ToxSci Advance Access originally published online on March 22, 2006
Toxicological Sciences 2006 91(2):532-539; doi:10.1093/toxsci/kfj172
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Imaging the Peripheral Benzodiazepine Receptor Response in Central Nervous System Demyelination and Remyelination
Molecular Neurotoxicology Laboratory, Department of Environmental Health Sciences, The Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland 21205
1 To whom correspondence should be addressed at Department of Environmental Health Sciences, The Johns Hopkins University, Bloomberg School of Public Health, 615 North Wolfe Street, Room E6622, Baltimore, MD 21205. Fax: (410) 502-2470. E-mail: tguilart{at}jhsph.edu.
Received December 19, 2005; accepted March 20, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We used a rodent model of cuprizone-induced demyelination to examine the peripheral benzodiazepine receptor (PBR) response during remyelination. C57BL/6J mice were fed a 0.2% cuprizone-containing or control diet for 3 weeks and then removed to allow for remyelination. Quantitative autoradiography of 3H-(R)-PK11195 binding to PBR in the corpus callosum showed increased levels at 3 weeks of demyelination and gradually decreased as a function of remyelination. PBR levels were associated with the degree of remyelination and activation of microglia and astrocytes. However, the temporal pattern suggests that the PBR signal during the late stages of remyelination was primarily associated with astrocytes. We also used small-animal positron-emission tomography (PET) imaging to determine if this technique could be used to monitor PBR levels in the brain of living mice. The results indicate that 11C-(R)-PK11195 levels are significantly elevated in the mouse brain during cuprizone-induced demyelination and normalize at a time in which remyelination is complete. These findings support the notion that PBR is a sensitive marker for the visualization and quantification of brain injury and recovery. Further, the in vivo imaging of the PBR response is now possible in the living rodent brain.
Key Words: peripheral benzodiazepine receptor (PBR); (R)-PK11195; demyelination; remyelination; cuprizone; microglia; astrocyte; positron-emission tomography (PET).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The peripheral benzodiazepine receptor (PBR) was first identified as an alternative binding site for benzodiazepines in peripheral organs (Braestrup and Squires, 1977
). PBR is highly expressed in peripheral organs that are involved in steroid production (see review Gavish et al., 1999; Papadopoulos et al., 1997) but are expressed at very low levels in the brain neuropil (Gavish et al., 1999). At the cellular level, PBR in the brain is detected primarily in glial cells (Chen et al., 2004; Kuhlmann and Guilarte, 1999, 2000), and one of its most studied functions is to regulate the transport of cholesterol into the mitochondria for the synthesis of pregnenolone, the parent compound to all steroids (Brown and Papadopoulos, 2001).
A large number of studies have shown that PBR levels markedly increase following brain injury, and this increase is associated with activation of glial cells. Thus, the PBR has been used as a marker of glial cell activation and indirectly as a marker of brain injury and inflammation. Increased PBR levels have been described in models of chemical-induced neurotoxicity (Chen et al., 2004; Guilarte et al., 1995; Kuhlmann and Guilarte, 1997, 1999, 2000), ischemic stroke (Gerhard et al., 2000, 2005; Pappata et al., 2000; Stephenson et al., 1995), physical trauma (Miyazawa et al., 1995; Raghavendra Rao et al., 2000), central nervous system (CNS) degenerative diseases (Cagnin et al., 2001a, 2004; Gerhard et al., 2003; Henkel et al., 2004), and CNS inflammatory disease (Banati et al., 2000; Debruyne et al., 2003; Mankowski et al., 2003; Ouchi et al., 2005; Vowinckel et al., 1997).
