Brain Advance Access originally published online on April 6, 2004
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Brain, Vol. 127, No. 6, 1379-1392, 2004
© 2004 Guarantors of Brain
doi: 10.1093/brain/awh161
Peripheral benzodiazepine receptor imaging in CNS demyelination: functional implications of anatomical and cellular localization
Molecular Neurotoxicology Laboratory, Department of Environmental Health Sciences, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD, USA
Correspondence to: Tomás R. Guilarte, PhD, Department of Environmental Health Sciences, Johns Hopkins University, Bloomberg School of Public Health, 615 North Wolfe Street, Room W2001, Baltimore, MD 21205, USA. E-mail: tguilart{at}jhsph.edu
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The peripheral benzodiazepine receptor (PBR) has been used as a sensitive marker to visualize and measure glial cell activation associated with various forms of brain injury and inflammation. Previous studies have shown that increased PBR levels following brain injury are specific to areas expressing activated glial cells. However, the contribution of glial cell types responsible for the increases in PBR levels following brain injury is not well defined. In the present study, we used a murine model of cuprizone-induced demyelination to broaden the application of PBR as a marker of brain injury and to validate the relationship between PBR levels and glial cell types. C57BL/6J mice were maintained on a cuprizone-containing or control diet and sacrificed at specific time points after initiation of treatment. Quantitative autoradiography of the PBR-selective ligand [3H]-(R)-PK11195 and [125I]-(R)-PK11195 showed that increased PBR levels were associated with the degree of demyelination assessed by Black–Gold histochemistry and activation of glial cells assessed by glial fibrillary acidic protein (GFAP) immunohistochemistry for astrocytes and CD11b (Mac-1) for microglia. Our findings indicate that brain PBR levels increased as a function of dose and duration of cuprizone treatment and it was detectable prior to observable demyelination. Increased PBR levels were associated with the degree of demyelination and temporal activation of glial cell types in different anatomical regions. In the corpus striatum, we found a close anatomical correlation between microglial activation and increased PBR levels in demyelinating fibre tracts. In the deep cerebellar nuclei, the temporal increases in PBR paralleled demyelination and microglia and astrocyte activation. On the other hand, in the corpus callosum there was an apparent temporal shift in the increase in PBR levels by different glial cell types from an early and predominantly microglial contribution to a late microglial and astrocytic response. High-resolution emulsion autoradiography of [3H]-(R)-PK11195 binding to PBR coupled with GFAP or Mac-1 immunohistochemistry showed that demyelination-induced increases in PBR levels were co-localized to both microglia and astrocytes. These findings support the notion that PBR is a sensitive and specific marker for the in vitro and in vivo visualization and quantification of neuropathological changes in the brain.
Key Words: peripheral benzodiazepine receptor (PBR); (R)-PK11195; demyelination; cuprizone; microglia; astrocyte
Abbreviations: GFAP = glial fibrillary acidic protein; PBR = peripheral benzodiazepine receptor; PLP = paraformaldehyde–lysine–periodate; RCA-1 = Ricinus communis agglutinin lectin-1
Received October 28, 2003. Revised January 13, 2004. Accepted February 7, 2004.
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Introduction |
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Glial cells play an important role in brain injury and neurodegenerative processes in the CNS (Unger, 1998
). Reactive gliosis, including activation of microglia and astrocytes, is the hallmark response of the CNS to injury and inflammation (O’Callaghan, 1991, 1993; Norton et al., 1992; Streit et al., 1999; Streit, 2000; McGraw et al., 2001). The peripheral benzodiazepine receptor (PBR) is a glial protein that has been used as a sensitive marker of reactive gliosis and inflammation associated with chemical-induced neurotoxicity (Guilarte et al., 1995; Kuhlmann and Guilarte, 1997, 1999, 2000), ischaemic stroke (Stephenson et al., 1995; Pappata et al., 2000), physical trauma (Miyazawa et al., 1995) and CNS inflammatory disease (Vowinckel et al., 1997; Banati et al., 2000; Cagnin et al., 2001a, b; Mankowski et al., 2003). Increased PBR levels following brain injury are specific to areas expressing activated glial cells and the receptor can be visualized and quantified using in vitro and in vivo imaging techniques (Kuhlmann and Guilarte, 1997, 1999, 2000; Banati et al., 2000; Banati, 2002; Cagnin et al., 2001a, b; Mankowski et al., 2003; Versijpt et al., 2003). Therefore, this approach offers great potential for the in vivo imaging of a wide variety of neuropathological conditions. Further, recent work has demonstrated that administration of PBR-specific ligands can alter expression of inflammatory cytokines (Choi et al., 2002) and promotion of neuronal survival and repair (Ferzaz et al., 2002). Therefore, PBR may also play an important role in the inflammatory response of the brain to injury and it may be amenable to modulation by therapeutic intervention (Choi et al., 2002; Ferzaz et al., 2002).
