Elsevier

Life Sciences

Volume 80, Issue 4, 2 January 2007, Pages 345-355
Life Sciences

Functional and morphological study of cultured pancreatic islets treated with cyclosporine

https://doi.org/10.1016/j.lfs.2006.09.034Get rights and content

Abstract

Cyclosporine A (CsA), a potent immunosuppressive drug, has been found to induce glucose intolerance through its toxic effect on the endocrine pancreas. It is not exactly known whether CsA has a direct effect on the endocrine pancreas or induces its effect indirectly. The present study was therefore undertaken to examine the function and morphology of isolated pancreatic islets when they are directly exposed in vitro to CsA. Pancreatic islets were isolated from adult male Lewis rats using collagenase ductal perfusion technique. The islets were separated with the discontinuous Ficoll gradient technique and further purified by hand picking of the non-islet tissue. The islets were cultured in RPMI-1640, pH 7.4 and maintained at 37 °C in a humid atmosphere of 5% (v/v) carbon dioxide in air. Cyclosporine was added to the culture medium to give a final concentration of 1 μg/ml (therapeutic dose), 5 μg/ml (toxic dose), or vehicle (control). Islets were harvested at 1, 4 and 10 days of culture and processed for functional or histological study. The functional study of the islets cultured with 1 μg/ml CsA showed insulin and C-peptide contents similar to those of the control islets. The islets cultured with 5 μg/ml CsA showed a marked decrease in insulin and C-peptide contents. Glucose-dependent insulin release was variable. C-peptide release was lower than that of the control following both the therapeutic and toxic doses of CsA. Phase contrast microscopy showed that the islets cultured with 1 μg/ml CsA were mostly normal looking with a well-defined regular periphery; a few islets had ill-defined or irregular peripheries. The islets cultured with 5 μg/ml CsA had ill-defined irregular peripheries at 1 day, and were dense and forming clumps at 4 and 10 days following culture. There was a decrease in the islet number following the therapeutic dose; the decrease was more following the toxic dose of CsA. The islet diameters increased after the therapeutic dose, but slightly decreased following the toxic dose of CsA. Islets showed a weakly positive immunoperoxidase reaction for insulin that was weaker following the toxic dose of CsA. It is concluded that CsA has a direct effect on B-cells that was proved by the functional and morphological changes seen in the pancreatic islets cultured in vitro.

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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