Beitr. Path. Bd. 156, 117-121 (1975)
From the Departments of Pharmacology and Experimental Pathology and Toxicology at C. H. Boehringer Sohn, Ingelheim/Rhein
Detection of Aerosols in the Respiratory Tract Nachweis von Aerosolen im Respirationstrakt G. MUACEVIC and T. TILOV With 4 Figures· Received May 14, 1975 . Accepted in revised form June 6, 1975
Key words: Aerosol - Detection - Fluorescent reagents - Inhalation Respiratory tract
Summary A fluorescence method for the detection of inhaled aerosols in the respiratory tract has been described. The most suitable fluorescence indicator for the histological examination was ethidium bromide.
If an inhalation method for the use of pharmaceutical agents suspended in aerosols is to be regarded as successful, detection of the agent in the lungs should be possible. Detection of the inhaled substances is of particular importance in animal experiments for the testing of aerosols for medical treatment, cosmetics and plant protection. Lithium (Habel et aL, 1972), coal dust (Rhoades, 1972) isotopes (Goerg and Locher, 1972) and p-aminohipuric acid (Franklin, 1950) have been used to prove respiratory desposition, the later substance being detectable in the urine following inhalation. It was our aim to develop a quick and uncomplicated method to prove respiratory deposition in animal experiments by using fluorescent agents in an aerosol. In these tests we looked for fluorescent reagents which would mark the different parts of the respiratory tract and would bind firmly with chemical structures of the tissues. The best results in our experiments have been obtained with ethidium bromide. 9 Beitr. Path. Bd. 156
118 . G. Muacevic and T. Tilov
Material and Method The experiments were carried out on rats and dogs. The animals inhaled one of the nebulized 0.2-1.0010 aqueous solutions of sodium fluorescein, haematoporphyrin, orange G, eosin scariet, thiazine red R, 2-toluidinonaphtyl-6-sulfphonic acid-K-salt (2,6-TNS), and acetone solutions (acetone: water, 60 : 40) of ethidium bromide, for 15-30 minutes. For nebulisation of the solutions we used a Draeger nebuliser (Aerolette Type) and a Pari nebuliser (Pari Primus Type) . The aerosol particles had an average size of 0.55 11m (according to instruction of the producers). Each substance was administered to 3 male rats (Chbb : THOM (SPF)), weighing 200 g. in an aerosol chamber of 2 I capacity. A group of further 3 rats served as control. The inhalation of ethidium bromide aerosol was carried out additionally on one dog (Chbi-beagle, male, weighing 10 kg), a further dog was used as control. In order to prove the reproducibility of the method seven test repetitions were performed with ethidium bromide and haematoporphyrin on rats (altogether 21 animals). For the ide,ntification of fluorescence lung and trachea sections were prepared and examined as follows: Immediately after death lungs and trachea were fixed for 24 hours by immersion in buffered 7.5010 formol (Merck, Darmstadt); subsequently frozen (C02) 15 /lm and paraffin sections 5 11m thick were prepared. Cryostat sections (7 11m thickness) of the non fixed tissue (frozen immediately after death with C02) were also prepared. These three kinds of histological sections were investigated using a Zeiss-photo microscope with transmitted fluorescence device. A metal - halide - CSF - 250 Ware lamp served as UV-light source. The optimal filter combinations were the Zeiss BG-Il or BG-3 excitation filters and the Zeiss 47, 50 or 53 barrier filters.
Results and Discussion Among the above mentioned fluorochromes, sodium fluorescein, haematoporphyrin and ethidium bromide were selected for further tests. With our combination of UV~light-sorce and filter the greenish-yellow fluorescence of sodium fluorescein could hardly be discerned from the native yellowish-green fluorescence of the lungs. For this reason, sodium fluorescein was found to be an unfavourable indicator under the described conditions. The more intensive red fluorescence of haematoporphyrin, however, could easily be distinguished from the native fluorescence of the lungs and is thus more suitable for our purposes than sodium fluorescein, but the substance is water soluble an'd not very stable (as fluorescenting agent in the tissue). The most suitable fluorescence indicator in our experiments was ethidiurn bromide. With RNA and DNA it forms stable compounds with strong fluorescence (Le Pecq and Paoletti, 1966). The compounds of ethidium bromide and tissue have a highly intensive golden-orange fluorescence which is stronger than that of ethidium bromide itself (Fig. 1-4).
