Characterizing infarction and selective neuronal loss following temporary focal cerebral ischemia in the rat: A multi-modality imaging study
Highlights
► Tissue outcome post stroke includes infarction but also selective neuronal loss (SNL) ► It would be useful to have an in vivo surrogate for this range of tissue outcomes ► We obtained T2 MR, 11C-flumazenil PET and post mortem 28 days after MCAo in rats ► PET, but not MR, detected both infarction and SNL, and appears a valid surrogate.
Introduction
According to the dominant “core/penumbra” model of anterior circulation stroke, the acutely ischemic brain comprises both a core of rapidly irreversibly damaged tissue and a less severely hypoperfused, mainly cortical area termed the ischemic penumbra, which is still viable but will inevitably undergo complete tissue infarction (i.e., “pan-necrosis” Auer, 2008, Garcia et al., 1996a, Garcia et al., 1996b), unless rapidly reperfused (Astrup et al., 1981, Baron, 1999, Moskowitz et al., 2010). Given that final infarct volume strongly impacts neurological outcome (Brott et al., 1989), rescuing the penumbra has major clinical relevance (Baron, 1999, Furlan et al., 1996) and accordingly acutely administered intravenous thrombolysis strongly improves functional outcome (Lees et al., 2010).
Despite this undisputed operational validity of the core/penumbra model, studies in rodents have demonstrated that following temporary middle cerebral artery occlusion (tMCAo) tissue outcome is not dichotomic but can range from pan-necrosis right through to subtle changes in dendritic arborization, with selective neuronal loss (SNL) and partial infarction as intermediate stages (Auer, 2008, DeGirolami et al., 1984, Garcia et al., 1996a, Garcia et al., 1996b, Heiss and Rosner, 1983, Ito et al., 2007, Jones et al., 1981, Li et al., 1999). Of particular interest is SNL involving the penumbra rescued from infarction (Garcia et al., 1996b, Garcia et al., 1997, Hughes et al., 2010, Li et al., 1999, Li et al., 2000, Sicard et al., 2006a). Although SNL does impact early behavioral performance in rodent models (Sicard et al., 2006a, Sicard et al., 2006b), its long-term effects, particularly if cortical areas are affected, are less clearly established. Specifically, given the major role of peri-infarct plastic processes (Furlan et al., 1996, Jaillard et al., 2005), it is likely that SNL impedes optimal plasticity post-stroke and hence delays or dampens behavioral recovery. In turn, SNL may represent a novel therapeutic target in the post-acute phase of ischemic stroke (Baron, 2005).
In order to explore the pathophysiology of ischemic stroke, determine potential therapeutic targets and monitor effects of therapy, in vivo imaging surrogates of post-tMCAo histopathological tissue outcomes, including SNL, applicable in the clinical setting would be extremely useful. For instance, using T2-weighted MRI in the chronic post-stroke stage, hyperintense signal is considered a reliable surrogate for tissue infarction, based on studies in rodents (Jacobs et al., 2000, Ringer et al., 2001, van Lookeren Campagne et al., 1999, Wagner et al., 2012), and accordingly is the standard in the clinical setting (or its FLAIR variant, which however may be over-sensitive). Noteworthy, SNL is not known to be associated with abnormal signal on follow-up T2-weighted imaging in the rodent (Sicard et al., 2006a, Sicard et al., 2006b). In the clinical setting, peri-infarct SNL in the chronic post-stroke stage has been inferred time and again from observations of mildly reduced specific binding of the neuron-specific PET ligand 11C-flumazenil (FMZ) — or of its SPECT derivatives — as compared to markedly reduced within infarcted areas (Dong et al., 1997, Guadagno et al., 2008, Hatazawa et al., 1995, Nakagawara et al., 1997, Saur et al., 2006, Yamauchi et al., 2007, Yamauchi et al., 2009). However, the inference that reduced FMZ binding is a valid surrogate of chronic post-stroke ischemic damage including SNL has not received histopathological confirmation so far.
In the present study, we assessed the long-term (28 days post-stroke) sequelae of temporary focal cerebral ischemia in the rat using T2-weighted MRI and FMZ-PET as in vivo imaging markers, against post mortem immunohistochemistry (IHC) as a gold standard to characterize neuronal and tissue damage, from SNL to frank infarction. Even though technically challenging relative to standard proximal MCAo models, we implemented here the distal clip MCAo model (Buchan et al., 1992, Kaplan et al., 1991), given our specific interest in cortical pathology; another advantage of the clip model relative to e.g., the filament model is that it allows direct verification of restoration of blood flow through the MCA following clip removal. Likewise, despite their higher cost, we elected to use spontaneously hypertensive rats (SHRs) in order to enhance our study's clinical relevance (Duverger and MacKenzie, 1988). Our choice of 28 days for the PET and IHC assessments was dictated by our interest here in established, as opposed to potentially still evolving, ischemic lesions, particularly given occasional reports of delayed “infarct maturation” up to 3 weeks after the ischemic insult in SHRs (Buchan, 2001, Lehrmann et al., 1997).
Section snippets
Materials and methods
Eight adult male SHRs, 10–12 weeks of age, were used. All studies were conducted in accordance with the UK Animals Scientific Procedures Act 1986 and The University of Cambridge Ethical Review Panel. Importantly, to comply with these regulations, the study was designed so as to keep the number of animals used to a minimum, yet sufficient to obtain meaningful results. All rats were subjected to 45 min distal clip tMCAo, followed by 11C-FMZ PET and MRI 28 days later. Immediately after completion of
Histopathology and Immunohistochemistry
Fig. 1, Fig. 2, Fig. 3 illustrate typical exemples of pan-necrosis, partial infarction and SNL in rats 1, 6 and 3, respectively. Fig. 4 illustrates the patterns of lesions across the 8 rats, and Table 1 summarizes the occurrence and topography of these changes in each rat. Pan-necrosis was observed in all subjects except rats 3 and 5; partial infarction in all rats except 3; and SNL also in all rats, but in isolation in rat 3 (see Fig. 3).
When SNL was associated with infarction, it had the
Discussion
The aim of this study was to characterize the long-term sequelae of 45 min distal tMCAo in SHRs with emphasis on SNL vs infarction, using IHC, T2-weighted MRI and FMZ-PET, all obtained in the same animals. Three main findings emerged. First, histopathological cortical changes as depicted by NeuN IHC included all three subtypes of SNL (patchy or band-like), partial infarction and frank infarction, variably associated across subjects. Second, the MRI findings differed between SNL and complete or
Conclusion
In this study, we characterized the long-term sequelae of 45 min distal tMCAo in SHRs with emphasis on SNL vs infarction, using multi-modality in vivo imaging against post mortem neuron-specific IHC. Histopathology disclosed a mix of SNL, partial infarction and frank infarction variably associated across subjects, with one rat only showing pure SNL. These findings differ from those from our previous study where brains were collected at Day 14 that showed mainly SNL, suggesting slow infarct
Acknowledgments
This study was funded by an EU grant (EUSTROKE Health-F2-2008-2022131). TA was supported by a Marshall Fellowship.
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These two authors contributed equally to this work.