Name: oleic acid-injection in pigs as a model for acute respiratory distress syndrome
Uploaded: 2018-10-26T12:30:00
Description: Watch this Scientific Journal Video about Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome at JoVE.com

The authors want to thank Dagmar Dirvonskis for excellent technical support.

All animal experiments described here have been approved by the institutional and state animal care committee (Landesuntersuchungsamt Rheinland-Pfalz, Koblenz, Germany; approval number G14-1-077) and were conducted in accordance with the guidelines of the European and German Society of Laboratory Animal Sciences. The experiments were conducted in anesthetized male pigs (sus scrofa domestica) of 2-3 months age, weighing 27-29 kg.
1. Anesthesia, Intubation and Mechanical Ventilation
<ol>
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<ol>
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This article describes one method of oleic acid-induced lung injury as a model for studying various aspects of severe ARDS. There are also other protocols with different emulsions, different injection sites, and different temperatures of the emulsion23,24,25,26,27,28,29. Our method offers a ...

The acute respiratory distress syndrome (ARDS) is an intensive care syndrome that has been extensively studied since its first description about 50 years ago1. This body of research led to a better understanding of the pathophysiology and causes the development of ARDS resulting in improved patient care and outcome2,3. Nevertheless, the mortality in patients suffering from ARDS remains very high with about 35-40%4,5,6. The fact that about 10% of ICU admissions and 23% of ICU patients who require mechanical ventilation is due to ARDS underscores the relevance for further research in this field.
Animal models are widely used in research to examine pathophysiologic changes and potential treatment modalities for different kinds of diseases. Due to the complexity of ARDS, there is no single animal model to mimic this disease, but different models representing different aspects7. One well-established model is oleic acid injection (OAI)-induced lung injury. This model has been used in a wide array of animals, including mice8, rats9, pigs10, dogs11, and sheep12. Oleic acid is an unsaturated fatty acid and the most common fatty acid in the body of healthy humans13. It is present in human plasma, cell membranes, and adipose tissue13. Physiologically, it is bound to albumin while it is carried through the bloodstream13. Increased levels of fatty acids in the blood stream are associated with different pathologies and the severity of some diseases correlates with serum fatty acid levels13. The oleic acid ARDS-model was developed in an attempt to reproduce ARDS caused by lipid embolism as seen in trauma patients14. Oleic acid has direct effects on innate immune receptors in the lungs13 and triggers neutrophil accumulation15, inflammatory mediator production16, and cell death13. Physiologically, oleic acid induces rapidly progressing hypoxemia, increase in pulmonary arterial pressure and accumulation of extravascular lung water. Furthermore, it induces arterial hypotension and myocardial depression7. The disadvantages of this model are the necessity for central venous access, the questionable mechanistic relevance and the potential lethal progress caused by rapid hypoxemia and cardiac depression. The advantage of this model compared to other models is the usability in small and large animals, the valid reproducibility of the pathophysiological mechanisms in ARDS, the acute onset of ARDS after injection of oleic acid, and the possibility to study isolated ARDS without systemic inflammation like in many other sepsis models7. In the following article, we give a detailed description of the oleic acid-induced lung injury in pigs and provide representative data to characterize the stability of the compromises in lung function. There are different protocols for OAI-induced lung injury. The protocol provided here is able to reliably induce acute lung injury.

<table >
	<tbody>
		<tr >
			<td >3-way-stopcock blue</td>
			<td >Becton Dickinson Infusion Therapy AB Helsingborg, Sweden</td>
			<td >394602</td>
			<td ></td>
		</tr>
		<tr >
			<td >3-way-stopcock red</td>
			<td >Becton Dickinson Infusion Therapy AB Helsingborg, Sweden</td>
			<td >394605</td>
			<td ></td>
		</tr>
		<tr >
			<td >Atracurium</td>
			<td >Hikma Pharma GmbH , Martinsried</td>
			<td>4262659</td>
			<td ></td>
		</tr>
		<tr >
			<td >Canula 20 G</td>
			<td >Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td>
			<td >301300</td>
			<td ></td>
		</tr>
		<tr >
			<td >Datex Ohmeda S5</td>
			<td >GE Healthcare Finland Oy, Helsinki, Finland</td>
			<td></td>
			<td ></td>
		</tr>
		<tr >
			<td >Desinfection</td>
			<td >Sch&#252;lke &#38; Mayr GmbH, Germany</td>
			<td>104802</td>
			<td ></td>
		</tr>
		<tr >
			<td >Endotracheal tube</td>
			<td >Teleflex Medical Sdn. Bhd, Malaysia</td>
			<td >112482</td>
			<td ></td>
		</tr>
		<tr >
			<td >Endotracheal tube introducer</td>
			<td >R&#252;sch</td>
			<td>5033062</td>
			<td ></td>
		</tr>
		<tr >
			<td >Engstr&#246;m Carestation</td>
			<td >GE Heathcare, Madison USA</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Fentanyl</td>
			<td >Janssen-Cilag GmbH, Neuss</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Gloves</td>
			<td >Paul Hartmann, Germany</td>
			<td>9422131</td>
			<td ></td>
		</tr>
		<tr >
			<td >Incetomat-line 150 cm</td>
			<td >Fresenius, Kabi Germany GmbH</td>
			<td >9004112</td>
			<td ></td>
		</tr>
		<tr >
			<td >Ketamine</td>
			<td >Hameln Pharmaceuticals GmbH</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Laryngoscope</td>
			<td >Teleflex Medical Sdn. Bhd, Malaysia</td>
			<td >671067-000020</td>
			<td ></td>
		</tr>
		<tr >
			<td >Logical pressure monitoring system</td>
			<td >Smith- Medical Germany GmbH</td>
			<td>MX9606</td>
			<td ></td>
		</tr>
		<tr >
			<td >Logicath 7 Fr 3-lumen 30cm</td>
			<td >Smith- Medical Germany GmbH</td>
			<td>MXA233x30x70-E</td>
			<td ></td>
		</tr>
		<tr >
			<td >Masimo Radical 7</td>
			<td >Masimo Corporation Irvine, Ca 92618 USA</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Mask for ventilating dogs</td>
			<td >Henry Schein, Germany</td>
			<td >730-246</td>
			<td ></td>
		</tr>
		<tr >
			<td >Neofox Kit</td>
			<td >Ocean optics Largo, FL USA</td>
			<td>NEOFOX-KIT-PROBE</td>
			<td ></td>
		</tr>
		<tr >
			<td >Norepinephrine</td>
			<td >Sanofi- Aventis, Seutschland GmbH</td>
			<td >73016</td>
			<td ></td>
		</tr>
		<tr >
			<td >Oleic acid</td>
			<td >Applichem GmbH Darmstadt, Germany</td>
			<td>1,426,591,611</td>
			<td ></td>
		</tr>
		<tr >
			<td >Original Perfusor syringe 50ml Luer Lock</td>
			<td >B.Braun Melsungen AG, Germany</td>
			<td>8728810F</td>
			<td ></td>
		</tr>
		<tr >
			<td >PA-Katheter Swan Ganz 7,5 Fr 110cm</td>
			<td >Edwards Lifesciences LLC, Irvine CA, USA</td>
			<td>744F75</td>
			<td ></td>
		</tr>
		<tr >
			<td >Percutaneous sheath introducer set 8,5 und 9 Fr, 10 cm with integral haemostasis valve/sideport</td>
			<td >Arrow international inc. Reading, PA, USA</td>
			<td>AK-07903</td>
			<td ></td>
		</tr>
		<tr >
			<td >Perfusor FM Braun</td>
			<td >B.Braun Melsungen AG, Germany</td>
			<td>8713820</td>
			<td ></td>
		</tr>
		<tr >
			<td >Potassium chloride</td>
			<td >Fresenius, Kabi Germany GmbH</td>
			<td>6178549</td>
			<td ></td>
		</tr>
		<tr >
			<td >Propofol 2%</td>
			<td >Fresenius, Kabi Germany GmbH</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Saline</td>
			<td >B.Braun Melsungen AG, Germany</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Sonosite Micromaxx Ultrasoundsystem</td>
			<td >Sonosite Bothell, WA, USA</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Stainless Macintosh Size 4</td>
			<td >Teleflex Medical Sdn. Bhd, Malaysia</td>
			<td >670000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterofundin</td>
			<td >B.Braun Melsungen AG, Germany</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Stresnil 40mg/ml</td>
			<td >Lilly Germany GmbH, Abteilung Elanco Animal Health</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Syringe 10 mL</td>
			<td >Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td>
			<td>309110</td>
			<td ></td>
		</tr>
		<tr >
			<td >Syringe 2 mL</td>
			<td >Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td>
			<td>300928</td>
			<td ></td>
		</tr>
		<tr >
			<td >Syringe 20 mL</td>
			<td >Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td>
			<td >300296</td>
			<td ></td>
		</tr>
		<tr >
			<td >Syringe 5 mL</td>
			<td >Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td>
			<td>309050</td>
			<td ></td>
		</tr>
		<tr >
			<td >venous catheter 22G</td>
			<td >B.Braun Melsungen AG, Germany</td>
			<td>4269110S-01</td>
			<td ></td>
		</tr>
	</tbody>
</table>

oleic acid-injection in pigs as a model for acute respiratory distress syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

PaO2/FiO2-ratio decreases after fractionated application of oleic acid (Figure 1). In the presented study, 0.185 &#177; 0.01 ml kg-1 oleic acid was necessary for the induction of lung injury. All animals showed an impaired oxygenation after the induction of lung injury, with varieties in the further time course. In animal 1 and 3, it remained at one level with little fluctuations; in animal 2, we observe an initial increase, f...

