Morfologiniai kumelių gimdos gleivinės pokyčiai endometrito ir endometriozės atvejais ; Morphological changes in equine endometrium in cases of endometritis and endometrosis
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LITHUANIAN VETERINARY ACADEMY The work was done in the Department of Infectious Diseases of Lithuanian Veterinary Academy and in the Department of Pathobiology of Utrecht University (NL). The part of this research was supported through the Euro-pean Community Marie Curie Fellowship. Research supervisor – Assoc. prof. dr. Albina Aniulien ė (Biomedical sciences, Veterinary medici- ne – 12B, Lithuanian Veterinary Academy). Research adviser – Prof. habil. dr. Henrikas Žilinskas (Biomedical sciences, Veterinary medici- ne – 12B, Lithuanian Veterinary Academy). Chairman of Veterinary medicine science council – Prof. habil. dr. Antanas Sederevi čius (Biomedical sciences, Veterinary me-Jūrat ė Sabeckien ė dicine – 12B, Lithuanian Veterinary Academy); Members: Prof. habil. dr. Vaiva Lesauskait ė (Biomedical sciences, Medicine – 07B, Kaunas University of Medicine); Prof. habil. dr. Ramutis Klimas (Biomedical sciences, Zootechny – 13B, Šiauliai University); Prof. habil. dr. J ūrat ė Šiugždait ė (Biomedical sciences, Veterinary medicine – 12B, Lithuanian Veterinary Academy); MORPHOLOGICAL CHANGES Assoc. prof. dr. Eugenijus Aniulis (Biomedical sciences, Veterinary medici-IN EQUINE ENDOMETRIUM IN CASES OF ne – 12B, Lithuanian Veterinary Academy). ENDOMETRITIS AND ENDOMETROSIS Opponents: Prof. habil. dr. Algimantas Matusevi čius (Biomedical sciences, Veterinary medicine – 12B, Lithuanian Veterinary Academy); Prof. habil.
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| Publié par | lithuanian_veterinary_academy |
| Publié le | 01 janvier 2006 |
| Nombre de lectures | 69 |
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LITHUANIAN VETERINARY ACADEMY
J ū rat ė Sabeckien ė MORPHOLOGICAL CHANGES IN EQUINE ENDOMETRIUM IN CASES OF ENDOMETRITIS AND ENDOMETROSIS Summary of doctoral dissertation Biomedical sciences, Veterinary medicine (12 B) Kaunas 2006
The work was done in the Department of Infectious Diseases of Lithuanian Veterinary Academy and in the Department of Pathobiology of Utrecht University (NL). The part of this research was supported through the Euro-pean Community Marie Curie Fellowship. Research supervisor Assoc. prof. dr. Albina Aniulien ė (Biomedical sciences, Veterinary medici-ne 12B, Lithuanian Veterinary Academy). Research adviser Prof. habil. dr. Henrikas ilinskas (Biomedical sciences, Veterinary medici-ne 12B, Lithuanian Veterinary Academy). Chairman of Veterinary medicine science council Prof. habil. dr. Antanas Sederevi č ius (Biomedical sciences, Veterinary me-dicine 12B, Lithuanian Veterinary Academy); Members: Prof. habil. dr. Vaiva Lesauskait ė (Biomedical sciences, Medicine 07B, Kaunas University of Medicine); Prof. habil. dr. Ramutis Klimas (Biomedical sciences, Zootechny 13B, iauliai University); Prof. habil. dr. J ū rat ė iugdait ė (Biomedical sciences, Veterinary medicine 12B, Lithuanian Veterinary Academy); Assoc. prof. dr. Eugenijus Aniulis (Biomedical sciences, Veterinary medici-ne 12B, Lithuanian Veterinary Academy). Opponents: Prof. habil. dr. Algimantas Matusevi č ius (Biomedical sciences, Veterinary medicine 12B, Lithuanian Veterinary Academy); Prof. habil. dr. Dalia Pangonyt ė (Biomedical sciences, Medicine 07B, Kaunas University of Medicine). Public defence of doctoral dissertation in Veterinary medicine science coun-cil will take place at the Lithuanian Veterinary Academy I auditorium on 23 rd November, 2006. Address: Til ė s 18, LT-47181 Kaunas, Lithuania. The summary of doctoral dissertation has been sent on 23 rd of October, 2006 according to the confirmed address list. The dissertation is available at the libraries of the Lithuanian Veterinary Academy and LVA Veterinary Institute.
