Regional spinal cord blood flow Jn primates ALAN N. SANDLER,B.Sc., M.B., CH.B., AND CHARLES H. TATOR, M.D., PH.D., F.R.C.S. (C) Department o f Surgery, Division o f Neurosurgery, University o f Toronto, Toronto, Canada

Spinal cord blood flow (SCBF) was measured in the primate thoracic spinal cord using the "C-antipyrine autoradiographic technique that allowed clear differentiation between white and gray matter blood flow. Individual SCBF values were obtained for 0.1-sq mm areas of the thoracic cord cross section. White matter blood flow was homogeneous throughout with a mean value of 10.3 • 0.2 ml/100 gm/min. Graymatter flow was more variable with lower values in the dorsal horns and higher values in the central gray and anterior horns. Mean gray-matter flow was 57.6 • 2.3 ml/100 gm/min. Arterial pO2 was 123 • 2 torr, pCO2 was 40.2 • 0.5 torr and pH was 7.327 • 0.010. Mean arterial blood pressure was 113 • 3 mm Hg and core temperature was 36.4~ • 0.1 ~ C. KEY WORDS autoradiography

9

spinal cord blood flow

HERE have been few attempts to quantitate spinal cord blood flow (SCBF), 4,6,1~176 and only four of these have achieved the resolution necessary to differentiate gray and white matter SCBF. Landau, et al.fl ~ used the gas, tri-fluoro-iodomethane 18q(CF3 18~I), and autoradiography to measure regional cerebral blood flow and regional cervical SCBF simultaneously in the cat. Blood flow in the cord white matter was shown to be 14.0 ml/100 gm/min and in the cord gray matter 63.0 ml/100 gm/min. Griffiths ~ employed the xenon-133 (~33Xe) washout technique in which l " X e in saline was injected into the dog spinal cord and SCBF was estimated from its subsequent washout into the systemic circulation. He found that the white-matter flow was 15.7 ml/100 gm/min and the gray-matter flow was 48.4 ml/100 gm/min. Kobrine, et al., 2~ used the hydrogen clearance method to measure SCBF in

T

J. Neurosurg. / Volume 45 / December, 1976

9 "C-antipyrine

9

primates. The rate at which inhaled hydrogen was washed out of the cord was measured by changes in potential in electrodes inserted into the cord substance. By analysis of the clearance curve they could determine the SCBF: the white-matter flow was 17.5 ml/100 g m / m i n and the "central" cord flow, presumably g r a y - m a t t e r flow, was 14.0 ml/100 gm/min. Griffiths and coworkers la also measured SCBF using the hydrogen clearance technique. In dogs the blood flow in the white matter was 11.5 ml/100 gm/min and in the gray matter only 10.8 ml/100 gm/min; in baboons the white-matter flow was 13.7 ml/100 gm/min and the gray-matter flow was only 16.5 ml/100 gm/min. Thus, both Kobrine, et al.fl ~ and Griffiths and his associates la found very low values for graymatter flow with the hydrogen clearance technique in contrast to the values obtained by Landau, et al.)* with the radioactive gas and 647

A. N. S a n d i e r a n d C. H. T a t o r autoradiographic technique, and by Griffiths TM in his earlier work with the 13SXeclearance technique. Griffiths~,ls has thus obtained different values for gray-matter flow in the same species (dogs) with these two different methods. The only method allowing high resolution of spinal cord structures in terms of regional blood flow without traumatizing the organ is the autoradiographic technique. We have adapted and modified this technique as developed by Landau, et a1.,22 and Reivich, et a1.,23 to allow evaluation and quantitation of SCBF in tissue volumes of 100 # • 1 0 0 . • 30 #. Clear differentiation between gray- and white-matter flow was possible as well as differentiation between smaller areas within the gray or white matter. Methods

Theory o f the Method The theoretical basis for the use of an inert diffusible substance to measure local blood flow has been extensively reviewed by KetyY The technique is based on the Fick' principle, which for a biologically inert, diffusible tracer substance states that the time rate of change of the concentration of tracer within the tissue is equal to the difference between the rate at which the substance is brought to the tissue in the arterial blood and removed from it in the venous blood. Kety~e,18has further shown that this can be expressed mathematically as C, (T) = X k~fT (Ca)e-k'(T-t'ot, where Cl (T) = the concentration of the tracer substance in the tissue at the time T; ), = the tissue-blood partition coefficient for the tracer material; ki = the rate of blood flow per unit weight of tissue multiplied by the reciprocal of the partition coefficient for that tissue; Ca = the concentration of tracer substance in the arterial blood; e = the base for natural logarithms (2.71828); and t = each time interval at which arterial blood sampling occurs during the tracer infusion. Thus, to calculate the blood flow to a given homogeneous region of the spinal cord, three parameters are required: 1) the tissue-blood partition coefficient of the tracer substance for that region of the spinal cord; 2) the concentration of the tracer substance in that region of the spinal cord at some time T, which was 1 minute after the start of the 648

tracer infusion; and 3) the time course of the change in arterial tracer concentration.

