Traumatic brain injury is characterized by initial tissue damage, which then can lead to secondary processes such as cell death and blood-brain-barrier disruption. Clinical and preclinical studies of traumatic brain injury typically employ anatomical imaging techniques and there is a need for new molecular imaging methods that provide complementary biochemical information. Here, we assess the ability of a targeted, near-infrared fluorescent probe, named PSS-794, to detect cell death in a brain cryolesion mouse model that replicates certain features of traumatic brain injury. In short, the model involves brief contact of a cold rod to the head of a living, anesthetized mouse. Using noninvasive whole-body fluorescence imaging, PSS-794 permitted visualization of the cryolesion in the living animal. Ex vivo imaging and histological analysis confirmed PSS-794 localization to site of brain cell death. The nontargeted, deep-red Tracer-653 was validated as a tracer dye for monitoring blood-brain-barrier disruption, and a binary mixture of PSS-794 and Tracer-653 was employed for multicolor imaging of cell death and blood-brain-barrier permeability in a single animal. The imaging data indicates that at 3 days after brain cryoinjury the amount of cell death had decreased significantly, but the integrity of the blood-brain-barrier was still impaired; at 7 days, the blood-brain-barrier was still three times more permeable than before cryoinjury.

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.

The ability to assess in near-real time the tumor cell killing efficacy of chemotherapy regimens would improve patient treatment and survival. An ineffective regimen could be abandoned early in favor of a more effective treatment. We sought to noninvasively image treatment-related tumor cell death in mice using an optically labeled synthetic heat shock protein-90 (Hsp90) alkylator, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid (GSAO). The Hsp90 chaperone is an important element in oncogene addiction and tumor cell survival, and its expression is enhanced by chemotherapy. These factors were predicted to favor the detection of tumor cell death using GSAO. GSAO specifically labeled apoptotic and necrotic tumor cells in culture and cells of comparable morphology in subcutaneous human pancreatic carcinoma tumors in mice. A near-infrared fluorescent conjugate of GSAO was used to noninvasively image cyclophosphamide-induced tumor cell death in murine orthotopic human mammary tumors. The GSAO conjugate did not accumulate in healthy organs or tissues in the mouse, and unbound compound was excreted rapidly via the kidneys. There was a significant increase in the GSAO fluorescence signal in the treated tumors measured either in vivo or ex vivo, and the fluorescence signal colocalized with apoptotic cells in sectioned tumors. The favorable biodistribution of optically labeled GSAO, the nature of its tumor cell target, and its capacity to noninvasively detect tumor cell death should facilitate the application of this compound in studies of the efficacy of existing and new chemotherapeutics.

Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 103 and 5 × 104 cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging.FigOptical imaging using TurboLuc fusion reporter protein

BackgroundObesity is associated with a hypercoagulable state and increased risk for thrombotic cardiovascular events.ObjectiveEstablish the onset and reversibility of the hypercoagulable state during the development and regression of nutritionally-induced obesity in mice, and its relation to transcriptional changes and clearance rates of coagulation factors as well as its relation to changes in metabolic and inflammatory parameters.MethodsMale C57BL/6J mice were fed a low fat (10% kcal as fat; LFD) or high fat diet (45% kcal as fat; HFD) for 2, 4, 8 or 16 weeks. To study the effects of weight loss, mice were fed the HFD for 16 weeks and switched to the LFD for 1, 2 or 4 weeks. For each time point analyses of plasma and hepatic mRNA levels of coagulation factors were performed after overnight fasting, as well as measurements of circulating metabolic and inflammatory parameters. Furthermore, in vivo clearance rates of human factor (F) VII, FVIII and FIX proteins were determined after 2 weeks of HFD-feeding.ResultsHFD feeding gradually increased the body and liver weight, which was accompanied by a significant increase in plasma glucose levels from 8 weeks onwards, while insulin levels were affected after 16 weeks. Besides a transient rise in cytokine levels at 2 weeks after starting the HFD, no significant effect on inflammation markers was present. Increased plasma levels of fibrinogen, FII, FVII, FVIII, FIX, FXI and FXII were observed in mice on a HFD for 2 weeks, which in general persisted throughout the 16 weeks of HFD-feeding. Interestingly, with the exception of FXI the effects on plasma coagulation levels were not paralleled by changes in relative transcript levels in the liver, nor by decreased clearance rates. Switching from HFD to LFD reversed the HFD-induced procoagulant shift in plasma, again not coinciding with transcriptional modulation.ConclusionsChanges in dietary fat content rapidly alter the mouse plasma coagulation profile, thereby preceding plasma metabolic changes, which cannot be explained by changes in relative expression of coagulation factors or decreased clearance rates.