MICROVASCULAR
The
RESEARCH
Structure
(1976)
11,361-379
of Scorbutic Regenerating Skeletal Muscle Wounds’
Capillaries
in
RALPH V. MCKINNEY, JR. Departments of Oral Pathology and Oral Biology, School of Dentistry, Medical College of Georgia, Augusta, Georgia 30902 Received
January
9, 1976
The purpose of this investigation was to characterize scorbutic regenerating capillaries in skeletal muscle wounds and to compare their structure with that of normal regenerating capillaries and mature (nonwound) scorbutic capillaries. Wounds were created in the cremaster muscle of scorbutic guinea pigs. The capillary fringe areas of the wounds were examined at intervals of 3 to 16 days postoperatively by light and electron microscopy. Nonscorbutic animals served as controls. No significant change was noted between the perivascular collagen of scorbutic and nonscorbutic wounds. Organelle changes were similar to those reported in nonwound scorbutic endothelium. Specific structural effects noted in the scorbutic wound capillaries were a slower rate of ingrowth of the capillary fringe into a wound; decreased sprouting and proximal anastomosing of the growing vessels; a bulbousshaped capillary tip with numerous thin spots in the endothelium; ready ingress of tracer substances to the capillary tip; and, the presence of pale endothelial cells of unknown function. The capillary basement membrane surrounded all portions of the endothelium and did not exhibit any discontinuities except around cytoplasmic extrusions. This study showed that scorbutic regenerating wound capillaries differ significantly in their structure from normal regenerating capillaries and mature (nonwound) scorbutic capillaries. Application of the law of Laplace (T= P x R) to the bulbous scorbutic capillary tips allows us to theorize that increased endothelial wall tension accounts for their fragility.
INTRODUCTION Spontaneouspetechial hemorrhagesand delayed wound healing have beenrecognized as clinical signsof ascorbic acid deficiency for many years, yet the mechanismsfor these phenomena are still not entirely clear. Beginning with the classicalstudiesof Wolbach and Howe (1926), microscopists have suggestedseveral possible explanations for the so-called “fragility” of the mature microvasculature in scurvy. Gersh and Catchpole (1949) suggesteddisaggregation of the glycoprotein of capillary basementmembrane on the basisof decreasedperiodic acid-Schiff staining intensity. Stolman et al. (1961) felt the predominant defect was a breakdown or degeneration of collagen fibers outside the endothelial wall. Using electron microscopy, Gore et al. (1968) describedendothelial cell disjunction, cytoplasmic disruption, and discontinuities of the basementmembrane as being responsiblefor the increasedpermeability of scorbutic capillaries. Interestingly, the regenerating capillaries in wounds of vitamin C-deficient animals have received limited attention even though Wolbach and Howe (1926) commented on a 1 This study was supported in part by USPHS Grant No. DE-03486. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
361
362
RALPH
V. MCKINNEY,
JR.
tendency for the regenerating scorbutic endothelium to “accumulate in clumps.” Penny and Balfour (1949) and Dunphy et al. (1956) also noted abnormalities in the newly formed scorbutic vessels but did not elaborate on their observations. Others have reported the delay in the growth rate of scorbutic vessels into a wound (Ross and Benditt, 1961; Wolbach and Howe, 1926). The closest study relating to the ultrastructure of scorbutic growing capillaries was that of Friederici and associates (1966) who examined the capillaries of inflammed synovia in scorbutic guinea pigs. These authors described “rarified” endothelial cells and a “tenuous basement membrane” as prominent ultrastructural features of these vessels. The objective of this investigation was to characterize regenerating capillaries in scorbutic animals, particularly those in skeletal muscle wounds, in order to compare their structure with normal regenerating capillaries and mature (nonwound) scorbutic capillaries. Emphasis was placed on the characteristics of the endothelium, the basement membrane, and the surrounding perivascular collagen fibers since changes in these structures have been implicated in the fragility of nonregenerating scorbutic vessels. In addition, intravascular injection of tracers was carried out in an attempt to delineate the permeability characteristics of the scorbutic regenerating capillaries. MATERIALS
AND
METHODS
Animals and Wounding A total of 29 adult male guinea pigs weighing 550-1000 grams were used in this investigation. Nineteen guinea pigs were allowed an adlibitum diet ofvitamin C-deficient guinea pig chow and water. Ten animals were used as controls. Twelve of the animals on the scorbutic diet received a commercial preparation of the Reid-Briggs ascorbic acid-deficient diet (Nutritional Biochemicals Corp., Cleveland, Ohio 44128). The other seven scorbutic animals received a normal pelleted Purina rabbit chow that had the vitamin C inactivated by heating the chow to 100°F for 30 to 60 min. The animals were maintained on the scorbutogenic diet 8 days prior to wounding. At the end of the 8 days, a circular wound of 2-4 mm in diameter was created in the cremaster muscle of the scorbutic guinea pigs employing a previously described wounding procedure (McKinney and Panner, 1972; Schoefl, 1963). The animals were then continued on the scorbutogenie diet after wounding and biopsies were taken of the wounds at 3, 7, 8, 9, 14, or 16 days. The 10 control animals were fed a normal pelleted guinea pig chow containing vitamin C and subjected to a similar surgical procedure and biopsy interval. Biopsy Procedure At the time of biopsy, light ether anesthesia was used to allow a cutdown procedure to the saphenous vein of the guinea pig. Pentobarbital solution (65 mg per ml of NaPentobarbital solution), 0.05 ml per 100 grams of body weight, was injected intravascularly to maintain a suitable level of anesthesia. Five minutes prior to biopsy the animal was injected intravenously with colloidal carbon (Biological Ink, Koh-I-Noor, Inc., Bloomsbury, New Jersey), 0.1 ml per 100 grams of body weight, or with horseradish (Sigma Type II, Sigma Chemical Co., St. Louis, Missouri) 10 mg per 100 grams of body weight dissolved in 1 ml of 0.9% sterile saline solution. The balance of the biopsy procedure has been previously described (McKinney and Panner, 1972).
SCORBUTIC
REGENERATING
CAPILLARIES
363
Ascorbic Acid Determination Samples of the animal’s lung, adrenal, spleen, and liver were removed for ascorbic acid analysis. Visual titration of the homogenized tissue sample was carried out using 2,6-dichlorophenolindophenol according to the modified Tillman’s method (Gyorgy and Pearson, 1967). Data was expressed as milligrams of ascorbic acid per 100 grams (wet weight) of tissue sample.
FIG. 1. Fringe of normal (right side of figure). Some carbon while other capillary of the capillary tips. Whole
(nonscorbutic) capillary sprouts growing toward the center of the wound of the larger capillaries and a few of the tips are blackened with injected tips do not contain carbon (arrows). Note the cylindrical or tapered shape mount of an I-day nonscorbutic wound edge. Unstained, x420.
Light and Electron Microscopy The muscletissuewas fixed with glutaraldehyde-paraformaldehyde fixative in 0.2 M cacodylate buffer at pH 7.2 for 2-4 hr, either at room temperature or 4°C. The tissue was then rinsed in 0.2 A4 cacodylate buffer three times, trimmed, and placed in buffer solution on a slide for light photomicroscopy of the fringe of growing capillaries at the wound edge(Figs. 1 and 2). Subsequently, the tissuewas trimmed, postfixed in osmium tetroxide, and embeddedaswe have previously detailed (McKinney and Panner, 1972). Thin sectionswere cut on an LKB Ultratome III using 50” angle glassand diamond knives. Sections were stained for 15-25 min with aqueous uranyl acetate and 3-5 min with lead citrate, then examined in a Hitachi HU-1 1A or RCA EMU4C electron microscopeat 75 kV. One-micron-thick sectionswere cut and stained with Paragon stain for light microscopic orientation.
364
RALPH V. MCKINNEY, JR.
Histochemistry Some tissuesampleshad the electron histochemical stain ruthenium red (Luft, 1971) added to the fixative solutions to enhance the demonstration of glycosaminoglycuronoglycans. We have previously reported the methodsand modifications we usedfor this procedure (McKinney and Panner, 1972).
FIG. 2. Fringe of scorbutic capillary sprouts growing toward the center of the wound (right of picture). The capillary tips are saccular or bulbous and appear black because of the injected carbon. Some of the bulbous tips are bent at 90-degree angles to the more proximal portion of the capillary (arrows). There is also a decrease in the number of anastomosing capillaries proximal from the growing fringe when compared with Fig. 1. Whole mount of an 8-day scorbutic wound edge. Unstained, x 420.
Following initial fixation, the tissue samplesinjected with horseradish peroxidase (HRP) were washed overnight in 0.1 A4 cacodylate buffer at 4°C. These were then cut into small piecesand transferred to 0.05 A4Tris buffer, pH 7.6, and incubated for 30 min
365
SCORBUTIC REGENERATING CAPILLARIES
in the incubation medium of Graham and Karnovsky (1966). Following incubation the tissue was washed three times in distilled water and postfixed in 1% 0~0,. The methenamine silver histochemical reaction was applied to some selectedtissue samplesin an attempt to demonstrate collagen or collagenlike fibers in the basement membrane and extravascular space. Following initial fixation the tissue was taken up to 70 % ethanol, then silver impregnation was carried out according to the method of Churg et al. (1958). It wasfound that 24-hr incubation in the silver methenaminesolution was sufficient for impregnation of the thin cremaster muscle.Following incubation, the tissuewas rinsed three times in distilled water, once in cacodylate buffer, and postfixed in 1% 0~0,. Although the mechanismsof action for the ionic binding of silver grains on collagen or collagenlike fibrils is not completely understood, it has been shown that the reduced silver grains selectively deposit on collagen fibers in a periodic fashion and are not eliminated by hyaluronidase treatment (Grassman and Hofman, 1957). RESULTS Gross Observations The scorbutic guinea pigs recovered well from the wounding procedure and the abdominal incision always healed uneventufully. Most animals remained active and fairly healthy up to 14 days on the scorbutogenic diet. After 14-16 days on the scorbutic TABLE 1 MEAN ASCORBICAcre LEVELS OF GUINEA Lung
Adrenal
PIG TISSUE”
Spleen
Liver
Control animals
27.5 f 2.12 (10)
125.5 _+ 7.56 (JO)
47.4 -+ 5.48 (9)
21.2 _+3.12 (10)
g-Day scorbutic animals
5.17 + 1.25 (6) P < 0.001
31.0 * 4.14 (5) P < 0.001
8.83 + 2.01 (6) P < 0.001
6.17 + 1.74 (6) P < 0.005
15-, 16-, and 1‘J-Day scorbutic animalsb
4.33 f 0.84 (6) P < 0.001
12.80 i 3.31 (5) P < 0.001
7.00 + 0.68 (6) P < 0.001
4.00 + 0.55 (5)
3.60 + 0.68 (5)
8.80 i. 0.66 (5) P < 0.001
6.60 + 0.40 (5)
4.50 + 0.96 (4) P < 0.01
22- and 24Day scorbutic animal?
P
The
RESEARCH
Structure
(1976)
11,361-379
of Scorbutic Regenerating Skeletal Muscle Wounds’
Capillaries
in
RALPH V. MCKINNEY, JR. Departments of Oral Pathology and Oral Biology, School of Dentistry, Medical College of Georgia, Augusta, Georgia 30902 Received
January
9, 1976
The purpose of this investigation was to characterize scorbutic regenerating capillaries in skeletal muscle wounds and to compare their structure with that of normal regenerating capillaries and mature (nonwound) scorbutic capillaries. Wounds were created in the cremaster muscle of scorbutic guinea pigs. The capillary fringe areas of the wounds were examined at intervals of 3 to 16 days postoperatively by light and electron microscopy. Nonscorbutic animals served as controls. No significant change was noted between the perivascular collagen of scorbutic and nonscorbutic wounds. Organelle changes were similar to those reported in nonwound scorbutic endothelium. Specific structural effects noted in the scorbutic wound capillaries were a slower rate of ingrowth of the capillary fringe into a wound; decreased sprouting and proximal anastomosing of the growing vessels; a bulbousshaped capillary tip with numerous thin spots in the endothelium; ready ingress of tracer substances to the capillary tip; and, the presence of pale endothelial cells of unknown function. The capillary basement membrane surrounded all portions of the endothelium and did not exhibit any discontinuities except around cytoplasmic extrusions. This study showed that scorbutic regenerating wound capillaries differ significantly in their structure from normal regenerating capillaries and mature (nonwound) scorbutic capillaries. Application of the law of Laplace (T= P x R) to the bulbous scorbutic capillary tips allows us to theorize that increased endothelial wall tension accounts for their fragility.
