Short courses of mechanical ventilation with high-O 2 levels in elderly rat lungs

Purpose: To evaluate the effects of mechanical ventilation (MV) of high-oxygen concentration in pulmonary dysfunction in adult and elderly rats. Methods: Twenty-eight adult (A) and elderly (E), male rats were ventilated for 1 hour (G-AV1 and G-EV1) or for 3 hours (G-AV3 and G-EV3). A and E groups received a tidal volume of 7 mL/kg, a positive end-expiratory pressure of 5 cm H2O, respiratory rate of 70 cycles per minute, and an inspiratory fraction of oxygen of 1. We evaluated total protein content and malondialdehyde in bronchoalveolar lavages (BAL) and performed lung histomorphometrical analyses. Results: In G-EV1 animals, total protein in BAL was higher (33.0±1.9 μg/mL) compared with G-AV1 (23.0±2.0 μg/mL). Upon 180 minutes of MV, malondialdehyde levels increased in elderly (G-EV3) compared with adult (G-AV3) groups. Malondialdehyde and total proteins in BAL after 3 hours of MV were higher in elderly group than in adults. In G-EV3 group we observed alveolar septa dilatation and significative increase in neutrofiles number in relation to adult group at 60 and 180 minutes on MV. Conclusion: A higher fraction of inspired oxygen in short courses of mechanical ventilation ameliorates the parameters studied in elderly lungs.


Introduction
Elderly patients have become an increasingly prevalent proportion of the intensive care unit (ICU) population.Outcomes of patients with acute respiratory distress syndrome (ARDS) have been significantly improved in recent years 1 .
In the very elderly population (85 years and older), the acute severity of illness is the most significant predictor of mortality after an ICU admission associated with a significant functional deterioration 2,3 .The senescence process itself decrease of the muscular strength and aerobics capacity, vasomotor instability, reduction of the bone mass, reduction of the lung ventilation etc. 4 .Regarding the mortality rate post-ICU, a study by Gordo et al. 5 showed that among patients over 75 years the age can be associated with mortality as an independent factor, whereas Iapichino et al. 6 demonstrated that after discharging ICU the mortality rate was increased in patients over 78 years.
Artificial ventilation is one of the most important techniques in the ICU, besides being an important tool in the approach of the acute lung injury/acute respiratory distress syndrome (ALI/ARDS) of any nature or in other cases 7,8 .In many cases of respiratory diseases, the use of ventilatory support is required.Besides that, a ventilatory support is needed in other situations, including sepsis and sepsis shock; neuromuscular disease; during postoperative state; and in cases of altered mental status with loss of consciousness 9,10 .
Several studies consider the age as a factor influencing the clinical prognosis of a patient needing mechanical ventilation 11,12 .These are in disagreement with others that consider, in addition to the age, other factors like functional status and the seriousness of the acute process [11][12][13][14] , and others yet which consider age as an associated factor [15][16][17][18] .Although no consistent data exist in this scenario, it is presumable that aged individuals have greater susceptibility to the harmful effects of mechanical ventilation than adults.
Animal studies the effects of hyperoxia have employed very high levels of inspired oxygen fraction (FiO 2 > 0.95) for extended periods of time.These experimental models produce severe lung injury and death in 48-72 h 19 .Patients without lung dysfunction requiring such high concentrations of supplemental oxygen, however, often receive concurrent ventilatory support.The deleterious effects of hypoxia are well known and physicians may be mostly concerned about avoiding hypoxia and giving additional oxygen 'to remain on the safe side 20,21 .
Significant resistance to hyperoxia-induced oxidative stress has been demonstrated in pulmonary endothelial cells overexpressing manganous superoxide dismutase (Mn-SOD) 22 .Limited literature, however, exists on the role of reactive oxygen species (ROS) in modulating the inflammatory response during early or late phase of acute inflammation [23][24][25] .
The present study tested in an animal model the effect of two different periods of mechanical ventilation and we hypothesize that elderly subjects are more susceptible than adults to the deleterious effects of a large period of mechanical ventilation using a higher fraction of inspired oxygen and to the accompanying oxidative stress.

