A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

Summary

Here, we present a reproducible intensive care unit-oriented endotoxin model in rats.

Abstract

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ranges between 20 and 30%. Novel treatment approaches in order to reduce sepsis-related multiorgan failure and death are urgently needed. Robust animal models allow for one or multiple treatment approaches as well as for testing their effect on physiological and molecular parameters. In this article, a simple animal model is presented.

First, general anesthesia is induced in animals either with the use of volatile or by intraperitoneal anesthesia. After placement of an intravenous catheter (tail vein), tracheostomy, and insertion of an intraarterial catheter (tail artery), mechanical ventilation is started. Baseline values of mean arterial blood pressure, arterial blood oxygen saturation, and heart rate are recorded.

The injection of lipopolysaccharides (1 milligram/kilogram body weight) dissolved in phosphate-buffered saline induces a strong and reproducible inflammatory response via the toll-like receptor 4. Fluid corrections as well as the application of norepinephrine are performed based on well-established protocols.

The animal model presented in this article is easy to learn and strongly oriented towards clinical sepsis treatment in an intensive care unit with sedation, mechanical ventilation, continuous blood pressure monitoring, and repetitive blood sampling. Also, the model is reliable, allowing for reproducible data with a limited number of animals in accordance with the 3R (reduce, replace, refine) principles of animal research. While animal experiments in sepsis research cannot easily replaced, repetitive measurements allow for a reduction of animals and keeping septic animals anesthetized diminishes suffering.

Introduction

Sepsis and its more severe form, septic shock, are syndromes on the ground of an infection, resulting in an overshooting inflammatory reaction with the release of cytokines, leading to physiological and biochemical changes with a suppressed immune defense and fatal results^1,2. This unbalanced inflammatory reaction results in organ dysfunction and organ failure in various vital organs such as lung, kidney, and liver. With 37%³, sepsis is one of the most common reasons for a patient to be admitted to an intensive care unit (ICU). Mortality of sepsis currently ranges around 20-30%

Protocol

All experiments presented in this protocol were approved by the Veterinary Authorities of the Canton Zurich, Switzerland (approval numbers 134/2014 and ZH088/19). Moreover, all steps performed in this experiment were in accordance with the Guidelines on Experiments with Animals by the Swiss Academy of Medial Sciences (SAMS) and Guidelines of the Federation of European Laboratory Animal Science Associations (FELASA).

1. Anesthesia induction and animal monitoring

1. Keep male Wistar rats with a weight of 250-300 gram (g) in ventilated cages under pathogen-free conditions. Provide a 12-12-hour light/dark cycle at an ambient temperatur....

Results

The system presented allows for endotoxemia with hemodynamically stable animals as reported previously⁹. While the mean arterial pressure remains stable in animals with, and without LPS stimulation LPS treated animal develop characteristics of sepsis such as a negative base excess and a strong inflammatory reaction measured by plasma cytokines (6 hours after application) such as CINC-1 (867 ng/mL), MCP-1 (5027 ng/mL), and IL-6 (867 ng/mL)⁸, Figure 5

Discussion

The protocol described here allows for a highly reproducible, yet simple to learn sepsis model, which can be adapted according the research question. Essential in vivo data referring to organ function such as heart rate, blood pressure, and peripheral arterial oxygen saturation may be collected continuously, and blood sampling may be performed repetitively throughout the experiment. In addition, modifications with regard to fluid replacement protocols and vasopressor support can be installed. Given the hemodynamic stabil.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-0 silk sutures	Ethicon, Sommerville, NJ	K833	Standard surgical
26 intravenous catheter	Becton Dickinson, Franklin Lakes, NJ	391349	Standard anesthesia equipment
6-0 LOOK black braided silk	Surgical Specalities Corporation, Wyomissing, PA	SP114	Standard surgical
Alaris Syringe Pump	Bencton Dickinson
Betadine	Mundipharma, Basel, Switzerland	7.68034E+12	GTIN-number
Curved fine tips microforceps	World precision instruments (WPI), Sarasota, FL	504513	Facilitates vascular preparation
Fine tips microforceps	World precision instruments (WPI), Sarasota, FL	501976	Tips need to be polished regularly
Infinity Delta XL Anesthesia monitoring	Draeger, Lübeck, Germany
Isoflurane, 250 mL bottles	Attane, Piramal, Mumbai, India	LDNI 22098	Standard vet. equipment
Ketamine (Ketalar)	Pfitzer, New York, NY
Lipopolysaccharide (LPS) from Escherichia coli, serotype 055:B5	Sigma, Buchs, Switzerland
Q-tips small	Carl Roth GmbH, Karlsruhe, Germany	EH11.1	Standard surgical
Ringerfundin	Bbraun, Melsungen, Germany
Tec-3 Isofluorane Vaporizer	Ohmeda, GE-Healthcare, Chicago, IL	not available anymore	Standard vet. equipment
Xylazine (Xylazin Streuli)	Streuli AG, Uznach, Switzerland