acquired using a custom-written interface using Kmax software and ... Clinical application of cell-specific molecular imaging probes such as cFLFLF may permit ...
Noninvasive imaging of inflammation after lung transplantation using SPECT and a formyl peptide receptor ligand cFLFLF The University of Virginia, Department of Surgery Eric Charles, Mahendra D. Chordia, Ashish K. Sharma, J. Hunter Mehaffey, Yi Zhang, David Glover, Irving Kron, Dongfeng Pan, Victor E. Laubach 99mTc-cFLFLF
Background
• Ischemia-reperfusion
injury (IRI) leads to primary graft dysfunction (PGD) and early mortality after lung transplantation.
A)
Transverse Plane
SPECT imaging of lung IRI in mice Coronal Plane
B)
Cy7-cFLFLF fluorescence imaging
C) A) In vivo
• Current methods to diagnose PGD are limited to x-ray, CT, and functional tests, none of
B) Ex vivo Sham
which specifically address inflammation or immune cell activation; key components of IRI.
• The development of noninvasive, cell-specific molecular imaging methods to provide early
Sham
IR
IR (2 hr)
Left
C) Intensity IR (2 hr)
Right
Left
12 1.20E+09
• The synthetic peptide cFLFLF binds formyl peptide receptor 1 (FPR1), which is up-regulated on activated neutrophils during inflammation.
Avg. radiance (p/s/cm2/sr) x108
diagnosis of IRI would permit timely and targeted therapeutic interventions.
•
2hr
99mTc-cFLFLF
successfully targets neutrophils to identify inflammation by SPECT in murine models (Mol Imaging Biol 17:337-44, 2015; J Nucl Med 50:790-7, 2009).
• We hypothesize that targeting neutrophil activation via
99mTc-cFLFLF
Left lung
Right
Sham
SPECT will provide
sensitive and accurate diagnosis of lung IRI.
Right lung
10 1.00E+09
8
8.00E+08
6
6.00E+08
4
4.00E+08
2
2.00E+08
0
0.00E+00
Sham
IR
Methods Mouse lung IRI: An in vivo hilar occlusion model of IRI was used in C57BL/6 mice. Left lung ischemia was initiated after left thoracotomy and suture ligation of the left hilum, and reperfusion was initiated upong suture removal to allow in vivo reperfusion for 2, 12, or 24 hrs. Sham animals received the same surgery without hilar occlusion.
IR
12hr
Analysis of lung IRI: After reperfusion, animals were re-anesthetized and pulmonary function was measured using an isolated mouse lung system. Cytokines were measured in bronchoalveolar lavage fluid using multiplex cytokine bead-array assay (Bio-Rad Laboratories).
Sham
Mouse SPECT/CT: SPECT/CT was performed in the laboratory of Dr. Dongfeng Pan as previously described using a microSPECT/CT scanner designed and built at UVA (Bioconjug Chem 21:1788-93, 2010). Mice were injected with 99mTc-cFLFLF 2 hrs prior to SPECT imaging. SPECT was performed with a gamma camera on a 10cm×10cm field of view. SPECT data are acquired using a custom-written interface using Kmax software and reconstructed using a custom-written maximum-likelihood expectation-maximization algorithm. CT images are reconstructed with a Feldkamp algorithm and fused with an Interactive Data Language program. Porcine SPECT: Images were acquired as previously described (Circulation 106:592-8, 2002) using a Siemens Orbiter SPECT camera. Image datasets were reconstructed using an OSEM software algorithm, and 99mTc-cFLFLF probe uptake is quantified using the 3D region-of-interest tool available in the image analysis software (VivoQuant, InVicro, Inc.). Near-infrared fluorescence imaging: Ex vivo lungs were imaged using an IVIS Spectrum Fluorescence Imaging system. Mice were injected with Cy7-cFLFLF 2 hrs prior to imaging.
Airway Resistance (cm H2O/μl/s)
Lung Compliance (µL/cm H2O)
*
*
* *#
99mTc-cFLFLF
SPECT after of pig lung transplantation
IR L
R
R
L
R
L
24hr
Sham
Lung inflammation was visualized by SPECT after sham or IR at 2, 12, and 24 hours of reperfusion. A) Representative SPECT images are shown. B) Maximum signal intensity ratios in left lungs after IR were significantly higher than sham mice after 2 and 12 hours of reperfusion but not after 24 hours. C) 99mTc-cFLFLF uptake (via gamma counts of whole ex vivo lungs) in left lungs decreased over time.
Lung IRI peaks at 2 hrs and resolves by 24 hrs *#
Near-infrared fluorescence imaging of cFLFLF-Cy7 in live (shaved), anesthetized mice (A) and in ex vivo lungs (B) demonstrating left lung IR injury (elevated fluorescence). C) Fluorescence intensity in ex vivo lungs was 4-fold greater in left lung after IR vs. right or sham lungs.
K
K
K
SPECT imaging of 99mTc-cFLFLF uptake after porcine left lung ischemia, transplantation, and 4 hrs reperfusion. Robust inflammation is visible in the left (L) lung vs. right (R). Three different Maximum Intensity Projection views from the 360° orbit are shown. Clearance by the kidneys (K) is visible, which is expected.
#
Organ biodistribution of 99mTc-cFLFLF in mice 6
Sham
IR (2hr)
IR (12hr)
*
PA Pressure (cm H2O)
Sham
IR (24hr) 160
*#
*
IR (2hr)
IL-17
IR (12hr)
IR (24hr)
IL-10
#
pg/ml
120
* *
80 40
*
*
0
Sham
IR (2hr)
IR (12hr)
IR (24hr)
Sham IR IR IR (2) (12) (24)
Sham IR IR IR (2) (12) (24)
Rel. radioactivity (ID/body wt) x106
6.E+06
Sham
IR
Conclusions
4
4.E+06
• SPECT 2
2.E+06
•
*
• 0
0.E+00
IRI peaks at 2 hrs reperfusion and resolves by 24 hrs. Lung function (compliance, airway resistance, PA pressure) is significantly worse vs. sham after 2 and 12 hrs reperfusion but improved after 24 hrs. IRI entails progressive reduction of pro-inflammatory cytokines (IL-17) and production of antiinflammatory cytokines (IL-10). *p
