A new in vitro model for force measurements at the isolated entire rat diaphragm

Introduction

Several diseases of different origin as well as prolonged mechanical ventilation result in diaphragmatic dysfunction and atrophy. To date most in vitro experiments on the mechanics of diaphragm muscles are performed at isolated muscle strips. Since the improvement of the knowledge about the diaphragm functionality is lacking in view of the position and curvature, a model of the whole diaphragm appears necessary. Here we present a new in vitro model of an isolated whole rat diaphragm inside a bioreactor [1].

Methods

The bioreactor consists of a pressure chamber on which a highly flexible membrane is attached. A rat diaphragm is fixed on this membrane. By application of a gas volume into the pressure chamber, a defined deflection of the diaphragm is achieved. The highly flexible membrane adapts a given shape thus allowing the diaphragm to take up its in vivo profile. Electrical stimulation results in a contraction of the diaphragm. These diaphragm-twitches generate pressure pulses inside the pressure chamber. Rat diaphragms were excised rapidly and kept at room temperature in Krebs–Henseleit solution bubbled with oxygen. By application of one of two different initial pressures (P1: 7 mbar; P2: 12 mbar) the diaphragms were set to a defined degree of deflection. Using a platinum wire electrode, the diaphragms were electrically stimulated at impulses of 6 V. The stimulation train duration was set to 500 ms or 1 second. The pulse duration was set to 50 ms at a frequency of 50 Hz. The pressure pulses resulting as the response on muscle contraction were measured inside the pressure chamber.

Results

An increase of the initial pressure led to an increased pressure caused by the muscle contraction. An enlarged muscle regeneration time led to an increase of the diaphragmatic twitch-induced pressure.

Conclusion

Our new in vitro model of an isolated whole rat diaphragm allows the performance of mechanical and physiological investigations on the entire diaphragm. A dependency of pressure development of the diaphragm on its deflection state could be demonstrated. Furthermore, we found effects of diaphragm muscle fatigue. Our new model could be used in numerous types of investigations, such as releasing factors of diaphragmatic dysfunction or respiratory muscle fatigue.