Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.
Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 °C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.
Islet transplantation is a promising approach to treat type 1 diabetes mellitus1. The first attempt to transfer a sheep pancreatic fragment into a diabetic patient was performed by Watson Williams in 1893. However, a major breakthrough in clinical islet transplantation was achieved with the Edmonton protocol, and thereafter, a series of national programs were developed2. In the past, several sites, such as the bone marrow, omental pouch, intramuscular region, gastric mucosal surface, spleen, and kidney capsule, were explored in preclinical models, but the portal venous system is considered as one of the suitable and effective site for clinical programs3,4,5,6.
Exogenous administration of insulin can be substituted by islet infusion into the portal vein. This is considered a preferred site because the oxygen supply is comparable to that in the native pancreas due to the location downstream of the confluence of the portal artery and vein. Moreover, there is a large surface area, so that the three-dimensional structure of the islets may be preserved, and vascularization could be facilitated5. In a recipient diabetic mouse, 2,000 human or porcine islet equivalents or 350 mouse islets are adequate to reverse the hyperglycemic state7,8. Euglycemic levels were reported for 15 days in the mouse recipient model of xenogeneic islets and in the allogeneic mouse-to-mouse model, and for more than 120 days in the syngeneic mouse-to-mouse transplantation.
Factors pertinent to the efficacy of islet transplantation via portal vein are appropriate anesthesia, method of puncture, and hemostasis9. Anesthesia can be induced by inhalation (5% isoflurane) or injection (ketamine, xylazine, or pentobarbital), and the substances are often combined. For control of the depth and time of anesthesia, attention should be paid to the state of the mouse, such as the color of the mucous membrane, eye lid, corneal reflex, respiratory rate, and body temperature. It is important to ensure that the animal is not struggling and that it survives the operation. The temperature can be maintained by different heating devices, such as heating pads, red light bulbs, etc. A warm thermal plate has been used to maintain a temperature of 25-30 °C between the mouse and the operation table, which prevents the occurrence of hypothermia. The dosages of the anesthetics are important, as all of them are metabolized by the liver, and hepatic function may be transiently disordered by the infusion of the islet suspension. The ideal puncture point for the portal vein is the position between the first and second tributary vein.
The number of islets and the intended injection volume also influence the outcome of the transplant, as an excessive volume may increase the shear stress. A 0.2-mL volume is considered appropriate for islet transplantation into the portal vein. The puncturing wound (26-gauge needle) induces bleeding from the portal vein, which needs to be stopped in a timely and effective manner. Sterile gauze or a finger can be applied with minimal pressure at the site of the puncture for about 6 min to stop the bleeding. Taken together, portal islet injection is effective and provides regulation over blood glucose levels in chemically induced diabetic mouse models.
All procedures in this protocol have been approved by German Animal welfare law and guidelines. Consider using 10- to 12-week-old NMRI nude mice and C57Bl/6 mice (donor: old female; recipient: male) throughout the experiments. Use NMRI nude mice for islet isolation. Keep the mice under defined conditions according to local animal facility regulations.
1. Induction of Diabetes by Streptozotocin
2. Isolation of Pancreatic Islets from Mice
3. Preparation of Animals for Islet Transplantation
4. Islet Transplantation via the Intraportal Route
The competency of transplanted islets was studied in chemically induced diabetic mice. The islets were transplanted via the portal venous system in diabetic mice model. The donor-recipient ratio was 2:1. In the syngeneic mouse model, two donor mice were utilized to obtain 350 islets. The islets were then transplanted into diabetic mice. A reduction in blood glucose level was observed on day 1 after transplantation, suggesting a role of transplanted pancreatic islets in reducing the blood glucose level. Streptozotocin increased the blood glucose level up to about 22 mmol/L. A drop in the blood glucose level to 4.6 mmol/L (day 1) was observed after islet transplantation (Figure 1A). Normoglycemia was maintained for up to 121 days, as shown in Figure 1A. The body weight and blood glucose level roughly followed the same pattern. After the streptozotocin injection and surgery, the body weight decreased. From day 2 onward, it tended to rise towards normal, from 25.3 g (day 4) to 29.7 g (day 121), as shown in Figure 1B.
