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Microscopic radioautography of adult rat brain cholesterol. Problem of the blood-brain barrier
EXPERIMENTAL
NEUROLOGY
Microscopic
44, 1-9 (1974)
Radioautography Problem of the
of Adult Blood-Brain F.
C.SBROUCNEAND Laboratoire
Jartuary
Cholesterol.
CHEVALLIER]
dr Physiologie de la Nutrition, BBtimwt 447, 91405 Orsay, Rrceived
Rat Brain Barrier
Universitk (Frame)
de Paris-Sold,
22,1974
Microscopic radioautographs of adult rat brain obtained by intraventricular injection of ‘H-mevalonate or by ingestion of ‘H-cholesterol were performed at various time intervals after administration of the labeled compound. The same sites were labeled when cholesterol was synthesized irz situ or when transferred from plasma. The most active sites are those which contain the highest cholesterol concentrations, viz. myelin and nerve endings. These results suggest that both cholesterol synthesized ilz situ as well as that transferred from plasma are mixed in all the cerebral structures; this was confirmed by recent kinetic studies. No specific cells could be shown to synthesize cholesterol, suggesting that all cerebral cells are capable of this synthesis. Plasma cholesterol turned over much faster in the cerebral capillary walls than in the cerebral tissue. This was demonstrated when capillary walls were labeled 1 day after aH-cholesterol ingestion whereas the cerebral structures were almost unlabeled. Thus, the hypothesis of a “blood-brain barrier” represented by the cerebral capillaries is not valid for cholesterol.
INTRODUCTION As shown by previous work in our laboratory, one fraction of the cerebral cholesterol of the adult rat derives from plasma cholesterol (6, 8) while another fraction is synthesized in situ (8). Studies to localize cholesterol from synthetic and plasma origins were carried out by use of a macroscopic technique (7, 14). Brain cholesterol from both sources was found in all of the myelinated areas. The sameresult was obtained, for cerebral cholesterol from plasma origin, by Torvik and Sidman (18). In this report, the problem of localization of cholesterol from synthetic and plasma origins is examined,
at a cellular
level, with
a microscopic
technique.
1 Supported by grants from D.G.R.S.T. (no. 72.7.0132), from C.N.R.S. (ERA no. 415) and from C.E.A. (no. 11 512 II/B@. We are grateful to Dr. Droz for his interest and advice.
Copyright@ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
SltROUGNE
MATERIALS
AND
AND
CHEVALLIER
METHODS
In order to label the cerebral cholesterol from plasma origin, three albino male rats of adult age, weighing about 350 g, were fed with a semisynthetic diet containing 100 mCi of 3H-cholesterol (0.5 Ci/mmole). The first rat ingested the labeled diet in 1 day and was killed the next. The two other rats received 100 mCi of 3H-cholesterol in the diet over a 2mo period and were killed 2 and 6 mo thereafter. The animals were killed by aortic puncture, and the vascular system was rinsed, in sitzr, three or four times, with an isotonic saline solution. The brains were removed, frozen with solid CO2 and put in a Dittes cryostat regulated for -20 to -30 C. The radioautographic technique was performed as described by Appleton (1). Frozen sections (10 PC>were cut in the cryostat which was kept in a darkroom and mounted directly onto cold microscope slides (O’C) coated with Kodak AR-10 stripping film with the emulsion upward. The slides were kept in light-tight boxes at -20 C with a desiccant (PzO5) for the time needed for suitable exposure. After 10 min in methanol, the fixed sections were developed for 4 min in Drs Kodak, fixed for 6 min in 30% Na thiosulfate and washed for 3-4 min. After drying, the sections were stained with 0.1% toluidine blue for 5-10 sec. The labeling of synthetic cholesterol in the brain of adult rats was obtained by the injection of 10 mCi of oL-3H-mevalonic acid lactone in saline solution (70 ~1) in a lateral ventricle of the brain, using a previously described technique (5). Six animals received such an injection and were killed at 30 min, 20 and 26 hr, 4, 14, and 31 days after, respectively. Sacrifices were performed as described above. Brains were removed, frozen with solid COz and put in the cryostat. The whole technique described for brains labeled by plasma cholesterol was carried out for brains of rats killed 4, 14. and 31 days after injection. A special procedure was applied to the other three rats. Sections of brain were carried out, in the light, in the cryostat and removed on microscope slides. These slides were then washed five times in baths of distilled water for 10 min each, in order to remove water-soluble radioactive compounds. The radioactivity lost in the water baths was measured in a scintillation spectrometer (Intertechnique). Sections were then dried and coated, in the dark, with Kodak AR-10 stripping film. After drying, sections coated with emulsion were kept in light-tight boxes at o-4 C with a desiccant (PZOS) until suitable exposure. The development, fixation, and washing of the radioautographs were performed as above. In order to visualize the cerebral capillaries, a gelatine solution (1%)) colored with India ink, was injected via the abdominal aorta of a rat after exsanguination by aortic puncture. After 5-10 min of retention of this solution in the vascular system, the rat was killed and the brain removed. Frozen sections were carried out in the cryostat.
