Investigation of Effects of Anesthesia and Age on Aspiration in Mice through LacZ Gene Transfer by Recombinant E1-deleted Adenovirus Vectors SHINJI TERAMOTO, TAKESHI MATSUSE, TERUAKI OKA, HIDEKI ITO, YOSHINOSUKE FUKUCHI, and YASUYOSHI OUCHI Departments of Geriatrics and Pathology, Tokyo University Hospital; Division of Endocrinology and Metabolism, Tokyo Metropolitan Geriatric Hospital; and Department of Respiratory Medicine, Juntendo University, Tokyo, Japan
To examine the role of disturbed upper airway reflexes in aspiration, we administered 20 ml of the adenovirus (Ad) vector Ad–CMV–LacZ or 20 ml of phosphate buffered saline (PBS) intranasally to C57 black mice. We investigated expression of the LacZ gene by this Ad vector in the nostrils of each mouse, with or without anesthesia. Under anesthesia, LacZ gene expression was detected in the lungs of every mouse given the Ad vector. However, no LacZ gene expression was found in the trachea or lungs of mice given the Ad vector without anesthesia. In mice given PBS or wild-type adenovirus transnasally during anesthesia, there was no LacZ gene expression in the nostrils, trachea, or lungs, suggesting that with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) staining, bluestained cells indicated transferred LacZ gene expression. These results suggested that aspiration of intranasal solution into lower airways was caused by disturbed upper airway reflexes during anesthesia. This process can be analyzed by the distribution of LacZ gene expression in airways. We next examined the effect of age on anesthesia-induced aspiration. Twenty-six-mo-old mice exhibited more LacZ gene expression in their lungs than did 6-mo-old mice at a concentration of 0.5 to 4.0% halothane in 100% oxygen. This suggests that light anesthesia may depress upper airway reflexes and cause aspiration in older animals. This novel model of aspiration, generated with the Ad–CMV–LacZ vector, may be useful for elucidating the mechanism of development of aspiration pneumonia in relation to age-related impairment of upper airway reflexes. Teramoto S, Matsuse T, Oka T, Ito H, Fukuchi Y, Ouchi Y. Investigation of effects of anesthesia and age on aspiration in mice through LacZ gene transfer by recombinant E1-deleted adenovirus vectors. AM J RESPIR CRIT CARE MED 1998;158:1914–1919.
Bacterial and viral pneumonia are major causes of death in later life (1–3). Conversely, advanced age has become a wellrecognized risk factor for death in patients with pneumonia. Recent studies have revealed that silent aspiration does not rarely occur in healthy subjects (4, 5), and that pneumonia caused by aspiration is of clinical importance in normal elderly persons and patients with cerebrovascular diseases (CVD) (1, 6–11). Because pneumonia is in principle prevented by such defense mechanisms as upper airway reflexes, mucociliary clearance, and phagocytosis by alveolar macrophages (AM), age-dependent declines in upper airway reflexes may be a component of the pathophysiology of aspiration pneumonia in older subjects (12–14). Furthermore, aspiration pneumonia
(Received in original form January 30, 1998 and in revised form June 5, 1998) Supported by grants from the Japan Research Foundation for Chronic Diseases and Rehabilitation (RFCDR-Japan) and the Culture Ministry of Japan. Correspondence and requests for reprints should be addressed to Shinji Teramoto, M.D., Department of Geriatrics, Tokyo University Hospital, 7-3-1 Hongo Bunkyo-ku, Tokyo, 113-8655, Japan. E-mail:
[email protected] Am J Respir Crit Care Med Vol 158. pp 1914–1919, 1998 Internet address: www.atsjournals.org
often develops in patients with CVD, who exhibit disturbed airway reflexes. In conjunction with the foregoing findings, anesthesia, unconsciousness, and depression of airway reflexes by sedatives are considered to produce the utmost risk for aspiration pneumonia (5, 15–19). Several investigators, using radioisotope techniques, found that pharyngeal content was aspirated into the lungs in humans (4, 7, 18, 19). Although these studies clearly demonstrated that silent aspiration occurred often in humans, they did not prove that aspirated solution caused infection or inflammation. Because the radiolabeled solution used in the studies did not interact with or infect airway epithelial cells, the use of radio-labeled solution may not duplicate the mechanism of aspiration pneumonia. Appropriate animal models of aspiration pneumonia may be required for studying the mechanism of aspiration and aspiration-induced pneumonia. For the purpose of elucidating the role of disturbed upper airway reflexes in aspiration, we administered an adenovirus (Ad) vector consisting of E1-deleted recombinant adenovirus carrying the Escherichia coli LacZ gene intranasally to mice with or without anesthesia. Because the Ad vector is known to infect airway cells and to express the E. coli LacZ gene in epithelial cells, the distribution in airways of intranasally adminis-
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Figure 1. Nostril and tracheal histology findings in 3-mo-old C57BL mice after intranasal administration of E1-deleted adenoviral vector (Ad–CMV–LacZ) with or without pentobarbital anesthesia. Nostril and tracheal histology findings in 3-mo-old C57BL mice after intranasal administration of wild-type adenovirus (Ad dl309) with pentobarbital anesthesia. Awake mice given PBS in place of pentobarbital sodium; anesthetized mice given pentobarbital sodium (5 mg/100 g body weight) intraperitoneally. Histologic sections were stained with X-gal and H&E. Original magnification; lower magnification: 340; higher magnification 3200. Blue-stained cells were considered to express LacZ gene.
