Rate of Turnover of the Stratum Corneum in Hairless Mice*

Vol. 49, No.4 Printed in U.S.A.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Copyright

1967 by The Williams & Wilkios Co.

RATE OF TURNOVER OF THE STRATUM CORNEUM IN HAIRLESS MICE* ALAN M. DOWNES, M.Sc.,t A. GEDEON MATOLTSY, M.D. AND THOMAS M. SWEENEY, M.D.

labelled glycine both intradermally and intraThere have been several recent studies of the venously to pigs and determined the subserate of renewal of stratified squamous epithe- quent location of the label by means of autoha, largely as a result of the introduction of radiography. By this method, he found the tritiated thymidine as a label for tbe germi- turnover time of pig stratum corneum to be native cells which are in the DNA-synthesizing about sixteen days. Rothberg, Crounse and Lee (14) scraped normal stratum corneum at phase (1—7). With appropriate radioautographic technics the turnover time of the germinative various times from the backs of two patients cell population or of the whole population of who had received glycine-2-'4C intravenously. INTRODUCTION

malpighian cells can be determined. Little infor- The glycine in the proteins of the collected

mation is available, however, on the renewal material contained detectable radioactivity after the 13th day, and the authors suggested rate of the stratum corneum alone (7). In a study of the fate of tritiated thymidine that the 13-day period repersented the turnin the epidermis of hairless mice Skjaeggestad over time of the stratum comeum.

(8) concluded that all of the tritium incorporated into the nuclei of the basal cells ultimately was removed with the desquamated

cells of the horny layer. He found that the turnover time of the whole epidermis in this species was 4—41/2 days and that the turnover

We have studied the turnover time of the stratum corneum of hairless mice following the administration of L-cystine-1S. In one set of experiments, the skin was stripped in vivo on successive days and the amount of labelled material appearing in the outermost

time of the horny layer alone was probably layers of the stratum corneum was determined. one day. However, other evidence from studies In another set of experiments, repeated stripof the epidermis of hairless mice (9) and of ping was performed on excised skin from anithe wool follicle (10) suggests that the label mals sacrificed at varying time intervals foldisappears and is probably largely resorbed lowing the administration of the labelled as the nuclei break up during keratinization. cystine. In this manner, the changing pattern If this is true, then valid measurements of of labelling of successive layers of the stratum the rate of turnover of the stratum corneum corneum was observed. could not be obtained directly with labelled MATERIALS AND METHODS

thymidine.

A better approach would be to use labelled

Hairless mice, (strain HRS/J, from the Jack-

lamino acids, which are incorporated, at son Laboratories, Bar Harbor, Maine) aged 8—16 and weighing 15—26 grams, were used. least to some extent, into proteins in all layers weeks Stripping of the stratum corneum in vivo was of the epidermis below the stratum corneum accomplished by means of pressing Scotch brand

within one hour of an intraperitoneal injec- cellophane tape onto the skin as evenly as possible. tion (11—13). Weinstein (7) administered The tape was then removed and the stripped area measured. Stripping of excised skin was performed

This investigation was supported in part by immediately after the mice were sacrificed. The research grant AM 05924 and graduate training skin was placed dermis down on a block of lucite grant Tl-AM 5295, National Institute of Arthritis

(4 x 7 x 0.5 cm). A lucite frame was placed over

Health Service. Received for publication May 18, 1967. * From the Department of Dermatology, Boston

phane tape was then placed on the epidermis and pressure was applied by moving a hand roller over the tape six times with as uniform a pressure as possible before each stripping. The tape was then

and Metabolic Diseases, United States Public the block to hold the skin firmly in place. Cello-

University School of Medicine, and Evans Memorial Department of Clinical Research, University Hospital, Boston University Medical Center, removed slowly. Boston, Massachusetts. For these studies, 9.2 mg of L-cystine-MS (12 t Present address: C.S.I.R.O., Division of Ani- mc/mMole), obtained from Schwarz Bioresearch mal Physiology, Parramatta, N.S.W., Australia. Inc., Orangeburg, New York, was dissolved ia 04 400

TURNOVER TIME OF STRATUM CORNEUM

ml of 1 N IIC1 and diluted to 10 ml with water. This solution was stored frozen until just before use. Each animal was injected intraperitoneally with 0.2 ml (9 icc) of this solution. The toluene scintillation solution contained 0.4%

(w/v) 2,5 diphenyloxazole and 0.01% (w/v) 1,4bis-[2-(5-phenyloxazolyl)j-benzene.

The toluene-methanol scintillation solution consisted of 7 parts of the toluene solution pius 3 parts of methanol by volume. Radioassays: All samples were assayed at room temperature in a Nuclear Chicago Liquid Scintillation Counter, Model #701. At least 4000 counts were registered for each sample and the net counting rates were corrected for decay. Each piece of cellophane tape with the adherent stratum corneum

was placed in a counting vial with 10 ml of the toluene solution and radioassayed. After standing in the solution for 1—2 hours, the stripped material

had fallen from the tape. The tapes were then removed and the material from each set of strippings

from each mouse was transferred to a centrifuge tube to determine the dry weight for the calculation of the specific activities. After centrifugation and decanting of the supernatant, the sediment was

washed twice with ethanol and once with ether. The residue was then transferred to a weighing vessel, dried overnight in an oven at 500 C and

160

r. 140 Cd

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zD 0

-9,

0 z 0.

a.