The cellular and subcellular nature of the PBR response to brain injury and inflammation is not fully understood. Studies using ischaemic models have suggested that activated microglia and astrocytes are responsible for increased PBR levels (Benavides et al., 1991). However, subsequent studies have suggested that elevated PBR levels are consistently correlated with activated microglia rather than astrocytes using ischaemic models (Stephenson et al., 1995), axotomy (Banati et al., 1997), and multiple sclerosis and experimental autoimmune encephalomyelitis rodent models (Vowinckel et al., 1997; Banati et al., 2000). On the other hand, in situ co-localization of PBR levels has been demonstrated in activated microglia and/or astrocytes using double-labelling fluorescence immunohistochemistry in a chemical model of brain injury (Kuhlmann and Guilarte, 2000), in the brain of jimpy and shiverer mice expressing mutations in genes associated with myelination (Le Goascogne et al., 2000), and in temporal lobe epilepsy in humans (Sauvageau et al., 2002). The most plausible explanation for the differences amongst these studies is that glial cells have different temporal profiles of increases in PBR levels following injury. Therefore, it is necessary to examine different models of brain injury and the temporal profile of glial cell activation in order to extend our understanding of the cellular sources and the function of the PBR response to injury.
Cuprizone (bis-cyclohexanone-oxaldihydrazone), a copper chelator, has been used to induce CNS demyelination in mice (Carlton, 1966; Blakemore, 1973; Johnson and Ludwig, 1981; Komoly et al., 1992; Hiremath et al., 1998; Matsushima and Morell, 2001). Cuprizone-induced CNS demyelination is reproducible and reversible and has a relatively simple immunological response with an intact blood–brain barrier (Matsushima and Morell, 2001). Previous studies using cuprizone have suggested an early appearance of microglia/macrophages in the corpus callosum prior to observed demyelination and the concurrent activation of astrocytes with demyelination (Hiremath et al., 1998). In the present study, we used the murine model of cuprizone-induced demyelination to further validate the relationship between PBR levels and activation of glial cell types. Our findings indicate that the temporal increase in [3H]-(R)-PK11195 binding to PBR in the mouse brain precedes histological evidence of demyelination and is in direct correlation with the severity of demyelination in specific anatomical regions. Further, high-resolution microautoradiography coupled with immunohistochemical labelling demonstrates that increased [3H]-R-PK11195 binding to PBR is associated with both activated microglia and astrocytes.
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Animal model and tissue preparation
C57BL/6 male mice (8 weeks old; Charles River, Wilmington, MA, USA) were continuously fed a powdered diet containing 0, 0.1, 0.2 or 0.3% cuprizone [bis-cyclohexanone oxaldihydrazone] (w/w) (Sigma, St Louis, MO, USA) (Hiremath et al., 1998
). Mice were sacrificed 11 days, 2 weeks, 3 weeks or 4 weeks following diet administration and euthanized by either decapitation to obtain fresh-frozen brain tissue for receptor autoradiography or by transcardiac perfusion for immunohistochemistry. Brain tissue was harvested and frozen on dry ice and stored at –80°C until used. For transcardiac perfusion, animals were deeply anaesthetized with urethane (1.5 g/kg) and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4 at 4°C) or a paraformaldehyde–lysine–periodate (PLP) solution that consisted of 2% paraformaldehyde, 75 mM L-lysine and 10 mM sodium metaperiodate in 37 mM phosphate buffer (pH 7.4, 4°C). The PLP solution was used to perfuse animals whose brains were used for the [3H]-(R)-PK11195 emulsion autoradiography in conjunction with glial fibrillary acidic protein (GFAP) or CD11b (Mac-1) immunohistochemistry (see sections below). Brains from perfused animals were postfixed overnight in the same fixative, cryoprotected with 20% sucrose for at least 48 h, snap-frozen in dry ice-cooled isopentane, and stored at –80°C until used. All animal studies were reviewed and approved by the Johns Hopkins University Animal Care and Use Committee.
