Introduction: Cell death is one of the pathophysiological hallmarks after stroke. Markers to image death of brain cells in vivo are highly desirable. One of the candidate targets is phosphatidylserine (PS). Where-as PS is usually located on the inner leaflet of the plasma membrane bilayer facing the cytosol, it is translocated to the surface of the cell during cell death. This makes PS accessible for markers based on Annexin A5 (AnxA5) characterized by a high affinity and specific binding to PS. The aim of this thesis was to investigate whether AnxA5 can be used to specifically visualize cell death of brain cells after ex-perimental stroke.
Methodology: In this thesis, AnxA5 was explored using three different approaches. First, we investigat-ed a fluorescently labeled AnxA5 for in vivo and ex vivo near-infrared fluorescence imaging in experi-mental stroke in the mouse. We compared it to a non-binding control of AnxA5 and to other established cell death markers. In the second study, we evaluated a dual-labeled AnxA5 in the same animal model using single-photon emission computed tomography (SPECT), ex vivo activity measurements, and auto-radiography. As a positive control, we used ethanol-induced cell death in the femur muscle. In the third study, we developed a long-circulating version of AnxA5, called XTEN-AnxA5. We examined its binding affinity using camptothecin-induced cell death in Jurkat T cells and specific accumulation inside chemo-therapy-induced cell death in tumors.
Results: We showed that fluorescently labeled AnxA5 can be used to visualize cell death after experi-mental stroke in vivo. Only for functional, but not for non-binding AnxA5 control, increased signal intensi-ties in the ipsilateral compared with contralateral hemisphere were found. AnxA5 specifically bound to dead or dying cells as confirmed by immunohistochemistry where the vast majority of cells were also positive for other cell death markers. However, we did not detect dual-labeled AnxA5 in the brain using SPECT, whereas it was observed in ethanol-induced cell death in the femur muscle. Our newly devel-oped long-circulating XTEN-AnxA5 prolonged blood half-life to about 1 h and improved further imaging properties. We showed that XTEN-AnxA5 bound specifically to dead or dying cells in culture and dis-played an increased accumulation inside the tumors compared to wild-type AnxA5.
Conclusions: Whereas AnxA5 seems to bind specifically to dead or dying cells, its applicability for brain imaging needs to be further investigated using compounds designed to overcome the blood-brain barrier and reach their target.
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