All animal experiments were performed in accordance with the approval of the Institutional Animal Care and Use Committee of Columbia University. Male wild-type (WT) C57BL/6 mice (n = 20) were obtained (Jackson Laboratories, Bar Harbor, ME, USA). Diabetic mice (n = 8) and non-diabetic mice (n = 8) were injected with radiolabeled anti-RAGE F(ab’)2. Probe control non-diabetic mice (n = 4) were injected with non-immune IgG F(ab’)2.
Induction of diabetes
At 6 weeks of age, mice were treated with streptozotocin (STZ; Sigma-Aldrich Corporation, St. Louis, MO, USA). Animals were injected with five consecutive daily doses of STZ dissolved in citrate buffer (55 mg/kg, pH 4.5) via the intraperitoneal route. One week after the first dose, blood glucose levels were assessed using a glucometer. The criterion of two consecutive glucose levels >250 mg/dL was used to indicate diabetes. If glucose levels were <250 mg/dL, then the mice received two additional doses of STZ. Both diabetic and non-diabetic mice were followed for 2 months to allow the diabetes to stabilize before femoral artery ligation.
Femoral artery ligation
The hair on the abdominal wall and pelvis and the upper legs was shaved using an electronic shaver after the mouse was under anesthesia with 4% isoflurane induction and 1% maintenance. The skin was prepped using povidone-iodine (5%) followed by three alcohol preps. A skin incision was made on the upper thigh of the mouse. The inguinal ligament and the upper half of the femoral artery are exposed in both legs. On the left leg, the femoral artery was ligated with two sterile 8/0 non-absorbable silk sutures below the inguinal ligament proximally and just above the bifurcation into the superficial and deep femoral arteries distally. The vascular bundle on the right leg was isolated without further intervention. The skin incision was closed with sterile 5/0 nylon suture.
Preparation of radiotracer
Monoclonal anti-RAGE antibody was developed as previously described . For 99mTc labeling, the antibody was fragmented into F(ab’)2 using a pepsin digestion kit (Pierce, Rockford, IL, USA). Approximately 1 mg of the F(ab’)2 was conjugated with 5 M excess of diethylene triamine pentaacetic acid (DTPA; Sigma). The reaction mixture was incubated at room temperature for 30 min followed by overnight dialysis at 4°C in phosphate-buffered saline (PBS) (0.15 M NaCl, 0.05 M NaHCO3, pH 7.6). To 50 to 100 μg of anti-RAGE F(ab’)2, 50 μg of SnCl2 in 0.1 N HCl (flushed in N2 gas for 15 min) and 30 to 50 mCi 99mTc were added and incubated at room temperature for 45 min. The 99mTc-labeled antibody fragments were separated from free 99mTc using a PD-10 column pre-equilibrated with 0.1 M PBS (pH 7.4). The mean radiopurity was 97 ± 0.5% by instant thin-layer chromatography. Control non-specific mouse IgG F(ab’)2 was similarly conjugated with DTPA for 99mTc labeling as described above.
In vivo imaging
Five days after femoral artery ligation, each mouse was anesthetized with isofluorane (4% to induce, 1% to maintain) for placement of a jugular vein catheter (Braintree Scientific, Braintree, MA, USA) and was injected with 0.41 ± 0.12 mCi 99mTc-anti-RAGE F(ab’)2 or control non-specific mouse IgG F(ab’)2 and 4 to 5 h later (blood pool clearance) underwent single-photon emission computed tomography/computed tomography (SPECT/CT) imaging on nanoSPECT/CT (Bioscan, Washington, DC, USA).
