The American Journal of PATHOLOGY November 1977
-
Volume 89, Number 2
Endothelial Proliferation in Inflammation I. Autoradiographic Studies Following Thermal Injuiry to the Skin of Normal Rats Milton M. Sholley, PhD, Tito Cavallo, MD, and Ramzi S. Cotran, MD
Endothelial proliferation was studied in sites of acute inflammation induced by necrotizing (60 C for 20 seconds) or mild (54 C for 20 seconds) thermal injury to the skin of rats. DNA synthesis in endothelial cells was assayed 6 hours to 10 days following injury by quantitation of the 3H-thymidine labeling indices on 2-M Epon section autoradiographs. In lesions induced at 60 C for 20 seconds, increase in DNA synthesis in small vessels around the necrotic tissue began at 1 day and became significant at 2 and 3 days (10 to 12% for endothelial cells, 9% for perivascular cells). This increased endothelial replication resulted in the formation of new blood vessels by 5 to 7 days. Endothelial labeling diminished progressively after 3 days, as the epidermis regenerated. Foci completely covered by new epidermis consistently showed lower labeling indices than those which were not reepithelialized. Mild thermal injury (54 C for 20 seconds) also resulted in significant increases in endothelial labeling (6%), but the labeling was present mainly in superficial vessels and was not followed by neovascularization. The findings with mild injury are consistent with data that vascular leakage from superficial vessels is due to direct, albeit delayed, endothelial damage. Electron microscopic studies confirmed labeling in endothelial cells and indicated that ultrastructural alterations that were previously ascribed to activation, recovery, or regenerative transformation of endothelium represent, in the main, endothelial proliferation. (Am J Pathol 89:277-296, 1977)
ALTHOLUGH ADULT V-ASCULAR ENDOTHELIUMN normally has a low rate of turnover, 14 endothelial proliferation occurs during diverse pathologic processes involving both large and small vessels. Interest in growth control and proliferation of endothelial cells has been stimulated by at From the Departments of Pathology. Peter Bent Brigham Hospital and Harvard Mtedical Sch(xl. Boston. Massachusetts. Stipprted hb Grant HL-08231 from the National Institutes of Health and 3 1 T32 HL-07066-01 from the U'S Puiblic Health Service. Accepted for ptiblication Jtine 28. 1977. Address reprint requests to Dr. Ramzi S. Cotran. Department of Patholotsy. Peter Bent Brihzham Hospital. 721 Huntington Avenue. Boston. \I A 02113. 277
278
SHOLLEY ET AL.
American Journal of Pathology
least two current lines of investigation in a number of laboratories: a) the role of endothelial injury and regeneration in the evolution of atheromatous plaque 5 and b) the possible importance of neovascularization in such diverse pathologic processes as the growth of tumors, diabetic retinopathy, and psoriasis.6 There have been, for example, detailed studies of the possible mediators of tumor neovascularization,7 the kinetics of endothelial proliferation in the microvasculature of tumors,8'9 and the relationship of vascularization to malignant potential.6'10"1 Endothelial proliferation is a constant feature of the local reaction to injury and is a component of granulation tissue induced by wounding or other stimuli of the inflammatory response. Although there have been numerous excellent studies on the gross and histologic features,'2-16 ultrastructure,15-17 and permeability characteristics 15,16,18-21 of new vessels in wound healing, the factors responsible for endothelial growth under these conditions are poorly understood. We have thus initiated a series of studies on the time-sequence and degree of endothelial proliferation in immunogenic and nonimmunogenic models of the inflammatory response and examined the relationship of increased endothelial replication to other components of inflammation, especially leukocytic infiltration. In this paper, we investigated the endothelial proliferative response induced by two types of thermal injury in the skin of rats, models which have been used in our laboratory22-25 and by others 26-29 to study the vascular response in inflammation. Thermal injury at 60 C for 20 seconds induces a clearly necrotizing lesion, which is associated with immediate and sustained increase in vascular permeability and severe endothelial damage; 54 C for 20 seconds induces a milder form of injury, with a typical delayed-prolonged vascular response and ultrastructural evidence of mild endothelial damage. Endothelial proliferation was assessed by (quantitative light microscopic autoradiography and electron microscopic autoradiography after administration of tritium-labeled thymidine. In a subsequent paper, the relationship of endothelial proliferation to infiltrating mononuclear cells and neutrophils is examined in irradiated, leukopenic animals. Materials and Methods Animals
In a pilot set of experiments, 12 male CD rats (Sprague-Dawley derived, Charles River Breeding Laboratories, Wilmington, Mass.), weighing 300 to 450 g, were used. In later studies, 3.3 female CD rats, weighing 170 to 280 g, were used. The rats were fed standard laboratorv chow and water ad libitum.
