THE ANATOMICAL RECORD 256:165–176 (1999) Regional Difference of Lipid Distribution in Brain of Apolipoprotein E Deficient Mice MASAO MATO,1,2* SHIGEO OOKAWARA,2 TOSHIHIRO MASHIKO,2 ATSUSHI SAKAMOTO,2 TAKASHI K. MATO,2 NOBUYO MAEDA,3 AND TATSUHIKO KODAMA4 1Center of Medical Education, International University of Health and Welfare, Tochigi, 324-0011 Japan 2Department of Anatomy, Jichi Medical School, Tochigi, 329-0498, Japan 3Department of Pathology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7525 4Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, 153-0041, Japan ABSTRACT According to recent knowledge, apolipoprotein E (apo E) plays a significant role in the homeostasis of intracellular cholesterol level in various tissues. Apo E deficient mice develop hyperlipidemia, and suffer from atherosclerosis in extracerebral blood vessels and neurodegeneration in the central nervous system. Furthermore, Walker et al. (Am. J. Path., 1997;151:1371–1377) demonstrated cerebral xanthomas of various sizes in the brain of apo E deficient mice. In the present study, it is illustrated that in the homozygous apo E deficient mice of advancing age, a great number of foamy macrophages extravasate from microvessels in thalamus and fimbria hippocampi, and scatter in the perivascular regions and migrate toward the ependyma, fimbria hippocampi, hippocampus, and thalamus. Here, it must be pointed out that under hyperlipidemia, although foamy macrophages made clusters in the perivascular region, the cerebral microvessels did not develop atherosclerosis. On the other hand, in the other cerebral regions such as cerebral cortex, caudoputamen, globus pallidus, and substantia nigra, macrophages did not appear and microvessels retained normal shapes, but the fluorescent granular perithelial (in short, FGP) cells accompanied by these vessels contained a certain amount of lipids. That is, in the cerebral cortex and caudoputamen, lipid components are detected in FGP cells and microglia, while in the globus pallidus and substantia nigra, they are mainly localized in astrocytes. The reason why the astrocytes in such defined regions contain, specifically, a high quantity of lipid components remains unsettled. Axonal degenerations are often represented in thalamus, globus pallidus, and substantia nigra. On the other hand, in the specimens of Wild-type mice, lipid components were observed only in FGP cells, and the vascular architecture took a normal profile. Any lipid laden macrophages and the axonal degenerations could not be detected through the cerebral parenchyma. Furthermore, it is also a noticeable finding that immunohistochemically, the FGP cells express a positive reaction against the antibody of apo E in the Wild-type mice, but those of homozygous apo E deficient mice are immunonegative. FGP cells are not only provided with the scavenger receptor, but also contribute to the lipid metabolism in the brain. Anat Rec 256:165–176, 1999. r 1999 Wiley-Liss, Inc. Key words: apolipoprotein E; astrocyte; atherosclerosis; brain; cholesterol metabolism; fluorescent granular perithelial cell; macrophage *Correspondence to: Prof. Masao Mato, MD, Department of Anatomy, Jichi Medical School, Tochigi, 329-0498, Japan. Received 21 December 1998; Accepted 16 June 1999 r 1999 WILEY-LISS, INC. 166 MATO ET AL. According to Mahley (1998), apolipoprotein (apo) E is distributed widely in various tissues of the body, and plays an important role in a lipid transport and cholesterol homeostasis. Boyles et al. (1985) and Pitas et al. (1987) already reported that apo E in the brain was synthesized in astrocytes, but not in neurons, oligodendrocytes, microglia, ependymal cells, and choroidal cells. Furthermore, Boyles et al. (1990), Masliah et al. (1994), Nathan et al. (1994), and Poirier et al. (1993) published excellent papers concerning the role of apo E in the development, remodeling, and regeneration of the nervous system, and Masliah et al. (1995) reported the reduction of synaptophysinimmunoreactive nerve terminals and microtubule associated protein 2-immunoreactive dendrites in the neocortex and hippocampus of the apo E deficient mice. Homozygous apo E deficient mice created by Zhang et al. (1992) and Plump et al. (1992) had been regarded as animal models of the familial type III hypercholesterolemia. The genetic disorder was characterized by the elevation of total cholesterol and triglycerides, and by the reduction of high density lipoprotein levels in plasma. As evidenced by Reddick et al. (1994), the deficient mice developed atherosclerosis in the cardiac sinus, coronary arteries and ascending aorta, even if fed a normal diet. According to the previous studies of the present authors (Mato et al., 1981, 1985, 1986a,b, 1989, 1996, 1997, 1998; Hikishima et al., 1990; Sakamoto et al., 1992), under the physiological condition, only Mato’s fluorescent granular perithelial (FGP) cells are provided with the scavenger receptor and engaged in the scavenging of foreign substances administered in the cerebral tissue, while in the pathological conditions, adding to the FGP cells, extravasated macrophages, and microglia are also facilitated to remove excessive foreign substances and degenerating cell debris in the brain. In this report, the present authors intend to demonstrate i) the structural alteration of cerebral microvessels, ii) the abnormal deposition-sites of lipid components in brain of apo E deficient mice and iii) the appearance of apo E in the FGP cells surrounding cerebral microvessels. MATERIALS AND METHODS Thirteen homozygous apo E deficient mice at the age of 8- to 10-months-old, and six Wild-type mice (control) of the same age, were employed in this study. Apo E deficient mice were delivered from the University of North Carolina and fed on the standard mice chow (CE-2) purchased by CLEA Inc., Tokyo, Japan. Under ether anesthesia, control (Wild-type mice) and apo E deficient mice were perfused via the heart with cold physiological saline. In order to obtain stretch specimens for the light microscopical observations of FGP (fluorescent granular perithelial) cells, the cerebral cortices of both groups (control and apo E deficient mice) were prepared with the method reported previously (Mato and Ookawara, 1979). The other mice, after perfusion of physiological saline, were again perfused with 4% paraformaldehyde buffered with 0.1M phospate. Just after decapitation, the brains of both groups of mice were quickly removed, and cut coronally with the blade at about 1 mm in thickness. The serial slice specimens of the brains were divided into two groups for light and electron microscopic observations. For the light microscopic observation, slice specimens were cut with cryostats after cryoprotection with 18% sucrose, and frozen with liquid nitrogen, and then stained with oil red O or sudan black B for revealing lipid components. For the immunostaining, the other stretch specimens and the cryostats sections were refixed with cold methanol for 10 minutes and immersed in 0.1% iodate peroxide solution for the prevention of endogenous peroxidase. Then, these specimens were treated with BM8 (Biomedical Lab., monoclonal antibody for mouse macrophage), F4/80 (Serotec LTD, Oxford, England, monoclonal antibody for microglia) or with apo E polyclonal antibody (Santa Cruz Biochemical Ltd.) for 15 hr, and with a Vectastain ABC kit and visualized with 3-38 diaminobenzidine reagent. For the control of immunostain, the primary antibody was omitted, and then the specimens were treated in the same manner as mentioned above. For the electron microscopic observation, the remaining slice specimens of the brain were again fixed with the mixture of 2% paraformaldehyde and 2.5% glutaraldehyde buffered with 0.1M phosphate for 10 hr, and then with 1% osmium tetroxide buffered with 0.1M phosphate for 2 hr at 4°C. After dehydration with ethanol, they were embedded in Epon 812, and cut with RMC MT-7 ultramicrotome. Following the staining with uranyl acetate and lead hydroxide, the specimens were observed with a JEM 2000EX electron microscope. RESULTS Light Microscopic Observation The serial coronal sections of brains of control and apoE deficient mice are depicted in Figures 1A to 5. Figures 1 to 3 indicate the distribution of lipid components stainable Fig. 1. A: Fimbria hippocampi (FH) and globus pallidus (GP) of control mice brain stained with oil red O. Corpus callosum and nerve bundles in the globus pallidus are moderately stained. There is some artifact in it. B: The same region of A of apo E deficient mice stained with oil red O. The fimbria hippocampi and the globus pallidus are intensely stained, but the caudoputamen (CP) is free from this staining. CC, cerebral cortex. Counterstain, hematoxylin. ⫻15. Fig. 2. Figure 2 is obtained from the region posterior to Figure 1. A: The fimbria hippocampi (FH) and corpus callosum are moderately stained with oil red O, but the hippocampus (HP) is not stained. Neurons are regularly arranged in CA2, CA3, and dentate nucleus. B: Obtained from apo E deficient mice, the fimbria hippocampi and adjacent areas to it (hippocampus