Radioligand synthesis
(R)-1-(2-iodophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline (R-N-desmethyl PK11195), the precursor for the radioisotope labelled R-PK11195, was purchased from ABX (Advanced Biochemical Compounds, Dresden, Germany). [3H]-(R)-PK11195 (85.5 Ci/mmol) was radiolabelled and purified by NEN Life Science Products (Boston, MA, USA). Synthesis of [125I]-(R)-PK11195 was accomplished by a modification of the method of Gildersleeve et al. (1989) using direct displacement of aromatic chlorine in the solid-state condition. Briefly, [125I][NaI] in NaOH (aq) (0.1 N, 20 – 40 µl) was added to a vial containing (NH4)2SO4 (6 mg), R-PK11195 (100 µg), EtOH/H2O (1 : 2) (300 µl) and 3 mm glass beads. The reactions contained 3 mCi of [125I][NaI]. The reaction mixture was evaporated at 230°C and then 10 ml of air was slowly injected into the vial. Heating at 230°C was continued for 20 min. After the reaction mixture had cooled to room temperature, it was extracted with EtOH/H2O (1 : 2, 3 x 100 µl). The [125I]-R-PK11195 contained in the combined crude extracts was purified by reversed phase HPLC (0.8 x 10 cm NovaPak C-18 radial Pak column, 50 : 50 CH3CN/H2O containing 0.1% trifluoroacetic acid, TFA). The radiochemical yield was 40–60%. The purified [125I]-R-PK11195 contained in the eluate was extracted into ethanol by solid phase extraction techniques using C-18 SepPak. The specific activity of [125I]-R-PK11195 was 2200 Ci/mmol.
Quantitative receptor autoradiography
Fresh-frozen brains were sectioned (20 µm) on a freezing cryostat in the horizontal plane. Brain sections were thaw-mounted onto poly-L-lysine-coated slides (Sigma) and stored at –20°C until used. [3H] and [125I]-(R)-PK11195 autoradiography to measure PBR levels were performed on adjacent brain sections using the same procedures. Slides were thawed and dried at 37°C for 30 min and prewashed in 50 mM Tris–HCl buffer (pH 7.4) for 5 min at room temperature. Sections were then incubated in 1 nM [3H]-(R)-PK11195 or 0.5 nM [125I]-(R)-PK11195 in buffer for 30 min at room temperature. For non-specific binding, adjacent sections were incubated in the presence of 10 µM racemic PK11195. The reaction was terminated by two 3-min washes in cold buffer (4°C) and two dips in cold deionized water (4°C). Sections incubated with [3H]-R-PK11195 were air-dried and apposed to Hyperfilm-[3H] (Amersham, Arlington Heights, IL, USA) with [3H]-Microscales (Amersham) for 2 weeks. Sections incubated with [125I]-(R)-PK11195 were apposed to Kodak Bio-Max MR films with [125I]-Microscales (Amersham) for 1 h. Images were acquired using the Inquiry system (Loats Associate, Westminster, MD, USA) and quantified using NIH Image v1.62.
[3H]-R-PK11195 binding and Scatchard analysis
Individual cerebral cortices from mice exposed to 0.2% cuprizone for 4 weeks or from control mice were homogenized using a Polytron in 100 volume of 50 mM Tris–HCl buffer (pH 7.4, 4°C) and centrifuged at 40 000 g for 30 min. The resulting pellet was resuspended in buffer and used for the radioligand-binding assay. Total binding of [3H]-R-PK11195 was determined for concentrations ranging from 0.25 to 15 nM. Non-specific binding was assessed for each [3H]-R-PK11195 concentration in the presence of 50 µM unlabelled PK11195. Binding reactions were incubated for 60 min at 4°C in a total volume of 500 µl. The reaction was terminated by vacuum filtration through GF/B filters, followed by four washes with 5 ml of ice-cold buffer. Radioactivity trapped in the filters was measured by liquid scintillation spectrometry. Protein concentrations of tissue homogenates were determined by the Bradford protein assay. Binding parameters were estimated using the LIGAND in KELL version 6 program for Windows (Biosoft, Cambridge, UK).