LIETUVOS VETERINARIJOS AKADEMIJA J ū rat ė Sabeckien ė MORFOLOGINIAI KUMELI Ų GIMDOS GLEIVIN Ė S POKY Č IAI ENDOMETRITO IR ENDOMETROZ Ė S ATVEJAIS Daktaro disertacijos santrauka Biomedicinos mokslai, veterinarin ė medicina (12 B) Kaunas 2006
Disertacija rengta 2002 2006 metais Lietuvos veterinarijos akademijos Ukre č iam ų j ų lig ų katedroje ir Utrechto universiteto (Nyderlandai) Patobio-logijos katedroje. Mokslinio darbo vadov ė Doc. dr. Albina Aniulien ė (Lietuvos veterinarijos akademija, biomedicinos mokslai, veterinarin ė medicina 12B). Mokslinio darbo konsultantas Prof. habil. dr. Henrikas ilinskas (Lietuvos veterinarijos akademija, bio-medicinos mokslai, veterinarin ė medicina 12 B). Veterinarin ė s medicinos mokslo krypties disertacijos gynimo taryba Pirmininkas Prof. dr. Antanas Sederevi č ius (Lietuvos veterinarijos akademija, biomedi-cinos mokslai, veterinarin ė medicina 12 B); Nariai: Prof. habil. dr. Vaiva Lesauskait ė (Kauno medicinos universitetas, biomedi-cinos mokslai, medicina 07 B); Prof. habil. dr. Ramutis Klimas (iauli ų universitetas, biomedicinos moks-lai, zootechnika 13 B); Prof. dr. J ū rat ė iugdait ė (Lietuvos veterinarijos akademija, biomedicinos mokslai, veterinarin ė medicina 12 B); Doc. dr. Eugenijus Aniulis (Lietuvos veterinarijos akademija, biomedicinos mokslai, veterinarin ė medicina 12 B). Oponentai: Prof. habil. dr. Algimantas Matusevi č ius (Lietuvos veterinarijos akademija, biomedicinos mokslai, veterinarin ė medicina 12 B); Prof. habil. dr. Dalia Pangonyt ė (Kauno medicinos universitetas, biomedici-nos mokslai, medicina 07 B). Disertacija bus ginama vieame Veterinarin ė s medicinos mokslo krypties tarybos pos ė dyje 2006 m. lapkri č io 23 d. 14 val. Lietuvos veterinarijos aka-demijos I auditorijoje. Adresas: Til ė s 18, 47181 Kaunas, Lietuva. Disertacijos santrauka isiuntin ė ta 2006 m. spalio 23 d. pagal patvirtint ą adres ų s ą ra ą . Disertacij ą galima peri ū r ė ti Lietuvos veterinarijos akademijos ir LVA Ve-terinarijos instituto bibliotekose.
1. INTRODUCTION Endometrial pathology for a long time has been recognized as a major cause of infertility in broodmares. Two most commonly encountered its conditions are: persistent infectious endometritis and chronic degenerative endometritis endometrosis (Bracher, 1992; Schoon H.-A et al.,1999; Tu-nón, 1999; Evans et al.,2001). Endometritis is characterized mainly by ac- cumulation of inflammatory cells, whereas endometrosis is based on chronic histopathological changes in the endometrium. The latter include diffuse endometrium fibrosis, formation of dilated glands or glandular nests, lym-phatic lacunae and, in severe cases, endometrium atrophy (Morrow, 1986; Bracher, 1992; Tunón, 1999; Walter et al., 2001, 2003). The pathogenesis of this disease is still unknown, but some authors associated it with the age of the affected animal and/or number of foals (Ricketts, 1991; Schoon H-A. et al., 1999; Evans et al., 2000; Walter et al., 2001; Cadario et al., 2002). Studying degenerative changes in mares endometrium (endometrosis), myofibroblasts were found situated around normal and dilated glands (Ev-ans et al., 1998; Walter et al., 2001). Walter (2001) found, that some mares endometrium fibroblasts showed features of myocytes and, apart from fi-bronectin, laminin, collagen type IV and tenascin, started to produce also α -smooth muscle actin, desmin and tropomyosin (Walter et al., 2001). These findings gave new features in uterine tissue remodelling during normal oes-trus cycle and pathological conditions. Endometrosis is inseparable from endometritis, so it is vital to notice the first signs of inflammation, thus acute phase proteins can be the markers for this reason. In spite of that, there were very few attempts to study them in equines (Pepys et al., 1989; Hulten, 1999; Hulten et al., 2002). Uterine blood vessels and blood flow stability have huge affect on ma-res fertility. Alterations of the vessels walls, affected by higher age and parity, can influence uterus blood flow and oxygen saturation. The latter induce development of degenerative changes and higher number of abor-tions (Oikawa et al., 1993; Voss et al., 1994; Grüninger et al., 1998; Schoon D. et al., 1999). To this moment mares endometrium and uterine blood vessels pathol-ogy and its outspread were not examined in Lithuania. The aim of the work: To determine changes in mares endometrium, blood vessels and synthe-sis of serum amyloid A in cases of endometritis and endometrosis.
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Goals of the work: 1. To evaluate frequency of endometritis and endometrosis in mares; 2. To evaluate the prevalence of myofibroblasts and tenascin in endo-metrium in cases of endometritis and endometrosis. 3. To determine serum amyloid A synthesis in mares endometrium dur-ing endometritis and endometrosis. 4. To determine the changes of the extra- and intrauterine blood vessels walls thickness and the amount of elastic fibers (elastin), regarding mares age and endometrium pathology. The novelty of the work: • The display of Lithuanian mares endometritis and endometrosis was investigated; • The prevalence of myofibroblasts and tenascin was determined dur-ing endometritis and endometrosis. • An acute phase protein serum amyloid A studies in mares endo-metrium were carried out. • The changes of the vessels walls thickness and the amount of elastic fibers, due to mares age and endometrial pathology, were evaluated using morfometric analysis. 2. MATERIALS AND METHODS During 2003-2006, in the Department of Infectious Diseases of Lithua-nian Veterinary Academy and in the Department of Pathobiology of Utrecht University (The Netherlands), 82 samples from uteruses of mares (3 to 30 years of age) were studied. Uterine biopsy samples (n=41), samples from horses at necropsy (n=18) and samples from archive (n=23) were collected for this study. There were 75 purebred (Trakehner, Lithuanian Heavy Drafts, KWPN, Friesian, Arabian, various pony breed) mares and 7 mongrels. Re-productive organs were grossly examined clinically and, where possible, at necropsy; stages of the oestrus cycle were determined according to changes in the ovaries and uterus. The anamnesis concerning mares age, parity, live foals and reproductive disorders was collected. The mares were divided into 3 age groups: young 3-8 years (n=26), middle aged 9-14 years (n=30) and old 15-30 years (n=26). The control group consisted of 5 from 2 to 3 years old fillies which belonged to the first age group. Mares were grouped in four categories: I, IIa, IIb and III, according Kenney and Doig (1986) classification system.
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Single uterine biopsy samples were obtained per animal and three sam-ples per animal were taken during necropsy. Samples of extra-uterine blood vessels were obtained from ovarian and uterine arteries, 1-0.5 cm above ramification in the broad ligament. Samples for histological study were fi-xed in Bouins solution for 12-24 h, whereas samples for immunohisto-chemical and histochemical examination were fixed in 10% neutral formal-dehyde for 24 h. After fixation all the samples were dehydrated and embed-ded in paraffin wax (Paraplast) using automated embedding equipment. Serial sections were cut at 4 μ m, placed on poly-l-lysine-coated glass slides and dried at 53 ° C overnight. Histochemical and immunohistochemical ex-aminations were carried out. Hematoxylin & eosin stained samples were categorized according to Kenney and Doig (1986) classification system. Histochemical methods were used to show special structures. Connective tissue was stained in van Gieson and azan; elastic fibbers were showed with resorcine - fuchsine and Lawson solution, mucins were determinated with periodic acid and alcian blue. Amyloid was identified using Congo red dye and polarization filters. Acute phase protein serum amyloid A and endometrium matrix compo-nents were identified using immunohistochemical imunoperoxidase reaction of avidin-biotin complex. Primary monoclonal antibodies: anti vimentin (Biogenex, San Ramon, CA, USA), anti α -smooth muscle actin (Biogenex, San Ramon, CA, USA), anti desmin (clone D33) (Monosan, The Netherlands), anti human muscle actin (clone HHF35) (Dako Cytomation, Danmark), anti tenascin-C (clone HF10TT) (IBL (EGF line), Malaysia) and anti serum amyloid A (SAA) (Tridelta, Dortmund, Germany) were used. Secondary antibodies: marked with biotin horse IgG against mouse serum IgG. Control sections were treated with mouse IgG-1 or PBS instead of primary antibody. The sections were incubated with ABC/PO complex (Vector) or using Envision/PO method. Visualisation was performed with DAB substrate (10 mg 33-diaminobenzidine in 50 ml of 0.05 mol Tris buf-fer (pH 7.8) (Sigma, St.Louis, MO, USA) and 0.033% H 2 O 2 for 10 min at room temperature. Nuclei were stained with Mayers hematoxylin (Merck, Germany). The slides were dehydrated and mounted with Eukitt (Kindler GmBh & Co, Freiburg, Germany). All incubations were performed at room temperature in a humid chamber. Antibody against vimentin was used to identify fibroblasts; myofibroblasts were identified with antibodies against α -smooth muscle actin, desmin (clone D33), human muscle actin (clone HHF35) and vimentin. Pathological morphological and morfometrical evaluation of extra-and intra-uterine arteries . Pathological morphological examinations of 82 mares extra- and intra-uterine arteries were carried out. Changes of the
