setting up the perfusion chamber to measure contractility in isolated, pressurized lymph vessels

实时分析离体淋巴管中的钙和收缩力

The lymphatic vasculature is found in many organ systems including the brain, heart, lungs, kidney, and mesentery1,2,3,4,5,6, and operates by propelling fluid (lymph) from the interstitial spaces to the central venous ducts to maintain fluid homeostasis7,8,9,10. It starts with blind-ended lymphatic capillaries within the vascular capillary beds that drain into collecting lymph vessels (LVs). Collecting LVs are made of two layers of cells: a layer of endothelial cells encompassed by a layer of lymphatic muscle cells (LMCs)10,11. Lymph fluid transport is achieved through both extrinsic forces (e.g. new lymph formation, arterial pulsations, central venous pressure fluctuations) and intrinsic forces12.
The intrinsic force for lymph transport is the spontaneous rhythmic contraction of collecting LVs, which is the focus of the majority of studies investigating lymphatic function. This intrinsic lymphatic pump is principally regulated by the cyclic rise and fall of cytosolic, free Ca2+ ([Ca2+]i). Spontaneous depolarization of the plasma membrane in LMCs activates voltage-gated &#34;L-type&#34; Ca2+ (Cav1.x) channels triggering Ca2+ influx and subsequent LV rhythmic contraction8,9,10. This role was demonstrated by blocking Cav1.x with specific agents, like nifedipine, which inhibited LV contractions and caused vessel dilation13,14. The transient rise in [Ca2+]i or &#34;Ca2+ spike&#34; in the LMCs mediated by Cav1.x channels also may mobilize intracellular Ca2+ stores by activating inositol triphosphate (IP3) receptors and ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR)15,16,17,18. Current evidence suggests IP3 receptors contribute more Ca2+ required for normal LV contractions compared to RyRs15,16,19,20,21; however, RyRs may play a role during pathology or in response to pharmaceutical intervention17,18. Additionally, the activation of Ca2+-activated K+ channels22 and ATP-sensitive potassium (KATP) channels23,24 can hyperpolarize the LMC membrane and inhibit spontaneous contractile activity.
There are many other ion channels and proteins that may regulate Ca2+ dynamics in collecting LVs. Utilizing methods to study changes in Ca2+ and vessel contractility in response to pharmacological agents in real time is important to understand these potential regulators. An earlier method using Fura-2 to measure relative changes in LV [Ca2+]i has been described25. Because the dissociation constant for Fura-2 and Ca2+ is known26, it is possible to calculate actual concentrations of Ca2+, which broadens the application of this method and provides additional insight into Ca2+ signaling, membrane excitability, and contractility mechanisms27, as well as allowing for baseline comparisons between experimental groups. This latter approach has been used in cardiomyocytes28, and therefore, can be adapted to LVs. This paper presents an improved method that combines these two approaches to measure and calculate changes in absolute [Ca2+]i as well as vessel contractility/rhythmicity continuously in real time in isolated, pressurized LVs. We also provide representative results for LVs treated with nifedipine.

作者聚焦：淋巴水肿和高血压研究的创新方法

实时评估隔离、加压淋巴管中的绝对、胞质、游离 Ca2+ 和相应的收缩力

设置灌注室以测量孤立的加压淋巴管中的收缩力

首先，取从商业来源购得的硼硅酸盐玻璃微量移液器。将微量移液器切割并抛光至约一至两厘米的长度，以便在隔离的血管灌注室中使用。将血管灌注室中每个安装的玻璃套管连接到独立的压力传感器，这些压力传感器与独立的重力进气压力调节器对齐，并使用聚乙烯管连接。用生理盐溶液回填输液室、玻璃微量移液器和重力进料压力调节器以及聚乙烯管，然后夹紧压力以确保插管不受压。

setting-up-perfusion-chamber-to-measure-contractility-isolated

Research

JoVE Journal

Immunology and Infection

Setting Up the Perfusion Chamber to Measure Contractility in Isolated, Pressurized Lymph Vessels

208 次观看. 首先，取从商业来源购得的硼硅酸盐玻璃微量移液器。将微量移液器切割并抛光至约一至两厘米的长度，以便在隔离的血管灌注室中使用。将血管灌注室中每个安装的玻璃套管连接到独立的压力传感器，这些压力传感器与独立的重力进气压力调节器对齐，并使用聚乙烯管连接。用生理盐溶液回填输液室、玻璃微量移液器和重力进料压力调节器以及聚乙烯管，然后夹紧压?...

视频: 设置灌注室以测量孤立的加压淋巴管中的收缩力

Real-time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i).
This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i&#160;and diameter measurements continuously in real time.
The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

This protocol describes a method to simultaneously measure cytosolic, free calcium [Ca2+]i and vessel diameter in contracting lymph vessels in real time and then calculate absolute Ca2+ concentrations as well as contractility/rhythmicity parameters. This protocol can be used to study Ca2+ and contractile dynamics across a variety of experimental conditions.

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical ...