Protocols in Current Issue
Protocols in Past Issues
0 Q&A 3963 Views Apr 5, 2021

Sepsis is a dysregulated hyperinflammatory disease caused by infection. Sepsis leads to multiple organ dysfunction syndrome (MODS), which is associated with high rates of mortality. The cecal ligation and puncture (CLP) model has been widely used in animals and has become the gold-standard method of replicating features of sepsis in humans. Despite several studies and modified CLP protocols, there are still open questions regarding the multifactorial determinants of its reproducibility and medical significance. In our protocol, which is also aimed at mimicking the sepsis observed in clinical practice, male Wistar rats are submitted to CLP with adequate fluid resuscitation (0.15 M NaCl, 25 ml/kg BW i.p.) immediately after surgery. At 6 h after CLP, additional fluid therapy (0.15 M NaCl, 25 ml/kg BW s.c.) and antibiotic therapy with imipenem-cilastatin (single dose of 14 mg/kg BW s.c.) are administered. The timing of the fluid and antibiotic therapy correspond to the initial care given when patients are admitted to the intensive care unit. This model of sepsis provides a useful platform for simulating human sepsis and could lay the groundwork for the development of new treatments.

0 Q&A 2504 Views Nov 20, 2020

Maternal immune activation (MIA) is increasingly well appreciated as an environmental risk factor for some psychiatric disorders. Administration of proinflammatory compounds such as the synthetic double-stranded RNA molecule polyinosinic-polycytidylic acid (polyI:C) to pregnant rodents results in the release of proinflammatory cytokines in the maternal circulation. Various behavioural and brain changes are produced in the offspring that are associated with psychiatric disorders such as autism and schizophrenia. This protocol describes the steps necessary for inducing MIA in pregnant rat dams, which will allow for investigations into the mechanisms in the dam and offspring that mediate the long-term effects of exposure to inflammation while in utero. Increasing our understanding of these mechanisms may provide new insights for the diagnosis, treatment, and prevention of psychiatric disorders. This protocol has been developed and improved over the years by various researchers in Dr. Howland’s laboratory at the University of Saskatchewan.

0 Q&A 10822 Views Jul 5, 2019
Skin wound healing is a complex process involving different events such as blood coagulation, inflammation, new blood vessels formation, and extracellular matrix deposition. These events can be observed by using histology techniques. However, the lack of the standardization of such parameters impacts on the reproducibility of results. Here, we describe a protocol to perform macroscopic and microscopic analyses of the events that occur during skin wound healing using the experimental model of excisional wounds in rats.
0 Q&A 8327 Views Nov 20, 2015
Left ventricular (LV) remodeling occurs in many patients after myocardial infarction (MI). LV remodeling is characterized by progressive ventricular dilatation and contractile dysfunction, consequently to cardiomyocyte hypertrophy and fibrosis. Despite reperfusion therapies, this pathophysiological process is the main cause of cardiac evolution toward heart failure. Moreover, the outcome of patients after MI is largely dependent on the initial cardiac injury. Thus, this is of major clinical interest to develop new pharmacological strategies to limit infarct size and prevent or reverse left ventricular remodeling. Such preclinical cardiovascular treatments are often tested in rodents. The rat model of myocardial infarction is commonly used. In this model, the permanent ligation of the left anterior descending coronary artery is performed (Bousquenaud et al., 2013a).

After being used to this surgical technique and experimented, the operator will need 20 min per rat from the anesthesia to the rat recovering.
0 Q&A 10461 Views Oct 20, 2015
Angiogenesis is a multifactorial event which requires the migration, proliferation, differentiation and structure rearrangement of endothelial cells. This angiogenic process has been commonly studied using in vitro assays such as Boyden chamber assay, wound healing assay and tube formation assay. These assays mainly use monolayers of endothelial cells which are modified by repeated passages and are fully proliferative, a situation far away from physiology. In addition, not only endothelial cells are involved in this process but surrounding cells (such as pericytes, smooth muscle cells, fibroblasts) and the supporting matrix are also major players.

The three-dimensional ex vivo aortic ring model recapitulates the complexities of angiogenesis and combines the advantages of in vitro and in vivo models. The aortic ring is cultivated in a chemically defined culture environment. Microvessels which grow in this system are lumenized vessels with surrounding supporting cells and are essentially indistinguishable from microvessels formed during angiogenesis in vivo. The efficacy of pro-or anti-angiogenic factors can be determined in the absence of serum molecules which may otherwise interfere with the substances being tested (Nicosia and Ottinetti, 1990). However, this system requires access to fresh rat tissue but several samples can be prepared from one aorta.
0 Q&A 19027 Views Nov 20, 2013
The procedures described below are for producing gastric aspiration pneumonitis in mice with alterations for rats and rabbits described parenthetically. We use 4 different injury vehicles delivered intratracheally to investigate the inflammatory responses to gastric aspiration:
1) Normal saline (NS) as the injury vehicle control
2) NS + HCl, pH = 1.25 (acid)
3) NS + gastric particles, pH ≈ 5.3 (part.)
4) NS + gastric particles + HCl, pH = 1.25 (acid + part.)
The volume, pH, and gastric particle concentration all affect the resulting lung injury. In mice, we generally use an injury volume of 3.6 ml/kg (rat: 1.2 ml/kg, rabbit: 2.4 ml/kg), an injury pH (for the acid-containing vehicles) of 1.25, and a gastric particulate concentration (in the particulate-containing vehicles) of 10 mg/ml (rat: 40 mg/ml). In our hands this results in a maximal, non-lethal lung injury with ≤ 10% mortality for the most injurious vehicle (i.e., acid + part.) The maximum tolerable particulate concentration needs to be determined empirically for any new strains to be used, especially in genetically-altered mice, because an altered inflammatory response may have detrimental affects on mortality.
We have extensive experience utilizing these procedures in the outbred strain, CD-1, as well as many genetically-altered inbred stains on the C57BL/6 background. Choice of strain should be carefully considered, especially in terms of strain-specific immune bias, to assure proper data interpretation. The size of the mouse should be ≥ 20 g at the time of injury. Smaller mice can be attempted, if necessary, but the surgical manipulation becomes increasingly more difficult and the surgery survival rate decreases substantially. There are no size or strain constraints for rat and rabbit models, but we generally use Long-Evans rats at 250-300 g and New Zealand White rats at ≈ 2 kg at the time of initial injury.
0 Q&A 9862 Views Apr 20, 2013
Intracerebral infusion of kainic acid (KA) by a microdialysis probe induces a focal swelling in the brain-perfused area which promotes inflammation (Compan et al., 2012; Oprica et al., 2003). The microdialysis technique allows the local in vivo perfusion of KA and the simultaneous collection of inflammatory mediators, and other neuroactive substances, released in the injured brain. This protocol also allows the perfusion of different solutions in each cerebral hemisphere at the same time. By perfusing KA in isotonic solution of Krebs-Ringer Bicarbonate (KRB) (280-290 mOsm) in one hippocampus and KA in hypertonic KRB solution (1,400-1,500 mOsm) in the contralateral side, we can evaluate in vivo the efficiency of hypertonic solutions in preventing inflammation induced by swelling after KA infusion. Once the inflammatory response has been induced, it is possible to infuse through the microdialysis probe a biotinylated specific inhibitor of caspase-1 allowing the detection of the brain regions and cells involved in IL-1 production in response to the injury (Oprica et al., 2003).

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