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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 m....

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 sect....

Representative 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.......

Disclosures

The authors declare that they have no competing interests.

Acknowledgements

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 "the Fundamental Research Funds for the Central Universities" to TJ.

....

Materials

NameCompanyCatalog NumberComments
4% ParaformaldehydeBiosharpBL539ATissue Fixative
4',6-diamidino-2-phenylindoleMCEHY-D0814Nucleic Dyes
Alexa-647 Fluor Conjugated Isolectin B4ThermoI32450Binding Microvessels
Anti-fluorescent Tablet SealerAbcamAB104135Sample Fixation
Antigen Repair FluidBiosharpBL151ARepair of Antigenic Sites
Biopsy CassetteActivFlo39LC-500-1Fixing and Positioning Tissue Samples
Bovine Serum AlbuminSigmaB2064-50GSealing Solution
Cold PlateLeicaHistoCore Arcadia H Freezing Samples
Constant Temperature Electric Drying OvenTaisite101-0ABHigh Temperature Repair
Disposable Microtome BladeLeica14035838383Cutting Tissue Samples to Prepare Sections
Embedding MoldsShitai26155166627Fixing Tissue Samples
EthanolKelongCAS 64-17-5Tissue Dehydration Solution
Heated Paraffin Embedding StationLeicaEG1150Embedding Tssue Samples in Paraffin
HistoCore Water BathLeicaHI1220Flatten and Fix Tissue Samples
ImageJ (Fiji) NIH1.54fQuantitative Tool
Immunohistochemistry PensBiosharpBC004Water-blocking Agent
Medical ForcepsShanghai Medical EquipmentN/AGrasping, Manipulating, or Moving tissue samples
MicroscopeNikonTs2Imaging Device
Mounting MediaJiangyuanTastelessFixing and Preserving Tissue Sections
Paraffin WaxSCHLEDEN80200-0014Fixing Tissue Structure
PBSBeyotimeC0221AWash Buffer
Pentobarbital SodiumBeijing Chemical Reagent CompanyQ/H82-F158-2002Anesthetic
Rotary MicrotomeBiobaseBk-2258Preparing Slices
Sterile ScissorsShanghai Medical EquipmentN/Asegmenting Tissue Samples
Surgical ScalpelShanghai Medical EquipmentN/ACutting Tissue Samples
Triton SolarbioT8200Permeabilization Solution
Wash-Free SlidePLATINUM PROPRO-04Fixing Samples for Staining
XyleneSUMXK13-011-00031Tissue De-waxing Solution

References

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