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, 183 (3), 692-708

Differences in the Degree of Cerulein-Induced Chronic Pancreatitis in C57BL/6 Mouse Substrains Lead to New Insights in Identification of Potential Risk Factors in the Development of Chronic Pancreatitis

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Differences in the Degree of Cerulein-Induced Chronic Pancreatitis in C57BL/6 Mouse Substrains Lead to New Insights in Identification of Potential Risk Factors in the Development of Chronic Pancreatitis

Barbara Ulmasov et al. Am J Pathol.

Abstract

A frequently used experimental model of chronic pancreatitis (CP) recapitulating human disease is repeated injection of cerulein into mice. C57BL/6 is the most commonly used inbred mouse strain for biomedical research, but widespread demand has led to generation of several substrains with subtly different phenotypes. In this study, two common substrains, C57BL/6J and C57BL/6NHsd, exhibited different degrees of CP, with C57BL/6J being more susceptible to repetitive cerulein-induced CP as assessed by pancreatic atrophy, pancreatic morphological changes, and fibrosis. We hypothesized that the deficiency of nicotinamide nucleotide transhydrogenase (NNT) protein in C57BL/6J is responsible for the more severe C57BL/6J phenotype but the parameters of CP in NNT-expressing transgenic mice generated on a C57BL6/J background do not differ with those of wild-type C57BL/6J. The highly similar genetic backgrounds but different CP phenotypes of these two substrains presents a unique opportunity to discover genes important in pathogenesis of CP. We therefore performed whole mouse genome Affymetrix microarray analysis of pancreatic gene expression of C57BL/6J and C57BL/6NHsd before and after induction of CP. Genes with differentially regulated expression between the two substrains that might be candidates in CP progression included Mmp7, Pcolce2, Itih4, Wdfy1, and Vtn. We also identified several genes associated with development of CP in both substrains, including RIKEN cDNA 1810009J06 gene (trypsinogen 5), Ccl8, and Ccl6.

