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The use of peroxidase as a tracer in studies of alterations in the blood-brain barrier
Journal of the neurological Sciences
205
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
The Use of Peroxidase as a Tracer in Studies of Alterations
in the Blood-Brain Barrier A. H I R A N O , N. H. BECKER A N D H. M. Z I M M E R M A N Laboratoo, Dit,ision, Montefiore Hospital and Medical Center, Bronx, N.Y. (U.S.A .) (Received 17 March, 1969)
INTRODUCTION
One of the major contributions of the fine structural studies of the pathology of the central nervous system has been the elucidation of the nature and site of inflammatory edema in the brain (ZIMMERMAN 1964; BAKAV AND LE~ 1965; KLATZO AND SEITELBERGER 1967). It is, by now, generally agreed that, at least in the acute stage of inflammation, abnormal amounts of fluid accumulate in the extracellular spaces of the brain, especially in the white matter. The subsequent fate of this fluid and the various cellular reactions to it are the subject of a number of studies (H1RANO et al. 1967: HIRANO 1969). With this information in hand, we are now in a position to ask another question. How does the fluid pass from the blood vessels into the parenchyma? Another way of putting the same question is: What changes in the blood-brain barrier occur under conditions of edema that permit the excessive flow of material into the brain? At least one part of the anatomical seat of the blood-brain barrier has been reported to be the endothelium of the cerebral vessels. Using the protein tracer, horseradish peroxidase, REESE AND KARNOVSKV (1967) and BRIGHTMAN AND REESE (1968) have shown that, under normal conditions, the tracer does not penetrate the endothelial layer in the brain as it does in other tissues (KARNOVSKY 1967; BRIGHTMAN 1967; BECKER el al. 1968; HASHIMOTO AND HAMA 1968; BODENHEIMER AND BRIGHTMAN 1968). It is the purpose of the present study to examine the penetration of the same tracer under various conditions in which the blood-brain barrier is altered. Some preliminary reports of this work have been published elsewhere (BECKER et al. 1968, 1969: HIRANO et al. 1969). This report reviews some of the highlights of the previous publications and adds certain new information which has only recently been obtained. Paper presented at a symposium on The Blood-Brain Barrier, held 11 September, 1968, in New York, N.Y.(U.S.A.). This investigation was supported by Grant NB 05257 from the U.S. Public Health Service. J. neurol. Sei.,1970, I0:205-213
2(16
x. till/ANt), ;,4. 1t. BE('KliR, I1. M. ZIMMERMAN
~lA H':RImS A~D
MvJ
ltODS
Young, adL,lt albino rats. of mixed sex, each weighing about 200 251i ~4 were u~ed throughout this study. Twenty-five mg of horseradish peroxidase (Sigma Type [I) in I ml of physiological saline were injected into the femoral vein. Cerebral edema was induced by stab wounds in the brain, electroconvulsive shocks (BEcK~-;R~'/a/. 1969), or by experimental allergic encephalomyelitis (EAE) induced by a method described elsewhere (LEV|NE AND WENK 1965) ~. At various intervals after administration of the tracer (usually within i h), mos~ of the brains and spinal cords were lixed by vascular perfusion with 5 o~., giutaraldehyde in 0.066 M phosphate buffer, p H 7.4 (see H]RANO et al. 1954, 1965 for details) or by immersion fixation. After fixation and overnight rinsing in buffer, 10 15/~ frozen sections were cut on a freezing microtome. The frozen sections were incubated for peroxidase activity using the method of GRAHAMAND KARNOVSKY(1966), postfixed in osmic acid and embedded for electron microscopy by a technique described elsewhere (BECKER ~'t a/. 1968). Thin sections were cut and were examined, unstained, in a Siemens IA electron microscope. RESULTS
