Click on an contribution preview the abstract content.
Indocyanine Green labeling for optical and photoacoustic imaging of Mesenchymal Stem Cells after in vivo transplantation. (#389)
F. Garello1, M. Filippi1, F. Arena1, P. Giustetto1, C. Pasquino2, E. Terreno1
1 University of Torino, Molecular and Preclinical Imaging Centers, Department of Molecular Biotechnology and Health Sciences, Torino, Italy
Cell therapy using Mesenchymal Stem Cells (MSCs) holds great promise for the treatment of many diseases, and poses the challenge to monitor the cell fate in vivo. Photoacoustic imaging (PAI) is a good candidate. So far, cell imaging by PAI has been performed loading cells with gold and carbon-based nanoparticles.1 Despite the good results obtained, some episodes of adverse reactions were reported. The clinically approved Indocyanine Green (ICG) is a valid alternative. This study aims at assessing the use of ICG as cell tracer to label and track murine bone marrow-derived MSCs by PAI.
MSCs were isolated from the bone marrow of male C57BL/6J mice, cultured, and incubated with 0.25 mg/mL of ICG for variable times, ranging from 2 minutes to 6 hours. Cell viability and phenotype preservation were checked at each time point. The cell uptake of ICG was estimated by fluorimetry. Comparison among free ICG, ICG in presence of cells and ICG internalized within cells was carried out by OI (IVIS Spectrum, Perkin Elmer) and PAI (VisualSonics Vevo 2100 LAZR). 3x105 MSCs were then labeled upon optimized incubation conditions, suspended in 100 µL of PBS, and intramuscularly injected into the right hindlimb of C57BL/6J mice. Transplanted cells were monitored by both OI and PAI for 7 days.
In vitro assays, carried out on MSCs, demonstrated that viability and phenotype were preserved upon 1 hour incubation, with good internallization efficiency (1.71×1010 ± 3.88×109 internalized molecules/cell). PA spectra acquired on samples containing pure ICG at different concentrations (15µM-1mM), ICG mixed with MSCs cells or ICG-labeled cells (final dye concentration 85 µM) demonstrated that the PA sensitivity dramatically increased when the fluorescent dye was confined into MSCs (Figure 1), resulting into a clearly detectable signal. When 3x105 MSCs labeled cells were transplanted in the right hindlimb the engraftment could be monitored until 3 days p.i. both by PAI and OI (Figure 2). Increasing the number of cells to 1x106 made cell tracking possible until 7 days p.i.
In summary, ICG was successfully used as PA-FLI dual-mode contrast agent to label MSCs both in vitro and in vivo. Since the number of MSCs involved in several experimental circumstances is usually similar or higher than that used here,2 we conclude that in a forward-looking vision this technique retains a considerable potential for transplantation-focused research and therapy by providing the in vivo cell fate surveillance with safety, real time content, good endogenous contrast among soft tissues and improved spatial resolution.
Keywords: Cell tracking, ICG, Photoacoustic Imaging, Stem Cells
Exploring alternative radiolabelling strategies for sialic acid binding Ig-like lectin 9 peptide: [68Ga]Ga-NOTA-SIglec-9 and [18F]AlF-NOTA-Siglec-9 (#356)
O. Moisio1, R. Siitonen1, E. Suomela1, X. - G. Li1, S. Jalkanen2, A. Roivainen3
1 University of Turku, Turku PET Centre, Turku, Finland
Amino acid residues 283-297 from sialic acid binding Ig-like lectin 9 (Siglec-9) form a cyclic peptide ligand targeting vascular adhesion protein-1 (VAP-1). VAP-1 is associated with the transfer of leukocytes from blood to tissues upon inflammation . Therefore, positron emitting radioactive analogues of Siglec-9 peptide are good candidates for visualizing inflammation. The PET ligand [68Ga]-DOTA-Siglec-9 has been evaluated extensively for this purpose [2-5]. Here, we explored two alternative strategies for radiolabeling the Siglec-9 peptide using a NOTA-chelator to bind 68Ga and Al18F.
After setting up radiosynthesis, the ligands were evaluated by in vivo PET imaging and ex vivo gamma counting of turpentine-induced sterile skin inflammation in Sprague-Dawley rats. The rats were subcutaneously injected with 50 µL turpentine oil (Sigma-Aldrich) into right shoulder area to induce inflammation, which was allowed to develop for 24 hours prior to PET imaging. From the PET images, time-activity curves were determined from selected regions of interest using CARIMAS software (Turku PET Centre).
