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. 2006 Oct;6(10):2321-31.
doi: 10.1111/j.1600-6143.2006.01469.x. Epub 2006 Jul 25.

Intraoperative detection of cell injury and cell death with an 800 nm near-infrared fluorescent annexin V derivative

S Ohnishi  1 , J-L Vanderheyden, E Tanaka, B Patel, A M De Grand, R G Laurence, K Yamashita, J V Frangioni
Affiliations

Intraoperative detection of cell injury and cell death with an 800 nm near-infrared fluorescent annexin V derivative

S Ohnishi et al. Am J Transplant. 2006 Oct.

Abstract

The intraoperative detection of cell injury and cell death is fundamental to human surgeries such as organ transplantation and resection. Because of low autofluorescence background and relatively high tissue penetration, invisible light in the 800 nm region provides sensitive detection of disease pathology without changing the appearance of the surgical field. In order to provide surgeons with real-time intraoperative detection of cell injury and death after ischemia/reperfusion (I/R), we have developed a bioactive derivative of human annexin V (annexin800), which fluoresces at 800 nm. Total fluorescence yield, as a function of bioactivity, was optimized in vitro, and final performance was assessed in vivo. In liver, intestine and heart animal models of I/R, an optimal signal to background ratio was obtained 30 min after intravenous injection of annexin800, and histology confirmed concordance between planar reflectance images and actual deep tissue injury. In summary, annexin800 permits sensitive, real-time detection of cell injury and cell death after I/R in the intraoperative setting, and can be used during a variety of surgeries for rapid assessment of tissue and organ status.

