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Apolipoprotein E and Apolipoprotein D Expression in a Murine Model of Singlet Oxygen-Induced Cerebral Stroke
Biochemical and Biophysical Research Communications 268, 835– 841 (2000) doi:10.1006/bbrc.2000.2205, available online at http://www.idealibrary.com on
Apolipoprotein E and Apolipoprotein D Expression in a Murine Model of Singlet Oxygen-Induced Cerebral Stroke Vuong N. Trieu* and Fatih M. Uckun† *Department of Cardiovascular Biology and †Department of Molecular Epidemiology, Parker Hughes Institute, 2665 Long Lake Road, St. Paul, Minnesota 55113
Received January 17, 2000
Apolipoprotein E (apoE)-deficient mice exhibit neuronal abnormalities similar to those in Alzheimer’s disease and enhanced sensitivity to stroke-associated injuries. Here, we show that apoE deficiency results in impaired microglia/macrophage recruitment and accumulation after cerebral infarct. Astrogliosis and apolipoprotein D (apoD) expression are unaffected, suggesting that the neurological abnormalities of apoE-deficient mice could be due to impaired microglia/macrophage recruitment/ accumulation, which is important for the clearance of neurodegenerative products via reverse cholesterol transport. To our knowledge, the results presented herein provide the first experimental evidence that brain microglia/macrophage recruitment/accumulation is affected by apoE deficiency. The insights gained from this study should facilitate the elucidation of the role of apoE in neurological disorders such as dementia with stroke and Alzheimer’s disease. © 2000 Academic Press
Stroke is an important health problem as it is the third leading cause of death in the United States, affecting 600,000 Americans each year with a 25–34% mortality rate within the first year (1). Percutaneous transluminal coronary angioplasty (PTCA) and the use of the tissue plasminogen activator (tPA) facilitate the restoration of blood flow and reduce the ischemic brain injury following stroke. In addition, new neuroprotective agents are being developed to salvage the penumbra exposed to Ca 2⫹ influx, free radicals, and excitatory amino acids (2, 3). Recently, it has been shown that apoE4 is an adverse prognostic factor in various neuropathologic conditions, such as the Alzheimer’s disease and dementia (4, 5). ApoE4 is also associated with geriatric dementia (6), dementia with stroke (7), other dementing neurological disorders (8), and neurologic deficits in high exposure boxers (9). The co-occurence of apoE4 and cerebrovascular disease results in greater
brain atrophy and white matter hyperintensities, suggesting that apoE4 contributes to increased susceptibility to or impaired repair of brain injury (10, 11). The elucidation of the role(s) of apoE in Alzheimer’s disease and dementia with stroke would help us design effective prevention and treatment programs for these neurologic conditions. ApoE is a protein of 34 kDa which is found on lipoprotein particles from both plasma and cerebrospinal fluid (12). It facilitates the hepatic removal of cholesterol from plasma by acting as a ligand for either the LDL (Low Density Lipoprotein) receptor (LDL-R) or the LDL related receptor (LDLR-R). ApoE, whether free or HDL (High Density Lipoprotein)-bound, exhibits reverse cholesterol transport activity, an antiatherosclerotic activity by which cholesterol is moved from extrahepatic tissues (i.e., vascular wall) to the liver. ApoE exhibits three common isoforms (E2, E3, and E4) due to differences at amino acid residues 112 and 158 (12). In comparison to apoE3, the most common apoE isoform, apoE4 exhibits decreased affinity for HDL (13), impaired reverse cholesterol transport (14), and increased risk of coronary artery disease (15– 17). Consistently, apoE4 transgenic mice exhibit dyslipidemia and enhanced atherosclerotic plaque size in comparison to apoE3 transgenic mice (18). ApoE plays a central role in the mobilization and redistribution of cholesterol and phospholipid during nerve generation and degeneration. Both astrocytic glia of the central nervous system and nonmyelinating glia of the peripheral nervous system have been shown to secrete apoE (19). Injury to rat sciatic and optic nerves causes overexpression of apoE by cells in the nerve sheath, distal to the site of injury (20). And apoE-deficient mice exhibit neuronal abnormalities resembling those of Alzheimer’s disease (21–24). These abnormalities could account for the enhanced sensitivity of apoE-deficient mice to global ischemia, in terms of mortality, lesion size, and neuronal damage (25–27).
