Human pancreatic tissue was obtained at autopsy from 42 individuals with type 1 diabetes (24 male, 18 female; age 40.5±12.5 years; BMI 22.2±5.1 kg/m2; glucose 14.7±5.8 mmol/l). Potential cases were identified by retrospective analysis of the Mayo Clinic autopsy database. To be included, cases were required to have had: (1) a full autopsy within 12 h of death; (2) a general medical examination, including at least one blood glucose measurement documented within the year before death; and (3) pancreatic tissue stored that was of adequate size and quality. Subjects were excluded if pancreatic tissue had undergone autolysis or showed evidence of acute pancreatitis.
The diagnosis of type 1 diabetes was established clinically by independent chart review by three physicians (P. C. Butler, R. A. Rizza and J. J. Meier) according to the following criteria: (1) ketosis at diabetes onset; (2) young age of onset (most were aged 10–18 years); (3) absence of a family history of type 2 diabetes; and (4) immediate insulin requirement after diabetes onset, with sustained requirement. Clinical diagnosis was supported by pathological evaluation of pancreas (marked beta cell deficit, absence of islet amyloid and absence of pancreatitis). Eleven of the 42 cases of type 1 diabetes had received a renal transplant and subsequent immunosuppressive therapy. Pancreatic specimens from six non-diabetic subjects (three male, three female; age 49.8±15.3 years, BMI 27.7±6.5 kg/m2, glucose 5.1±0.94 mmol/l) were obtained from tumour-free pancreas removed at surgery for adenocarcinoma of the biliary tree or pancreatic adenomas, and from eight non-diabetic subjects (four male, four female; age 37±10 years; BMI 20.2±3.0 kg/m2, glucose 5.1±0.3 mmol/l) at the time of autopsy. None of the latter group of eight non-diabetic subjects was known to have any disease affecting the pancreas prior to death.
Neither glycosylated haemoglobin nor home blood glucose monitoring values were available for the diabetic subjects, as these were not commonly used at the time of death of these individuals. Therefore, to obtain the best possible estimate of glycaemic control, all laboratory-measured blood glucose concentrations documented in the medical record (average recorded values 8.3±0.6, range 2–23 mmol/l) were used to calculate a mean glucose value, which was then compared with beta cell abundance.
The number of pancreatic tissue blocks available per diabetic subject was as follows: one block in 27 cases, two blocks in ten cases, three blocks in one case, four blocks in one case and six blocks in three cases. Of the three diabetic subjects in which no beta cells could be detected, two had one block and one had two blocks. Of the four patients with the most insulin-positive islets, three had one block and one had four blocks. In this latter case, one block revealed multiple islets with insulin-positive cells and numerous scattered individual beta cells; one block had one islet with insulin-positive cells, but no individual scattered beta cells; the third and fourth blocks had numerous individual scattered beta cells, but the islets contained no insulin-positive cells.
Pancreatic tissue processing
Pancreas was fixed in formaldehyde and embedded in paraffin for subsequent analysis as previously described . Five sequential 5-μm sections were stained as follows: (1) for insulin (peroxidase staining) and haematoxylin for light microscopy; (2) insulin and cleaved caspase-3 combined (immunofluorescence); (3) insulin, terminal deoxynucleotidyl transferase biotin-dUTP nick end-labelling (TUNEL) and 4′,6-diamidino-2-phenylindole (DAPI; immunofluorescence); (4) insulin, Ki67 and DAPI combined (immunofluorescence); and (5) insulin, CD3 and CD68 combined (immunofluorescence).
The following primary antibodies were used for immunofluorescence: guinea-pig anti-insulin, 1:200 (Dako, Grostrup, Denmark); mouse Ki67, 1:200 (MIB-1, Dako); rabbit cleaved caspase-3, 1:50 (Biocare Medical, Concord, CA, USA); rabbit CD3, 1:50 (Dako); mouse CD68, 1:100 (Dako). Secondary antibodies labelled with Cy3, fluorescein-5-isothiocyanate (FITC) and [6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid] (AMCA) were obtained from Jackson Laboratories (West Grove, PA, USA) and used at dilutions of 1:100 to 1:200. For TUNEL staining, the in situ cell death detection kit TMR Red from Roche Diagnostics (Mannheim, Germany) was used.