[68Ga]Ga-NOTA-Siglec-9 and [18F]AlF-NOTA-Siglec-9 were produced with high radiochemical quality (53 ± 2 % (n=3) for [68Ga] and 26 ± 3 % (n=3) for [18F]) and sufficient specific radioactivity (5.0 ± 0.2 GBq/nmol (n=3) with [68Ga] and 8.8 ± 0.9 GBq/nmol (n=3) with [18F]). Successful in vivo PET imaging of sterile inflammation was achieved with both tracers, and the results were further confirmed by γ-counting of excised tissue sections. γ-counted Inflamed tissue-to-muscle activity ratios were 10.8 ± 1.7 to 1 and 11.2 ± 2.7 to 1, respectively.
In conclusion, [68Ga]- and Al[18F]-NOTA-Siglec-9 were produced with high radiochemical quality and suffiecieny specific radioactivity to achieve successful preclinical in vivo PET imaging of sterile skin inflammation. Imperatively, low bone uptake was indicative of the absence of in vivo radiofluorination which can be a concern with 18F-labelled ligands.
1. Tohka, S.; Laukkanen, M.; Jalkanen, S.; Salmi, M. Vascular adhesion protein 1 (VAP-1) functions as a molecular brake during granulocyte rolling and mediates recruitment in vivo. FASEB J. 2001, 15, 373–382, doi:10.1096/fj.00-0240com.
2. Virtanen, H.; Autio, A.; Siitonen, R.; Liljenbäck, H.; Saanijoki, T.; Lankinen, P.; Mäkilä, J.; Käkelä, M.; Teuho, J.; Savisto, N.; Jaakkola, K.; Jalkanen, S.; Roivainen, A. 68Ga-DOTA-Siglec-9 – a new imaging tool to detect synovitis. Arthritis Res. Ther. 2015, 17, 308, doi:10.1186/s13075-015-0826-8.
3. Silvola, J. M. U.; Virtanen, H.; Siitonen, R.; Hellberg, S.; Liljenbäck, H.; Metsälä, O.; Ståhle, M.; Saanijoki, T.; Käkelä, M.; Hakovirta, H.; Ylä-Herttuala, S.; Saukko, P.; Jauhiainen, M.; Veres, T. Z.; Jalkanen, S.; Knuuti, J.; Saraste, A.; Roivainen, A. Leukocyte trafficking-associated vascular adhesion protein 1 is expressed and functionally active in atherosclerotic plaques. 2016, doi:10.1038/srep35089.
4. Ahtinen, H.; Kulkova, J.; Lindholm, L.; Eerola, E.; Hakanen, A. J.; Moritz, N.; Söderström, M.; Saanijoki, T.; Jalkanen, S.; Roivainen, A.; Aro, H. T. 68Ga-DOTA-Siglec-9 PET/CT imaging of peri-implant tissue responses and staphylococcal infections. EJNMMI Res. 2014, 4, 45, doi:10.1186/s13550-014-0045-3.
5. Retamal, J.; Sörensen, J.; Lubberink, M.; Suarez-Sipmann, F.; Borges, J. B.; Feinstein, R.; Jalkanen, S.; Antoni, G.; Hedenstierna, G.; Roivainen, A.; Larsson, A.; Velikyan, I. Feasibility of (68)Ga-labeled Siglec-9 peptide for the imaging of acute lung inflammation: a pilot study in a porcine model of acute respiratory distress syndrome. Am. J. Nucl. Med. Mol. Imaging 2016, 6, 18–31.
This research was conducted within the Finnish Centre of Excellence in Cardiovascular and Metabolic Diseases supported by the Academy of Finland, University of Turku, Turku University Hospital, and Åbo Akademi University. The research leading to these results was further supported by funding from the Academy of Finland (#258814), the State Research Funding (#13856), and the Sigrid Jusélius Foundation.
Representative PET images of [86Ga]- and Al[18F]-NOTA-Siglec-9 distribution in rats. Regions of interest: 1) inflamed area, 2) control area, 3) kidneys, 4) liver 5) urinary bladder. "> Representative PET images of tracer distribution in rats.
Representative PET images of [86Ga]- and Al[18F]-NOTA-Siglec-9 distribution in rats. Regions of interest: 1) inflamed area, 2) control area, 3) kidneys, 4) liver 5) urinary bladder.