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FIG. 1. Impaired microglia/macrophage accumulation in apoE-deficient mice. Cellular accumulation around the lesion site (L) is clearly demonstrated by Trichrome staining at 100⫻ (A and B) and 200⫻ magnification (C and D). The lesions from apoE-deficient mice (B and D) lack lipid-rich foam cells present in the lesions from wild-type mice (A and C). Representative foam cells are indicated by the arrows in C. The cellular accumulation stained positively for microglia/macrophages in lesions from wild-type mice (E) and negatively in lesions from apoE-deficient mice (F), shown at 200⫻.
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FIG. 2. ApoE and apoD immunostaining. ApoE immunostaining is shown as extracellular and adjacent to the lesion (L) in wild-type mice (A). No apoE immunostaining was observed in apoE-deficient mice (B), at 200⫻ magnification. ApoD immunostaining is shown as cellular and distal to the lesion (L) in both wild-type (C and E) and apoE-deficient mice (D and F), at 100⫻ magnification (C and D) and 200⫻ magnification (E and F).
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Neurodegeneration and infarct size were reduced by forced overexpression of human apoE3 (but not by human apoE4) in apoE-deficient mice, suggesting that apoE deficiency in mice mimics the human apoE4 status (28, 29). In the present study, we compared apoE-deficient mice with wild-type mice in a previously reported noninvasive murine model of stroke which employs rose bengal to photochemically induce focal cerebral ischemia and cortical infarction (30). In this model, the reaction between the intravenously administered, photosensitive dye, rose bengal and transcranial green light produces oxygen singlets which cause focal cerebral ischemia and stroke by inducing microthrombi, followed by necrosis and death of the cranial tissue (23). The responses of apolipoprotein E-deficient mice and wild-type mice to cerebral stroke were evaluated in terms of microglia/macrophage accumulation and astrogliosis around the damaged area. ApoD and apoE expression were also examined. In our hands, apoE-deficiency caused impaired microglia/macrophage recruitment and accumulation around the site of injury. ApoE was detected in the extracellular space adjacent to the microglia/macrophages and probably participated in the recruitment of microglia/macrophages to the site of injury. ApoD expression was astrocytic and independent of the apoE status. Astrogliosis, determined by apoD and Glial Fibrillary Acidic Protein (GFAP) immunostains, was also unaffected. MATERIALS AND METHODS A murine model of stroke employing photochemically induced focal cerebral ischemia. The model was described previously (30). C57Bl/6 wild-type mice (Taconic, Germantown, NY) and apoEdeficient mice (Jackson Labs, Bar Harbor, Maine) were kept in microisolator cages and fed regular rodent chow. Mice were injected with 300 l of a 3 mg/ml solution of rose bengal in sterile phosphatebuffered saline (PBS) via their tail vein. Mice were anesthetized with a Ketamine/Xylazine solution (200 mg Ketamine/kg and 5 mg Xylazine/kg) and illumination of the skull with cold green light (300 Watt Xenon arc lamp equipped with a 550 nm broadband interference filter having a 70 nm bandwidth which effectively blocks all heat generating infrared, Oriel Scientific, Stratford, CT) was performed on the shaved scalp by placing a 1.6 mm glass fiber optic light guide directly onto the scalp for 5 min. Thirty minutes prior to elective euthanasia, 300 l of a 1 mg/ml solution of Evans Blue in PBS was administered intravenously. The animals were then anesthetized and perfused with PBS (for 5 min) followed by 4% phosphate buffered formalin. PBS and formalin were pumped through the left ventricle and allowed to exit through a 3 mm incision through the anterior wall of the right ventricle. After perfusion, the brain was removed from the cranial vault, post-fixed in 4% phosphate buffered formalin overnight, and processed for Masson’s trichrome staining or immunostaining. For lesion assessment, images were collected using a Pixera camera (Pixera Corp., CA) and the lesion size in mm 2 was quantitated using the NIH Image 1.61 program. Cross sections of the lesions were examined for cellular necrosis, microthrombi, and matrix disintegration. All numerical data are shown as mean ⫾ SEM.