Determination o f Partition Coefficient

Spinal

Cord-Blood

Nine male rhesus monkeys weighing between 5.5 and 7.6 kg (mean weight 6.6 + 0.6 kg) were used to determine the partition coefficients between spinal cord white and gray matter and blood under steady state conditions. The animals were premedicated with intramuscular injections of atropine plus a fentanyl-droperidol mixture.* They were then anesthetized with intravenous thiopentone, intubated with an oral-endotracheal cuffed tube, and maintained on a 30% 02/70% N20 gas mixture delivered via a Harvard respirator.f Muscular relaxation was achieved with intravenous pancuronium:]: and supplemental analgesia was provided by intravenous doses of fentanylwif indicated during the procedure by the increase of blood pressure and cardiac rate. Approximately 200 to 300 ml of normal saline were given intravenously during the operation. The cardiac rate was monitored with an esophageal stethoscope and the blood pressure via a PE100 polyethylene catheter in the right femoral artery connected to a pressure transducer and multichannel recorder.** Temperature was recorded with an esophageal thermistor probe connected to a telethermometer.tf An infrared heating lamp maintained the monkeys' body temperature at 36 ~ to 37 ~ C during the procedure. The left femoral artery was cannulated to allow the collection of blood samples for the measurement of PaO2, PaCO2, pH, and tracer concentration during the equilibration *Innovar-Vet manufactured by Pitman-Moore Ltd., Don Mills, Ontario, Canada. tHarvard respirator manufactured by Harvard Apparatus Company, Inc., 150 Dover Road, Millis, Massachusetts 02054. ~Pavulon manufactured by Organon, Canada, Ltd., West Hill, Ontario, Canada. w manufactured by McNeil Laboratories (Canada) Ltd., Don Mills, Ontario, Canada. **Transducer and recorder manufactured by Statham Instruments Inc., 2230 Statham Boulevard, Oxnard, California 93030. ffThermistor probe manufactured by Yellow Springs Instruments, Inc., Yellow Springs, Ohio 45387.

J. Neurosurg. / Volume 45 / December, 1976

Regional spinal cord blood flow in primates

Fxc. 1. Graph showing the time course of the concentration of l'C-antipyrine in blood after a bolus injection of the tracer. With prevention of excretion or degradation of"C-antipyrine, blood radioactivity is constant after 20 to 30 minutes. S2A, S2B, $2C, etc. = blood samples from the nine monkeys in which the partition coefficients for "C-antipyrine between spinal cord and blood were determined. ta to prevent access to the liver by the tracer. Both renal arteries were ligated at the renal pedicles to prevent urinary excretion of the tracer. A laminectomy was then performed from T-4 to L-1 after which 0.1 to 0.3 ~Ci/gm of "C-antipyrine was injected intravenously and the animals maintained at a steady state for approximately 2 hours (mean time, 114 4- 20 mins). During this period of equilibration arterial blood samples were drawn for the measurement of PaO,, PaCO2, pH, and blood "C-antipyrine concentration. At the end of the equilibration period, cardiac arrest was induced with intravenous KCI, and the exposed spinal cord was removed and immediately frozen in 2-methylbutane chilled in liquid nitrogen. The mean values 4- SEM for the PaO,, PaCO~, pH, mean arterial blood pressure, and temperature immediately prior to KCI injection were 140 4- 6 torr, 35.6 4- 1.0 torr, 7.349 4- 0.29, 115 4- 4 mm Hg, and 36.7 ~ 4- 0.2* C respectively. The amount of $$Reading microelectrodes manufactured by radioactivity in each blood sample taken durEschweiler, Kiel, West Germany, and Radiometer, 72 Emdrupvej, DK 2400, Copenhagen, Denmark. ing the equilibration period was determined ~Gas mixtures obtained from Gas Dynamics, as described below and then plotted as a function of time for each animal (Fig. 1). In all Toronto, Ontario, Canada.

period. For the measurement of the blood gases and pH approximately 100 #1 of blood was drawn anaerobically into glass capillary tubes and the arterial pO2, pCO2, and pH were then measured with direct reading microelectrodes$$ operated at 37* C, and the values were corrected for body temperature. 24 The electrodes were calibrated by use of three gas mixtures of known CO2 and O~ content~j and controlled by measurement of tonometered blood. The pH was referred to National Bureau of Standards phosphate buffers, pH 7.383 and 6.481. The tracer used was "C-antipyrine as described for the measurement of regional cerebral blood flow by Reivich, et aL 28 Antipyrine is excreted unchanged in the urine ~ and metabolized in the liver? To maintain a constant level of 14C-antipyrine during the equilibration period the celiac trunk and the superior and inferior mesenteric arteries were ligated as they arose from the abdominal aor-

J. Neurosurg. / Volume 45 / December, 1976

649

A. N. Sandier and C. H. Tator nine animals there was negligible change in the 14C-antipyrine concentration in the blood 30 minutes after the injection, indicating equilibrium had been reached. With chromatographic techniques, Kennedy ~s found that 14C-antipyrine was not degraded, and that the ~'C label was not split from the antipyrine molecule while in the circulation if access to the liver was denied the tracer. Thus, it is probable that all blood and tissue radioactivity measured in the present experiments represented ~4C-antipyrine.

Radioactivity Estimations Spinal Cord Samples

in Blood and

All blood samples were counted in triplicate. Approximately 50-mg aliquots of blood were placed in preweighed scintillation vials containing 1 ml of a quarternary ammonium solubilizing agent, Soluene-350,* and then reweighed. The vials were heated in a water bath at approximately 50 ~ C for 30 minutes by which time all the blood was in solution. Color quenching was reduced by bleaching the samples with the addition of 0.2 ml hydrogen peroxide (30%) followed by 0.2 ml isopropanol. The vials were reheated at 50 ~ C for 30 minutes with the screw caps closed loosely to allow excess hydrogen peroxide to escape. After cooling, 10 ml of a commercially prepared scintillation cocktail, Dimilume,* was added and the samples counted in a liquid scintillation counterf by the channels ratio method s to measure the counter's efficiency. A sample of nonradioactive blood treated in a similar fashion to the radioactive samples served as a blank and was counted at the same time as the radioactive samples. Counts from the radioactive samples were only accepted when the counts obtained from the blank were minimal (usually < 30 disintegrations/min) indicating minimal chemiluminescence and complete dark adaptation. The channels ratio versus efficiency plot used to measure the counting efficiency for the blood samples was produced based upon a set of six samples prepared in a similar fashion to the blank but with a known amount of ~'C-toluene added. Increased quenching was achieved by adding increased quantities of carbon tetrachloride to the six samples. *Soluene-350 and Dimilume obtained from Packard Instrument Company, Inc., 200 Warrensville, Downers Grove, Illinois. 650

Each of the nine spinal cords was divided into four segments approximately 1.5 cm long in a cryostat at - 2 0 ~ C. Three of these segments were used to directly measure the ~4Cantipyrine concentration in the white matter of the cord. With a dissecting microscope mounted in the cryostat, approximately 50 mg of white matter of each segment of cord was dissected and placed in weighed vials containing 1 ml of Soluene-350. The vials were reweighed and the same procedure followed for scintillation counting as for the blood samples except for the omission of the bleaching step. It was not possible to obtain large enough quantities of gray matter by microdissection, and therefore the gray matter ~Cantipyrine concentration was measured using autoradiographic means as described below. To measure the counting efficiency for the cord samples, a set of six samples was made up in the same way as for the blood except nonradioactive spinal cord samples were used to produce the correct channels ratio versus efficiency plot.