Vol. 89, No. 2 November 1977
ENDOTHEUAL PROUFERATION IN INFLAMMATION
279
ThFmIn-
Before induction of thermal injury, the caudal half of the dorsolateral skin of the back was closely clipped on one side, depilated with Nair (Carter Hair Products, Inc., N.Y.), rinsed with lukewarm water, and dried. Thermal injurv was pmduced by a standard method,'" utilizing an apparatus similar to that of Wilhelm and Mason.' A copper disk 9 mm in diameter was kept at a constant temperature by circulating water heated at either 60 or 54 C. Lesions were induced on the depilated skin of lightly etherized animals by firm contact with the disk for 20 seconds. Two to six lesions were applied to each animal; 160 lesions were studied. Lesions were studied histologically and/or by light microscopic autoradiographv at 6 hours and 1, 2, 3, 5, 7, and 10 days following 60 C injury and at 3 and 7 days following 54 C injury. Control skin was obtained from animals depilated with Nair as well as from animals subjected only to hair clipping (detailed results reported separately).4 Selected lesions were additionally studied by electron microscopy and ultrastructural autoradiographv.
TrytiaeTldmi_ie Labeki Sties DNA synthesis by endothelial and perivascular cells was assessed by studying the incorporation of 'H-methyl thymidine (3H-TdR) (New England Nuclear Corporation, Boston, Mass., specific activity 6.7 Ci/mmole). In pilot experiments, lesions were carefully removed en bloc from etherized animals and were incubated with constant agitation for 1 hour at 37 C in medium 199, containing 10% calf serum (Microbiological Associates, Bethesda, Md.) and 25 pCi of 'H-TdR. This in vitro incubation resulted in dense labeling in most cases, but in some lesions there was uneven penetration of the 3H-TdR, Therefore, for the quantitative studies, 'H-TdR (0.5 pCi/g) was injected intraperitoneally 30 minutes before the animals were sacrificed by blunt occipital trauma. In addition to skin, segments of jejunum were removed from each animal to serve as "hot" controls for the autoradiography.
Vascua Traces To localize the sites of vascular injury, colloidal carbon (Pelikan Special Ink, Batch C11/1431A, The Morilla Co., Long Island City, N.Y.) was injected (0.1 ml/100 g) 2 or 3 hours after injurv via the tail vein. This procedure "labels" leaking vessels 30 at the site of thermal injurv. The colloidal carbon was filtered through Whatman No. 1 filter paper immediatelv before injection.
Trs
Preparai
Squares of skin bearing the lesions were gently sliced across the direction of the hair into strips 1 mm wide. For paraffin sectioning, whole strips (approximately 14 mm long) were fixed overnight in 10% neutral buffered formalin, washed in running water to remove unbound 3H-TdR, embedded in paraffin, and sectioned at 5 p. For Epon embedding and sectioning, the strips were bisected, fixed in cold half-strength Karnovsky's fixative 3' for 18 hours, washed in 0.1 M cacodylate buffer, postfixed in osmium tetroxide, dehvdrated through ethanols, and embedded flat in Epon. Sections were cut from the entire lengths of the bisected strips at a thickness of 2 to 3 p on a Porter-Blum MT2-B ultramnicrotome. In selected lesions, one strip was initiallv diced into blocks approximatelv 1 X 1 X 2 mm and embedded in Epon as above, and thin sections were cut for electron microscopic autoradiography. Paraffin sections were stained with hematoxvlin and eosin; thick Epon sections with 0.25% Azure II-0.25% methylene blue in 0.25% borax (modified from Richardson et al. );'* and thin sections with uranyl acetate and Revnold's lead citrate."
