scientificprotocols authored over 7 years ago
Authors: Dong-sheng Li, Jianqiang Hao, Ya-Hong Yuan, Se Hak Yun, Jing-Bo Feng, Long-Jun Dai & Garth L. Warnock
Mouse islet subcapsule transplantation is widely used in diabetes-related studies. Reliable and reproducible transplantation is essential to the success of those types of investigations. The present protocol presents detailed procedures on the mouse diabetic model and islet implantation. The blood glucose tracings under varied transplant circumstances are presented, covering syngeneic, allogeneic and xenogeneic islet transplantations. This protocol is straightforward and has been proven to be practical and reproducible.
Islet transplantation has become a very progressive field in diabetes studies in the recent few decades. It took almost 30 years from the first islet transplantation in rats to the first successful islet allotransplantation in patients with type 1 diabetes 1,2. Islet transplantation as an important procedure is widely used in almost all diabetes-related investigations, including diabetic therapy, anti-rejection drug selection, xeno-islet searching, and stem cell-based islet replacement. An ideal islet transplantation protocol is a basic requirement for reliable diabetic studies. As a concomitant technique of the protocol for islet isolation from mouse pancreas 3, we present a practical and reliable protocol for kidney subcapsular islet transplantation in mice.
Step 1: Diabetic model was not induced in due course
The sensitivity of mice to STZ varies with their age, gender and species. To our knowledge, C57BL/6J mice require lower dose of STZ than Balb/C mice. So, it is a good practice to take a small-scaled trial for the given species before starting the formal experiment. Animal age should be limited if possible to a small range.
Step 8: The incision toward kidney was mislocated
After cutting the skin, the spleen is readily recognized through the thin layer of the muscle. The incision should be made along the lower edge of the spleen.
Step 12: Kidney capsule was broken by PE-50 tubing
The following two tips may be helpful in avoiding the problem: (1) while inserting the tubing into the pouch, make sure to position the tube along the curve of the kidney surface; (2) always keep the kidney surface wet with saline.
As illustrated in Fig. 3, STZ-induced hyperglycemia was promptly corrected by syngeneic islet transplantation. The corrected euglycemia was reversed to hyperglycemia when islet graft-bearing kidney was removed, thereby confirming the function of transplanted islets. A short time rebound of blood glucose after islet transplantation is not a rare phenomenon. It might be due to operation-related stress and/or manipulation-induced islet instability. Hyperglycemia following nephrectomy in STZ-induced diabetic mice can be corrected again by the second transplantation on the other kidney (Fig. 4). The second islet transplantation may be a useful option for some investigations. The immunohistochemistry of islet-graft was not given in the present protocol. However, interested investigators may refer our recent report 16. The time course of islet-graft being rejected is mainly determined by the conformity of histology between donor and recipient. Figure 5 displayed the different patterns under different transplant combinations. Syngeneic islet transplantation showed no rejection during the observation period (Fig. 5a). Evident rejection started on days 10-14 after transplantation on allogeneic islet transplantation group (Fig. 5b). For xenogeneic islet transplantation, however, the rejection took place almost instantly after transplantation (Fig. 5c). This information can be referenced during implement of certain investigations.
This study was supported by National Natural Science Fund of China (81070614), Provincial Natural Science Fund of Hubei (2008CDA044), Canadian Institute of Health Research (CIHR, mop-79414), and Juvenile Diabetes Research Foundation (JDRF, 1-2008-474). We are grateful to Crystal Robertson for her assistance on the manuscript preparation.
Figure 1: Packaging islets into transplantation tubing.
Figure 1. Packaging islets into transplantation tubing. Given amount of islets for each mouse were kept with an eppendorff tube. After the islets settled down, they were collected into PE-50 tubing with the aid of microsyringe. The islet-containing tubing was folded and inserted into a 200 μl pipet tip, then, put into a 15 ml conical centrifuge tube. An islet pellet was formed after centrifugation. The islet pellet containing tubing was mounted onto the microsyringe for transplantation.
Figure 2: Illustration of renal subcapsule pouch making and islet transplantation.
Figure 2. Illustration of renal subcapsule pouch making and islet transplantation. The corresponding step-wise flow chat is described as follows: punch a hole on the top pole of the kidney → insert the glass rod and make a subcapsular pouch → insert the islet-containing tubing with the aid of the glass rod → move the tubing back and forth as indicated by arrows → release islet into the bottom of the pouch with the aid of the microsyringe.
Figure 3: Blood glucose monitoring in diabetic mice.
Figure 3. Blood glucose monitoring in diabetic mice. STZ-induced diabetic mice were maintained in the institutional animal center with free access to water and chow. Blood glucose concentration was continuously assessed and presented on control animals (n = 4, gray lines) and islet-transplanted animals (n = 7, black lines) respectively. Female C57BL/6J mice were used as recipients and male C57BL/6J as donors. 400 islets were subcapsularly transplanted. Tx: islet transplantation; Nc: nephrectomy.
Figure 4: Blood glucose monitoring in diabetic mice with two times of islet transplantation.
Figure 4. Blood glucose monitoring in diabetic mice with two times of islet transplantation. STZ-induced diabetic C57BL/6J mice (n = 7) were transplanted on the left kidney with 400 syngeneic islets at day 3. The left kidney was removed on day 65 and the second islet transplantation on the right kidney at day 70. Tx: islet transplantation; Nc: nephrectomy.
Figure 5: Blood glucose monitoring in diabetic mice with different type of islet transplantation.
Figure 5. Blood glucose monitoring in diabetic mice with different type of islet transplantation. The left panels show the distributions of individual blood glucose detections. The right panels represent the trends of blood glucose changes with mean ± S.E.. (a) Syngeneic islet transplantation (n = 11). 400 syngeneic islets were transplanted into C57Bl/6J mice. The time course is chosen to make it consistent with other groups. However, the actual observation time was up to 120 days in this group. (b) Allogeneic islet transplantation (n = 28). C57Bl/6J mice were transplanted with 400 islets isolated from Balb/C mice. (c) Xenogeneic islet transplantation (n = 15). C57Bl/6J mice were transplanted with 3000 I.E. human islets provided from Ike Barber Human Islet Transplant Laboratory at University of British Columbia. Tx: islet transplantation.
Dong-sheng Li, Jianqiang Hao, Ya-Hong Yuan, Se Hak Yun, Jing-Bo Feng & Long-Jun Dai, Unaffiliated
Garth L. Warnock, Warnock & Li's Labs, University of British Columbia, Hubei University of Medicine
Correspondence to: Garth L. Warnock ([email protected])
Source: Protocol Exchange (2011) doi:10.1038/protex.2011.221. Originally published online 17 March 2011.