In vivo analysis of angiogenesis in endometriosis-like lesions by intravital fluorescence microscopy

In vivo analysis of angiogenesis in endometriosis-like lesions by intravital fluorescence microscopy Matthias W. Laschke, M.D.,a Antje Elitzsch, D.V.M.,a Brigitte Vollmar, M.D.,b and Michael D. Menger, M.D.a a

Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany; and Experimental Surgery, University of Rostock, Rostock, Germany

b

Department of

Objective: To establish a novel endometriosis model that allows for repetitive in vivo analyses of angiogenesis in ectopic endometrial tissue. Design: Intravital fluorescence microscopic study. Setting: Institute for Clinical and Experimental Surgery, University of Saarland. Animal(s): Female Syrian golden hamsters equipped with skinfold chambers. Intervention(s): Large (0.5 mm2) and small (0.1 mm2) endometrial fragments were mechanically isolated and transplanted autologously into skinfold chambers of untreated hormonally synchronized or bilaterally ovariectomized hamsters. Main Outcome Measure(s): Angiogenesis, vascularization, and microhemodynamics were analyzed over a 14-day period. Result(s): In untreated controls, endometrial fragments developed complete microvascular networks during the experimental observation period. Interestingly, microvascular blood flow was higher in large than in small fragments. Histologic examinations revealed proliferating endometriosis-like lesions with dilated endometrial glands surrounded by a richly vascularized stroma. Vascularization of endometrial fragments in synchronized animals did not differ from that of untreated controls. In contrast, endometrial fragments in ovariectomized animals showed a delay in angiogenesis and a significantly decreased blood perfusion, indicating the essential role of ovarian estrogens for ectopic vascularization and perfusion of endometrial tissue. Conclusion(s): This novel model of endometrial tissue transplantation is a useful experimental approach, not only to focus on the in vivo pathogenesis of endometriosis but also to develop antiangiogenic strategies for the treatment of this disease. (Fertil Steril威 2005;84(Suppl 2):1199 –209. ©2005 by American Society for Reproductive Medicine.) Key Words: Endometriosis, angiogenesis, Syrian golden hamster, dorsal skinfold chamber, transplantation, intravital fluorescence microscopy, synchronization, ovariectomy, microcirculation

Endometriosis, defined by the presence and proliferation of functional endometrial glands and stroma outside the uterine cavity, is a frequent gynecologic disease, which affects approximately 10%–15% of all women in reproductive age and 40%–50% of all infertile women (1, 2). The patients suffer from severe pelvic pain during their menstrual cycle and bear an increased risk of developing ovarian cancer (3). Despite the fact that endometriosis is one of the most investigated diseases in gynecology, many questions about its etiology and pathogenesis are still unanswered, which is due, in part, to the fact that endometriotic lesions often reveal contradictory biologic characteristics (4). The most widely accepted theory for the development of endometriosis is retrograde menstruation (i.e., reflux of endometrial fragReceived January 17, 2005; revised and accepted May 8, 2005. This work was supported by a grant of the Wilhelm Sander-Foundation (Nr. 2002.008.01) and the research program of the Medical Faculty of the University of Saarland, HOMFOR A/2004/09. Reprint requests: Michael D. Menger, M.D., Institute for Clinical and Experimental Surgery, University of Saarland, D-66421 Homburg/Saar, Germany (FAX: ⫹49-6841-162-6553; E-mail: [email protected]).

0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2005.05.010

ments in the fallopian tubes during menstruation and their implantation and growth in the peritoneal cavity) (5). This theory is supported by several studies that demonstrate by histology that endometrial tissue grafted to ectopic sites is able to implant and develop endometriosis-like lesions with functional endometrial glands and stroma (6 – 8). A major prerequisite for successful engraftment is the process of angiogenesis (i.e., the development of new capillary blood vessels from the pre-existing vasculature) to sufficiently supply the endometrial implants with oxygen and essential nutrients. The expression of several angiogenic factors is increased in endometriotic lesions and in the peritoneal fluid of patients with endometriosis including basic fibroblast growth factor (9, 10), vascular endothelial growth factor (VEGF) (11, 12), interleukin-8 (13), and plateletderived endothelial cell growth factor (14). Moreover, former studies have shown that larger endometriotic lesions grow in areas with a rich blood supply, and that the most active lesions are those that are highly vascularized (15). Thus, endometriosis can be assigned to the group of angiogenic diseases, which includes cancer, proliferative diabetic retinopathy, rheumatoid arthritis, and psoriasis (16, 17).