Double labelling of GFAP or Mac-1 immunohistochemistry and high-resolution [3H]-R-PK11195 emulsion autoradiography
In order to obtain information on the co-localization of PBR with glial cell types, PLP-perfused brain tissue was used. We have previously shown that there are no differences in [3H]-R-PK11195 binding to PBR between fresh-frozen and PLP-fixed brains (Kuhlmann and Guilarte, 1999). PLP fixed brains were sectioned (20 µm) on a freezing cryostat in the horizontal plane. Brain sections were thaw-mounted onto poly-L-lysine-coated slides and stored at –20°C until used. Immunohistochemistry (Mac-1 or GFAP) was performed as described below. After processing of 3,3'-diaminobenzidine (DAB) stain, slides were immediately processed for [3H]-(R)-PK11195 (1 nM concentration) receptor autoradiography performed as described above. Tissue sections were air-dried, coated with photographic emulsion (EM-1; Amersham) and exposed in the dark for 4 weeks. Kodak D-19 developer and Rapid-fix were used for photographic processing.
GFAP and Mac-1 (CD11b) immunohistochemistry
Free-floating brain sections from paraformaldehyde perfused animals were sectioned (30 µm) using a freezing microtome (Leica). Sections were incubated in 0.6% H2O2 for 30 min followed by blocking solution with either 5% normal goat serum (GFAP) or 5% normal rabbit serum (Mac-1) containing 0.2% Triton X-100 for 1 h. Sections were incubated with rabbit anti-GFAP antibody (1 : 1000, Dako, Carpinteria, CA, USA) or rat anti-mouse CD11b antibody (1 : 100, BD PharMingen, San Diego, CA, USA) at 4°C overnight. Sections were then incubated with biotinylated goat anti-rabbit antibody for GFAP (1 : 1000, ICN, Costa Mesa, CA, USA) or biotinylated rabbit anti-rat antibody for Mac-1 (1 : 300, Vector, Burlingame, CA, USA) for 60 min. Immunoreactivity was visualized using ABC elite, an avidin–biotin–horseradish peroxidase (HRP) complex (Vector Burlingame, CA, USA), with 3, 3'-diaminobenzidine (DAB) as the chromogen.
Black–Gold histochemistry for myelin
Paraformaldehyde-fixed brain sections (30 µm) were mounted on gelatin-coated slides, dried at 50°C for 30 min and rehydrated in deionized water for 2 min. The sections were incubated in 0.2% Black–Gold (Histo-Chem, Jefferson, AR, USA) at 60°C for 18 min, rinsed for 2 min in deionized water, fixed for 3 min in a 2% sodium thiosulphate solution, and rinsed in tap water for 15 min (Schmued and Slikker, 1999). Sections were dehydrated through graded alcohols, cleared in xylene, and coverslipped with DPX mounting medium.
Statistical analysis
Multiple means comparison of [3H]-(R)-PK11195 autoradiography results was done by one-way analysis of variance (ANOVA), followed by the Student–Newman–Keuls post hoc test where significance was indicated. A linear regression model was used for comparison between [3H]-(R)-PK11195 and [125I]-(R)-PK11195 autoradiography. Student’s t-test was used for the comparison of [3H]-(R)-PK11195 binding kinetics between control and cuprizone-treated mice. For all tests, the significance level was set at P < 0.05.
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PBR levels following cuprizone treatment
Quantitative film autoradiography of the PBR-selective ligand [3H]-(R)-PK11195 was used to assess PBR levels in different brain regions during the process of cuprizone-induced demyelination. It should be noted that high levels of PBR are normally expressed in the ependymal cells of the ventricles and in the choroid plexus (Fig. 1). Temporal response of PBR levels to demyelination was examined using the 0.2% cuprizone treatment group, because at this level of cuprizone treatment mice do not have liver toxicity and are able to survive for several weeks (Hiremath et al., 1998
). Increased [3H]-(R)-PK11195 binding to PBR was measured in most white matter and in some grey matter regions in the brains of cuprizone-treated mice. Significant increases (P < 0.05) in PBR levels at 2 weeks of cuprizone treatment were present in the corpus callosum (143 ± 9%), hippocampus (71 ± 19%), frontal cortex (123 ± 22%), temporal cortex (74 ± 18%) and entorhinal cortex (73 ± 20%) relative to controls (Fig. 2). A greater number of brain regions and higher levels of PBR were measured at 3 and 4 weeks of cuprizone treatment. These included the corpus callosum (382 ± 24 and 655 ± 59%), hippocampus (133 ± 17 and 95 ± 21%), frontal cortex (253 ± 50 and 183 ± 43%), temporal cortex (140 ± 29 and 99 ± 24%), entorhinal cortex (159 ± 26 and 110 ± 23%), thalamus (435 ± 67 and 467 ± 84%), deep cerebellar nuclei (144 ± 33 and 275 ± 45%), globus pallidus (384 ± 48 and 441 ± 54%), striatum (240 ± 47 and 207 ± 45%), intermediate white layer superior colliculus (385 ± 90 and 653 ± 99%), frontoparietal cortex (208 ± 39 and 155 ± 38%) and striate cortex (142 ± 34 and 92 ± 32%) (Fig. 2). In all brain regions examined, PBR levels reached maximal levels at either 3 or 4 weeks of cuprizone treatment. However, it is possible that higher PBR levels could have been achieved in some brain regions if longer treatment times had been examined.
