Visualisierung der Clearance von roten Blutkörperchen und Immunzellen mittels 19F-Magnetresonanztomographie nach experimenteller Subarachnoidalblutung

Subarachnoid hemorrhage (SAH) to bleeding in the subarachnoid space (SAS), followed by secondary brain injury and inflammatory events originating from extravasated blood and immune cells. Intravenously injected nanoemulsions prepared from perfluoro-5-crown-15-ether (PFCE) have been shown to preferably label circulating inflammatory cells. We aimed to visualize immune cell and erythrocyte clearance after SAH using Rhodamine-labelled PFCE in combination with 19F MRI.

A filament perforation surgery was performed to induce SAH in C57BL/6 mice and Sham operation was done for the matching control group (ntotal=40). Immediately after surgery, mice received an intravenously injected PFCE nanoemulsion (150 μL, 40% v/v). Accumulation of 19F particles was visualized at different time points in vivo by 1H/19F 7T MRI, registered to Allen Brain Atlas and a custom SAS atlas and evaluated by volume-of-interest analysis and incidence maps. Immunofluorescence staining for nuclei, arachnoid cells, microglia, macrophages, and neutrophils was analyzed by confocal imaging to elucidate the histological location of rhodamine labeled PFCEs (Fig. 1A). Characterization of 19F particle phagocyting immune cells was performed using flow cytometry.

Comparing integrated signal-to-noise ratio (SNR) and subtractive incidence maps, 1H/19F MRI revealed increased 19F signal in the hypothalamus and basal brain parenchyma bordering the perforated vessel, as well as leptomeningeal infiltration of PFCEs into the ipsilateral inferior and superior parts of the SAS after SAH (Sham day 1 vs. SAH day 1: 6,092.2 ±2,848.7 vs. 47,110.7 ±11,269.4; p=0.001; Fig. 1B-C). Over time, clearance of PFCE particles from the rostral and occipital SAS was evident in both subtractive incidence maps and analysis of integrated SNR, elucidating lymphatic drainage of the SAS via detection and clearance of leptomeningeal PFCEs following SAH (SAH day 1 vs. SAH day 7: 17,165.6 ±3,859.0 vs. 4,396.2 ±2,214.4; p=0.008). Confocal imaging verified the accumulation of PFCEs in the ipsilateral basal SAS and basal brain parenchyma, and showed spatial affinity of 19F particles to microglia, macrophages, and neutrophils after SAH (Fig. 1E-G).

PFCE nanoemulsions generate positive 19F MRI contrast for visualization of immune cell and blood accumulation, imaging of SAH-associated inflammation and elucidating lymphatic drainage of the SAS following SAH. PFCEs may enable monitoring of immune cell activation and blood clearance after SAH.