This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting.
This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting. Critically ill patients require intensive medical support in order to survive. While the majority of patients will recover within a few days, about a quarter of the patients need prolonged intensive care and are at high risk of dying from non-resolving multiple organ failure. Furthermore, the prolonged phase of critical illness is hallmarked by profound muscle weakness, and endocrine and metabolic changes, of which the pathogenesis is currently incompletely understood. The most widely used animal model in critical care research is the cecal ligation and puncture model to induce sepsis. This is a very reproducible model, with acute inflammatory and hemodynamic changes similar to human sepsis, which is designed to study the acute phase of critical illness. However, this model is hallmarked by a high lethality, which is different from the clinical human situation, and is not developed to study the prolonged phase of critical illness. Therefore, we adapted the technique by placing a central venous catheter in the jugular vein allowing us to administer clinically relevant supportive care, to better mimic the human clinical situation of critical illness. This mouse model requires an extensive surgical procedure and daily intensive care of the animals, but it results in a relevant model of the acute and prolonged phase of critical illness.
Critical illness is a disease state in which the function of one or more organ systems is hampered to the extent that the patient will die, unless intensive medical support is administered. Whereas the initial cause for admission to the intensive care unit (ICU) can vary, ranging from trauma, complicated surgery, burns, disease exacerbations to sepsis, all critically ill patients suffer from cellular damage, caused by hypoperfusion, hypoxia and excessive inflammation among others, which leads to organ failure. Most patients survive their acute insult, but an important fraction of the patients do not immediately recover and need prolonged intensive care. They are at high risk of death due to non-resolving multiple organ failure. Furthermore, the prolonged phase of critical illness is hallmarked by profound muscle weakness and endocrine and metabolic change, of which the pathogenesis is currently incompletely understood.
Several rodent models are being used in the critical care research setting. The two mostly used models are the exogenous administration of lipopolysaccharide (LPS) and cecal ligation and puncture (CLP). Both models are developed to mimic the acute phase of sepsis, defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection, and one of the primary reasons for admission to the ICU worldwide1,2. The LPS model has several disadvantages as it only transiently affects the release of cytokines and the hemodynamic status of the animal3. Unlike humans, rodents are also particularly resistant to endotoxin and the use of 'high doses' of endotoxin is necessary to produce hypotension and mortality, hereby further raising concerns of the validity of this method4,5. The other model, cecal ligation and puncture model (CLP) features a ligation of a portion of the cecum followed by a needle puncture through-and-through. This procedure causes a polymicrobial abdominal infection with tissue damage, followed by a translocation of the bacteria into the blood compartment. This will trigger a systemic inflammatory response and the development of sepsis. The CLP model has been extensively recognized as an animal model of acute critical illness that reproduces the main features of sepsis: hyperinflammation, vasodilation, hypotension and increased cardiac output6,7. However, this model does not allow study of non-resolving multiple organ failure, muscle wasting, and endocrine and metabolic changes, which are typical for the prolonged phase of critical illness. Furthermore, recently the validity of mouse models for critical illness have been questioned, since findings from mouse models cannot always be translated to the human setting8,9,10. A possible explanation might be that the supportive care that is provided to critically ill human patients differs substantially from the care that is provided to critically ill mice.
Therefore, to resemble the human setting more closely and to allow investigation of the prolonged phase of critical illness, we developed a mouse model that mimics the acute intensive care as given to humans, such as extensive intravenous fluid administration and antibiotic treatment, and which allows to administer supportive care in order to survive the prolonged phase of critical illness, such as nutritional support. For this purpose, we adapted the mouse model of CLP-induced sepsis, being the golden standard for sepsis, and placed a central venous line which enables the administration of fluids, nutrition and drugs.
The protocol was approved by the University of Leuven Ethical Review Board for Animal Research.
