This study was supported by the National Natural Science Foundation of China (NSFC 82100034), Guangzhou Science and Technology Planning Project (202102010168).

All animal experiment procedures were approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (20230656). Adult male specific-pathogen-free C57BL/6 mice, aged 8-10 weeks and weighing 20-25 g, were used in this study. The details of the reagents and equipment used are listed in the&#160;Table of Materials.
1. Chemical reagent preparation
<ol>
	<li>Citric acid (CA) solution preparation
	<ol>
		<li>For cough assessment, prepare a 0.4 M solution by dissolving 3.84 g of citric acid powder in normal saline to a final volume of 50 mL.</li>
		<li>For animal treatment, prepare a 0.1 M solution by dissolving 9.6 g of citric acid powder in normal saline to a final volume of 500 mL.</li>
	</ol>
	</li>
	<li>Capsaicin solution preparation
	<ol>
		<li>Dissolve 30.5 mg of capsaicin powder in PBS containing 10% ethanol and 10% Tween-80 to make a 10 mM stock solution.</li>
		<li>Dilute the stock solution 1:100 in normal saline to make a 100 &#181;M working solution.</li>
	</ol>
	</li>
	<li>Methacholine solution preparation
	<ol>
		<li>Dissolve 100 mg of methacholine powder in PBS to a final volume of 2 mL to make a 50 mg/mL solution.</li>
		<li>Dilute this solution with PBS to obtain concentrations of 25 mg/mL, 12.5 mg/mL, 6.25 mg/mL, and 3.125 mg/mL.</li>
	</ol>
	</li>
</ol>
2. Animal preparation
<ol>
	<li>Housing conditions
	<ol>
		<li>House mice in cages with free access to food and water, maintained at a temperature of 22 &#177; 1 &#176;C with a standard 12-h light/dark cycle. Experimental manipulations were performed after 1 week of acclimation to the feeding environment.</li>
	</ol>
	</li>
	<li>Acclimation
	<ol>
		<li>Allow mice to acclimate to the feeding environment for 1 week before conducting experimental manipulations.</li>
	</ol>
	</li>
</ol>
3. Development of the model
<ol>
	<li>Group assignment
	<ol>
		<li>Randomly divide the mice into a model group and a control group, with 8 mice in each group.</li>
	</ol>
	</li>
	<li>Exposure setup
	<ol>
		<li>Place the mice in two independent chambers (as shown in&#160;Figure 1), each connected to an ultrasonic nebulizer.</li>
		<li>Expose the model group to 0.1 M citric acid (CA) aerosol and the control group to 0.9% normal saline (NS) aerosol at an atomization rate of 3 mL/min for 2 h per day over 2 weeks (as shown in Figure 2). Return the mice to their cages after each exposure.</li>
	</ol>
	</li>
	<li>Cough sensitivity assessment
	<ol>
		<li>After the last exposure, assess cough sensitivity by evaluating reflexive cough (challenged with NS, CA (0.4 M), and capsaicin (100 &#181;M)) and spontaneous cough (without any challenge). 
		NOTE: To monitor dynamic changes in cough sensitivity during model development, assess cough sensitivity on days 0, 3, 7, and 10 using a CA (0.4 M) challenge.</li>
	</ol>
	</li>
</ol>
4. Cough sensitivity assessment
<ol>
	<li>Instrument preparation
	<ol>
		<li>Use a non-invasive whole-body plethysmography (WBP) system to measure cough sensitivity. Connect the chambers, flow transducers, bias flow, and other components according to the application manual (see Table of Materials).</li>
		<li>Perform a calibration process to ensure accurate measurements by clicking on the Calibrate button. A status screen will display the progress, and once the status bar reaches 100%, the calibration is complete.</li>
	</ol>
	</li>
	<li>Cough detection
	<ol>
