Hum. Reprod. Advance Access originally published online on November 17, 2005
Human Reproduction 2006 21(3):810-817; doi:10.1093/humrep/dei387
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Quantification of endometriotic lesions in a murine model by fluorimetric and morphometric analyses
1 Department of Gynaecology, Université Catholique de Louvain, Avenue Hippocrate 10, 1200 Brussels and 2 CELL Unit, Université Catholique de Louvain and Christian de Duve Institute of Cellular Pathology, 1200 Brussels, Belgium
3 To whom correspondence should be addressed. E-mail: donnez{at}gyne.ucl.ac.be
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Abstract |
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BACKGROUND: In animal models of endometriosis, the identification and quantification of lesions originating from human endometrium is often hampered by the small size of the implants and their embedding in murine tissue. The purpose of the present study was to develop two new methods of quantifying endometriosis-like lesions in a nude mouse model: fluorimetry and morphometry. METHODS: Human menstrual endometrium was labelled using a fluorescent tracker, carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE), and transplanted into the pelvic cavity of mice by injection through the peritoneum after performing a cutaneous incision. After 5 days, lesions were recovered by laparotomy. The fluorescence of the recovered endometriotic lesions was measured. Endometrial stroma and glands were immunostained in lesion sections with anti-CD10 and anti-CK22 antibodies, and their surface area was evaluated by morphometric analysis. RESULTS: Fluorescent labelling allows identification of lesions not visible macroscopically. A good correlation was observed between fluorimetry and morphometry (r = 0.88) applied for lesion quantification. CONCLUSIONS: Fluorimetric evaluation combined with morphometric analysis of endometriosis-like lesions allows objective and reliable recording of endometriosis development in a nude mouse model. This quantification method could therefore be useful for future pharmacological and toxicological studies.
Key words: carboxyfluorescein diacetate, succinimidyl ester/endometriosis/lesion quantification/menstrual endometrium/nude mouse model
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Introduction |
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Endometriosis is a common gynaecological disorder characterized by the proliferation of endometrial glands and stroma outside the uterine cavity. Retrograde menstruation, peritoneal adhesion of shed endometrial tissue, and outgrowth of these endometrial cells are essential steps in the pathogenesis of endometriosis according to the implantation theory of Sampson (1927
). The aetiology of this multifactorial disease remains elusive despite an increasing number of studies on its physiopathology. In order to study the mechanisms underlying ectopic endometriotic implant formation and to test new therapeutic approaches, several animal models have been developed.
Monkey models are undoubtedly the most physiologically relevant since primates are the only mammals developing endometriosis spontaneously (D’Hooghe and Debrock, 2002). Nevertheless, due to practical constraints, most groups have concentrated their research on smaller mammalian models such as rabbits (Donnez et al., 1987), mice (Cummings and Metcalf, 1995; Foster et al., 1997; Somigliana et al., 1999; Rossi et al., 2000; Dabrosin et al., 2002; Fang et al., 2004; Efstathiou et al., 2005) and rats (Vernon and Wilson, 1985; Sharpe-Timms, 2002). However, such studies usually imply the use of autologous animal endometrium, which is dissimilar to human endometrium. Indeed, many phylogenetic and biochemical differences exist between human and animal endometrium. The estrous cycle is shorter than the menstrual cycle and is characterized by endocrinological differences (Awwad et al., 1999). Significant interspecies differences in histological observations were also reported (Zaino et al., 1985).
Several groups have therefore developed endometriosis models in immunodeficient mouse strains, such as severe combined immunodeficient (SCID) mice that have a combined congenital deficiency in T- and B-lymphocyte function (Awwad et al., 1999; Aoki et al., 1994; Grümmer et al., 2001), nude mice with congenital thymus aplasia, resulting in a deficient T-lymphocyte system (Zamah et al., 1984; Zaino et al., 1985; Bergqvist et al., 1985; Bruner et al., 1997; Tabibzadeh et al., 1999; Nisolle et al., 2000; Grümmer et al., 2001; Beliard et al., 2002; Fortin et al., 2003; Hull et al., 2003; Nap et al., 2003) and transgenic RAG-2/
(c)KO mice (Greenberg and Slayden, 2004). Use of mouse models that lack normal immune reactions may represent a limitation of these models, due to the fact that the immune system appears to play an important role in the aetiopathogenesis of endometriosis. It does, on the other hand, allow for the utilization of human endometrium without the risk of graft rejection. Indeed, in 2000, we published a paper on the induction of endometriosis-like lesions after injection of menstrual endometrium that closely mimics the process of retrograde menstruation (Nisolle et al., 2000).
In murine models, quantification of lesions is based on criteria such as the number of induced lesions (Awwad et al., 1999; Illera et al., 2000; Bruner et al., 2002; Hull et al., 2003; Nap et al., 2004; Van Langendonckt et al., 2004; Eggermont et al., 2005), the size of macroscopic lesions (Zamah et al., 1984; Awwad et al., 1999; Efstathiou et al., 2005; Harris et al., 2005; Hull et al., 2005), and their weight and macroscopically evaluated surface area (Somigliana et al., 2001). However, the process of identification and measurement of lesions deriving from human endometrium is often impeded, given the small size of the implants and their embedding in murine tissue.
The purpose of the present study was to set up two new methods to quantify human endometriosis-like lesions in a nude mouse model.
The first method involved labelling endometrial tissue with a fluorescent tracker prior to injection into mice and subsequent fluorimetric quantification of the endometriosis-like lesions. Fluorimetric approaches have proved to be very useful in identifying and visualizing endometriotic transplants (Tabibzadeh et al., 1999; Fortin et al., 2003; Hirata et al., 2005) and fluorimetry has recently been used as a tool to quantify recovered lesions (Hirata et al., 2005). In the present study, we chose carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE), a cell permeant activable fluorescein ester classically used for in vivo tracking (Spinelli et al., 2002).
The second method consisted of morphometric measurement of the stromal and glandular surface area in a section of the lesion. Immunohistochemical staining with specific markers for human endometrial stroma and glands ensures accurate identification of these cell types.
Both methods were applied and compared to quantify endometriosis-like lesions in nude mice.
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Materials and methods |
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Human endometrial tissue
The use of human tissue for this study was approved by the Institutional Review Board of the Université Catholique de Louvain.
Menstrual endometrium was obtained from 15 women aged from 22 to 47 years (mean age 33.5±7.9 years) undergoing surgery for benign conditions, by aspiration with a syringe without any anticoagulants. A sample of the biopsy was fixed in 4% buffered formaldehyde and embedded in paraffin for histological confirmation of the menstrual phase using established criteria (Noyes et al., 1950) and for immunohistochemical controls. The rest of the tissue was injected into nude mice, as described below.
Fluorescent labelling of endometrium
Before transplantation, human menstrual endometrium was minced into microfragments able to pass through a 19G needle (maximal size = 750 µm) and labelled with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) (Molecular Probes, Eugene, USA). In order to label the tissue, the minced menstrual endometrium was incubated in a solution of 8 µmol/l CFDA-SE in phosphate-buffered saline (PBS; Gibco, UK). After 15 min incubation in a 37°C water bath, the labelled tissue was washed in fresh PBS and viewed by fluorescence microscopy to ensure labelling. The viability of the endometrial fragments after staining was checked by the Trypan Blue dye exclusion test (Sigma; Fornelli et al., 2004). Preliminary experiments have shown that there is no CFDA-SE toxicity, even at higher concentrations and increased incubation times (data not shown).
CFDA-SE is a cell-permeant, non-polar lipophilic molecule. After cell internalization, CFDA-SE is converted into anionic CFSE by cleavage of the acetyl groups by intracellular esterases, producing a markedly fluorescent derivative. Amine-reactive coupling of CFSE to proteins results in stable long-term intracellular retention.
The stability of the fluorescent staining in vivo was confirmed by injecting one mouse with labelled tissue and checking for the presence of fluorescent endometrial implants after 2 weeks (data not shown). Moreover, Becker et al. (2004) showed that the fluorescence intensity of CFDA-SE-labelled cells initially declines, but then stabilizes after 4 h, enabling detection weeks after labelling.
Transplantation of menstrual endometrium into nude mice and lesion recovery
The guidelines for animal welfare were approved by the Committee on Animal Research of the Université Catholique de Louvain.
