Understanding and interfering with resident microglia activation following subarachnoid hemorrhage (SAH)

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In our previous work, we have characterized CNS immune response after subarachnoid hemorrhage (SAH) as an activation of the innate immune system. Blood in the subarachnoid compartment creates a pro-inflammatory environment, leads to an activation of macrophages at the brain base and induces an inflammatory response at the perivascular space. This subsequently activates exclusively the resident CNS-specialized microglia (as shown by chimera experiments) in an outside-in fashion. Furthermore, we have demonstrated that secondary neuro-axonal injury after SAH is linked to microglia activation. Tumor-necrosis factor- α (TNF-α) has been identified as a primary candidate for mediating this inflammatory response. Besides, our preliminary work indicates that extracellular RNA (eRNA) and DNA accumulate in and around the brain after SAH and may mediate TNF-α liberation from microglia. eRNA has been shown to become released from cells under diverse pathological conditions (outside the CNS) and induces pro-inflammatory and vessel permeability-inducing processes. Thus, eRNA is a target for modulating myeloid cell effector functions. In the herein presented project, we first aim at understanding the phenotype and function of activated microglia after SAH in more detail. Therefore, we will characterize their inflammatory phenotype and further study the impact of these effector cells on the pathological hallmarks of SAH, i.e. early loss of blood-brain barrier (BBB) function and delayed neuro-axonal injury. This will be achieved by a combination of expression profiling, in vitro cellular, electrophysiological and histoanalytical experiments. Secondly, we will assess the dynamics and mechanisms of eRNA accumulation in the subarachnoid space and brains after SAH. Here, we will stain mouse and human autopsy specimens for quantified content of eRNA and will identify the source of eRNA in a set of in vivo and in vitro experiments. In the third part, we will study study the mechanism of TNF-α liberation by microglia and its downstream signaling in vitro. We hypothesis that eRNA, released after SAH, is capable of stimulating the release of TNF-α from CNS macrophages/microglia and exerting effector functions, such as BBB damage and neuro-axonal cell death. In the fourth part, we will interfere with eRNA functions in vivo and will evaluate RNase1 and RNase Inhibitor as modulators of vascular homeostasis to control the function of eRNA after brain injury following SAH. To test this, we will use exogenous RNase1/RNase Inhibitor or transgenic EC-specific conditional knockouts of Rnase1 and RNase Inhibitor. Finally, we will evaluate novel therapeutic strategies to interfere with the activation of the innate immune system and the subsequent secondary brain injury following experimental SAH. Here, we will put our emphasis on pharmacological strategies that target (i) microglial cells and (ii) TNF-α, which have the potential to be rapidly translated into a clinical setting.