Our laboratory has been interested in determining the degree of damage necessary in order to produce an increase in PBR levels following brain injury from exposure to a variety of neurotoxicants. The results indicate that the PBR response to injury is directly associated with the degree of damage ranging from frank neuronal cell loss (Guilarte et al., 1995; Kuhlmann and Guilarte, 1997, 2000) to subtle injury to neuronal terminals (Guilarte et al., 2003; Kuhlmann and Guilarte, 1999) and the loss of myelination (Chen et al., 2004). These studies also showed that the PBR is a much more sensitive indicator in detecting brain damage than histological techniques (Chen et al., 2004; Kuhlmann and Guilarte, 1997). Further, increased levels of PBR are also measured in secondary areas of brain injury resulting from the primary lesion, providing a more extensive assessment of damage associated with neural networks (Banati et al., 2000; Cagnin et al., 2001b; Kuhlmann and Guilarte, 1999; Turner et al., 2004). An important advantage of the PBR as a biomarker of brain injury is that it can be visualized and quantified using ex vivo quantitative autoradiography and noninvasive in vivo imaging techniques (Banati et al., 2000; Cagnin et al., 2001a,b; Chen et al., 2004; Gerhard et al., 2005; Gerhard et al., 2005; Guilarte et al., 1995, 2003; Kuhlmann and Guilarte, 1997, 1999, 2000; Mankowski et al., 2003; Pappata et al., 2000; Versijpt et al., 2000). Therefore, this approach offers great potential for the in vivo imaging of a wide variety of neuropathological conditions.
While the majority of studies during the last decade have focused on the PBR as a marker of brain injury, emerging evidence suggests that the administration of PBR-specific ligands can alter the expression of inflammatory cytokines, reduce the level of reactive gliosis (Choi et al., 2002; Ryu et al., 2005; Veiga et al., 2005), and promote neuronal survival and repair (Ferzaz et al., 2002; Ryu et al., 2005; Veiga et al., 2005). 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 using PBR-selective ligands (Choi et al., 2002; Ferzaz et al., 2002; Ryu et al., 2005; Veiga et al., 2005). However, the functional significance of the PBR response to brain injury and inflammation is still not fully understood. Moreover, there are currently a limited number of studies on the PBR response during recovery from brain injury.
We have used a murine model of cuprizone-induced demyelination to demonstrate that the temporal increase in 3H-(R)-PK11195 binding to PBR in the mouse brain is inversely associated with the level of myelin in specific anatomical regions (Chen et al., 2004). We also confirmed that increased 3H-(R)-PK11195 binding to PBR is associated with activated microglia and astrocytes using high-resolution emulsion microautoradiography coupled with immunohistochemical labeling (Chen et al., 2004). Because cuprizone-induced demyelination is reversible if the animal is removed from the cuprizone exposure (Matsushima and Morell, 2001), we can use the same model to assess the PBR response during recovery or remyelination.
In the present study, we investigate the temporal and cellular patterns of PBR expression during remyelination. We found that high PBR expression is present in the demyelinated corpus callosum, but PBR levels decreased gradually in the remyelination phase. The gradual decrease in PBR levels in the corpus callosum was associated with an increase in myelin content and a lower level of glial cell activation. Lastly, we used small-animal positron-emission tomography (PET) imaging to demonstrate in vivo changes in PBR levels in the same animal during demyelination and remyelination. To our knowledge, this is the first report on the use of small-animal PET imaging of the PBR in the living mouse brain to monitor brain injury and recovery during demyelination and remyelination.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal model and tissue preparation.
C57BL/6J male mice (8 weeks old; Charles River, Wilmington, MA) were continuously fed a powdered diet containing either 0.0% or 0.2% cuprizone [bis-cyclohexanone oxaldihydrazone] (wt/wt) (Sigma, St Louis, MO) for 3–4 weeks (Chen et al., 2004
; Hiremath et al., 1998). After cuprizone exposure for 3 (ex vivo) or 4 weeks (in vivo), animals were fed a control diet in order for remyelination to occur. For the ex vivo studies, mice were sacrificed at 3 weeks after feeding a 0.0% or 0.2% cuprizone diet or 3 and 6 weeks of remyelination by transcardiac perfusion (Fig. 1). Briefly, animals were deeply anesthetized with urethane (1.5 g/kg) and perfused with a paraformaldehyde-lysine-periodate (PLP) solution that consisted of 2% paraformaldehyde, 75mM L-lysine, and 10mM sodium metaperiodate in 37mM phosphate buffer (pH 7.4, 4°C). Brains from perfused animals were postfixed overnight in PLP fixative, cryoprotected with 20% sucrose for at least 48 h, snap frozen in dry ice–cooled isopentane, and stored at – 80 °C until used. For the in vivo imaging studies, animals were imaged at 4 weeks of demyelination and at 9–10 weeks of remyelination and compared to controls. Two important comments need to be made in regards to the selection of time used for cuprizone-induced demyelination for the ex vivo autoradiography and in vivo studies. First, we have previously shown that exposure to 0.2% cuprizone in the diet produces a time-dependent increase of the PBR response in the corpus callosum (Chen et al., 2004). One of the problems with 0.2% cuprizone exposure for 4 weeks is that in our hands there is a high degree of mortality. Thus, to reduce the loss of animals and since at 3 weeks of exposure there is a significant amount of demyelination as well as increased PBR response, this time of exposure was selected. For the in vivo studies, a different time point for demyelination and remyelination was used due to scheduling for small-animal imaging. This is a highly used instrument, and we obtain time when possible, thus the different time points. For the in vivo studies, the main point was to show the feasibility of the approach. All animal studies were reviewed and approved by the Johns Hopkins University Animal Care and Use Committee.
