Name: Video: Author Spotlight: Advances in Quantifying Microvascular Density in Aging Murine Lungs
Uploaded: 2025-01-03T12:40:00.000+00:00
Duration: 10 min
Description: 2.7K Views. Sichuan University. 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 authors express their gratitude for the invaluable support received from the public experimental platform at West China School of Pharmacy. Special appreciation is extended to Wendong Wang for providing critical and highly valuable advice on pathology. This research has been made possible through the funding from the Science and Technology Department of Sichuan Province (grants 2023NSFSC0130 and 2023NSFSC1992) and &#34;the Fundamental Research Funds for the Central Universities&#34; to TJ.

All the experiments were carried out following the ethical guidelines of the Sichuan University Animal Research Committee (No K2023023).
1. Preparation of paraffin sections for mouse lung lobes
<ol>
	<li>Acquire lung tissue from the mouse.
	<ol>
		<li>Choose male C57Bl/6 mice at 6 weeks and 16 months to assess the microvascular density in mouse lungs across different age groups.</li>
		<li>Administer 100 mg/kg of pentobarbital sodium via intraperitoneal injection in mice. Euthanize the mice by cervical dislocation after anesthesia. Open the chest cavity and obtain lung tissue.</li>
	</ol>
	</li>
	<li>Prepare paraffin sections.
	<ol>
		<li>Fix the tissue by placing it in a 4% paraformaldehyde solution that is 10 times the volume of the tissue to preserve its morphology and structure and prevent degradation. 
		NOTE: A recent new method was developed to inflate air during vascular perfusion-fixation in murine lungs. This method is reported to preserve better morphology and location of the airway, alveolar cells, and interstitium, making them more suitable for lung histologic studies. This method is recommended for laboratories with the corresponding equipment and consumables with in-house-made instruments8.</li>
		<li>Cut the lung lobe to determine the embedding direction (Figure 1).
		<ol>
			<li>After 3 h of fixation, separate the five pulmonary lobes (under direct observation of the eyes) and embed the cranial, middle, and accessory lobes of the right lung separately, with sections oriented in the direction of the vertical lobe-to-bronchus connection.</li>
			<li>Cut the caudal lobe of the right lung into 2 pieces at 4 mm from left to right in the direction perpendicular to the lung lobe-bronchus connection, and embed the slices in a wax block with the cut surface facing upward.</li>
			<li>Make three cuts in the left lung lobe. Make the first cut transversely above the main bronchial junction, 2 mm below the top. Make the second cut transversely above the main bronchial junction, 5 mm below the top. Make the third cut at the end of the lobe, 8 mm below the top. Discard the uppermost piece of tissue and encase the remaining three pieces in a wax block with the cut surface facing upward. 
			NOTE: When embedding multiple lung pieces together, a microscope wiping paper can be used to wrap them before placing them in a tissue embedding box to continue the dehydration process.</li>
			<li>Re-fix the tissue in the fixative for 24 h.</li>
		</ol>
		</li>
		<li>Place the tissue block in a container and rinse it under flowing water for 15 min.</li>
		<li>Sequentially place the tissue in the following solutions: 65% ethanol for 30 min, 75% ethanol for 30 min, 85% ethanol for 30 min, 95% ethanol I for 60 min, 95% ethanol II for 60 min, absolute ethanol I for 60 min, absolute ethanol II for 60 min, 70% ethanol for 120 min, 80% ethanol for 120 min, 90% ethanol for 120 min, 95% ethanol for 120 min, absolute ethanol I for 120 min, and absolute ethanol II for 120 min.</li>
		<li>Place the tissue in xylene I for 30 min and xylene II for 30 min.</li>