280
SHOLLEY ET AL.
American Journal of Pathology
Autoradiography
Autoradiographs were prepared by dipping in NTB-2 emulsion (Eastman Kodak, Rochester, N.Y.) diluted 1:1 with distilled water. After exposure at 4 C for 21 days (paraffin sections) or 42 days (thick Epon sections), the autoradiographs were developed for 4.5 minutes at 15 C in half-strength Kodak D-19 developer and then treated with Kodak Ektaflo Fixer. The developed autoradiographs were stained as described above. Thin section autoradiography was carried out using Ilford L-4 emulsion (Ilford Ltd., Ilford, England) and the basic method of Caro et al.m4 Quantitation of Labeling Indices The 3H-TdR labeling index (percentage of labeled nuclei in a population of cells) was quantitated only on thick Epon sections. Autoradiography of Epon-embedded sections permitted accurate identification of endothelial and perivascular cells (pericytes and adventitial cells in close approximation to the endothelium); the large size and identical orientation of each section facilitated the counting of nuclei from vessels in topographically equivalent areas. On each section, all endothelial and associated perivascular nuclei from capillaries and venules in skin layers above the panniculus carnosus were counted by rectilinear scanning under oil immersion (X 1000) from a point 1 mm from the lesion center. A cell was considered labeled if five or more silver grains were located over its nucleus. Three to six sections were quantitated from each lesion; the average number of cells counted per lesion or control site was 1004 ± 482 in the case of endothelial cells and 401 ± 164 in the case of perivascular cells. Four to twelve separate lesions were quantitated for each group and time studied. For lesion times up to 3 days, mean labeling indices were calculated from the composite index of each lesion. However, at 7 and 10 days, varying stages of healing from section to section on each lesion made it necessary to group the sections according to the stage of reepidermalization; section means were then calculated (see Table 3). In order to evaluate differences between various mean labeling indices, the Student t test for unpaired data was
employed.
Results Endothelial Labeling in Normal Skin
The 3H-thymidine labeling index of endothelial cells from capillaries and small venules in normal rat skin ranged from 0.08% to 2.87%, with a mean of 1.27 ± 0.41 %. As reported previously,4 labeling indices near the higher end of this range were found in skin containing active hair follicles, while indices near the lower end of the range were found in skin containing inactive hair follicles. Skin from dorsolateral body areas prepared by clipping and depilation with Nair 72 hours prior to sampling had a labeling index of 1.16 ± 0.55%, indicating that preparation of the skin for thermal injury did not alter the control amount of endothelial proliferation. Perivascular cells associated with the endothelial populations just described had similar labeling indices (Table 1).
