Regional cerebral blood flow in acute experimental allergic encephalomyelitis

Brain Research, 363 (1986) 272-278

272

Elsevier BRE 11376

Regional Cerebral Blood Flow in Acute Experimental Allergic Encephalomyelitis M. JUHLER and O.B. PAULSON

Department of Neurology, Rigshospitalet, Copenhagen (Denmark) (Accepted May 14th, 1985)

Key words: experimental allergic encephalomyelitis- - regional cerebral blood flow - [14C]iodoantipyrine - - quantitative autoradiography - - spatial resolution

Regional cerebral blood flow was studied in Lewis rats with fulminant acute experimental allergic encephalomyelitis (EAE). [14C]iodoantipyrine was used as a tracer. By employing a short experimental time and an infusion schedule producing an increasing arterial tracer concentration, the spatial resolution of the method was fine enough to detect focal increases in blood flow in the small central nervous system lesions (lymphocytic accumulations). An increase of flow of 100% in the lesions and a decrease of 50% in the cerebral cortex of EAE animals was statisticallysignificant. In all other regions studied (deep cerebral structures, cerebellum), blood flow in EAE animals did not differ from the control values. The flow increase corresponding to the lesions may be due to inflammatory hyperemia. The cortical decrease in flow may be secondary to sensory motor impairment. INTRODUCTION Experimental allergic encephalomyelitis ( E A E ) is induced by sensitization to central nervous system (CNS) components4,10,18, 32. Besides general symptoms of malaise, one of the first changes to be observed is focal b l o o d - b r a i n barrier (BBB) damage to the CNS veins5,15-17,30,31. Several days later, focal perivenular lymphocytic accumulations occur coinciding with clinical disease, which is characterized by an acute, ascending, flaccid paresisS,ts,30,32, followed by slow remission36. The traditional approach to E A E is either neuropathologica121-23 or immunologicaP, 39. Besides a large number of reports on the BBB in E A E 15-17, only a few studies have focused on o t h e r physiological phenomena6, 35, and only one study has dealt with blood flow in the CNS in E A E 35. These authors were unable to detect any abnormalities in b l o o d flow in E A E animals by using [14C]antipyrine as a tracer and tissue sampling from macroscopically defined regions. As the lesions in E A E are small and often highly focal 17, regional differences in cerebral b l o o d flow and other physiologic processes, may be obscured by the m a j o r i t y of surrounding normal tissue 17.

The purpose of the present study was to investigate regional cerebral blood flow (rCBF) in E A E by quantitative a u t o r a d i o g r a p h y ( Q A R ) which offers both a high regional resolution and a direct correlation to histological changes 3. Because of the small size of the lesions, diffusion of tracer into the adjacent tissue during the e x p e r i m e n t a l period and during p r e p a r a t i o n of the tissue may greatly increase the measured spatial extent and decrease the m e a s u r e d magnitude of focal changes in any physiological variable 17. Therefore a short experimental period could be essential for detecting focal flow abnormalities associated with the lesions. A second purpose of the present study was therefore to evaluate the validity of r C B F m e a s u r e m e n t s by Q A R using a shorter than normal experimental time (20 s). MATERIALS AND METHODS

Induction of E A E Guinea pig spinal cord h o m o g e n i z e d in isotonic saline (1:1 w/v) was emulsified in an equal volume of complete F r e u n d ' s adjuvant fortified with 10 mg/ml M y c o b a c t e r i u m Tuberculosis H 37 R A . E A E was induced in four 200-g male Lewis rats by inoculating 0.1 ml of the emulsion intradermally in both hindfoot

Correspondence: M. Juhler, Department of Neurology 2081, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

273 pads. On occurrence of severe clinical disease (tail and hindlimb paralysis, incontinence, and in 3 animals also forelimb ataxia) 14-16 days after inoculation, the animals were used for the study. Three sexand age-matched rats served as controls.

rCBF tracer [14C]iodo-antipyrine in ethanol, 0.1 mC m1-1, 54.5 Ci mol< (New England Nuclear), was evaporated and redissolved in isotonic saline to a volume activity of 100 mCi m1-1. Radiotracer purity was greater than 99% determined by chromatography.

Experimental procedures The animals were anaesthetized, relaxed and ventilated in a 70/30 N20/O z mixture. During insertion of catheters into both femoral arteries and one femoral vein, 0.6% halothane was added to the gas mixture. After surgery, halothane was discontinued, and the animals were heparinized and used for the study when a satisfactory physiological state was obtained: mean arterial blood pressure (MABP) 123 mm Hg + 16 (S.D.), pO 2 126 mm Hg + 14 (S.D.), pCO2 38.2 mm Hg + 2.8 (S.D.), body temperature 36.5-38 °C. One femoral artery was used for continuous recording of MABP during the experimental period, the other was shortened to 2-3 cm to reduce dead space and used for sampling of arterial blood. The catheter in the femoral vein was connected to an infusion pump. At the beginning of the experiment, infusion of [14C]iodoantipyrine was started at a con-

.c~/

FC 8

stant rate of 2 ml min q , i.e. 70 pCi/20 s. This infusion schedule invariably produced on arterial curve as shown in Fig. 1. Arterial blood was sampled every 2 s into Eppendorf tubes. At the end of the 20 s experimental period, the infusion was discontinued, the animal was rapidly decapitated and the brain removed and frozen at -60 °C in isopentane cooled in acetone/dry-ice. The time from decapitation to freezing was 45-60 s. In addition, both olfactory bulbs were sampled and prepared for liquid scintillation counting together with the blood samples Is. The blood samples were immediately stirred, and 10 #1 whole blood from each was counted (Packard Tricarb) and corrected for quenching. The arterial integral (nCi rain m1-1) was calculated. The brains were cut at -20 °C into 20 pm thick sections which were dried rapidly at 60-70 °C. They were exposed for two weeks to Kodak MR-1 film together with a series of 9 plastic standards calibrated against brain tissue of known activity34,39. After development of the films (Kodak RP X-O-mat), the sections were fixed and stained by Klfiwer-Barrera for histological examination.

Data analys& Autoradiograms were analyzed by computerized densitometry through a video camera (Leitz TAS Plus). On the stained sections, regions of interest were selected, and subsequently served as templates for measurement of optical density in the corresponding autoradiogram. The units of tissue activity were calculated by the computer using a standard curve based on the plastic standards 34. CBF was then calculated by iteration using the formula derived by Sakurada et al. 33.

PLASMA lAP-INTEGRAL

Regions studied J

0

5

10

15

t SEC 20

Fig. 1. In all experiments, a constant infusion rate of 210/~Ci min-1 during the 20-s experimental period produced an arterial curve with an upslope and a plateau. A typical example is shown.

In the forebrain, autoradiographic measurements were made in the parietal cortex, corpus callosum, hippocampus (gyrus dentatus and Ammon's horn), ventral medial nucleus of the thalamus, hypothalamus, and in the internal capsule. In the brainstem, measurements were made in the cerebellar cortex, cerebellar peduncules, the nuclei at the floor of the fourth ventricle, nucleus solitarius, and in the tractus solitarius. The olfactory bulbs were studied using a tissue sampling technique. Statistical analysis was performed using Student's t-test.

274

Fig. 2. A: histological section of the brainstem and corresponding autoradiogram from an EAE animal. Lesions are clustering around the fourth ventricle and extending into the cerebellar peduncules. Less dense accumulations of lesions are also seen in the right side of the brainstem. B: corresponding autoradiogram; severely hyperemic areas are seen especially corresponding to the lesions in the cerebellar peduncules and in the nuclei at the floor of the fourth ventricle. Note also the hyperemic area in the right side of the brainstem,

Fig. 3. A: histological section from the forebrain of an EAE animal, through parietal cortex, caudate nucleus and ventral medial thalamic nucleus. Lesions are seen above and below the lateral ventricles in the corpus callosum and extending into the capsula interna, B: corresponding autoradiogram; slight increases in flow are seen overlying the lesions, especially in the capsula interna. In the cortex, a columnar flow pattern is seen.

ventricles (Fig. 3). Lesions w e r e also seen in the hyp o t h a l a m i c area. N o lesions w e r e seen outside t h e s e RESULTS

areas.

