Research ReportC-reactive protein expression in a rodent model of chronic cerebral hypoperfusion
Highlights
► Chronic cerebral hypoperfusion causes white matter damage in Long Evans rats. ► C-reactive protein (CRP) was detected at the protein and mRNA level in the brain of rats subjected to chronic cerebral hypoperfusion. ► CRP mRNA and protein was detected in cultured rat brain microglia.
Introduction
White matter lesions (WML) are a manifestation of cerebral small vessel disease, which is one of the common causes of vascular dementia (Black, 2005). They appear as hyper intense signals on T2-weighted magnetic resonance images (MRI) and occur in 94% of the elderly population aged 64 and over (Simpson, JE et al., 2007). With the widespread use of this imaging technique it has become apparent that many individuals can have a significant amount of white matter change without having had a clinically recognized transient ischemic attack (TIA) or complete stroke (Pohjasvaara et al., 2000). In addition, the presence of more severe white matter lesions increases stroke risk (Bokura et al., 2006).
Recent research has been focused on identifying parameters which may have a correlation with WML. To this effect C-reactive protein (CRP) has been proposed as a candidate, however, its definite relation has yet to be elucidated. C-reactive protein (CRP), named for its capacity to precipitate the somatic C-polysaccharide of Streptococcus pneumonia (Tillett and Francis, 1930), was the first acute-phase protein to be described (Hirschfield and Pepys, 2003). Plasma CRP is produced only by hepatocytes (Pepys and Hirschfield, 2003). Extra hepatic synthesis of CRP has also been reported in neurons in Alzheimer patients (Yasojima et al., 2000), atherosclerotic plaques (Jialal et al., 2004), lymphocytes (Kuta and Baum, 1986), alveolar macrophages (Dong and Wright, 1996), aortic endothelial cells (Venugopal et al., 2005), coronary artery smooth muscle cells (Calabro et al., 2003), and adipocytes (Calabro et al., 2005). Induction of CRP in hepatocytes is principally regulated at the transcriptional level by the cytokine interleukin-6 (IL-6), an effect which can be enhanced by interleukin-1β (IL-1β) (Kushner et al., 1995). More recently some population based studies have explored the relation between serum CRP and WML and, while some reported an association between the two (Fornage et al., 2008, van Dijk et al., 2005), others did not find an apparent correlation (Schmidt et al., 2005, Wada et al., 2008). Reasons for these controversial results were attributed to differences in the participant's health status (Schmidt et al., 2005) and to the ethnic status (Wada et al., 2008). It is clear that the findings from the population-based studies need to be explored further. To the best of our knowledge the study of CRP in an animal model of chronic cerebral hypoperfusion manifesting WML has not been previously documented.
Microglia, the surveillance cells of the brain, have been shown to attain an activated state in association with WML in rat brain (Farkas et al., 2004) and Simpson et al. (2007) have reported the presence of activated microglia in both periventricular and deep subcortical WML in human brain. The direct relation between microglial activity and WML suggests an important role for the known and “unknown” effectors of microglia in these processes. White matter injury and microglial activation have been reported to occur in a rodent of chronic cerebral hypoperfusion (Farkas et al., 2004, Wakita et al., 1994). Previous studies using this model, where both common carotid arteries are permanently ligated, have reported WM rarefaction (vacuolation), axonal disruption, gliosis and microglial activation in lesional areas (Simpson et al., 2007, Wakita et al., 1994, Wakita et al., 2002). In the present study we have used this model to study the association between microglial activation, WML and CRP expression.
Section snippets
Characteristics of WM injury following BCCAL
H&E staining of paraffin embedded sections showed an increase in WM vacuolation in the optic tract as early as 3 days after BCCAL (Fig. 1C). Vacuolation increased throughout the time period of the study and at 30 days vacuoles occupied 9.62 ± 0.70% of the total area of the optic tract seen in paraffin sections, while the area occupied by vacuoles in sham operated rats was 0.83 ± 0.13% (Fig. 1C). In the corpus callosum vacuolation was not as severe as that found in the optic tract but was evident from
Discussion
The aim of the present study was to assess the expression of the acute phase protein CRP in brain tissue in a rodent model of chronic cerebral hypoperfusion. The vast majority of studies using the rodent model of BCCAO have used the Sprague Dawley or Wistar strains of rat. In the present study the Long Evans strain was used and the characteristic features of white matter rarefaction (vacuolation), astro- and microgliosis and axonal damage were evident, as previously reported in the literature
Establishment of permanent bilateral common carotid artery ligation (BCCAL) model
All experiments conformed to the guidelines set forth by the Canadian Council for the Use and Care of Animals in Research (CCAC) with approval from the Ottawa Health Research Institute Animal Care Facility. An animal model of chronic hypoperfusion, which experimentally induces WML, was reproduced through BCCAL procedure (Wakita et al., 1994) conducted on Long Evans rats (weighing 350–450 g, Charles River, Saint-Constant, Quebec, Canada). Animals were anesthetized with isoflurane (5% induction,
Acknowledgments
Funds for this study were provided by the Heart and Stroke Foundation of Ontario Centre for Stroke Recovery and the Canadian Stroke Network. The authors would like to thank Kim Wong, Department of Cellular and Molecular Medicine, University of Ottawa and Louise Pelletier and Li Dong, Department of Pathology, Faculty of Medicine, University of Ottawa, for technical assistance.
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