Vol. 89, No. 2 November 1977
ENDOTHELIAL PROLIFERATION IN INFLAMMATION
281
Table 1-Endothelial and Perivascular 3H-Thymidine Labeling Indices in Normal Rat Skin
Hair clipped only Nair depilation
Endothelial labeling index (mean ± SE)
Perivascular labeling index (mean ± SE)
1.27 ± 0.41% 1.16 ± 0.55%
1.29 ± 0.36% 1.37 ± 0.71%
ErddFheI Labeig After Themal lnIwy at 60 C for 20 Sonds General Characteristics of the Lesion
Heating rat skin at 60 C for 20 seconds uniformly resulted in coagulation necrosis of the epidermis and most of the dermis (Figure 1). In some lesions, particularlv those applied to the thinner skin containing inactive hair follicles,Ss,N the necrotic area extended into the hvpodermis and occasionally into the panniculus carnosus centrallv. Previous studies from our laboratorv 2324 have shown that this injurn causes an immediate and sustained increase in vascular permeability. In the superficial layer of the dermis, leakage occurs between and through extensively damaged endothelial cells in capillaries and small venules and continues for approximately 3 hours, until widespread stasis ensues.24 Vessels in the deeper laver of the dermis, the hypodermis, and, in deeper lesions, the panniculus carnosus continue to leak for about 24 hours, as evidenced bv their ability to be labeled with colloidal carbon.23'24 By 24 to 48 hours, the necrotic tissue grossly formed a thick, black eschar. At 72 hours, some lesions already displayed a modest amount of epidermal regeneration; short tongues of epidermis projected into a partially liquified region of numerous degenerating neutrophils under the eschar. By 7 to 10 days (Figure 2), increasingly larger circumferential regions of the circular eschar had become undermined by regenerating epidermis, which itself was underlain by a highly vascularized layer of new connective tissue. Light Microscopic Autoradiography
The vessels from which labeling indices were quantitated were capillaries and venules (Figures 3 and 4), these being the types of vessels which contained the great majority of labeled cells. However, endothelial labeling and mitoses were also observed occasionallv in small arterioles (Figure 5). Endothelial and perivascular mitoses were seen frequently at 48 and 72 hours (Figures 3 and 5).
282
American Journal of Pathology
SHOLLEY ET AL.
At 6 hours after injury, the cells of the epidermis and most of the dermis already displayed coagulation necrosis, but there were very few inflammatory cells present in the extravascular space. Endothelial and perivascular labeling indices at this time were similar to controls (Table 2). By 24 hours, most degeneration had occurred in the epidermis and dermis, and the hypodermis of many sections displayed moderate but variable numbers of infiltrating neutrophils and mononuclear cells. At this time, endothelial and perivascular labeling indices were increased over controls, but the increase was not statistically significant (Table 2). At 48 hours, however, both endothelial (12.12%) and perivascular (9.23%) labeling indices had increased sharply and significantly over both controls and earlier lesions (Table 2, Figures 3-5). The lesions at this time displayed a heavy band of fresh and degenerated neutrophils above the interface of dead and viable tissue, and the hypodermis usually exhibited moderate to heavy infiltration of mononuclear cells. The inflammatory infiltrate was similar at 72 hours (Figure 1), as were the labeling indices of endothelial and perivascular cells (Table 2). At these times of peak labeling, vessels having proliferating cells were found not only deep to the necrotic tissue, but also throughout an approximately 1-mm-wide ring of viable skin adjacent to the necrotic area. By 7 to 10 days, the earlier vigorous endothelial turnover had resulted in newly formed vessels (Figure 6) which were embedded in a layer of immature connective tissue (Figures 2 and 6). The vascular labeling at these later times varied from section to section and was correlated with the degree of reepidermalization (Table 3). Sections which were not completely covered by new epidermis had higher overall labeling indices than those which were completely recovered (Table 3). This phenomenon also could be detected regionally on each section. For example, on one section at 10 days the endothelial labeling index in the area under the new epidermis was 2.00%, while that in the central nonepidermalized area was Table 2-Endothelial and Perivascular 3H-Thymidine Labeling Indices After Necrotizing Thermal Injury (60 C, 20 seconds) to the Skin
Perivascular
Time after injury
Endothelial labeling index (mean ± SE)
labeling index (mean ± SE)
6 hours 1 day 2 days 3 days
0.83 ± 0.36% 3.02 ± 0.65% 12.12 ± 0.68%* 10.07 ± 0.48%*
1.21 ± 0.64% 2.06 ± 0.54% 9.23 ± 0.68%* 8.72 ± 1.01 %*
* Value significantly different (P
-
Volume 89, Number 2
Endothelial Proliferation in Inflammation I. Autoradiographic Studies Following Thermal Injuiry to the Skin of Normal Rats Milton M. Sholley, PhD, Tito Cavallo, MD, and Ramzi S. Cotran, MD