Fertility and Sterility姞 Vol. 84, Suppl 2, October 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.

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Because angiogenesis seems to play a major role in the pathogenesis of endometriosis, application of antiangiogenic agents might be a future therapeutic strategy in the treatment of this disease. Therefore, experimental models are needed to improve the knowledge about blood vessel development within endometriotic lesions and the influence of angiogenesis inhibitors on the course of endometriosis. Because angiogenesis is a complex dynamic process, characterized by capillary budding and sprouting, capillary network formation, and remodeling of microhemodynamics over time, the aim of our study was to establish an endometriosis model, which allows for the first time the repetitive in vivo analysis of the microcirculation in ectopic endometrial tissue. For this purpose, we transplanted mechanically isolated endometrial fragments of different size into the dorsal skinfold chamber of untreated, hormonally synchronized and bilaterally ovariectomized Syrian golden hamsters and analyzed morphologic and microhemodynamic parameters of the grafts’ microcirculation using the technique of intravital fluorescence microscopy. MATERIALS AND METHODS Animals The experiments were conducted in accordance with the German legislation on protection of animals and the NIH Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, Washington, DC) and were approved by the local governmental animal care committee. Eight- to 10-week-old female Syrian golden hamsters with a body weight of 60 – 80 g were used for the study. The animals were housed one per cage and had free access to tap water and standard pellet food (Altromin, Lage, Germany) throughout the experiment. Preparation of the Dorsal Skinfold Chamber The dorsal skinfold chamber preparation contains one layer of striated muscle, subcutaneous tissue, and skin and allows for intravital microscopic observation of the microcirculation in the awake animal over a period of time of up to 3 weeks (Fig. 1A). The chamber technique and its implantation procedure have been described previously in detail (18). Briefly, under sodium pentobarbital anesthesia (50 mg/kg body weight IP), two symmetrical titanium frames were implanted on the extended dorsal skinfold of the hamsters so that they sandwiched the double layer of skin. One layer of skin was then completely removed in a circular area of ⬃15 mm in diameter, and the remaining layers (consisting of striated skin muscle, subcutaneous tissue, and skin) were covered with a removable coverslip incorporated into one of the titanium frames. After the preparation, the animals were allowed to recover from anesthesia and surgery for at least 48 hours. 1200

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Isolation and Transplantation of Endometrial Fragments For isolation of endometrial fragments, each hamster equipped with a skinfold chamber was anesthetized with pentobarbital sodium (50 mg/kg body weight IP). After laparotomy, one uterus horn was aseptically removed and placed in a 30-mmdiameter Falcon plastic Petri dish filled with 37°C warm Dulbecco’s modified Eagle’s medium (10% fetal calf serum, 0.1 mg/mL gentamicin) and the fluorescent dye bisbenzimide H33342 (200 ␮g/mL; Sigma, Deisenhofen, Germany). The specific fluorescence/background fluorescence ratio is high enough to precisely delineate the stained endometrial tissue from the nonstained surrounding host tissue after transplantation into the dorsal skinfold chamber (19). In addition, bisbenzimide, which stains the nuclear structure of the cells, can be used to study apoptotic cell death in vivo by analyzing condensation, fragmentation, and margination of chromatin (20). The uterus horn was opened longitudinally and the endometrium was dissected from the uterine muscle under a stereo microscope. Then the endometrium was transferred into 37°C warm bisbenzimide H33342-free Dulbecco’s modified Eagle’s medium and microdissected into endometrial fragments of different size. For autologous transplantation of endometrial fragments, the cover glass of the dorsal skinfold chamber was removed and four endometrial fragments were placed on the striated muscle within the chamber. During closure of the chamber, one or the other of the grafts was flushed away. Therefore, not all chambers finally contained the four endometrial fragments for analysis. In general, the endometrial fragments were grouped according to their initial size at the time point of isolation and transplantation into (a) small grafts with a tissue area of ⬃0.1 mm2 and (b) large grafts with a tissue area of ⬃0.5 mm2. Because the grafts were placed with a maximal distance to each other (size of the host tissue within the observation window: 95 mm2), the angiogenic process and the microvascular perfusion could be considered unaffected by the neighboring grafts. A first group of graft recipient animals (n ⫽ 8) was hormonally synchronized (i.e., in the same stage of the 4-day estrus cycle in the hamster), and endometrial fragments were transplanted on estrus. Synchronization was performed according to the method of Gross by administration of two subcutaneous injections of 7.5 ␮g/135 g body weight of estradiol (E2) (Sigma), given 24 hours apart, followed by one injection of 1.0 mg/135 g body weight of progesterone (P) (Sigma), given 20 hours after the last E2 injection (21). To study the role of estrogens in angiogenesis of endometriotic lesions, a second group of animals comprised bilaterally ovariectomized hamsters (n ⫽ 6), which were allowed to recover from ovariectomy for at least 1 week before the preparation of the dorsal skinfold chamber and endometrium transplantation. A third group of untreated animals (n ⫽ 5) served as controls. Vol. 84, Suppl 2, October 2005

FIGURE 1 (A) Titanium chamber (weight ⬃4 g) implanted into the dorsal skinfold of a Syrian golden hamster. The observation window provides access for intravital microscopic studies of the microcirculation of striated muscle and subcutaneous tissue. Scale bar: 7.5 mm. (B–D) Intravital fluorescence microscopy of a large endometrial graft (borders indicated by arrows) directly (B), 2 days (C), and 10 days (D) after autologous transplantation into the dorsal skinfold chamber of an untreated Syrian golden hamster. Note the lack of nutritive capillaries within the freshly isolated and transplanted graft (B). At day 2 after transplantation (C), newly formed microvessels create a network of capillaries, although a substantial part of the endometrial graft still lacks vascularization (asterisk). At day 10 after transplantation (D), the endometrial graft exhibits a complete glomerulum-like microvasular network. Blue-light epi-illumination with contrast enhancement by 5% FITC-labeled dextran 150,000 IV. Scale bars: 200 ␮m.

Laschke. Angiogenesis in endometriosis. Fertil Steril 2005.

Intravital Fluorescence Microscopy For in vivo microscopic observation, the animals were immobilized in a Plexiglas tube and the dorsal skinfold preparation was attached to the microscopic stage (22). After IV injection of 0.1 mL of 5% fluorescein isothiocyanate (FITC)labeled dextran 150,000 (contrast enhancement by intravascular staining of plasma) and 0.1 mL of 0.1% rhodamine 6G (Sigma) (direct in vivo staining of white blood cells), intravital fluorescence microscopy was performed using a modified Leitz Orthoplan microscope with a 100-W mercury lamp attached to a Ploemo-Pak illuminator with blue, green, and ultraviolet filter blocks (Leitz, Wetzlar, Germany) for epi-illumination (23). The microscopic images were recorded by a charge-coupled device video camera (CF8/1 FMC; Kappa, Gleichen, Germany) and transferred to a video system for off-line evaluation. With the use of 4⫻, 6.3⫻, 10⫻, and 20⫻ long-distance objectives (Leitz), magnificaFertility and Sterility姞

tions of ⫻86, ⫻136, ⫻216 and ⫻432 were achieved on a 14-inch video screen (PVM 1444; Sony, Tokyo, Japan). Microcirculatory Analysis Quantitative off-line analysis of the videotapes was performed by means of the computer-assisted image analysis system CapImage (Zeintl, Heidelberg, Germany) and included the determination of the size of the transplanted endometrial fragments (mm2), the size of the blood perfused microvascular networks (given in percent of the size of the grafts), the microvessel density (i.e., the length of red blood cell (RBC)-perfused microvessels per observation area) (cm/ cm2), the diameters of the microvessels (␮m), and the centerline RBC velocity VRBC (␮m/sec). Volumetric blood flow (VQ) of individual microvessels was calculated from VRBC and diameter (d) for each microvessel as VQ ⫽ ␲ ⫻ (d/2)2 ⫻ VRBC/K (pL/sec), where K (⫽1.3) represents the Baker/ 1201