Keywords: VAP-1, Siglec-9, PET-imaging, sterile skin inflammation, gallium-68, aluminum- fluoride, fluorine-18
Omics profiling of inflammatory immune response in GPI-induced arthritis. (#562)
M. A. Jarboui1, K. Fuchs1, M. A. Neveu1, J. Schwenck1, N. Mucha1, B. J. Pichler1, M. Kneilling1
1 Werner Siemens Imaging Center, Eberhard Karls University Tuebingen, Tuebingen, Baden-Württemberg, Germany
Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease marked by extensive synovitis and joint damage. Despite advances in RA diagnosis and management, RA pathogenesis is not yet fully elucidated. The systemic effect mediated by immune cells activation correlate with metabolic changes. Comprehensive analysis of omics signature in major immune lymphoid, inflammation sites and plasma could provide valuable insights into the systemic inflammatory response. Also, understand how subjects suffering from chronic inflammatory disease are more prone to develop tumor metastasis.
Using the well-established glucose-6-phosphate (GPI) antibody (Ab) induced RA mouse model, we isolated the spleen, the bone marrow (BM) and the synovial fluid at the peak of joint inflammation (6 days post GPI injection) and isolated myeloid-derived suppressor cells (MDSCs) using the MACS isolation kit. As control, we used control Ab injected healthy mice. Additionally, we collected serum from GPI or control Ab injected mice. Next, we analyzed the proteomic signature using shot-gun proteomics on high-resolution LTQ Orbitrap Fusion. Acquired MS spectra were further analyzed using label-free quantification algorithm. Moreover, we analyzed the metabolomic profiles of the mice serum using the targeted metabolomic platform from Biocrates where metabolites were detected by LC-MS analysis.
Comparative proteomic analysis of different MDSCs strongly correlates with their organ of origin and inflammatory state. Unbiased PCA analysis of the proteomic signature exhibited a clear separation between the synovial fluid (SF-MDSCs), the bone marrow (BM-MDSCs) and the spleen MDSCs (S-MDSCs) population. Arg1, a key player in immune response, was more abundant in the BM-MDSCs and SF-MDSCs of AR mice. As the integral hallmark in RA is angiogenesis, we identified several markers from the S100 proteins family. Accordingly, S100a6 and S100a8-S100a11 were highly abundant in the SF-MDSCs population of AR mice. Interestingly, S100a8/S100a9 heterodimer is considered a potential biomarker for acute RA (1,2). Plasma metabolomic analysis showed increased abundance of polyunsaturated fatty acids (PUFA) and the biogenic amines: carnosine, serotonin and polyamines. Interestingly, PUFA abundance, associated with oxidative stress, leading to an increase in carnosine, described as a ROS scavenger.
Altogether, our MDSCs proteomic and plasma metabolomic analysis of GPI induced RA model revealed the coordinated multiplex systemic immune response and the molecular changes leading to the establishment of chronic inflammation. Moreover, we identified potential biomarkers that could indicate the severity of the inflammatory process and possible molecular targets that could modulate inflammation. Interestingly, Zn-carnosine (polaprezinc) is used as an inhibitory drug of H Pylori- induced inflammation. As our analysis was carried out in one time point, we plan continuative longitudinal studies.
1. Perera C et al, CL, Immunol Cell Biol. 2010 Jan; 88(1):41-9,
2. Van Lent PL et al Arthritis Rheum. 2008 Dec; 58(12):3776-87.
Keywords: Inflammation, arthritis, autoimmune, metabolomics, proteomics, metastasis
Novel µCT-derived biomarkers for longitudinal evaluation of silica-induced pulmonary fibrosis in a mouse model (#197)
K. Dekoster1, S. Van Den Broucke2, T. Decaesteker3, N. Berghen4, A. Krouglov1, J. Wouters1, R. Lories4, P. Hoet3, J. Vanoirbeek2, G. Vande Velde1
1 KU Leuven, Biomedical MRI unit/MoSAIC, Department of Imaging and Pathology, Leuven, Belgium
Pulmonary fibrosis, the formation of excess connective lung tissue, is a life-threatening condition for which effective treatment is still lacking1,2. This shortage of effective drugs exposes a gap in our knowledge regarding the disease mechanism and potential treatment targets in lung fibrosis. As we need to rethink the methodology used to evaluate lung fibrosis and its therapy in the lung research field, we introduced an in vivo 4D-respiratory gated low dose µCT protocol. This allows longitudinal assessment of an improved silica-induced lung fibrosis model in free-breathing mice.
