Multimodal Transgastric Local Pancreatic Hypothermia Reduces Severity of Acute Pancreatitis in Rats and Increases Survival

Abstract

Background & Aims: Acute pancreatitis (AP) of different etiologies is associated with activation of different signaling pathways in pancreatic cells, posing challenges to development of targeted therapies. We investigated whether local pancreatic hypothermia, without systemic hypothermia, can reduce severity of AP, induced by different methods in rats.Methods: A urethane balloon, with two polyurethane tubes was placed inside the stomach of the rats. AP was induced in Wistar rats by administration of caerulein or glyceryl-trilinoleate (GTL). Cold water was then infused into the balloon to cool the pancreas. Pancreatic temperatures were selected based on those found to reduce acinar cell injury. An unperfused balloon was used as a control. Pancreatic and rectal temperatures were monitored, and an infrared lamp and/or heating pad were used to avoid generalized hypothermia. We collected blood, pancreas, kidney, and lung tissues and analyzed them by histology, immunofluorescence, immunoblot, cytokine and chemokine magnetic bead, and DNA damage assays. The effect of hypothermia on signaling pathways initiated by caerulein and GTL was studied in acinar cells.Results: Rats with pancreatic cooling developed less severe GTL-induced AP compared with rats that received the control balloon. In acinar cells, cooling reduced the lipolysis induced by GTL, increased the micellar form of its fatty acid, reduced the increase in cytosolic calcium, prevented loss of mitochondrial membrane potential (by 70%-80%), a and resulted in a 40%-50% reduction in uptake of a fatty acid tracer. In rats with AP, cooling reduced pancreatic necrosis by 48%, reduced serum levels of cytokines and markers of cell damage, decreased markers of lung and renal damage. The pancreatic cooling increased the proportions of rats surviving 6 hrs after

Ringer’s Lactate Prevents Early Organ Failure by Providing Extracellular Calcium

“…Stained cells were suspended in a UV/VIS-transparent cuvette containing a stir bar and placed in a F2100 Hitachi Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan). Fura-2 ratio (340/380 nm excitation, 510 nm emission) and JC-1 ratio (510 nm excitation, 530/590 nm emission) were collected every 15 s with stirring and temperature control as previously described [25].…”

“…Linoleic acid (LA) was administered intraperitoneally at 0.1% body weight to adult CD-1 mice (30-40 g) as described previously [25]. These were monitored for vitals daily before and after LA administration as described below.…”

“…The biochemical assays (amylase, lipase, BUN [blood urea nitrogen], Calcium; Pointe Scientific, Canton, MI, USA) and colorimetric total free fatty acids using Non-Esterified Fatty Acid kit (FUJIFILM Wako Diagnostics, Mountain View, CA, USA) were done as per the manufacturer protocol and described previously [22][23][24][25]30]. Serum CRP level was measured by using Mouse C-Reactive Protein/CRP Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) as per manufacturer's protocol.…”

“…We next tested whether hypocalcemia may be relevant to the pathogenesis of NEFA-induced organ failure. Both oleic acid [32][33][34] and linoleic acid [25,30] can cause organ failure, as may happen in severe pancreatitis [22,25]. We thus analyzed the relevant parameters after administration of linoleic acid (0.1% body weight) in a cohort of mice we have previously studied [25].…”

“…Fatty acids are elevated during critical illnesses [15][16][17][18][19] such as severe acute pancreatitis in humans [20,21] and rodents [22][23][24][25], but not during mild disease [22,23]. Previous studies have shown hypocalcemia to result from fatty acid administration [26] and calcium to be present in the (peri) pancreatic fat necrosis associated with pancreatitis [24], consistent with saponification [24,27,28].…”