Morphological and mechanical changes in juxtarenal aortic segment and aneurysm before and after open surgical repair of abdominal aortic aneurysms.

OBJECTIVE: The aim of study was to assess how the ultrastructure of the wall of aortic aneurysms, sac and neck influences aortic wall distensibility and proximal dilatation 2 years after open repair. METHODS: Biopsies for electron microscopy were taken from aneurysmal sac and neck of 30 patients. Patients were assessed by computed tomography (CT) and ultrasound for aneurysm diameter and distensibility (M-mode ultrasonography). RESULTS: Postoperative CT of the aortic stump distinguished two groups. Group I (n = 11) with little enlargement, median 1 mm (1-3 mm) and group II (n = 19) with significant aortic enlargement, median 5.2 mm (4-12 mm). In group II, changes in elastic fibres in the aneurysm neck were comparable to, but as extreme as in the aneurysm sac. For group I, the distensibility of the aneurysmal sac was significantly lower than in the neck or at the renal arteries. For group II, the distensibility in both the neck and sac was significantly lower than at the juxtarenal segment (p = 0.01). The biopsies of group II patients showed the extensive degeneration of normal architecture, which was associated with altered wall distensibility in both the aneurysmal neck and sac. CONCLUSIONS: Disorganisation and destruction of normal aortic architecture at the ultrastructural level are associated with decreasing aortic distensibility. Low aortic neck distensibility is associated with proximal aortic dilatation at 2 years postoperatively.

Detection of DNA, mRNA and early antigen of the human cytomegalovirus using the immunomax technique in autopsy material of children with intrauterine infection.

The present study focuses on the immunomax technique in association with the avidin-biotin-peroxidase complex (ABC) technique and a non-isotopic variation of in situ hybridisation (ISH) for optimal microscopical detection of human cytomegalovirus (HCMV). The studies were performed on an archival paraffin material originating from five children deceased due to intrauterine infection. The results of immunocytochemical and hybridocytochemical studies, with or without amplification using biotinylated tyramine, were compared with the routine histopathological results and results obtained using the polymerase chain reaction (PCR). Early antigen (EA)-HCMV was demonstrated in approximately twice as many cells as detected in the routine staining and also in cells that seemed morphologically intact. The hybridocytochemical studies confirmed the presence of HCMV DNA in cells that were positive in the immunocytochemical tests and, in addition (using the ISH-immunomax technique), in cell nuclei of intact myocardial myocytes. In general, fewer cells manifested the presence of HMCV mRNA than the presence of HCMV DNA. The immunomax technique was found to be more sensitive than the techniques of classical immunocytochemistry or of ISH. The former technique permitted the documentation of a higher number of HCMV replication sites than could be detected using the latter techniques. However, the clinical course of HCMV infection or the cause of death of the children was not directly related to the intensity of HCMV expression in tissues.

Additional cell damage after transpupillary thermotherapy in choroidal malignant melanoma.

The aim of this study was to evaluate the effectiveness of transpupillary thermotherapy (TTT) in generating tumour necrosis by light and electron microscopy, as well as to evaluate additional cell damage in the area directly adherent to the necrotic zone. Four eyes of four patients diagnosed with intraocular malignant melanoma of the uvea were treated experimentally with diode laser TTT. In all cases a standard technique was used. All eyes were enucleated: one eye the day after TTT, two eyes 2 days after TTT, and one eye 6 weeks after TTT. Immediately after enucleation the eyes were immersed in standard Karnovsky's fixative with cocodylate buffer and prepared for light and electron microscopy. In the treated area of all four melanomas we found a dense band of necrotic tissue (zone A) consisting of an amorphous mass of dead cells sharply demarcated from the rest of the neoplastic tissue. Next to this zone was a more eosinophilic and also sharply demarcated band (zone B) that consisted of similar but less intensive changes. In the next band (zone C), marked injury to the cellular membrane and subcellular structures were seen on electron microscopy. The next band (zone D) consisted of changes mainly observed only within the cytoplasm of neoplastic cells and significantly less intensive than those in zone C. Outside zone D tumour cells that were normal in appearance were seen. No scleral alterations induced by heat were found. We concluded that after TTT the cytotoxic effect gradually decreases in proportion to the distance from the central point of the diode laser spot, with additional cell damage in the area adjacent to the necrotic zone. The interval between TTT and enucleation had no influence on the histological results.

