1 Department of Obstetrics and Gynaecology, Medical University Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, 2 Department of Obstetrics and Gynaecology, Medical University Ulm and 3 Institut für Lasertechnologien in der Medizin und Messtechnik, Ulm, Germany
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Abstract |
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Key words: 5-aminolaevulinic acid/chorioallantoic membrane/endometriosis/fluorescence diagnosis/photodynamic diagnosis
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Introduction |
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We therefore sought to visualize such non-pigmented peritoneal changes more accurately. To achieve this, we first conducted an in-vivo study evaluating the fluorescence diagnosis of endometriosis using 5-aminolaevulinic acid (ALA). The experiments were done using endometrial implants on the chorioallantoic membrane of fertilized chicken eggs. The results obtained have to be validated on patients in a subsequent pilot study.
The enhanced visualization of certain cancerous and non-cancerous tissues after treatment with a specific photosensitizer has been previously reported. The light-induced fluorescence after treatment with ALA is an experimental model used in the early diagnosis of cancerous disorders in urology (Kriegmair et al., 1993, 1994
; Baumgartner et al., 1994
) and pulmonology (Hung et al., 1991
). In the field of dermatology, ALA is also used for photodynamic therapy (Peng et al., 1997
).
ALA is a precursor of protoporphyrin IX (Pp IX) in the haem pathway. It has no inherent photosensitizing activity. The rate-limiting step in the haem synthesis is the step of converting Pp IX to haem (Fehr et al., 1996). Exogenous ALA therefore induces an excess of Pp IX which accumulates in the cell and has a strong photosensitizing effect. It can therefore be used for fluorescence diagnosis by exposure to light at a given wavelength.
In the field of gynaecology, ALA has previously been used as a diagnostic and therapeutic tool in tumour cell lines (Ishiwata et al., 1988; Rossi et al., 1996
) and endometrium (Fehr et al., 1996
; Steiner et al., 1996
; Yang et al., 1996
). A pilot study on the fluorescence diagnosis of endometriosis using ALA in patients has previously been published by our group (Malik et al., 1998
)
A simple and reliable in-vivo model of endometriosis was needed to show the selective uptake of the photosensitizer in ectopic endometrium and subsequent fluorescence in this tissue.
We chose the chorioallantoic membrane (CAM) of the chicken embryo, a model that has been known since 1887 (Gerlach, 1887) and used extensively for the culture of all kinds of tissues and cell lines (Rubovits and Abrams, 1951
; Hall, 1978
; Kunzi-Rapp et al., 1992
).
Our experiments on the CAM should enable us to demonstrate (i) whether it is possible to attain selective fluorescence of ectopic endometrium using ALA and (ii) the time interval before maximum fluorescence of the endometrium is attained and hence the best one for fluorescence diagnosis.
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Materials and methods |
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Fertilized chicken eggs were purchased from a breeding station (Zeh KG, Laichingen, Germany). They were stored blunt end up in an incubator at 60% humidity and 37°C until day 5 or 6 after fertilization. A circular window ~3 cm in diameter was then made in the sharp end of the shell using an electrical engraving tool and scissors. The window was then covered with a lid and incubation was continued until the egg was used. As the immune system of the embryo is known not to be sufficiently developed until day 1617 of incubation, CAM culture could be performed to this point (Ausprunk et al., 1975). When grafting was performed between day 6 and 10, the window in the sharp end of the egg was widened and tissue fragments were placed on the CAM near the Y branch of a large blood vessel. Implants were left on the CAM for 37 days. Viable implants appeared white or pink while rejected tissue was found to be black within 1 or 2 days after grafting. The circulation of the CAM vessels could readily be evaluated. Implants were then explanted and stained with haematoxylineosin for histological evaluation of viability. All specimens were evaluated by an experienced gynaecological pathologist on the basis of the following criteria: (i) was there any glandular tissue in the preparation and was this tissue intact or in dissociation?; (ii) what was the quantity and the quality of fibroblasts in the preparation?; (iii) were there any signs of necrobiosis in the preparations? and (iv) was there any sign of an inflammatory reaction?
The decision whether an implant was rated viable or non-viable was based on a scoring system shown in Table I. A maximum of 11 points could be achieved. Preparations with a score of
6 were rated viable. All preparations with a score <6 were rated non-viable together with all preparations with complete necrobiosis.
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Prior to the topical application, a silicone ring was placed around the graft on the CAM surface. 20 µl of the photosensitizer were then applied inside the ring using a micropipette. Fluorescence was assessed after 60 and 120 min and then every 120 min up to 36 h. After each measurement, the egg was covered with a lid and returned to the incubator.
