Name: improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and x-gal staining of lacz-tagged tumor cells
Uploaded: 2012-08-21T13:00:00
Duration: 8 min 47 s
Description: Watch this Scientific Journal Video about Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tum

The authors would like to thank Dr. Lubor Borsig (Institute of Physiology, University of Zurich) for his advice on the technique of lung perfusion.
This work was supported by grants from the Krebsliga of the Kanton Zurich, the Walter L. and Johanna Wolf Foundation, Zurich, the Lydia Hochstrasser Foundation, Zurich, the Swiss National Science Foundation, SNF, Switzerland, the Schweizerischer Verein Balgrist, and the University of Zurich.

1. In-situ Lung Perfusion and Fixation
<ol>
 <li>Anesthetize mice by i.p. injection of ~150 &mu;l phosphate buffered saline (PBS) containing 112.5 mg/kg (body weight) ketamine, 16.5 mg/kg xylazine, and 15 mg/kg acepromazine (or by another anesthesia containing appropriate pain killers).</li>
 <li>When reflexes of the mouse are no longer observed, fix it on the operating table back-down and wet the fur with 70% Ethanol to slick the hairs down.</li>
 <li>Open the skin from the abdomen up to the neck and pull it to the sides or remove it.</li>
 <li>Open the peritoneum and the thorax to an ample size. Prevent ruptures of big vessels (e.g. the vena jugularis) to avoid reduced efficiency of the subsequent perfusion.</li>
 <li>Cut the vena cava caudalis underneath the liver.</li>
 <li>Use a 20 ml syringe equipped with a 21G - 24G needle to inject slowly 10-15 ml PBS into the right ventricle of the beating heart until the lung is completely white and the heart stops beating (asystole).</li>
 <li>Pinch off the blood vessels above the liver and the diaphragm with a vascular clamp.</li>
 <li>Use a 10 ml syringe equipped with a 21G - 24G needle to inject ~2 ml of 3% paraformaldehyde (PFA) in PBS into the right ventricle.</li>
 <li>Uncover the trachea outside of the thorax. Use the same syringe to inject ~3 ml of 3% PFA in PBS cranial (outside of thorax) into the trachea until the lung is inflated. Immediately after removal of the needle, pinch off the trachea caudal of the puncture with a vascular clamp and wait for 10 min to allow fixation.</li>
 <li>Transect the blood vessels above the vascular clamp, remove the clamp and inject ~2 ml PBS into the right ventricle to remove excess PFA.</li>
 <li>Puncture the lower parts of all lung lobes with a needle, remove the vascular clamp at the trachea and inject 3-5 ml of a solution of PolyFreeze embedding medium/PBS 1:1 into the trachea caudal of the first puncture until the PFA in the lung lobes is replaced by the solution.</li>
 <li>Remove the lung carefully by pulling the heart with a forceps and by transecting connective tissue ligaments, blood vessels and the trachea. Keep the heart connected to the lungs.</li>
 <li>Depending on how to proceed with the lung tissue analysis continue with the protocols outlined in sections 2 or 3. If you want to do both analyses, remove one lung lobe and proceed as described under 3. Process the remaining lobes connected to the heart as described under 2.</li>
</ol>
2. Visualization of lacZ-tagged Metastatic Tumor Cells in Entire Lung Lobes
<ol>
 <li>Place the lung in a plastic cup with tight closing lid and fix it with 2% formaldehyde in PBS at room temperature for 30-60 min.</li>
 <li>Remove fixation solution and wash thoroughly 3 times with PBS.</li>
 <li>Add at least 10 ml freshly prepared 5-Bromo-4-chloro-3-indolyl-&beta;-D-galactoside (X-Gal) staining solution (X-Gal stock solution [40 mg/ml in Dimethylformamide] 1:40 diluted in Basic Staining Solution; pH 7.1 (Table 1); protect from light).</li>
 <li>Put a piece of gauze on top of the swimming lung to keep it completely in the solution and place the lid only loosely on the pot to allow exchange of air.</li>
 <li>Incubate at 37 &deg;C for 3-5 hr protected from light.</li>
 <li>Remove X-Gal solution, rinse once with PBS to remove residual X-Gal solution (optional) and add 4% PFA.</li>
</ol>
3. Visualization of lacZ-tagged Metastatic Tumor Cells in Lung Cryosections
<ol>
 <li>Prefill a labeled embedding mold to 1/3 with undiluted PolyFreeze embedding medium. Put the lung lobe on top and fill up with embedding medium until the lobe is completely covered. Try to prevent bubbles.</li>
 <li>Incubate the embedded lungs at 4 &deg;C for 20 min.</li>
 <li>Then freeze the embedded lungs slowly in a mixture of dry ice and Isopentane and store at -80 &deg;C.</li>
