Radiotherapy and Oncology, 22 (1991) 68-70 © 1991 Elsevier Science Publishers B.V. All rights reserved 0167-8140/91/$03.50
68 R A D I O N 00887
Short Communication
A s e m i - a u t o m a t e d m e t h o d for breathing rate m e a s u r e m e n t in the m o u s e s. P. Lockhart 1, D. Hill 2, Stuart King 2 and J. D. Down 1Radiotherapy Research Unit and 2Electronics Department, Institute of Cancer Research, Clifton Avenue, Belmont, Sutton, Surrey SM2 5PX, U.K. (Received 2 August 1989, revision received 21 June 1991, accepted 5 July 1991)
Key words: Semi-automated system; Breathing rate measurement; Lung damage
However, despite these advantages the measurement of breathing rate by whole plethysmography is a time-consuming method and an effort has been made to improve the speed at which the measurement can be performed.
Summary
Methods
A semi-automated system of breathing rate measurement for the assessment of lung damage in mice has been developed. Based on the plethysmographic method, a BBC B microcomputer plus Torch CPM examines four plethysmographs at any given time, looking for periods of quiet breathing and measuring the breathing rate during rest. This requires both machine code and Microsoft Basic programs, linked using Microsoft Link 80. The results correlated closely with results obtained using the well-validated measurement of chart recorder assessed breathing rate. Using the computer method, up to 40 animals could be examined per hour.
Pressure changes within the plethysmograph were detected by an electret condenser microphone (RS components). The signal was then passed through a DC pre-amplifier to a low pass filter (break frequency, fo, 9.5 Hz above which signals cut off at 12 dB/octave) and converted to a clipped output by a zero-crossing detector. For chart recorder measurements this was passed through a rate-meter whose output was recorded on a two-channel chart recorder (type BS-272, Bryans Southern Instruments Ltd., Mitcham, U.K.). This allowed two mice to be examined at one time. The record was visually examined for periods of quiet breathing, where the breathing rate could be read from the chart. Drift within the recorder required this to be calibrated against a 5 and 10 Hz signal at the beginning of each series of animals and resetting at the 5 Hz level between each mouse. For semi-automated recording, the clipped output was passed to a BBC B microcomputer (Acorn computers), with a Torch 780 Disc Pack (Torch Computers Ltd., Great Shelford, Cambridge) with an Epson RX-80 F/T + printer (Epson U K Ltd., Wembley). A program was developed using a machine code component and a Basic component linked using the Link 80 program (Microsoft). The machine code component repeatedly examined the four channels in rapid succession. It recorded the time of each breath in terms of the number of times it had examined the channel before a breath occurred. This time was recorded in the RAM in two bytes. The first breath was stored in A~o (low byte) and A~o + 1 (high byte). Breath x in channel y was stored in A~o + (2x - 2) + (y - 1) (low byte) and A~o + (2x - 1) + (y - 1) (high byte). In a 16-bit computer only one byte would need to be used. The Basic program called up this series of results. The times of the first six breaths were reconstituted (40-90 below) and the lengths of the first five interbreath intervals calculated (11-120). So that only a complete set of results was examined, the result was set at 0 if any result was unavailable (60,130). The values for the intervals were converted to rate in breaths per second (140). In order to ensure that results were only taken during quiet breathing, results were examined to see if each breath differs from the previous one
Introduction Changes in breathing rate measured by whole body plethysmography have become established as a marker for pulmonary damage to irradiation [7] or to drugs [8] in rodents. Much work on pulmonary radiation damage was initially performed using mortality at around 40 weeks as a measure of pulmonary damage, but this technique is of limited value after wider field irradiation where damage to other organs may lead to death, in interaction studies where the other agent may cause death independently of lung damage and also after irradiation of narrower fields such as hemithoracic fields when animals may not die despite developing lung damage in the irradiated lung [2]. In each of these circumstances, changes in breathing rate allow assessment of lung damage as damage to other tissues causes either no change or a fall in breathing rate [4] and breathing rate rises after hemithoracic irradiation to parallel radiological, histological and lung weight changes indicating lung damage [2]. Moreover, mortality as late as 40 weeks may include deaths from pleural effusions whereas changes in breathing rate up to about 20 weeks have been demonstrated to be due to pneumonitis [1]. Studies involving interactions may alter the rate at which lung damage develops [6] and so a serial functional assay is preferable to an invasive assay which can only be performed at one time point for each animal.