Surgical procedure
All procedures were approved by the Institutional Research Committee at Federal University of Sao Paulo, in accordance with the National Institutes of Health (NIH) Guidelines Regarding Animal Experimentation and the principles of the Brazilian College on Animal Experimentation.
Twenty eight male Wistar rats (Rattus norvegicus albinus) from which 14 were adult (4 month-old, body weight [bw] -320-360 g) and 14 were elderly (24 month-old, bw 430-465 g), were divided in four groups (n = 7 animals each) as follows: adult (G-AV1) and elderly (G-EV1) rats ventilated for 1 hour; and adult (G-AV3) and elderly (G-EV3) rats ventilated for 3 hours.The animals were individually housed, fed rat chow and water ad libitum, and were maintained on a 12-h light/dark cycle for 3 weeks prior to initiation of the experiments.

Mechanical ventilation (MV)
Rats were anesthetized with an intraperitoneal (ip) bolus injection of sodium thiopental (50 mg/kg; Thionembutal ® , Abbott, SP, Brazil).After reaching a surgical plane of anesthesia and after tracheostomy, a 14G cannula was inserted into the trachea.Upon tracheostomy, a sterile PE50 catheter was inserted into the right carotid artery for blood gases sampling and another one into the left carotid artery for mean arterial pressure (MAP) control (Pressure transducer, Gould-Statham, CA, USA).Muscle relaxation was performed with ip succinylcholine chloride (0.8 mg/kg; Uniao Quimica Farmaceutica Nacional, SP, Brazil).
The animals were immediately connected to a rodent ventilator (Harvard Apparatus, MA, USA) and ventilated during 1 or 3 h using a tidal volume (V T ) of 7 mL/kg, a positive endexpiratory pressure (PEEP) of 5 cmH 2 O, a fraction of inspired oxygen (FiO 2 ) of 1 and a respiratory rate (RR) of 70 cycles per minutes (cpm).Body temperature was maintained at nearest 37 °C by the use of a heating blanket and controlled by a rectal thermometer (YSI Inc., OH, EUA).

Arterial blood gases measurements
Arterial blood gases were collected in sterile vented plastic syringes (PICO 70, Radiometer, Copenhagen, Denmark) and measured at different times: in the groups ventilated for 1 hour at the times 0 min and 60 min; the ventilated groups for 3 h were evaluated at the times 0 min, 60 min and 180 min, using an automatic analyzer (AVL-Compact3, Roche Diagnostic, Germany).

Bronchoalveolar lavage (BAL)
At the end of experiment, the animals were euthanized (ip doses of 1 mL/100 g body weight; T-61 Euthanasia Solution, Schering-Plough, SP, Brazil).A sternotomy was performed and the lungs and structures were removed and weighed.The left main stem bronchus lung was tied and the right lung was washed three times with 10 mL/kg of sterile saline solution.Lavage fluids were kept separate and centrifuged (400 x g for 10 min, 4 ºC) and the supernatants were immediately frozen on dry ice and stored at -80ºC.

Total cell countings and total protein determinations in BAL
The total number of cells in BAL was counted in a hemocytometer (Neubauer chambers) under 100x magnification.Total protein content was measured (in duplicate) in 1-mL aliquot of BAL by using the method of Lowry and colleagues 26 .

Thiobarbituric acid-reactive substances (TBARS)
Products of lipid and protein oxidation were determined in the homogenate of the left lung by using the method of Das 27 .The concentrations of lipid peroxidation (LPO) products (thiobarbituric acid-reactive substances, TBARS) were determined through the absorbance at 532 nm and expressed as mmol/mg protein.