 <li>Cut 7-10 &mu;m cryosections on a cryostat and mount them on SuperFrostPlus microscope slides.</li>
 <li>Incubate the sections immediately with X-Gal staining solution at 37 &deg;C for 24 hr in a humidified chamber.</li>
 <li>Rinse the slides in PBS 2 times for 5 min and then briefly in distilled water.</li>
 <li>Counter-stain with nuclear fast red for 10-30 sec, rinse in distilled water and mount the slides with Immu-Mount.</li>
</ol>
4. Representative Results
Asai et al. reported in the original article on the Dunn-derived LM8 OS mouse model that s.c. primary tumors derived from the parental Dunn cells, different from those derived from the highly metastatic LM8 sub cell line, do not spontaneously form detectable lung metastases in syngeneic C3H mice14. With the here reported techniques, we reinvestigated the formation of metastases in the Dunn/LM8 mouse OS models. We took advantage of stable lacZ-transduced Dunn and LM8 cells and of a protocol for in-situ lung perfusion and fixation in mice. Representative images of perfused and non-perfused lungs of mice subcutaneously injected with lacZ-transduced and non-transduced control Dunn and LM8 cells are shown in Figure 1. In mice injected with control Dunn cells, macroscopic and microscopic metastases remained undetectable in non-perfused and perfused lungs (Figure 1A, i-iv). But, interestingly, in mice injected with Dunn-lacZ cells, X-gal staining revealed blue micrometastatic foci of single cells or small cell clusters (&lt; 0.1 mm) on the surface of non-perfused lungs (Figure 1A, vi). In-situ perfusion and fixation of the lungs further improved the detectability of Dunn-lacZ micrometastases (Figure 1A, viii). However, outgrowth to macroscopic foci was not observed (Figure 1A, v, vii).
In mice injected with control LM8 cells, translucent, barely detectable macrometastatic foci larger than 0.1 mm in diameter were recognized in non-perfused lungs (Figure 1B, i). Perfusion of the lung (Figure 1B, iii) did not improve the detection of the foci. However, in mice injected with LM8-lacZ cells multiple X-Gal stained blue macro- (Figure 1B, v) and micrometastases (Figure 1B, vi) were detected on the surface of non-perfused organs. Moreover, perfusion of the lungs further improved the detectability of macro- and micrometastases (Figure 1B, vii-viii). Consequently, micro- and macrometastases became visible at a higher density and a larger number, mainly due to the translucency of the perfused tissue in which foci underneath the organ surface became also visible.
An additional histological analysis using cryosections of lung tissue confirmed the improved detectability of lung metastases in mice injected with lacZ-transduced Dunn and LM8 cells. In mice with primary tumors derived from Dunn-lacZ or LM8-lacZ cells, unlike in mice with primary tumors of the respective control cells, micrometastases or even single cell foci were recognized in lung sections (Figure 2). Moreover, macrometastases were also more clearly visible in mice injected with LM8-lacZ cells than in animals injected with the control LM8 cells (Figure 2C, D).
<img alt="Figure 1" src="/files/ftp_upload/4162/4162fig1.jpg" /> 
 Figure 1. Detection of spontaneous metastases of non-transduced (i-iv) and lacZ-transduced (v-viii) Dunn (A) and LM8 (B) cells in non-perfused (i, ii, v, vi) and perfused (iii, iv, vii, viii) lungs in C3H mice. Metastases in representative X-Gal-stained whole lungs (v, vii in A and B) and respective close-ups (vi, viii in A and B) appear blue. Macrometastatic foci (&gt; 0.1 mm) in the organs of mice injected with non-tagged LM8 cells (i-iv in B) are indicated by asterisks. Areas shown in close-ups are representative for entire lungs. Scale bars indicate 1 mm in images of whole lungs and 0.1 mm in close-ups. <a href="https://www.jove.com/files/ftp_upload/4162/4162fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/4162/4162fig2.jpg" /> 
 Figure 2. Detection of Dunn and LM8 micrometastases in lung cryosections. Sections of O.C.T. embedded lung tissue were incubated in X-Gal staining solution at 37 &deg;C for 24 hr in a humidified chamber and then counterstained with nuclear fast red. Arrows point to micrometastatic foci recognized in lung sections of mice injected with Dunn-lacZ (B) or LM8-lacZ cells (D). Micrometastases remained undetectable in lung sections of mice injected with non-transduced Dunn (A) or LM8 cells (C). Scale bars indicate 0.1 mm.