C57BL/6 mice were instilled endotracheally with either crystalline silica (to induce lung fibrosis, n = 6) or saline (control, n = 4). After instillation, animals were scanned weekly for a period of 5 weeks. Respiratory gated µCT images were obtained using a small-animal µCT scanner (SkyScan 1278, Bruker microCT). To assess whether inflammation and fibrosis occurred in the silica-induced mice, we evaluated four different lung biomarkers derived from the scans: total lung volume, lung tissue volume, aerated lung volume and mean lung density3. After the last scan, at day 35, lung function measurements were obtained (FlexiVent 7.3, SCIREQ) and data was cross-validated with ex vivo gold standard techniques.
Silica instillation resulted in a significant increase of tissue volume, corresponding with tissue fibrosis, visible after 7 days and remaining stable up until 5 weeks (Fig. 1). Total lung volume increased after 14 days. Remarkably, the aerated lung volume dropped after 7 days to gradually increase again. For the mean lung density there was no significant difference between silica and control conditions. Imaging results were mostly consistent with pulmonary function test.
Our findings indicate that low-dose µCT is an excellent technique to longitudinally evaluate silica-induced pulmonary fibrosis in mice, a novel approach that allows evaluating host response, disease progression and stabilization over several weeks.
1. Gabrielli, A., Avvedimento, E. V & Krieg, T. Scleroderma. N. Engl. J. Med. 360, 1989–2003 (2009).
2. Volkmann, E. R. & Tashkin, D. P. Treatment of systemic sclerosis-related interstitial lung disease: A review of existing and emerging therapies. Ann. Am. Thorac. Soc. 13, 2045–2056 (2016).
3. Vande Velde, G. et al. Longitudinal micro-CT provides biomarkers of lung disease and therapy in preclinical models, thereby revealing compensatory changes in lung volume. Dis Model Mech 9, 91–98 (2016).
This research was supported by KU Leuven Internal Funds (C24/17/061).
Keywords: Silica-induced pulmonary fibrosis, µCT-derived biomarkers
Uncovering the essential reactive oxygen and nitrogen species (ROS/RNS) source with a ROS-sensitive chemiluminescence optical imaging (OI) probe in mice with differently impaired ROS/RNS production (#355)
R. Mehling1, J. Schwenck1, 2, B. Zhou1, D. Hartl3, 4, M. Röcken5, B. J. Pichler1, M. Kneilling1, 5
1 Eberhard Karls University of Tübingen, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Baden-Württemberg, Germany
ROS as well as RNS play an important immunomodulatory role during T-cell driven cutaneous delayed-type hypersensitivity reactions (DTHR) associated with recruitment of myeloperoxidase (MPO) expressing neutrophils and NADPH oxidase and MPO expressing macrophages. Aim was to uncover the dominant ROS/RNS source (NADPH oxidase, MPO, iNOS) non-invasively in vivo with the ROS-sensitive chemiluminescence optical imaging (OI) probe L-012 in mice with differently impaired ROS/RNS production during an acute and chronic trinitrochlorobenzene (TNCB) induced cutaneous DTHR.
Wild-type mice (WT) and MPO (MPO-/-), NADPH oxidase (gp91phox-/-), as well as inducible nitric oxide synthase (iNOS-/-) deficient mice were sensitized with TNCB at the abdomen and challenged at the right ear 7 days later (acute CHSR). To induce chronic CHSR we challenged mice every 48h up to 5 times. We determined ear-swelling (ES) responses and conducted in vivo OI 0h, 4h, 12h, and 24h after the 1st, 3rd and 5th challenge (Ch) and obtained ear tissue for H&E-histology and draining lymph nodes and spleen for dihydrorhodamin 123 (DHR) flow cytometry analysis.
MPO-/-, gp91phox-/- as well as iNOS-/- mice exhibited enhanced ES responses during acute and chronic CHSR when compared to WT mice. Non-invasive in vivo OI measurements revealed an enhanced L-012 signal intensity (SI) in ears of WT mice after the 1st, 3rd and 5th Ch, indicating high ROS/RNS levels. In contrast, inflamed ears of gp91phox-/- mice yielded a completely abrogated ROS/RNS expression during acute and chronic DTHR. In line with this, DHR flow cytometry analysis revealed abrogated ROS expression in leucocytes of gp91phox-/- even after phorbol-12-myristat-13-acetat stimulation. OI analysis of the MPO-/- mice showed a strongly up to 70% decreased L-012 SI in ears with acute and chronic DTHR when compared to WT mice. DHR flow cytometry analysis of MPO-/- leucocytes revealed similar results. In sharp contrast, we observed no significant difference in L-012 SI in inflamed ears of iNOS-/- mice and in the DHR flow cytometry analysis of iNOS-/- leucocytes when compared to WT mice.
The analysis of mice with differently impaired ROS production revealed that MPO-mediated hypochlorous acid production is the dominant reactive oxygen species during acute and chronic DTHR. However, MPO relies on the production of hydrogen peroxide as a substrate, which is determined by dismutation of the superoxide anion produced by NADPH oxidase. Consequently, the L-012 SI was completely abrogated in inflamed ears of gp91phox-/- mice.
CT assessment of mouse pulmonary fibrosis in response to a novel drug-delivery system (#208)
G. Garaulet1, C. Penalba1, S. Leal1, G. Visdomine1, T. Álvarez1, M. Rovira2, D. Muñoz-Espín3, M. Serrano2, F. Mulero1
1 CNIO, Molecular Imaging Unit, Madrid, Spain
Computerised tomography (CT) is a useful technique to assess the evolution of different pulmonary conditions in pre-clinical studies. Here we wondered whether the response to a galactose-encapsulated nanoparticle (GalNP) loaded with a cytotoxic drug, could be evaluated in a bleomycin-induced pulmonary fibrosis mouse model by CT.
To induce pulmonary fibrosis, 8- to 10-week-old C57BL/6 wild-type mice were intubated intratracheally with a 24GA catheter. Then, bleomycin was inoculated in at 1.5 U/kg of body weight. 10 days later, mice were treated (i.v.) daily with free drug or with GalNP(drug) (1 mg/kg) for ~2.5 weeks.
CT scans were performed at both initial and final points of the study. Mice were anesthetized with a continuous flow of 1% to 3% isoflurane/oxygen mixture (2 L/min), and the chest area was able to be imaged at one time using the GE eXplore Locus micro-CT scanner (GE Healthcare, London, Canada). The isotropic resolution of this instrument is 45 µm. The reconstructed slices were output in the CT manufacturer's raw and analyzed using MicroView analysis + version 2.2 software (GE Healthcare).
Mice were scanned pre- and post-treatment, at days 11 and 29 respectively, after bleomycin inoculation. In order to obtain the fibrosis volume, a region of interest (ROI) was drawn manually over the lung “slice by slice” on the areas that showed abnormal increase of density, corresponding to fibrotic tissue (Figure 1). Finally, a 3-dimensional ROI was generated providing the fibrotic volume. The image analysis quantification was performed by an independent and well-trained researcher in a blinded manner.
Quantification of the fibrotic lesions (Figure 2) indicated a significant reduction (p<0.01) in the lung volume affected by inflammation and fibrosis in GalNP(drug)-treated mice (mean relative change of 0.51) compared with the free drug-treated group (mean relative change of 1.16).
CT scan allows the evaluation of pulmonary fibrosis, in response to treatment with drug-delivery systems, in a bleomycin-induced longitudinal mouse model.
Keywords: CT, Pulmonary fibrosis
Non invasive X-ray based lung function for the detection of subtle variations in different mouse models experimental asthma (#498)
A. M. Markus1, J. Albers2, F. Alves1, 2, 3, C. Dullin2
1 Max-Plank-Institute for Experimental Medicine, Translational Molecular Imaging, Goettingen, Germany
X-ray based lung function (XLF) is a powerful method to assess the lung function in mice as it includes both functional and anatomical features of the diseased lung 1. In a long-term mouse model of asthma we previously showed that XLF is able to measure alterations in lung function due to a persistent loss of lung tissue elasticity in mice recovered from acute allergic airway inflammation (AAI) 2. Our aim was to test if this CT based imaging method is sensitive enough to distinguish between subtle differences in the long term effect of AAI in three different mouse models of AAI.
The following models were used in BALB/c mice: i) a severe ovalbumin (OVA) induced AAI using 4x250µg OVA challenges, ii) a mild OVA induced AAI with 2x100µg OVA challenges, and iii) a house dust mite (HDM) AAI with 2x50µg HDM challenges. Lung function was assessed before challenge (AAI-BC) as well as 2 days (AAI-AC) and 4 months after challenge (AAI-R), using in vivo XLF. Airway remodeling was evaluated in AAI-R by phase contrast synchrotron radiation µCT (pSRµCT) as well as diaphragm motion analysis from the acquired XLF images. Lung sections were histologically stained to evaluate the amount of collagen, elastic fibers and alpha smooth-muscle actin expression was assessed by immunohistochemistry.
XLF revealed that recovered mice AAI-R of the three different asthma models still show changes in some lung function parameters and can thereby be distinguished from each other. Assessment of structural alterations by pSRµCT showed a significantly reduced soft-tissue volume ratio in recovered mice, suggesting that in comparison to controls the lungs of these mice contained a higher air volume. Different parameters of possible causes such as air trapping as well as histology of the lung are assessed and will be presented. Our results show that subtle changes in pulmonary function in different mouse models of allergic inflammation after long term recovery can be evaluated by XLF.