Thirty-seven nude (Swiss nu/nu) 6–10 week old female mice (Charles River Laboratories, Wilmington, USA) were used for the present study. Handling and maintenance were implemented as previously described (Nisolle et al., 2000).
The mice were anaesthetized with an (intraperitoneal) i.p. injection of ketamine (75 mg/kg, Anesketin®; Eurovet, Heusden-Zolder, Belgium) and medetomidine (1 mg/kg, Domitor®; Pfizer, Cambridge, MA, USA). After surgery, the anaesthesia was reversed by injection of atipamezole (1 mg/kg, Antisedan®; Pfizer).
In order to mimic retrograde menstrual discharge, menstrual endometrium was injected i.p. A ventral incision of 0.5 cm was made in the skin, keeping the abdominal wall intact, followed by an i.p. injection of the fluorescent menstrual endometrium, under visual control (Figure 1). A thin needle (19G) was used to avoid i.p. organ injury or damage to the peritoneal layer. The skin was then sutured with one or two stitches.
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Five days after injection, when typical endometriotic lesions were noted, as defined previously (Nisolle et al., 2000), the mice were killed by cervical dislocation according to the standard euthanasia guidelines for rodents from the Institutional Animal Care and Use Committee (IACUC) (http://www.iacuc.ucsf.edu/Policies/awGlEuthR.asp). Implanted endometrial lesions were recovered by laparotomy. Macroscopic lesions (visible to the eye) were recognized by their nodular aspect distinct from normal murine tissues and by their adhesion to peritoneum and i.p. organs. All the murine abdominal tissues and organs were then dissected and checked under an inverted fluorescence microscope (DMIL; Leica, Heerbrugg, Switzerland) for the presence of fluorescent microscopic lesions on the apparently healthy pelvic surfaces.
Quantification of endometriosis-like lesions by fluorescence
Samples were analysed by a Kodak 2000MM image station. Fluorescence of CFDA SE was detected at lex465/lem535. Numerical values of regions of interest (ROI) were obtained by means of Kodak 1D image analysis software (version 3.6), using the ‘net intensity’ function and expressed with arbitrary units as the sum of the background-subtracted pixel values within the ROI.
In order to validate the quantitative aspect of this method, preliminary experiments were conducted with non-grafted endometrium, correlating the weight (ranging from 2 to 50 mg) with the fluorescent signal.
In this study, this technique was used to quantify the lesions induced in mice. For each recovered lesion, the fluorescence was measured immediately after dissection. The lesions were then fixed in 4% buffered formaldehyde and embedded in paraffin for histological examination.
Quantification of endometriosis-like lesions by immunohistochemical morphometric analysis
Immunohistochemical morphometric analysis allows measurement of specific constituents of endometriosis-like lesions: epithelial glands and stroma (Figure 2).
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To this end, paraffin-embedded lesions were cut into semi-serial sections of 5 µm. Every fifth slide was stained by haematoxylin–eosin and the section with the largest endometriotic lesion surface area was selected. The two next serial sections were stained immunohistochemically.
Immunostaining was performed on these sections with a labelled immunoperoxidase method using mouse monoclonal antibody to human CD10 (clone 56C6; Novocastra, Newcastle, UK) and to cytokeratin cocktail CK22 (Biomeda, Foster City, CA, USA). CD10 is an immunohistochemical marker of endometrial stroma (Toki et al., 2002), which has been used to confirm diagnosis of endometriosis (Sumathi and McCluggage, 2002; Potlog-Nahari et al., 2004). Endometrial glands selectively express CK22. Briefly, endogenous peroxidase activity quenching, heat epitope retrieval and blocking of non-specific staining were performed. The specimens were then incubated overnight at 4°C with primary antibodies (dilutions: 1:800 for CK22 and 1:200 for CD10), followed by incubation with secondary antibody conjugated to peroxidase (EnVision+TM, Dako, CA, USA). The presence of peroxidase was revealed using 3,3'-diaminobenzidine (Dako) and specimens were counterstained with Mayer’s haemalum solution (Merck, Darmstadt, Germany).
CD10- and CK22-stained slides were examined with a microscope (Zeiss, Munich, Germany) at 100x magnification and all fields were digitalized using a Leica DFC320 camera and IM50 program (Leica).
The ‘ImageJ’, a freely available image processing and analysis program developed at the National Institutes of Health (http://rsb.info.nih.gov/ij/), was used to delimit all glandular and stromal structures of the lesions and to measure their surface area.
In order to validate the quantitative aspect of this method, we measured non-grafted endometrial fragments by immunohistochemical morphometry and weight. For each fragment, the morphometry was related to the wet weight.
Experimental design
The study was divided into two parts.
The first set of experiments was designed to localize endometriosis-like lesions within the pelvic cavity of the mice using the fluorescent tracker. Menstrual endometrium was obtained from eight donors and injected into 24 nude mice (three mice per donor; two mice died during surgery). Macroscopic and microscopic endometriosis-like lesions were identified and recovered, as described above. Lesion localization was recorded and classified as: peritoneal, perivesical fat, intestinal and mesenteral, hepatic, splenic, stomachal or pancreatic. When a lesion was attached to several sites (two or three), all the sites were taken into account.
In the second part, experiments were carried out to quantify the formation of endometriosis-like lesions. Menstrual endometrium was obtained from six donors and transplanted into 13 nude mice. Endometrium collected from a single donor was used per experiment involving one reference mouse receiving 100 µl, and a second mouse receiving 200 µl, of menstrual endometrium suspension, homogenized and labelled as described above. If enough endometrial tissue was available, a third mouse was injected with 400 µl. The amount of human endometrium was measured using a 1 ml syringe. Preliminary tests indicate that the volume of injected suspension corresponds to the weight of injected tissue.
On day 5, the endometriosis-like lesions were recovered by laparotomy and immediately quantified by fluorimetry, as described above. The lesions were then fixed in 4% buffered formaldehyde, embedded in paraffin and cut into semi-serial sections of 5 µm for histological confirmation and immunohistochemical morphometry evaluation, as previously described.
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Results |
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In the present study, we mimicked the ectopic implantation of human menstrual endometrium. As previously shown, endometrium is able to implant onto intact mesothelium and reorganize itself into structured glands and stroma, forming endometriosis-like lesions on peritoneal and organ surfaces in athymic mice (Nisolle et al., 2000
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First set of experiments: endometriotic lesion recovery
In the first series of experiments, 76 endometriotic lesions were recovered from 22 mice (mean number 3.45 ± 2.18 lesions per mouse). Fifteen per cent of the lesions were not visible macroscopically but could be identified by fluorescent labelling.
Anti-human CD10 and CK22 immunohistochemical staining proved the human origin of these lesions, confirming that they originated from the injected tissue.
Among the 76 lesions, 26 (34.2%) were bound to perivesical fat, 23 (30.3%) to peritoneum, 16 (21.1%) to the pancreas, nine (11.8%) to the intestine or mesentery, six (7.9%) to the spleen, six (7.9%) to the stomach and five (6.6%) to the liver. Only one lesion (1.3%) was recovered from the injection site at the level of the scar.
These lesions were fixed in formol and embedded in paraffin. The histological characteristics of endometriosis were confirmed after haematoxylin–eosin staining (Figure 3). Lesions were classified as follows: (A) median lesions (0.5–3 mm); (B) very large lesions (>3 mm) with extensive necrosis; and (C) small lesions (<0.5 mm) with only one gland and a thin layer of stroma that were not macroscopically visible and only revealed by fluorescence (Figure 4). The incidence of lesions A, B and C was 72, 13 and 15% respectively.
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Quantification by fluorescence
As illustrated in Figure 5a, preliminary experiments were performed with non-grafted endometrium and showed that the fluorescent signal is proportional to the weight of the labelled tissue (ranging from 2 to 50 mg) (r = 0.86, P < 0.01, n = 23).
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Quantification by immunohistochemical morphometric analysis
As illustrated in Figure 5b, the quantitative aspect of immunohistochemical morphometric analysis was confirmed by measuring non-grafted endometrial fragments by morphometry and weight. For each fragment, the results obtained by morphometry were related to the weight (r = 0.98, P < 0.01, n = 7).
Second set of experiments: quantification of recovered endometriosis-like lesions
In this set of six experiments, menstrual endometrium was obtained from six donors. Endometrium collected from a single donor was used per experiment involving one reference mouse receiving 100 µl, and a second mouse 200 µl, of tissue. In experiment 2, a third mouse was injected with 400 µl of tissue.