and thalamus [TH]) are markedly stained. Round cells laden with lipid components and cholesterol clefts disperse around the fimbria hippocampi and adjacent areas. Counterstain, hematoxylin. ⫻32. Fig. 3. In the cross-section through substantia nigra and cerebral peduncle of apo E deficient mice, ependyma (EP) and substantia nigra (SN, arrow) are stained intensely with oil red O. CP, choroid plexus. Counterstain, hematoxylin. ⫻32. Fig. 4. Microvessels (BV, arrows) of thalamus obtained from control (A) and apo E deficient mice (B). In the perivascular regions of B, the cells including fat droplets make clusters, but in that of control, any lipid laden cells are not seen. ⫻130. Fig. 5. Microvessels in the cerebral cortex. Arrows indicate the immunopositive processes of microglia reacted with F4/80 close to the vessels. Counterstain, hematoxylin. ⫻120. Fig. 6. Fimbria hippocampi (FH, A) and substantia nigra (SN, B) of apo E deficient mice after immunostaining with BM8. In A, the immunopositive cells are scattered (arrows), but in B, the immunopositive cells are not found. Counterstain, hematoxylin. ⫻130. Fig. 7. Stretch specimens of cerebral cortices of control and apo E deficient mice treated with the antibody against apolipoprotein E. The FGP cell of control mice shows the positive reaction (A, arrow), but that of apo E deficient mice is negative (B, arrow). Counterstain; methygreen (A), hematoxylin (B). ⫻350. Figures 1–7. 168 MATO ET AL. with oil red O or sudan black B methods. As both methods for lipid stain afforded almost similar findings, in the present paper, the specimens stained with oil red O are only illustrated. Figure 1A,B indicate oil red O-stained regions of fimbria hippocampi and globus pallidus obtained from control and apo E deficient mice, respectively. Contained within, myelinated nerve fibers in corpus callosum and basal ganglia are moderately stained, but in Figure 1B, intensely stained lipid components appear in fimbria hippocampi and globus pallidus, different from those of Figure 1A. In Figure 2A (control mice) and B (apo E deficient mice), myelinated nerve fibers in hippocampus and thalamus were also stained moderately, similar to Figure 1A,B. However, specifically, intensely stained regions can be detected in fimbria hippocampi, and some regions of hippocampus and thalamus adjoining to the fimbria as shown in Figure 2B. In the closer observations, cholesterol clefts and lipid granule laden cells were found in these regions. A similar feature has already been reported by Walker et al. (1997) in the xanthoma. In serial sections, as demonstrated in Figure 3, the substantia nigra in apo E deficient mice is also stained intensely with oil red O similar to the globus pallidus. However, with regard to controls, such stainable substances could not be seen. The walls of blood vessels distributing in the thalamus of control mice looked thin and was demarcated distinctly from the neural tissue (Fig. 4A), but, as demonstrated in Figure 4B, the vascular walls in the same area of apo E deficient mice looked thickened owing to the clustering of extravasated cells containing oil red O stainable droplets. These findings mentioned above were different from each individual apo E deficient mouse. Xanthoma appeared in the ratio of 4:13, lipid droplets in globus pallidus and substantia nigra appeared in ratio of 6:13 and 7:13, respectively. FGP cells and ependymal cells always contained lipid droplets in all specimens, although they were various in quantity. Immunohistochemically, in the cerebral cortex, microglia occasionally appear close to microvessels and react against F4/80 (Fig. 5). Figure 6A,B indicate the immunoreaction of fimbria hippocampi in the same region of Figure 2A and substantia nigra in the same region of Figure 3. Figure 6A shows the distribution of BM8 positive cells in fimbria hippocampi. In the closer observation, the positive cells are large and take polygonal shapes, and they were identified as macrophages. However, any immunopositive cells against BM8 antibody could not be observed in substantia nigra (Fig. 6B) and globus pallidus (data not shown). Figure 7A,B are the stretch specimens of brains of Wild-type and apo E deficient mice treated with the antibody against apo E. The FGP cells in the cerebral cortex of Wild-type mice showed the positive reaction (Fig. 7A), while as depicted in Figure 7B, the FGP cells in the same region of the apo E deficient mice tested negative for this immunostaining. When the primary antibody was omitted, the immunoreactions for BM8, F4/80, and