		<li>Place the tissue in soft wax below 54 &#176;C for 1 h, hard wax I at 58 &#176;C for 1 h, and hard wax II at 58 &#176;C for 30 min.</li>
		<li>Select a suitable mold size, fill it with an appropriate amount of liquid paraffin, and use forceps to position the tissue in the embedding frame in the center of the mold in the correct direction. Place the mold and tissue in the freezing area of the embedding machine and gently press the tissue with forceps.</li>
		<li>When the paraffin starts to whiten slightly, quickly position the uncovered embedding frame on top of the mold and perform the second wax injection, sealing the holes at the bottom of the embedding frame. Transfer the entire mold to the freezing table for molding.</li>
		<li>After securing the wax block onto the slicer and revealing the smooth and flat cut surface through rough and fine trimming, rotate the microtome&#39;s wheel crank to cut slices with a thickness of 4 &#181;m.</li>
		<li>Smoothly place the removed sections onto the water surface in sequential order. Once the sections are fully extended, separate the broken pieces at the junctions between each section.</li>
		<li>Tilt a glass slide underneath the section and, upon contact, slowly and evenly lift the slide out of the water surface in a vertical motion. The sections will adhere to the glass slide.</li>
	</ol>
	</li>
</ol>
2. Immunofluorescence staining for detection of pulmonary microvasculature
<ol>
	<li>Place the paraffin-embedded slices in a 65 &#176;C oven for 60 min.</li>
	<li>Place the slices on a slide rack and carry out the following steps in staining jars: immerse xylene 1, xylene 2, and xylene 3 for 15 min each, followed by immersion in xylene/ethanol (1:1) for 5 min, 100% ethanol #1, 100% ethanol #2, 85% ethanol, 75% ethanol, and distilled water for 5 min each.</li>
	<li>Dilute the modified sodium citrate antigen retrieval solution 50-fold with deionized water. Preheat the microwave on high power until boiling, then place the slide in the antigen extract. Allow the slide to boil in the antigen extract for 15 min and then cool naturally to room temperature.</li>
	<li>Wash the slides twice with PBS for 5 min each. Permeabilize with 0.25% Triton twice for 10 min each.</li>
	<li>Encircle the tissue with an immunohistochemistry pen and incubate that with 5% BSA at room temperature (RT) for 1 h. Wash the slides once with PBS for 5 min.</li>
	<li>Perform Alexa-647 Fluor conjugated IB4 and DAPI staining.
	<ol>
		<li>Keep the IB4 on ice and dilute it 1:50 with 1% BSA. Remove excess water around the circle, add 50-100 &#181;L of the diluted IB4 to each tissue section, and incubate overnight at 4 &#176;C (from this step onwards, perform all procedures in the dark).</li>
		<li>Wash the slides three times with PBS for 5 min each. Permeabilize with 0.25% Triton for 10 min.</li>
	</ol>
	</li>
	<li>Dilute DAPI (10 &#181;g/mL) with PBS at a ratio of 1:10 and add 50-100 &#181;L to each tissue section at RT for 5 min.</li>
	<li>Wash the slides three times with PBS for 5 min each.</li>
	<li>Apply a drop of anti-fade mounting medium to the slides, avoiding light exposure. Air dry the slide, then observe and capture images of the nucleus and target signals under a fluorescence microscope.</li>
</ol>
3. Quantification of pulmonary microvascular density
<ol>
	<li>Acquire images.
	<ol>
		<li>Observe lung microvascular density signals and DAPI signals under a 10x objective for each lung lobe in both the young and old age groups.</li>
		<li>Standardize the light source intensity and exposure time for each channel based on the observation results. Capture the dual-channel images covering the entire area of each lung lobe. Save the image in ND2 format with two channels (Figure 2).</li>
	</ol>
	</li>
	<li>Quantify using Image J (Supplementary Figure 1).
	<ol>
		<li>Launch ImageJ software.</li>
