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1.  Novel Methods for Delivery of Cell Based Therapies 
Pulmonary hypoplasia (PH) is found in 15-20% of all neonatal autopsies, accounting for 2,850 deaths yearly. Development of engineered tissue substitutes that could functionally restore damaged tissue remains a unique opportunity for biotechnology. Recently, we isolated and characterized murine fetal pulmonary cells (FPC) and engineered 3-dimensional pulmonary tissue constructs in vitro. Our goal is to devise a reliable and reproducible method for delivering FPC into a live animal model of PH.
Materials and Methods
Three methods of delivery were explored: intraoral, intratracheal and intrapulmonary injection. Adult Swiss Webster mice were anesthetized and fluorescent labeled microspheres (20μm diameter) were delivered by intraoral and intratracheal injection. Subsequently, labeled FPC (Cell Tracker, CMPTX) were delivered by the same methods. In addition, direct transpleural intrapulmonary injection of FPC was performed.
Outcome analysis included survival, reproducibility, diffuse vs. confined location of the injected substance, and adequacy of delivery. Routine histological examination, fluorescent microscopy, and immunostaining were performed.
We demonstrated reproducible, diffuse instillation via tracheotomy into the distal alveoli. Intraoral delivery appeared less reliable compared to direct intratracheal injection.
Intratracheal injection was a reliable method of delivery. Labeled FPC showed transepithelial migration after 7 days of in vivo culture. Intrapulmonary injection led to local accumulation of cells in sites of injection.
We demonstrate that delivery of FPC is feasible with intratracheal injection giving the most reliable, diffuse delivery throughout the lung. This represents the first step towards translational research with site-specific delivery for a cell-based therapeutic approach towards PH and similar pulmonary diseases.
PMCID: PMC2373425  PMID: 17686493
Cell-based therapy; pulmonary hypoplasia; murine fetal pulmonary cells; intratracheal injection; intraoral injection; intrapulmonary injection; transepithelial migration
2.  Pulmonary toxicity of components of textile paint linked to the Ardystil syndrome: intratracheal administration in hamsters. 
OBJECTIVES: It was hypothesised from an epidemiological investigation that a formula change from Acramin FWR (a polyurea) to Acramin FWN (a polyamide-amine) had led to severe pulmonary disease in textile printing sprayers in SPAIN AND ALGERIA. To verify this, the pulmonary toxicity of the components of the paint systems involved was assessed in experimental animals. METHODS: Individual components and relevant mixtures, diluted in phosphate buttered saline, were given by intratracheal instillation of 2 ml/kg to hamsters. Pulmonary toxicity was assessed on days 3, 7, 14, 28, and 92 after a single intratracheal instillation, by histology and by measuring wet and dry lung weight, protein concentration, the activities of lactate dehydrogenase, alkaline phosphatase, beta-N-acetyl-glucosaminidase, and gamma-glutamyltransferase, inflammatory cell number and distribution in bronchoalveolar lavage fluid (BALF), and hydroxyproline content in dried lung tissue. RESULTS: Based on the doses that killed 50% of the animals (LD50s), the various components were found to be 10 to 1250 times more toxic when given intratracheally than when given orally (according to reported oral LD50s in rats). Acramin FWN, Acramin FWR, Acrafix FHN, or their mixtures caused lung damage. Protein concentration, enzyme activities, total cell number, and percentage of polymorphonuclear neutrophils were increased in BALF during the first week after intratracheal instillation. Lung weights remained high for at least a month. Histology showed inflammatory cell infiltration and subsequent fibrosis with collagen deposition. This finding was confirmed by an increased hydroxyproline content in dried lung tissue. Acramoll W did not show toxic effects. CONCLUSIONS: The study suggests that there is no major difference, in hamsters, between the acute intratracheal toxicity of Acramin FWR and that of Acramin FWN. Consequently, there is no simple toxicological explanation for the epidemiological hypothesis. However, the pulmonary toxicity of these non-irritant polymeric compounds is surprisingly high. The Ardystil disaster and these results should serve as a strong warning that conventional toxicity testing of chemicals does not necessarily protect workers against respiratory toxicity.
PMCID: PMC1128797  PMID: 9245943
3.  Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. 
Journal of Clinical Investigation  1994;93(3):1298-1306.
Keratinocyte growth factor (KGF) administered as a single intratracheal injection causes a prominent dose-dependent proliferation of type II alveolar epithelial cells in the lungs of adult rats. The increase in mitotically active alveolar cells histologically appears as a micropapillary epithelial cell hyperplasia after 2 d and peaks after 3 d in the form of monolayers of cuboidal epithelial cells lining alveolar septae. Proliferating cell nuclear antigen immunohistochemistry confirmed the profound proliferative response induced by KGF. The hyperplastic alveolar lining cells contain immunoreactive surfactant protein B and are ultrastructurally noted to contain lamellar inclusions characteristic of surfactant-producing type II pneumocytes. Mild focal bronchiolar epithelial hyperplasia is noted but is much less striking than the proliferation of type II pneumocytes. Large airways are unaffected by KGF. Daily intravenous injection of KGF is also able to cause pneumocyte proliferation. The normal adult rat lung constitutively expresses both KGF and KGF receptor mRNA, suggesting that endogenous KGF may be implicated in the paracrine regulation of the growth of pneumocytes. In conclusion, KGF rapidly and specifically induces proliferation and differentiation of type II pneumocytes in the normal adult lung.