Statistical differences among groups were performed by Student t test using Instat, GraphPad Software (San Diego, CA). Immunohistochemistry. Immunohistochemistry was performed as described (31, 32). Parrafin embedded sections were washed three times at 10 min each in Hemo De (Fisher Scientific, Pittsburgh, PA), rinsed in absolute ethanol, then treated with 0.5% hydrogen peroxide in methanol for 30 min. The slides were then washed in water, incubated in PBS-Tween (PBS with 0.1% Tween 20) for 10 min, followed by blocking in 10% FBS in DMEM for 1 h. For visualization of the microglia/macrophages, the sections were incubated with a 1/100 dilution of rabbit polyclonal antisera against mouse macrophage (Accurate Chemical & Scientific Corp., Westbury, NY). Immunopositive microglia/macrophages also stained positively with FITC-labeled lectin from Lycopersicon esculentum. For visualization of apolipoprotein D and apolipoprotein E, the sections were incubated with 1/100 dilutions of sheep polyclonal antisera against rat apoD and rabbit polyclonal antisera against rat apoE, respectively. Like other apolipoproteins, there is 92.0% homology between rat and mouse apolipoprotein E cDNAs and 92.6% homology between rat and mouse apolipoprotein D cDNAs (33–36). Antibodies against apoD and apoE have been characterized previously and were shown to cross react with mouse apoD and mouse apoE, respectively (37–39). For visualization of GFAP, the sections were incubated with a 1/100 dilutions of mouse ascites fluid containing G-A-5 monoclonal antibody against GFAP (Sigma, St. Louis, MO). After 1 h, the sections were washed twice in PBS-Tween for 5 min each, then incubated an additional hour with a 1:100 dilution of appropriate horseradish peroxidase conjugated secondary antibodies (Pierce, Rockford, IL). Sections were washed PBS-Tween and incubated for 10 min in the substrate solution (100 l of a stock solution of 3-amino-9ethylcarbazole in N⬘N⬘-dimethyl formamide at 2.4 mg/ml, 1 ml of acetate buffer, pH 5.2, and 5 l of 30% w/w hydrogen peroxide). Sections were counterstained with Mayer’s Hematoxylin and mounted using Crystal Mount (BioMeda Corp., Foster City, CA). The immunostained slides were scored using a scoring system of: negative (0), weak positive (1), positive (2), and strong positive (3). Astrocyte density was calculated from images captured with a 3CCD camera (DAGE-MTI Inc., Michigan City, USA) using IMAGE Pro Plus software (Media Cybernetics, Silver Spring, MD). Confocal laser scanning microscopy. Dual-color immunofluorescence was employed to study the co-expression of GFAP and apoD among astrocytes as described (40). GFAP immunoreactivity was visualized by rhodamine-labeled anti-mouse-IgG, apoD immunoreactivity was visualized by FITC-labeled anti-sheep-IgG, and the coverslips were mounted with Vectashield (Vector Labs, Burlingame, CA). Sections were viewed with a confocal laser scanning microscope (Bio-Rad MRC 1024) mounted in a Nikon Labhophot upright microscope. Digital images were saved on a Jaz disk and processed with Adobe Photoshop software (Adobe systems, Mountain View, CA).
RESULTS Impaired microglia/macrophage accumulation in apoE-deficient mice. Cerebral stroke was induced in apoE deficient mice and wild type mice by intracranial photoactivation of rose bengal dye which releases tissue damaging oxygen singlets. Twenty four hours postirradiation, the damage was determined by extravasation of Evan’s blue. Lesion size was similar between apoE-deficient mice and wild-type mice. ApoE deficient mice had a mean lesion size of 16.9 ⫾ 24 mm 2, (N ⫽ 10)
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which was not significantly different from that of wildtype mice (19.4 ⫾ 1.7 mm 2, N ⫽ 15, P ⫽ 0.4). At 7 days post-irradiation (N ⫽ 10), there was a substantial cellular accumulation around the damaged area (N ⫽ 10) (Fig. 1). Cellular accumulation was not observed at 24 h (N ⫽ 5) or 72 h post-irradiation (N ⫽ 9). Immunostaining by anti-macrophage antibody revealed a large number of macrophages where the cellular accumulation was observed (Fig. 1). The mean microglia/ macrophage immunostaining score increased from 0 ⫾ 0 (N ⫽ 5) at 24 h, to 0.8 ⫾ 0.1 (N ⫽ 9) at 72 h, and to 3.4 ⫾ 0.2 (N ⫽ 10) at 7 days post-irradiation (P ⬍ 0.0001, 7 days vs. 72 h). The cellular accumulation in apoEdeficient mice did not stain positively for macrophages (Fig. 1). ApoE-deficient mice exhibited a mean macrophage immunostaining score of 0.7 ⫾ 0.3 (N ⫽ 3), which was significantly lower than the mean macrophage immunostaining score of wild-type C57Bl/6 mice (P ⬍ 0.0001). ApoE and apoD expression following cerebral stroke. The same tissues examined above were also immunostained for apoD and apoE. ApoD expression was intracellular and distant from the lesion, whereas apoE expression was extracellular and adjacent to the lesion (Fig. 2). The specificity of our antibody against apoE was evidenced by the lack of apoE immunostaining in tissues obtained from apoE-deficient mice (Fig. 2). ApoE deficiency did not affect the expression of apoD in response to cerebral stroke (Fig. 2). The apoDimmunostained cells were identified as astrocytes by dual immunolabeling with an anti-GFAP antibody, which is a specific marker for astrocytes. The immunostaining patterns of anti-apoD and anti-GFAP antibodies showed an almost complete overlap (Fig. 3D). The progressive increase in apoD and GFAP immunostaining in response to cerebral stroke was accounted for by increased recruitment/accumulation of apoD/GFAP positive astrocytes (Fig. 3). The average number of astrocytes per 100 X field increased from 61 ⫾ 23 (N ⫽ 3) at 24 h to 152 ⫾ 22 (N ⫽ 7) at 72 h (P ⫽ 0.04, 24 h vs. 72 h), to 505 ⫾ 63 (N ⫽ 8) at 7 days post-injury (P ⫽ 0.0003, 7 days vs. 72 h). The number of astrocytes in apoE-deficient mice 7 days post-irradiation were not significantly different from that of wild type mice (413 ⫾ 106, N ⫽ 3). DISCUSSION In this report, we demonstrated that apoE deficiency is associated with impaired accumulation of brain microglia/macrophages surrounding the cerebral infarct, 7 days following the initial insult. Although the molecular mechanism for this defect remains to be deciphered, it is likely to contribute to the observed
neurological abnormalities in apoE deficient mice (21– 27). Impaired brain microglia/macrophage accumulation provides a cogent explaination for the delayed clearance of axonal degeneration products rich in cholesterol esters and phospholipid, because both apoE and microglia/macrophages are needed for the clearance of these lipids via reverse cholesterol transport (41). The accumulation of degeneration products could explain the enhanced sensitivity of apoE-deficient mice to neurological injuries (21–29). In fact, cholesterol is required for the formation of seeding amyloid betaprotein, which catalyzes the fibrillogenesis of soluble beta amyloid (41). This suggests that formation of misfolded amyloid beta protein is dependent on presence of cholesterol-rich microdomains and is consistent with the high affinity of beta amyloid for cholesterol (3.24 ⫻ 10 ⫺9 M) (42). In apoE-deficient mice, the accumulated cholesterol from neurodegeneration could present cholesterol-rich microdomains necessary for the formation of precipitating -amyloid leading to neurological abnormalities similar to that of the Alzheimer’s disease. Another apolipoprotein, apolipoprotein D (apoD), also participates in neuronal repair. ApoD expression increases by 500-fold during regeneration of the crushed rat sciatic nerve (43). ApoD is a 27–31 kDa glycoprotein, first isolated as a minor lipoprotein from High Density Lipoprotein (HDL) (44). Subsequently, apoD has been identified as the major protein component of breast cyst fluid (45) and cerebrospinal fluid (CSF) (46). CSF apoD level is elevated in Alzheimer’s disease (47). And apoD exhibits the highest level of expression in the central nervous system, primarily among the astrocytes (48, 49). Due to its similarity to the lipocalin superfamily of proteins that transport small hydrophobic molecules including cholesterol, sterols, bilirubin, porphyrin, steroid hormones and arichidonic acid, it has the potential to bind to and remove the cholesterol and phospholipids from the lesion site, via reverse cholesterol transport (50). Therefore, we also examined apoD expression in this study. Immunodetection of astrocytes by antibodies raised against GFAP and apoD demonstrated that astrogliosis and apoD expression are not affected by apoE deficiency. Though the timecourse for astrogliosis and microglia/ macrophage accumulation were similar among wild type mice, the unimpaired astrogliosis versus the impaired microglia/macrophage accumulation among apoE-deficient mice suggests that these two cell types are responding to different stimuli. This notion is further supported by the topographic differences in accumulation for astrocytes and microglia/macrophage. The location of apoD in relation to the site of injury also suggests that apoD is not directly participating in the clearance of neurodegenerative products from the site of injury.
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FIG. 3. Normal astrogliosis in apoE-deficient mice. The progressive astrogliosis in wild-type mice is shown as accumulation of GFAPpositive astrocytes at 24 h (A), 72 h (B), and 7 days (C) after injury. These astrocytes express both apoD and GFAP, as shown by the complete overlap of the red GFAP immunofluorescence staining and the green apoD immunofluorescence staining at 200⫻, denoted as yellow (D). The inset is shown at 1000⫻ magnification.
