Early assessment of apoptosis in isolated islets of Langerhans. Transplantation 2001 Apr 15;71(7):857-62
Date
05/15/2001Pubmed ID
11349716DOI
10.1097/00007890-200104150-00006Scopus ID
2-s2.0-0035870568 (requires institutional sign-in at Scopus site) 60 CitationsAbstract
BACKGROUND: There is substantial evidence to link early graft loss after islet transplantation to isolation-induced islet cell apoptosis. Measurement of caspase 3 activity and detection of the lost cell membrane asymmetry, revealed by annexin V binding, are newly available assays that allow the analysis of early events of apoptosis.
METHODS: In this study, we compared these tests with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and analysis of DNA fragmentation after gel electrophoresis in freshly isolated islets obtained from rats, before and after treatment with interleukin-1 beta, interferon gamma, and tumor necrosis factor a, cytokines known to induce islet cell damage.
RESULTS: A measurable level of apoptosis was observed the day after isolation when caspase 3 activity and annexin V binding were used as assays, although no substantial DNA fragmentation was detected with TUNEL assay and DNA gel electrophoresis. Baseline caspase 3 activity was 0.8+/-0.3 U/100 islet equivalents and it increased to 1.4+/-0.45 U/100 islet equivalents 3 hr after cytokine stimulation (P<0.05 vs. unstimulated islets). The baseline level of apoptosis, as detected by annexin V binding, was 21.1%+/-5.8%, and it increased to 27.5%+/-8.1% 6 hr after addition of the cytokine cocktail (P<0.01 vs. unstimulated islets). An increase in the number of TUNEL-positive nuclei was detected 24 hr after stimulation and peaked at 48 hr. DNA laddering was also evident 24 hr after cytokine treatment.
CONCLUSION: These data suggest that measurement of caspase 3 activity and annexin V binding analysis might represent reliable markers of early events of islet cell apoptosis.
Author List
Cattan P, Berney T, Schena S, Molano RD, Pileggi A, Vizzardelli C, Ricordi C, Inverardi LAuthor
Stefano Schena MD, PhD Associate Professor in the Surgery department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsAnnexin A5
Apoptosis
Biomarkers
Caspase 3
Caspases
DNA Fragmentation
Electrophoresis, Polyacrylamide Gel
In Situ Nick-End Labeling
In Vitro Techniques
Islets of Langerhans
Rats
Rats, Inbred Lew