Address for correspondence: Dr. S. P. Lockhart, Clinical Development Group, Lederle Laboratories, Fareham Road, Gosport, Hants PO13 0AS, U.K.
69 by + 4 % . This value was e s t a b l i s h e d empirically as a greater tolerance led to erratic results being p r o d u c e d during periods o f animal m o v e m e n t a n d less tolerance led to no results being prod u c e d even w h e n a n i m a l s were clearly at rest. If t h e series of b r e a t h s was within this limit t h e n a result was given as a value to two decimal places (230, 270-280). If the limits were b r e a c h e d t h e n the p r o g r a m looped to e x a m i n e t h e next series o f six b r e a t h s (starting only one b r e a t h on from the previous e x a m i n a t i o n ) a n d c o n t i n u e d to ripple t h r o u g h the 64 b r e a t h s (240-250), after w h i c h if no result was available, the result w a s set at 0 (250-260). lO 20 30 40 50 60 70 80 90 100 1 I0 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
A% = 0 I=0:CHAN0=0:D=25 CALL MOUSE (A%) A1 = P E E K ( A % + C H A N 0 ) : A 2 = P E E K ( A % + ( C H A N 0 + 1 ) ) : I S T = A1 + ( A 2 , 2 5 6 ) A1 = P E E K ( A % + ( C H A N 0 + 2 ) ) : A 2 = P E E K ( A % + ( C H A N 0 + 3)): S C N 0 = AI + (A2 * 256) IF S C N D = 0 T H E N 260 A1 = P E E K ( A % + ( C H A N 0 + 4 ) ) : A 2 = P E E K ( A % + ( C H A N 0 + 5)) : T H I R D = A1 + (A2 * 256) A1 = P E E K ( A % + ( C H A N 0 + 6 ) ) : A 2 = P E E K ( A % + ( C H A N 0 + 7)) : F O U R T H = A1 + (A2 * 256) A1 = P E E K ( A % + ( C H A N 0 + 8 ) ) : A 2 = P E E K ( A % + ( C H A N 0 + 9)): F I F T H = A1 + (A2 * 256) A1 = P E E K ( A % + ( C H A N 0 + 1 0 ) ) : A 2 = P E E K ( A % + ( C H A N 0 + 11)): S I X T H = A1 + ( A 2 * 256) RES0 = SCND-IST : RES 1 = THIRD-SCND : RES2 = FOURTH-THIRD RES3 = F I F T H - F O U R T H : R E S 4 = S I X T H - F I F T H IF R E S 3 = 0 T H E N 260 E L S E IF R E S 4 = 0 T H E N 260 RES0 = RES0/880:RES1 = RES1/880:RES2 = RES2/880: RES3 = RES3/880 : R E S 4 = RES4/880 IF R E S 1 > R E S 0 + R E S 0 / D T H E N 240 IF R E S 1 < R E S 0 - R E S 0 / D T H E N 240 IF R E S 2 > RES1 + R E S 1 / D T H E N 240 IF R E S 2 < R E S I - R E S 1 / D T H E N 240 IF R E S 3 > R E S 2 + R E S 2 / D T H E N 240 IF R E S 3 < R E S 2 - R E S 2 / D T H E N 240 I F R E S 4 > R E S 3 + R E S 3 / D T H E N 240 IF R E S 4 < R E S 3 - R E S 3 / D T H E N 240 G O T O 270 I = I + 1 :CHAN0 = CHAN0 + 2 IF I < 64 T H E N 40 RES1 = 0 R E S = R E S 1 • 100 : R E S = I N T ( R E S ) : R E S = R E S / 1 0 0 P R I N T R E S ; " b r e a t h s per second".
second), the c o m p u t e r w a s p r o g r a m m e d to reject the result a n d ask for it to be repeated. To a s s e s s the possible effect o f n o n - n o r m a l i t y of results in each animal the m e a n a n d m e d i a n values were c o m p a r e d on 68 recordings on treated a n d u n t r e a t e d animals. T h e n u m b e r o f e a c h a n i m a l a n d cage n u m b e r were r e q u e s t e d at the start o f e a c h r u n a n d p r i n t e d out with results. T h e m e a n a n d s t a n d a r d error in each g r o u p w a s calculated at the e n d of e a c h session, as well as the n u m b e r of a n i m a l s in e a c h g r o u p exceeding the control group by 15, 20, 30 a n d 5 0 % , to allow d o s e - r e s p o n s e curves to be calculated [2]. Relative r e s p o n s e times were c o m p a r e d b e t w e e n p a p e r recorder a n d c o m p u t e r using artificial signals at 5 a n d 10 Hz. F o r this p u r p o s e , the p a p e r s p e e d w a s accelerated from the u s u a l 3 c m / m i n to 1 cm/s. In 66 a n i m a l s , including b o t h treated a n d u n t r e a t e d animals, t h e results by c h a r t recorder were correlated with the results by c o m p u t e r .
Results T h e r e s p o n s e time o f t h e c h a r t recorder w a s 4 s (equivalent to 20 b r e a t h s ) to r e a c h 9 5 % o f the true value from 0 to 5 H z , a n d 5.5 s (equivalent to 55 b r e a t h s ) from 0 to 10 Hz. It w a s t h e n n e c e s s a r y for the resting b r e a t h i n g to c o n t i n u e for at least 10 s (50 a n d 100 breaths, respectively) for a clear period o f quiet b r e a t h i n g to be a p p a r e n t over 0.5 c m on the trace with a p a p e r speed o f 3 c m / m i n . T h e c o m p u t e r s y s t e m registered a result after 5 resting b r e a t h s regardless o f the rate. This short period n e e d e d to be r e p e a t e d 7 times for a result to be p r o d u c e d . T h e m e d i a n a n d m e a n results for a given animal on the c o m p u t e r were very closely correlated (r = 0.995, m e d i a n = 0.985 × m e a n + 0.037) a n d so the m e a n w a s u s e d for each animal in the p r e s e n t a t i o n o f results. T h e c h a r t recorder a n d c o m p u t e r results were closely correlated (r = 0.978, c h a r t recorder result = 1.042 x c o m p u t e r result - 0.392, see Fig. 1).
10o/ /
-j
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0
\
O/
0
¢n ,c
/ /
8 8-
oo
,....
/
CV
08o I-..
O0 /
0
7 Q:
This c a n be m a d e to e x a m i n e four c h a n n e l s in s e q u e n c e by substituting lines 10-40 with the following: 10 20 30 31 32 33 34
A% = 0 I = 0 : C H A N 0 = 0 : C H A N 1 = 128 384 : D = 25 CALL MOUSE (A%) G O S U B 40 C H A N 0 = C H A N 1 :I = 0 : G O S U B CHAN0 = CHAN2"I = 0:GOSUB CHAN0 = CHAN3:I = 0:GOSUB
6
o
: C H A N 2 = 256 : C H A N 3 =
(:3 (j
~('o
o
o %,
Q. /
5
o
o
/
40 40 40
T h e p r o g r a m w a s t h e n e x t e n d e d to repeat this operation 20 times for e a c h channel. If there were less t h a n seven n o n - z e r o results, or if the scatter was too wide (confidence interval > 0.75 b r e a t h s per
/
/,
/.
o /
[ 5
I
I
I
6 7 8 CHART RECORDER RESULTS ( breaths/sec )
I
I
9
10
Fig. 1. The correlation between breathing rate measured by the chart recorder compared to measurement by computer. The dashed line is the best fit.
70 The breathing rates of about 40 animals could be recorded in an hour using this system.
Discussion This semi-automated method of breathing rate measurement allows rapid asessment of functional damage to the lungs of mice, which will be of value in the assessment of radiation [7] and drug damage [8], and particularly in the study of drug irradiation interactions [6] where lethality may be due to a number of factors. Breathing rate measurement allows toxicity to other organs to be distinguished from that due to lung damage [4]. In combination treatment the time course of the response to irradiation may be altered (for example cyclophosphamide accelerates and BCNU delays pneu-
monitis [5]) it is therefore important to serially assess breathing rate rather than to measure it at a fixed time. Caution should be taken not to rely solely upon breathing rate measurement as a rise in breathing rate at a late stage, from about 24 weeks onward after irradiation alone in CBA mice, may be due to pleural effusions rather than pneumonitis [3]. As this system allows 40 mice to be examined it means that large numbers of animals can be examined in this way. This system also has the advantage that it is objective, and that data can be analysed without further data entry. In conclusion, this method allows rapid objective measurement of breathing rate in mice. This will assist the identification of potentially important drug-radiation interactions in the mouse lung, as well as facilitating the study of both radiation and drug toxicity in the lung.
References 1 Down, J.D. The nature and relevance of late lung pathology following localised irradiation of the thorax in mice and rats Br. J. Cancer 53, Suppl VIII: 330-332, 1986. 2 Down, J.D., Nicholas, D. and Steel, G.G. Lung damage after hemithoracic irradiation: dependence on mouse strain. Radiother. Oncol. 6: 43-50, 1986. 3 Down, J. D. and Tarbell, N.J. Pitfalls in the assessment of late lung damage in irradiated mice: complications related to pleural effusion. Int. J. Radiat. Biol. 55: 473-478, 1989. 4 Hakkinen, P.J., Frankel, R., Morse, C. and Witschi, H. Effect of lung, liver, and kidney toxicants on respiratory rate in the mouse. Toxicology 26: 181-192, 1983.
5 Lockhart, S.P. The pharmacological modification of lung damage. DM Thesis, University of Oxford, 1988. 6 Lockhart, S. P., Down, J. D. and Steel, G.G. The effect of low dose-rate and cyclophosphamide on the radiation tolerance of the mouse lung. Int. J. Radiat. Oncol. Biol. Phys. 12: 1437-1440, 1986. 7 Travis, E. L., Vojnovic, B., Davies, E. E. and Hirst, D.G. A plethysmographic method for measuring function in locally irradiated mouse lung. Br. J. Radiol. 52: 67-74, 1979. 8 Travis, E. L., Brightwell, D., Aiken, M. and Boyd, M.R. Whole body plethysmography as a noninvasive assay of toxic lung injury in mice: studies with the pulmonary alkylating agent and cytotoxin, 4-ipomeanol. Toxicol. Appl. Pharmacol. 66: 193-200, 1982.
68 R A D I O N 00887
Short Communication
A s e m i - a u t o m a t e d m e t h o d for breathing rate m e a s u r e m e n t in the m o u s e s. P. Lockhart 1, D. Hill 2, Stuart King 2 and J. D. Down 1Radiotherapy Research Unit and 2Electronics Department, Institute of Cancer Research, Clifton Avenue, Belmont, Sutton, Surrey SM2 5PX, U.K. (Received 2 August 1989, revision received 21 June 1991, accepted 5 July 1991)
Key words: Semi-automated system; Breathing rate measurement; Lung damage
However, despite these advantages the measurement of breathing rate by whole plethysmography is a time-consuming method and an effort has been made to improve the speed at which the measurement can be performed.
Summary
Methods
A semi-automated system of breathing rate measurement for the assessment of lung damage in mice has been developed. Based on the plethysmographic method, a BBC B microcomputer plus Torch CPM examines four plethysmographs at any given time, looking for periods of quiet breathing and measuring the breathing rate during rest. This requires both machine code and Microsoft Basic programs, linked using Microsoft Link 80. The results correlated closely with results obtained using the well-validated measurement of chart recorder assessed breathing rate. Using the computer method, up to 40 animals could be examined per hour.
