- Email: [email protected]
Dermo-Epidermal Separation with Suction
Vol. 48, No. 5
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1987 by The Williams & Wilkins Co.
DERMO-EPIDERMAL SEPARATION WITH SUCTION ELECTRON MICROSCOPIC AND HISTOCHEMICAL STUDY OF INITIAL EVENTS OF BLISTERING ON HUMAN SKIN*
U. KIISTALA, M.D.t AND K. K. MUSTAKALLIO, M.D.
The natural adherence of the epidermis to osmium tetroxide. After dehydration in graded the corium is impressive. It has been impos- ethanol series the specimens were embedded in
812 (8). Thin sections for electron microssible to create a blister by injecting fluid Epon copy and 0.5 e thick sections for light microscopy
under maximal manual pressure directly into were cut with glass knives using a Porter-Blum the eorium (1), neither does the conspicuous MT-i microtome. The thin sections were stained dermal edema of an urtica result in blistering with lead citrate (9) and examined with a Siemens (2).
Even if human epidermis and dermis have
resisted practically any physical effort to
Elmiskop I. The 0.5 ji sections were stained with toluidine blue (10).t The following standard histochemical methods (ii) were applied to 10 e thick frozen sections cut immediately after removal of the biopsy specimens: PAS reaction for diastase-resistant non-sul-
separate them, this can be achieved by prolonged suction (3). Suction blistering occurs fated mueopolysaccharides; acid and alkaline phos-
at the dermo-epidermal junction Without pro- phatases according to Gomori; non-specific esterase found alterations in the microscopic structure using both alpha-naphthyl acetate and naph(3) or vitality (4) of the epidermis. At the thol-AS acetate as the substrate; leucine aminoultrastructural level the dermo-epidermal sep- peptidase; lactate dehydrogenase; succinate deglucose-6-phosphate dehydrogenase, aration was found to occur between the basal hydrogenase; and DOPA oxidase. cell membrane and the basement membrane beginning without any electron microscopic or OBSERVATIONS histochemical evidence of damage.
Mocrosopie blistering was preceded by minimal edema in the entire suction area.
MATERIAL AND METHOD5
The edema disappeared within a few minutes
The suction blisters studied were produced on the mid-volar forearm skin of six healthy young after discontinuation of suction. Petechiae men. The diameter of the suction cup of the de- were not formed. vice Dermovac® was 25 mm (5). To avoid damage,
During the suction, water-clear vesicles ap-
the pressure within the cup was maintained at 150 peared within 150 minutes first around the mmllg below the atmospheric pressure. To capture epidermal appendages. If suction is continued, the initial events of suction blistering, suction was discontinued instantly when the first tiny vesicles the ecerine sweat gland duets disrupt usually
became visible through the window of the suc- at the level of the upper dermis (Fig. 1). The tion cup. The required suction time averaged 120 hair follicles become either disrupted, demm. Immediately after removal of the cup, punch tached without sebaceous glands, or left behind biopsy specimens were taken without anesthesia attached to the blister base (Fig. 2).
Under light microscope the initial events
from sites with and without visible blistering. Control biopsy specimens were obtained from the cor-
of blistering were seen at sites devoid of overt vesiculation. The microscopic blisters ap-
responding area of the contralateral forearm. Altogether, 18 specimens were subjected for study. peared first on tops of dermal papillae (Fig. Small fragments of the biopsy specimens were fixed for one hour in 1 per cent osmium tetroxide 3). Only occasional flat basal cells remained adjusted to pH 7.4 with veronal-acetate (6). Al- attached to the dermis. The basal layer of the ternate fragments were fixed in 2 per cent gluta- detached epidermis often retained its normal
raldehyde for two hours (7) and post-fixed in
columnar pattern. Here, even the intercellular spaces appeared unwidened. In contrast, the * From the Electron Microscope Laboratory, still adherent epidermis at tips of rete ridges, University of Helsinki, and from the Department of Dermatology, University Central Hospital, where the individual keratinocytes and melSnellmaninkatu 14, Helsinki 17, Finland. t Assistant of the Finnish Medical Research t We are indebted to Miss Tellervo Huima for Received for publication October 3, 1966.
