Improved Yield and Functional Parameters of Rat Pancreas Islets Isolated under Intramuscular Anesthesia

Abstract

Intraperitoneal (IP) anesthesia is commonly used for laboratory animal experiments including rat islet isolation. However, the direct effects of anesthetics on pancreatic islets have been neglected. This study compared the islet function and recovery yield from rats that were anesthetized using IP and intramuscular (IM) injection. In addition, the lag time required to lose deep pain was measured in the following anesthetics combinations. Lewis rats were anesthetized using ketamine and xylazine (K/X) or zoletil and xylazine (Z/X). A glucose challenge test was performed on each group of prepared islets. The effect of the anesthetic agents (e.g., ketamine, zoletil, xylazine alone, and the combination of K/X and Z/X) on cell lines (rat insulinoma; RIN-5F) was investigated by determining their effect on the cell viability, the amount of insulin, and insulin mRNA expression levels of RIN-5F. The time needed for deep anesthesia in IM anesthesia was significantly shortened in comparison to IP [K/X (IM: 313 ± 66 s, IP: 371 ± 84 s) and Z/X (IM: 206 ± 76 s, IP: 245 ± 92 s)]. In addition, number of isolated islet yield by IM anesthesia was significantly improved [K/X (IM: 1530 ± 242, IP: 1245 ± 149) and Z/X (IM: 1136 ± 226, IP: 511 ± 154)]. The functions of fresh islets, indicated by the stimulation index, acquired under IM anesthesia was better preserved than that of IP. The viability and the insulin secretion of RIN-5F were decreased at 24 and 48 h. Insulin gene expression levels were decreased at 24 h as well. Anesthetics may be absorbed through the pancreas surface to the islets and have a direct effect, resulting in islet exposure and deterioration during isolation. In conclusion, for rodent islet isolation, IM anesthesia is simpler and safer in comparison to IP anesthesia.

Adenine Nucleotide Levels in a Closed Enzymatic Digestion System for Porcine Islet Isolation

“…Obtaining a higher mean islet mass from a pancreatic graft is crucial for a successful outcome in the islet isolation procedure (7,25). Although several methods for obtaining a higher islet mass have been reported (12), the optimal timing at which to terminate the collagenase digestion and start dilution usually depends on the appearance and number of islets in the serially digested samples (4,13,25). Further objective parameters are therefore needed to determine the digestion termination point and promote successful outcomes (4).…”

“…However, it is still difficult to recover the entire quantity of islets contained in a pancreas (16). Given that islet isolation is crucial for the success of this transplantation procedure, many attempts to obtain more viable islets have been reported in the literature (1,12,18). In rodent models, collagenase digestion can be performed for a specific period of time to obtain a peak islet yield; however, in humans, the timing of dilution is empirically determined by human expertise and experience (1,16–18).…”

Pancreatic Islets

“…Rat islets appear to provide greater challenge, as there appears to be greater variance in yield between strains, ages and condition of donor rats 33,34 . Overall, the approach of intra-ductal inflation and sheer-force disruption of the digested tissue are consistent with mouse protocols.…”

Taking a Lesson from the past in Organ Biology

“…Diabetes continued to be a major focus with three papers evaluating different aspects of islet transplantation. Ikemoto et al (4) looked at islet quality following international shipping, while Kim et al (8) reported on the improved yield and functional parameters of rat pancreas islets under intramuscular anesthesia. Noguchi et al (18) compared modified Celsior solution and M-Kyoto solution for human pancreas preservation.…”