PMCID: PMC294086  PMID: 8132770
In the preceding pages we have submitted evidence which shows that a simple intratracheal injection of a solution in a normally breathing rabbit penetrates within a few seconds to the alveoli, chiefly those of the left lower lobe; that absorption is rapid and well maintained; and that the procedure may be repeated effectively a number of times even with a substance like adrenalin which decreases absorption. It was also shown that absorption of adrenalin from the lung could be obtained at a time when double the dose given intramuscularly exerted no blood pressure effect whatever, and that absorption could still take place after the development of pulmonary edema, when there was an undoubted dilution of the injected solution with a serum-containing liquid and when a diminution of the absorptive field had occurred. The solution injected, after reaching the alveoli, is probably largely taken up by the capillaries of the pulmonary veins. This is indicated by the great rapidity with which an intratracheal injection of adrenalin may cause a rise of blood pressure. In numerous instances, for example, the pressure began to rise less than 5 seconds after the completion of an injection, equaling and even surpassing in rapidity of effect an intramuscular injection. Absorption by the lymphatics probably plays a secondary part, an assumption rendered all themore likely if we consider that lymph nodes are interpolated in the lymphatic pulmonary path, where the bed of the lymph stream becomes greatly widened and the current slowed. Injection into the lungs, however, offers another advantage due to the vascular arrangement of the absorbing field which could be of value therapeutically. Absorption of liquids injected into the lung probably takes place largely through the capillaries of the pulmonary veins; to a slight extent possibly through the capillaries of the bronchial veins which empty partly into the pulmonary veins, partly into the azygos veins; and probably some absorption occurs also through the lymphatics. By far the larger proportion of the absorbed material will thus be rapidly delivered to the left auricle and then to the left ventricle. At each succeeding systole, as long as absorption continues, a fraction of the drug will be driven into the coronary arteries and be able to affect the musculature of the cardiac pump. This fact ought to render the procedure of intratracheal injection a valuable method when it becomes imperative to stimulate a suddenly failing heart as promptly as possible by drugs of the digitalis group. Intratracheal injection is perhaps better under the conditions mentioned than the intravenous route, for the surface veins cannot always be entered with promptness and certainty even under fairly normal conditions, and in cases of cardiac weakness the difficulties will be measurably increased, while an intratracheal injection can be carried out with ease. Moreover, it is legitimate to expect that some absorption will take place from the lung alveoli as long as the heart-lung circulation persists, no matter how feebly, and that thus some of the drug will reach the heart to act on this structure itself more promptly perhaps than when the drug is administered successfully through surface veins. As far as the intramuscular route is concerned, we have shown that the intratracheal injection of adrenalin gives prompt though diminished absorption at a time when double the dose intramuscularly exerts no blood pressure effect whatever. The technical difficulties of giving an intratracheal injection in animals are slight. Tracheotomy as practised by us in the present series of experiments is not necessary, for the injection may be given into the intact trachea without exposure of the trachea. The hypodermic needle is inserted through the skin about 1 cm. below the larynx in a slanting caudad direction; the entrance of the needle into the trachea is readily felt. The injection should not be so rapid that the injected solution fills the entire tracheal lumen, but it should flow down the sides of the trachea. If the lumen is entirely filled, an expiration may drive some of the injected liquid into the larynx causing cough. In our experiments each injection of about 0.5 cc. consumed approximately 5 seconds. In the human subject no data are available as far as our knowledge goes, but a priori it would seem that an intratracheal injection is almost as simple as in the lower animals. The free hypodermic needle could be inserted into the tracheal lumen immediately below the cricoid cartilage. The needle itself should preferably be connected with the syringe by a short length of rubber tubing to minimize the danger of breaking the needle by a sudden move of the patient. The amount of the solution should not be too small, so that at least a fraction of it may reach the alveoli as promptly as possible; 3 to 5 cc. probably would suffice. Insertion of the needle in the locality mentioned would puncture the isthmus of the thyroid, but this is of no significance, especially when the procedure is employed in cases of cardiac failure where the gravity of the condition would warrant incurring much heavier risks than a slight bleeding from the thyroidal isthmus. In conclusion it may be said that the incorporation of drugs by intratracheal injection, while not as generally applicable as other methods, nevertheless has advantages which warrant its use also in human therapeutics.
PMCID: PMC2125455  PMID: 19868021
5.  Intravenous and Intratracheal Mesenchymal Stromal Cell Injection in a Mouse Model of Pulmonary Emphysema 
COPD  2013;11(3):310-318.
The aim of this study was to characterize the evolution of lung function and -structure in elastase-induced emphysema in adult mice and the effect of mesenchymal stromal cell (MSC) administration on these parameters. Adult mice were treated with intratracheal (4.8 units/100 g bodyweight) elastase to induce emphysema. MSCs were administered intratracheally or intravenously, before or after elastase injection. Lung function measurements, histological and morphometric analysis of lung tissue were performed at 3 weeks, 5 and 10 months after elastase and at 19, 20 and 21 days following MSC administration. Elastase-treated mice showed increased dynamic compliance and total lung capacity, and reduced tissue-specific elastance and forced expiratory flows at 3 weeks after elastase, which persisted during 10 months follow-up. Histology showed heterogeneous alveolar destruction which also persisted during long-term follow-up. Jugular vein injection of MSCs before elastase inhibited deterioration of lung function but had no effects on histology. Intratracheal MSC treatment did not modify lung function or histology. In conclusion, elastase-treated mice displayed persistent characteristics of pulmonary emphysema. Jugular vein injection of MSCs prior to elastase reduced deterioration of lung function. Intratracheal MSC treatment had no effect on lung function or histology.
PMCID: PMC4046870  PMID: 24295402
MSC; elastase; histology; morphometry; jugular vein
6.  Comparative role of complement in pneumococcal and staphylococcal pneumonia. 
Infection and Immunity  1982;37(3):1270-1277.
To evaluate the role of complement in pneumococcal and staphylococcal pneumonia, we decomplemented rats with cobra venom factor and inoculated them intratracheally with Staphylococcus aureus or type 25 pneumococci. S. aureus produced a patchy bronchopneumonia in normal Sprague-Dawley or Lewis rats, and decomplementation did not increase the severity of staphylococcal infection in either rat strain as judged by quantitative cultures of the lungs and blood at 6, 24, and 48 h after inoculation. In contrast, decomplementation markedly increased the severity of pneumonia caused by type 25 pneumococci in Sprague-Dawley and Lewis rats. In Sprague-Dawley rats, decomplementation significantly increased the number of bacteria in the lungs at 3, 6, and 24 h of infection. Bacteremia developed early in decomplemented Sprague-Dawley rats, but the higher pulmonary bacterial counts did not appear to be caused by bacteremic seeding of the lungs. Decomplemented Sprague-Dawley rats inoculated intravenously with pneumococci failed to develop the very high levels of bacteria in the lungs that were observed when the rats were inoculated intratracheally. Moreover, decomplemented Lewis rats inoculated intratracheally with pneumococci developed significantly increased numbers of pneumococci in the lungs early in infection (3 and 6 h) when they had no detectable bacteremia. These data indicate that in murine models complement plays a major protective role against type 25 pneumococci in the lung, whereas complement is not important to host defense in staphylococcal pneumonia.
PMCID: PMC347674  PMID: 6982230
7.  Toxicological investigations on silicon carbide. 2. In vitro cell tests and long term injection tests. 
Silicon carbide (SiC) dust and other dusts for comparison were injected intratracheally at a high dose (50 mg) into rats and the response of the lungs and the lymph nodes was studied after an appropriate experimental period. The indices studied were: histological changes in the lung and lymph nodes, organ weights, the formation of collagenous fibres, and the appearance of quartz typical areas. According to several epidemiological investigations and previous experimental animal studies, SiC produces silicogenic (fibrogenic) effects. No changes in the tissues studied in terms of damaging fibrogenic effects could be found after eight months (first series) and three and 12 months (second series). In particular, the histological findings and the absence of quartz typical areas as well as the quantitative determination of collagen fibres show that SiC had no harmful effects on tissues. Based on these results, the extent to which other exposures during the production of SiC can be responsible for the established radiological alterations is discussed. Without doubt the following may be confounders: SiC fibres, crystalline SiO2 (quartz, cristobalite, tridymite), and possibly gaslike emissions (SO2). From the hygienic medical point of view the workplaces during SiC manufacture should be examined carefully. The substance SiC dust as such can be considered as inert from the experimental results based on qualitative and extremely sensitive procedures. A revision of the present threshold value for SiC in ther German MAK list is called for.