Council.
technical assistance.
466
467
DERMO-EPIDERMAL SEPARATION WITH SUCTION
Fia. 1. A stump of an ecerine sweat gland duct protruding from the underside of a suc-
tion blister roof. X 10.
ft
'ft
—i
I
FIG. 2. Hair follicle remaining attached to the suction blister base. Note the capillary
basket. X 10.
anocytcs protrude deeper in the dermis, showed
mained fixed on the dermis. These alterations
before detachment cellular stretching nnd were accentuated at the areas of epidermal widening of the intercellular spaces (Fig. 3). appendages where the dermo-epidermal anWhen the separation had proceeded over these choring is as strongest. The eeerine sweat sites, isolated basal cells not infrequently re- gland duets tended to become disrupted before
468
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
S
S
S
I"
Pt
-4'.
a
S —•
qi',
p
a
-
A
I,-
I..
•-:
spL
.4S
.',.,-t :- —.
—
- ,._ il4
- ..
— -2.
¶ 4.
II,
- .-.. S
IL
.
V S.,
-e 4'
54, S
• • --''I---.
- ft V
-a
Fm. 3. Two suction microblisters (02 mm in diameter) on tops of dermal papillae. The rete ridge in the center is almost released, that at left still tightly anchored. A few fiat epidermal cells remain attached to the dermis, which has a normal appearance without cxtravasation of blood-borne cells. A toluidine blue-stained epoxy section. X 180.
their entrance into the epidermis, but some- the basement membrane was still intact, aptimes also intracpidermally even at the level parently because of their true half-desmosomes. of the granular cell layer. Before actual blistering, the basal keratinoNeither the detached epidermis nor the un- cytes displayed inward herniation along the derlying dermis showed any histochemieally basal plasma membrane between the halfdetectable alterations. The PAS-stainability of desmosomes, which still anchored the cells to the topmost dermis was not lost. The distribu- the basement membrane (Fig. 6). Ultimately tion and activity of the hydrolases and the half-desmosomes were released from the dehydrogenases studied was normal (Fig. 4). basement membrane, and the epidermis was DOPA positivity was displayed not only by separated from the dermis in the plane between the melanoeytes of the epidermal roof but also the basal cell membranes and the basement by most of the few cells remaining adherent membrane proper. onto the blister base. In the roof of the primordial blisters, formed Under the electron microscope the initial on tops of dermal papillae, the epidermal cells events of dermo-epidermal separation were best showed little if any evidence of ultrastruc-
seen in the vicinity of microscopic blister- tural damage (Fig. 7). As a rule, the indiing. The first cell type involved in primordial vidually resolvable tonofilaments retained conblistering seemed to be the basal melanocyte tact with the intact-looking desmosomes and lacking true half-desmosomal contact to the cell membranes. basement membrane. It showed small spherical At rete ridges and around epidermal apinbulgings along the basal plasma membrane pendages the suction stretch often caused facing the basement membrane (Fig. 5). At widening of the intercellular spaces. Here the
this stage, the contact of the keratinocytes to tonofilaments sometimes were released from
469
DERMO-EPIDERMAL SEPARATION WITH SUCTION
the cell membranes, but the desmosomal con-
perimental blistering but also in several
tacts between the keratinoeytes usually re- diseases of the skin. There is evidence that in sisted the tension (Fig. 8). Occasionally, how- most junctionally blistering diseases the sep-
ever, some isolated basal keratinocytes re- aration is secondary either to epidermal damage (12, 13) or to some weakness in the mained anchored into the dermis.
A few keratinocytes, especially in the dermis (12—14). In blisters caused by friction stretched areas, showed a peculiar crescentic (15), thermal damage (16), or certain chemidisplacement of the otherwise normal-looking cals (13) the separation is probably secondary nucleus (Fig. 8). Such cavitation between the to epidermal damage. Against this background the mechanism of outer and inner nuclear membranes was never suction blistering is unique because the clean seen in the control specimens. The melanocytes differed from the other epi- intermembraneous separation seems to be of dermal cells in their response to suction, Not primary nature. There was neither inflammainfrequently melanocytes were left lying on tory nor autolytic (17) activation of lysosothe blister base despite the fact that inbulgings mal hydrolases. In the absence of any evidence along their basal cell membrane were the earliest of proteolysis the infrequently encountered detectable alterations induced by suction (Fig. partial dissolution of collagen may be of non9). This is conceivable because melanocytes lack enzymatic nature. The essentially non-inflamintercellular bridges with flanking keratino- matory suction blisters are ideally suited for cytes. Langerhans cells, on the contrary, were the study of cell dynamics in induced inflamnever seen attached to the blister base. Even mation (18). the intrabasal Langerhans cells, which lie in a basket, as if upon interlacing processes of basal
keratinocytes, were withdrawn with the epidermal blister roof.
In the dermis underlying the suction blisters only minor ultrastructural alterations were encountered. The detachment of the epidermis did not break the continuity of the basement membrane (Fig. 10). In tops of dermal papil-
lae the space between collagen fibrils was sometimes widened, and infrequently some col-
lagen fibrils showed splitting and rarefaction (Fig. 11). Deeper in the dermis the collagen bundles had a normal appearance.
The lymphatics encountered appeared distended but the papillary capillaries showed little if any evidence of dilatation (Fig. 10). Marked pinocytotic vesiculation was seen both
on the luminal and antiluminal face of the endothelial cells. Extravascular erythrocytes were seen only exceptionally during these initial stages of suction blistering, and there was neither margination nor diapedesis of leukocytes. Even the few pcricapillary mast cells encountered had retained their granules (Fig. 10).
.
DT5OU55ION
The mechanism by which the dermo-epidermal coherence is maintained is of more than academic interest, because the dermoFm. 4. Histochemically demonstrable lactate doepidermal junction is the site of major in- hydrogenase activity appears to be normal both volvement not only in certain types of cx- in the blister roof and in its base. X 90.
470
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
I'
W'T%L
S
•4
___
• ••4 }/
P.
E•
tt
FIG. 5. Initial events of dermo-epidermal separation at subcellular level. Fluid has accumulated through the basement membrane leading to inbulgings of the basal cell membrane of the melanocyte at the left. The haif-desmosomes of the keratinocyte at the right
still resist the pressure. X 53,000.
DERMO-EPIDERMAL SEPARATION WITH SUCTION
a Ftc. 6. Primordial herniation of the basal plasma membrane between baif-desmosomes
of a keratinocyte. X 24,000.
471
FIG. 7. Completed dermo-epidermal separation. Note the normal appearance of intracellular organdIes, tonofilaments, cell membranes, and desmosomes. A suprabasal Langerhans cell is also seen. This field is from a section adjacent to that depicted in Fig. 3. X 8,300.
7
4
hS
H
0
H
L"i
H
H
0
z
0
H
473
DERMO-EPIDERMAL SEPARATION WITH SUCTION
1'
•
"1kt?
b
'I
r Ft
I
FIG. 8. Spinous cells of a rete ridge stretched by suction. Note the crescentic displacement of nuclei and the release of tonofilaments from the plasma membrane and desmo-
somes. X
12,000.
474
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
a;
I,
t4ftI Vtm
Fm. 9. Partly detached melanocyte remaining in contact to the normal looking dermis.
Note the axon immediately below the melanocyte. >( 12,000.
Apparently the fluid accumulating between shown that circulatory and hydrostatic presthe basement membrane and the cell mem- sures enhance the rapidity of blistering. Apbranes of epidermal cells causes the junctional plication of a tourniquet proximally from the blistering. Some preliminary experiments have suction area or elevation of the extremity
;P 1:
..
