Archives of

Arch. Dermatol. Res. 266, 219-225 (1979)

ical Deseermatqlog arch

9 Springer-Verlag 1979

Original Works Animal Experiments Investigations on the Action of Topical Corticoids on the Normal Guinea Pig Skin Peter Altmeyer and Christiane Sprengart Dermatology Clinic of the University of Saarland (Director: Prof. Dr. F. N6dl), D-6650 Homburg/Saar, Federal Republic of Germany

Summary. By means of the test procedure described, the action of topical corticosteroids on the untreated skin of guinea pigs may be exactly assessed and quantified. Of significance is the statistically verifiable observation that the steroid-induced epidermal hypoplasia is completely reversible after the withdrawal of the corticosteroid preparation. Moreover, the onset of a reactive epidermal sPreading occurs, which exceeds the initial value to a small extent. Key: words: Corticosteroids - Epidermal atrophy - Healing phase

Zusammenfassung, Die Wirkung von Corticoidexterna auf die unbehandelte Haut yon Meerschweinchen 1/~l]t sich mittels des geschilderten Testverfahrens exakt erfassen und quantifizieren. Von Bedeutung ist die statistisch zu sichernde Beobachtung, dab die steroidinduzierte Epidermishypoplasie nach Absetzen des Corticoidpr~parates vollst~ndig reversibel ist. Eine einsetzende reaktive Epidermisverbreiterung tibertrifft dartiber hinaus in geringem Umfang den Ausgangswert.

Schliisseiwiirter: Corticosteroide - EPidermisatrophie - Erholungsphase The influence of topical corticosteroids on both the unstimulated [2, 3, 14, 16] and the hyperplastic epidermis of research animals [I 0 - 13, 15] is a pharmacodynamic mechanism which has been the starting point of numerous dermatopharmacological experiments [10]. The most important research results relevant to this were obtained on the guinea pig skin [19]. From the available results on the analogous reactivity of human and guinea pig skin [13], knowledge of the reactivity of the human skin under similar research conditions may be inferred. Long-term topical application of synthetic corticoids produces an atrophy of the epithelial layer, with partly considerable histologically demonstrable alteration of the epidermal cells [1]. In the first place, the interesting question here is how far the corticoid-conditioned atrophy of the epithelium is reversible. To answer this we availed ourselves of a research model in which corticoids were applied to the normal, unstimulated skin of guinea pigs. 0340-3696/79/0266/0219/$1.40

220

P. Altmeyer and Ch. Sprengart

With an adequate research procedure, a measurable effect on the epithelial structure must be interpreted as a sign of corticosteroid-conditioned hypoplasia (atrophy) of the normal epidermis. An epidermal thickening appearing after the interruption of topical steroid application to the experimental animal would evidence the healing phase of the epithelium.

Material and Methods Experimental Animals Male albino guinea pigs, o f the Pirbright strain (Versuchstierzucht Winkelmann, Borchen near Paderborn) were used. At the beginning of the investigation they were about 13 weeks old, and their weight was between 400 and 600 g. They were kept in Makrolon cages, always 2 animals per cage, at a room temperature of 20 ~C and an air humidity between 55 - 75 %, under daylight conditions. They were fed with Alma research animal food, and received drinking water ad libitum.

Methodology 1. Preparation After a preliminary phase of 4 weeks (acclimatization time), with regular observation of weight and general condition, mechanical depilation of the healthy-skinned animals was carried out. The size o f the depilation fields was 10 x 5 cm extending dorsolaterally over the thorax and lumbo-abdominal region and they were separated from one another by a 2 cm broad skin strip. The depilation was repeated weekly. Marking of the fields of application was carried out with a circular stamper (diameter = 1 cm, area = 0.78 cm2). Tincture of iodine was used as marking fluid. This left a light yellow impression on the skin which was outlined by a felt pen (Ball Pental blue).

