Morphological findings in the tracheal epithelium of dogs exposed to the inhalation of poorly conditioned gases under use of an endotracheal tube or laryngeal mask airway

PuRPOsE: To study morphological findings in the tracheal epithelium of dogs exposed to the inhalation of poorly conditioned gases under use of an endotracheal tube (ET) or laryngeal mask airway (LMA). METHOds: Twelve dogs randomly were allocated to two groups: ET group (n-6) and LMA group (n-6), anaesthetized and mechanically ventilated, without CO2 reabsorption. Haemodynamic and ventilatory parameters, tympanic temperature, temperature, relative and absolute humidity of the ambient and inhaled gases were analyzed during three hours. The animals were submitted to euthanasia and biopsies were carried out along the tracheal segment to morphological study. Three healthy dogs were used to morphological control. REsuLTs: Inhaled gas temperature was maintained between 24oC and 26oC, relative humidity between 10% and 12%, and absolute humidity between 2 3 mg H2O.L -1 with no significant differences between groups. In both groups, histological analysis showed epithelial inflammation and congestion in the corion and scanning electron microscopy showed ciliary grouping and disorganization. Transmission electron microscopy showed higher alterations in ET group than LMA group as widening of cell junctions, ciliary disorientation, cytoplasmic vacuolization, nuclear abnormalities, picnosis and chromatin condensation. CONCLusION: LMA determined less pronounced changes in the tracheal epithelium in dogs exposed to the inhalation of poorly conditioned gases.


Introduction
During respiration, inspired air gains approximately 75% of heat and humidity as it passes through the nose and rhinopharynx, due to the high vascularization and a large contact surface 1 .In the remainder airway segments (pharynx, larynx, trachea and bronchi), the conditioning process is completed.
Nasal obstruction, tracheotomy, intubation or laryngectomy are conditions that the passage of inspired air through the nasal cavities is impaired requiring high participation of remaining airway segments in the humidity and heat exchanges 2 .
The characteristics of the respiratory epithelium are important in the conditioning of inhaled air.The respiratory epithelium is sensitive to the action of inspired irritative factors.
Experimental researches have reported respiratory epithelial injuries of varying degrees in consequence of the inhalation of cold and dry gases as decreased mucociliary clearance; increased mucus viscosity and dryness; ciliary disorganization, grouping or devastation; mucosa erosion, ulceration and necrosis; inflammation and even metaplasia of the tracheal epithelium in chronic processes [3][4][5] .
In a previous experiment carried by our group, the real participation of the laryngotracheal segment in air conditioning was quantitatively demonstrated.Significantly, higher tracheal air absolute humidity was observed in dogs exposed to the inhalation of high fresh gas flow with inadequate temperature and humidity through a laryngeal mask airway (LMA) (23 mg H 2 O.L -1 ) than in those ventilated through an endotracheal tube (ET) (14 mg H 2 O.L -1 ).These satisfactory results were attributed to the positioning of the LMA, which is placed higher on the airway (supraglottic region) keeping the laryngotracheal segment free from contact with the inspired gases, and allowing its exposure to heat and humidity exchanges 6 .However, the performance of tracheotomy, necessary for the introduction of a thermohygrometer to monitor the temperature and humidity of the tracheal gases, did not allow morphological analyses of the tracheal epithelium.Thus, in order to complement our previous investigation, this study aimed at describing the morphological particularities of the tracheal epithelium in dogs exposed to the inhalation of gases with low temperature and humidity to confirm the advantages of using a LMA in relation to ET.