Preparation o f A utoradiographs and Calibration o f A utoradiographic Standards The fourth segment of each spinal cord was mounted on microtome chucks with Cryoform and then sectioned at 30 u in the cryostat at - 2 0 ~ C. Sections were picked up from the knife with microscope slides at room temperature and immediately placed on a hot plate at 60 ~ C. The sections dried within 7 seconds. In the darkroom sections were placed face down on Kodak No-Screen medical x-ray film and the slides were taped to the film to prevent any movement during exposure. Six sheets of methyl methacrylate 1 mm thick were commercially prepared with ~4Cbenzoic acid evenly dispersed in the methacrylate in concentrations ranging from 0.01 tzCi/gm to 0.55 #Ci/gm.~ These sheets were divided into 20 squares measuring 1 • 1 cm. In this way 20 sets of six plastic standards each were available, which allowed up to 20 autoradiographs to be prepared simultaneously. One set of six plastic standards was taped onto each film in addition to the slides bearing the cord sections. From these tUnilux II liquid scintillation counter manufactured by Nuclear Chicago Corp., 2000 Nuclear Drive, Des Plaines, Illinois 60018. 1:Preparation by New England Nuclear, Boston, Massachusetts.

J. Neurosurg. / Volume 45 / December, 1976

Regional spinal cord blood flow in primates plastic standards a calibration curve was generated relating percentage light transmission (PLT) to 14C concentration as described below. The film, the slides, and the standards were then placed together between two glass plates, kept in position with bulldog clips, and put in a light-proof cardboard box at room temperature for the 7-day exposure period. After exposure, the film was removed, separated from the slides and standards, developed in Kodak liquid x-ray developer for 5 minutes at 20 ~ C, rinsed for 30 seconds in a 28% acetic acid stop bath, fixed for 8 minutes in Kodak rapid fixer, and then washed for 20 minutes in clean running water. All solutions were maintained within 2 ~ to 3 ~ C of the developer temperature. The autoradiographs of the cords and standards were mounted in cardboard 35-mm slide mounts. After the autoradiographs were made, the cord sections were stained with luxol fast blue and counterstained with hematoxylin and eosin. The PLT values of the autoradiographs were analyzed with a Zeiss scanning microscope photometer.w The variable square diaphragm stop positioned in front of the photomultiplier and the magnification in the optical pathway were adjusted to admit light and measure the PLT from a 100 t~ X 100 # area of the autoradiographs. We define PLT as the ratio of the light passing through the autoradiographs to the light passing through an equal area of the background fog present in each film X 100. As the intensity of light passing through the background fog was always adjusted to 100% PLT by varying the voltage to the photomultiplier of the densitometer used, direct values of PLT for the autoradiographs were obtained. The set of plastic standards on each film also corrected for any differences in exposure and developing that occurred between films. The scanning stage can be set to move in steps of 100 ~t with ranges of motion of up to 75 X 25 mm. The number of steps in an X or Y direction can be controlled by a stage control unit. As the autoradiographs of the spinal cord are approximately 5 mm in diameter the stage was set to move 80 steps on the X axis (8 ram). After the completion of each line scan, the stage was set to shift one step (100 ~) on the Y axis, and then w Scanning Microscope Photometer Type SMP 05 manufactured by Carl Zeiss, Oberkochen, West Germany. J. Neurosurg. / Volume 45 / December, 1976

scan the next subjacent line. After 70 steps (7 mm) on the Y axis the program was terminated. Thus 100-sq ~z areas of the autoradiographs lying adjacent to one another were scanned in sequence, with no overlapping, in a zigzag pattern. The PLT values for each 100sq u area of the autoradiographs were recorded on magnetic tape using a Kennedy incremental tape recorder after signal processing. The process is entirely automatic; the only intervention required is the changing of autoradiographs. A program was written for the Xerox Sigma 5 computer to present the recorded data in a map form, with the PLT values for the autoradiographs coded as shown in Fig. 2. Thus the background area of the film (100 PLT) was represented by 9 and areas of low tracer concentration (white matter) were represented by high numbers whereas areas of high tracer concentration (gray matter) were represented by low numbers. The real PLT values for each 100-sq u area scanned were printed out below each map, line by line. Examination of the numerical map in Fig. 2 shows that the gray and white matter are clearly differentiated in terms of the coded PLT values. A microscope eyepiece reticule was used to partition off the corresponding 30 u thick spinal cord section of each scanned autoradiograph so that any rectangular area of the cord could be located on the autoradiograph by its X, Y coordinates in terms of 100 lz steps. Each area chosen could be directly transposed to the numerical map as each number represented a distance of 100 u in an X or Y direction. Areas encompassing the dorsal columns, the right and left lateral white columns, and the central gray matter were routinely chosen from all the autoradiographs scanned (Fig. 2). The computer was used to calculate the means and standard deviations for the PLT values for each selected area from the raw data on the magnetic tapes. As the PLT values for the areas selected from the dorsal columns and right and left white matter areas were not significantly different for individual animals, they were pooled and a semilogarithmic plot was made (Fig. 3) of the pooled PLT value for white matter of each animal versus the measured 14C-antipyrine concentration as determined by liquid scintillation counting of microdissected samples of white matter from the same animals. By linear regression analysis, the values for the l~C-antipyrine 651

A. N. S a n d i e r

a n d C. H . T a t o r

gray or white m a t t e r at equilibrium/14C-anti pyrine concentration in blood at equilibrium. To correct for differences in the absorption characteristics between the radioactive plastic standards and the 30-/~ spinal cord sections, a semilogarithmic plot of the P L T values versus l~C-concentration for the plastic standards was compared to the semilogarithmic plot of pooled P L T values versus '4C-concentration for the white matter of the nine spinal cords (Fig. 3). The ratio of the slope of the plot for the white matter to the slope of the plot for the plastic standards provided a correction factor that was subsequently applied to future plastic standard calibration curves and this corrected for the differences in the absorption characteristics.

