liritish lourml of Dermatology {\99i) 125.419-425.
Sequence of morphological events during topical application of retinoic acid on the rhino mouse skin FRANCOISE BERNERD. M.DEMARCHHZ. J.-P.ORTONNE* AND j.CZERNlELEWSKI Centre liilcnuithiinl ilf Rci'lii'nhcs Dfrnuilolofikiiies Cinlderma. VtiUmivu: France *Dcpiiiti-mcKl ih- Dcniiiiti'loiiii', Hopitiil l\isleur. Nice. Fnmce Accepted for putiliL-atiun 2 I M;iy
Summary
in a study of the comedolytic action of retinoids on the epidermal pseudocomedones of rhino mouse skin, the earliest changes and the sequence of events induced by the treatment were investigated. Rhino mice were treated topically with all-(rrt?(s retinoic acid and at various time intervals from day 0 to 3 weeks, histological and ultrastructural studies as well as quantititation of mast cells were performed. The earliest changes occurred in the dermis after 2 days of treatment and were characterized by degranulation of mast cells, clumping and association of I,angerhans cells and lymphocytes. During the second week of treatment there was hyperplasia. hypergranulosis and an increase in the number of membrane coating granules with disorganization of the horny layer both of the epidermis and the folllcular epithelium. These changes in the proliferation and the differentiation of keratinocytes resulted in a comedolytic effect. Associated with these epidermal changes there were changes in the dermis with an increased number of mast cells and a decreased number of Langerhans cells.
The skin of the adult rhino mouse (iir"^'' hr'"'') is characterized by the utriculi that are derived from the infundibular zone of the initial folllcular units.' These are histologically similar to retentional acneic lesions, such as comedones. These rhino mice are used as an in-vivo model to evaluate the potential comedolytic action of new drugs for the treatment of acne.' ^ Retinoids produce a good comedolytic effect by reducing the number, size and diameter of the pseudocomedones.- ' We have shown that this comedolytic effect is related to changes in the proliferation and the differentiation of keratinocytes situated both in the epidermis and in the epithelial wall of pseudocomedones."^ It is now well known that retinoic acid influences keratinocyte differentiation and proliferation.^ ^' Some authors have also reported effects on the dermal extracellular matrix.'''" and others have examined modifications of the dermal inllammatory components in disorders such as psoriasis.' ' However, no kinetic experiments have so far been reported. The aim of this study was to determine the sequent'e of morphological events which occur in the different compartments of the rhino mouse skin, during treatment with a topical retinoid. Correspondence: l)r F. [iernerd, Centre Inleriiiilioniil dc Recherchfs Dermalologiques Cialderina. Sophia Aniipolis. Ofi^fiS Valbonne. Friiiice.
Methods Animals and treatment Three groups of 6-week-okl male and female rhino mice (hr'^'' hr^'') (CNRS. La Source, 45()()O Orleans I-rance) were used. In Groups 1 and 2 the animals were treated over the entire dorsal surface with SO /ji of either the drug preparation (all-tr(ins retinoic acid gel at 0 0 2 5 % w/w (Aberel® gel, Cilag Laboratories) or acetone, once a day. 5 days per week. Group 3 was composed of nontreated controls.
Biopaies
After 2. 5. 5.8. 10. 12. 15, 17and 19 days of treatment, three animals in each group were sacrificed by cervical dislocation. Punch biopsies (6 mm) were taken from the treated zones and cut into three pieces which were immersed in tormalin. Karnovsky's solution, or frozen in liquid nitrogen.
Histohgij After fixation in formalin, samples were embedded in paraftin and 4-/im vertical skin sections were stained with haematoxylin-phloxin-safran. 419
420
F. BKRNHRU et «/.
Ekctron microscopy Biopsies were fixed for 4 h at room temperature in Karnovsky's solution (2% w/v para form aldehyde. 2-5% w/v glutaraldehyde in 0-1 M cacodylate buffer. pH 7 4). then washed in 5% w/v sucrosed cacodylate bufler, postfixed in 1% Os()4. dehydrated and embedded in Epon 812. Semithin and ultrathin sections were cut and mounted on grids which were then observed with a Jeol 1200 EX electron microscope operating at 80 kV. Some samples were prepared for scanning-electron microscopy (SKM). After fixation and dehydration, they were critical-point dried and coated with gold. Specimens were observed with Jeol T300 scanning-electron microscope operating at 25-i() kV.
4% w/v paraformaldehyde solution in phosphate-bufiered saline (PBS. pH 7-2) for 10 min, washed with PBS. incubated with 1 /SOO FITC-avidin (Miles-Yeda I,T1)| for 60 min as previously described,'- washed tiircc times in PBS and mounted. Slides were examined by lluorescence microscopy with an Axiophtit Zeiss microscope. The number of cells was determined using an ocular grid at X 20 magnification, counting the mast cells in five {)-5mm- fields in each section. Three alternate serial sections were observed for each animal, and the mean value calculated. The standard error of the mean was evaluated for each group of three animals.
Results In acetone-treated skin, no significant modifications were observed.
Quantification of dermal Langerhans cells ihe number of Langerhans cells per 50-/mi- area of dermis was determined in sections from six non-treated animals and from five animals treated with the drug. Statistical analysis was performed using the Wilcoxon two-sided test for comparison of treated and non-treated skin. Mast-cell labelliiiif
Frozen samples were embedded in Tissue Tek (Miles Laboratories). Vertical cryosections (5 fim) were fixed in
After 5-8daysoftreatment the first effects were noted in the epidermis and in the epithelial wall of pseudocomedont'S with nn enlargement of the intercellular space which disappeared after day 12. Simultaneously there was comedolysis and this increased up to day 17. when there was a decrease in the size and diameter of the utriculi and expulsion of follicular plugs occurred {Fig. 1). Between days 5 and 12. hyperplastic features were
(a)
»'
l-iynii' I . I -iMii'iliin
-
••'.: ••;• • •
•; . r i [ > M i l ) i r l i [ i o k - M - \ k \ . u i I N u i i - i i v d l f d I i i i u o n u m s e s k i l l , I ' X ' . e p i d e r m u l r o m e d o . ( b I A f t e r 5 d a y s o f t r e a t m e n t
the
epidermis shows a hypLTpinstic response and an Increased granuifir layer (arrow), (c) After I 7 days of Ireatnionl there is comedolysis wiih a decrease in the size and diiimeler of the (•iiineiio. There is no further atviimulation of horny material in the folliculiir lanal I x S2(l).
EFFECTS OF TOPICAL HA ON RHINO MOUSE SKIN
noted in the epidermis and epithelial wall of the comedones (Fig. lb) associated with an increased nLimluT of epidermyl granular cell layers. i'.h'ctron nticroscopy
Tlic earliest uUrastructural changes were observed in
ligure 2. Rhino iiictuso skin after 2 days of treatment with reHnoic at iil %) showing degranulated masi cfll in iht- clorinis Ilmr 2 /im).
h'igure i. AlttT i ttays ofrctinoic acid Irciilmc'tit llitTt.' is dose (.•(«it;ii"l bctwtrn sevtTiil Liinyerhiatis cells |i.l'| willi typical Rirheck granules (arrows and irisetsj and lyniptuKytfs (I,y) (bar 2 [tin).
