Adult male inbred Lewis rats, purchased from Charles River Laboratories (Sulzfeld, Germany), were used. The animals were kept in a specific non-pathogen environment and had free access to water and pelleted food throughout the course of the experiment. ‘Principles of laboratory animal care’ (National Institutes of Health, Bethesda, MD, USA; publication no. 85–23, revised 1985; http://grants1.nih.gov/grants/olaw/references/phspol.htm, accessed 1 January 2012) were followed, as well as specific national laws where applicable. All experimental procedures were approved by the Animals Ethics Committee of Uppsala University.
The rats were anaesthetised by an i.p. injection of 120 mg/kg body weight thiobutabarbital sodium (Inactin; Sigma-Aldrich, St Louis, MO, USA) and placed on a thermostat-controlled heating plate to maintain a core body temperature of 38°C. The trachea was detached from surrounding tissues and a polyethylene catheter was inserted to secure free airways during the experiment. Polyethylene catheters were also inserted into the ascending aorta through the right carotid artery for blood pressure measurement and subsequent microsphere injection, and into the femoral artery for reference blood sample collection. After the surgical procedure, the blood pressure was allowed to stabilise for 10–15 min; 1 ml of 30% (wt/vol.) d-glucose was then injected into the femoral vein 10 min before fluorescent microsphere injection (10 μm, FluoSpheres Polystyrene Microspheres; Molecular Probes, Eugene, OR, USA) [16, 17]. A reference blood sample was collected during 1 min, starting 5 s before the microsphere injection, in order to determine the blood flow each microsphere represented. The pancreas was removed for islet isolation and the adrenal glands were examined to evaluate adequate mixing of microspheres in the systemic circulation. Only in animals with equal amounts of microspheres in the adrenal glands (<10% difference), indicating a successful mix of microspheres with the arterial blood, was the pancreas processed for islet isolation.
Islets were isolated by collagenase digestion, as described previously , and thereafter hand-picked. Islets were cultured free-floating and dispersed in 50 mm Petri dishes (100 islets per dish; Sterilin, Newport, UK) in RPMI 1640 medium supplemented with l-glutamine (Sigma-Aldrich), benzylpenicillin (100 U/ml; Roche Diagnostics Scandinavia, Bromma, Sweden), streptomycin (0.1 mg/ml; Sigma-Aldrich) and 10% (vol./vol.) FCS (Sigma-Aldrich) for 3 days. Using a fluorescence microscope the islets were then sorted dichotomously into two groups, with or without microspheres. Each experiment was if possible performed on islets from one rat. However, pooling of islets from more than one rat was often necessary, and then each rat contributed with the same percentage of microsphere- and non-microsphere-containing islets.
Cellular stress in vitro
In each experiment 100 microsphere-containing or non-microsphere-containing islets were assigned to each of two groups. This resulted in a total of 200 analysed islets per experiment. The microsphere-containing islets were in each experiment matched by size (median diameter) and number with non-microsphere-containing islets from the same rat. Visible islet aggregations were excluded. The islets were incubated in 20% or 1.5% O2 in a humidified airtight chamber for 4 h at 37°C. In separate experiments, the islets were incubated with or without cytokines (50 U/ml IL-1β, 1,000 U/ml IFN-γ, 1,000 U/ml TNF-α; PeproTech, London, UK) for 24 h. In the former experiments, gas mixtures were produced by a certified manufacturer (Air Liquid Gas, Stockholm, Sweden), and the oxygen levels were continuously monitored with an oxygen sensor (Dräger Pac III; Dräger, Lübeck, Germany) in order to maintain correct oxygen tension (pO2) exposure during the experiments. Viability of the islets was evaluated by staining with propidium iodide (PI) (10 μg/ml; Sigma-Aldrich) and bisbenzimide (20 μg/ml, Hoechst 33342; Sigma-Aldrich). Fluorescence was analysed in a Kodak Image Station 4000 MM (Kodak, New Haven, CT, USA). The ratio of PI to bisbenzimide was taken as a relative measure of islet viability.
The rats were anaesthetised by an i.p. injection of 60 mg/kg body weight of pentobarbital sodium (Apoteket, Umeå, Sweden) and placed on a thermostat-controlled heating plate to maintain a core body temperature of 38°C. One hundred microsphere- and 100 non-microsphere-containing islets were transplanted separately at a distance 10–15 mm apart beneath the left renal capsule. Two days or 1 month post transplantation the graft-bearing kidneys were removed. Two hours before removal of the 2-day-old grafts the hypoxia marker pimonidazole hydrochloride (60 mg/kg body weight, Hypoxyprobe-1; HPI, Burlington, MA, USA) was intravenously injected. The grafts were fixed in 10% (vol./vol.) buffered formalin, dehydrated and embedded in paraffin. Embedded tissues were consecutively sectioned (5 μm thick) and mounted on Polysine slides (Thermo Scientific, Braunschweig, Germany).