Wayland factor (24), considering the parabolic velocity profile of blood in microvessels. Rhodamine 6G–stained leukocytes were classified in accordance to their interaction with the endothelium of newly formed microvessels (25). Rolling cells were defined as cells moving with a velocity less than two-fifths of the centerline velocity. Adherent cells were defined as cells that did not move or detach from the endothelial lining during an observation period of ⬎20 seconds. Experimental Protocol A total of 17 endometrial fragments were transplanted into the dorsal skinfold chambers of five untreated female hamsters. A total of 19 endometrial fragments per group were transplanted into the dorsal skinfold chambers of eight hormonally synchronized and six bilaterally ovariectomized hamsters. The macroscopic appearance of the skinfold chamber preparations and the implanted grafts were documented daily. Intravital fluorescence microscopic analysis of the microcirculation was performed on days 0, 2, 4, 7, 10, and 14 after transplantation of the endometrial fragments. Measurements on vascular density and microhemodynamics included only newly formed microvessels that could be clearly distinguished by their glomerulum-like arrangement from the autochthonous host striated muscle microvessels, which display the typical parallel arrangement of muscle capillaries (22). Microvessel density was measured within three regions of interest per graft and observation time point. Microvascular diameters and microhemodynamic parameters were determined by analyzing ten microvessels per region of interest, randomly chosen among those that crossed a vertical line drawn over the center of the video screen. In all microvessels selected, both vessel diameter and VRBC were determined for subsequent calculation of VQ. At the end of the in vivo experiments (i.e., day 14 after transplantation of endometrial fragments), the animals were killed with an overdose of pentobarbital, and the dorsal skinfold chamber preparations were processed for hematoxylin-eosin staining and immunohistochemistry. Histology and Immunohistochemistry For light microscopy, formalin-fixed specimens of the dorsal skinfold chamber were embedded in paraffin. Four-␮m-thick sections were cut and stained with hematoxylin and eosin according to standard procedures. For immunohistochemical detection of proliferating cells within the endometrial grafts, proliferative cell nuclear antigen (PCNA) staining was performed by a mouse monoclonal anti-PCNA antibody as primary antibody (1:50; Dako Cytomation, Hamburg, Germany). This was followed by a goat antimouse antibody (1:200; Amersham, Freiburg, Germany). 3,3=-diaminobenzidine was used as chromogen. The sections were counter1202

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stained with hemalaun and examined by light microscopy (BX60; Olympus, Hamburg, Germany). Statistics Data were first analyzed for normal distribution and equal variance. Differences between groups were then calculated by ANOVA followed by the appropriate post hoc test compensating for multiple comparisons. To test for time effects within each experimental group, ANOVA for repeated measures was applied. This was followed by a post-hoc paired comparison, including correction of the ␣-error according to Bonferroni probabilities for repeated measurements (SigmaStat; Jandel Corporation, San Rafael, CA). All data are given as mean ⫾ SEM. Statistical significance was accepted for P⬍.05. RESULTS In general, isolated endometrial fragments were able to induce angiogenesis and to vascularize after transplantation into the dorsal skinfold chamber regardless of whether they had been grafted in untreated, synchronized, or ovariectomized hamsters. After isolation and transplantation, large endometrial grafts presented with a comparable initial size of 0.55 ⫾ 0.04 mm2 in untreated, 0.55 ⫾ 0.06 mm2 in synchronized, and 0.50 ⫾ 0.04 mm2 in ovariectomized hamsters. The large grafts significantly differed from the small grafts with initial sizes of 0.11 ⫾ 0.01 mm2, 0.13 ⫾ 0.01 mm2, and 0.11 ⫾ 0.01 mm2 in the respective groups. Sequential analysis of the grafts’ sizes over the 14-day observation period revealed an increase of ⬃60%–70% in large grafts and ⬃80%–90% in small grafts without statistical differences between the groups, indicating appropriate growth and viability after transplantation. Analysis of bisbenzimide nuclear staining did not indicate manifestation of significant apoptotic cell death in the endometrial fragments of animals of either of the groups. In untreated animals, angiogenesis was already observed at day 2 after transplantation, characterized by bud formation, sprout protrusion, and sinusoidal sacculations. During the further time course, the sprouts interconnected with each other and finally developed complete microvascular networks at day 7. These networks presented with a microvessel density of ⬃300 – 400 cm/cm2. During the remainder of the experiment, microvessel density showed some variations, which, however, did not prove to be significant over time (Figs. 1B–D and Fig. 2; Table 1). Owing to their glomerulum-like appearance, the newly formed microvascular networks could easily be distinguished from the parallelly arranged striated muscle capillaries of the dorsal skinfold chamber (22). At day 14, the size of the vascularized area almost matched the size of the graft (Fig. 2). In endometrial grafts transplanted in synchronized animals, the process of angiogenesis and microvascular network formation did not significantly differ from that of grafts in Vol. 84, Suppl 2, October 2005