apo E became negative. Electron Microscopic Observation Control specimens. In order to compare the ultrastructure of microvessels, microglia, neurons, and astrocytes of Wild-type and apo E deficient mice, several regions concerned have been analyzed: In the cerebral cortex of control mice, the vascular walls of microvessels are composed of flat endothelium, smooth muscle cells rich in fine myofilaments, and FGP cells which enclose round inclusions of moderate density (Fig. 8). The FGP cells were clearly defined with the glia limitans and, in general, were lined with astrocytes. The astrocytes, as demonstrated in Figure 8, possess pale cytoplasm, and small amounts of mitochondria, endoplasmic reticula, and some glial filaments. The perikaryon of neurons is occupied by round nucleus with a distinct nucleolus, and the cytoplasm contains Nissl bodies, mitochondria, ribosomes, and lysosomes. The neurons in the C3 region of hippocampus contain also a relatively large and oval nucleus with a distinct nucleolus and a certain number of mitochondria (Fig. 9). In the cytoplasm, lipofuscin granules could be rarely observed. The blood vessel in hippocampus showed the similar shape as in the cerebral cortex. Astrocytes, axons, and microvessels in the globus pallidus and caudoputamen are displayed in Figures 10 and 11. The vessel in Figure 10, consisting of endothelium and smooth muscle cells, is surrounded by a pale astrocyte with an oval nucleus. The astrocyte contains a small amount of glial filaments and dark mitochondria, but not vacuoles. As demonstrated in Figure 11, the FGP cells surround the microvessels and are rich in inclusions of various intensity and smooth surfaced endoplasmic reticula. Astrocytic processes subjacent to the FGP cells and among axons are pale and not so swollen. Microglia sometimes appear close to the microvessels. Apo E deficient mice. In the cerebral cortex, neurons retained normal shape and degenerating signs could rarely be observed in them. However, microvessels in this region take unusual profiles; that is, the electron opacity of endothelium and smooth muscle cells was moderately enhanced as compared to control mice, and the luminal surface of endothelium is not smooth (Figs. 12 and 13). The FGP cells, as demonstrated in Figure 12, possess round inclusions of various shapes and intensity, and some of the inclusions are larger and denser, and contain lipid components. Astrocytes subjacent to the FGP cells are swollen and are not always pale owing to the scattering of rosettes of ribosomes. However, some FGP cells look pale and include a small number of inclusions (Fig. 13). As demon- Fig. 8. The vascular wall in cerebral cortex is composed of endothelium (E), smooth muscle cell (SM), and FGP cell (F). The intensity of FGP cell is moderate and provided with relatively pale inclusions (IB), endoplasmic reticula, and mitochondria. In the periphery of inclusions, a few and small dense bodies are seen (arrows). The FGP cells is lined with the astrocytic process (As). ⫻21,000. Fig. 9. The electron micrograph shows some neurons (N) in CA3 of hippocampus of control mice. They possess polysomes, endoplasmic reticula, and mitochondria. Nuclei of them are relatively large and take round shapes. ⫻3,700. Fig. 10. The electron micrograph shows a blood vessel and astrocyte in globus pallidus. Endothelium (E) and smooth muscle cells are surrounded by astrocytes (As). The astrocyte contains oval nucleus, mitochondria, and a small amount of glial filaments (arrows). ⫻11,000. Fig. 11. The electron micrograph of blood vessel is obtained from caudoputamen. The blood vessel composing endothelium (E) and smooth muscle cells (SM) is lined with FGP cell (F). The FGP cell contain vacuoles, mitochondria, many intense inclusions, and smooth surfaced endoplasmic reticula. Subjacent to it, astrocytic processes (As) are seen. ⫻21,000. REGIONAL DIFFERENCE OF LIPID DEPOSITION IN BRAIN Figures 8–11. 