Our findings suggest that XLF is sensitive enough to distinguish between different types of experimental asthma even month after acute inflammation of the lung. This method therefore has the potential to non-invasively evaluate subtle changes and differences in parameters that may occur in different types and grade of severity of experimental asthma.
1. Dullin, C. et al. X-Ray based Lung Function measurement-a sensitive technique to quantify lung function in allergic airway inflammation mouse models. Sci. Rep. 6, 36297 (2016).
2. Markus, M. A. et al. X-ray based lung function measurement reveals persistent loss of lung tissue elasticity in mice recovered from allergic airway inflammation. Am. J. Physiol. Lung Cell. Mol. Physiol. ajplung.00136.2017 (2017). doi:10.1152/ajplung.00136.2017
We thank Sarah Garbode, Bärbel Heidrich, Bettina Jeep and Sabine Wolfgramm for excellent technical assistance.
Keywords: x-ray based lung function, asthma, allergic inflammation
Preclinical imaging of integrin avb3 in rheumatoid arthritis. (#31)
F. Clarke1, M. T. Ma2, T. J. Peel1, N. K. Ramakrishnan2, K. Sunassee2, J. K. Bordoloi2, G. H. Cornish1, A. P. Cope1, S. Y. A. Terry2
1 King's College London, Centre for Inflammation Biology and Cancer Immunology, London, England, United Kingdom
Rheumatoid arthritis (RA) is an autoimmune disease leading to chronic synovial joint inflammation. Monitoring response to therapies by molecular imaging techniques could influence therapy regimens and enhance therapeutic outcome. Integrin avb3 is found on osteoclasts, macrophages and newly formed blood vessels. Here, we investigated the ability of radiolabelled RGD peptide 68Ga(HP3-RGD3), which targets integrin avb3, to image RA.
Preclinical studies using the K/BxN serum transfer model of arthritis were carried out. Control groups were PBS alone or co-injection with a blocking dose of c(RGDfK).
C57Bl/6 male and female mice were injected with a 2-fold dilution of arthritogenic serum (n=10-20/group). Mice were imaged by PET/CT with 9-16MBq/4 mg 68Ga(HP3-RGD3) at 1h post injection on day 6. Mice used for biodistribution only were injected with 1.5-6MBq/4 mg 68Ga(HP3-RGD3).
In a separate study C57Bl/6 male mice were injected with arthritogenic serum, diluted 2-fold (severe arthritis) or 8-fold (moderate arthritis). On day 8, animals (n=3 per group) were injected with 8–13 MBq/4 mg 68Ga(HP3-RGD3), imaged by PET/CT 1h later, culled and used for biodistribution studies.
Mice imaged at day 6 did not have external signs of arthritis nor could PET/CT visualise early signs of arthritis. In biodistribution studies, a difference in joint uptake of 68Ga(HP3-RGD3) was seen between mice receiving arthritogenic serum and PBS. For example, tracer uptake in the feet and ankles was 0.84±0.16 %ID and 0.67±0.22 %ID (p=0.007), respectively, indicating integrin avb3 was upregulated in mice receiving arthritogenic serum at this early time point.
At day 8, imaging could differentiate between severely arthritic joints (increased diameter >0.5mm) and joints with little inflammation, even within the same mouse. In biodistribution data, tracer uptake in severely arthritic joints was 0.26±0.02 %ID in wrists and 0.68±0.18 %ID in ankles, whereas wrists measured 0.14±0.02 %ID and ankles 0.29±0.03 %ID in non-arthritic mice. In blockade studies, radiotracer uptake in severely arthritic joints measured 0.08±0.01 %ID in wrists and 0.24±0.02 %ID in ankles (p = 0.0005).
Radiolabelled RGD, here, 68Ga(HP3-RGD3, is an excellent and specific tracer to image rheumatoid arthritis by PET but only where disease is established. Studies of imaging disease onset are on-going.
Funding for the project was in part provided by the Rosetrees Trust.
Imaging integrin avb3 in arthritic mice
PET/CT maximum intensity projections of mice administered [68Ga(HP3-RGD3)] (8 - 13 MBq) 1 h PI (PET images scaled from 0.08 to 8.0 %ID g-1): (a) a healthy C57Bl/6 mouse; (b) a C57Bl/6 mouse with severe rheumatoid arthritis, arrows indicate joints where increase in swelling > 0.5 mm; (c) a C57Bl/6 mouse with severe rheumatoid arthritis coadministered c(RGDfK).