FIGURE 2 (A, B) Intravital fluorescence microscopy of small endometrial grafts 4 days after autologous transplantation into the dorsal skinfold chamber of an untreated (A) and a bilaterally ovariectomized (B) Syrian golden hamster. In the untreated animal (A) the major area of the endometrial graft shows an appropriately perfused microvasculature. In contrast, the endometrial graft of the ovariectomized hamster (B) exhibits only a small area with microvascular perfusion (asterisk). Note that beneath these perfused microvessels some additional vascular segments can be observed, which, however, appear dark in the intravital microscopic imaging owing to the lack of FITC-dextran– labeled blood perfusion (B, arrows). Blue-light epi-illumination with contrast enhancement by 5% FITC-labeled dextran 150,000 IV. Scale bars: 125 ␮m. (C, D) Vascularized area (%) of large (C) and small (D) endometrial grafts after autologous transplantation into the dorsal skinfold chambers of untreated (open circles), hormonally synchronized (closed circles), and bilaterally ovariectomized (closed triangles) Syrian golden hamsters, as assessed by intravital fluorescence microscopy and computer-assisted image analysis. Means ⫾ SEM. *P⬍.05 vs. synchronized animals at corresponding time points; #P⬍.05 vs. untreated animals at corresponding time points; aP⬍.05 vs. day 0; bP⬍.05 vs. days 0 and 2; cP⬍.05 vs. days 0, 2, and 4.

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untreated controls (Fig. 2; Table 1). In contrast, endometrial fragments grafted in ovariectomized animals revealed a delay in vascularization. Large grafts showed a significantly decreased vascularized area and microvessel density at day 2 Fertility and Sterility姞

(Fig. 2C; Table 1). Small grafts revealed an even more pronounced delay in vascularization, in that the formation of new blood vessels could first be observed at day 4 after transplantation, and mean values of the vascularized area at 1203

TABLE 1 Microvessel density (cm/cm2, mean ⴞ SEM) of newly formed microvascular networks within endometrial fragments transplanted into dorsal skinfold chambers of untreated, synchronized, and ovariectomized Syrian golden hamsters.

Large endometrial grafts: Untreated Synchronized Ovariectomized Small endometrial grafts: Untreated Synchronized Ovariectomized

Day 2

Day 4

Day 7

Day 10

Day 14

231 ⫾ 35 282 ⫾ 15 115 ⫾ 40*

330 ⫾ 44a 370 ⫾ 24a 270 ⫾ 44a

416 ⫾ 22a 374 ⫾ 18a 405 ⫾ 37b

363 ⫾ 24a 411 ⫾ 17a 388 ⫾ 30b

360 ⫾ 22a 410 ⫾ 34a 343 ⫾ 24b

306 ⫾ 29 198 ⫾ 40 0 ⫾ 0*#

375 ⫾ 26 346 ⫾ 34a 301 ⫾ 82a

362 ⫾ 34 333 ⫾ 36a 405 ⫾ 47a

284 ⫾ 36 346 ⫾ 19a 376 ⫾ 46a

383 ⫾ 34 380 ⫾ 21a 361 ⫾ 44a

* P⬍.05 vs. synchronized animals at corresponding time points. # P⬍.05 vs. untreated animals at corresponding time points. a P⬍.05 vs. day 2. b P⬍.05 vs. days 2 and 4. Laschke. Angiogenesis in endometriosis. Fertil Steril 2005.

day 14 reached only ⬃70% of the size of the grafts (Fig. 2B and D; Table 1). In all groups studied, the diameters of the newly formed capillaries within both large and small endometrial grafts were found reduced from ⬃12–17 ␮m at day 2 to ⬃9 –11 ␮m at day 14 after transplantation, indicating maturation of the microvessels (Table 2). In contrast, centerline red blood cell velocity progressively increased in grafts of untreated and synchronized hamsters throughout the observation period up to values of ⬃400 ␮m/sec (large grafts) and ⬃260