Pressure changes within the plethysmograph were detected by an electret condenser microphone (RS components). The signal was then passed through a DC pre-amplifier to a low pass filter (break frequency, fo, 9.5 Hz above which signals cut off at 12 dB/octave) and converted to a clipped output by a zero-crossing detector. For chart recorder measurements this was passed through a rate-meter whose output was recorded on a two-channel chart recorder (type BS-272, Bryans Southern Instruments Ltd., Mitcham, U.K.). This allowed two mice to be examined at one time. The record was visually examined for periods of quiet breathing, where the breathing rate could be read from the chart. Drift within the recorder required this to be calibrated against a 5 and 10 Hz signal at the beginning of each series of animals and resetting at the 5 Hz level between each mouse. For semi-automated recording, the clipped output was passed to a BBC B microcomputer (Acorn computers), with a Torch 780 Disc Pack (Torch Computers Ltd., Great Shelford, Cambridge) with an Epson RX-80 F/T + printer (Epson U K Ltd., Wembley). A program was developed using a machine code component and a Basic component linked using the Link 80 program (Microsoft). The machine code component repeatedly examined the four channels in rapid succession. It recorded the time of each breath in terms of the number of times it had examined the channel before a breath occurred. This time was recorded in the RAM in two bytes. The first breath was stored in A~o (low byte) and A~o + 1 (high byte). Breath x in channel y was stored in A~o + (2x - 2) + (y - 1) (low byte) and A~o + (2x - 1) + (y - 1) (high byte). In a 16-bit computer only one byte would need to be used. The Basic program called up this series of results. The times of the first six breaths were reconstituted (40-90 below) and the lengths of the first five interbreath intervals calculated (11-120). So that only a complete set of results was examined, the result was set at 0 if any result was unavailable (60,130). The values for the intervals were converted to rate in breaths per second (140). In order to ensure that results were only taken during quiet breathing, results were examined to see if each breath differs from the previous one
Introduction Changes in breathing rate measured by whole body plethysmography have become established as a marker for pulmonary damage to irradiation [7] or to drugs [8] in rodents. Much work on pulmonary radiation damage was initially performed using mortality at around 40 weeks as a measure of pulmonary damage, but this technique is of limited value after wider field irradiation where damage to other organs may lead to death, in interaction studies where the other agent may cause death independently of lung damage and also after irradiation of narrower fields such as hemithoracic fields when animals may not die despite developing lung damage in the irradiated lung [2]. In each of these circumstances, changes in breathing rate allow assessment of lung damage as damage to other tissues causes either no change or a fall in breathing rate [4] and breathing rate rises after hemithoracic irradiation to parallel radiological, histological and lung weight changes indicating lung damage [2]. Moreover, mortality as late as 40 weeks may include deaths from pleural effusions whereas changes in breathing rate up to about 20 weeks have been demonstrated to be due to pneumonitis [1]. Studies involving interactions may alter the rate at which lung damage develops [6] and so a serial functional assay is preferable to an invasive assay which can only be performed at one time point for each animal.
Address for correspondence: Dr. S. P. Lockhart, Clinical Development Group, Lederle Laboratories, Fareham Road, Gosport, Hants PO13 0AS, U.K.
69 by + 4 % . This value was e s t a b l i s h e d empirically as a greater tolerance led to erratic results being p r o d u c e d during periods o f animal m o v e m e n t a n d less tolerance led to no results being prod u c e d even w h e n a n i m a l s were clearly at rest. If t h e series of b r e a t h s was within this limit t h e n a result was given as a value to two decimal places (230, 270-280). If the limits were b r e a c h e d t h e n the p r o g r a m looped to e x a m i n e t h e next series o f six b r e a t h s (starting only one b r e a t h on from the previous e x a m i n a t i o n ) a n d c o n t i n u e d to ripple t h r o u g h the 64 b r e a t h s (240-250), after w h i c h if no result was available, the result w a s set at 0 (250-260). lO 20 30 40 50 60 70 80 90 100 1 I0 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
A% = 0 I=0:CHAN0=0:D=25 CALL MOUSE (A%) A1 = P E E K ( A % + C H A N 0 ) : A 2 = P E E K ( A % + ( C H A N 0 + 1 ) ) : I S T = A1 + ( A 2 , 2 5 6 ) A1 = P E E K ( A % + ( C H A N 0 + 2 ) ) : A 2 = P E E K ( A % + ( C H A N 0 + 3)): S C N 0 = AI + (A2 * 256) IF S C N D = 0 T H E N 260 A1 = P E E K ( A % + ( C H A N 0 + 4 ) ) : A 2 = P E E K ( A % + ( C H A N 0 + 5)) : T H I R D = A1 + (A2 * 256) A1 = P E E K ( A % + ( C H A N 0 + 6 ) ) : A 2 = P E E K ( A % + ( C H A N 0 + 7)) : F O U R T H = A1 + (A2 * 256) A1 = P E E K ( A % + ( C H A N 0 + 8 ) ) : A 2 = P E E K ( A % + ( C H A N 0 + 9)): F I F T H = A1 + (A2 * 256) A1 = P E E K ( A % + ( C H A N 0 + 1 0 ) ) : A 2 = P E E K ( A % + ( C H A N 0 + 11)): S I X T H = A1 + ( A 2 * 256) RES0 = SCND-IST : RES 1 = THIRD-SCND : RES2 = FOURTH-THIRD RES3 = F I F T H - F O U R T H : R E S 4 = S I X T H - F I F T H IF R E S 3 = 0 T H E N 260 E L S E IF R E S 4 = 0 T H E N 260 RES0 = RES0/880:RES1 = RES1/880:RES2 = RES2/880: RES3 = RES3/880 : R E S 4 = RES4/880 IF R E S 1 > R E S 0 + R E S 0 / D T H E N 240 IF R E S 1 < R E S 0 - R E S 0 / D T H E N 240 IF R E S 2 > RES1 + R E S 1 / D T H E N 240 IF R E S 2 < R E S I - R E S 1 / D T H E N 240 IF R E S 3 > R E S 2 + R E S 2 / D T H E N 240 IF R E S 3 < R E S 2 - R E S 2 / D T H E N 240 I F R E S 4 > R E S 3 + R E S 3 / D T H E N 240 IF R E S 4 < R E S 3 - R E S 3 / D T H E N 240 G O T O 270 I = I + 1 :CHAN0 = CHAN0 + 2 IF I < 64 T H E N 40 RES1 = 0 R E S = R E S 1 • 100 : R E S = I N T ( R E S ) : R E S = R E S / 1 0 0 P R I N T R E S ; " b r e a t h s per second".
second), the c o m p u t e r w a s p r o g r a m m e d to reject the result a n d ask for it to be repeated. To a s s e s s the possible effect o f n o n - n o r m a l i t y of results in each animal the m e a n a n d m e d i a n values were c o m p a r e d on 68 recordings on treated a n d u n t r e a t e d animals. T h e n u m b e r o f e a c h a n i m a l a n d cage n u m b e r were r e q u e s t e d at the start o f e a c h r u n a n d p r i n t e d out with results. T h e m e a n a n d s t a n d a r d error in each g r o u p w a s calculated at the e n d of e a c h session, as well as the n u m b e r of a n i m a l s in e a c h g r o u p exceeding the control group by 15, 20, 30 a n d 5 0 % , to allow d o s e - r e s p o n s e curves to be calculated [2]. Relative r e s p o n s e times were c o m p a r e d b e t w e e n p a p e r recorder a n d c o m p u t e r using artificial signals at 5 a n d 10 Hz. F o r this p u r p o s e , the p a p e r s p e e d w a s accelerated from the u s u a l 3 c m / m i n to 1 cm/s. In 66 a n i m a l s , including b o t h treated a n d u n t r e a t e d animals, t h e results by c h a r t recorder were correlated with the results by c o m p u t e r .
Results T h e r e s p o n s e time o f t h e c h a r t recorder w a s 4 s (equivalent to 20 b r e a t h s ) to r e a c h 9 5 % o f the true value from 0 to 5 H z , a n d 5.5 s (equivalent to 55 b r e a t h s ) from 0 to 10 Hz. It w a s t h e n n e c e s s a r y for the resting b r e a t h i n g to c o n t i n u e for at least 10 s (50 a n d 100 breaths, respectively) for a clear period o f quiet b r e a t h i n g to be a p p a r e n t over 0.5 c m on the trace with a p a p e r speed o f 3 c m / m i n . T h e c o m p u t e r s y s t e m registered a result after 5 resting b r e a t h s regardless o f the rate. This short period n e e d e d to be r e p e a t e d 7 times for a result to be p r o d u c e d . T h e m e d i a n a n d m e a n results for a given animal on the c o m p u t e r were very closely correlated (r = 0.995, m e d i a n = 0.985 × m e a n + 0.037) a n d so the m e a n w a s u s e d for each animal in the p r e s e n t a t i o n o f results. T h e c h a r t recorder a n d c o m p u t e r results were closely correlated (r = 0.978, c h a r t recorder result = 1.042 x c o m p u t e r result - 0.392, see Fig. 1).
10o/ /
-j
/ 9 /
0
\
O/
0
¢n ,c
/ /
8 8-
oo
,....
/
CV
08o I-..
O0 /
0
7 Q:
This c a n be m a d e to e x a m i n e four c h a n n e l s in s e q u e n c e by substituting lines 10-40 with the following: 10 20 30 31 32 33 34
A% = 0 I = 0 : C H A N 0 = 0 : C H A N 1 = 128 384 : D = 25 CALL MOUSE (A%) G O S U B 40 C H A N 0 = C H A N 1 :I = 0 : G O S U B CHAN0 = CHAN2"I = 0:GOSUB CHAN0 = CHAN3:I = 0:GOSUB
6
o
: C H A N 2 = 256 : C H A N 3 =
(:3 (j
~('o
o
o %,
Q. /
5
o
o
/
40 40 40
T h e p r o g r a m w a s t h e n e x t e n d e d to repeat this operation 20 times for e a c h channel. If there were less t h a n seven n o n - z e r o results, or if the scatter was too wide (confidence interval > 0.75 b r e a t h s per
/
/,
/.
o /
[ 5
I
I
I
6 7 8 CHART RECORDER RESULTS ( breaths/sec )
I
I
9
10
Fig. 1. The correlation between breathing rate measured by the chart recorder compared to measurement by computer. The dashed line is the best fit.
70 The breathing rates of about 40 animals could be recorded in an hour using this system.
Discussion This semi-automated method of breathing rate measurement allows rapid asessment of functional damage to the lungs of mice, which will be of value in the assessment of radiation [7] and drug damage [8], and particularly in the study of drug irradiation interactions [6] where lethality may be due to a number of factors. Breathing rate measurement allows toxicity to other organs to be distinguished from that due to lung damage [4]. In combination treatment the time course of the response to irradiation may be altered (for example cyclophosphamide accelerates and BCNU delays pneu-
monitis [5]) it is therefore important to serially assess breathing rate rather than to measure it at a fixed time. Caution should be taken not to rely solely upon breathing rate measurement as a rise in breathing rate at a late stage, from about 24 weeks onward after irradiation alone in CBA mice, may be due to pleural effusions rather than pneumonitis [3]. As this system allows 40 mice to be examined it means that large numbers of animals can be examined in this way. This system also has the advantage that it is objective, and that data can be analysed without further data entry. In conclusion, this method allows rapid objective measurement of breathing rate in mice. This will assist the identification of potentially important drug-radiation interactions in the mouse lung, as well as facilitating the study of both radiation and drug toxicity in the lung.
References 1 Down, J.D. The nature and relevance of late lung pathology following localised irradiation of the thorax in mice and rats Br. J. Cancer 53, Suppl VIII: 330-332, 1986. 2 Down, J.D., Nicholas, D. and Steel, G.G. Lung damage after hemithoracic irradiation: dependence on mouse strain. Radiother. Oncol. 6: 43-50, 1986. 3 Down, J. D. and Tarbell, N.J. Pitfalls in the assessment of late lung damage in irradiated mice: complications related to pleural effusion. Int. J. Radiat. Biol. 55: 473-478, 1989. 4 Hakkinen, P.J., Frankel, R., Morse, C. and Witschi, H. Effect of lung, liver, and kidney toxicants on respiratory rate in the mouse. Toxicology 26: 181-192, 1983.
5 Lockhart, S.P. The pharmacological modification of lung damage. DM Thesis, University of Oxford, 1988. 6 Lockhart, S. P., Down, J. D. and Steel, G.G. The effect of low dose-rate and cyclophosphamide on the radiation tolerance of the mouse lung. Int. J. Radiat. Oncol. Biol. Phys. 12: 1437-1440, 1986. 7 Travis, E. L., Vojnovic, B., Davies, E. E. and Hirst, D.G. A plethysmographic method for measuring function in locally irradiated mouse lung. Br. J. Radiol. 52: 67-74, 1979. 8 Travis, E. L., Brightwell, D., Aiken, M. and Boyd, M.R. Whole body plethysmography as a noninvasive assay of toxic lung injury in mice: studies with the pulmonary alkylating agent and cytotoxin, 4-ipomeanol. Toxicol. Appl. Pharmacol. 66: 193-200, 1982.
