes more accurately. (4) If the whole image of the pial vessel is recorded on the videotape, repeated measurements of the velocity and diameter of any other vessels within the field can be performed later. It was shown by Busija et al. (1981) that a good correlation existed between changes in regional ce rebral blood flow calculated from the cross sectional area of the vessel x flow velocity, and from microspheres. Thus, calculation of the blood flow in a single pial vessel by our method should yield useful information on-line about serial changes in the regional cerebral blood flow. In summary, our video camera system may pro vide a useful and reliable tool for investigating the cerebral circulation, and its application may shed light on new aspects of the hemodynamic properties of the brain. Acknowledgment: The authors wish to thank Miss M. Masuda for her assistance in the preparation of the manu script.

REFERENCES Auer L. Haydn F (1979) Multichannel videoangiometry for con tinuous measurement of pial microvessels. Acta Neurol Scand 60(Suppl 72):208-209 Baez S (1966) Recording of microvascular dimensions with an

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image-splitter television microscope. J Appl Physiol 21:299-301 Bayliss LE (1959) The axial drift of the red cells when blood flows in a narrow tube. J Physiol (Lond) 149:593-613 Busija DW, Heistad DD, Marcus ML (1981) Continuous mea surement of cerebral blood flow in anesthetized cats and dogs. Am J Physiol 241:H228-H234 Charm SE, Kurland GS (1972) Blood rheology. In: Cardiovas cular Fluid Dynamics, Vol 2 (Bergel DH, ed), London, Academic Press, pp 157-203 Forbes HS (1928) The cerebral circulation. I. Observation and measurement of pial vessels. Arch Neurol Psychiatry 19:751-761 Gotoh F, Muramatsu F, Fukuuchi Y, Amano T (1975) Dual con trol of cerebral circulation: Separate sites of action in vas cular tree in autoregulation and chemical control. In: Cere bral Circulation and Metabolism (Langfitt TW, McHenry LC Jr, Reivich M, Wollman H, eds), New York, Springer Verlag, pp 43-45 Gotoh F, Fukuuchi Y, Shimazu K, Amano T, Tanaka K, Komat sumoto S (1980) Contribution of autonomic nervous activity to dutoregulation of cerebral blood flow. In: Advances in Physiological Sciences, Vol. 9: Cardiovascular Physiology. Neural Control Mechanisms (Kovach AGB, Sandor P, Kollai, M, eds), London, Pergamon Press, pp 127-136 Gotoh F, Fukuuchi Y, Shimazu K, Amano T, Tanaka K, Komat sumoto S (1981) Blockade of autonomic nervous system at different sites and autoregulation of cerebral blood flow. J Cereb Blood Flow Metabol l(Suppl 1):S331-S332 Gurdjian ES, Webster JE, Martin FA, Thomas LM (1958) Cinephotomicrography of the pial circulation. Arch Neurol Psychiatry 80:418-435 Harper AM, MacKenzie ET (1977) Effects of 5-hydroxy tryptamine on pial arteriolar calibre in anesthetized cats. J Physiol (Lond) 271:735-746 Heiss W-D (1981) What method to choose for quantification of cerebral blood flow in experimental research. Stroke 12:555-557 Intaglietta M, Silverman NR, Tompkins WR (1975) Capillary flow velocity measurements in vivo and in situ by television methods. Microvasc Res 10:165-179

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Lassen NA (1981) Regional cerebral blood flow measurements in stroke: The necessity of a tomographic approach. Editorial. J Cereb Blood Flow Metabol 1:141-142 Ma YP, Kao A, Kwan HC, Cheng KK (1974) On line measure ment of the dynamic velocity of erythrocytes in the cerebral microvessels in the rat. Microvasc Res 8: 1-13 Okayasu H, Gotoh F, Muramatsu F, Fukuuchi Y, Amano T, Tanaka K (1979) A method for continuous measurement of pial vessel diameter by means of a vidicon camera system. Acta Neurol Scand 60(Suppl 72):256-257 Purves MJ (1972) The Physiology of the Cerebral Circulation. Cambridge, Cambridge University Press Purves MJ (1978) Do vasomotor nerves significantly regulate ce rebral blood flow? Circ Res 43:485-493 Rosenblum WI (1969) Erythrocyte velocity and a velocity pulse in minute blood vessels on the surface of the mouse brain. Circ Res 24:887-892 Rosenblum WI (1970) Effects of blood pressure and blood vis cosity on fluorescein transit time in the cerebral microcir culation in the mouse. Circ Res 27:825-833 Rosenblum WI (1972) Ratio of red cell velocities near the vessel wall to velocities at the vessel center in cerebral microcir culation, and an apparent effect of blood viscosity on the ratio. Microvasc Res 4:98-101 Russel RWR, Simcock JP (1966) A new technique for examining the leptomeningeal circulation. Lancet 2:942-943 Tanaka K, Gotoh F, Muramatsu F, Fukuuchi Y, Amano T, Okayasu H, Suzuki N (1980) Effects of nimodipine (Bay e 9736) on cerebral circulation in cats. Arzneimitte/forsch 30:1494-1497 Tanaka K, Gotoh F, Fukuuchi Y, Amano T, Suzuki N (1981) Effects of inhibition of axonal conduction on autoregulation of cerebral blood flow. In: 12th World Congress of Neurol ogy, International Congress Series No. 548, Amsterdam, Excerpta Medica. Wahl M, Kuschinsky W, Bosse 0, Thurau K (1973) Dependency of pial arterial and arteriolar diameter on perivascular os molarity in the cat. Circ Res 32:162-169 Wiederhielm CA (1963) Continuous recording of arteriolar di mensions with a television microscope. J Appl Physiol 18:1041-1042