Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart
July 29th, 2020
5.3K views
Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.
Overview
This protocol outlines a method to study coronary microcirculation in living murine heart tissue through ex vivo monitoring of arterial perfusion pressure and flow. It focuses on vascular tree components, including capillary beds and pericytes, during the cannulation and pressurization of the septal artery.
Key Study Components
Area of Science
- Cardiovascular physiology
- Microcirculation
- Vascular biology
Background
- Understanding coronary microcirculation is crucial for cardiac health.
- Pericytes play a significant role in vascular stability and function.
- Ex vivo models allow for controlled experimental conditions.
- Monitoring arterial perfusion pressure is essential for assessing microcirculatory function.
Purpose of Study
- To investigate the dynamics of coronary microcirculation in murine hearts.
- To analyze the role of pericytes in vascular function.
- To measure vascular diameter and arteriole luminal pressure.
Methods Used
- Ex vivo monitoring of arterial perfusion pressure and flow.
- Cannulation of the septal artery using custom-made micropipette cannulae.
- Preparation of cannulae with specific dimensions for optimal insertion.
- Flushing of cannulae with Tyrode's solution to ensure patency.
Main Results
- The method successfully allows for the assessment of coronary microcirculation.
- Pericyte function can be evaluated in the context of vascular health.
- Arterial perfusion pressure and flow can be accurately monitored.
- Vascular diameter measurements provide insights into microcirculatory dynamics.
Conclusions
- This protocol is a valuable tool for studying coronary microcirculation.
- It enhances understanding of pericyte roles in vascular biology.
- The method can be adapted for various experimental conditions.
Frequently Asked Questions
What is the significance of studying coronary microcirculation?
Studying coronary microcirculation is vital for understanding heart function and the role of vascular components in health and disease.
How are the cannulae prepared for the experiment?
Cannulae are prepared using a micropipette puller to create long thin tips, followed by cutting and fire polishing to achieve the desired diameter.
What solutions are used during the cannulation process?
Tyrode's solution is used to flush the cannula and ensure it is free of obstructions.
Can this method be applied to other models?
Yes, while this protocol focuses on murine hearts, it can be adapted for other experimental models.
What measurements can be taken using this protocol?
The protocol allows for measurements of arterial perfusion pressure, flow, vascular diameter, and arteriole luminal pressure.
Executive Industry Relevance
This method enables direct measurement of coronary microcirculation dynamics in murine heart tissue, providing a physiologically relevant platform to evaluate vascular responses to pharmacological agents. By simultaneously monitoring arterial perfusion pressure, flow, and vascular diameter changes in arterioles and capillaries, it supports mechanistic de-risking of cardiovascular targets. The ability to visualize pericytes and assess their functional contribution adds translational value for target validation in heart failure and ischemic disease programs.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses on coronary tone regulation by measuring arteriolar and capillary diameter changes under controlled perfusion conditions.
- Operational Value: Enables functional validation of vascular targets such as pericytes and ion channels in a near-physiological, beating tissue preparation.
Screening & Assay Development
- Scientific Value: Prepares validated vascular networks for compound screening by enabling real-time imaging of lumen diameter and pressure responses.
- Operational Value: Supports assay standardization through reproducible cannulation and perfusion protocols, facilitating cross-laboratory consistency in microcirculation assessment.
Translational & Preclinical Research
- Scientific Value: Links discovery-phase vascular phenotypes to preclinical outcomes by monitoring pericyte-mediated capillary regulation in disease-relevant genetic models.
- Operational Value: Provides quantitative, imaging-based endpoints for go/no-go decisions in cardiovascular lead optimization.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target engagement to functional validation, particularly for cardiovascular programs focused on microvascular health and pericyte biology.
- Discovery Biology: Supports pathway clarification by linking agonist/antagonist exposure to real-time vascular diameter and pressure changes in arterioles and capillaries.
- Screening: Enables assay readiness through stable, long-term imaging of vascular responses under constant flow or pressure conditions.
- Analytics: Generates quantitative readouts of luminal pressure, flow rate, and vessel diameter that allow objective comparison of compound effects.
- Translational Research: Connects microcirculatory findings to preclinical continuity via pericyte visualization in NG2-DsRed transgenic models, supporting biomarker-aligned target validation.
- Enterprise Reuse: Establishes a reusable platform for assessing cardiovascular compounds across multiple projects by standardizing vascular preparation and imaging parameters.
Operational & Enterprise Impact
- Scientific Value: Enhances predictive confidence in target validation by reducing mechanistic ambiguity in coronary microcirculation regulation.
- Operational Value: Promotes reproducibility through standardized cannulation, perfusion, and imaging workflows applicable across murine strains and disease models.
- Strategic Value: Improves capital efficiency by enabling early identification of compounds with off-target vascular effects, reducing late-stage attrition.
- Portfolio Impact: Informs risk-adjusted advancement decisions by providing direct evidence of target engagement in physiologically relevant microvascular beds.
Implementation Considerations
- Requires expertise in microsurgery, vascular cannulation, and confocal microscopy for successful tissue preparation and imaging.
- Depends on specialized instrumentation including pressure myograph chambers, servo pumps, and high-speed confocal systems capable of Z-stack imaging.
- Necessitates cross-team standardization between surgical, perfusion, and imaging teams to maintain consistent arterial pressure and flow settings.
- Involves adaptation considerations when applying the method to different mouse models, including transgenic lines expressing fluorescent reporters in pericytes or endothelial cells.
- Limited by the need for meticulous bubble-free tubing preparation and stable superfusion conditions to avoid artifacts in pressure and diameter measurements.
Biopharma FAQs
Why does null hypothesis testing matter for target validation in coronary microcirculation studies?
Null hypothesis testing ensures that observed changes in arteriolar or capillary diameter are statistically significant and not due to random variation, supporting confident target validation decisions.
How does independent variable isolation fit the discovery pipeline for vascular compound screening?
Isolating independent variables such as agonist concentration or perfusion pressure allows researchers to attribute vascular responses directly to the compound under test, improving target specificity in early discovery.
What quantitative dependent variable measurements enable mechanistic de-risking in cardiovascular target validation?
Quantitative measurements of luminal pressure, flow rate, and vessel diameter provide objective, reproducible endpoints to assess compound effects on microcirculatory function.
Why do replication requirements matter for cross-functional collaboration in microcirculation research?
Replication ensures that vascular responses are consistent across experiments and teams, building confidence in assay reliability for multi-site preclinical programs.
What statistical analysis capabilities are required before implementing this method in a discovery workflow?
The method requires baseline normalization, variance analysis, and significance testing to distinguish true vascular responses from noise, ensuring data integrity for go/no-go decisions.
Chapters in this video
0:04
Introduction
0:41
Cannula Preparation
1:39
Mouse Heart Extraction
2:33
Septal Artery Preparation and Cannulation
3:47
Preparation Stabilization
4:38
Fluorescence-Tagged Wheat-Germ Agglutinin (WGA) Loading and Confocal Arteriole and Capillary Imaging
5:42
Results: Representative Capillary and Arteriole Imaging and Luminal Pressure and Atrial Diameter Assessment
6:42
Conclusion