PMCID: PMC1061313  PMID: 8398874
8.  The Toxicity of Precipitated Silica 
The proportion of respirable particles in dust clouds generated from three samples of precipitated silica has been shown to range between one-quarter and one-third by weight.
After a single intratracheal dose of the silicas to rats, chemical analysis shows a progressive disappearance of silica from the lungs, though it is still detectable after 12 months. Some silica appears in the liver and kidneys but in two of the three samples none remains after 12 months.
The nature and duration of the lung lesions produced in rats after a single intratracheal injection are described. A mild degree of fibrosis was observed which showed a steady regression with time and was to some extent influenced by the nature of the silica injected. The lesions showed little resemblance to those arising from quartz and were more akin to those produced by non-fibrogenic dusts.
Recommendations are made for the precautions to be taken during the industrial handling of these dusts.
PMCID: PMC1038244  PMID: 13875292
By the intratracheal injection into monkeys of unfiltered nasopharyngeal washings from cases of measles in the preeruptive and early eruptive stages of the disease a relatively constant group of symptoms was induced which closely resemble those of measles in man. Of seven monkeys inoculated intratracheally with unfiltered nasopharyngeal washings from seven cases of measles, five developed the symptoms. The same group of symptoms was induced in one monkey by inoculation of the mucous membrane of the nose and mouth with unfiltered nasopharyngeal washings from a case of measles. In these experiments a variety of organisms, largely saprophytic inhabitants of the nasopharynx and mouth, were present in the material inoculated. There is sufficient evidence, however, that these organisms were in no way responsible for the reaction, since the same group of symptoms was induced in two monkeys by the intratracheal injection of nasopharyngeal washings from three cases of measles after the washings had been freed from ordinary organisms of the mouth flora by filtration through Berkefeld N filters. The characteristic group of symptoms which follows the inoculation of monkeys with the nasopharyngeal washings from patients with measles has been. successfully carried through six passages by intratracheal injection of saline emulsions of the skin and buccal mucous membranes of monkeys killed from 2 to 6 days after the onset of the reaction. From the fourth passage monkey the reaction was also successfully induced in three monkeys by means of citrated whole blood injected intravenously. This experiment showed the blood to be capable of inciting the reaction from at least the 7th to 13th days after intratracheal inoculation of the donor monkey, but incapable of inducing it from the 2nd to 4th days. Cultures of the blood showed no growth. The group of symptoms induced has been constant and definite in character. After an incubation period of 6 to 10 days the animal becomes listless and drowsy, the conjunctivæ become injected, and small, discrete, hyperemic macules appear on the labial mucous membrane. These spots increase in number and may eventually coalesce in the course of 2 to 4 days to form a diffuse, red, granular rash. This rash is usually limited to the labial mucous membrane but may extend to the inside of the cheeks. The individual macules may or may not show the minute bluish white center characteristic of Koplik spots. From one to several days after onset an eruption of small, discrete, red maculopapules appears on the skin, usually coming out first on the face. The rash progressively increases in the number and size of the individual lesions and may in the course of 2 to 3 days extend to the skin of the neck, shoulders, upper arms, chest, abdomen, and thighs. It is constant in character but varies considerably in extent in different animals. By the time the exanthem is fully developed, the rash on the mucous membranes has begun to fade and soon disappears. The exanthem in turn progressively fades, sometimes with a branny desquamation, sometimes without. There may be moderate pigmentation. By the 6th to the 10th day after onset all symptoms have disappeared and the animal again appears well. Coincident with this group of symptoms there is a constant and definite reduction in the total leucocyte count, frequently constituting a true leucopenia. Other symptoms of irregular occurrence are photophobia, diarrhea, and fever. Symptoms of rhinitis and bronchitis have not been noted. Histological sections of the lesions of the skin show an exudative and proliferative lesion about the capillaries of the corium in which endothelial leucocytes are the predominating cells. Mitotic cells are not infrequently present in these areas. The endothelial leucocytes may be seen migrating toward and occasionally invading the epithelial layers of the hair follicles, sebaceous glands, and epidermis. In places the epithelial cells appear edematous and vacuolated, and there is evidence of minute vesicle formation. The lesions of the labial mucous membrane are similar in character. Minute vesicle formation in the epithelium is more frequent and the vesicles occasionally assume a more pustular appearance. Similar lesions are found in histological sections of the tongue. Cultures of the blood made both during the incubation period and during the course of the reaction in a variety of media, aerobic and anaerobic, have consistently shown no growth. The close similarity of the symptoms and pathological lesions of the reaction to the symptoms and pathological lesions of measles, the successful transmission of the reaction from monkey to monkey, and the elimination of ordinary bacteria as a possible source of error in the interpretation of the results, warrant the belief that the reaction is caused by the inciting organism of measles.
PMCID: PMC2128193  PMID: 19868504
10.  Prevention of LPS-Induced Acute Lung Injury in Mice by Mesenchymal Stem Cells Overexpressing Angiopoietin 1 
PLoS Medicine  2007;4(9):e269.
The acute respiratory distress syndrome (ARDS), a clinical complication of severe acute lung injury (ALI) in humans, is a leading cause of morbidity and mortality in critically ill patients. ALI is characterized by disruption of the lung alveolar–capillary membrane barrier and resultant pulmonary edema associated with a proteinaceous alveolar exudate. Current specific treatment strategies for ALI/ARDS are lacking. We hypothesized that mesenchymal stem cells (MSCs), with or without transfection with the vasculoprotective gene angiopoietin 1 (ANGPT1) would have beneficial effects in experimental ALI in mice.
Methods and Findings
Syngeneic MSCs with or without transfection with plasmid containing the human ANGPT1 gene (pANGPT1) were delivered through the right jugular vein of mice 30 min after intratracheal instillation of lipopolysaccharide (LPS) to induce lung injury. Administration of MSCs significantly reduced LPS-induced pulmonary inflammation, as reflected by reductions in total cell and neutrophil counts in bronchoalveolar lavage (BAL) fluid (53%, 95% confidence interval [CI] 7%–101%; and 60%, CI 4%–116%, respectively) as well as reducing levels of proinflammatory cytokines in both BAL fluid and lung parenchymal homogenates. Furthermore, administration of MSCs transfected with pANGPT1 resulted in nearly complete reversal of LPS-induced increases in lung permeability as assessed by reductions in IgM and albumin levels in BAL (96%, CI 6%–185%; and 74%, CI 23%–126%, respectively). Fluorescently tagged MSCs were detected in the lung tissues by confocal microscopy and flow cytometry in both naïve and LPS-injured animals up to 3 d.
Treatment with MSCs alone significantly reduced LPS-induced acute pulmonary inflammation in mice, while administration of pANGPT1-transfected MSCs resulted in a further improvement in both alveolar inflammation and permeability. These results suggest a potential role for cell-based ANGPT1 gene therapy to treat clinical ALI/ARDS.
Using a mouse model of acute respiratory distress syndrome, Duncan Stewart and colleagues report that rescue with mesenchymal stem cells expressing human angiopoietin 1 can avert lung injury from lipopolysaccharide.
Editors' Summary
Critically ill people who have had an injury to their lungs, for example through pneumonia, trauma, or an immune response to infection, may end up developing a serious complication in the lung termed acute respiratory distress syndrome (ARDS). In ARDS, inflammation develops in the lung, and fluid builds up in the alveoli (the air sacs resembling “bunches of grapes” at the ends of the network of tubes in the lung). This buildup of fluid prevents oxygen from being carried efficiently from air into the blood; the individual consequently experiences problems breathing and can develop further serious complications, which contribute significantly to the burden of illness among people in intensive care units. The death rate among individuals who do develop ARDS is very high, upward of 30%. Normally, individuals with ARDS are given extra oxygen, and may need a machine to help them breathe; treatments also focus on addressing the underlying causes in each particular patient. However, currently there are very few specific treatments that address ARDS itself.
Why Was This Study Done?
The researchers here wanted to work toward new treatment options for individuals with ARDS. One possible approach involves cells known as mesenchymal stem cells (MSCs). These cells are typically found in the bone marrow and have a property shared by very few other cell types in the body; they are able to carry on dividing and renewing themselves, and can eventually develop into many other types of cell. The researchers already knew that MSCs could become incorporated into injured lungs in mice and develop there into the tissue layers lining the lung. Some interesting work had also been done on a protein called angiopoeitin 1 (ANGPT1), which seemed to play a role in protecting against inflammation in blood vessels. Therefore, there was a strong rationale for carrying out experiments in mice to see if MSCs engineered to produce the ANGPT1 protein might “rescue” lung injury in mice. These experiments would be an initial step toward developing possible new treatments for humans with ARDS.
What Did the Researchers Do and Find?
The researchers used a mouse model to mimic the human ARDS condition. This involved injecting the windpipe of experimental mice with lipopolysaccharide (a substance normally found on the outer surface of bacteria that brings about an immune reaction in the lung). After 30 minutes, the mice were then injected with either salt solution (as a control), the MSCs, or MSCs producing the ANGPT1 protein. The researchers then looked at markers of lung inflammation, the appearance of the lungs under a microscope, and whether the injected MSCs had become incorporated into the lung tissue.
The lipopolysaccharide brought about a large increase in the number of inflammatory cells in the lung fluid, which was reduced in the mice given MSCs. Furthermore, in mice given the MSCs producing ANGPT1 protein, the number of inflammatory cells was reduced to a level similar to that of mice that had not been given lipopolysaccharide. When the researchers looked at the appearance under the microscope of lungs from mice that had been given lipopolysaccharide, they saw signs of inflammation and fluid coming out into the lung air spaces. These signs were reduced among both mice treated with MSCs and those treated with MSCs producing ANGPT1. The researchers also measured the “leakiness” of the lung tissues in lipopolysaccharide-treated mice; MSCs seemed to reduce the leakiness to some extent, and the lungs of mice treated with MSCs producing ANGPT1 were no more leaky than those of mice that had never been injected with lipopolysaccharide. Finally, the MSCs were seen to be incorporated into lung tissue by three days after injection, but after that were lost from the lung.
What Do These Findings Mean?
Previous research done by the same group had shown that fibroblasts producing ANGPT1 could prevent lung injury in rats later given lipopolysaccharide. The experiments reported here go a step further than this, and suggest that MSCs producing ANGPT1 can “rescue” the condition of mouse lungs that had already been given lipopolysaccharide. In addition, treatment with MSCs alone also produced beneficial effects. This opens up a possible new treatment strategy for ARDS in humans. However, it should be emphasized that the animal model used here is not a precise parallel of ARDS in humans, and that more research remains to be done before human studies of this sort could be considered.
Additional Information.
Please access these Web sites via the online version of this summary at
Medline Plus entry on acute respiratory distress syndrome, providing basic information about what ARDS is, its effects, and how it is currently managed
ARDS Network from the US National Heart, Lung, and Blood Institute of the National Institutes of Health; the site provides frequently asked questions about ARDS as well as a list of clinical trials conducted by the network
Information about stem cells from the US National Institutes of Health, including information about the potential uses of stem cells
Wikipedia page about mesenchymal stem cells (note: Wikipedia is an internet encyclopedia anyone can edit)
PMCID: PMC1961632  PMID: 17803352
11.  Emission-particle-induced ventilatory abnormalities in a rat model of pulmonary hypertension. 
Environmental Health Perspectives  2004;112(8):872-878.
Preexistent cardiopulmonary disease in humans appears to enhance susceptibility to the adverse effects of ambient particulate matter. Previous studies in this laboratory have demonstrated enhanced inflammation and mortality after intratracheal instillation (IT) and inhalation (INH) of residual oil fly ash (ROFA) in a rat model of pulmonary hypertension induced by monocrotaline (MCT). The present study was conducted to examine the effects of ROFA in this model on ventilatory function in unanesthetized, unrestrained animals. Sixty-day-old male CD rats were injected with MCT (60 mg/kg) or vehicle (VEH) intraperitoneally 10 days before IT of ROFA (8.3 mg/kg) or saline (SAL) (control) or nose-only INH of ROFA [15 mg/m3 for 6 hr on 3 consecutive days or air (control)]. At 24 and 72 hr after exposure, rats were studied individually in a simultaneous gas uptake/whole-body plethysmograph. Lungs were removed at 72 hr for histology. Pulmonary test results showed that tidal volume (VT) decreased 24 hr after IT of ROFA in MCT-treated rats. Breathing frequency, minute volume (VE), and the ventilatory equivalent for oxygen increased in MCT- and VEH-treated rats 24 hr after IT or INH of ROFA and remained elevated 72 hr post-IT. O2 uptake (VO2) decreased after IT of ROFA in MCT-treated rats. Carbon monoxide uptake decreased 24 hr after IT of ROFA, returning to control values in VEH-treated rats but remaining low in MCT-treated rats 72 hr post-IT. ROFA exposure induced histologic changes and abnormalities in several ventilatory parameters, many of which were enhanced by MCT treatment.
PMCID: PMC1242015  PMID: 15175175
12.  Role of infective, immunological, and chronic irritative factors in the development of silicosis. 
The effect of infective, immunological, and irritative factors on the onset and development of silicosis after intratracheal inoculation with 50 mg of tridymite was investigated on 220 specific pathogen free (SPF) female Sprague-Dawley rats. Even after 12 months the rats, always kept in SPF conditions after intratracheal injection of the dust, showed mainly granulomas with little tendency to confluence or to fibrohyalinosis. Chronic infective stimulation was obtained by keeping groups of SPF animals injected with tridymite for three, six, or 12 months in a conventional animal house, where they were exposed to the endemic bacterial flora. In these animal silicosis developed much more rapidly and produced much more severe confluent lesions than in rats always kept in SPF conditions. Horseradish peroxidase and ferritin given by intratracheal injection and by inhalation were histochemically shown mainly in the dust granulomas but did not accelerate the development of silicosis. Exposure to ozone increased the prevalence of lung infections and thus enhanced the silicosis in conventionally kept animals, without modifying the evolution of silicosis in SPF animals. These experiments showed that the presence of bacterial flora, and particularly bronchopulmonary infections, accelerated the development of silicosis and led to the suggestion that individuals subject to frequent bronchopulmonary infections are unfit for occupations necessitating exposure to silica dust.
PMCID: PMC1009019  PMID: 7093151
The present study was undertaken in order to determine whether animals exhibiting cutaneous hypersensitiveness to pneumococcus would show an acute inflammatory reaction in the lungs when pneumococcus autolysate was brought into contact with the pulmonary tissues and, if so, whether the pulmonary reaction might be shown to be due to the allergic state of the animal, rather than to intrinsic properties of the autolysate. Twenty young rabbits were sensitized to pneumococcus by various procedures and their degree of hypersensitiveness determined at frequent intervals over varying periods of time by means of intracutaneous injections of pneumococcus autolysate standardized on the basis of nitrogen content and so treated as to be devoid of the known toxic principles. Twenty-four hours after the last skin test each rabbit was injected intratracheally with the same pneumococcus autolysate. Seven non-sensitive controls were similarly injected intratracheally. Twenty-four hours after intratracheal injection the rabbits were killed. The lungs were removed, a portion was cultured, and the rest was examined histologically. Of the twenty sensitized rabbits, three that showed no cutaneous sensitivity and three that were only slightly skin-sensitive at the time of intratracheal injection exhibited no detectable pulmonary reaction to the autolysate; eleven of fourteen that showed moderate to extreme cutaneous hypersensitiveness were found to have an acute exudative inflammation of the lungs. The exudate consisted largely of polymorphonuclear leukocytes and serum. It varied in extent from a slight focal exudate lining the bronchioles and adjacent alveoli to a very marked diffuse involvement of considerable portions of the lung. In all but one of the eleven the cultures of the lungs were sterile. The single animal showing a positive culture (B. lepisepticum) has been excluded. None of the seven non-sensitive controls showed any pulmonary reaction to the autolysate. From these results it may be concluded that there is in rabbits a fairly close parallelism between cutaneous and pulmonary hypersensitiveness to pneumococcus autolysate and that the inflammatory response of the pulmonary tissue resulting from contact with the autolysate depends upon the allergic state of the animal rather than upon inherently injurious substances in the autolysate. The observations are in harmony with the theory that allergy may play a part in the pathogenesis of pneumococcus pneumonia in man.
PMCID: PMC2131885  PMID: 19869783
14.  Inhibition of poly (adenosine diphosphate-ribose) polymerase attenuates lung-kidney crosstalk induced by intratracheal lipopolysaccharide instillation in rats 
Respiratory Research  2013;14(1):126.
Acute respiratory distress syndrome (ARDS) is a severe form of lung injury that frequently occurs during pneumonia and sepsis. Lung inflammation in ARDS patients may have deleterious effects on remote organs such as the kidney. The nuclear enzyme poly(adenosine diphosphate-ribose) polymerase (PARP) enhances the nuclear factor (NF)-κB-dependent transcription of inflammatory cytokines. This study was conducted to elucidate two questions: first, whether the activation of PARP and NF-κB mediates the renal inflammation secondary to the lipopolysaccharide (LPS)-induced acute lung inflammation; second, whether a PARP inhibitor, 3-aminobenzamide (3-AB), attenuates lung and kidney inflammation by inhibiting NF-κB-dependent proinflammatory cytokines.
Male Sprague–Dawley rats were anesthetized, ventilated, and divided into three groups; a control group (n = 8); an LPS group (n = 12) intratracheally instilled with LPS (16 mg/kg), and an LPS + 3-AB group (n = 12) given the same dose of LPS by the same method followed by an intravenous injection of 3-AB (20 mg/kg). Hemodynamics, arterial blood gas, and the plasma levels of lactate, creatinine and potassium were measured at 0,1,2,3, and 4 h after treatment. The lung wet/dry ratio was measured at 4 h. The mRNA expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in the lung and kidney were measured by TaqMan real-time PCR. PARP and NF-κB in the lung and kidney were histologically examined by immunostaining and assigned expression scores.
LPS induced metabolic acidosis, hypotension, hypoxemia, increased the lung wet/dry ratio, increased the plasma levels of creatinine and potassium, and increased the cytokine mRNA expressions in the lung and kidney. All of these effects were associated with strong expression of PARP and NF-κB. Treatment with 3-AB prevented the LPS-induced metabolic acidosis and hypotension, reduced the plasma levels of lactate, creatinine and potassium, reduced the cytokine mRNA expressions, reduced the expression of PARP and NF-κB, improved pulmonary edema and oxygenation and preserved renal function.
The PARP inhibition attenuated lung-kidney crosstalk induced by intratracheal LPS instillation, partly via an inhibition of NF-κB dependent proinflammatory cytokines.
PMCID: PMC3833186  PMID: 24229378
Acute respiratory distress syndrome (ARDS); 3-aminobenzamide (3-AB); Cytokines; Lipopolysaccharide; Nuclear factor (NF)-κB; Lung and kidney; Poly (adenosine-diphosphate ribose) polymerase (PARP)
15.  The antigen-presenting activities of Ia+ dendritic cells shift dynamically from lung to lymph node after an airway challenge with soluble antigen 
The Journal of Experimental Medicine  1995;181(4):1275-1283.