tered solution containing Ad vectors can be identified by blue staining of LacZ with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) (20). The present study was designed to examine the relationship between aspiration and the depression of upper airway reflexes by anesthesia or age. In initial studies, we examined the effect of anesthesia on aspiration in young mice. We then examined the effect of age on anesthesia-induced aspiration under various degrees of anesthesia.
METHODS Animals In our study, we used 150 male C57 black mice (C57 BL). In the initial studies we used 50 C57 BL mice of 3 mo of age, weighing from 28 to 30 g. In the subsequent examination of the effect of age on impairment of upper airway reflexes, we used 50 young mice of 6 mo of age, weighing from 34 to 42 g, and 50 mice of 26 mo of age, weighing from 30 to 36 g (21, 22). The mice were specific-pathogen-free and housed in an air-conditioned, temperature controlled (22 6 28 C) colony room at the Tokyo Metropolitan Institute of Gerontology (21, 22). The mice were maintained with free access to food (CE-2; Nihon CLEA, Tokyo, Japan) and water ad libitum.
Adenoviral Vector A replication-defective adenoviral vector based on the human Ad 5 serotype (hAd5) was used for the study. In the hAd5–CMV–LacZ vector, E1 and E3 sequences were deleted and replaced with a minigene containing the CMV promoter, and a cytoplasmic LacZ gene
was inserted at the site of E1 deletion in the Ad genome (23, 24). The Ad vector was propagated in 293 cells, purified by CsCl gradient ultracentrifugation, and stored at 2708 C until used for infection of endothelial cells. Ad vector titers (in transducing units [TU]/ml) were determined from the number of LacZ gene-expressing 293 cells per milliliter through histochemical X-gal staining (23, 24). The experiments were done with vectors at a titer of 1 3 1012 TU/ml. The ratio of TU to viral particle number (as measured by optical density [OD] at 260 nm) was approximately 1;20. Heat-inactivated Ad vectors were also used in this study. The Ad vectors were inactivated by incubation in water at 658 C for 1 h. The wild-type adenovirus Ad dl309 was used as a sham control for the vector. The titer of Ad dl309 was 1 3 1013 particles/ml.
Intranasal Administration of Ad Vector In the initial studies we administered a total volume of 20 ml of the Ad–CMV–LacZ vector or 20 ml of phosphate buffered saline (PBS) intranasally to 3-mo-old C57 black mice with or without intraperitoneal anesthesia (pentobarbital sodium [Abbott Laboratories, North Chicago, IL], 5 mg/100 g body weight). For the sham control, Ad vectors were administered after intraperitoneal administration of PBS in place of pentobarbital sodium. Additionally, to determine whether the blue cells visualized with X-gal staining represented LacZ gene expression by Ad vectors or upregulation of endogenous b-galactosidase, we administered intranasally both wild-type Ad dl309 and heat-inactivated Ad vectors carrying the LacZ gene. To test the effect of age on the impairment of upper airway reflexes, we used halothane (Takeda Chemical Industries, LTD., Osaka, Japan), a volatile anesthetic, in this study. Young (6-mo-old) and aged
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Figure 2. Lung histology of 3-mo-old C57BL mice after intranasal administration of Ad-CMV-LacZ vector with or without pentobarbital anesthesia. Lung histology of 3-mo-old C57BL mice after intranasal administration of wild-type adenovirus (Ad dl309) with pentobarbital anesthesia. Other abbreviations are as in Figure 1. (26-mo-old) C57BL mice were placed in airtight clear glass chambers that measured 30 cm wide 3 30 cm deep 3 17.5 cm high. Halothane in 100% oxygen (O2) (fresh gas flow, 4 L/min), delivered from a dedicated, calibrated vaporizer, was introduced into the chamber at one end and was vented at the other end. After 15 min of administration of halothane (0.5 to 4%) in 100% O2 in the chamber, a steady-state level of anesthesia was obtained and animals were given Ad vectors through the nostrils. A total of 20 ml of Ad vectors was administered intranasally to both the young and aged mice. Further, to test the effect of age alone on the susceptibility of the airway epithelia to adenoviral transduction, we administered the Ad vectors intranasally to young and aged mice without anesthesia.
