Islet transplantation has become an attractive option for the treatment of insulin-dependent diabetes mellitus. For the improvement of islet transplantation, mouse models have been widely used. The classical procedure of islet isolation is based on collagenase digestion of pancreas and separation of islets (1–4), with the common bile duct (CBD) as the puncture site to inject the collagenase solution. Here, a novel puncture route of in situ pancreas perfusion is described, in which we cannulate into the splenic vein (SV). This protocol is feasible to be mastered and especially suitable for the conditions in which CBD method does not work.
In SV protocol, the pancreas was swelled via injection of collagenase solution (0.5 mg/mL in Hank balanced salt solution) into the SV. As illustrated in Figure 1A, phrenicosplenic and gastrosplenic ligament are severed to free the spleen. Splenic pancreas is separated from descending colon and rectum by severing any ligament between them. Then the spleen and splenic pancreas are flipped over to the right to expose the spleen vein on the dorsal surface of the splenic pancreas. The distal end of the splenic vein close to the spleen is clamped with a hemostatic forceps. The diaphragm is incised and the heart is transected to terminate the pancreatic blood circulation. Under a dissecting microscope, SV is punctured with a 24G needle to a point just at the junction where it joins with the superior mesenteric vein. Then 1 to 2 mL of collagenase was injected into the splenic vein until the splenic pancreas is fully inflated. The inflated splenic pancreas is dissected free of the remaining gastric and duodenal pancreas lobes and any other attachments, and placed in the 15-mL tube containing 1 mL of collagenase solution. Then the inflated pancreas was digested and the islets were purified just like in the CBD method as previously described (2). Finally, islets were harvested by hand under microscopy.
Compared to common bile duct, the SV is longer, thicker, and stronger. In addition, the anatomical structure of splenic vein is stable. In young mice (such as 2-week-old mice), the increased difficulty of puncture and perfusion often leads to failure by CBD protocol. In obese mice (such as high-fat-diet-induced or ob/ob mouse model) or pregnant mice, the distal part of pancreas is exclusively not easy to be inflated by CBD protocol. By SV protocol, we isolated islets from 6-month-old ob/ob mice and 2-week-old mice with a higher success rate (Fig. 1D, E). Moreover, the SV protocol is more effective when the bile duct is blocked or damaged in some mouse models. The pancreatic duct ligation (PDL) mouse model is widely used to investigate pancreatitis and islet regeneration. We successfully isolated islets from the PDL mouse model (Fig. 1F) by the SV protocol. The quantity of islets and success rate is not limited by duct blockade in these mice. The isolated islets by the SV protocol can be cultured and used for further experiments and analysis (Fig. 1G). Different dyes are used to determine the viability of islets. Acridine orange (AO) incorporates into healthy cells and fluoresces green. Propidium iodide (PI) is a membrane-penetrated red fluorescent dye, which enters only dead or dying cells. Figure 1H and I demonstrated that SV protocol did not increase cell death significantly. For further examination of β-cell function in vivo, we performed subcapsular kidney islet transplantation in streptozotocin-induced diabetes mouse models. The transplantation of 400 islets isolated by the SV protocol could improve hyperglycemia effectively.
When we isolated islets from normal mice, the CBD protocol is the first choice for us. However, if the puncture of CBD failed, the SV protocol could be performed to avoid unnecessary sacrifice of mice. In some mice (∼10%), the splenic ducts joins gastric duct and opens into the duodenum with an accessory papilla, so the collagenase solution cannot inflate the splenic and gastric pancreatic lobes through the CBD method. Moreover, the common bile duct is not suitable for puncture in several models, such as obesity and PDL model. Our data showed that the SV protocol is practicable and reproducible for islet isolation in these mouse models. Gotoh et al. (5) reported rat islets could be isolated with portal vein (PV) as the collagenase injection route. However, the procedure of PV method is complicated and is not very feasible for mouse model.
In conclusion, the SV is a complementary site for collagenase perfusion in the process of islet isolation. The SV protocol extends the feasibility of collagenase perfusion in multiple mouse models and reduces the number of sacrificed mice.
1 Shanghai Clinical Center for
Endocrine and Metabolic Diseases
Department of Endocrinology and
Metabolism, Rui-Jin Hospital
Affiliated to Shanghai Jiao-Tong
University School of Medicine
2 Laboratory of Endocrinology and
Metabolism, Institute of Health Sciences
Shanghai Institutes for Biological
Sciences (SIBS), Chinese Academy of
Sciences (CAS) & Shanghai Jiao Tong
University School of Medicine (SJTUSM)
The authors thank Yang Ping for technical assistance.
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