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Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

This method can help answer key questions about the pathophysiologic changes that occur during ARDS for the development and evaluation of new treatment options for this disease. The main advantage of this technique is that it reproduces the basic pathologic changes observed in human ARDS. For ultrasound guided instrumentation, place an ultrasound probe on the right inguinal ligament of the experimental pig and scan for femoral vessels.Turn the probe 90 degrees to fully visualize the femoral artery in the long axis and use a Seldinger's needle to cannulate the right femoral artery under in-line ultrasound visualization. When pulsating bright blood flows out, introduce the guidance wire and retract the needle. Check to make sure that the positioning of the Seldinger's wire is correct by visualizing it with ultrasound.Visualize the femoral vein and cannulate the vein under in-line ultrasonography in continuous aspiration with the needle. When venous blood is able to be aspirated, insert Seldinger's wire and retract the needle. Use Seldinger's technique to insert a 5-French arterial introducer sheath and essential venous catheter and turn the corresponding three-way stopcocks back.Before inserting the pulmonary artery catheter, check that it is working properly and insert it through the left venous introducer sheath. When the pulmonary artery catheter has passed through the introducer sheath, inflate the balloon with one milliliter of air and advance the pulmonary artery catheter while monitoring the typical waveforms. Then deflate the balloon and check if blood can be aspirated through all of the ports of the pulmonary artery catheter.Then insert the probe for ultrafast measurement of the partial oxygen pressure through the right arterial introducer sheath. To induce lung injury, load one 20 milliliter syringe with 0.1 milliliters per kilogram of oleic acid and a second 20 milliliter syringe with two milliliters of freshly drawn blood. Add saline to the syringe with blood, to a total volume of 20 milliliters in both syringes and connect both syringes to a three-way stopcock.Connect a norepinephrine syringe pump to one port of the central venous catheter and begin an ultrafast partial oxygen pressure measurement. Record the baseline values of all of the relevant parameters and set the fraction of inspired oxygen to 1.0. Conduct a lung recruitment maneuver and connect the three-way stopcock to the proximal port of the pulmonary artery catheter.Then inject the oleic and blood saline mixtures, repeatedly from one syringe into the other, via the three-way stopcock, to thoroughly mix the solutions. When a homogeneous emulsion has been achieved, inject two milliliters of the emulsion into the central venous catheter and continue mixing. Closely monitor the hemodynamics after the injection, giving norepinephrine as 10 to 100 microgram bolus injection as necessary to keep the mean arterial pressure above 60 millimeters of mercury.Repeat the two milliliters oleic acid solution injection every three minutes, until the arterial partial pressure of oxygen fraction of inspired ratio is below 200 millimeters of mercury. Wait 30 minutes and reevaluate the partial pressure of oxygen fraction of inspired ratio. If the ratio is still over 200 millimeters of mercury, repeat the injections until the partial pressure of oxygen fraction of inspired ratio falls below 200 millimeters of mercury.Then set the ventilation according to the suggestions from the Acute Respiratory Distress Syndrome network. The partial oxygen pressure fraction of inspired oxygen ratio decreased after the fractionated application of oleic acid. Despite a typical impairment of oxygenation after the induction of lung injury, frequent variations in oxygen impairment are observed between animals across the impairment timecourse.Necessitating a close monitoring of the partial oxygen pressure fraction of inspired oxygen ratio while delivering the oleic acid. The decrease in partial oxygen pressure fraction of inspired oxygen ratio is paralleled by an increase in pulmonary arterial pressure which typically remains elevated for the rest of the experiment, with some fluctuation also observed between animals. Lung injury is visually detectable in lungs harvested six hours after lung injury induction, and histological analysis reveals alveolar edema and hemorrhage.After watching this video, you should have a good understanding of how to induce an acute lung injury in pigs by essential venous injection of oleic acid. Don't forget that working with oleic acid can be extremely hazardous and take precautions such as wearing gloves, and safety classes should always be taken while performing this procedure.

oleic-acid-injection-pigs-as-model-for-acute-respiratory-distress

Research

JoVE Journal

Medicine

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e., medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Purpose. Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroCardiographic evaluatiOn of ischeMia comParing invAsive to phaRmacological trEatment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Method. Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future therapies. Severe lung injury with a reproducible deterioration of pulmonary gas exchange and hemodynamics can be induced in anesthetized pigs using repeated lung lavages with warmed 0.9% saline (50 ml/kg body weight). Including standard respiratory and hemodynamic monitoring with clinically applied devices in this model allows the evaluation of novel therapeutic strategies (drugs, modern ventilators, extracorporeal membrane oxygenators, ECMO), and bridges the gap between bench and bedside. Furthermore, induction of lung injury with lung lavages does not require the injection of pathogens/endotoxins that impact on measurements of pro- and anti-inflammatory cytokines. A disadvantage of the model is the high recruitability of atelectatic lung tissue. Standardization of the model helps to avoid pitfalls, to ensure comparability between experiments, and to reduce the number of animals needed.

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

Repeated pulmonary lavages in anesthetized pigs induce lung injury resembling major aspects of human acute respiratory distress syndrome (ARDS). For this purpose the lungs are repeatedly lavaged with 0.9% saline at 37 &#176;C. The goal of the protocol is a reproducible mitigation of gas exchange and hemodynamics for research in ARDS.

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...