Exploring the impact of toxic gut-lymph on mitochondrial function in multiple organ dysfunction syndrome using a model of acute pancreatitis

Abstract

Critical acute disease is caused by various pathological conditions and has two common features: intestinal ischemic injury and a consistent pattern of organ failure resulting in Multiple Organ Dysfunction Syndrome (MODS). MODS is the leading cause of death in acute critical illness and may be linked to mitochondrial dysfunction. Mitochondria produce cellular energy and their dysfunction often leads to cell injury or death. Intestinal ischemic injury suffered during shock promotes the release of various toxic factors such as cytokines, damage associated molecular patterns and reactive oxygen species into the gut lymph. This could result in mitochondrial dysfunction in key organs as gut-lymph enters the bloodstream at the subclavian vein proximate to the heart and lungs, effectively bypassing liver detoxification. The progressive failure of organs in MODS follows the circulation of gutlymph, therefore, lymph toxicity induced by shock could be a causal feature of MODS. The impact of toxic gut-lymph on mitochondrial function was explored in acute pancreatitis using multiple surgical models performed on male Sprague Dawley rats. Acute pancreatitis (AP) was induced using a 5% taurocholic acid infusion at the pancreatic duct. To evaluate the impact of toxic gut-lymph one model of acute pancreatitis included lymph drainage at the mesenteric duct before entry to circulation. A lymph drainage and non-drainage sham model were used respectively as controls. During surgery exhaled carbon dioxide, breaths per minute, heart-rate, body temperature, mean arterial blood pressure and systolic blood pressure were monitored. Serum was collected at the end of surgery via cardiac puncture and a comprehensive biochemical profile was obtained to determine the impact of surgery upon the general health of the animals. A detailed respiratory analysis of the toxic effects of gut-lymph on mitochondria and cellular function was conducted ex vivo on the permeabilised heart and lung tissues from rats assigned to the different surgical groups. This involved the use of high-resolution oxygraphs coupled with O2k-fluorometry to measure mitochondrial function and ROS production. Commercial rodent L2 lung cells were cultured and incubated on 96 well plates with gut-lymph gathered from various rodent models of acute diseases to determine the cytotoxic impact of lymph in vitro. Toxic impact was evaluated through high content screening via the use of various fluorescent dyes pertinent to mitochondrial function. These dyes included NucBlueTM, MitotrackerTM Deep Red, MitoSOXTM Red, CellROXTM Deep Red, CellROXTM Green and JC- 10. Differences observed in the cytotoxicity of lymph from AP and sham models both in vivo and in vitro were limited; this suggests that the surgery alone was considerably stressful for the animals and may have confounded results. This could be addressed by using a model of AP that does not require major surgery, such as a caerulein method which only requires a subcutaneous injection. Significant dysfunction of Complex I (CI) was detected in the permeabilised lung tissue of rats induced with AP. Furthermore, the drainage of lymph in AP rats improved CI output on average by 82%, indicating that there is a pathophysiological connection between gut-lymph and mitochondrial dysfunction in distant organs. Lymph drainage also appeared to improve Complex IV function in permeabilised cardiac fibres by ~45% for sham models and ~48% AP treatments. This indicates that both the trauma of surgery and acute illness increases lymph toxicity to cardiac mitochondria, most likely through the iNOS immune response. Additional support for improved respiratory function in drainage models was provided by an increase in exhaled carbon dioxide in the absence of hypoventilation. Furthermore, heart rate was significantly elevated in sham and AP models suggesting that both surgery and AP induced a stress response, which appeared to be mitigated by lymph drainage. The reduction in hyperglycemia with lymph drainage provides further evidence that drainage alleviated some of the stress of surgery and disease induction. Treatment of L2 cells with gut-lymph from both sham and AP rats resulted in significant nuclear swelling and mitochondrial fragmentation, which are associated with cell death via oncosis. This potentially explains the excessive inflammatory damage inflicted upon the lungs during acute illness and severe trauma, as oncosis could promote an overactive immune response due to the release of cellular debris into surrounding tissues. Therefore, lymph drainage could be a valuable treatment option for MODS and may even be a useful procedure to manage the impact of trauma inflicted by major surgery in high-risk patients.