ExcerptThe brain is a heterogeneous

Excerpt
The brain is a heterogeneous tissue composed of various highly interconnected cell types. Each type has a particular pattern of expression and is differentially located in the brain regions. In some pathological situations such as brain injury, cascades of metabolic, cellular and molecular events ultimately lead to brain cell death, tissue damage and atrophy. To better understand these cellular processes it is important to obtain information regarding the spatial and temporal patterns of gene expression. In this chapter, we will describe the method of intracardial perfusion of a mouse prior to brain removal. We will then describe the detailed methodology for the detection of proteins and mRNA utilizing immunohistochemistry (IHC) and in situ hybridization (ISH) respectively in brain sections; respectively. The brain is composed of areas of gray and white matter and consists of various regions, including the cerebral cortex, the thalamus, the hypothalamus, the brain stem, and the cerebellum. The sensory areas of the cerebral cortex are involved in perception of sensory information: motor areas control execution of voluntary movements and association areas deal with complex integrative functions such as memory, personality traits, and intelligence. The limbic system promotes a range of emotions including pleasure, pain, affection, fear, and anger. The thalamus relays almost all sensory input to the cerebral cortex: it contributes to motor functions by transmitting information from the cerebellum and basal nuclei to motor areas of the cerebral cortex. It also plays a role in maintaining consciousness. The hypothalamus controls and integrates activities of the autonomic nervous system: it regulates emotional and behavioral patterns and circadian rhythms. The cerebellum smoothes and coordinates contractions of skeletal muscles, regulates posture and balance, and may have a role in cognition and language processing. The brain is a heterogeneous tissue that contains neurons, neuroglia, and other cell types that vary among anatomical regions. Nonneuronal cell types are broadly categorized into (1) astrocytes, (2) radial glia, (3) oligodendrocytes, (4) ependymal cells, and (5) microglia. The role of each cell type is well defined; moreover, their interaction is essential for the neuronal function of the central nervous system. The cellular communication is substantially involved in the establishment of the majority of neurological disorders (Pham and Gupta, 2009). Brain function is determined by the communication between electrically excitable neurons and the surrounding glial cells, which perform many tasks in the brain. Oligodendrocytes are one type of glial cell that form an insulating protective myelin sheath around the axons of neurons that enables saltatory nerve conduction. A loss of myelin in defined areas of brain leads to an impairment of axonal conductance. This is what happens in many forms of myelin disorders, such as multiple sclerosis, and it results in a permanent loss of neuron impulse transmission. It is evident that the demyelinated region contains inflammatory cells such as infiltrating lymphocytes and macrophages and activated microglia. These cells might potentiate or even initiate a damage cascade leading to continuous neurodegeneration. Microglia are the resident phagocytic cells in the brain, taking part in immune-mediated defense mechanisms and clearing damaged cell debris (Ransohoff and Cardona, 2010; Ransohoff and Perry, 2009). Previously, it was thought that microglia, in their resting state, are relatively quiescent. More recent work suggests that microglia are constantly active and surveying their surroundings (Hughes, 2012; Nimmerjahn et al., 2005). Microglia are now implicated in synapse pruning during both development and throughout adulthood, and therefore play a role in regulating homeostatic synaptic plasticity (Schafer et al., 2012). Together with astrocytes, another type of glial cell, microglia can release neuromodulatory chemicals that influence neuronal firing and intracellular signaling. When first described, astrocytes were seen merely as structural scaffolding to support and fill the gaps between neurons. However, recent evidence suggests that astrocytes serve as much more than a nutrient supply or supportive scaffolding to protect neural networks (Nedergaard et al., 2003). Astrocytes are highly secretory cells, participating in rapid brain communication by releasing factors that modulate neurotransmission (Haydon and Carmignoto, 2006; Huang et al., 2004; Pascual et al., 2012) and more recently have been suggested to possess their own repertoire of gliotransmitters (Bezzi et al., 2004; Cali et al., 2008; Cali and Bezzi, 2010; Domercq et al., 2006; Jourdain et al., 2007; Prada et al., 2011; Santello et al., 2011). Astrocytes also express a wide variety of functional neurotransmitter receptors essential for sensing neuronal activity (Verkhratsky et al., 1998). When a local inflammatory reaction is triggered in the brain, the increased levels of proinflammatory mediators such as tumor necrosis factor-alpha and prostaglandin 2 can deeply alter the properties of glial network and thus of neuronal network (Bezzi and Volterra, 2001). The important roles played by glial cells in normal and pathological brain functioning are growing, and a more complete picture of neuron–glia interactions is beginning to emerge. Cellular behaviors such as proliferation, differentiation, migration, and cell death are studied during brain development and in pathological situations. To better understand the developmental processes involved, it is important to obtain information regarding the spatial and temporal patterns of gene expression. Over the past three decades, animal models have been developed to replicate the various aspects of human brain injury to better understand the underlying pathophysiology and to explore potential treatments. Among more recent models for traumatic brain injury, four specific models are widely used in research: fluid percussion injury (Dixon et al., 1987), controlled cortical impact injury (Dixon et al., 1991; Lighthall, 1988), weight drop impact acceleration injury (Marmarou et al., 1994), and blast injury (Cernak et al., 1996; Leung et al., 2008). Rodents are mostly used in traumatic brain injury research because of their modest cost, small size, and standardized outcome measurements. In this chapter, we will describe the method of intracardial perfusion and an appropriate method to dissect and remove the brain of a mouse. We will then describe methods for the detection of protein (immunohistochemistry, IHC) and messenger RNA (mRNA) (in situ hybridization, ISH) in brain sections.