In vivo analysis of angiogenesis in endometriosis-like lesions by intravital fluorescence microscopy  Matthias W. Laschke, M.D., Antje Elitzsch, D.V.M., Brigitte Vollmar, M.D., Michael D. Menger, M.D.  Fertility and Sterility  Volume 84, Pages 1199-1209 (October 2005) DOI: 10.1016/j.fertnstert.2005.05.010 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

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 μm. Laschke. Angiogenesis in endometriosis. Fertil Steril 2005. Fertility and Sterility 2005 84, 1199-1209DOI: (10.1016/j.fertnstert.2005.05.010) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

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 μm. (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. Laschke. Angiogenesis in endometriosis. Fertil Steril 2005. Fertility and Sterility 2005 84, 1199-1209DOI: (10.1016/j.fertnstert.2005.05.010) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Centerline red blood cell velocity (μm/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. Laschke. Angiogenesis in endometriosis. Fertil Steril 2005. Fertility and Sterility 2005 84, 1199-1209DOI: (10.1016/j.fertnstert.2005.05.010) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

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 μm; C: 230 μm; D: 70 μm; E: 120 μm; F: 200 μm. Laschke. Angiogenesis in endometriosis. Fertil Steril 2005. Fertility and Sterility 2005 84, 1199-1209DOI: (10.1016/j.fertnstert.2005.05.010) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions