INTRODUCTION

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Endotracheal intubation is one of the most common techniques to assist ventilation in intensive care units. Although life-saving, this invasive procedure can result in laryngeal injury (1, 2). The onset of inflammatory signs usually occurs as early as within the first hour after extubation and is characterized by glottic and subglottic edema, ulceration, and stenosis (3). The injury can result from a direct mechanical stretching and rubbing of the tube and/or from mucosal ischemia induced by the tube or its cuff pressing against the laryngeal mucosa (3, 4). In this regard, it has been demonstrated that the accumulation in the submucosal vessels of marginated inflammatory cells after short-term intubation of the larynx is similar to the vascular histologic features seen in ischemia and reperfusion injury of other tissues (5).

Recently, reactive oxygen and nitrogen species have been implicated as mediators of the disruption of the epithelial barrier in inflammatory airway diseases (6). As highly reactive molecules, radicals and oxidants may indiscriminately attack DNA, causing oxidative modification and strand breakage. The DNA strand breaks activate the nuclear enzyme poly (ADP-ribose) synthetase (PARS), which results in a rapid depletion of intracellular NAD⁺ and ATP energetic pools, leading to cellular dysfunction and death (10). This suicide cellular phenomenon driven by PARS activation has been demonstrated in several cell types, including pulmonary epithelial cells, endothelial cells, macrophages, smooth muscle cells, fibroblasts, and cardiac myoblasts (13).

Recent studies have established that pharmacologic or genetic inhibition of PARS activation may exert beneficial effects ameliorating the metabolic changes, but not the development of DNA damage, in inflammation such as pleurisy and ischemia/reperfusion processes such as cardiovascular shock, multiple organ failure, myocardial infarction, and neural damage during stroke (13).

Considering the importance of the PARS pathway in inflammation, we investigated whether 3-aminobenzamide, a specific inhibitor of PARS, may interfere with the development of laryngeal injury. To this aim, we developed an animal model for laryngeal mechanical injury by short-term intubation followed by extubation. Furthermore, by performing immunohistochemistry for nitrotyrosine (a marker of nitrogen-derivates) in the laryngeal mucosa, we investigated whether the inflammatory process subsequent to extubation is associated with nitrosative stress and whether 3-aminobenzamide treatment may affect this process.

	METHODS

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Animals

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23 revised 1985) and with the approval of the Institutional Animal Care and Use Committee. Male Wistar rats (Charles River Laboratories, Wilmington, MA), weighing 300 to 350 g, were housed in a room with controlled temperature (22° C) and 12-h light/dark and allowed food and water ad libitum.

Induction of Laryngeal Injury by Intubation

Animals were anesthetized with thiopentone sodium (120 mg/kg intraperitoneally). Laryngeal injury was induced by a retrograde intubation technique. A tracheotomy was performed at the third tracheal ring and the trachea was cannulated. A small incision was made between the first and second tracheal rings and a 4F embolectory catheter (Baxter Healthcare Corp., Irvine, CA) was inserted retrogradedly through the tracheal incision and pulled out through the mouth. This was used as a guide for endotracheal intubation with 2 mm (internal dimension) tube, which is an oversized tube in relation to the size of the rat larynx. The catheter was then removed. The animals were intubated for 1 h and then the tube was removed. At 1 hour after extubation, the larynx was excised and snap frozen in liquid nitrogen. The animals were randomly distributed into four groups (eight rats for each group). Group 1: a control group of animals that underwent tracheotomy and were treated with saline solution, but were not intubated (sham+vehicle); Group 2: a control group of animals that underwent tracheotomy and were treated with 3-aminobenzamide (10 mg/kg intraperitoneally), but were not intubated (sham+3-AB). Group 3: a group of animals that underwent the traumatic laryngeal intubation and were treated with saline solution (LI+vehicle). Group 4: a group of animals that underwent the traumatic laryngeal intubation and were treated with 3-aminobenzamide (10 mg/kg intraperitoneally) 15 min prior to extubation (LI+3-AB).

Histopathologic Analysis and Damage Score

Tissue were fixed in 10% formalin solution and embedded in paraffin. Several sections were cut every 40 µm beginning at the inferior margin of the cricoid cartilage. Sections were stained with hematoxylin-eosin for histologic evaluation of tissue damage. In order to have a quantitative estimation of laryngeal damage, sections were scored by two independent observers blinded to the experimental protocol. The following morphologic criteria were considered: Score 0, no damage; Score 1 (mild), interstitial edema and focal mucosal damage; Score 2 (moderate), diffuse mucosal damage and gland swelling; Score 3 (severe), diffuse mucosal damage, gland swelling and neutrophil infiltrate; Score 4 (highly severe), diffuse mucosal damage, gland swelling, neutrophil infiltrate, and hemorrhage.