		<li>Create a cough study for mice and configure the parameters for 1 min of acclimation, 10 min of response time, and 10 min of chemical solution delivery.</li>
		<li>Place the conscious mice into individual chambers, ensuring there is only one mouse per chamber. 
		NOTE: Make sure the mouse is completely inside the chamber before closing the lid to avoid damaging its tail and digits.</li>
		<li>Add 1 mL of the chemical solutions (NS, CA, or capsaicin) to the nebulizer. 
		NOTE: The number of cough events-each consisting of three phases: inspiratory, compressive, and expulsive15 (as shown in Figure 3)-will be recorded once the study begins. After 10 min of recording, cough detection will be complete. Clean the chamber after detecting each animal to avoid cross-contamination.</li>
	</ol>
	</li>
</ol>
5. Airway Hyperresponsiveness (AHR) measurement
<ol>
	<li>Instrument preparation
	<ol>
		<li>Prepare instruments as described in step 4.1.</li>
	</ol>
	</li>
	<li>AHR measurement
	<ol>
		<li>Create a dose-response study for mice, configuring the measurement types, parameters, and task sequence: 1 min for acclimation, 30 s for methacholine solution delivery, 3 min for response time, and 1 min for recovery.</li>
		<li>Place the conscious mice into individual chambers, ensuring there is only one mouse per chamber.</li>
		<li>Add 50 &#181;L of the chemical solutions (PBS or methacholine) to the nebulizer and initiate the study.</li>
		<li>Record the value of Penh, an indicator of bronchoconstriction, in response to different concentrations of methacholine (0 mg/mL, 3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL, 25 mg/mL, 50 mg/mL).</li>
	</ol>
	</li>
</ol>
6. Bronchoalveolar lavage collection
<ol>
	<li>Sacrifice
	<ol>
		<li>Sacrifice the mice using hyperanesthesia with pentobarbital sodium (50 mg/kg) administered via intraperitoneal injection (following institutionally approved protocols).</li>
	</ol>
	</li>
	<li>Sample collection
	<ol>
		<li>Collect blood from the orbital sinus of the mouse into a 1.5 mL microcentrifuge tube, then place the tube on ice. Centrifuge the blood at 3000 x g for 10 min at 4&#176;C. Use a pipette to collect the supernatant, aliquot it, and store it at -80 &#176;C.</li>
		<li>Collect bronchoalveolar lavage fluid (BALF) by opening the chest to expose the trachea, inserting a 22 G indwelling needle into the trachea, then lavaging three times with 0.5 mL of pre-cooled PBS and collecting the fluid.</li>
		<li>After collection, centrifuge the BALF at 500 x g for 10 min at 4 &#176;C. Collect the supernatant, aliquot it, and store it at -80 &#176;C. Resuspend the pellet in 100 &#181;L of PBS and prepare cell smears for further analysis.</li>
		<li>Collect lung tissues by opening the chest to expose the lungs, removing the lung tissues, and placing them into cryogenic vials. Store the vials at -80 &#176;C for future studies.</li>
	</ol>
	</li>
</ol>
7. Quantitative RT-PCR
<ol>
	<li>Extract total RNA from lung tissue using TRIzol reagent. Perform cDNA synthesis using a First-strand cDNA synthesis kit, following the manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Conduct PCR in a real-time quantitative PCR detection system using SYBR green fluorescence. Normalize the quantification of relative SP and CGRP mRNA expression to the expression of &#946;-Actin9.</li>
</ol>
8. Statistical analysis
<ol>
	<li>Present data as the mean &#177; SEM. Use a t-test to compare data between two groups, considering P &#60; 0.05 as statistically significant. Conduct all statistical analyses using statistical and graphing software.</li>
</ol>

