Light Emission Requires Exposure to the Atmosphere in Ex Vivo Bioluminescence Imaging

Abstract

The identification of organs bearing luciferase activity by in vivo bioluminescence imaging (BLI) is often difficult, and ex vivo imaging of excised organs plays a complementary role. This study investigated the importance of exposure to the atmosphere in ex vivo BLI. Mice were inoculated with murine pro-B cell line Ba/F3 transduced with firefly luciferase and p190 BCR-ABL. They were killed following in vivo BLI, and whole-body imaging was done after death and then after intraperitoneal air injection. In addition, the right knee was exposed and imaged before and after the adjacent bones were cut. Extensive light signals were seen on in vivo imaging. The luminescence disappeared after the animal was killed, and air injection restored the light emission from the abdomen only, suggesting a critical role of atmospheric oxygen in luminescence after death. Although no substantial light signal at the right knee was seen before bone cutting, light emission was evident after cutting. In conclusion, in ex vivo BLI, light emission requires exposure to the atmosphere. Bone destruction is required to demonstrate luciferase activity in the bone marrow after death.

Introduction

In vivo bioluminescence imaging (BLI) enables one to evaluate the intensity and distribution of expression of the luciferase gene in an intact laboratory animal and is used increasingly for various purposes, such as monitoring gene therapy and cell trafficking, investigating transcriptional regulation, and evaluating protein–protein interactions [1]. Commonly, animals expressing firefly luciferase are injected with d-luciferin for in vivo BLI. Light photons are produced through the oxidation of d-luciferin in the presence of oxygen, adenosine triphosphate, and magnesium [2] and are detected by using a sensitive charge-coupled device (CCD) camera. Wholebody, quantitative assessments of luciferase expression can be performed repetitively in a given animal.

Bioluminescence imaging provides projectional images, not tomographic images, and, in addition, the scattering of light photons impairs the spatial resolution. These factors reduce the ability of in vivo BLI to identify the organs with luciferase activity. Using a CCD camera, one can easily determine the luciferase-expressing organs by imaging the excised organs after d-luciferin injection, an imaging procedure called ex vivo BLI [3–8]. Ex vivo imaging enhances the detectability of luciferase expression [7,9], and the quantitative accuracy of ex vivo imaging has been validated by using a conventional luciferase assay of organ homogenates as a standard [3]. Although ex vivo imaging is invasive and does not allow repetitive assessments of an individual animal, it serves as a valuable adjunct to in vivo imaging for detailed evaluation.

It has been reported that the luminescence of the liver of a mouse having luciferase-expressing intrahepatic tumors and hemorrhagic ascites disappeared after sacrifice and recovered after excision of the liver [10]. The authors attributed the increase in the light signal after excision to the increased oxygen availability on exposure to ambient air. However, light photons are severely attenuated by the tissues and blood, and the increase in the detected light signal might be due to the elimination of attenuation by the abdominal wall and hemorrhagic ascites. In this study, we investigated the importance of exposure to the atmosphere in ex vivo BLI to obtain insight into the ex vivo technique.

Materials and Methods

Cell Lines

The interleukin-3 (IL-3)-dependent murine pro-B cell line Ba/F3 was transfected with both the firefly luciferase gene and the wild-type p190 BCR-ABL fusion gene retrovirally as described previously [11], and the established cells were termed Ba/F3-Luc/Wt cells. The cDNA encoding the firefly luciferase was excised from the pGL3-basic vector (Promega, Madison, WI), and the long-term stability of luciferase expression in the established cells was confirmed in vitro. The p190 BCR-ABL fusion gene is important in the development of acute lymphoblastic leukemia (ALL) [12], and Ba/F3 cells transformed with p190 BCR-ABL show IL-3-independent, autonomous proliferation [13]. The Ba/F3-Luc/Wt cells were maintained in RPMI 1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS, JRH Biosciences, Lenexa, KS) and 1% penicillin/streptomycin (Invitrogen), in the absence of IL-3. Cultures were incubated at 37°C in 5% CO₂.

Animals

Five 8-week-old female BALB/c nu/nu mice were inoculated with 2 × 10⁶ Ba/F3-Luc/Wt cells intravenously via the tail vein. The mice were obtained from SLC Japan (Tokyo, Japan) and were handled according to the guidelines of the Institute of Medical Science, University of Tokyo. The experiments were approved by the committee for animal research at the institution.

Imaging Procedures

About 4 weeks after cell inoculation, we performed BLI with a cooled CCD camera system (IVIS Imaging System 100, Xenogen, Alameda, CA). The mice received an intraperitoneal injection of 150 mg/kg d-luciferin, and photographic and luminescent images in the dorsal, left lateral, ventral, and right lateral projections were acquired under isoflurane anesthesia (in vivo BLI). Additional ventral images were obtained 20 min after injection. Subsequently, the mice were killed by cervical dislocation and then imaged repetitively. About 10 min after death, 4 mL of air was injected intraperitoneally, and imaging was performed again. The imaging parameters used to acquire these whole-body luminescent images were an exposure time of 10 sec and binning of 8. A region of interest covering the entire mouse except the tail was placed on the ventral image, and the whole-body signal intensity was quantified by using the Living Image software (version 2.50, Xenogen).

Following the whole-body imaging, ex vivo imaging of the excised organs was performed. The liver, spleen, intestine, ovary, uterus, kidney, lung, and heart were imaged using the CCD camera system. In addition, the right knee was exposed, and ventral images of the body remaining after removing the internal organs were obtained. The imaging was repeated after cutting the right distal femur and right proximal tibia. A region of interest was placed over the right knee, and the signal intensity was compared before and after cutting. The imaging parameters used to obtain the ex vivo luminescent images were an exposure time of 10 sec and binning of 4 or 8.

Results

In vivo BLI demonstrated extensive light signals, indicating proliferation of the implanted cells in various regions, including the head and neck, chest, abdomen, and limbs (Figure 1). After sacrifice, the light signal decreased rapidly and essentially disappeared within several minutes. The whole-body signal intensities in the ventral projection just before sacrifice, 2 min after death, and 5 min after death were 3.49 × 10⁸ ± 9.10 × 10⁷ (mean ± SD), 1.43 × 10⁶ ± 7.28 × 10⁵, and 2.25 × 10⁵ ± 1.06 × 10⁵ p s⁻¹, respectively. Intraperitoneal air injection definitely increased the signal (6.62 × 10⁷ ± 2.65 × 10⁷ p s⁻¹ 5 min after injection), while the light sources were localized only in the abdomen.

Ex vivo imaging of the excised organs showed intense light signals for the liver and spleen. Signals were also observed for the lung, intestine, and gynecologic organs, except for the absence of intestinal signals in one mouse. Imaging of the body remaining after removal of the internal organs showed a small bright focus in the paraaortic region in two mice. Enlarged paraaortic lymph nodes were removed and imaged together with the remaining body, and the lymph nodes were proven to be the light sources. In two mice, light emission from the anterior thorax near the thoracotomy incision was shown. For the right knee, no substantial luminescent signal was found before bone cutting. The light signal increased dramatically after bone cutting (Figure 2). The signal intensities for the right knee before and 1 min after cutting were 1.72 × 10⁵ ± 5.75 × 10⁴ and 3.51 × 10⁷ ± 2.66 × 10⁷ p s⁻¹, respectively.

Figure 1.

Whole-body bioluminescence images. The pseudocolor luminescent image is overlaid on the grayscale photographic image. Shown are ventral images before sacrifice (left), 5 min after death (middle), and 5 min after intraperitoneal air injection (right). The same color scale was used in the three panels. The light signal disappeared after death and was recovered only for the abdomen after air injection.

Figure 2.

Bioluminescence images before (left) and 1 min after (right) cutting around the right knee. The same color scale was used for both panels. A light signal at the right knee was seen only after cutting.

Discussion

Patients with Philadelphia chromosome-positive ALL frequently express the p190 BCR-ABL fusion protein and have a poor prognosis [12]. Although the BCR-ABL tyrosine kinase inhibitor, imatinib mesylate (Novartis Pharmaceuticals, Basel, Switzerland), is effective, resistance to this drug develops rapidly and novel therapeutic strategies need to be explored [14]. Ba/F3-Luc/Wt cells show IL-3-independent, autonomous proliferation and stable luciferase expression, and mice injected with the cells intravenously may be used to evaluate treatments for Philadelphia chromosome-positive ALL by in vivo BLI.

The mice implanted with Ba/F3-Luc/Wt cells showed extensive light emission in vivo. This light signal disappeared after death, as described previously [10], and the intraperitoneal air injection restored light emission from the abdomen only. Light photons should be attenuated similarly before and after air injection, and these observations support the hypothesis that luminescence after sacrifice requires exposure to the atmosphere. Luminescence from cells transduced with firefly luciferase relies on the oxidation of d-luciferin catalyzed by luciferase. Oxygen in the atmosphere appears to play a critical role in the oxidation after death.

Although the identification of the involved organs was difficult from in vivo images, ex vivo imaging showed the proliferation of implanted cells in the liver, spleen, lung, intestine, gynecologic organs, and lymph nodes. However, signals from these organs could not fully explain those observed on in vivo images. Proliferation in the bone marrow is expected based on the nature of Ba/F3 cells. Although some in vivo signals appeared to originate from the skeletal system, light signals were essentially absent on imaging the remaining body after removing the internal organs and involved lymph nodes, except for the signal from the anterior thorax near the thoracotomy incision in two mice. We evaluated luminescence at the right knee further. Whereas in vivo BLI suggested luminescence at the site, imaging of the dead body did not show a substantial light signal, even after removal of the overlying tissues. Subsequent cutting around the knee restored the light emission dramatically. The transfer of oxygen from the atmosphere to cells within the bone marrow cavity appeared to be blocked by the bone cortex. The signal from the anterior thorax was probably attributable to luminescence in the incised sternum. Our results indicate that destruction of the bone cortex is required to assess luciferase expression in the bone marrow.

Although the need for oxygen in luciferase reaction is well known, to the best of our knowledge, the crucial role of exposure to atmospheric oxygen in ex vivo imaging has not been clearly demonstrated. In particular, the knowledge that bone destruction is required to assess luciferase activity in the bone marrow would be important in experiments using models of bone metastasis and blood cell transplantation. If a researcher picks up a bone, taking care not to damage the bone, and image it, he or she may miss luciferase activity in the bone marrow cavity. If a researcher does not know the need for bone destruction, he or she may image the whole skeleton after removing skin and soft tissues to assess luciferase activity in the bone marrow, resulting in failure of detection.

In summary, we demonstrated that light emission in ex vivo BLI needs exposure to the atmosphere. Researchers should be aware that bone destruction is required to elucidate luciferase activity in the bone marrow cavity after sacrifice.