When cerebral edema was induced by stab wounds, the tracer was found permeating the extracellular spaces most obviously in the white matter but in the grey matter as well (Fig. 1). The greatest accumulation of peroxidase was in the immediate area of the stab. From that region, it spread diffusely through the extracellular spaces especially between cell processes in the white matter, for considerable distances from lhe lesion. Special attention was, of course, paid to the blood vessels and the perivascular areas. The tracer filled the perivascular areas and permeated the endothelial basement membrane. Pinocytotic vesicles were found both on the luminal and basal surfaces if immersion fixation was used. Perfusion fixation usually washed the luminal surface free of reaction product. A few endothelial cells, or large parts of them, were presumably damaged and evidenced a diffuse infiltration of the tracer into the cellular ground substance. The junctions between endothelial cells were, for the most part, filled with reaction product only part of the way. Occasionally, however, the tracer formed a continuous dense zone between adjacent endothelial cells over most of the cell junction (Fig. 2). Sometimes such zones extended from the lumen all the way to the perivascular space (HtRANO et al. 1969). [n most cases, after electroshocks great enough to induce convulsions, the fine structure of the areas of the brain examined appeared normal. No cerebral edema was evident and no tracer was found in the brain parenchyma. The tracer filled the vascular lumina and apparently penetrated between adjacent endothelial cells only for short distances before it was stopped at the tight junctions (Fig. 3). Tracer-filled pinocytotic ~ E A E - a n i m a l s were kindly contributed by Dr. S e y m o u r Levine, New York Medical College, for this study. J. neurol. Sci., 1~)70, 10:205 21
ALTERATION OF THE BLOOD-BRAIN BARRIER
207
Fig. 1. The extracellular spread of peroxidase after a stab wound is seen in this section of the cerebrum. ;:< 64,000.
vesicles in the endothelial cells were small, rare and confined to the luminal surfaces. In a few cases, however, at higher electroshock doses, a n d especially in the midline area near the third ventricle, tracer could be f o u n d in the extracellular p a r e n c h y m a t o u s spaces. The most p r o m i n e n t finding in these cases was the diffuse a n d very extensive infiltration o f some endothelial cells (Fig. 4) which sometimes were i m m e d i a t e l y adj a c e n t to a p p a r e n t l y intact non-infiltrated endothelial cells (Fig. 5). The presence o f dense peroxidase reaction p r o d u c t t h r o u g h o u t the g r o u n d substance o f the infiltrated cells m a d e it very difficult to determine whether or not the reaction p r o d u c t was J. neurol. Sci., 1970, 10:205 213
208
,x. H I R A N ( ) .
N. It. BE( K E R . H. M. / I M M E R M ~ ' ,
Fig. 2. An endothelial junction separaled by a narrow, continuous band of tracer extending from Iht: lumen (arrow) in an animal in.iecled intravenously with peroxidase and sub ecied i~ a cerebral stah ~,~,o t l n d .
(~6,000
Fig. 3. A peroxidase-filled lumen in the cerebrum of a rat subjected to electroshock ,\n intercellular junction is only partially filled with the tracer farrou'). 96,00{I,
p r e s e n t b e t w e e n the cells since the p l a s m a m e m b r a n e s w e r e a l m o s t i m p o s s i b l e to d e l i n e a t e (Fig. 5). F r e q u e n t l y , pericytes a n d p e r i v a s c u l a r as well as o t h e r a s t r o c y t i c processes were also diffusely infiltrated (Fig. 6). T h e rats in which e x p e r i m e n t a l allergic e n c e p h a l o m y e l i t i s had been r e d u c e d also J. nettrol. ,S('i.. 1~70. lO: 205 213
ALTERATION OF THE BLOOD-BRAIN BARRIER
209
showed s p r e a d o f the tracer protein from the vasculature into the extracellular spaces o f the l u m b o s a c r a l spinal cord. The extravascular peroxidase was especially p r o m i n e n t a r o u n d venules and in the adjacent extracellular spaces (Fig. 7). The endothelial cells o f the vessels contained peroxidase-filled pinocytotic vesicles which, for the most part,
Fig. 4. Diffusely-stained endothelial cells near the third ventricle in a rat subjected to electroshock. ~." 6000.