Dendritic cells (DC) are widely distributed in the lung where they are distinguished by their morphology and class II major histocompatibility complex (Ia) antigen expression. Although a role for DC as pulmonary antigen-presenting cell (APC) has been suggested, little is currently known concerning how these cells respond to inhaled antigens in vivo. Hen-egg lysozyme (HEL) was injected intratracheally into Lewis rats; DC were subsequently purified from the lung and regional lymph nodes (LN) at intervals of up to 14 d and examined for their ability to stimulate the proliferation of HEL-immune T cells in vitro in the absence of added HEL. Pulmonary DC displayed APC activities at 3 h and for up to 7 d after the injection of antigen. Dendritic cells in the draining hilar LN showed APC activities that appeared at 24 h, peaked at day 3, and then diminished progressively. After the primary sensitization, HEL- immune T cells were detected in hilar LN but not in the lung. A second airway challenge with HEL at day 14 yielded an antigen-specific pulmonary immune response, characterized histologically by the accumulation of mononuclear cells around lung venules. We conclude that APC activities shift from lung to lymph node during the response to inhaled antigen.
PMCID: PMC2191960  PMID: 7699319
16.  Administration of Bleomycin via the Oropharyngeal Aspiration Route Leads to Sustained Lung Fibrosis in Mice and Rats as Quantified by UTE-MRI and Histology 
PLoS ONE  2013;8(5):e63432.
Pulmonary fibrosis can be experimentally induced in small rodents by bleomycin. The antibiotic is usually administered via the intratracheal or intranasal routes. In the present study, we investigated the oropharyngeal aspiration of bleomycin as an alternative route for the induction of lung fibrosis in rats and mice. The development of lung injury was followed in vivo by ultrashort echo time magnetic resonance imaging (UTE-MRI) and by post-mortem analyses (histology of collagen, hydroxyproline determination, and qRT-PCR). In C57BL/6 mice, oropharyngeal aspiration of bleomycin led to more prominent lung fibrosis as compared to intranasal administration. Consequently, the oropharyngeal aspiration route allowed a dose reduction of bleomycin and, therewith, a model refinement. Moreover, the distribution of collagen after oropharyngeal aspiration of bleomycin was more homogenous than after intranasal administration: for the oropharyngeal aspiration route, fibrotic areas appeared all over the lung lobes, while for the intranasal route fibrotic lesions appeared mainly around the largest superior airways. Thus, oropharyngeal aspiration of bleomycin induced morphological changes that were more comparable to the human disease than the intranasal administration route did. Oropharyngeal aspiration of bleomycin led to a homogeneous fibrotic injury also in rat lungs. The present data suggest oropharyngeal aspiration of bleomycin as a less invasive means to induce homogeneous and sustained fibrosis in the lungs of mice and rats.
PMCID: PMC3646779  PMID: 23667616
17.  Inflammogenic effect of well-characterized fullerenes in inhalation and intratracheal instillation studies 
We used fullerenes, whose dispersion at the nano-level was stabilized by grinding in nitrogen gas in an agitation mill, to conduct an intratracheal instillation study and an inhalation exposure study. Fullerenes were individually dispersed in distilled water including 0.1% Tween 80, and the diameter of the fullerenes was 33 nm. These suspensions were directly injected as a solution in the intratracheal instillation study. The reference material was nickel oxide in distilled water. Wistar male rats intratracheally received a dose of 0.1 mg, 0.2 mg, or 1 mg of fullerenes and were sacrificed after 3 days, 1 week, 1 month, 3 months, and 6 months. In the inhalation study, Wistar rats were exposed to fullerene agglomerates (diameter: 96 ± 5 nm; 0.12 ± 0.03 mg/m3; 6 hours/days for 5 days/week) for 4 weeks and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. The inflammatory responses and gene expression of cytokine-induced neutrophil chemoattractants (CINCs) were examined in rat lungs in both studies.
In the intratracheal instillation study, both the 0.1 mg and 0.2 mg fullerene groups did not show a significant increase of the total cell and neutrophil count in BALF or in the expression of CINC-1,-2αβ and-3 in the lung, while the high-dose, 1 mg group only showed a transient significant increase of neutrophils and expression of CINC-1,-2αβ and -3. In the inhalation study, there were no increases of total cell and neutrophil count in BALF, CINC-1,-2αβ and-3 in the fullerene group.
These data in intratracheal instillation and inhalation studies suggested that well-dispersed fullerenes do not have strong potential of neutrophil inflammation.
PMCID: PMC2848185  PMID: 20226088
1. The intratracheal injection of egg albumin or pneumococcus protein induces an inflammatory reaction in the lungs of rabbits previously inoculated with the respective antigen. 2. A similar reaction occurs following intratracheal injection of pneumococcus protein into the lungs of rabbits previously inoculated with heat-killed suspensions of the bacteria. 3. This reaction appears to be related to the presence of circulating antibody and to have the nature of the Arthus reaction. 4. A study of the reaction of the lung of rabbits to infection caused by intravenous injections of Pneumococcus reveals that (a) reactions occur irregularly in the lung; (b) in the lungs in which reactions do occur, the histological changes are not different in normal rabbits and in rabbits made resistant by previous intravenous or intracutaneous injections of pneumococci. 5. Intratracheal injection of pneumococcus protein followed by intravenous injection of virulent pneumococci on the next day does not alter the course and character of the infection in resistant rabbits. 6. The experiments reported in this paper bring no evidences to support the view that the lesions in the lungs of rabbits following the intravenous injection of pneumococci are modified by any previous state of sensitivity.
PMCID: PMC2132136  PMID: 19870031
19.  Use of immunoblotting to detect Aspergillus fumigatus antigen in sera and urines of rats with experimental invasive aspergillosis. 
Journal of Clinical Microbiology  1990;28(7):1575-1579.
Immunoblotting was used to detect Aspergillus fumigatus antigen in sera and urines of immunosuppressed rats experimentally infected with A. fumigatus. Organisms were administered by both intravenous and intratracheal injections. Intravenously infected rats developed disseminated aspergillosis, but intratracheally infected rats developed pulmonary disease only. Fungal cultures of blood and urine samples from infected rats were negative. In the urines of intravenously infected rats, antigen was detected 24 to 48 h after infection; in the urines of intratracheally infected animals, antigen was detected on days 4 to 5 after infection. Antigen in serum was detected later than antigen in urine was. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of serum and urine samples, the most strongly reacting antigenic materials were found in the 88-, 40-, 27-, and 20-kilodalton regions. These dominant antigens appeared to be the same as those of control antigens prepared from A. fumigatus grown in vitro. Rabbit antiserum to Aspergillus filtrate antigen was found to be more immunoreactive than antiserum to mycelial or conidial antigen. No mycelium-specific antigens were detected.
PMCID: PMC267991  PMID: 2199519
20.  The effects of salbutamol on epithelial ion channels depend on the etiology of acute respiratory distress syndrome but not the route of administration 
Respiratory Research  2014;15(1):56.
We investigated the effects of intravenous and intratracheal administration of salbutamol on lung morphology and function, expression of ion channels, aquaporin, and markers of inflammation, apoptosis, and alveolar epithelial/endothelial cell damage in experimental pulmonary (p) and extrapulmonary (exp) mild acute respiratory distress syndrome (ARDS).
In this prospective randomized controlled experimental study, 56 male Wistar rats were randomly assigned to mild ARDS induced by either intratracheal (n = 28, ARDSp) or intraperitoneal (n = 28, ARDSexp) administration of E. coli lipopolysaccharide. Four animals with no lung injury served as controls (NI). After 24 hours, animals were anesthetized, mechanically ventilated in pressure-controlled mode with low tidal volume (6 mL/kg), and randomly assigned to receive salbutamol (SALB) or saline 0.9% (CTRL), intravenously (i.v., 10 μg/kg/h) or intratracheally (bolus, 25 μg). Salbutamol doses were targeted at an increase of ≈ 20% in heart rate. Hemodynamics, lung mechanics, and arterial blood gases were measured before and after (at 30 and 60 min) salbutamol administration. At the end of the experiment, lungs were extracted for analysis of lung histology and molecular biology analysis. Values are expressed as mean ± standard deviation, and fold changes relative to NI, CTRL vs. SALB.
The gene expression of ion channels and aquaporin was increased in mild ARDSp, but not ARDSexp. In ARDSp, intravenous salbutamol resulted in higher gene expression of alveolar epithelial sodium channel (0.20 ± 0.07 vs. 0.68 ± 0.24, p < 0.001), aquaporin-1 (0.44 ± 0.09 vs. 0.96 ± 0.12, p < 0.001) aquaporin-3 (0.31 ± 0.12 vs. 0.93 ± 0.20, p < 0.001), and Na-K-ATPase-α (0.39 ± 0.08 vs. 0.92 ± 0.12, p < 0.001), whereas intratracheal salbutamol increased the gene expression of aquaporin-1 (0.46 ± 0.11 vs. 0.92 ± 0.06, p < 0.001) and Na-K-ATPase-α (0.32 ± 0.07 vs. 0.58 ± 0.15, p < 0.001). In ARDSexp, the gene expression of ion channels and aquaporin was not influenced by salbutamol. Morphological and functional variables and edema formation were not affected by salbutamol in any of the ARDS groups, regardless of the route of administration.
Salbutamol administration increased the expression of alveolar epithelial ion channels and aquaporin in mild ARDSp, but not ARDSexp, with no effects on lung morphology and function or edema formation. These results may contribute to explain the negative effects of β2-agonists on clinical outcome in ARDS.
PMCID: PMC4026154  PMID: 24886221
Salbutamol; Acute respiratory distress syndrome; Elastance; Alveolar epithelial cells; Epithelial sodium channel; Edema
21.  Evaluation of novel particles as pulmonary delivery systems for insulin in rats 
AAPS PharmSci  2003;5(2):10-20.
The purpose of the study was to evaluate the influence of calcium phosphate (CAP) and polyethylene glycol (PEG) particles on the systemic delivery of insulin administered by the pulmonary route. Two methods of pulmonary delivery were employed: intratracheal instillation and spray instillation. Insulin-CAP-PEG particles in suspension (1.2 U/kg, 110–140 μL) were administered to the lungs of fasted rats by intratracheal instillation (INCAPEG) or spray instillation (SINCAPEG). Control treatments consisted of insulin solution (1.2 U/kg) by intratracheal instillation, spray instillation, and subcutaneous administration (SC). Plasma concentrations of insulin and glucose were determined by chemiluminescence and colorimetric methods, respectively. Data were analyzed by compartmental and non-compartmental methods, and pharmacokinetic (PK) and pharmacodynamic (PD) parameters of insulin disposition were determined. PK analysis suggested that insulin administered in particles had a longer half-life, a longer mean residence time, and a smaller rate of elimination than insulin in solution. In addition, insulin bioavailability after SINCAPEG was 1.8-fold that of insulin solution administered SC. PD analysis showed that smaller areas under the effect curve and, conversely, larger areas above the effect curve were obtained after INCAPEG in comparison to insulin solution. The magnitude of this effect was increased after SINCAPEG. The presence of CAP-PEG particles appears to positively influence the disposition of insulin administered to the lungs of Sprague-Dawley rats. Spray instillation appears to be a more efficient method of delivering insulin to the lungs of rats than intratracheal instillation.
PMCID: PMC2751517  PMID: 12866936
pulmonary delivery; insulin; CAP-PEG particles; pharmacokinetics; pharmacodynamics
22.  Intratracheal Delivery of CX3CL1-Expressing Mesenchymal Stem Cells to Multiple Lung Tumors 
Molecular Medicine  2009;15(9-10):321-327.
The lung is one of the organs to which cancers from solid tumors frequently metastasize. Multiple tumors in the lung are usually treated by systemic chemotherapy because of the lack of efficient methods of targeting antitumor agents to the lung. Although intratracheal administration is an ideal route for targeting multiple lung tumors, antitumor agents are often harmful to the organ or induce inflammation. Mesenchymal stem cells (MSCs), nonhematopoietic stem cells capable of differentiating into various mesoderm-type cells, have a propensity to migrate to and proliferate in tumor tissues after systemic administration. We intratracheally injected MSCs expressing CX3CL1 (MSC/RGDFKN) into the lung of lung tumor–bearing mice with multiple metastases of C26 or Lewis lung carcinoma (LLC). Antitumor effects were evaluated by counting the number of lung metastases and survival. We demonstrated the tropism of mouse MSCs to lung tumor tissues after intratracheal administration of GFP-positive MSCs. Intratracheal injection of MSC/RGDFKN strongly inhibited growth of lung metastases of C26 or LLC, and thus prolonged survival. Intratracheal injection of MSC/RGDFKN did not induce an inflammatory reaction in the lung. These results suggest that MSCs expressing antitumor agents can be delivered intratracheally into multiple lung tumor tissues without causing inflammation.