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uterine blood vessels walls (elastosis, fibrosis, angiitis) were evaluated by using histological and histochemical experiments. Vascular degeneration index (VDI) was calculated using Ludwigs (2001) formula: VDI = MEI + I + (M x d) + A, where MEI is membrane elastica interna, I intima, M media, A adventitia and d destructive alterations factor (Ludwig et al., 2001). Morfometrical uterine vessels evaluation was performed using com-puter image analysis system Leica QWin Standart V 2.7 (Leica Microsys-tems Imaging Solutions, Cambridge, UK) and programs, prepared by J.L.M.A. Lammertink and under supervision of prof. dr. Erik Gruys and A.M. van Ederen of the Department of Pathobiology of Utrecht University. 4 µm slices stained with resorcine-fuchsine and Lawson solution were pre-pared for this study. From each necropsy and biopsy material, 10 arteries from endometrium mucosa and muscle layers were measured. Arteries from the both layers were divided into two groups, depending to the area of their walls: less then 10 000 μ m 2 (n=60) and over 10 000 μ m 2 (n=37). From the each artery the area of all layers (%) and the amount of elastin (%) were measured three times. There were made 2910 initial measurements. Arteries (less and over 10 000 μ m 2 ) from five 2 to 3 years old fillies uteruses were used as the controls. Statistical analysis. Analyses were performed using Microsoft Excel 2000 and Microsoft Excel + Analyse-it (ANOVA) software packages. The coefficient of the reliability of difference between groups was considered as statistically reliable, if p<0.005. The arithmetic mean, mean square deviation and correlation coefficient were calculated. 3. RESEARCH RESULTS 3.1. Frequency, nature and category of endometrium lesions Using Kenney and Doig classification system, mares were divided into four categories. The majority of samples were classified as category IIa (n=42, 51%) and IIb (n=22, 27%), where mild and average endometrium lesions were found. Only few samples were categorized as category III (n=4, 5%). Large number of mares (n=14, 17%) were sound (category I). Mares age groups and categories were compared together. There were more sound mares and less endometrium lesions in the first age group (3-8 years), com-paring with others (fig. 1). Majority of the mares were sound (category I) or had mild endometrium lesions (category IIa). Average and severe endo-metrium lesions were diagnosed only in few mares (categories IIb and III) (p<0.005). Mild and average lesions (categories IIa and IIb) were diagnosed in the second age group (9-14 years). The number of the sound mares (cate-gory I) was lesser comparing with the first age group, but the number of the
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severe affected endometrium (category III) stayed the same in this age group (p<0.005) (fig. 1). The categories distribution in the last age group (15-30 years) was the same: mild and average endometrium lesions (categories IIa and IIb) dominated over severe alterations (category III). There were no sound mares in this category. proc.58 6053 46 50 42 37 38 40 30 20 10 8 7 3 0 4 4 0 3-8 yrs. 9-14 yrs 15-30 yrs I IIa IIb III Fig. 1. The distribution of mares in age groups according to Kenney and Doig (1986) classification system (p<0.005) . 1 pav. Kumeli ų paskirstymas pagal Kenney ir Doig (1986) kategorijas priklausomai nuo amiaus grup ė s (p<0,005) . As expected, the majority of young (3-8 years) mares belonged to cate-gory I, while older (9-23 years) mares belonged to the categories IIa and IIb. There was no notable correlation between category III and age of the mares: mares of 7, 8, 12 and 30 years belonged there. The chance of delivering a live foal directly depends on category. From the 82 mares used in our study, major part (68%) fulfills requirements of the broodmares (category I and IIa, 90-50% fertility). For the delivering a live foal they need only minimal routine veterinary supervision. 27% of mares (category IIb) need veterinary supervision before and after covering or arti-ficial insemination, and their chance to deliver a live foal is only 10-50%. The rest of the mares had chance of only 1 10%. -
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Of the 82 mares, endometritis was diagnosed in 55 cases (67%) and en-dometrosis in 30 (37%). There were 17 cases (21%) where endometrosis occurred together with endometritis and 14 mares (17%) were sound. Difference of endometrium pathologies was in all three age groups (fig. 2). Majority of the sound (n=12, 46%) mares and mares with endometritis (n=12, 46%) were in the first age group (3-8 years) (p>0.005), for few of them (n=2, 2%) endometritis and endometrosis were diagnosed together. There was no endometrosis without endometritis in this age group. In the second age group (9-14 years) the number of the sound mares was lesser (n=2, 7%), there was an increase of endometritis (n=16, 53%), endometritis together with endometrosis (n=7, 23%) and endometrosis (n=5, 17%) (p<0.005). There were no sound mares in the last age group (15-30 years), the cases of other pathology were almost equal: 38% (n=10) of endometritis, 31% (n=8) of endometritis with endometrosis and 31% (n=8) of endometro-sis (p>0.005). proc. 53 60 46* 46* 50 38 40 31+ 31+ 30 17 23 208 10 7 0 0 0 3-8 yrs. 9-14 yrs. 15-30 yrs. Sound Endometrosis Endometritis/ Endometrosis Endometritis Fig. 2. The distribution of endometrial pathologies in age groups (p<0.005, except marked ones). 2 pav. Kumeli ų gimdos gleivin ė s patologij ų pasiskirstymas pagal ami ų (skirtumai statistikai patikimi, iskyrus paym ė tas grupes (p < 0,005)).
