ELECTRONIC SUPPLEMENTARY MATERIAL Intensive Care Medicine
“Time course of metabolic activity and cellular infiltration in a murine model of acid-induced lung injury”
Vanessa Zambelli1,2*, Giuseppe Di Grigoli3,4,6*, Margherita Scanziani1,5, Silvia Valtorta4,6,7, Maria Amigoni1,5, Sara Belloli4,6, Cristina Messa3,4,6, Antonio Pesenti1,5, Ferruccio Fazio3,6, Giacomo Bellani1,5, Rosa Maria Moresco3,4,6.
* The two authors equally contributed to this article.
1 Department of Experimental Medicine, University of Milan-Bicocca, Monza (MB), Italy
2 Department of Cardiovascular Research, Istituto di Ricerche Farmacologiche Mario Negri, Milan (MI), Italy.
3 Tecnomed Foundation, Foundation of University of Milano-Bicocca, Milan (MI), Italy.
4 IBFM, CNR, Italy
5 Department of Perioperative Medicine and Intensive Care, San Gerardo Hospital, Monza (MB), Italy
6 Nuclear Medicine Department and PET Centre, San Raffaele Scientific Institute, Milan (MI), Italy
7 Fellowship of the Doctorate School of Molecular Medicine, University of Milan, Milan (MI), Italy
Corresponding author: Giacomo Bellani, M.D., Ph.D.
Department of Experimental Medicine (DIMS), University of Milan-Bicocca
Via Cadore 48 20090 Monza (MB) Italy.
Phone ++39-039-233.3293 Fax ++39-039-233.2297
Computed Tomography (CT) imaging and analysis. We manually outlined, on each transverse section, the right and left lung’s profile separately, excluding main bronchi, heart and vena cava. Based on images obtained with the same procedure in six control (untreated) mice we defined the threshold for hypoaeration as the value of the 95th percentile of the frequency histogram of voxels’ densities [corresponding to - 140 Hounsfield Units (HU)]. This was necessary since the density of mice lungs measured by CT scan are lower that that of humans, preventing the use of “standard” definition of “normal”, “poor” or absent aeration. The weight of each voxel is computed as the product of voxel size and density, which is in turn computed as (CTvox+1000)/1000, where CTvox is the CT number of each voxel expressed in HU. The relative weight of hypoaerated compartments were computed as the sum of the weight of the voxels belonging to each compartment normalized by the sum of the weight of the voxels of the entire lung.
Positron emission tomography (PET) imaging. To better obtain correlation between CT and PET images and thus improve region of interest analysis, the same reference system was adopted to place animals in both CT and PET scanners. The coordinates of the middle chest were evaluated using CT image. Considering mouse positioned prone in the scanner bed we considered 40 mm in head-tail direction starting from mouse ears (z=0). In the Field of View (FOV) we calculated in millimeters the lungs size in z axis and then obtain the organ middle point; this point was centered in the scanner FOV with a laser system pointers.
Normalization of [18F]FDG values by the calculated lung tissue. Tissue density can influence tissue uptake particularly in an heterogeneous tissue like lungs where normal aerated, collapsed or edematous tissue may be present. The normalization for lung tissue did not modify the shape of [18F]FDG uptake time course. However, differently from what observed from ex-vivo cells analysis, normalized values of [18F]FDG uptake (Fig. E7) in the right side did not significantly differ from those of the left side at 24 and 48 hours (p>0.05). To explore whether normalization for tissue density could overcorrect [18F]FDG concentration or values, a correlation analysis between CT and [18F]FDG data was performed. Results of the analysis showed only a slight correlation between tissue density and [18F]FDG uptake in the right lung ( R2=0.22) and no correlation in the left side. These results are in line with what reported by Bellani et al in ARDS patients [1], indicating that cluster of inflammatory cells are present also in lung region with no structural modifications. In addition, in our case, the limited spatial resolution of small animal dedicated scanner, does not allow to separate normally aerated, collapsed and edematous tissue in sub-regions of the same lung due to the presence of spillover effects from surrounding regions.
Images analysis. To obtain comparable volume, PET and CT images were manually co-registered using PMod 2.7 software. Mean activity for gram of tissue was determined in the injured right lung by drawing Regions Of Interest (ROIs). In order to visualize in the same time co-registered PET and CT images we used Analyze 5.0 software. ROIs (Area=7.49mm2) were drawn in ten transaxial slices, in the dorsal region of the right damaged lung and on the untreated left lung following the anatomical borders visible in CT image. ROIs include approximately 25 % of the right lung area and 20% of left lung area. The same ROI analysis was conducted also in the control groups to obtain baseline [18F]FDG uptake values.
Assessment of pressure-volume (PV) curves. After a recruitment maneuver (at 30 cmH2O for 30 seconds), three steps of inspiratory volumes were delivered, starting from functional residual capacity to a total volume of 0.9 ml. For each step, the compliance was calculated as the ratio between the delivered volume and the pressure variation. The three compliance values were averaged (Cstat).
Hydroxyproline assay. Lungs were homogenized in 1 ml of PBS, and a 0.5ml aliquot was hydrolyzed in 6N HCl at 110°C for 12 h. Twenty five microliter aliquots were added to 0.5ml of 1.4% chloramine T (Sigma, St. Louis, MO), 10% n-propanol, and 0.5M sodium acetate. After 20 min of incubation at room temperature, 0.5ml of Erlich's solution (1M p-dimethylaminobenzaldehyde [Sigma] in 70% n-propanol, 20% perchloric acid) was added and a 15 min incubation at 65°C performed. Absorbance was measured at 550nm and the amount of hydroxyproline was determined against a standard curve.
Immunohistochemical staining. Staining for leukocyte-specific esterase, Naphthol AS-D chloroacetate esterase (Sigma Aldrich): slides were incubated in a solution of sodium nitrate, Fast Red Violet BL base solution, TRIZMAL 6.3 buffer, and Naphthol AS-D chloroacetate solution in deionized water for 3 hours at 37°C. After rinsing, slides were counterstained with Gills hematoxylin solution and coverslipped. Staining for macrophages was performed with rhodaminated Griffonia simplicifolia Lectin 1 (RL-1102 Vector Laboratories). Stained lung sections were examined microscopically for morphology and positively stained cells. Staining for macrophages was done on 5μm thick paraffin embedded sections. Nuclei were stained with bisbenzimide. To establish the total number of neutrophils (esterase-positive) or macrophages (Lectin 1-positive) present in the sample, tissue sections were randomly screened (right lung: 10 fields/slide; left lung: 6 fields/slide) at X1000 (0.01mm2/field) [2] and cells were counted. Both results were expressed as number of cells per high-power field (HPF, 0.2mm2). All histological analyses were performed blinded to group assignment.
Myocardial uptake. As observed also in other models of lung inflammation, myocardial uptake was present also in our model, as indicated by the visual inspection of the entire set of [18F]FDG PET images derived from injured animals [3]. In particular at 6 hours and 7 days from acid instillation, 92% and 78% of mice respectively, presented a clear sign of radiotracer uptake in the myocardium. A lower number of mice showed heart signal at 24 hours and 48 hours after injury (47% and 42% respectively). In order to better understand the potential role of physical artifacts (spillover from heart to lung) on left lung radioactivity in HCl exposed mice, a group of fed normal mice was included in the analysis. In fact, it is well known that myocardial tissue utilizes glucose during feeding condition or under hypoxia condition as is the case of our model [4]. In longitudinal study, when compared with controls, right lungs of injured mice displayed higher levels of radioactivity concentration than right side of both fast and feeding control mice at all time frames. Acid instillation produced an increase of radioactivity concentration at all time frames also in the left side as can be seen from comparison with control mice acquired in fasting condition. On the contrary, when compared with feeding condition, we observed an increased in radioactivity concentration at 6 hours, one of the two time frame (the other was 7 days) in which the majority of injured mice displayed clear signs of radioactivity uptake in the myocardium and when the spill-over effect from heart to lung could promote confounding results. The significant increase observed in the left side that reached values above those observed in feeding mice, clearly indicated that at early time, in presence of radioactivity uptake in the heart, the inflammation phenomenon is spread to both lungs and is not simply caused by a physical artifact. On the other hand, the presence of a spill-over effect from myocardium to surrounding lung may cause an overestimation of the level of radioactivity concentration in the left side also at 7 days. Consequently, the loss of significantly difference between [18F]FDG uptake in the lungs observed at 7 days could be due to the heart spill-over effect that masks a residual presence of inflammation.