Keywords: RGD, integrin avb3, Ga-68, THP, arthritis
Selective detection of liver fibrosis: Perfusion through hypercapnia challenge rather than T1 mapping. (#136)
J. J. Connell1, T. A. Roberts1, M. Zaw-Thin1, P. S. Patrick1, D. J. Stuckey1, R. Ramasawmy1, M. Chouhan2, L. Ressel3, M. F. Lythgoe1, T. L. Kalber1
1 University College London, Centre for Advanced Biomedical Imaging, London, United Kingdom
Quantitative T1 mapping has been identified as a potential tool for liver fibrosis staging1,2. Although inflammation is an important part of the underlying pathology of liver fibrosis, it is also associated with many other liver diseases and is a major component of pathological increases in T1. Also, response to novel liver fibrosis cell therapies include an inflammatory component, further confounding the implications of changes in T1 signal. The aim of this study was to develop a quantitative and non-invasive MRI parameter that was selective for liver fibrosis rather than liver inflammation.
Using mouse models of 6 weeks of carbon tetrachloride induced liver fibrosis and acute lipopolysaccharide induced liver inflammation, quantitative measures of T1 and the change in perfusion in response to a CO2 gas challenge were acquired. A multislice flow sensitive alternating inversion recovery arterial spin labelling (FAIR-ASL) technique3 was used with respiration triggering and retrospective respiration gating with 100 Look-Locker gradient echo readouts (TR/TE 3.11/1.16ms, 3x1mm slices, 1 avg, data matrix 128x128, 30x30mm, ≈6min) with both slice selective (3mm) and global (200mm) inversion pulses. Two perfusion maps were acquired before and 5min after the onset of a hypercapnia challenge through 10% CO2 in the anaesthesia carrier gas prior to analysis with in-house MATLAB scripts.
T1 was significantly higher in mice with either liver fibrosis or acute liver inflammation compared to their age-matched controls ((Figure 1 A-E) n=16 fibrosis: 1492 ±32.9 ms vs. n=8 control: 1435 ±27.8 ms; *** P = 0.0004; n=5 inflammation 1416 ±12.6 ms vs. n=7 control 1382 ±23.3 * P = 0.0437). However, mice with liver fibrosis displayed a significantly lower change in perfusion in response to CO2 gas challenge compared to controls, while mice with acute inflammation did not ((Figure 1 F-J) fibrosis: -0.254 ±0.052 ml/g/min vs. controls: 0.269 ±0.074 ml/g/min **** P <0.0001; inflammation: 0.140 ±0.008 ml/g/min vs. controls: 0.165 ±0.16 ml/g/min n.s.d. P=0.884). Histological tissue staining (Figure 2) of fibrotic liver indicated a high level of perivascular collagen and activated perivascular hepatic stellate cells (α-smooth muscle actin) absent in control and acute inflammation livers.
These results indicate that while T1 mapping does distinguish fibrotic livers from controls, the cause of this change could likely come as a result of the inflammatory environment that is also present. The novel method presented here of hypercapnia induced perfusion change has developed a quantitative measure of disease that is not present in hepatic inflammation. The perivascular pathology is therefore hypothesised to directly impair the ability of liver vasculature to vasodilate. These results encourage a more cautious approach to interpreting hepatic T1 map data in liver fibrosis.
1. Banerjee R, Pavlides M, Tunnicliffe EM, Piechnik SK, Sarania N, Philips R, et al. Multiparametric magnetic resonance for the non-invasive diagnosis of liver disease. J Hepatol. 2014 Jan;60(1):69–77.
2. Pavlides M, Banerjee R, Sellwood J, Kelly CJ, Robson MD, Booth JC, et al. Multiparametric magnetic resonance imaging predicts clinical outcomes in patients with chronic liver disease. J Hepatol. 2016 Feb;64(2):308–15.
3. Ramasawmy R, Campbell-Washburn AE, Wells JA, Johnson SP, Pedley RB, Walker-Samuel S, et al. Hepatic arterial spin labelling MRI: an initial evaluation in mice. NMR Biomed. 2015 Feb;28(2):272–80.