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We also examined dose–response relationships between cuprizone-induced demyelination and PBR levels in the corpus callosum. We selected this brain region because it is one of the most widely studied in this model of demyelination (Komoly et al., 1992
; Hiremath et al., 1998; Mason et al., 2001; Matsushima and Morell, 2001). Since there was a significant mortality rate in mice exposed to cuprizone at doses higher than 0.2% for more than 2 weeks, we used two time points: 11 days for cuprizone doses up to 0.3% and 4 weeks of treatment for cuprizone doses up to 0.2%. At 11 days of continuous cuprizone treatment, PBR levels increased significantly in the corpus callosum in the 0.1, 0.2 and 0.3% cuprizone-treated groups (65 ± 12, 74 ± 9 and 63 ± 11%) compared with control. However, no significant differences were detected amongst the treatment groups at this time point (Fig. 3A). The lack of a dose–response relationship at 11 days of cuprizone treatment may be due to insufficient time for the resolution of a full glial response. This is consistent with the fact that at 4 weeks of treatment there is a clear dose–response relationship between control and the different levels of cuprizone treatment (Fig. 3B). That is, PBR levels as assessed by [3H]-(R)-PK11195 binding in the corpus callosum of 0.1 and 0.2% cuprizone-treated mice increased significantly (432 ± 39 and 684 ± 29%) from control levels and differed significantly from each other (Fig. 3B).
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[3H]-R-PK11195 binding and Scatchard analysis in cerebral cortex
Scatchard analysis of [3H]-(R)-PK11195 binding isotherms to cerebral cortex homogenates was used to determine if increased [3H]-(R)-PK11195 binding to PBR in cuprizone-treated brain resulted from changes in receptor number (Bmax) or binding affinity (Kd). Estimates of [3H]-(R)-PK11195 binding parameters of 0.2% cuprizone-treated cerebral cortex or control tissue at 4 weeks showed that the increase in [3H]-R-PK11195 binding was a result of an increase in the apparent number of binding sites with no changes in receptor binding affinity (Fig. 4).
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PBR levels correlate with the degree of demyelination and glial cell activation
Black–Gold histochemistry for myelin was used to demonstrate the degree of CNS demyelination in the cuprizone–treated brain. The intensity of red colour in brain slices represents the level of myelin content (Fig. 5, panels 2A–D). In the corpus callosum and deep cerebellar nuclei, small or undetectable changes in demyelination were present in the 0.2% cuprizone group at 2 weeks, progressing to moderate demyelination at 3 weeks and virtually complete demyelination at 4 weeks of continuous treatment (Fig. 5, panels 2A–D and Fig. 7, panel 2A–D). A similar pattern of demyelination was observed in the hippocampus, striatum, globus pallidus, entorhinal cortex, frontal cortex and temporal cortex (not shown). We selected the striatum for detailed examination because it exhibits myelinated fibre bundles in which demyelination can be easily assessed. Figure 8 (panels 2A–D) shows that, as in the other brain regions, the degree of demyelination in the striatum increased with time of cuprizone treatment. In the corpus callosum, deep cerebellar nuclei and striatum, increased PBR levels were closely associated with the degree of demyelination (compare Fig. 5, panels 1A–D and 2A–D, Fig. 7, panels 1A–D and 2A–D and Fig. 8, panels 1A–D and 2A–D).
