Novel Animal Model of Chronic Pancreatitis

Pancreatitis is the inflammation of the pancreas, which is characterized by auto-digestion of the pancreas catalyzed by the enzyme pancreas protease. The types of pancreatitis include acute, recurrent acute, and chronic pancreatitis. Symptoms of pancreatitis include nausea, fever, vomiting, abdominal pain, and diarrhea.

Previous studies demonstrated that the development of acute pancreatitis and subsequent advancement to chronic pancreatitis is associated with mutations in genes that encode for proteases or protease inhibitors. Pathogenic mutations in SPINK1 (Kazal-type serine protease inhibitor 1), PRSS1 (cationic trypsinogen), and CTRC (pancreatic rennin C) promote the transformation of trypsinogen into more aggressive pancreatic proteases. The transformation occurs either by spontaneous activation of pancreatic proteases or due to impaired protective mechanisms. 

Spontaneous transformation of trypsinogen into pancreatic protease in the pancreas is known to have a direct pathogenic effect. This statement is supported by genetic and biochemical pieces of evidence. However, the lack of animal models concerning spontaneous chronic pancreatitis or mutation-driven self-activation of trypsinogen has limited preclinical trials of therapeutic formulations that target intrapancreatic trypsin.

In order to fill the gap, Andrea Geisz and Miklos Sahin-Toth at Boston University developed a T7D23A knock-in mouse model. The results demonstrated that the mouse serves as an excellent model for both study and intervention owing to a heterozygous mutation (p.D23A) in the cationic trypsinogen gene (T7 isoform). Important findings of this study were published in Nature Communications.

The Development of T7D23A Knock-in Mice

Twenty trypsinogen genes are found in the mouse genome. Out of the twenty genes, T7, T8, T9, and T209 isoforms are expressed at relatively higher levels. The T7 isoform attributes to 40-50% of all the trypsinogen encoded by the mouse genome. The gene is rapidly and spontaneously activated to produce greater levels of trypsinogen. 

To develop T7 mutation and spontaneous T7 activation in the mouse model, the researchers induced mutation (p.D23A) in the activating peptide. In order to evaluate the activation properties of this mutant gene and protein, the researchers purified the mutant and recombinant wild-type T7 trypsinogen, followed by an assessment of spontaneous activation. 

The researchers observed that T7 p.D23A mutation led to a 50-fold rise in the spontaneous activation of trypsinogen as opposed to the wild-type T7 trypsinogen. They also evaluated the effect of histones B and L or lysosomal cysteine proteases on the p.D23A mutant. The researchers observed no alterations in the histone-mediated breakage of this mutant in contrast to the wild-type T7. The p.D23A mutant gene alters the self-activation of trypsinogen. However, the function of histones and lysosomal cysteine proteases remains unaffected.  

Assessment of mouse genome mutation was followed by the preparation of T7D23A knock-in mice by Cyagen: the development of this model involves induction of the p.D23A mutation of the mouse genome via homologous recombination of the C57BL/6 embryonic stem cells. The novel strain generated, T7D23A, reproduced normally and had no obvious phenotypic alterations. DNA sequencing revealed two equivalent peaks at the site of p.D23A mutation. Thus, the expression of mutant and wild-type allele levels was comparable.

T7D23A Mice Develop Spontaneous Acute and Chronic Pancreatitis 

During the same study, the pancreatic morphology of mutant mouse models remained normal 2-3 weeks after birth. However, at 4-5 weeks, the pancreatic morphology became more pathologically variable. The size of the pancreas reduced significantly after 2 months. Histological examination of the pancreas revealed that the onset of acute pancreatitis initiates from week 3, with the frequency rising at weeks 4-5 (~ 40%). Edema disrupts the pancreatic tissue structure along with infiltration of inflammatory cells and central localized necrosis of lobules. (Figure 2.). 

Significant progression of pancreatitis was observed during 4-5 weeks in the majority of the gene modified mice population. Pancreatic sections of these mice demonstrate regeneration as well as signs of early chronic pancreatitis. These signs include ductal complexes, diffuse interstitial fibrosis, and dilation and distortion of pancreatic ducts. Fat begins to accumulate, whereas ductal complexes and inflammatory cells tend to decline during the period of 2 months to 6-12 months. Diffuse interstitial fibrosis is usually found around the distorted and dilated pancreatic ducts. Fibrosis may be filled with eosinophil material that marks advanced chronic pancreatitis.

Pancreas Weight in T7D23A Mice 

As compared to C57BL/6N control mice, the researchers found a significant increase in the pancreatic weight at 4-5 weeks in 40% of T7D32A mice. This finding is consistent with the observation of acute edematous pancreatitis in pancreatic tissue sections. 

On the contrary, 50% of T7D32A mice population demonstrated a decline in the pancreatic weight, indicating early chronic pancreatitis at 4-5 weeks. The entire T7D32A mice population demonstrated pancreatic atrophy and associated advanced chronic pancreatitis during 2-12 months of age. This is accompanied by a 70% decline in pancreatic weight in T7D23A mice compared with the control mice.  

Pancreatic Amylase Activity Assay in T7D23A Mice 

Amylase is the most common diagnostic marker for acute pancreatitis. The researchers evaluated the activity of plasma amylase in all T7D23A mice. High amylase values were observed in 40% of mice during 4-5 weeks, indicating acute pancreatitis. However, the researchers were unable to determine acute pancreatitis indicators in all the mice. Underlying reasons include the age of onset, time of sampling, and absence of acute pancreatitis in certain mice. 

Study of Intrapancreatic Trypsin Activity in T7D23A Mice 

The researchers measured the activity of trypsin in mice of different ages. At 3 weeks, a rise in trypsin activity was not observed when compared to the control group. However, higher enzyme activity was seen in 4 weeks-old mice. Increased enzyme activity seen at 2 months signify that the activation of intrapancreatic trypsin persists throughout the course of pancreatitis. 

T7D23A, K24G Mice Did Not Develop Pancreatic Disease 

A double-mutant mouse strain, p.D23A, K24G, has a heterozygous T7D23A allele and mutated Lys24 trypsinogen activated site. 5.5 months old mice with double-mutant did not demonstrate spontaneous pathologic changes. This proves that trypsinogen misfolding does not support the characteristic T7D32A phenotype. 

Characterization of T7D23A Mice Compared with Human Pancreatitis 

The T7D32A mouse model demonstrates the clinical features of pancreatitis in humans, initiating as acute pancreatitis and progressing into irreversible chronic pancreatitis. The researchers, however, failed to observe the onset of recurrent acute pancreatitis in the mouse model. The probable cause is the mutation of the trypsinogen used in this study. This is the first preclinical animal model of chronic pancreatitis driven by a rise in spontaneous activation of pancreatic trypsinogen. The researchers conclude that pancreatic trypsin can serve as the potential therapeutic target for chronic pancreatitis.