Induction of diabetes in small rodents by -cell toxins or partial surgical pancreas resection has been followed by regeneration of -cell mass and reversal of diabetes (4,40). conversion factors will permit development of models to evaluate -cell turnover in fixed pancreas tissue. Keywords:Ki67, TdT-mediated dUTP nick-end labeling, insulin, Tandutinib (MLN518) conversion factor both type 1 and type 2 diabetesare characterized by a deficiency of -cells (5,6,22,30). Pancreas transplantation restores glycemic control in type 1 and type 2 diabetes (10,26). However, given the insufficient number of pancreases available for transplantation and the risks of prolonged immunosuppression, restoration of -cells by transplantation is not a viable option for Rabbit Polyclonal to GK2 most people with diabetes. A potential option strategy is restoration of -cells through Tandutinib (MLN518) endogenous regeneration. Induction of diabetes in young rodents by -cell toxins or partial surgical pancreas resection has been followed by regeneration of -cell mass and reversal of diabetes (4,40). The origin of these -cells has been actively debated: some have proposed duplication of existing -cells, as well as others have suggested formation of new -cells from a variety of sources (7). Although ongoing lineage studies will provide insights into the origins of -cells in mice (3,11,12,27,37), this approach is not available in humans and does not provide guidance as to the rate of -cell formation that might be anticipated in humans. Thus, although there is usually indirect evidence that there might be ongoing -cell formation in adult humans with type 1 diabetes (22), there is no information as to rate of -cell turnover in human pancreas. Although methods do exist for detection of -cell replication and -cell apoptosis, these methods cannot yet be used to compute the rate of -cell replication or apoptosis in humans. Incorporation of labeled nucleotides [bromodeoxyuridine (BrdU)-labeling index], which has been used to estimate the -cell replication rate in rodents (15,38), cannot be used in humans, since it requires administration of BrdU (a toxic and mutagenic material) before pancreas collection. Moreover, BrdU can disturb the cell cycle, and its use cannot distinguish cell replication from DNA repair (7). Immunostaining of the nuclear protein Ki67 is an alternative method for quantification of the frequency of cell replication. Ki67 is usually expressed in all phases of the cell cycle, except the resting phase (G0) and for a short period at the beginning of the G1phase (7). Ki67 is not detected during DNA repair. Therefore, although Ki67 labeling is usually more sensitive and specific as an estimate of the frequency of -cell replication than use of BrdU (7), it does not provide a rate of replication. TdT-mediated dUTP nick-end labeling (TUNEL) determined by immunohistochemistry is the most widely used approach to quantify cell death and has the advantage in this regard, in that it detects apoptosis and necrosis in tissue samples (9,18). In common with Ki67 staining, TUNEL permits evaluation of the frequency, but not the rate, of cell death in a tissue of interest. The goal of the present studies was to establish conversion factors that would provide1) the rate of -cell replication from the frequency of -cell replication determined by Ki67 and insulin immunostaining and2) the rate of -cell apoptosis from the frequency of apoptosis detected by TUNEL and insulin immunostaining. These goals are important, because they must be accomplished before models intended to quantify Tandutinib (MLN518) -cell turnover in humans can be developed. == MATERIALS AND METHODS == == == == Study design. == Our strategy involved use of time-lapse video microscopy (TLVM) to directly quantify the rate of -cell apoptosis or replication and then fix that tissue and employ immunohistochemistry to determine the corresponding frequency of -cell replication or apoptosis. The requirements for this strategy are as follows. First, we established conditions in which steady-state rates of -cell replication or -cell apoptosis were present for 24 h in pilot experiments using INS-1 cells and isolated islets. Second, we established conditions in which we could reproducibly identify -cells during TLVM from the subsequent immunostaining of islets (for insulin). Third, we needed to observe -cell replication and apoptosis at a variety of rates to establish a relationship between these two parameters. This was particularly challenging for -cell replication in islets, since we discovered (as have others recently) that, even in rodent islets, -cell replication is usually rare in adults. The challenge Tandutinib (MLN518) was overcome by use of islets from rats at 1 mo of age, when the postnatal growth of -cell numbers through -cell replication is still occurring. Having established these conditions, we studied juvenile rat islets under steady-state conditions of -cell replication and -cell apoptosis and then immunostained.