		<li>Import indicated images to ImageJ, then proceed to load and select them. Split the channels accordingly. Load a file in ND2 format for both DAPI and IB4 channels.</li>
		<li>Navigate to Analyze &#62; Set Scale &#62; Configure the Image Parameters as 0.48&#160;&#181;m/pixel (depending on the photography parameters) &#62; Global &#62; OK.</li>
		<li>Set the same display range to adjust the image presentation effect. Select Image&#62; Adjust &#62; Brightness/Contrast &#62; Set. Set display range for each of the two channels separately, IB4: 0-10000; DAPI: 0-20000.</li>
		<li>To eliminate the influence of background signals, proceed with the following steps: Click Process &#62; Math &#62; Subtract; Subtract the background signal based on the signal values detected by the Magic Wand tool on the background, IB4: 3000; DAPI:300.</li>
		<li>To select the threshold value that best aligns with the original image, go to Image &#62; Duplicate.</li>
		<li>Select both original and duplicate Image &#62; Type &#62; 8 bit.</li>
		<li>Select an appropriate threshold value to precisely identify the lung microvasculature. Click on Image &#62; Adjust &#62; Threshold &#62; Intermodes&#160;(choose the most realistic threshold against the original diagram), tick &#62; Dark Background &#62; Apply.</li>
		<li>Eliminate IB4 positive signals from large blood vessels in the mouse lung, including arteries and veins: Use the Magic Wand tool to select larger blood vessels compared to the duplicated image, then click Delete&#160;(repeat that until the IB4 positive signals in the endothelial cells of larger blood vessels are removed). 
		NOTE: IB4 staining signals are detected in endothelial cells of arteries and veins in the mouse lung. To achieve a specific examination of pulmonary microvasculature occurrences, it is recommended to uniformly remove the signal foci in endothelial cells of larger blood vessels.</li>
		<li>Count the number of target signals, click Analyze &#62; Set Measurements, and tick Area, Limit to Threshold, and Display Label. Then select Analyze Particles and set Size: 0-infinity; Circularity: 0.00-1.00; Show: Overly Masks; tick Summarize and click OK.</li>
		<li>Choose Summarize Results &#62; File &#62; Save as.</li>
	</ol>
	</li>
	<li>Data analysis and statistics
	<ol>
		<li>Match and organize the number of IB4 positive foci and DAPI positive foci, respectively, at the same image into a unified table.</li>
		<li>Summarize the total number of IB4 positive foci and DAPI positive foci for each lobe in indicated young or age groups.
		<ol>
			<li>Calculate the percentage of IB4 positive foci (%) by dividing the total number of IB4 positive foci by the total number of DAPI staining foci in each lobe by different groups. Present the number of IB4 positive foci, the number of DAPI staining foci, and the percentage of IB4 positive foci (%) as shown in Figure 3A.</li>
		</ol>
		</li>
		<li>Launch the data analysis software and create a new column table. 
		NOTE: GraphPad Prism 9 was used here for statistical analysis.</li>
		<li>Sequentially name them from Group A to Group J as &#34;Young cranial lobe&#34;, &#34;Old cranial lobe&#34;, &#34;Young middle lobe&#34;, &#34;Old middle lobe&#34;, &#34;Young caudal lobe&#34;, &#34;Old caudal lobe&#34;, &#34;Young accessory lobe&#34;, &#34;Old accessory lobe&#34;, &#34;Young left lobe&#34;, &#34;Old left lobe&#34;. Then, input the corresponding IB4 positive foci (%) into the table (Supplementary Table 1).</li>
		<li>Utilize unpaired t-tests for pairwise comparison to assess significant differences in the corresponding regions between the two age groups. Conduct a total of five comparisons for five lobes.</li>
		<li>Create a graph for result visualization (Figure 3B). Opt for a bar chart and include statistical analysis in the figure.</li>
	</ol>
	</li>
</ol>

