This work was supported by the Guangzhou Science and Technology Planning Project (202002030151), the Major Project of Guangzhou National Laboratory (GZNL2024A02001), and the grant of State Key Laboratory of Respiratory Disease (SKLRD-Z-202202).

All the procedures were approved by the Animal Care and Use Committee of Guangzhou Medical University (20240248) and were performed in strict accordance with approved guidelines. Male specific pathogen-free C57BL/6 mice weighing 20-25 g were used in this study. All mice were housed under controlled temperature (22 &#177; 2 &#176;C), humidity (50% &#177; 20%), and lighting (6:30 AM to 6:30 PM) in solid bottom cages with food and water available ad libitum. The time line of the protocol is shown in Figure 1.
1. Establishment of a mouse model of cough
<ol>
	<li>Use influenza A/California/7/2009 (H1N1) virus. Determine the lethal dose of the H1N1 virus in mice.
	<ol>
		<li>Briefly, anesthetize mice (n = 10 per group) with 2.5% isoflurane and then intranasally infect with a 10-fold serially diluted virus.</li>
		<li>Monitor the death over a period of 15 days. The survival rates and body weights of mice were recorded daily. Calculate the median lethal dose (LD50) by the Reed-Muench method26. 
		NOTE: Mice were painlessly sacrificed with an overdose of pentobarbital sodium when they reached or exceeded 30% body weight loss.</li>
	</ol>
	</li>
	<li>Infect the mouse with the H1N1 virus.
	<ol>
		<li>Dissolve 0.8 x LD50 of H1N1 virus in 50 &#181;L of phosphate-buffered saline (PBS).</li>
		<li>Anesthetize the mice with 2.5% isoflurane.</li>
		<li>When the mice were deeply anesthetized, place the mouse in a supine position with the nostrils facing upward.</li>
		<li>Instill 5-10 &#181;L of H1N1 virus solution or PBS solution (as a control) intranasally into one nostril of mice using a pipette (Figure 2A).</li>
		<li>Then, hold the mouse&#39;s mouth closed with the thumb to make its nose inhale hard so that the H1N1 virus solution in the nasal cavity is completely inhaled into the lungs (Figure 2B).</li>
		<li>Repeat steps 1.2.4 and 1.2.5 in the other nasal cavity of the mice.</li>
		<li>When all H1N1 virus prepared in step 1.2.1 is intranasally instilled into the mouse&#39;s lungs, place it in the supine position for rest (Figure 2C).</li>
	</ol>
	</li>
	<li>Measure the cough sensitivity of the mouse after model establishment using the Buxco small animal whole-body plethysmography system. On day 21, intraperitoneally anesthetize the mouse with pentobarbital sodium (150 mg&#183;kg-1). Collect and process blood, spleen, BALF, lung, and trachea tissues (Figure 1).</li>
</ol>
2. The cough sensitivity measurement
<ol>
	<li>Prepare citric acid (0.4 M): Place 0.1537 mg of citric acid into a 5 mL centrifuge tube and add normal saline to a volume of 2 mL.</li>
	<li>Instrument inspection
	<ol>
		<li>Check the channels: Connect the whole-body plethysmograph chambers according to the manufacturer&#39;s instructions and click the calibration button-the calibration button changes from orange to green, indicating that calibration is successful (Figure 3A).</li>
		<li>Calibrate the nebulizer:
		<ol>
			<li>After connecting the nebulizer, add 500 &#181;L of normal saline into the nebulizer and click the nebulization button.</li>
			<li>When the liquid in the nebulizer is completely aerosolized, click the nebulization button again to see the power of the nebulizer, which is generally about 0.3 mL/min. Click the nebulization button again to accept the current nebulized power.</li>
		</ol>
		</li>
		<li>After checking the channels, ensure the error value is less than 0.5%.</li>
	</ol>
	</li>
	<li>Setting parameters
	<ol>
		<li>Click Create New Study, select the Cough option, select Mouse in Species, and click Next.</li>
		<li>Select the CCnt parameters: Set the duration of the acclimation period to 1 min, the response time to 10 min, the aerosol volume to 1 mL, and the delivery duration to 10 min.</li>
		<li>Place conscious unrestrained mouse in individual plastic transparent whole-body plethysmograph chambers. Input the Weight and Subject ID of the mouse and click Next. 
		NOTE: Mice are thigmotaxic and do not need to habituate to the small chambers; hence, habituation is not performed before the experiment.</li>
		<li>Add 1 mL of citric acid (0.4 M) solution to the nebulizer and observe real-time changes in CCnt (Figure 3B).</li>
		<li>After citric acid is completely used, click File and End Session to complete the experiment.</li>
	</ol>
	</li>
</ol>
3. Harvesting of blood, spleen, BALF, lung, and trachea tissues of mouse (Figure 4)
<ol>
	<li>Collect blood.
	<ol>
		<li>After cough detection, anesthetize the mouse with pentobarbital sodium (150 mg&#183;kg-1) by intraperitoneal injection. Collect blood from the orbits of the anesthetized mouse. Mix 1 mL of blood in 0.1 mL of anticoagulant buffer (9.9 mg/mL heparin sodium dissolved in PBS) at 4 &#176;C (Figure 4A). 
		NOTE: The method of retroorbital blood harvesting is simple and can get a large volume of blood.</li>
		<li>Shake the blood collection tube to fully mix the blood and anticoagulant to prevent blood coagulation.</li>
		<li>Centrifuge the collected blood at 800 x g for 5 min at 4 &#176;C. Collect the supernatant, store it at -80 &#176;C (used for cytokine measurement), and resuspend the pellet in 1 mL of D-Hank&#39;s solution. Spread 10 &#181;L of the blood cell suspension on the glass slide to determine cell profiles.</li>
	</ol>
	</li>
	<li>Harvest the spleen.
	<ol>
		<li>Open the mouse&#39;s chest, collect the blood, and exsanguinate via the artery (Figure 4B).</li>
		<li>Remove the left auricle, and then perfuse the pulmonary and systemic circulation with 5 mL of normal saline (Figure 4C, D). Remove the whole spleen from the mouse using surgical forceps (Figure 4E).</li>
		<li>After measuring the weight of the spleen, cut it into two halves. Fix the first half with 4% paraformaldehyde at room temperature (RT) for histopathological analysis. Store the second half at -80 &#176;C for cytokine measurement.</li>
	</ol>
	</li>
	<li>Bronchoalveolar lavage
	<ol>
		<li>Make the bronchoalveolar lavage tube using a Pasteur pipet. Heat the Pasteur pipette using an alcohol lamp. When it becomes soft, lengthen it to form a thin tube. Glue the catheter portion of a slip-tip catheter to the thin tube. The length of the bronchoalveolar lavage tube is 5 cm. The upper diameter is 5 mm, and the lower diameter is 1 mm (Figure 5). 
		NOTE: This bronchoalveolar lavage tube is a viable tool for BALF collection.</li>
		<li>For BALF collection, separate the right lung by ligating at the right mainstem bronchus. Collect the BALF from the left lungs by lavaging three times with 0.5 mL of PBS pre-cooled on ice. The recovery ratio of BALF is more than 80% (Figure 4F).</li>
		<li>Centrifuge the collected BALF at 800 x g for 5 min at 4 &#176;C. Collect the supernatant and measure the total protein concentration, uric acid concentration, LDH activity, and cytokine concentrations. 
		NOTE: The inflammatory markers, including total protein concentration, the uric acid concentration, and the LDH activity in the BALF supernatant, are detected using the assay kit according to the manufacturer&#39;s guidelines24.</li>
		<li>Resuspend the pellet in 200 &#181;L of PBS, and count the leukocytes in BALF with the help of a counting slide.</li>
		<li>To determine cell profiles by differential counts, smear 50 &#181;L of the cell suspension onto the glass slides and allow it to dry.</li>
		<li>Fix the slide with 4% paraformaldehyde overnight, and then stain with the hematoxylin-eosin (HE). Classify at least 400 cells into neutrophils, macrophages, lymphocytes, or eosinophils per slide.</li>
	</ol>
	</li>
	<li>Harvesting the lung and trachea tissues for histopathological analysis
	<ol>
		<li>After BALF collection, remove and fix half of the right lung (Figure 4G) and trachea (Figure 4H) with 4% paraformaldehyde at RT for histopathological analysis. Store the other half at -80 &#176;C for western blotting, Real-time quantitative polymerase chain reaction (qPCR), and Enzyme-linked immunosorbent assay (ELISA).</li>
	</ol>
	</li>
</ol>

