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Bronchoalveolar lavage of the live anaesthetised sheep
Research in Veterinary Science, 41. I09-l/2
Bronchoalveolar lavage of the live anaesthetised sheep C. BURRELLS, J. T. WILLIAMS Moredun Research Institute, 408 Gilmerton Road,
Edinburgh EHI7 7JH
Segmental lavage was performed on anaesthetised adult sheep using either endoscopic catheterisation or fibreoptic bronchoscopy. Cellular and humoral data were compared with results obtained from gross lavage of whole excised lungs. Both techniques proved rapid, convenient and reproducible, providing sufficient material for qualitative and quantitative assays of respiratory cells and fluid. Although total cells per unit volume of fluid obtained b)' catheterisation or by bronchoscopy were decreased compared with whole lung lavage, differential cell populations, fluid recovery and immunoglobulin and complement (C 3 ) concentrations were comparable.
employed in several investigations (Gorin et al 1979, Rola-pleszczynski et al 1981). There has, however, been little definition of the technical aspects of these procedures or consideration of the area of lung irrigated by the lavage fluid. The purpose of this study was the examination of both techniques of bronchoalveolar lavage in live sheep, the definition of the area of lung irrigated by the procedure, the comparison of several cellular and humoral parameters in samples obtained by different methods and consideration of the effects of the procedure on the animals.
IMMUNITY in the lower respiratory tract of sheep has been studied in three different ways; (a) by the analysis of lymph draining the respiratory tract (Gorin et al 1979), (b) by immunohistochemical examination of fixed tissue sections (Alley et al 1980) or (c) by examination of samples obtained by bronchoalveolar lavage (Gorin et al 1979, Rolapleszczynski et al 1981). The use of fibreoptic bronchoscopy to obtain samples has enabled studies to be made of cellular and humoral immune elements in the lower respiratory tract of humans (Reynolds and Newball 1976, Hunninghake et al 1979). Cell types and lymphocyte subpopulations in the lower respiratory tract of monkeys have been examined using lavage samples obtained by endotracheal tubes (Kastello et al 1979) or by a fibreoptic bronchoscope (Kazmierowski et al 1976). Endotracheal catheters have been used in the study of pulmonary immunoglobulins of dogs (Kaltreider and Chan 1976) and pigs (Morgan et al 1980). Similar work has been carried out on bovine pulmonary lavage samples obtained either by fibreoptic bronchoscopy. (Wilkie and Markham 1979) or by gross lavage of whole excised lungs (Walker et al 1980a, b). Examinations of ovine pulmonary cellular and humoral immune mechanisms have been undertaken using the technique of whole lung lavage (Davies and Penwarden 1981, Burrells 1985). Endoscopic catheterisation or fibreoptic bronchoscopy, however, provide the means of obtaining repeated and sequential samples from ovine lungs and have been
Materials and methods
Animals Fifteen two-year-old Blackface ewes were used in this study. Four of these were utilised to determine the area of lung irrigated by the lavage procedures (two by endotracheal catheterisation and two by fibreoptic bronchoscopy). Lung wash fluids for examination were collected from four sheep using a catheter and from seven sheep using a bronchoscope.
Anaesthesia Food was withheld for 24 to 36 hours before anaesthesia which was achieved by intravenous administration of pentobarbitone sodium (Sagatal; May & Baker) at the rate of 32 mg kg - 1 bodyweight. After induction of anaesthesia, an endotracheal tube (10 or II mm diameter) was. passed into the trachea, tied in place and the cuff inflated.
Lavage procedures Endotracheal catheterisation A Folatex balloon catheter (14G) extended to a length of 80 ern was used in the early part of this study. Anaesthetised sheep were placed in left lateral recumbency and the catheter was fed down the endotracheal tube. The catheter entered the left principal bronchus and was slowly inserted until the tip wedged in one of the smaller bronchioles. The cuff was inflated with 2 to
109
110
C. Burrells, J. T. Williams
3 ml of saline, thereby isolating a small area of the lung. A volume (50 ml) of sterile Hanks' basic salt solution (HBSS) was infused through the catheter from a 50 ml syringe and this was followed immediately by 10 to 20 ml of air. As much fluid as possible was gently withdrawn using the same syringe and the lavage procedure was repeated three times.
Fibreoptic bronchoscopy This technique was carried out using an Arnolds VFS-2 fibreoptic bronchoscope (Arnolds Veterinary Products). With the animals in left lateral recumbency, the bronchoscope tube was passed down the trachea into the left principal bronchus until it lodged in the diaphragmatic lobar bronchus. A 50 ml volume of HBSS was introduced into the lung through the bronchoscopic channel and negative pressure was applied immediately (Mityvac pump-3, Arnolds Veterinary Products) to aspirate the fluid. The lavage procedure was repeated with a second 50 ml volume of HBSS and the bronchoscope was withdrawn. Determination of the area of the lungs irrigated by lavage Endotracheal catheterisation To determine the extent of penetration into the lungs, a radio-opaque catheter was inserted into the lungs of one sheep and X-ray photographs were taken. To assess the area of lung irrigated, 50 ml of a radio-opaque fluid (Isopaque 440; Nyegaard) were infused through a catheter into the lungs of a second sheep and X-ray photographs were again taken. Fibreoptic bronchoscopy Evans blue dye (I per cent in phosphate buffered saline) was injected through the bronchoscope into the lungs of two sheep (one sheep receiving 2 ml of the dye and the other receiving
FIG 1: X-ray photograph of sheep lungs showing the extent of penetration of a radio-opaque catheter (arrowed) into the pulmonary airways
10 ml), The animals were immediately killed with excess pentobarbitone and the lungs were removed for examination.
Radiography X-ray photographs were obtained using a Philips Praktix 90120 X-ray source at an anode-film distance of 28 inches (70 ern). Films were exposed for 0·08 seconds at 90 kVP at 20 rnA.
Treatment of lavage samples Lavage samples were treated as described previously (Burrells 1985) to assay total and differential cell counts and concentration of immunoglobulin isotypes and the C 3 component of complement.
Statistical analysis Cellular and humoral data obtained from animals in this study were compared with similar data obtained previously from whole lung lavage samples from normal sheep of a similar age (Burrells 1985). Intergroup comparisons were made using the Student test. Figures are expressed as mean ± standard error of the mean. Results Bronchoalveolar lavage of each anaesthetised sheep was carried out in 10 to 15 minutes and all animals made uneventful recoveries from the experimental procedures.
Area of lung irrigated by the procedure The X-ray photographs (Figs I and 2) indicate that the small diameter catheters entered the left principal
FIG 2: X-ray photograph of sheep lungs after insertion of an endotracheal catheter (C) and instillation of 50 ml of radio-opaque fluid (RF). The inflatable cuff (lC) can be seen lodged in the dorsal basal segment of the diaphragmatic lobe
Bronchoalveolar lavage of sheep
III
TABLE 1: Total cell counts and differential leucocyte proportions in ovine bronchoalveolar lavage fluids obtained by different techniques Cell type
Whole lung* lavage (mean±SEM)
Endotracheal catheter (mean±SEMI
Fibreoptic bronchoscope (mean±SEMI
Total cells
4'5±0'6' (n=23)
0'6±0'2 (n=4)
1'3±0'6 (n=7)
70·3±1·Tt
NT NT NT NT NT
68·7±5·3 2O·9±6·1 1·4±0·5 8·0±3·0 1'0±0'6 (n =7)
Macrophages Lymphocytes Neutrophils Eosinophils Basophils
26·6±2·2 0'6±0'3 2'1±1'4 0'4±0'4 (n= 18)
• Total cells expressed as lOS ml ' of lavage fluid Differential cell proportions expressed as per cent of total leucocytes NT Not tested * Data taken from Burrells (19851
t
of individual leucocyte types did not differ significantly in lung washes from bronchoscopy or whole lung lavage (Table I). Such counts were not performed on catheterisation samples.
