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Diabetes mellitus type I also called insulin-dependent diabetes is a chronic autoimmune disease characterized by the destruction of cells in the islets of Langerhans in the pancreas. The disease has both genetic and environmental components and an age of onset between 4-6 and 10-14 years. Î²-cells are important in maintaining glucose homeostasis by producing the hormone insulin in response to alterations in blood glucose levels. Destruction of more than 90% of these cells will lead to an impairment in the maintenance of the glucose homeostasis and the need for life-long insulin therapy. Insulin therapy has improved life expectancy of patients, but it is not as tightly regulated as in healthy persons and many patients have wide fluctuations in blood glucose levels leading to secondary complications of diabetes such as ketoacidosis, kidney failure, cardiovascular disease, stroke and blindness which hampers the quality of life and increases mortality and morbidity. Furthermore, insulin therapy increases the risk of severe hypoglycemic episodes by 3-fold, much of the time unnoticed by patients. Therefore, many diabetes mellitus type I patients undergo hypoglycemia unawareness, which can cause loss of consciousness or inability to awake from sleep (also called "dead-in-bed" syndrome), and this accounts for up to 10% of mortality.1-3
Whole pancreas transplantation is done in patients with a high risk of secondary complications and/or a life-threatening hypoglycemic unawareness. The goal of whole pancreas transplantation is to restore blood glucose levels back to normal.2,4,5 However, whole pancreas transplantation is a major surgery with several complications such as acute graft rejection, exocrine leakage (leading to damage to surrounding tissue) and graft thrombosis. Furthermore, recipients must have life-long immunosuppression in order to prevent graft rejection, but this increases susceptibility to infections, renal dysfunction, hyperlipidemia, anemia, mouth ulcers and an increased risk of cancer.4-6
The largest part of the pancreas is composed of exocrine tissue that is responsible for the production of digestive enzymes, while the insulin-producing Î²-cells compose only 2% of the pancreas. Therefore, islet transplantation should be a better approach than whole pancreas transplantation. Islet transplantation is less invasive and is associated with fewer complications.6,7 Furthermore, it has been shown that islet transplantation has an approximate 20-fold lower morbidity risk than whole pancreas transplantation. A major disadvantage at the moment is that for 1 recipient about 2-4 donor pancreases are needed.1 The first islet transplantation was done in 1989 and lasted just a few days. In 2000, the Edmonton protocol was introduced in which steroid-free immunosuppression was achieved, as steroids amplify the disease. The Edmonton center reported 80% insulin independence at 1 year, 30-40% at 3 years and 10% at 5 years post-transplantation. Furthermore, about 80% of all recipients had to undergo a second islet transplantation. However, many of these recipients required less insulin and hypoglycemia occurred less frequent, suggesting meaningful cell survival of the transplant.2,3,7
Although the pancreas should be the best place for islet transplantation, due to the difficult surgery, high risk of complications due to enzyme leakage from exocrine tissue and the autoimmune reaction of diabetes mellitus type I, islets are injected into the portal vein of the patient to settle in the liver.8 Complications associated with this procedure are: bleeding, portal vein thrombosis, hypercholesterolemia and instant blood-mediated inflammatory reaction (IBMIR). Due to the latter, amongst other things, recipients must have life-long immunosuppression in order to prevent graft rejection.4,8
In order to prevent graft rejection animal experiments were done with encapsulated islet transplantation. Moreover, encapsulation can also inhibit immune-mediated destruction of the transplanted islets without requiring life-long immunosuppression. Encapsulation of islets provides a semi-permeable barrier which allows bi-directional diffusion of small molecules such as oxygen, carbon dioxide, cellular nutrients and waste products, ions, growth factors and insulin; but it also prevent macrophages, antibodies and complement factors from reaching the islets and inducing damage. One of a few islets (microcapsulation) are encapsulated in a spherical membrane of alginate coated with poly-L-lysine (PLL) to improve mechanical stability. Then a thin layer of alginate is placed on top to prevent the PLL to initiate a proinflammatory response in the recipient. Microcapsules are mechanically stable for years in animals (and humans), are simple to construct, have a high surface area-to-volume ratio, enabling efficient diffusion, and can be implanted without major surgery.5-7,9
Justification state of art
At the moment, maintenance of glucose homeostasis by islet transplantation is monitored by glucose levels (fasted and stimulated), oral glucose tolerance testing, C-peptide levels (a byproduct of endogenous insulin synthesis), and insulin secretion. However, abnormalities in these parameters are mostly seen when most of the islets are already destroyed.10 Furthermore, in experimental animals every data point requires sacrificing the animal through which following a time course in an animal is impossible.11 Therefore, noninvasive in vivo imaging techniques would be an outcome to monitor graft function after transplantation.