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3.2. The data of histological and histochemical examination of mares endometrium After the histological examination it was found that endometritis was major lesion (n=55, 67%). Chronic endometritis was found in 62% (n=51) of mares, 5% (n=4) were acute cases. Infiltration of inflammatory cells in the mucosa, their migration towards lumen and accumulation near or around blood vessels were typical features of endometritis. Endometrium edema was found in 57.5 % of mares, however majority of samples were taken in estrus. In the majority of samples (67.5%) inflammatory cells were scattered through endometrium mucosa, while formed localized foci in the rest of samples (32.5%). As expected, majority of inflammatory cells were found in the stratum compactum (64%). Margination of inflammatory cells through the epithelium was found in 56% of samples. Large accumulations of eosi-nophils (32% of samples) in the stratum compactum were found almost in the every case of endometritis. Siderophages were found in 40% of all cases. Mild endometritis (n=40, 60%) was found in the majority of samples, aver-age endometritis was diagnosed in 28% (n=19) of samples, and severe le-sions were found in 12% (n=8) of samples. We found that endometrosis was second (n=30, 37%) large lesion of mares endometrium. In cases of endometrosis, formation of fibroblasts and myofibroblasts circles around normal and dilated glands, glandular nests and diffuse endometrium fibrosis, dilation of glands with/without epithelium lesion and lymphatic lacunae were found. Mostly fibroblastic rings sur-rounded dilated glands, but in a few cases they were found around normal glands. Depending of the degree of alteration, there were from 1-2 to 5-6 layers of fibroblasts or myofibroblasts around normal, dilated glands and glandular nests. The part of dilated glands (n=13) showed no signs of fibro-sis. In the 37.5% of samples, diffuse mild (n=11) and average (n=8) endo-metrium fibrosis was found. Lymphatic lesions and lacunae were found in 36% of samples (n=30). Minor accumulation of glandular secretion material can be found in normal endometrium, yet increased amount is considered as pathology and is referred as one of the sings of endometrosis. While histo-logical examination (n=82), eosinophylic material (n=69, 84%) was found in the glandular lumen. Solid structures with basophilic centre were found in some glands (n=24, 29%). There were three samples (4%) with totally calci-fied structures. All material in glandular lumen was negative for amyloid. Positive periodic acid and alcian blue reaction in the endometrium and glan-dular epithelial cells showed intensive synthesis of mucopolysacharides. Especially strong positive reaction and accumulation of secrete were found in the dilated individual glands or glandular nests. The strongest reaction
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was found in the apical part of cell, yet whole cells were positive in 14% (n=11) of cases. 3.3. The data of immunohistochemical examination of mares endo-metrium Part of the samples (n=30) with signs of inflammation were prepared for acute phase protein serum amyloid A ( SAA) study. All samples were stained with Congo red to avoid reaction with amyloid A, and all samples were negative. Positive reaction for SAA was found only in four samples near inflammation areas in the endometrium matrix liquids and in side blood vessels. Endometrium cells were negative for SAA. Fibroblasts were identified with antibodies against vimentin, while myo-fibroblasts against vimentin, α -smooth muscle actin, desmin (clone D33), human muscle actin (clone HHF35) and tenascin-C (clone HF10TT). Fibroblasts were found in all samples (n=60, 100%). Myofibroblasts were identified in 91% (n=51) samples of mares endometrium, 27% (n=15) of them had signs of endometrosis, 62% (n=35) of endometritis and 2% (n=1) of samples were without obvious sings of any pathology. In cases without endometrium fibrosis, myofibroblasts were found mostly around dilated glands.
Fig. 3. Positive reaction of pathological glands epithelium to anti-body against vimentin (x200) . 3 pav. Teigiama paeist ų liauk ų epitelini ų l ą steli ų vimentino imunohis-tochemin ė reakcija (x200) .
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Plenty of fibroblasts were found around dilated (n=35, 67%) and normal (n=40, 77%) glands, also in the glandular nests and around them (n=28, 54%). Positive reaction to antibody against vimentin was found in the en-dometrium (n=15, 29%) and glandular (n=21, 40%) epithelial cells. Major part of the positive reaction in the endometrium was in the basal part of the cells (n=12, 80%), in one case (6.7%) cell was positive in the apical part and two time positive reaction were in the whole cell (13.3%) (fig. 3). The epithelial and connective tissue cells reaction to vimentin had no direct con-nection with category, mares age and endometrium lesions.
