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In vivo imaging of endogenous enzyme activities using luminescent 1,2-dioxetane compounds.

Tseng JC, Kung AL - J. Biomed. Sci. (2015)

Bottom Line: In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity.In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

View Article: PubMed Central - PubMed

Affiliation: Lurie Family Imaging Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA. jct223@gmail.com.

ABSTRACT

Background: Here we present a non-invasive imaging method for visualizing endogenous enzyme activities in living animals. This optical imaging method is based on an energy transfer principle termed chemically initiated electron exchange luminescence (CIEEL). The light energy is provided by enzymatic activation of metastable 1,2-dioxetane substrates, whose protective groups are removed by hydrolytic enzymes such as β-galactosidase and alkaline phosphatase. In the presence of a nearby fluorescent recipient, the chemical energy within the activated substrate is then transferred via formation of a charge-transfer complex with the fluorophore, a mechanism closely related to glow stick chemistry.

Results: Efficient CIEEL energy transfer requires close proximity between the trigger enzyme and the fluorescent recipient. Using cells stained with fluorescent dialkylcarbocyanines as the energy recipients, we demonstrated CIEEL imaging of cellular β-galactosidase or alkaline phosphatase activity. In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.

Conclusions: In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity. In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

No MeSH data available.


Related in: MedlinePlus

CIEEL energy transfer imaging of elevated alkaline phosphatase activity during tissue inflammation. a Splenocytes were harvested and cultured in the presence of LPS, a potent stimulant for inflammatory responses. The cells were then stained with DiR and subjected to CIEEL imaging with Galacton-plus. Untreated cells were used as negative control. The cells were scanned from 520–800 nm to establish their luminescence emission profiles. b CIEEL imaging of endogenous phosphatase activity in the peritoneal cavity of normal mice. Animals received i.p. injection of CSPD in the presence or absence of DiR, and were then subject to sequential luminescence acquisition from 520 to 800 nm. c NCr nude mice were i.p. treated with LPS to induce inflammatory responses in the peritoneal cavity. CIEEL imaging was performed 24 h later. Untreated control mice were used as imaging control. d Mice were pretreated with dexamethasone, a potent anti-inflammatory drug, and then challenged by i.p. LPS injection. CSPD-DiR CIEEL imaging was performed 24 h after LPS challenge. Error bar = SEM
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Fig6: CIEEL energy transfer imaging of elevated alkaline phosphatase activity during tissue inflammation. a Splenocytes were harvested and cultured in the presence of LPS, a potent stimulant for inflammatory responses. The cells were then stained with DiR and subjected to CIEEL imaging with Galacton-plus. Untreated cells were used as negative control. The cells were scanned from 520–800 nm to establish their luminescence emission profiles. b CIEEL imaging of endogenous phosphatase activity in the peritoneal cavity of normal mice. Animals received i.p. injection of CSPD in the presence or absence of DiR, and were then subject to sequential luminescence acquisition from 520 to 800 nm. c NCr nude mice were i.p. treated with LPS to induce inflammatory responses in the peritoneal cavity. CIEEL imaging was performed 24 h later. Untreated control mice were used as imaging control. d Mice were pretreated with dexamethasone, a potent anti-inflammatory drug, and then challenged by i.p. LPS injection. CSPD-DiR CIEEL imaging was performed 24 h after LPS challenge. Error bar = SEM

Mentions: We then determined if this energy transfer strategy can be used for imaging endogenous leukocyte alkaline phosphatase (LAP) activity in mouse splenocytes [20]. In this set of studies, we used a potent stimulant lipopolysaccharide (LPS) to upregulate LAP activity in splenocytes (Fig. 6a). LPS is an important component of bacterial membrane and is known for its capability to upregulate LAP activity in neutrophils [20]. Splenocytes were cultured in the presence of LPS to stimulate intracellular LAP activity. Next day, we used DiR to stain the cell membrane and then added CSPD to provide chemical energy for imaging. LPS treatment significantly increased the CSPD-DiR energy transfer luminescence in the range of 720–800 nm (Fig. 6a).Fig. 6


In vivo imaging of endogenous enzyme activities using luminescent 1,2-dioxetane compounds.

