Effective targeting of microglial P2X7 following intracerebroventricular delivery of nanobodies and nanobody-encoding AAVs.

The P2X7 ion channel is a key sensor for extracellular ATP and a key trigger of sterile inflammation. Intravenous injection of nanobodies that block P2X7 has shown to be beneficial in mouse models of systemic inflammation. P2X7 has also emerged as an attractive therapeutic target for inflammatory brain diseases. However, little is known about the ability of nanobodies to cross the BBB. Here we evaluated the ability of P2X7-specific nanobodies to reach and to block P2X7 on microglia following intravenous or intracerebral administration. For this study, we reformatted and sequence-optimized P2X7 nanobodies for higher stability and elevated isoelectric point. Following injection of nanobodies or nanobody-encoding adeno-associated viral vectors (AAV), we monitored the occupancy and blockade of microglial P2X7 in vivo using ex vivo flow cytometry. Our results show that P2X7 on microglia was within minutes completely occupied and blocked by intracerebroventricularly injected nanobodies, even at low doses. In contrast, very high doses were required to achieve similar effects when injected intravenously. The endogenous production of P2X7-antagonistic nanobodies following intracerebral or intramuscular injection of nanobody-encoding AAVs resulted in a long-term occupancy and blockade of P2X7 on microglia. Our results provide new insights into the conditions for the delivery of nanobodies to microglial P2X7 and point to AAV-mediated delivery of P2X7 nanobodies as a promising strategy for the treatment of sterile brain inflammation.

Interleukin-10 improves stroke outcome by controlling the detrimental Interleukin-17A response.

BACKGROUND: Lymphocytes have dichotomous functions in ischemic stroke. Regulatory T cells are protective, while IL-17A from innate lymphocytes promotes the infarct growth. With recent advances of T cell-subtype specific transgenic mouse models it now has become possible to study the complex interplay of T cell subpopulations in ischemic stroke. METHODS: In a murine model of experimental stroke we analyzed the effects of IL-10 on the functional outcome for up to 14 days post-ischemia and defined the source of IL-10 in ischemic brains based on immunohistochemistry, flow cytometry, and bone-marrow chimeric mice. We used neutralizing IL-17A antibodies, intrathecal IL-10 injections, and transgenic mouse models which harbor a deletion of the IL-10R on distinct T cell subpopulations to further explore the interplay between IL-10 and IL-17A pathways in the ischemic brain. RESULTS: We demonstrate that IL-10 deficient mice exhibit significantly increased infarct sizes on days 3 and 7 and enlarged brain atrophy and impaired neurological outcome on day 14 following tMCAO. In ischemic brains IL-10 producing immune cells included regulatory T cells, macrophages, and microglia. Neutralization of IL-17A following stroke reversed the worse outcome in IL-10 deficient mice and intracerebral treatment with recombinant IL-10 revealed that IL-10 controlled IL-17A positive lymphocytes in ischemic brains. Importantly, IL-10 acted differentially on αß and γδ T cells. IL-17A producing CD4+ αß T cells were directly controlled via their IL-10-receptor (IL-10R), whereas IL-10 by itself had no direct effect on the IL-17A production in γδ T cells. The control of the IL-17A production in γδ T cells depended on an intact IL10R signaling in regulatory T cells (Tregs). CONCLUSIONS: Taken together, our data indicate a key function of IL-10 in restricting the detrimental IL-17A-signaling in stroke and further supports that IL-17A is a therapeutic opportunity for stroke treatment.

Monitoring Intracranial Cerebral Hemorrhage Using Multicontrast Real-Time Magnetic Particle Imaging.

Magnetic particle imaging (MPI) is an innovative radiation-free tomographic imaging method providing excellent temporal resolution, contrast, sensitivity, and safety. Mobile human MPI prototypes suitable for continuous bedside monitoring of whole-brain perfusion have been developed. However, for the clinical translation of MPI, a crucial gap in knowledge still remains: while MPI can visualize the reduction in blood flow and tissue perfusion in cerebral ischemia, it is unclear whether MPI works in intracranial hemorrhage. Our objective was to investigate the capability of MPI to detect intracranial hemorrhage in a murine model. Intracranial hemorrhage was induced through the injection of collagenase into the striatum of C57BL/6 mice. After the intravenous infusion of a long-circulating MPI-tailored tracer consisting of superparamagnetic iron oxides, we detected the intracranial hemorrhage in less than 3 min and could monitor hematoma expansion in real time. Multicontrast MPI can distinguish tracers based on their physical characteristics, core size, temperature, and viscosity. By employing in vivo multicontrast MPI, we were able to differentiate areas of liquid and coagulated blood within the hematoma, which could provide valuable information in surgical decision making. Multicontrast MPI also enabled simultaneous imaging of hemorrhage and cerebral perfusion, which is essential in the care of critically ill patients with increased intracranial pressure. We conclude that MPI can be used for real-time diagnosis of intracranial hemorrhage. This work is an essential step toward achieving the clinical translation of MPI for point-of-care monitoring of different stroke subtypes.