The ratio of the beta cell area:exocrine area was quantified as previously described . The frequency of scattered single beta cells unrelated to islets or pancreatic ducts was counted manually in each subject and expressed relative to pancreas area. The frequency of insulin-positive cells in exocrine ducts (∼60 ducts per subject) was determined manually.
Beta cell apoptosis and replication
Beta cell apoptosis in type 1 diabetic patients and non-diabetic control subjects was identified using TUNEL staining as previously described . Since TUNEL detects DNA-strand breaks, it is theoretically possible that the frequency of TUNEL-positive cells in a pancreatic specimen obtained at autopsy is artefactually increased due to post-mortem changes. Therefore, cleaved caspase-3, which represents the final activation step in the apoptotic signalling cascade, was used as an additional marker for beta cell apoptosis. Moreover, the frequency of cleaved caspase-3-positive beta cells was compared between non-diabetic individuals with a pancreatic specimen obtained at autopsy (n=8) and those with a specimen obtained at surgery (n=6). The frequency of beta cell apoptosis by cleaved caspase-3 was similar in these tissue samples (2.8±0.2 vs 1.9±0.4%, NS). To compute a meaningful relative frequency of either beta cell replication or apoptosis in type 1 diabetic patients vs non-diabetic control subjects, sufficient beta cells per subject are required. There were insufficient beta cells in most subjects with type 1 diabetes to allow this computation. We therefore used the values for the four type 1 diabetic patients with the greatest number of detectable beta cells and compared these with the values for the eight non-diabetic control subjects. In these 12 subjects, slides were co-stained for insulin (FITC) and cleaved caspase-3 (Cy3). Ten islets from each of these 12 cases were examined in detail at ×200 magnification (×20 objective, ×10 ocular). The number of beta cells was counted and the number of beta cells positive for caspase-3 in these ten islets documented. A total of 2,690 and 6,679 beta cells were evaluated thus in the four type 1 diabetic patients and the eight autopsy control subjects, respectively. Slides from the same subjects co-stained for insulin (FITC), Ki67 (Cy3) and DAPI were evaluated for the frequency of beta cell replication, using a method similar to that used to assess apoptosis.
As the frequency of replication in human islets is extremely low (in comparison with murine islets), we performed several positive controls to ensure that Ki67 is a valid marker for replication in the paraffin-embedded tissue obtained at autopsy, and that this well-documented marker for cell replication provides a reliable measure of beta cell replication in humans. Therefore, samples of human tissue (spleen, pancreas, liver [autopsy] and pancreatic insulinoma [surgery]), fixed and processed under identical conditions in the same pathology laboratory (Mayo Clinic), were similarly stained for Ki67. Ki67-positive cells were observed to be scattered similarly in exocrine tissue from both type 1 diabetic patients and control subjects. As expected, splenic white pulp was characterised by a high frequency of Ki67-positive cells, clearly indicating cell division. Ki67-positive cells were also present in liver. In addition, Ki67-positive dividing beta cells were evident in a human insulinoma.
To quantify the extent of fibrosis, haematoxylin- and insulin-stained slides from each subject were evaluated by light microscopy. Each subject was scored using an arbitrary scale of 0 to 4, where 0 indicates no fibrosis present; 1, minimal fibrosis in the periductal area; 2, moderate fibrosis in the periductal area; 3, marked fibrosis around ducts and in the interlobular area; and 4, severe extensive fibrosis around ducts and in the interlobular area.
Subject characteristics are reported as means±SD and results are presented as means±SEM. Statistical calculations were carried out by one-way ANOVA and Duncan’s post hoc test using Statistica, Version 6.0 (Statsoft, Tulsa, OK, USA). Categorical variables were analysed by Fisher’s exact test using GraphPad Prism 4 (San Diego, CA, USA). A p value of less than 0.05 was considered statistically significant.