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Electron microscopic investigations of the cryolesion
3
Electron Microscopic Investigations of the Cryolesion
Eckhard W. Breitbart, MD, and Gertrud Schaeg
From the Department of Dermatology, University of Hamburg, Hamburg, Federal Republic of Germany
An understanding of the morphologic changes that occur after cryosurgery is important in considering the applications of cryosurgery in dermatology. Because our histologic investigations provided little information concerning the actual cell destruction, only the electron microscopic findings will be discussed here. We have found that cold-induced changes do not become visible using light microscopy before 6 hours; however, electron microscopic studies show that by this time there is clear evidence of complete cytolysis. The characteristic cold-induced findings have already been described. Reports of Breitbart (1978), Uyeda et al (1977) and Weiss and Armstrong (1960) are in agreement that because visible histologic changes do not occur until the second freeze-thaw cycle, earlier changes can only be visualized by electron microscopy. Method Directly following biopsy, the tissue was fixed in a cooled 1% solution of osmium acid in 0.1 m cacodylate buffer (ph 7.5). The samples were then washed in buffer solution, dehydrated with alcohol, and embedded in Epon. The ultrathin sections were contrasted with uranyl acetate and lead citrate. An EM 9Ss Zeiss microscope was used. Results
Arter second Freezemow cycle For normal skin prior to cryosurgery, see Figures 3-l-3-4. After the second freeze-thaw cycle, clear morphologic changes are visible under the electron microscope. Cell swelling begins in the epidermis (Fig. 3-5). The cytoplasm is more homogeneous, cell organelles disappear, and the fragmented tonofibrils move to the edge of the cell. The nucleus is partially deformed and the nuclear membrane shows an osmiophilic thickening (Fig. 3-6). Isolated clumping of the chromatin is observed. The nuclear membrane is ruptured and karyoplasm flows out (Figs. 3-7 and 3-8). 30
January-March Volume 8 Number 1
1990 Electronic
skin FIG. 3-l. Normal before cryosurgery: Stratum spinosum. Nuclei (K) are round, chromatin distribution is homogeneous. Regular formation of tonofibrils (Tf) and desmosomes (D) and melanocytes (M). (X 3,600)
FIG. 3-2. Normal skin before cryosurgery: melanocyte, melanosome (M), Golgi zone (G), mitochondria (Mi). (X 19,ooo)
FIG. 3-3. Normal skin before cryosurgery: basement membrane, membrane of keratinocyte with half desmosomes (arrows) and lamina densa (top of arrows). (X 21,400)
Microscopic
lnvestigatlons
31
Clinics in
32
E. W. Breitbart
and G. Schaeg
Dermatology
skin FIG. 3-4. Normal before cryosurgery: collagen fibers in different sections; elastic fibers (E). (X 19,000)
FIG. 3-5. Second freeze-thaw cycle: the upper strafum spinosum, stratum granulosum. Perinuclear cell edema (stars), interruption and osmiophilia of the nuclear membrane. Intercellular spaces are not expanded. (X 3,600)
FIG. 3-6. Second freeze-thaw cycle: unidentified cutaneous cell. Deformation of the nucleus and osmiophilic condensation of the cell wall; mitochondria (Mi), Golgi zone (G). Fragmented tonofibrils (Tf) in the surrounding keratinocytes. (X 9,400)
January-March Volume 8 Number 1
1990 Electronic
Microscopic
Investigations
33
FIG. 3-7. Second freeze-thaw cycle: cytolysis of keratinocytes in the lower stratum spinosum. Clumped chromatin and beginning of nuclear membrane hyperchromatosis. With rupture of the nuclear membrane, the karyoplasm flows out (arrow); nucleus (K), ribosomes (R), desmosomes (D) intact. (X 9,400)
In the lower epidermis, more cells are obviously affected than in the upper epidermis (stratum ~U&OSUT$. The unidentified cells demonstrate pathologic changes earlier than the keratinocytes (Fig. 3-6). The desmosomes are intact and the intercellular spaces are not expanded (Fig. 3-5). The basement membrane appears totally unchanged (Fig. 3-7). On the whole, the vascular endothelium is inconspicuous (Fig. 3-10). Inter-vascular cell debris is observed. Threadlike parts of connective tissue are inconspicuous (Fig. 3-9). 6 Hours Alter Cryosurgery Distinct cell destruction
in the epidermis
FIG. 3-6. Second freeze-thaw cycle: nuclear material flows out into the cytoplasma (arrows); nucleus (K), rough endoplasmatic reticulum = RER (ER). (X 56,OCKI)
and corium is visible after 6 hours (Fig. 311). Advanced cytolysis is evident through the strangulation of numerous matrix vesicles (Fig. 3-12). Particular destruction is evident in the lower epidermal area. The desmosomes appear intact and detach as complete structures from the cell membranes (Fig. 3-13). The intercellular spaces are open, and massive cell debris is observed (Fig. 3-12). The fragmented tonofibrils build myelin figures. The basement membrane appears to be intact (Fig. 3-13). Nuclear destruction of different degrees occurs, for example, clumped chromatin/karyolysis. The strong osmiophilic character of the nuclear membrane disappears. The vascular endothelium (Fig. 3-15)
Cllnfcs in
34
E. W. Breitbart
and G. Schaeg
Dermatology
FIG. 3-9. Second freeze-thaw cycle: fibrocyte in the upper corium. Early stages of cytolysis as seen by cytoplasmic vesicles (arrow). Highly osmiophilic nuclear membrane and osmiophilic ribosomes on the RER (ER). (X 11,300)
FIG. 3-10. Second freeze-thaw cycle: blood vessel. Cells of the endothelium (En) appear unchanged; basal lamina (BL) shown intact; shadow of erythrocytes (E); intravascular debris (arrow). (X 12,gOO)
FIG. 3-11. Six after hours cryosurgery: Stratum basale, advanced evidence of nuclear cytolysis; destruction. Basement membrane (BM). (X 3.600)
January-March 19% Volume 8 Number 1
Electronic Microscopic Investigations
FIG. 3-12. Six hours after cryosurgery: Stratum base/e. Expanded intercellular spaces (stars). Desmosomes (D) are intact but detached. Strangulation of matrix vesicles in the intercellular spaces (fop of arrows). (X WW
FIG. 3-13. Six hours after cryosurgery: basement membrane. Intact lamina densa (top of arrows). Focal interruption of the keratinocyte membrane (arrow). Myelin figures (My). Strangulation of matrix vesicles in the expanded intercellular spaces (stars). (X 19,ooo)
FIG. 3-14. Six hours after cryosurgery: damaged fibrocyte (F) in the upper corium. Condensed cytoplasm. Cytoplasmic vesiculation (arrows). (X 9,400)
35
E. W. Breitbari
36
Clinics in Dermatology
and G. Schaeg
FIG. 3-15. Six hours after cryosurgery: vessel. Destruction and vesiculation (V) of the endothelium. Basal lamina (BL) intact. Damaged cells (Z) intravascular. (X 19,000)
and connective tissue are severely damaged and vesiculation and vesicle formation are seen (Figs. 3-14 and 3-16). Collagen remains unchanged. 24 HoursNter
cryosurgery
Extensive blistering of the stratum basale is seen, although the lamina densa of the basement membrane is preserved. The keratinocyte cell membrane, containing half desmosomes, detaches in the interior of the blister (Fig. 3-17). The epidermis detaches as a whole (Fig. 3-18). The connective tissue cells show a similar degree of degeneration 6 hours after cryosurgery. The capillary wall structure has almost disintegrated. Lyosomes are
present, both inter- and extracellularly (Fig. 3-19). Collagen is swollen, although the periodicity of collagen is preserved.
48 and 72 Hours After Cryosurgery Biopsies taken after 48 and 72 hours show no substantial changes in comparison with the above findings, with only gradual alterations in degree of necrosis.
Discussion From the electron microscopic findings, the effect of intracellular crystallization is appar-
FIG. 3-16. Six hours after cryosurgery: vessel. Nuclear hyperchromasia of the endothelium cells. Aggregation of erythrocytes (E). Numerous cell debris (stars). Fibrocyte (F). Collagen (Co). (X 4,600)
January-March Volume 8 Number 1
IWO
Electronic Microscopic Investigations
37
ent with the rupture of nuclear membrane followed by a loss of karyoplasm. Cell edema is a consequence of cell death and is evident in the homogeneous cytoplasm, the disappearance of organelles, fragmentation of tonofibrils, and clumping of chromatin. Similar changes have been described by Schattenberg et al (19’78) and Helpap (1980) upon freezing the liver. The transitory osmiophilic thickening of the nuclear membrane (described by Lovelock (1957), as a consequence of the denaturation of membrane lipoproteins remains unsettled. Furthermore, the destruction of the unidentified cells occurs earlier than that of fibroblasts and keratinocytes. This is strongly suggested by our own observations. We observed hardly any changes in the basement membrane and desmosomes, although distinct alterations of the blood vessels could be seen. This confirms earlier reports by Uyeda et al (1977,1978).
Conclusion Our investigations for the practical application of cryosurgery demonstrate that the damage of all cell structures can be seen after the second freeze-thaw cycle.