Immunohistochemical Staining for Nitrotyrosine

Tyrosine nitration was detected in laryngeal sections by immunohistochemistry (14). Frozen sections 5 µm thick were fixed in 4% paraformaldehyde and incubated for 2 h with a blocking solution (0.1 M phosphate-buffered saline containing 0.1% Triton X-100 and 2% normal goat serum) in order to minimize nonspecific adsorption. Sections were then incubated overnight with 1:500 dilution of primary antinitrotyrosine antibody or with control solutions. Controls included buffer alone or nonspecific purified rabbit IgG. Specific labeling was detected by incubating for 30 min with a biotin-conjugated goat antirabbit IgG and amplified with avidin-biotin peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) after quenching endogenous peroxidase with 0.3% H₂O₂ in 100% methanol for 15 min. Diaminobenzidine was used as a chromogen. To quantitate the degree of nitrotyrosine staining, a 0 to 4 grading system was used: 0 indicated no staining, 1 to 3 indicated increasing degrees of intermediate staining, 4 indicated extensive staining. In each experimental group, five to six sections were evaluated by two independent observers blinded to the experimental protocol.

Myeloperoxidase Activity

Myeloperoxidase activity was determined as an index of neutrophil accumulation, as previously described (14). Tissues collected 1 h after extubation were homogenized in a solution containing 0.5% hexa- decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH, 7) and centrifuged for 30 min at 20,000 × g at 4° C. An aliquot of the supernatant was allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H₂O₂. The rate of change in absorbance was measured by spectrophotometry at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 µmol of hydrogen peroxide/min at 37° C and expressed in milliunits per milligram tissue.

Materials

Primary antinitrotyrosine antibody was purchased from Upstate Biotech (Saranac Lake, NY). Reagents and secondary and nonspecific IgG antibodies for immunohistochemical analysis were from Vector Laboratories. All other chemicals were from Sigma/Aldrich (St. Louis, MO).

Data Analysis

All values in the figures and text are expressed as mean ± SEM of n observations, where n represents the number of rats (n = 8 animals for each group). The results were examined by analysis of variance followed by Bonferroni's correction post hoc t test. A p value less than 0.05 was considered significant.

	RESULTS

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In Vivo Treatment with 3-Aminobenzamide Attenuates the Postintubation Damage

In all of the laryngeal sections from intubated animals, a variable loss of mucosal epithelium was noted. The damage was scored 3.4 ± 0.7 and consisted of edema, mucosal necrosis, infiltration of inflammatory cells, and dilated submucosal gland. In contrast, the histologic features of intubated rats treated with 3-aminobenzamide were typical of mild architectural alterations characterized by reduced edema and few localized necrotic areas. The damage score for 3-aminobenzamide-treated rats was significantly reduced (1.3 ± 0.3) when compared with vehicle-treated rats (p < 0.05) (Figures 1 and 2).

View larger version (79K): [in this window] [in a new window]   Figure 1.   Histology of the larynx of rats subjected to intubation- injury with and without treatment with 3-aminobenzamide. (A) Representative section from a control rat shows a normal architecture of the larynx. (B) Mucosal injury was produced by 1 h intubation followed by 1 h extubation and was characterized by absence of epithelium, a massive mucosal and submucosal infiltration of inflammatory cells, and gland swelling. (C ) Treatment with 3-aminobenzamide corrected the disturbances in morphology and reduced neutrophil infiltration. Original magnification: ×100. Figures show representative patterns. A similar pattern was seen in n = 4 to 6 different tissue sections in each group.

View larger version (15K): [in this window] [in a new window]   Figure 2.   Effect of 3-aminobenzamide (3-AB) treatment on damage score after laryngeal injury (LI) induced by 1 h intubation followed by 1 h extubation. Drug treatment (10 mg/kg, intraperitoneally) was given 15 min prior to extubation. Results are mean ± SEM of eight rats for each group. *Significantly different from control rats (p < 0.05). #Significantly different from untreated rats subjected to laryngeal injury (p < 0.05).