␮m/sec (small grafts), whereas large as well as small endometrial fragments in ovariectomized animals presented with significantly lower red blod cell velocities of ⬃140 ␮m/sec at day 14 (Fig. 3A and C). Accordingly, endometrial fragments in untreated and synchronized animals revealed a significantly higher volumetric blood flow of ⬃25 pL/sec (large grafts) and ⬃12–15 pL/sec (small grafts) when compared with ⬃8pL/sec in fragments of ovariectomized animals (Fig. 3B and D). Of interest, irrespective of the treatment modality, the values of red blood cell velocity and

TABLE 2 Microvessel diameters (␮m, mean ⴞ SEM) of newly formed capillaries within endometrial fragments transplanted into dorsal skinfold chambers of untreated, synchronized, and ovariectomized Syrian golden hamsters. Day 2 Large endometrial grafts: Untreated Synchronized Ovariectomized Small endometrial grafts: Untreated Synchronized Ovariectomized

Day 4

Day 7

Day 10

Day 14

16.9 ⫾ 1.3 16.4 ⫾ 1.4 14.6 ⫾ 0.2

14.5 ⫾ 1.4 14.2 ⫾ 0.9 14.1 ⫾ 0.9

10.9 ⫾ 0.3b 12.3 ⫾ 0.7b 10.2 ⫾ 0.5*b

10.6 ⫾ 0.5b 11.7 ⫾ 0.2b 10.1 ⫾ 0.4*b

10.9 ⫾ 0.6b 10.9 ⫾ 0.3d 10.3 ⫾ 0.3b

12.0 ⫾ 1.0* 17.1 ⫾ 1.9 —

11.7 ⫾ 0.7 12.5 ⫾ 0.6a 11.2 ⫾ 0.5

9.2 ⫾ 0.5*b 12.2 ⫾ 0.6a 10.6 ⫾ 0.6

9.0 ⫾ 0.6b 10.3 ⫾ 0.3c 10.7 ⫾ 0.5

9.7 ⫾ 0.8 10.0 ⫾ 0.2c 9.9 ⫾ 0.6

* P⬍.05 vs. synchronized animals at corresponding time points. a P⬍.05 vs. day 2. b P⬍.05 vs. days 2 and 4. c P⬍.05 vs. days 2, 4, and 7. d P⬍.05 vs. days 2, 4, 7, and 10. Laschke. Angiogenesis in endometriosis. Fertil Steril 2005.

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FIGURE 3 Centerline red blood cell velocity (␮m/sec) (A, C) and volumetric blood flow (pL/sec) (B, D) of large (A, B) and small (C, D) endometrial grafts after autologous transplantation into the dorsal skinfold chambers of untreated (open circles), hormonally synchronized (closed circles), and bilaterally ovariectomized (closed triangles) Syrian golden hamsters, as assessed by intravital fluorescence microscopy and computer-assisted image analysis. Means ⫾ SEM. *P⬍.05 vs. synchronized animals at corresponding time points; #P⬍.05 vs. untreated animals at corresponding time points; aP⬍.05 vs. day 2; bP⬍.05 vs. days 2 and 4; cP⬍.05 vs. days 2, 4 and 7; dP⬍.05 vs. days 2, 4, 7, and 10.

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volumetric blood flow of individual microvessels in large grafts were markedly higher than those measured in small grafts (Fig. 3). In all groups, the analysis of rhodamine 6G–stained leukocytes showed that almost all the leukocytes (⬎95%) passed the newly formed microvascular networks without any transient tethering or rolling interactions. Moreover, adherent leukocytes could only rarely be observed, indicating absence of inflammation in this model of endometrial tissue transplantation (Fig. 4A and B). Histologic examination of the dorsal skinfold preparations at day 14 after transplantation revealed typical endometriotic lesions, which consisted of cyst-like dilated endometrial glands surrounded by a richly vascularized endometrial stroma (Fig. 4C and D). Immunohistochemical detection of PCNA proved cell proliferation within the glandular epithelium, the endometrial stroma, and the endothelium of newly developed blood vessels (Fig. 4E). Because the fluorescent dye rhodamine 6G accumulated in the glandular epithelium, Fertility and Sterility姞

the cysts could also be identified by intravital fluorescence microscopy using green-light epi-illumination (Fig. 4F). Interestingly, some of these cysts showed a tendency to grow throughout the observation period, indicating secretory activity of the glandular epithelium. DISCUSSION In the present study, we could establish a new endometriosis model in rodents, which allows for the first time the repetitive in vivo analysis of angiogenesis, vascularization, and microhemodynamics in ectopic endometrial tissue during a time period of 2 weeks using intravital fluorescence microscopy. The major findings from our study are that [1] newly formed blood vessels of large endometriotic lesions show a higher red blood cell velocity and individual volumetric blood flow compared with that of small lesions, and [2] the lowering of estrogen levels by ovariectomy significantly delays the process of vascularization and reduces endometrial graft perfusion. 1205