'k. C
Fin. 10. Base of a suction blister with two flat epidermal cells. The basement membrane and the entire dermis, even the pencapillary mast cell, have a normal appearance. This field is from a section adjacent to those depicted in Fig. 3 and 7. x 2,900.
k' H C
cc
H
C
C
476
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Fm. 11. Limited area of topmost papillary dermis showing partial dissolution of collagen
fibrils. The longest flbnl has retained the 640 A periodicity. X 50,000.
slowed down the rate of blistering. With a ment membrane and the plasma membrane of prolonged suction junctional blisters could be epidermal cells. At rete ridges and around epiproduced also on corpse skin. dermal appendages suction stretch often caused widening of intercellular spaces. There, some sUMMARY melanocytes and even keratinocytes remained
Suction pressure of 150 mm Hg caused, adherent to the blister base. within three hours, dermo-epidermal separation of healthy human skin. Suction blister-
ing started on tops of dermal papillac. The cleavage occurred in the plane between the
In blister roof, the cells and their constituents generally had a normal ultrastructural appearance. As a rule, the dermis showed negligible alterations, and there was neither morphologic nor histochemical evidence of in-
basement membrane and the basal cell layer. The half-desmosomes of basal keratinocytes flammation or autolysis. were released from the dermis but the desREFERENCES mosomal cell to cell contact resisted the tension
of the fluid accumulating between the base-
1. Stoughton, R. B.: Mechanisms of blister formation. Arch. Derm. (Chicago), 76: 584, 1957.
DERMO-EPIDERMAL SEPARATION WITH SUCTION
2. Herzberg, J. J.: Die Genese der Blasenbildung. Arch. Kim. Exp. Derm., 218: 583, 1964.
3. Kiistala, U. and Mustakallio, K. K.: In-vivo separation of epidermis by production of suction blisters. Lancet, I: 1444, 1964.
4. Ingemansson-Nordqvist, B., Kiistala, U. and Rorsman, H.: Culture of adult human epi-
dermal cells obtained from roofs of suction blisters. Acta Dermatovener. (Stockholm), in press.
5. Kiistala, U.: Suction blister device for separation of viable epidermis from dermis, to be published.
6. Pnlade, G. E.: A study of fixation for electron microscopy. J. Exp. Med., 95: 285, 1952.
7. Sabatini, D. D., Bensch, K. and Barrnett, J.:
Cytochemistry and electron microscopy. The
preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17: 19, 1963. 8. Luft, J. H.: Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409, 1961.
9. Reynolds, E. S.: The use of lead citrate at
high p11 as an electron opaque stain in electron microscopy. J. Cell Biol., 17: 208, 1963.
10. Trump, B. F., Smuckler, E. A. and Benditt, E. P.: A method for staining epoxy sections. J. Ultrastruct. Res., 5: 343, 1961.
477
11. Pearse, A. G. E.: Histochemistry, Theoretical
and Applied, 2nd ed., London, J. & A. Churchill, Ltd., 1960.
12. Pearson, 11. W.: Studies on the pathogenesis of epidermolysis bullosa. J. Invest. Derm., 39: 551, 1962. 13. Pearson, R. W.: Some observations on epidermolysis bullosa and experimental blister-
ing, in The Epidermis, p. 613. Montagna, W. and Lohitz, W., Jr., editors, New York, Academic Press, 1964.
14. Caulfield, J. B. and Wilgram, G. F.: An electron microscopic study of blister formation in erythema multiforme. J. Invest. Derm., 39: 307, 1962.
15. Naylor, P. F. D.: Experimental friction blisters. Brit. J. Derm., 67: 327, 1955. 16. Pearson, 11. W.: Response of human epidermis
to graded thermal stress. Arch. Environ. Health (Chicago) 11: 498, 1965.
17. Braun-Falco, 0. and Winter, W.: Untersuchungen fiber die Autolyse der Haut. II. Histochemische Verknderungen. Arch. Kim. Exp. Derm., 220: 417, 1964.
18. Kiistala, U., Mustakalhn, K. K. and Rorsman, H.: Suction blisters in the study of cellular dynamics of inflammation. Acta Dermatovener. (Stockholm), in press.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1987 by The Williams & Wilkins Co.
DERMO-EPIDERMAL SEPARATION WITH SUCTION ELECTRON MICROSCOPIC AND HISTOCHEMICAL STUDY OF INITIAL EVENTS OF BLISTERING ON HUMAN SKIN*
U. KIISTALA, M.D.t AND K. K. MUSTAKALLIO, M.D.
The natural adherence of the epidermis to osmium tetroxide. After dehydration in graded the corium is impressive. It has been impos- ethanol series the specimens were embedded in
812 (8). Thin sections for electron microssible to create a blister by injecting fluid Epon copy and 0.5 e thick sections for light microscopy
under maximal manual pressure directly into were cut with glass knives using a Porter-Blum the eorium (1), neither does the conspicuous MT-i microtome. The thin sections were stained dermal edema of an urtica result in blistering with lead citrate (9) and examined with a Siemens (2).
Even if human epidermis and dermis have
resisted practically any physical effort to
Elmiskop I. The 0.5 ji sections were stained with toluidine blue (10).t The following standard histochemical methods (ii) were applied to 10 e thick frozen sections cut immediately after removal of the biopsy specimens: PAS reaction for diastase-resistant non-sul-
separate them, this can be achieved by prolonged suction (3). Suction blistering occurs fated mueopolysaccharides; acid and alkaline phos-
at the dermo-epidermal junction Without pro- phatases according to Gomori; non-specific esterase found alterations in the microscopic structure using both alpha-naphthyl acetate and naph(3) or vitality (4) of the epidermis. At the thol-AS acetate as the substrate; leucine aminoultrastructural level the dermo-epidermal sep- peptidase; lactate dehydrogenase; succinate deglucose-6-phosphate dehydrogenase, aration was found to occur between the basal hydrogenase; and DOPA oxidase. cell membrane and the basement membrane beginning without any electron microscopic or OBSERVATIONS histochemical evidence of damage.
Mocrosopie blistering was preceded by minimal edema in the entire suction area.
MATERIAL AND METHOD5
The edema disappeared within a few minutes
The suction blisters studied were produced on the mid-volar forearm skin of six healthy young after discontinuation of suction. Petechiae men. The diameter of the suction cup of the de- were not formed. vice Dermovac® was 25 mm (5). To avoid damage,
During the suction, water-clear vesicles ap-
the pressure within the cup was maintained at 150 peared within 150 minutes first around the mmllg below the atmospheric pressure. To capture epidermal appendages. If suction is continued, the initial events of suction blistering, suction was discontinued instantly when the first tiny vesicles the ecerine sweat gland duets disrupt usually
became visible through the window of the suc- at the level of the upper dermis (Fig. 1). The tion cup. The required suction time averaged 120 hair follicles become either disrupted, demm. Immediately after removal of the cup, punch tached without sebaceous glands, or left behind biopsy specimens were taken without anesthesia attached to the blister base (Fig. 2).
Under light microscope the initial events
from sites with and without visible blistering. Control biopsy specimens were obtained from the cor-
of blistering were seen at sites devoid of overt vesiculation. The microscopic blisters ap-
responding area of the contralateral forearm. Altogether, 18 specimens were subjected for study. peared first on tops of dermal papillae (Fig. Small fragments of the biopsy specimens were fixed for one hour in 1 per cent osmium tetroxide 3). Only occasional flat basal cells remained adjusted to pH 7.4 with veronal-acetate (6). Al- attached to the dermis. The basal layer of the ternate fragments were fixed in 2 per cent gluta- detached epidermis often retained its normal
raldehyde for two hours (7) and post-fixed in
columnar pattern. Here, even the intercellular spaces appeared unwidened. In contrast, the * From the Electron Microscope Laboratory, still adherent epidermis at tips of rete ridges, University of Helsinki, and from the Department of Dermatology, University Central Hospital, where the individual keratinocytes and melSnellmaninkatu 14, Helsinki 17, Finland. t Assistant of the Finnish Medical Research t We are indebted to Miss Tellervo Huima for Received for publication October 3, 1966.
