Birte Ohm is supported by the Berta-Ottenstein-Program for Clinician Scientists, Faculty of Medicine, University of Freiburg. Steffen U Eisenhardt is a Heisenberg Professor of the German Research Foundation (DFG) and supported this work with personal grants. Furthermore, we would like to thank Sheena Kreuzaler for her technical assistance. Figure 1 was created with the help of Biorender.com.&#160;Imaging was performed at the Lighthouse Core Facility, which is funded in part by the Medical Faculty, University of Freiburg (Project Numbers 2023/A2-Fol; 2021/B3-Fol), the DKTK, and the DFG (Project Number 450392965).

All animal protocols comply with the ethical principles of the 3Rs for humane animal research and were approved by the local veterinary ethical committee (Veterin&#228;ramt Kanton Z&#252;rich, Switzerland, Study number 45/2014). Likewise, readers should obtain permission from the relevant institutions before performing any procedures on laboratory animals.
1. Sample acquisition
<ol>
	<li>Conduct animal experiments according to individual needs and respective ethical approval. A single microsurgeon performs all surgeries without any additional prolonged ischemia times. 
	NOTE:&#160;For a reproduction of our representative results, obtain specific pathogen-free adult male C57BL/6J, C57BL/10J, and BALB/c mice weighing 27-30 g from Charles River Laboratories. For isografts, use C57BL/6J animals as both donors and recipients. In the minor mismatched model, transplant C57BL/10J donor lungs to C57BL/6J recipients. In the major mismatched model, transplant BALB/c donor lungs to C57BL/6J recipients and postoperatively inject cyclosporine (10 mg/kg/day) and methylprednisolone (1.6 mg/kg/day) subcutaneously daily5. Use saline solution for perfusion and retrieve lungs after 8 weeks (56 days).</li>
	<li>For euthanasia, intubate the animal with a 20G catheter and ventilate it with O2 supplemented with 2%-3% isoflurane. Confirm sufficient depth of anesthesia by the absence of a response to a paw pinch.</li>
	<li>Perform a median laparosternotomy by incising the abdominal wall with surgical scissors and cutting the length of the sternum to expose the heart. Identify the inferior vena cava and the left atrial appendage by their anatomical location.</li>
	<li>In quick succession, cut the inferior vena cava and the left atrial appendage with surgical scissors and perfuse the animal with 3-5 mL of 0.9% NaCl through the root of the pulmonary artery with a syringe to remove all blood from the pulmonary circulation. 
	NOTE: Euthanasia should be performed according to the respective ethical approval. Other methods can be equally applied. It is recommended that blood from the pulmonary circulation be thoroughly removed, which can also be performed by perfusion after circulatory arrest.</li>
	<li>Remove the perfused lungs using scissors.</li>
	<li>For conservation, incubate the samples in 4% formalin. Inject the formalin solution directly into the bronchus and pulmonary vasculature. Submerge the samples in formalin and let them incubate for at least 6 h. 
	NOTE: For inflation of the lung with formalin, a standardized procedure should be applied to obtain comparable results. Example protocols for such procedures are published elsewhere10,11.</li>
	<li>Trim the organ according to experimental needs. Transversally cut the lungs in half to capture all parts of the bronchial tree in a section.</li>
	<li>Transfer samples to an automatic tissue processor for dehydration in alcohol sequence, clearing in xylol and paraffin coating as follows: 70% ethanol, 70% ethanol, 80% ethanol, 96% ethanol, 100% ethanol (1 h each), 100% ethanol, 100% ethanol, xylol (2 h each), xylol (1 h), and paraffin 3x for 2 h.</li>
	<li>Melt paraffin and embed samples in the correct orientation in Polyoxymethylen embedding cassettes. Take care to embed the samples with the cut surface facing downwards. Use a rotary microtome to cut 5 &#181;m sections consecutively. Let the slides dry for 24 h at 37 &#176;C before further processing.</li>
</ol>
2. Deparaffinization and hydration of paraffin slides
<ol>
	<li>Remove paraffin by incubating the samples 2x in 100% xylol for 5 min each. 
	CAUTION: Xylol is flammable and hazardous to health. Only use under the fume hood.</li>