Fig. 5. Higher magnification of an area similar to Fig. 4. Portions of 2 endothelial cells are visible. One, whose luminal surface is indicated by the small arrows, is diffusely stained with peroxidase. The adjacent endothelial cell is not. The junction between the cells is indicated by the large arrow. The perivascular space is diffusely infiltrated by the tracer, x 70,000.
were localized at the basal surface (Fig. 8). A t the intercellular junctions, the tracer usually filled the lower two-thirds but occasionally extended a l m o s t to the l u m i n a which were clear due to perfusion fixation. The perivascular spaces were diffusely and densely infiltrated with reaction p r o d u c t . J. neurcd. Sci., 1970, 10:205-213
210
A. HIRANO, N. H. BECKER, H. M. Z!MMERMAN
Fig. 6. A n area similar to Fig. 4. The endothelial cells, as well as astrocytic processes, are diffusely infiltrated with the tracer. ".: 8000.
Fig. 7. A venule in the lumbar spinal cord o f an animal subjected to EAE. The perivascular spaces are diffusely infiltrated with reaction product, x 6000. ,I. neurol. Sci., 1970, I0:205-213
ALTERATION OF THE BLOOD-BRAIN BARRIER
211
Fig. 8. Higher magnification of a n area similar to Fig. 7 illustrating the presence of peroxidase-filled pinocytotic vesicles in the endothelium, m o s t of which are at the basal surface. Nearby extracellular spaces are faintly m a r k e d by the presence of reaction product, × 27,000.
DISCUSSION
When one compares the results of the stab wound experiment with the uninjured control, there is no doubt that the experimental procedure resulted in clear-cut changes in the endothelial cells even some distance from the lesion. Among these changes were some in which direct communication could be observed between the vascular lumen and the extracellular space in the brain parenchyma. The relationship, however, between these communications and the blood-brain barrier is unclear. One might argue that all of these phenomena are the result of the reaction of an intact endothelium to the presence of excess extracellular fluid caused by the leakage of materials from blood vessels grossly damaged by the stab wound. We may, in fact, be observing the return of the tracer from the parenchyma to the vasculature through a secondarily-altered endothelium. In other words, these phenomena may be the secondary reflections of the breakdown of the blood-brain barrier and not its primary cause. To overcome this objection, we resorted to the use of animals in which the bloodbrain barrier was altered but in which the blood vessels were not subjected to gross, mechanical trauma; namely, rats subjected to electroconvulsive shock or experimental allergic encephalomyelitis (EAE). As described above, similar results, namely extracellular infiltration of tracer, indicating a breakdown in the blood-brain barrier, were obtained in these animals as well. Among the rats subjected to electroconvulsive shock, however, numerous endothelial cells appeared diffusely-stained and therefore probably damaged. These, then, might act much as the grossly-altered blood vessels in the stab wound experiments. In the EAE animals, on the other hand, such diffusely-stained endothelial cells were only rare. Nevertheless, as described above, the extracellular spaces, especially around the vessels, were densely filled with reaction product. Some workers have reported that pinocytosis was the route by which material moved from the circulation to the parenchyma in cerebral edema (TAM AND EVANS 1965; RAIMONDI 1966). Other workers, however, have already shown that in EAE animals leukocytes (BuBIS AND LUSE 1964; LEVlNEet al. 1965; LAMPERTAND CARPENTER 1965 ; LAMPERT1967), thorotrast (LAMPERTAND CARPENTER 1965 ; LAMPERT 1967 ; LA~PERT et al. [967) and hematogenous fluid (LEvINE et al. 1965) can pass between adjacent endothelial cells. On the other hand, in experimental allergic neuritis, J. neurol. Sci., 1970, 10 : 205-213
212
A. |tlRAN(), N. tl. BE(?KER, It. M. ZIMMERMAN
lymphocytes have been reported to move through individual endothelial cells (Asvk¢)~t et al. 1968). Further work attempting to delineate more clearly the preci~c route of peroxidase from the blood vessels into the parenchyma during EAE is needed and i~ the subject of current research in our laboratory.
