Name: intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation
Uploaded: 2016-03-02T15:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

We thank Dr. Carson Harrod for critical reading of the manuscript. We also thank Mr. Minghui Zeng and Drs. Mahesh Kathania and Prashant Khare for their contributions. This work was supported by grants from the American Cancer Society (Research Scholar grant, 122713-RSG-12-260-01-LIB) and the Sammons Cancer Center (Pilot Project grant) to K. Venuprasad.

All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Baylor Research Institute.
1. Culturing Non-typeable Haemophilus influenzae (NTHi) and Preparing the Inoculum
<ol>
	<li>Plate NTHi on a chocolate agar plate and keep the plate upside down in a humidified CO2 incubator overnight at 37 &#176;C and 5% CO2. The following day, culture the bacteria in brain heart infusion broth. Then, add 1 ml of the broth into a sterile 1.5 ml tube.</li>
	<li>Centrifuge at 4,000 x g for 5 min at room temperature. Then, discard the supernatant and re-suspend the pellet in 1 ml of sterile 1x PBS. Repeat this step once.</li>
	<li>Transfer 100 &#181;l of re-suspended solution into 900 &#181;l of 1x PBS and measure the absorbance of the diluted culture at 600 nm wavelength in a spectrophotometer.</li>
	<li>Determine the viability and colony forming units (CFUs) of the inoculum by culturing 1:10 serial dilutions on chocolate agar plates and incubating overnight at 37 &#176;C and 5% CO2.</li>
	<li>Based on the viability and CFUs determined from the previous step, adjust the inoculum to a final concentration of 20 x 106 CFU/ml.</li>
</ol>
2. Intra-tracheal (i.t) Instillation
<ol>
	<li>Inject the mouse intraperitoneally (i.p) with 0.1 ml/10 g body weight of mouse with a 150 mg/ml ketamine + 20 mg/ml xylazine solution.&#160;The procedure should be done in the hood with a light source to keep the animal warm.</li>
	<li>Pinch the interdigital space of the mouse to verify that the mouse is adequately anesthetized.</li>
	<li>Place the mouse on its back. Shave the ventral area of the neck and disinfect with chlorhexidine and 70% ethyl alcohol.</li>
	<li>Lift the skin of the upper ventral part of the neck using rat tooth forceps. Make a small incision (0.5-1.0 cm) above the thymus using a scalpel and carefully dissect the muscle to expose the trachea.
	<ol>
		<li>Using a 1 ml syringe with a 27 G needle, slowly inject 0.05 ml air, followed by 0.05 ml NTHi obtained from Section 1 or saline as a control, followed by another 0.05 ml air. Keep animal vertical for around 1 min.</li>
	</ol>
	</li>
	<li>Close the incision with surgical glue. Keep the mouse laterally recumbent and warm for about 10 min.</li>
</ol>
3. BALF Collection
<ol>
	<li>Sacrifice the mouse by cervical dislocation 24 hr after infection.</li>
	<li>Open the abdominal cavity of the mouse with anatomical scissors and cut the ventral aorta to bleed. Expose the ventral part of the lung.</li>
	<li>Expose the trachea and intubate it with a 20 gauge catheter. Instill 0.8 ml of BALF buffer (1x PBS, 0.1% dextrose, 10 U/ml heparin) and collect the lavage by gentle aspiration. Repeat this procedure three times.</li>
	<li>Count the total number of cells in 100 &#181;l of BALF suspension using a hemocytometer under 10X magnification with Trypan Blue staining.</li>
	<li>Prepare slides for modified Giemsa staining. Aliquot 100 &#181;l of BALF into the appropriate wells of the cytospin and centrifuge at 500 x g for 5 min. Dry the slides for 10 min and stain the cells using modified Giemsa stain according to the manufacturer&#39;s protocols.</li>
	<li>Use the remaining cells for subsequent analysis, such as florescence activated cell sorting (FACS), confocal microscopy, gene expression and western blot11-13.</li>
</ol>
4. Histopathology Preparation
<ol>
	<li>For histopathology analysis, infect mice with NTHi as described in step 2.4. After 24 hr of infection, sacrifice the animals by cervical dislocation. Cut open the thoracic cavity with anatomical scissors to expose the lungs and the trachea as mentioned before. Then, insert the 20 gauge catheter into the trachea.</li>
