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Updated: Oct 5, 2025
Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse
Published on: March 8, 2013
Quantitative Three-Dimensional Analysis of the Lymphatic Vasculature in the Postnatal Mouse Heart
Konstantinos Klaourakis1, Paul R Riley2, Joaquim Miguel Vieira3
1Burdon-Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, and British Heart Foundation-Oxbridge Centre of Regenerative Medicine, CRM, University of Oxford, Oxford, UK.
Insights
This study presents a new imaging protocol to analyze lymphatic vessel development in neonatal mouse hearts. The method quantifies lymphatic branching, length, and endpoints, aiding research into cardiac lymphatic function.
Area of Science:
- Cardiovascular Biology
- Lymphatic System Development
- Developmental Biology
Background:
- Lymphatic vasculature is crucial for organ function; its disruption causes severe phenotypes like myocardial edema.
- Cardiac lymphatic malfunction leads to reduced cardiac output and persistent inflammation.
- Understanding cardiac lymphatic development from fetal stages to adulthood is vital.
Purpose of the Study:
- To describe a protocol for whole-mount, multi-view imaging and quantification of lymphatic vessel parameters in the murine postnatal heart.
- To characterize the expression patterns of lymphatic markers in the neonatal heart.
- To provide an adaptable image analysis pipeline for studying lymphatic systems.
Main Methods:
- Whole-mount, multi-view imaging of neonatal mouse hearts.
- Quantification of lymphatic vessel parameters: junction number, vessel length, and endpoints.
- Utilizing antibodies against lymphatic endothelial cell (LEC) markers: LYVE1, VEGFR3, PDPN, and NRP2.
Main Results:
- A detailed protocol for imaging and quantifying lymphatic vasculature in neonatal murine hearts is established.
- Expression patterns of LYVE1, PDPN, and NRP2 were characterized in neonatal heart tissues.
- VEGFR3 was identified as a suitable marker for imaging the sub-epicardial lymphatic network in neonatal hearts.
Conclusions:
- The developed protocol enables comprehensive analysis of cardiac lymphatic development.
- The study provides insights into the expression of key lymphatic markers in the neonatal heart.
- The image analysis pipeline can be adapted for studying other organ systems, including blood vasculature and the nervous system.
Abstract:
The development and maturation of the lymphatic vasculature are essential for organ function with disruption leading to severe phenotypes. For example, malfunction of cardiac lymphatics results in myocardial oedema, persistent inflammation and reduced cardiac output. Thus, it is important to study the process of cardiac lymphatic formation and growth from the early stages of fetal development to adulthood. In the murine heart the lymphatics continue to develop and expand postnatally with extensive growth and patterning occurring up to at least 2 weeks after birth. Here, we describe a protocol for whole-mount, multi-view imaging and quantification of lymphatic vessel parameters, including vessel junction number (i.e., branching density), vessel length, and number of vessel end points in the murine postnatal heart. This protocol is based on the use of reliable antibodies against key markers of lymphatic endothelial cells (LECs), specifically the glycoprotein lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), the vascular endothelial growth factor receptor 3 (VEGFR3; also known as Fms-related receptor tyrosine kinase 4, FLT4), the mucin-type protein podoplanin (PDPN), and the co-receptor neuropilin 2 (NRP2). For imaging and quantitative analysis of the sub-epicardial network in neonatal hearts, VEGFR3 was selected given its exclusive expression in the lymphatic endothelium. In addition to LECs, LYVE1 expression was detected in tissue-resident macrophages, PDPN in the epicardium, and NRP2 in the autonomic nervous system of the heart. Overall, we characterized the expression patterns of commonly used lymphatic markers in the context of the neonatal heart and provide an image analysis pipeline that can be adapted to study other organs and systems (e.g., blood vasculature and nerve system).