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FU,

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in

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0

then to constant weight in a desiccator.

The following additional procedures were per-

formed on a series of representative samples in order to check the accuracy of this method of assay.

401

TIME AFTER INJECTION (DAYS)

Fm.

°°S content in the outermost layer of the

stratum corneum stripped in vivo at various

1. To determine if there was any difference in times after administration of L-Cystine-°°S to four the counting rate when the stratum corneum was hairless mice. adherent to the tape or fallen free, radioassay was performed immediately after the tape was placed and each sample was again assayed. The results into the scintillation solution and again after showed that assay of the digested material conenough time had elapsed for the stratum corneum sistently gave counting rates about 10% higher to fall from the tape. There was no significant dif- than assay of the undigested material in the presference in the counting rate whether the stratum ence of the tapes. It was, therefore, decided that corneum was attached to or fallen from the tape. accurate relative results for different specimens 2. Since it was necessary to weigh the material could be obtained by radioassay of undigested which remained in the counting vials after re- stratum corneum. moval of the tapes for determination of specific activities, it was of importance to determine if any RESULTS

labelled material was removed with the tapes. Therefore, radioassay was performed prior and

subsequent to removal of the tapes. A comparison of the counting rates showed that less than 1% of the labelled material had remained on the tapes. 3. All counting rates used for the calculation of experimental results were obtained from specimens consisting of undigested stripped stratum corneum in the presence of cellophane tape. Therefore, it was necessary to determine if any inconsistency in counting rates resulted from the tape's presence in the counting vial or from unequal distribution of the label due to its location within undigested cells. In order to investigate this possibility, the material which bad been recovered for weighing was digested with 1(011 (0.5 ml, 125 N in 50% ethanol)

at 70° C for three hours. To this digested material 20 ml of the toluene-methanol solution was added

Detection of °8 in the Outermost Layer of the Epidermis Four hairless mice were injected with the labelled cystine solution. On either the first or the third day following the injection, an area of stratum corneum measuring approximately 1.25 x 2.0 cm was stripped with cellophane tape and the 355 content of the stripped material was determined. On five to seven sub-

sequent days, similar samples of stratum corneum were obtained, each from a different region of the body. The results, shown in Fig.

1, indicate that a small amount of S was al-

402

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ready present in the outermost layer of the then stripped with cellophane tape four times. stratum eorneum 24 hours after the injection. Each tape was placed in a counting vial for The maximum concentration of °5S in the radioassay. Microscopic investigation of secoutermost layer was present 5—6 days after the tions cut from control and stripped skin reinjection, after which time the concentration vealed that four successive strippings were sufficient to remove practically all of the fell rapidly. stratum corncum and practically none of the in the Distribution of underlying malpighian layer of epidermis. The Stratum Corneum strip method, as applied to excised skin in Eight animals were sacrificed at varying this study, removes relatively even layers of intervals between one hour and six days after the stratum corneum; in the sections studied the administration of labelled cystine. The only a few deeply or superficially stripped reskin was removed immediately and three areas,

4 x 1.25 cm on each skin specimen, were

gions were apparent (Fig. 2).

A determination of the radioactivity present

Jig—

a Rio. 2. Microphotographs showing sections of normal and stripped skin of the hairless

mouse. The numbers in the lower right corner indicate the number of strippings.

403

TURNOVER TIME OF STRATUM CORNEUM

TABLE I in the successive strippings obtained one hour after the administration of the label revealed Amounts of material recovered from Scotch tape

very little S. The distribution of 55S in the successive strippings of the 1—6 day samples is shown in Fig. 3A. These results show that after one day most of the 55S was in the third and fourth strippings and that there was also

a significant amount in the first and second strippings. During the 4—5 day period after the injection, the S moved to the outer layers

strip pings of hairless mouse skin Stripping

Mass of dried material (mg/rm' skin)

% of total stripped material

1

0.137

2

0.099

.022 .020

3 4

0.050

.012

31 16

0.034

.010

11

43

of the stratum corneum (first and second stripcounts/mm/cm2 for each set of strippings pings). The amounts of the material recovered from (Fig. 3A), divided by the corresponding mass

the tapes are shown in Table 1. The total of the material recovered from the tapes are

shown in Fig. 3B. These calculated results must (B)6

(A)6

H-k 5

5 201

be approximately proportional to the specific activities of the various samples. They show

that the material from the fourth stripping had the highest specific activity in the oneday specimens. After three days the material

from the second stripping had the highest specific activity and after the longer periods (4—6 days) the material from the first two

t

01.

strippings had the highest specific activities.

