Naunyn-Schmiedeberg's Arch. Pharmaeol. 291, 385--394 (1975) 9 by Springer-Verlag 1975
Pupillometric Studies in the Beagle Dog* I. Weisse, H. Ganz, a n d F. K n a p p e n with the technical assistance of M. R o d e Abteilung fiir experimentelle Pathologie and Toxikologie C. H. Boehringer Solm, Ingelheim am Rhein Received April 24 / Accepted October 13, 1975 Summary. A method of measuring pupillary diameter in the dog using a Kowa RC-2 fundus camera, to which an eyepiece graticule had been fitted, is described. Pupil diameter was measured either directly by reading off from the calibrated gratieule or from photographs. The pupillary diameters of 105 (53~, 529) untreated English beagles aged between 8 and 32 months were measured under light intensities of 150--200 lux and 1000-- 1400 lux. Mydriatie and miotic pupillary diameters were measured in 62 male and 80 female dogs. Time-effect curves were plotted for two groups of 5 beagles receiving 75 ~g/kg or 175 [zg/kg atropine and 100 ~g/kg or 300 ~g/kg propantheline intravenously. The dose-effect curve for propantheline was found to be linear. A dose of 109 (97--121) ~g/kg propantheline increased pupillary diameter by 3 ram, 20 min after administration. The time- and dose-effect curves, the significant sex differences and age dependency seen in this study indicate both a high degree of accuracy and the suitability of both of these methods for use in pharmacological and toxicological studies. Key words: Dog -- Pupil -- Dose- and time-response relationship -- Atropine -Propantheline. I n clinical practise pupillary measurement is carried out either to determine static pupillary diameter, or to analyse pupillary motility. I n h u m a n o p h t h a l m o l o g y the m o s t elaborate equipment is used to continuously register pupil m o v e m e n t b y c i n e m a t o g r a p h y (Lowenstein a n d Friedman, 1942) a n d photo-electric pupillography (Ctippers, 1954). Pupillometrie techniques for the measurement of static pupillary diameter are c o m m o n l y less complicated. H a a b ' s scale (1903) or the hole pupillometer allow the observer to read off pupillary diameter according to the marks or holes on the scale. Kasatskij's pupillometer (1956), allows the pupillary diameter to be read f r o m a triangular aperture when two sides of the triangle have been b r o u g h t to bear tangentially on the pupillary circumference. Despite the considerable margin of error inSend o]]print requests to: I. Weisse, Abteilung ftir experimentelle Pathologie und Toxikologie, C.H. Boehringer Sohn, D-6507 Ingelheim am Rhein, Federal Republic of Germany. * Presented in part at the 16th Spring Meeting of the German Pharmacological Society in Mainz, March 4--7, 1975. 26*
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v o l v e d these t e c h n i q u e are r e g u l a r l y e m p l o y e d in t h e i n v e s t i g a t i o n o f anisocoria in m a n b o t h before a n d a f t e r a p p l i c a t i o n of c o m p o u n d s affecting t h e p u p i l (Schrader, 1970). P h o t o g r a p h i c techniques a r e also u s e d in h u m a n p u p i l l o m e t r y . E l l i o t a n d W a y (1961), J a s i n s k i a n d M a r t i n (1967) a n d others h a v e d e s c r i b e d such techniques. N u m e r o u s c o m m u n i c a t i o n s h a v e a p p e a r e d on p u p i l l o m e t r i c studies in e x p e r i m e n t a l animals. T h e size of t h e p u p i l is a sensitive i n d i c a t o r o f t h e a c t i v i t y o f a c o m p o u n d on t h e a u t o n o m i c n e r v o u s system. I n this field, m e t h o d s a n d results h a v e b e e n p r e s e n t e d b y : P u l e w k a (1932), N e m e c et al. (1969) (mouse); Brodie (1966), Weisse et al. (1971) (rat); W e r n e r (1965), B u r n s et al. (1970) ( r a b b i t ) ; H e u b n e r (1926), U e k i a n d Oono (1963) (eat) ; a n d T S r n q v i s t (1969), L e a d e r s a n d F o r t e n b e r r y (1971) (monkey). H a a b ' s scale was f o u n d to be u n s u i t a b l e for d e t e r m i n i n g changes in p u p i l l a r y d i a m e t e r in l o n g - t e r m toxicological studies in dogs for t w o r e a s o n s : firstly t h e scope o f t h e scale was n o t wide e n o u g h a t m a x i m u m myclriasis a n d s e c o n d l y t h e t e m p e r a m e n t o f t h e i n d i v i d u a l a n i m a l s d e t e r m i n e s t h e a c c u r a c y of t h e values o b t a i n e d . The a n t e r i o r p a r t s o f t h e b u l b u s c a n be m e a s u r e d w i t h a high degree of a c c u r a c y w i t h a K o w a 1~C-2 f u n d u s camera. W e therefore a d a p t e d this c a m e r a s l i g h t l y for p u p i l l o m e t r i e p u r p o s e s in t h e dog.
Materials and Methods a) Animals. The animals used were 217 (101 ~, 116 9) clinically healthy English beagles, aged 8--32 months. They were housed singly in kennels with covered runs and having an ambient temperature of at least 15~ The mean light intensity inside the kennels lay between 150 and 200 lux and in the covered runs between 1000 and 1400 lux. The dogs received 500 g food (type 42H, Zahn, Hockenhelm) per day and water ad libitum. b) Measurement o] the Static Pupillary Diameter. A standard Kowa 1~C-2 fundns camera (Kowa Co. Ltd., Tokyo), with a calibrated hair-crossplate graticule incorporated into the eyepiece, was used 1. The camera was fixed to the laboratory bench at eye-level for the seated observer. Height and angle of the camera were adjustable. A constant distance between lens and pupil was ensured by setting the diopter scale at -- 20 diopters. Focus was thus 65.6 mm in front of the lens (normal focal length 41.82 ram). The depth of focus was 64--67 ram. The margin of error arising therefrom is • 0.07 ram/1 mm pupillary diameter. These limits of depth of focus and margin of error are tolerable, and were determined from a sliding millimeter grid in front of the lens. The graduated units on the graticule were calibrated using the same method. The following two procedures proved to be the most practical: The maximum dilation and constriction of the pupil were determined using the graduated graticule. Two halogen lamps (Stoff, Lemgo, 250 watt) were focussed on the eye from a distance of 80 cm. measurements were taken at 3000--6000 lux. The built in observation light was not used. 1 Our thanks are due to Zeiss, Oberkochen, for its kind cooperation.
