New lab builds on imaging, genetic progress of past decade
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Microglia are the resident immune cells of the brain and spinal cord that form the first line of defense when the CNS or the blood-brain barrier (BBB) is compromised. Microglia can be protective when they contain injuries and remove dead cells or pathogens — or damaging when their activation exacerbates pathologies.
Historically, microglia were mostly studied in the context of CNS disease, but over the past decade microglia have been continuously linked with new functions that are essential for normal brain function, like sculpting neuronal networks during development and possibly even regulating neuronal plasticity in the adult brain. The advent of cutting-edge imaging technologies and novel genetic tools to detail and manipulate microglial behavior in living animal models promises an even more exciting future for microglial research.
In the physiological brain, microglia were previously thought to be sessile and were hence termed resting microglia. When we imaged microglia in the living mouse brain for the first time, we found that they continuously survey the CNS by extending and retracting their fine processes on a time scale of seconds (Figure 1).1 This unexpected finding inspired numerous studies aimed at understanding the mechanisms and the significance of this novel microglial function for neuronal plasticity, function and dysfunction.
Figure 1. Microglia perform tissue surveillance by constantly extending and retracting processes (A, circles), rapidly responding to localized brain injury (B) in an ATP-dependent manner (C). Adapted from Davalos et al. in Nature Neuroscience.1
We also found that microglia have an impressive ability to detect small injuries in the brain and contain them with their processes within only a few minutes (Figure 1). Moreover, we identified that adenosine triphosphate (ATP), a molecule that gives energy to every living cell, can function as a danger signal that makes microglia aware of injury in their vicinity. When cells in the brain are physically injured, they spill ATP in the surrounding tissue. Since ATP is normally not present in the extracellular space, it is immediately detected by astrocytes in the vicinity of the injury, and the astrocytes release more ATP locally, which guides microglial processes to find and contain brain damage.1
These unexpected findings introduced a new category of microglial responses that are much faster than their previously known inflammatory activation. The findings also established a new paradigm for studying microglia in physiology and after injury, by combining two-photon microscopy with laser- or mechanically induced injury and micropharmacology in the living brain, in real time.
More recently, we studied microglial responses following BBB disruption using the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). We identified microglia as the first cells to show signs of activation by clustering around blood vessels. By performing repetitive in vivo imaging in the same mice, we found that perivascular clustering of microglia starts before disease onset and continues throughout the course of EAE (Figure 2).2
Figure 2. (A) Microglia (green) cluster around blood vessels (red) throughout experimental autoimmune encephalomyelitis (EAE), starting before disease onset. (B) Clusters form in perivascular areas with fibrin deposition. Axonal damage (cyan), including bending, swelling and fragmentation (arrowhead), occurs only within such fibrin-rich areas (red) with microglial clusters. Adapted from Davalos et al. inNature Communications.2
Also, by imaging microglia and axons in real time, we found that axons undergo severe morphological alterations within microglial clusters that often result in axonal fragmentation and uptake by microglia (Figure 2).2 These microglial clusters form at sites of BBB disruption where the blood protein fibrinogen leaks out of blood vessels and is converted to insoluble fibrin. Fibrin is the end product of the coagulation cascade and is extensively deposited in EAE and MS lesions.
By combining pharmacological or genetic inhibition approaches with correlated histology of spinal cord areas previously imaged in vivo, we found that fibrin(ogen) binding to the integrin receptor CD11b/CD18 (also known as Mac-1, αMβ2 or complement receptor 3 [C3R]) is required for microglial clustering and axonal damage.2 Moreover, by showing that, upon extravasation, fibrinogen induces reactive oxygen species release by microglia, we identified a link between BBB disruption, microglial activation and axonal damage, the main culprit in neuroinflammatory disease.
At our newly established lab in the Department of Neurosciences in Cleveland Clinic’s Lerner Research Institute, we are pursuing two main projects designed to characterize the cellular responses and molecular pathways involved with microglial-neuronal and microglial-vascular interactions in the healthy CNS and in the context of neurological disease.
We use novel genetic tools to perform microglia-specific pathway manipulation in combination with in vivo imaging in models of neuroinflammation to decipher how microglia and peripheral immune cells might be involved with processes regulating BBB integrity and neuronal damage. We are also investigating the sequence of events and the molecular pathways that drive microglial responses in the ischemic brain. In particular, we study the real-time responses of microglia, in relation to vascular and neuronal disruption during and after ischemic injury, by longitudinal in vivo imaging. Also, by using genetic models to manipulate fibrinogen, we aim to decipher the molecular links between vascular rupture or ischemic insult and microglial responses, especially in relation to neuronal degeneration after stroke.
Our in vivo imaging studies have the potential to provide target validation for the activating effects of blood proteins and other danger signals on microglia. These studies should prove instrumental for the initiation of drug discovery efforts and for development of screening and validation assays for novel therapeutic targets for diseases with BBB disruption, such as MS and stroke.
Dr. Davalos is an assistant staff member in the Department of Neurosciences in Cleveland Clinic Lerner Research Institute.
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