[1] Bellani G, Messa C, Guerra L, Spagnolli E, Foti G, Patroniti N, Fumagalli R, Musch G, Fazio F, Pesenti A (2009) Lungs of patients with acute respiratory distress syndrome show diffuse inflammation in normally aerated regions: A [18F]-fluoro-2-deoxy-D-glucose PET/CT study. Crit Care Medicine 37:2216-2222
[2] Lomas-Neira J, Chung CS, Perl M, Gregory S, Biffl W and Ayala A (2006) Role of alveolar macrophage and migrating neutrophils in hemorrhage-induced priming for ALI subsequent to septic challenge. Am J Physiol Lung Cell Mol Physiol 290:L51–L58
[3] Zhou Z, Kozlowksi J, Goodrich AL, Markman N, Chen DL, Schuster DP (2005) Molecular imaging of lung glucose uptake after endotoxin in mice. Am J Physiol Lung Cell Mol Physiol 289:L760–L768
[4] Phelps ME, Hoffman EJ, Selin C, Huang SC, Robinson G, MacDonald N, Schelbert H, Khul DE (1978) Investigation of [18F]FDG for the measure of myocardial glucose metabolism. J Nucl Med 19:1311-1319
E1 Experimental design: the two set of experiments.
E2 Single-point exp: Histological and immunohistochemical sections. a) and b) hematoxylin and eosin staining (100X and 400X respectively). c.) Lectin 1 staining for macrophages (600X). d) Naphthol staining for neutrophils (600X).
E3 Single-point exp: time course of neutrophil-specific esterase+ cells (solid circle), macrophages lectin 1-positive cells (empty circle) and PET signal (dotted line) in right (a) and left (b) lung tissue sections. PMN and macrophages counts were expressed n/HPF and [18F]FDG uptake as % ID/g. Values were expressed as mean ± S.D. (n=4-5/group).
E4 Single-point exp: percentage of hypoaerated areas in right (solid line) and left (dotted line) lung. Values were expressed as mean ± S.D. (n=5-17-5-5-4/group).
E5 Single-point exp: correlation between [18F]FDG uptake (expressed as % ID/g) and % of hypoaerated tissue at all time points (6, 24, 48 hours and 7 days) in the right (solid circle) and left (empty circle) (n=19).
E6 Single-point exp: time course of PET signal, represented as non-normalized (dotted line) and as density normalized (solid line) in right (a) and left (b) lung. [18F]FDG uptake was expressed as % ID/g and as (% ID/g)/density. Values were expressed as mean ± S.D. (n=4-5/group).
Time point / Uncorrected data(%ID/g) / Tissue density
(d) / Normalized data
((%ID/g)/tissue density)
RL / LL / RL / LL / RL / LL
6h / mean / 2,56 / 1,61 / 0,69 / 0,55 / 3,75 / 2,92
SD / 0,26 / 0,38 / 0,11 / 0,14 / 0,46 / 0,10
24h / mean / 3,49 / 2,18 / 0,75 / 0,49 / 4,70 / 4,57
SD / 0,53 / 0,42 / 0,11 / 0,06 / 0,95 / 1,20
48h / mean / 2,94 / 1,48 / 0,77 / 0,55 / 3,95 / 2,73
SD / 0,82 / 0,10 / 0,11 / 0,07 / 1,48 / 0,30
7d / mean / 1,87 / 1,70 / 0,56 / 0,55 / 3,38 / 3,11
SD / 0,21 / 0,12 / 0,00 / 0,03 / 0,39 / 0,19
E7 Single-point exp: Time course of [18F]FDG uptake at each time point expressed as %ID/g and normalized data in right (RL) and left (LL) lung (n=4-5/group).