Summary

Правого желудочка систолическое давление измерений в сочетании с Harvest легких и иммунной образцы тканей у мышей

Published: January 16, 2013
doi:

Summary

Конкретных и быстрых протоколов одновременно исследовать функцию правого сердца, воспаление легких, и иммунный ответ описывается как инструмент обучения. Видео и цифры описывают физиологию и микродиссекции методы в организованном командой подход, который адаптирован для использования в малых и больших размеров исследований.

Abstract

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.

Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.

Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.

The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

Protocol

1. Подготовка Подготовьте следующие решения и трубы (таблица 1) следующим образом: Хэнкс решение, не кальций, магний или индикатора с пенициллина (100 ЕД / мл) / стрептомицин (100 мкг / мл). Фосфатным буферным раствором (PBS), 1x, ни кальций, ни магний. Этанол, 70%, состав?…

Representative Results

Основным результатом для получения права кривые давления сердца достигается правильное положение правой катетер сердца. Форма кривых давления время имеет решающее значение, потому что правильное размещение катетера в правом желудочке приведет к давлению плато (рис. 4). Spiky кр…

Discussion

Экспериментальные потока изложенные здесь обеспечивает быстрое и одновременное измерение давления правого желудочка систолическое и сбор образцов для анализа ответов в легких, сердца и иммунной системы у мышей. Процедура сочетает в себе сердце физиологии измерений, микро-рассечени?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Эта работа финансировалась Национальным институтом здоровья 1R21HL092370-01 (GG), 1R01 HL095764-01 (GG); R01HL082694 (JW), Американской ассоциации сердца, Учредителей филиал (0855943D, GG); Stony Wold – Герберт фонда, Нью-Йорк (SHP).

Materials

Name Company Catalogue number Comments (optional)
Reagents
2-Methyl-2-butanol Sigma-Aldrich 152463
2,2,2-Tribromoethanol Sigma-Aldrich T48402
disinfectant soap (Coverage Spray TB plus Steris) Fisher Scientific 1629-08
Ethyl Alcohol, 200 Proof, Absolute, Anhydrous ACS/USP Grade PHARMCO-AAPER 111000200 Dilute to 70 % with distilled water
Formaldehyde solution Sigma-Aldrich F1635-500ML Dilute to a 7-10 % formaldehyde concentration at a PBS concentration of 1x using PBS stock solution and water
Hanks solution, no calcium, magnesium Fisher Scientific 21-022-CV
O.C.T Tissue-Tek 4583
Penicillin (10,000 U/ml) / Streptomycin (10,000 mg/ml) solution Thermo Scientific SV30010
Phosphate buffered saline (PBS), no calcium, no magnesium, 1x and 10x solutions Fisher Scientific
Sodium pentobarbital 26% Fort Dodge Animal Health NDC 0856-0471-01
Labware
Plates 12, 24, 96 well Falcon
Transfer Pipet Fisher Scientific 13-711-9BM
Tube, EDTA coated Sarstedt 2013-08
Tubes 0.65 ml and 1.7 ml micro-centrifuge VWR
Tubes 12 x 75 mm polypropylene Fisher Scientific 14-956-1D
Tubes, various sizes, polypropylene Fisher Scientific
Instruments
Forceps, Dumon #5 Fine Fine Science Tools 11254-20
Forceps, extra fine graefe -0.5 mm tips curved Fine Science Tools 11152-10
Forceps, extra fine graefe -0.5 mm tips straight Fine Science Tools 11150-10
Cannula 18 ga, 19 ga BD Precision Glide Needles Cut to optimal length, blunted and outside rasped to create a rough outside surface.
Scissors, Dissector scissors-slim blades 9 cm Fine Science Tools 14081-09
Suture for BAL, braided silk suture, 4-0 Fine Science Tools SP116
Suture for right heart catheterization, braided silk suture, 6-0 Teleflex medical 18020-60
Syringe, 1 ml BD 309659
Equipment
Amplifier, PowerLab 4/30 ADInstrument Model ML866
Catheter, pressure F1.4 Millar Instruments, Inc 840-6719
Dissecting Microscope Variscope
Forceps, Vannas spring scissors-2 mm blades Fine Science Tools 15000-00
Halogen Illuminated Desk Magnifier Fisher Scientific 11-990-56
Laptop computer Asus Model number A52F i5 processor; 15 inch
Light Source Amscope HL-250-A
Pressure Control Unit Millar Instruments, Inc PCU-2000
Software, Labchart-Pro V.7 AD Instruments

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Cite This Article
Chen, W., Park, S., Hoffman, C., Philip, C., Robinson, L., West, J., Grunig, G. Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice. J. Vis. Exp. (71), e50023, doi:10.3791/50023 (2013).

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