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Current Respiratory Medicine Reviews, 2012, 8, 448-453

The Effects of Erythropoietin on the Respiratory Function: Measurements of Respiratory Mechanics in the Rat Alessandro Rubini1, Gerado Bosco1, Andrea Par magnani1, Daniele del Monte2 and Vincenzo Catena2 1

Department of Biological Sciences, Section Physiology, University of Padova, Italy

2

Department of Emergency and Intensive Care, ULSS 2, Feltre (BL), Italy Abstract: Data reported in the literature indicate that erythropoietin (EPO) influences mammals respiratory function, for example stimulating pulmonary ventilation. Direct experimentations about possible effects of EPO on respiratory mechanics are lacking. In the present report, the endinflation occlusion method was applied in control and EPO-treated anaesthetised and positive-pressure ventilated rats to assess respiratory mechanics. The method involves a sudden flow arrest after a constant flow inflation, and allows to measure the ohmic airway resistance and the respiratory system elastance. A significant decrement of the ohmic airway resistance after 20 and 30 minutes from i.p. EPO administration was observed in experimental animals, which was not seen in control animals. The elastic characteristics of the respiratory system did not change over time in both groups. Hypothesis about the mechanism(s) explaining the results and potential applications to humans are addressed. In particular, further data were obtained by performing additional experiments suggesting that the observed airway resistance decrement may be related to an EPO-induced increased nitric oxide production, a rather well known bronchodilator agent. Literature and present results indicate that the spontaneous increments of plasmatic EPO concentrations, such as those happening in hypoxia and/or blood loss, may be associated with airway resistance decrement. It is suggested that erythropoietin, beside the well known effect on haematopoiesis, may activate complex physiological responses including haematological, circulatory and respiratory adaptations to hypoxia in mammals.

Keywords: Airway resistance, end-inflation occlusion method, erythropoietin, hypoxia, rat, respiratory mechanics. 1. INTRODUCTION Erythropoietin (EPO) is known to stimulate differentiation and growth of erythroid precursor, but recently some non-haematopoietic effects have also been reported. As indicated by the expression of EPO and its receptors in normal healthy human lung tissues [1], those effects may include influences on the respiratory system functions. If verified, these influences could suggest that EPO-mediated responses to hypoxia and/or blood loss represent a complex scenario of physiological mechanisms of adaptation, including not only haematological but also circulatory and respiratory responses. However, few data are available regarding this subject. 2. REPORTED EFFECTS OF EPO ON RESPIRATORY FUNCTION It has been reported that EPO can increase pulmonary ventilation by increasing both breathing rate and tidal volume in patients with renal failure [2], and a similar effect has also been detected in anaesthetised rats after intracisternal administration [3].

*Address correspondence to this author at the Department of Biological Sciences, Section Physiology, University of Padova, Via Marzolo, 3, 35100 Padova, Italy; Tel: ++390498275310; Fax: ++390498275301; E-mail: [email protected] 1875-6387/12 $58.00+.00

Moreover, EPO probably has some effect on airway resistance: it has been reported to cause inhibition of carbachol-induced bronchoconstriction in mice [4], and to induce increments in forced vital capacity and in peak expiratory flow rate in humans [2]. Similar effects may also ensue because of beneficial effects of EPO on inflamed lung tissues [5, 6], counteracting, for example, the increment in airway resistance caused by inflammatory cytokines such as IL-6 [7]. In addition, EPO affects vascular smooth muscle tone causing relaxation [8, 9], suggesting a possible similar effect on airway smooth muscle tone too. The relaxing activity has been, in fact, related to increased nitric oxide (NO) production [10-12], a well known bronchodilator [13-16]. Studies investigating the possible direct EPO’s effects on respiratory mechanics parameters and airway resistance in healthy mammals are lacking in the literature. However, the effects of EPO administration on respiratory system mechanics were recently assessed in rats using the endinflation occlusion method [17]. This made it possible to investigate both the ohmic, Newtonian airway resistance, as predicted by Poiseuille's law, and respiratory system elastance [18-20]. Purpose of the study was to elucidate if variations in EPO blood concentration occurring in spontaneous conditions, such as blood loss or hypoxia, may affect respiratory system mechanics parameters. © 2012 Bentham Science Publishers

Erythropoietin and Respiratory Mechanics

3. DESCRIPTION OF EPO’S EFFECTS RESPIRATORY MECHANICS: METHODS.

Current Respiratory Medicine Reviews, 2012, Vol. 8, No. 6

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Animals These experiments were performed on 20 albino rats of both sexes (mean weight 307 ± 23 g., 10 males). To investigate the effects of EPO on respiratory mechanics eight animals were used, and six additional rats with similar characteristics were studied as control animals. Six others rats served for the experiments testing the hypothesis that EPO administration may increase NO production (see below). The experimental protocol was approved by the local Ethical Committee (CEASA, University of Padova, ref. N° 48/2011). The housing and the management of the experimental animals were in keeping with the Italian law on animal experimentation (L. 116/92) and with the European Council (EC) provision 86/609/EEC. Experimental Procedure Rata were anaesthetized (50 mg /100 gr ip chloralose) and laid on a heated operating table. Following tracheostomy, a polyethylene tracheal cannula (2mm ID, 5 cm long) was inserted and secured in place. ECG was derived from limb probes. After the start of positive pressure ventilation, the animals was paralyzed (cisatracurium 1 mg/100 gr ip). Positive pressure ventilation used 10 ml/Kg tidal volume and a 60/min breathing frequency (PEEP 3 cmH2O) (Rodent Ventilator 7025, Basile, Italy), and these parameters were maintained constantly throughout the experiment.

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For each rat, measurements by the end-inflation occlusion method were performed at different times (10, 20, and 30 minutes) after the ip injection of 1000 U Kg-1 rat recombinant EPO (SIGMA, St. Louis, Missouri, USA) dissolved in 100 μl PBS. Literature indicates that the adopted mechanical ventilation parameters are not injurious to the respiratory system [7, 16]. Nevertheless, additional six (a number considered sufficient for this purpose) control animals were studied to verify if the variations observed in the experimental group (see below) could be due to a timedependent effect. The control rats received 100 μl PBS ip and respiratory mechanics were studied according to the same procedure described for the experimental rats receiving EPO. Data Calculation On magnified recordings (Fig. 1), the static elastic pressure of the respiratory system achieved after the inflations (Pel,rs), and the resistive pressure drop due to the flow interruption (Pmin,rs), were measured. Pmin,rs was identified as the pressure drop between Pdyn, max, the maximum value of the tracheal pressure at the end of the inflations, and the pressure value immediately after the flow was arrested (P1, see Fig. 1). In the magnified pressure tracings, P1 separated the sudden Newtonian pressure drop due to airflow frictional forces in the airway (Pmin,rs) from the subsequent slower, nearly exponential, pressure decay which is due to the respiratory system visco-elastic characteristics, i.e. stress relaxation [19, 20, 23, 24]. According to the literature [19, 20, 23, 24], the overall pressure decay, including Pmin,rs and the subsequent viscoelastic component, was termed Pmax,rs (Fig. 1).

After 5 minutes, mechanical ventilation and PEEP were discontinued and the end-inflation occlusion method applied to measure respiratory system mechanics [18-20], as follows. A constant flow pump (SP 2000 Series Syringe Pump sp210iw, World Precision Instruments, USA) delivered an inflation volume (VT) of 3 ml according to a square wave flow (F) of 4 ml/sec through the tracheal cannula. The time taken for the arrest of inflation flow was approximately 30 msec. The pump settings were carefully checked during separate measurements performed before the experiments. According to the literature, to avoid significant blood gas changes the time the mechanical ventilation was suspended during the constant-flow inflations was kept very short (less than 30 sec.) [21]. The pressure in the tracheal cannula was laterally monitored (142 pc 01d, Honeywell, USA) and recorded (1326 Econo Recorder, Biorad, Italy). In the ventilatory circuit abrupt changes in the diameters of tubing were not present, so that errors in flow resistance measurements, such as those previously described [22], were avoided. The frequency-response characteristics of the transducer and of the pressure measuring circuit were checked by sinusoidal forcing. The frequency response was found to be flat up to 20 Hz which, according with literature data [23, 24], allowed to avoid significant mechanical artefacts in pressure signal recording.

Fig. (1). An example of lateral tracheal pressure tracing on flow interruption. The pressures used for the definition of respiratory system mechanics are reported: the maximal pressure at end inflation (Pdyn max), the pressure immediatly after airflow interruption (P1), the static elastic pressure of the respiratory system after inflation (Pel,rs), the nearly instantaneous pressure drop due to the ohmic respiratory system resistance (Pmin,rs) and the total pressure drop including the effects of the visco-elastic characteristics of the respiratory system’ tissues, i.e. stress relaxation (Pmax,rs).

To limit the effect of visco-elastic pressure components was in Pmin,rs, P1 values were determined by extrapolating

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the pressure tracings to the instant the flow stopped, as previously described in the literature [25].

Est,rs

The data obtained from 3 to 5 inflations for each rat were used in the calculation of the respiratory system static elastance (Est,rs = Pel,rs/VT) and of the ohmic inspiratory resistance to airflow offered by frictional forces in the airways (Rmin,rs =Pmin,rs/F).

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Est,rs %

The value of the equipment resistance (Req), which included the tracheal cannula and a standard three-way stopcock, was separately measured. Measurements were performed at the same flow rate of 4 ml/sec which was used in the experiments. Req resulted 0.0575 cmH2O ml-1 s-1 and was subtracted from the results, which thus represent intrinsic values.

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Statistics The mean values of respiratory system mechanics parameters before and following EPO were statistically compared (ANOVA). Data are here reported as mean values ± SE (n=8). 4. DESCRIPTION OF EPO’S EFFECTS RESPIRATORY MECHANICS: RESULTS

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The mean values of respiratory mechanics parameters before EPO administration resulted as follows: Rmin,rs = 0.14±0.02 cmH2O ml-1 sec-1, Est,rs= 1.95±0.07 cmH2O ml-1. The time-related percentage variations following EPO and PBS intraperitoneal administration in the experimental and control rats are outlined in Figs. (2, 3). No significant timerelated changes were detected in the control animals after ip injections of PBS, while in the experimental rats significant decrements in Rmin,rs were observed after EPO. Est,rs mean values did not change significantly neither in the control nor in the experimental animals. Heart rate values did not vary significantly during the experiments (Table 1).

Rmin,rs 125

*p