ACKNOWLEDGEMENTS
The authors wish to express appreciation to Dr. Herbert Dembitzer, Laboratory Division, Montefiore Hospital and Medical Center. for his invaluable assistance.
SUMMARY
The distribution of peroxidase in the brain and spinal cord was examined after stab wounds, electroshock-induced convulsions, and experimental allergic encephalomyelitis. In all cases, the tracer was found to have moved from the vasculature into the parenchymatous extracellular spaces. The routes to that site may be via pinocytosis. between altered endothelial junctions or through diffusely-infiltrated endothelial cells.
REFERENCES
/~STROM,K.
E., H. deF. WEBSTER AND B. G. ARNASON (1968') The initial lesions in experimental allergic neuritis. A phase and electron microscopic study, J. exp. Med., 128 : 469496. BAKAY, L. AND J. C. LEE (1965) Cerebral Edema, Thomas, Springfield, I11. BECKER, N. H., A. HIRANO AND H. M. ZIMMERMAN(1968) Observation of the distribution of exogenous peroxidase in the rat cerebrum, J. Neuropath. exp. Neurol., 27: 439-452. BECKER, N. H., A. HIRANO AND U. M. ZIMMERMAN(1969) The cerebral endothelial barriers to peroxidase, Amer. J. Pathol., 55 : 52a (abstract). BECKER, N. H., A. B. NOVIKOFF AND H. M. ZIMMERMAN(1968) Fine structure observations of the uptake of intravenously injected peroxidase by the rat choroid plexus, J. Histochem. C~,tochem., 15 : 160-165. BODENHEIMER, T. S. AND M. W. BRIGHTMAN(1968) A blood-brain barrier to peroxidase in capillaries surrounded by perivascular spaces, Amer. J. Anat., 122: 249-268. BRIGHTMAN, M. W. (1967) The intracerebral movement of proteins injected into blood and cerebrospinal fluid of mice. In: A. LAJTHAAND D. H. FORD (Eds.), Brain Barrier Systems (Pro.~,ress in Brain Research, Vol. 29), Elsevier, Amsterdam, pp. 19-37. BRIGHTMAN, M. W. AND T. S. REESE (1968) The role of intercellular junctions in the movement of substances within the brain. In: Program o f the 44th Annual Meeting, American Association o f Ne,lropathologists, June 13-15, Washington Hilton, Washington, D. C., p. 29. BUBIS, J. J. AND S. A. LUSE (1964) An electron microscopic study of experimental allergic encephalomyelitis in the rat, Amer. J. Pathol., 44: 299-317. GRAHAM,R. C. JR. AND M. J. KARNOVSKY(1966) The early stages of absorptionofinjected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14: 291-302. HASH1MOTO, P. H. AND K. HAMA (1968) An electron microscope study on protein uptake into brain regions devoid of the blood-brain barrier, Med. J. Osaka Univ., 18:331-346. HIRANO, A. (1969) The fine structure of brain in edema. In: G. H. BOURNE(Ed.), Structure and Function o f the Nervous system. Vol. 2, Academic Press, New York, pp. 69-135. HIRANO, A., N. H. BECKER AND H. M. ZIMMERMAN(1969) Pathological alterations in the cerebral endothelial cell barrier to peroxidase, Arch. Neurol. (Chic.), 20: 300-308. H1RANO, A., H. M. ZIMMERMANAND S, LEVINE(1964.') Thefine structure of cerebral fluid accumulation, Part 3 (Extracellular spread of cryptococcal polysaccharide in the acute stage), Amer. J. Patho[., 45: 1-19.