BRAIN
CHOLESTEROL
3
FIG. 1. Radioautographs from brain slices of rats labeled by aH-mevalonate injection (A, ‘C, E) or by sH-cholesterol in#gestion (B, D, F). Autopsy was performed 4 days (E) and 31 days (A, C) after injection, or 6 mo after ingestion (B, D, F)
RESULTS Specific Observations for Syntlzetic Cholesterol, The observations previously made on macroscopic radioautographs in the rat brain (14) were confirmed on the microscopic ones. A blackening gradient from the site of mevalonate injection (left lateral ventricle) to other regions of the brain is
4
SdROUGNE
AND
CHEVALLIER
labelin g : radioautographs FIG. 2. Some examples of myelin and capsula interna (B) of rats injected with sH-mevalonate or 4 days (A, B) after injection.
of cerebellum (A, C) and killed 20 hr (C)
seen on microscopic radioautographs from brains of rats killed 30 min, 20, 26 hr, 4 (Fig. lE), 14, and 31 days (Fig. lA, C) after injection. The intensity of this gradient decreases when the time interval between injection and autopsy increases. A second observation concerns the radioautographs of brain from rats killed 30 min and 20 and 26 hr after injection. Strong labeling appears in the granular layer of the cerebellum (Fig. 2C). But, 4 days after mevalonate injection, the granular layer is less labeled compared to the myelin and the molecular layer (Fig. 1A). Specific Observations for Transfer Cholesterol. The results obtained from
BRAIN
CHOLESTEROL
5
radioautographs of FIG. 3. Some examples of cellular and nerve endings labeling: three brain areas, Purkinje cells, hippocampal cells, and cerebellum myelin, when rats were injected with “H-mevalonate (A, C, E) or ingested 3H-cholesterol (B, D, F). Animals were killed 4 days (A, C), 31 days after injection, or 2 mo (B) or 6 rn’o (D, F) after ingestion.
the rat killed immediately after ingestion of 3H-cholesterol for 1 day are presented in Fig. 4. At a slight magnification, we can see a background labeling and some very strong concentrations of blackening in points and tracks (B). Similarity is noted between this disposition (B) and that of
6
SdROUGNE
AND
CHEVALLIER
of brain slices from rats labeled by ingestion of sHFIG. 4. Radioautographs cholesterol for 1 day and killed the next day: B, D, E, F. Brain slices from rats injected with India ink: A, C.
cerebral capillaries (A). At a higher magnification, many endothelial cells are distinct (C, D) when capillaries are sectioned longitudinally. When they are sectioned transversely, only packs of radioactivity are detectable (E) . The walls of large vesselsare labeled (F) . Common Results for Synthetic and Transfer Cholesterol. The radioautographs presented in this paper show that once cholesterol is synthesized
BRAIN
CHOLESTEROL
7
in situ or transferred from plasma to brain, it becomes distributed in the same cerebral regions. The most labeled regions are the myelinated ones. Some examples are seen in the white matter of the cerebellum (Fig. lA, B) , fimbria of the hippocampus (Fig. lE), fiber tracts of the capsula interna (Fig. lC, D ; Fig. 2B). Another example concerns the granular layer of the cerebellum where myelinated fibers passing through this layer are strongly labeled (Fig. 2A). All these observations can be made at a slight magnification. At a higher magnification, a weaker but definite labeling is found in the cytoplasm of all cells, nerve and glial. Nuclei are much less labeled. Some examples are seen in Fig. 3, in the Purkinje cells of the cerebellum (A, B) and in the hippocampus (C, D). It is noted that the cytoplasmic labeling is heterogeneous and present little packs of blackening. An heterogeneous labeling is also observed in the myelinated areas like white layer of the cerebellum, corpus callosum, fimbria, and so on. Little black packs occur in these areas. An example is given on Fig. 3 (E, F) in the white matter of the cerebellum. DISCUSSION Previous studies have shown that 1 day after mevalonate injection, 8991% of the radioactivity in the brain is in the unsaponifiable fraction (precursor sterols and cholesterol) (5). F or b rains of rats killed 1 hr after injection, we demonstrated that the washing of the slices removed almost all of the radioactivity which is not in the unsaponifiable fraction (14). This result was confirmed with brain slices from rats killed 30 min, and 20 and 26 hr after injection. So, we can consider that the blackening observed on the radioautographs represents the only cholesterol or precursor sterols (at the shortest times) localization. We have to note that the blackening gradient observed on radioautographs of brain slices from rats labeled by synthetic cholesterol is only induced by the technique of injection. No particular cells were seen to synthesize cholesterol in the brain. We think that, if a particular type of cell was the only synthetic site for cholesterol, it should be detectable 30 min after mevalonate injection. Because such a localization is not revealed and because all the cell cytoplasmas are labeled, our conviction is that all the cerebral cells synthesize cholesterol. Since no cholesterol catabolism occurs in the rat brain, the only labeled compound recovered in the brain when animals ingested 3H-cholesterol is this substance itself. The capillaries labeling raises a question about the nature of the labeled structure. One would think that it should be red blood cells which are labeled in the capillaries. But red blood cells have not been observed in the large vessels of the brain (Fig. 4F). Only the walls of these
8
&ROUGNE
AND
CHEVALLIER
vessels are labeled. Moreover, Figure 4D shows that only the capillary walls are labeled since the blackening is quite uniform along the capillary. So, the washing of the vascular system is complete. These radioautographic results suggest that cholesterol of the capillary walls has a relatively high rate of turnover. The existence of such a compartment has been advanced in a previous work of Chevallier (4) and recently confirmed (unpublished kinetic study). As compared to cholesterol exchanges between cerebral capillaries and plasma, those between cerebral structures and capillaries are relatively slower. It has been shown by previous experiments that the exchanges of cholesterol between blood and brain are much slower than those between blood and the other organs (6). The presence of a “bloodbrain barrier” has been and is still extensively discussed. Several interpretations for the nature of this barrier have been proposed. Some authors localize this barrier on the cerebral capillary walls (2, 3, 15, 19). The fact that the passage of cholesterol from blood to capillary walls can be seen on radioautographs suggests that this step of cholesterol entry into the brain is relatively easy. Some other authors believe that the “blood-brain barrier” is located in the astroglia (12) or that no real argument exists in favor of one or the other hypothesis (10). We believe, as Dobbing (13) that the “blood-brain barrier” is an inadequate term “until you can show that there is a physical structure interposed between the blood and the brain” which limits the rate of access of substances to the brain. Dobbing regarded the slow entry of cholesterol as a reflection of its metabolism in the brain. We believe that this slow entry of cholesterol into the brain is the result of the relative slow mobility of cholesterol in the brain compared to that in the other organs (6). The identity of cellular localization of cholesterol in the brain after it has been synthesized in sitzl or transferred from the plasma to the brain is in agreement with the similitude of fate of synthetic and plasma cholesterol in the brain which has been demonstrated in a recent work (16). Thus, cerebral cholesterol is not an inert compound but is in continual transfer within the brain, likely exchanging between the different cerebral structures. We observe that the highest labelings are located in the regions that have the highest cholesterol concentrations. The myelin is known to have the highest cholesterol amount in the rat brain : 36% (9, 11). Then, the nerve endings contain 19.370 of the total cerebral cholesterol and microsomes 11.3% (9). It is likely that labeling packs found in cell cytoplasms and in heavily myelinated areas are over synapses but they could also be located on glial endings. No information is actually furnished for the cholesterol content of glial endings. Our present results are in agreement with those of Spohn and Davison (17) who showed that “once radioactive cholesterol enters the brain . . . it becomes distributed throughout all sub-
BRAIN
cellular structures the brain.”