Noninvasive Real-Time Whole-Body Plethysmography for Cough Sensitivity in Mice

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The authors have nothing to disclose.

Some chronic refractory and postinfectious coughs are common conditions associated with respiratory virus infection27. In order to better evaluate cough sensitivity and airway inflammation, a mouse model of cough was established using the H1N1 virus in this study. Appropriate mouse cough models should be selected for other studies according to the purpose of the study. Most previous studies used the guinea pig as an animal model in mechanistic studies or new drug trials for cough...

Cough is an important defense behavior to maintain airway patency and protect the lungs from potentially harmful substances. However, when dysregulated, cough becomes a pathological condition1. Chronic cough, usually defined as lasting eight or more weeks, is one of the most frequent symptoms requiring medical attention2. Because chronic cough frequently persists for years, patients suffer from a huge socioeconomic burden and a marked decrement in quality of life3,4,5. Chronic cough is widely considered a cough hypersensitivity syndrome and is characterized by troublesome coughing often triggered by low levels of thermal, mechanical, or chemical exposure6. The occurrence of cough hypersensitivity is closely related to airway inflammation7. However, the pathophysiological mechanisms underlying the modulation of cough sensitivity need to be further elucidated.
Animal models can mimic the complex pathophysiology of the cough and are important tools for cough research8,9. Previous studies have found that viral infection, intrapulmonary Interferon-&#947; (IFN-&#947;) instillation, esophageal perfusion of hydrochloric acid, pollutant exposure, cigarette smoke, and citric acid can induce cough in animals10,11,12,13,14,15,16,17. In order to better evaluate cough and airway inflammation, a mouse model of cough was established using a nonlethal dose of the H1N1 virus in this study. For the detection of cough, some cough measurement tools have been established clinically to measure cough, including subjective and objective methods18. The subjective evaluation tools to assess the cough severity primarily include a visual analog scale, the cough score, and quality of life questionnaires, etc19,20. However, they are unlikely to be used to assess cough in animals. In addition, cough can be objectively assessed through a cough challenge test and cough frequency monitoring. The cough challenge test using a whole-body plethysmography (WBP) system is an objective method widely used in animal studies to measure cough sensitivity and reveal the underlying mechanisms of cough13,16. Based on the neuroanatomical characteristics of the cough reflex, citric acid, capsaicin, adenosine 5&#39;-triphosphate (ATP), allyl isothiocyanate (AITC), and inflammatory mediator bradykinin are commonly used as the tussive agents to induce cough21,22. Citric acid is one of the earliest and the most widely used tussive agents triggering cough re&#64258;exes, which has been validated for measuring cough sensitivity. Besides, the citric acid challenge has good safety, feasibility, and tolerability and is suggested to assess cough reflex sensitivity in response to cough therapies23. Therefore, this article will describe the method for measuring cough sensitivity in response to citric acid in mice using a noninvasive and real-time WBP system.
The studies of the pathophysiology of cough require test samples, including samples from the blood, bronchoalveolar lavage fluid (BALF), and lung and trachea tissues to verify changes in the levels of key factors24. Currently, there is a lack of normative procedures for harvesting tissue samples of mice, and related studies employ different approaches that complicate the assessment of airway inflammation. Bronchoalveolar lavage is an important method to evaluate airway inflammation in respiratory diseases25. Different methods of bronchoalveolar lavage will lead to a lack of comparability between related studies. Moreover, different bronchoalveolar lavage methods have an impact on inflammatory cells and inflammatory cytokines in BALF. Therefore, this article will describe the establishment of a mouse model of cough with a nonlethal dose of the H1N1 virus, the measurement of cough using a WBP system, and a reliable, safe, and highly successful bronchoalveolar lavage method of mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% paraformaldehyde&#160;</td>
			<td>Biosharp</td>
			<td>BL539A</td>
			<td></td>
		</tr>
		<tr>
			<td>Buxco Small Animal Whole Body Plethysmography System&#160;</td>
			<td>DSI</td>
			<td>&#8212;</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium-free and magnesium-free Hank&#8217;s Balanced Salt Solution</td>
			<td>Beyotime</td>
			<td>C0219</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C2404</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin-Eosin</td>
			<td>BASO Biotechnology</td>
			<td>BA-4098</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium&#160;</td>
			<td>Alfa Aesar</td>
			<td>A16198</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A/California/7/2009 (H1N1) virus</td>
			<td>ATCC</td>
			<td>VR-1894</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>R510-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactate dehydrogenase assay kit</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A020-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>Guangzhou Zhongbo Biotechnology</td>
			<td>1234-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet</td>
			<td>NEST</td>
			<td>318415</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Merck</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160;</td>
			<td>Meilunbio</td>
			<td>MA0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Total protein assay kit</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A045-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Uric acid assay kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A22181</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for detecting cough and airway inflammation in mice

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge socioeconomic burden and a marked decrement in quality of life. Animal models can mimic the complex pathophysiology of the cough and are important tools for cough research. The detection of cough sensitivity and airway inflammation is of great significance for studying the complex pathological mechanism of cough. This article describes the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples (including blood, lung, spleen, and trachea) of mice. It introduces some methods to assess airway inflammation, including pathological changes in hematoxylin and eosin (HE)-stained lung and trachea sections, the total protein concentration, the uric acid concentration, and the lactate dehydrogenase (LDH) activity in the supernatant of bronchoalveolar lavage fluid (BALF), and the leukocytes and differential cell counts of BALF. These methods are reproducible and serve as valuable tools to study the complex pathophysiology of cough.

Figure 6 shows representative images of pathological changes in HE-stained lung (Figure 6A,B), trachea (Figure 6C,D), and spleen (Figure 6E,F). H1N1 virus infection resulted in inflammatory changes in mouse lungs, including edema and many lymphocytes and neutrophil infiltration. H1N1 virus infection also induced inflammatory changes in mouse tracheae, i...

Methods for Detecting Cough and Airway Inflammation in Mice

Here, we describe the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples of mice and introduce some methods to assess airway inflammation.

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge ...

methods-for-detecting-cough-and-airway-inflammation-in-mice

Research

JoVE Journal

Medicine

471 Views.  The First Affiliated Hospital of Guangzhou Medical University. Here, we describe the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples of mice and introduce some methods to assess airway inflammation.

Video: Methods for Detecting Cough and Airway Inflammation in Mice

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.

This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances.
Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a &#39;sepsis-like&#39; state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations.

Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...