Functional and morphological study of cultured pancreatic islets treated with cyclosporine
Introduction
Cyclosporine A (CsA) is a cyclic undecapeptide, which has been known as a very potent immunosuppressive agent. The potent immunosuppressive properties of CsA have made it the drug of choice in many transplantation procedures, including transplantation of segments of pancreas or isolated pancreatic islets (Rynaziewicz et al., 1982, Gunnarsson et al., 1983, Gunnarsson et al., 1984, Pozza et al., 1983, Sutherland et al., 1984, Di Landro et al., 1996, Nakagawa et al., 1999, Stratta et al., 2001, Vantyghem et al., 2003, Dieterle et al., 2004, Squifflet et al., 2004). It has also become an interesting therapeutic alternative for immunosuppression therapy in early diagnosed insulin dependent type I diabetes, based on the assumption that the disease is of autoimmune nature (Nerup and Larnmark, 1981, Stiller et al., 1984, Assan et al., 1985, Feutren et al., 1986, Carel et al., 1996, De Filippo et al., 1996).
Following the clinical use of CsA, a number of adverse effects were recorded. One of these was glucose intolerance that was first reported by Gunnarsson et al. (1983). This was supported by similar findings of Merrell et al. (1984), Engfeldt et al. (1986), Hahn et al., 1986a, Hahn et al., 1986b, Tyden et al. (1987), Menegazzo et al. (1998), Dean et al. (2002) and Dieterle et al. (2004). Concomitant decrease in insulin release was reported by Basadonna et al. (1988), Chandrasekar and Mukherjee (1988), Muller et al. (1988), Gillson et al. (1989), Bani-Sacchi et al. (1990), Shi et al. (1993), Sai et al. (1994), Menegazzo et al. (1998), Dufer et al. (2001), Dean et al. (2002), Paty et al. (2002), Yu et al. (2003) and Dieterle et al. (2004).
The mechanism of CsA toxicity on the endocrine pancreas is not fully identified. It is not exactly known whether CsA has a direct effect on the pancreatic B-cells or induces its effect indirectly. This could be elucidated by in vitro study on isolated and cultured pancreatic islets. These islets, which are free of nerve and counter-regulatory factors, are suitable for evaluation of the direct effect of CsA by adding the drug to the culture medium. The present study was therefore designed to elucidate the in vitro effect of CsA on isolated cultured pancreatic islets. The study aims also at correlating the functional metabolic effects of CsA with its effect on the morphology of the pancreatic islet cells.
Section snippets
Animals
Adult male Wistar rats weighing 220–250 g were obtained from the Animal House, King Fahd Medical Research Center, King Abdulaziz University, Jeddah. The breeding and experimental use of the rats were reviewed and monitored by the Animal Ethics Committee of King Fahd Medical Research Center. Blood glucose was measured before isolation of the islets to ensure that the experimental rats are normoglycemic.
Isolation of the pancreatic islets
The pancreatic islets were isolated using the ductal perfusion method of Sutton et al. (1986)
Functional evaluation
The isolated islets cultured with the therapeutic dose of CsA showed insulin and C-peptide contents (Fig. 1A and B) more or less similar to those of the islets cultured with the vehicle (control group). The isolated islets cultured with the toxic dose of CsA had insulin and C-peptide contents that were significantly lower than those of the control group (P < 0.05).
Islets cultured with the therapeutic or the toxic dose of CsA showed variable insulin release in response to glucose challenge.
Immunohistochemical examination
Islets cultured with vehicle (control) showed normal looking healthy cells after 1, 4, and 10 days culture. Sections stained with immunoperoxidase for insulin showed that most of the islet cells were positively stained (Fig. 6A and B). Several islets were large in diameter. The peripheral cells of some islets showed strong positive reaction, whereas the centrally located cells showed a relatively weak positive reaction (Fig. 6A).
Islets cultured with 1 μg/ml CsA (therapeutic dose) showed more or
Discussion
With the recent development of the techniques of isolation of the pancreatic islets, it became possible to get relatively purified endocrine pancreatic islets, which could be used to assess the biochemical function of the isolated islets in response to various conditions. The ductal collagenase technique, used in this study, is the standard method of isolation of islets, which is widely used for harvesting islets from the rat (Sutton et al., 1986, Hara et al., 1989, James et al., 1989, Van Der
Acknowledgement
This work was supported by grant no. 011/419 from King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
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