In Vivo Treatment with 3-Aminobenzamide Attenuates Nitrosative Damage

The architectural derangement was associated with the appearance of a positive immunohistochemical staining for nitrotyrosine, a tyrosine nitration product of nitrogen derivatives, in the injured laryngeal tissue (Figure 3). However, nitrotyrosine staining was virtually abolished in the larynx from rats treated with 3-aminobenzamide. On a scale of 0 to 4 the intensity of staining was, in fact, 0.7 ± 0.2 in laryngeal sections of 3-aminobenzamide-treated rats, significantly lower than the intensity of staining of sections of vehicle-treated rats (3.6 ± 0.3; p < 0.05) (Figure 4).

View larger version (76K): [in this window] [in a new window]   Figure 3.   Immunohistochemical staining of nitrotyrosine of the larynx of rats subjected to intubation-induced injury with and without treatment with 3-aminobenzamide. (A) Representative section from a control rat showing no staining for nitrotyrosine within a normal laryngeal architecture. (B) Representative section from a rat subjected to 1 h intubation followed by 1 h extubation showed a massive nitrotyrosine staining in the area of infiltrated cells in the mucosa and submucosa. (C ) Treatment with 3-aminobenzamide markedly reduced nitrotyrosine. Original magnification: ×100. Figures show representative patterns. A similar pattern was seen in n = 4 to 6 different tissue sections in each group.

View larger version (15K): [in this window] [in a new window]   Figure 4.   Effect of 3-aminobenzamide (3-AB) treatment on score of immunostaining of nitrotyrosine after laryngeal injury (LI) induced by 1 h intubation followed by 1 h extubation. Drug treatment (10 mg/kg, intraperitoneally) was given 15 min prior to extubation. Results are mean ± SEM of eight rats for each group. *Significantly different from control rats (p < 0.05). #Significantly different from untreated rats subjected to laryngeal injury (p < 0.05).

In Vivo Treatment with 3-Aminobenzamide Attenuates Neutrophil Infiltration

Laryngeal injury induced by intubation was also characterized by an increase of myeloperoxidase activity, indicative of neutrophil infiltration in inflamed tissue, confirming the enhanced leukocytes infiltration seen at the histologic inspection. Treatment with 3-aminobenzamide reduced myeloperoxidase activity, thus suggesting a reduction in leukocyte infiltration (Figure 5).

View larger version (18K): [in this window] [in a new window]   Figure 5.   Effect of 3-aminobenzamide (3-AB) treatment on myeloperoxidase activity after laryngeal injury (LI) induced by 1 h intubation followed by 1 h extubation. Drug treatment (10 mg/kg, intraperitoneally) was given 15 min prior to extubation. Results are mean ± SEM of eight rats for each group. *Significantly different from control rats (p < 0.05). #Significantly different from untreated rats subjected to laryngeal injury (p < 0.05).

	DISCUSSION

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In the postintubation period, laryngeal injury can be the cause of worsening obstruction that may require reintubation and consideration of tracheotomy (1, 17, 18). Because of the high morbidity of reintubation and tracheotomy, major efforts have recently been focusing on identifying the pathogenetic mechanisms of the tissue injury and on developing anti-inflammatory drugs that can prevent postintubation complications (19). Recently, certain inhibitors of PARS activation have been described as potent anti-inflammatory drugs with therapeutical efficacy in experimental inflammation such as arthritis, pleurisy, and paw edema and in ischemia and reperfusion injury of myocardium and splanchnic tissue (13).

In the present study, we were able to develop a rat model using large endotracheal intubation. This experimental procedure closely mimicked the complications of extubation seen in human conditions (3). In this model signs of inflammation occurred as early as within the first hour and included subglottic edema, mucosal necrosis, and submucosal infiltration of inflammatory cells, as indicated by the histopathologic analysis and increase of myeloperoxidase activity. Under these conditions, we found that PARS activation may have a pathologic role since treatment with 3-aminobenzamide markedly suppressed tissue injury and attenuated leukocyte infiltration. 3-Aminobenzamide is a prototypical PARS inhibitor that has been used in a large number of investigations to inhibit the catalytic activity of PARS when DNA single strand breakage was triggered by oxyradicals or by peroxynitrite. In these studies, 3-aminobenzamide provided cytoprotective effects but did not interfere with the development of oxidant-induced DNA strand breakage and did not scavenge oxyradicals or peroxynitrite (10, 23). Several in vivo reports have also demonstrated that treatment with 3-aminobenzamide exerted protective effects in several experimental models of inflammation and reperfusion injury in correlation with inhibition of PARS activity (13).