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Apolipoprotein E and Apolipoprotein D Expression in a Murine Model of Singlet Oxygen-Induced Cerebral Stroke Vuong N. Trieu* and Fatih M. Uckun† *Department of Cardiovascular Biology and †Department of Molecular Epidemiology, Parker Hughes Institute, 2665 Long Lake Road, St. Paul, Minnesota 55113
Received January 17, 2000
Apolipoprotein E (apoE)-deficient mice exhibit neuronal abnormalities similar to those in Alzheimer’s disease and enhanced sensitivity to stroke-associated injuries. Here, we show that apoE deficiency results in impaired microglia/macrophage recruitment and accumulation after cerebral infarct. Astrogliosis and apolipoprotein D (apoD) expression are unaffected, suggesting that the neurological abnormalities of apoE-deficient mice could be due to impaired microglia/macrophage recruitment/ accumulation, which is important for the clearance of neurodegenerative products via reverse cholesterol transport. To our knowledge, the results presented herein provide the first experimental evidence that brain microglia/macrophage recruitment/accumulation is affected by apoE deficiency. The insights gained from this study should facilitate the elucidation of the role of apoE in neurological disorders such as dementia with stroke and Alzheimer’s disease. © 2000 Academic Press
Stroke is an important health problem as it is the third leading cause of death in the United States, affecting 600,000 Americans each year with a 25–34% mortality rate within the first year (1). Percutaneous transluminal coronary angioplasty (PTCA) and the use of the tissue plasminogen activator (tPA) facilitate the restoration of blood flow and reduce the ischemic brain injury following stroke. In addition, new neuroprotective agents are being developed to salvage the penumbra exposed to Ca 2⫹ influx, free radicals, and excitatory amino acids (2, 3). Recently, it has been shown that apoE4 is an adverse prognostic factor in various neuropathologic conditions, such as the Alzheimer’s disease and dementia (4, 5). ApoE4 is also associated with geriatric dementia (6), dementia with stroke (7), other dementing neurological disorders (8), and neurologic deficits in high exposure boxers (9). The co-occurence of apoE4 and cerebrovascular disease results in greater
brain atrophy and white matter hyperintensities, suggesting that apoE4 contributes to increased susceptibility to or impaired repair of brain injury (10, 11). The elucidation of the role(s) of apoE in Alzheimer’s disease and dementia with stroke would help us design effective prevention and treatment programs for these neurologic conditions. ApoE is a protein of 34 kDa which is found on lipoprotein particles from both plasma and cerebrospinal fluid (12). It facilitates the hepatic removal of cholesterol from plasma by acting as a ligand for either the LDL (Low Density Lipoprotein) receptor (LDL-R) or the LDL related receptor (LDLR-R). ApoE, whether free or HDL (High Density Lipoprotein)-bound, exhibits reverse cholesterol transport activity, an antiatherosclerotic activity by which cholesterol is moved from extrahepatic tissues (i.e., vascular wall) to the liver. ApoE exhibits three common isoforms (E2, E3, and E4) due to differences at amino acid residues 112 and 158 (12). In comparison to apoE3, the most common apoE isoform, apoE4 exhibits decreased affinity for HDL (13), impaired reverse cholesterol transport (14), and increased risk of coronary artery disease (15– 17). Consistently, apoE4 transgenic mice exhibit dyslipidemia and enhanced atherosclerotic plaque size in comparison to apoE3 transgenic mice (18). ApoE plays a central role in the mobilization and redistribution of cholesterol and phospholipid during nerve generation and degeneration. Both astrocytic glia of the central nervous system and nonmyelinating glia of the peripheral nervous system have been shown to secrete apoE (19). Injury to rat sciatic and optic nerves causes overexpression of apoE by cells in the nerve sheath, distal to the site of injury (20). And apoE-deficient mice exhibit neuronal abnormalities resembling those of Alzheimer’s disease (21–24). These abnormalities could account for the enhanced sensitivity of apoE-deficient mice to global ischemia, in terms of mortality, lesion size, and neuronal damage (25–27).
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FIG. 1. Impaired microglia/macrophage accumulation in apoE-deficient mice. Cellular accumulation around the lesion site (L) is clearly demonstrated by Trichrome staining at 100⫻ (A and B) and 200⫻ magnification (C and D). The lesions from apoE-deficient mice (B and D) lack lipid-rich foam cells present in the lesions from wild-type mice (A and C). Representative foam cells are indicated by the arrows in C. The cellular accumulation stained positively for microglia/macrophages in lesions from wild-type mice (E) and negatively in lesions from apoE-deficient mice (F), shown at 200⫻.