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In order to understand the cellular sources of the PBR response to the cuprizone-induced demyelination, we used Mac-1 immunohistochemistry to assess microglia and GFAP for astrocytes. In the corpus callosum, microglia appeared activated 2 weeks after cuprizone treatment, peaked at 3 weeks, and appeared to be reduced by 4 weeks of treatment (Fig. 5, panels 3A–D and Fig. 6, panels 1A–D). The degree of microglial activation at 3 and 4 weeks of treatment was so pronounced that it was difficult to distinguish microglia morphology (Fig. 6, panels 1C and D). On the other hand, GFAP levels in the corpus callosum were low after 2 weeks of treatment, increased at 3 weeks, and were markedly expressed after 4 weeks of treatment (Fig. 5, panels 4A–D). In general, increased PBR levels in the corpus callosum at 2 and 3 weeks appeared to have a significant microglial component, but at 4 weeks increased PBR levels appeared to be associated with both activated microglia and astrocytes (Fig. 6).
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In both the deep cerebellar nuclei and the corpus striatum, a different temporal pattern of glial cell activation was apparent. Microglia and astrocytes seemed to track the temporal increases in PBR levels as a result of demyelination (Figs 7 and 8). In the corpus striatum, PBR levels appeared to have a closer anatomical association with microglia than with astrocytes. For example, Fig. 8 shows that at 4 weeks of treatment activated microglia were closely associated with demyelinating fibre bundles (compare panels 1D, 2D and 3D in Fig. 8). GFAP staining was also present within the demyelinating fibre bundles, but there was more generalized GFAP staining within the striatal neuropil (Fig. 8, panel 4D). In general, it appears that the early PBR response to demyelination is primarily from microglia but in some brain regions both microglia and astrocytes are responsible for the increased PBR levels at later time points. It should be noted that, in these figures, Mac-1 and GFAP immunohistochemistry is not a quantitative measure; thus, a direct correlation with PBR levels cannot be made. The Mac-1 and GFAP immunohistochemistry represents a relative measure of glial cell activation.
Co-localization of [3H]-R-PK11195 binding with activated microglia and astrocytes
We performed high-resolution emulsion autoradiography of [3H]-(R)-PK11195 binding to PBR. The silver grain density represents the levels of [3H]-(R)-PK11195 binding. Similar to the results of film autoradiography, there were dramatic increases in the silver grain density in brain regions undergoing demyelination after 4 weeks of 0.2% cuprizone treatment (Fig. 9). The accumulation of silver grain density in the high-resolution emulsion autoradiography provided a clear delineation of glial cell bodies and processes in the cuprizone-treated tissue (Fig. 9B). On the other hand, in the control tissue a more homogeneous distribution of silver grains was present in the brain neuropil (Fig. 9A). When GFAP or Mac-1 immunohistochemistry was combined in the tissue section with high-resolution emulsion autoradiography of [3H]-(R)-PK11195 binding to PBR, there was an excellent in situ co-localization of [3H]-(R)-PK11195 binding with both GFAP immunostaining of astrocytes (Fig. 9C-F) and Mac-1 immunostaining of microglia (Fig. 9G–J). Cellular localization of silver grain density was observed in GFAP- and Mac-1-positive cells at a low density in control tissue (Fig. 9D and H). However, a greater density of silver grains and a greater number of GFAP- and Mac-1-positive cells with overlaying silver grains were present in the cuprizone-treated tissue (Fig. 9F and J). It should be noted that in Fig. 9 panels C, E, G and I are at a slightly different focal plane to observe the immunostaining of the same cell in panels D, F, H and J, which are at a different focal plane in order to observe the silver grain density generated by the binding of [3H]-(R)-PK11195 to PBR. These images provide unequivocal evidence that both activated astrocytes and microglia were responsible for the enhanced [3H]-(R)-PK11195 binding to PBR in brain areas undergoing demyelination.
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Correlation between [125I]-R-PK11195 and [3H]-R-PK11195 autoradiography for PBR
We examined whether [125I]-(R)-PK11195 quantitative receptor autoradiography could provide the same degree of sensitivity as [3H]-(R)-PK11195 autoradiography in measuring PBR levels. The rationale for this comparison is that the iodinated compound considerably reduces the amount of time required to obtain autoradiograms. Our findings indicated that, in general, there was very good overall correlation between the quantitative [125I]-(R)-PK11195 and [3H]-(R)-PK11195 autoradiography in measuring PBR levels in the same animals and anatomical regions (Fig. 10) (R2 = 0.8687, P < 0.01).
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The present study demonstrates the regional, temporal and cellular patterns of PBR levels during the progression of cuprizone-induced demyelination in the mouse brain. The results reveal that demyelination involved microglia and astrocyte activation with distinct temporal profiles in different brain regions, providing evidence that both glial cell types are responsible for increased PBR levels. The ability to perform high-resolution [3H]-(R)-PK11195 emulsion autoradiography with GFAP or Mac-1 immunohistochemistry in the same brain sections provides unequivocal evidence that both activated microglia and astrocytes are responsible for enhanced PBR levels (Fig. 9). However, the degree of contribution of increased PBR levels from microglia and astrocytes differs at time points following injury. For the most part, it appears that the earliest PBR response to demyelination has a predominant microglial component while increases at later time points are due to both microglia and astrocytes. These findings are consistent with previous observations on the relationship between increased PBR levels and glial cell activation (Kuhlmann and Guilarte, 2000
). Further, it follows the staging of graded responses to injury by microglia and astrocytes (Raivich et al., 1999)
Demyelination-induced increase in [3H]-(R)-PK11195 binding to PBR in the mouse brain was the result of an increase in the maximal number of [3H]-(R)-PK11195 binding sites (Bmax) with no change in receptor affinity (Kd) (Fig. 4). It is apparent from the [3H]-(R)-PK11195 high-resolution emulsion autoradiograms that increases in the number of binding sites were the result of an increase in both the number of PBR per glial cell and the number of glial cells with increased PBR levels (Fig. 9). In this model of demyelination, PBR levels are significantly increased at 11 days of cuprizone treatment, a time point at which demyelination is not readily apparent using histochemical methods. This finding confirms and extends previous observations that the PBR is a sensitive marker of brain pathology (Guilarte et al., 1995; Kuhlmann and Guilarte, 1997, 1999, 2000).
Previous studies using C57BL/6 mice and continuous exposure to 0.2% cuprizone show complete demyelination in the corpus callosum 4–6 weeks after treatment (Hiremath et al., 1998). Cell counting of Ricinus communis agglutinin lectin-1 (RCA-1)-positive microglia and GFAP-positive astrocytes reached plateau levels at 4 weeks (Hiremath et al., 1998). In the present study, the enhanced PBR levels corresponded well with the levels of activated microglia and astrocytes in the corpus callosum. However, we see a slightly different temporal pattern of activated microglia from previous studies examining the corpus callosum, since in our study the apparent peak microglial response was at 3 weeks and levels appeared to subside by 4 weeks. A possible explanation for this discrepancy may be the difference in characteristics between RCA-1-positive and Mac-1-positive cells. The Mac-1 antibody specifically detects the surface marker CD11b or complement receptor type 3 (CR3) on microglia/macrophage while the RCA-1 detects ß-D-galactosyl residues. It is possible that these two different microglial markers have different temporal expression profiles. Importantly, the temporal pattern of astrocyte activation in the corpus callosum observed in our study is identical with that observed in previous studies (Hiremath et al., 1998).
In the corpus striatum, activated microglia reached ‘peak’ levels 4 weeks after treatment and expressed a closed anatomical correlation with increased PBR levels in demyelinating fibre tracts. In fact, it appears that activated microglia are in close contact with demyelinating fibres (panel 3D in Fig. 8). In the deep cerebellar nuclei there was a close temporal correlation between PBR levels and activated glial cells (Fig. 7). In general, it appears that both activated microglia and astrocytes contribute to the increased levels of PBR in affected brain regions, based on the time following injury.
Recent studies have favoured the notion that microglia are primarily responsible for the enhanced PBR levels following brain injury (Stephenson et al., 1995; Vowinckel et al., 1997; Banati et al., 1997, 1999, 2000; Banati, 2002). The basis for this conclusion was the close anatomical association between microglia immunohistochemistry with [3H]-PK11195 film autoradiography (Stephenson et al., 1995; Vowinckel et al., 1997) and high-resolution [3H]-(R)-PK11195 microautoradiography (Banati et al., 1997, 2000; Banati, 2002). Although our present findings support an early microglial contribution to increased PBR levels, the present study supports the notion that astrocytes also increase their level of PBR at later times following brain injury (Le Goascogne et al., 2000; Kuhlmann and Guilarte, 2000).