Histological analysis

rCBF

In all of the E A E animals, the brain and b r a i n s t e m

O n a typical a u t o r a d i o g r a m f r o m a control a n i m a l

w e r e studied and p e r i v a s c u l a r l y m p h o c y t i c infiltrations typical of E A E 22,23,36 w e r e found. In all 4 ani-

a h e t e r o g e n e o u s , ' s t r i p e d ' or p a t c h y flow p a t t e r n is s e e n in the cortex. H e t e r o g e n e i t y , but to a lesser de-

mals, the g r e a t e s t density of lesions was f o u n d in the

gree, is also s e e n in the t h a l a m u s . All o t h e r r e g i o n s

b r a i n s t e m , especially in the a r e a a r o u n d the f o u r t h

p r e s e n t a h o m o g e n e o u s distribution of t r a c e r and

ventricle (Fig. 2). In 3 of the 4 E A E animals the le-

thus of flow, w i t h i n a n a t o m i c a l l y d e f i n e d regions.

sions e x t e n d e d into the c e r e b e l l u m a l o n g the c e r e b e l -

T h e values of r C B F in s e l e c t e d regions in b o t h con-

lar p e d u n c u l e s . In the s a m e 3 animals a few lesions

trol and E A E animals are s h o w n in T a b l e I. G r a y

w e r e also f o u n d in the f o r e b r a i n in the c o r p u s callo-

m a t t e r areas ( c o r t e x , t h a l a m u s , nuclei) s h o w a high

sum and in the i n t e r n a l capsule close to t h e lateral

s t a n d a r d d e v i a t i o n . This variability is seen n o t only as

275 TABLE I Values of rCBF (ml g-1 rnin J) +_ S.D. in the studied regions of the CNS in control and E A E animals

rCBF in lesion-free areas in the EAE animals is significantly different from control values in parietal cortex (P < 0.05) and brainstem nuclei (P < 0.025) rCBF in the actual lesions (lymphocytic accumulations) differs significantly in the cerebellar peduncules (P < 0.001). See text for further details. Region

Control

EAE animals Lesion-free areas

Parietal cortex Corpus callosum Ventral medial nucleus of thalamus Hippocampus, gyrus dentatus Hippocampus, Ammon's horn Internal capsule Hypothalamus Cerebellum, cortex Cerebellum, peduncules Brainstem, nuclei Brainstem, tracts

2.68 ± 0.92 0.53 + 0.06 1.40 _+0.22 0.91 + 0.07 0.84 + 0.01 0.45 + 0.04 0.79 + 0.02 0.84 + 0.05 0.37 + 0.04 1.25 ± 0.30 0.41 + 0.06

1.46 + 0.44 0.52 + 0.11 1.54 + 0.36 1.20 + 0.24 1.28 + 0.28 0.48 + 0.12 1.25 + 0.40 0.99 _+0.24 0.46 _+0.16 1.51 ± 0.29 0.46 + 0.16