On day 5, the endometriosis-like lesions were recovered. A total of 34 lesions were recovered from 13 mice. They were located in similar areas to those in the first series of experiments (data not shown). Each lesion was then quantified by fluorimetry [results expressed as arbitrary units (AU)] and by morphometry (results expressed as µm2).
As the results of the fluorimetric analysis are expressed in AU, they are not comparable between experiments. On the contrary, the results obtained by immunohistochemical morphometric evaluation are expressed in µm2, and can therefore be compared between experiments. In the present study, pooled experimental lesions were normalized relative to the pooled reference mouse lesions.
Results from the quantitative analyses are summarized in Figure 5c: the surface area of the pooled lesions correlates with the fluorescence of these lesions.
A good correlation was observed (r = 0.88, P < 0.01, n = 7) between the results obtained by morphometry and those measured by fluorescence. Indeed, in most cases, fluorescence measurements correlated with morphometric surface area evaluation, as well as the quantity of injected tissue. However, in some cases, apparent discrepancies were observed between the results obtained with the two methods. This may be explained by the occurrence of necrosis or the presence of murine tissue, as observed histologically in some lesions recovered in these cases.
Indeed, in lesions (similar to the one shown in Figure 3b) that contained necrotic tissue, we observed that fluorescent measurements were overestimated due to the brighter staining of necrotic tissue, compared to viable tissue. We also noted that some murine organs, such as the pancreas, showed a certain degree of autofluorescence. This did not hinder identification of the lesions, but could potentially lead to an overestimation of the fluorimetric measurements when murine tissue is dissected together with endometriotic tissue.
Consequently, this fluorescence quantification method must be complemented by (immuno)histological analysis of the sample to confirm the histological characteristics of endometriosis-like lesions and detect the presence of necrotic or murine tissue.
Immunohistochemical labelling with anti-human CD10 antibodies specific for stromal cells, and anti-human CK22 antibodies specific for glandular epithelial cells, confirms the human origin of endometrial tissue and enables clear differentiation of endometrial cells from murine tissue. Morphometric measurement of the immunostained area allows quantitative evaluation of the endometriotic lesion surface area on a representative slide. In lesions induced in nude mice, a direct relationship was observed between morphometric data and the amount of endometrial tissue transplanted. Moreover, the results obtained by morphometric analysis correlated with the results obtained by fluorimetric analysis (Figure 5c).
The advantages and drawbacks of fluorimetric and morphometric analyses are compared in Table I.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The aim of this study was to set up a method to quantify endometriosis-like lesions by a combination of morphometric and fluorimetric approaches in a nude mouse model. Human menstrual endometrium was used to induce endometriosis, since this tissue appears to be the most appropriate to mimic menstrual effluent discharge (Nisolle et al., 2000
). However, the labelling and quantification procedures described in the present manuscript can also be applied to other animal models of endometriosis, using endometrial tissue from other cycle phases or other species.
In our study, fluorescent labelling of human tissue before injection allowed the recovery of macroscopically non-visible lesions. Indeed, we were able to detect some very small lesions, consisting of just one gland surrounded by one layer of stromal cells, representing 15% of all recovered lesions. In classic animal models of endometriosis, transplanted endometrium was not previously labelled. Only large, macroscopically visible adhesions and lesions could be identified, but smaller or microscopic implants of human endometrial cells on the peritoneal surface or embedded in murine tissue could not be detected. As highlighted by Evers et al. (2005) failure to detect microscopic lesions, that appear to play a role in subsequent growth, proliferation, and interaction with the surrounding environment, may contribute to an underestimation of the disease.
Fluorescent tagging of the endometrium before transplantation thus facilitates the detection of human tissue grafted into mice, which can be clearly distinguished from unstained murine tissue. This is consistent with studies by Tabibzadeh et al. (1999) who used a membranous fluorescent dye, DiO, Fortin et al. (2003) who transfected human endometrial cells with the green fluorescent protein (GFP) cDNA and Hirata et al. (2005), who induced endometriosis in wild-type C57/B6 mice by injection of murine endometrium from transgenic mice that ubiquitously express GFP.
