Experimental protocol
All animal experiments were performed in accordance with the approval of the Institutional Animal Care and Use Committee of Columbia University. Homozygous male RAGE null (RAGE-/-) mice (backcrossed >10 generations into C57BL/6) were generated as described previously [9]. Male wild-type (WT) C57BL/6 mice were obtained (Jackson Laboratories). At age 6 weeks, half of the WT and half of the RAGE-/- mice were treated with streptozotocin (STZ; Sigma). Two months later, all mice underwent femoral artery (FA) ligation.
Induction of diabetes
Mice were treated 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, glucose levels were assessed by glucometer. The criteria 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 (55 mg/kg).
Femoral artery ligation
Under isoflurane anesthesia, the hair on the abdominal wall and pelvis and both upper legs was shaved and the skin prepped with iodine and alcohol. An incision was made on the upper thigh of both the left and right legs of each mouse. The inguinal ligament and the upper half of the femoral artery were exposed. On the left side, the vascular bundle was isolated from below the inguinal ligament proximally to just above the bifurcation into the superficial and deep femoral arteries distally. The femoral artery was dissected free, and two ligatures were placed around it with 8/0 non-absorbable sutures and tied. Both skin incisions were closed with sterile 5/0 nylon suture.
Preparation of radiotracer
Aliquots of 5 μg of HYNIC-RGD were incubated with 0.5 ml of tricine solution (70 mg/ml in distilled water) and approximately 0.5 ml of 99mTcO4
- solution (50 mCi = 1,850 MBq) and 20 μl of tin(II) solution (10 mg of SnCl2·2H2O in 10 ml of nitrogen-purged 0.1 N HCl for 20 min) at room temperature. To test the specificity of the HYNIC-RGD, cyclo [Arg-Ala-Asp-D-Phe-Lys (HYNIC)] (Peptides International, Louisville, KY, USA) was similarly radiolabeled and used as control peptide. Radiochemical purity was >94% by Tec-control chromatography (Biodex, Shirley, NY, USA).
Injection of radiotracer and imaging
Under isoflurane anesthesia (1.5% isoflurane at a flow rate of 0.5% L/min oxygen per mouse), a cutdown was made over the jugular vein and a specially designed vascular catheter was placed (Braintree Scientific, Braintree, MA, USA). Mice in each of four groups were injected with 99mTc-HYNIC-RGD and imaged 3 or 7 days after FA ligation: WT without diabetes (n = 14), WT with diabetes (n = 14), RAGE-/- without diabetes (n = 16), RAGE-/- with diabetes (n = 14), and five WT without diabetes were injected with control peptide. All mice were injected through the jugular vein catheter with 1 ± 0.2 mCi of 99mTc-HYNIC-RGD in 0.05 to 0.1 ml (corresponding to 1 μg of peptide) or control peptide. Blood pool clearance was measured in five mice injected with 99mTc-HYNIC-RGD. By 60 to 75 min after injection, residual blood pool activity was below 10% of peak. Whole-body planar gamma images in the anteroposterior view were acquired on a high-resolution high-sensitivity dedicated small animal camera with parallel hole collimator (provided by Jefferson Lab, Newport News, VA, USA). The camera is based on a 5-in. Hamamatsu position sensitive photomultiplier type R3292 with an active field of view of about 95 mm diameter. The scintillator sensor is 1.6-mm-step 6-mm-thick pixelated NaI(Tl) scintillator array. The photo peak was set at 140 keV with a 15% energy window.
Ex vivo tissue counting
At completion of the imaging session, each animal was euthanized by an intraperitoneal injection of pentobarbital (100 mg/kg). The anterior tibialis muscles were dissected, weighed, and counted in a gamma counter (Wallac Wizard 1470, PerkinElmer, Waltham, MA, USA) for determination of the percent injected dose of radiotracer per gram (%ID/g) tissue. The radiotracer activity in the samples was corrected for background, decay time, and tissue weight. Limb counting was performed in 28 animals. The remaining animals were used for immunohistochemistry.
Histopathology
For immunohistochemical analyses, tibialis anterior muscles were harvested and fixed in 10% formalin for 48 h. Specimens were embedded in paraffin, and tissue slices (5 μm in thickness) were prepared. Serial sections were stained with hematoxylin and eosin (H&E) for morphology. Immunostaining was performed for capillary sprouting using biotinylated Griffonia Bandeiraea Simplicifolia Isolectin I (Vector Laboratories, Burlingame, CA, USA) for β3 (1:50; Abcam, Cambridge, MA, USA.) and for αν (1:100; Millipore, Temecula, CA, USA). Serial sections were also stained for RAGE using a monoclonal antibody against RAGE (50 μg/ml). Secondary stains were performed using avidin-biotin visualization systems (Vectastain ABC Kit, Vector Laboratories). All brown staining capillaries were counted for each of 5 to 6 sections for both the left and right anterior tibialis muscles for each experiment and then were averaged. The average number of capillaries for the left anterior tibialis muscle was divided by the average number for the right (control) anterior tibialis muscle. RAGE staining was quantified as area staining positive for the brown chromagen per 100× field.
Immunofluorescence
Dual immunofluorescent studies were undertaken to determine the cell types expressing αν integrin. Serial sections (5 μm in thickness) obtained from the ischemic hindlimb were deparaffinized in xylene and incubated with αν (rat anti-mouse integrin αν, 1:100) and co-stained with endothelial cell marker (FVIII, 1:200) or macrophage marker (Mac-3, 1:50). Secondary fluorescent antibodies were Texas Red anti-rabbit and FITC anti-mouse. The images were captured and processed using confocal fluorescence microscope (Nikon, Tokyo, Japan) and SPOT imaging software (Diagnostic Instruments, Inc., Sterling Heights, MI, USA).
Image analysis
Radiotracer counts in the ischemic hindlimb were determined from the in vivo scans by using the region of interest (ROI) method in the mini gamma camera image using public domain Image J software (NIH, Bethesda, MD, USA). A region was drawn around the focal uptake, and the mean counts were determined. Radioactivity in the contralateral control limb was similarly determined using a comparable ROI (same anatomic location and the number of pixels). The counts from each of these areas were used to determine the ischemic to non-ischemic ratios.
Statistical analysis
Continuous variables were expressed as mean ± standard deviation. Normality was assessed using the Shapiro-Wilk test. Comparisons between two groups were made using the Student's t test. Correlation was assessed using the Pearson product-moment correlation coefficient. All statistical tests were two-tailed, with P < 0.05 denoting significance. All statistical analyses were performed using STATA 10.1 (StataCorp, College Station, TX, USA).