Imaging Microglia in the Central Nervous System: Past, Present and Future
3 Challenges/Perspective: Imaging Microglia in Animal Models of CNS Disease
52 D. Davalos and K. Akassoglou
scale of several hours to days in accordance with typical immunological response paradigms. The constant motility of microglial processes in unperturbed conditions implies a mechanism that is acutely regulated within the brain tissue.
Nimmerjahn et al. explored the possibility that neuronal or synaptic activity could be regulating the baseline microglial dynamics. By using the Na+-channel blocker tetrodotoxin (TTX) to reduce neuronal activity they found no significant effect on microglial activity. On the other hand, enhancing synaptic activity by using the GABA-receptor blocker bicuculline had a slightly stimulating effect on microglial processes. However, since bicuculline also elicits seizure activity, the motility-enhancing effect that was observed could have also been due to the incipi-ent cortical damage (Raivich, 2005). Davalos et al. reasoned that the directional convergence of microglial processes towards the trauma implies the presence of a gradient of one or more highly diffusible and abundant molecules that can mediate this phenomenon. Using a combination of inhibitory and activating approaches while imaging microglial responses in vivo they demonstrated that extracellular ATP and activation of P2Y receptors are necessary for the rapid microglial response towards injury. Interestingly, the baseline activity of microglial processes was shown to be also affected by pharmacological inhibitors of purinergic signaling, indicating that there could be a uniform mechanism in place that controls microglial motility under both normal and injury conditions. Moreover, these results appear to suggest a role of the surrounding tissue in preserving and regenerating the signals that fuel the observed microglial response to damage. Although neurons, oli-godendrocytes and endothelial cells are likely to release large amounts of ATP upon injury and thereby contribute to microglial reaction, they provided evidence to suggest that astrocytes may play an important role in mediating the rapid and widespread microglial response (Davalos et al., 2005). The study of this phenome-non directly inside the intact brain, where all the potential players are present and able to perform their roles as they are faced with an experimental challenge, allowed the identification of a very intriguing cell-cell interaction between astro-cytes and microglia in an orchestrated attempt to respond to the incurred tissue damage (Davalos et al., 2005).
3 Challenges/Perspective: Imaging Microglia in Animal
Imaging Microglia in the Central Nervous System: Past Present and Future 53
The technical ability to relocate and re-image a specific area in the living brain revealed the progression of microglia responses, which is particularly important in understanding the mechanisms of disease in the nervous system.
In vivo imaging can be further applied to study the involvement of microglia in multiple animal models of disease pathogenesis. Microglia have been shown to be involved in a variety of diseases, such as Alzheimer’s disease (AD), multiple scle-rosis (MS), HIV and other infections of the central nervous system. Overall, their prolonged intervention has been found to be exacerbating rather than improving the tissue condition (Gonzalez-Scarano and Baltuch, 1999; Stoll and Jander, 1999).
Breakthrough studies have applied in vivo two-photon microscopy in AD, ischemia and seizures (for review see (Misgeld and Kerschensteiner, 2006) ). For example in AD, the contribution of amyloid plaques to neurodegeneration has been documented in a pioneering study using mice with fluorescently labeled neurons and transcranial two-photon imaging (Tsai et al., 2004). In this study it was shown that dendrites passing through or near amyloid deposits undergo spine loss, and nearby axons develop large varicosities, leading to neurite breakage and permanent disruption of neuronal connections (Tsai et al., 2004). The ability to repetitively image the brain in vivo allowed for the first time to study the responses of neurons to a toxic stimulus over time. Although correlations between neuronal damage and beta-amyloid had been previously made with conventional histopa-thology, the use of two-photon microscopy allowed the assessment of the dynamics between neurodegeneration and a toxic stimulus in the CNS. It has been proposed that similar studies could be performed to observe and correlate micro-glial activation in AD and other neurodegenerative models (Misgeld and Kerschensteiner, 2006).
Microglial activation is a hallmark of neuroinflammatory diseases such as MS. MS lesions are heterogeneous and characterized by infiltration of immune cells into the CNS parenchyma, destruction of myelin and axonal damage (Lassmann et al., 2001; 2007). Microglia play a central role in this processes, due to their ability to phagocytose myelin via the CD11b/CD18 integrin receptor (van der Laan et al., 1996) and secrete proinflammatory cytokines that are actively involved in demyelination (Platten and Steinman, 2005). Recently, the blood protein fibrino-gen was identified as the CD11b/CD18 ligand that drives microglial activation and phagocytosis (Adams et al., 2007). Genetic or pharmacologic inhibition of the bind-ing of fibrinogen to the microglial CD11b/CD18 integrin suppressed microglial activation and relapsing paralysis in experimental autoimmune encephalomyelitis (EAE), an established animal model of MS (Adams et al., 2007). These studies proposed fibrinogen as a “danger signal” that triggers microglial activation after BBB disruption (Adams et al., 2007). Interestingly, in human MS microglial activation in areas of demyelinating plaques correlates with areas of deposition of fibrinogen due to BBB disruption (Gay et al., 1997), which is one of the earliest histopathologic alterations in MS lesions (Vos et al., 2005). Recent studies have demonstrated that microglia not only act as phagocytes, but they are also involved in the onset of inflammatory demyelination in CNS autoimmune disease (Heppner et al., 2005). Overall, these studies suggest that signals, such as fibrinogen,
54 D. Davalos and K. Akassoglou
that may initiate microglial activation could be crucial for the onset of disease pathogenesis in the CNS.
Although several imaging studies have been applied in different animal models of neurodegenerative disease, in vivo imaging of neuroinflammation using two-photon microscopy has not been performed. Imaging in animal models of inflam-matory demyelination such as EAE, has been either limited to the vasculature with the use of intravital microscopy (Vajkoczy et al., 2001), or performed ex vivo using tissue explants (Kawakami et al., 2005; Nitsch et al., 2004). Inflammatory demy-elination is a complex pathological setting where microglia can exchange signals with both neuronal and immune cells (Carson, 2002). Given the immunologic prop-erties of microglia and their complex interactions with cells on both sides of the BBB, imaging their functions in EAE can reveal crucial information regarding their dual identity as immune cells that reside within the central nervous system. For example, imaging microglia in EAE could identify the link between microglial activation and the onset of inflammatory demyelination. Overall, establishing an in vivo imaging technique in animal models of inflammatory demyelination could reshape our understanding of the pathophysiological role of microglia within demyelinating lesions and reveal the cell-cell interactions that could develop during an inflammatory attack that leads to nervous system damage.
From an imaging perspective, two-photon microscopy in demyelinated areas presents several challenges. First, myelin – and as a result inflammatory demyelina-tion – is located in deep layers of the brain. In EAE, a “hot spot” for demyelinademyelina-tion in the brain is the cerebellar white matter. Therefore, by contrast to animal models of AD where the pathology is mostly localized in the mouse cortex, inflammation in animal models of MS is localized deep into the brain tissue. Given the depth limitations of even two-photon microscopy, it would be extremely challenging to apply this technique for imaging demyelination (Svoboda and Yasuda, 2006).
Second, although demyelinating lesions in the spinal cord would be an ideal site to study microglial responses in vivo, limited work has been performed on imaging the living spinal cord to date, due mainly to the movement artifacts generated by the heartbeat and the breathing of the animal. Regarding the brain, alternative approaches, such as intravital microscopy or regeneratively amplified two-photon microscopy (Helmchen and Denk, 2005; Theer et al., 2003) could possibly be effi-cacious in imaging microglia located within the demyelinated white matter.
Regarding the spinal cord, the development of novel techniques that would allow the stable and repetitive imaging of the mouse spinal cord using two-photon micro-scopy would be essential to decipher the role of microglia within the demyelinated lesions. Therefore, due to the distinct characteristics of the location of neuroimmu-nologic lesions, their imaging requires further advances in both microscopy and surgery. Future research should take into account the specific features of inflamma-tory demyelination and aim towards the development of novel methodologies to access inflammatory foci in the CNS in ways that would allow their study by two-photon microscopy. Performing in vivo imaging of microglia in the presence of neuronal and inflammatory stimuli can further correlate microglial functions with the progression of inflammation, demyelination and neuronal damage. Moreover,
Imaging Microglia in the Central Nervous System: Past Present and Future 55
the application of such novel imaging approaches to study neuroinflammatory lesions can be crucial for identifying and testing potential pharmacological targets and regulate microglial activation within demyelinating lesions.
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