In the present study, fluorescent labelling of the endometrium was performed with the lipophilic dye, CFDA-SE, prior to its i.p. injection. Preliminary data indicate that this fluorochrome is non-toxic to human endometrial cells and stable for 
2 weeks at the selected incubation time and concentration. In addition, labelling with CFDA-SE is very quick and easy, and does not require prolonged culture, transfection of cells with GFP, or the use of transgenic animals.
This fluorescent labelling approach allows precise screening of the localization of endometriotic lesions. The recovered lesions were mainly attached to the abdominal side wall, to perivesical fat and to the pancreas. The difference in lesion implantation sites between the human (mainly the pelvic region) and the mouse can be explained by the different directions of gravity action between the quadruped mouse and the upright biped human (Grümmer et al., 2001).
The surgical procedure applied in this study, avoiding incision of the abdominal wall and peritoneum (Figure 1), strongly reduces the possible bias of scar inflammation as only one lesion was recovered from the injection site. In studies inducing endometriosis by means of ‘transplantation by laparotomy’, trauma, inflammatory responses and adhesions are provoked by surgery and lesions are often discovered in or next to the scar (Tabibzadeh et al., 1999). This confounds quantitative investigations using these models.
In addition, visual control during the procedure ensures precise injection of tissue as opposed to a blind i.p. injection, which may result in subcutaneous implantation or cause damage to organs, as pointed out by Awwad et al. (1999).
These advantages support the administration of this type of endometrial injection for endometriosis-like lesion induction in a murine model. Furthermore, fluorescent labelling ensures swift quantification of the whole lesion.
In preliminary experiments, a direct relationship was demonstrated between the weight and intensity of fluorescent staining of non-grafted endometrium (Figure 5a), as well as the weight and morphometric measurement of non-grafted endometrium (Figure 5b). In the murine model, in most cases, fluorescence measurements correlated with the morphometric evaluation of the surface area (Figure 5c), as well as the amount of endometrial tissue transplanted.
However, the present investigation highlights the limitations of fluorimetric and morphometric approaches. Indeed, the drawback of fluorimetric quantification is that, contrary to immunohistochemical morphometric analysis, it does not distinguish between necrotic and viable tissue. The occurrence of necrotic tissue in large lesions appears to be a typical feature of early stage lesions. It is probably due to the delay in vascularization, causing ischaemia in ectopic endometrial tissue, as pointed out by Aoki et al. (1994). Such necrotic tissue might turn into fibrosis in older lesions, as suggested by the presence of fibrotic areas in the centre of lesions collected after 3 weeks (Zamah et al., 1984).
Fluorimetry may also take into account the autofluorescence of some murine tissue, dissected together with endometriotic tissue. Consequently, this precludes in vivo quantification of lesions. Immunohistochemical morphometric analysis, however, using anti-human CD10 and CK22 antibodies, is able to confirm the human origin of endometrial tissue and allow clear differentiation of endometrial cells from murine tissue.
In conclusion, our nude mouse model, involving a cutaneous incision followed by i.p. injection of CFDA-SE-labelled human menstrual endometrial tissue, minimizes the number of lesions developing at the level of the scar and damage to organs. At the end of the experiment, the fluorescent lesions can be easily detected, even when they are microscopic or embedded in adipose tissue. Quantification of the recovered lesions was performed by a combination of fluorimetry and immunohistochemical morphometry. These two methods, used together, provide a more precise evaluation of the quantity and severity of endometriosis than conventional methods based on lesion counting and gross surface area evaluation. This may well prove to be a very useful quantification method for pharmacological and toxicological studies.
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Acknowledgments |
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The authors thank Philippe Befahy, MD, Marie-Madeleine Dolmans, MD, Belen Martínez, VMD, PhD, Department of Gynaecology, for their assistance in the handling of mice and Mira Hryniuk, BA, for reviewing the manuscript. We also thank the Department of Anatomo-pathology for endometriotic lesion embedding and haematoxylin–eosin staining. The present study was supported by grant no. 3.4552.00 from the Fonds National de la Recherche Scientifique de Belgique and from the Fonds Spécial de la Recherche de l’Université Catholique de Louvain.
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Submitted on July 14, 2005; resubmitted on October 4, 2005; accepted on October 7, 2005.
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