The experiments were carried out on eight Wistar albino rats of both sexes (mean weight 327 ± 26 g., four males).
The animals were housed and treated in accordance with the Italian law on animal experimentation (L. 116/92) and with the European Council (EC) provision 86/609/EEC, which received the World Medical Association Declaration of Helsinki.
Rats were anesthetized with 50 mg /100 g i.p. chloralose and laid on a heated operating table. After a tracheostomy, a small polyethylene cannula (2 mm i.d., 5 cm long) was inserted through an incision in the second tracheal ring and firmly secured in place.
Positive pressure ventilation with a 10 ml/kg tidal volume and a 60 per min breathing frequency (PEEP 3 cm H2O) (Rodent Ventilator 7025, Basile, Italy) was begun, and constantly maintained throughout the experiment (apart from the short time necessary to measure respiratory mechanics, see below).
Limb ECG probes were placed, and the rats were paralyzed (cis-atracurium 1 mg/100 g i.p.). Positive pressure ventilation was maintained for 5 min, and respiratory mechanics were then measured using the end-inflation occlusion method.
The ventilator was disconnected, PEEP was discontinued, and the tracheal cannula was connected to a constant flow pump (SP 2000 Series Syringe Pump SP210iw, World Precision Instruments, USA) set to deliver a tidal volume (VT
) of 3 ml with a square wave flow (F
) of 4 ml/s. The time for the rise and the fall of the flow was approximately 30 ms. The pump setting was carefully checked by directly taking measurements before beginning the experiments. For each inflation, the time that the ventilator remained disconnected was about 10–15 s, so that determinant arterial blood gas changes were avoided.[18
The lateral tracheal pressure proximal to the tracheal cannula was monitored (142 pc 01d, Honeywell, USA) and continuously recorded (1326 Econo Recorder, Biorad, Italy). Because of abrupt changes in diameters were not present in the circuit, errors in flow resistance measurements, such as those previously reported,[19
] were avoided. The frequency response of the transducer and the pressure measuring system was tested by sinusoidal forcing and found to be flat up to 20 Hz. In accordance with the literature,[6
] this frequency response was adequate to avoid mechanical artefacts in the pressure signal records.
The entire experimental procedure lasted less than 1 h. Data in the literature indicate that mechanical ventilation parameters here adopted are not injurious to the respiratory system for at least 1 h,[8
] so that the results we obtained are not affected by mechanical ventilation-linked respiratory system injury.
Respiratory mechanics parameters measurements were obtained as below described in the following conditions: (a) in control conditions (supine position) after 5 min of mechanical ventilation; (b) some minutes after the imposition of Tnd (about 20–25° head-down tilt); (c) some minutes after Pnp induction and Tnd resolution (supine position); and (d) some minutes after Tnd restoring while Pnp was maintained.
Pnp was induced by a small bore needle (22G) inserted through the abdominal wall and air insufflation up to a pressure of about 12 mmHg. At the end of the experiments, the animals were killed by a lethal i.p. injection (Tanax® 0.3 ml/kg).
The end-inflation occlusion method was utilized to determine the parameters of respiratory mechanics: the static elastic pressure of the respiratory system (Pel,rs
) and the sudden Newtonian resistive pressure drop at flow interruption (Pmin,rs
) were measured on adequately magnified tracings . Pmin,rs
was calculated as the difference between Pdyn,max,
the maximum value of pressure at end inflation, and P1,
the pressure value immediately after flow interruption . The sum of Pmin,rs
and of the slower, nearly exponential, pressure drop following flow interruption due to respiratory system viscoelastic behaviour, i.e. stress relaxation,[5
] is named Pmax,rs
. Our tracings allowed to identify P1
, which separates the pressure drop due to the frictional forces developed in the movement of airflow in the airway (Pmin,rs
) from the following nearly exponential viscoelastic pressure drop which represents the effects of stress relaxation.
Figure 1 Example of tracing recorded upon constant flow inflation arrest. The maximum pressure achieved at end inflation (Pdyn,max), the pressure drop due to frictional forces in the airway (Pmin,rs), and the overall resistive pressure drop (Pmax,rs), including (more ...)
To avoid a viscoelastic pressure component in Pmin, rs,
values were identified by extrapolating the pressure tracings to the time the flow stopped.[20
The mean pressure data obtained from two to three inflations for each rat were used to calculate the respiratory system static elastance (Est,rs = Pel,rs/VT) and the ohmic inspiratory resistance to airflow offered by the airways and the movement of respiratory system tissues (Rmin,rs = Pmin,rs/F). The overall inspiratory pressure drop (Pmax,rs) measurement allowed us to calculate Rmax,rs = Pmax,rs/F, which includes the ohmic airways resistance and the viscoelastic component here named Rvisc,rs = (Pmax,rs - Pmin,rs)/F.
The equipment resistance, including the tracheal cannula and the standard three-way stopcock, was measured separately at a flow rate of 4 ml/s and amounted to 0.0575 cm H2O ml-1 s-1 (Req). All inflations were performed at a fixed flow rate of 4 ml/s, and Req was subtracted from the results, which thus represent intrinsic values.
The mean values of the measured and calculated respiratory system mechanics parameters obtained in the four tested experimental conditions (see above) were calculated and statistically compared each other. A nonparametric text (Wilcoxon) was applied because of the rather small sample size. Data are expressed as mean ± SD (n = 8).