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Quantitative receptor autoradiography.
The PBR-selective radioligand 3H-(R)-PK11195 (85.5 Ci/mmol) was radiolabeled and purified by NEN Life Science Products (Boston, MA). Brains fixed in PLP 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-(R)-PK11195 autoradiography to measure PBR levels was performed on adjacent brain sections using the same procedures. Slides were thawed and dried at 37°C for 30 min and prewashed in 50mM Tris-HCl buffer (pH 7.4) for 5 min at room temperature. Sections were then incubated in 1nM 3H-(R)-PK11195 in buffer for 30 min at room temperature. For nonspecific binding, adjacent sections were incubated in the presence of 10µM 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) with [3H]-Microscales (Amersham) for 2 weeks. Images were acquired using the Inquiry System (Loats Associates, Westminster, MD) and quantified using NIH Image v1.62.
Glial fibrillary acidic protein or Mac-1 (CD11b) immunohistochemistry.
PLP-fixed brain sections (20 µm) were incubated in 0.6% H2O2 for 30 min followed by blocking solution with either 5% normal goat serum (glial fibrillary acidic protein, 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) or rat anti-mouse CD11b antibody (1:100, BD PharMingen, San Diego, CA) at 4°C overnight. Sections were then incubated with biotinylated goat anti-rabbit antibody for GFAP (1:250, Vector, Burlingame, CA) or biotinylated rabbit anti-rat antibody for Mac-1 (1:300, Vector) for 60 min. Immunoreactivity was visualized using ABC elite, an avidin-biotin-horseradish peroxidase complex (Vector), with 3,3'-diaminobenzidine as the chromogen.
Black-Gold histochemistry for myelin.
PLP-fixed brain sections (20 µm) were 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) at 60°C for 18 min, rinsed for 2 min in deionized water, fixed for 3 min in a 2% sodium thiosulfate 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 media (Flukia, Bucks, Switzerland).
Synthesis of 11C-(R)-PK11195.
(R)-1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide (R-N-desmethyl PK11195), the precursor for the radioisotope-labeled (R)-PK11195, was purchased from ABX (Radeberg, Germany). The synthesis of 11C-(R)-PK11195 was accomplished by a modification of the method of Camsonne et al. (1984). Briefly, 11C-methyl iodide (11C-CH3I) was transferred by a stream of nitrogen at a flow rate of 10 ml/min into the reaction medium containing 1.2 µmol of R-N-desmethyl PK11195 previously dissolved in 120 µl dimethylsulfoxide and 20 mg potassium hydroxide. The reaction began at room temperature. The bubbling of 11C-CH3I was continued over 6–10 min. Purification was carried out by high pressure liquid chromatography using a reversed-phase C18 column and an ethanol/water (70/30) mixture as the mobile phase. Radiochemical yields were consistently over 50%. 11C-(R)-PK11195 was obtained with a specific radioactivity of over 814 GBq/µmol (22,000 Ci/mmol).
In vivo 11C-(R)-PK11195 PET imaging in mice.
C57BL/6J mice were anesthetized by an ip injection of a mixture of acepromazine maleate (10 mg/ml) (Phoenix Pharmaceutical Inc., St Joseph, MO), Ketaject (ketamine HCl, 100 mg/ml) (Phoenix Pharmaceutical Inc.), and normal saline (0.9%) (ratio 1:1:2, dosage 2.3 µl/g). Approximately 37 MBq (1 mCi) 11C-(R)-PK11195 (0.2-ml injection volume) was administered by tail vein injection. Anesthetized mice were then fixed on a platform (prone position) in the gantry of the ATLAS small-animal PET scanner (NIH, Bethesda, MD), which has an 11.8-cm ring diameter, an 8-cm aperture, a 6-cm effective transverse field of view, and a 2-cm axial field of view. The imaging system comprised of 18 depth-of-interaction detector modules that surround the mouse. Radial and tangential resolutions of the reconstructed image (pixel size = 0.56 mm) using a three-dimensional ordered subset expectation maximum (3D OSEM) algorithm (10 iterations) were 1.36 mm at the center and 1.96 (radial) and 2.13 (tangential) at 2-cm radial offset. Sensitivity was > 2.0 % after correcting for position escape. Maximal noise equivalent count rate was 10.3 kcps at the rate of 52.2 MBq (1.41 mCi) total activity for the rat phantom. Ten minutes after 11C-(R)-PK11195 injection, static images were acquired for 20 min x 4 in two bed positions covering from nose to chest (10 min for each position). The animals then underwent computed tomography (CT 20-min acquisition) in the same bed position immediately after PET using a dedicated small-animal single-photon emission computed tomography camera (Gamma Medica X-SPECT, Gamma Medica, Northridge, CA) for anatomical imaging. PET images were reconstructed by 3D OSEM using the ATLAS software interface. Regions of interest were drawn manually over the whole brain, and averaged signal intensity per pixel was measured using the Image J software interface (NIH).
Statistical analysis.
Multiple means comparison of 3H-(R)-PK11195 autoradiography results were done by one-way ANOVA followed by Student-Newman-Keuls post hoc test where significance was indicated. The results of 11C-(R)-PK11195 PET were analyzed by one-way ANOVA with repeated measure followed by least-squares means post-hoc test. Significance level was set at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PBR Levels in the Corpus Callosum in Demyelination and Remyelination
Quantitative film autoradiography of the PBR-selective ligand 3H-(R)-PK11195 was used to measure PBR levels in the corpus callosum (see Fig. 2-1). 3H-(R)-PK11195 binding to PBR was markedly increased (412 ± 40%) following a 3-week exposure to the 0.2% cuprizone diet relative to controls (Figs. 2-1A, 2-1B, and 2-2). Once the animals were removed from the cuprizone exposure, PBR levels in the corpus callosum decreased gradually after 3 and 6 weeks of remyelination (Figs. 2-1C, 2-1D, and 2-2). However, even at 6 weeks of remyelination, PBR levels in the corpus callosum remained elevated relative to controls, suggesting that recovery was not complete at this time (Figs. 2-1D and 2-2).
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PBR Levels Correlate with the Levels of Myelin and Glial Cell Activation
Black-Gold histochemistry was used as a qualitative method of assessing myelin content in the corpus callosum at the end of the demyelination and in the remyelination phase. The intensity of red color in brain slices represents the level of myelin content (Fig. 3). Severe demyelination was noted at 3 weeks with a partial improvement present at 3 weeks of remyelination, and myelin content appeared to increase at 6 weeks of remyelination. However, even at 6 weeks of recovery, there was a significant level of myelin loss remaining relative to the control condition (Fig. 3). Comparing the level of myelination with PBR levels in the corpus callosum indicates an inverse relationship between the PBR response and myelin content (compare Figs. 2-1 and 3). That is, PBR levels are dramatically increased at the time point of maximal demyelination and decrease as remyelination occurred.
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In order to understand the cellular sources of the PBR response during the remyelination phase, we used Mac-1 immunohistochemistry to assess microglia and GFAP for astrocytes. Figure 4 shows the temporal expression of microglia and astrocyte activation in the corpus callosum of control at 3 weeks of demyelination and at 3 and 6 weeks of remyelination. Microglia in the control brain express a "resting" morphology with small cell bodies and highly ramified processes (Fig. 4-1A). Microglia activation is dramatically increased at 3 weeks of demyelination relative to the control brain (Fig. 4-1B). Microgliosis comprises an increase in the number of microglia, formation of microglia clusters, enhanced expression of surface marker (Mac-1) with strong immunostaining, and changes in morphology with "bushy"-appearing cells as well as round phagocytic cells (see Fig. 4-1B). After 3 weeks of remyelination, the microglial response in the corpus callosum was dramatically decreased (Fig. 4-1C) with a limited number of activated microglia, and their appearance was virtually normal at 6 weeks of remyelination (Fig. 4-1D). On the other hand, astrocyte activation is dramatically increased at 3 weeks of demyelination, but it appears to have a more gradual decline of their activation state during remyelination (Fig. 4-2A–4-2D). In contrast to microglia, there are still a relatively greater number of activated astrocytes after 6 weeks of remyelination (Fig. 4-2D).
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Small-Animal PET Imaging of Global PBR Levels in the Mouse Brain
In order to apply the PBR as a marker of glial cell activation in the living mouse brain, we performed 11C-(R)-PK11195 PET using a dedicated small-animal PET imaging system. We show that in vivo imaging is able to visualize increased PBR levels in the brain after 0.2% cuprizone treatment for 4 weeks relative to control (Fig. 5-1). The quantification of PBR levels in the whole brain showed a 73–175% increase of 11C-(R)-PK11195 accumulation in the cuprizone-exposed brain at different time points after tracer injection relative to control values (Fig. 5-2). The same animals exposed to cuprizone were removed from the exposure and assessed again at 9–10 weeks of recovery. Figure 5-1 indicates that the level of PBR expression was significantly decreased to those measured in control animals, suggesting that at this time a full recovery had occurred consistent with the known pattern of remyelination (Matsushima and Morell, 2001
). Unfortunately, we were not able to obtain the small-animal imaging results at the same time points as the in vitro autoradiography studies due to scheduling difficulties for the instrumentation and the radioligand synthesis. However, the results indicate that small-animal PET imaging provides a new and important approach to visualize and measure PBR expression in the living brain during demyelination and remyelination.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study demonstrates the temporal and cellular patterns of the PBR response in the corpus callosum during the remyelination phase of cuprizone-induced demyelination. Consistent with our previous findings (Chen et al., 2004
), our present results also show that PBR levels track the degree of glial cell activation not only during demyelination but also during remyelination. Importantly, during recovery from demyelination we noted a very rapid decline in the number of activated microglia relative to astrocytes in the corpus callosum. For example, at 6 weeks of remyelination, microglia are not only reduced in number but the remaining cells expressed a more normal morphology than at 3 weeks of remyelination (Fig. 4-1D). On the other hand, the number of activated astrocytes remains at a higher level (Fig. 4-2D). This pattern of microglia and astrocyte activation during remyelination has been observed by other investigators in this same model (Matsushima and Morell, 2001; Plant et al., 2005). These findings suggest that during remyelination the microglial contribution to the PBR response decays rapidly, and at the later time points of recovery, the majority of the 3H-(R)-PK11195 binding to PBR is associated with astrocytes.
These findings, together with previous studies (Balaban et al., 1988; Chen et al., 2004; Kuhlmann and Guilarte, 2000), support the notion that both glial cell types are responsible for changes in PBR levels during brain injury and recovery, but their contribution to the PBR signal changes with time. This pattern of glial response to neuropathological events appears to be a programmed response of the CNS to injurious events. One potential explanation to the different patterns of glial cell activation and their temporal contribution to the PBR signal is that they serve different functions in brain injury and recovery.
At present, there is a lack of knowledge on the functional significance of increased PBR levels in glial cells following neuronal injury or in recovery and whether the enhanced PBR expression in microglia serves similar functions in astrocytes. For example, microglia constantly survey their environment and have the ability to respond to brain injury within minutes by directing their ramifications to the site of damage (Nimmerjahn et al., 2005). Microglia are also able to proliferate and migrate to the sites of brain injury (Kreutzberg, 1996; Streit, 2000; Streit et al., 1988, 1999), characteristics that are not possessed by astrocytes (Norton et al., 1992). Previous studies indicate that PBR ligands can influence both the rate of DNA synthesis and the chemotactic potential of breast cancer cell lines (Hardwick et al., 1999), gliomas (Miccoli et al., 1999) and hepatic tumor cell lines (Corsi et al., 2005). Further, PBR ligands have been shown to modulate chemotaxis and phagocytosis in peripheral monocytes and neutrophils (Cosentino et al., 2000; Marino et al., 2001; Ruff et al., 1985). Since microglia are the monocytes/macrophages of the brain (Kreutzberg, 1996; Streit, 2000; Streit et al., 1988, 1999), it is possible that injury-induced upregulation of PBR levels in microglia may be associated with the proliferative, migratory, and phagocytic capacity of microglia, characteristics that are essential for the microglial response to injury. Another potential role of increased PBR expression in microglia may be related to the secretion of inflammatory cytokines. Choi et al. (2002) have shown that the PBR antagonist PK11195 can inhibit lipopolysaccharide-induced increases in cyclooxygenase-2 and tumor necrosis factor-
levels in cultured human microglia (Choi et al., 2002). Further, PK11195 can reduce the expression of proinflammatory cytokines and neuronal death in the quinolinic acid–injected rat striatum (Ryu et al., 2005).
Glial cells also increase PBR level following injury to increase neurosteroid synthesis at the sites of damage. 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 neurotropic and neuroprotective activities (Le Goascogne et al., 2000; Schumacher et al., 2000; Veiga et al., 2005). Relevant to a potential difference in the function of the PBR in microglia and astrocytes, it has been noted that astrocytes but not microglia are capable of synthesizing neurosteroids in culture (Cascio et al., 2000).
Emerging evidence suggests that the administration of PBR-selective ligands may be useful in the treatment of inflammatory conditions (Torres et al., 2000) as well as attenuation of seizures and brain injury (Ferzaz et al., 2002; Ryu et al., 2005; Veenman et al., 2002; Veiga et al., 2005). The exact mechanisms by which these protective effects of PBR-specific ligands confer protection are not precisely known. However, associations between PBR activation and stimulation of neurosteroid synthesis have been noted (Lacapere and Papadopoulos, 2003). Cascio et al. (2000) have shown a correlation between PBR expression, steroid synthesis, myelination, and oligodendrocyte differentiation. Thus, PBR activation may assist in the recovery from injury that produces demyelination. Consistent with this hypothesis, Lacor et al. (1999) have shown that PBR levels and DBI (diazepam binding inhibitor—the putative endogenous ligand of PBR) are increased after peripheral nerve injury. Following regeneration, PBR and DBI levels decreased to normal levels, and in the absence of regeneration, PBR and DBI levels remained elevated. In the same studies, they also showed that PBR activation by an exogenous ligand Ro5-4864 increased pregnenolone levels in the injured tissue. These findings strongly suggest that PBR plays an important role in regeneration and that neurosteroid synthesis may be a trophic factor in recovery from nerve injury.
In the present study we also demonstrate the potential usefulness of small-animal PET imaging in monitoring "global" PBR levels in the living mouse brain in this model of demyelination and remyelination. While PBR levels have been visualized and measured in several human neurodegenerative disorders (Banati et al., 2000; Cagnin et al., 2001a,b; Versijpt et al., 2000), there is a lack of studies showing its utility in small animals such as rodents. The use of PBR PET imaging in the living mouse brain provides a significant advance in monitoring brain injury and recovery during demyelination and remyelination in the same animal. This approach may be useful in studying the function of the PBR in animal models of chemical-induced neurotoxicity, providing a useful in vivo biomarker of neurotoxicity. The use of small-animal imaging provides a novel approach to examine the effects of neurotoxicants on the brain on a longitudinal fashion since the same animal can be imaged repeatedly over time. Further, the use of small-animal imaging significantly reduces the number of animals needed since the same animal can be used at multiple time points.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant ES07062 to T.R.G. The authors would like to thank Mr James Fox and Dr Martin Pomper for their assistance with the small-animal PET imaging studies. The work was performed in partial fulfillment of doctoral degree requirements for M.-K.C.
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