Visualization and Assessment of LacZ Gene Expression by Ad Vectors in Airways To assess the distribution of LacZ gene expression by Ad vectors in airways, X-gal staining was done on tissues from nostrils, trachea, and lungs. LacZ gene-expressing cells were detected in situ as blue-stained cells by X-gal staining (23, 24). One day after transnasal administration of Ad vectors under anesthesia, animals were killed by exsanguination of the abdominal aorta, and their nostrils, tracheae, and lungs were removed. Lungs were inflated with 2% paraformaldehyde (PFA) in PBS for 1 h and then sectioned in the frontal plane at the depth of the hilum. Nostrils and tracheae were also fixed with 2% PFA in PBS for 1 h. After fixation, tissues were washed twice with cold PBS with 1 mM MgCl2, and were then stained with X-gal for 4 h at 378 C. Tissue sections were counterstained with hematoxylin and eosin (H&E) after X-gal staining. Positive, LacZ gene-expressing sections from the nostrils, tracheae, and lungs were defined by more than 1% of the cells expressing the LacZ gene in 20 randomly selected fields as seen microscopically. For the more quantitative analysis of LacZ gene expression in the lung, we measured the frequency of LacZ gene expression in the lung of each mouse that underwent halothane anesthesia. The frequency of LacZ gene expression in the lung was determined as the percentage of LacZ gene expressed per field of
each tissue section as seen microscopically (Microphot EPI-FL3; Nikon, Tokyo, Japan). More than 50 fields were counted in each section under the microscope.
Statistical Analysis Data are presented as mean 6 SE. The chi-square test was used to compare the frequency of LacZ gene expression in the airway tissues of mice. Analysis of variance (ANOVA) with Fisher’s protected least significant difference method was used for comparing the data for the frequency of LacZ gene expression per field in young and aged mice under anesthesia. The analyses were made with the Stat View 4.0 software package (Abacus Concepts, Inc., Berkeley, CA). A value of p , 0.05 was considered statistically significant.
RESULTS LacZ gene expression in the nostrils was investigated in mice with or without anesthesia (Figure 1). However, LacZ gene expression in the trachea and lungs was different in anesthetized and unanesthetized mice. Under intraperitoneal anesthesia with pentobarbital sodium, LacZ gene expression, identified as blue X-gal-stained cells in the lungs, was found in every young mouse given the Ad vector (Figures 1 and 2). Further, half of the anesthetized mice exhibited LacZ gene expression in the trachea. However, no gene expression was found in the tracheae or lungs of mice given the Ad vector without intraperitoneal anesthesia. In mice given PBS without Ad vector transnasally, there was no LacZ gene expression in the nostrils, tracheae, or lungs. The results for LacZ gene expression after intranasal administration of Ad vector or PBS alone are summarized in Table 1. All mice given Ad vector under anesthesia exhibited LacZ gene expression in the lungs, but none of the mice given Ad vectors without anesthesia
Teramoto, Matsuse, Oka, et al.: Effects of Anesthesia and Age on Aspiration in Mice TABLE 1 FREQUENCY OF LacZ EXPRESSION OF THE TISSUE IN MICE Anesthesia Administration
Nostril Trachea Lungs
(1)
(2)
(1)
(1)
PBS
Ad–LacZ
Ad–LacZ
Ad dl309
(1) Heat-inactivated Ad–LacZ
0/10 (0%) 0/10 (0%) 0/10 (0%)
10/10 (100%)* 0/10 (0%) 0/10 (0%)
10/10 (100%)* 5/10 (50%)* 10/10 (100%)*
0/10 (0%) 0/10 (0%) 0/10 (0%)
0/10 (0%) 0/10 (0%) 0/10 (0%)
Definition of abbreviations: Ad dl309 5 wild-type adenovirus dl309; Ad-LacZ 5 adenovirus vector carrying LacZ gene; PBS 5 phosphate-buffered saline. * p , 0.01 compared with mice given PBS.
showed LacZ-gene-positive cells in their lungs, indicating that disturbed upper airway reflexes by anesthesia cause aspiration resulting in possible penetration of intranasal solution to the lower airways. Further, no LacZ gene expression was found in the nostrils, tracheae, or lungs of mice given the wild-type Addl 309 or the heat-inactivated Ad–CMV–LacZ vector (Table 1). Thus, LacZ gene expression was not due to the upregulation of endogenous sources, but to the LacZ gene transferred by the Ad vector. In the nostrils, LacZ gene expression was measured in every mouse, with or without anesthesia and irrespective of age (Figure 3). In lungs, no LacZ gene expression was found in
Figure 3. Relationship between percentage of animals expressing LacZ gene in the nostrils and lungs and degree of halothane anesthesia in young (6-mo-old) and aged (26-mo-old) C57BL mice. Each value represents the percentage of animals expressing LacZ gene in the lungs (n 5 8) at specified concentration of halothane anesthesia.
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any young or aged mouse without anesthesia (Figure 3). Therefore, age alone apparently had no effect on susceptibility of the airway epithelia to adenoviral transduction. However, LacZ gene expression in lungs was found in both young and aged mice with halothane anesthesia. There was a relationship between the depth of anesthesia and the ratio of the number of mice expressing the LacZ gene in the lungs to the total number of mice among both young and aged mice (Figure 3). LacZ gene expression was found in the lungs of every mouse anesthetized with 4% halothane in oxygen irrespective of age. However, a higher percentage of LacZ gene expression in the lungs was found in aged mice than in young mice at a lower concentration of halothane in oxygen (Figure 3). For the more quantitative analysis of LacZ gene expression in the lung, we measured its frequency in the lungs of each mouse and compared the percentage of LacZ gene-expressing fields per tissue section as seen microscopically. The percentage of LacZ gene-expressing fields was significantly greater in aged mice than in young mice with 1% to 3% of halothane in 100% O2 (Figure 4). This suggests that lighter anesthesia depressed upper airway reflexes in aged rather than in young mice, and that it may cause the aspiration of nasopharyngeal contents into lower airways in aged mice.
DISCUSSION Swallowing disorders and aspiration are often found in elderly subjects and patients with pulmonary disease (4–7, 25). We have recently demonstrated that recurrent silent aspiration causes a chronic inflammation of bronchioles accompanying a foreign body reaction (8). Although many pulmonologists and geriatricians have recognized that silent aspiration and swallowing disorder might be very important for the pathogenesis of aspiration pneumonia and nosocomial pneumonia in older patients (1, 5, 7, 10, 11), the precise neural mechanism of swallowing, the relative importance of the effect of age on aspiration, and the effect of central depressants on swallowing disor-
Figure 4. Relationship between percentage of LacZ gene expressed per field in sections of lungs in young and aged mice and degree of halothane anesthesia. Each value represents the mean value of percentage of LacZ gene expressed per field in tissue sections of lungs from eight mice at specified concentration of halothane anesthesia; *p , 0.05 compared with value for young mice at same halothane concentration.
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der have not been fully elucidated. In addition, an appropriate animal model for examination of the mechanism of silent aspiration (but not of massive aspiration such as Mendelson’s syndrome) has not been established. Several investigators have shown that aspiration of radiolabeled oropharyngeal secretions into the lungs occurs in humans (4, 7, 18, 19). These studies clearly indicated that pharyngeal aspiration extended to the lower airways, but did not reveal the pathologic interaction between the aspirate and airway epithelia or airway cells. Unfortunately, the studies demonstrated only one aspect or one process in the development of aspiration pneumonia caused by swallowing disorder. Because silent aspiration does not always cause pneumonia, aspiration alone does not provide sufficient information for studying the mechanism of aspiration pneumonia. Our animal model of aspiration, using recombinant E1-deleted Ad vectors, may be advantageous relative to earlier models for assessing the development of aspiration pneumonia in association with disturbed upper airway reflexes, since DNA virus infection of bronchiolar epithelial cells in the lower respiratory tract can be assessed by the localization and intensity of LacZ gene expression in situ. In the current study, animals under appropriate degrees of anesthesia exhibited aspiration of nasopharyngeal contents, and the fate of the aspirate was identified through blue staining in broad areas of airways. However, unanesthetized animals did not show abnormal swallowing, and LacZ gene expression was never found in their tracheae or lungs. These results suggest that aspiration is often caused by disturbed upper airway reflexes during anesthesia. The process of aspiration of an intranasally administered solution into the trachea and lower airways can be analyzed by the distribution of LacZ gene-expressing epithelial cells in airways. However, there is a possibility that blue staining of cells visualized through X-gal staining was not merely due to LacZ gene expression by Ad vectors, but also came from upregulation of endogenous b-galactosidase. We therefore also administered the wild-type Ad dl309 adenovirus, and the