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Increased peripheral benzodiazepine receptors in arterial plaque of patients with atherosclerosis: An autoradiographic study with [3H]PK 11195
Atherosclerosis 201 (2008) 108–111
Short communication
Increased peripheral benzodiazepine receptors in arterial plaque of patients with atherosclerosis: An autoradiographic study with [3H]PK 11195 Yota Fujimura a,∗ , Paul M. Hwang b , Hugh Trout III c , Louis Kozloff c , Masao Imaizumi a , Robert B. Innis a , Masahiro Fujita a a b
Molecular Imaging Branch, National Institute of Mental Health, Bethesda, MD, USA Cardiology Branch, National Heart, Lung and Blood Institution, Bethesda, MD, USA c Suburban Hospital, Bethesda, MD, USA
Received 21 November 2007; received in revised form 26 December 2007; accepted 25 February 2008 Available online 14 March 2008
Abstract Inflammation in atherosclerotic plaques makes them unstable and can cause thrombosis. Therefore, it is important to detect macrophage activity for clinical management of atherosclerosis. Peripheral benzodiazepine receptor (PBR) is expressed in various tissue and organs including macrophages. In this study, we tested whether inflammation characterized by macrophage infiltration can be detected by PBR binding. Six patients diagnosed as carotid atherosclerosis underwent endarterectomy. Using the fresh frozen sections, presence of PBRs and macrophages was examined by in vitro autoradiography using [3 H]PK 11195 and immunohistochemical staining of CD68, respectively. All sections showed specific binding of [3 H]PK 11195, and the staining with CD68 indicating macrophage infiltration. Density and distribution of PBR detected by [3 H]PK 11195 autoradiography were consistent with those of the immunohistochemical staining. In conclusion, this study demonstrated that macrophage and inflammatory activity in atherosclerotic plaque can be imaged specifically by the binding of PBR indicating future application of PET imaging for PBR. Published by Elsevier Ireland Ltd. Keywords: Atherosclerosis; Macrophage; Peripheral benzodiazepine receptor; [3 H]PK 11195; Imaging; Nuclear medicine; CD68
1. Introduction The rupture of atherosclerotic plaque and subsequent formation of thrombi are the main factors responsible for myocardial and cerebral infarctions [1,2]. Plaque at risk of rupture (i.e., “vulnerable” plaque) is characterized by inflammation and infiltration of macrophages [3]. X-ray contrast angiography is currently the standard technique to image atherosclerosis, but it assesses only morphology without detecting inflammation [4]. The purpose of this study was to ∗ Corresponding author at: Molecular Imaging Branch, National Institute of Mental Health, Building 31, Room B2-B37, 31 Center Drive, Bethesda, MD 20892, USA. Tel.: +1 301 594 5661; fax: +1 301 480 3610. E-mail address: [email protected] (Y. Fujimura).
0021-9150/$ – see front matter. Published by Elsevier Ireland Ltd. doi:10.1016/j.atherosclerosis.2008.02.032
examine the feasibility of measuring inflammation in vulnerable plaque by imaging a biomarker of macrophage activity. The biomarker was the peripheral benzodiazepine receptor (PBR), a mitochondrial protein that is highly expressed in phagocytic inflammatory cells, both activated macrophages in the periphery and microglia in the brain [5–8]. Recently, [18 F]-fluorodeoxyglucose (18 F-FDG) positron emission tomography (PET) has been used to image vulnerable plaques by detecting inflammatory activity [9,10]. This method may provide only an indirect measure of inflammation because glucose uptake may not reflect inflammation well. A key component of inflammation, macrophage expresses PBR [6–8]. Recently, we have developed PET radioligands for PBR. These radioligands provide high specific signal
Y. Fujimura et al. / Atherosclerosis 201 (2008) 108–111
[11,12] and may be able to image vulnerable plaque in vivo. As an initial test of feasibility, we performed in vitro receptor autoradiography for PBR on surgical samples from patients who had undergone carotid endarterectomy.
2. Materials and methods 2.1. Subjects Surgical samples were obtained from six patients (79 ± 7.8 years old, mean ± S.D.; range 65–88 years, 3 males and 3 females) who underwent carotid endarterectomy for stenosis greater than 70%. We have no in vivo evidence that the plaque was unstable. These samples were obtained from anonymous patients, for whom we knew only age and sex. Fresh frozen sections of 20 and 5 m thickness were processed for receptor autoradiography and immunohistochemistry, respectively.
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cific binding was 88% ± 8% of total binding. In addition, the specificity of the immunostaining was confirmed by the lack of staining in adjacent sections incubated without the primary antibody (Fig. 1D). Binding of [3 H]PK 11195 and immunostaining for CD68 was distributed heterogeneously within each sample. The intensity and the distribution of [3 H]PK 11195 binding correlated with CD68 immunoreactivity, indicating that PBR binding reflected the presence of macrophages. To calculate the density (Bmax ) of PBR in plaque, we used the Scatchard binding equation, the mean specific binding from the six subjects, and the published value of the radioligand’s dissociation constant (KD = 18 nM) for human macrophages [15]. The mean specific binding of [3 H]PK 11195, determined from one total and one nonspecific section per subject was 0.084 ± 0.031 pmol/mg tissue. Based on these values, the density of PBRs in the carotid artery samples was estimated to be ∼1.6 pmol/mg tissue.
2.2. Autoradiography
4. Discussion
Sections were stored at −70 ◦ C and thawed on the day of assay to room temperature. PBRs were labeled with [3 H]PK 11195 (specific activity 84.8 Ci/mmol; 1 mCi/mL, PerkinElmer, Inc.) using a method adapted from the literature [13]. In brief, sections were consecutively incubated at room temperature in buffer (50 mM Tris–HCl, pH 7.4) for 15 min and then in buffer containing 1 nM [3 H]PK 11195 for 30 min. Nonspecific binding was determined on serial sections in the presence of excess nonradioactive PK 11195 (20 M). Sections were then washed for 2 min × 6 min in ice-cold buffer, dipped in ice-cold water, and dried. Tissue sections and 3 Hstandards (Amersham) were exposed to a tritium-sensitive imaging plate for 7 days. The imaging plates were scanned with the BAS 5000 device and analyzed with Multi Gauge V3.0 (Fujifilm, Tokyo, Japan). A standard curve based on 3 H-standards was used to measure regional radioactivity.
Because inflammation plays an important role in the pathophysiology of atherosclerosis [1–3], biomarkers of inflammation may be more useful than morphological measurements of arterial obstruction to identify plaque that is vulnerable to rupture. As a preliminary test of the utility of in vivo imaging, we studied whether macrophages in atherosclerotic plaque can be imaged in vitro by radioligand binding to PBR. In fact, all six sequentially obtained human carotid artery samples showed high densities of PBRs labeled with [3 H]PK 11195, and the distribution of binding paralleled that of macrophages stained with an antibody to CD68. The density (Bmax ) of PBRs was estimated to be ∼1.6 pmol/mg tissue, which is several fold greater than the density in normal human brain tissue but similar to that in glioma [16], which has been successfully imaged with an older PET radioligand for PBRs. Considering their high density, PBRs on macrophages in atherosclerotic plaques may be able to be imaged with PET radioligands that we have recently developed [11,12]. Nevertheless, the feasibility of imaging PBRs in plaque is difficult to predict, because it will depend on other variables such as the size of the plaque, the density of PBR, and the clearance of background radioactivity from blood. Another promising PET technique to image inflammation in atherosclerotic plaque is the increased uptake of a radiolabeled analog of glucose, [18 F]-fluorodeoxyglucose [9,10]. In fact, macrophages in inflammation contribute to the increased glucose uptake in inflammation. Thus, if future PET studies show that PBRs can be imaged in vivo in human atherosclerotic plaque, this technique should be compared to [18 F]-fluorodeoxyglucose to determine relative sensitivity and whether either technique provides clinically useful information for carotid plaque risk stratification and patient management.
2.3. Immunohistochemical staining Using sections adjacent to those for autoradiography, macrophages were stained with an antibody to CD68, a protein marker specific to macrophages. CVPath, Inc. (Gaithersburg, MD, USA) performed the immunohistochemical staining [14].
3. Results All six samples showed high densities of PBRs labeled with [3 H]PK 11195, and the distribution of receptor binding paralleled that of macrophages stained with an antibody to CD68 (Fig. 1). The specific binding of [3 H]PK 11195 was calculated by subtracting residual (nonspecific) binding in the presence of excess PK 11195 (20 M) (Fig. 1B). Spe-
110
Y. Fujimura et al. / Atherosclerosis 201 (2008) 108–111
Fig. 1. Peripheral benzodiazepine receptors (PBRs) labeled with autoradiography and macrophages labeled with immunohistochemistry in carotid endarterectomy tissue from a single patient. (A) Total binding of [3 H]PK 11195 by autoradiography. (B) Residual (or nonspecific) binding of [3 H]PK 11195 was determined by co-incubation with excess nonradioactive PK 11195 (20 M). Specific binding is defined as total minus nonspecific and represented the majority (∼90%) of total binding shown in panel A. (C) Immunohistochemical staining of macrophages labeled with an antibody to CD68. (D) Control section was processed without the primary antibody to CD68 and showed negligible staining.
Acknowledgements This research was supported by the Intramural Program of NIMH (project # Z01-MH-002852-03). We thank David Mozley, MD (Merck Research Laboratory) for suggesting the potential utility of PBR imaging as a marker of inflammation in vulnerable plaque. References [1] Schroeder AP, Falk E. Vulnerable and dangerous coronary plaques. Atherosclerosis 1995;118(Suppl.):S141–9. [2] Lendon C, Born GV, Davies MJ, Richardson PD. Plaque fissure: the link between atherosclerosis and thrombosis. Nouvelle revue francaise d’hematologie 1992;34:27–9. [3] Libby P, Geng YJ, Aikawa M, et al. Macrophages and atherosclerotic plaque stability. Curr Opin Lipidol 1996;7:330–5. [4] Crisby M, Nordin-Fredriksson G, Shah PK, et al. Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaques: implications for plaque stabilization. Circulation 2001;103:926–33. [5] Papadopoulos V, Lecanu L, Brown RC, Han Z, Yao ZX. Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis, neuropathology and neurological disorders. Neuroscience 2006;138:749–56.
[6] Benavides J, Dubois A, Dennis T, Hamel E, Scatton B. Omega 3 (peripheral type benzodiazepine binding) site distribution in the rat immune system: an autoradiographic study with the photoaffinity ligand [3 H]PK 14105. J Pharmacol Exp Ther 1989;249:333–9. [7] Schlumpf M, Parmar R, Lichtensteiger W. Prenatal diazepam induced persisting downregulation of peripheral (omega 3) benzodiazepine receptors on rat splenic macrophages. Life Sci 1993;52:927– 34. [8] Zavala F, Haumont J, Lenfant M. Interaction of benzodiazepines with mouse macrophages. Eur J Pharmacol 1984;106:561–6. [9] Rudd JH, Warburton EA, Fryer TD, et al. Imaging atherosclerotic plaque inflammation with [18 F]-fluorodeoxyglucose positron emission tomography. Circulation 2002;105:2708–11. [10] Ogawa M, Ishino S, Mukai T, et al. (18)F-FDG accumulation in atherosclerotic plaques: immunohistochemical and PET imaging study. J Nucl Med 2004;45:1245–50. [11] Fujimura Y, Ikoma Y, Yasuno F, et al. Quantitative analyses of 18 FFEDAA1106 binding to peripheral benzodiazepine receptors in living human brain. J Nucl Med 2006;47:43–50. [12] Imaizumi M, Briard E, Zoghbi SS, et al. Kinetic evaluation in nonhuman primates of two new PET ligands for peripheral benzodiazepine receptors in brain. Synapse 2007;61:595–605. [13] Guilarte TR, Kuhlmann AC, O’Callaghan JP, Miceli RC. Enhanced expression of peripheral benzodiazepine receptors in trimethyltinexposed rat brain: a biomarker of neurotoxicity. Neurotoxicology 1995;16:441–50.
Y. Fujimura et al. / Atherosclerosis 201 (2008) 108–111 [14] Kunisch E, Fuhrmann R, Roth A, et al. Macrophage specificity of three anti-CD68 monoclonal antibodies (KP1, EBM11, and PGM1) widely used for immunohistochemistry and flow cytometry. Ann Rheum Dis 2004;63:774–84. [15] Venneti S, Wang G, Wiley CA. The high affinity peripheral benzodiazepine receptor ligand DAA1106 binds to activated and infected brain
111
macrophages in areas of synaptic degeneration: Implications for PET imaging of neuroinflammation in lentiviral encephalitis. Neurobiol Dis 2008;29:232–41. [16] Takada A, Mitsuka S, Diksic M, Yamamoto YL. Autoradiographic study of peripheral benzodiazepine receptors in animal brain tumor models and human gliomas. Eur J Pharmacol 1992;228:131–9.