In case of major histocompatibility (MHC) mismatched mice, 350 islets from two C57Bl/6 (H2d) mice were transplanted into diabetic BALB/c (H2b) mice. After streptozotocin injection, the blood glucose increased up to 26.6 mmol/L. After islet transplantation, the blood glucose level dropped to 4.4 mmol/L (day 1; Figure 2A). Normoglycemia (4.44 to 7.2 mmol/L) was observed for up to 10 days, but not after 15 days (20.1 mmol/L). The body weight of diabetic mice dropped from 26 g (day -3) to 23 g (day 0) and further to 21.5 g (day 1). After transplantation, the body weight gradually recovered from 23 g (day 2) to 25 g (day 10). Due to MHC-mismatch, the blood glucose level increased and the body weight decreased 10 days after islet transplantation (Figure 2B).
Figure 1: Effect of syngeneic islet transplantation. (A) The blood glucose level was measured at different time points in streptozotocin-induced diabetic mice (n = 3). A single dose of streptozotocin (180 mg/kg) was injected before the transplantation of 350 islets to each recipient (day 0). The blood glucose level (mmol/L) was measured for up to 121 days (day -3: 17 ± 2.3; day 0: 22.6 ± 1; day 1: 4.6 ± 0.3; day 5: 7.5 ± 0.4; and day 121: 5.3 ± 0.09). (B) Increase in body weight after islet transplantation. The data above represents the mean ± the standard deviation. Please click here to view a larger version of this figure.
Figure 2: Effect of allogeneic islet transplantation. (A) The blood glucose level was measured at different time points in streptozotocin-induced diabetic mice (n = 2). A single dose of streptozotocin (180 mg/kg) was injected before the transplantation of 350 pancreatic islets (day 0). The blood glucose level (mmol/L) was measured for up to 15 days (day -1: 25.5 ± 5.3; day 0: 26.6 ± 1.4; day 2: 8 ± 0.9; day 10: 6.2 ± 0.08; and day 15: 20.1 ± 1.6). Normoglycemic state was maintained for up to 10 days, and subsequently, blood glucose concentrations increased. (B) Follow-up of body weight before and after islet transplantation. The data represent the mean ± the standard deviation. Please click here to view a larger version of this figure.
Figure 3: Representative immunostaining picture of transplanted pancreatic islets into liver: Wash the liver section (5 µm) with PBS, block with 10% donkey serum followed by overnight incubation at 4 °C with insulin primary antibody (1:500) and platelet endothelial cell adhesion molecule PECAM-1 primary antibody (1:100). Next day, stain with secondary antibodies for 1 h, wash with PBS, counter stain with Hoechst and capture the images with Leica fluorescent microscope. Please click here to view a larger version of this figure.
In this study, the intraportal route of islet transplantation is explored. This protocol is highly efficient in relieving diabetic stress and provides an in-depth opportunity to explore islet transplantation studies. This protocol confirms that about 350 murine islets are capable of reversing the hyperglycemic state. Moreover, none of the mice died during the procedure, and glycemic control was achieved in all recipients. Body weight and blood glucose levels were regularly recorded to reflect the competence, standard, and functionality of transplanted islets in reversing hyperglycemia. In syngeneically transplanted mice, blood glucose levels were measured every day for the first week and once a week thereafter. After islets transplantation, hypoxic condition and others factors trigger the endothelial cells to secrete pro-angiogenic factors such as VEGF after 14 days and thereby increase the vascular engraftment in the liver as shown in Figure 3. In allogeneic mice, blood glucose levels were measured thrice a week for up to 15 days. After 15 days, transplanted islets were rejected in the allogeneic model due to an immune attack, unless immunosuppressive drugs are administered
There are several islet isolation protocols published in literature10,11. The most straightforward is to pick islets manually on the basis of their size and morphology after collagenase digestion. This is one of the most crucial steps to select only islets and to avoid exocrine cells, debris, etc. Selected islets should be maintained at 37 °C overnight in an incubator to allow them to recover from mechanical stress encountered during the digestion procedure. The selection of the streptozotocin dose and the intraportal islet transplantation are also key to this protocol. The body temperature should be regulated and maintained using a thermal plate throughout the surgical procedure. Sterile gauze should be applied with minimal pressure for 6 min at the site of puncture to avoid bleeding. Mice should be kept under observation until they regain sufficient consciousness.