J. nem'ol. S~'i,, 1970. t0 : 205 2 t 3
ALTERATION OF THE BLOOD-BRAIN BARRIER
213
HIRANO, A., H. M. ZIMMERMANAND S. LEVINE(.1965) The fine structure of cerebral fluid accumulation, Part 9 (Edema following silver nitrate implantation), Amer. J. Pathol., 47: 537-548. HIRANO, A., H. M. ZIMMERMANAND S. LEVINE(1967) The fine structure of cerebral fluid accumulation, Part 10 (A review of experimental edema in white matter). I n : 1. KLATZOAND F. SE~TELBERGER (Eds.), Brain Edema, Springer, New York, pp. 569-589. KARNOVSI(Y, M. (1967) The ultrastructural basis of capillary permeability studied with peroxidase as a tracer, J. Cell Biol., 35 : 213-236. KLATZO,1. AND F. SEITELBERGER(1967) Brain Edema, Springer, New Yrok. LAMPERT, P. (1967) Electron microscopic studies on ordinary and hyperacute experimental allergic encephalomyelitis, Acta neuropath. ( Berl.), 9: 99-126. LAMPERT, P. AND S. CARPENTER 0965) Electron microscopic studies on the vascular permeability and the mechanism of demyelination in experimental allergic encephalomyelitis, J. Neuropath. exp. Neurol. 24 : 11-24. LAMPERT, P., E. GARRO AND A. PENTSCHEW (1967) Lead encephalopathy in suckling rats, An electron microscopic study. In: I. KLATZO AND F. SEITELBERGER(Eds.), Brain Edema, Springer, New York, pp. 207 222. LEVlNE, A. AND E. J. WENK (1965) Hyperacute form of allergic encephalomyelitis, Amer. J. Pathol., 47 : 61-68. LEVINE S., A. HIRANO AND H. M. ZIMMERMAN(1965) Hyperacute allergic encephalomyelitis, Electron microscopic observations, Amer. J. Pathol., 47; 209-221. RAIMONDI, A. J. (1966) Experimental cerebral edema. Fine structure and pathogenesis, in: W. F. CAVENESS AND F. E. WALKER(Eds.), Head Injury (Conference Proceedings), Lippincott, Philadelphia, Pa., pp. 300-320. REESE, T. S. AND U. J. KARNOVSKY(1967) The structural localization of a brain barrier to exogenous peroxidase, J. Cell Biol., 34:207-217. TANI, E. AND J. P. EVANS(1965) Electron microscopic studies of cerebral swelling, Part 1 (Studies on the permeability of brain capillaries, using ferritin molecules as tracers), Acta neuropath. (Berl.), 4: 507-526. ZIMMERMAN, H. M. (1964) Some contributions of electron microscopy to problems in pathology Bull, N. Y.Acad. Med., 40: 831-862.