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FIG. 2. ApoE and apoD immunostaining. ApoE immunostaining is shown as extracellular and adjacent to the lesion (L) in wild-type mice (A). No apoE immunostaining was observed in apoE-deficient mice (B), at 200⫻ magnification. ApoD immunostaining is shown as cellular and distal to the lesion (L) in both wild-type (C and E) and apoE-deficient mice (D and F), at 100⫻ magnification (C and D) and 200⫻ magnification (E and F).
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Neurodegeneration and infarct size were reduced by forced overexpression of human apoE3 (but not by human apoE4) in apoE-deficient mice, suggesting that apoE deficiency in mice mimics the human apoE4 status (28, 29). In the present study, we compared apoE-deficient mice with wild-type mice in a previously reported noninvasive murine model of stroke which employs rose bengal to photochemically induce focal cerebral ischemia and cortical infarction (30). In this model, the reaction between the intravenously administered, photosensitive dye, rose bengal and transcranial green light produces oxygen singlets which cause focal cerebral ischemia and stroke by inducing microthrombi, followed by necrosis and death of the cranial tissue (23). The responses of apolipoprotein E-deficient mice and wild-type mice to cerebral stroke were evaluated in terms of microglia/macrophage accumulation and astrogliosis around the damaged area. ApoD and apoE expression were also examined. In our hands, apoE-deficiency caused impaired microglia/macrophage recruitment and accumulation around the site of injury. ApoE was detected in the extracellular space adjacent to the microglia/macrophages and probably participated in the recruitment of microglia/macrophages to the site of injury. ApoD expression was astrocytic and independent of the apoE status. Astrogliosis, determined by apoD and Glial Fibrillary Acidic Protein (GFAP) immunostains, was also unaffected. MATERIALS AND METHODS A murine model of stroke employing photochemically induced focal cerebral ischemia. The model was described previously (30). C57Bl/6 wild-type mice (Taconic, Germantown, NY) and apoEdeficient mice (Jackson Labs, Bar Harbor, Maine) were kept in microisolator cages and fed regular rodent chow. Mice were injected with 300 l of a 3 mg/ml solution of rose bengal in sterile phosphatebuffered saline (PBS) via their tail vein. Mice were anesthetized with a Ketamine/Xylazine solution (200 mg Ketamine/kg and 5 mg Xylazine/kg) and illumination of the skull with cold green light (300 Watt Xenon arc lamp equipped with a 550 nm broadband interference filter having a 70 nm bandwidth which effectively blocks all heat generating infrared, Oriel Scientific, Stratford, CT) was performed on the shaved scalp by placing a 1.6 mm glass fiber optic light guide directly onto the scalp for 5 min. Thirty minutes prior to elective euthanasia, 300 l of a 1 mg/ml solution of Evans Blue in PBS was administered intravenously. The animals were then anesthetized and perfused with PBS (for 5 min) followed by 4% phosphate buffered formalin. PBS and formalin were pumped through the left ventricle and allowed to exit through a 3 mm incision through the anterior wall of the right ventricle. After perfusion, the brain was removed from the cranial vault, post-fixed in 4% phosphate buffered formalin overnight, and processed for Masson’s trichrome staining or immunostaining. For lesion assessment, images were collected using a Pixera camera (Pixera Corp., CA) and the lesion size in mm 2 was quantitated using the NIH Image 1.61 program. Cross sections of the lesions were examined for cellular necrosis, microthrombi, and matrix disintegration. All numerical data are shown as mean ⫾ SEM.