to give uniform
9
CHOLESTEROL
specific activity
of the lipid throughout
REFERENCES 1. APPLETON, T. C. 1964. Auto’radiography of soluble labelled compounds. J. Roy. Microsc. Sot. 83 : 277-281. 2. BODENHEIMER, T. S., and M. W. BRIGHTMAN. 1968. A blood-brain barrier to peroxidase in capillaries surrounded by perivascular spaces. Amer. J. Amat. 122 : 249-268. 3. BROMAN, T. 1949. “The Permeability of the Cerebrospinal Vessels in Normal and Pathological Conditions,” pp. 7-19. E. Munksgaard, Copenhagen. 4. CHEVALLIER, F. 1969. Etude des transferts de choleste?ol d’origine plasmatique dans le systeme nerveux du rat adulte et en croissance. Ne~wopatol. Pol. 7: 261-269. 5. CHEVALLIER, F., and C. GAUTHERON. 1969. A method for the study of cholesterol biosynthesis in the central nervous system. J. Nenrochem. 16 : 323-331. 6. CHEVALLIER, F., and F. GIRAUD. 1966. Renouvellement par transfert du cholest&ol chez les rats adultes et en croissance. Bull. Sot. Ckim BioE. 48: 787-801. 7. CHEVALLIER, F., and L. PETIT. 1966. Incorporation of cholesterol into the central nervous system and its autoradiographic localization. Exp. Neural. 16: 250254. 8. CHEVALLIER, F., F. D’HOLLANDER, and F. SIMONNET. 1968. Renouvellement, par synthese et par transfert, du cholesterol libre et esterifie. Grandeur des compartiments chez le rat adulte et leur repartition tissulaire. Biochinz. Biophys. Acta 164 : 339-356. 9. CUZNER, M. L., and A. N. DAVISON. 1968. The lipid composition of rat brain myelin and subcellular fractions during development. Biochem. J. 106: 29-34. 10. DAVSON, H. 1967. “The Blood-Brain Barrier. Physiology of the Cerebrospinal Fluid,” pp. 82-103. Churchill, London. 11. DAVISON, A. N. 1970. p. 90. In “Myelination.” A. N. Davison and A. Peters [Eds.] Thomas, Springfield, Ill. 12. DE ROBERTIS, E., and M. M. GERSHENFELD. 1961. Submicroscopic mot-phology and function of glial cells. Int. Rev. Neurol. 3 : l-65. 13. DOBBING, J. 1972. p. 71. In Lipids, Malnutrition and the Developing Brain. Ciba Found. Symp. Elsevier. 14. GAUTHERON, C., L. PETIT, and F. CHEVALLIER. 1969. Synthesis of cholesterol into the central nervous system and its radioautographic localization. Exp. Neurol. 25: 18-23. 15. REESE, T. S., and M. J. KARNOVSKY. 1967. Fine structural localization of a bloodbrain barrier to exogenous peroxidase. J. Cell BioZ. 34: 207-217. 16. SEROUGNE-GAUTHERON C., and F. CHEVALLIER. 1973. Time course of biosynthetic cholesterol in the adult rat brain. Biochim. Bioghys. Acta 316: 244-250. 17. SPOHN M., and A. N. DAVISON. 1972. Cholesterol metabolism in myelin and other subcellular fractions of rat brain. J. Lipid Res. 1.3: 563-570. 18. TORVIK, A., and R. L. SIDMAN. 1962. Autoradiographic studies on lipid synthesis in the mouse brain during postnatal development. J. Neurochem. 9 : 421-425. 19. TSCHIRGI, R. D. 1952. “Blood-Brain Barrier. The Biology of Mental Health and Disease,” pp. 34-46 Hoeber-Harper.
NEUROLOGY
Microscopic
44, 1-9 (1974)
Radioautography Problem of the
of Adult Blood-Brain F.
C.SBROUCNEAND Laboratoire
Jartuary
Cholesterol.
