measurement of the absolute concentrations of &#91;ca2+&#93;i in isolated, pressurized lymph vessels

Real-Time Analysis of Calcium and Contractility in Isolated Lymph Vessels

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.

Author Spotlight: Innovative Methods in Lymphedema and Hypertension Research

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

We study mechanisms of lymph vessel contractions, and lymph flow to better understand the underlying pathology of cancer-related lymphedema, as well as the connection between the lymphatic system and hypertension. Our goal is to identify therapeutic targets within the lymphatic vasculature so that we can develop novel treatments for lymphatic diseases. Recent discoveries of different ang channels in lymphatic muscle and endothelial cells, which contribute to the underlying calcium dynamics have proved instrumental in moving the field forward.Isolated vessels and intravital microscope have been crucial techniques for studying lymphatic vessel functions. More recently, there has been an upward trend of using organ on a chip and micro-fabricated vessels to investigate many aspects of lymphatic biology. The very small tissue size challenges, studying the lymphatic vasculature, requiring a lot of time, patience, and the development of new technologies.The unavailability of lymphatic muscle cell specific markers is another hurdle for the whole scientific community. The advantage of this protocol is the ability to measure lymphatic contractions and absolute calcium concentrations within the same vessel. This provides additional insight into calcium signaling, mechanisms of contraction, as well as the ability to measure baseline comparisons between treatment groups.Compared to other techniques that maybe rely on relative changes in calcium or paralyzed vessels.

Measurement of the Absolute Concentrations of &#91;Ca2+&#93;i in Isolated, Pressurized Lymph Vessels

measurement-absolute-concentrations-ca2-i-isolated-pressurized-lymph

Research

JoVE Journal

Immunology and Infection

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 ...

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

To begin, take borosilicate glass micro-pipettes acquired from a commercial source. Cut and polish the micro-pipettes to a length of approximately one to two centimeters for use in the isolated vessel perfusion chamber. Connect each mounted glass cannula in the vessel perfusion chamber to independent pressure transducers aligned with independent gravity-fed pressure regulators and connected using polyethylene tubing.Backfill the profusion chamber, glass micro-pipettes, and the gravity-fed pressure regulator along with the polyethylene tubing with the physiological salt solution, then clamp the pressure to ensure the cannulas are not pressurized.