Short communication
Increased peripheral benzodiazepine receptors in arterial plaque of patients with atherosclerosis: An autoradiographic study with [3H]PK 11195 Yota Fujimura a,∗ , Paul M. Hwang b , Hugh Trout III c , Louis Kozloff c , Masao Imaizumi a , Robert B. Innis a , Masahiro Fujita a a b
Molecular Imaging Branch, National Institute of Mental Health, Bethesda, MD, USA Cardiology Branch, National Heart, Lung and Blood Institution, Bethesda, MD, USA c Suburban Hospital, Bethesda, MD, USA
Received 21 November 2007; received in revised form 26 December 2007; accepted 25 February 2008 Available online 14 March 2008
Abstract Inflammation in atherosclerotic plaques makes them unstable and can cause thrombosis. Therefore, it is important to detect macrophage activity for clinical management of atherosclerosis. Peripheral benzodiazepine receptor (PBR) is expressed in various tissue and organs including macrophages. In this study, we tested whether inflammation characterized by macrophage infiltration can be detected by PBR binding. Six patients diagnosed as carotid atherosclerosis underwent endarterectomy. Using the fresh frozen sections, presence of PBRs and macrophages was examined by in vitro autoradiography using [3 H]PK 11195 and immunohistochemical staining of CD68, respectively. All sections showed specific binding of [3 H]PK 11195, and the staining with CD68 indicating macrophage infiltration. Density and distribution of PBR detected by [3 H]PK 11195 autoradiography were consistent with those of the immunohistochemical staining. In conclusion, this study demonstrated that macrophage and inflammatory activity in atherosclerotic plaque can be imaged specifically by the binding of PBR indicating future application of PET imaging for PBR. Published by Elsevier Ireland Ltd. Keywords: Atherosclerosis; Macrophage; Peripheral benzodiazepine receptor; [3 H]PK 11195; Imaging; Nuclear medicine; CD68
1. Introduction The rupture of atherosclerotic plaque and subsequent formation of thrombi are the main factors responsible for myocardial and cerebral infarctions [1,2]. Plaque at risk of rupture (i.e., “vulnerable” plaque) is characterized by inflammation and infiltration of macrophages [3]. X-ray contrast angiography is currently the standard technique to image atherosclerosis, but it assesses only morphology without detecting inflammation [4]. The purpose of this study was to ∗ Corresponding author at: Molecular Imaging Branch, National Institute of Mental Health, Building 31, Room B2-B37, 31 Center Drive, Bethesda, MD 20892, USA. Tel.: +1 301 594 5661; fax: +1 301 480 3610. E-mail address: [email protected] (Y. Fujimura).
0021-9150/$ – see front matter. Published by Elsevier Ireland Ltd. doi:10.1016/j.atherosclerosis.2008.02.032
examine the feasibility of measuring inflammation in vulnerable plaque by imaging a biomarker of macrophage activity. The biomarker was the peripheral benzodiazepine receptor (PBR), a mitochondrial protein that is highly expressed in phagocytic inflammatory cells, both activated macrophages in the periphery and microglia in the brain [5–8]. Recently, [18 F]-fluorodeoxyglucose (18 F-FDG) positron emission tomography (PET) has been used to image vulnerable plaques by detecting inflammatory activity [9,10]. This method may provide only an indirect measure of inflammation because glucose uptake may not reflect inflammation well. A key component of inflammation, macrophage expresses PBR [6–8]. Recently, we have developed PET radioligands for PBR. These radioligands provide high specific signal
Y. Fujimura et al. / Atherosclerosis 201 (2008) 108–111
[11,12] and may be able to image vulnerable plaque in vivo. As an initial test of feasibility, we performed in vitro receptor autoradiography for PBR on surgical samples from patients who had undergone carotid endarterectomy.
2. Materials and methods 2.1. Subjects Surgical samples were obtained from six patients (79 ± 7.8 years old, mean ± S.D.; range 65–88 years, 3 males and 3 females) who underwent carotid endarterectomy for stenosis greater than 70%. We have no in vivo evidence that the plaque was unstable. These samples were obtained from anonymous patients, for whom we knew only age and sex. Fresh frozen sections of 20 and 5 m thickness were processed for receptor autoradiography and immunohistochemistry, respectively.
109
cific binding was 88% ± 8% of total binding. In addition, the specificity of the immunostaining was confirmed by the lack of staining in adjacent sections incubated without the primary antibody (Fig. 1D). Binding of [3 H]PK 11195 and immunostaining for CD68 was distributed heterogeneously within each sample. The intensity and the distribution of [3 H]PK 11195 binding correlated with CD68 immunoreactivity, indicating that PBR binding reflected the presence of macrophages. To calculate the density (Bmax ) of PBR in plaque, we used the Scatchard binding equation, the mean specific binding from the six subjects, and the published value of the radioligand’s dissociation constant (KD = 18 nM) for human macrophages [15]. The mean specific binding of [3 H]PK 11195, determined from one total and one nonspecific section per subject was 0.084 ± 0.031 pmol/mg tissue. Based on these values, the density of PBRs in the carotid artery samples was estimated to be ∼1.6 pmol/mg tissue.
2.2. Autoradiography
4. Discussion
Sections were stored at −70 ◦ C and thawed on the day of assay to room temperature. PBRs were labeled with [3 H]PK 11195 (specific activity 84.8 Ci/mmol; 1 mCi/mL, PerkinElmer, Inc.) using a method adapted from the literature [13]. In brief, sections were consecutively incubated at room temperature in buffer (50 mM Tris–HCl, pH 7.4) for 15 min and then in buffer containing 1 nM [3 H]PK 11195 for 30 min. Nonspecific binding was determined on serial sections in the presence of excess nonradioactive PK 11195 (20 M). Sections were then washed for 2 min × 6 min in ice-cold buffer, dipped in ice-cold water, and dried. Tissue sections and 3 Hstandards (Amersham) were exposed to a tritium-sensitive imaging plate for 7 days. The imaging plates were scanned with the BAS 5000 device and analyzed with Multi Gauge V3.0 (Fujifilm, Tokyo, Japan). A standard curve based on 3 H-standards was used to measure regional radioactivity.
Because inflammation plays an important role in the pathophysiology of atherosclerosis [1–3], biomarkers of inflammation may be more useful than morphological measurements of arterial obstruction to identify plaque that is vulnerable to rupture. As a preliminary test of the utility of in vivo imaging, we studied whether macrophages in atherosclerotic plaque can be imaged in vitro by radioligand binding to PBR. In fact, all six sequentially obtained human carotid artery samples showed high densities of PBRs labeled with [3 H]PK 11195, and the distribution of binding paralleled that of macrophages stained with an antibody to CD68. The density (Bmax ) of PBRs was estimated to be ∼1.6 pmol/mg tissue, which is several fold greater than the density in normal human brain tissue but similar to that in glioma [16], which has been successfully imaged with an older PET radioligand for PBRs. Considering their high density, PBRs on macrophages in atherosclerotic plaques may be able to be imaged with PET radioligands that we have recently developed [11,12]. Nevertheless, the feasibility of imaging PBRs in plaque is difficult to predict, because it will depend on other variables such as the size of the plaque, the density of PBR, and the clearance of background radioactivity from blood. Another promising PET technique to image inflammation in atherosclerotic plaque is the increased uptake of a radiolabeled analog of glucose, [18 F]-fluorodeoxyglucose [9,10]. In fact, macrophages in inflammation contribute to the increased glucose uptake in inflammation. Thus, if future PET studies show that PBRs can be imaged in vivo in human atherosclerotic plaque, this technique should be compared to [18 F]-fluorodeoxyglucose to determine relative sensitivity and whether either technique provides clinically useful information for carotid plaque risk stratification and patient management.
2.3. Immunohistochemical staining Using sections adjacent to those for autoradiography, macrophages were stained with an antibody to CD68, a protein marker specific to macrophages. CVPath, Inc. (Gaithersburg, MD, USA) performed the immunohistochemical staining [14].
3. Results All six samples showed high densities of PBRs labeled with [3 H]PK 11195, and the distribution of receptor binding paralleled that of macrophages stained with an antibody to CD68 (Fig. 1). The specific binding of [3 H]PK 11195 was calculated by subtracting residual (nonspecific) binding in the presence of excess PK 11195 (20 M) (Fig. 1B). Spe-
110
Y. Fujimura et al. / Atherosclerosis 201 (2008) 108–111
Fig. 1. Peripheral benzodiazepine receptors (PBRs) labeled with autoradiography and macrophages labeled with immunohistochemistry in carotid endarterectomy tissue from a single patient. (A) Total binding of [3 H]PK 11195 by autoradiography. (B) Residual (or nonspecific) binding of [3 H]PK 11195 was determined by co-incubation with excess nonradioactive PK 11195 (20 M). Specific binding is defined as total minus nonspecific and represented the majority (∼90%) of total binding shown in panel A. (C) Immunohistochemical staining of macrophages labeled with an antibody to CD68. (D) Control section was processed without the primary antibody to CD68 and showed negligible staining.
