Angiogenesis is crucial to full-thickness skin graft survival. 1 Improvement of graft vascularization and maintenance of adequate microvascular perfusion contribute to the success of transplantation.Vascularization of free full-thickness skin grafts is achieved through early anastomoses between the original small vessel of the graft and the bed. 2 Research has shown that the large arteries in rabbits survive full-thickness skin autograft and become permanently joined at the periphery of the grafts to adjacent severed arteries in the host. 3
Prostaglandin E2 (PGE2) is among the molecules that promote angiogenesis. Investigators have reported that PGE2 enhances angiogenesis in wound healing of soft tissue. 4 In addition, studies show that PGE2 stimulates angiogenesis in mechanical spinal cord injury; 5 gastric ulcer healing; 6,7 and gastric, breast, head and neck, and colorectal carcinoma. 6,8–10 PGE2 also induces vascularization during bone formation by stimulating vascular endothelial growth factor (VEGF) 11 and acting as an inflammatory angiogenesis factor. 12
Infusion of PGE2 has been used to enhance the preservation of lung and skin transplantation. 13,14 PGE2 elicits a dose-dependent increase in femoral blood flow. 15 Furthermore, evidence exists that PGE2 may have a role in prevention of allograft rejection. 16,17 However, only limited quantitative work on this subject is available.
Given these considerations, the goal of the present study was to determine whether PGE2 improves angiogenesis in full-thickness skin graft. By using stereologic methods, the authors examined how much of the unit volume of the grafts induced by PGE2 is occupied by the vessels.
Twenty male Dutch rabbits, weighing between 1800 and 2100 grams, were divided randomly into 2 groups—experimental and control. Each group was then randomly divided into 2 groups again, yielding 2 experimental and 2 control groups. Principles of laboratory care established by the National Institutes of Health 18 were followed.
The left posterior sides of the rabbits’ necks were shaved. After the induction of anesthesia by intramuscular administration of ketamine/xylosin (44 mg/kg and 8 mg/kg, respectively), a single full-thickness skin autograft of 2 cm in diameter was removed, rotated 180º, and reattached on the same animal at the same site. The wound was closed with a continuous 3/0 Dafilon suture and dressed by tie-over graft.
The same dressing, treated with tetracycline, was used on all rabbits. Solutions of 0.2 mL of PGE2 in triacetin (glycerol triacetate 19 0.1 mg/mL) were prepared and injected locally into the graft site, between the graft and its bed. Rabbits were housed individually, with free access to food and water, in a room with a constant temperature of 22º to 24º C and a humidity of 55%. The rabbits were exposed to 12-hour cycles of light and darkness.
Skin transplantations were allowed to heal in 1 experimental and 1 control group for 5 days, with injections of 0.1 mL of the same solution of PGE2 continued daily up to day 5. The other groups (1 experimental and 1 control) were allowed to heal for 10 days, receiving the same injections as the former groups.
At 5 and 10 days after transplantation, grafts were judged for graft viability on the basis of gross appearance, texture and adherence, and histologic criteria. Skin grafts were harvested at days 5 and 10, respectively.
The harvested skin samples were fixed in neutral buffered formaldehyde, dehydrated, and embedded in paraffin blocks. Each skin sample was cut perpendicular to the skin surface into a series of 30 sections that were each 7 microns thick, separated by 1 mm of the skin that was cut and excluded by a microtome (Leika, Japan). This procedure was repeated until the entire area of the graft was prepared.
With random systematic sampling, 5 sections (7-micron thick) were selected from each collection of 30 sections with a known distance of “t,”then stained with Heidenhain’s azan. 20 These stereologic estimates are independent of the orientation of the set of sections and the shape or orientation of various structures (eg, vessels). 21
Morphometric study was done using a projecting microscope (Visupan, Austria) equipped with a circular screen. On each sampled section, an average of 5 microscopic, nonoverlapping fields were selected by moving in equal distances the microscope’s stage in X and Y. Selection of the nonoverlapping fields helps avoid sampling error by giving every part of the section an equal chance of being sampled.
The fractional volume of the vessels, Vv (vessel), was calculated by the point counting method. 21 A point test system was used, consisting of 100 points at final magnification of x800, according to the Deless principle. 21 The number of points hitting the vessels’ profiles (p) and total points hitting the reference space (P) of the section were counted, and the fractional volume of vessels was estimated using the formula: Vv = Pp.100. The percent of vessels was calculated as a proportion of the nonvascular background tissue.
The references volume,V (ref), was estimated with Cavalierie’s principle by using the formula: V(ref) = ∑p.a(p).t. The “p” is the total points hitting the reference space,“a(p)”is the area associated with each point at the microscopic level, and “t” is the known distance between the selected sections.
The absolute volume of the vessels was estimated in the reference volume of the grafted dermis by multiplying the fractional volume and the reference space; interpretation of fractional volume individually may lead to incorrect conclusion that has been called “reference trap”in stereologic text. 21
Finally, absolute volume of the vessels (mm3) in the unit volume (1 mm3) of the dermis was calculated. At the selected microscopic field, lumen diameter of the vessel was measured at right angle to the maximum width of the vessel profile. 22
A Mann-Whitney test was used for comparison between the different groups. A probability level of α< 0.05 was selected as statistically significant.