PMCID: PMC2710294  PMID: 19603106
23.  Coal dust alters β-naphthoflavone-induced aryl hydrocarbon receptor nuclear translocation in alveolar type II cells 
Many polycyclic aromatic hydrocarbons (PAHs) can cause DNA adducts and initiate carcinogenesis. Mixed exposures to coal dust (CD) and PAHs are common in occupational settings. In the CD and PAH-exposed lung, CD increases apoptosis and causes alveolar type II (AT-II) cell hyperplasia but reduces CYP1A1 induction. Inflammation, but not apoptosis, appears etiologically associated with reduced CYP1A1 induction in this mixed exposure model. Many AT-II cells in the CD-exposed lungs have no detectable CYP1A1 induction after PAH exposure. Although AT-II cells are a small subfraction of lung cells, they are believed to be a potential progenitor cell for some lung cancers. Because CYP1A1 is induced via ligand-mediated nuclear translocation of the aryl hydrocarbon receptor (AhR), we investigated the effect of CD on PAH-induced nuclear translocation of AhR in AT-II cells isolated from in vivo-exposed rats. Rats received CD or vehicle (saline) by intratracheal (IT) instillation. Three days before sacrifice, half of the rats in each group started daily intraperitoneal injections of the PAH, β-naphthoflavone (BNF).
Fourteen days after IT CD exposure and 1 day after the last intraperitoneal BNF injection, AhR immunofluorescence indicated that proportional AhR nuclear expression and the percentage of cells with nuclear AhR were significantly increased in rats receiving IT saline and BNF injections compared to vehicle controls. However, in CD-exposed rats, BNF did not significantly alter the nuclear localization or cytosolic expression of AhR compared to rats receiving CD and oil.
Our findings suggest that during particle and PAH mixed exposures, CD alters the BNF-induced nuclear translocation of AhR in AT-II cells. This provides an explanation for the modification of CYP1A1 induction in these cells. Thus, this study suggests that mechanisms for reduced PAH-induced CYP1A1 activity in the CD exposed lung include not only the effects of inflammation on the lung as a whole, but also reduced PAH-associated nuclear translocation of AhR in an expanded population of AT-II cells.
PMCID: PMC2732588  PMID: 19650907
24.  Therapy with un-engineered naïve rat umbilical cord matrix stem cells markedly inhibits growth of murine lung adenocarcinoma 
BMC Cancer  2010;10:590.
Lung cancer remains the leading cause of cancer-related mortality despite continuous efforts to find effective treatments. Data from the American Cancer Society indicate that while the overall incidence of lung cancer is declining, it continues to rise in women. Stem cell-based therapy has been an emerging strategy to treat various diseases. The purpose of this paper is to determine the efficacy of an intrinsic anti-cancer effect of rat umbilical cord matrix stem cells (UCMSCs) on lung cancer.
A mouse syngeneic lung carcinoma model was used to test the basic ability of UCMSCs to control the growth of lung cancer. Lung tumors were experimentally induced by tail vein administration of Lewis lung carcinoma (LLC) cells derived from the lung of C57BL/6 mouse. Rat UCMSCs were then administered intratracheally five days later or intravenously on days 5 and 7. The tumor burdens were determined by measuring lung weight three weeks after the treatment.
Co-culture of rat UCMSCs with LLC significantly attenuated the proliferation of LLC cells as monitored by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a tetrazole cell proliferation assay, thymidine uptake, and direct cell counts. In vitro colony assays with rat UCMSCs as feeder layers markedly reduced LLC colony size and number. Co-culture of rat UCMSCs with LLCs causes G0/G1 arrest of cancer cells. This is evident in the decrease of cyclin A and CDK2 expression. The in vivo studies showed that rat UCMSC treatment significantly decreased tumor weight and the total tumor mass. Histological study revealed that intratracheally or systemically administered rat UCMSCs homed to tumor areas and survived for at least 3 weeks without any evidence of differentiation or adverse effects.
These results indicate that rat UCMSCs alone remarkably attenuate the growth of lung carcinoma cells in vitro and in a mouse syngeneic lung carcinoma graft model and could be used for targeted cytotherapy for lung cancer.
PMCID: PMC2988749  PMID: 21029413
25.  Mechanisms of cigarette smoke induced increased airspace permeability. 
Thorax  1996;51(5):465-471.
BACKGROUND: Increased epithelial permeability of the airspaces occurs commonly in the lungs of cigarette smokers. It is likely to be important in augmenting the inflammatory response in the airspaces and hence may have a role in the pathogenesis of emphysema. It has previously been shown that intratracheal instillation of cigarette smoke condensate induces increased epithelial permeability in vivo in rats and in vitro in epithelial cell monolayers, associated with a disturbance in the lung antioxidant, glutathione (GSH). The aim of this study was to assess the role of neutrophils, GSH, and tumour necrosis factor (TNF) in the increased epithelial permeability following intratracheal instillation of cigarette smoke condensate. METHODS: Epithelial permeability of the airspaces was measured in rat lungs as the passage of intratracheally instilled 125-iodine labelled bovine serum albumin (BSA) into the blood. The permeability of a monolayer of human type II alveolar epithelial cells to 125I-BSA was also measured. RESULTS: Cigarette smoke condensate produced a 59.7% increase in epithelial permeability over control values peaking six hours after instillation and returning to control values by 24 hours. Depletion of neutrophils and, to a lesser extent, macrophages by an intraperitoneal injection of antineutrophil antibody did not influence the increased epithelial permeability induced by cigarette smoke condensate. Although instillation of human recombinant TNF alpha produced an increase in epithelial permeability in the rat lung from 0.62 (0.61)% to 1.27 (0.08)%, only a trivial amount of TNF alpha was detected in bronchoalveolar lavage (BAL) fluid in vivo or in culture medium from BAL leucocytes obtained from animals treated with cigarette smoke condensate (94.9 (28.8) units/ml). Furthermore, antiTNF antibody did not abolish the increased epithelial permeability produced by cigarette smoke condensate. The role of GSH was assessed by measuring the changes in both the reduced (GSH) and oxidised form (GSSG) in lung tissue and in BAL fluid. One hour after instillation of cigarette smoke condensate there was a marked fall in the GSH content in the lung (from 809.8 (31.8) to 501.7 (40.5) nmol/g) in association with increased GSSG levels (from 89.8 (2.7) to 148.7 (48.8) nmol/g). This was followed by a return of GSH levels to control values, with a concomitant decrease in GSSG levels six hours after instillation. GSH levels in BAL fluid fell dramatically following cigarette smoke condensate (from 2.56 (0.30) to 0.31 (0.21) nmol/ml) and this fall was sustained up to six hours after instillation of cigarette smoke condensate. CONCLUSIONS: These studies suggest that neutrophils and TNF do not have a major role in the increased epithelial permeability induced by cigarette smoke condensate. However, the data support a role for the depletion of the antioxidant glutathione in the increased epithelial permeability caused by cigarette smoke condensate.
PMCID: PMC473589  PMID: 8711672

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