Fig. 4. Positive reaction of dilated glands with and without fibrosis to antibody against α -smooth muscle actin (x400) . 4 pav. Teigiama α -lygi ų j ų raumen ų aktino imunohistochemin ė reak-cija aplink isipl ė tusias liaukas su fibroz ė s poymiais ir be j ų (x400). Myofibroblasts (n=51, 85%) were found around normal (n=41, 80%) and dilated (n=43, 84%) glands, and in case of glandular nests formation (n=48, 94%) (fig. 4). Positive reaction was also found around dilated glands with-out fibrosis. The majority of samples (n=44, 86%) positive for myofibro-blasts were found in the deeper layer of endometrium, near the glandular
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trees. Glandular nest were surrounded with 1-2-3 layers of myofibroblasts in the 58% of samples (n=28), the remaining part (n=17, 36%) was surrounded with 4-6 layers. Especially many layers (6-8) were found in three samples (6%). Positive reaction (n=22, 43%) to α -smooth muscles actin was also found in the stratum compactum, far from glands and their nests. These cells formed thick longitudinal layer near the basal membrane (fig. 5). Such myo-fibroblasts were spread in the whole endometrium in seven cases (32%), and formed local foci in the rest part of samples. Myofibroblasts were scattered in endometrium matrix in nine samples (18%).
Fig. 5. Positive reaction of endometrium stratum compactum to an-tibody against α -smooth muscle actin (x400) 5 pav. Teigiama gimdos kompaktinio sluoksnio gleivin ė s α -lygi ų j ų rau-men ų aktino imunohistochemin ė reakcija (x400). Myofibroblasts positive for tenascin (n=25, 53%) were found around di-lated (n=7, 28%) and normal (n=5, 20%) glands, around glandular nests (n=6, 24%) and in the longitudinal layer in the stratum compactum, near the basal membrane (n=20, 80%). Positive reaction for tenascin was found in 32% (n=15), of mares with endometrosis, while 26% of mares (n=12) were with endometritis, and 4% (n=2) were sound.
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3.4. The data of pathological morphological and morphometrical ex-amination of intra- and extrauterine blood vessels After morphological examination of intrauterine (n=79) and extrauterine (n=14) blood vessels vascular degeneration index (VDI) was estimated. In the major part of the samples (n=42, 53%) mild vascular lesions were esti-mated, 44% of mares were sound. There were no severe vascular lesions in our samples. Majority of the young mares (3-8 years) had minor lesions or were sound, and 7 mares (29%) had average lesions. Older mares (second and third age group) had higher number (81% ir 52%) of lesions and they were more severe (fig. 6). proc. 90 81 80 71 70 60 52 50 41 40 29 30 19 20 7 10 0 0 0 0 0 0 3-8 yrs. 9-14 yrs. 15-30 yrs. None Mild Averige Severe Fig. 6. The distribution of vascular lesions according to vascular de-generation index (VDI) in age groups (p<0.005). 6 pav. Kumeli ų gimdos kraujagysli ų paeidim ų (pagal VDI) pasiskir-stymas priklausomai nuo amiaus grup ė s (p<0,005). Severity of vascular lesions depended on category (p<0,005). Minor le-sions were found in I and III categories, while major lesions were in IIa and IIb categories (p<0,005). In cases of average and severe endometrosis (IIb and III categories), vascular elastosis in intima and adventitia was found. There were duplication, splitting and irregularity in membrane elastica in-terna. Fibrosis of media and adventitia was found. In three cases angiitis with mononuclear cell infiltration were found. Vascular lesions were the same in all parts of endometrium. 14 samples of extrauterine arteries were
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examined and in 13 samples irregularity and duplication of membrane elas-tica interna was found. Elastosis or fibrosis was not found, while degenera-tion of media was found in four samples. It was marked by loss of myocytes uniformity and integrity and appearance of connective tissue. We analyzed individual intrauterine arteries wall layers area (%) and amount of elastic fibrils (%) by using morfometrical study. Control group was from five young (2-3 years) fillies. The thickness of small vessels (less then 10 000 μ m 2 ) layers depended on pathology. The slight reduction in media size in endometritis and higher reduction in endometrosis and in en-dometrosis together with endometritis can be seen (p<0,005) (fig. 7). Area of intima of the pregnant mares arteries was higher then in control group. The changes of the media were smaller; the highest difference was between control and sound mares of different age. Adventitia has not changed in endometritis, but was visible thicker in endometrosis and in cases of endo-metrosis together with endometritis. The area of adventitia was reduced in pregnant and sound mares (fig. 8). 25 20 15 10 5 0
Fig. 7. The differences between the thickness of small vessels (less then 10 000 μ m 2 ) intima area (%) according to physiological and patho-logical changes (median, lower and upper quartiles, confidence interval around the median) (p<0.005). 7 pav. Iki 10000 μ m 2 kraujagysli ų vidinio sluoksnio plotas (%) priklau-somai nuo fiziologin ė s kumel ė s b ū kl ė s ir vyraujan č ios patologijos (medi-ana, patikima medianos riba, kvartilio ribos) (p<0,005).