	<li>Fill the lung with 2% warm agarose solution prepared in 4% formalin. Once the lungs are fully inflated, place ice on top of the lungs to solidify the agarose solution.</li>
	<li>Then, carefully remove the thoracic plug and put it in a 50 ml solution of 4% formalin in 1x PBS until it is processed for hematoxylin and eosin (H&#38;E) sections.</li>
</ol>
5. FACS Staining Cells in the BALF
<ol>
	<li>Put 1 x 105 cells from the BALF fluid in a 96-well V-bottom micro titer plate.</li>
	<li>Wash cells with 300 &#181;l of cold 1x PBS and spin at 4,000 x g for 3 min at 4 &#176;C.</li>
	<li>Stain the cells with cell stain solution in 100 &#181;l 1x PBS (1:1,000 dilution) and keep at room temperature for 15 min.</li>
	<li>Wash cells with 300 &#181;l of cold 1x PBS at 4,000 x g for 3 min at 4 &#176;C.</li>
	<li>Keep cells in 100 &#181;l of FACS wash buffer [Wash buffer (1x PBS, 2% FBS, 1 mM EDTA, 0.1% sodium azide] containing 1:100 diluted Fc-Block for 15 min at 4 &#176;C.</li>
	<li>Centrifuge cells at 4,000 x g for 3 min and discard supernatant.</li>
	<li>Stain cells with 100 &#181;l surface staining cocktail (1:100 dilution) in FACS wash buffer for 30 min at 4&#176;C.</li>
	<li>Wash cells thrice with 300 &#181;l of FACS wash buffer at 4,000 x g for 3 min at 4 &#176;C.</li>
	<li>Fix cells in 100 &#181;l of 4% paraformaldehyde for 15 min at room temperature.</li>
	<li>Wash cells twice with 300 &#181;l 1x PBS at 400 x g for 3 min at 4 &#176;C and re-suspend in 300 &#181;l of FACS wash buffer.</li>
	<li>Make single-color staining controls using flow cytometry beads.
	<ol>
		<li>Add one drop of beads into the 96-well V-bottom microliter plate and wash with 200 &#181;l 1x PBS.</li>
		<li>Add 1 &#181;l of staining antibody to 100 &#181;l 1x PBS and beads and incubate at room temp for 5 min.</li>
		<li>Wash beads twice with 300 &#181;l 1x PBS.</li>
	</ol>
	</li>
	<li>Analyze single-stained controls and cells by flow cytometry14.</li>
</ol>
6. Homogenization of Lung Tissue to Isolate RNA
<ol>
	<li>After infection, collect a small piece of lung (20-40 mg) and keep it in RNA stabilizing reagent at 4 &#176;C overnight. Then, store it at -80 &#176;C until the next step.</li>
	<li>Wash the lung tissues in cold 1x PBS to get rid of the RNA stabilization reagent.</li>
	<li>Place the tissue into a 1.5 ml centrifuge tube containing lysis buffer (1 ml lysis buffer + 10 &#181;l &#946;-mercaptoethanol).</li>
	<li>Chop the tissue into small pieces using pointed scissors and homogenize using a cordless motor pellet pestle for 20-40 sec.</li>
	<li>Keep the lysate on ice for 5 min and proceed with step 7.1.</li>
</ol>
7. RNA Isolation from the Lung
<ol>
	<li>Centrifuge the lysate at 18,000 x g for 3 min. Remove the supernatant by pipetting and carefully transfer it to a new 1.5 ml tube.</li>
	<li>Add 1 volume of 70% ethanol to the lysate. Mix immediately by pipetting and wait for 2-4 min.</li>
	<li>Transfer up to 700 &#181;l of lysate to a spin column placed in a 2 ml collection tube.</li>
	<li>Centrifuge at 9,600 x g for 15 sec and discard the flow-through.</li>
	<li>Add 350 &#181;l stringent wash buffer and centrifuge at 9,600 x g for 15 sec. Discard the flow-through.</li>
	<li>Add 80 &#181;l DNase I directly to the spin column membrane and keep it at room temperature for 15 min.</li>
	<li>Add 350 &#181;l stringent wash buffer to the spin column and centrifuge at 9,600 x g for 15 sec. Discard the flow-through.</li>
	<li>Add 500 &#181;l RNA wash buffer and centrifuge at 9,600 x g for 15 sec. Discard the flow-through.</li>
	<li>Add 500 &#181;l RNA wash buffer and spin at 9,600 x g for 2 min. Discard the flow-through.</li>
	<li>Place the spin column in a new 2 ml collection tube and centrifuge at 9,600 x g for 1 min. Discard the flow-through.</li>
	<li>Add 30-50 &#181;l RNase-free water directly to the spin column and centrifuge at 9,600 x g for 1 min. Store RNA at -80 &#176;C.</li>
	<li>Perform RNAseq analysis14.</li>
</ol>