1. Preparation of the Venous Line
Figure 1: Construction of Venous Line. Venous lines are prepared by stretching the MRE tubing to a small diameter and connecting it via polyethylene tubing to a rodent swivel device. See Table 1 for instructions on lengths of the different parts. Please click here to view a larger version of this figure.
Legend | Length | Volume |
1) Luer stub needle 22 G | ||
2) PE50 – polyethylene .023" x .038" | 5 cm | 13 µL |
3) PE10 – polyethylene .011" x .024” | 50 cm | 30 µL |
4) Rodent Swivel 20 G | ||
5) PE-10 | 15 cm | 9 µL |
6) PE-50 (connector) | 5 cm | 13 µL |
7) MRE025 Tip stretched microrenathane .025" x .012" | 30 cm | 27 µL |
Total sum | 105 cm | 92 µL |
Table 1: Construction of Venous Line. This table provides a legend for Figure 1.
2. Anesthesia and Pre-surgery Handling
3. Placement of a Chronic Indwelling Catheter
4. Cecal Ligation and Puncture
5. Post-surgical Treatment and Fluid Resuscitation
6. Intensive Care
7. End of the Experiment
NOTE: Approval of and recommendations on the level of severity of the model and guidelines and policies for human endpoints should be sought from the local Institutional Ethical Review Board for Animal Research.
NOTE: In case of a nonfunctional venous line such as blocked catheter, delocalization of the catheter, problems with the syringe pump, the animal is excluded from the study and euthanized.
C57BL/6 mice were made critically ill as described above. We performed two experiments to assess post-CLP survival until two time points: survival until day 5 (n = 15) and survival until day 7 (n = 22) post-CLP. Survival curves of the two experiments were not significantly different (compared until day 5), indicating the reproducibility of the experimental setup. Non–surviving animals were found death or euthanized due to reaching human endpoints. Ligation of 50% of the cecum in combination with antibiotics, fluid resuscitation and total parenteral nutrition via a venous catheter in the vena jugularis, as described, resulted in a 13% mortality after 1 day of critical illness, 24% mortality after 3 days of critical illness, 27 – 31% mortality after 5 days of critical illness and 36 % after 7 days of critical illness. The healthy pair-fed mice, which were caloric restricted to the nutrient intake of the critically ill mice, did not show any mortality.
Figure 2: Survival Curves after 5 or 7 Days of Critical Illness. There was no mortality in the group of healthy animals (A, B, dashed line – healthy animals without surgery). Five days after surgery (A), mortality rate was 27% (solid line). Seven days after induction of sepsis (B), the mortality rate was 36%. Mice with leaking or dislodged catheters were excluded from the experiment (15%). Please click here to view a larger version of this figure.
We developed a more clinically relevant mouse model of critical illness, by combining the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration. This experimental setup is reproducible, allows to study the prolonged phase of critical illness and results in a stable mortality rate, hereby mimicking the human clinical situation1.
Cecal ligation and puncture has been extensively recognized as an animal model of acute critical illness that reproduces the main features of sepsis6,7. However, this model of acute critical illness does not allow the study of non-resolving multiple organ failure, muscle wasting, and endocrine and metabolic changes, which are typical for the prolonged phase of critical illness. Furthermore, preclinical findings with this model often fail to translate to the clinical human setting8,9,10. Therefore, we developed a model where placing a central venous line allows the investigator to administer extensive fluid resuscitation, drugs and total parenteral nutrition. These supportive measures are of vital importance for critically ill patients in order to survive the acute phase of critical illness. Despite the placement of this central venous line, the animals are still able to freely move with relatively minor discomfort. Daily pain medication helps alleviate suffering, as assessed by the mouse grimace scale of pain11. This model also incorporates other essential aspects of supportive care for critically ill patients, such as antibiotic treatment and pain medication.