Detection of Aerosols in the Respiratory Tract·
119
Fig. I. Lung of the rat: native green autofluorescence of lung tissue. Frozen section; X 64, Zeiss excitation filter BG-3 and Zeiss barrier filter 53.
Fig. 2. Lung of rat after inhalation of ethidium bromide aerosol. Ethidium bromide in combination with RNA and DNA of the bronchialmucosal cells and alveolar wall produces a yellow-orange fluorescence compared with the green autofluorescence of the lung. Frozen section; X 64, filters as in fig. I.
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Fig. 3. Trachea of the dog: orange-yellow fluorescence of the mucosal ciliated epithelium after inhalation of ethidium bromide aerosol. Green autofluorescence of the submucosa. Frozen section; X roo, filter as in fig. I.
120 .
G. Muacevic and T. Tilov
Fig. 4. Lung of the dog: yellow-orange fluorescence after the inhalation of ethidium bromide aerosol in the bronchial and alveolar epithelium. Frozen section; X 64, filters as in fig. I.
Another adavantage of ethidium-RNA or ethidium-DNA compounds was owing to the fact that they could not be washed out or destroyed during fixation and during the above-described histological processing. The investigations on cryostat- and, in particular, paraffin sections showed no advantages. The frozen sections prepared from fixed lung tissue were adequate for histological detection of the substance inhaled. The fluorescence of the preparations with ethidium bromide continued to be highly intensive for more than six months after histological processing. This stability of ethidium bromide is in accordance with the literature (Le Pesq and Paoletti, 1966; T. Tao et al., 1970). The above-described histochemical method is uncomplicated and reproducible. It allows detailed morphological examination and microphotographic documentation. The described experiments showed that ethidium bromide was the most suitable indicator for the detection of aerosols in the respiratory tract because the good contrast and stable fluorescenting properties.
Zusammenfassung Es wird eine Fluoreszenzmethode fur den Nachweis inhalierter Aerosole im Respirationstrakt beschrieben. Ethidiumbromid erweist sich als der beste Fluoreszenzindikator bei der histologischen Untersuchung. Acknowledgement For technical assistance we are indebted to Miss Ute Pietzonka, Mr. Leonidas Nikolaidis, Mrs. Wilma GeuB and Miss Mioko Fukui.
Detection of Aerosols in the Respiratory Tract .
121
Literature Franklin, W.: Pulmonary retention of aerosols. J. elin. invest. 29, 1I77-IIS1 (1950) Goerg, R., und Locher, J. Th.: Vorteile der Dusenzerstaubung fur die Aerosoltherapie. Schweiz. med. Wschr. 101, 1838-1843 (1972) Hobel, M., Maroske, D., Glanzmann, Ch., und Eichler, 0.: Resorption von Wirkungssubstanzen aus Aerosolen in Abhangigkeit vom Atmungstyp. Arch. into Pharmacodyn. 198,76-84 (197 2) Le Pecq, J. B., and Paoletti, C.: A new f1uorometric method for RNA and DNA determination. Anal. Biochem. 17, 100-107 (1966) Rhoades, R. A.: Effect of inhaled carbon on surface properties of rat lungs. Life Sci. II, 39-42 (1972) Tao, T., Nelson, J. H., and Con tor, Ch. R.: Conformational Studies on Transfer Ribonucleic Acid. Fluorescence Lifetime and Nanosecond Depolarization Measurements on Bound Ethidium Bromide. Biochem. 9, (18), 3514-3524 (1970) Dr. Gojko Muacevic, C. H. Boehringer Sohn, Abt. Pharmakologie, D-6507 Ingelheiml Rhein
From the Departments of Pharmacology and Experimental Pathology and Toxicology at C. H. Boehringer Sohn, Ingelheim/Rhein
Detection of Aerosols in the Respiratory Tract Nachweis von Aerosolen im Respirationstrakt G. MUACEVIC and T. TILOV With 4 Figures· Received May 14, 1975 . Accepted in revised form June 6, 1975
Key words: Aerosol - Detection - Fluorescent reagents - Inhalation Respiratory tract
Summary A fluorescence method for the detection of inhaled aerosols in the respiratory tract has been described. The most suitable fluorescence indicator for the histological examination was ethidium bromide.
