Micro-Autoradiographic Assessment of Cell Types Contributing to 2-Deoxy-2-[18F]Fluoro-d-Glucose Uptake During Ventilator-Induced and Endotoxemic Lung Injury

B World Molecular Imaging Society, 2012 Published Online: 3 July 2012 Mol Imaging Biol (2013) 15:19Y27 DOI: 10.1007/s11307-012-0575-x RESEARCH ARTICLE Micro-Autoradiographic Assessment of Cell Types Contributing to 2-Deoxy-2-[18F]FluoroD-Glucose Uptake During Ventilator-Induced and Endotoxemic Lung Injury Dalia Saha,1 Kazue Takahashi,2 Nicolas de Prost,1 Tilo Winkler,1 Miguel Pinilla-Vera,3 Rebecca M. Baron,3 Marcos F. Vidal Melo1 1 Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, 55 Fruit St., Boston, MA 02114, USA 2 Program of Developmental Immunology, Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 3 Department of Medicine (Pulmonary and Critical Care), Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Abstract Purpose: The aim of the study was to use micro-autoradiography to investigate the lung cell types responsible for 2-deoxy-2-[18F]fluoro-D-glucose (FDG) uptake in murine models of acute lung injury (ALI). Procedures: C57/BL6 mice were studied in three groups: controls, ventilator-induced lung injury (VILI), and endotoxin. VILI was produced by high tidal volumes and zero end-expiratory pressure and endotoxin ALI, by intranasal administration. Following FDG injection, the lungs were processed and exposed to autoradiographic emulsion. Grain density over cells was used to quantify FDG uptake. Results: Neutrophils, macrophages, and type 2 epithelial cells presented higher grain densities during VILI and endotoxin ALI than controls. Remarkably, cell grain density in specific cell types was dependent on the injury mechanism. Whereas macrophages showed high grain densities during endotoxin ALI, similar to those exhibited by neutrophils, type 2 epithelial cells demonstrated the second highest grain density (with neutrophils as the highest) during VILI. Conclusions: In murine models of VILI and endotoxin ALI, FDG uptake occurs not only in neutrophils but also in macrophages and type 2 epithelial cells. FDG uptake by individual cell types depends on the mechanism underlying ALI. Key Words: Micro-autoradiography, 2-Deoxy-2-[18F]fluoro-D-glucose, Positron emission tomography, Ventilator-induced lung injury, Acute lung injury, Endotoxin lung injury, Neutrophil, Macrophage, Type 2 epithelial cell, Lung Introduction A cute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are important causes of morbidity Correspondence to: Marcos F. Vidal Melo; e-mail: and mortality in critically ill patients [1]. Sepsis is a major etiologic factor for ALI/ARDS [1]. Management of patients usually involves mechanical ventilation, and ventilator-induced lung injury (VILI) is an increasingly recognized condition that can produce and exacerbate lung inflammation and contribute to mortality [2, 3]. Lung inflammation is a key feature of ALI, the degree of which is believed to impact the clinical course of the illness [3, 4]. For these reasons, there is 20 increasing interest in the application of positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-D-glucose (FDG) to assess the degree of pulmonary inflammation during ALI/ARDS non-invasively, regionally and in vivo [5–9]. Studies in humans and animals suggest that FDG-PET imaging may be a valuable tool to study the mechanisms underlying ALI/ARDS [7–10], predict severe respiratory failure [11], and evaluate the effects of therapeutic interventions [12]. Previous studies speculated that the FDG signal was localized to neutrophils [7, 13, 14]. Thus, in the acutely injured non-tumoral lung, FDG-PET has been interpreted to reflect neutrophilic inflammation. However, studies characterizing the source of the pulmonary FDG signal in the nontumoral lung were based either on bronchoalveolar lavage samples [7], models of fibrotic lung disease [13], or indirect deductions from PET imaging of humans with pneumonia or bronchiectasis [14]. Consequently, there is no information regarding the pulmonary FDG signal from direct assessment of FDG uptake in lung parenchymal cells, which are likely to contribute to the signal. Micro-autoradiography is a semi-quantitative method to assess the spatial distribution of radioisotope uptake in tissue [15, 16]. No previous study has addressed the contribution of different pulmonary cell types to FDG uptake during endotoxin exposure and VILI, which are key models of ALI/ ARDS. Furthermore, ALI experiments in mouse and sheep indicated the presence of FDG uptake during significant neutropenia [2, 17], suggesting the contribution of cell types other than neutrophils to the FDG signal. These cell types could play distinct roles in the propagation of ALI. Neutrophils are among the earliest immune cells to be recruited to the injury site, whereas macrophages and type 2 epithelial cells provide important signaling for neutrophil chemoattraction during ALI [18]. Consequently, characterization of the cell types contributing to the FDG uptake signal during ALI/ARDS is essential to accurately understand and interpret experimental and clinical data. In the present study, we used FDG-based micro-autoradiography in mouse models of acute lung injury due to endotoxin exposure and VILI in order to (1) identify cell types participating in the FDG signal and (2) semiquantitatively assess the contribution of those cell types to the FDG uptake measurement. Materials and Methods All experiments were performed under an approved protocol by the Subcommittee on Research Animal Care at the Massachusetts General Hospital, Boston, MA. Acute Lung Injury Models Male and female C57/BL6 mice (8–12 weeks old, 20–25 g) were divided into three experimental groups: controls, endotoxin, and VILI. Mice in all groups were fasted for 18 h prior to D. Saha et al.: FDG Uptake During Acute Lung Injury administration of FDG. For the endotoxin and VILI groups, anesthesia was induced with a combination of intraperitoneal injection of ketamine (120 μg/g) and xylazine (40 μg/g). In the endotoxin group (n05), following anesthesia, intranasal endotoxin (250 μg/g in 40 μl, Escherichia coli O55:B5, SigmaAldrich) was administered. Mice were then allowed to recover from anesthesia and breathe spontaneously for 18 h before tracer administration (described below). In the VILI group (n04), following anesthesia, mice underwent tracheotomy using a 20G angiocatheter. General anesthesia was maintained with additional boluses of ketamine and xylazine, and muscle relaxation was achieved with pancuronium (0.08 μg/kg). VILI was induced by mechanically ventilating the animals with initial peak airway pressures at 22 cmH2O, progressively increased to 30 cmH2O, FIO2 01.0, zero end-expiratory pressure, and respiratory rate of 75–115 breaths/min (Harvard Model 687 mechanical ventilator, Boston, MA, USA). During mechanical ventilation, intravascular volume status of the mouse was assessed by evaluation of paw capillary refill, with (...truncated)