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Figures

Figure 1
Figure 1. Synthesis and Optical Properties of Annexin800
A) Absorbance of annexin800 (labeling ratio = 1.2) in PBS compared to 20% methanol (left ordinate). Fluorescence of annexin800 in 20% methanol (right ordinate). B) The labeling ratio as a function of protein/fluorophore mixing ratio. C) Per fluorophore fluorescence, as a function of labeling ratio, compared to free CW800-CA (1 μM) in PBS (open circles). Per molecule total fluorescence yield calculated from the labeling ratio and per fluorophore fluorescence, and compared to the same dye concentration (1 μM) of free CW800-CA in PBS (closed circles). D) MALDI-TOF analysis of annexin800 (labeling ratio = 2.2) compared to annexin V.
Figure 1
Figure 1. Synthesis and Optical Properties of Annexin800
A) Absorbance of annexin800 (labeling ratio = 1.2) in PBS compared to 20% methanol (left ordinate). Fluorescence of annexin800 in 20% methanol (right ordinate). B) The labeling ratio as a function of protein/fluorophore mixing ratio. C) Per fluorophore fluorescence, as a function of labeling ratio, compared to free CW800-CA (1 μM) in PBS (open circles). Per molecule total fluorescence yield calculated from the labeling ratio and per fluorophore fluorescence, and compared to the same dye concentration (1 μM) of free CW800-CA in PBS (closed circles). D) MALDI-TOF analysis of annexin800 (labeling ratio = 2.2) compared to annexin V.
Figure 2
Figure 2. Predicted Three-Dimensional Structure of Annexin800
The conjugation sites of CW800 to annexin V (white) were established using trypsin digestion and ES-TOF LC/MS peptide analysis, and are shown overlaid with the crystal structure (31). The amino acid substitution sites are Lys 208 (red) and Lys 286 (green). Annexin V's calcium binding sites are shown in yellow.
Figure 3
Figure 3. Biopotency of Annexin800
The biopotency of annexin V and annexin800 measured as the binding capacity to PS in vitro using plasmon surface resonance. EC50 of annexin V and annexin800 (labeling ratio = 1.2) is 0.28 mM and 0.21 mM, respectively. RU; resonance unit.
Figure 4
Figure 4. Detection of Cell Injury and Death in Vitro
U937 leukemic cells were treated with or without 50 μM etoposide for 6 hrs, then stained with PBS (control) or 50 nM protein of either annexin800 (labeling ratio = 1.2), annexin800 (labeling ratio = 2.2), or CW800 conjugated to human serum albumin (HSA800; labeling ratio = 3.0).
Figure 5
Figure 5. Detection of Cell Injury and Death after Ischemia/Reperfusion of Rat Liver
A) Right hepatic artery and corresponding portal vein were clamped for 1 hr, and reperfused for 2 hrs. Imaging was performed at 30 min post-injection of 1.2 mg/kg of annexin800 protein (labeling ratio = 1.2; 40 nmol/kg of fluorophore total). I: ischemic segments; N: normal segments; Arrows mark right hepatic lobe subjected to I/R. B) SBR (mean ± SEM; n = 6 animals) of ischemic versus normal liver post-injection of annexin800 as described in (A). C) Frozen sections obtained from the liver in 5A were analyzed by H&E (top row), DAPI staining of all nuclei (second row), TUNEL staining (third row), and NIR fluorescence (bottom row). Shown are representative central (left) and portal (right) fields.
Figure 5
Figure 5. Detection of Cell Injury and Death after Ischemia/Reperfusion of Rat Liver
A) Right hepatic artery and corresponding portal vein were clamped for 1 hr, and reperfused for 2 hrs. Imaging was performed at 30 min post-injection of 1.2 mg/kg of annexin800 protein (labeling ratio = 1.2; 40 nmol/kg of fluorophore total). I: ischemic segments; N: normal segments; Arrows mark right hepatic lobe subjected to I/R. B) SBR (mean ± SEM; n = 6 animals) of ischemic versus normal liver post-injection of annexin800 as described in (A). C) Frozen sections obtained from the liver in 5A were analyzed by H&E (top row), DAPI staining of all nuclei (second row), TUNEL staining (third row), and NIR fluorescence (bottom row). Shown are representative central (left) and portal (right) fields.
Figure 6
Figure 6. Detection of Cell Injury and Death after Ischemia/Reperfusion of Rat Intestine
A) A branch artery of the superior mesenteric artery and its corresponding vein (arrowhead), and both sides of the occupying intestine (arrows) were clamped for 1 hr, and reperfused for 2 hrs. Imaging was performed at 60 min post-injection of 1.2 mg/kg of annexin800 protein (labeling ratio = 1.2; 40 nmol/kg of fluorophore total). B) SBR (mean ± SEM; n = 6 animals) of ischemic versus normal intestine post-injection of annexin800 as described in (A). C) Frozen sections obtained from the intestine in 6A were analyzed by H&E (top row), DAPI staining of all nuclei (second row), TUNEL staining (third row), and NIR fluorescence (bottom row).
Figure 6
Figure 6. Detection of Cell Injury and Death after Ischemia/Reperfusion of Rat Intestine
A) A branch artery of the superior mesenteric artery and its corresponding vein (arrowhead), and both sides of the occupying intestine (arrows) were clamped for 1 hr, and reperfused for 2 hrs. Imaging was performed at 60 min post-injection of 1.2 mg/kg of annexin800 protein (labeling ratio = 1.2; 40 nmol/kg of fluorophore total). B) SBR (mean ± SEM; n = 6 animals) of ischemic versus normal intestine post-injection of annexin800 as described in (A). C) Frozen sections obtained from the intestine in 6A were analyzed by H&E (top row), DAPI staining of all nuclei (second row), TUNEL staining (third row), and NIR fluorescence (bottom row).
Figure 7
Figure 7. Detection of Cell Injury and Death after Ischemia/Reperfusion of Canine Heart
A) A branch of the left anterior descending artery (arrowhead) was occluded for 2 hrs, and reperfused for 2 hrs. Imaging was performed at 30 min post-injection of 0.3 mg/kg of annexin800 protein (labeling ratio = 1.2; 10 nmol/kg of fluorophore total). Arrow marks territory at risk. B) The heart was sectioned 60 min after injection of annexin800 and stained with TTC. NIR fluorescence was concordant with TTC staining (arrows). C) The kidney was sectioned 60 min after injection of annexin800, revealing high annexin800 accumulation in the renal cortex.
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