Male C57BL/6J mice (Charles River, Frankfurt, Germany) were housed under a 12 h light–dark cycle with free access to food and water. Experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research. The study was approved by the Regional Commission in Karlsruhe, Germany.
Eight-week-old streptozotocin (STZ)-induced diabetic mice (diabetic control [DC]), 8-week-old STZ-induced diabetic mice receiving isogenic pancreatic islet transplantation (Tx) after 6 weeks of diabetes (DC+Tx) and age-matched controls (non-diabetic control [NC]) were analysed after 6 weeks (6W) and 12 weeks (12W). Use of isogenic animals and sterile conditions ensured the absence of immune responses and surgical complications. All mice were euthanised under general anaesthesia.
Diabetes was induced by intraperitoneal injection of STZ (160 mg/kg body weight; Sigma-Aldrich, Munich, Germany) in 8-week-old mice. Stable hyperglycaemia was confirmed 7 days after injection by blood glucose >16.7 mmol/l. Body weight and blood glucose were measured throughout the experiment (BGStar; Sanofi-Aventis, Frankfurt am Main, Germany; limited to 33.3 mmol/l). HbA1c was measured using affinity chromatography (In2it; Biorad, Munich, Germany).
Transplantation of pancreatic islet cells was performed by the Clinical Research Unit, Giessen (TL). Islets were taken from 8-week-old male C57BL/6J mice (Janvier Labs, Saint-Berthevin, France). Pancreatic islet-cell isolation was performed using pancreatic collagenase digestion and handpicked selection . About 300 islets were injected below the kidney capsule of the recipient mice after 6 weeks of diabetes induction. To support a basal insulin release of the islet graft, insulin-releasing pellets (LinBit; LinShin Canada, Toronto, ON, Canada), each releasing insulin at 0.1 U/24 h, were placed subcutaneously below the mid dorsal skin. The number of pellets placed in each recipient mouse, estimated by the targeted reduction of blood glucose levels (< 13.9 mmol/l), was two to four.
Retinas were isolated after overnight fixation of frozen eyes (−80°C) in 4% buffered formalin and were digested using a trypsin-based digestion method. Four to six retinas from each group were analysed morphometrically. In each retina, ten fields were randomly selected using ×400 magnification and CellF analysing software (Olympus, Hamburg, Germany). The cell numbers were normalised to relative capillary density (cell number/mm2 capillary area).
Frozen eyes were dissected and the retinas were extracted and immediately suspended in Trizol reagent (Invitrogen, Carlsbad, CA, USA). Zirconium oxide beads (1 mm, RNase-free; Next Advance, Averill Park, NY, USA) and a Bullet Blender (Next Advance) were used for retinal homogenisation. RNA was extracted using Trizol. RNA quality was verified using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and quantity measured by spectroscopy using an Infinite200 PRO NanoQuant System (Tecan, Männedorf, Switzerland).
Gene expression profiling
Gene expression profiling (n = 5 or 6 per group) was performed using Affymetrix GeneChip Mouse Gene 2.0 ST Array. Biotinylated antisense cRNA was then prepared in accordance with a standard labelling protocol. All protocols and equipment were from Affymetrix (High Wycombe, UK).
A custom CDF Version 19 with Entrez-based gene definitions was used to annotate the arrays. The raw fluorescence intensity values were normalised applying quantile normalisation and robust multi-array average (RMA) background correction. ANOVA was performed to identify differentially expressed genes (SAS JMP10 Genomics v6; SAS Institute, Cary, NC, USA). A false-positive rate of a = 0.05 with correction for false discovery rate was taken as the level of significance (GEO: GSE87433). Metabolic memory genes were identified, if they were regulated positively or negatively, in both NC12W → DC12W and NC12W → DC+Tx@12W. Analysis of the resulting gene list was performed using DAVID bioinformatics database (version 6.7, https://david-d.ncifcrf.gov/) using the categories UP_TISSUE and GOTERM_CC_FAT.
Metabolic data and pericyte numbers are expressed as mean ± SEM and were analysed using one-way ANOVA followed by Tukey’s multiple comparison post hoc test. Animals were assigned to experimental groups by simple randomisation. Experimenters were blind to group assignment and outcome assessment. No data, samples or animals were excluded from the reported results.