Determination o f the Normal Spinal Cord Blood Flow

FIG. 2. Densitometric analysis of autoradiographs. The numerical map below the autoradiograph is made up of coded values representing the PLT values for corresponding 0.1 sq mm areas of the autoradiograph. 9 = 90-100 PLT and represents the background fog of the x-ray film, 8 = 80-90, 7 = 70-80, 6 = 60-70, and so on to 0 = 0-10 PLT. Differentiation of gray and white matter on the numerical map is clearly seen: gray matter has PLT values of 1 to 7 and white matter 5 to 8. Areas encompassing dorsal and lateral white and gray matter similar to those outlined in Fig. 4 were averaged and allowed the construction of the PLT versus radioactivity plot shown in Fig. 3. concentration for gray matter could be derived from the observed P L T value for gray matter for each animal. In this way the partition coefficients for gray and white matter listed in Table 1 could be calculated from the equation: ~, = x~C-antipyrine concentration in 652

Spinal cord blood flow was measured in a series of 22 male rhesus monkeys weighing between 5.8 and 9.8 kg. In two of the monkeys the spinal cord was not injured and normal S C B F was measured in the thoracic cord at T9-10. In the remainder, SCBF was measured after varying degrees of trauma to the cord at T9-10. In these, SCBF was measured at the injury site and also in cord segments proximal and distal to it. In the uninjured segments that were at least 3 cm from the injured area, S C B F was not significantly different from the S C B F in the two uninjured animals and will be included here. The effects of injury on S C B F will be presented in a separate report. The same a n e s t h e t i c p r o c e d u r e was followed as described above for the measurement of the ~C-antipyrine partition coefficients between cord and blood. Cardiac rate, blood pressure, b o d y core t e m p e r a t u r e , arterial pO2, pCO2 and p H were all monitored as described above. Immediately before each blood flow measurement an arterial sample was drawn and analyzed for pO2, pCO~, and pH. The mean values + S E M for the PaO2, PaCO2, pH, mean arterial blood pressure, and temperature immediately before blood flow measurement were 123 + 2 torr, 40.2 4- 0.5 torr, 7.327 4- 0.010, 113 4- 3 mm Hg, and 36.4 ~ 4-0.1 ~ C respectively. P E I 0 0 polyethylene catheters were inserted into the right and left femoral arteries and the right femoral vein. The catheter in the right

J. Neurosurg. / Volume 45 / December, 1976

Regional spinal cord blood flow in primates femoral artery was connected to the blood pressure monitoring system and the catheter in the left femoral artery was used to obtain arterial samples for PaO2, PaCO2, and pH analysis as well as samples of arterial blood during the infusion of the tracer. The catheter in the right femoral vein was connected to an infusion pump for delivery of the tracer. This method of measuring S C B F requires determination of the curve of the arterial concentration of the isotope from time zero to the time the tissue concentration is measured. To prevent distortion of the curve, the venous infusion catheter was less than 50 mm in diameter and the arterial collection catheter less than 40 mm. Thus, correction for smearing of the arterial curve 2s was not required. A laminectomy between T-7 and T-12 was performed before measurement of SCBF. At the time of flow measurement 100/~Ci/kg of I~Cantipyrine dissolved in normal saline was infused at a constant rate via the cannula in the right femoral vein. During the infusion arterial blood samples were taken in glass capillary tubes at 10-second intervals from the short catheter in the left femoral artery. After 1 minute the heart was stopped with intravenous KCI. The blood samples were immediately transferred to preweighed liquid scintillation vials and processed and counted as described above. The spinal cord was rapidly r e m o v e d and frozen in 2methylbutane chilled in liquid nitrogen. The frozen cords were sectioned at 30 u thickness in the cryostat and autoradiographs of the sections were made as described above. The P L T values for 100-sq u areas of the cord autoradiographs were obtained using the Zeiss S M P 05 scanning m i c r o s c o p e photometer as described above. A program was written for the Xerox Sigma 5 computer to combine the necessary parameters and compute blood flow in the following way. First, the P L T values o f the autoradiographs of the set of six plastic standards on each film were recorded on magnetic tape before recording the P L T values obtained from scanning the autoradiographs of the spinal cord sections on the same film. The calibration curve of P L T versus radioactivity for the standards was calculated by the computer. The slope of this curve was then corrected using the correction factor previously determined to correct for the different absorption characteristics of the plastic and the J. Neurosurg. / Volume 45 / December, 1976

FIG. 3. Semilogarithmic plot of PLT versus radioactivity. The closed circles are values for white matter obtained by microdissection of the frozen spinal cords. The open circles are values for the set of plastic standards placed on the same xray film as the cord sections. The ratio of the slope of the white-matter plot to the plastic standard plot provided a factor which was used to correct for the differing absorption characteristics between the plastic standards and the cord sections. Values for the gray matter were obtained by reading off the radioactivity for the corresponding PLT value for gray matter obtained by densitometry.

