A separate group of adult rats (n= 7) received an intraperitoneal overdose of the previously described anaesthetic cocktail. The thorax was immediately opened through a wide mid-sternal thoracotomy to expose the whole diaphragm which was then excised by carefully micro-dissecting the tendineous fibres around the phrenic centre and the muscular fibres along the external peripheral costal and vertebral margins. The excised diaphragm was first rinsed and then maintained in 4°C PBS. Tissue strips were cut from fresh diaphragm within 2–3 h of the anaesthesia overdose. Four tissue strips (∼8 mm long and ∼3 mm wide) were excised from: (1) the ventral hemi-diaphragm, dissected in the direction perpendicular to diaphragmatic fibres axis; (2) the ventral hemi-diaphragm, dissected in the direction parallel to fibres axis; (3) the dorsal hemi-diaphragm, dissected in the direction parallel to diaphragmatic fibres axis; and (4) the dorsal hemi-diaphragm, dissected in the direction perpendicular to fibres axis, as schematically depicted in Fig. 2A. Each sample was gripped with a testing machine (Fig. 2B; Enduratec ELF3200, Bose Corporation, Eden Prairie, MN, USA, equipped with a load cell of 22 N) and underwent a series of tension tests. The length between the grips was measured by a calliper after imposing a pre-load of 0.05 N and was defined as the initial sample length. To avoid dehydration, the samples were immersed in saline during the test. After 20 pre-conditioning cycles up to 20% strain, the specimens were exposed to the standard stress–relaxation test. Each relaxation test was performed by applying a fixed stretch corresponding to the desired strain and measuring the resulting force. Force and displacements were recorded during the test by the machine software and then processed to obtain strains (ɛ) and stresses (σ). The strain is defined as:
where l and l0 are the specimen length after elongation and the initial length, respectively. With the initial specimen resistant area (Ao), given by the product of specimen width (Wo) times thickness (ho), the stress, according to the Lagrangial description, is defined as:
where F is the measured force returned by the machine (Fig. 2B). Specimen width was measured by a calliper, whereas its thickness was measured, before the test sequence, by placing the sample between the machine grips and applying a small compressive load (5 mN) in the direction of the sample thickness. The step-wise test was defined as a series of four strain ramps, each 5% of the sample initial length, at a velocity of 10% s−1, followed by stress relaxation to equilibrium. The elastic modulus of the tissue (Et) for all of the tested strain levels was calculated from the stress–relaxation test as:
where Δσ and Δɛ are, respectively, variation of equilibrium stress (i.e. at the end of stress–relaxation) and variation of strain between successive ramps. After the last ramp, the samples were returned to the initial length and a constant velocity elongation of 10% s−1 was applied until specimen failure (Fig. 2C). The elastic modulus before failure (Et,f) is defined as the linear portion of the curve preceding the yield stress, the latter being the value of σ where the stress–strain curve bends before failure. As graphically indicated in Fig. 2C, in our experiments this linear portion was between 80 and 90% of the maximum stress.
Figure 2. Tension tests on diaphragmatic tissue strips A, schematic drawing of the diaphragm with indication of the site of excision of the tissue strips: 1, ventral zone, transversal to major fibres axis; 2, ventral zone, longitudinal with respect to fibres axis; 3, dorsal zone, longitudinal to major fibres axis; 4, dorsal zone, transversal to fibres axis. B, schema of the apparatus used for tension test on diaphragmatic tissue strips. F, force measured by the machine; Ao, initial sample resistant area; σ, resulting stress. C, typical stress (σ)–strain (ɛ) plot until failure (arrow) of a diaphragmatic tissue strip. The elastic modulus before failure, Et,f, was graphically represented by the slope of the tangential line to the stress–strain curve at a stress of 80 to 90% of the failure stress (Failure = 100% on the right Y axis). Mathematically, Et,f was obtained as: .
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