Methods
The study was approved by the Animal Research Ethics Committee of Botucatu Medical School -UNESP.Twelve adult mixed-breed male dogs, weighing 15-27 kg, underwent venous anaesthesia with sodium pentobarbital and with mechanical ventilation through a system without CO 2 reabsorption for three hours.The animals were randomly allocated into the two following groups, according to the method used for airway maintenance: ET group (endotracheal group; n -6), using ET Portex n o 8.5 (England)   and LMA group (Laryngeal Mask Airway; n -6), using LMA Classic TM n o 4 (Singapore).A biomonitor (AS3, Datex Engstron) was used for monitoring and recording temperature, ventilation, oxygenation and haemodynamic variables.
After 12 hours of fasting, a 20-G catheter was placed in the femoral vein.The dogs received a bolus injection of 25 mg/ kg pentobarbital, followed by intravenous infusion maintenance of 5 mg/kg per hour, and infusion of 5 mL/kg per hour of lactated Ringer's solution for hydration, using a two channel infusion pump (Anne, Abbott).The animals were placed in dorsal recumbence with the head extended, and maintained under mechanical ventilation in a system without CO 2 reabsorption with 3 L/min ambient air and 2 L/min O2 (K.TAKAOKA, model 2600, Series Nikkei).Neuromuscular block was induced by alcuronium chloride at an initial dose of 0.2 mg/kg, followed by maintenance doses of 0.06 mg/kg every 60 minutes.We used a tidal volume of 20 mL/kg and a respiratory rate of 12-14 breaths/min to maintain an end-tidal CO 2 (PetCO 2 ) concentrations at 4.66-5.33kPa.The left femoral artery was cannulated with polyethylene catheter (P240) to measure mean arterial pressure.A three-lead electrocardiogram (DII lead) was installed and a pulse oximetry sensor was located on the animal's tongue.The temperature and relative humidity of the inspiratory gas were measured using an electronic digital thermo-hygrometer (hygrotermo 95, Gulton), connected by a Tplaced between the inspiratory branch and ET or LMA.Absolute humidity was calculated from relative humidity and temperature according to a specific table.The formula used was: Haemodynamic parameters (mean arterial pressure, heart rate), ventilatory parameters (respiratory rate, tidal volume, peripheral oxyhaemoglobin saturation -SpO 2 , P ET CO 2 , inspiratory pressure), tympanic temperature, temperature and both relative and absolute humidity of the inhaled gases were measured after 10 minutes of mechanical ventilation installation, and at 60, 120 and

Morphological findings in the tracheal epithelium of dogs exposed to the inhalation of poorly conditioned gases under use of an endotracheal tube or laryngeal mask airway
After three hours, the animals were euthanized by pentobarbital overdose.The laryngotracheobronchial segment was removed, and in ET group the tracheal tube was maintained to identify the exact position of its distal extremity.Biopsies measuring 0.5 X 1 cm were performed below the 5 th tracheal ring in both groups, on the inner surface of the anterior portion of the trachea.Thus, the following sites of biopsies were pre determined: (PT -proximal trachea, MT -medial trachea and DT -distal trachea, Figure 1).The fragments were examined under light and electron microscopy (scanning and transmission).The pathologist was blinded to the origin of the samples.
Three healthy male dogs were euthanized by pentobarbital overdose.The laryngotracheobronchial segments were removed and biopsies were done in the same areas to morphological control of light and electron microscopy.

Light microscopy
The histopathological analysis included the epithelium Significance was set at p<0.05.

Haemodynamic and ventilation parameters remained constant over
time, with no significant differences between groups (p>0.05).
Tympanic temperature was kept between 37 o C and 38.5 o C, with no differences between groups (p=0.69).Inhaled gas temperature was between 24 o C and 26 o C with no significant differences between groups (p=0.46),relative humidity was maintained between 10% and 12%, while absolute humidity was maintained between 2 mg H 2 O.L -1 and 3 mg H 2 O.L -1 with no significant difference between groups (p=0.22 and p=0.62, respectively).

Morphological assessment
The morphological analysis of all tracheal specimens of the control animals showed normal aspect.In light microscopy the pseudostratified columnar ciliated epithelium was composed by ciliated cells, goblet cells and basal cells.No edema, congestion or inflammation were identified in the lamina propria (Figure 2).However, in the animals of ET and LMA groups, histological changes were observed in some areas that included acute inflammation, polymorphonuclear neutrophil infiltration in the epithelium, and congestion in the lamina propria (Figure 3).These findings were slightly more accentuated in ET Acta Cirúrgica Brasileira -Vol.26 ( 5) 5011 -361 Morphological findings in the tracheal epithelium of dogs exposed to the inhalation of poorly conditioned gases under use of an endotracheal tube or laryngeal mask airway group, though no statistical difference was reached (p>0.05).
Scanning electron microscopy (SEM) showed abundant ciliated cells on tracheal epithelium of the control animals and goblet cells lied between them (Figure 4).In ET and LMA groups were observed ciliary grouping and disorganization, without difference between groups (p=0.58)(Figure 5).Transmission electron microscopy (TEM) demonstrated similar aspect with ciliated, goblet and basal cells.Numerous cilios could be identified on the apical portion of ciliated cells.
Intracytoplasmatic organelles were disposed with a distinct polarity and a large nucleus occupying about half the cell volume.
The goblet cells were filled with vesicles containing material of variable density.The cells are connected by desmosomes and interdigitations of the cell membrane (Figure 6).In ET and LMA groups, TEM demonstrated some important alterations, particularly among the dogs under ET ventilation (p=0.01).These alterations included widening of cell junctions, ciliary disorientation, cytoplasmic vacuolization, nuclear abnormalities, picnosis and chromatin condensation (Figure 7).Neither epithelial erosion nor disruptions in basement membrane integrity were observed in any of the groups.

discussion
The main finding of this study was that ventilation via LMA gave less pronounced changes in the tracheal epithelium than ventilation via an ET in dogs exposed to the inhalation of poorly conditioned gases.Then, our study is the first one to demonstrate that the LMA seems to protect tracheal epithelium during inhalation of dry air.A system without CO 2 reabsorption with high FGF was used in an attempt to reproduce the same methods employed in our previous experiment by keeping the humidity and heat of inspired air at low levels 6 .Some investigators Morphological findings in the tracheal epithelium of dogs exposed to the inhalation of poorly conditioned gases under use of an endotracheal tube or laryngeal mask airway have shown that high FGF has a reducing effect on humidity and temperature.Henriksson et al. 8 assessed humidity and temperature at different gas flows (<1 L.min -1 , 2 L.min -1 , and 5 L.min -1 ), and found lower heat and humidity levels at a FGF of 5 L.min -1 , which was also used in our study.
The dog trachea has about forty cartilaginous rings that are mostly located in the cervical region facilitating access to biopsy sites.In this study, morphological evaluation was performed on biopsy fragments of the proximal, medial and distal trachea, located beneath the distal edge of the tube, avoiding the tracheal areas where there was contact with the cuff.
Damages to the respiratory epithelium secondary to the inhalation of poorly conditioned gases via an ET have been reported in several experimental studies 3,9,10 .During anaesthesia, the lowest absolute humidity of the inspired gases is recommended to be 23 mg H 2 O.L -1 to reduce the risk of dehydration of the respiratory tract 11 .It is not infrequent the inhalation of gases with inadequate humidity during anaesthesia, even if low FGF (1 L.min -1 ) is utilized 12 .Thus, the levels of inspired gas humidity used in this study were lower than those considered as critical.Under these conditions, the most remarkable histological alterations observed during the short 3-hour period of exposure investigated were those related to acute inflammatory processes, such as migration of polymorphonuclear neutrophils (PMN) to the lamina propria and congestion.These findings were observed in both groups, but were slightly more intense in the group of animals ventilated via an ET.
Perhaps, extending the study period, and consequently the time of mucosa exposure to such conditions, could provide more relevant histological results in the ET group.Inflammation is a complex reaction consisting of vascular responses, migration and activation of leukocytes.Vasodilatation leads to fluid accumulation and leukocyte migration to extravascular tissues under the influence of chemotactic agents.The inflammatory response is essentially a defense mechanism whose primary goal is to eliminate the initial cause of cell lesion and its consequences (tissue repair process).
No animal in any of the study groups showed more intense histological damages, such as epithelial hemorrhage and erosion.
These findings are more commonly reported in more severe epithelial injuries caused by intubation trauma and in sites where the mucosa is in contact with cuffs 13,14 , but these areas were not examined here.
Scanning electron microscopy most commonly revealed ciliary grouping and disorganization on the epithelial surface in both groups.These alterations, attributed to low inspired gas humidity, may be observed when the mucus is thicker and dehydrated, leading to cilial adherence as described by others authors 3 .In this study, the prevalence of these alterations in the group ET could be attributed to the less favorable conditioning of the gases inspired.However, the analysis of the semiquantitative scores once more demonstrated no significant difference, not allowing the establishment of a causal relationship among variables.In our previous study 6 , significantly higher tracheal air absolute and relative humidity were verified in dogs exposed to the inhalation of high FGF through a LMA (23 mg H 2 O.L -1 and 80%, respectively) than in those ventilated through an ET (14 mg H 2 O.L -1 and 50%, respectively).
The relative humidity of the gas is also important, even if the absolute humidity was sufficient, a low relative humidity of about 50% dried the secretions and the condensed secretions might stick in the ET of the patients in a critical care unit 15 .Thus, the low relative humidity of the inhaled gas in both groups could to explain the lack of significant differences between the results obtained under SEM in the groups.
Only TEM showed significant differences between groups.The animals ventilated via an endotracheal tube displayed more intense morphological alterations, such as disorganized ciliary positioning, widening of cell junctions, inflammatory cell infiltrates in the epithelium, cytoplasmic vacuolization, and abnormal nuclear morphology including irregular picnotic nuclei with condensed chromatin.The basement membrane seemed normal in all animals.Some of the ultrastructural alterations, such as cytoplasmic vacuolization and changes in nuclear shape, were exclusively observed in the group ET.The inclusion of TEM examination in this study was crucial to detect early tracheal epithelium alterations.Otherwise, these morphological signs of cell suffering and death would not be detected.
Some further limitations of this study need to be discussed.As with any animal model, data cannot be directly transposed to humans.The principal advantage of an animal model is that ensures identical standardized treatments and allows post-mortem histological studies.In the study, we utilized LMA or ET for three hours.However, LMA is usually used for surgery not exceeding 1 -2 hours.This could difficult the extrapolation of our reported results to a realistic clinical scenario.On other hand, the use or a LMA during prolonged surgery is increased 16,17 .

Conclusion
In comparison with ventilation via an endotracheal tube, the use of a laryngeal mask airway determines less pronounced changes in the tracheal epithelium in dogs exposed to the inhalation of poorly conditioned gases.

(
photographed at increasing magnitudes under a scanning electron microscope (model QUANTA 200, FEI, Czech Republic).Each fragment was semiquantitatively scored as follows: 0 (no ciliary alteration, completely intact epithelium), 1 (focal ciliary grouping and/or disorientation), 2 (extensive ciliary grouping and/or disorientation, without epithelial ulceration), 3 (extensive ciliary rarefaction, without epithelial ulceration) and 4 (extensive ciliary rarefaction and epithelial ulceration).For TEM, the specimens were fixed in 2.5% glutaraldehyde, flushed in 0.1M phosphate buffer, pH 7.3, and added to 1% tannic acid in order to facilitate extracellular matrix visualization.Subsequently, they were sectioned into 3mm x 1mm slices on pink dental wax, wetted in 2.5% glutaraldehyde fixing solution, fixed by 1% osmium acid and 0.1M phosphate buffer, pH 7.3, dehydrated in an increasing series of acetone solution (50%, 70%, 90% and 100%), and dipped in a mixture of acetone and araldite resin (Polysciences, Inc.).The fragments were removed from this mixture and embedded in an araldite resin block in an oven at 37 o C. Semi-fine 0.5-μm sections were cut and dyed with

FIGuRE 4 -
FIGuRE 4 -Control dog.Distal trachea.Scanning electron microscopy showing goblet cells (gc) lie between ciliated cells (A, x 4,820), and abundance of the ciliated cells and normal cilia structure (B -x 4,055, C -x 4,630 and d -x 6,840).

FIGuRE 6 -
FIGuRE 6 -Control dog.Medial trachea.Transmission electron microscopy showing abundance of the ciliated cells and normal aspect of the cilia (A -x 17,000); goblets cells and ciliated cells (B -x 13,250 ; C -x 5,750); intact and continuous basement membrane (arrow; d -x 17,000).