FIGURE 4 (A, B) Intravital fluorescence microscopy of a completely vascularized endometrial graft (asterisk) located beneath a larger venule (V) of the host tissue at day 10 after autologous transplantation into the dorsal skinfold chamber of an untreated Syrian golden hamster. Note that only few white blood cells that interact with the endothelium of the newly formed capillaries can be observed (arrows). A: Blue-light epi-illumination with contrast enhancement by 5% FITC-labeled dextran 150,000 IV; B: Green-light epi-illumination for visualization of rhodamine 6G–stained white blood cells. (C, D) Hematoxylin-eosin–stained cross-section of a large endometrial graft at day 14 after transplantation onto the striated muscle (C, arrows) of the dorsal skinfold chamber of an untreated Syrian golden hamster. Higher magnification (D) reveals typical signs of an endometriotic lesion such as cyst-like dilated endometrial glands (asterisk) with an intact glandular epithelium surrounded by a richly vascularized endometrial stroma (arrowheads). (E) Immunohistochemical detection of PCNA shows cell proliferation within the glandular epithelium (arrows), the endometrial stroma (arrowheads), and the endothelium of newly developed blood vessels (double arrow), indicating growth and viability of the endometrial graft. (F) Intravital fluorescence microscopy of a large endometrial graft 14 days after autologous transplantation into the dorsal skinfold chamber of a bilaterally ovariectomized Syrian golden hamster. Using green-light epi-illumination, the borders of endometrial cysts (arrows) can exactly be identified because the fluorescent dye rhodamine 6G accumulates in the cyst-lining glandular epithelial cells. Scale bars: A, B: 125 ␮m; C: 230 ␮m; D: 70 ␮m; E: 120 ␮m; F: 200 ␮m.

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It is well known that the occurrence of spontaneous endometriosis is dependent on menstruation and thus restricted to humans and subhuman primates. However, in agreement with our findings previous studies reported that isolated endometrial fragments are able to implant and to develop endometriosis-like lesions when transplanted to ectopic sites such as the chorioallantoic membrane (8, 26 –28) and the peritoneal cavity (6, 7, 29, 30) and subcutaneous tissue of mice (31–33). Therefore, transplantation of endometrial tissue in rodents is considered an accepted method for the experimental induction of endometriosis. In our study, we used the dorsal skinfold chamber of Syrian golden hamsters as the host site for autologous endometrial tissue transplantation, which bears several advantages when compared to previously described endometriosis models. By the use of different fluorescent dyes for ex vivo and in vivo staining, the dorsal skinfold preparation in combination with intravital microscopy allows for a systematic quantitative analysis of dynamic processes during endometrial graft vascularization, including capillary sprouting and network formation, microvascular blood perfusion, and leukocyte– endothelial cell interaction. The observation window of the chamber provides easy access to the implantation site for repeated measurements. In contrast to rats and mice, the preparation of the skinfold chamber in hamsters causes less tissue trauma because the retractor muscle can more easily be removed. In addition, the tissue layers of the hamster are thinner than those of the mouse and the rat, resulting in better translucency and microscopic image quality. Finally, the hamster’s 4-day estrous cycle is extremely regular and can easily be synchronized by hormonal treatment (21). We demonstrated that isolated endometrial fragments are able to vascularize and to develop endometriosis-like lesions after transplantation into the dorsal skinfold chamber. The untreated animals showed some fluctuations in vascular density during the time course of observation. These fluctiations, however, did not prove to be significant. Although these fluctuations may just reflect a physiological variation, it cannot be excluded that they are associated with the stage of estrous cycle, particularly because the fluctuations in microvessel density of the synchronized animals were smaller. Although the stage of the estrous cycle of the untreated animals during the observations was not determined, different stages in the individual animals have to be assumed. In fact, to exclude those differences in stage of estrous cycle, the additional group of synchronized animals was included. The growth and morphology of the endometrial fragments were not dependent on the availability of ovarian hormones. This finding coincides with the results of previous studies describing that the growth of endometrium was unaffected by the estrus stage of the animal (33), and that no difference could be observed in the development of necrosis of endometrial grafts in unsupplemented and E2-supplemented mice (29). However, the comparison of untreated and synchronized hamsters vs. ovariectomized animals revealed that Fertility and Sterility姞

