Background & Aims
Acute pancreatitis (AP) is the most common diagnosis of patients admitted for gastroenterological diseases, with pain being the primary reason they present to the emergency department.1–4 As of 2024, treatments remain inconsistent, and there is still no known cure.5 Reports of sex differences vary in their conclusions and are still being investigated. There is limited literature on pain in AP in humans, despite abdominal pain being the most common symptom.6–8 While many animal models of AP claim to mimic the human condition, few have been evaluated for presentation of pain and/or altered nociception.8 The models that have been used to investigate abdominal pain typically use only male mice.9 The goal of this study was to investigate the presence of sex differences in the most commonly used mouse model of AP, repeated cerulein injection. Cerulein, a cholecystokinin-pancreozymin analogue, is thought to induce AP by directly and indirectly (vagal reflex) driving enzymatic hypersecretion.
Methods
12-week-old C57BL/6J mice were given 8 hourly injections of cerulein or saline (50mg/kg, ip) on two consecutive days. Animals were sacrificed either 1 or 12 hours after the final injection. Abdominal von Frey was performed using the percent response method out of 10 trials. Rearing behavior was performed in a Plexiglas tube for 5 minutes and total time rearing and number of bouts of rearing were measured using Boris software. Limbo is our novel abdominal pain assay where mice in a clear chamber are posed the opportunity to escape to a dark chamber if they compress themselves under a glass bar. Arterial blood was collected, and serum separated to measure circulating digestive enzymes. Pancreata were wet weighed and percent of total body weight was calculated. Pancreata were then either snap frozen to measure myeloperoxidase activity or fixed in 4% PFA for H&E analysis. T8-T13 dorsal root ganglia (DRG) were collected and snap-frozen for qPCR to assess for ATF3, a marker of nerve injury.
Results
Sex differences in the cerulein model of AP are apparent. At 1 hour after the final injection, male mice exhibit an increase in serum amylase but not lipase activity whereas females have the opposite pattern of activity. At 12 hours after the final injection, both male and female mice have elevated lipase levels, pancreas weight, and myeloperoxidase activity, but preliminary studies suggest only females have obvious edema upon histology. Behaviorally, both male and female mice show increased latency to limbo as well as mechanical allodynia. Consistent with chronic abdominal diseases, male mice showed a significant reduction in rearing behavior, however, female mice showed an increase in rearing. Interestingly, male mice alone exhibit an upregulation of the neuronal injury marker Atf3 mRNA in thoracic DRG.
Conclusions
The most striking difference is that circulating digestive enzymes are elevated at 1hr in a sex-specific manner. While female mice have increased inflammation and edema in the pancreas, they do not appear to have nerve injury at the 12-hour mark. Both male and female mice exhibit pain behaviors throughout the time course, so it is possible that the sex difference in Atf3 mRNA suggests differential underlying mechanisms and may relate to differences in pain behaviors. We believe this to be indicative of an underlying mechanistic difference in how the disease progresses in both sexes. Future studies will further investigate the preliminary observations as well as examine longer-time courses to evaluate the resolution of pain in males and females, an area that has not been studied in animal models of AP.
References
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