Although it is difficult to establish from the in vivo data which is the prevalent pathologic pathway of laryngeal injury and, therefore, which is the protective action of 3-aminobenzamide in the amelioration of the damage, several mechanisms may contribute. Although the cuff of the endotracheal tube has in some cases been shown to lead to mechanical mucosal damage, several investigators have demonstrated that an important factor in mucosal injury is capillary perfusion (3, 4). When pressure from the firm walls of the endotracheal tube is greater than mucosal capillary pressure, ischemia occurs (3, 4). The circumference of the subglottic space is especially vulnerable in infants and small children because of its relatively small caliber (19). Degree of necrosis of laryngotracheal tissue after intubation has also been directly related to the pressure of overinflated endotracheal tube and cuff and duration of intubation (24, 25). It is noteworthy that the histopathologic characteristics of post-intubation-induced damage in humans and our experimental model such as infiltration of neutrophils and release of oxidative species are similar to those seen after reperfusion in previously ischemic tissues. Therefore, it is conceivable that the ischemic damage produced by intubation may be exacerbated by the extubation, which may allow reperfusion and the related inflammatory response.

Intracellularly generated reactive species of both oxygen and nitrogen have been implicated in signaling responses in airway epithelial and in mediating tissue damage in several inflammatory conditions of the upper and lower airways. Several reports from clinical research or experimental animal studies support this concept in respiratory distress syndrome and trachea hyperresponsiveness (6). One of the proposed inflammatory mechanisms induced by peroxynitrite, hydroxyl radical, oxidants, and other free radicals includes DNA damage and activation of the nuclear enzyme PARS. The occurrence of this process has been demonstrated to lead to a rapid depletion of the intracellular NAD⁺ and ATP energetic pools in pulmonary and intestinal epithelial cells, macrophages, myocytes, smooth muscle, and endothelial cells (13). Furthermore, in vivo experiments have demonstrated that in addition to its involvement in the loss of intracellular energetics, PARS plays a critical role in the regulation of the expression of endothelial adhesion molecules such as intercellular adhesion molecule-1 and P-selectin and infiltration of inflammatory cells into the injured area (26, 27).

It has recently been proposed that during an inflammatory process nitrotyrosine may be formed as a result of a concerted action of several oxidants and may be considered a sign of a sustained nitrosative stress. There is, in fact, evidence that nitrosylation of tyrosine residues may be produced by peroxynitrite, the potent oxidant generated by the reaction of nitric oxide and superoxide anion. In addition, other reactions such as the reaction of nitrite with hypochlorous acid or, in the presence of neutrophils, the reaction of nitrite with myeloperoxidase and hydrogen peroxide may yield nitrotyrosine (28). In our experiments we found a positive immunohistochemical staining for nitrotyrosine in the injured larynx, thus suggesting the formation of peroxynitrite and other nitrogen-derived oxidants. To our knowledge, this is the first report showing that an intense nitrosative damage may occur in the upper airways after intubation. Treatment with 3-aminobenzamide markedly reduced the nitrotyrosine staining in the laryngeal tissue, which corresponded with the amelioration of the architectural derangement and reduction of infiltrated inflammatory cells. Therefore, our data strongly suggest that the postintubation laryngeal injury is a result of a pathologic cycle where early production of oxidants after extubation (i.e., during reperfusion) leads to PARS activation, endothelial and epithelial dysfunction, neutrophil infiltration, and, consequently, more production of reactive species.

Our data suggest that postintubation laryngeal injury is a result of ischemia and reperfusion injury driven by PARS activation and characterized by infiltration of inflammatory cells and nitrotyrosine formation into the injured tissue. Treatment with 3-aminobenzamide suppresses the course of tissue damage by inhibiting the recruitment of neutrophils and the subsequent generation of nitrogen-centered oxidants. Our hypothesis is in agreement with multiple studies demonstrating that pharmacologic or genetic inhibition of PARS exerts beneficial effects in several experimental models of inflammation and ischemia and reperfusion-related diseases (13, 26, 27). Although further studies are needed to provide definitive answers about the ischemia and reperfusion phenomenon occurring during postintubation and the role of PARS in human conditions, the rat model described in the present study may represent a suitable experimental tool for the investigation of laryngeal injury.