CHEVALLIER]
dr Physiologie de la Nutrition, BBtimwt 447, 91405 Orsay, Rrceived
Rat Brain Barrier
Universitk (Frame)
de Paris-Sold,
22,1974
Microscopic radioautographs of adult rat brain obtained by intraventricular injection of ‘H-mevalonate or by ingestion of ‘H-cholesterol were performed at various time intervals after administration of the labeled compound. The same sites were labeled when cholesterol was synthesized irz situ or when transferred from plasma. The most active sites are those which contain the highest cholesterol concentrations, viz. myelin and nerve endings. These results suggest that both cholesterol synthesized ilz situ as well as that transferred from plasma are mixed in all the cerebral structures; this was confirmed by recent kinetic studies. No specific cells could be shown to synthesize cholesterol, suggesting that all cerebral cells are capable of this synthesis. Plasma cholesterol turned over much faster in the cerebral capillary walls than in the cerebral tissue. This was demonstrated when capillary walls were labeled 1 day after aH-cholesterol ingestion whereas the cerebral structures were almost unlabeled. Thus, the hypothesis of a “blood-brain barrier” represented by the cerebral capillaries is not valid for cholesterol.
INTRODUCTION As shown by previous work in our laboratory, one fraction of the cerebral cholesterol of the adult rat derives from plasma cholesterol (6, 8) while another fraction is synthesized in situ (8). Studies to localize cholesterol from synthetic and plasma origins were carried out by use of a macroscopic technique (7, 14). Brain cholesterol from both sources was found in all of the myelinated areas. The sameresult was obtained, for cerebral cholesterol from plasma origin, by Torvik and Sidman (18). In this report, the problem of localization of cholesterol from synthetic and plasma origins is examined,
at a cellular
level, with
a microscopic
technique.
1 Supported by grants from D.G.R.S.T. (no. 72.7.0132), from C.N.R.S. (ERA no. 415) and from C.E.A. (no. 11 512 II/B@. We are grateful to Dr. Droz for his interest and advice.
Copyright@ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
SltROUGNE
MATERIALS
AND
AND
CHEVALLIER
METHODS
In order to label the cerebral cholesterol from plasma origin, three albino male rats of adult age, weighing about 350 g, were fed with a semisynthetic diet containing 100 mCi of 3H-cholesterol (0.5 Ci/mmole). The first rat ingested the labeled diet in 1 day and was killed the next. The two other rats received 100 mCi of 3H-cholesterol in the diet over a 2mo period and were killed 2 and 6 mo thereafter. The animals were killed by aortic puncture, and the vascular system was rinsed, in sitzr, three or four times, with an isotonic saline solution. The brains were removed, frozen with solid CO2 and put in a Dittes cryostat regulated for -20 to -30 C. The radioautographic technique was performed as described by Appleton (1). Frozen sections (10 PC>were cut in the cryostat which was kept in a darkroom and mounted directly onto cold microscope slides (O’C) coated with Kodak AR-10 stripping film with the emulsion upward. The slides were kept in light-tight boxes at -20 C with a desiccant (PzO5) for the time needed for suitable exposure. After 10 min in methanol, the fixed sections were developed for 4 min in Drs Kodak, fixed for 6 min in 30% Na thiosulfate and washed for 3-4 min. After drying, the sections were stained with 0.1% toluidine blue for 5-10 sec. The labeling of synthetic cholesterol in the brain of adult rats was obtained by the injection of 10 mCi of oL-3H-mevalonic acid lactone in saline solution (70 ~1) in a lateral ventricle of the brain, using a previously described technique (5). Six animals received such an injection and were killed at 30 min, 20 and 26 hr, 4, 14, and 31 days after, respectively. Sacrifices were performed as described above. Brains were removed, frozen with solid COz and put in the cryostat. The whole technique described for brains labeled by plasma cholesterol was carried out for brains of rats killed 4, 14. and 31 days after injection. A special procedure was applied to the other three rats. Sections of brain were carried out, in the light, in the cryostat and removed on microscope slides. These slides were then washed five times in baths of distilled water for 10 min each, in order to remove water-soluble radioactive compounds. The radioactivity lost in the water baths was measured in a scintillation spectrometer (Intertechnique). Sections were then dried and coated, in the dark, with Kodak AR-10 stripping film. After drying, sections coated with emulsion were kept in light-tight boxes at o-4 C with a desiccant (PZOS) until suitable exposure. The development, fixation, and washing of the radioautographs were performed as above. In order to visualize the cerebral capillaries, a gelatine solution (1%)) colored with India ink, was injected via the abdominal aorta of a rat after exsanguination by aortic puncture. After 5-10 min of retention of this solution in the vascular system, the rat was killed and the brain removed. Frozen sections were carried out in the cryostat.
BRAIN
CHOLESTEROL
3
FIG. 1. Radioautographs from brain slices of rats labeled by aH-mevalonate injection (A, ‘C, E) or by sH-cholesterol in#gestion (B, D, F). Autopsy was performed 4 days (E) and 31 days (A, C) after injection, or 6 mo after ingestion (B, D, F)
RESULTS Specific Observations for Syntlzetic Cholesterol, The observations previously made on macroscopic radioautographs in the rat brain (14) were confirmed on the microscopic ones. A blackening gradient from the site of mevalonate injection (left lateral ventricle) to other regions of the brain is
4
SdROUGNE
AND
CHEVALLIER
labelin g : radioautographs FIG. 2. Some examples of myelin and capsula interna (B) of rats injected with sH-mevalonate or 4 days (A, B) after injection.
of cerebellum (A, C) and killed 20 hr (C)
seen on microscopic radioautographs from brains of rats killed 30 min, 20, 26 hr, 4 (Fig. lE), 14, and 31 days (Fig. lA, C) after injection. The intensity of this gradient decreases when the time interval between injection and autopsy increases. A second observation concerns the radioautographs of brain from rats killed 30 min and 20 and 26 hr after injection. Strong labeling appears in the granular layer of the cerebellum (Fig. 2C). But, 4 days after mevalonate injection, the granular layer is less labeled compared to the myelin and the molecular layer (Fig. 1A). Specific Observations for Transfer Cholesterol. The results obtained from
BRAIN
CHOLESTEROL
5
radioautographs of FIG. 3. Some examples of cellular and nerve endings labeling: three brain areas, Purkinje cells, hippocampal cells, and cerebellum myelin, when rats were injected with “H-mevalonate (A, C, E) or ingested 3H-cholesterol (B, D, F). Animals were killed 4 days (A, C), 31 days after injection, or 2 mo (B) or 6 rn’o (D, F) after ingestion.
the rat killed immediately after ingestion of 3H-cholesterol for 1 day are presented in Fig. 4. At a slight magnification, we can see a background labeling and some very strong concentrations of blackening in points and tracks (B). Similarity is noted between this disposition (B) and that of
6
SdROUGNE
AND
CHEVALLIER
of brain slices from rats labeled by ingestion of sHFIG. 4. Radioautographs cholesterol for 1 day and killed the next day: B, D, E, F. Brain slices from rats injected with India ink: A, C.
cerebral capillaries (A). At a higher magnification, many endothelial cells are distinct (C, D) when capillaries are sectioned longitudinally. When they are sectioned transversely, only packs of radioactivity are detectable (E) . The walls of large vesselsare labeled (F) . Common Results for Synthetic and Transfer Cholesterol. The radioautographs presented in this paper show that once cholesterol is synthesized
BRAIN
CHOLESTEROL
7
in situ or transferred from plasma to brain, it becomes distributed in the same cerebral regions. The most labeled regions are the myelinated ones. Some examples are seen in the white matter of the cerebellum (Fig. lA, B) , fimbria of the hippocampus (Fig. lE), fiber tracts of the capsula interna (Fig. lC, D ; Fig. 2B). Another example concerns the granular layer of the cerebellum where myelinated fibers passing through this layer are strongly labeled (Fig. 2A). All these observations can be made at a slight magnification. At a higher magnification, a weaker but definite labeling is found in the cytoplasm of all cells, nerve and glial. Nuclei are much less labeled. Some examples are seen in Fig. 3, in the Purkinje cells of the cerebellum (A, B) and in the hippocampus (C, D). It is noted that the cytoplasmic labeling is heterogeneous and present little packs of blackening. An heterogeneous labeling is also observed in the myelinated areas like white layer of the cerebellum, corpus callosum, fimbria, and so on. Little black packs occur in these areas. An example is given on Fig. 3 (E, F) in the white matter of the cerebellum. DISCUSSION Previous studies have shown that 1 day after mevalonate injection, 8991% of the radioactivity in the brain is in the unsaponifiable fraction (precursor sterols and cholesterol) (5). F or b rains of rats killed 1 hr after injection, we demonstrated that the washing of the slices removed almost all of the radioactivity which is not in the unsaponifiable fraction (14). This result was confirmed with brain slices from rats killed 30 min, and 20 and 26 hr after injection. So, we can consider that the blackening observed on the radioautographs represents the only cholesterol or precursor sterols (at the shortest times) localization. We have to note that the blackening gradient observed on radioautographs of brain slices from rats labeled by synthetic cholesterol is only induced by the technique of injection. No particular cells were seen to synthesize cholesterol in the brain. We think that, if a particular type of cell was the only synthetic site for cholesterol, it should be detectable 30 min after mevalonate injection. Because such a localization is not revealed and because all the cell cytoplasmas are labeled, our conviction is that all the cerebral cells synthesize cholesterol. Since no cholesterol catabolism occurs in the rat brain, the only labeled compound recovered in the brain when animals ingested 3H-cholesterol is this substance itself. The capillaries labeling raises a question about the nature of the labeled structure. One would think that it should be red blood cells which are labeled in the capillaries. But red blood cells have not been observed in the large vessels of the brain (Fig. 4F). Only the walls of these
8
&ROUGNE
AND
CHEVALLIER
vessels are labeled. Moreover, Figure 4D shows that only the capillary walls are labeled since the blackening is quite uniform along the capillary. So, the washing of the vascular system is complete. These radioautographic results suggest that cholesterol of the capillary walls has a relatively high rate of turnover. The existence of such a compartment has been advanced in a previous work of Chevallier (4) and recently confirmed (unpublished kinetic study). As compared to cholesterol exchanges between cerebral capillaries and plasma, those between cerebral structures and capillaries are relatively slower. It has been shown by previous experiments that the exchanges of cholesterol between blood and brain are much slower than those between blood and the other organs (6). The presence of a “bloodbrain barrier” has been and is still extensively discussed. Several interpretations for the nature of this barrier have been proposed. Some authors localize this barrier on the cerebral capillary walls (2, 3, 15, 19). The fact that the passage of cholesterol from blood to capillary walls can be seen on radioautographs suggests that this step of cholesterol entry into the brain is relatively easy. Some other authors believe that the “blood-brain barrier” is located in the astroglia (12) or that no real argument exists in favor of one or the other hypothesis (10). We believe, as Dobbing (13) that the “blood-brain barrier” is an inadequate term “until you can show that there is a physical structure interposed between the blood and the brain” which limits the rate of access of substances to the brain. Dobbing regarded the slow entry of cholesterol as a reflection of its metabolism in the brain. We believe that this slow entry of cholesterol into the brain is the result of the relative slow mobility of cholesterol in the brain compared to that in the other organs (6). The identity of cellular localization of cholesterol in the brain after it has been synthesized in sitzl or transferred from the plasma to the brain is in agreement with the similitude of fate of synthetic and plasma cholesterol in the brain which has been demonstrated in a recent work (16). Thus, cerebral cholesterol is not an inert compound but is in continual transfer within the brain, likely exchanging between the different cerebral structures. We observe that the highest labelings are located in the regions that have the highest cholesterol concentrations. The myelin is known to have the highest cholesterol amount in the rat brain : 36% (9, 11). Then, the nerve endings contain 19.370 of the total cerebral cholesterol and microsomes 11.3% (9). It is likely that labeling packs found in cell cytoplasms and in heavily myelinated areas are over synapses but they could also be located on glial endings. No information is actually furnished for the cholesterol content of glial endings. Our present results are in agreement with those of Spohn and Davison (17) who showed that “once radioactive cholesterol enters the brain . . . it becomes distributed throughout all sub-
BRAIN
cellular structures the brain.”
to give uniform
9
CHOLESTEROL
specific activity
of the lipid throughout
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