ovariectomy results in delayed graft vascularization with a significantly reduced vascularized area and microvessel density during the first days after transplantation. The altered vascularization is most probably due to the lack of ovarian estrogens, which have been shown to be involved in angiogenesis by up-regulating VEGF mRNA expression in primary cultures of human uterine stromal cells (11, 34) and in the rat uterus (35, 36). Our in vivo results now support the view that ovarian steroids contribute to blood vessel development in endometrial implants and the observation that down-regulating doses of GnRH analogs decrease the vascularity of endometriotic lesions (37). Besides the hormonal regulation of angiogenesis, hypoxic stress with subsequent induction of VEGF (38) has to be considered as an additional unspecific driving force for the development of new blood vessels because in our model, the endometrial grafts lacked an initial vascular supply and thus were solely dependent on oxygen diffusion initially after transplantation. Therefore, it was to be expected that endometrial grafts in ovariectomized hamsters also exhibited microvascular network formation at day 14 after transplantation. The large percentage increase of vascularization in the ovariectomized animals from day 2 to day 4 has to be explained by the fact that hypoxia, which triggers VEGF, was markedly more pronounced at day 2 in these grafts when compared with that of the other two groups, as indicated by a significantly lower microvessel density and vascularized area. In fact, in all transplanted and healing tissues, hypoxia has to be considered as a major determinant for VEGF induction and thus tissue vascularization. The function of estrogen in endometrial tissue has to be considered additive, accelerating the vascularization process. Red blood cell velocity and volumetric blood flow in endometrial grafts of ovariectomized animals were found significantly reduced. This might be best interpreted as a decreased cellular activity due to the lack of estrogen stimulation, which is known to result in a lower functional demand for oxygen and essential nutrients. In line with this view, the large grafts, which are expected to have a higher metabolic demand because of their tissue mass, presented with an increased microvascular perfusion when compared with that of the small fragments. Because the activation of the immune system has been proposed to be involved in angiogenic processes during the development of endometriosis (39), we also investigated leukocyte-endothelial cell interaction. However, increased numbers of white blood cells tethering or firmly adhering to the endothelial lining of the vessel wall could rarely be observed in the newly developed microvascular networks of the endometrial grafts and the surrounding host microvasculature of the dorsal skinfold chamber. Thus, we exclude inflammation or immune response with release of leukocytic angiogenic factors as a significant inducer of endometrial graft vascularization in our model. 1207

Under physiologic conditions, the uterine endometrium undergoes predictable stereotypic changes throughout the ovarian cycle, including blood perfusion. However, this is not the case for endometriotic lesions. As previously shown by immunhistochemistry, the density of estrogen receptors in endometriotic lesions is highly variable and does not undergo cyclic changes throughout the estrus cycle (40, 41). This may be due to the implantation site, which offers a paracrine environment markedly different than that of the uterus (41). Correspondingly, we have demonstrated in vivo that the time course and the extent of vascularization are comparable in untreated and synchronized animals. Moreover, although the hamster’s estrus cycle lasts only 4 days, there were no cyclic changes in microhemodynamic parameters over the 14-day observation period. In conclusion, our novel model of endometrial tissue transplantation into the dorsal skinfold chamber of Syrian golden hamsters allows for the repetitive in vivo analysis of regulatory mechanisms, which are involved in angiogenesis of endometriotic lesions. Thus, it may be a useful experimental approach not only to focus on the pathogenesis of endometriosis but also to develop future antiangiogenic strategies in the treatment of this frequent gynecologic disease. Acknowledgments: We are grateful for the technical assistance of Janine Becker (Institute for Clinical and Experimental Surgery, Homburg/Saar) and Dorothea Frenz (Department of Experimental Surgery, Rostock).

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