A microPET study of the regional distribution of [11C]-PK11195 binding following temporary focal cerebral ischemia in the rat. Correlation with post mortem mapping of microglia activation
Highlights
► Microglia activation (MA) has both neurotoxic and prorepair effects after stroke. ► Does the regional distribution of 11C-PK11195 correlate with in vitro MA post stroke? ► In rats we found significant correlation between Day 14 11C-PK11195 and OX42 staining. ► Statistically significant 11C-PK11195 binding increases were also present at Day 2. ► These results further validate the use of 11C-PK11195 PET for MA imaging after stroke.
Introduction
After a stroke, microglial activation (MA) may be neurotoxic but may also facilitate neuroplasticity in the peri-infarct zone (del Zoppo et al., 2001). MA is therefore a potential therapeutic target, and as such an important pathophysiological process to investigate after stroke. Using selective immunohistochemistry (IHC) markers such as OX42, an antibody which binds to CD11, a protein selectively expressed by activated microglia, MA can be documented post mortem after MCA occlusion (MCAo) in rodents in both the infarct and the peri-infarct areas (Hughes et al., 2010, Lehrmann et al., 1997, Myers et al., 1991, Schroeter et al., 2009). MA can also be studied using radioligands specific to the translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor, PBR), which is also massively expressed by activated microglia (Banati, 2002). Previous work in an axotomy model has demonstrated the association between in vitro CD11 and TSPO binding (Pedersen et al., 2006). Using the reference ligand 3H-[R]-PK11195 (Chauveau et al., 2008), which co-localizes to resident activated microglia as shown by emulsion autoradiographic (ARG) studies (Banati et al., 1997), seminal rodent MCAo studies documented several-fold increases in TSPO binding relative to normal brain (Benavides et al., 1990, Dubois et al., 1988, Myers et al., 1991).
PK11195 was subsequently labeled with 11C for PET applications (Chauveau et al., 2008). Interestingly, although PET studies using 11C- PK11195 have consistently reported low specific binding in the normal brain across species, 3H-PK11195 binding is substantial in vitro (e.g. Bmax range in gray matter structures ~ 200–400 pmol/g in the mouse and 200–600 pmol/g in the pig) (Cumming et al., 2006, Pedersen et al., 2006), suggesting some state difference in the TSPO between the in vivo and in vitro conditions (Cumming et al., 2006). Increased 11C-PK11195 binding has been consistently reported after stroke in both non-human primates and man (Gerhard et al., 2000, Gerhard et al., 2005, Price et al., 2006, Ramsay et al., 1992, Sette et al., 1993, Thiel et al., 2010), and similar findings have been recently reported using microPET in rodents (Fukumoto et al., 2011, Rojas et al., 2007, Schroeter et al., 2009).
In order to fully exploit the potential of 11C-PK11195 as an in vivo marker of MA in stroke, it is important to ensure that its binding as assessed with PET does reflect MA, as expected from the above studies linking CD11 to TSPO binding (Pedersen et al., 2006). In vitro or ex vivo studies using 3H-PK11195 in rodent models of cerebral ischemia have reported co-localization of the radiotracer with resident microglia and macrophages (Myers et al., 1991, Stephenson et al., 1995) as well as a topographical correlation with IHC markers of MA but not GFAP (Myers et al., 1991). In two recent PET studies, 11C-PK11195 uptake showed overall topographical congruence with post mortem OX 42 at the microscopic level, but a direct correlation using the same regions-of-interest (ROIs) in the same subjects was not reported (Fukumoto et al., 2011, Rojas et al., 2007), while another study found congruence in the peri-infarct areas but not in the core (Schroeter et al., 2009). Thus, no study so far has directly investigated the relationship between the regional distribution of 11C-PK11195 binding assessed with PET, and that of MA assessed post mortem using IHC, in the same ROIs and subjects and at the spatial resolution of PET, after experimental stroke.
Using OX42, we recently documented the occurrence of extensive MA two weeks after 45 min temporary MCAo (tMCAo) in spontaneously hypertensive rats (SHRs), involving both the infarct and non-infarcted areas acutely affected by penumbral hypoperfusion (Hughes et al., 2010). Here we report the regional distribution of 11C-PK11195 assessed in the same series of SHRs scanned at Day 14 post-MCAo, an optimal time to detect MA based on ARG studies (Benavides et al., 1990, Cremer et al., 1992), and investigate the correlation between 11C-PK11195 binding and post mortem OX42 throughout the whole MCA territory using ROIs of size consistent with PET resolution. To address this aim, we employed our previously validated semi-quantitative method for assessing IHC within a template of ROIs spanning the MCA territory, designed to be comprehensive, allow correlation with PET data, and at the same time avoid operator bias as far as possible (Hughes et al., 2010). In addition, PET was also performed in the same subjects at day 2 to address discrepancies in the literature regarding 11C-PK11195 findings at this very early time point (Fukumoto et al., 2011, Myers et al., 1991, Ramsay et al., 1992, Rojas et al., 2007, Sette et al., 1993).
Section snippets
Materials and methods
To comply with UK Home Office regulations, this study was designed so the number of animals used be as small as possible yet adequate for scientifically robust results. Thus, the planned 11C-PK11195-OX42 relationship was to be tested not by group comparison but within animals. Accordingly, a sample of six rats was judged adequate. All animals were treated in accordance with the UK Animals Scientific Procedures Act 1986 and Cambridge University Ethical Review Panel.
According to a longitudinal
Results
Due to unreliable synthesis of 11C-PK11195, the number of successful PET studies was n = 5 at day 2 (rats 1, 3, 4, 5 and 6) and n = 4 at day 14 (rats 2, 3, 5 and 6). Thus, three rats (rats 3, 5 and 6) had successful PK scans at both time-points.
Discussion
We report a strong correlation in the expected biological direction between in vivo PKday14 binding measured across 44 ROIs spanning the affected hemisphere and post-mortem OX42 binding across the same ROIs and same rats. This provides further evidence, for the first time at the PET resolution level, of the validity of 11C-PK11195 as an imaging marker of MA following stroke, previously documented at the cellular and microscopic levels only. We also found that 11C-PK11195 allows mapping of MA
Acknowledgments
Work supported by MRC grant G0001219, EU-funded EUSTROKE grant Health-F2-2008-2022131 and an equipment grant from Pfizer Ltd to JSB. JLH was funded by an MRC Studentship. PSJ and DKM were supported by the Cambridge Biomedical Research Centre. The help of Tulasi Marrapu with the illustrations is also warmly acknowledged.
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These two authors contributed equally to this work.