Endothelial proliferation was studied in sites of acute inflammation induced by necrotizing (60 C for 20 seconds) or mild (54 C for 20 seconds) thermal injury to the skin of rats. DNA synthesis in endothelial cells was assayed 6 hours to 10 days following injury by quantitation of the 3H-thymidine labeling indices on 2-M Epon section autoradiographs. In lesions induced at 60 C for 20 seconds, increase in DNA synthesis in small vessels around the necrotic tissue began at 1 day and became significant at 2 and 3 days (10 to 12% for endothelial cells, 9% for perivascular cells). This increased endothelial replication resulted in the formation of new blood vessels by 5 to 7 days. Endothelial labeling diminished progressively after 3 days, as the epidermis regenerated. Foci completely covered by new epidermis consistently showed lower labeling indices than those which were not reepithelialized. Mild thermal injury (54 C for 20 seconds) also resulted in significant increases in endothelial labeling (6%), but the labeling was present mainly in superficial vessels and was not followed by neovascularization. The findings with mild injury are consistent with data that vascular leakage from superficial vessels is due to direct, albeit delayed, endothelial damage. Electron microscopic studies confirmed labeling in endothelial cells and indicated that ultrastructural alterations that were previously ascribed to activation, recovery, or regenerative transformation of endothelium represent, in the main, endothelial proliferation. (Am J Pathol 89:277-296, 1977)
ALTHOLUGH ADULT V-ASCULAR ENDOTHELIUMN normally has a low rate of turnover, 14 endothelial proliferation occurs during diverse pathologic processes involving both large and small vessels. Interest in growth control and proliferation of endothelial cells has been stimulated by at From the Departments of Pathology. Peter Bent Brigham Hospital and Harvard Mtedical Sch(xl. Boston. Massachusetts. Stipprted hb Grant HL-08231 from the National Institutes of Health and 3 1 T32 HL-07066-01 from the U'S Puiblic Health Service. Accepted for ptiblication Jtine 28. 1977. Address reprint requests to Dr. Ramzi S. Cotran. Department of Patholotsy. Peter Bent Brihzham Hospital. 721 Huntington Avenue. Boston. \I A 02113. 277
278
SHOLLEY ET AL.
American Journal of Pathology
least two current lines of investigation in a number of laboratories: a) the role of endothelial injury and regeneration in the evolution of atheromatous plaque 5 and b) the possible importance of neovascularization in such diverse pathologic processes as the growth of tumors, diabetic retinopathy, and psoriasis.6 There have been, for example, detailed studies of the possible mediators of tumor neovascularization,7 the kinetics of endothelial proliferation in the microvasculature of tumors,8'9 and the relationship of vascularization to malignant potential.6'10"1 Endothelial proliferation is a constant feature of the local reaction to injury and is a component of granulation tissue induced by wounding or other stimuli of the inflammatory response. Although there have been numerous excellent studies on the gross and histologic features,'2-16 ultrastructure,15-17 and permeability characteristics 15,16,18-21 of new vessels in wound healing, the factors responsible for endothelial growth under these conditions are poorly understood. We have thus initiated a series of studies on the time-sequence and degree of endothelial proliferation in immunogenic and nonimmunogenic models of the inflammatory response and examined the relationship of increased endothelial replication to other components of inflammation, especially leukocytic infiltration. In this paper, we investigated the endothelial proliferative response induced by two types of thermal injury in the skin of rats, models which have been used in our laboratory22-25 and by others 26-29 to study the vascular response in inflammation. Thermal injury at 60 C for 20 seconds induces a clearly necrotizing lesion, which is associated with immediate and sustained increase in vascular permeability and severe endothelial damage; 54 C for 20 seconds induces a milder form of injury, with a typical delayed-prolonged vascular response and ultrastructural evidence of mild endothelial damage. Endothelial proliferation was assessed by (quantitative light microscopic autoradiography and electron microscopic autoradiography after administration of tritium-labeled thymidine. In a subsequent paper, the relationship of endothelial proliferation to infiltrating mononuclear cells and neutrophils is examined in irradiated, leukopenic animals. Materials and Methods Animals
In a pilot set of experiments, 12 male CD rats (Sprague-Dawley derived, Charles River Breeding Laboratories, Wilmington, Mass.), weighing 300 to 450 g, were used. In later studies, 3.3 female CD rats, weighing 170 to 280 g, were used. The rats were fed standard laboratorv chow and water ad libitum.