INTRODUCTION Spontaneouspetechial hemorrhagesand delayed wound healing have beenrecognized as clinical signsof ascorbic acid deficiency for many years, yet the mechanismsfor these phenomena are still not entirely clear. Beginning with the classicalstudiesof Wolbach and Howe (1926), microscopists have suggestedseveral possible explanations for the so-called “fragility” of the mature microvasculature in scurvy. Gersh and Catchpole (1949) suggesteddisaggregation of the glycoprotein of capillary basementmembrane on the basisof decreasedperiodic acid-Schiff staining intensity. Stolman et al. (1961) felt the predominant defect was a breakdown or degeneration of collagen fibers outside the endothelial wall. Using electron microscopy, Gore et al. (1968) describedendothelial cell disjunction, cytoplasmic disruption, and discontinuities of the basementmembrane as being responsiblefor the increasedpermeability of scorbutic capillaries. Interestingly, the regenerating capillaries in wounds of vitamin C-deficient animals have received limited attention even though Wolbach and Howe (1926) commented on a 1 This study was supported in part by USPHS Grant No. DE-03486. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
361
362
RALPH
V. MCKINNEY,
JR.
tendency for the regenerating scorbutic endothelium to “accumulate in clumps.” Penny and Balfour (1949) and Dunphy et al. (1956) also noted abnormalities in the newly formed scorbutic vessels but did not elaborate on their observations. Others have reported the delay in the growth rate of scorbutic vessels into a wound (Ross and Benditt, 1961; Wolbach and Howe, 1926). The closest study relating to the ultrastructure of scorbutic growing capillaries was that of Friederici and associates (1966) who examined the capillaries of inflammed synovia in scorbutic guinea pigs. These authors described “rarified” endothelial cells and a “tenuous basement membrane” as prominent ultrastructural features of these vessels. The objective of this investigation was to characterize regenerating capillaries in scorbutic animals, particularly those in skeletal muscle wounds, in order to compare their structure with normal regenerating capillaries and mature (nonwound) scorbutic capillaries. Emphasis was placed on the characteristics of the endothelium, the basement membrane, and the surrounding perivascular collagen fibers since changes in these structures have been implicated in the fragility of nonregenerating scorbutic vessels. In addition, intravascular injection of tracers was carried out in an attempt to delineate the permeability characteristics of the scorbutic regenerating capillaries. MATERIALS
AND
METHODS
Animals and Wounding A total of 29 adult male guinea pigs weighing 550-1000 grams were used in this investigation. Nineteen guinea pigs were allowed an adlibitum diet ofvitamin C-deficient guinea pig chow and water. Ten animals were used as controls. Twelve of the animals on the scorbutic diet received a commercial preparation of the Reid-Briggs ascorbic acid-deficient diet (Nutritional Biochemicals Corp., Cleveland, Ohio 44128). The other seven scorbutic animals received a normal pelleted Purina rabbit chow that had the vitamin C inactivated by heating the chow to 100°F for 30 to 60 min. The animals were maintained on the scorbutogenic diet 8 days prior to wounding. At the end of the 8 days, a circular wound of 2-4 mm in diameter was created in the cremaster muscle of the scorbutic guinea pigs employing a previously described wounding procedure (McKinney and Panner, 1972; Schoefl, 1963). The animals were then continued on the scorbutogenie diet after wounding and biopsies were taken of the wounds at 3, 7, 8, 9, 14, or 16 days. The 10 control animals were fed a normal pelleted guinea pig chow containing vitamin C and subjected to a similar surgical procedure and biopsy interval. Biopsy Procedure At the time of biopsy, light ether anesthesia was used to allow a cutdown procedure to the saphenous vein of the guinea pig. Pentobarbital solution (65 mg per ml of NaPentobarbital solution), 0.05 ml per 100 grams of body weight, was injected intravascularly to maintain a suitable level of anesthesia. Five minutes prior to biopsy the animal was injected intravenously with colloidal carbon (Biological Ink, Koh-I-Noor, Inc., Bloomsbury, New Jersey), 0.1 ml per 100 grams of body weight, or with horseradish (Sigma Type II, Sigma Chemical Co., St. Louis, Missouri) 10 mg per 100 grams of body weight dissolved in 1 ml of 0.9% sterile saline solution. The balance of the biopsy procedure has been previously described (McKinney and Panner, 1972).
SCORBUTIC
REGENERATING
CAPILLARIES
363
Ascorbic Acid Determination Samples of the animal’s lung, adrenal, spleen, and liver were removed for ascorbic acid analysis. Visual titration of the homogenized tissue sample was carried out using 2,6-dichlorophenolindophenol according to the modified Tillman’s method (Gyorgy and Pearson, 1967). Data was expressed as milligrams of ascorbic acid per 100 grams (wet weight) of tissue sample.
FIG. 1. Fringe of normal (right side of figure). Some carbon while other capillary of the capillary tips. Whole
(nonscorbutic) capillary sprouts growing toward the center of the wound of the larger capillaries and a few of the tips are blackened with injected tips do not contain carbon (arrows). Note the cylindrical or tapered shape mount of an I-day nonscorbutic wound edge. Unstained, x420.