2. Test Exposures As test preparation, a 1% solution of fluorandrenolone was employed (FA). A mixture of pure acetone (analytical grade) and absolute ethanol in the proportion of 1/1 (ACE) served as a vehicle. Two control fields on the left flank remained untreated (Co) or were treated with the solvent mixture (ACE) only (CAts). Application of the steroid solution or the vehicle (ACE) was carried out by simple dropping of the fluid into the test area, using a microliter pipette (Eppendorf 50 ~tl) in a volume of 2 • 50 ~tl per test circle, which was strictly centered in the test field. Bandages were not applied. The volume of individual doses amounted to lO0/pl/test field/day. The individual surface dose of corticoid was calculated at 1 mg per test field and 1.27 mg/cm 2, The test was Carried out in two series, using 12 animals, respectively. In series I, we applied the corticoid solution to the right flank of the guinea pigs during research days 1 - 2 8 , Monday through Friday, altogether 20 times per case. On the left flank, there remained an untreated control field (Co), and a further field was treated 10 times during 14 days with acetone/ethanol mixture (CAcE)- On days 15 and 29, a biopsy was taken from the center of a steroid-treated test field in each animal. On day 15 additional biopsies were taken from the contralateral control fields (Co, CAts). From research day 29, the steroid application was discontinued, and on days 43 and 57 control biopsies were taken from the previously steroid-treated test areas. In Research series II, the test phase lasted 70 days altogether. The steroid solution was applied 30 times totally on days 1 - 42 according to the previously mentioned method. We took skin samples from the steroid or control fields on research days 22 and 43. After termination of the corticoid treatment, biopsy samples were taken on days 57 and 71, i.e., 14 and 28 days after withdrawal of the topical corticoids.

3. Excision Method The animals were narcotized for the taking of skin samples. Following this, biopsies were taken from the centers of the test fields by a hand-punch (Skin biopsy punch/Stiefel, diameter 6 mm). The biopsy

Steroid-induced Epidermal Hypoplasia

221

wounds were closed by 2 stitches and treated with antibiotic powder. The stitches were removed after 8 to 10 days.

4. Histological Preparations It is possible to obtain distortion-free tissue fixation by applying the excised samples to rectangular paper sheets and placing section and paper together in the fixative solution (Bouin's solution: picric acid 1.2 ~, formalin 4 ~ , acetic acid, in the proportions 15:2:1). Embedding was carried out after 24-h fixation at room temperature by the standard paraffin wax method. Staining then followed by Van Gieson's method.

5. Measurement of Ep#helial Thickness by Ocular Micrometer We carried out the measurement by an ocular screw micrometer (Carl Zeiss). The scale in the ocular is a transmission measure, which is used in conjunction with an objective micrometer, and must be suitable for the microscope (Zeiss photomicroscope III) and magnification in question (1 ocular unit = 31x10 - 6 m ) , Over the entire length of the section one measuring point per field was surveyed in each case in consecutive fields of view. Only the interfollicular epithelium was measured, i. e., the point which comes to lie exactly between two follicles. For delimitation of the two measuring lines, the upper boundary was taken as that between the stratum granulosum and the stratum corneum, and the lower boundary as that between the basal cell layer and the corium. The measured line is the perpendicular level joining the outermost to the innermost limit of the measuring points, i. e., it lies strictly perpendicular to the general stratification of the epidermis. After adjustment of the line-marking in the ocular to the top and bottom object limits, the number associated with the object is read off on the vernier. After this, the section is laterally displaced by an entire field of view, the new measuring point as specified is focussed, and the thickness is again read off. The following parameters, corresponding to the data in the literature, were read off [11]: 1. Valuesobtainedbybiopsyof80individualmeasuredvalues = epithelial thickness in avisnalfield (GEB); 2. the arithmetic mean of 80 individual values = mean epithelilal thickness (MEB); 3. the arithmetic mean from the mean epithelial thickness of all animals in a research series = (grand) average epithelial thickness (DEB).

6. Evaluation of Activity Variance, standard deviation, and coefficient of variation of the visa1 field epithelial thickness (GEB) as well as the mean epithelial thickness (MEB) were calculated. For the estimation of the statistical significance and the significance levels of the activity differences, the calculation of 95 ~o confidence limits was used, and for the mean values the t-test. Difference which were established with a probability of error of 5 ~ (P < 0.05) were considered to be highly significant. When the probability of error was higher than 5 ~o (P > 0.05), we considered the probability of a true difference of activity as not proven.

Results 1. T a b l e 1 s h o w s a r e v i e w o f t h e first r e s e a r c h series, w h i c h w a s b a s e d o n 12 e v a l u a b l e a n i m a l s . Co a n d CArE d e n o t e t h e u n t r e a t e d o r a c e t o n / e t h a n o l m i x t u r e t r e a t e d c o n t r o l fields, $14 t o Ss6 t h e s t e r o i d - t r e a t e d t e s t fields. F i r s t o f all, it is c l e a r t h a t t h e d a i l y a p p l i c a t i o n o f v e h i c l e b r o u g h t a b o u t a n a d a p t i v e e p i t h e l i a l h y p e r p l a s i a o f 26.6 ~ . T h e a t r o p h y - p r o m o t i n g a c t i o n o f t h e s y n t h e t i c c o r t i c o s t e r o i d is a p p a r e n t f r o m t h e S 14 a n d $28 v a l u e s . A s c o n t r a s t e d w i t h t h e c o n t r o l fields s i m p l y t r e a t e d w i t h t h e s o l v e n t o n l y (CARE) a d i f f e r e n c e a m o u n t i n g t o 21 ~ is a p p a r e n t f o r S2s. I n t h e t - t e s t t h e d i f f e r e n c e s w e r e h i g h l y s i g n i f i c a n t i n 11 o f 12 c a s e s ( P < 0.05).

222

P. Altmeyer and Ch. Sprengart

Tabelle 1. Review of the 1st research series Animal N o

Co

CAc~

$14

828

$42

S56

1 2 3 4 5 6 7 8 9 10 11 12

0.97 0.88 0.94 0.79 0.83 0.85 0.92 0.87 0.94 0.81 0.87 0.77

1.08 0.97 1.05 1.33 1.12 1.31 1.15 0.86 1.03 1.21 1.13 0.97

1.06 0.75 0.86 0.75 0.93 0.72 0.83 0.79 0.92 0.92 0.86 0.94

0.95 0.62 0.81 0.74 1.15 1.01 0.76 0.79 0.98 0.89 0.88 0.85

0.88 0.97 0.96 0.99 1.03 1.33 0.89 0.95 1.03 0.75 1.07 1.01

1.24 1.21 1.39 1.23 1.41 1.30 1.10 1.37 1.11 0.99 1.20 1.22

DEB

0.87

1.10

0.86

0.87

0.99

1.23

C O = untreated control field CAe E = control field treated with acetone/ethanol mixture S x 4 - $56 = steroid-treated test fields DEB = average epithelial thickness (units)

Tabelle 2. Review of the 2. research series Animal N o

CO

CA~E

Szl

$42

$56

Svo

1 2 3 4 5 6 7 8 9 10 11 12

0.97 0.96 0.94 0.75 0.82 0.92 0.85 0.86 0.89 0.81 0.78 0.85

1.45 1.33 1.10 0.92 1.00 1.07 1.02 1.15 1.02 1.09 0.97 1.04

0.87 0.83 0.88 0.79 0.69 0.82 0.83 0.86 0.68 0.83 0.86 0.81

0.79 0.76 0.81 0.68 0.67 0.76 0.96 0.84 0.58 0.76 0.78 0.69

1.31 1.08 0.98 0.96 0.90 1.08 1.07 0.91 1.20 1.21 1.11 1.08

1.72 1.37 1.36 1.46 1.04 1.16 0.98 1.34 1.36 1.25 1.48 1.40

DEB

0.86

1.10

0.81

0.76

1.07

1.33

Co = Cacz $21DEB

untreated control field = control field treated with acetone/ethanol mixture $7o = steroid-treated test-fields = average epithelial thickness (units)

Fourteen days after withdrawal of the topical steroid, an epithelial spreading in comparison to S2s which amounted to 13.7% was demonstrable ($42). Twentyeight days later a difference of 41.4 % ($56/$2s) was established. This value proved to be highly significant in all cases (P < 0.05). A comparison of the control values Co/CAcE and of the values after withdrawal ($56) yielded differences of 41.2 % of

