Abstract
Traumatic brain injury is a major public health concern and is characterised by both apoptotic and necrotic cell death in the lesion. Anatomical imaging is usually used to assess traumatic brain injuries and there is a need for imaging modalities that provide complementary cellular information. We sought to non-invasively image cell death in a mouse model of traumatic brain injury using a near-infrared fluorescent conjugate of a synthetic heat shock protein-90 alkylator, 4-(N-(S-glutathionylacetyl) amino) phenylarsonous acid (GSAO). GSAO labels both apoptotic and necrotic cells coincident with loss of plasma membrane integrity. The optical GSAO specifically labelled apoptotic and necrotic cells in culture and did not accumulate in healthy organs or tissues in the living mouse body. The conjugate is a very effective imager of cell death in brain lesions. The optical GSAO was detected by fluorescence intensity and GSAO bound to dying/dead cells was detected from prolongation of the fluorescence lifetime. An optimal signal-to-background ratio was achieved as early as 3 h after injection of the probe and the signal intensity positively correlated with both lesion size and probe concentration. This optical GSAO offers a convenient and robust means to non-invasively image apoptotic and necrotic cell death in brain and other lesions.
Highlights
An established model of traumatic brain injury in mice is the cryolesion
The specificity and sensitivity of labelling of dying/dead cells by AF750-tagged GSAO was examined in in vitro models of apoptotic and necrotic (4T1-luc[2] mouse breast cancer cells treated with dry ice) cell death
Staurosporine is a microbial alkaloid and broad spectrum protein kinase inhibitor that triggers mitochondrial-mediated apoptotic cell death, whereas snap freezing of cells ruptures the plasma membrane resulting in necrotic cell death
Summary
An established model of traumatic brain injury in mice is the cryolesion. A sharp delineated area of neuronal cell death is induced in the mouse brain by applying a liquid nitrogen cooled rod to the skull.[5]. Intracellular ice crystals rupture the cell membrane causing direct cell damage.[6] Second, injury to endothelial cell junctions results in increased vascular permeability, oedema, thrombosis and failure of the microcirculation, leading to a secondary ischaemic assault.[7] the centre of the lesion is characterised by tissue necrosis, apoptosis is observed in the periphery as early as 12-h post injury.[5] The latter is thought to occur via mitochondrial permeabilisation following increased expression of Bax.[8] Upregulation of genes involved in neuronal survival or regeneration is observed.[9,10] This lesion bears many features of neurotrauma.[6,7] Here we have non-invasively imaged the cell death in these lesions using a heat shock protein-90 (Hsp-90) alkylator.. GSAO-AF750 + DMP detect cell death in mouse brain cryolesions.[15,16,17,18,19] GSAO was conjugated to the near-infrared fluorophore, Alex Flour 750 (GSAO-AF750), to maximise tissue penetration of the fluorescent signal and to minimise the complications of tissue auto-fluorescence.[20]
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