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100 90 80 70 60 50 40
Fig. 8. The differences between the thickness of small vessels (less then 10 000 μ m 2 ) adventitia area (%) according to physiological and pathological changes (median, lower and upper quartiles, confidence inter-val around the median) (p<0.005). 8 pav. Iki 10000 μ m 2 kraujagysli ų iorinio sluoksnio plotas (%) prik-lausomai nuo fiziologin ė s b ū kl ė s ir vyraujan č ios patologijos (mediana, patikima medianos riba, kvartilio ribos) (p<0,005). The area of the bigger vessels (more then 10 000 μ m 2 ) slightly differed due to the pathology. The area of intima increased in all pathologies, espe-cially in endometrosis and in pregnant mares (p>0.05). The area changes in media were smaller; the bigger difference was between control group and in cases of endometrosis with endometritis (p<0.005). Adventitia was thicker in endometritis and in endometritis together with endometrosis (p<0.05). Marked changes of the vascular layers were found in the different age groups. The area of intima and media of smaller vessels (less then 10 000 μ m 2 ) decreased with increasing mares age (p<0.05) (fig. 9). The area of adventitia increased with the increasing age (p<0.001) (fig. 10). The changes of the bigger vessels (more then 10 000 μ m 2 ) differed from smaller ones. The area of media increased with the age, while the changes of area of me-dia and adventitia did not depend on mares age.
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14 12 10 8 6 4 2 0 3-8 yrs. 9-14 yrs. 15-30 yrs. Fig. 9. The differences between the thickness of small vessels (less then 10 000 μ m 2 ) intima area (%) according to age group (median, lower and upper quartiles, confidence interval around the median) (p<0.005). 9 pav. Iki 10000 μ m 2 kraujagysli ų vidinio sluoksnio plotas (%) priklau-somai nuo amiaus grup ė s (mediana, patikima medianos riba, kvartilio ribos) (p<0,05). 90 80 70 60 3-8 yrs. 9-14 yrs. 15-30 yrs. Fig. 10. The differences between the thickness of small vessels (less then 10 000 μ m 2 ) adventitia area (%) according to age group ((median, lower and upper quartiles, confidence interval around the median) (p<0.005)). 10 pav. Iki 10000 μ m 2 kraujagysli ų iorinio sluoksnio plotas (%) prik-lausomai nuo amiaus grup ė s (mediana, patikima medianos riba, kvartilio ribos) (p<0,001)). 18
30 25 20 15 10 5 0 3-8 yrs. 9-14 yrs. 15-30 yrs. Fig. 11. The differences between the amount of elastin (%) in small vessels (less then 10 000 μ m 2 ) intima according to age group (median, lower and upper quartiles, confidence interval around the median) (p<0.005). 11 pav. Iki 10000 μ m 2 ploto kraujagysli ų vidiniame sluoksnyje esan č i ų elastini ų skaidul ų kiekis (%) priklausomai nuo amiaus (mediana, pati-kima medianos riba, kvartilio ribos) (p<0,005) . 45 35 25 15 5 -5 3-8 yrs. 9-14 yrs. 15-30 yrs. Fig. 12. The differences between the amount of elastin (%) in small vessels (less then 10 000 μ m 2 ) adventitia according to age group (median, lower and upper quartiles, confidence interval around the median) (p<0.005). 12 pav. Iki 10000 μ m 2 ploto kraujagysli ų ioriniame sluoksnyje esan-č i ų elastini ų skaidul ų kiekis (%) priklausomai nuo amiaus (mediana, patikima medianos riba, kvartilio ribos) (p>0,005)). 19
The amount of elastic fibers (%) was measured in the intima and adven-titia of the intrauterine endometrium arteries. In the smaller arteries, the amount of elastic fibers in intima differed slightly due to pathologies. Marked difference was found in adventitia in cases of endometritis. Smaller amounts of elastin were found in cases of endometritis with endometrosis and in endometrosis. The amount of elastic fibers in the intima of bigger vessels was lower then in control group. There was no marked difference of elastin amount in adventitia in different groups; increased amount was found in sound mares of different age. In smaller vessels, the amount of elastic fibers in intima (fig. 11) was similar in all age groups, but there was marked difference in adventitia (p<0.005) (fig. 12). In this layer, the amount of elastin increased with in-creasing age of mares. The amount of elastin in intima and adventitia of bigger vessels did not depend on the mares age (p>0.005). 4. CONCLUSIONS 1. Average endometrium lesions (78%) (category IIa ir IIb) were domi-nant for older mares (9-14 years and 15-30 years). 2. The major lesion of endometrium was endometritis (67%); endomet-rosis was more rare (37%). Endometrosis was more often for older mares (p<0.01), while the incidence of endometritis did not depend from the age. 3. Myofibroblasts were identified in the endometrium of mares in the case of endometritis (62%) and endometrosis (27%). The most often they were found in endometrium around pathological glands and in the stratum compactum. 4. Positive reaction for tenastin was found in the cases of endometritis (32%) and endometrosis (26%). Tenascin together with myofibroblasts was found in stratum compactum of endometrium (43%). 5. In the cases of endometritis and endometrosis, the synthesis of serum amyloid A in endometrium was not found. 6. Using vascular degeneration index (VDI), it was estimated that 53% of intrauterine and extrauterine blood vessels had average lesions (p<0.05). 7. The area of intima of smaller vessels (less then 10 000 μ m 2 ) de-creased with increasing mares age, and in cases of endometritis and endo-metrosis, while the area of adventitia increased (p<0.01). The changes of the bigger vessels (more then 10 000 μ m 2 ) layers area did not depend on mares age and examined endometrium pathologies. 8. The amount of elastic fibers in adventitia of smaller vessels (less then 10 000 μ m 2 ) increased with increasing mares age and in cases of endo-metritis and endometrosis. (p<0.005). 20
5. THE LIST OF ARTICLES 1. Sabeckiene J., Ederen A-M van, Zeinstra E., Aniulien ė A., Gruys E. Uterine myofibroblastst in mares. Medycyna Weterynaryjna, Lublin. 2005. Vol. 61. Nr. 12. P. 1347-1350; 2. Sabeckien ė J., Tarasauskas S., Aniulien ė A. Endometrosis in Lithua-nian Mares. Veterinarija ir zootechnika. Kaunas. 2005. T 30 (52). P. 30-33.
