Postmortem diffusion of autoradiographic blood flow tracers

Brain Research 842 Ž1999. 184–191 www.elsevier.comrlocaterbres

Research report

Postmortem diffusion of autoradiographic blood flow tracers Joel H. Greenberg ) , Calogera LoBrutto, Kathleen M. Lombard, Jergin Chen Department of Neurology, CerebroÕascular Research Center, UniÕersity of PennsylÕania, 429 Johnson PaÕilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6063, USA Accepted 13 July 1999

Abstract The heterogeneity of blood flow in the brain under normo- and pathophysiological conditions, as well as during functional activation, has stimulated an interest in the use of autoradiography as a technique for the measurement of local cerebral blood flow. w14Cxiodoantipyrine is the most prevalent tracer for the autoradiographic measurement of local cerebral blood flow since it is inert, nonvolatile, and is readily diffusible through the blood–brain barrier. The ability to diffuse freely in cerebral tissue, however, can lead to significant errors if the time duration between when the animal is sacrificed and when the tissue is frozen becomes appreciable, leading to significant postmortem diffusion of the tracer. Using an in vitro technique, the bulk diffusion coefficient for w14Cxiodoantipyrine was measured in brain tissue Ž2.1 = 10y6 cm2rs.. Cerebral blood flow was measured with w14Cxiodoantipyrine in anesthetized rats. At the end of the radiotracer infusion, the brain was freeze-captured using a device consisting of two rapidly spinning stainless steel blades that were pneumatically driven through the head, freezing the tissue several hundred milliseconds following sacrifice. Autoradiograms from these brains exhibit considerable heterogeneity in blood flow. Computer simulations of the effect of tracer diffusion on these autoradiograms show significant degradation of the images highlighting the importance of very rapid postmortem freezing. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Cerebral blood flow; Iodoantipyrine; Tracer diffusion; Autoradiography

1. Introduction Development of in situ autoradiographic techniques has demonstrated the significant heterogeneous distribution of blood flow in the brain w8,13,20,21x. The differences in blood flow between cerebral regions is probably related to the heterogeneous distribution of cerebral glucose metabolism since cerebral blood flow and cerebral glucose metabolism appear to be coupled w12,19x. The tracer most frequently used to measure the local distribution of cerebral blood flow in laboratory animals is w14 Cxiodoantipyrine. This is a highly diffusible compound whose distribution in the tissue, at least at physiological blood flow rats, is flow- and not diffusion limited. Although w14 Cxiodoantipyrine is not as diffusible a tracer as the inert gas w131 Ix-labeled trifluoroiodomethane, originally used for the measurement of local cerebral blood flow w3x, it diffuses more freely through the blood–brain barrier than w14 Cxantipyrine, which was in common usage prior to the introduction of w14 Cxiodoantipyrine w21x. )

Corresponding author. Fax: [email protected]

q 1-215-349-5629;

E-mail:

Although unrestricted diffusion through the blood–brain barrier is an important property for a compound used as a tracer for the autoradiographic measurement of local cerebral blood flow, this ability to diffuse freely in cerebral tissue can lead to significant errors if the time duration between the cessation of cerebral blood flow Ždeath. and tissue freezing is appreciable w23x. During this period the w14 Cxiodoantipyrine can diffuse from areas of high flow into regions of low flow obscuring the actual heterogeneity of the blood flow distribution. We have undertaken simulations to examine the diffusion of w14 Cxiodoantipyrine post-mortem and show that this diffusion is not negligible even if the freezing time is as short as 30 s.

2. Materials and methods 2.1. Cerebral blood flow studies These studies were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Five Sprague–Dawley rats were anesthetized by an i.p.

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J.H. Greenberg et al.r Brain Research 842 (1999) 184–191

injection of pentobarbital sodium ŽAbbott Laboratories, North Chicago, IL, USA. Ž40 mgrkg.. A catheter was placed into a femoral vein for the infusion of w14 Cxiodoantipyrine Ž14 C-IAP. ŽDu Pont NEN, Boston, MA, USA.. The head was shaved and the rats were placed into the Plexiglas holding chamber of a rapid slicing instrument modified after the design of Quistorff w17,18x. Twenty to twenty-five mCi of 14 C-IAP was infused into the venous catheter using a programmed pump that delivered the tracer at a steadily increasing rate so as to produce a nearly linear increase in arterial 14 C-IAP concentration. Forty-five seconds after the start of the tracer infusion, the animal was sacrificed with the rapid slicing instrument. This instrument consisted of two rapidly spinning stainless steel blades spaced 10 mm apart that were pneumatically driven laterally through the head of the rat in a coronal plane roughly parallel to the orbital–meatal line. The tissue caught between the blades was clamped by two aluminum blocks that had been supercooled in liquid nitrogen and the clamped tissue immediately immersed in a liquid nitrogen bath. Less than 100 ms elapsed between the time the blades began to cut into the head and the time when the tissue block was immersed into the liquid nitrogen. To prevent cracking, 10 s later the tissue was transferred to a container of powdered dry ice for 5–10 min and then stored at y708C until dissected. The dissection procedure involved removing the brain tissue from the tissue slice which, besides brain, consisted of frozen muscle, bone, skin, and connective tissue. This dissection was done in a cold room on a metal block cooled with dry ice, and took less than 5 min. The brain was then placed into a cryostat ŽBright Instrument, Huntington, England. at y228C, and 20 mm thick brain sections were cut and thaw mounted onto glass coverslips. These sections were placed in apposition to autoradiographic film ŽBiomax MR1-Eastman Kodak, Rochester, MN, USA. for 5–7 days along with calibrated w14 Cxmethylmethacrylate standards, and the films hand developed. The autoradiograms from the blood flow Ž14 C-IAP. studies were digitized using an image analysis system comprised of a video camera interfaced to a frame grabber on a Quadra 800 computer ŽApple, Cupertino, CA, USA. running Brain V2.0 image analysis software w15x. The digital image produced was a 256 by 256 array and each pixel represented a 25 mm = 25 mm region of the film. A set of six standards that had been calibrated in units of 14 C concentration for 20 mm thick brain sections were also scanned, and the autoradiographic images were converted to tracer concentration values using a 3rd order polynomial fit and a look-up table. 2.2. Determination of diffusion coefficient Rats Ž n s 5. were sacrificed with an overdose of Nembutal Ž150 mgrkg i.p.., and the brain was quickly removed

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from the skull. It was placed into a rodent brain matrix ŽASI, Warren, MI. and two coronal cuts made in the brain, at bregma and at 4.0 mm posterior to bregma. This 4 mm tissue slab was placed anterior face down onto a small plastic weighing tray and 2–4 mCi 14 C-IAP in 50 ml was pipetted onto the posterior surface of the slab. Two minutes later, the excess fluid was carefully removed with filter paper and the plastic tray containing the brain slab was floated onto the surface of water in a dish at 378C. A top was placed over the dish and it was placed into an oven maintained at 378C. This procedure prevented the brain from drying out during the incubation period. Three to four hours later, the dish was removed from the oven and the brain placed onto a siliconized glass slide. Another glass slide was placed over the brain with spacers inserted on either side of the brain to prevent it from being compressed or distorted. The glass–brain–glass sandwich was immersed into a bath of isopentane cooled to y508C with dry ice. After the brain was totally frozen it was placed into a cryostat and 20 mm thick serial sections obtained parallel to the flat brain surface starting from the posterior face of the slice and continuing throughout the slice thickness. Following thaw mounting, these sections were placed onto autoradiographic film as described above. The images of the serial sections were digitized as described above. Homogeneous regions of interest were identified on the images and the w14 Cx concentrations obtained from all of the serial sections. The diffusion coefficient, D, was calculated from a plot of the log of the w14 Cx concentration against distance as determined by section number and section thickness w4x.

2.3. Simulations Diffusion of IAP in the brain was simulated using the two dimensional diffusion equation: EC Et

sD

ž

E2 C Ex2

E2 C q

E y2

/

Ž 1.

where C is the concentration of the 14 C-IAP in the brain tissue, and D is the diffusion coefficient of IAP. An iterative technique was used to solve this equation and simulate diffusion of the 14 C-IAP in the image:

Ý Ž C j y Ci . DQ i s D

j

d

SD t

Ž 2.

where DQ i is the net flux of tracer into or out of voxel i from neighboring voxels j over a given time duration D t, d is the diffusion distance between voxels, and S is the area of the boundary between two voxels. Assuming cubic voxels of dimensions equal to the pixel size of the image

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Fig. 1. Distribution of w14 Cxiodoantipyrine in a two-tissue slab model at various times Ž t s 0 s, t s 5 s, t s 20 s, t s 45 s, t s 1.5 min, and t s 2 min. after the start of diffusion. Prior to the start of diffusion, the concentration is set at unity to the left of the interface Žnegative distances., and zero to the right of the interface Žsolid line.. The diffusion coefficient of iodoantipyrine used in the simulation was 2.1 = 10y6 cm2rs.

