Imaging Microglia in the Central Nervous System: Past, Present and Future
2 Imaging of Microglia In vivo .1 Microglial Subtypes
2.3 Studies of Microglial Activation In vivo
2.3.1 Challenging the Concept of “Resting” Microglia
Microglia display a highly branched morphology in the unperturbed cerebral cortex, with each cell soma decorated by long processes with fine termini. For decades, this morphological phenotype has been considered completely sessile;
its functional potential was found limited, compared to the activated type, which in the adult was only observed following tissue trauma (Davis et al., 1994; Stoll and Jander, 1999; Streit et al., 1988). Based solely on similarities in receptor and cytokine expression profiles with phagocytic macrophages, microglia were char-acterized as “resting” and expected to perform tissue surveillance in the CNS.
Two recent studies by Davalos et al. and Nimmerjahn et al. have challenged the term of “resting” microglia. By combining transcranial two-photon imaging (Grutzendler et al., 2002) and CX3CR1GFP/+ mice (Jung et al., 2000) in which brain microglia selectively express the enhanced green fluorescent protein (EGFP), they showed in real time a physical demonstration of their tissue sur-veillance function. It is important to emphasize that the use of two-photon microscopy is ideal for the study of microglia, since it minimizes photodamage to the living tissue which could by itself cause activation of microglia. In these studies, ramified microglia demonstrated a highly motile behavior of their higher order processes while their main branches and cell bodies remained at relatively fixed positions in the tissue (Fig. 4.1). Through cycles of small exten-sions and retractions of only their finer termini, microglia were able to patrol their territory without perturbing the densely packed and mostly stable neuronal network (Grutzendler et al., 2002). This thorough scanning of the extracellular space of the brain allows microglia to sample the tissue’s integrity on a continu-ous basis and be in a constant state of readiness to respond to any challenge, whenever it should occur.
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2.3.2 Microglial Responses to Localized Trauma In vivo
As mentioned above, studies in brain slices are limited by the fact that the slicing procedure can inherently activate microglia, induce their transformation into a mor-phologically distinct and highly reactive state (Koshinaga et al., 2000; Petersen and Dailey, 2004; Stence et al., 2001) and thereby obscure potentially important dynamic processes (Davalos et al., 2005). In order to challenge these cells in a more localized manner in vivo, both Davalos et al. and Nimmerjahn et al. took advantage of the focal properties of the two photon laser and introduced a very confined injury
0 2 4 6 8 10
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Length (μm)
Process 1 Process 2 Process 3 Process 4
Fig. 4.1 Transcranial two-photon imaging shows rapid dynamics of fine microglial processes. (Top) Time-lapse imaging of the same microglial branches demonstrates rapid extension and retraction of fine microglial processes over seconds. Circles and rounded box indicate four representative processes that change in length and shape over time. (Bottom) Length changes of the four processes marked in (a) as a function of time. Scale bar, 5 µm. Reproduced from Davalos et al. (2005). For timelapse imaging of baseline microglial dynamics see Supplementary Video 1 online at: http://
www.nature.com/neuro/journal/v8/n6/suppinfo/nn1472_S1.html.
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in the mouse cortex. They both observed a very rapid response of neighboring microglial cells within only minutes of laser-induced injury (Fig. 4.2). The micro-glial processes moved directly towards the site of injury and appeared to surround and contain it while keeping their cell bodies at their original positions. Mechanical damage of the brain tissue introduced with a glass electrode through a small crani-otomy demonstrated the traumatic nature of the laser ablation, while an ablation of two sites very close to each other showed that individual microglial processes of even the same cell can differentially respond to challenges in their close proximity (Davalos et al., 2005). When the laser ablation was performed on a blood vessel in the brain, microglia responded again in a very rapid manner and appeared to con-tain the hemorrhage by wrapping their processes around the damaged vessel wall (Nimmerjahn et al., 2005). Although it is unclear if the actual damage of the vessel wall and the cellular processes around it caused the microglial response towards the ablated vessel, it is indeed an attractive hypothesis that the disruption of the blood-brain barrier (BBB) and/or the release of blood factor(s) in the brain parenchyma can potentially activate and attract microglial processes.
Fig. 4.2 Microglial processes move rapidly towards the site of injury induced by the two-photon laser. (a–f) After creating a localized ablation inside the cortex (∼15µm in diameter) with a two-photon laser (b), nearby microglial processes respond immediately with bulbous termini (b) and extend toward the ablation until they form a spherical containment around it (c–f). At the same time, the same cells retract those of their processes that lay in directions opposite to the site of injury (arrows in d and e). Scale bar, 10 µm. Reproduced from Davalos et al. (2005). For timelapse imaging of the rapid microglial responses towards a localized injury in the living mouse brain see Supplementary Video 2 online at: http://www.nature.com/neuro/journal/v8/n6/suppinfo/nn1472_
S1.html.
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2.3.3 Challenging the Concept of Microglial “Reactive Transformation”
It is important to note that in all injury paradigms performed in both studies, the onset of microglial responses showed significant differences to activation previ-ously described in ex vivo setups. There was no microglial proliferation or migra-tion observed toward the sites of injury for at least 10 h (Davalos et al., 2005), nor did pre-existing microglial processes need to retract in order for new motile ones to form and respond to the damage. On the contrary, the so far believed inactive microglial processes showed signs of morphological activation within seconds after they were challenged, and started to extend toward each site of injury without delay.
Moreover, the ability of even individual microglial processes to identify, extend and eventually contain any tissue damage in their vicinity occurs without a morphologi-cal transformation to a reactive state and in a much faster timesmorphologi-cale than ever described before (Davalos et al., 2005). It seems that small-size damage that can be contained locally, does not require the massive mobilization described in brain slice experiments and thus the behavior of these cells in the in vivo setting is quite dif-ferent from what was previously predicted.
Finally, the studies of microglia in the living brain revealed no morphological differences between microglial subtypes in the healthy, intact or locally injured mouse cortex. Individual microglial cells exhibited similar dynamic behaviors in all the in vivo experiments, namely baseline motility, laser ablation, mechanical injury of the brain parenchyma or the vasculature (Davalos et al., 2005; Nimmerjahn et al., 2005). The distinction between perivascular and other parenchymal microglia may also be simply conditional; although some cell bodies appeared in closer proximity (or even in direct contact) with the vasculature compared to others, there was no apparent behavioral distinction between cells situated closer or further away from blood vessels, in both baseline and following injury microglial responses.
2.3.4 Studying Signaling Mechanisms in Microglia In vivo
Many studies performed on microglia in culture have attempted to correlate their molecular properties with their functional roles, often leading to conflicting conclu-sions. Since gene expression profiles in cultured microglia differ significantly among cells that were extracted by using different isolation methods, and often even more so when compared to microglia in vivo, the interpretation of these results towards a functional prediction warrants extreme caution (Melchior et al., 2006).
The surprising features of the observed microglial behaviors in vivo created the need to readdress the role of these cells by studying their molecular properties and signaling mechanisms in their natural habitat, namely directly inside the living brain. Questions raised focused upon the mechanism that regulates their tissue sur-veillance function and the molecules involved in attracting microglial processes towards sites of damage. Several molecules have been shown to activate microglia in vitro, induce morphological changes and attract them in a concentration gradient dependent manner. However, most studies have addressed these issues on a time
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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).