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1. Į VADAS Gimdos gleivin ė s paeidimai yra viena i pagrindini ų kumeli ų nevaisin-gumo prieas č i ų . Iskiriami du daniausiai pasitaikantys gimdos paeidimai: l ė tinis infiltracinis endometritas ir l ė tinis degeneracinis gimdos gleivin ė s paeidimas endometroz ė (Bracher, 1992; Schoon H.-A et al., 1999; Tu-nón, 1999; Evans et al., 2001). Endometrito atveju vyrauja udegimini ų l ą steli ų sankaupos, o endometroz ė pasireikia l ė tiniais gimdos gleivin ė s pakitimais. Pastariesiems b ū dinga gimdos gleivin ė s fibroz ė , liauk ų isipl ė -timas, vadinam ų j ų liauk ų lizd ų formavimas, limfagysli ų isipl ė timas (limfo-lak ū nomis) ir, sunkiais atvejais, gimdos gleivin ė s atrofija (Morrow, 1986; Bracher, 1992; Tunón, 1999; Walter et al., 2001, 2003). io susirgimo pato-genez ė pilnai neinoma, ta č iau kai kurie autoriai endometroz ę sieja su ku-mel ė s amiumi ir/ar atvest ų kumeliuk ų skai č iumi (Ricketts, 1991; Schoon H-A. et al., 1999; Evans et al., 2000; Walter et al., 2001; Cadario et al., 2002). Tyrin ė jant kumeli ų gimdos gleivin ė s degeneracinius pakitimus (endo-metroz ę ), aptikti miofibroblastai, isid ė st ę aplink normalias bei isipl ė tusias gimdos liaukas (Evans et al., 1998; Walter et al., 2001). Walter su bendraau-toriais (2001) nustat ė , kad gimdos gleivin ė s fibroblastai į gyja miocit ų savy-bes ir, be fibronektino, laminino, IV tipo kolageno ir tenscino, pradeda ga-minti α -lygi ų j ų raumen ų aktin ą , desmin ą , tropomiozin ą . ie atradimai sutei-k ė nauj ų ini ų apie gimdos gleivin ė s kitimus rujos ir patologini ų b ū kli ų metu. Endometroz ė neatsiejama nuo endometrito, tod ė l labai svarbu laiku pas-teb ė ti pirmus udegimo poymius. iuo tikslu gal ė t ų b ū ti naudojami ū mios udegimo faz ė s protein ų tyrimai, ta č iau arkli ų lig ų atvejais jie tyrin ė ti labai maai (Pepys et al., 1989; Hulten, 1999; Hulten et al., 2002). Didel ė s reikm ė s kumeli ų vaisingumui turi gimdos kraujagysl ė s bei kraujotakos stabilumas. Amiaus ar didelio kumelingumo skai č iaus į takoti kraujagysli ų sieneli ų paeidimai sutrikdo gimdos kraujotak ą ir jos apr ū pi-nim ą deguonimi, taip veikdami patologini ų pakitim ų vystym ą si gimdoje ir padid ė jus į abort ų skai č i ų (Oikawa et al., 1993; Voss ir k.t, 1994; Grüninger et al., 1998; Schoon D. et al., 1999). Lietuvoje duomen ų apie kumeli ų gimdos gleivin ė s ir jos kraujagysli ų pa-tologijas bei j ų paplitim ą n ė ra. Darbo tikslas į vertinti kumeli ų gimdos gleivin ė s ir kraujagysli ų poky- č ius bei serumo amiloido A sintez ę gimdoje endometrito ir endometroz ė s atvejais.
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