Statistical differences among groups were performed by Student t test using Instat, GraphPad Software (San Diego, CA). Immunohistochemistry. Immunohistochemistry was performed as described (31, 32). Parrafin embedded sections were washed three times at 10 min each in Hemo De (Fisher Scientific, Pittsburgh, PA), rinsed in absolute ethanol, then treated with 0.5% hydrogen peroxide in methanol for 30 min. The slides were then washed in water, incubated in PBS-Tween (PBS with 0.1% Tween 20) for 10 min, followed by blocking in 10% FBS in DMEM for 1 h. For visualization of the microglia/macrophages, the sections were incubated with a 1/100 dilution of rabbit polyclonal antisera against mouse macrophage (Accurate Chemical & Scientific Corp., Westbury, NY). Immunopositive microglia/macrophages also stained positively with FITC-labeled lectin from Lycopersicon esculentum. For visualization of apolipoprotein D and apolipoprotein E, the sections were incubated with 1/100 dilutions of sheep polyclonal antisera against rat apoD and rabbit polyclonal antisera against rat apoE, respectively. Like other apolipoproteins, there is 92.0% homology between rat and mouse apolipoprotein E cDNAs and 92.6% homology between rat and mouse apolipoprotein D cDNAs (33–36). Antibodies against apoD and apoE have been characterized previously and were shown to cross react with mouse apoD and mouse apoE, respectively (37–39). For visualization of GFAP, the sections were incubated with a 1/100 dilutions of mouse ascites fluid containing G-A-5 monoclonal antibody against GFAP (Sigma, St. Louis, MO). After 1 h, the sections were washed twice in PBS-Tween for 5 min each, then incubated an additional hour with a 1:100 dilution of appropriate horseradish peroxidase conjugated secondary antibodies (Pierce, Rockford, IL). Sections were washed PBS-Tween and incubated for 10 min in the substrate solution (100 l of a stock solution of 3-amino-9ethylcarbazole in N⬘N⬘-dimethyl formamide at 2.4 mg/ml, 1 ml of acetate buffer, pH 5.2, and 5 l of 30% w/w hydrogen peroxide). Sections were counterstained with Mayer’s Hematoxylin and mounted using Crystal Mount (BioMeda Corp., Foster City, CA). The immunostained slides were scored using a scoring system of: negative (0), weak positive (1), positive (2), and strong positive (3). Astrocyte density was calculated from images captured with a 3CCD camera (DAGE-MTI Inc., Michigan City, USA) using IMAGE Pro Plus software (Media Cybernetics, Silver Spring, MD). Confocal laser scanning microscopy. Dual-color immunofluorescence was employed to study the co-expression of GFAP and apoD among astrocytes as described (40). GFAP immunoreactivity was visualized by rhodamine-labeled anti-mouse-IgG, apoD immunoreactivity was visualized by FITC-labeled anti-sheep-IgG, and the coverslips were mounted with Vectashield (Vector Labs, Burlingame, CA). Sections were viewed with a confocal laser scanning microscope (Bio-Rad MRC 1024) mounted in a Nikon Labhophot upright microscope. Digital images were saved on a Jaz disk and processed with Adobe Photoshop software (Adobe systems, Mountain View, CA).
RESULTS Impaired microglia/macrophage accumulation in apoE-deficient mice. Cerebral stroke was induced in apoE deficient mice and wild type mice by intracranial photoactivation of rose bengal dye which releases tissue damaging oxygen singlets. Twenty four hours postirradiation, the damage was determined by extravasation of Evan’s blue. Lesion size was similar between apoE-deficient mice and wild-type mice. ApoE deficient mice had a mean lesion size of 16.9 ⫾ 24 mm 2, (N ⫽ 10)
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which was not significantly different from that of wildtype mice (19.4 ⫾ 1.7 mm 2, N ⫽ 15, P ⫽ 0.4). At 7 days post-irradiation (N ⫽ 10), there was a substantial cellular accumulation around the damaged area (N ⫽ 10) (Fig. 1). Cellular accumulation was not observed at 24 h (N ⫽ 5) or 72 h post-irradiation (N ⫽ 9). Immunostaining by anti-macrophage antibody revealed a large number of macrophages where the cellular accumulation was observed (Fig. 1). The mean microglia/ macrophage immunostaining score increased from 0 ⫾ 0 (N ⫽ 5) at 24 h, to 0.8 ⫾ 0.1 (N ⫽ 9) at 72 h, and to 3.4 ⫾ 0.2 (N ⫽ 10) at 7 days post-irradiation (P ⬍ 0.0001, 7 days vs. 72 h). The cellular accumulation in apoEdeficient mice did not stain positively for macrophages (Fig. 1). ApoE-deficient mice exhibited a mean macrophage immunostaining score of 0.7 ⫾ 0.3 (N ⫽ 3), which was significantly lower than the mean macrophage immunostaining score of wild-type C57Bl/6 mice (P ⬍ 0.0001). ApoE and apoD expression following cerebral stroke. The same tissues examined above were also immunostained for apoD and apoE. ApoD expression was intracellular and distant from the lesion, whereas apoE expression was extracellular and