J. neurol. Sci., 1970, 10:205--213
205
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
The Use of Peroxidase as a Tracer in Studies of Alterations
in the Blood-Brain Barrier A. H I R A N O , N. H. BECKER A N D H. M. Z I M M E R M A N Laboratoo, Dit,ision, Montefiore Hospital and Medical Center, Bronx, N.Y. (U.S.A .) (Received 17 March, 1969)
INTRODUCTION
One of the major contributions of the fine structural studies of the pathology of the central nervous system has been the elucidation of the nature and site of inflammatory edema in the brain (ZIMMERMAN 1964; BAKAV AND LE~ 1965; KLATZO AND SEITELBERGER 1967). It is, by now, generally agreed that, at least in the acute stage of inflammation, abnormal amounts of fluid accumulate in the extracellular spaces of the brain, especially in the white matter. The subsequent fate of this fluid and the various cellular reactions to it are the subject of a number of studies (H1RANO et al. 1967: HIRANO 1969). With this information in hand, we are now in a position to ask another question. How does the fluid pass from the blood vessels into the parenchyma? Another way of putting the same question is: What changes in the blood-brain barrier occur under conditions of edema that permit the excessive flow of material into the brain? At least one part of the anatomical seat of the blood-brain barrier has been reported to be the endothelium of the cerebral vessels. Using the protein tracer, horseradish peroxidase, REESE AND KARNOVSKV (1967) and BRIGHTMAN AND REESE (1968) have shown that, under normal conditions, the tracer does not penetrate the endothelial layer in the brain as it does in other tissues (KARNOVSKY 1967; BRIGHTMAN 1967; BECKER el al. 1968; HASHIMOTO AND HAMA 1968; BODENHEIMER AND BRIGHTMAN 1968). It is the purpose of the present study to examine the penetration of the same tracer under various conditions in which the blood-brain barrier is altered. Some preliminary reports of this work have been published elsewhere (BECKER et al. 1968, 1969: HIRANO et al. 1969). This report reviews some of the highlights of the previous publications and adds certain new information which has only recently been obtained. Paper presented at a symposium on The Blood-Brain Barrier, held 11 September, 1968, in New York, N.Y.(U.S.A.). This investigation was supported by Grant NB 05257 from the U.S. Public Health Service. J. neurol. Sei.,1970, I0:205-213
2(16
x. till/ANt), ;,4. 1t. BE('KliR, I1. M. ZIMMERMAN
~lA H':RImS A~D
MvJ
ltODS
Young, adL,lt albino rats. of mixed sex, each weighing about 200 251i ~4 were u~ed throughout this study. Twenty-five mg of horseradish peroxidase (Sigma Type [I) in I ml of physiological saline were injected into the femoral vein. Cerebral edema was induced by stab wounds in the brain, electroconvulsive shocks (BEcK~-;R~'/a/. 1969), or by experimental allergic encephalomyelitis (EAE) induced by a method described elsewhere (LEV|NE AND WENK 1965) ~. At various intervals after administration of the tracer (usually within i h), mos~ of the brains and spinal cords were lixed by vascular perfusion with 5 o~., giutaraldehyde in 0.066 M phosphate buffer, p H 7.4 (see H]RANO et al. 1954, 1965 for details) or by immersion fixation. After fixation and overnight rinsing in buffer, 10 15/~ frozen sections were cut on a freezing microtome. The frozen sections were incubated for peroxidase activity using the method of GRAHAMAND KARNOVSKY(1966), postfixed in osmic acid and embedded for electron microscopy by a technique described elsewhere (BECKER ~'t a/. 1968). Thin sections were cut and were examined, unstained, in a Siemens IA electron microscope. RESULTS
When cerebral edema was induced by stab wounds, the tracer was found permeating the extracellular spaces most obviously in the white matter but in the grey matter as well (Fig. 1). The greatest accumulation of peroxidase was in the immediate area of the stab. From that region, it spread diffusely through the extracellular spaces especially between cell processes in the white matter, for considerable distances from lhe lesion. Special attention was, of course, paid to the blood vessels and the perivascular areas. The tracer filled the perivascular areas and permeated the endothelial basement membrane. Pinocytotic vesicles were found both on the luminal and basal surfaces if immersion fixation was used. Perfusion fixation usually washed the luminal surface free of reaction product. A few endothelial cells, or large parts of them, were presumably damaged and evidenced a diffuse infiltration of the tracer into the cellular ground substance. The junctions between endothelial cells were, for the most part, filled with reaction product only part of the way. Occasionally, however, the tracer formed a continuous dense zone between adjacent endothelial cells over most of the cell junction (Fig. 2). Sometimes such zones extended from the lumen all the way to the perivascular space (HtRANO et al. 1969). [n most cases, after electroshocks great enough to induce convulsions, the fine structure of the areas of the brain examined appeared normal. No cerebral edema was evident and no tracer was found in the brain parenchyma. The tracer filled the vascular lumina and apparently penetrated between adjacent endothelial cells only for short distances before it was stopped at the tight junctions (Fig. 3). Tracer-filled pinocytotic ~ E A E - a n i m a l s were kindly contributed by Dr. S e y m o u r Levine, New York Medical College, for this study. J. neurol. Sci., 1~)70, 10:205 21
ALTERATION OF THE BLOOD-BRAIN BARRIER
207
Fig. 1. The extracellular spread of peroxidase after a stab wound is seen in this section of the cerebrum. ;:< 64,000.
vesicles in the endothelial cells were small, rare and confined to the luminal surfaces. In a few cases, however, at higher electroshock doses, a n d especially in the midline area near the third ventricle, tracer could be f o u n d in the extracellular p a r e n c h y m a t o u s spaces. The most p r o m i n e n t finding in these cases was the diffuse a n d very extensive infiltration o f some endothelial cells (Fig. 4) which sometimes were i m m e d i a t e l y adj a c e n t to a p p a r e n t l y intact non-infiltrated endothelial cells (Fig. 5). The presence o f dense peroxidase reaction p r o d u c t t h r o u g h o u t the g r o u n d substance o f the infiltrated cells m a d e it very difficult to determine whether or not the reaction p r o d u c t was J. neurol. Sci., 1970, 10:205 213
208
,x. H I R A N ( ) .
N. It. BE( K E R . H. M. / I M M E R M ~ ' ,
Fig. 2. An endothelial junction separaled by a narrow, continuous band of tracer extending from Iht: lumen (arrow) in an animal in.iecled intravenously with peroxidase and sub ecied i~ a cerebral stah ~,~,o t l n d .
(~6,000
Fig. 3. A peroxidase-filled lumen in the cerebrum of a rat subjected to electroshock ,\n intercellular junction is only partially filled with the tracer farrou'). 96,00{I,
p r e s e n t b e t w e e n the cells since the p l a s m a m e m b r a n e s w e r e a l m o s t i m p o s s i b l e to d e l i n e a t e (Fig. 5). F r e q u e n t l y , pericytes a n d p e r i v a s c u l a r as well as o t h e r a s t r o c y t i c processes were also diffusely infiltrated (Fig. 6). T h e rats in which e x p e r i m e n t a l allergic e n c e p h a l o m y e l i t i s had been r e d u c e d also J. nettrol. ,S('i.. 1~70. lO: 205 213
ALTERATION OF THE BLOOD-BRAIN BARRIER
209
showed s p r e a d o f the tracer protein from the vasculature into the extracellular spaces o f the l u m b o s a c r a l spinal cord. The extravascular peroxidase was especially p r o m i n e n t a r o u n d venules and in the adjacent extracellular spaces (Fig. 7). The endothelial cells o f the vessels contained peroxidase-filled pinocytotic vesicles which, for the most part,
Fig. 4. Diffusely-stained endothelial cells near the third ventricle in a rat subjected to electroshock. ~." 6000.
Fig. 5. Higher magnification of an area similar to Fig. 4. Portions of 2 endothelial cells are visible. One, whose luminal surface is indicated by the small arrows, is diffusely stained with peroxidase. The adjacent endothelial cell is not. The junction between the cells is indicated by the large arrow. The perivascular space is diffusely infiltrated by the tracer, x 70,000.
were localized at the basal surface (Fig. 8). A t the intercellular junctions, the tracer usually filled the lower two-thirds but occasionally extended a l m o s t to the l u m i n a which were clear due to perfusion fixation. The perivascular spaces were diffusely and densely infiltrated with reaction p r o d u c t . J. neurcd. Sci., 1970, 10:205-213
210
A. HIRANO, N. H. BECKER, H. M. Z!MMERMAN
Fig. 6. A n area similar to Fig. 4. The endothelial cells, as well as astrocytic processes, are diffusely infiltrated with the tracer. ".: 8000.