Morphology and morphometry
Lungs were removed en bloc and inflated at a pressure of 20 cmH 2 O with 4% paraformaldehyde in phosphate solution (100 mM phosphate-buffered saline [PBS] pH 7.4) at room temperature for fixation during 12 h.Fragments of the left lung were washed in 50mM phosphate buffer, pH 7.4, dehydrated in graded concentrations of ethanol, and then embedded in paraffin.From every lung, 4-µm sections were obtained and stained with hematoxylin and eosin (HE) to evaluate lung morphology and morphometry.Neutrophils counts were performed in the alveolar septae by image capture with a light microscope using 400X magnification (Axiolab Standart 2.0, Carl Zeiss) coupled to a video camera (AxionCam, Carl Zeiss).

Statistical analyses
Data are presented as mean ± standard deviation (SD).Statistical intergroup comparisons of total cell, total protein, TBARS, and lung neutrophil were performed using one-way ANOVA.Statistical comparisons of mean arterial blood pressure, and blood gas analyses among the groups were performed using two-way ANOVA.Differences among groups were tested by the Tukey's multiple comparisons test.A P value less than 0.05 was considered significant.Statistical analyses were performed using a standard computer software (GraphPad Prisma, GraphPad Software, CA, USA).

Discussion
We investigated whether bronchoalveolar levels of malondialdehyde, total protein and neutrophil influx in elderly rats submitted to mechanical ventilation for 1 hour and 3 hours.
In a recent study, Nin et al. 28 ventilated young and elderly animals during 60 min using respiratory rate 70 cpm, FiO 2 0.35, and a protective lung strategy (V T = 9 mL/kg and PEEP = 5 cmH 2 O) or with an injurious strategy (V T = 35 mL/kg and PEEP = 0 cmH 2 O, overventilated).In this study elderly rats showed increased susceptibility to injurious mechanical ventilationinduced pulmonary injury, vascular dysfunction, and systemic inflammation in lungs using a lower FiO 2 .The higher FiO2 in our study was chosen because the high FiO 2 used under certain circumstances may help to cause redistribution of pulmonary blood flow and to decrease vasoconstriction in atelectatic or nonventilated areas 29 .Our study showed that higher FiO 2 promoted an increase of Pa O 2 in adult and elderly animals ventilated for 1 hour, but Pa O 2 decreased and Pa CO 2 increased after 180 min in elderly compared with adults mechanically ventilated.
The use of higher FiO 2 was determinate by unclear whether such high FiO 2 will increase oxygen free radical activity.The higher FiO 2 can induce a concomitant increase in free radical activity and the lipid peroxidation causes tissue damage 21,[30][31][32][33] .Moreover, lipid peroxide end-products of tissue damage can disrupt cellular function by formation of disulphide bridges across nucleotide or amino acid chains 32 and inhibits lymphocytic activity 33 .Whether MV per se initiates pulmonary inflammation in patients with non-injured lungs is still unclear 34 , although we have shown that a lung protective MV strategy attenuates intrapulmonary coagulation caused by a more conventional MV strategy 35 .
Some studies have assessed the changes occurring in lungs of aged rats.First, the older lungs might be subjected to greater mechanical stress due to a difference in the alveolar septae, which may result in a greater susceptibility to stretch-induced lung injury.
Second, for any given mechanical stress, the aged lung may have a more vigorous inflammatory response.This potential mechanism would be in line with studies showing that, after certain stimuli, aged animals and humans show a more marked response after an inflammatory challenge 29,36 .Although oxygen toxicity is a well known entity 37 , FiO 2 s up to 0.5 are commonly considered 'safe' by physicians 20 .It appears that physicians are more concerned about avoiding hypoxia and ischaemia than about the risks of hyperoxia.In The Netherlands, no formal guidelines for oxygenation targets are available.This may be related to the fact that the influence of oxygenation targets has never been studied making it impossible to provide evidence-based recommendations.Based on other observational studies, it may well be that also in other countries actual PaO 2 s in ICU patients are higher than recommended 38,39 .
Our study, using protective strategy and higher FiO 2 , expanded the results to demonstrate the increase in total protein and malondialdehyde in BAL in elderly animals ventilated for 180 min when compared with adults.In 1-h ventilated elderly animals, the differences in the biochemical analyses were augmented when compared with adults, but this effect was neither followed by arterial oxygen decrease nor was the alveolar structure affected.
This study has limitations.Animals submitted to MV and PEEP were ventilated with a 100% inspired oxygen fraction, although subsequent hyperoxia may have influenced lung results.However, this protocol was used to avoid any possibility of hypoxia.In fact, hypoxia has been associated with rapid increases in vascular permeability and in leukocyte adherence 40 .Secondly, in our study the tracheal peak pressure was not evaluated.Thirdly, ventilatory strategies using a moderate tidal volume of 10 mL/kg and PEEP levels from zero up to 10 cmH 2 O have been used with surgical patients 41,42 .

