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A Method for the Separation of the Epidermis from the Dermis
PRELIMINARY AND SHORT REPORTS A METHOD FOR THE SEPARATION OF THE EPIDERMIS FROM THE DERMIS* IRVIN H. BLANK, PH.D. AND OWEN 0. MILLER, M.D.
As one part of a study of the ability of the skin to transmit water, we attempted to pull water through a piece of skin by the use of a vacuum. A piece of human abdominal skin, obtained at the autopsy table, was tightly wired across the top of a heavy-walled thistle tube so that the dermal side was outside. The tube was inverted and the dermal side of the skin was allowed to dip into water, but the edge of the piece of skin was kept above the Water level. The tube was then attached to a vacuum pump. It is seen that in this arrangement any passage of water is in a direction corresponding to a passage of water from the inside of the body out through the skin. .1
3cm.
a.,—
Fin. I. BULLAR FomuEn DURING AN ATTEMPT TO PULL WATER THROUGH THE SKIN Wr MEANS Oa A VACUUM.
No visible amount of water passed entirely through the skin. After a period of time, however, bullae began to form on the skin. When the pressure within the thistle tube was held at approximately 150 mm. of mercury (a pressure difference of approximately 610 mm. tending to force water through the skin), the time required for the bullae to form varied from one to two hours for different pieces of skin. One such piece of skin is shown in Figure I. When the pressure was reduced to 10 mm. of mercury (pressure difference of approximately 750 mm.), bullae sometimes formed in from 15 to 20 minutes. From the histological section of one of these bullae, as shown in Figure II, it can be seen that the separation occurs at the dermal-epidermal junction. This technic therefore becomes a method whereby small pieces of epidermis may be obtained free from the der*From the Department of Dermatology, Harvard University Medical School, Massachusetts General Hospital, Boston, Massachusetts. Received for publication February 5, 1950. 9
10
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
mis. It has the advantage over methods previously used for making this separation in that it is unnecessary to subject the skin to heat, chemicals, enzymes or alteration of pH. These bullae form probably as a result of a pressure differential at different points in the skin due to a variation in the permeability of the different layers of skin to water. The least permeable layer is in the epidermis. When the epidermis is scratched or broken, water passes through the skin and the bullac do not form because the pressure differentials do not arise.
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—
•
'a'I'.--.
''4 —
-
t
a
I'
—-
—
I-
—
——
Fin. II. RrsToLoorcAL SECTION OF THE BULLAE SHOWN IN FIOTJEE I.
We are unable to obtain this epidermal separation when rabbit skin is used. Rabbit skin does not transmit visible amounts of water at 150 nun, pressure, but the pressure differentials which occur within the skin seem to be insufficient to separate the epidermis from the dermis when the epidermis is more firmly held to the dermis by the frequent invagination of hair follicles. At a pressure of 10 mm. some water is transmitted by rabbit skin after about five minutes but no bullae are formed. Histological sections from the pieces of rabbit skin which transmit water show breaks in the epidermis that may have been caused by the stretching of the skin by the vacuum.
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.
As one part of a study of the ability of the skin to transmit water, we attempted to pull water through a piece of skin by the use of a vacuum. A piece of human abdominal skin, obtained at the autopsy table, was tightly wired across the top of a heavy-walled thistle tube so that the dermal side was outside. The tube was inverted and the dermal side of the skin was allowed to dip into water, but the edge of the piece of skin was kept above the Water level. The tube was then attached to a vacuum pump. It is seen that in this arrangement any passage of water is in a direction corresponding to a passage of water from the inside of the body out through the skin. .1
3cm.
a.,—
Fin. I. BULLAR FomuEn DURING AN ATTEMPT TO PULL WATER THROUGH THE SKIN Wr MEANS Oa A VACUUM.
No visible amount of water passed entirely through the skin. After a period of time, however, bullae began to form on the skin. When the pressure within the thistle tube was held at approximately 150 mm. of mercury (a pressure difference of approximately 610 mm. tending to force water through the skin), the time required for the bullae to form varied from one to two hours for different pieces of skin. One such piece of skin is shown in Figure I. When the pressure was reduced to 10 mm. of mercury (pressure difference of approximately 750 mm.), bullae sometimes formed in from 15 to 20 minutes. From the histological section of one of these bullae, as shown in Figure II, it can be seen that the separation occurs at the dermal-epidermal junction. This technic therefore becomes a method whereby small pieces of epidermis may be obtained free from the der*From the Department of Dermatology, Harvard University Medical School, Massachusetts General Hospital, Boston, Massachusetts. Received for publication February 5, 1950. 9
10
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
mis. It has the advantage over methods previously used for making this separation in that it is unnecessary to subject the skin to heat, chemicals, enzymes or alteration of pH. These bullae form probably as a result of a pressure differential at different points in the skin due to a variation in the permeability of the different layers of skin to water. The least permeable layer is in the epidermis. When the epidermis is scratched or broken, water passes through the skin and the bullac do not form because the pressure differentials do not arise.
'S
—
•
'a'I'.--.
''4 —
-
t
a
I'
—-
—
I-
—
——
Fin. II. RrsToLoorcAL SECTION OF THE BULLAE SHOWN IN FIOTJEE I.
We are unable to obtain this epidermal separation when rabbit skin is used. Rabbit skin does not transmit visible amounts of water at 150 nun, pressure, but the pressure differentials which occur within the skin seem to be insufficient to separate the epidermis from the dermis when the epidermis is more firmly held to the dermis by the frequent invagination of hair follicles. At a pressure of 10 mm. some water is transmitted by rabbit skin after about five minutes but no bullae are formed. Histological sections from the pieces of rabbit skin which transmit water show breaks in the epidermis that may have been caused by the stretching of the skin by the vacuum.
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.