Fig. 7. A venule in the lumbar spinal cord o f an animal subjected to EAE. The perivascular spaces are diffusely infiltrated with reaction product, x 6000. ,I. neurol. Sci., 1970, I0:205-213
ALTERATION OF THE BLOOD-BRAIN BARRIER
211
Fig. 8. Higher magnification of a n area similar to Fig. 7 illustrating the presence of peroxidase-filled pinocytotic vesicles in the endothelium, m o s t of which are at the basal surface. Nearby extracellular spaces are faintly m a r k e d by the presence of reaction product, × 27,000.
DISCUSSION
When one compares the results of the stab wound experiment with the uninjured control, there is no doubt that the experimental procedure resulted in clear-cut changes in the endothelial cells even some distance from the lesion. Among these changes were some in which direct communication could be observed between the vascular lumen and the extracellular space in the brain parenchyma. The relationship, however, between these communications and the blood-brain barrier is unclear. One might argue that all of these phenomena are the result of the reaction of an intact endothelium to the presence of excess extracellular fluid caused by the leakage of materials from blood vessels grossly damaged by the stab wound. We may, in fact, be observing the return of the tracer from the parenchyma to the vasculature through a secondarily-altered endothelium. In other words, these phenomena may be the secondary reflections of the breakdown of the blood-brain barrier and not its primary cause. To overcome this objection, we resorted to the use of animals in which the bloodbrain barrier was altered but in which the blood vessels were not subjected to gross, mechanical trauma; namely, rats subjected to electroconvulsive shock or experimental allergic encephalomyelitis (EAE). As described above, similar results, namely extracellular infiltration of tracer, indicating a breakdown in the blood-brain barrier, were obtained in these animals as well. Among the rats subjected to electroconvulsive shock, however, numerous endothelial cells appeared diffusely-stained and therefore probably damaged. These, then, might act much as the grossly-altered blood vessels in the stab wound experiments. In the EAE animals, on the other hand, such diffusely-stained endothelial cells were only rare. Nevertheless, as described above, the extracellular spaces, especially around the vessels, were densely filled with reaction product. Some workers have reported that pinocytosis was the route by which material moved from the circulation to the parenchyma in cerebral edema (TAM AND EVANS 1965; RAIMONDI 1966). Other workers, however, have already shown that in EAE animals leukocytes (BuBIS AND LUSE 1964; LEVlNEet al. 1965; LAMPERTAND CARPENTER 1965 ; LAMPERT1967), thorotrast (LAMPERTAND CARPENTER 1965 ; LAMPERT 1967 ; LA~PERT et al. [967) and hematogenous fluid (LEvINE et al. 1965) can pass between adjacent endothelial cells. On the other hand, in experimental allergic neuritis, J. neurol. Sci., 1970, 10 : 205-213
212
A. |tlRAN(), N. tl. BE(?KER, It. M. ZIMMERMAN
lymphocytes have been reported to move through individual endothelial cells (Asvk¢)~t et al. 1968). Further work attempting to delineate more clearly the preci~c route of peroxidase from the blood vessels into the parenchyma during EAE is needed and i~ the subject of current research in our laboratory.
ACKNOWLEDGEMENTS
The authors wish to express appreciation to Dr. Herbert Dembitzer, Laboratory Division, Montefiore Hospital and Medical Center. for his invaluable assistance.
SUMMARY
The distribution of peroxidase in the brain and spinal cord was examined after stab wounds, electroshock-induced convulsions, and experimental allergic encephalomyelitis. In all cases, the tracer was found to have moved from the vasculature into the parenchymatous extracellular spaces. The routes to that site may be via pinocytosis. between altered endothelial junctions or through diffusely-infiltrated endothelial cells.
REFERENCES
/~STROM,K.
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