Bioluminescence imaging (BLI) of luciferase reporters in small animal models offers an attractive approach to monitor regulation of gene expression, signal transduction, and protein-protein interactions, as well as following tumor progression, cell engraftment, infectious pathogens, and target-specific drug action. Conventional BLI can be repeated within the same animal after bolus reinjections of a bioluminescent substrate. However, intervals between image acquisitions are governed by substrate pharmacokinetics and excretion, therefore restricting temporal resolution of reinjection protocols to the order of hours, limiting analyses of processes in vivo with short time constants. To eliminate these constraints, we examined use of implanted microosmotic pumps for continuous, long-term delivery of bioluminescent substrates. Pump-assisted D-luciferin delivery enabled BLI for >7 days from a variety of luciferase reporters. Pumps allowed direct repetitive imaging at , 5-minute intervals of the pharmacodynamics of proteasome- and IKK-inhibiting drugs in mice bearing tumors stably expressing ubiquitin-firefly luciferase or IκBα-firefly luciferase fusion reporters. Circadian oscillations in the olfactory bulbs of transgenic rats expressing firefly luciferase under the control of the period1 promoter also were temporally resolved over the course of several days. We conclude that implanted pumps provide reliable, prolonged substrate delivery for high temporal resolution BLI, traversing complications of repetitive substrate injections.
Imaging inhibition of IKK activity in vivo. HeLa(IκBα-FLuc) or HeLa(FLuc) tumor xenografts were established in NCr nu/nu mice. D-Luciferin loaded pumps (release rate = 0.5 µL/h) were implanted ~3 weeks postinoculation of the reporter cells. (A) Representative bioluminescence images of a HeLa(IκBα-FLuc) tumor-bearing mouse, taken 2 hours before and at the indicated time points after oral administration of the IKK inhibitor PS-1145 (50 mg/kg body weight). (B) Temporal changes in bioluminescence in vivo (-2 to 46 hours) of HeLa(IκBα-FLuc) tumors induced by treatment with PS-1145 (50 mg/kg; closed circles; arrow) or vehicle (open circles), as well as temporal changes in bioluminescence of control HeLa(FLuc) tumors treated with PS-1145 (50 mg/kg; triangles). Data are normalized to initial (-2 hours) values.
Imaging oscillations of the per1 gene in vivo.D-Luciferincontaining micro-osmotic pumps were used to monitor daily rhythms in clock gene (per1) expression in the olfactory bulb (OB) of live transgenic rats [J Neurosci 2005; 25: 8620-6]. One day prior to imaging, two osmotic pumps (release rate = 1 µL/h), each loaded with 78 mg/mL of D-luciferin, were implanted into the peritoneal cavity of a per1-->FLuc transgenic rat. Clock gene expression in the OB was imaged through an agarose/glass window in the skull (see the dorsal view of a rat's head in the upper inset, OB window marked by a white circle). Pseudocolor images of 1-minute exposures, taken every 4 to 8 hours for 48 hours, show bioluminescence emitted from the rat's head (see the scale next to the images), particularly from the bare skin and the OB. Bioluminescence emitted from the OB of blind rats peaks at the end of the light phase to which they were previously entrained (circadian time 11, marked by gray [light] and black [dark] bars on the x-axis), on 2 consecutive days. The peak to trough ratio is approximately 2:1. White numbers on the image strip indicate at which circadian time the image was taken (0 = lights on in the previous lightdark cycle, 12 = lights off in the previous light-dark cycle).
Gross S, Abraham U, Prior JL, Herzog ED, Piwnica-Worms D. Continuous delivery of D-luciferin by implanted micro-osmotic pumps enables true real-time bioluminescence imaging of luciferase activity in vivo. Molec Imaging 2007; 6(2): 121-30.