If an inhalation method for the use of pharmaceutical agents suspended in aerosols is to be regarded as successful, detection of the agent in the lungs should be possible. Detection of the inhaled substances is of particular importance in animal experiments for the testing of aerosols for medical treatment, cosmetics and plant protection. Lithium (Habel et aL, 1972), coal dust (Rhoades, 1972) isotopes (Goerg and Locher, 1972) and p-aminohipuric acid (Franklin, 1950) have been used to prove respiratory desposition, the later substance being detectable in the urine following inhalation. It was our aim to develop a quick and uncomplicated method to prove respiratory deposition in animal experiments by using fluorescent agents in an aerosol. In these tests we looked for fluorescent reagents which would mark the different parts of the respiratory tract and would bind firmly with chemical structures of the tissues. The best results in our experiments have been obtained with ethidium bromide. 9 Beitr. Path. Bd. 156
118 . G. Muacevic and T. Tilov
Material and Method The experiments were carried out on rats and dogs. The animals inhaled one of the nebulized 0.2-1.0010 aqueous solutions of sodium fluorescein, haematoporphyrin, orange G, eosin scariet, thiazine red R, 2-toluidinonaphtyl-6-sulfphonic acid-K-salt (2,6-TNS), and acetone solutions (acetone: water, 60 : 40) of ethidium bromide, for 15-30 minutes. For nebulisation of the solutions we used a Draeger nebuliser (Aerolette Type) and a Pari nebuliser (Pari Primus Type) . The aerosol particles had an average size of 0.55 11m (according to instruction of the producers). Each substance was administered to 3 male rats (Chbb : THOM (SPF)), weighing 200 g. in an aerosol chamber of 2 I capacity. A group of further 3 rats served as control. The inhalation of ethidium bromide aerosol was carried out additionally on one dog (Chbi-beagle, male, weighing 10 kg), a further dog was used as control. In order to prove the reproducibility of the method seven test repetitions were performed with ethidium bromide and haematoporphyrin on rats (altogether 21 animals). For the ide,ntification of fluorescence lung and trachea sections were prepared and examined as follows: Immediately after death lungs and trachea were fixed for 24 hours by immersion in buffered 7.5010 formol (Merck, Darmstadt); subsequently frozen (C02) 15 /lm and paraffin sections 5 11m thick were prepared. Cryostat sections (7 11m thickness) of the non fixed tissue (frozen immediately after death with C02) were also prepared. These three kinds of histological sections were investigated using a Zeiss-photo microscope with transmitted fluorescence device. A metal - halide - CSF - 250 Ware lamp served as UV-light source. The optimal filter combinations were the Zeiss BG-Il or BG-3 excitation filters and the Zeiss 47, 50 or 53 barrier filters.
Results and Discussion Among the above mentioned fluorochromes, sodium fluorescein, haematoporphyrin and ethidium bromide were selected for further tests. With our combination of UV~light-sorce and filter the greenish-yellow fluorescence of sodium fluorescein could hardly be discerned from the native yellowish-green fluorescence of the lungs. For this reason, sodium fluorescein was found to be an unfavourable indicator under the described conditions. The more intensive red fluorescence of haematoporphyrin, however, could easily be distinguished from the native fluorescence of the lungs and is thus more suitable for our purposes than sodium fluorescein, but the substance is water soluble an'd not very stable (as fluorescenting agent in the tissue). The most suitable fluorescence indicator in our experiments was ethidiurn bromide. With RNA and DNA it forms stable compounds with strong fluorescence (Le Pecq and Paoletti, 1966). The compounds of ethidium bromide and tissue have a highly intensive golden-orange fluorescence which is stronger than that of ethidium bromide itself (Fig. 1-4).