The authors acknowledge the following financial support: JJ Connell is funded by the UK Regenerative Medicine Platform. TL Kalber is funded by an EPSRC Early Career Fellowship (EP/L006472/1). MF Lythgoe receives funding from the Medical Research Council (MR/J013110/1); King’s College London and the UCL Comprehensive Cancer Imaging Centre CR-UK & EPSRC, in association with the MRC and DoH (England); the National Centre for the Replacement, Reduction and Refinement of Animal in Research (NC3Rs); UK Regenerative Medicine Platform Safety Hub (MRC: MR/K026739/1); Eli Lilly and Company.
Quantitative MRI of mouse livers.
(A-E) T1 was significantly increased in mice with 6 weeks of CCl4-induced liver fibrosis, or acute inflammation compared to age matched controls. (F-J) Hepatic perfusion was measured before and during a hypercapnia gas challenge, and the change in perfusion was quantified on a voxel-wise basis. Only mice with liver fibrosis showed a significant difference compared to age matched controls.
Keywords: MRI, liver inflammation, perfusion mri, T1, liver fibrosis
Magnetic Particle Imaging for Longitudinal Visualization of Traumatic Brain Injury Inflammation (#339)
R. Orendorff1, A. Peck2, B. Zheng1, S. Shirazi2, R. M. Ferguson3, A. Khandhar3, S. J. Kemp3, P. W. Goodwill4, G. Brooks2, D. Kaufer2, S. M. Conolly1, 5
1 University of California, Berkeley, Bioengineering, Berkeley, California, United States of America
To diagnose the effects of head trauma, doctors use CT or MRI to see hemorrhages caused by severe impacts. While these techniques work well for evaluation, many mild to medium grade impacts go undiagnosed or untreated ; in both preclinical and clinical settings, qualitative metrics are used to detect lower severity impacts. Both of these methods have significant downsides: CT and MRI can only detect severe injuries, while the qualitative metrics can change between researchers; MPI offers a direct way to visualize these traumatic events by visualizing tracer concentrations in the brain .
Two rats were anesthetized using 3% isoflurane and a catheter was inserted into a tail vein. A bolus of 2.5 mg/kg of LodeSpin LS-13 particles was injected into both animals. One animal was moved under anesthesia to a TBI impact device and a 450 gram weight was dropped onto the rat’s head from a one meter height, making impact through a hex nut. X-ray imaging of the impact was performed with a resolution of 10lp/mm.
Imaging was performed using a the Berkeley field free point (FFP) MPI scanner with a gradient strength of 7 by 3.5 by 3.5 T/m in the x, y, and z directions. The brain was imaged over a 10 minute acquisition period. The field of view was 4 cm by 3.75 cm by 8.75 cm (x, y, z). Images were then taken initially every three hours, and then every day after 24 hours for three days, followed by scans every two days.
The acquired MPI images indicate that SPIO particles accumulated quickly after impact at the inflammation site in the skull and persisted for a two week period. Additionally, X-ray scans of the TBI animal indicated a small skull fracture in an epsilon shape near lambda in X-ray images. The clearance rate of particles at the inflammation site and locations within certain regions of the head were significantly retarded in the TBI animal from the estimated six hour blood half-life of LS-13 particles; signal persisted for four days in the TBI animal while no signal persisted in the sham.
Our work represents the first visualization of TBI by MPI. Currently investigators and clinicians rely on neurological batteries, which primarily evaluate motor function and loss of reflexes, to evaluate injury severity [3,4]. We demonstrate that MPI can be used to determine site, severity, and depth of injury from a closed skull TBI model regardless of brain regions targeted and behavioral manifestation of injury. This quantitative information is useful for evaluating the severity of primary TBI and affected brain regions without performing terminal studies such as histological analyses.
 Lee B, Newberg A. NeuroRx. 2005.
 P.W. Goodwill et al. IEEE Trans. Med. Imaging. 2010
 Shohami, Novikov, and Bass 1995.
 Chen et al. 1996
We are grateful for funding support from the Keck Foundation Grant 009323, NIH 1R01EB019458, NIH 1R24MH106053, and a UC Discovery Grant. LodeSpin Labs is also grateful for NIH 2R42EB013520-02A1. Ryan Orendorff would like to thank the NSF GRFP for funding support.
Magnetic Particle Imaging of Traumatic Brain Injury
(Left) XZ MPI maximum intensity projection (MIP) of the TBI impact rat and a control rat initially after injection/impact (respectively). Note the large hemorrhage in the TBI rat. (Right) MPI MIP of the same rats after 3 days. Green circles indicate lymph nodes. The TBI rat has significant signal from the hemorrhage, as well as signal inside the lymph nodes, unlike the control.
Keywords: Traumatic Brain Injury, Magnetic Particle Imaging, Hemorrhage, Lymph Nodes