Parenchymal Tissue

We collectively refer to intra-lobular and interlobular fat as “intrapancreatic fat” or “pancreas fat” as MRI cannot distinguish between these two fat compartments. This terminology is used throughout this paper.
MRI data were acquired using a 3.0 Tesla Philips Achieva scanner (Philips, Best, The Netherlands) with a 6 channel cardiac array for signal detection. The protocol consisted of matched breath-held acquisitions of (i) a 3 point Dixon acquisition to quantify the intrapancreatic triglyceride and (ii) a balanced turbo field echo image to aid anatomical delineation of the pancreas[29 (link)]. Another 3-point Dixon acquisition was prescribed at the level of the L4-L5 intervertebral space to estimate subcutaneous and visceral fat areas in this slice. The 3 point Dixon method [30 (link)]acquires three gradient-echo scans during one breath-hold with adjacent out-of-phase and in-phase echoes (repetition time/echo times/averages/flip angle = 50ms/3.45, 4.60, 5.75ms/1/5°, bandwidth 435Hz/pixel). Field-of-view was set according to patient size (400-480x300mm), zero filled to give a resolution of 1.39x1.40mm. 12 sections of 5mm thickness were used to image the pancreas during two 17-second breath-holds, while one section was acquired at L4-L5 Custom MATLAB software was used to model the fat and water contributions to the gradient echo signals using a spectral model of fat with 6 peaks based on [31 (link)] and a single R2* component. Proton density fat fraction maps (the fat signal expressed as a percentage of the total signal) were constructed taking account of noise bias[32 (link)]. The anatomical delineation was performed on a matched balanced turbo field echo (BTFE) image. BTFE images contain a mix of T₁ and T₂ contrast, which distinguishes high signal intensity from vessels with visceral fat with lower intensity signals from the pancreas. It can therefore be used to clearly delineate the boundaries of the pancreas from adjacent structures, including the surrounding visceral fat, the splenic vein, the superior mesenteric vessels the inferior vena cava and duodenum. Twelve axial sections of 5mm thickness were imaged during an eight second breath-hold (repetition time/echo time/flip angle = 3.1ms/1.6ms/40°, turbo factor 95, parallel imaging factor 2, bandwidth 1156Hz per pixel). The field of view and zero filled resolution were matched to the 3 point Dixon imaging.The conventional method of freehand drawing round an area to be within the substance of the pancreas and a newly developed MR image ‘biopsy’ method (MR-opsy) were compared. For both methods, the regions of interest were selected to be within the parenchymal tissues and avoiding areas of visceral fat, main blood vessels.
For the conventional method, the ImageJ Polygon tool was used to select a region of interest in the parenchymal tissue of the pancreas head, body and tail. The region was selected to be as large as possible whilst being clear of the pancreas borders to avoid any possible contamination of surrounding visceral fat (Fig 1A).For MR-opsy, the Oval tool of ImageJ was used to select three regions of interest (~100 mm² each) to represent equally the pancreas head, body and tail, the size of selection was chosen after pilot studies to permit easy placement entirely within the pancreas considering the irregularity in pancreas morphology (Fig 1A and 1B) [16 (link)]. In view of potential uneven distribution of parenchymal fat between different regions of the pancreas observed in some [33 (link)–37 (link)] but not all studies [12 (link), 13 (link), 38 (link)–40 (link)], sampling regions were placed equally throughout the pancreas to avoid possible bias. Analysis of both study datasets using the conventional methodology as originally published was carried out by experts experienced in pancreas anatomy. This was performed blinded to glucose tolerance and all clinical and metabolic markers both in the original studies and the present comparative study. Visceral and subcutaneous fat areas at L4-L5 were calculated from the L4-L5 proton density fat fraction map by thresholding and watershed analysis [41 (link)].
A step-by-step description of the process is presented in the Supplementary Methods section.
Two representative slices were selected to be assessed by each method and pancreatic fat content was calculated as the average pancreatic fat fraction of both slices.