Several techniques are available for in vivo noninvasive imaging, such as magnetic resonance imaging (MRI), positron emission tomography (PET), single positron emission tomography (SPECT), bioluminescence and fluorescence. All of these techniques have both advantages and disadvantages. MRI has a high spatial resolution and penetration depth and there is no ionizing radiation. However, it cannot distinguish the transplanted islets from the surrounding tissue, for this a contrast agent is required. PET and SPECT have a high sensitivity, but a low spatial resolution and need radioactive probes with a short half-life for imaging. In comparison with MRI, PET and SPECT, bioluminescence and fluorescence cost less and have a higher throughput (as they have short processing times). However, bioluminescence is not applicable for signals coming from deep tissues due to light attenuation by tissue and luciferase substrate must always be added just before imaging.12,13 Therefore, fluorescence is the noninvasive in vivo imaging technique of choice.
Fluorescent proteins, such as GFP (green fluorescent protein) can be genetically linked with any protein of choice thereby providing a permanent and heritable label in live cells. Furthermore, the fluorophore of GFP is protected by the three-dimensional structure of the protein and is therefore relatively unaffected by the external environment. It has been shown that noninvasive in vivo whole-body imaging with fluorescent proteins can visualize cancer, gene expression, graft vs. host disease, angiogenesis and infectious disease in animal models. Furthermore, fluorescent proteins are available in different colors which each have characteristic excitation and emission spectra, so several processes can be visualized simultaneously.14-16
Although GFP is used in many whole-body imaging applications, it has been shown that whole-body imaging is more effective when using fluorescent proteins with longer wavelengths, such as far-red and near-infrared wavelengths (>600 nm). These wavelengths have a strong and stable signal and are shown to be less absorbed by tissue and molecules, cause less autofluorescence and are less scattered. In this way, better contrast and a higher sensitivity can be achieved.14,17,18 Experiments have shown that wavelengths such as far-red and near-infrared can propagate through tissues up to 15 centimeters, while visible wavelengths can only propagate a few millimeters.15,19,20 Fluorescent proteins can be visualized with fluorescence reflectance imaging (FRI) where the light source and the detector are located on the same side of the animal. FRI is now a commonly used experimental methods in living subjects as it is ideal for high-throughput imaging and relatively inexpensive. A high-sensitivity CCD camera is needed to capture the images, which has a short acquisition time, is portable and does not need much space.15,20
Although islet transplantation seems to be a better approach than whole pancreas transplantation, not much is known about the development and eventually death of the transplanted islet cells. At this moment, monitoring of the transplanted islets in vivo can only be done in an invasive way by sacrificing the experimental animals after certain time periods. Therefore, a way of monitoring the transplanted islets in vivo in a noninvasive way would be informative as real-time detection can provide more data of the progress of transplanted islets.
Therefore, a pilot study will be done researching the possibility of imaging transplanted islets noninvasively in vivo by means of fluorescence. Transplanted islets will emit GFP and the insulin gene will get a Cy5.5 (far-red) reporter. As the reporter will be encoded in the genome, the signal is not diluted by cell divisions.
Eight male Lewis rats are used, of which six are used as recipients of islet transplantation. Eighteen male GFP-LEW Tg rats are used as donors. These rats show strong and ubiquitously GFP in most of their organs, including the pancreas.21
Diabetes will be induced in the six male Lewis rats by injection of 75-90mg/kg of Streptozotocin via the tail vein. A week after diabetes-induction islets are isolated from the 18 male GFP-LEW Tg rats. The abdomen is opened under anesthesia after which the pancreas is retrieved. The pancreas will then be chopped and digested with collagenase (which does not digest the islets, only exocrine tissue). Islets are then separated from exocrine tissue by centrifugation and further purified by handpicking. Isolated islets are cultured overnight in petri dishes at 37Â°C in humidified air containing 5% CO2.22,23 After placing the islets on the petri dishes the islets are transfected with an adenovirus-expressing Cy5.5 gene that will be expressed under the transcriptional control of the promoter of the insulin gene and cultured for 2h at 37Â°C and 5% CO2 in serum-free medium. After 2h fetal bovine serum will be added to reach a final concentration of 10% and islets are cultured for 48h at 37Â°C in humidified air containing 5% CO2.24 After 48 h the islets are transplanted into the male Lewis recipient rats. Each recipient needs about 2000 islets for transplantation, which accounts for 3 donors per recipient. The rats are anesthetized and a small incision is made in the abdomen after which the islets are injected into the peritoneal cavity.23 As the transplantation is isogeneic, no immunosuppressive drugs are needed.
Plan for interpretation
The transplanted islets are not encapsulated, therefore it is predicted that they will adhere somewhere in the peritoneal cavity instead of floating in the peritoneal cavity.
The six male Lewis rats that received the transplantation are followed for 1 year. Twelve hours after transplantation fluorescence images are made and a glucose tolerance test is done of each rat. Thereafter fluorescence images are made and a glucose tolerance test is done each month for twelve months.
The fluorescence images are made by using a charge-coupled device (CDD) camera, which is capable of imaging live animals. A filter is used to reduce the autofluorescence of the skin. Furthermore, hair emits often a fluorescence signal, so maybe the hair of the rats has to be trimmed if it interferes with the signal. Three different fluorescence images are obtained. The first is the GFP expression, with emission at 520 nm. The second is NADPH expression, with emission at 470 nm. As these emission peaks are close together, they interfere with each other. By measuring also NADPH expression, the 'real' expression of GFP can be obtained.14,25 It was previously shown that there was an inverse correlation between fluorescent signal intensity and duration of the normoglycemic period in experimental animals.24 Therefore, GFP intensity can correlate with graft survival. The third fluorescence image that is obtained is the Cy5.5 expression at 694 nm. Cy5.5 expression was correlated with the insulin production as the Cy5.5 protein, transfected by means of adenovirus, was placed under the transcriptional control of the promoter of the insulin gene. A study has shown that adenoviral infection did not interfere with the health, insulin production and survival time of the Î²-cells.24 Therefore, expression of Cy5.5 is correlated with insulin production.
The glucose tolerance test is done with the 6 transplanted rats and 2 healthy rats, which serve as the control. The glucose tolerance test is done as follows: the rats are fasted overnight (approximately 16 hours) before the test. The next day blood glucose (time 0) is determined in a drop of blood by a glucometer. The drop of blood is obtained by cutting the tail tip with a pair of scissors. Then glucose solution (2,5 g D-glucose in 10 ml milliQ water) is injected intraperitoneally and blood glucose is determined at 10, 20, 30, 60, 90 and 120 minutes after the injection of glucose.
Encapsulated islets transplantation
This pilot study was done with islets that were not encapsulated. This is first necessary to see if the protocol works and to reduce unnecessary costs. If it is shown that the protocol works, it should be repeated with encapsulated islets as they are less prone to graft rejection.
At the moment, FRI is most commonly used to image fluorescence in vivo in live animals. However, this method has several disadvantages, such as penetration depths of only a few millimeters with visible wavelengths and blurred images from deeper depths. Furthermore, FRI is only capable of semiquantification as a high fluorescence signal in deeper tissue could yield the same appearance on the surface as a low fluorescence signal in superficial tissue. Recently, a new imaging technique was introduced, namely fluorescence molecular tomography (FMT), which can be used for quantification of fluorescence signals. FMT can obtain three-dimensional reconstructions of the fluorescence signal. In order to do so, the animal is illuminated at different sites and multiple measurements are collected. FMT is a practical and relatively inexpensive system of multiple detection channels using CCD cameras.15,19,20
Albino Oxford rats
In this pilot study, male Lewis rats are used as they have the highest number of islets yield. However, it was shown that the AO rats had a 2-fold higher number of islet yield than Lewis rats, although the function of the islets was in the two strains approximately the same.26 The reason that AO rats are not used is that, at the moment, they are not available at the supplier. When they are available again, a GFP-AO rat strain can be made to reduce the amount of donors needed for 1 recipient. This would be more ethical and would also reduce costs. The GFP-AO rat strain can be made according to instructions from the article of Inoue, H. et al (2005).21