الفهرس Only 14 pages are availabe for public view
 |  | from | 203 |  |  |
| | | from | 203 | | |
Abstract
V. Summary and Conclusion
Transplantation of normal isolated islets of Langerhans for the treatment of diabetes remains an elusive goal in clinical practice. Perhaps the major problem preventing clinical islet transplantation has been limited by the inability to prevent islet allograft or xenograft rejection. Various approaches and different sites have been suggested to prevent islet graft rejection and maintain long-term islet cell function.
One of these approaches is the protection of the transplanted islet from recipient immune system by enclosing them in membranes that prevent inward diffusion by immune mediators, but allow free exchange of glucose and insulin
Previous studies had demonstrated that islet can be entrapped in viable state in alginate capsules which are characterized by a shell of alginate –polyethyleneimine. The drawback of this technique is the fragile nature of such preparations and the instability of these microcapsules. When these microcapsules were placed interperitoneally they produced only temporary remission of hyperglycemia. Another study had shown that encapsulation of human islets in acrylic-copolymer fiber could prevent rejection of the grafts without immunosuppression for a period of 2 weeks.
This study was conducted to examine whether hamster islets enclosed in agarose microcapsules can survive in the peritoneal cavity of mice as xenograft transplantation. Also to examine whether these microcapsules can maintain normoglycemia and provide normal glucose tolerance test response. Also, This study was planned to evaluate the role of cyclosporin A on the biochemical parameters such as blood glucose, CyA level in a trial to determine the minimum level of cyclosporin which cause maximum suppression of immune system and minimal nephrotoxicity to achieve maximum survival of transplanted islets.
Healthy Golden syrian Hamsters weighing between 100 - 120 g were used as islet donors. Swiss mice were used as the recipients..A total of 80 streptozotocin induced mice were randomized to receive islets xenografts from golden syrian hamsters by 7 different approaches as follow :-
Group 1 :
Ten mice grafted with 1000 free islets in the abdominal cavity.
Group 2 :
Ten mice grafted with 1000 free islets and 1000 empty microcapsules in the abdominal cavity.
Group 3 :
Ten mice received 500 islets microcapsules in the abdominal cavity.
Group 4:
Ten mice received 1000 islets microapsulesin the abdominal cavity.
Group 5:
Ten mice received 1000 islets microcapsules that cultured in CMRL -1066 medium for 4 weeks at 37°C in the abdominal cavity.
Group 6:
Ten mice grafted with islets macrocapsulated in the abdominal cavity
Group 7:
Ten mice received islets in the renal subcapsular space and were given Cyclosporin A in a dose of 15 mg/kg/day for three days (group 7A) and ten mice received islets without Cyclosporin A (group 7B).
Hamster islet were isolated according to the method previously described by intraduct injection of collagenase solution followed by digestion and extensive purification on Ficoll gradients. Hand-picked islets were cultured in CMRL-1066 medium containing 10% heat inactivated fetal calf serum (FCS) and antibiotic-antimycotic solution (1 ml/ 100 ml). The islets were then incubated at 37 C for 3 days in a humidified atmosphere of 5% CO2. Islets were stained for viability by diphenylthiocarbazone (DTZ).
The main number of islets obtained from one pancreas using 0.7 mg/ml collagenase type XI was 41448 islets , without small islets (40-100um). Poorer yields were obtained using lower concentration while distraction of islets were occurred with higher concentration of collagenase. The microscopic appearance of the freshly isolated islets is shown in. The islets were spherical or oval, some of them had exocrine tissue attached to their surface. Morphologically the cultured islets were characteristic by golden brown with a distinct outer intact membrane.
The insulin release from fresh islets compared with cultured islets in response to low (2.8 mM) and high (16.7 mM) glucose concentration is showed that, there is no significant difference in insulin release from fresh and cultured islets in response to low glucose concentration (2.8 mM) was obtained. But, there was a tendency for 24-hour cultured islets to release
more insulin than fresh islets or islets cultured for a shorter period, when using high (16.7 mM). glucose concentration
Pancreatic islet microcapsules were prepared according to the method previously described. At transplantation, a midline incision (2 mm) was made in the abdominal cavity and islet microcapsules were injected through 1 ml syringe without needle.
The result of islet microcapsule transplantation are summarized as follow: Mice transplanted with agarose pancreatic microcapsules (group 3,4,5) became normoglycemic and remained so for 30 to 65 days with gain in body weight. To confirm that encapsulation of islets xenografts in microcapsules prevented islet graft rejection, free hamster islets were transplanted intraperitoneally in 10 mice (group 1). None of these mice became normoglycemic. Similarly, when the hamster free islets were transplanted together with empty agarose microcapsules10 mice (group 2), none of these mice became normoglycemic. To determine the ideal recommended number of islets required to achieve normoglycemia 500 or 1000 microcapsules were tried (10 mice / group). Although euglycemia was achieved in both groups, blood glucose readings were higher with fluctuated values when less than 1000 microcapsules were used.
The diabetic (10 mice) that were received (1000) gelfoam-agarose islet macrocapsules became normoglycemic within 16-20 days, the blood glucose level decreased from 482± 23.21 to 84.7±2.6.
The diabetic (10 mice) that were transplanted with free islets under the kidney capsule and treated with cyclosporine A (15 mg/kg/day) for 3 days (group7A), became normoglycemic and the mean graft survival was 11 ± 4 and remain normoglycemic as long as cyclosporine A was administrated. The mean glucose level decreased from 464 ± 56.9 to 89.2 ± 15.4 mg/dl. In this group rejection occurred after stopping cyclosporine A. But the The diabetic (10 mice) that were received 1000 islets under kidney capsule without cyclosporine A failed to achieve normoglycemia.
The microscopic examination of retained microcapsules 30 days after transplantation showed intact islet with neither tissue reaction nor adhesions. Moreover no evidence of inflammatory cells within or on the microcapsules were detected.
In conclusion, purified hamster islets prepared by collagenase digestion, were successfully transplanted in diabetic mice as xenograft transplantation. The results of these study indicates that it is time to proceed with the development of microcapsules suitable for transplantation of islet xenografts in larger animals and ultimately in humans.
In addition such microcapsules may be useful in treating conditions caused by an impaired functioning or the loss of other secretory cells. Such as disorders of a growth factor or any other hormone. Also, they may provide means for relatively long-term storage and preservation of islet prior to transplantation.
Cyclosporin was found to be a potent drug that resulted in a significant improvement in xenotransplantation results. It possible to create an immune privileged site under the kidney capsule by administration of cyclosporine-A daily in a dose of 15 mg/kg/day for three days .
It is hoped that the results of the present study could be applied to human of both sexes especially when agarose islet microcapsules were used gave encouraging results.