heat-inactivated Ad–CMV–LacZ vector into the noses of mice. Because no blue cells were seen in any nostril or lung with this procedure, the blue-stained cells were not due to the upregulation of endogenous b-galactosidase following adenovirus infection or to an immune reaction to proteins of the vectors, but rather to LacZ gene expression transferred by the active Ad vectors used in our study. Many features of the development of pneumonia after aspiration have not yet been determined. When aspiration causes pneumonia, the airway epithelium, which is the target for aspirated organisms, may exhibit inflammatory changes, and damaged epithelium should be repaired following infections. Although the mechanism of viral infection of airway epithelial cells is important for understanding the development of pneumonia, viral attachment to airway cells and internalization of virus into airway cells in the specifically affected area cannot be easily assessed with conventional virologic techniques in the vast area of the lung. Because the LacZ gene of the Ad vector introduced transnasally in our study was expressed in lower airway epithelial cells, the intranasally administered vector was considered to have become attached to airway epithelial cells and to have been internalized into these cells. The current study therefore provides a unique approach both to detecting the distribution of viral infection and the distribution of aspirated contents in the lung. We next examined the effect of age on aspiration in our model. We first tested the effect of age alone on susceptibility of the airway epithelia to Ad transduction with or without anesthesia. LacZ gene expression was found in the nostrils of every mouse with or without anesthesia and irrespective of age,
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whereas no LacZ gene expression was found in the lungs of any young or aged mouse without anesthesia. The susceptibility of the airway epithelia to Ad transduction was therefore not considerably affected by age alone. We also investigated the effect of age on aspiration with different degrees of anesthesia. Although LacZ gene expression in the lungs was found in every young and aged mouse concentrations of 4% and more of halothane in oxygen, the percentage of LacZ gene expression in the lungs at lower concentrations of halothane was greater in aged than in young mice (Figure 4). These results suggest that aspiration frequently occurred in young and aged animals under deeper anesthesia; however, lighter anesthesia, which did not cause aspiration in young animals, may have caused aspiration in older ones. The current study may confirm that aged animals are likely to aspirate oropharyngeal secretions during sedation and/or anesthesia. This is in agreement with our clinical experience, in which elderly patients were liable to have aspiration pneumonia. Although the current study did not examine mechanisms of susceptibility of older mice to disturbance of upper airway reflexes under light anesthesia, the new animal model of aspiration used in the study, involving transnasal administration of Ad–CMV–LacZ, may provide a means for examining various interventions in relation to age-related impairment of upper airway reflexes. Several limitations of the present study must be acknowledged. Although both wild-type Ad and E1-deleted Ad vectors are known to infect airway epithelial cells and to cause inflammation and immune responses (26–30), the differences in infection of airway epithelial cells with E1-deleted Ad vectors and wild-type Ad have not yet been fully elucidated. Several in vivo studies have shown that recombinant Ad vectors exhibit a low efficiency of infection of well-differentiated cells of the airway epithelium. Although the inefficient transduction efficiency of Ad vectors in differentiated airway cells may be a considerable problem for gene therapy, transduction by the Ad vectors used in our study was very efficient in lower airways. Because the site of pneumonic infiltration is mainly the lower rather than the upper airways, Ad vectors carrying the LacZ gene are among the candidate techniques for proving the occurrence of lower airway infection by small organisms in aspirated pharyngeal contents. Although the LacZ gene transduction effected by Ad vectors is transient, which is a major hurdle for the gene therapy with Ad vectors in airway disease, the transient expression was not a major problem in our study. Because the viral infection is basically self-limited and transient, the prognosis with such therapy is based mainly on the subject’s host defense capacity. However, the gene-transfer efficiency of Ad vectors differs among cells and species (20). Therefore, the data for mice do not solely predict results of Ad vector administration in humans. Although aspiration pneumonia can be caused by microorganisms colonizing the supraglottic area, bacteria rather than viruses are important for the development of such pneumonia. The effects of coinfection with bacteria or coadministration of lipopolysaccharides on Ad-associated airway inflammation should be further studied. In conclusion, we report the development of an animal model of aspiration produced with Ad vectors for the basic study of swallowing, and which may suggest many clinical implications of aspiration pneumonia in the elderly. We believe that our findings, in which older animals with central nervous depression were liable to aspiration, may raise further concern about the clinical significance of cerebral vascular diseases and use of sedatives in elderly patients. Acknowledgment : The authors thank Prof. J. P. Barron for reviewing this manuscript.
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