An important question that has gained recent interest is that of the functional implications of increased PBR levels in microglia and astrocytes in brain injury or inflammation. It is known that PBR is the rate-limiting step in the transport of cholesterol from the outer to the inner mitochondria membrane (Li and Papadopoulos, 1998; Lacapere and Papadopoulos, 2003). Cholesterol is then metabolized to pregnenolone by cytochrome p450scc (Lacapere and Papadopoulos, 2003) and pregnenolone serves as the parent precursor for the synthesis of neurosteroids in the brain (Brown and Papadopoulo, 2001). Studies have shown that PBR activation in glial cells promotes the synthesis of pregnenolone and progesterone (Le Goascogne et al., 2000), two neurosteroids that possess neurotrophic and neuroprotective activity (Le Goascogne et al., 2000). It is possible that increased PBR levels in glial cells during the process of demyelination are associated with the induction and secretion of substances such as neurosteroids that promote the repair and/or survival of oligodendrocytes. In this context, it is known that microglia secrete proinflammatory cytokines such as IL-1ß and TNF-
, which have recently been shown to promote proliferation of oligodendrocyte progenitors and remyelination in the cuprizone model (Arnett et al., 2001; Mason et al., 2001). Thus, it is possible that there is an association of increased levels of PBR and the levels of cytokines and nerve growth factors in the areas of damage. In fact, Bourdiol et al. (1991) have shown that the injection of IL-1 and TNF- plays an important role in the process that leads to increased PBR levels in the injured brain. Recent studies have shown that PBR ligands can inhibit lipopolysaccharide-induced increases in cyclooxygenase-2 and tumour necrosis factor- levels in cultured human microglia (Choi et al., 2002), two key proinflammatory cytokines. Importantly, PBR ligands can promote neuronal survival and repair in axotomy and neuropathy rodent models (Ferzaz et al., 2002) and prevent neuronal injury from chemical-induced seizures (Veenman et al., 2002). Therefore, it is possible that the use of PBR ligands may be a useful approach in the treatment of demyelination-associated diseases. Future studies should investigate whether a relationship exists between increased PBR levels in the injured brain and the production and secretion of cytokines and growth factors important in the inflammatory response.
The global discontinuation of tritium-sensitive film for autoradiography (3H-Hyperfim) has had a significant impact on research laboratories using receptor autoradiography methods. Commercially available alternatives, such as Kodak MR and MS films, are able to provide resolution similar to that given by 3H-Hyperfim, but they require approximately three times greater exposures times (personal experience). This translates to approximately 6 weeks of exposure time for [3H]-(R)-PK11195 autoradiography. Because the specific activity of [125I]-(R)-PK11195 (2200 Ci/mmol) is greater than that of [3H]-(R)-PK11195 (85 Ci/mmol), the exposure time for [125I]-(R)-PK11195 film autoradiography is much shorter than for [3H]-(R)-PK11195. Thus, a significant benefit of [125I]-(R)-PK11195 autoradiography is its extremely short exposure time (1–2 h) to obtain autoradiograms with acceptable anatomical resolution. In the present study, we demonstrate an excellent correlation between [125I]-(R)-PK11195 and [3H]-(R)-PK11195 autoradiography (Fig. 10). Thus, the use of [125I]-(R)-PK11195 quantitative autoradiography may serve as a rapid in vitro screening method to assess primary as well as secondary sites (Cagnin et al., 2001b) of brain inflammation and injury in post-mortem tissue.
In summary, the present studies support previous findings from our laboratory and those of others showing that PBR is a useful and sensitive marker for the visualization of neuropathological changes in the brain. The PBR could be used as an in vivo marker for the early detection and monitoring of a variety of neuropathological conditions, including demyelinating diseases, using non-invasive brain imaging techniques, as has been demonstrated for multiple sclerosis (Vowinckel et al., 1997; Banati et al., 2000).
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The authors wish to thank Dr Brian Schofield for assistance with microscopic imaging and the use of core facilities of the Johns Hopkins Center on Urban Environmental Health (ES03819). The work was performed in partial fulfilment of doctoral degree requirements for M.K.C. This work was supported by NIH grants ES07062 (to T.R.G.) and CA32845 (to K.B.).
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