FIG. 3-18. Twenty-four hours after cryosurgery: Stratum spinosum. Advanced cytolysis; large empty areas observed in the perinuclear zone. (X 3,600)
FIG. 3-17. Twenty-four hours after cryosurgery: formation of blister (BI). The lamina densa is intact (arrow). Cytolysis (Z); collagen (Co). (X 4,000)
38
E. W. Breitbari
Clinics in Dermatology
and G. Schaeg
FIG. 3-19.
/ f
References Breitbart EW. Neue Gesichtapunkte in der kryochirurgischen Behandlung von Neubildungen der Haut. In: Salfeld, ed. Operative Dermatologie. Berlin: Springer-Verlag, 1978~230-233. Helpap B. Der kryochirurgische Eingriff und seine Folgen. In: Doerr, Leonhardt, eds. Morphologische und zellkinetische Analyse. Stuttgart: George Thieme Verlay, 1980:65-70. Lovelock JE. The denaturation of lipid-protein complexes as a cause of damage by freezing. Pioc Roy Sot Med. 1957;147:427-433.
Address for correspondence: Eckhard Universitats-Hautklinik und -Poliklinik, Republic of Germany.
Twenty-four
hours
after
cryosurgery: vessel. Damaged endothelial cell (En);fragmentsof nuclei (K); intravascular debris and lyosomes (stars); erythrocytes (E). (X 5,500)
4. Schattenberg PJ, Totovic V, Helpap B, et al. Ultrastructure of early changes in circumscribed cryonecrosis. Path01 Res Pratt. 1978;163:334-352. 5. Uyeda K, Nakayasu K, Kishimoto S, et al. Electron microscopic studies of cryosurgery in the dermatological field. In: Proceedings of the XV International Congress in Dermatology, Mexico. 1977;565-567. 6. Uyeda K, Nakayasu K, Kishimoto S, et al. Electron microscopic observation of epidermal cells and infiltrated cells in the verrucous lesions after cryosurgery. J Clin Electron Microscopy. lS79;12:5-6. 7. Weiss L, Armstrong JA. Structural changes in mammalian cells associated with cooling to -76°C. J Biophys Biochem Cytol. 1960;7:673-677.
W. Breitbart, Martinistrasse
MD, Department of Dermatology, 52, D-2000, Hamburg 20, Federal
Electron Microscopic Investigations of the Cryolesion
Eckhard W. Breitbart, MD, and Gertrud Schaeg
From the Department of Dermatology, University of Hamburg, Hamburg, Federal Republic of Germany
An understanding of the morphologic changes that occur after cryosurgery is important in considering the applications of cryosurgery in dermatology. Because our histologic investigations provided little information concerning the actual cell destruction, only the electron microscopic findings will be discussed here. We have found that cold-induced changes do not become visible using light microscopy before 6 hours; however, electron microscopic studies show that by this time there is clear evidence of complete cytolysis. The characteristic cold-induced findings have already been described. Reports of Breitbart (1978), Uyeda et al (1977) and Weiss and Armstrong (1960) are in agreement that because visible histologic changes do not occur until the second freeze-thaw cycle, earlier changes can only be visualized by electron microscopy. Method Directly following biopsy, the tissue was fixed in a cooled 1% solution of osmium acid in 0.1 m cacodylate buffer (ph 7.5). The samples were then washed in buffer solution, dehydrated with alcohol, and embedded in Epon. The ultrathin sections were contrasted with uranyl acetate and lead citrate. An EM 9Ss Zeiss microscope was used. Results
Arter second Freezemow cycle For normal skin prior to cryosurgery, see Figures 3-l-3-4. After the second freeze-thaw cycle, clear morphologic changes are visible under the electron microscope. Cell swelling begins in the epidermis (Fig. 3-5). The cytoplasm is more homogeneous, cell organelles disappear, and the fragmented tonofibrils move to the edge of the cell. The nucleus is partially deformed and the nuclear membrane shows an osmiophilic thickening (Fig. 3-6). Isolated clumping of the chromatin is observed. The nuclear membrane is ruptured and karyoplasm flows out (Figs. 3-7 and 3-8). 30
January-March Volume 8 Number 1
1990 Electronic
skin FIG. 3-l. Normal before cryosurgery: Stratum spinosum. Nuclei (K) are round, chromatin distribution is homogeneous. Regular formation of tonofibrils (Tf) and desmosomes (D) and melanocytes (M). (X 3,600)
FIG. 3-2. Normal skin before cryosurgery: melanocyte, melanosome (M), Golgi zone (G), mitochondria (Mi). (X 19,ooo)
FIG. 3-3. Normal skin before cryosurgery: basement membrane, membrane of keratinocyte with half desmosomes (arrows) and lamina densa (top of arrows). (X 21,400)
Microscopic
lnvestigatlons
31
Clinics in
32
E. W. Breitbart
and G. Schaeg
Dermatology
skin FIG. 3-4. Normal before cryosurgery: collagen fibers in different sections; elastic fibers (E). (X 19,000)