Assessing Pulmonary Microvasculature with Isolectin B4 Staining in Mice

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The authors declare that they have no competing interests.

The study of pulmonary microvascular density holds significant implications for understanding pulmonary physiological processes and also for defining biomarker(s) for respiratory diseases. The pulmonary circulation boasts an extensive capillary surface area enveloped by a slender layer of endothelial cells. The harmonious juxtaposition of these cells and alveolar epithelial cells gives rise to a fragile alveolar-capillary membrane specifically designed to facilitate the intricate process of gas exchange...

Endothelial cells (ECs) are a special type of cell located on the inner lining of blood vessels, covering the entire arterial and venous tree and playing a crucial role in maintaining the stability of blood vessels and organs1. The lungs are highly vascularized organs and play essential physiological and pathological roles in the lungs, such as forming the vascular wall, regulating blood flow, facilitating gas exchange, modulating inflammatory responses, controlling platelet activity, secreting regulatory substances involved in vascular growth, repair, and maintaining coagulation balance.
Lung microvascular endothelial cells (LMECs) are specific endothelial cells of pulmonary tissue, particularly in the microvasculature (capillaries) of the lungs, distinguishing them from the more generalized arterial and venous endothelial cells in the lungs. These cells have various functions, including regulating vascular tone, controlling vascular permeability, participating in the regulation of inflammatory responses, and regulating thrombus formation. They play a crucial role in pulmonary circulation, regulating gas exchange and the transport of nutrients, and are involved in various physiological and pathological processes related to the lungs, likely for aging2. Moreover, the abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction3. Employing the conventional endothelial cell marker CD31 and spatial localization (specifically, the peripheral regions of the lungs), Larissa L.&#160;et al. observed a significant decrease in microvascular endothelial cell density in aged mice (18 months old) compared to their younger counterparts (4 months old)4. In the context of the pulmonary pathology associated with asthma, Makoto H. et al. demonstrated a substantial increase in vessel induction in bronchial biopsy specimens stained with anti-collagen IV from asthmatic patients in comparison to control subjects5. Recently, by introducing the techniques of transmission and scanning electron microscopy, Maximilian A. et al. reported a notable increase in the numeric density of features related to intussusceptive and sprouting angiogenesis in patients who died from Covid-19 or Influenza A (H1N1)6. Evidently, the abnormal microvascular genesis is linked with pulmonary dysfunction. However, there is currently no simple, economical method available for quantifying changes in microvascular density.
In murine models, the lungs are conventionally segmented into five distinct lobes: right cranial, right middle, right caudal, left cranial, and left caudal. Each lobe exhibits unique characteristics in terms of size, shape, location, and likely vascularization, contributing to efficient gas exchange and potentially synergistic regulation of pulmonary circulation. However, to the best of our knowledge, no methodologies account for the differences among these lung lobes when investigating Lung microvascular changes.
This study presents a new method for sectioning lobules in mice, utilizing IB4, a well-defined marker of lung micro-endothelial cells7, for unbiased quantitative assessment of pulmonary microvascular density. This innovative approach addresses the need for a more comprehensive understanding of microvascular alterations in murine lungs by considering the distinct properties of individual lobes of mice. As a demonstration, in aging&#160;mice, a significant reduction in pulmonary 
microvascular density is observed specifically&#160;within both the caudal lobe and left lobe.&#160;The protocol underscores the importance of integrating lobe-specific analyses into investigations of changes in the microvascular landscape of murine lungs. Notably, this method provides valuable research references for investigators seeking a comprehensive understanding of both physiological and pathological progression of lung developments and lesions, extending beyond angiogenesis.

<table><tbody><tr><td>4% Paraformaldehyde</td><td>Biosharp</td><td>BL539A</td><td>Tissue Fixative</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole</td><td>MCE</td><td>HY-D0814</td><td>Nucleic Dyes</td></tr><tr><td>Alexa-647 Fluor Conjugated Isolectin B4</td><td>Thermo</td><td>I32450</td><td>Binding Microvessels</td></tr><tr><td>Anti-fluorescent Tablet Sealer</td><td>Abcam</td><td>AB104135</td><td>Sample Fixation</td></tr><tr><td>Antigen Repair Fluid</td><td>Biosharp</td><td>BL151A</td><td>Repair of Antigenic Sites</td></tr><tr><td>Biopsy Cassette</td><td>ActivFlo</td><td>39LC-500-1</td><td>Fixing and Positioning Tissue Samples</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>B2064-50G</td><td>Sealing Solution</td></tr><tr><td>Cold Plate</td><td>Leica</td><td>HistoCore Arcadia H&amp;nbsp;</td><td>Freezing Samples</td></tr><tr><td>Constant Temperature Electric Drying Oven</td><td>Taisite</td><td>101-0AB</td><td>High Temperature Repair</td></tr><tr><td>Disposable Microtome Blade</td><td>Leica</td><td>14035838383</td><td>Cutting Tissue Samples to Prepare Sections</td></tr><tr><td>Embedding Molds</td><td>Shitai</td><td>26155166627</td><td>Fixing Tissue Samples</td></tr><tr><td>Ethanol</td><td>Kelong</td><td>CAS 64-17-5</td><td>Tissue Dehydration Solution</td></tr><tr><td>Heated Paraffin Embedding Station</td><td>Leica</td><td>EG1150</td><td>Embedding Tssue Samples in Paraffin</td></tr><tr><td>HistoCore Water Bath</td><td>Leica</td><td>HI1220</td><td>Flatten and Fix Tissue Samples</td></tr><tr><td>ImageJ (Fiji)&amp;nbsp;</td><td>NIH</td><td>1.54f</td><td>Quantitative Tool</td></tr><tr><td>Immunohistochemistry Pens</td><td>Biosharp</td><td>BC004</td><td>Water-blocking Agent</td></tr><tr><td>Medical Forceps</td><td>Shanghai Medical Equipment</td><td>N/A</td><td>Grasping, Manipulating, or Moving tissue samples</td></tr><tr><td>Microscope</td><td>Nikon</td><td>Ts2</td><td>Imaging Device</td></tr><tr><td>Mounting Media</td><td>Jiangyuan</td><td>Tasteless</td><td>Fixing and Preserving Tissue Sections</td></tr><tr><td>Paraffin Wax</td><td>SCHLEDEN</td><td>80200-0014</td><td>Fixing Tissue Structure</td></tr><tr><td>PBS</td><td>Beyotime</td><td>C0221A</td><td>Wash Buffer</td></tr><tr><td>Pentobarbital Sodium</td><td>Beijing Chemical Reagent Company</td><td>Q/H82-F158-2002</td><td>Anesthetic</td></tr><tr><td>Rotary Microtome</td><td>Biobase</td><td>Bk-2258</td><td>Preparing Slices</td></tr><tr><td>Sterile Scissors</td><td>Shanghai Medical Equipment</td><td>N/A</td><td>segmenting Tissue Samples</td></tr><tr><td>Surgical Scalpel</td><td>Shanghai Medical Equipment</td><td>N/A</td><td>Cutting Tissue Samples</td></tr><tr><td>Triton&amp;nbsp;</td><td>Solarbio</td><td>T8200</td><td>Permeabilization Solution</td></tr><tr><td>Wash-Free Slide</td><td>PLATINUM PRO</td><td>PRO-04</td><td>Fixing Samples for Staining</td></tr><tr><td>Xylene</td><td>SUM</td><td>XK13-011-00031</td><td>Tissue De-waxing Solution</td></tr></tbody></table>

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.

To distinguish between the lesions in the main bronchi and small airway branches, it is crucial to ensure that the continuous structure of lesions in these two types of airways is observed. This can be achieved by following the cutting and embedding procedures outlined in Figure 1. Given the numerous lung lobes in mice, which are oriented in various directions and possess a mesh-like structure, they are more susceptible to collapse compared to other solid tissues. To achieve a clear and intu...

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.

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 ...

quantifying-pulmonary-microvascular-density-in-mice-across-lobules

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2.7K Views.  Sichuan University. 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.

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

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Immunology and Infection

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

Developmental Biology

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Medicine

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. Assessment of RV function and morphology is complex, partly due to the complex shape of the RV. Currently, cardiac magnetic resonance (CMR) imaging is the golden standard for noninvasive assessment of RV function and morphology. The current protocol describes CMR imaging in a mouse model of RV pressure load induced by pulmonary artery banding (PAB). PAB is performed by placing a 6-0 suture around the pulmonary artery over a 23 G needle. The PAB gradient is determined using echocardiography at 2 and 6 weeks. At 6 weeks, the right and left ventricular morphology and function is assessed by measuring both end-systolic and end-diastolic volumes and mass by ten to eleven cine slices 1 mm thick using a 9.4 T magnetic resonance imaging scanner equipped with a 1,500 mT/m gradient. Representative results show that PAB induces a significant increase in RV pressure load, with significant effects on biventricular morphology and RV function. It is also shown that at 6 weeks of RV pressure load, cardiac output is maintained. Presented here is a reproducible protocol for the quantification of biventricular morphology and function in a mouse model of RV pressure load and may serve as a method for experiments exploring determinants of RV remodeling and dysfunction.

To understand the pathophysiology of right ventricular (RV) adaptation to abnormal loading, experimental models are crucial. However, assessment of RV dimensions and function is complex and challenging. This protocol provides a method to perform cardiac magnetic resonance imaging (CMR) as a noninvasive benchmark procedure in mice subjected to RV pressure load.

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Quantifying Pulmonary Microvascular Density in Mice Across Lobules

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Chapters in this video

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Determining Bile Duct Density in the Mouse Liver

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Measurement of the Pressure-volume Curve in Mouse Lungs

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies