Beta cell count instead of beta cell mass to assess and localize growth in beta cell population following pancreatic duct ligation in mice

Abstract

Background: Pancreatic-tail duct ligation (PDL) in adult rodents has been reported to induce beta cell generation and increase beta cell mass but increases in beta cell number have not been demonstrated. This study examines whether PDL increases beta cell number and whether this is caused by neogenesis of small clusters and/or their growth to larger aggregates.

Methodology: Total beta cell number and its distribution over small (<50 µm), medium, large (>100 µm) clusters was determined in pancreatic tails of 10-week-old mice, 2 weeks after PDL or sham.

Principal findings: PDL increased total beta cell mass but not total beta cell number. It induced neogenesis of small beta cell clusters (2.2-fold higher number) which contained a higher percent proliferating beta cells (1.9% Ki67+cells) than sham tails (<0.2%); their higher beta cell number represented <5% of total beta cell number and was associated with a similar increase in alpha cell number. It is unknown whether the regenerative process is causally related to the inflammatory infiltration in PDL-tails. Human pancreases with inflammatory infiltration also exhibited activation of proliferation in small beta cell clusters.

Conclusions/significance: The PDL model illustrates the advantage of direct beta cell counts over beta cell mass measurements when assessing and localizing beta cell regeneration in the pancreas. It demonstrates the ability of the adult mouse pancreas for neogenesis of small beta cell clusters with activated beta cell proliferation. Further studies should investigate conditions under which neoformed small beta cell clusters grow to larger aggregates and hence to higher total beta cell numbers.

Figure 1. Comparison of tissue composition in unligated (left) and ligated (right) pancreatic tails.

Two weeks after duct ligation the tissue is devoid of acini. It mainly consists of ducts with interspersed islets (arrows) and enlarged fibrous septa and adipose tissue (stars). It exhibits an inflammatory infiltration with numerous CD45-positive leucocytes in the septa (red arrowheads); these cells were virtually absent in unligated tissue. The degree of infiltration was less pronounced than after one week. Hematoxylin-eosin, scale bar 200 µm (upper panel) and immunohistochemistry for insulin (red) and CD45 (brown), scale bar 100 µm (lower panel).

Figure 2. Proliferation-activated beta cells in small beta cell clusters of PDL-mouse tails (left panel) and of human pancreas with inflammatory infiltration (right panel).

Two weeks after PDL pancreatic tails contain a larger pool of Ki67-positive beta cells in the small beta cell clusters (arrows in left panel) (391±84 cells versus 27±27 in unligated tails, p<0,01). They also present higher percentages of Ki67-positive cells in large and small ducts (arrowheads). A number of human donor organs were found to present a diffuse inflammatory infiltration . The small beta cell clusters in three consecutively analysed organs contained higher percentages of Ki67-positive beta cells (arrows in right panel) than those in age- and gender-matched controls without infiltration (3, 0.8 and 1.4% versus ≤0.1%). Immunohistochemistry for insulin (red) and Ki67 (brown), scale bar 100 µm.