FIG. 3-5. Second freeze-thaw cycle: the upper strafum spinosum, stratum granulosum. Perinuclear cell edema (stars), interruption and osmiophilia of the nuclear membrane. Intercellular spaces are not expanded. (X 3,600)
FIG. 3-6. Second freeze-thaw cycle: unidentified cutaneous cell. Deformation of the nucleus and osmiophilic condensation of the cell wall; mitochondria (Mi), Golgi zone (G). Fragmented tonofibrils (Tf) in the surrounding keratinocytes. (X 9,400)
January-March Volume 8 Number 1
1990 Electronic
Microscopic
Investigations
33
FIG. 3-7. Second freeze-thaw cycle: cytolysis of keratinocytes in the lower stratum spinosum. Clumped chromatin and beginning of nuclear membrane hyperchromatosis. With rupture of the nuclear membrane, the karyoplasm flows out (arrow); nucleus (K), ribosomes (R), desmosomes (D) intact. (X 9,400)
In the lower epidermis, more cells are obviously affected than in the upper epidermis (stratum ~U&OSUT$. The unidentified cells demonstrate pathologic changes earlier than the keratinocytes (Fig. 3-6). The desmosomes are intact and the intercellular spaces are not expanded (Fig. 3-5). The basement membrane appears totally unchanged (Fig. 3-7). On the whole, the vascular endothelium is inconspicuous (Fig. 3-10). Inter-vascular cell debris is observed. Threadlike parts of connective tissue are inconspicuous (Fig. 3-9). 6 Hours Alter Cryosurgery Distinct cell destruction
in the epidermis
FIG. 3-6. Second freeze-thaw cycle: nuclear material flows out into the cytoplasma (arrows); nucleus (K), rough endoplasmatic reticulum = RER (ER). (X 56,OCKI)
and corium is visible after 6 hours (Fig. 311). Advanced cytolysis is evident through the strangulation of numerous matrix vesicles (Fig. 3-12). Particular destruction is evident in the lower epidermal area. The desmosomes appear intact and detach as complete structures from the cell membranes (Fig. 3-13). The intercellular spaces are open, and massive cell debris is observed (Fig. 3-12). The fragmented tonofibrils build myelin figures. The basement membrane appears to be intact (Fig. 3-13). Nuclear destruction of different degrees occurs, for example, clumped chromatin/karyolysis. The strong osmiophilic character of the nuclear membrane disappears. The vascular endothelium (Fig. 3-15)
Cllnfcs in
34
E. W. Breitbart
and G. Schaeg
Dermatology
FIG. 3-9. Second freeze-thaw cycle: fibrocyte in the upper corium. Early stages of cytolysis as seen by cytoplasmic vesicles (arrow). Highly osmiophilic nuclear membrane and osmiophilic ribosomes on the RER (ER). (X 11,300)
FIG. 3-10. Second freeze-thaw cycle: blood vessel. Cells of the endothelium (En) appear unchanged; basal lamina (BL) shown intact; shadow of erythrocytes (E); intravascular debris (arrow). (X 12,gOO)
FIG. 3-11. Six after hours cryosurgery: Stratum basale, advanced evidence of nuclear cytolysis; destruction. Basement membrane (BM). (X 3.600)
January-March 19% Volume 8 Number 1
Electronic Microscopic Investigations
FIG. 3-12. Six hours after cryosurgery: Stratum base/e. Expanded intercellular spaces (stars). Desmosomes (D) are intact but detached. Strangulation of matrix vesicles in the intercellular spaces (fop of arrows). (X WW
FIG. 3-13. Six hours after cryosurgery: basement membrane. Intact lamina densa (top of arrows). Focal interruption of the keratinocyte membrane (arrow). Myelin figures (My). Strangulation of matrix vesicles in the expanded intercellular spaces (stars). (X 19,ooo)
FIG. 3-14. Six hours after cryosurgery: damaged fibrocyte (F) in the upper corium. Condensed cytoplasm. Cytoplasmic vesiculation (arrows). (X 9,400)
35
E. W. Breitbari
36
Clinics in Dermatology
and G. Schaeg
FIG. 3-15. Six hours after cryosurgery: vessel. Destruction and vesiculation (V) of the endothelium. Basal lamina (BL) intact. Damaged cells (Z) intravascular. (X 19,000)
and connective tissue are severely damaged and vesiculation and vesicle formation are seen (Figs. 3-14 and 3-16). Collagen remains unchanged. 24 HoursNter
cryosurgery
Extensive blistering of the stratum basale is seen, although the lamina densa of the basement membrane is preserved. The keratinocyte cell membrane, containing half desmosomes, detaches in the interior of the blister (Fig. 3-17). The epidermis detaches as a whole (Fig. 3-18). The connective tissue cells show a similar degree of degeneration 6 hours after cryosurgery. The capillary wall structure has almost disintegrated. Lyosomes are
present, both inter- and extracellularly (Fig. 3-19). Collagen is swollen, although the periodicity of collagen is preserved.
48 and 72 Hours After Cryosurgery Biopsies taken after 48 and 72 hours show no substantial changes in comparison with the above findings, with only gradual alterations in degree of necrosis.
Discussion From the electron microscopic findings, the effect of intracellular crystallization is appar-
FIG. 3-16. Six hours after cryosurgery: vessel. Nuclear hyperchromasia of the endothelium cells. Aggregation of erythrocytes (E). Numerous cell debris (stars). Fibrocyte (F). Collagen (Co). (X 4,600)
January-March Volume 8 Number 1
IWO
Electronic Microscopic Investigations
37
ent with the rupture of nuclear membrane followed by a loss of karyoplasm. Cell edema is a consequence of cell death and is evident in the homogeneous cytoplasm, the disappearance of organelles, fragmentation of tonofibrils, and clumping of chromatin. Similar changes have been described by Schattenberg et al (19’78) and Helpap (1980) upon freezing the liver. The transitory osmiophilic thickening of the nuclear membrane (described by Lovelock (1957), as a consequence of the denaturation of membrane lipoproteins remains unsettled. Furthermore, the destruction of the unidentified cells occurs earlier than that of fibroblasts and keratinocytes. This is strongly suggested by our own observations. We observed hardly any changes in the basement membrane and desmosomes, although distinct alterations of the blood vessels could be seen. This confirms earlier reports by Uyeda et al (1977,1978).
Conclusion Our investigations for the practical application of cryosurgery demonstrate that the damage of all cell structures can be seen after the second freeze-thaw cycle.
FIG. 3-18. Twenty-four hours after cryosurgery: Stratum spinosum. Advanced cytolysis; large empty areas observed in the perinuclear zone. (X 3,600)
FIG. 3-17. Twenty-four hours after cryosurgery: formation of blister (BI). The lamina densa is intact (arrow). Cytolysis (Z); collagen (Co). (X 4,000)
38
E. W. Breitbari
Clinics in Dermatology
and G. Schaeg
FIG. 3-19.
/ f
References Breitbart EW. Neue Gesichtapunkte in der kryochirurgischen Behandlung von Neubildungen der Haut. In: Salfeld, ed. Operative Dermatologie. Berlin: Springer-Verlag, 1978~230-233. Helpap B. Der kryochirurgische Eingriff und seine Folgen. In: Doerr, Leonhardt, eds. Morphologische und zellkinetische Analyse. Stuttgart: George Thieme Verlay, 1980:65-70. Lovelock JE. The denaturation of lipid-protein complexes as a cause of damage by freezing. Pioc Roy Sot Med. 1957;147:427-433.
Address for correspondence: Eckhard Universitats-Hautklinik und -Poliklinik, Republic of Germany.
Twenty-four
hours
after
cryosurgery: vessel. Damaged endothelial cell (En);fragmentsof nuclei (K); intravascular debris and lyosomes (stars); erythrocytes (E). (X 5,500)
4. Schattenberg PJ, Totovic V, Helpap B, et al. Ultrastructure of early changes in circumscribed cryonecrosis. Path01 Res Pratt. 1978;163:334-352. 5. Uyeda K, Nakayasu K, Kishimoto S, et al. Electron microscopic studies of cryosurgery in the dermatological field. In: Proceedings of the XV International Congress in Dermatology, Mexico. 1977;565-567. 6. Uyeda K, Nakayasu K, Kishimoto S, et al. Electron microscopic observation of epidermal cells and infiltrated cells in the verrucous lesions after cryosurgery. J Clin Electron Microscopy. lS79;12:5-6. 7. Weiss L, Armstrong JA. Structural changes in mammalian cells associated with cooling to -76°C. J Biophys Biochem Cytol. 1960;7:673-677.
W. Breitbart, Martinistrasse
MD, Department of Dermatology, 52, D-2000, Hamburg 20, Federal