169 Figures 12–15. REGIONAL DIFFERENCE OF LIPID DEPOSITION IN BRAIN 171 Fig. 16. Fimbria hippocampi corresponds to Figure 2B. A: Cholesterol cleft (arrow), macrophages (arrow heads), cell debrices, and erythrocyte with irregular shape. ⫻2,800. B: FGP cell (F), macrophages (M), and glia limitans (arrows). The FGP cell is filled with inclusions of various size and shape, and lined with a glia limitans. ⫻5,500. Fig. 17. CA3 of hippocampus which corresponds to Figure 2B. In this figure, neurons (N), cell debrices, and macrophages (M) are displayed. Neurons take irregular shapes and contain vacuoles and dense inclusions. The cells filled with lipid droplets are also observed in this area (arrow). They seem to be microglia. ⫻3,500. strated in Figure 13, microglia or their processes approach close to the microvessels and invade occasionally in the interstices between the FGP cell and astrocytes. Furthermore, microglia in the parenchyma are laden with a moderate quantity of lipid-like material (Fig. 14). Some- times, the smooth muscle cells in the cerebral cortex also contain lipid-like and dense bodies of various size, and astrocytes possess a weak contour of cytoplasmic membrane (Fig. 13). In addition, abnormal features of synapse appear in the parenchyma of the cerebral cortex (Fig. 15). In the fimbria hippocampi and hippocampus adjacent to the fimbria hippocampi, a great number of foamy macrophages, cell debris, and cholesterol clefts appear, and result in the destruction of the normal architecture (Figs. 16A,B and 17). Often, cytoplasmic membranes of macrophages were broken and the contents in macrophages (composed of vesicles and cell debris) were dispersed in the surrounding regions (Fig. 16A). The FGP cells are rich in inclusions and vesicles of moderate density, and tend to degenerate (Fig. 16B). Some neurons in CA3 of hippocampus close to the fimbria take irregular forms and contain irregularly shaped nuclei and vacuoles (Fig. 17). Microglia and FGP cells are not always identified owing to the taking of various forms and intensity. Most of them are predisposed to degeneration. Astrocytes look atrophic and occasionally involve only some membranous structures. Ependymal cells in the apo E deficient mice lost a normal appearance and were often swollen containing large inclusions with medium intensity and pale vacuoles, Figs. 12 and 13. These figures show blood vessel (BV), FGP cells (F), astrocytes (As), and neuron (N) in the cerebral cortex. The vascular cells—endothelium (E) and smooth muscle cells (SM)—are relatively high in intensity and cytoplasmic projections of endothelium give rise on their luminal surface. In smooth muscle cells, some dense inclusions (arrows) are presented in Figure 13. The FGP cell in Figure 12 contains opaque cytoplasm and large inclusions (arrows) of intense lipidic components, and takes normal profiles, while the FGP cell in Figure 13 is atrophic and contains a scanty of inclusions and cytoplasmic organelles. Astrocytes are swollen and not always pale in these figures. The FGP cell in Figure 13 is closely associated with the dark process of microglia (Mg). ⫻10,000 (Fig. 12), ⫻12,000 (Fig. 13). Figs. 14 and 15. These electron micrographs are also obtained from the cerebral cortex. The microglia (Mg) in Figure 14 contain various kinds of inclusions. Arrows indicate round vacuolated inclusions. Adding to them, intensely stained and irregularly shaped bodies appear in microglia. ⫻16,000. Fig. 15 shows the unusually distended postsynaptic bag (PS). In it, small number of vesicles, vacuoles, mitochondria and amorphous material are seen. ⫻32,000. 172 MATO ET AL. and sometimes atrophic (Fig. 18). In the deeper layers of the ependyma, occasionally foamy macrophages, cell debris, and cholesterol clefts are distributed (Fig. 18). In the globus pallidus and substantia nigra, there were two types of FGP cells; the one looked pale and swollen, and sometimes contained large pale vacuoles. The cytoplasmic organelles in them were sparse, while the other type of FGP cells possessed a somewhat opaque cytoplasm and several intense lysosomal inclusions as shown in Figure 19. The astrocytes and their cytoplasmic processes are somewhat swollen, and their cytoplasmic membranes are not always defined. They are distributed underlying the FGP cells or surrounding neurons, and are filled with many large pale vacuoles, a small amount of thin endoplasmic reticula, polysomes and glial filaments (Figs. 20 and 21). The intracellular pale vacuoles in astrocytes measure about 2 to 4 µm in diameter. The distribution of pale vacuoles of astrocytes and FGP cells in the globus pallidus and substantia nigra appears corresponding with that of the lipid droplets in the specimens stained with oil red O (Fig. 1B). However, foamy macrophages and microglia could hardly be detected in these regions, and neurons in these regions contain nuclei of unusual morphology and are surrounded by swollen astrocytes (Fig. 21). Degenerating axons were occasionally observed in globus pallidus, substantia nigra and thalamus. Within the axons, there are degenerating cell-organelles and the myelin sheaths enclosing axons are irregular in lamellar structures and thickness (Fig. 22). In the caudoputamen, in spite of the vicinity of globus pallidus, the astrocytes rarely contained pale vacuoles, and the neurons possessed normal morphology. Macrophages did not appear in this region either, and the profiles and contents of FGP cells and vascular wall resembled those in the cerebral cortex (Fig. 23). That is, the cytoplasm of FGP cells is filled with fine lipid-like substance or contain various kinds of inclusions. At some point, the boundary between FGP cells and astrocytes became obscure. Microglia increased in the vicinity of microvessels of cerebral cortex and caudoputamen. In the thalamus, lipid laden macrophages appeared occasionally, surrounding microvessels with a relatively large diameter. Degenerating neurons and axons were also found distributed close to microvessels. The microvessels consist of swollen or thin endothelium with small projections and smooth muscle cells with irregular shape and various electron density. When many foamy macrophages extravasated into the perivascular spaces, they formed clusters within the glia limitans (Fig. 24). In this situation, FGP cells and smooth muscle cells appeared to degenerate and fragments of degenerating smooth muscle cells were phagocytosed by extravasated macrophages (Fig. 25). In this region, any proliferation of smooth muscle cells and the formation of fibrous cap did not progress in the vascular wall. Owing to degenerative changes of FGP cells and microglia, it is impossible to clearly identify the FGP cells (Figs. 24 and 25). Astrocytes in the thalamus show a moderate density and contain a certain amount of glial filaments. In the other areas, apart from the microvessels damaged by foamy macrophages, neurons and glial cells did not suffer heavy damage. They retained normal morphology. DISCUSSION Apo E is composed of 299 amino acids and its molecular weight is 34,000. According to Pitas et al. (1987) and Boyles et al. (1985), apo E was synthesized not only in hepatocytes and macrophages, but also in brain astrocytes. In addition, it is demonstrated in this paper that apo E is also recognized in the FGP cells of Wild-type mice, but not in those of homozygous apo E deficient mice. The findings were consistent with that of Boyles et al. (1985). On the other hand, as mentioned before, FGP cells are provided with scavenger receptors under physiological conditions (Mato et al., 1996). Therefore, FGP cells are regarded as very important cells in the lipid metabolism of the central nervous system. In other words, the role of apo E in astrocytes and FGP cells might be closely related to the transport of lipids into and out of the nervous tissue. As previously reported by Walker et al. (1997), xanthoma developed in the brain of apo E deficient mice, and in the present paper, it is also confirmed that excessive lipid components are distributed with taking of various appearances in the fimbria hippocampi, globus pallidus, some regions of the hippocampus, and thalamus adjacent to the fimbria hippocampi, perivascular region, ependyma, and substantia nigra—different from those of control mice at the same age. Occasionally, some parts of the choroid plexus were also stainable with oil red O. However, the deposition-sites of lipid in the brain are different depending on the region in the brain and each individual. That is, most excessive lipids in the fimbria hippocampi, adjoining hippocampus, and thalamus are incorporated in macrophages extravasated from microvessels, as demonstrated immunohistochemically. Occasionally, foamy macrophages form the xanthoma as Walker et al. (1997) described. In the cerebral cortex and caudoputamen, lipid droplets are localized in FGP cells and microglia, and the microglia and their processes were also demonstrated with the antibody of F4/80 close to microvessels. This evidence shows that, where the foamy macrophages are predominant, the FGP cells and microglia are predisposed to be atrophic and degenerated, and, in general, astrocytes are rich in glial filaments. Subsequently, it seems possible that macrophage, FGP cells and microglia play the different role for removal of lipid components in the central nervous system. Here, the authors recalled that the FGP cells and extravasated macrophages behaved in a different manner even in the same specimen that suffered from cerebral bleeding (Mato et al., 1984). Furthermore, in globus pallidus and substantia nigra, lipids are mainly accumulated and stored Fig. 18. The ependyma of third ventricle. In the ependymal