Vol. 89, No. 2 November 1977
ENDOTHEUAL PROUFERATION IN INFLAMMATION
279
ThFmIn-
Before induction of thermal injury, the caudal half of the dorsolateral skin of the back was closely clipped on one side, depilated with Nair (Carter Hair Products, Inc., N.Y.), rinsed with lukewarm water, and dried. Thermal injurv was pmduced by a standard method,'" utilizing an apparatus similar to that of Wilhelm and Mason.' A copper disk 9 mm in diameter was kept at a constant temperature by circulating water heated at either 60 or 54 C. Lesions were induced on the depilated skin of lightly etherized animals by firm contact with the disk for 20 seconds. Two to six lesions were applied to each animal; 160 lesions were studied. Lesions were studied histologically and/or by light microscopic autoradiographv at 6 hours and 1, 2, 3, 5, 7, and 10 days following 60 C injury and at 3 and 7 days following 54 C injury. Control skin was obtained from animals depilated with Nair as well as from animals subjected only to hair clipping (detailed results reported separately).4 Selected lesions were additionally studied by electron microscopy and ultrastructural autoradiographv.
TrytiaeTldmi_ie Labeki Sties DNA synthesis by endothelial and perivascular cells was assessed by studying the incorporation of 'H-methyl thymidine (3H-TdR) (New England Nuclear Corporation, Boston, Mass., specific activity 6.7 Ci/mmole). In pilot experiments, lesions were carefully removed en bloc from etherized animals and were incubated with constant agitation for 1 hour at 37 C in medium 199, containing 10% calf serum (Microbiological Associates, Bethesda, Md.) and 25 pCi of 'H-TdR. This in vitro incubation resulted in dense labeling in most cases, but in some lesions there was uneven penetration of the 3H-TdR, Therefore, for the quantitative studies, 'H-TdR (0.5 pCi/g) was injected intraperitoneally 30 minutes before the animals were sacrificed by blunt occipital trauma. In addition to skin, segments of jejunum were removed from each animal to serve as "hot" controls for the autoradiography.
Vascua Traces To localize the sites of vascular injury, colloidal carbon (Pelikan Special Ink, Batch C11/1431A, The Morilla Co., Long Island City, N.Y.) was injected (0.1 ml/100 g) 2 or 3 hours after injurv via the tail vein. This procedure "labels" leaking vessels 30 at the site of thermal injurv. The colloidal carbon was filtered through Whatman No. 1 filter paper immediatelv before injection.
Trs
Preparai
Squares of skin bearing the lesions were gently sliced across the direction of the hair into strips 1 mm wide. For paraffin sectioning, whole strips (approximately 14 mm long) were fixed overnight in 10% neutral buffered formalin, washed in running water to remove unbound 3H-TdR, embedded in paraffin, and sectioned at 5 p. For Epon embedding and sectioning, the strips were bisected, fixed in cold half-strength Karnovsky's fixative 3' for 18 hours, washed in 0.1 M cacodylate buffer, postfixed in osmium tetroxide, dehvdrated through ethanols, and embedded flat in Epon. Sections were cut from the entire lengths of the bisected strips at a thickness of 2 to 3 p on a Porter-Blum MT2-B ultramnicrotome. In selected lesions, one strip was initiallv diced into blocks approximatelv 1 X 1 X 2 mm and embedded in Epon as above, and thin sections were cut for electron microscopic autoradiography. Paraffin sections were stained with hematoxvlin and eosin; thick Epon sections with 0.25% Azure II-0.25% methylene blue in 0.25% borax (modified from Richardson et al. );'* and thin sections with uranyl acetate and Revnold's lead citrate."
280
SHOLLEY ET AL.
American Journal of Pathology
Autoradiography
Autoradiographs were prepared by dipping in NTB-2 emulsion (Eastman Kodak, Rochester, N.Y.) diluted 1:1 with distilled water. After exposure at 4 C for 21 days (paraffin sections) or 42 days (thick Epon sections), the autoradiographs were developed for 4.5 minutes at 15 C in half-strength Kodak D-19 developer and then treated with Kodak Ektaflo Fixer. The developed autoradiographs were stained as described above. Thin section autoradiography was carried out using Ilford L-4 emulsion (Ilford Ltd., Ilford, England) and the basic method of Caro et al.m4 Quantitation of Labeling Indices The 3H-TdR labeling index (percentage of labeled nuclei in a population of cells) was quantitated only on thick Epon sections. Autoradiography of Epon-embedded sections permitted accurate identification of endothelial and perivascular cells (pericytes and adventitial cells in close approximation to the endothelium); the large size and identical orientation of each section facilitated the counting of nuclei from vessels in topographically equivalent areas. On each section, all endothelial and associated perivascular nuclei from capillaries and venules in skin layers above the panniculus carnosus were counted by rectilinear scanning under oil immersion (X 1000) from a point 1 mm from the lesion center. A cell was considered labeled if five or more silver grains were located over its nucleus. Three to six sections were quantitated from each lesion; the average number of cells counted per lesion or control site was 1004 ± 482 in the case of endothelial cells and 401 ± 164 in the case of perivascular cells. Four to twelve separate lesions were quantitated for each group and time studied. For lesion times up to 3 days, mean labeling indices were calculated from the composite index of each lesion. However, at 7 and 10 days, varying stages of healing from section to section on each lesion made it necessary to group the sections according to the stage of reepidermalization; section means were then calculated (see Table 3). In order to evaluate differences between various mean labeling indices, the Student t test for unpaired data was
employed.
Results Endothelial Labeling in Normal Skin
The 3H-thymidine labeling index of endothelial cells from capillaries and small venules in normal rat skin ranged from 0.08% to 2.87%, with a mean of 1.27 ± 0.41 %. As reported previously,4 labeling indices near the higher end of this range were found in skin containing active hair follicles, while indices near the lower end of the range were found in skin containing inactive hair follicles. Skin from dorsolateral body areas prepared by clipping and depilation with Nair 72 hours prior to sampling had a labeling index of 1.16 ± 0.55%, indicating that preparation of the skin for thermal injury did not alter the control amount of endothelial proliferation. Perivascular cells associated with the endothelial populations just described had similar labeling indices (Table 1).
Vol. 89, No. 2 November 1977
ENDOTHELIAL PROLIFERATION IN INFLAMMATION
281
Table 1-Endothelial and Perivascular 3H-Thymidine Labeling Indices in Normal Rat Skin
Hair clipped only Nair depilation
Endothelial labeling index (mean ± SE)
Perivascular labeling index (mean ± SE)
1.27 ± 0.41% 1.16 ± 0.55%
1.29 ± 0.36% 1.37 ± 0.71%
ErddFheI Labeig After Themal lnIwy at 60 C for 20 Sonds General Characteristics of the Lesion
Heating rat skin at 60 C for 20 seconds uniformly resulted in coagulation necrosis of the epidermis and most of the dermis (Figure 1). In some lesions, particularlv those applied to the thinner skin containing inactive hair follicles,Ss,N the necrotic area extended into the hvpodermis and occasionally into the panniculus carnosus centrallv. Previous studies from our laboratorv 2324 have shown that this injurn causes an immediate and sustained increase in vascular permeability. In the superficial layer of the dermis, leakage occurs between and through extensively damaged endothelial cells in capillaries and small venules and continues for approximately 3 hours, until widespread stasis ensues.24 Vessels in the deeper laver of the dermis, the hypodermis, and, in deeper lesions, the panniculus carnosus continue to leak for about 24 hours, as evidenced bv their ability to be labeled with colloidal carbon.23'24 By 24 to 48 hours, the necrotic tissue grossly formed a thick, black eschar. At 72 hours, some lesions already displayed a modest amount of epidermal regeneration; short tongues of epidermis projected into a partially liquified region of numerous degenerating neutrophils under the eschar. By 7 to 10 days (Figure 2), increasingly larger circumferential regions of the circular eschar had become undermined by regenerating epidermis, which itself was underlain by a highly vascularized layer of new connective tissue. Light Microscopic Autoradiography
The vessels from which labeling indices were quantitated were capillaries and venules (Figures 3 and 4), these being the types of vessels which contained the great majority of labeled cells. However, endothelial labeling and mitoses were also observed occasionallv in small arterioles (Figure 5). Endothelial and perivascular mitoses were seen frequently at 48 and 72 hours (Figures 3 and 5).
282
American Journal of Pathology
SHOLLEY ET AL.
At 6 hours after injury, the cells of the epidermis and most of the dermis already displayed coagulation necrosis, but there were very few inflammatory cells present in the extravascular space. Endothelial and perivascular labeling indices at this time were similar to controls (Table 2). By 24 hours, most degeneration had occurred in the epidermis and dermis, and the hypodermis of many sections displayed moderate but variable numbers of infiltrating neutrophils and mononuclear cells. At this time, endothelial and perivascular labeling indices were increased over controls, but the increase was not statistically significant (Table 2). At 48 hours, however, both endothelial (12.12%) and perivascular (9.23%) labeling indices had increased sharply and significantly over both controls and earlier lesions (Table 2, Figures 3-5). The lesions at this time displayed a heavy band of fresh and degenerated neutrophils above the interface of dead and viable tissue, and the hypodermis usually exhibited moderate to heavy infiltration of mononuclear cells. The inflammatory infiltrate was similar at 72 hours (Figure 1), as were the labeling indices of endothelial and perivascular cells (Table 2). At these times of peak labeling, vessels having proliferating cells were found not only deep to the necrotic tissue, but also throughout an approximately 1-mm-wide ring of viable skin adjacent to the necrotic area. By 7 to 10 days, the earlier vigorous endothelial turnover had resulted in newly formed vessels (Figure 6) which were embedded in a layer of immature connective tissue (Figures 2 and 6). The vascular labeling at these later times varied from section to section and was correlated with the degree of reepidermalization (Table 3). Sections which were not completely covered by new epidermis had higher overall labeling indices than those which were completely recovered (Table 3). This phenomenon also could be detected regionally on each section. For example, on one section at 10 days the endothelial labeling index in the area under the new epidermis was 2.00%, while that in the central nonepidermalized area was Table 2-Endothelial and Perivascular 3H-Thymidine Labeling Indices After Necrotizing Thermal Injury (60 C, 20 seconds) to the Skin
Perivascular
Time after injury
Endothelial labeling index (mean ± SE)
labeling index (mean ± SE)
6 hours 1 day 2 days 3 days
0.83 ± 0.36% 3.02 ± 0.65% 12.12 ± 0.68%* 10.07 ± 0.48%*
1.21 ± 0.64% 2.06 ± 0.54% 9.23 ± 0.68%* 8.72 ± 1.01 %*
* Value significantly different (P