Figures

Figure 1
Figure 1
Severity of cerulein-induced CP in B6N and B6J substrains. Mice were subjected to three episodes of cerulein treatment over 5 days to induce CP and were sacrificed at 3 days after the last treatment. A: Loss of pancreatic weight relative to total body weight indicates significant pancreatic atrophy. B6J mice have significantly more pancreatic atrophy, compared with B6N mice. B: Histological changes in H&E-stained pancreas of B6N and B6J mice after induction of CP. Pancreas from B6N and B6J mice after control saline treatment shows no abnormalities. After cerulein treatment, pancreas from both B6J and B6N mice shows disrupted acinar architecture, dedifferentiation to tubular complexes, and interstitial inflammation; all of these parameters were more severe in B6J than B6N mice (Table 2). Data are expressed as means ± SEM. n = 6 mice per group. ∗∗∗P < 0.001. Original magnification, ×200.
Figure 2
Figure 2
Collagen content in the pancreas assessed by Sirius Red staining after induction of CP by repetitive cerulein injections. A: Representative pancreatic sections of pancreas from saline-treated control B6N and B6J mice and from cerulein-treated B6N and B6J mice. Control mice exhibit some perilobular collagen, with no difference between substrains. Cerulein-treated mice demonstrate extensive interlobular and periacinar collagen staining, indicating robust fibrogenesis in this model of CP. Collagen staining was more pronounced in B6J than in B6N mice. B: Quantification by morphometric analysis showed that the relative amount of pancreatic collagen was greater in B6J than in B6N mice. Data are expressed as means ± SEM. n = 6. P = 0.0005. Original magnification, ×200.
Figure 3
Figure 3
Activation of PSCs after induction of CP. A: Western blotting of pancreatic extracts demonstrated increased expression of α-SMA, a marker of PCS activation, in both B6N and B6J mice. ERK1/2 was used as a loading control. B: Densitometric analysis of α-SMA protein expression after induction of CP. An increase in α-SMA expression was observed for B6J mice, compared with B6N mice, but the difference did not reach statistical significance (P = 0.9). Data are expressed as means ± SEM. n = 5 mice per group.
Figure 4
Figure 4
Parameters of acute pancreatic injury in B6J and B6N substrains. Mice were subjected to a single episode of acute pancreatitis (six hourly injections in one day) and were sacrificed at 9 hours after the first cerulein injection. Saline-injected mice were used as controls. A: Pancreatic edema, measured as the ratio of pancreatic weight to body weight, showed similar increases in B6N and B6J mice. B: Plasma amylase levels were also elevated similarly in B6N and B6J mice. C: Histological changes in the pancreas were evaluated by H&E staining. Pancreas from saline-treated mice appeared normal, whereas the pancreas from cerulein-treated mice showed acute injury including vacuolization, inflammatory cell infiltration, and necrosis. Data are expressed as means ± SEM. n = 6 mice per group. ∗∗P < 0.01. Original magnification, ×200.
Figure 5
Figure 5
B6J mice are nicotinamide nucleotide transhydrogenase (NNT) deficient. A: PCR analysis of genomic DNA from B6J and B6N mice confirmed that B6J mice lack the Nnt gene. B: Western blotting of pancreatic extracts demonstrated the presence of NNT protein in B6N and its absence in B6J mice. β-Actin was used as in internal control.
Figure 6
Figure 6
Western blot of pancreatic mitochondrial nicotinamide nucleotide transhydrogenase (NNT) protein, confirming absence of expression in B6J mice and similar expression in B6J Nnt+/+ transgenic mice and B6J mice.
Figure 7
Figure 7
Pancreatic Itih4 expression in B6J and B6N substrains. A: Itih4 mRNA expression in the pancreas after induction of CP was much lower in B6J mice than in B6N mice under control conditions of saline injections. After induction of CP, pancreatic Itih4 expression was markedly down-regulated in both substrains, but its expression was higher in B6N than in B6J. B: Itih4 mRNA expression in the pancreas 9 hours after the acute injury induced by one cerulein treatment. Acute injury did not decrease Itih4 mRNA expression. Itih4 mRNA expression was normalized to Rplp0 and expressed as fold increase over the level in B6N saline-treated control mice. Data are expressed as means with 95% confidence intervals. Confidence intervals were calculated using the ΔCT values before exponential transformation to fold increase in mRNA and are therefore asymmetric about the means. n = 6. P < 0.05.
Figure 8
Figure 8
Pancreatic T5 mRNA expression in B6J and B6N substrains. A: Pancreatic T5 expression in saline-injected control mice and in repetitive cerulein-injected mice after induction of CP. T5 expression was significantly induced after induction of CP in both B6N and B6J substrains. B: Pancreatic T5 expression in mice after acute cerulein-induced injury. Similar to the increase in CP, T5 mRNA expression was significantly induced in both substrains after acute injury. T5 mRNA expression was normalized to Rplp0 and expressed as fold increase over the level in B6N saline-treated control mice. Data are expressed as means with 95% confidence intervals. Confidence intervals were calculated using the ΔCT values before exponential transformation to fold increase in mRNA and are therefore asymmetric about the means. n = 6. P < 0.05.
Figure 9
Figure 9
Pancreatic Ccl8 expression in B6J and B6N substrains. A: Ccl8 expression in the pancreas after induction of CP. Ccl8 was strongly up-regulated in the pancreas of mice with CP, compared with control mice. The up-regulation in B6J mice was not significantly greater than in B6N mice (P = 0.02). B: Ccl8 expression in the pancreas after acute cerulein-induced injury. Acute injury induced Ccl8 mRNA increase approximately 2.5-fold in B6J mice, but not in B6N mice. Ccl8 mRNA expression was normalized to Rplp0 and expressed as fold increase over the level in B6N saline-treated control mice. Data are expressed as means with 95% confidence intervals. Confidence intervals were calculated using the ΔCT values before exponential transformation to fold increase in mRNA and are therefore asymmetric about the means. n = 6. P < 0.05.
Figure 10
Figure 10
Wdfy1 mRNA expression in PSCs isolated from B6N and B6J substrains. Wdfy1 mRNA expression was normalized to Rplp0 and expressed as fold change over the level in PSCs isolated from B6N mice. Data are expressed as means (n = 3) with 95% confidence intervals. Confidence intervals were calculated using the ΔCT values before exponential transformation to fold increase in mRNA and are therefore asymmetric about the means. P < 0.05.

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