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No conflicts of interest declared.

The here presented results in the Dunn/LM8 mouse OS model demonstrate the power of the newly established method that combines the X-Gal staining of lacZ-tagged tumor cells with the in-situ perfusion/fixation of lung tissue. This combination of the two techniques allows high sensitivity detection of micrometastatic lesions down to the single cell level and also improves the visualization of macrometastases on the lung surface (Figure 1) as well as in lung sections (Figure 2). While the X-Gal staining also allows the detection of (micro)metastases in other organs, in-situ perfusion/fixation improves the detectability of metastatic foci in tissues other than the lung only slightly because of the natural blood- and tissue-related color of these organs13. Even if the perfusion is directed to another organ, e.g. the liver, removal of the blood would only partially improve the contrast between X-Gal staining and the natural color of the organ. However, the method is applicable to any type of lacZ-tagged tumor cells, will dramatically improve the detectability of lung metastasis down to the level of dormant single-cell micrometastases and enables an easy and reliable quantification of macro- and micrometastases. A limitation of this method and all other techniques that are based on reporter genes, including luciferase and fluorescent proteins, is the stability of the transgene expression. As shown in Figure 2d, not all tumor cells within the macrometastatic foci are stained blue, indicating a lack of beta-galactosidase activity. This might be related to necrosis, but it is more likely due to a loss of transgene expression. We observed that the Dunn and LM8 cells are very effective in the down-regulation of lacZ and other transgenes even under continuous selection for expression. We therefore switched in recent studies to the murine K12 and K7M2 and the human HOS, 143B and SaOS-2 osteosarcoma cell lines, which all maintained stable lacZ expression in vitro as well as in vivo up to 100% over time.
Once stable lacZ-expression is guaranteed, this technique can be applied in studies with gene-manipulated tumor cells, e.g. to mechanistically investigate the process of tissue colonization13 as well as for the development and testing of new therapies aiming at eradication of metastatic lesions15,16. Furthermore, it can serve as a benchmark for the improvement of current radiological imaging techniques, such as PET, (micro)CT and MRI, used for early detection of metastatic lesions. In a recent PET study (unpublished) with different tracers we verified the in vivo detected lung metastases subsequently ex vivo with the described protocol. In an ongoing study with a new small animal micro-CT (SkyScan) we are so far able to detect lung metastases in vivo down to a size of 0.5 mm and ex vivo down to 0.3 mm, but we aim at a resolution of 0.1 mm. Interestingly, this is the size limit that we set to distinguish macro- from micrometastases with the combined method of in-situ perfusion and X-Gal staining. This underlines again the sensitivity and usefulness of this easy and cost-effective technique.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Narketan10 (ketamine)</td>
 <td>V&eacute;toquinol AG</td>
 <td></td>
 <td>100 mg/ml solution</td>
 </tr>
 <tr>
 <td>Xylazin (xylazine) </td>
 <td>Streuli Pharma AG</td>
 <td></td>
 <td>20 mg/ml solution </td>
 </tr>
 <tr>
 <td>Prequilan (acepromazine)</td>
 <td>Fatro S.p.A.</td>
 <td></td>
 <td>10 mg/ml solution</td>
 </tr>
 <tr>
 <td>Bulldog Type Serrefine vascular clamp</td>
 <td>Fine Science Tools</td>
 <td>18051-35</td>
 <td>curved, 35 mm</td>
 </tr>
 <tr>
 <td>Plastic cup with lid</td>
 <td>Semadeni</td>
 <td>2988</td>
 <td>25 ml cups with lids</td>
 </tr>
 <tr>
 <td>5-Bromo-4-chloro-3-indolyl-&beta;-D-galactoside (X-Gal); powder</td>
 <td>Axxora</td>
 <td>ALX-582-002-G005</td>
 <td>dissolve in N,N-Di-methylformamide to 40 mg/ml, store light-protected at -20 &deg;C </td>
 </tr>
 <tr>
 <td>Basic staining solution, equilibrated to pH 7.1, stored light-protected at 4 &deg;C for max. 1 month</td>
 <td>self-made</td>
 <td></td>
 <td>100 ml/L 10xPBS, 1.64 g/L K3Fe(CN)6, 2.1 g/L K4Fe(CN)6, 2 ml/L 1M MgCl2, 2 ml/L 10% NP40, 1 ml/L 10% Sodium-Deoxycholate</td>
 </tr>
 <tr>
 <td>PolyFreeze Embedding Medium</td>
 <td>Polysciences; swiss distributor: Brunschwig</td>
 <td>19636-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Embedding mold</td>
 <td>Polysciences</td>
 <td>18646A-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Leica CM1850 cryostat</td>
 <td>Leica Microsystems</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>SuperFrost slides</td>
 <td>Menzel</td>
 <td>J1800AMNZ</td>
 <td></td>
 </tr>
 <tr>
 <td>Nuclear-Fast Red - Aluminum sulfate solution</td>
 <td>Division Chroma Waldeck GmbH&amp;Co KG distributor: Medite</td>
 <td>84-0241-00</td>
 <td></td>
 </tr>
 <tr>
 <td>Immu-Mount</td>
 <td>Thermo Electron, swiss distributor: Histocom</td>
 <td>9990412</td>
 <td></td>
 </tr>
 </tbody>
</table>
Table 1. Reagents and Equipment.

improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and x-gal staining of lacz-tagged tumor cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Watch this Scientific Journal Video about Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tum

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

improved-visualization-lung-metastases-at-single-cell-resolution-mice

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Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

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The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

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

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

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

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

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

Innovative Lung Imaging Using Agarose-Embedded Vibratome Sections

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

Author Spotlight: Studying Spatial Protein Expression Using Agarose Embedded Lung Tissue Sections 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...