Council.
technical assistance.
466
467
DERMO-EPIDERMAL SEPARATION WITH SUCTION
Fia. 1. A stump of an ecerine sweat gland duct protruding from the underside of a suc-
tion blister roof. X 10.
ft
'ft
—i
I
FIG. 2. Hair follicle remaining attached to the suction blister base. Note the capillary
basket. X 10.
anocytcs protrude deeper in the dermis, showed
mained fixed on the dermis. These alterations
before detachment cellular stretching nnd were accentuated at the areas of epidermal widening of the intercellular spaces (Fig. 3). appendages where the dermo-epidermal anWhen the separation had proceeded over these choring is as strongest. The eeerine sweat sites, isolated basal cells not infrequently re- gland duets tended to become disrupted before
468
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
S
S
S
I"
Pt
-4'.
a
S —•
qi',
p
a
-
A
I,-
I..
•-:
spL
.4S
.',.,-t :- —.
—
- ,._ il4
- ..
— -2.
¶ 4.
II,
- .-.. S
IL
.
V S.,
-e 4'
54, S
• • --''I---.
- ft V
-a
Fm. 3. Two suction microblisters (02 mm in diameter) on tops of dermal papillae. The rete ridge in the center is almost released, that at left still tightly anchored. A few fiat epidermal cells remain attached to the dermis, which has a normal appearance without cxtravasation of blood-borne cells. A toluidine blue-stained epoxy section. X 180.
their entrance into the epidermis, but some- the basement membrane was still intact, aptimes also intracpidermally even at the level parently because of their true half-desmosomes. of the granular cell layer. Before actual blistering, the basal keratinoNeither the detached epidermis nor the un- cytes displayed inward herniation along the derlying dermis showed any histochemieally basal plasma membrane between the halfdetectable alterations. The PAS-stainability of desmosomes, which still anchored the cells to the topmost dermis was not lost. The distribu- the basement membrane (Fig. 6). Ultimately tion and activity of the hydrolases and the half-desmosomes were released from the dehydrogenases studied was normal (Fig. 4). basement membrane, and the epidermis was DOPA positivity was displayed not only by separated from the dermis in the plane between the melanoeytes of the epidermal roof but also the basal cell membranes and the basement by most of the few cells remaining adherent membrane proper. onto the blister base. In the roof of the primordial blisters, formed Under the electron microscope the initial on tops of dermal papillae, the epidermal cells events of dermo-epidermal separation were best showed little if any evidence of ultrastruc-
seen in the vicinity of microscopic blister- tural damage (Fig. 7). As a rule, the indiing. The first cell type involved in primordial vidually resolvable tonofilaments retained conblistering seemed to be the basal melanocyte tact with the intact-looking desmosomes and lacking true half-desmosomal contact to the cell membranes. basement membrane. It showed small spherical At rete ridges and around epidermal apinbulgings along the basal plasma membrane pendages the suction stretch often caused facing the basement membrane (Fig. 5). At widening of the intercellular spaces. Here the
this stage, the contact of the keratinocytes to tonofilaments sometimes were released from
469
DERMO-EPIDERMAL SEPARATION WITH SUCTION
the cell membranes, but the desmosomal con-
perimental blistering but also in several
tacts between the keratinoeytes usually re- diseases of the skin. There is evidence that in sisted the tension (Fig. 8). Occasionally, how- most junctionally blistering diseases the sep-
ever, some isolated basal keratinocytes re- aration is secondary either to epidermal damage (12, 13) or to some weakness in the mained anchored into the dermis.
A few keratinocytes, especially in the dermis (12—14). In blisters caused by friction stretched areas, showed a peculiar crescentic (15), thermal damage (16), or certain chemidisplacement of the otherwise normal-looking cals (13) the separation is probably secondary nucleus (Fig. 8). Such cavitation between the to epidermal damage. Against this background the mechanism of outer and inner nuclear membranes was never suction blistering is unique because the clean seen in the control specimens. The melanocytes differed from the other epi- intermembraneous separation seems to be of dermal cells in their response to suction, Not primary nature. There was neither inflammainfrequently melanocytes were left lying on tory nor autolytic (17) activation of lysosothe blister base despite the fact that inbulgings mal hydrolases. In the absence of any evidence along their basal cell membrane were the earliest of proteolysis the infrequently encountered detectable alterations induced by suction (Fig. partial dissolution of collagen may be of non9). This is conceivable because melanocytes lack enzymatic nature. The essentially non-inflamintercellular bridges with flanking keratino- matory suction blisters are ideally suited for cytes. Langerhans cells, on the contrary, were the study of cell dynamics in induced inflamnever seen attached to the blister base. Even mation (18). the intrabasal Langerhans cells, which lie in a basket, as if upon interlacing processes of basal
keratinocytes, were withdrawn with the epidermal blister roof.
In the dermis underlying the suction blisters only minor ultrastructural alterations were encountered. The detachment of the epidermis did not break the continuity of the basement membrane (Fig. 10). In tops of dermal papil-
lae the space between collagen fibrils was sometimes widened, and infrequently some col-
lagen fibrils showed splitting and rarefaction (Fig. 11). Deeper in the dermis the collagen bundles had a normal appearance.
The lymphatics encountered appeared distended but the papillary capillaries showed little if any evidence of dilatation (Fig. 10). Marked pinocytotic vesiculation was seen both
on the luminal and antiluminal face of the endothelial cells. Extravascular erythrocytes were seen only exceptionally during these initial stages of suction blistering, and there was neither margination nor diapedesis of leukocytes. Even the few pcricapillary mast cells encountered had retained their granules (Fig. 10).
.
DT5OU55ION
The mechanism by which the dermo-epidermal coherence is maintained is of more than academic interest, because the dermoFm. 4. Histochemically demonstrable lactate doepidermal junction is the site of major in- hydrogenase activity appears to be normal both volvement not only in certain types of cx- in the blister roof and in its base. X 90.
470
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
I'
W'T%L
S
•4
___
• ••4 }/
P.
E•
tt
FIG. 5. Initial events of dermo-epidermal separation at subcellular level. Fluid has accumulated through the basement membrane leading to inbulgings of the basal cell membrane of the melanocyte at the left. The haif-desmosomes of the keratinocyte at the right
still resist the pressure. X 53,000.
DERMO-EPIDERMAL SEPARATION WITH SUCTION
a Ftc. 6. Primordial herniation of the basal plasma membrane between baif-desmosomes
of a keratinocyte. X 24,000.
471
FIG. 7. Completed dermo-epidermal separation. Note the normal appearance of intracellular organdIes, tonofilaments, cell membranes, and desmosomes. A suprabasal Langerhans cell is also seen. This field is from a section adjacent to that depicted in Fig. 3. X 8,300.
7
4
hS
H
0
H
L"i
H
H
0
z
0
H
473
DERMO-EPIDERMAL SEPARATION WITH SUCTION
1'
•
"1kt?
b
'I
r Ft
I
FIG. 8. Spinous cells of a rete ridge stretched by suction. Note the crescentic displacement of nuclei and the release of tonofilaments from the plasma membrane and desmo-
somes. X
12,000.
474
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
a;
I,
t4ftI Vtm
Fm. 9. Partly detached melanocyte remaining in contact to the normal looking dermis.
Note the axon immediately below the melanocyte. >( 12,000.
Apparently the fluid accumulating between shown that circulatory and hydrostatic presthe basement membrane and the cell mem- sures enhance the rapidity of blistering. Apbranes of epidermal cells causes the junctional plication of a tourniquet proximally from the blistering. Some preliminary experiments have suction area or elevation of the extremity
;P 1:
..
'k. C
Fin. 10. Base of a suction blister with two flat epidermal cells. The basement membrane and the entire dermis, even the pencapillary mast cell, have a normal appearance. This field is from a section adjacent to those depicted in Fig. 3 and 7. x 2,900.
k' H C
cc
H
C
C
476
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Fm. 11. Limited area of topmost papillary dermis showing partial dissolution of collagen
fibrils. The longest flbnl has retained the 640 A periodicity. X 50,000.
slowed down the rate of blistering. With a ment membrane and the plasma membrane of prolonged suction junctional blisters could be epidermal cells. At rete ridges and around epiproduced also on corpse skin. dermal appendages suction stretch often caused widening of intercellular spaces. There, some sUMMARY melanocytes and even keratinocytes remained
Suction pressure of 150 mm Hg caused, adherent to the blister base. within three hours, dermo-epidermal separation of healthy human skin. Suction blister-
ing started on tops of dermal papillac. The cleavage occurred in the plane between the
In blister roof, the cells and their constituents generally had a normal ultrastructural appearance. As a rule, the dermis showed negligible alterations, and there was neither morphologic nor histochemical evidence of in-
basement membrane and the basal cell layer. The half-desmosomes of basal keratinocytes flammation or autolysis. were released from the dermis but the desREFERENCES mosomal cell to cell contact resisted the tension
of the fluid accumulating between the base-
1. Stoughton, R. B.: Mechanisms of blister formation. Arch. Derm. (Chicago), 76: 584, 1957.
DERMO-EPIDERMAL SEPARATION WITH SUCTION
2. Herzberg, J. J.: Die Genese der Blasenbildung. Arch. Kim. Exp. Derm., 218: 583, 1964.
3. Kiistala, U. and Mustakallio, K. K.: In-vivo separation of epidermis by production of suction blisters. Lancet, I: 1444, 1964.
4. Ingemansson-Nordqvist, B., Kiistala, U. and Rorsman, H.: Culture of adult human epi-
dermal cells obtained from roofs of suction blisters. Acta Dermatovener. (Stockholm), in press.
5. Kiistala, U.: Suction blister device for separation of viable epidermis from dermis, to be published.
6. Pnlade, G. E.: A study of fixation for electron microscopy. J. Exp. Med., 95: 285, 1952.
7. Sabatini, D. D., Bensch, K. and Barrnett, J.:
Cytochemistry and electron microscopy. The
preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17: 19, 1963. 8. Luft, J. H.: Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409, 1961.
9. Reynolds, E. S.: The use of lead citrate at
high p11 as an electron opaque stain in electron microscopy. J. Cell Biol., 17: 208, 1963.
10. Trump, B. F., Smuckler, E. A. and Benditt, E. P.: A method for staining epoxy sections. J. Ultrastruct. Res., 5: 343, 1961.
477
11. Pearse, A. G. E.: Histochemistry, Theoretical
and Applied, 2nd ed., London, J. & A. Churchill, Ltd., 1960.
12. Pearson, 11. W.: Studies on the pathogenesis of epidermolysis bullosa. J. Invest. Derm., 39: 551, 1962. 13. Pearson, R. W.: Some observations on epidermolysis bullosa and experimental blister-
ing, in The Epidermis, p. 613. Montagna, W. and Lohitz, W., Jr., editors, New York, Academic Press, 1964.
14. Caulfield, J. B. and Wilgram, G. F.: An electron microscopic study of blister formation in erythema multiforme. J. Invest. Derm., 39: 307, 1962.
15. Naylor, P. F. D.: Experimental friction blisters. Brit. J. Derm., 67: 327, 1955. 16. Pearson, 11. W.: Response of human epidermis
to graded thermal stress. Arch. Environ. Health (Chicago) 11: 498, 1965.
17. Braun-Falco, 0. and Winter, W.: Untersuchungen fiber die Autolyse der Haut. II. Histochemische Verknderungen. Arch. Kim. Exp. Derm., 220: 417, 1964.
18. Kiistala, U., Mustakalhn, K. K. and Rorsman, H.: Suction blisters in the study of cellular dynamics of inflammation. Acta Dermatovener. (Stockholm), in press.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.