Quantifying Pulmonary Microvascular Density in Mice Across Lobules

Summary

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.

Abstract

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.

Introduction

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. 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 mice, a significant reduction in pulmonary
microvascular density is observed specifically within both the caudal lobe and left lobe. 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.

Protocol

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

1. Acquire lung tissue from the mouse.
   1. Choose male C57Bl/6 mice at 6 weeks and 16 months to assess the microvascular density in mouse lungs across different age groups.
   2. 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.
2. Prepare paraffin sections.
   1. 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.
   2. Cut the lung lobe to determine the embedding direction (Figure 1).
      1. 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.
      2. 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.
      3. 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.
      4. Re-fix the tissue in the fixative for 24 h.
   3. Place the tissue block in a container and rinse it under flowing water for 15 min.
   4. 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.
   5. Place the tissue in xylene I for 30 min and xylene II for 30 min.
   6. Place the tissue in soft wax below 54 °C for 1 h, hard wax I at 58 °C for 1 h, and hard wax II at 58 °C for 30 min.
   7. 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.
   8. 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.
   9. After securing the wax block onto the slicer and revealing the smooth and flat cut surface through rough and fine trimming, rotate the microtome's wheel crank to cut slices with a thickness of 4 µm.
   10. 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.
   11. 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.

2. Immunofluorescence staining for detection of pulmonary microvasculature

1. Place the paraffin-embedded slices in a 65 °C oven for 60 min.
2. 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.
3. 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.
4. Wash the slides twice with PBS for 5 min each. Permeabilize with 0.25% Triton twice for 10 min each.
5. 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.
6. Perform Alexa-647 Fluor conjugated IB₄ and DAPI staining.
   1. Keep the IB₄ on ice and dilute it 1:50 with 1% BSA. Remove excess water around the circle, add 50-100 µL of the diluted IB₄ to each tissue section, and incubate overnight at 4 °C (from this step onwards, perform all procedures in the dark).
   2. Wash the slides three times with PBS for 5 min each. Permeabilize with 0.25% Triton for 10 min.
7. Dilute DAPI (10 µg/mL) with PBS at a ratio of 1:10 and add 50-100 µL to each tissue section at RT for 5 min.
8. Wash the slides three times with PBS for 5 min each.
9. 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.

3. Quantification of pulmonary microvascular density

1. Acquire images.
   1. Observe lung microvascular density signals and DAPI signals under a 10x objective for each lung lobe in both the young and old age groups.
   2. 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).
2. Quantify using Image J (Supplementary Figure 1).
   1. Launch ImageJ software.
   2. 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.
   3. Navigate to Analyze > Set Scale > Configure the Image Parameters as 0.48 µm/pixel (depending on the photography parameters) > Global > OK.
   4. Set the same display range to adjust the image presentation effect. Select Image> Adjust > Brightness/Contrast > Set. Set display range for each of the two channels separately, IB₄: 0-10000; DAPI: 0-20000.
   5. To eliminate the influence of background signals, proceed with the following steps: Click Process > Math > Subtract; Subtract the background signal based on the signal values detected by the Magic Wand tool on the background, IB₄: 3000; DAPI:300.
   6. To select the threshold value that best aligns with the original image, go to Image > Duplicate.
   7. Select both original and duplicate Image > Type > 8 bit.
   8. Select an appropriate threshold value to precisely identify the lung microvasculature. Click on Image > Adjust > Threshold > Intermodes (choose the most realistic threshold against the original diagram), tick > Dark Background > Apply.
   9. 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 (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.
   10. Count the number of target signals, click Analyze > 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.
   11. Choose Summarize Results > File > Save as.
3. Data analysis and statistics
   1. Match and organize the number of IB₄ positive foci and DAPI positive foci, respectively, at the same image into a unified table.
   2. Summarize the total number of IB₄ positive foci and DAPI positive foci for each lobe in indicated young or age groups.
      1. 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.
   3. Launch the data analysis software and create a new column table.
      NOTE: GraphPad Prism 9 was used here for statistical analysis.
   4. Sequentially name them from Group A to Group J as "Young cranial lobe", "Old cranial lobe", "Young middle lobe", "Old middle lobe", "Young caudal lobe", "Old caudal lobe", "Young accessory lobe", "Old accessory lobe", "Young left lobe", "Old left lobe". Then, input the corresponding IB₄ positive foci (%) into the table (Supplementary Table 1).
   5. 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.
   6. Create a graph for result visualization (Figure 3B). Opt for a bar chart and include statistical analysis in the figure.

Results

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...

Discussion

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^...

Materials

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