U

4

DIsCUsSION

rrm

It was shown by Bern, Harkncss and Blair (11) that the entire vaginal epithelium below

in I-.

the stratum corneum was radioactive four hours after the injection of 55S cystinc intraperitoneally into estrogen-treated female mice. Moreover, the greatest concentration of radioactivity was present immediately below the stratum corneum. Also, Fukuyama, Nakamura

and Bernstein (12) and Fukuyama and Epstein (13) administered various other labelled amino acids to newborn rats and found some

0

radioactivity in all layers of the epidermis below the stratum corneum within one hour.

Weinstein found a uniform labelling of all layers of the epidermis below the stratum corneum one hour after the intradermal administration of labelled glycine to pigs. It 2 STRIPPING NUMBER 1

can, therefore, be assumed that in our studies

the entire malpighian layer was labelled

within a very short time after administration 3A. Columns indicate the amount of S of the labelled cystine. In view of this, the in successive strippings of the stratumS corneum. FIG.

The number of days elapsed since administration

high specific activity of the material from

neum. The specific activities were calculated by dividing the results shown in Fig. 3A by the cor-

tion of S. If the high specific activity were due to contamination from the malpighian

of the label is indicated by the number above the fourth stripping of the mouse sacrificed each set of columns. FIG. 3B. Columns indicate the specific activities one day after the injection of labelled cystine of the successive strippings of the stratum cor- can be regarded as due to genuine incorporaresponding mass of recovered material.

404

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cells, then a similar high specific activity becoming labelled during the four-day period. would he expected in the fourth stripping from the one hour specimen. In fact the total strippings from the one hour specimen contained a

The subsequent fall in specific activity can be explained just as easily on the basis of resorp-

negligible amount of 855•

stratum corneum. It therefore seems much more likely that the turnover time of 4—41/s days reported by Skjaeggestad should refer to the differentiating cells alone and should not include

The presence of some a5 in each of the strippings from the one day specimens was probably due to the unevenness with which the stripping removes the stratum corneum, although the possibility of some contamination from excreta or of movement of some

tion as on passage of the label through the

the turnover of the horny layer. Baden (9) stated that his "data suggest that the basal

cells take about three days to reach the

free cystine through the stratum corneum can- stratum corneum" in hairless mouse skin, but not be excluded. it is not clear whether this represents the averThe results obtained from the studies on age turnover time of the differentiating cells excised skin indicate that the turnover time or the minimum time required for basal cells of the stratum eorneum of hairless mice is to reach the stratum corneum.

about 4—5 days. This supports the data ob-

The results of the in vivo study (Fig. 1)

tained by daily in vivo stripping of the suggest that information on the turnover rate

stratum corneum which also revealed maximal labelling of the outermost layers of the stratum corneum within four to six days following ad-

of the stratum corneum may be obtained rela-

tively easily by the stripping technic. This

technic, used in conjunction with intradermal ministration of the labelled material. These doses (15), could probably be applied to stud-

findings are very different from those of ies of the turnover of human stratum corneum. Ski aeggestad (8) who concluded that the turn- Epstein and Maibach (4) stated that "the over time of the differentiating cells together with those of the horny layer was —'/2 days in hairless mice 10—16 weeks old, and that the turnover time of the horny layer alone was

probably one day. As mentioned in the Introduction, this was based on a study of the fate of tritiated thymidine, in which it was assumed that all of the tritium incorporated into the nuclei of the basal cells ultimately passed through the horny layer and was removed in the desquamated cells. However, Skjaeggestad himself pointed out that the radioactivity which he detected in the "horny layer" during the fitst day after ad-

method utilized by Rothberg et al is theoretically ideal and should allow for accurate detailing of the first appearance of label on the sur-

face and any subsequent peaks". However, further work is needed to establish the accuracy with which such a technic can measure the turnover time of the stratum eorneum. The results of Rothberg, Crounse and Lee (14) are not convincing in this regard because the

samples which they obtained 13 or 14 days after administering glyeine-2-"C to humans gave counting rates very close to the background rate for the counter. If larger doses or a more sensitive detector had been used it is pos-

ministering tritiated thymidine was probably sible that radioactivity would have been dedue to contamination of the stratum corneum tected earlier than 13 days after the injection. with some malpighian cells. Newly labelled 5UMMAnY basal cells would not be expected to reach the stratum corneum so soon. The probable 1. The turnover time of the stratum eorneum contamination is not surprising in view of of hairless mice was investigated by means of

the method used to collect the "horny layer", cellophane tape stripping of the skin at varyby abrasion of the surface of the dried epi- ing time intervals following the intraperidermis. Skjaeggestad's observation of an in- toneal injection of L-cystine-S.

crease in the specific activity of the horny layer to a maximum on about the fourth day is

merely what would be expected if the main

contamination came from the upper malpighian cells, more and more of which were

2. In vivo stripping studies showed that the outermost layer of stratum corneum cells became maximally labelled after a period of 4—6 days.

3. Stripping of successive layers of the

TURNOVER TIME OF STRATUM CORNEUM

stratum eorneum of excised skin showed that the lowermost layers are heavily labelled after

one day and these layers gradually move outward within a period of 4—5 days.

4. The results obtained in both studies

suggest that the turnover time of the stratum corneum of hairless mice aged 8—16 weeks, is about 4—5 days.

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