In Vivo Pathology: Seeing with Molecular Specificity and Cellular Resolution in the Living Body

Department of Pediatrics, Radiology, and Microbiology & Immunology, Stanford University, Stanford, California 94305, USA.
Annual Review of Pathology Mechanisms of Disease (Impact Factor: 18.75). 02/2007; 2(1):277-305. DOI: 10.1146/annurev.pathol.2.010506.091930
Source: PubMed


The emerging tools of in vivo molecular imaging are enabling dynamic cellular and molecular analyses of disease mechanisms in living animal models and humans. These advances have the potential to dramatically change a number of fields of study, including pathology, and to contribute to the development of regenerative medicine and stem cell therapies. The new tools of molecular imaging, which have already had a tremendous impact on preclinical studies, hold great promise for bringing important and novel information to the clinician and the patient. These approaches are likely to enable early diagnosis, rapid typing of molecular markers, immediate assessment of therapeutic outcome, and ready measures of the extent of tissue regeneration after damage. However, the full impact of these new techniques will be determined by our ability to translate them to the clinic and to develop a general strategy that integrates them with other advances in molecular diagnostics and molecular medicine.

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    • "Firefly luciferase is one such protein, and it produces yellow-red light in the presence of its substrate, luciferin (Nakatsu et al., 2006). Mammalian systems readily tolerate luciferase and luciferin, and this system has been used in vivo to measure gene activity and track labeled cells (Contag, 2007). Notably, the emission spectrum of firefly luciferase overlaps highly with the action spectrum of halorhodopsin (Zhang et al., 2007), a bacterially derived, amber light photoreceptive chloride pump that inhibits neural activity. "
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    ABSTRACT: Optogenetics is an extremely powerful tool for selective neuronal activation/inhibition and dissection of neural circuits. However, a limitation of in vivo optogenetics is that an animal must be tethered to an optical fiber for delivery of light. Here, we describe a new method for in vivo, optogenetic inhibition of neural activity using an internal, animal-generated light source based on firefly luciferase. Two adeno-associated viruses encoding luciferase were tested and both produced concentration-dependent light after administration of the substrate, luciferin. Mice were co-infected with halorhodopsin- and luciferase-expressing viruses in the striatum, and luciferin administration significantly reduced Fos activity compared to control animals infected with halorhodopsin only. Recordings of neuronal activity in behaving animals confirmed that firing was greatly reduced after luciferin administration. Finally, amphetamine-induced locomotor activity was reduced in halorhodopsin/luciferase mice pre-injected with luciferin compared to controls. This demonstrates that virally encoded luciferase is able to generate sufficient light to activate halorhodopsin and suppress neural activity and change behavior. This approach could be used to generate inhibition in response to activation of specific molecular pathways.
    Frontiers in Behavioral Neuroscience 04/2014; 8:108. DOI:10.3389/fnbeh.2014.00108 · 3.27 Impact Factor
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    • "Thus, BLI has limited clinical use, and it is more suitable for small animal studies. Finally, owing to tissue attenuation and refraction, the eGFP of fluorescence imaging is only 2 mm [25], [26]. Because of interference by the fur and tissue of rats, thoracotomy is required before fluorescence imaging, as shown in Figure 1A. "
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    ABSTRACT: Non-invasive techniques to monitor the survival and migration of transplanted stem cells in real-time is crucial for the success of stem cell therapy. The aim of this study was to explore multimodality molecular imaging to monitor transplanted stem cells with a triple-fused reporter gene [TGF; herpes simplex virus type 1 thymidine kinase (HSV1-tk), enhanced green fluorescence protein (eGFP), and firefly luciferase (FLuc)] in acute myocardial infarction rat models. Rat myocardial infarction was established by ligating the left anterior descending coronary artery. A recombinant adenovirus carrying TGF (Ad5-TGF) was constructed. After transfection with Ad5-TGF, 5×106 bone marrow mesenchymal stem cells (BMSCs) were transplanted into the anterior wall of the left ventricle (n = 14). Untransfected BMSCs were as controls (n = 8). MicroPET/CT, fluorescence and bioluminescence imaging were performed. Continuous images were obtained at day 2, 3 and 7 after transplantation with all three imaging modalities and additional images were performed with bioluminescence imaging until day 15 after transplantation. High signals in the heart area were observed using microPET/CT, fluorescence and bioluminescence imaging of infarcted rats injected with Ad5-TGF-transfected BMSCs, whereas no signals were observed in controls. Semi-quantitative analysis showed the gradual decrease of signals in all three imaging modalities with time. Immunohistochemistry assays confirmed the location of the TGF protein expression was the same as the site of stem cell-specific marker expression, suggesting that TGF tracked the stem cells in situ. We demonstrated that TGF could be used as a reporter gene to monitor stem cells in a myocardial infarction model by multimodality molecular imaging.
    PLoS ONE 03/2014; 9(3):e90543. DOI:10.1371/journal.pone.0090543 · 3.23 Impact Factor
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    • "While this result represents a significant advance, the practical limitations of the imaging experimental procedures, including frequent substrate injection and anesthesia, present challenges in monitoring a large cohort of animals at short time intervals. In addition, it is well-known that the luminescence propagation through living tissue can cause compromised sensitivity, reduced spatial resolution and accuracy [40]. The ex vivo assessment of secreted Gluc in TCF and blood samples as reporter for bacterial burden addresses the above limitations and therefore has significant advantages (Figure 7 and 8). "
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    ABSTRACT: Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.
    PLoS ONE 03/2014; 9(3):e90382. DOI:10.1371/journal.pone.0090382 · 3.23 Impact Factor
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