Tseng JC, Kung AL - J. Biomed. Sci. (2015)

CIEEL energy transfer imaging of elevated alkaline phosphatase activity during tissue inflammation. a Splenocytes were harvested and cultured in the presence of LPS, a potent stimulant for inflammatory responses. The cells were then stained with DiR and subjected to CIEEL imaging with Galacton-plus. Untreated cells were used as negative control. The cells were scanned from 520–800 nm to establish their luminescence emission profiles. b CIEEL imaging of endogenous phosphatase activity in the peritoneal cavity of normal mice. Animals received i.p. injection of CSPD in the presence or absence of DiR, and were then subject to sequential luminescence acquisition from 520 to 800 nm. c NCr nude mice were i.p. treated with LPS to induce inflammatory responses in the peritoneal cavity. CIEEL imaging was performed 24 h later. Untreated control mice were used as imaging control. d Mice were pretreated with dexamethasone, a potent anti-inflammatory drug, and then challenged by i.p. LPS injection. CSPD-DiR CIEEL imaging was performed 24 h after LPS challenge. Error bar = SEM
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: CIEEL energy transfer imaging of elevated alkaline phosphatase activity during tissue inflammation. a Splenocytes were harvested and cultured in the presence of LPS, a potent stimulant for inflammatory responses. The cells were then stained with DiR and subjected to CIEEL imaging with Galacton-plus. Untreated cells were used as negative control. The cells were scanned from 520–800 nm to establish their luminescence emission profiles. b CIEEL imaging of endogenous phosphatase activity in the peritoneal cavity of normal mice. Animals received i.p. injection of CSPD in the presence or absence of DiR, and were then subject to sequential luminescence acquisition from 520 to 800 nm. c NCr nude mice were i.p. treated with LPS to induce inflammatory responses in the peritoneal cavity. CIEEL imaging was performed 24 h later. Untreated control mice were used as imaging control. d Mice were pretreated with dexamethasone, a potent anti-inflammatory drug, and then challenged by i.p. LPS injection. CSPD-DiR CIEEL imaging was performed 24 h after LPS challenge. Error bar = SEM
Mentions: We then determined if this energy transfer strategy can be used for imaging endogenous leukocyte alkaline phosphatase (LAP) activity in mouse splenocytes [20]. In this set of studies, we used a potent stimulant lipopolysaccharide (LPS) to upregulate LAP activity in splenocytes (Fig. 6a). LPS is an important component of bacterial membrane and is known for its capability to upregulate LAP activity in neutrophils [20]. Splenocytes were cultured in the presence of LPS to stimulate intracellular LAP activity. Next day, we used DiR to stain the cell membrane and then added CSPD to provide chemical energy for imaging. LPS treatment significantly increased the CSPD-DiR energy transfer luminescence in the range of 720–800 nm (Fig. 6a).Fig. 6

Bottom Line: In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity.In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

View Article: PubMed Central - PubMed

Affiliation: Lurie Family Imaging Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA. jct223@gmail.com.

ABSTRACT

Background: Here we present a non-invasive imaging method for visualizing endogenous enzyme activities in living animals. This optical imaging method is based on an energy transfer principle termed chemically initiated electron exchange luminescence (CIEEL). The light energy is provided by enzymatic activation of metastable 1,2-dioxetane substrates, whose protective groups are removed by hydrolytic enzymes such as β-galactosidase and alkaline phosphatase. In the presence of a nearby fluorescent recipient, the chemical energy within the activated substrate is then transferred via formation of a charge-transfer complex with the fluorophore, a mechanism closely related to glow stick chemistry.

Results: Efficient CIEEL energy transfer requires close proximity between the trigger enzyme and the fluorescent recipient. Using cells stained with fluorescent dialkylcarbocyanines as the energy recipients, we demonstrated CIEEL imaging of cellular β-galactosidase or alkaline phosphatase activity. In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.

Conclusions: In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity. In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

No MeSH data available.


Related in: MedlinePlus