A topogram (sequence of 2D side view X-ray projections) was used to determine the axial scan range for SPECT and CT imaging. CT images were acquired with an integrated CT scanner using an X-ray tube at 45 kVp and an exposure time of 1,000 ms per view. Following CT acquisition, helical SPECT scans were acquired using dual-headed detectors, each outfitted with nine pinhole apertures. Each pinhole has a diameter of 1.4 mm with each collimator providing a transaxial field-of-view (FOV) of 30 mm and an axial FOV of 16 mm, extendable through helical scanning to 270 mm. SPECT data were acquired with the following parameters: step and shoot rotation, 30° step in 360° rotation using 24 projections, 60 s per projection, 256 × 256 frame size with 1.0-mm pixels, and 140 keV with 10% energy window. The obtained projection data were reconstructed by ordered subsets expectation maximization algorithm with the subset and iteration number set to 16 and 8, respectively, and a voxel size of 300 μm and SPECT and CT datasets fused.
At the completion of in vivo imaging, mice were euthanized by an intraperitoneal injection of pentobarbital (100 mg/kg).
The scans were reconstructed and processed using InVivoScope software (Invicro, Boston, MA, USA). On serial 5-voxel-thick transverse sections from below hip joints to distal hind limbs, regions of interest were drawn and activity in mCi summed for each limb and subsequently divided by the injected dose (ID).
Gamma well counting
The anterior tibialis muscles were dissected and weighed, and the radioactivity was determined in a gamma well counter (Wallac Wizard 1470, PerkinElmer, Waltham, MA, USA) and expressed as the percentage of injected dose per gram (%ID/g) of tissue. The radiotracer activity in the samples was corrected for background, decay time, and tissue weight.
Three sets of explanted tibialis anterior muscles per group were fixed in 10% formalin for 48 h. Three serial sections (5 μm thick) per experiment from paraffin-embedded blocks were processed for hematoxylin and eosin (H&E) for morphological evaluation and immunohistochemical analysis.
Serial sections were deparaffinized and rehydrated followed by quenching of endogenous peroxidase activity with 0.3% hydrogen peroxide. Slides were then incubated overnight with monoclonal anti-RAGE antibody (50 μg/ml). Slides were incubated for 30 min with biotinylated secondary antibody. To identify capillary sprouting, staining was performed with biotinylated Griffonia (Bandeiraea) simplicifolia isolectin I (1:50; Vector Laboratories, Burlingame, CA, USA). Sections were treated for 30 min with VECTASTAIN ABC reagent (Vector Laboratories). Color reaction was visualized with 3′,3′-diaminobenzidine (DAB substrate kit, Dako, Mississauga, Ontario, Canada) and counterstained with Gill’s hematoxylin solution. All brown staining areas were counted for each of the three sections for both the left and right anterior tibialis muscles for each experiment, and averaged RAGE and lectin staining were quantified as area staining positive for the brown chromogen per 100× field.
Images were captured using a digital camera mounted on a Nikon microscope (Nikon Co., Tokyo, Japan) and analyzed using Image-Pro Plus software (Media Cybernetics Inc., Bethesda, MD, USA).
Dual fluorescent confocal microscopy studies were performed to identify the cell source of RAGE staining. Muscle sections (5 μm thick) were deparaffinized in xylene and incubated with monoclonal mouse anti-RAGE F(ab')2 (50 μg/ml; Texas Red) and co-stained with endothelial cells (FVIII, 1:200; fluorescein isothiocyanate), macrophages (Mac-3, 1:50; fluorescein isothiocyanate), and myocytes (anti-sarcomeric actin, 1:50; fluorescein isothiocyanate). The images were examined using a confocal fluorescence microscope (Nikon) and SPOT imaging software (Diagnostic Instruments, Inc., Sterling Heights, MI, USA).
Continuous variables were expressed as mean ± standard deviation. Normality was assessed using the Sharpiro-Wilk W test. Equality of variances was assessed using Levene’s test. Comparisons between groups were made using paired two-tailed Student’s t test or the Mann–Whitney U test, as appropriate with P < 0.05 denoting significance. Correlation was assessed using the Pearson product–moment correlation coefficient. Statistical analyses were performed using STATA 10.1 (StataCorp, College Station, TX, USA).