Animals and the induction of experimental diabetes
Diabetes was induced in 10-week-old male nude mice (C57BL/6; Harlan Laboratories, Indianapolis, IN, USA) or male mice null for Erα
] by a single i.p. injection of 200 mg/kg streptozotocin (STZ; Sigma Aldrich, St Louis, MO, USA). Blood glucose was monitored with a One Touch Ultra Glucose Monitor (LifeScan, Milpitas, CA, USA). Mice with fed blood glucose exceeding 20.8 mmol/l were used as recipients. All animal experiments were approved by Northwestern University Animal Care and Use Committee.
Human islet transplantation and in vivo oestrogen treatment
Human islets were obtained through the Integrated Islet Distribution Program and cultured for 48 h in phenol-red-free CMRL-1066 medium (Sigma Aldrich) containing 10% (vol./vol.) charcoal-stripped FBS (Gemini Bio, West Sacramento, CA, USA). For in vivo treatment, E2, 17α-oestradiol (17α-E2) and G1 (0.18 mg/pellet), propyl-pyrazole-triol (PPT) and diarylpropionitrile (DPN) (3.6 mg/pellet) 60-day-release pellets (Innovative Research of America, Sarasota, FL, USA) were implanted subcutaneously immediately before islet transplantation. The dose of E2 was chosen to obtain serum concentrations within physiological limits (ranging from oestrus to pregnancy). The doses of 17α-E2 and G1, PPT and DPN were chosen according to the affinity of each agonist for its ER to achieve similar activation of ERs as caused by E2. A marginal dose of 1,000 islet equivalents (IEQ) of human islets were transplanted under the kidney capsule of recipient mice.
Immunosuppression was achieved as described in the Edmonton protocol, following adaptation to the mouse [1
]. Sirolimus (rapamycin; LC Laboratories, Woburn, MA, USA) was administered via i.p. injection every other day at the dose of 0.1 mg/kg for 4 weeks, starting from the day of islet transplantation. Tacrolimus (FK506; Cayman, Ann Arbor, MI, USA) was administered i.p. daily at 1 mg/kg, starting on the day of islet transplantation. Control animals received daily vehicle injections.
Kidneys bearing islet grafts were fixed overnight in 4% (wt/vol) paraformaldehyde at 4°C. The tissues were immersed in 30% (wt/vol) sucrose and embedded in tissue-freezing medium (Tissue-Tek; Sakura Finetek, Torrance, CA, USA). Sections, 5–10 μm, were mounted on charged slides. For immunohistochemical studies, the following primary antibodies were used: guinea pig anti-human insulin (1:1,000; Linco Research, Saint Charles, MO, USA); rat anti-mouse CD31 (1:400; BD Biosciences, San Jose, CA, USA); rabbit anti-mouse Ki67 (1:400; Novocastra, Newcastle Upon Tyne, UK); rat anti-mouse F4/80 (1:200; AbD Serotec, Raleigh, NC, USA); mouse anti-ERα (1D5; 1:100; Zymed Laboratories, South San Francisco, CA, USA); and goat anti-ERβ (Y-19; 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies were: Cy3-conjugated donkey anti-guinea pig; Cy3-conjugated goat anti-rat; biotinylated goat anti-rat; FITC-conjugated goat anti-rat; and FITC-conjugated donkey anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Biotinylated goat anti-rat was visualised using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). For staining with ERα and ERβ, the Alexa 568 tyramide signal amplification kit (Molecular Probes, Eugene, OR, USA) was used. For nuclear staining, the sections were incubated with DAPI (Molecular Probes, Eugene, OR, USA). Images were obtained with a Nikon Eclipse E400 microscope (Nikon Instruments, Melville, New York, USA) or TissueGnostics High Throughput Imaging System (TissueGnostics, Vienna, Austria).
Measurement of apoptosis using TUNEL assay
Apoptotic cells were detected by TUNEL assay using a fluorescein in situ cell death detection kit (Roche, Indianapolis, IN, USA) 1 day after transplantation. Frozen tissue sections, 5 μm, were fixed at room temperature for 1 h in 4% (vol./vol.) PFA in PBS, pH
7.4. Samples were washed in PBS for 30 min. Following the manufacturer’s instructions, sections were permeabilised, washed, labelled, incubated and analysed. Sections were subsequently stained with guinea pig anti-human insulin as a primary antibody (1:1,000) and Cy3-conjugated donkey anti-guinea pig as a secondary antibody. Images were obtained with a Nikon Eclipse E400 microscope.
Measurement of oxygenation in transplanted islet graft
Islet oxygenation was investigated in the transplants, 1 day post-transplantation [26
]. Pimonidazole hydrochloride (hpi Hydroxyprobe-1; Omni Kit, Burlington, MA, USA) (60 mg/kg) was injected i.p. Mice were killed 3 h later, and their kidney containing the islet graft was processed for immunohistochemistry. Rabbit anti-pimonidazole antibody (hpi Hydroxyprobe-1; Omni Kit) was used to visualise pimonidazole hydrochloride accumulation in hypoxic cells of the graft.
Kidneys bearing islet grafts were sectioned in 10 μm thickness and four sections per tissue were randomly chosen for morphometric analysis. Anti-human-insulin and anti-mouse-CD31 antibodies were used to visualise beta cells and blood vessels, respectively. Morphometric analysis was conducted using the ImageJ 1.37v (rsb.info.nih.gov/ij/) program. In islet transplants, the beta cell area was calculated by dividing the insulin-positive area by the graft area. Blood vessel density was calculated by dividing the mouse-CD31-positive area by the graft area [27
]. The demarcation of an islet graft was considered to be the parenchyma of the surrounding kidney as described [10
Hormone and thiobarbituric acid reactive substances assays
Human insulin and mouse glucagon was measured in serum by ELISA and RIA, respectively (Linco, St Charles, MO, USA). Systemic oxidative stress was assessed by measuring the concentration of thiobarbituric acid reactive substances (TBARS) in plasma (Cayman) and kidney homogenates (ZeptoMetrix, Franklin, MA, USA) [28
Biotinylated tomato lectin (Vector Laboratories) was injected into the tail vein (200 μg) and allowed to circulate for 5 min before the mice were killed. The kidneys bearing the islet grafts were removed and prepared for histological study. Lectin staining was visualised using the Vectastain Elite ABC kit (Vector Laboratories). Functional blood vessel density was obtained by dividing the lectin-positive area by the graft area.
Data are presented as mean
SEM unless otherwise stated. For the rodent study, data were analysed by either the unpaired Student’s t
test or one-way ANOVA. A value of p
0.05 was considered statistically significant.