Conclusions
The present study showed that the association of a protective tidal volume and high FiO 2 may be harmful to microcirculation in the setting of elderly normal lungs during a short time of MV.Our findings indicate that aged rats ventilated for 3h and high inspired oxygen concentration developed more marked local changes compared with their adult, younger counterparts.This finding may have relevance in understanding the development of complications related to MV and the use of high fraction of inspired oxygen in elderly subjects.
O 2 and PaCO 2  at the time points 0 (zero), 60 and 180 minutes in the groups G-AV1, G-EV1, G-AV3 and G-EV3 (n=7 per group).Data are mean ± SD.G-AV1= adults ventilated for 1 h; G-AV3= adults ventilated for 3 h;G-EV1= elderly ventilated for 1 h; G-EV3= elderly ventilated for 3 h.a P=0.001 compared with G-AV1 at the time 60 min -Pa O 2; b P=0.001 compared with G-AV3 at the time 60 min -Pa O 2 ; c P=0.001 compared with G-AV3 at the time 180 min -Pa O 2 ; d P=0.01 compared with G-AV1 at the time 60 min -Pa CO 2 ; e P=0.01 compared with G-AV3 at the time 60 min -Pa CO 2 ; f P=0.01 compared with G-AV3 at the time 180 min -Pa CO 2 .

TABLE 3 -
Effects of mechanical ventilation on the mean arterial pressure (MAP) at the time points 0 (zero), 60 and 180 minutes in groups G-AV1, G-EV1, G-AV3 and G-EV3 (n=7 per group).Data are mean ± SD.
for 3 h.a P<0.05 compared with G-EV3 at the time 180 min.b P<0.05 compared with G-AV3 at the time 180 min.

FIGURE 1 -
FIGURE 1 -Effects of mechanical ventilation (MV) on total cells in bronchoalveolar lavage at the end of 1-h and 3-h MV.Data are mean ± SD.

FIGURE 2 -
FIGURE 2 -Effects of mechanical ventilation on total protein in bronchoalveolar lavage at the end of 1-h and 3-h MV.Data are expressed as mean ± SD.

FIGURE 3 -
FIGURE 3 -Effects of mechanical ventilation on markers of lipid oxidation (TBARS; in nmol/mg protein) in the bronchoalveolar lavage at the end of 1-h and 3-h MV.Data are expressed as mean ± SD.

FIGURE 4 -
FIGURE 4 -Effects of mechanical ventilation on neutrophil counting in lung tissue at the end of 1-h and 3-h MV.Data are expressed as mean ± SD.

FIGURE 5 -
FIGURE 5 -Representative photomicrographs of rat lung histological sections in G-AV1; G-EV1; G-AV3 and G-EV3.Presence of inflammatory cells (arrows) in the animals ventilated for 1 h compared with the 3-h ventilated groups.(HE, 400x).