a variation from animal to animal but is also present within the same animal as a heterogeneous flow pattern is seen on the a u t o r a d i o g r a m in these regions. The values obtained for r C B F correspond to those m e a s u r e d by others 21,34 except for the cortical areas where considerably lower flow values are o b t a i n e d when longer experimental times are used. A s flow in the frontal lobes was m e a s u r e d using a tissue uptake method, a comparison with other studies using this technique was possible. In the present study frontal flow in the control animals was 1.07 ml g-1 rain-1 +_ 0.07 ( S . E . M . ) in control animals c o m p a r e d to 1.29 ml g-a m i n - i _+ 0.07 measured by G j e d d e et al. 12. In the E A E animals, a similar general p a t t e r n was observed. In addition, corresponding to the lesions focal increases in r C B F were seen (Figs. 2 and 3). Focal flow increases accompanying lesions in white matter regions were more easily distinguished from the surrounding tissue than those corresponding to lesions in gray m a t t e r regions such as the h y p o t h a l a m u s and the nuclei at the floor of the fourth ventricle. Lesions in the cerebellar peduncules were best separated from the surrounding p a r e n c h y m a , and here the difference in blood flow between the actual lesions and the surrounding tissue was highly significant ( P < 0.001). In the other areas (corpus callosum, capsula interna, h y p o t h a l a m u s and the nuclei of the hrainstem), where lesions were not as easily distinguished from the surroundings, the difference in C B F between lesions and surrounding tissue ap-

EAE animals Lesions

0.52 + 0.05

0.63 + 0.19 1.19 ± 0.46 1.35 ± 0.10

proached, but did not reach the 5% significance level. Focal flow increases without corresponding histological lesions were occasionally found. They did not differ from increases with accompanying lesions. W h e n areas free of lesions and of focal flow changes in the E A E animals were c o m p a r e d to corresponding regions in control animals, significantly higher flow was found in the nuclei of the brainstem (P < 0.025). In the cortex, C B F was significantly lower in the E A E group than in the control group (P < 0.05). DISCUSSION Our modification of the existing methods for measurement of r C B F by shortening the tracer circulation time was made because we wanted to avoid overlooking small, focal C B F changes. In addition, the steadily increasing plasma concentration of the tracer and rapid removal/freezing of the brain served to minimize diffusion of the tracer in the p a r e n c h y m a and to maximize any contrast in tracer concentration between lesions and surrounding parenchyma. A n extensive discussion on the effect of tracer diffusion on measurements of small focal changes is p r e s e n t e d elsewhere 17, but briefly, diffusion of tracer away from small focal increases in tracer concentration m a y result in significant underestimations of tracer concentration in the lesion itself, and overestimations of the area where the change is present. The

276 chosen experimental time was short enough to make possible the detection of changes in CBF corresponding to the lesions and long enough to give reliable measurements in other parts of the brain. The studies by Ekel6f et al. 9 Sakurada et al. 33 and Eckmann et al. 7 emphasize the importance of the kinetics of the tracer for reliable measurements of CBF. B l o o d - b r a i n barrier permeability and blood flow are both expressed quantitatively in terms of clearance of a tracer from the circulation, i,e. ml g-1 min-L Whether one or the other is measured depends on the characteristics of the tracer. The entrance of the tracer across the b l o o d - b r a i n barrier into the CNS is determined by blood flow and by permeability: if the permeability is very low, then this becomes rate-limiting for entrance into the CNS and the tracer can be used to measure permeability. If permeability is high compared to flow, then flow is the limiting factor 11. When very short circulation times are used, any impediment in permeation of a flow tracer across the b l o o d - b r a i n barrier should become more pronounced and give lower flow values. As this is not the case comparing the measured values of rCBF (Table I, < 0 to those obtained by others20.33), it seems that iodoantipyrine has almost ideal kinetic characteristics for a flow tracer, and gives good regional resolution if diffusion of the tracer to neighbouring areas is minimized as in the present study. A shortening of the experimental period to 10 s was discarded because of: (1) the relatively larger influence of the time lag between injection of the tranCi/

FGPg. 10

P L A S M A rAP-INTEGRAL

~

-5

I SEC 10

Fig. 4. When a constant infusion rate was used for an experimental period of 10 s, only the upslope of the arterial curve is produced. The delay in appearance of the tracer in arterial blood at the sampling site (right femoral artery) is almost half of the experimental period, and the curve is based only on the remaining 3 time-points.