Light and Electron Microscopy The muscletissuewas fixed with glutaraldehyde-paraformaldehyde fixative in 0.2 M cacodylate buffer at pH 7.2 for 2-4 hr, either at room temperature or 4°C. The tissue was then rinsed in 0.2 A4 cacodylate buffer three times, trimmed, and placed in buffer solution on a slide for light photomicroscopy of the fringe of growing capillaries at the wound edge(Figs. 1 and 2). Subsequently, the tissuewas trimmed, postfixed in osmium tetroxide, and embeddedaswe have previously detailed (McKinney and Panner, 1972). Thin sectionswere cut on an LKB Ultratome III using 50” angle glassand diamond knives. Sections were stained for 15-25 min with aqueous uranyl acetate and 3-5 min with lead citrate, then examined in a Hitachi HU-1 1A or RCA EMU4C electron microscopeat 75 kV. One-micron-thick sectionswere cut and stained with Paragon stain for light microscopic orientation.
364
RALPH V. MCKINNEY, JR.
Histochemistry Some tissuesampleshad the electron histochemical stain ruthenium red (Luft, 1971) added to the fixative solutions to enhance the demonstration of glycosaminoglycuronoglycans. We have previously reported the methodsand modifications we usedfor this procedure (McKinney and Panner, 1972).
FIG. 2. Fringe of scorbutic capillary sprouts growing toward the center of the wound (right of picture). The capillary tips are saccular or bulbous and appear black because of the injected carbon. Some of the bulbous tips are bent at 90-degree angles to the more proximal portion of the capillary (arrows). There is also a decrease in the number of anastomosing capillaries proximal from the growing fringe when compared with Fig. 1. Whole mount of an 8-day scorbutic wound edge. Unstained, x 420.
Following initial fixation, the tissue samplesinjected with horseradish peroxidase (HRP) were washed overnight in 0.1 A4 cacodylate buffer at 4°C. These were then cut into small piecesand transferred to 0.05 A4Tris buffer, pH 7.6, and incubated for 30 min
365
SCORBUTIC REGENERATING CAPILLARIES
in the incubation medium of Graham and Karnovsky (1966). Following incubation the tissue was washed three times in distilled water and postfixed in 1% 0~0,. The methenamine silver histochemical reaction was applied to some selectedtissue samplesin an attempt to demonstrate collagen or collagenlike fibers in the basement membrane and extravascular space. Following initial fixation the tissue was taken up to 70 % ethanol, then silver impregnation was carried out according to the method of Churg et al. (1958). It wasfound that 24-hr incubation in the silver methenaminesolution was sufficient for impregnation of the thin cremaster muscle.Following incubation, the tissuewas rinsed three times in distilled water, once in cacodylate buffer, and postfixed in 1% 0~0,. Although the mechanismsof action for the ionic binding of silver grains on collagen or collagenlike fibrils is not completely understood, it has been shown that the reduced silver grains selectively deposit on collagen fibers in a periodic fashion and are not eliminated by hyaluronidase treatment (Grassman and Hofman, 1957). RESULTS Gross Observations The scorbutic guinea pigs recovered well from the wounding procedure and the abdominal incision always healed uneventufully. Most animals remained active and fairly healthy up to 14 days on the scorbutogenic diet. After 14-16 days on the scorbutic TABLE 1 MEAN ASCORBICAcre LEVELS OF GUINEA Lung
Adrenal
PIG TISSUE”
Spleen
Liver
Control animals
27.5 f 2.12 (10)
125.5 _+ 7.56 (JO)
47.4 -+ 5.48 (9)
21.2 _+3.12 (10)
g-Day scorbutic animals
5.17 + 1.25 (6) P < 0.001
31.0 * 4.14 (5) P < 0.001
8.83 + 2.01 (6) P < 0.001
6.17 + 1.74 (6) P < 0.005
15-, 16-, and 1‘J-Day scorbutic animalsb
4.33 f 0.84 (6) P < 0.001
12.80 i 3.31 (5) P < 0.001
7.00 + 0.68 (6) P < 0.001
4.00 + 0.55 (5)
3.60 + 0.68 (5)
8.80 i. 0.66 (5) P < 0.001
6.60 + 0.40 (5)
4.50 + 0.96 (4) P < 0.01
22- and 24Day scorbutic animal?
P