This model has several limitations. First, the extensive surgical procedure is demanding and requires sufficient training. Nevertheless, with sufficient training, on average 10% of the operated animals die during or immediately after the surgery. Furthermore, catheters that are not well-placed, can lead to leakage of resuscitation fluids or parenteral nutrition into the thorax of the animal. On average, 15% of surviving animals have to be excluded during the study due to these catheter related problems. Thus, when one calculates the required number of animals for an experiment, one has to take into account a 25% surplus due to surgery and catheter related losses. Second, with only one venous access point, antibiotics and pain medication still have to be administered subcutaneously twice daily. Indeed, compatibility of parenteral nutrition with antibiotics and pain medication drugs cannot be guaranteed, and therefore co-infusion should be avoided. Flushing of the line followed by a bolus injection is also technically not possible due to limited blood volume of the mice. Third, the anesthesia and surgery needed to place the catheter will induce a severe stress response by itself. We use healthy animals without surgery as controls, and consider the catheter related surgery as a part of the critical illness, comparable to what human surgical ICU patients have to endure. Fourth, whereas post-operative pain medication and antibiotics and the use of a central venous line to allow administration of parenteral nutrition increases the clinical relevance of the murine CLP model, our model still does not completely mimic the clinical human situation. Indeed, due to the small animal size it is challenging to introduce several advanced supportive techniques, such as renal replacement therapy. However, the use of this model allows genetic interference, vital in unraveling the pathogenesis of critical illness.
It has been shown that mortality and severity of the CLP procedure can be manipulated, if deemed necessary, by the portion of the cecum that is ligated, by the size and number of punctures, subcutaneous fluid resuscitation and daily antibiotic administration6,12. We choose a protocol in which we provided extensive fluid resuscitation at 10 mL/kg/h for the first 20 h, as it has been demonstrated previously that this improves mortality13. Our adaptations should be incorporated into the protocol if a model of prolonged critical illness is desired instead of a model of lethality. This mouse model uses a mid-grade CLP, intravenous fluid resuscitation and antibiotic treatment in order to create a more clinically relevant model of critical illness, as is shown by its prolonged survival curves which mimics the survival rate of human sepsis1. The ICU patient population, as the general population, is aging14. In order to mimic the human ICU setting even more closely, one can use mature mice (6 months), as used in this study, in order to enhance clinical relevance of the experimental model.
In conclusion, this mouse model requires an extensive surgical procedure and daily intensive care of the animals, but it results in a model of critical illness, which allows the investigator to study the aspects of the prolonged phase of critical illness.
The authors have nothing to disclose.
GVdB, via the University of Leuven (KU Leuven), receives long-term structural research support from the Methusalem Program funded by the Flemish Government (METH08/07) and holds a European Research Council Advanced Grant AdvG-2012-321670 from the Ideas Program of the European Union seventh framework program. SET received a Research Foundation-Flanders (FWO) Research Assistant Fellowship.
Buprenorphine | Ecuphar | Vetergesic 0,3mg/ml | |
C57BL/6 | Janvier labs | C57BL/6JRj | |
colloids | Fresenius Kabi | Volulyte 6% | |
crystalloids | Baxter | Plasmalyte a viaflo | |
Ethilon 3.0 | Ethicon | F3211 | |
Imipenem | MSD | Tienam 500mg powder for injection fluid | |
Isoflurane | Eurovet | Iso-vet | |
Ketamine | Eurovet | Nimatek 100mg/ml | |
LocTite Super glue3 all plastics | Rectavit | 119818 | |
Mersilk 3.0 | Ethicon | L192 | |
Mersilk 5.0 | Ethicon | F682 | |
Microrenathane .025 O.D." x .012 I.D." | Bioseb | MRE-025 | |
olimel N7E | Baxter | ||
PE10 – Polyethylene .011" x .024" | Instech Solomon | BTPE-10 | |
PE-50 tubing .023"x.038" | Instech Solomon | BTPE-50 | |
Rodent Swivel 20 G | Bioseb | RS-20G | |
Ropivacaïne | Astrazenica | Naropin 2mg/ml | |
Xylazine | VMD | Xylazine hydrochloride 2% |