2.2. In vivo studies of diabetic mice

To investigate the role of MMP9 in early atherosclerosis development in diabetes, KK. Cg-A^y /J mouse (KK mouse, Jackson Lab., USA), a T2DM mouse model, was used. The animal experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Singapore Health Services Pte Ltd, Singapore. All experimental and animal maintenance procedures were performed in accordance with the Animal Use Guidelines of the Singapore Health Services Pte Ltd and conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

KK mice at 3 months of age were screened for hyperglycemia by glucose tolerance test (GTT) as described (31–33). KK mice with hyperglycemia were randomly assigned into three animal groups: 1. KK mice with normal diet + saline injection (the ND + saline Group); 2. KK mice with high fat diet + saline injection (the HFD + saline Group); 3. KK mice with high fat diet + act-hMMP9 injection (the HFD + act-hMMP9 Group). The rodent high fat diet contains 60 Kcal% Fat (D12492, Research Diets, USA). Since no mouse act-MMP9 is available, act-hMMP9 was diluted at 1 µg/0.1 mL in saline for injection. 0.1 mL saline alone or containing 1 µg act-hMMP9 was retro-orbitally injected into mouse under anesthesia once every 2 days.

KK mice were euthanized 2 or 10 weeks after saline or act-hMMP9 treatment. Under anesthesia using inhaled 2% isoflurane, mice were intraperitoneally injected with 1000 U heparin and placed in supine position. After 5 min, the hearts were stopped by injection of 1 mL 10% KCl. Mice were perfused to remove blood. Briefly, 10 mL of 5 mM EDTA in PBS was slowly injected into left ventricle followed by 20 mL of ice-cold PBS. Next, 10 mL of ice-cold of 4% PFA in PBS was injected into the left ventricle and left for 10 min. Then, 10 mL of ice-cold 5% sucrose in PBS was injected into left ventricle to wash away the PFA. The left and right common carotid arteries (CCA) were isolated and embedded into OCT for cryo-sections.

To determine vessel wall thickness and inner circumference, cryo-sections of CCAs were stained with mouse anti-smooth muscle actin (SMA) conjugated with Cy3 (C6198, Sigma-Aldrich) and mouse anti-CD31 conjugated with FITC (sc-18916, Santa Cruz, USA). Vessels were imaged using Olympus IX73 microscope and Cell Sens Standard software (Olympus, Japan). Vessel wall thickness and inner circumference were calculated using photoshop (28, 34).

A stock Oil Red O (0.5%) isopropanol solution was prepared using Oil Red O (O-0625, Sigma-Aldrich) and stored in dark at 4°C. Oil red O working solution was freshly prepared by mixing 6 mL Oil Red O stock solution with 4 mL distilled H₂O and filtered. Vessel samples were air-dried and fixed with 4% PFA at room temperature for 10 min and washed with PBS twice. Samples were soaked in 60% isopropanol for 1 min followed with Oil Red O working solution for 8 min. Samples were washed with 60% isopropanol, PBS, and air-dried. Then, samples were mounted with 90% glycerol for imaging.

A Sudan IV working solution was freshly prepared by mixing 0.1 g Sudan IV (198102, Scientific Laboratory Supplies Ltd, UK) in 50 mL acetone and 50 mL 70% ethanol and filtered. Vessel samples were air-dried and fixed with 4% PFA at room temperature for 10 min, washed with PBS, and rinsed in 70% ethanol for 30 sec. Samples were soaked in Sudan IV staining solution for 45 sec at 57°C, washed with 70% ethanol and distilled H₂O, and air-dried. Then, samples were mounted with 90% glycerol for imaging.

To determine the infiltration of macrophages into mice CCAs, fluorescence immunostaining for Galectin-3 (Mac-2) (SC-32790-FITC, Santa Cruz) to identify macrophages was performed as described previously (34–37).