Fluorometric experiments were performed using a Zeiss Axiophot stereo-microscope (Zeiss, Oberkochen, Germany) coupled with a silicone-intensifying-target camera (SIT-camera 2400 Hamamatsu Photonics, Herrsching am Ammersee, Germany) able to detect even extremely weak fluorescence at low illumination (Schneckenburger et al., 1988). This camera was of special importance for the detection of the initial fluorescence prior to fluorescence diagnosis, as fast-reacting photosensitizers tend to wear out quickly. ALA-mediated fluorescence was induced using a special filter-block BP436/LP470 (Zeiss) for illumination that allowed only blue light of 436 nm wavelength to pass. An additional red filter (>490 nm) was attached to the SIT-camera since only the red portion of fluorescence was to be detected. An additional image-intensifying system (video frame memory C 1901 Mark II; Hamamatsu) enhanced the signal background relationship by adding 64 single pictures. The fluorescence of the specimen was transformed into 16-bit gray scales. A gray scale was used to detect the relative level of fluorescence.
Statistical analysis included Fisher's exact test for the evaluation of the optimal time of implantation and explantation, and analysis of variance for the evaluation of the fluorescence measurements. Using receiver operator curve analysis (ROC), we defined the optimum cut-off-point for the diagnosis of endometriosis. Using this cut-off-point, we detailed the validity of our test by calculating sensitivity and specificity values.
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Results |
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Preparations with the best score of viability were obtained when grafting was performed between day 7 and 9 of incubation and when implants were allowed on the CAM for 35 days (Table II). When tissue was grafted onto the CAM on day 7 of incubation and kept on the CAM for 35 days, a significantly higher score of viability was obtained than was the case with grafting on days 6, 8 or 10 and culture for 3, 5 or 7 days respectively (P < 0.05). There was no statistically significant difference in all the other combinations.
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Figure 2 depicts the invasion of the graft by CAM vessels. Figure 3
shows the strong vascularization adjacent to the graft.
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Exposure to light at a wavelength of 436 nm after topical ALA application revealed the selective uptake of the photosensitizer in ectopic endometrium.
The fluorescence of ectopic endometrium was measured in 81 preparations, each on an individual chicken egg for a total of 12 patients. Fluorescence of fimbriae was measured in 33 preparations from five patients and fluorescence of peritoneum in 21 preparations from four patients. The origin of the specimens was histologically confirmed in each case. The mean and standard deviation of the measured values were calculated.
Figure 4 shows the intensity of fluorescence in these three tissues quantified in arbitrary units. Only the red portion of fluorescence was detected.
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Discussion |
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In our model, 76.6% of the preparations were viable at histological examination and 23.4% showed signs of necrosis following various periods of incubation until grafting and various periods of incubation on the CAM.
Experiments with regard to the best time for grafting and explanting various tissues when CAM culture is performed have previously been published (Knighton et al., 1977; Petruzelli et al., 1993
; Kirchner et al., 1996
). Using endometrium as a graft, our experiments did not differ significantly from these data. The pre-incubation period has to be individually chosen depending on the development of the CAM. This is mainly due to the varying conditions of temperature and humidity in which the eggs are stored prior to delivery.
The topical application of ALA is simple, especially compared to i.v. injections in rats or rabbits. A rat model has been used to investigate the metabolism of ALA to Pp IX in experimentally induced endometriosis (Yang et al., 1996). This model compares with the cost-efficiency of the rabbit model used earlier (Manyak et al., 1990
).
Using our experimental model, we were able to show the selective uptake of ALA in ectopic endometrium. In various fields of medicine, numerous experiments have been conducted using ALA-induced fluorescence aiming in particular at photodynamic therapy. In the field of urology, most investigations have focused on bladder tumours (Kriegmair et al., 1994). Pigs (van Staveren et al., 1996
) and rats (Kriegmair et al., 1995
) were used as animal models. In gynaecology, ALA has been applied for the diagnosis and treatment of intrauterine endometrium. Maximum fluorescence of the endometrium was demonstrated after 24 h (Yang et al., 1996
). Intravenous injections of ALA yielded higher rates of fluorescence than oral application. Uteri from hysterectomized patients were used to show that a maximum of fluorescence is reached after 48 h and that fluorescence of the endometrium is 48 times stronger than that of the underlying myometrium (Fehr et al., 1996
).
Our experiments demonstrate a rapid increase in fluorescence of the endometrium in contrast to peritoneum and fimbriae. A maximum is reached after 1014 h (P < 0.01), at which point the endometrium displays a fluorescence twice as strong as that of other tissues.
Fimbriae, which are of special interest when ALA is applied via the transcervical, transuterine and transtubal routes, displayed up to 16 h the same fluorescence as the peritoneum. Only after 1618 h was an increase in fluorescence in fimbriae noted, differing markedly from that of normal peritoneum (P < 0.05). At this time, fluorescence diagnosis should not be performed. However, histological examination of the fimbriae after fluorescence diagnosis revealed no signs of physical damage, therefore making impairment of the Fallopian tubes by fluorescence diagnosis unlikely. On the basis of these results, we can conclude that endometrium implanted to the CAM is a simple and efficient model for experimentally induced endometriosis. Selective uptake of ALA in ectopic endometrium was demonstrated and maximum fluorescence after topical application was evaluated in an in-vivo model.
These results encouraged us to perform fluorescence diagnosis of endometriosis in patients. The results of our pilot study on the fluorescence diagnosis of endometriosis have previously been published (Malik et al., 1998, 1999).
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Acknowledgments |
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Notes |
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References |
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Submitted on May 20, 1999; accepted on November 22, 1999.