Gross appearance of the control and experimental grafts were the same. Qualitative histologic examination of successful grafts in experimental and control groups showed areas of viable epidermis with a negligible inflammatory infiltrate and moderate fibrosis. Blood cells were frequently seen in the vessels under investigation. Quantitative examination of the histologic slides showed that mean fractional volume (percent) and absolute volume of the vessels (mm3) per unit volume (mm3) of the grafted skin of experimental groups were significantly higher than in the control groups (Table 1).
The morphometric parameters showed that the fractional and absolute volume of the vessels were increased significantly in the experimental groups when compared with the control groups (67% and 53% in experimental groups 5 and 10 days after the surgery, respectively; P < 0.008). No significant differences were found between the 2 experimental groups harvested 5 and 10 days after surgery; the same was true of the 2 control groups (Figure 1). This finding may be related to the decrease in angiogenesis rate and the discontinuation of PGE2 injections after day 5.
Lumen diameter of the vessels in the grafted dermis of the control and experimental groups were measured. No significant differences were found between the caliber of the vessels in the experimental and control groups (Table 2).
Significant evidence supports the involvement of PGE2 in angiogenesis. 5-9 Research also shows that its inhibitors suppress angiogenesis. 23,24 However, the exact mechanisms by which PGE2 can promote angiogenesis are still unclear. Several molecules have been hypothesized, primarily based on studies of angiogenesis in different types of tumors and wound healing. 4-6,8,9
Various growth factors, such as VEGF, 5,10 epidermal growth factor (EGF), 25 and fibroblast growth factor (FGF), 4 are angiogenetic factors that promote endothelial cell proliferation during development and repair. PGE2 may participate in angiogenesis and healing mechanisms in soft tissue by means of induction of basic fibroblast growth factor (bFGF) 4 and VEGF production. 5,8,10 The literature suggests that an important collaborative interaction of transforming growth factor–ß1 and EGF signaling occurs in the induction of cyclooxygenase and its product, PGE2, in some cell lines. 7,26
Oral administration of specific cyclooxygenase–2 inhibitors lowers the expression of potent angiogenetic factors such as VEGF and FGF, thus reducing angiogenesis and growth. 27 Decreased PGE2 production by cyclooxygenase–2 inhibition is associated with an increase in apoptosis and a decrease in proliferation of endothelial cells. 28 This may also delay reepithelialization in the early phase of wound healing and inhibit angiogenesis. 29
Despite this evidence, the relationship between PGE2 and growth factors has not been confirmed in all studies. For example, some studies indicate that angiogenesis is significantly inhibited by indomethacin (inhibitor of PGE2 production) but VEGF production is not reduced. This suggests that inhibition of angiogenesis in indomethacin-delayed ulcer healing cannot be explained by VEGF expression. 9,24
Other studies focus on tissues free of tumors. A single, local injection of PGE2 into soft tissue surrounding the femoral vein induced a sudden and intense angiogenesis with vascular sprouts arising from endothelial cell in the intima of the vein. 19
Data from the present study show that the fractional and absolute volume of vessels in the graft dermis of PGE2-injected groups is greater than in the control groups within a short period after transplantation (5 and 10 days). This may indicate either an increased number of blood vessels (angiogenesis) or an increased number of turns in the same number of blood vessels coursing through the tissue. Further research is needed to resolve this question and to explore the effects of PGE2 on full-thickness skin graft take. Quantitative studies of promoters and potential promoters of angiogenesis in full-thickness skin grafts were also performed. For example, bFGF increased vessel profile count in a high dose (5000 ng) in cryo-injected grafts. 30
Some quantitative studies were conducted to investigate the effect of human peripheral blood mononuclear cells 31 and endothelial cell culture on angiogenesis response in skin graft.32 Research has shown that microvessel counts were reduced in tumors after inhibiting PGE2,9 however, no quantitative studies have shown how much of PGE2-treated grafts were occupied by vessels when compared with the control groups.
PGE2 causes a vasodilatating effect. 5 Vasodilatation, increasingly tortuous or “wrinkled”vessels, or increasing the number of blood vessels (angiogenesis) could increase the absolute amount of vessels in the containing or “reference”space. However, estimation of the lumen diameter of vessels showed no significant difference between the control and experimental animals.
It can be concluded that either angiogenesis or increasing numbers of vascular turns or wrinkles are the main factors for an increase in absolute volume of the vessels. Local injection of PGE2 can increase the fractional and absolute volume of vessels in full-thickness skin graft and can be explored as a possible agent to improve angiogenesis of the grafts.
The authors thank the research deputy of Shiraz University of Medical Sciences for supporting the project by grant no. 79-1088, personnel of the university’s animal house, and F. Pirsalami for the preparation of histologic specimens.
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