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Development of factors to convert frequency to rate for β-cell replication and apoptosis quantified by time-lapse video microscopy and immunohistochemistry

¹Larry Hillblom Islet Research Center, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California; and ²Department of Information Engineering, University of Padova, Padua, Italy

Submitted 15 August 2008 ; accepted in final form 16 October 2008

An obstacle to development of methods to quantify β-cell turnover from pancreas tissue is the lack of conversion factors for the frequency of β-cell replication or apoptosis detected by immunohistochemistry to rates of replication or apoptosis. We addressed this obstacle in islets from 1-mo-old rats by quantifying the relationship between the rate of β-cell replication observed directly by time-lapse video microscopy (TLVM) and the frequency of β-cell replication in the same islets detected by immunohistochemistry using antibodies against Ki67 and insulin in the same islets fixed immediately after TLVM. Similarly, we quantified the rate of β-cell apoptosis by TLVM and then the frequency of apoptosis in the same islets using TdT-mediated dUTP nick-end labeling and insulin. Conversion factors were developed by regression analysis. The conversion factor from Ki67 labeling frequency (%) to actual replication rate (%events/h) is 0.025 ± 0.003 h^–1. The conversion factor from TdT-mediated dUTP nick-end labeling frequency (%) to actual apoptosis rate (%events/h) is 0.41 ± 0.05 h^–1. These conversion factors will permit development of models to evaluate β-cell turnover in fixed pancreas tissue.

Ki67; TdT-mediated dUTP nick-end labeling; insulin; conversion factor