cell, many vacuoles (arrow heads) and large lipidic inclusion (arrow) are seen. Subjacent to it, macrophages (M) and cholesterol cleft (CF) are demonstrated. ⫻4,400. Fig. 19. In the perivascular space, pale and moderately intense FGP cells (F1 and F2) are displayed. The former is shrunk, while the latter look healthy. In pale FGP cells, cytoplasmic organelles decrease and look to be degenerated. Subjacent to them, astrocytes (As) including large vacuoles (P) is displayed. E, endothelial cell; SM, smooth muscle cell. ⫻18,000. Fig. 20. The astrocyte in this figure contains many pale vacuoles (P) and a small amount of fibers (arrow). The nucleus of it is somewhat indented. ⫻5,200. Fig. 21. Neuron (N) possesses atypical nucleus, polysomes, endoplasmic reticula, and some number of lipofuscin granules. The astrocytes including many pale vacuoles (P) surround the neuron. ⫻4,400. Figures 18–21. Figures 22–25. REGIONAL DIFFERENCE OF LIPID DEPOSITION IN BRAIN in astrocytes, and there are no lipid laden macrophages, but the FGP cells close to the microvessels were swollen and the degeneration of axons was clearly demonstrated. The degeneration of axons and neurons reported in this paper might be directly caused by the excessive oxidized lipid components in the brain and/or by hydrolytic enzymes secreted by extravasated macrophages. The other mechanism of neural degeneration might attribute to the dysfunction of synaptic complexes in the brain of apo E deficient mice as reported by Masliah et al. (1995). The lipid components in the pale lipid vacuoles of macrophages, FGP cells and astrocytes in the apo E deficient mice seem to be composed of triglycerides and cholesterol judging from oil red O or sudan black B staining, and from the shape and opacity in the electron micrographs. The final discussion is concerned with the atherosclerotic change of cerebral microvessels in the apo E deficient mice. As already reported by several researchers, apo E deficient mice suffered from atherosclerosis in the extracerebral blood vessels such as coronary arteries and ascending aorta, but, as demonstrated in the results of the present paper, the vascular walls of cerebral microarteries in some regions of the thalamus and fimbria hippocampi carried out only so-called atheromatous changes owing to the extravasation of macrophages, and the proliferation of vascular cells did not occur in the intima of cerebral microvessels. That is, the reactivity of the vascular smooth muscle cells in cerebral microvessels for excessive lipid is different from that of extracerebral blood vessels, although the mechanism is yet to be determined. From the evidences mentioned above, it is concluded that excessive lipid components in the brain developed in apo E deficient mice are scavenged by macrophages, astrocytes and FGP cells respectively depending on the location of cerebral parenchyma, and afford severe neural and vascular damage. Here, it must be pointed out that apo E plays a significant role in the lipid metabolism of the central nervous system. ACKNOWLEDGMENTS We are grateful to Prof. Charles K. Dobbs at the International University of Health and Welfare for his kind criticism of English usage. Supported in part by The Fund of the Ministry of Welfare (Project of Longevity) of Japan, The Science Research Promotion Fund of Japan Private School Promo- Fig. 22. This figure shows the axonal degeneration (DA) in globus pallidus. Myelin sheath is partially damaged and degenerated structures are seen in it. ⫻8,200. Fig. 23. The blood vessel in this figure is obtained from caudoputamen. The endothelium (E) is thin and the vascular wall is lined with FGP cell (F). In the FGP cell, there are many inclusions (IB) of various intensity and size and some mitochondria. The cytoplasm of it is not pale, and the cytoplasmic membrane is not always smooth. The astrocyte (As) subjacent to it is swollen. ⫻7,000. Figs. 24 and 25. These figures are obtained from thalamus. In these perivascular spaces (Fig. 24), foamy macrophages (M) and cell debrices are scattered and the FGP cells are not recognized. The perivascular space is lined with glia limitans (arrows). In the perivascular space of Figure 25, foamy macrophages and cholesterol cleft (CF) are displayed. Degenerated smooth muscle cells (DSM) are phagocytosing by macrophage. BV, blood vessel; E, endothelial cell. ⫻3,500 (Fig. 24), ⫻5,200 (Fig. 25). 175 tion Foundation, and The Fund of Research Promotion Program of International University of Health and Welfare. 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