Steroid-induced EpidermalHypoplasia

223

11.8 % in favor of 856. This difference was established as highly significant in 8 of 12 animals. 2. Research series II (n -- 12) differed from the first group only in that the period of treatment extended to 42 days. Similarly, the observation time after withdrawal of the steroid amounted to 2 x 14 days ($56/$7o). A 30-fold local steroid treatment extending over 42 days, as compared to the solvent-treated controls (CAts), leads to an epithelial atrophy of 31.0% (Table2). Fourteen days after conclusion of the corticoid treatment, $56 and the CAcE differed only minimally from each other (0.03 units). $7o, on the contrary, exceeded the control value CAr~ by 20.9 %, and the value of the untreated skin field by 51.2%. On submitting the individual results to a statistical analysis in which the following alternative hypotheses were tested: (1) CAc E > $42 ] (2) $7o > $42 ; and (3) $7o > CAeEwe found in 1 and 2, 11/12 cases, in 3, 10/12 cases, in which the results were highly significant (P < 0.05).

Conclusion

The aim of this work was less to demonstrate the well-known epithelial atrophying effect of topically applied corticosteroids [1, 4, 5, 8] but rather to seek an answer to the as yet unsolved problem of the extent to which a steroid-induced epidermal atrophy is reversible. As a research animal we chose the albino guinea pig of the Pirbright race. The anatomy of the skin of this animal species has been thoroughly investigated [18]. The already available experience of several authors on the analogies of human and guinea pig skin gives cause to surmise that under analogous research conditions and a correspondingly protracted treatment period essentially similar conditions would also be found in the human skin [9]. The present treatment period of 4 or 6 weeks is theoretically comparable in human life with 104 or 156 weeks [17]. According to our present clinical experience, protracted corticosteroid treatment of that kind is certainly no rarity [1]. It should be pointed out that the research results submitted here are limited to particular solutions which can be applied to the skin without rubbing in, and which on account of their volatility ensure a rapid fixation of the drug to the horny layer of the epidermis. In the use of creams, ointments, or pastes as carrier media it would be necessary to consider additional effects [19]. As test substance employed fluorandrenolone in a 1% formulation. The concentration of this preparation, which lies substantially above the clinical therapeutic optimum of 0.05 %, was chosen because the conclusion can be drawn from the work of other authors [6, 7, 9, 13] that a clearly determinable inhibition of the artificial epidermal hyperplasia in guinea pigs generally requires a higher corticoid concentration than is required for the attainment and measurement of anti-inflammatory actions in other animals and in man. For reference values we chose the epithelial thickness in a control field which was treated for 14 days with the vehicle mixture (CAts). Thus, we found that the known interactions of the basal formulation on the epithelium, demonstrated here, largely did not apply in our calculations.

224

P.

Altmeyer and Ch. Sprengart

A

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research series. Distinct epithelial atrophy after local steroid treatment (Sa4 , Szs). After with drawal of the corticosteroids (S~z, $56) reactive epidermal spreading. Epithelial thickness in ocular units (u). CAr = control field

Fig. 1 . 1 .

t~

tll

.ta

I --. \

9. . . . . . .... ~\\ QI

!~:i9 :,:;,ii

o.J

i?9 u

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x

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Fig. 2 . 2 research series. DEB-values of a similar kind as in the first research series. C A c E = control field; S2t = 21 days steroid treatment; $42 = 42 days steroid treatment; Ss6 = 14 days after the last steroid applications; $7o = 28 days after the last steroid applications

In the evaluation of the results one can speak of values of a similar kind in both research series. After 20 to 30-fold steroid application, a highly significant epidermal atrophy occurred (Figs. 1 and 2). The maximal effect in research group I is 21.0 ~ and in group II, 31.0 ~ of the average epithelial thickness of the control field (CAcE). According to this it is established that unter the research conditions chosen here a steroid treatment led to a distinct epithelial atrophy; this effect is statistically verified with a probability of error of less than 0.5 ~ (P < 0.05), and therefore we regard it as highly significant. After discontinuation of the topical corticosteroid treatment in both groups there occurred a continual increase in the mean epithelial breadth (MEB). In both research series, this statement was statistically confirmed in 19/24 animals. The measured values of mean epithelial breadth after the conclusion of the observation period ($56 or $70) exceeded the CAcE value by about 11.8 ~ or 20.9 ~ . Compared to the untreated control field (Co), we demonstrated a difference of 41.4 or 53.2 ~ . In both research groups these results are statistically highly significant. Therefore, it can be regarded as proven by animal experimentation that a steroid-induced