Detection of Aerosols in the Respiratory Tract·
119
Fig. I. Lung of the rat: native green autofluorescence of lung tissue. Frozen section; X 64, Zeiss excitation filter BG-3 and Zeiss barrier filter 53.
Fig. 2. Lung of rat after inhalation of ethidium bromide aerosol. Ethidium bromide in combination with RNA and DNA of the bronchialmucosal cells and alveolar wall produces a yellow-orange fluorescence compared with the green autofluorescence of the lung. Frozen section; X 64, filters as in fig. I.
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I ry
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Fig. 3. Trachea of the dog: orange-yellow fluorescence of the mucosal ciliated epithelium after inhalation of ethidium bromide aerosol. Green autofluorescence of the submucosa. Frozen section; X roo, filter as in fig. I.
120 .
G. Muacevic and T. Tilov
Fig. 4. Lung of the dog: yellow-orange fluorescence after the inhalation of ethidium bromide aerosol in the bronchial and alveolar epithelium. Frozen section; X 64, filters as in fig. I.
Another adavantage of ethidium-RNA or ethidium-DNA compounds was owing to the fact that they could not be washed out or destroyed during fixation and during the above-described histological processing. The investigations on cryostat- and, in particular, paraffin sections showed no advantages. The frozen sections prepared from fixed lung tissue were adequate for histological detection of the substance inhaled. The fluorescence of the preparations with ethidium bromide continued to be highly intensive for more than six months after histological processing. This stability of ethidium bromide is in accordance with the literature (Le Pesq and Paoletti, 1966; T. Tao et al., 1970). The above-described histochemical method is uncomplicated and reproducible. It allows detailed morphological examination and microphotographic documentation. The described experiments showed that ethidium bromide was the most suitable indicator for the detection of aerosols in the respiratory tract because the good contrast and stable fluorescenting properties.
Zusammenfassung Es wird eine Fluoreszenzmethode fur den Nachweis inhalierter Aerosole im Respirationstrakt beschrieben. Ethidiumbromid erweist sich als der beste Fluoreszenzindikator bei der histologischen Untersuchung. Acknowledgement For technical assistance we are indebted to Miss Ute Pietzonka, Mr. Leonidas Nikolaidis, Mrs. Wilma GeuB and Miss Mioko Fukui.
Detection of Aerosols in the Respiratory Tract .
121
Literature Franklin, W.: Pulmonary retention of aerosols. J. elin. invest. 29, 1I77-IIS1 (1950) Goerg, R., und Locher, J. Th.: Vorteile der Dusenzerstaubung fur die Aerosoltherapie. Schweiz. med. Wschr. 101, 1838-1843 (1972) Hobel, M., Maroske, D., Glanzmann, Ch., und Eichler, 0.: Resorption von Wirkungssubstanzen aus Aerosolen in Abhangigkeit vom Atmungstyp. Arch. into Pharmacodyn. 198,76-84 (197 2) Le Pecq, J. B., and Paoletti, C.: A new f1uorometric method for RNA and DNA determination. Anal. Biochem. 17, 100-107 (1966) Rhoades, R. A.: Effect of inhaled carbon on surface properties of rat lungs. Life Sci. II, 39-42 (1972) Tao, T., Nelson, J. H., and Con tor, Ch. R.: Conformational Studies on Transfer Ribonucleic Acid. Fluorescence Lifetime and Nanosecond Depolarization Measurements on Bound Ethidium Bromide. Biochem. 9, (18), 3514-3524 (1970) Dr. Gojko Muacevic, C. H. Boehringer Sohn, Abt. Pharmakologie, D-6507 Ingelheiml Rhein