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The authors have nothing to disclose.

Herein, we describe a unique and minimally invasive procedure to inoculate the lungs of mice with a bacterial lung pathogen. We demonstrate that this procedure can be used to study the function of different genes using mice that are deficient in genes of inflammatory signaling pathways. This procedure can also be used to study the inflammatory responses to viral and fungal lung infections. The advantages of this procedure over other methods such as intranasal or aerosol inhalation are (1) in this procedure, the pathogeni...

Inflammation is a fundamental immune mechanism of defense against infectious agents. It promotes pathogen eradication and repair of damaged tissue. It also facilitates the recovery to a normal healthy state1. However, dysregulated inflammation often leads to chronic inflammatory diseases2. Airway inflammation is an initial trigger for different pulmonary diseases such as chronic obstructive pulmonary disease (COPD), asthma and pulmonary fibrosis3.
The non-typeable (unencapsulated) Haemophilus influenzae (NTHi) is associated with chronic upper and lower lung inflammatory diseases4,5. It is the dominant species isolated from the lower airways of children and adults with chronic obstructive pulmonary disease4,6,7. The inflammatory response after NTHi infection is characterized by the upregulation of proinflammatory cytokines (such as TNF and IL-1&#946;), and it is mediated by mitogen activated protein kinase (MAPK) and nuclear factor-&#954;B (NF-&#954;B) through toll-like receptors (TLRs)8.
Mouse models are very useful tools for analyzing the underlying pathology of lung inflammatory disease because of the availability of different gene-deficient lines. Several methods have been used to inoculate live/attenuated bacteria and bacterial products, including intranasal instillation and aerosolized inhalation9,10. Here, we demonstrate intra-tracheal instillation. Although used less frequently, this approach is more efficient and highly reproducible because of the direct delivery of the inoculum to the lower respiratory tract.

<table><tbody><tr><td>Chocolate agar plate</td><td>Fisher Scientific</td><td>CAS50-99-7</td><td></td></tr><tr><td>Dextrose Anhydrous</td><td>Themo Scientific</td><td>R01300</td><td></td></tr><tr><td>Heparin</td><td>Hospira,Inc</td><td>RL-3010</td><td></td></tr><tr><td>Deft quick solution</td><td>Sigma</td><td>GS500-500ML</td><td></td></tr><tr><td>Syringe needle 20/26G&amp;nbsp;</td><td>BD</td><td>(REF305115/175)</td><td></td></tr><tr><td>Iml syringe</td><td>BD</td><td>REF 309602</td><td></td></tr><tr><td>Catheter 20GA</td><td>BD</td><td>REF 381433</td><td></td></tr><tr><td>Dissecting Scissors, straight, 10 cm long</td><td>kentscientific</td><td>INS600393</td><td></td></tr><tr><td>Iris Forceps, serrated, 10cm long</td><td>kentscientific</td><td>INS650915</td><td></td></tr><tr><td>Tweezer #5 Stainless steel, 11cm long</td><td>kentscientific</td><td>INS600095</td><td></td></tr><tr><td>10% Formalin</td><td>Fisher Scientific</td><td>CAS 67-56-1</td><td></td></tr><tr><td>Agarose</td><td>peqlab</td><td>35-1020</td><td></td></tr><tr><td>5ml polystyrene round-bottom tubes</td><td>BD</td><td>REF 352058</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tubes</td><td>Light Labs</td><td>A-7001-R</td><td></td></tr><tr><td>Reasy Mini&amp;nbsp; kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Pellet pestile motor (Tissue homoginizer)</td><td>Sigma</td><td>Z359971-1EA</td><td></td></tr><tr><td>96 well microtiter plates V bottom</td><td>Thermo</td><td>2605</td><td></td></tr><tr><td>1X PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111-42</td><td></td></tr><tr><td>CD45.2-APC</td><td>eBioscience</td><td>17-0454-81</td><td>Working dilution 1:100</td></tr><tr><td>Ly-6G-eFlor 450</td><td>eBioscience</td><td>48-5931-82</td><td>Working dilution 1:100</td></tr><tr><td>BSA</td><td>HyClone</td><td>SH30574.03</td><td></td></tr><tr><td>RBC Lysis Buffer (10X)</td><td>Biolegend</td><td>420301</td><td></td></tr><tr><td>Live/Dead fixable aqua dead cell stain kit</td><td>Invitrogen</td><td>L-34957</td><td></td></tr><tr><td>EDTA (0.5M)</td><td>lifetechnologies</td><td>15575-020</td><td></td></tr><tr><td>CD16/CD32 FcBlock</td><td>BD</td><td>553142</td><td></td></tr><tr><td>Facs tubes polystyrene round bottom tube</td><td>BD</td><td>352052</td><td></td></tr><tr><td>Formaldehyde</td><td>Polyscience</td><td>4018</td><td></td></tr></tbody></table>

intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal instillation resulted in a markedly increased number of leukocytes in the BALF (Figure 1A, left panel) than installation with saline. The differential count analysis of the leukocytes clearly showed increased neutrophil infiltration (Figure 1, right panel). The FACS analysis of the cells in the BALF further confirmed the increased number of neutrophils (Figure 1B). Histological analysis of H&#38;E-stained sections of the lu...

Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

intra-tracheal-administration-haemophilus-influenzae-mouse-models-to

Research

JoVE Journal

Immunology and Infection

10.1K Views.  Baylor Research Institute. We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models. 

Video: Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Medicine

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

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Chapters in this video

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

An Improved Method for Rapid Intubation of the Trachea in Mice

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice