Strain influences on inflammatory pathway activation, cell infiltration and complement cascade after traumatic brain injury in the rat
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► Genetic heterogeneity influences the inflammatory response and neurodegeneration in traumatic brain injury.
Introduction
Traumatic brain injury (TBI) is one of the leading factors of neurological disability and death among young individuals worldwide. Trauma to the brain results in a primary mechanical damage characterized by disruption of the brain parenchyma and blood vessels, which initiates a complex cascade of molecular events and physiological processes. Some of these processes that take place hours and days after the primary insult may lead to further injury on nerve cells and axons, i.e. secondary brain damage. Neurological disability and clinical outcome depends on the combined extent of both acute and subacute injury processes. This provides a potential therapeutic window for efforts directed at reducing secondary neuronal damage.
In the last decades, many studies have explored the role of the immune system in TBI and it is now recognized that inflammation plays a major role in secondary injury, not only immediately after the trauma, but perhaps even after months and years (Ramlackhansingh et al., 2011). The initial impact leads to immediate cell death, which together with disruption of blood brain barrier (BBB) integrity leads to an inflammatory reaction. This reaction is characterized by activation of local glial cells, with up-regulation and release of inflammatory molecules such as complement factors, chemokines and cytokines, and infiltration of blood derived immune cells into the brain tissue (Kumar and Loane, 2012, Shlosberg et al., 2010). However, the inflammatory response is complex and both detrimental and protective aspects of diverse pathways and molecules have been described in previous studies (Morganti-Kossmann et al., 2002, Morganti-Kossmann et al., 2007).
Studies in human diseases such as multiple sclerosis and rheumatoid arthritis, where the immune system plays an instrumental role, has generated the term “complex diseases” in which disease risk is dependent on a complex interplay between environmental and genetic factors (Kim and Moudgil, 2009, McElroy and Oksenberg, 2011). Not surprisingly most of the genetic polymorphisms regulating disease susceptibility identified so far affect immune related molecules and pathways. The identification of these polymorphisms has been achieved by association studies in humans aided by the use of inbred strains and forward genetics in animal disease models (Marta et al., 2010, McElroy and Oksenberg, 2011). Although comparative strain differences have been used for years to elucidate the mechanisms regulating susceptibility to autoimmune conditions such as EAE, so far only limited efforts have been made to test if this approach is feasible also in primarily non-autoimmune disease models, such as traumatic nerve injuries (Kornblum, 1968). We have found considerable rat strain differences in the inflammatory response and outcome in previous studies on nerve, spinal cord and brain trauma, suggesting that genetic factors acting on the inflammatory response are important for outcome (Al Nimer et al., 2011, Bellander et al., 2010, Birdsall Abrams et al., 2007, Dominguez et al., 2008, Lidman et al., 2003).
In recent years there is an increasing awareness that genetic factors that vary between individuals may affect outcome of TBI (Cederberg and Siesjo, 2010, Jordan, 2007, Reid et al., 2010, Sun and Jiang, 2008, Zhou et al., 2008). Genetic variability may influence pathophysiological processes triggered by TBI, in turn of relevance for outcome and possibly also response to treatment (Dardiotis et al., 2010, Maas et al., 2010, McAllister, 2010, Zhou et al., 2008). DA (RT1av1) and PVG-RT1av1 are two inbred rat strains that share MHC haplotypes, but differ substantially in neuronal survival following TBI and ventral root avulsion (Al Nimer et al., 2012, Strom et al., 2012). We here hypothesized that genetic heterogeneity would affect the pathophysiological mechanisms and nerve cell death in a standardized brain contusion model and that it would be possible to determine quantitative and qualitative differences between DA (RT1av1) and PVG-RT1av1.
Using global expression profiling we found several differentially regulated functional networks, out of which the strongest were related to inflammation, such as inflammatory cell influx and complement activation. Further characterization of the immune cell influx and local cell activation demonstrated that the DA strain displayed greater influx of neutrophils, monocytes/macrophages, and NK cells, as well as more intense microglia activation and higher local expression of complement factors such as C1, C3 and complement receptor 2 (CR2). Higher levels of neurofilament-light in cerebrospinal fluid (CSF) suggested that increased inflammatory activation in the DA strain was associated with increased secondary nerve injury. The results strengthen the notion that genetic heterogeneity affects inflammatory regulation in primarily non-autoimmune traumatic disease models and should be further explored in order to understand the pathophysiology of TBI.
Section snippets
Animals and surgery
The DA (RT1av1) strain was originally provided by Professor Hans Hedrich (Medizinische Hochschule, Hannover, Germany), while the MHC congenic PVG-RT1av1 (hereafter called PVG) strain was obtained from Harlan UK Ltd. (Blackthorn, UK). All animals were bred in our in-house breeding facility with 12 h light/dark cycles and fed standard rodent chow and water ad libitum. Experimental traumatic brain contusion was performed in male animals weighing approximately 230–300 g, at an age of 10–14 weeks,
Global expression profiling after TBI
The pericontusional area of 5 DA and 5 PVG rats one day post-injury (dpi), as well as the corresponding area of 4 DA and 4 PVG naïve rats were analyzed with microarrays. The data were analyzed in Partek Express in order to calculate the impact of noise, TBI alone, strain alone or TBI and strain together on mRNA transcript regulation. Differences in transcript regulation were accounted by 59.1% for TBI, 24.1% for strain, 10.2% for strain regulation of TBI and 6.6% for noise; showing that
Discussion
In this study a detailed characterization of the response to an experimental brain contusion in two inbred rat strains was performed. Global expressional profiling of injured brains showed that variation in the expression of around one third of the more than 27,000 examined transcripts could be accounted to strain, if differences between naïve animals were also included. Functional classification of the microarray data showed that inflammation was one of the top biological functions activated
Acknowledgments
This study was supported by the 6th Framework Program of the European Union, NeuroproMiSe, LSHM-CT-2005-018637, EURATools, LSHG-CT-2005019015, and the 7th Framework Program of the European Union, EURATrans, HEALTH-F4-2010-241504, by the Swedish Research Council, the Swedish Brain Foundation and the Swedish Association of Persons with Neurological Disabilities.
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