cer and its appearance in the arterial samples; and (2) the scarcity of data points (Fig. 4). Methods based on counting the uptake of tracer into different, dissectable regions of the brain (tissue uptake methods) yield a certain degree of spatial resolution but 'contamination' of a sample from a defined area with adjacent tissue (which sometimes has an entirely different flow) must take place. This could explain the generally lower flow values obtained by such methods compared to the measurements by Q A R 8,9,12,26,29. The values obtained by gross tissue sampling in the present study are comparable to previously reported values 9,12. This is further evidence in favour of the selected of experimental conditions. Our results show heterogeneity of CBF in high flow areas, i.e. cortex, and to a lesser degree thalamus. It is seen both as an inter-animal variation (large standard deviations, Table I) and as an intraanimal variation (heterogeneous pattern in Fig. 3). A similar pattern in cortical CBF has previously been reported using a similar time-frame 14, whereas the phenomenon is not seen when the experimental period is extended 2°.25,33. A possible explanation might be alternating perfusion in neighbouring areas. A time constant of about 6 min ] has been suggested from electrode measurements of pO 2 oscillations 34. After a number of cycles, the columns will not be seen, because all areas will have been 'turned' on and off several times. However: (1) the same pattern is seen in functional mapping of the brain using 2-deoxy-glucose, which does not diffuse and which requires much longer experimental times38; and (2) vasomotor oscillations in pial vessels have been observed directly 2. Therefore, it could seem more likely that the columns are stationary in space, at least within the time-frame in question, but oscillations do occur within the same area 24. The reason that the pattern is not seen with flow tracers using longer experimental times could then simply be diffusion. The present study shows that there are considerable, focal increases in CBF in E A E . Characteristically, the increases are found corresponding to histological lesions (perivascular lymphocytic accumulations), but they may also be seen without accompanying lesions. Simmonds et al. did not find any alterations in CBF in E A E 35. This could be explained by their use of a tissue uptake method, where small, 10-

277 calized changes can ' d r o w n ' in the majority of surrounding normal tissue. F u r t h e r m o r e , the r e m o v e d neural tissue was frozen within its bony coverings, and significant diffusion, and thus smearing of focal changes may have taken place before the temperature in the neural tissue was low enough to slow diffusion considerably. F o r this reason the spinal cord, which takes several minutes to remove from the vertebral canal, was not examined in the present study. Increased concentrations of lactate in the lesions have been m e a s u r e d , and it has been p r o p o s e d that this could be caused by focal ischaemia35,36. The present study does not support this theory, but it is possible that the increase in flow is smaller relative to focal changes in glucose metabolism which have been reported 6. F u r t h e r elucidation of the m a t t e r awaits studies connecting r C B F , regional glucose m e t a b o lism and regional pH. A t which time during the evolution of E A E the focal abnormalities in C B F occur, cannot be answered from the present data, but their presence in some places in the cerebellar peduncules without accompanying inflammatory changes suggests that they m a y arise as an early event before any evidence of histopathological changes or clinical disease. A n o t h e r question which can be raised is how these focal dis-

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turbances are produced. T h e y may represent focal, inflammatory h y p e r e m i a resulting from the release of vasoactive substances released by the infiltrating cells. This explanation, however, does not seem to cover flow increases in the absence of cellular inflammation, unless it can be p r o d u c e d by a few unnoticed lymphocytes in the p a r e n c h y m a or by cells adherent to the luminal e n d o t h e l i u m 2s,37. A n o t h e r possible explanation is h y p e r e m i a resulting from antibodies reacting with neural antigen whereby vasoactive substances, e.g. c o m p l e m e n t split products, may be formeda3,31. M o r e generalized disturbances in C B F are also present. Neither in the present study nor in other studies have lesions been found in the cerebral cortex 15,17,19,30. The observed decrease in cortical flow to about 50% therefore cannot be connected directly with the inflammatory changes. It could reflect interference with regulatory neurovascular mechanisms. A more likely explanation seems to be a functional decrease in blood flow secondary to the severe sensory m o t o r impairment of the animals. Similar r e m o t e decreases in r C B F have been r e p o r t e d in connection with other focal abnormalities in the CNS, e.g. the cerebellar diaschisis seen in hemispheric brain infarcts 27.

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