Immunoglobulins and C) in lavage fluid
FIG 3: Sheep lungs excised after the instillation of (al (above) 2 ml and (b) (below) 10 ml of Evans blue dye using a fibreoptic bronchoscope
bronchus, continued down the diaphragmatic lobar bronchus and lodged in the dorsal basal segment of the diaphragmatic lobe (Fig I). When a cuffed catheter was used and 50 ml of fluid injected, a discrete area of the dorsal basal segment was irrigated (Fig 2). The larger diameter bronchoscope lodged in the lateral dorsal segment of the diaphragmatic lobe. When dye was injected down the bronchoscope and the animals immediately killed, it was seen from the excised lungs that a similar discrete area of the dorsal basal segment of the diaphragmatic lobe was irrigated (Fig 3a, b).
Proportions of lavage fluid recovered in each procedure were similar (43 to 49 per cent). Concentrations of IgG and IgA in samples obtained by catheter and bronchoscope were greater than those obtained by whole lung lavage (Table 2), but as a result of small numbers of segmental lavage samples and wide individual variation, these differences were not statistically significant. Differences in immunoglobulin classes as percentages of the total immunoglobulins were likewise not statistically significant. No IgM was detected in either of the two
TABLE 2: Immunoglobulins (lgsl and complement 1C31 in ovine bronchoalveolar lavage fluids obtained by different techniques Ig
IgG IgA IgM
Total and differential cell counts 105
I)
Total cell counts in catheter (0' 6 x ml- and bronchoscope (I' 3 x lOS ml-') lavage fluids were significantly lower (P<0'05) than counts obtained by whole lung lavage (4' 5 x 105 ml-') (Table I). Differential cell counts indicated that proportions
Whole lung§ lavage (mean±SEM) (n=26)
Endotracheal catheter .(mean±SEM) (n=2)
Fibreoptic bronchoscope (mean±SEM) (n=5)
249·2±35·r
402· 6±56' 7 (47' 2±0' 5) 452' 0±72' (52'8±0' 5) (0)
335' 6±76· 3 (46' 7±6' 3) 411'7±122'2 (54'2±7'9) 6·8±1·4 (1'0±0'3)
23'2±13'6 (n=2)
NT (n=5)
(49' 4±2'
srr
264'3±47'1 (50' 2±2' 4) 4·7±2·2 (Q'B±O' 21
27' 2±4'o* (n=261
a
a
• Igs expressed as mg litre -1 of lavage fluid Igs expressed as percentages of total immunoglobulins * C3 expressed as a percentage in relation to a serum standard NT Not tested § Data taken from Burrells (1985)
t
112
C. Burrells, J. T. Williams
catheter samples but amounts and proportions of this isotype were comparable in whole lung lavage and bronchoscope samples. Similar concentrations of C 3 were found in whole lung lavage samples and in segmental lavage samples obtained by catheterisation. Discussion Segmental lavage in anaesthetised sheep using endobronchial catheters or a fibreoptic bronchoscope proved to be a convenient, reliable and reproducible procedure for obtaining lung wash fluid. Although it has been reported for cattle (Fogarty 1984) and sheep (Begin et al 1981, Rola-pleszczynski et a11981) that bronchopulmonary lavage can be performed on conscious unanaesthetised animals, it has been observed that sheep do not readily tolerate the passage of catheters or bronchoscope tubes into the nasal passages, trachea or bronchioles. Even when a topical anaesthetic was sprayed on to the back of the throat, animals experienced involuntary coughing and 'gagging' with the introduction of tubes into the respiratory passages (J. T. Williams and C. Burrells, unpublished observations). This invariably caused rupture of small blood vessels resulting in the presence of blood in the first lavage harvest. Contamination with blood was considered unacceptable in samples subsequently to be examined for elements of local cellular and humoral immunity. Pentobarbitone sodium was used in preference to general gaseous anaesthetics such as halothane which can inhibit certain cellular functions of macrophages and lymphocytes (Adam and Dudszus 1980, Bruce 1980). Although the proportion of lavage fluid recovered by catheter or bronchoscope was similar to that recovered by whole lung lavage, the cellular content per unit volume was significantly lower in samples obtained by segmental lavage. This may result from the fact that segmental lavage involves the instillation of fluid into the lungs and immediate withdrawal. The method of whole lung lavage, on the other hand, which includes gentle massage of the lungs, would provide a more thorough flushing of the airways and thus increase cell harvests. Despite the difference in total cell concentrations between segmental and whole lung lavage, no statistically significant differences were observed in the proportions of differential leucocyte populations and macrophage: lymphoid cell ratios were similar. Amounts and proportions of individual immunoglobulin classes and C 3 in fluids obtained by the different techniques were also similar. It is concluded that segmental lavage of the lungs of anaesthetised adult sheep is a rapid and reliable tech-
nique for the collection of respiratory cells and fluid. The technique yields samples which provide data essentially comparable to data obtained by gross lavage of whole excised lungs, and sampling is achieved with minimal discomfort to or lasting effect upon the animals. The two techniques compared favourably in terms of speed and ease, but the facility to visually direct the entry of the bronchoscope was an obvious advantage. However, it may be necessary to use the much narrower diameter catheter when obtaining samples from younger, smaller animals. Acknowledgements The authors gratefully acknowledge the technical expertise of Mr G. U. Aitcheson and Mr G. Newlands in producing the X-ray plates.
References ADAM H. Von & DUDSZUS, W. (1980) Anaesthesiologie und Reanimation 5, 26-31 ALLEY, M. R., WELLS, P. W., SMITH, W. D. & GARDINER, A. C. (1980) Veterinary Pathology 17,372-380 BEGIN, R., ROLA-PLESZCZYNSKI, M., SIROIS, P., MASSE. S., NADEAU, D. & BUREAU, M. A. (1981) Journal of Applied Physiology 50, 665-671 BRUCE, D. L. (1980) Federation Proceedings 39, 1592-1594 BURRELLS, C. (1985) Veterinary Immunology and Immunopathology 10, 225-243 DAVIES, D. H. & PENWARDEN, R. A. (1981) Veterinary Microbiology 6, 183-189 FOGARTY, U. (1984) PhD thesis, University of Dublin GORIN, A. B., STEWART, P. & GOULD, J. (1979) Research in Veterinary Science 26, 126-128 HUNNINGHAKE, G. W., GADEK, G. E., KAWANAMI, 0., FERRANS, V. J. & CRYSTAL, R. F. (1979) American Journal of Pathology 97, 149-198 KALTREIDER, H. B. & CHAN, M. K. L. (1976) Journal of Immunology 116, 423-429 KASTELLO, M. D., EMMERT, A. D., DENSON, R. F. & KISHIMOTO, R. A. (1979) American Journal of Veterinary Research 40,271-273 KAZMIEROWSKI, J. A., FAUCI, A. S. & REYNOLDS, H. Y. (1976) Journal of Immunology 116, 615-618 MORGAN, K. L., HUSSEIN, A. M., NEWBY, T. J. & BOURNE, F. J. (1980) Immunology 41, 729-736 REYNOLDS, H. Y. & NEWBALL, H. H. (1976) The Lung in Health and Disease. Vol I. Eds C. H. Kirkpatrick and H. Y. Reynolds. New York, Basel, Marcel-Dekker. pp 3-27 ROLA-PLESZCZYNSKI, M., SIROIS, P. & BEGIN, R. (1981) Lung 159,91-99 WALKER, R. D., CORSTVET, R. E. & PANCIERA, R. J. (I 980a) American Journal of Veterinary Research 41, 1008-1014 WALKER, R. D., CORSTVET, R. E., LESSLEY, B. A. & PANCIERA, R. J. (l980b) American Journal of Veterinary Research 41,1015-1023 WILKIE, B. N. & MARKHAM, R. J. F. (1979) American Journal of Veterinary Research 40,1690-1693
Accepted April 19, 1986
Bronchoalveolar lavage of the live anaesthetised sheep C. BURRELLS, J. T. WILLIAMS Moredun Research Institute, 408 Gilmerton Road,
Edinburgh EHI7 7JH
Segmental lavage was performed on anaesthetised adult sheep using either endoscopic catheterisation or fibreoptic bronchoscopy. Cellular and humoral data were compared with results obtained from gross lavage of whole excised lungs. Both techniques proved rapid, convenient and reproducible, providing sufficient material for qualitative and quantitative assays of respiratory cells and fluid. Although total cells per unit volume of fluid obtained b)' catheterisation or by bronchoscopy were decreased compared with whole lung lavage, differential cell populations, fluid recovery and immunoglobulin and complement (C 3 ) concentrations were comparable.
employed in several investigations (Gorin et al 1979, Rola-pleszczynski et al 1981). There has, however, been little definition of the technical aspects of these procedures or consideration of the area of lung irrigated by the lavage fluid. The purpose of this study was the examination of both techniques of bronchoalveolar lavage in live sheep, the definition of the area of lung irrigated by the procedure, the comparison of several cellular and humoral parameters in samples obtained by different methods and consideration of the effects of the procedure on the animals.
IMMUNITY in the lower respiratory tract of sheep has been studied in three different ways; (a) by the analysis of lymph draining the respiratory tract (Gorin et al 1979), (b) by immunohistochemical examination of fixed tissue sections (Alley et al 1980) or (c) by examination of samples obtained by bronchoalveolar lavage (Gorin et al 1979, Rolapleszczynski et al 1981). The use of fibreoptic bronchoscopy to obtain samples has enabled studies to be made of cellular and humoral immune elements in the lower respiratory tract of humans (Reynolds and Newball 1976, Hunninghake et al 1979). Cell types and lymphocyte subpopulations in the lower respiratory tract of monkeys have been examined using lavage samples obtained by endotracheal tubes (Kastello et al 1979) or by a fibreoptic bronchoscope (Kazmierowski et al 1976). Endotracheal catheters have been used in the study of pulmonary immunoglobulins of dogs (Kaltreider and Chan 1976) and pigs (Morgan et al 1980). Similar work has been carried out on bovine pulmonary lavage samples obtained either by fibreoptic bronchoscopy. (Wilkie and Markham 1979) or by gross lavage of whole excised lungs (Walker et al 1980a, b). Examinations of ovine pulmonary cellular and humoral immune mechanisms have been undertaken using the technique of whole lung lavage (Davies and Penwarden 1981, Burrells 1985). Endoscopic catheterisation or fibreoptic bronchoscopy, however, provide the means of obtaining repeated and sequential samples from ovine lungs and have been
Materials and methods
Animals Fifteen two-year-old Blackface ewes were used in this study. Four of these were utilised to determine the area of lung irrigated by the lavage procedures (two by endotracheal catheterisation and two by fibreoptic bronchoscopy). Lung wash fluids for examination were collected from four sheep using a catheter and from seven sheep using a bronchoscope.
Anaesthesia Food was withheld for 24 to 36 hours before anaesthesia which was achieved by intravenous administration of pentobarbitone sodium (Sagatal; May & Baker) at the rate of 32 mg kg - 1 bodyweight. After induction of anaesthesia, an endotracheal tube (10 or II mm diameter) was. passed into the trachea, tied in place and the cuff inflated.
Lavage procedures Endotracheal catheterisation A Folatex balloon catheter (14G) extended to a length of 80 ern was used in the early part of this study. Anaesthetised sheep were placed in left lateral recumbency and the catheter was fed down the endotracheal tube. The catheter entered the left principal bronchus and was slowly inserted until the tip wedged in one of the smaller bronchioles. The cuff was inflated with 2 to
109
110
C. Burrells, J. T. Williams
3 ml of saline, thereby isolating a small area of the lung. A volume (50 ml) of sterile Hanks' basic salt solution (HBSS) was infused through the catheter from a 50 ml syringe and this was followed immediately by 10 to 20 ml of air. As much fluid as possible was gently withdrawn using the same syringe and the lavage procedure was repeated three times.
Fibreoptic bronchoscopy This technique was carried out using an Arnolds VFS-2 fibreoptic bronchoscope (Arnolds Veterinary Products). With the animals in left lateral recumbency, the bronchoscope tube was passed down the trachea into the left principal bronchus until it lodged in the diaphragmatic lobar bronchus. A 50 ml volume of HBSS was introduced into the lung through the bronchoscopic channel and negative pressure was applied immediately (Mityvac pump-3, Arnolds Veterinary Products) to aspirate the fluid. The lavage procedure was repeated with a second 50 ml volume of HBSS and the bronchoscope was withdrawn. Determination of the area of the lungs irrigated by lavage Endotracheal catheterisation To determine the extent of penetration into the lungs, a radio-opaque catheter was inserted into the lungs of one sheep and X-ray photographs were taken. To assess the area of lung irrigated, 50 ml of a radio-opaque fluid (Isopaque 440; Nyegaard) were infused through a catheter into the lungs of a second sheep and X-ray photographs were again taken. Fibreoptic bronchoscopy Evans blue dye (I per cent in phosphate buffered saline) was injected through the bronchoscope into the lungs of two sheep (one sheep receiving 2 ml of the dye and the other receiving
FIG 1: X-ray photograph of sheep lungs showing the extent of penetration of a radio-opaque catheter (arrowed) into the pulmonary airways
10 ml), The animals were immediately killed with excess pentobarbitone and the lungs were removed for examination.
Radiography X-ray photographs were obtained using a Philips Praktix 90120 X-ray source at an anode-film distance of 28 inches (70 ern). Films were exposed for 0·08 seconds at 90 kVP at 20 rnA.
Treatment of lavage samples Lavage samples were treated as described previously (Burrells 1985) to assay total and differential cell counts and concentration of immunoglobulin isotypes and the C 3 component of complement.
Statistical analysis Cellular and humoral data obtained from animals in this study were compared with similar data obtained previously from whole lung lavage samples from normal sheep of a similar age (Burrells 1985). Intergroup comparisons were made using the Student test. Figures are expressed as mean ± standard error of the mean. Results Bronchoalveolar lavage of each anaesthetised sheep was carried out in 10 to 15 minutes and all animals made uneventful recoveries from the experimental procedures.
Area of lung irrigated by the procedure The X-ray photographs (Figs I and 2) indicate that the small diameter catheters entered the left principal
FIG 2: X-ray photograph of sheep lungs after insertion of an endotracheal catheter (C) and instillation of 50 ml of radio-opaque fluid (RF). The inflatable cuff (lC) can be seen lodged in the dorsal basal segment of the diaphragmatic lobe
Bronchoalveolar lavage of sheep
III
TABLE 1: Total cell counts and differential leucocyte proportions in ovine bronchoalveolar lavage fluids obtained by different techniques Cell type
Whole lung* lavage (mean±SEM)
Endotracheal catheter (mean±SEMI
Fibreoptic bronchoscope (mean±SEMI
Total cells
4'5±0'6' (n=23)
0'6±0'2 (n=4)
1'3±0'6 (n=7)
70·3±1·Tt
NT NT NT NT NT
68·7±5·3 2O·9±6·1 1·4±0·5 8·0±3·0 1'0±0'6 (n =7)
Macrophages Lymphocytes Neutrophils Eosinophils Basophils
26·6±2·2 0'6±0'3 2'1±1'4 0'4±0'4 (n= 18)
• Total cells expressed as lOS ml ' of lavage fluid Differential cell proportions expressed as per cent of total leucocytes NT Not tested * Data taken from Burrells (19851
t
of individual leucocyte types did not differ significantly in lung washes from bronchoscopy or whole lung lavage (Table I). Such counts were not performed on catheterisation samples.
Immunoglobulins and C) in lavage fluid
FIG 3: Sheep lungs excised after the instillation of (al (above) 2 ml and (b) (below) 10 ml of Evans blue dye using a fibreoptic bronchoscope
bronchus, continued down the diaphragmatic lobar bronchus and lodged in the dorsal basal segment of the diaphragmatic lobe (Fig I). When a cuffed catheter was used and 50 ml of fluid injected, a discrete area of the dorsal basal segment was irrigated (Fig 2). The larger diameter bronchoscope lodged in the lateral dorsal segment of the diaphragmatic lobe. When dye was injected down the bronchoscope and the animals immediately killed, it was seen from the excised lungs that a similar discrete area of the dorsal basal segment of the diaphragmatic lobe was irrigated (Fig 3a, b).
Proportions of lavage fluid recovered in each procedure were similar (43 to 49 per cent). Concentrations of IgG and IgA in samples obtained by catheter and bronchoscope were greater than those obtained by whole lung lavage (Table 2), but as a result of small numbers of segmental lavage samples and wide individual variation, these differences were not statistically significant. Differences in immunoglobulin classes as percentages of the total immunoglobulins were likewise not statistically significant. No IgM was detected in either of the two
TABLE 2: Immunoglobulins (lgsl and complement 1C31 in ovine bronchoalveolar lavage fluids obtained by different techniques Ig
IgG IgA IgM
Total and differential cell counts 105
I)
Total cell counts in catheter (0' 6 x ml- and bronchoscope (I' 3 x lOS ml-') lavage fluids were significantly lower (P<0'05) than counts obtained by whole lung lavage (4' 5 x 105 ml-') (Table I). Differential cell counts indicated that proportions
Whole lung§ lavage (mean±SEM) (n=26)
Endotracheal catheter .(mean±SEM) (n=2)
Fibreoptic bronchoscope (mean±SEM) (n=5)
249·2±35·r
402· 6±56' 7 (47' 2±0' 5) 452' 0±72' (52'8±0' 5) (0)
335' 6±76· 3 (46' 7±6' 3) 411'7±122'2 (54'2±7'9) 6·8±1·4 (1'0±0'3)
23'2±13'6 (n=2)
NT (n=5)
(49' 4±2'
srr
264'3±47'1 (50' 2±2' 4) 4·7±2·2 (Q'B±O' 21
27' 2±4'o* (n=261
a
a
• Igs expressed as mg litre -1 of lavage fluid Igs expressed as percentages of total immunoglobulins * C3 expressed as a percentage in relation to a serum standard NT Not tested § Data taken from Burrells (1985)
t
112
C. Burrells, J. T. Williams
catheter samples but amounts and proportions of this isotype were comparable in whole lung lavage and bronchoscope samples. Similar concentrations of C 3 were found in whole lung lavage samples and in segmental lavage samples obtained by catheterisation. Discussion Segmental lavage in anaesthetised sheep using endobronchial catheters or a fibreoptic bronchoscope proved to be a convenient, reliable and reproducible procedure for obtaining lung wash fluid. Although it has been reported for cattle (Fogarty 1984) and sheep (Begin et al 1981, Rola-pleszczynski et a11981) that bronchopulmonary lavage can be performed on conscious unanaesthetised animals, it has been observed that sheep do not readily tolerate the passage of catheters or bronchoscope tubes into the nasal passages, trachea or bronchioles. Even when a topical anaesthetic was sprayed on to the back of the throat, animals experienced involuntary coughing and 'gagging' with the introduction of tubes into the respiratory passages (J. T. Williams and C. Burrells, unpublished observations). This invariably caused rupture of small blood vessels resulting in the presence of blood in the first lavage harvest. Contamination with blood was considered unacceptable in samples subsequently to be examined for elements of local cellular and humoral immunity. Pentobarbitone sodium was used in preference to general gaseous anaesthetics such as halothane which can inhibit certain cellular functions of macrophages and lymphocytes (Adam and Dudszus 1980, Bruce 1980). Although the proportion of lavage fluid recovered by catheter or bronchoscope was similar to that recovered by whole lung lavage, the cellular content per unit volume was significantly lower in samples obtained by segmental lavage. This may result from the fact that segmental lavage involves the instillation of fluid into the lungs and immediate withdrawal. The method of whole lung lavage, on the other hand, which includes gentle massage of the lungs, would provide a more thorough flushing of the airways and thus increase cell harvests. Despite the difference in total cell concentrations between segmental and whole lung lavage, no statistically significant differences were observed in the proportions of differential leucocyte populations and macrophage: lymphoid cell ratios were similar. Amounts and proportions of individual immunoglobulin classes and C 3 in fluids obtained by the different techniques were also similar. It is concluded that segmental lavage of the lungs of anaesthetised adult sheep is a rapid and reliable tech-
nique for the collection of respiratory cells and fluid. The technique yields samples which provide data essentially comparable to data obtained by gross lavage of whole excised lungs, and sampling is achieved with minimal discomfort to or lasting effect upon the animals. The two techniques compared favourably in terms of speed and ease, but the facility to visually direct the entry of the bronchoscope was an obvious advantage. However, it may be necessary to use the much narrower diameter catheter when obtaining samples from younger, smaller animals. Acknowledgements The authors gratefully acknowledge the technical expertise of Mr G. U. Aitcheson and Mr G. Newlands in producing the X-ray plates.
References ADAM H. Von & DUDSZUS, W. (1980) Anaesthesiologie und Reanimation 5, 26-31 ALLEY, M. R., WELLS, P. W., SMITH, W. D. & GARDINER, A. C. (1980) Veterinary Pathology 17,372-380 BEGIN, R., ROLA-PLESZCZYNSKI, M., SIROIS, P., MASSE. S., NADEAU, D. & BUREAU, M. A. (1981) Journal of Applied Physiology 50, 665-671 BRUCE, D. L. (1980) Federation Proceedings 39, 1592-1594 BURRELLS, C. (1985) Veterinary Immunology and Immunopathology 10, 225-243 DAVIES, D. H. & PENWARDEN, R. A. (1981) Veterinary Microbiology 6, 183-189 FOGARTY, U. (1984) PhD thesis, University of Dublin GORIN, A. B., STEWART, P. & GOULD, J. (1979) Research in Veterinary Science 26, 126-128 HUNNINGHAKE, G. W., GADEK, G. E., KAWANAMI, 0., FERRANS, V. J. & CRYSTAL, R. F. (1979) American Journal of Pathology 97, 149-198 KALTREIDER, H. B. & CHAN, M. K. L. (1976) Journal of Immunology 116, 423-429 KASTELLO, M. D., EMMERT, A. D., DENSON, R. F. & KISHIMOTO, R. A. (1979) American Journal of Veterinary Research 40,271-273 KAZMIEROWSKI, J. A., FAUCI, A. S. & REYNOLDS, H. Y. (1976) Journal of Immunology 116, 615-618 MORGAN, K. L., HUSSEIN, A. M., NEWBY, T. J. & BOURNE, F. J. (1980) Immunology 41, 729-736 REYNOLDS, H. Y. & NEWBALL, H. H. (1976) The Lung in Health and Disease. Vol I. Eds C. H. Kirkpatrick and H. Y. Reynolds. New York, Basel, Marcel-Dekker. pp 3-27 ROLA-PLESZCZYNSKI, M., SIROIS, P. & BEGIN, R. (1981) Lung 159,91-99 WALKER, R. D., CORSTVET, R. E. & PANCIERA, R. J. (I 980a) American Journal of Veterinary Research 41, 1008-1014 WALKER, R. D., CORSTVET, R. E., LESSLEY, B. A. & PANCIERA, R. J. (l980b) American Journal of Veterinary Research 41,1015-1023 WILKIE, B. N. & MARKHAM, R. J. F. (1979) American Journal of Veterinary Research 40,1690-1693
Accepted April 19, 1986