Acknowledgements This research was supported by the Intramural Program of NIMH (project # Z01-MH-002852-03). We thank David Mozley, MD (Merck Research Laboratory) for suggesting the potential utility of PBR imaging as a marker of inflammation in vulnerable plaque. References [1] Schroeder AP, Falk E. Vulnerable and dangerous coronary plaques. Atherosclerosis 1995;118(Suppl.):S141–9. [2] Lendon C, Born GV, Davies MJ, Richardson PD. Plaque fissure: the link between atherosclerosis and thrombosis. Nouvelle revue francaise d’hematologie 1992;34:27–9. [3] Libby P, Geng YJ, Aikawa M, et al. Macrophages and atherosclerotic plaque stability. Curr Opin Lipidol 1996;7:330–5. [4] Crisby M, Nordin-Fredriksson G, Shah PK, et al. Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaques: implications for plaque stabilization. Circulation 2001;103:926–33. [5] Papadopoulos V, Lecanu L, Brown RC, Han Z, Yao ZX. Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis, neuropathology and neurological disorders. Neuroscience 2006;138:749–56.
[6] Benavides J, Dubois A, Dennis T, Hamel E, Scatton B. Omega 3 (peripheral type benzodiazepine binding) site distribution in the rat immune system: an autoradiographic study with the photoaffinity ligand [3 H]PK 14105. J Pharmacol Exp Ther 1989;249:333–9. [7] Schlumpf M, Parmar R, Lichtensteiger W. Prenatal diazepam induced persisting downregulation of peripheral (omega 3) benzodiazepine receptors on rat splenic macrophages. Life Sci 1993;52:927– 34. [8] Zavala F, Haumont J, Lenfant M. Interaction of benzodiazepines with mouse macrophages. Eur J Pharmacol 1984;106:561–6. [9] Rudd JH, Warburton EA, Fryer TD, et al. Imaging atherosclerotic plaque inflammation with [18 F]-fluorodeoxyglucose positron emission tomography. Circulation 2002;105:2708–11. [10] Ogawa M, Ishino S, Mukai T, et al. (18)F-FDG accumulation in atherosclerotic plaques: immunohistochemical and PET imaging study. J Nucl Med 2004;45:1245–50. [11] Fujimura Y, Ikoma Y, Yasuno F, et al. Quantitative analyses of 18 FFEDAA1106 binding to peripheral benzodiazepine receptors in living human brain. J Nucl Med 2006;47:43–50. [12] Imaizumi M, Briard E, Zoghbi SS, et al. Kinetic evaluation in nonhuman primates of two new PET ligands for peripheral benzodiazepine receptors in brain. Synapse 2007;61:595–605. [13] Guilarte TR, Kuhlmann AC, O’Callaghan JP, Miceli RC. Enhanced expression of peripheral benzodiazepine receptors in trimethyltinexposed rat brain: a biomarker of neurotoxicity. Neurotoxicology 1995;16:441–50.
Y. Fujimura et al. / Atherosclerosis 201 (2008) 108–111 [14] Kunisch E, Fuhrmann R, Roth A, et al. Macrophage specificity of three anti-CD68 monoclonal antibodies (KP1, EBM11, and PGM1) widely used for immunohistochemistry and flow cytometry. Ann Rheum Dis 2004;63:774–84. [15] Venneti S, Wang G, Wiley CA. The high affinity peripheral benzodiazepine receptor ligand DAA1106 binds to activated and infected brain
111
macrophages in areas of synaptic degeneration: Implications for PET imaging of neuroinflammation in lentiviral encephalitis. Neurobiol Dis 2008;29:232–41. [16] Takada A, Mitsuka S, Diksic M, Yamamoto YL. Autoradiographic study of peripheral benzodiazepine receptors in animal brain tumor models and human gliomas. Eur J Pharmacol 1992;228:131–9.