421
the dermis after 2-i days of treatment. I rom day 2 to day 5 the mast cells showed evidence of degranuhition (Fig. 2). but by day K continuing degranulation of these cells was not observed. Dermal Langerhans cells with typical Birbeck granules, which were numerous in the nou-trcatcd rhino mouse skin us well us in the acetone-treated skin, were rapidly affected by the topical treatment with the retinoid. After i days of application some chunping of these cells was observed (I'ig. S) and close contact between lymphocytes and Langerhans cells was observed both in thcdcrmis (I'ig. 5) and epidermis. Some Langerhans cells were also observed projecting through the basement membrane. These features persisted until day 10 of treatment. Cytoplasmic and memhraiie alterations of dermal and epidermal Langeriiaus cells were then seen from days 10-19 ol treatment. The number of dermal Langerhans cells showed a significant decrease after i weeks of treatment (Table 1). After S-S days of treatment, some alterations in the koratinocytes with intracellular vacuolization. detachment of the nuclear envelope and an cnlargcmenl of the intercellular space were seen in the epidermis and epithelial wall of the comedones. These changes disappeared by day 1 2. After the first week, correlated with the comedolytic effect observed on histology, the ultrastructural changes primarily concerned proliferation and later differentia-
422
F. BERNERD el ai
Table I. yurintiHciilion of dermal l,iingerhans cells before and after J vvt'cks ol't(>pit';il iippliailions ofiilt-lrdns rctinoic acid
Mciin viiluf Treatment Nnn-trcated
Animal i
2 J 4 5 ft
LC
No.ofy.
} 9
2^
6 11 4 H
l« 21 12 16
LC/U
(±SD)
I)i7i ()'36() 0-313 0-524 O-iJi 0-500
0-4O±0-09 Retinoic-acidtreated mice
7
1
S 9 10
4 i 2 7
11
1i 14 16 29 2H
(1-091 0-2K(i 0-18S ()'()fi9 0-iSO
o-is±o-i(r
the iollicular epithelium (Fig. 5). Large quantities of extracellular multilamellar material were also observed in the intcrcL-lliilar space of t!ie stratum corneum (l-'iy. 5b). Using SHM. the shape and surface of the corneocytes were seen to be changed. Numerous features of premature desquamation were observed, including total detachment olthe stratum corneum (Fig. 4b). mainly in the infundibular zone of the epithelium wall of comedones. Mast-ceil quantification The avidin-FITCIabellingofmast cells is shown in Figure 6. From day 2 until day S. tlie number of these cells decreased but by day 8 the number of mast cells had returned to the levels found in acetone-lrealed skin. During the third week of treatment, the number of mast cells was greatly increased (Fig. 7).
•i'
Sequence of morphological events during topical application of retinoic acid on the rhino mouse skin FRANCOISE BERNERD. M.DEMARCHHZ. J.-P.ORTONNE* AND j.CZERNlELEWSKI Centre liilcnuithiinl ilf Rci'lii'nhcs Dfrnuilolofikiiies Cinlderma. VtiUmivu: France *Dcpiiiti-mcKl ih- Dcniiiiti'loiiii', Hopitiil l\isleur. Nice. Fnmce Accepted for putiliL-atiun 2 I M;iy
Summary
in a study of the comedolytic action of retinoids on the epidermal pseudocomedones of rhino mouse skin, the earliest changes and the sequence of events induced by the treatment were investigated. Rhino mice were treated topically with all-(rrt?(s retinoic acid and at various time intervals from day 0 to 3 weeks, histological and ultrastructural studies as well as quantititation of mast cells were performed. The earliest changes occurred in the dermis after 2 days of treatment and were characterized by degranulation of mast cells, clumping and association of I,angerhans cells and lymphocytes. During the second week of treatment there was hyperplasia. hypergranulosis and an increase in the number of membrane coating granules with disorganization of the horny layer both of the epidermis and the folllcular epithelium. These changes in the proliferation and the differentiation of keratinocytes resulted in a comedolytic effect. Associated with these epidermal changes there were changes in the dermis with an increased number of mast cells and a decreased number of Langerhans cells.
The skin of the adult rhino mouse (iir"^'' hr'"'') is characterized by the utriculi that are derived from the infundibular zone of the initial folllcular units.' These are histologically similar to retentional acneic lesions, such as comedones. These rhino mice are used as an in-vivo model to evaluate the potential comedolytic action of new drugs for the treatment of acne.' ^ Retinoids produce a good comedolytic effect by reducing the number, size and diameter of the pseudocomedones.- ' We have shown that this comedolytic effect is related to changes in the proliferation and the differentiation of keratinocytes situated both in the epidermis and in the epithelial wall of pseudocomedones."^ It is now well known that retinoic acid influences keratinocyte differentiation and proliferation.^ ^' Some authors have also reported effects on the dermal extracellular matrix.'''" and others have examined modifications of the dermal inllammatory components in disorders such as psoriasis.' ' However, no kinetic experiments have so far been reported. The aim of this study was to determine the sequent'e of morphological events which occur in the different compartments of the rhino mouse skin, during treatment with a topical retinoid. Correspondence: l)r F. [iernerd, Centre Inleriiiilioniil dc Recherchfs Dermalologiques Cialderina. Sophia Aniipolis. Ofi^fiS Valbonne. Friiiice.
Methods Animals and treatment Three groups of 6-week-okl male and female rhino mice (hr'^'' hr^'') (CNRS. La Source, 45()()O Orleans I-rance) were used. In Groups 1 and 2 the animals were treated over the entire dorsal surface with SO /ji of either the drug preparation (all-tr(ins retinoic acid gel at 0 0 2 5 % w/w (Aberel® gel, Cilag Laboratories) or acetone, once a day. 5 days per week. Group 3 was composed of nontreated controls.
Biopaies
After 2. 5. 5.8. 10. 12. 15, 17and 19 days of treatment, three animals in each group were sacrificed by cervical dislocation. Punch biopsies (6 mm) were taken from the treated zones and cut into three pieces which were immersed in tormalin. Karnovsky's solution, or frozen in liquid nitrogen.
Histohgij After fixation in formalin, samples were embedded in paraftin and 4-/im vertical skin sections were stained with haematoxylin-phloxin-safran. 419
420
F. BKRNHRU et «/.
Ekctron microscopy Biopsies were fixed for 4 h at room temperature in Karnovsky's solution (2% w/v para form aldehyde. 2-5% w/v glutaraldehyde in 0-1 M cacodylate buffer. pH 7 4). then washed in 5% w/v sucrosed cacodylate bufler, postfixed in 1% Os()4. dehydrated and embedded in Epon 812. Semithin and ultrathin sections were cut and mounted on grids which were then observed with a Jeol 1200 EX electron microscope operating at 80 kV. Some samples were prepared for scanning-electron microscopy (SKM). After fixation and dehydration, they were critical-point dried and coated with gold. Specimens were observed with Jeol T300 scanning-electron microscope operating at 25-i() kV.
4% w/v paraformaldehyde solution in phosphate-bufiered saline (PBS. pH 7-2) for 10 min, washed with PBS. incubated with 1 /SOO FITC-avidin (Miles-Yeda I,T1)| for 60 min as previously described,'- washed tiircc times in PBS and mounted. Slides were examined by lluorescence microscopy with an Axiophtit Zeiss microscope. The number of cells was determined using an ocular grid at X 20 magnification, counting the mast cells in five {)-5mm- fields in each section. Three alternate serial sections were observed for each animal, and the mean value calculated. The standard error of the mean was evaluated for each group of three animals.
Results In acetone-treated skin, no significant modifications were observed.
Quantification of dermal Langerhans cells ihe number of Langerhans cells per 50-/mi- area of dermis was determined in sections from six non-treated animals and from five animals treated with the drug. Statistical analysis was performed using the Wilcoxon two-sided test for comparison of treated and non-treated skin. Mast-cell labelliiiif
Frozen samples were embedded in Tissue Tek (Miles Laboratories). Vertical cryosections (5 fim) were fixed in
After 5-8daysoftreatment the first effects were noted in the epidermis and in the epithelial wall of pseudocomedont'S with nn enlargement of the intercellular space which disappeared after day 12. Simultaneously there was comedolysis and this increased up to day 17. when there was a decrease in the size and diameter of the utriculi and expulsion of follicular plugs occurred {Fig. 1). Between days 5 and 12. hyperplastic features were
(a)
»'
l-iynii' I . I -iMii'iliin
-
••'.: ••;• • •
•; . r i [ > M i l ) i r l i [ i o k - M - \ k \ . u i I N u i i - i i v d l f d I i i i u o n u m s e s k i l l , I ' X ' . e p i d e r m u l r o m e d o . ( b I A f t e r 5 d a y s o f t r e a t m e n t
the
epidermis shows a hypLTpinstic response and an Increased granuifir layer (arrow), (c) After I 7 days of Ireatnionl there is comedolysis wiih a decrease in the size and diiimeler of the (•iiineiio. There is no further atviimulation of horny material in the folliculiir lanal I x S2(l).
EFFECTS OF TOPICAL HA ON RHINO MOUSE SKIN
noted in the epidermis and epithelial wall of the comedones (Fig. lb) associated with an increased nLimluT of epidermyl granular cell layers. i'.h'ctron nticroscopy
Tlic earliest uUrastructural changes were observed in
ligure 2. Rhino iiictuso skin after 2 days of treatment with reHnoic at iil %) showing degranulated masi cfll in iht- clorinis Ilmr 2 /im).
h'igure i. AlttT i ttays ofrctinoic acid Irciilmc'tit llitTt.' is dose (.•(«it;ii"l bctwtrn sevtTiil Liinyerhiatis cells |i.l'| willi typical Rirheck granules (arrows and irisetsj and lyniptuKytfs (I,y) (bar 2 [tin).
421
the dermis after 2-i days of treatment. I rom day 2 to day 5 the mast cells showed evidence of degranuhition (Fig. 2). but by day K continuing degranulation of these cells was not observed. Dermal Langerhans cells with typical Birbeck granules, which were numerous in the nou-trcatcd rhino mouse skin us well us in the acetone-treated skin, were rapidly affected by the topical treatment with the retinoid. After i days of application some chunping of these cells was observed (I'ig. S) and close contact between lymphocytes and Langerhans cells was observed both in thcdcrmis (I'ig. 5) and epidermis. Some Langerhans cells were also observed projecting through the basement membrane. These features persisted until day 10 of treatment. Cytoplasmic and memhraiie alterations of dermal and epidermal Langeriiaus cells were then seen from days 10-19 ol treatment. The number of dermal Langerhans cells showed a significant decrease after i weeks of treatment (Table 1). After S-S days of treatment, some alterations in the koratinocytes with intracellular vacuolization. detachment of the nuclear envelope and an cnlargcmenl of the intercellular space were seen in the epidermis and epithelial wall of the comedones. These changes disappeared by day 1 2. After the first week, correlated with the comedolytic effect observed on histology, the ultrastructural changes primarily concerned proliferation and later differentia-
422
F. BERNERD el ai
Table I. yurintiHciilion of dermal l,iingerhans cells before and after J vvt'cks ol't(>pit';il iippliailions ofiilt-lrdns rctinoic acid
Mciin viiluf Treatment Nnn-trcated
Animal i
2 J 4 5 ft
LC
No.ofy.
} 9
2^
6 11 4 H
l« 21 12 16
LC/U
(±SD)
I)i7i ()'36() 0-313 0-524 O-iJi 0-500
0-4O±0-09 Retinoic-acidtreated mice
7
1
S 9 10
4 i 2 7
11
1i 14 16 29 2H
(1-091 0-2K(i 0-18S ()'()fi9 0-iSO
o-is±o-i(r
the iollicular epithelium (Fig. 5). Large quantities of extracellular multilamellar material were also observed in the intcrcL-lliilar space of t!ie stratum corneum (l-'iy. 5b). Using SHM. the shape and surface of the corneocytes were seen to be changed. Numerous features of premature desquamation were observed, including total detachment olthe stratum corneum (Fig. 4b). mainly in the infundibular zone of the epithelium wall of comedones. Mast-ceil quantification The avidin-FITCIabellingofmast cells is shown in Figure 6. From day 2 until day S. tlie number of these cells decreased but by day 8 the number of mast cells had returned to the levels found in acetone-lrealed skin. During the third week of treatment, the number of mast cells was greatly increased (Fig. 7).
•i'