Central (bronchial wall) and peripheral transbronchial (parenchymal tissue) biopsies were collected at different time points after transplantation (range 3 months–16 years), and biopsies were preferentially taken from the right lower lobe. Lung function was measured by standard spirometry and BOS grades were defined according to the International Society for Heart and Lung Transplantation guidelines (see online supplementary table S1).20 (link) All patients gave their written informed consent to participate in the study (2005-560). Fresh lung biopsies were cut into smaller pieces and then further dissociated by enzymatic digestion with collagenase type I, 300 U/mL (Gibco BRL, Paisley, USA), hyaluronidase, 1 mg/mL (Fisher Scientific) and DNAse (Qiagen, Solna, Sweden) in Dulbecco’s phosphate buffered saline (DPBS). After washing, lung cells were seeded in NH expansion medium (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 1% antibiotic antimycotic solution (Sigma Aldrich, Stockholm, Sweden) at 37°C, 5% CO₂ for generation of lung-derived MSC. Medium was changed after 3 days and weekly thereafter. MSC were passaged with 0.05% trypsin-EDTA (Invitrogen, Lidingö, Sweden) at 70–90% confluence.

Synthetic hydroxyapatite particles (10 µM average size) were purchased from Fluidinova (article 501203, Moreira da Maia, Portugal). Mouse microliths were isolated from the lungs of 20- to 28-week-old male Npt2b^−/− mice after euthanasia by intratracheal instillation of 3 ml of dispase (50 caseinolytic units/ml, Corning, NY), submersion of the organ in 1 ml of dispase for 45 min at room temperature, and transfer to a culture dish containing water. The parenchymal lung tissue was gently teased from the bronchi, then homogenized. For some experiments, mouse microliths were isolated without dispase treatment. Human PAM microliths were isolated from the lung explant of a PAM infant undergoing lung transplant, by gently teasing the lung parenchyma apart with forceps in a culture dish containing water. Microliths were collected by centrifugation, and washed 5 times with cell culture grade water.

Mice were sacrificed by intraperitoneal injection of Euthasol, and lungs were perfused with 10 ml of sterile normal saline via the pulmonary artery. The airway was cannulated via tracheostomy with a 20-gauge metallic angiocatheter, and 3 ml of dispase (50 caseinolytic units/ml, Corning) was instilled, followed by 0.5 ml of 1% low-melt agarose (warmed to 45 °C). Lungs were rapidly cooled on ice for 2 min, submerged in 1 ml of dispase for 45 min at room temperature, and transferred to a culture dish containing deoxyribonuclease I (100 U/ml) (Worthington Biochemicals, Malvern, PA). The parenchymal lung tissue was gently teased from the bronchi, and homogenized. Cell suspensions were filtered, collected by centrifugation, and panned over prewashed 100-mm tissue culture plates coated with CD45 (#553076, monoclonal, 1:100) and CD16/32 (#553142, monoclonal, 1:100) antibodies (BD Biosciences, San Jose, CA). After incubation for 60 min at 37 °C in a 5% CO₂ atmosphere to promote adherence of contaminating macrophages and fibroblasts, the AT2 were gently decanted from the plate, collected by centrifugation, and counted. For the Npt2b^−/− animals, differential centrifugation was used to separate microliths from the cells. Cell viability determined with trypan blue staining was routinely >90%, and cell purity determined by SP-C staining ranged from 75 to 90%.

Tissue distribution studies in mice with SCID are required in anti-liver cancer activity to determine the distribution and concentration of a drug within the body after administration (54 (link)). This information is important for understanding how the drug is metabolized and cleared by the body, as well as for determining appropriate dosing regimens. Additionally, tissue distribution studies can help to identify any potential toxicities or side effects associated with the drug in specific organs or tissues. In the case of anti-liver cancer activity, knowing the distribution of the drug in the liver would be particularly important to ensure the drug is reaching its target site and effectively inhibiting the growth of cancer cells. The tissue distribution of a bolus dosage of 20 mg/kg i.v. Cabozantinib (CNB), CNB-PLGA-PSar-NPs was documented. The greatest concentration of CNB was found in liver parenchymal hepatocyte tissue. The presence of CNB in the tissues of other essential organs, such as the heart and kidney, suggests that CNB distribution is indirectly dependent on the blood flow and perfusion rate of the organs. The increased affinity of CNB in the liver and pancreas enables the medication molecule to effectively treat liver cancer. However, limited expression of CNB in brain endothelial cells suggests that polymeric nanoparticles are unable to traverse the blood-brain barrier without difficulty.