adjacent to the lesion (Fig. 2). The specificity of our antibody against apoE was evidenced by the lack of apoE immunostaining in tissues obtained from apoE-deficient mice (Fig. 2). ApoE deficiency did not affect the expression of apoD in response to cerebral stroke (Fig. 2). The apoDimmunostained cells were identified as astrocytes by dual immunolabeling with an anti-GFAP antibody, which is a specific marker for astrocytes. The immunostaining patterns of anti-apoD and anti-GFAP antibodies showed an almost complete overlap (Fig. 3D). The progressive increase in apoD and GFAP immunostaining in response to cerebral stroke was accounted for by increased recruitment/accumulation of apoD/GFAP positive astrocytes (Fig. 3). The average number of astrocytes per 100 X field increased from 61 ⫾ 23 (N ⫽ 3) at 24 h to 152 ⫾ 22 (N ⫽ 7) at 72 h (P ⫽ 0.04, 24 h vs. 72 h), to 505 ⫾ 63 (N ⫽ 8) at 7 days post-injury (P ⫽ 0.0003, 7 days vs. 72 h). The number of astrocytes in apoE-deficient mice 7 days post-irradiation were not significantly different from that of wild type mice (413 ⫾ 106, N ⫽ 3). DISCUSSION In this report, we demonstrated that apoE deficiency is associated with impaired accumulation of brain microglia/macrophages surrounding the cerebral infarct, 7 days following the initial insult. Although the molecular mechanism for this defect remains to be deciphered, it is likely to contribute to the observed
neurological abnormalities in apoE deficient mice (21– 27). Impaired brain microglia/macrophage accumulation provides a cogent explaination for the delayed clearance of axonal degeneration products rich in cholesterol esters and phospholipid, because both apoE and microglia/macrophages are needed for the clearance of these lipids via reverse cholesterol transport (41). The accumulation of degeneration products could explain the enhanced sensitivity of apoE-deficient mice to neurological injuries (21–29). In fact, cholesterol is required for the formation of seeding amyloid betaprotein, which catalyzes the fibrillogenesis of soluble beta amyloid (41). This suggests that formation of misfolded amyloid beta protein is dependent on presence of cholesterol-rich microdomains and is consistent with the high affinity of beta amyloid for cholesterol (3.24 ⫻ 10 ⫺9 M) (42). In apoE-deficient mice, the accumulated cholesterol from neurodegeneration could present cholesterol-rich microdomains necessary for the formation of precipitating -amyloid leading to neurological abnormalities similar to that of the Alzheimer’s disease. Another apolipoprotein, apolipoprotein D (apoD), also participates in neuronal repair. ApoD expression increases by 500-fold during regeneration of the crushed rat sciatic nerve (43). ApoD is a 27–31 kDa glycoprotein, first isolated as a minor lipoprotein from High Density Lipoprotein (HDL) (44). Subsequently, apoD has been identified as the major protein component of breast cyst fluid (45) and cerebrospinal fluid (CSF) (46). CSF apoD level is elevated in Alzheimer’s disease (47). And apoD exhibits the highest level of expression in the central nervous system, primarily among the astrocytes (48, 49). Due to its similarity to the lipocalin superfamily of proteins that transport small hydrophobic molecules including cholesterol, sterols, bilirubin, porphyrin, steroid hormones and arichidonic acid, it has the potential to bind to and remove the cholesterol and phospholipids from the lesion site, via reverse cholesterol transport (50). Therefore, we also examined apoD expression in this study. Immunodetection of astrocytes by antibodies raised against GFAP and apoD demonstrated that astrogliosis and apoD expression are not affected by apoE deficiency. Though the timecourse for astrogliosis and microglia/ macrophage accumulation were similar among wild type mice, the unimpaired astrogliosis versus the impaired microglia/macrophage accumulation among apoE-deficient mice suggests that these two cell types are responding to different stimuli. This notion is further supported by the topographic differences in accumulation for astrocytes and microglia/macrophage. The location of apoD in relation to the site of injury also suggests that apoD is not directly participating in the clearance of neurodegenerative products from the site of injury.
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FIG. 3. Normal astrogliosis in apoE-deficient mice. The progressive astrogliosis in wild-type mice is shown as accumulation of GFAPpositive astrocytes at 24 h (A), 72 h (B), and 7 days (C) after injury. These astrocytes express both apoD and GFAP, as shown by the complete overlap of the red GFAP immunofluorescence staining and the green apoD immunofluorescence staining at 200⫻, denoted as yellow (D). The inset is shown at 1000⫻ magnification.
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