Pancreatic injury induces β-cell regeneration in axolotl

Abstract

Background: Diabetes is a condition characterized by a loss of pancreatic β-cell function, which results in the dysregulation of insulin homeostasis. Using a partial pancreatectomy model in axolotl, we aimed to observe the pancreatic response to injury.

Results: Here we show a comprehensive histological characterization of pancreatic islets in axolotl. Following pancreatic injury, no apparent blastema-like structure was observed. We found a significant, organ-wide increase in cellular proliferation post-resection in the pancreas compared to sham-operated controls. This proliferative response was most robust at the site of injury. Further, an increase in nuclear density was observed, suggesting compensatory congestion as a mechanism of regeneration. We found that β-cells actively contributed to the increased rates of proliferation upon injury. β-Cell proliferation manifested in increased β-cell mass in injured tissue at 2 weeks post-injury. At 4 weeks post-injury, we found organ-wide proliferation to be extinguished while proliferation at the injury site persisted, corresponding to pancreatic tissue recovery. Similarly, total β-cell mass was comparable to sham after 4 weeks.

Conclusions: Our findings suggest a non-blastema-mediated regeneration process takes place in the pancreas, by which pancreatic resection induces whole-organ β-cell proliferation without the formation of a blastemal structure. This process is analogous to other models of compensatory congestion in axolotl.

Keywords: compensatory congestion; diabetes; insulin; pancreatectomy; proliferation; resection.

Figure 1.. Transcript level pancreatic islet morphology in axolotl

A. HCR-FISH of Gcg, Ins, and Sst transcripts within pancreatic islets. B. HCR-FISH of Ins, Pdx1, and Nkx6–1 transcripts. Scale bars are 50 μm.

Figure 2.. Pancreatic resection surgery model.

A. Cartoon depicting sham and pancreatic resection surgery. B. Morphology of sham and pancreatic resection surgery at 0, 14 and 28 dpi. The pancreas is outlined in blue while black arrows point to the duodenum. 0 dpi image of the resection condition was taken immediately following 20% removal of pancreas by length. The pancreas extends into the abdominal cavity where it connects to the liver (not visible in Figure B). In each image, the orientation from left to right follows a rostral to caudal direction. Black scale bars in the bottom right corner of each image are 2 mm.

Figure 3.. Pancreatic resection initially provokes substantial proliferative responses throughout the entire organ, but cell proliferation becomes restricted local to the site of injury later in the process.

A. EdU and insulin (IHC) stain of coronal sections of pancreas samples at 14 dpi (sham n=5, resection n=6). B. Organ-wide proliferation is increased in resection samples at 14 dpi (p=0.0162). C. Proliferation local to the site of injury is increased at 14 dpi (p=0.0012). D. Proliferation within resection samples is higher local to the site of injury at 14 dpi (p=0.0032).E. Nuclear density is increased locally in resection samples. F. EdU and insulin stain of coronal sections of pancreas samples at 28 dpi (sham n=5, resection n=5). G. Organ-wide proliferation in resection samples is non-significant in comparison to sham at 28 dpi (p=0.8459). H. Proliferation local to the site of injury is increased at 28 dpi (p=0.0079).I. At 28 dpi, proliferation local to the site of injury is higher than the organ-wide proliferation within resection samples (p=0.0085). J. Nuclear density is not significantly different between sham and resection at 28 dpi. K. Organ-wide proliferation is decreased between resection samples from 14 to 28 dpi (p=0.0009). L. Local proliferation is decreased between resection samples from 14 to 28 dpi (p=0.0005). M. Cartoon representation of pancreatic resection, color coded based on condition described in statistical analysis. Scale bars are 100 μm.

Figure 4.. β–cell proliferation is observed at 14 days post injury but subsides by 28 days.

A. Representative image of a coronal section of regenerating pancreatic tissue at 14 dpi. White arrows point to proliferating β–cells. B. High resolution image of a proliferating β-cell (marked by white arrow), 90x magnification. C. A significant increase in β–cell proliferation was observed at 14 dpi (p = 0.0463) (sham n=5, resection n=6). D. A significant increase in β–cell population was observed at 14 dpi (p = 0.005) (sham n=5, resection n=6). E. No significant difference in β–cell proliferation was observed at 28 dpi (p = 0.0936) (sham n=5, resection n=5). F. No significant difference in β–cell population was observed at 28 dpi (p = 0.3181) (sham n=5, resection n=5). Scale bar is 200 μm in panel A, 20 μm in panel B.

Figure 5.. Nanopore sequencing data reveals transcriptomic changes in response to injury.

A. Bar plot detailing reads per sample after demultiplexing. Unclassified reads and reads with a qscore less than 8 were filtered out. B. Histogram detailing aligned read identity vs total read counts. C. Heatmap of a subset of differentially expressed genes at 14 dpi D. Volcano plot of differentially expressed genes at 14 dpi.

Figure 6:. Genes previously implicated in regeneration exhibit upregulation in axolotl pancreatic injury model.

A. HCR-FISH showing differential expression of Ins and Marco transcripts. Ctrb2 was not found to be differentially expressed; however, it provides structural context for exocrine tissue. Marco+ cells shown with arrows. B. HCR-FISH showing differential expression of Ins and Cirbp transcripts. Scale bars are 50 μm.