In vitro muscle contraction force measurements on isolated and entire rat diaphragms

Introduction

Inactivity of the diaphragmatic muscles during mechanical ventilation leads to atrophy and contractile dysfunction. Up to now, in vitro force measurements were performed only on single diaphragmatic muscle strips. Our intention was to find out how mechanical and electrical stimulation influences the condition of the diaphragm as a whole organ. To determine the status of the diaphragm, muscle contraction forces were measured on entire rat diaphragms.

Methods

We used an earlier described bioreactor [1] as the cultivation and measurement device for the whole rat diaphragm. The bioreactor consists of a pressure chamber and a supply chamber that are separated by a very flexible and soft membrane [2]. On this membrane the sample diaphragm is placed. By application of certain gas volumes (0 to 1.5 ml) in the pressure chamber, diaphragms are deflected to various levels of pretension. Diaphragms were electrically stimulated at each deflection level 10 times (750 ms duty cycle, 100 ms stimulation time, 5 ms pulse width, 200 Hz frequency). Pressure changes caused by muscle contraction were recorded inside the pressure chamber and muscle contraction forces were calculated. After initial force measurements, diaphragms were exposed for 6 hours to one of four different treatments: nonstimulated storage (control), cyclic mechanical deflection, electrical stimulation every 20 minutes, combination of cyclic deflection and electrical stimulation. After 6 hours another force measurement was performed. Supernatants were collected after 6 hours and investigated for IL-6 activity.

Results

Depending on the level of deflection of the diaphragms, muscle contraction force increased from 0.1 N (volume 0.6 ml) to 0.7 N (volume 1.5 ml). A larger pretension of the diaphragm resulted in larger muscle contraction force. After treatment, muscle contraction force decreased in all groups. Muscle contraction force was smallest in the passive control group (0.05 N), larger and similar in the electrically stimulated (0.1 N) and combination (0.09 N) groups and largest in the mechanically deflected group (0.15 N). IL-6 activity increased after 6 hours of treatment.

Conclusions

We conclude that it is possible to perform force measurements on whole rat diaphragms in our in vitro model. Additionally, the diaphragms can kept alive for >6 hours to apply different stimulation treatments. The diaphragmatic muscle force generation depends on the pretension level of the diaphragm as well as on the treatment history.