Autoimmunity After Ischemic Stroke and Brain Injury.

Ischemic Stroke is a major cause of morbidity and mortality worldwide. Sterile inflammation occurs after both stroke subtypes and contributes to neuronal injury and damage to the blood-brain barrier with release of brain antigens and a potential induction of autoimmune responses that escape central and peripheral tolerance mechanisms. In stroke patients, the detection of T cells and antibodies specific to neuronal antigens suggests a role of humoral adaptive immunity. In experimental models stroke leads to a significant increase of autoreactive T and B cells to CNS antigens. Lesion volume and functional outcome in stroke patients and murine stroke models are connected to antigen-specific responses to brain proteins. In patients with traumatic brain injury (TBI) a range of antibodies against brain proteins can be detected in serum samples. In this review, we will summarize the role of autoimmunity in post-lesional conditions and discuss the role of B and T cells and their potential neuroprotective or detrimental effects.

Genome-Wide Analysis of the Circulating miRNome After Cerebral Ischemia Reveals a Reperfusion-Induced MicroRNA Cluster.

BACKGROUND AND PURPOSE: Circulating microRNAs (miRNAs) are emerging biomarkers for stroke because of their high stability in the bloodstream and association with pathophysiologic conditions. However, the circulating whole-genome miRNAs (miRNome) has not been characterized comprehensively in the acute phase of stroke. METHODS: We profiled the circulating miRNome in mouse models of acute ischemic and hemorrhagic stroke by next-generation sequencing. Stroke models were compared with sham-operated and naive mice to identify deregulated circulating miRNAs. Top-ranked miRNAs were validated and further characterized by quantitative reverse transcription polymerase chain reaction. RESULTS: We discovered 24 circulating miRNAs with an altered abundance in the circulation 3 hours after ischemia, whereas the circulating miRNome was not altered after intracerebral hemorrhage compared with sham-operated mice. Among the upregulated miRNAs in ischemia, the top-listed miR-1264/1298/448 cluster was strongly dependent on reperfusion in different ischemia models. A time course experiment revealed that the miR-1264/1298/448 cluster peaked in the circulation around 3 hours after reperfusion and gradually decreased thereafter. CONCLUSIONS: Alteration of the miRNome in the circulation is associated with cerebral ischemia/reperfusion, but not hemorrhage, suggesting a potential to serve as biomarkers for reperfusion in the acute phase. The pathophysiological role of reperfusion-inducible miR-1264/1298/448 cluster, which is located on chromosome X within the introns of the serotonin receptor HTR2C, requires further investigation.

Leukocyte invasion of the brain after experimental intracerebral hemorrhage in mice.

BACKGROUND AND PURPOSE: Neuroinflammatory processes contribute to secondary neuronal damage after intracerebral hemorrhage. We aimed to characterize the time course of brain immigration of different leukocyte subsets after striatal injection of either autologous blood or collagenase in mice. METHODS: Intracerebral hemorrhage was induced by injection of either autologous blood (20 µL) or collagenase (0.03 U) in C57Bl/6J mice. Hematoma volumetry was performed on cryosections. Blood volume was measured by hemoglobin spectrophotometry. Leukocytes were isolated from hemorrhagic hemisphere 1, 3, 5, and 14 days after intracerebral hemorrhage, stained for leukocyte markers, and measured by flow cytometry. Heterologous blood injection from CD45.1 mice was used to investigate the origin of brain-invading leukocytes. RESULTS: Collagenase injection induced a larger hematoma volume but a similar blood content compared with blood injection. Cerebral leukocyte infiltration in the hemorrhagic hemisphere was similar in both models. The majority of leukocytes isolated from the brain originated from the circulation. CD4+ T lymphocytes were the predominant brain leukocyte population in both models. However, cerebral granulocyte counts were higher after collagenase compared with blood injection. CONCLUSIONS: Brain infiltration of systemic immune cells is similar in both murine intracerebral hemorrhage models. The pathophysiological impact of invading leukocytes and, in particular, of T cells requires further investigation.