sections. All P L T values for the cord autoradiographs were then converted to the corresponding tissue radioactivity values CI(T) using the corrected calibration curve. Second, the values of CI(T) for various values of K1 from 0 to 3.0 m l / g m / m i n were calculated by the computer by numerical integration and from this information blood flow for each Ci(T) for each 100-sq u area of the cord was determined. The scan of each a u t o r a d i o g r a p h was printed out as a numerical map as was described above except that for the S C B F experiments the coded numbers represented blood flow values as 653

A. N. S a n d i e r a n d C. H . T a t o r shown in Fig. 4. The real blood flow values were also printed out below the map for each line scanned. Third, selection of X, Y co-ordinates for any rectangular area from the numerical map

allowed the computer to average the blood flow values for that area. Blood flow was calculated routinely for areas encompassing the dorsal columns, the dorsal and ventral lateral white matter and the central gray matter (Fig. 4). Results

FIG. 4. Normal values for SCBF. The values in the autoradiograph (upper) represent the mean SCBF (ml/100 gm/min) in the areas outlined. D.C. = dorsal columns; RLD = right dorsolateral white matter; RLV = right ventrolateral white matter; LLD = left dorsolateral white matter; L L V - - l e f t ventrolateral white matter; and CG = central gray matter. The computergenerated numerical map (lower) gives coded values for blood flow with each number representing SCBF for the corresponding 0. l-sq mm area of the autoradiograph. 0 = 0 flow and represents the x-ray film background; * = 1-10, 1 = 10-20, 2 = 20-30, 3 = 30--40 and so on to 9 = 90 or more ml/100 gm/min. The map allowed accurate localization of regional SCBF. 654

The mean partition coefficient for gray matter was 1.13 + 0.22 S E M and for white matter was 0.79 4-0.09 S E M (Table 1). These values are significantly different from each other (p < 0.001). This is illustrated in Fig. 5, which shows autoradiographs from three different monkeys of spinal cord sections in which increasing amounts of l'C-antipyrine were administered for the determination of the partition coefficients. In all three animals there is more 14C-antipyrine in gray matter than in white matter at equilibrium independent of the quantity given. Blood flow m e a s u r e m e n t s in n o r m a l thoracic cord in 22 monkeys are summarized in Table 2. The values obtained for blood flow in the dorsal column white matter, the left lateral dorsal and ventral white matter, and the right lateral dorsal and ventral white matter were not significantly different and the mean values were therefore combined to give a value for the mean flow in thoracic cord white matter of 10.3 4- 0.2 ml/100 gm/min. The mean S C B F in thoracic cord gray matter was 57.6 4- 2.3 ml/100 g m / m i n . Gray-matter flow was significantly greater than whitematter flow (p < 0.001). The autoradiograph of a 30-~ section of one of the cords and the corresponding numerical map with the coded blood flow values for each 100-sq tz area of the section are shown in Fig. 4. It can be clearly seen that whereas white-matter flow was much the same in all areas, ranging from less than 10 ml/100 g m / m i n to 10-20 ml/100 g m / m i n , gray-matter flow was less homogeneous. The dorsal gray horns had higher flows than white matter, usually 20 to 40 ml/100 g m / m i n , whereas central graymatter flow and anterior horn flow values were between 20 and 90 ml/100 g m / m i n . In addition, the high resolution of the technique is further demonstrated by the higher flow values in the dorsal root entry zone where small arteries enter the cord and in the region of the a n t e r i o r spinal a r t e r y and its penetrating sulcal branches.

J. Neurosurg. / Volume 45 / December, 1976

Regional spinal cord blood flow in primates TABLE 1

Partition coefficients between blood and spinal cord gray and white matter (X) for ~4C-antipyrine Monkey No.

~4C-Antipyrine C o n c e n t r a t i o n ~,Ci/gm Blood W h i t e Matter* G r a y M a t t e r t

S2A S2B $2C S2D S2E S2F S2G S2H $2I m e a n =~ S E M

0.1632 0.2249 0.2664 0.1631 0.2347 0.1724 0.2368 0.3023 0.2428

0.1090 0.1841 0.2310 0.1293 0.2315 0.1222 0.1820 0.2241 0.1918

X White Matter Gray Matter

0.1578 0.2917 0.3181 0.2262 0.3343 0.1709 0.2902 0.2478 0.2164

0.67 0.82 0.87 0.79 0.99 0.71 0.77 0.74 0.79 0.79 • 0.09

0.97 1.29 1.19 1.38 1.42 0.99 1.22 0.81 0.89 1.13 • 0.22

* W h i t e m a t t e r r a d i o a c t i v i t y was m e a s u r e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g o f m i c r o d i s s e c t e d s a m p l e s o f w h i t e m atter. t G r a y m a t t e r r a d i o a c t i v i t y was c a l c u l a t e d f r o m d e n s i t o m e t r i c m e a s u r e m e n t s o f t he a u t o r a d i o g r a p h s .

TABLE 2

Normal spinal cord blood flow in 22 monkeys (ml/lO0 gm/min)* Monkey No. NFB t NFC NFD NFE NFF NFG NFH NFI NFJ NFK NFL NFM NFN NFP NFR NFUt NFV NFW NFY NFZ N2D N2F mean • SEM

Dorsal Columns 10.6 I1 .0 9.9 9.2 10.0 7.3 8.6 8.2 13.0 9.0 7.3 9.6 11.0 7.2 11.5 7.6 12.7 9.7 6.6 8.4 12.5 13.5

• ~= • =~ • • =~ e= • • =~ ~= ~= a= a= =~ ~= ~= • • :~ ~=

9.8 •

Thoracic White Matter Left Lateral Left L a t e r a l Right Lateral Dorsal Ventral Dorsal

0.3 0.2 0.1 0.1 0.2 0.1 0.1 0.2 0.2 0.1 0.2 0.3 0.3 0.3 0.2 0.2 0.4 0.2 0.2 0.3 0.2 0.5

10.6 11.3 9.2 9.9 10.4 7.7 10.9 8.5 12.2 11.5 9.0 10.5 10.6 7.1 13.1 8.9 9.5 8.7 12.6 9.5 13.5 8.7

:~ 0.3 • 0.2 a= 0.1 • 0.1 a= 0.2 a= 0.2 e= 0.2 • 0.2 =~ 0.3 ~= 0.2 • 0.3 • 0.3 :~ 0.3 ~= 0.4 • 0.2 ~= 0.3 • 0.4 =~ 0.3 =~ 0.4 • 0.3 • 0.2 ~= 0.3

0.4

10.2 n= 0.4

10.6 14.4 9.1 9.1 11.1 7.3 9.4 9.2 12.2 11.4 10.5 11.2 10.8 6.2 13.5 9.8 13.5 8.9 7.7 10.5 15.5 11.8

a: 0.4 a= 0.3 • 0.2 a= 0.1 =L 0.3 • 0.2 :~ 0.2 ~: 0.3 a: 0.3 :~ 0.2 :~ 0.4 • 0.4 • 0.3 :~ 0.4 =~ 0.2 • 0.3 • 0.5 =~ 0.3 ~= 0.3 • 0.3 • 0.3 =~ 0.3

10.6 •

0.5

11.1 11.7 10.1 10.9 10.5 7.6 10.3 9.0 12.0 8.0 10.1 10.6 10.7 8.1 13.3 8.2 10.9 11.6 9.2 8.6 12.3 8.2

a= 0.4 ~= 0.3 • 0.1 a= 0.2 =~ 0.2 a= 0.2 =~ 0.2 ~= 0.2 a= 0.4 =~ 0.2 ~= 0.3 a= 0.3 a= 0.3 ~= 0.4 • 0.2 a= 0.3 a= 0.4 a= 0.3 ~= 0.3 • 0.2 ~= 0.2 a= 0.3

10.2 •

0.5

Right Lateral Ventral 11.1 12.8 10.1 9.8 10.9 7.6 10.7 8.9 13.6 9.4 9.5 13.5 11.0 7.2 14.6 9.6 13.7 10.0 10.6 9.5 13.3 8.5

n= 0.5 =~ 0.3 ~ 0.2 • 0.2 a= 0.2 ~= 0.2 • 0.2 ~- 0.2 • 0.3 • 0.2 ~= 0.4 ~= 0.4 ~= 0.3 • 0.4 :~ 0.2 ~= 0.3 ~- 0.4 a= 0.3 a= 0.4 ~- 0.3 :~ 0.2 • 0.3

10.7 -" 0.4

Thoracic Gray Matter Central Gray 46.1 69.0 39.3 52.7 60.5 60.2 61.1 55.8 47.6 66.0 66.5 79.5 61.8 52.8 72.3 52.5 70.5 66.1 47.6 50.1 46.6 42.4

=~ 0.6 • 0.6 • 0.4 a= 0.4 e= 0.9 • 0.9 a= 0.8 =~ 1.0 a= 0.7 -'- 1.3 • 0.9 =u 1.9 ~= 1.4 • 1.0 a= 0.9 =~ 0.6 • 1.3 a= 1.2 ~= 0.9 • 1.0 • 0.6 a= 0.9

57.6 ~= 2.3

*Values a r e m e a n • S E M for each a n i m a l . In each a n i m a l a n a v e r a g e o f t hre e a u t o r a d i o g r a p h s w e r e used. F o r each o f t h e five w h i t e - m a t t e r a r e a s a n d the g r a y - m a t t e r a r e a o n e a c h a u t o r a d i o g r a p h , t h e m e a n S C B F was c o m p u t e d f r o m a p p r o x i m a t e l y 100 to 300 0.1-sq m m areas. T h e r e is no s i gni fi c a nt difference b e t w e e n a n y o f the w h i t e - m a t t e r values. T h e m e a n o f the five m e a n whi t e m a t t e r S C B F v a l u e s w a s t h e r e f o r e t a k e n as r e p r e s e n t i n g w h i t e - m a t t e r flow a n d was 10.3 :~ 0.2 S E M . t N o injury.

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655

A. N. Sandier and C. H. Tator

FIG. 5. Autoradiographs from three different monkeys in which the partition coefficients between spinal cord gray matter and white matter and blood were measured. Increasing quantities of ~4C-antipyrine were infused from left to right. In all cases there is more radioactivity in gray than in white matter indicating a larger value for the partition coefficient for gray matter. Discussion

The finding that the partition coefficient for antipyrine between primate cord gray matter and blood was greater than that between white matter and blood is at variance with the only other measurements of the partition coefficients for antipyrine in the central nervous system which have been made in rat brain gray and white matter? 3 In the rat the values for gray and white matter were found to be 1.00 and 0.97 respectively. Antipyrine has its greatest solubility in water (100 gm/100 ml) ~ and, as gray matter has a higher water content than white matter, I' it might be expected that the partition coefficient for antipyrine would be greater in gray than in white matter as was found in these experiments. This difference was clearly seen in the thoracic spinal cord with its relatively small volume of gray matter. Figure 5 shows autoradiographs of sections from three of the nine animals in this series and demonstrates that the concentration of 14C-antipyrine was always greater in gray matter than in the white matter regardless of the quantity of isotope given. The flow values we obtained in white matter were somewhat lower than those described by previous investigators (Table 3). Our blood flow values for the gray matter were similar to those described by Landau, et al., 22 for the cervical cord in cats and by Griffiths 12for the thoracic cord in dogs. However, our gray-matter values differed markedly from those found by Kobrine, et al., 2~ for the thoracic cord of rhesus monkeys and also from those found by Griffiths and coworkers ~a for the gray matter of the canine and primate spinal cords. The reasons for these latter differences may be related to methodology as 656

in their experiments both Kobrine, et al., 2~ and Griffiths and his associates iS used the hydrogen clearance technique to measure SCBF and found gray-matter flows four to five times smaller than those demonstrated by the autoradiographic method using either tri-fluoro-iodomethane-131122 or 14C-antipyrine. (Table 3). It is also interesting that Griffiths found gray-matter flow to be four to five times greater in dogs with the 138Xeclearance technique TM than he found with the hydrogen clearance technique. The finding of similar values for white- and gray-matter flow using hydrogen clearance may be explained by the very rapid diffusion of hydrogen through tissues. Thus, a probe in the central gray matter of the cord would be measuring hydrogen washout in the gray matter and a large part of the surrounding white matter. The fast component of the washout curve reflecting rapid gray-matter flow would be obscured by the prolonged slow component reflecting the lower white-matter flow. This places a severe restriction on the usefulness of the hydrogen clearance technique when it is desired to achieve resolution of flow in very small areas as is required in SCBF. In contrast to the results found with other methods of measuring SCBF in animals, the l'C-antipyrine autoradiographic technique has clearly shown that gray-matter flow is approximately five times greater than white-matter flow. In addition, this technique has other advantages. There is no trauma to the cord. Flow can be measured in very small areas of the cord (0.1 sq mm). This ability to measure truly regional SCBF has demonstrated heterogeneity of gray-matter flow (Fig. 4). The SCBF can be measured in any part of any one segment of the spinal cord or in all segments at the same J. Neurosurg. / Volume 45 / December, 1976

Regional spinal cord blood flow in primates TABLE 3 Quantitative studies of normal spinal cord blood flow

Method

Author

Species and Cord Segment

tri-fluoro-iodomethane (CFa~3q) uptake -b autoradiography

Landau, et al. (1955)

cats, cervical

~4C-antipyrine uptake -k autoradiography

Sandler & Tator (1976)

rhesus monkeys, thoracic

mXe washout

Smith, et al. (1969) Ducker & Perot (1972) Grifliths ( 1973)

goats, thoracolumbar dogs, thoracolumbar dogs, thoracic

Ducker & Garrison Argon washout + intracord vacuum mass spectrometer probe (1972) hydrogen clearance

Kobrine, et al. (1974, 1975)

primates, thoracolumbar rhesus monkeys, thoracic

Griffiths & Crawford dogs, (1975) segment not given baboons, segment not given particle distribution

Flohr, et al. (1969)

time and the location of the m e a s u r e m e n t is precisely known. I f it is desirable to observe changes in S C B F produced by physiological or pathological events over a period of time, the disadvantage of having to sacrifice the animal at the time of m e a s u r e m e n t m a y be overcome by performing experiments on groups of animals at differing time intervals after changing the particular parameter, for instance, arterial pCO~ or spinal injury. It is acknowledged that the use of "C-antipyrine to measure blood flow in the central nervous s y s t e m has been criticized by Eckman, et al., 6 who have c o m p a r e d experimentally derived flow values using either "C-antipyrine or CFsI TM against theoretically derived flow values g e n e r a t e d for a systematically varied range of blood flow and capillary permeability values using a digital computer. Errors in the calculated flow values were greatest for high rates of blood flow and low permeability coefficient-surface area products. The authors concluded that "C-antipyrine uptake into the cat brain was J. Neurosurg. / Volume 45 / December, 1976

cats, cervical thoracic lumbar

Spinal Cord Blood Flow (ml/100 gin/rain) white matter gray matter

14.0 63.0

white matter gray matter

10.3 57.6

white q- gray matter

16.2

white d- gray matter 15.6 white matter 15.7 gray matter 48.4 white q- gray matter

15.0

white matter gray matter

17.5 14.0

white matter gray matter

11.5 10.8

white matter gray matter white d- gray matter white q- gray matter white q- gray matter

13.7 16.5 20.3 16.5 23.7

limited by both blood flow and capillary permeability and that it was unwise to use ~4C-antipyrine for the m e a s u r e m e n t o f regional cerebral flow under conditions in which increased flow was being measured. However, Eklof, et al., 8 have also evaluated the a c c u r a c y o f cerebral b l o o d flow measurements using several tracers including "C-antipyrine and have provided data that support our use of this method for measuring SCBF. They measured the blood flow in rat brain using " C - a n t i p y r i n e , "C-ethanol, allwater, and lS3Xe, and the values obtained were c o m p a r e d to rat brain cortical flow values obtained using the Kety-Schmidt technique 19 with xaSXe substituted for N~O. 7 The period of tracer infusion was varied f r o m 30 to 120 seconds in all cases. For " C - a n t i pyrine at n o r m a l pCO2 levels, using the 60second infusion period, cortical flow was 90 + 5 m l / 1 0 0 g m / m i n whereas using the Kety-Schmidt technique cortical flow was 100 + 3 m l / 1 0 0 g m / m i n . As the thoracic SCBF in the gray m a t t e r in our experiment 657

A. N. Sandler and C. H. Tator References was less than 100 ml/100 gm/min, the results 1. Blair RDG, Waltz AG: Regional cerebral of Eklof, et al., support our view that 14Cblood flow during acute ischemia. Correlation antipyrine provides accurate values for SCBF of autoradiographic measurements with obserunder normal conditions. In addition, Eklof, vations of cortical microcirculation. Neuret al.,8 by theoretically varying C1, ~, and the ology 20:802-808, 1970 infusion times in conjunction with a typical 2. Brodie BB, Axelrod J: The fate of antipyrine experimentally derived arterial concentration in man. J Pharmacol Exp Ther 98:97-104, curve, demonstrated that varying ~ from 0.8 1950 to 1.2 produced insignificant changes in flow 3. Bush ET: General applicability of the channels ratio method of measuring liquid scintillation values for an infusion time of 60 seconds and counting efficiencies. Anal Chem 35: for flow values of 100 ml/100 gm/min. Thus, 1024-1029, 1963 although we used a value of ~, = 0.79 to 4. Ducker TB, Garrison WB: Experimental calculate white-matter flow and the value spinal cord trauma. II. Monkeys who were = 1.13 to calculate gray-matter flow in our paraplegic, paraparetic, or normal. In experiments, the effect of this variation in preparation on blood flow values less than 100 ml/100 5. Ducker TB, Perot PL Jr: Spinal cord blood g m / m i n as found in the spinal cord is flow compartments. Trans Am Neurol Assoc negligible. 96:229-231, 1972 The finding of a S C B F ratio between gray 6. Eckman WW, Phair RD, Fenstermacher JD, et al: The influence of capillary permeability and white matter of about 5: 1 is similar to the limitations on the measurement of regional gray-white flow ratio for the brain, although cerebral blood flow, in Langfitt TW, values for cerebral blood flow are somewhat McHenry LC Jr, Reivich M, et al (eds): higher than SCBF. For example, Reivich, et Cerebral Circulation and Metabolism. New al.? s using the x4C-antipyrine autoradioYork/Heidelberg/Berlin: Springer-Verlag, graphic technique in cats found cortical 1975, pp 129-131 gray-matter flow to be 122 ml/100 g m / m i n 7. Ekl6f B, Lassen NA, Nilsson L, et al: Blood whereas subcortical white matter was 21 flow and metabolic rate for oxygen in the ml/100 g m / m i n . Similarly Eklof, et al., 8 cerebral cortex of the rat. Acta Physiol Scand measured regional cerebral blood flow in the 88:587-589, 1973 cortex of rats using four indicators, l~C-anti8. EklSf B, Lassen NA, Nilsson L, et al: Regional cerebral blood flow in the rat pyrine, '4C-ethanol, 3H-water, and 133Xe, and measured by the tissue sampling technique; a found cortical gray-flow values between 95 critical evaluation using four indicators: C 14and 127 m l / 1 0 0 g m / m i n . In squirrel antipyrine, Cl'-ethanol, H 3 water and monkeys, Blair and Waltz ~ have recorded Xenon ~33. Acta Physioi Scand 91:1-10, 1974 mean values of 118 ml/100 g m / m i n for gray9. Fick A: Ober die Messung des Blutquantums matter flow and 49 ml/100 g m / m i n for in den Herzventrikeln. Quoted in its entirety white-matter flow using the ~C-antipyrine by Hoff HE, Scott H J: Physiology. N Engl J autoradiographic technique. Med 239:120-126, 1948 In conclusion, it is pertinent to emphasize 10. Flohr H, Brock M, P611 W: Spinal cord blood that white matter S C B F in the thoracic cord flow in anesthetized cats. Pfluegers Arch 312:R31, 1969 (Abstract 38; Ger) is of a very small magnitude (10 ml/100 gm/min) with little reserve, and thus any ll. Greenberg LA: Antipyrine, A Critical Bibliographic Review. New Haven: Hillhouse compromise of S C B F m a y be of vital imporPress, 1950 tance for the function and integrity of the 12. Griffiths IR: Spinal cord blood flow in dogs. I. spinal cord white m a t t e r that contains the allThe "normal" flow. J Neuroi Neurosurg important ascending and descending tracts. Psychiatry 36:34-41, 1973 13. Griffiths IR, Rowan JD, Crawford RA: Flow in the grey and white matter of the spinal cord Acknowledgments measured by a hydrogen clearance technique, Grateful acknowledgment is made to Mrs. L. in Harper AM, Jennet WB, Miller JD, et al Marmash, Miss V. Edmonds, R.N., and Mrs. J. (eds): Blood Flow and Metabolism in The Yang, M.Sc., for technical assistance and to Dr. V. Brain. Edinburgh: Churchill Livingstone, R. MacMillan, for performing the blood gas 1975, ch 4, pp 20-21 analyses. Part of the computer program was 14. Katzman R, Pappius HM: Brain Electrolytes supplied by Drs. D. Robertson and R. Hass, and Fluid Metabolism. Baltimore: Williams & Queen's University, Kingston, Ontario, Canada. Wilkins, 1973, pp 1-13

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Regional spinal cord blood flow in primates 15. Kennedy C J: Personal communication, June 1975 16. Kety SS: Measurement of local blood flow by the exchange of an inert, diffusible substance. Methods Med Res 8:228-236, 1960 17. Kety SS: The theory and applications of the exchange of inert gas at the lungs and tissues. Pharmaeol Rev 3:1-41, 1951 18. Kety SS: Theory of blood-tissue exchange and its application to measurement of blood flow. Methods Med Res 8:223-227, 1960 19. Kety SS, Schmidt CF: The determination of cerebral blood flow in man by the use of nitrous oxide in low concentration. Am J Physiol 143:53-66, 1945 20. Kobrine AI, Doyle TF, Martins AN: Local spinal cord blood flow in experimental traumatic myelopathy. J Neurosurg 42: 144-149, 1975 21. Kobrine AI, Doyle TF, Martins AN: Spinal cord blood flow in the rhesus monkey by the hydrogen clearance method. Surg Neurol 2:197-200, 1974 22. Landau WM, Freygang WH Jr, Roland LP, et al: The local circulation of the living brain;

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values in the unanesthetized and anesthetized cat. Trans Am Neurol Assoc 80:125-129, 1955 23. Reivich M, Jehle J, Sokoloff L, et al: Measurement of regional cerebral blood flow with antipyrine-14C in awake cats. J Appl Physiol 27:296-300, 1969 24. Severinghaus JW: Blood gas concentration, in Fenn WO, Rahn H (eds): Handbook of Physiology. Section 3: Respiration, Volume 2. Washington, DC: American Physiological Society, 1965, pp 1475-1487 25. Smith AL, Pender JW, Alexander SC: Effects of PCO2 on spinal cord blood flow. Am J Physiol 216:1158-1163, 1969 This work was supported by Medical Research Council of Canada Grant MT-4046. Dr. Sandler was a Research Fellow of the Medical Research Council of Canada at the time of this study. Address reprint requests to: Charles H. Tator, M.D., Acute Spinal Cord Injury Unit, Sunnybrook Medical Center, 2075 Bayview Avenue, Toronto M4N 3M5, Canada.

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