Citric Acid-Induced Mouse Model for Cough Hypersensitivity

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<li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+cough+hypersensitivity+with+tracheal+trpv1+activation+and+neurogenic+inflammation+in+a+novel+guinea+pig+model+of+citric+acid-induced+chronic+cough%5BTitle%5D)+AND+%22J+Int+Med+Res%22%5BJournal%5D)">Association of cough hypersensitivity with tracheal trpv1 activation and neurogenic inflammation in a novel guinea pig model of citric acid-induced chronic cough.</a> J Int Med Res. 46 (7), 2913-2924 (2018).</li>
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<li>Zhang, C., Lin, R. L., Hong, J., Khosravi, M., Lee, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cough+and+expiration+reflexes+elicited+by+inhaled+irritant+gases+are+intensified+in+ovalbumin-sensitized+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Regul+Integr+Comp+Physiol%22%5BJournal%5D)">Cough and expiration reflexes elicited by inhaled irritant gases are intensified in ovalbumin-sensitized mice.</a> Am J Physiol Regul Integr Comp Physiol. 312 (5), R718-R726 (2017).</li>
<li>Amato, A., Becci, A., Beolchini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Citric+acid+bioproduction%3A+The+technological+innovation+change%5BTitle%5D)+AND+%22Crit+Rev+Biotechnol%22%5BJournal%5D)">Citric acid bioproduction: The technological innovation change.</a> Crit Rev Biotechnol. 40 (2), 199-212 (2020).</li>
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The authors have nothing to disclose.

This study successfully established a mouse model with cough hypersensitivity through continuous inhalation of citric acid (CA). This model demonstrated a reliable increase in cough sensitivity for both spontaneous coughs and reflexive coughs elicited by citric acid and capsaicin. Citric acid and capsaicin are widely used to assess cough reflex sensitivity16.
Several critical steps in this protocol ensure...

Cough is a crucial defensive reflex that helps clear respiratory secretions or foreign materials from the airway. However, it is also one of the most common symptoms of many respiratory diseases, often prompting patients to seek medical attention1. Chronic cough, defined as a persistent cough lasting more than 8 weeks in adults, significantly impacts the quality of life, causing issues such as incontinence, insomnia, reflux, and other unpleasant experiences, along with a substantial economic burden2,3,4. It is widely believed that increased cough sensitivity is a pathophysiological hallmark of chronic cough, where low levels of thermal, mechanical, and chemical irritants can trigger coughing5. Cough hypersensitivity is associated with airway inflammation6, remodeling of airway sensory nerves7, and alterations in the central nervous system8, though the precise molecular mechanisms remain unclear and require further elucidation through suitable animal models.
Various animals, including guinea pigs, cats, rabbits, dogs, and pigs, have been used to study the mechanisms of cough9. Guinea pigs have traditionally been recognized as the most suitable model for studying cough mechanisms and the efficacy of antitussive drugs9,10,11,12. However, these models have several experimental limitations, including high costs, a lack of transgenic tools, and a scarcity of commercial reagents. Additionally, guinea pigs often exhibit poor environmental tolerance and high mortality when exposed to stimuli. In contrast, mice are smaller, easier to maintain, more cost-effective, and amenable to genetic manipulation, making them more suitable for mechanistic investigations. Previous studies on cough models have primarily focused on cough induced by airway inflammation, mainly used to evaluate the efficacy of antitussive drugs and peripheral mechanisms13,14. There is currently a lack of animal models for cough hypersensitivity.
In response, we introduce a method for establishing a mouse model of cough hypersensitivity&#160;via&#160;continuous inhalation of citric acid (CA). This model is simpler, easier to construct, and more feasible compared to other animal models.

<table><tbody><tr><td>0.9% normal saline</td><td>Biosharp</td><td>BL158A</td><td></td></tr><tr><td>Capsaicin</td><td>Cayman chemical</td><td>92350</td><td></td></tr><tr><td>Citric Acid</td><td>Sigma-Aldrich</td><td>C2404</td><td></td></tr><tr><td>Ethanol</td><td>Guangzhou chemical reagent factory</td><td>GSHB15-AR-0.5L</td><td></td></tr><tr><td>First-strand cDNA synthesis kit</td><td>TransGen Biotech</td><td>AT341</td><td></td></tr><tr><td>Methacholine</td><td>Sigma-Aldrich</td><td>A2251</td><td></td></tr><tr><td>Non-invasive whole-body plethysmography (WBP) system</td><td>DSI</td><td>601-1400-001</td><td></td></tr><tr><td>Pentobarbital sodium</td><td>Merk</td><td>P3761</td><td></td></tr><tr><td>PerfectStart Green qPCR SuperMix</td><td>TransGen Biotech</td><td>AQ601</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Meilunbio</td><td>MA0015</td><td></td></tr><tr><td>Real-time&amp;nbsp;quantitative&amp;nbsp;PCR&amp;nbsp;detecting&amp;nbsp;system</td><td>Bio-rad&amp;nbsp;</td><td>CFX&amp;nbsp; Connect</td><td></td></tr><tr><td>TRIzol reagent</td><td>Invitrogen</td><td>15596026CN</td><td></td></tr><tr><td>Tween-80</td><td>Solarbio</td><td>T8360-100</td><td></td></tr><tr><td>Ultrasonic nebulizer</td><td>Yuwell</td><td>402AI</td><td></td></tr></tbody></table>

establishment of a mouse model with cough hypersensitivity via inhalation of citric acid

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable economic burden. Increased cough sensitivity is a pathophysiological hallmark of chronic cough. It has been observed that cough hypersensitivity is related to airway inflammation, remodeling of airway sensory nerves, and alterations in the central nervous system. However, the precise molecular mechanisms remain unclear and require further elucidation using suitable animal models. Previous studies have utilized guinea pigs as models for studying cough, but these models present several experimental limitations, including high costs, a lack of transgenic tools, and a scarcity of commercial reagents. In addition, guinea pigs typically exhibit poor environmental tolerance and high mortality when exposed to stimuli. In contrast, mice are smaller, easier to maintain, more cost-effective, and amenable to genetic manipulation, making them more suitable for mechanistic investigations. In this study, we established a mouse model with cough hypersensitivity&#160;via&#160;continuous inhalation of citric acid (CA). This model is straightforward to operate and yields reproducible results, making it a valuable tool for further studies on the mechanisms and potential novel treatments for chronic cough.

As shown in&#160;Figure 4A, cough sensitivity in the model group (CA group) significantly increased after 1 week of exposure compared to the control group (NS group), and this heightened sensitivity persisted throughout the exposure period. Neither the control group nor the model group mice experienced mortality during the modeling process (Figure 4B).&#160;Figure 4C and Figure 4D demonstrate that the number of spontaneous cough...

Establishment of a Mouse Model with Cough Hypersensitivity via Inhalation of Citric Acid

This protocol describes the development of a mouse model with cough hypersensitivity, which can serve as an ideal model for studying the mechanisms of chronic cough.

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable ...

establishment-mouse-model-with-cough-hypersensitivity-via-inhalation

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Medicine

443 Views.  Guangzhou Medical University. Dit protocol beschrijft de ontwikkeling van een muismodel met hoestovergevoeligheid, dat als ideaal model kan dienen voor het bestuderen van de mechanismen van chronische hoest.

Video: Auteur Spotlight: Onderzoek naar de mechanismen van chronische hoest en overgevoeligheid voor hoesten: een kosteneffectief en genetisch aanpasbaar hulpmiddel voor 

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Establishment of a Clinic-based Biorepository

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is expected that there will be a parallel demand for cutaneous tumor samples for biomedical research studies. Tissue availability, however, is limited due the cost of establishing a biorepository and the lack of protocols available for obtaining clinical samples that do not interfere with clinical operations. A protocol was established to collect and process cutaneous tumor and associated blood and saliva samples that has minimal impact on routine clinical procedures on the date of a Mohs surgery. Tumor samples are collected and processed from patients undergoing their first layer of Mohs surgery for biopsy-proven cutaneous malignancies by the Mohs histotechnologist. Adjacent normal tissue is collected at the time of surgical closure. Additional samples that may be collected are whole-blood and buccal swabs. By utilizing tissue samples that are normally discarded, a biorepository was generated that offers several key advantages by being based in the clinic versus the laboratory setting. These include a wide range of collected samples; access to de-identified patient records, including pathology reports; and, for the typical donor, access to additional samples during follow-up visits.

Cutaneous tumors are often discarded following Mohs micrographic surgery. A protocol is described here that enables clinical support staff to effectively process and store cutaneous tumor (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) samples for downstream laboratory applications without interfering with clinical operations.

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and treatment of silicosis in humans have all been extensively studied using the mouse silicosis model. By repeatedly making mice inhale CSD into their lungs, the mice can mimic the clinical symptoms of human silicosis. This methodology is practical and efficient in terms of time and output and does not cause mechanical injury to the upper respiratory tract due to surgery. Furthermore, this model can successfully mimic acute/chronic transformation process of silicosis. The main procedures were as follows. The sterilized 1-5 &#181;m CSD powder was fully ground, suspended in saline, and dispersed in an ultrasonic water bath for 30 min. Mice under isoflurane-induced anesthesia switched from shallow rapid breathing to deep, slow aspiration for approximately 2 s. The mouse was placed in the palm of a hand, and the thumb tip gently touched the lip edge of the mouse's jaw to straighten the airway. After each exhalation, the mice breathed in the silica suspension drop by drop through one nostril, completing the process within 4-8 s. After the mice's breathing had stabilized, their chest was stroked and caressed to prevent the inhaled CSD from being coughed up. The mice were then returned to the cage. In conclusion, this model can quantify CSD along the typical physiological passage of tiny particles into the lung, from the upper respiratory tract to the terminal bronchioles and alveoli. It can also replicate the recurrent exposure of employees due to work. The model can be performed by one person and does not need expensive equipment. It conveniently and effectively simulates the disease features of human silicosis with high repeatability.

This protocol describes a method for establishing a mouse model of silicosis through repeated exposure to silica suspensions via a nasal drip. This model can efficiently, conveniently, and flexibly mimic the pathological process of human silicosis with high repeatability and economy.

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and ...

Modified Arteriovenous Fistula Surgery to Establish the RV Volume Overload Mouse Model

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

Author Spotlight: Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV ...

Development of an Effective Mice Model for Studying Oronasal Fistulas

Establishment of an Oronasal Fistula Mice Model

This study presents a method utilizing heated ophthalmologic cautery to develop a viable model for investigating oronasal fistulas. C57BL/6 mice were used to establish the oronasal fistula (ONF) model. To create the ONF, the mice were anesthetized, immobilized, and their hard palates were exposed. During the surgical procedure, a 2.0 x 1.5 mm full-thickness mucosal injury was induced in the midline of the hard palate using ophthalmologic cautery. It was crucial to control the size of the ONF and minimize bleeding in order to ensure the success of the experiment. Verification of the ONF model's effectiveness was conducted on the 7th-day post-operation, encompassing both anatomical and functional assessments. The presence of the nasal septum within the oral cavity and the outflow of sterile water from the nostrils upon injection into the oral cavity confirmed the successful establishment of the ONF model. The model demonstrated a practical and successful oronasal fistula, characterized by a low mortality rate, significant weight changes, and minimal variation in ONF size. Future studies may consider adopting this methodology to elucidate the mechanisms of palate wound healing and explore novel treatments for oronasal fistulas.

Author Spotlight: Investigating Wound Healing in Mice Models of Oronasal Fistulas 

This article outlines a step-by-step procedure for establishing a mice model with an oronasal fistula. The oronasal fistula was created by employing heated ophthalmologic cautery to damage the midline portion of the hard palate, resulting in the formation of an opening between the oral and nasal cavities.

This study presents a method utilizing heated ophthalmologic cautery to develop a viable model for investigating oronasal fistulas. C57BL/6 mice were used ...

Establishment of a Mouse Model with Cough Hypersensitivity via Inhalation of Citric Acid

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