Certain steps in this protocol can be modified. For example, the optimization of the digestion time and the volume of collagenase may increase the yield of islets in different mice strains. Instead of the rapid induction of diabetes (180 mg/kg streptozotocin), slowly increasing hyperglycemia can be induced using a lower dose over an extended period of time (e.g., 40 mg/kg/day streptozotocin for 5 consecutive days). This could provide better engraftment of transplanted islets. Other sites, such as the bone marrow, omental pouch, intramuscular region, gastric mucosal surface, spleen, and kidney can be explored as alternatives to the intraportal vein.
Poor engraftment of transplanted islets is still a major concern in clinical trials. Others include a shortage of donors, inconsistencies in maintaining blood glucose levels after transplantation, immunosuppression, and blood-mediated reactions12,13. Lack of methods to evaluate proper engraftment is another obstacle. Tracking transplanted islets by magnetic resonance or bioluminescence imaging could possibly replace classical methods such as the glucose tolerance test or c-peptide. In the future, stem cells transplanted together with islets might provide nutritional and immunomodulating support14.
The authors have nothing to disclose.
We would like to acknowledge Gundula Hertl for her support.
C57Bl/6 mice | Charles River (Sulzfeld, Germany) | (10-12 weeks) | |
BALB/c mice | Charles River (Sulzfeld, Germany) | (10-12 weeks) | |
NMRI nude mice | Charles River (Sulzfeld, Germany) | (10-12 weeks) | |
Ketamine | Bela-pharm | ||
Xylazine | CEVA TIERGESUNDHEIT GmbH | ||
Syringe, 23G and 26G needle | BD | ||
Petri Dish | Falcon | 303800 | |
NaCl | Carl Roth | 351007 | |
Sterile Gauze | Fuhrmann | 7647-14-5 | |
Shaking Water bath | Kotterman | 1501 | |
Scissors | Braun | 1501 | |
Glucose Meter | One Touch | ||
Warm Thermal Plate | Thermo Fisher | ||
Braunol (povidone-iodide solution) | B.Braun Melsungen AG | 3864154 | |
Liposic Edo – Sprinkled Ointment (eye ointment) | Dr. G Mann Chem.-Pharm. GmbH | ||
Incubator | Fa. Roth | ||
Flow Bench | Herdus | ||
Ficoll | Sigma-Aldrich | 10771 | |
5-0 Silk Suture Vicryl | Braun | (7.5 *1.75mm) | |
Michel Clamps (clips) | Aesculap | BN507R | |
P/FCS Solution | Gibco | 21180-021 | |
TCM-199 (10x) | Gibco | 21180-021 | |
FCS | Biowest | S1810-500 | |
Penicillin-streptomycin | Gibco | 15140-122 | |
HEPES (1 M) | Biochrom | L 1613 | |
Gentamycin | Ratiopharm | ||
Ciprofloxacin | Fresenius Kabi | ||
Hank's Solution | Gibco | 14060-040 | |
Collagenase | Roche | 11213865001 | |
Streptozotocin | Calbiochem | 572204 | |
OPSITE-spray (wound healer) | Smith and nephew | 66004978 | |
Reugular food | Altromin | ||
Head light LED 16042 (magnifying glass) | Eschenbach | 16042 | |
Rat anti-mouse PECAM-1(CD31) monoclonal primary antibody | Millipore, Chemicon | CBL1337 | Dilution (1:100) |
Donkey anti-rat secondary antibody | Dianova | 712095153 | Dilution (1:400) |
Polyclonal guinea pig anti-insulin primay antibody | Dako | A0564 | Dilution (1:500) |
Donkey anti-guinea pig secondary antibody | Dianova | 706-259-148 | Dilution (1:400) |