slice thickness of 1.0 mm and a temporal resolution of 22 seconds. Seventeen dynamic acquisitions were performed with a minimum of 2 images done prior to ...
ONLINE APPENDIX
Clinical and Experimental Pancreatic Islet Transplantation to Striated Muscle: Establishment of a Vascular System Similar to that in Native Islets Gustaf Christoffersson, Johanna Henriksnäs, Lars Johansson, Charlotte Rolny, Håkan Ahlström, Jose Caballero-Corbalan, Ralf Segersvärd, Johan Permert, Olle Korsgren, Per-Ola Carlsson, Mia Phillipson
SUPPLEMENTAL METHODS Magnetic resonance imaging of auto-transplanted islet grafts The subjects were scanned three to six months post-transplantation using a 1.5 T clinical MRscanner (Gyroscan Intera, Philips Medical Systems). The body coil was used for RFtransmission and a 45 mm circular linear coil for RF-reception. High resolution axial images were obtained using a T1-weighted 3D gradient echo acquisition with 32 slices and an inplane resolution of 200 x 200 μm2 and a slice thickness of 1.0 mm (TR/TE/flip=16/5/10). A dynamic contrast enhanced study was performed in two of the three subjects using a single axial slice (2D) positioned at a representative place through the graft. A dose of 0.1 mmol/kg body weight of contrast agent Gd-DTPA (Magnevist, Bayer Medical) was injected at 2 ml/s. The dynamic study (TR/TE/flip=19/5/20) had an in-plane resolution of 0.23 x 0.23 mm2, a slice thickness of 1.0 mm and a temporal resolution of 22 seconds. Seventeen dynamic acquisitions were performed with a minimum of 2 images done prior to contrast agent arrival serving as baseline data. A two-compartment kinetic model was applied to the dynamic study giving fractional plasma volume (Vp) maps as output (1). Following the ultra-high resolution acquisition of the third subject, an acquisition yielding fat-water separated images (2) was performed in order to assess if any lipid deposition was developed in the musculature. Visualization of intra-islet blood flow Intra-islet blood flow was visualized by intra-arterial injections of fluorescent dextran (FITC, 70 kDa, Sigma-Aldrich). Fluorescence intensity in islet capillaries at different time-points and parts of the islet was measured in ImageJ (NIH). In vivo visualization of transplanted islet mass in mice The presence of fluorescently labeled (Celltrace Far Red DDAO, Invitrogen) islets transplanted to the abdominal muscle was confirmed through non-invasive imaging of grafts by IVIS Spectrum and Living Image software (Caliper Life Sciences). Glucose tolerance test Functionality of islets transplanted into diabetic mice was tested by intraperitoneal glucose load (2.5 g/kg bw). Blood glucose concentrations were measured with a glucose monitor (FreeStyle, Abbott) at 0, 15, 30, 60 and 120 min after injection. Vessel diameter measurements Intra-islet vessel diameter measurements were done in confocal recordings of Alexa Fluor 488-SBA-lectin (Invitrogen) perfused muscle and pancreata (see Fig. 2 A and B for examples). ©2010 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/cgi/content/full/db10‐0205/DC1.
Determination of the specificity of the lectin BS-1 to endothelium Cryosections of pancreata were double-stained with the lectin Bandeiraea (Griffonia) Simplicifolia-1 (BS-1, Sigma-Aldrich), developed with Alexa Fluor 488-streptavidin (Invitrogen) and Alexa Fluor 555-anti-CD31-mAb (Invitrogen/eBioscience). See Supplemental Fig. 2. Digital enhancement of images Images have been digitally enhanced by means of brightness and contrast equally across entire images for increased clarity. REFERENCES TO SUPPLEMENTAL METHODS 1. Tofts PS, Brix G, Buckley DL, Evelhoch JL, Henderson E, Knopp MV, Larsson HB, Lee TY, Mayr NA, Parker GJ, Port RE, Taylor J, Weisskoff RM: Estimating kinetic parameters from dynamic contrast-enhanced T(1)-weighted MRI of a diffusable tracer: standardized quantities and symbols. J Magn Reson Imaging 10:223-232, 1999 2. Weis J, Astrom G, Vinnars B, Wanders A, Ahlstrom H: Chemical-shift micro-imaging of subcutaneous lesions. MAGMA 18:59-62, 2005
SUPPLEMENTAL TABLE 1. Islet Characterization Cytokine expression (pmol/µg DNA)
Insulin Islet Purity Volume Subject Yield (%) (µL) (IE)
Stimulation Content ADP/ index (ng/ng ATP (16.7/1.67 DNA) ratio mmol/l)
TF
MCP-1
IL-6
IL-8
1
286957
80
825
11.8
9,945
0,050 0,073
0,012
0,0004
0,031
2
202174
26
2725
>25
0,541
0,050 0,020
0,012
0,0006
0,030
3
250435
32
1800
>25
0,743
0,060 0,100
0,023
0,0018
0,042
TF, tissue factor; MCP-1, monocyte chemoattractant protein-1; IL, interleukin.
©2010 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/cgi/content/full/db10‐0205/DC1.
Supplemental Figure 1
Stainings of islets transplanted to muscle and liver, and islets in intact pancreas. Parafin sections (5 µm) stained with the endothelial marker BS‐1 and for insulin with insulin antibody. Bars correspond to 50 µm. Supplemental Figure 2
Confirmation of the specificity of the biotinylated lectin BS‐1 to endothelium. Image shows cryosectioned islet of Langerhans in pancreas stained for CD31 (red, Alexa Fluor 555‐CD31‐mAb), BS‐ 1 (green, biotinylated BS‐1 Bandeiraea (Griffonia) simplicifolia lectin/Alexa Fluor 488‐streptavdin) and Hoechst 33342 (blue), where the two endothelial stains overlap in the islet. Bar is 10 µm.
©2010 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/cgi/content/full/db10‐0205/DC1.
Supplemental Figure 3
©2010 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/cgi/content/full/db10‐0205/DC1.
Supplemental Figure 4
Supplemental Figure 5 A 3D surface reconstruction of an islet graft in the brachioradialis muscle of a human subject is displayed in red. ©2010 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/cgi/content/full/db10‐0205/DC1.