Steroid-induced Epidermal Hypoplasia

225

epidermal atrophy is not only completely reversible after the interruption of topical corticosteroid application, but also that a reactive epidermal spreading begins which exceeds the initial value. References 1. Altmeyer, P. : Ein Beitrag zur Histologie der von Kortikoidexterna verursachten Hautver/inderungen. Hautarzt 28, 8 3 - 8 8 (1977) 2. Baker, B. L., Whitacker, W. L.: Zit. nach Castor and Baker (1950). Anat. Rec. 102, 333 (1948) 3. Castor, C. W., Baker, B. L. : The local action of adrenocortical steroids on epidermis and connective tissue of the skin. Endocrinology 47, 234-241 (1950) 4. Gschwandtner, W. R. : Qber die perkutane Anwendung der Kortikosteroide und ihre Nebenwirkungen. Wien. Klin. Wschr. 85, 4 9 - 5 4 (1973) 5. Gschwandtner, W. R.: Striae cutis atrophicae nach Lokalbehandlung mit Corticosteroiden. Hautarzt 24, 7 0 - 7 3 (1973) 6. Heite, H. J., Rommel, F. : Uber den EinfluB yon Hydroeortison bzw. Prednisolon auf die experimentelle Akanthose. Derm. Wschr. 138, 1005-1014 (1953) 7. Paillard, R., Brun, R. : Experimentelles zur Frage der Wirkung von Hormonen auf die Haut. In: Aktuelle Probleme der Dermatologie, Schuppli, pp. 107-122 Basel: Karger 1959 8. Sch6pf, E.: Nebenwirkungen externer Corticosteroidtherapie. Hautarzt 23, 295-301 (1972) 9. Tronnier, H. : fiber die experimentelle Prfifung von entzfindungshemmenden Steroiden an der menschlichen Haut. Berufsdermatosen 8, 3 - 24 (1960) 10. Weirich, E. G., Schenk, C. : Die reaktive Epidermoplasie der Meerschweinchenhaut als Prfifmethode ffir Dermatika. Dermatologica 139 (Suppl. 2), 1 - 3 6 (1969) 11. Weirich, E. G., Longauer, J.: Beeinflussung der epidermalen Proliferationsaktivitfit durch Corticoidexterna und deren Grundlagen. Arch. Dermatol. Forsch. 241, 245-254 (1971) 12. Weirich, E. G., Longauer, J. : Tierexperimentelle Priifung des epidermal-hypoplastischen Effektes von Externcorticoiden. Arztl. Forsch. 25, 292-298 (1971) 13. Weirich, E. G., Longauer, J. : fiber die Wirkungsprtifung yon Externcorticoiden im Epidermoplasietest. Dermatologiea 142, 245-262 (1971) 14. Weirich, E. G., Longauer, J. : Tierexperimentelle Vergleichsuntersuchungen fiber die Epidermishypoplasie durch Externcorticoide. Z. Hautkr. 49, 9 1 - 1 0 6 (1973) 15. Weirich, E. G., Longauer, J. : Vergleichende Prfifung des epidermal antihyperplastischen Effektes von Externeorticoiden im Tierversuch. Arch. Dermatol. Forsch. 248, 179-189 (1973) 16. Weirich, E. G., Longauer, J. : Tierexperimentelle Vergleichsuntersuehungen fiber die Epidermishypoplasie durch Externkortikoide. Z. Hautkr. 49, 91 - 106 (1974) 17. Weirieh, E. G., Longauer, J. : Hypotrophieeffekt yon Externkortikoiden aufdas Hautbindegewebe. Res. exp. Med. 163, 229-239 (1974) 18. Rufli, Th., Longauer, J. K. : Formationsmuster des Hautepithels bei Mensch und Meerschweinchen. Dermatologica 152, 263-269 (1976) 19. Weirich, E. G. : Tierexperimentelle Untersuchungen fiber Salicyls/iurewirkung auf die normale Haut. )~rztl. Kosmetol. 8, 242--245 (1978) Received June 13, 1979