Sections from the islet grafts were deparaffinised with xylene and rehydrated in a series of graded ethanol (100% to 70%) followed by distilled water, or deparaffinised by Rodent Decloaker (Biocare Medical, Concord, CA, USA) in a pressure cooker (2100 Retriever; Prestige Medical, Blackburn, UK). Islet grafts retrieved 2 days post transplantation were stained for apoptosis and hypoxia markers, whereas islet grafts retrieved 1 month post transplantation were stained for insulin and endothelial cells. Pretreatment for hypoxia staining was performed with Pronase (0.01%; Roche Diagnostics, Mannheim, Germany), for apoptosis staining modified citrate buffer (pH 6.1; Target Retrieval Solution, Dako, Glostrup, Denmark), and for endothelial staining a heat-induction and neuraminidase solution (0.1 U/ml, Neuroamidase Type V; Sigma-Aldrich) was used , followed by peroxidase blocking in 3% hydrogen peroxidase (hydrogen peroxide 30%; Merck, Darmstadt, Germany) and protein blocking (TNB solution, TSA Biotin System Kit [Perkin Elmer, Waltham, MA, USA] or Background Sniper [Biocare Medical]). Apoptosis was detected by a cleaved caspase-3 antibody (1:100; Cell Signaling Technology, Danvers, MA, USA). The signal was amplified by a TSA Biotin System Kit (Perkin Elmer) according to the manufacturer’s protocol and detected by 3,3′-diaminobenzidine (Sigma-Aldrich). Rat pancreases were used as controls in order to exclude non-specific antibody binding. Hypoxyprobe-1 MAb1 (1:25; HPI) was used for hypoxia/pimonidazole hydrochloride staining, and biotinylated lectin from Bandeirea simplicifolia agglutinin-1 (1:100; Sigma-Aldrich) for staining of endothelial cells, followed by incubation with TrekAvidin-AP Label (Biocare Medical) and detection by Vulcan Fast Red (Biocare Medical). Insulin antibody (1:3,000, polyclonal anti-guinea pig insulin; Fitzgerald, Acton, MA, USA) was detected by MACH 3 Rabbit HRP-Polymer Detection (Biocare Medical) and visualised by 3,3′-diaminobenzidine. All sections were counterstained with haematoxylin before mounting.
Scanning and analysis were performed using a laser microdissection microscope (Leica LMD6000, Leica Microsystems, Wetzlar, Germany) or after digital image acquisition by Image J (National Institutes of Health). Necrotic graft area with structural disintegration of cells (pyknotic nuclei, cellular debris) was assessed by morphological evaluation, where on average a total graft area of 0.65 ± 0.1 mm2 per animal was evaluated in the analysis. Apoptosis rate was defined as the percentage of cells positively stained for cleaved caspase-3, where on average 2,841 ± 258 islet cells per group of islets and animal were evaluated. Hypoxic area was defined as the area positively stained for the hypoxia marker pimonidazole per total endocrine tissue area, where an endocrine area of 0.63 ± 0.1 mm2 per animal was evaluated. Vascular density was defined as the area of stained endothelial cells per insulin-positive area. The total insulin-positive area analysed per animal was 0.54 ± 0.06 mm2. Connective tissue area was evaluated by morphometry and defined as non-endocrine tissue per total graft area, where 1.42 ± 0.1 mm2 total graft area per animal was evaluated.
Measurements of islet graft blood flow and pO2
The animals were anaesthetised and surgically prepared according to a protocol similar to that used for microsphere administration. Additional procedures included the insertion of a femoral vein catheter for substitution of body fluid loss with Ringer’s solution (Apoteket), and exposure of the graft-bearing kidney by a left subcostal flank incision. The kidney was thereafter placed in a plastic cup and embedded in surgical cotton pads soaked in saline (154 mmol/l NaCl) in order to stabilise and maintain humidity and temperature of the organ. The blood flow and pO2 in the islet grafts and adjacent renal parenchyma were then recorded by laser Doppler flowmetry and Clark microelectrodes, respectively . Multiple (three or more) measurements of blood flow by laser Doppler flowmetry (Transonic BLF21 Series, probe 1.2 mm; Transonic, Ithaca, NY, USA) were performed in each of the two grafts and in the kidney cortex. Similarly, the pO2 in each of the islet grafts and adjacent kidney was investigated by repeated measurements (three or more in each of the locations) by Clark microelectrodes (outer tip diameter <5 μm; Unisense, Aarhus, Denmark). The mean of blood flow or pO2 values in each location and animal was then calculated and considered to be one experiment. Since it is difficult to calibrate the laser Doppler flowmetry instrument in physical units of blood flow, all blood flow values are given as arbitrary tissue perfusion units.
All values are given as means ± SEM. Differences between two groups of parametric data were analysed by unpaired or paired two-tailed Student’s t test, whereas for multiple comparisons of parametric data a two-way ANOVA with Bonferroni’s or Dunnett’s (comparisons only to control) post hoc test was applied; p values <0.05 were considered statistically significant.