	<li>Hydrate the samples in 100% ethanol, 100% ethanol, 95% ethanol, 70% ethanol, and 50% ethanol for 2 min each. Then, wash for 2 min in distilled water.</li>
</ol>
3. Staining with Picrosirius Red
<ol>
	<li>Stain slides for 8 min in Meyer&#39;s hemalum solution. Wash slides for 10 min under running tap water.</li>
	<li>Incubate slides for 1 h in Picrosirius Red solution. For Picrosirius Red solution, either use a premixed staining solution or use 0.1% of Direct Red 80 or Sirius Red F 3B (CI 35780) diluted in saturated picric acid. 
	CAUTION: Picric acid is flammable and highly explosive and should be treated carefully. 
	NOTE: There are many different suppliers for Picrosirius Red staining kits available, as listed in the Table of Materials. Be aware of differences in price and staining methods, and always adhere to suppliers&#39; instructions. It is recommended to only use staining kits with the specific color index for Sirius Red F 3B (CI 35780).</li>
	<li>Dip the slides 5x-10x in an acetic acid solution (0.5%). Gently shake the slides to physically remove the solution.</li>
	<li>Dehydrate the slides in 95% ethanol, 100% ethanol, 100% ethanol, 100% xylol, and 100% xylol for 5 min each.</li>
	<li>Embed by adding one drop of mounting medium on each sample and cover with a coverslip. Let slides dry under a fume hood.</li>
</ol>
4. Digitalization and image processing
<ol>
	<li>Focus the samples under a light microscope with appropriate imaging software until the picture becomes clear and sharp. Add a polarizing filter and adjust the degree of polarization until the background is completely dark or black. When the background is as dark as possible, the correct polarization is achieved. 
	NOTE: It is also possible to visualize Picrosirius Red staining with conventional transmitted light and without a polarizing filter. For this purpose, it is recommended to use Sirius Red with Fast Green, as it has been shown to be more sensitive than Sirius Red alone12,13. However, polarizing microscopy is necessary to distinguish between thick and thin collagen fibers.</li>
	<li>Scan all samples completely under 20x magnification and export as .tiff. Transfer to Fiji software14. Make sure to clean slides properly in advance and use standardized settings regarding exposure time and light source intensity.</li>
	<li>Surround the sample manually and cut it out in the software using Edit &#62; Clear Outside. Measure the total surface area by clicking Analyze &#62; Measure. 
	NOTE: The selection of the compartment to measure is a critical step in creating reliable and comparable results. By digitalizing and selecting the entire sample, a good cross-section is displayed, and heterogeneous tissues are considered as a whole. However, it is also possible to confine oneself to specific areas within the samples, such as pleura or individual bronchi. Keep in mind to always select compartments to compare in a standardized fashion. Should the samples show a large variability in ventilation, it is advisable to exclude the airspace area from the analyzed compartment15.</li>
	<li>Measure total collagen content per sample using color threshold by clicking Image &#62; Adjust &#62; Color Threshold and adjust the settings by clicking Analyze &#62; Analyze Particles. Set Size = 1 - Infinity, Clear, Summarize. Brightness = 35 - 255. Hue = 2 - 130 
	NOTE: A reasonable setting of the color thresholds is critical to achieve reliable results.</li>
	<li>Repeat for thick and thin collagen fibers by adjusting Hue as Red <img alt="Equation 1" src="/files/ftp_upload/66395/66395eq01.jpg" style="margin: auto;" /> Thick Collagen Fibers (Hue 2-30) and Yellow-Green <img alt="Equation 1" src="/files/ftp_upload/66395/66395eq01.jpg" style="margin: auto;" /> Thin Collagen Fibers (Hue 31-130). The result will be the area of stained pixels.</li>
	<li>Configure Macro&#160;for automated processing by clicking Plugins &#62; Macros &#62; Record and repeat with all samples.</li>
	<li>To receive final values standardized to the cross-section&#39;s surface area, divide the area of stained pixels for all parameters by the total surface area as measured in steps 4.3 and 4.5 for each sample, respectively.</li>
</ol>

Collagen Assessment in Murine Pulmonary Fibrosis Using Picrosirius Red Staining

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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+lung+transplant+research:+A+consensus+statement+from+the+National+Heart,+Lung,+and+Blood+Institute+workshop.">Models of lung transplant research: A consensus statement from the National Heart, Lung, and Blood Institute workshop.</a> JCI Insight. 2 (9), e93121 (2017).</li><li>Martinu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+chronic+lung+allograft+pathology+in+a+mouse+minor-mismatched+orthotopic+lung+transplant+model.">Spectrum of chronic lung allograft pathology in a mouse minor-mismatched orthotopic lung transplant model.</a> Am J Transplant. 19 (1), 247-258 (2019).</li><li>de Vleeschauwer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+rejection+pathology+after+orthotopic+lung+transplantation+in+mice:+The+development+of+a+murine+BOS+model+and+its+drawbacks.">Chronic rejection pathology after orthotopic lung transplantation in mice: The development of a murine BOS model and its drawbacks.</a> PLoS One. 7 (1), e29802 (2012).</li><li>Yamada, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+airway+fibrosis+in+orthotopic+mouse+lung+transplantation+models-An+experimental+reappraisal.">Chronic airway fibrosis in orthotopic mouse lung transplantation models-An experimental reappraisal.</a> Transplantation. 102 (2), e49-e58 (2018).</li><li>Whittaker, P., Kloner, R. 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The authors have nothing to disclose.

Standard histologic methods such as Hematoxylin and Eosin, and Masson&#39;s Trichrome staining are widely used to detect fibrotic changes in murine lungs in a spatially resolved manner16,19. However, additional methods are often necessary to quantify these changes and evaluate the tissue&#39;s collagen composition.
Picrosirius Red staining was first described in 1964 to identify collagen: under transmitted light microscopy, collagen ap...

Lung transplantation is the definite therapeutic option for patients suffering from end-stage lung disease. However, long-term survival is hampered by chronic rejection, affecting 50% of recipients within the first five postoperative years1. Fibrotic remodeling of the small airways and the lung parenchyma is the histologic hallmark underlying the progressive loss of pulmonary function in chronic lung allograft rejection2. Experimentally, chronic lung allograft rejection can be modeled with murine orthotopic lung transplantation between MHC-mismatched mouse strains. Different strain combinations have been proposed to achieve the phenotype of chronic rejection3. Among them, the transplant combination of the minor MHC mismatched C57BL/10 donor and C57BL/6 recipient is frequently used4. Alternatively, BALB/c donor lungs can be transplanted to C57BL/6 recipients receiving daily immunosuppressive treatment5. These models result in histologically different degrees of fibrotic changes6. These changes are often reported qualitatively using the standard Hematoxylin and Eosin, and Masson&#39;s Trichrome staining.
The standard histological staining with Hematoxylin and Eosin allows for an overview of the specimen as it stains nuclei blue, while cytoplasm and collagen fibers appear red. With Masson&#39;s Trichrome staining, nuclei are stained black, collagen fibers are stained green to blue, and the background, including cytoplasm, fibrin, and muscles, are red. Because Masson&#39;s Trichrome stain may lead to underestimated values, using Picrosirius Red stain is considered to be beneficial7. Picrosirius Red is a linear anionic dye that associates with long cationic collagen fibers and provides contrast-rich red staining. Furthermore, Picrosirius Red staining enhances the natural birefringence of collagen fibers under cross-polarized light8. This way, collagen fiber thickness and packing can be evaluated. Using a polarizing filter, the background turns dark, and distinguishing the thickness of deposited collagen becomes possible. While thick collagen fibers appear red, thin collagen fibers emerge green under polarization. A direct association between birefringence and collagen subtype is frequently described in the literature, with red birefringence assigned to collagen I and green birefringence to collagen III9.
This protocol describes the use of Picrosirius Red staining to evaluate fibrosis in murine lung allografts. In addition to conventional histological staining, it allows for a semiquantitative evaluation of fibrotic change in murine models of chronic rejection, provides a means of distinction between thick and thin collagen fibers in only one staining, is cost-efficient, and is easy to perform. This method can equally be applied to other murine experimental models characterized by fibrotic remodeling of the lung parenchyma.

<table><tbody><tr><td>Acetic acid</td><td>Honeywell, Charlotte, USA</td><td>33209</td><td></td></tr><tr><td>Axio Observer</td><td>Zeiss, Oberkochen, Germany</td><td>4633000956 (serial number)</td><td></td></tr><tr><td>Coverslip 1.5</td><td>Roth, Karlsruhe, Germany</td><td>KCY5.1</td><td></td></tr><tr><td>Formaldehyde 37%</td><td>Fisher Scientific, Leicestershire, UK</td><td>F/1501/PB15</td><td></td></tr><tr><td>Meyer&#039;s hemalum solution</td><td>Merck, Darmstadt, Germany</td><td>109249</td><td></td></tr><tr><td>Picrosirius Red Solution</td><td>Morphisto, Offenbach am Main, Germany</td><td>13422</td><td>alternatives that can be used: ab150681, abcam, Cambridge, UK; SRS250 ScyTek Laboratories, Logan City US</td></tr><tr><td>Polarizing filter</td><td>Zeiss, Oberkochen, Germany</td><td>000000-1121-813</td><td></td></tr><tr><td>Rotary microtome, HistoCore AUTOCUT</td><td>Leica, Wetzlar, Germany</td><td>149AUTO00C1, 14051956472</td><td></td></tr><tr><td>ROTI Histokitt mounting medium</td><td>Roth, Karlsruhe, Germany</td><td>6638.1</td><td></td></tr><tr><td>ROTI Plast Paraffin</td><td>Roth, Karlsruhe, Germany</td><td>6642.5</td><td></td></tr><tr><td>Rotilabo-embedding cassettes, POM</td><td>Roth, Karlsruhe, Germany</td><td>K113.1</td><td></td></tr><tr><td>Superfrost Plus Adhesion Microscope slide</td><td>epredia, Portsmouth, UK</td><td>J1800AMNZ</td><td></td></tr><tr><td>Tissue Processor&amp;nbsp;</td><td>Leica, Wetzlar, Germany</td><td>TP 1020</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fiji software version 2.14.0/1.54f</td><td>Open Source</td><td></td><td></td></tr><tr><td>Imaging Software ZEN 3.4.91</td><td>Zeiss, Oberkochen, Germany</td><td></td><td></td></tr></tbody></table>

picrosirius red staining for semiquantitative histopathologic evaluation of collagen deposition in murine models of chronic lung allograft rejection

Fibrosis is the pathophysiologic hallmark of chronic rejection after lung transplantation and the foremost hurdle to long-term recipient survival. Several murine lung transplantation models are available for the study of chronic rejection. However, they display heterogeneous results regarding fibrotic changes of the graft, and the histologic extent of fibrosis is mostly reported qualitatively. Therefore, a spatially resolved approach that allows for statistical analysis can aid in evaluating fibrosis in these models. This study presents Picrosirius Red staining for a semiquantitative evaluation of collagen organization in murine lung allografts and compares it to standard Hematoxylin and Eosin, Masson's Trichrome, and Herovici's stains. Staining was performed on sections from two different murine transplantation models based on minor and major histocompatibility complex (MHC) mismatches. The method was established for semiquantitative analysis of collagen organization in whole lung sections. Thus, it can serve as a tool for murine experimental models of fibrotic lung diseases.

The protocol described above allows for an objective semiquantitative evaluation of collagen deposition in murine lung tissue. Fibrotic remodeling is the pathophysiological hallmark of chronic rejection after lung transplantation. Therefore, Picrosirius Red staining was applied in chronic lung allograft rejection models using left-sided murine orthotopic lung transplantation. The major MHC-mismatched lung transplantation from a BALB/c donor to a C57BL/6 recipient under mild immunosuppression results in fibrotic changes c...

Author Spotlight: Analyzing Fibrosis Development in Chronic Lung Allograft Rejection Using Picrosirius Red Staining in Mouse Models

Picrosirius Red staining is a semiquantitative method for objectively assessing collagen deposition in murine pulmonary fibrotic remodeling in a spatially resolved manner. Apart from evaluating total collagen deposition, Picrosirius Red staining allows for differentiating collagen fibers of different thicknesses.

Picrosirius Red Staining for Semiquantitative Histopathologic Evaluation of Collagen Deposition in Murine Models of Chronic Lung Allograft Rejection

Fibrosis is the pathophysiologic hallmark of chronic rejection after lung transplantation and the foremost hurdle to long-term recipient survival. Several ...

picrosirius-red-staining-for-semiquantitative-histopathologic

Research

JoVE Journal

Biology

304 Views.  University of Freiburg. Ons onderzoek richt zich op het analyseren van de ontwikkeling van fibrose bij chronische afstoting van longtransplantaten. We zijn daarom geïnteresseerd in methoden die het fibrotische weefselcompartiment karakteriseren. Om transplantatiegerelateerde ziekten te reproduceren, gebruiken we het muismodel van enkelvoudige longtransplantatie dat we enkele jaren geleden hebben geïntroduceerd.In dit model is aangetoond dat de transplantatie van een long tu...

Video: Auteur Spotlight: Analyse van de ontwikkeling van fibrose bij chronische afstoting van longtransplantaten met behulp van Picrosirius Red-kleuring in muismodellen

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin &#38; eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

Medicine

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

Immunology and Infection

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Exploring Obliterative Airway Disease in Lung Transplants

Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially reported by our team, this model has gained use in the study of OAD due to its high technical reproducibility and suitability for investigating immunological behaviors and therapeutic interventions.
In the IPTT model, a rodent tracheal graft is directly inserted into the recipient's lung through the pleura. This model is distinct from the heterotopic tracheal transplantation (HTT) model, wherein grafts are transplanted into subcutaneous or omental sites, and from the orthotopic tracheal transplantation (OTT) model in which the donor trachea replaces the recipient's trachea. 
Successful implementation of the IPTT model requires advanced anesthetic and surgical skills. Anesthetic skills include endotracheal intubation of the recipient, setting appropriate ventilatory parameters, and appropriately timed extubation after recovery from anesthesia. Surgical skills are essential for precise graft placement within the lung and for ensuring effective sealing of the visceral pleura to prevent air leakage and bleeding. In general, the learning process takes approximately 2 months.
In contrast to the HTT and OTT models, in the IPTT model, the allograft airway develops airway obliteration in the relevant lung microenvironment. This allows investigators to study lung-specific immunological and angiogenic processes involved in airway obliteration after lung transplantation. Furthermore, this model is also unique in that it exhibits tertiary lymphoid organs (TLOs), which are also seen in human lung allografts. TLOs are comprised of T and B cell populations and characterized by the presence of high endothelial venules that direct immune cell recruitment; therefore, they are likely to play a crucial role in graft acceptance and rejection. We conclude that the IPTT model is a useful tool for studying intrapulmonary immune and profibrotic pathways involved in the development of airway obliteration in the lung transplant allograft.

Author Spotlight: Investigating the Key Factors of Obliterative Bronchiolitis After Lung Transplantation

The murine intrapulmonary tracheal transplantation (IPTT) model is valuable for studying obliterative airway disease (OAD) after lung transplantation. It offers insights into lung-specific immunological and angiogenic behavior in airway obliteration after allotransplantation with high reproducibility. Here, we describe the IPTT procedure and its expected results.

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

Assessing Pulmonary Microvasculature with Isolectin B4 Staining in Mice

Quantifying Pulmonary Microvascular Density in Mice Across Lobules

The abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction and is deeply linked to vascular wall integrity, blood flow regulation, and gas exchange. In murine models, lung lobes exhibit significant differences in size, shape, location, and vascularization, yet existing methods lack consideration for these variations when quantifying microvascular density. This limitation hinders the comprehensive study of lung microvascular dysfunction and the potential remodeling of microvasculature circulation across different lobules. Our protocol addresses this gap by employing two sectioning methods to quantify pulmonary microvascular density changes, leveraging the size, shape, and distribution of airway branches across distinct lobes in mice. We then utilize Isolectin B4 (IB4) staining to label lung microvascular endothelial cells on different slices, followed by unbiased microvascular density analysis using the freely available software ImageJ. The results presented here highlight varying degrees of microvascular density changes across lung lobules with aging, comparing young and old mice. This protocol offers a straightforward and cost-effective approach for unbiased quantification of pulmonary microvascular density, facilitating research on both physiological and pathological aspects of lung microvasculature.

Author Spotlight: Advances in Quantifying Microvascular Density in Aging Murine Lungs

Here, we describe a simple and economical protocol to perform unbiased quantification of pulmonary microvascular density for whole mice lung tissue using single staining of Isolectin B4.

The abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction and is deeply linked to vascular wall integrity, blood ...

Picrosirius Red Polarization Staining: A Technique To Detect Fibrosis in Cardiac Tissue Sections Using Bright-Field Microscopy

Source: Ku, H. C. et al., <a href="https://www.jove.com/v/54818">A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats.</a> J. Vis. Exp. (2016).&#160;
This video describes the picrosirius red staining of fibrotic tissues to detect excessive collagen deposition in fibrotic tissue sections. The staining technique helps assess collagen expression in connective-tissue diseases.

Picrosirius Red Staining for Semiquantitative Histopathologic Evaluation of Collagen Deposition in Murine Models of Chronic Lung Allograft Rejection

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