The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

Summary

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

Abstract

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap – previously cast to match the average size of the rat heart – is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 µL/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

Introduction

Heart failure (HF) is the leading cause of death in humans worldwide¹. HF often occurs because of myocarditis, ischemic insults to the myocardium, and left ventricular remodeling, leading to the progressive deterioration of cardiac contractile function and patients' quality of life. Although advances in cardiology and cardiac surgery have remarkably lowered HF mortality, they merely serve as transient "delayers" of an inevitably progressive disease process that carries significant morbidity. Therefore, the current lack of effective treatment underscores the need to identify novel molecular targets that can prevent or even reve....

Protocol

All experiments were approved by the local regulatory agency (LANUV of Nordrhein-Westfalen, Germany) and were performed according to the guidelines of animal use. Animals were fed with a standard chow diet and received tap water ad libitum. All equipment and chemicals necessary to each step of the experiment are available in the Table of Materials.

1. Preparation of the Latex Cap and Intraventricular Balloon

1. Make an aluminum mold using a milling machine that matches the average size of the rat heart (bodyweight of 300-350 g). Polish the mold with superfine (10/0) emery paper.
   NOTE: The d....

Results

The reversed-heart model enables the collection of cardiac interstitial transudate in an isolated, retro-perfused rat heart (Figure 1A-C). When perfused at a constant pressure of 100 cmH₂O, the rate of interstitial fluid formation ranged between 17 and 147 µL/min, amounting to 0.1-1% of the coronary venous effluent in the isolated heart.

The protein content of the CT, as .......

Discussion

The reversed-heart model is based on the well-established Langendorff heart perfusion technique¹² and is performed by simply inverting the heart into an upside-down position and holding this position using a rigid intra-ventricular balloon catheter. In such a way, cardiac interstitial transudate can be physically separated from coronary venous effluent perfusate, dripping by gravity from the base of the heart⁹. The CT can be continuously collected by means of a thin and fle.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Latex Solution	ProChemie	Z-Latex LA-TZ	http://kautschukgesellschaft.de/%E2%80%A8z-latex-la-tz
Aluminum Mold	Home made	-	Reverse heart model
Universal Ovens	Memmert	UNB 400	Reverse heart model
Latex Balloon	Hugo Sachs	Size 4	Reverse heart model
Milling Machine	Proxxon	MF70	Reverse heart model
Sodium Chloride	Sigma	SZBD0810V	Chemicals
Sodium Hydrogen Carbonate	Roth	68852	Chemicals
Potassium Chloride	Merck	49361	Chemicals
Magnesium Sulphate Heptahydrate	Merck	58861	Chemicals
Potassium Dihydrogen Phosphate	Merck	48731	Chemicals
D(+)-Glucose Anhydrous	Merck	83371	Chemicals
Calcium Chloride Dihydrate	Fluka	21097	Chemicals
Balance	VWR	SE 1202	Weighing chemicals
Double Distilled Water	Millpore	-	Disolving chemicals
Medical Pressure Transducer	Gold	-	Langendorff apparatus
Medical Flow Probe	Transonic	3PXN	Langendorff apparatus
Heating Circulating Bath	Haake	B3 ; DC1	Langendorff apparatus
Laboratory and Vaccum Tubing	Tygon	R-3603	Langendorff apparatus
Animal Research Flowmeters	Transonic	T206	Langendorff apparatus
PowerLab Data Acquisition Device	AD Instruments	Chart 7.1	Langendorff apparatus
LabChart Data Acquisition Software	AD Instruments	Chart 7.1	Langendorff apparatus
Peristaltic Pump	Glison	MINIPULS 3	Langendorff apparatus
Glass Water Column	home made	-	Langendorff apparatus
Water Bath Protective Agent	VWR	462-7000	Langendorff apparatus
Sterile Disposable Filters (0.2 µm)	Thermo Scientific	595-4520	Langendorff apparatus
Blood gas analyzers	Radiometer	ABL90 FLEX PLUS	Gas analyzer
70% ethanol	VWR	UN1170	Cleaning  tubings
100% ethanol	Merck	64-17-5	Cleaning tubings
Wistar Rats	Janvier	-	Animals
Stainless Scissors	AESCULAP	BC702R	Surgical Instruments
Stainless Scissors	AESCULAP	BC257R	Surgical Instruments
Big Forceps	AESCULAP	-	Surgical Instruments
8m/m Stainless Forceps	F.S.T	11052-10	Surgical Instruments
superfine (10/0) emery paper	3M	051111-11694	Reverse heart model