Ž25 mm = 25 mm., then S s d 2 , and Q s Crd 3. The new concentration in a pixel after time D t will thus be Cik s Ciky1 q

DD t d2

4

žÝŽ 1

C j y Ciky1 .

/

Ž 3.

where Cik is the 14 C-IAP concentration in the ith pixel after k iterations, and C j y Ciky 1 represents the communi-

cation between pixel i and the four pixels surrounding pixel i. Using Eq. Ž3., images were obtained of the 14 C-IAP concentration in the tissue at various time points Ž10 s, 20 s, 40 s, 80 s, 120 s. following the start of diffusion. The time interval of iteration Ž D t . was chosen to be 0.2 s after preliminary simulations indicated that the data did not change when a smaller time interval was used.

Fig. 2. Simulation of the blood flow distribution in a structure of diameter 1.5 mm of high flow Ž150 ml 100 gy1 miny1 . surrounded by tissue with a low flow Ž75 ml 100 gy1 miny1 . as a function of time between sacrifice and freezing of tissue. The distance scale is from the center of the high flow region. Even at a diffusion time of 4 min, the calculated blood flow in the center of the region is 99.5% of the true flow, and the average flow in the entire structure is 90% of the true flow.

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2.4. Analysis of simulated images The effect of simulated diffusion on image contrast was determined by examining the changes in shape of a line profile, 250 mm in width, drawn through the cortex 0.5 mm beneath the cortical surface. This line cuts though cortical columns evident in the original autoradiograms and yielded a sinusoidal-type pattern that was damped by diffusion. The distortion of this line profile due to simulated diffusion was calculated as the difference between the activity in a cortical column Ždark band., and that in the light area between the cortical columns, normalized by the activity in the cortical column. Another measure of the effect of diffusion was obtained by calculating the coefficient of variation of a region of interest Ž0.6 mm = 6 mm. in the cortex as a function of time. An estimate of diffusion of blood flow tracers in tissue can also be obtained by solving Eq. Ž1. explicitly in the special case of a simple one dimensional system with well defined boundary conditions. Considering a model system with an extended initial distribution such that C s C0 at Ž x - 0, y` - y - q`, t s 0,., and C s 0 at Ž x ) 0, y` - y - q`, t s 0., the solution to Eq. Ž1. becomes: x C Ž x ,t . s 1r2C0 erfc Ž 4. ' 2 Dt

ž

/

This describes a model system of two large flat pieces of tissue in contact with each other. At time zero one piece has a homogeneous 14 C-IAP concentration distribution

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equal to C0 , and in the other tissue piece, the 14 C-IAP concentration is zero. An examination of the effect on quantitation of cerebral blood flow due to diffusion from a relatively large region with a high blood flow Žsuch as the superior colliculus. surrounded by tissue was much lower flow was also made. The region was assumed to have a blood flow of 150 ml 100 gy1 miny1 , to be 1.5 mm in diameter, and to be surrounded by tissue with a flow of 75 ml 100 gy1 miny1 w21x. The flow profile was determined following 10 s, 30 s, 1 min, 2 min and 4 min of diffusion. A similar simulation was undertaken to examine the effect of delay in freezing on computed blood flow in the cortical columns. The cortical columns were taken to be alternating light and dark columns, each 400 mm across. These simulations were also undertaken at times of diffusion ranging from 10 s to 4 min.

3. Results 3.1. Diffusion coefficient The autoradiographic images obtained from the brain tissue slab were relatively homogeneous with only a few regions that appeared darker andror lighter than the remainder of the section. Only regions that exhibited good homogeneity and that were far from dark or light spots were used for the diffusion coefficient determinations.

Fig. 3. Simulation of the blood flow distribution in the cortical columns as a function of time between sacrifice and tissue freezing. Blood flow in the center of the column is assumed to be 220 ml 100 gy1 miny1 , blood flow between columns 100 ml 100 gy1 miny1 , and the columns are 400 mm across. When the diffusion time is only 1 min, the calculated blood flow in the column is appreciably understand. The black bars indicate location of the cortical columns.

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Table 1 Calculated blood flow in cortical columns Blood flow expressed as a percent of flow in the column Ž400 mm wide. prior to diffusion. b

50 mm 100 mmb 200 mmb 400 mmb

10 s a

30 s a

1 mina

2 mina

4 mina

99.8 99.7 99.1 93.0

95.7 95.3 93.8 87.8

88.6 88.3 87.1 82.9

80.0 79.9 79.4 77.4

74.3 74.2 74.1 73.7

a

Diffusion time. b Width of analysis area, centered at middle of cortical column.

There was a hint of a difference in optical density between the gray matter and the white matter. Since the volume of

the white matter was small, no attempt was made to measure the diffusion coefficient in it. The value obtained for the diffusion coefficient in the gray matter was 2.1 = 10y6 " 0.3 = 10y6 cm2rs Žmean " S.D... This value was used in all of the simulations presented below. 3.2. Simulations The distribution of 14 C-IAP in the two-tissue slab model at various times after the start of simulated diffusion can be seen in Fig. 1. Even after only a few seconds, the 14 C-IAP has appreciably diffused from the ‘hot’ tissue slab into the ‘cold’ slab. By 45 s, a region in the ‘cold’ slab 200 mm from the interface already has a concentration of

Fig. 4. Effect of postmortem diffusion on cerebral blood flow images obtained with w14 Cxiodoantipyrine. The top panel is from a coronal section from a rat brain frozen with a freeze clamp device several hundred milliseconds after cessation of blood flow, while the remaining panels are computer simulations of the blood flow distribution after 20 s, 45 s, 1.5 min, and 3 min of tracer diffusion. The cortical columns seen in the original image are progressively altered as the diffusion time increases and are not visually apparent by 3 min of simulated diffusion.

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7.3% that in the ‘hot’ slab, while by 120 s, the concentration has risen to almost 20%. Simulation of diffusion from a relatively large region with a high blood flow such as the superior colliculus, surrounded by a region with a flow 50% less, shows that the blood flow in the high flow region will be progressively underestimated as the postmortemrpre-freezing time increases ŽFig. 2.. The degree of flow underestimation also depends on the how much of the anatomic structure is analyzed in the autoradiogram. If only the central pixels are examined, there is no error, even at freezing times as large as 4 min, while if the entire structure is examined, then the degree of underestimation of blood flow is approximately 10%. Regions in which only a small area Žor volume. has a high blood flow, and this area is surrounded by an area with low flow, will show greater degradation. In the case of simulated cortical columns 400 mm wide separated by 400 mm with tissue in which the flow is only

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45.5% as large, the flow, even in the center of the column, is underestimated by 4.3% at 30 s, and 25.7% at 4 min ŽFig. 3; Table 1.. The blood flow underestimation is greater if the measurement area encompasses more of the ‘dark’ column. The appearances of the 14 C-IAP autoradiograms are noticeably altered as a result of the simulated IAP diffusion, primarily at the latter times. The radial cortical columns that exist in the original autoradiograms are still evident after a simulated diffusion time of 45 s, but are absent after 3 min ŽFig. 4.. The white matter, although not as distinct as in the original, is still very much evident, however. The difference between the activity in the cortical columns Ždark bands. and that in the regions between the columns Žlight bands. is attenuated by the simulated 14 CIAP diffusion. The activity in the dark bands decreases, while that in the light bands increases, so that by 2 min of diffusion, the difference is markedly depressed. In a typical dark–light band pair, the concentration ratio, computed as the activity in the dark band divided by the activity in the light band, decreases from 2.2 in the original autoradiogram to 1.5 after only 30 s of simulated diffusion ŽFig. 5A.. The coefficient of variation in a 3.6 mm2 Ž0.6 mm = 6 mm. region of interest in the cortex decreases similarly as a function of diffusion time ŽFig. 5B..

4. Discussion

Fig. 5. Effect of simulated w14 Cxiodoantipyrine diffusion on the cortical columns. ŽA. The ratio of the w14 Cxiodoantipyrine concentration in the dark cortical bands to the concentration in the light cortical bands as a function of time. This ratio fell from 2.2 in the original image to 1.5 at 30 s, and 1.13 at 2 min. ŽB. The coefficient of variation in a region of interest 0.6 mm by 6 mm aligned parallel to the cortical surface.

In order to properly examine the effect of diffusion of the tracer on autoradiographic images, it is necessary to obtain an autoradiogram from an animal in which postmortem diffusion is negligible. The freeze-trapping device that was used in this study immerses the brain tissue into liquid nitrogen in less than 100 ms, and the first several hundred microns freezes Žbelow 08C. at a rate of approximately 100 mm per 100 ms w18x. Since the tissue used for these simulations came from the outer 500 mm of the tissue slab, the time between animal sacrifice Žthe end of cerebral perfusion. and the freezing of the tissue was less than 600 ms. Reference to Fig. 1 indicates that the diffusion of 14 C-IAP is negligible in this period of time, so that the autoradiogram seen in Fig. 4 Ž0 s. is an excellent representation of the actual tissue distribution of 14 C-IAP just prior to sacrifice. The very basis of the technique for the measurement of local cerebral blood flow using diffusible tracers is that the tracer must be capable of freely diffusing across the blood–brain barrier and through extravascular tissues w10x. This requirement was met by the inert gas trifluoroiodomethane originally labeled with 131 I for the measurement of regional cerebral blood flow w3,11,13x. w131 Ixtrifluorod-iodomethane, however, suffered from a number of disadvantages as a practical tracer for the measurement of local cerebral blood flow. It was volatile,

190

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making autoradiographic processing cumbersome. In addition, the short half-life of 131 I Ž8 days. required that w131 Ixtrifluorodiodomethane be synthesized frequently, and with considerable difficulty. The introduction of w14 Cxantipyrine as a tracer for the autoradiographic measurement of cerebral blood flow simplified the technique considerably, since w14 Cxantipyrine is nonvolatile, and the long half-life of 14 C meant that the tracer could be made in large batches and stored for considerable periods of time w20x. It soon became apparent that the use of this tracer led to an underestimation of local cerebral blood flow due to a limitation of its diffusion through the blood–brain barrier w1,2x. The higher partition coefficient of w14 Cxiodoantipyrine than w14 Cxantipyrine between organic solvents and water suggested that w14 Cxiodoantipyrine may be a more suitable tracer, and blood flow values obtained with w14 Cxiodoantipyrine are higher than with w14 Cxantipyrine, and are comparable to those obtained with w131 Ixtrifluorodiodo-methane w21x. It is the ability of the tracer to diffuse across the blood–brain barrier and through the extravascular tissue that leads to the potential for significant postmortem diffusion. Investigators who routinely measure local cerebral blood flow autoradiographically generally recognize that the brain must be frozen as rapidly as possible following sacrifice. This is particularly important in small animals w6,7x. In the mouse, for example, Jay et al. w7x noted a significant lack of heterogeneity in brains frozen 1 min after sacrifice, whereas at 30 s the autoradiograms appeared similar to that seen in larger animals. The large difference in contrast between the 30-s animals and the 1-min animals indicates that significant diffusion of w14 Cxiodoantipyrine occurs between 30 s and 1 min post sacrifice, suggesting that significant diffusion also occurs in the 30 s between time of death and the 30 s image. Similar data was obtained in the gerbil in which autoradiograms from brains obtained by immersing whole heads in liquid nitrogen immediately after decapitation were compared to autoradiograms from brains removed from the skull and frozen in cold isopentane 90–95 s following sacrifice w6x. Using thermocouples inserted into the brain, it was estimated that tissue up to 3 mm beneath the skull was frozen Žbelow 08C. in 20–25 s in the animals in which the brain was frozen in situ with liquid nitrogen. Regional differences in optical densities of the autoradiograms from these brains were more distinct than in the autoradiograms from the brains that were removed from the skull prior to freezing. The importance of freezing time when using w14 Cxiodoantipyrine as an autoradiographic blood flow tracer has also been investigated in the rat w23x. In these studies, rats were decapitated 10 s following an intravenous bolus injection of w14 Cxiodoantipyrine and the head was immersed in chlorodifluoromethane at y408C either immediately following decapitation or 3 min after decapitation. With this procedure, the cerebral cortex takes be-

tween 30 s and 1 min to freeze w9,16,22x. Thus, the cortex from the brains that were immersed immediately was not frozen for at least 30 s, whereas in the animals in which immersion was delayed, freezing did not occur until more than 3.5 min following decapitation. The resulting autoradiograms exhibited a dramatic loss of heterogeneity in the brains frozen after the 3-min delay as compared to the autoradiograms from the quick frozen brains, in line with that predicted by our simulations. As expected, blood flows in highly perfused regions of the brain, such as the choroid plexus, calculated from the delayed frozen brains was significantly lower than in the quick frozen brains due to the diffusion of the w14 Cxiodoantipyrine out of regions of high flow. The degradation of the images due to simulated diffusion of the w14 Cxiodoantipyrine tracer Žsee Fig. 4., is of course dependent on the value of the diffusion coefficient. An overestimate of this parameter will produce a greater apparent loss of heterogeneity of blood flow. The value for the diffusion coefficient obtained in this study and used in these simulations, 2.1 = 10y6 cm2rs, is smaller than that measured in vitro in endothelial cells w4x. They obtained a diffusion coefficient of 7.7 = 10y6 cm2rs in the extracellular fluid, and 4.4 = 10y6 cm2rs in the intracellular fluid. It should be noted that these data pertain to endothelial cells, which may differ significant from brain tissue. The technique used in the present study directly measures IAP bulk diffusion in brain tissue, whereas in the study of Garrick et al. w4x, diffusion was measured in isolated endothelial cells and erythrocytes. Simulations using a diffusion coefficient comparable to that obtained by Garrick et al. w4x yields autoradiograms after 1 min of postmortem diffusion Ža relatively short period of time for removal of a rat brain from the skull. that is inconsistent with autoradiograms obtained by laboratories using w14 Cxiodoantipyrine as a tracer for the measurement of cerebral blood flow. The rather dramatic difference in contrast between the original autoradiogram ŽFig. 4. and even the first simulated image, after 20 seconds of diffusion, is due to the significant micro-heterogeneity of autoradiograms. This heterogeneity is evident when examining single small pixels and is due to inherent noise in film autoradiograms; it derives from statistical noise due to low counts in the pixel. Standard films that are used for in situ autoradiography have a low dynamic range and yield good optical densities Žat about midrange. when the product of tissue concentration and exposure time is approximately 30–40 mCi hrg Ži.e., 200 nCirg over 1 week.. With typical 20 mm thick sections, film of an area equal to one of our pixels Ž25 mm = 25 mm. receives radioactivity from only 12.5 ng of tissue. Assuming a solid angle of 2p , the film pixel is exposed to only 25–33 total disintegrations at mid range of exposure, and in some regions, such as white matter, less than 10 counts. Since radioactive decay is a stochastic process, the error involved in each pixel will be large, but

J.H. Greenberg et al.r Brain Research 842 (1999) 184–191

following several diffusion cycles, this micro-heterogeneity will be very much reduced. Consequently, the diffusion images, even at the early times, appear much smoother than the original. Given the potential for post-mortem movement of diffusible autoradiographic flow tracers, it is important to recognize that the apparent blood flow distribution may be altered in the minute or so it takes to remove and freeze the brain. If a precise blood flow distribution is important, other tracers must be used that do not diffuse in the time between cessation of perfusion and freezing of the tissue. A number of agents have been developed for use in single photon emission computerized tomography ŽSPECT. that act as ‘chemical microspheres’ w5,14,24x. These agents are locally trapped in the tissue by a variety of metabolic mechanisms and do not exhibit any postmortem diffusion. Although this trapping mechanism may be altered under pathophysiological conditions, these agents are better suited than are diffusible tracers for mapping the precise localization of blood flow in the brain. In summary, these simulations dramatically demonstrate the potential for diffusion of w14 Cxiodoantipyrine between the time of cessation of blood flow and the time when the tissue of interest is frozen. This diffusion is, of course dependent on this time delay, and can be appreciable when fine spatial resolution of the blood flow image is of importance.

w5x

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w9x w10x w11x

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Acknowledgements Supported by National Institutes of Health grants R01NS33785 and R01-NS32017. The authors thank Anna Rosen for technical assistance.

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References w20x w1x W.W. Eckman, R.D. Phair, J.D. Fenstermacher, C.S. Patlak, C. Kennedy, L. Sokoloff, Permeability limitation in estimation of local brain blood flow with w14 Cxantipyrine, Am. J. Physiol. 229 Ž1975. 215–221. w2x B. Eklof, N.A. Lassen, L. Nilsson, K. Norberg, B.K. Siesjo, P. Torlof, Regional cerebral blood flow in the rat measured by the tissue sampling technique; a critical evaluation using four indicators C14-antipyrine, C14-ethanol H3-water and xenon, Acta Physiol. Scand. 91 Ž1974. 1–10. w3x W.H. Freygang, L. Sokoloff, Quantitative measurement of regional circulation in the central nervous system by the use of radioactive inert gas, Adv. Biol. Med. Phys. 6 Ž1958. 263–279. w4x R.A. Garrick, U.S. Ryan, V. Bower, W.O. Cua, F.P. Chinard, The diffusional transport of water and small solutes in isolated endothe-

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