Pupillometric Studies in the Beagle Dog
387
Time effect and dose-effect curves were obtained by photographing the pupils on a diffused light intensity of 1000--1400 lux (flash intensity 5, Ilford 18 DIN). The pupillary diameters were measured on the developed negative. The first eye photographed alternated between the right and left, and an interval of at least 90 sec was allowed between taking the first and second eyes. The same photographic method was used to determine pupillary diameter of the animals housed in the kennels (150--200 lux) and those in the covered runs (1000--1400 lux). The relevant light intensities were provided by two infinitely variable halogen spotlamps (Staff, Lemgo, 250 watt) and a broad-beam unit (Novalux, Cologne, 500 watt). The camera observation lamp was not used. The three light units were arranged to provide uniform illumination of a white wail. The thus diffused light was then reflected into the eye of the test animal. The light intensity within the focal limits of the camera was repeatedly checked with a standard light meter (Gossen, Erlangen). I n both cases the papillary diameter was read in the horizontal plane and the readings were converted to millimeters. ~) Studies. The pupillary diameters of the control animals were determined at the various light intensities by simulating the light conditions in the kennels and covered runs with the above-mentioned lamps (150--200 lux and 1000--1400 lax) and allowing 30 min for adaptation before each measurement. 53 male and 52 female animals were used. Mydriasis was measured in 62 male and 80 female beagles after local application to both eyes of 2--3 drops of Mydriaticum Roche | (5 mg tropic acid-N-ethyl-N(y-picolyl)-amide and 0.01 mg phenylmercurie nitrate in 1 ml aqueous solution, Hoffmann La-Roche, Grenzach/Baden). Miosis was measured on 47male and 50female animals after similar treatment with Miotic ~ (0.1 g carbamylcholine chloride and 0.3 g dimethyleorbamoxitrimethyl ammonium methyl sulfate per 10 ml in isotonic autosterile solution with boric acid and a wetting agent, Mann, Berlin). Measurements were taken 30--60 rain after administration of the appropriate preparations at a light intensity of 3000--6000 lax. Time-effect curves were plotted for two groups each of 5 beagles initially treated with either 75 ttg/kg atropine or 100 ~g/kg propantheline, and subsequently with 175 ~g/kg atropine or 300 ~g/kg propantheline. The compounds were made up in aqueous solution (0.1 ml/kg body weight) and administered via the cephalic vein. Pupillary diameters were photographically determined prior to administration and subsequently at 5, 10, 15, 20, 30, 40, 50, and 60 min, and 2, 4, 6, 8 and 24 hrs. I n a study using propantheline intravenously at 35, 70, 105, 140, 175, 2t0, 280 and 350 ~g/kg in beagles, the same 10 animals were used for each dosage level and a dose-effect curve was plotted. A period of 1 week was allowed between dosage application. Pupillary diameters were determined prior to administration and 20 min thereafter. The procedure was based on a Youden design with randomised doselevels. d) Biometric Evaluation. The individual samples are designed as the arithmetical mean value (~), standard deviation (s) and sample size (n). The values for the left and right eyes were compared by the t-test for pair differences and t h e / - t e s t for independent samples was used to evaluate sex differences. The statistically significant limits are indicated together with the results. The time-effect curves were determined mathematically by an iterative process for non-linear regression analysis. Of the biomathematical models tested, the following was preferred because of its least residual sum of the squares: Y=Pl~-p2texp(pat)+pacosb+pssinb+p6cos2b+p~sin2b where: t ~ time after administration (h) b = 2 ~/24t-~
388
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This model is a modified and extended version of those used in glucose tolerance tests (Brown et al., 1969). Polynomials are less aptly able to describe time-effect relationships of this kind (Keller, 1969). Analysis of the effects of age-dependency on pupillary diameter and the determination of the dose-effect relationship were effected by fitting polynomials to the values obtained in the studies by applying the method of least squares. The polynomials were successively degraded. The contributions of the individual degradation steps were tested for significance by analysis of variance. P-values less than 0.05 were regarded as statistically significant. Linearity was demonstrated and the method described by l~inney (1971) was used te determine the ED (3 mm dilation) and the relevant confidence interval for P = 0.95.
Results T a b l e 1 shows the d a t a o b t a i n e d from the controls u n d e r the various
light intensities. The average value for the left eye of the females in 150--200 lux group was significantly higher than that for the left of the males (P < 0.05). A sex difference is thus to be found for mean values of both eyes (P < 0.05). I~o statistically significant dependency was seen (P > 0.05).
the eye the age
T a b l e 2 shows t h e d a t a o n t h e m y d r i a t i e a n d miotie pupils. No
statistically significant sex differences and age dependency could be detected in the latter group. However, the pupillary diameters in the males in the mydriatic group were significantly larger than those in the females (P < 0.05). Moreover, statistically significant positive relationships were obtained for this group in respect of age and pupillary diameter (r = 0.30 for males, r ---- 0.34 for females). A linear relationship for age dependency Table 1. Pupillary diameter (ram) of untreated dogs in various light intensities; mean value @), standard deviation (s), sample size (n) and level of significance (t-test, two-tailed) Sex
150--200 lux
Male s n Famale 8
Comparison male vs. female
1000-- 1400 lux
Right eye Left eye
Mean of Right eye Left eye Mean of both eyes both eyes
9.38 1.08 53
9.45 1.26 53
9.41 1.04 53
7.51 1.28 53
9.76 1.50 52
10.02 1.47 52
9.89 1.34 52
7.45 1.50 51
P>0.05
P 0.05 < 0.10
389
Pupillometrie Studies in the Beagle Dog
Table 2. Pupillary diameter (mm) after local application of Mydriaticum t~oche| or Miotic| mean value (2), standard deviation (s), sample size (n) and level of significance (t-test, two tailed) Sex
Mydriatic pupils
Miotic pupils
Right eye Left eye
Mean of l~ight eye Left eye Mean of both eyes both eyes
Male
2 s n
11.03 0.87 62
11.02 0.85 62
11.02 0.83 62
1.02 0.32 47
1.02 0.31 46
1.02 0.32 45
Female
2 s n
1.0.68 0.83 80
10.69 0.84 80
10.69 0.82 80
1.02 0.43 49
1.00 0.40 50
1.01 0.41 49
P
Comparison male vs. female
0.05
mm
12.6 12.4 12.2 12,0 11.8 11.6 11,411.2 11.0t
10,81 IO.S1 10.41 10,2J
10.0"
i
8
|
i
,
i
,
|
i
,
i
,
,
,
,
,
|
,
i
,
,
,
~
9
,
,
9 10 11 12 13 14 15 1G 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Age (months) Fig. 1. Age-dependency of mydriatie pupil diameters in Beagle dogs, treated with Mydriatieum Roche| Calculated regression lines (straight lines) and confidence belts for P -= 0.95 (hyperbolas)
was f o u n d i n b o t h sexes (P < 0.05 for males, P < 0.01 for females). The regression lines r u n parallel. There is a significant sex difference i n t h e distance b e t w e e n these parallel lines (Fig. 1, P < 0.01).
390
I. Weisse et al.
Table 3. Right-left differences of pupillary diameters (ram) of dogs; mean value (~), standard deviation (s), sample size (n) and level of significance (paired t-test, twotailed) Treatment
Lux
Sex
$
8
n
Level significance
Untreated Untreated Untreated Untreated l~ydriatie ~ydriatie Miotic Miotic
150-- 200 150-- 200 1000--1400 1000--1400 3000--6000 3000--6000 3000--6000 3000--6000
c~ ~ c~ ~ c~ ~ c~ ~
--+ + + --
1.082 1.286 1.174 1.160 0.434 0.270 0.074 0.176
53 52 53 51 62 80 45 49
P P P P P P P P
0.068 0.258 0.029 0.447 0.016 0.012 0.000 + 0.015
> > > < > > > >
...... E
8
0.05 0.05 0.05 0.01 0.05 0.05 0.05 0.05
7 5 j J g / k 9 i.v.
175 ~ 9 1 k 9 i.v.
7 E o "o
Q.
6 5
3 2
E o
I 0 0
Time (hours)
Fig. 2. Calculated time-effect curves for atropine on pupil diameter. The standard deviations about the regression are 0.39 and 1.13for 75 ~g/kg and 175 ~g/kg respectively
T a b l e 3 shows the results of the right-left comparison for b o t h the t r e a t e d a n i m a l s a n d the controls o n the basis of the various light i n t e n sities. O n l y the values for the u n t r e a t e d females i n the 1000--1400 l u x group showed a highly significant right-left difference (P < 0.01). The differences i n p u p i l l a r y d i a m e t e r i n the other groups were w i t h i n the r a n d o m d e v i a t i o n (P > 0.05.) The time-effect curves for the dosage levels of a t r o p i n e a n d prop a n t h e l i n e are shown i n Figs.2 a n d 3. The curves are based on the m e a n values for b o t h eyes of 5 a n i m a l s i n each case. The m a x i m u m
Pupillometrie Studies in the Beagle Dog
? E
391
8 7
....
I O O p g / k g i.v.
- -
3 0 0 p g / k g i.v.
6 E n~
5 /.
D
3 I
2
g o
! 0
;
i
i
i Time
;
i
~
;
(hours}
Fig.3. Calculated time-effect curves for propantheline on pupil diameter. The standard deviations about the regression are 0.36 and 0.6[ for 100 [~g/kg and
300 ~zg/kgrespectively
pupillary diameters were reached after the following time intervals (mean and extreme values) : atropine
75 ~zg/kg -= 12.1 (8.4--15.5) rain 175 Ezg/kg = 14.5 (8.5--22.6) rain
propantheline
100 tzg/kg = 17.0 (6.0--28.1) rain 300 ~zg/kg = 16.2 (10.1--21.3) min.
The dose-effect relationship for propantheline, given in Fig.4, was determined from the data obtained during the maximum mydriatie effect, about 20 rain after administration. The curve was found to be linear in a semilogarithmic plot for all dosages (P < 0.001). The lowest dose tested (35 ~g/kg) did not result in a significant change in pupillary diameter (P > 0.05). Characteristic of the highest dose (350 ~g/kg) was the fact that the anatomic maximum pupillary diameter was achieved. 109 (97--121) ~zg/kg propantheline increased the pupillary diameter by 3 ram. Discussion To our knowledge, apart from the statement by Magrane (1965) that the dog's pupil is larger than that in man, there has been no information published in recent years on pupillary size in the dog. The present study thus had as its object the development of a reliable technique for the repeated measurement of the static pupillary diameter in the conscious dog.
392
I. Wcisse
etal.
6.0
,//,~1/
6.5
II [l/~
5,0 4.5.
//
/,.0, E
3,5 3.0, o_ "10 >, 0
2.5. 2.0.
Q.
/ / ',,"/,}" //
1,5. 1,0.
& c o=
0.5-
(.3
O, -0,5 -
I
.(X,/ j"
i/I
//
-
1,0
-1..5
/ 3's/
50
"I
10,0
7b
losj,
150
200
1,o r)s 21o
300 280 350
lO8.g
Oose
~g/kg i.v.
Fig.4. Calculated dose-response curve for propantheline on pupil diameter with confidence belt for P = 0.95
The incorporation of the calibrated graticule in the camera was only considered usefut in the determination of the diameters of mydriatie and miotie pupils. Photographs were preferable for measuring the pupil at varying light intensities for the time-effect and dose-effect curves. At the 150--200 lux range it was possible to focus the iris but not to assess the diameter of the pupil precisely. Physiological pupillary excitation also made it difficult to read the calibrations on the graticule accurately. With the exception of the untreated females in the 1000--1400 lux group, the right/left differences in pupillary diameter which were seen in these photographic determinations lay within the limits imposed b y the technique and the biological variation. This exception is probably a result of the fact t h a t while the lighting was as diffuse as possible, the test animals were not constantly looking at the illuminated wall. From
Pupillometric Studies in the Beagle Dog
393
this it is clear that variations in light intensity and direct light reactions of the pupil were inevitably included in the measurements. Consensual pupillary reaction, which might have seemed to be a point for consideration particularly in the females in the 1000--1400 lux group, cannot be held responsible for the variation in pupillary diameter since the measurements were made on alternate eyes. Apart from this the interval between the two photographs was always long enough to allow the brightness reflex triggered b y the flash to return to normal. Evidence of the sensitivity of the two methods used is seen firstly in the detection of a sex difference by the calibrated graticule method. Secondly the time-effect curves for propantheline and atropine, and the linearity of the propantheline dose-effect relationships show that the method of photographic registration of pupillary diameter is capable of detecting variations resulting from the administration of drugs affecting pupillary size. I n view of the precision of the data obtained and the ease with which the fundus camera can be used, this technique is not only well suited for pharmacological studies but also for repeated observations on conscious dogs in long term toxicological studies. Reliable results can however only by expected if the animals are not excited and the observations are carried out under evenly diffused illumination.
References Brodie, D. A. : A comparison of anticholinergic drugs on gastric secretion, gastric emptying, and pupil diameter in the rat. Gastroenterology 50, 45--50 (1966) Brown, G., Quarrington, B., Stanler, H. C. : The mathematical analysis of glucose tolerance curves obtained by in vivo antoanalysis. Comp. Biomed. Res. 2, 558--567 (1969) Burns, J., ttorlington, M., Robson, D. : Positive correlation between initial pupil diameter and amplitude of miotic to piloearpine in rabbits and to carbachol in man. Brit. J. Pharmaeol. 40, 524--525 (1970) Clippers, C. : Die fortlaufende Registrierung der direkten und der konsensuellen Pupillenreaktion. Albrecht v. Graefes Arch. Ophthal. 155, 588--616 (1954) Elliot, H. W., Way, E. L. : Effect of narcotic antagonists on the pupil diameter of nonaddiets. Clin. Pharmaeol. Ther. 2, 713--721 (1961) Finney, D. J. : Statistical Method in Biological Assay. 2nd ed., 2nd impr. London: Charles Griffin & Co., Ltd. 1971 Haab, 0. : Der I-Iirnrindenreflex der Pupille. Arch. Augenheilk. 46, 1--29 (1903) Heubner, W.: Quantitative Bestimmnng yon Atropin neben Papaverin in einer Arznei. Klin. Wschr. 84, 1571 (1926) Jasinski, D. R., Martin, W. R.: Evaluation of a new photographic method for assessing pupil diameters. Clin. Pharmaeol. Ther. 8, 271--272 (1967) Kasatskij, B. B.: Vestn. Oftal. 69, 91 (1956) Koller, S.: Diskussionsbemerkung zum Vortrag yon W. Kosswig: ,,Beitrag zur Analyse yon Zeitreihen", 16. Biometr. Coll. der Dtsch. Region der Internat. Biometr. Ges., 20.--22. Febr. 1969 in Bad Nauheim
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Leaders, F. E., Fortenberry, B . R . : The effects of dl-l-(4-hydroxyphenyl)-2-isopropylaminoethanol HC1 (A1 842) on intraocular pressure and pupil size of rhesus monkeys (35460). Prec. Soc. exp. Biol. (N. Y.) 186, 1211--1215 (1971) Lowenstein, O., Friedman, E. D.: Pupillographic studies: I. Present state of pupillography, its method and diagnostic significance. Arch. Ophthal. 27, 969--993 (1942) Magrane, W. G.: Canine 0phthalmology, p. 135. Philadelphia: Lea and Febiger 1965 Nemec, J., Hradil, F., Wildt~ S.: Pupillenweite der Albinomaus des H-Stammes. Z. Versuchstierk. 11, 312--319 (1969) Pulewka, P.: Das Auge der ~aus als pharmakologisches Testobjekt. NaunynSchmiedebergs Arch. exp. Path. Pharmak. 168, 307--318 (1932) Schrader, K. E.: Untersuchungsmethoden der normalen und pathologiseh ver~nderten Pupille, ihrer Reaktionen und ReaktionsstSrungen. In: Die ophthalmologischen Untersuchungsmethoden, L Bd., Hrsg.: W. Straub. Stuttgart: F. Enke 1970 T6rnqvist, G.: Effect of reserpine on accomodation and pupil diameter in monkeys (macaca irns). Acta physiol, scand. 75, 565--567 (1969) Ueki, S., Oono, S. : A new method for continous recording of the pupil size by means of a phototransistor. Arch. int. Pharmacodyn. 142, 339--348 (1963) Weisse, I., StStzer, H., Knappen, F., Walland, A.: The effect of Clonidine on the pupil diameter and the retina in rats, assessed in relation to the intensity of light. Arzneimittel-Forseh. (Drug Res.) 21, 821--825 (1971) Werner, G.: Fermentdefekte als Ursache untersehiedlicher Wirksamkeit yon Atropin und Cocain bei Kaninchen. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 251, 320--334 (1965)
Pupillometric Studies in the Beagle Dog* I. Weisse, H. Ganz, a n d F. K n a p p e n with the technical assistance of M. R o d e Abteilung fiir experimentelle Pathologie and Toxikologie C. H. Boehringer Solm, Ingelheim am Rhein Received April 24 / Accepted October 13, 1975 Summary. A method of measuring pupillary diameter in the dog using a Kowa RC-2 fundus camera, to which an eyepiece graticule had been fitted, is described. Pupil diameter was measured either directly by reading off from the calibrated gratieule or from photographs. The pupillary diameters of 105 (53~, 529) untreated English beagles aged between 8 and 32 months were measured under light intensities of 150--200 lux and 1000-- 1400 lux. Mydriatie and miotic pupillary diameters were measured in 62 male and 80 female dogs. Time-effect curves were plotted for two groups of 5 beagles receiving 75 ~g/kg or 175 [zg/kg atropine and 100 ~g/kg or 300 ~g/kg propantheline intravenously. The dose-effect curve for propantheline was found to be linear. A dose of 109 (97--121) ~g/kg propantheline increased pupillary diameter by 3 ram, 20 min after administration. The time- and dose-effect curves, the significant sex differences and age dependency seen in this study indicate both a high degree of accuracy and the suitability of both of these methods for use in pharmacological and toxicological studies. Key words: Dog -- Pupil -- Dose- and time-response relationship -- Atropine -Propantheline. I n clinical practise pupillary measurement is carried out either to determine static pupillary diameter, or to analyse pupillary motility. I n h u m a n o p h t h a l m o l o g y the m o s t elaborate equipment is used to continuously register pupil m o v e m e n t b y c i n e m a t o g r a p h y (Lowenstein a n d Friedman, 1942) a n d photo-electric pupillography (Ctippers, 1954). Pupillometrie techniques for the measurement of static pupillary diameter are c o m m o n l y less complicated. H a a b ' s scale (1903) or the hole pupillometer allow the observer to read off pupillary diameter according to the marks or holes on the scale. Kasatskij's pupillometer (1956), allows the pupillary diameter to be read f r o m a triangular aperture when two sides of the triangle have been b r o u g h t to bear tangentially on the pupillary circumference. Despite the considerable margin of error inSend o]]print requests to: I. Weisse, Abteilung ftir experimentelle Pathologie und Toxikologie, C.H. Boehringer Sohn, D-6507 Ingelheim am Rhein, Federal Republic of Germany. * Presented in part at the 16th Spring Meeting of the German Pharmacological Society in Mainz, March 4--7, 1975. 26*
386
L Weisse et al.
v o l v e d these t e c h n i q u e are r e g u l a r l y e m p l o y e d in t h e i n v e s t i g a t i o n o f anisocoria in m a n b o t h before a n d a f t e r a p p l i c a t i o n of c o m p o u n d s affecting t h e p u p i l (Schrader, 1970). P h o t o g r a p h i c techniques a r e also u s e d in h u m a n p u p i l l o m e t r y . E l l i o t a n d W a y (1961), J a s i n s k i a n d M a r t i n (1967) a n d others h a v e d e s c r i b e d such techniques. N u m e r o u s c o m m u n i c a t i o n s h a v e a p p e a r e d on p u p i l l o m e t r i c studies in e x p e r i m e n t a l animals. T h e size of t h e p u p i l is a sensitive i n d i c a t o r o f t h e a c t i v i t y o f a c o m p o u n d on t h e a u t o n o m i c n e r v o u s system. I n this field, m e t h o d s a n d results h a v e b e e n p r e s e n t e d b y : P u l e w k a (1932), N e m e c et al. (1969) (mouse); Brodie (1966), Weisse et al. (1971) (rat); W e r n e r (1965), B u r n s et al. (1970) ( r a b b i t ) ; H e u b n e r (1926), U e k i a n d Oono (1963) (eat) ; a n d T S r n q v i s t (1969), L e a d e r s a n d F o r t e n b e r r y (1971) (monkey). H a a b ' s scale was f o u n d to be u n s u i t a b l e for d e t e r m i n i n g changes in p u p i l l a r y d i a m e t e r in l o n g - t e r m toxicological studies in dogs for t w o r e a s o n s : firstly t h e scope o f t h e scale was n o t wide e n o u g h a t m a x i m u m myclriasis a n d s e c o n d l y t h e t e m p e r a m e n t o f t h e i n d i v i d u a l a n i m a l s d e t e r m i n e s t h e a c c u r a c y of t h e values o b t a i n e d . The a n t e r i o r p a r t s o f t h e b u l b u s c a n be m e a s u r e d w i t h a high degree of a c c u r a c y w i t h a K o w a 1~C-2 f u n d u s camera. W e therefore a d a p t e d this c a m e r a s l i g h t l y for p u p i l l o m e t r i e p u r p o s e s in t h e dog.
Materials and Methods a) Animals. The animals used were 217 (101 ~, 116 9) clinically healthy English beagles, aged 8--32 months. They were housed singly in kennels with covered runs and having an ambient temperature of at least 15~ The mean light intensity inside the kennels lay between 150 and 200 lux and in the covered runs between 1000 and 1400 lux. The dogs received 500 g food (type 42H, Zahn, Hockenhelm) per day and water ad libitum. b) Measurement o] the Static Pupillary Diameter. A standard Kowa 1~C-2 fundns camera (Kowa Co. Ltd., Tokyo), with a calibrated hair-crossplate graticule incorporated into the eyepiece, was used 1. The camera was fixed to the laboratory bench at eye-level for the seated observer. Height and angle of the camera were adjustable. A constant distance between lens and pupil was ensured by setting the diopter scale at -- 20 diopters. Focus was thus 65.6 mm in front of the lens (normal focal length 41.82 ram). The depth of focus was 64--67 ram. The margin of error arising therefrom is • 0.07 ram/1 mm pupillary diameter. These limits of depth of focus and margin of error are tolerable, and were determined from a sliding millimeter grid in front of the lens. The graduated units on the graticule were calibrated using the same method. The following two procedures proved to be the most practical: The maximum dilation and constriction of the pupil were determined using the graduated graticule. Two halogen lamps (Stoff, Lemgo, 250 watt) were focussed on the eye from a distance of 80 cm. measurements were taken at 3000--6000 lux. The built in observation light was not used. 1 Our thanks are due to Zeiss, Oberkochen, for its kind cooperation.
Pupillometric Studies in the Beagle Dog
387
Time effect and dose-effect curves were obtained by photographing the pupils on a diffused light intensity of 1000--1400 lux (flash intensity 5, Ilford 18 DIN). The pupillary diameters were measured on the developed negative. The first eye photographed alternated between the right and left, and an interval of at least 90 sec was allowed between taking the first and second eyes. The same photographic method was used to determine pupillary diameter of the animals housed in the kennels (150--200 lux) and those in the covered runs (1000--1400 lux). The relevant light intensities were provided by two infinitely variable halogen spotlamps (Staff, Lemgo, 250 watt) and a broad-beam unit (Novalux, Cologne, 500 watt). The camera observation lamp was not used. The three light units were arranged to provide uniform illumination of a white wail. The thus diffused light was then reflected into the eye of the test animal. The light intensity within the focal limits of the camera was repeatedly checked with a standard light meter (Gossen, Erlangen). I n both cases the papillary diameter was read in the horizontal plane and the readings were converted to millimeters. ~) Studies. The pupillary diameters of the control animals were determined at the various light intensities by simulating the light conditions in the kennels and covered runs with the above-mentioned lamps (150--200 lux and 1000--1400 lax) and allowing 30 min for adaptation before each measurement. 53 male and 52 female animals were used. Mydriasis was measured in 62 male and 80 female beagles after local application to both eyes of 2--3 drops of Mydriaticum Roche | (5 mg tropic acid-N-ethyl-N(y-picolyl)-amide and 0.01 mg phenylmercurie nitrate in 1 ml aqueous solution, Hoffmann La-Roche, Grenzach/Baden). Miosis was measured on 47male and 50female animals after similar treatment with Miotic ~ (0.1 g carbamylcholine chloride and 0.3 g dimethyleorbamoxitrimethyl ammonium methyl sulfate per 10 ml in isotonic autosterile solution with boric acid and a wetting agent, Mann, Berlin). Measurements were taken 30--60 rain after administration of the appropriate preparations at a light intensity of 3000--6000 lax. Time-effect curves were plotted for two groups each of 5 beagles initially treated with either 75 ttg/kg atropine or 100 ~g/kg propantheline, and subsequently with 175 ~g/kg atropine or 300 ~g/kg propantheline. The compounds were made up in aqueous solution (0.1 ml/kg body weight) and administered via the cephalic vein. Pupillary diameters were photographically determined prior to administration and subsequently at 5, 10, 15, 20, 30, 40, 50, and 60 min, and 2, 4, 6, 8 and 24 hrs. I n a study using propantheline intravenously at 35, 70, 105, 140, 175, 2t0, 280 and 350 ~g/kg in beagles, the same 10 animals were used for each dosage level and a dose-effect curve was plotted. A period of 1 week was allowed between dosage application. Pupillary diameters were determined prior to administration and 20 min thereafter. The procedure was based on a Youden design with randomised doselevels. d) Biometric Evaluation. The individual samples are designed as the arithmetical mean value (~), standard deviation (s) and sample size (n). The values for the left and right eyes were compared by the t-test for pair differences and t h e / - t e s t for independent samples was used to evaluate sex differences. The statistically significant limits are indicated together with the results. The time-effect curves were determined mathematically by an iterative process for non-linear regression analysis. Of the biomathematical models tested, the following was preferred because of its least residual sum of the squares: Y=Pl~-p2texp(pat)+pacosb+pssinb+p6cos2b+p~sin2b where: t ~ time after administration (h) b = 2 ~/24t-~
388
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This model is a modified and extended version of those used in glucose tolerance tests (Brown et al., 1969). Polynomials are less aptly able to describe time-effect relationships of this kind (Keller, 1969). Analysis of the effects of age-dependency on pupillary diameter and the determination of the dose-effect relationship were effected by fitting polynomials to the values obtained in the studies by applying the method of least squares. The polynomials were successively degraded. The contributions of the individual degradation steps were tested for significance by analysis of variance. P-values less than 0.05 were regarded as statistically significant. Linearity was demonstrated and the method described by l~inney (1971) was used te determine the ED (3 mm dilation) and the relevant confidence interval for P = 0.95.
Results T a b l e 1 shows the d a t a o b t a i n e d from the controls u n d e r the various
light intensities. The average value for the left eye of the females in 150--200 lux group was significantly higher than that for the left of the males (P < 0.05). A sex difference is thus to be found for mean values of both eyes (P < 0.05). I~o statistically significant dependency was seen (P > 0.05).
the eye the age
T a b l e 2 shows t h e d a t a o n t h e m y d r i a t i e a n d miotie pupils. No
statistically significant sex differences and age dependency could be detected in the latter group. However, the pupillary diameters in the males in the mydriatic group were significantly larger than those in the females (P < 0.05). Moreover, statistically significant positive relationships were obtained for this group in respect of age and pupillary diameter (r = 0.30 for males, r ---- 0.34 for females). A linear relationship for age dependency Table 1. Pupillary diameter (ram) of untreated dogs in various light intensities; mean value @), standard deviation (s), sample size (n) and level of significance (t-test, two-tailed) Sex
150--200 lux
Male s n Famale 8
Comparison male vs. female
1000-- 1400 lux
Right eye Left eye
Mean of Right eye Left eye Mean of both eyes both eyes
9.38 1.08 53
9.45 1.26 53
9.41 1.04 53
7.51 1.28 53
9.76 1.50 52
10.02 1.47 52
9.89 1.34 52
7.45 1.50 51
P>0.05
P 0.05 < 0.10
389
Pupillometrie Studies in the Beagle Dog
Table 2. Pupillary diameter (mm) after local application of Mydriaticum t~oche| or Miotic| mean value (2), standard deviation (s), sample size (n) and level of significance (t-test, two tailed) Sex
Mydriatic pupils
Miotic pupils
Right eye Left eye
Mean of l~ight eye Left eye Mean of both eyes both eyes
Male
2 s n
11.03 0.87 62
11.02 0.85 62
11.02 0.83 62
1.02 0.32 47
1.02 0.31 46
1.02 0.32 45
Female
2 s n
1.0.68 0.83 80
10.69 0.84 80
10.69 0.82 80
1.02 0.43 49
1.00 0.40 50
1.01 0.41 49
P
Comparison male vs. female
0.05
mm
12.6 12.4 12.2 12,0 11.8 11.6 11,411.2 11.0t
10,81 IO.S1 10.41 10,2J
10.0"
i
8
|
i
,
i
,
|
i
,
i
,
,
,
,
,
|
,
i
,
,
,
~
9
,
,
9 10 11 12 13 14 15 1G 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Age (months) Fig. 1. Age-dependency of mydriatie pupil diameters in Beagle dogs, treated with Mydriatieum Roche| Calculated regression lines (straight lines) and confidence belts for P -= 0.95 (hyperbolas)
was f o u n d i n b o t h sexes (P < 0.05 for males, P < 0.01 for females). The regression lines r u n parallel. There is a significant sex difference i n t h e distance b e t w e e n these parallel lines (Fig. 1, P < 0.01).
390
I. Weisse et al.
Table 3. Right-left differences of pupillary diameters (ram) of dogs; mean value (~), standard deviation (s), sample size (n) and level of significance (paired t-test, twotailed) Treatment
Lux
Sex
$
8
n
Level significance
Untreated Untreated Untreated Untreated l~ydriatie ~ydriatie Miotic Miotic
150-- 200 150-- 200 1000--1400 1000--1400 3000--6000 3000--6000 3000--6000 3000--6000
c~ ~ c~ ~ c~ ~ c~ ~
--+ + + --
1.082 1.286 1.174 1.160 0.434 0.270 0.074 0.176
53 52 53 51 62 80 45 49
P P P P P P P P
0.068 0.258 0.029 0.447 0.016 0.012 0.000 + 0.015
> > > < > > > >
...... E
8
0.05 0.05 0.05 0.01 0.05 0.05 0.05 0.05
7 5 j J g / k 9 i.v.
175 ~ 9 1 k 9 i.v.
7 E o "o
Q.
6 5
3 2
E o
I 0 0
Time (hours)
Fig. 2. Calculated time-effect curves for atropine on pupil diameter. The standard deviations about the regression are 0.39 and 1.13for 75 ~g/kg and 175 ~g/kg respectively
T a b l e 3 shows the results of the right-left comparison for b o t h the t r e a t e d a n i m a l s a n d the controls o n the basis of the various light i n t e n sities. O n l y the values for the u n t r e a t e d females i n the 1000--1400 l u x group showed a highly significant right-left difference (P < 0.01). The differences i n p u p i l l a r y d i a m e t e r i n the other groups were w i t h i n the r a n d o m d e v i a t i o n (P > 0.05.) The time-effect curves for the dosage levels of a t r o p i n e a n d prop a n t h e l i n e are shown i n Figs.2 a n d 3. The curves are based on the m e a n values for b o t h eyes of 5 a n i m a l s i n each case. The m a x i m u m
Pupillometrie Studies in the Beagle Dog
? E
391
8 7
....
I O O p g / k g i.v.
- -
3 0 0 p g / k g i.v.
6 E n~
5 /.
D
3 I
2
g o
! 0
;
i
i
i Time
;
i
~
;
(hours}
Fig.3. Calculated time-effect curves for propantheline on pupil diameter. The standard deviations about the regression are 0.36 and 0.6[ for 100 [~g/kg and
300 ~zg/kgrespectively
pupillary diameters were reached after the following time intervals (mean and extreme values) : atropine
75 ~zg/kg -= 12.1 (8.4--15.5) rain 175 Ezg/kg = 14.5 (8.5--22.6) rain
propantheline
100 tzg/kg = 17.0 (6.0--28.1) rain 300 ~zg/kg = 16.2 (10.1--21.3) min.
The dose-effect relationship for propantheline, given in Fig.4, was determined from the data obtained during the maximum mydriatie effect, about 20 rain after administration. The curve was found to be linear in a semilogarithmic plot for all dosages (P < 0.001). The lowest dose tested (35 ~g/kg) did not result in a significant change in pupillary diameter (P > 0.05). Characteristic of the highest dose (350 ~g/kg) was the fact that the anatomic maximum pupillary diameter was achieved. 109 (97--121) ~zg/kg propantheline increased the pupillary diameter by 3 ram. Discussion To our knowledge, apart from the statement by Magrane (1965) that the dog's pupil is larger than that in man, there has been no information published in recent years on pupillary size in the dog. The present study thus had as its object the development of a reliable technique for the repeated measurement of the static pupillary diameter in the conscious dog.
392
I. Wcisse
etal.
6.0
,//,~1/
6.5
II [l/~
5,0 4.5.
//
/,.0, E
3,5 3.0, o_ "10 >, 0
2.5. 2.0.
Q.
/ / ',,"/,}" //
1,5. 1,0.
& c o=
0.5-
(.3
O, -0,5 -
I
.(X,/ j"
i/I
//
-
1,0
-1..5
/ 3's/
50
"I
10,0
7b
losj,
150
200
1,o r)s 21o
300 280 350
lO8.g
Oose
~g/kg i.v.
Fig.4. Calculated dose-response curve for propantheline on pupil diameter with confidence belt for P = 0.95
The incorporation of the calibrated graticule in the camera was only considered usefut in the determination of the diameters of mydriatie and miotie pupils. Photographs were preferable for measuring the pupil at varying light intensities for the time-effect and dose-effect curves. At the 150--200 lux range it was possible to focus the iris but not to assess the diameter of the pupil precisely. Physiological pupillary excitation also made it difficult to read the calibrations on the graticule accurately. With the exception of the untreated females in the 1000--1400 lux group, the right/left differences in pupillary diameter which were seen in these photographic determinations lay within the limits imposed b y the technique and the biological variation. This exception is probably a result of the fact t h a t while the lighting was as diffuse as possible, the test animals were not constantly looking at the illuminated wall. From
Pupillometric Studies in the Beagle Dog
393
this it is clear that variations in light intensity and direct light reactions of the pupil were inevitably included in the measurements. Consensual pupillary reaction, which might have seemed to be a point for consideration particularly in the females in the 1000--1400 lux group, cannot be held responsible for the variation in pupillary diameter since the measurements were made on alternate eyes. Apart from this the interval between the two photographs was always long enough to allow the brightness reflex triggered b y the flash to return to normal. Evidence of the sensitivity of the two methods used is seen firstly in the detection of a sex difference by the calibrated graticule method. Secondly the time-effect curves for propantheline and atropine, and the linearity of the propantheline dose-effect relationships show that the method of photographic registration of pupillary diameter is capable of detecting variations resulting from the administration of drugs affecting pupillary size. I n view of the precision of the data obtained and the ease with which the fundus camera can be used, this technique is not only well suited for pharmacological studies but also for repeated observations on conscious dogs in long term toxicological studies. Reliable results can however only by expected if the animals are not excited and the observations are carried out under evenly diffused illumination.
References Brodie, D. A. : A comparison of anticholinergic drugs on gastric secretion, gastric emptying, and pupil diameter in the rat. Gastroenterology 50, 45--50 (1966) Brown, G., Quarrington, B., Stanler, H. C. : The mathematical analysis of glucose tolerance curves obtained by in vivo antoanalysis. Comp. Biomed. Res. 2, 558--567 (1969) Burns, J., ttorlington, M., Robson, D. : Positive correlation between initial pupil diameter and amplitude of miotic to piloearpine in rabbits and to carbachol in man. Brit. J. Pharmaeol. 40, 524--525 (1970) Clippers, C. : Die fortlaufende Registrierung der direkten und der konsensuellen Pupillenreaktion. Albrecht v. Graefes Arch. Ophthal. 155, 588--616 (1954) Elliot, H. W., Way, E. L. : Effect of narcotic antagonists on the pupil diameter of nonaddiets. Clin. Pharmaeol. Ther. 2, 713--721 (1961) Finney, D. J. : Statistical Method in Biological Assay. 2nd ed., 2nd impr. London: Charles Griffin & Co., Ltd. 1971 Haab, 0. : Der I-Iirnrindenreflex der Pupille. Arch. Augenheilk. 46, 1--29 (1903) Heubner, W.: Quantitative Bestimmnng yon Atropin neben Papaverin in einer Arznei. Klin. Wschr. 84, 1571 (1926) Jasinski, D. R., Martin, W. R.: Evaluation of a new photographic method for assessing pupil diameters. Clin. Pharmaeol. Ther. 8, 271--272 (1967) Kasatskij, B. B.: Vestn. Oftal. 69, 91 (1956) Koller, S.: Diskussionsbemerkung zum Vortrag yon W. Kosswig: ,,Beitrag zur Analyse yon Zeitreihen", 16. Biometr. Coll. der Dtsch. Region der Internat. Biometr. Ges., 20.--22. Febr. 1969 in Bad Nauheim
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Leaders, F. E., Fortenberry, B . R . : The effects of dl-l-(4-hydroxyphenyl)-2-isopropylaminoethanol HC1 (A1 842) on intraocular pressure and pupil size of rhesus monkeys (35460). Prec. Soc. exp. Biol. (N. Y.) 186, 1211--1215 (1971) Lowenstein, O., Friedman, E. D.: Pupillographic studies: I. Present state of pupillography, its method and diagnostic significance. Arch. Ophthal. 27, 969--993 (1942) Magrane, W. G.: Canine 0phthalmology, p. 135. Philadelphia: Lea and Febiger 1965 Nemec, J., Hradil, F., Wildt~ S.: Pupillenweite der Albinomaus des H-Stammes. Z. Versuchstierk. 11, 312--319 (1969) Pulewka, P.: Das Auge der ~aus als pharmakologisches Testobjekt. NaunynSchmiedebergs Arch. exp. Path. Pharmak. 168, 307--318 (1932) Schrader, K. E.: Untersuchungsmethoden der normalen und pathologiseh ver~nderten Pupille, ihrer Reaktionen und ReaktionsstSrungen. In: Die ophthalmologischen Untersuchungsmethoden, L Bd., Hrsg.: W. Straub. Stuttgart: F. Enke 1970 T6rnqvist, G.: Effect of reserpine on accomodation and pupil diameter in monkeys (macaca irns). Acta physiol, scand. 75, 565--567 (1969) Ueki, S., Oono, S. : A new method for continous recording of the pupil size by means of a phototransistor. Arch. int. Pharmacodyn. 142, 339--348 (1963) Weisse, I., StStzer, H., Knappen, F., Walland, A.: The effect of Clonidine on the pupil diameter and the retina in rats, assessed in relation to the intensity of light. Arzneimittel-Forseh. (Drug Res.) 21, 821--825 (1971) Werner, G.: Fermentdefekte als Ursache untersehiedlicher Wirksamkeit yon Atropin und Cocain bei Kaninchen. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 251, 320--334 (1965)