Quantitative morphometry--an easy and inexpensive method for comparative studies of antigen expression during development.

The morphometric studies due to the development of computerized techniques became a very suitable tool for quantitative analysis. In the present paper we describe an easy and relatively inexpensive method useful for comparative studies on changes of expression of examined molecules during development of tissue. We used with a success this method for studies on the expression of CD31 adhesion molecule during the fetal and newborn lung development. We found that expression of CD31 is regulated during the perinatal period. It might be related to the dynamic changes in developing vascular bed in rat lung.

The entrance of surface active material to pulmonary capillaries in the lungs of newborn rats.

In newborn rats we investigated the entrance of surfactant to the pulmonary capillaries a few minutes after the birth. Time is crucial for the process which can be called "drying" of the lung, when a large amount of intraalveolar fluid enters the pulmonary circulation. Ultrastructural studies have revealed bulk transport and endocytosis of surfactant via alveolar septa to the blood vessels, but it is still questionable whether the entrance of surfactant to pulmonary capillaries is related to its clearance or to "drying" of lung's alveoli.

The expression of PDGF, its receptors and capillary morphometry in the developing lung: quantitative studies.

The aim of the present study was the evaluation of the expression levels of PDGF-A and B, and PDGFR-alpha, andchanges of capillary cross-sectional areas during terminal vascular network formation in the developing lung. Pathogen-free fetuses and newborns used for rats were used. Lungs were processed for frozen section immunohistochemical staining and parallel tissue specimens were prepared for transmission electron microscopy. The expression levels of PDGF-A and B, and PDGFR-alpha were obtained using quantitative image analysis. Additionally. the morphometry of capillary cross-sectional area was performed. We found that the expression levels of the studied antigens fluctuated, and the highest values were found in21-day old fetuses (p < 0.0001). Although there were no significant differences of the area of "a single" capillary, the area of the endothelium decreased significantly in perinatal period (p <0.0001). We conclude that the statistically significant changes of the studied antigens' expression as well as the of capillary cross-sectional area occur in the perinatal period,

Ultrastructural studies on selected elements of the extracellular matrix in the developing rat lung alveolus.

As the respiratory system adapts to the extrauterine life, the extracellular matrix (ECM) plays an important structural and regulatory role during its development. Therefore the purpose of this investigation was to analyze ultrastructurally several elements of extracellular connective tissue during the process of rat lung development. Morphological observations were mainly focused on the terminal part of respiratory system. To outline different components of connective tissue network, several ultrastructural techniques were used (both histochemical and immunohistochemical). The distribution and amount of the following proteins were studied: laminin, collagen type IV, collagen fibrils (CFs), elastic fibers (EFs) and fibronectin (FN). Additionally localization of glycosaminoglycans (GAGs) was examined. The present study deals with four periods of lung development: pseudoglandular, canalicular, saccular and alveolar. In all these stages localization and amount of ECM components change rapidly. In early periods of lung development, the amount of connective tissue fibers was low, basement membranes (BMs) were incomplete, and FN was distributed nearly uniformly. Later when the process of lung alveoli formation begins, the number and thickness of both CFs and EFs rapidly increased, BMs became complete, the content and distribution of FN were irregular. In all stages of lung development GAGs were distributed in BMs and among connective tissue fibers. The results described in the present study summarize morphologically ECM changes occurring during formation of lung alveolus.

CD56(NCAM) antigen in glandular epithelium of human thyroid: light microscopic and ultrastructural study.

CD56 antigen, an isoform of the neural cell adhesion molecule (NCAM) was previously found by us in human thyroid by APAAP immunohistochemistry in light microscopy on frozen tissue sections. In the current study, it was attempted to trace the antigen in question using another light microscopic immunohistochemical procedure and to validate the results at the ultrastructural level. For light microscopy, cryostat sections of 12 surgical samples of human thyroid were subjected to ABC (preformed avidin-biotin-peroxidase complex) method. For immunoelectron microscopy, immunoperoxidase reaction was carried out on prefixed, small thyroid tissue blocks. Following preliminary inspection of semithin sections, ultrathin sections were examined in the transmission electron microscope. ABC reaction revealed distinct specific CD56 staining of thyrocyte cell membranes. The staining was weak or absent in thyroid papillary carcinoma cells. The results were confirmed in semithin sections by indirect immunoperoxidase. The latter reaction in ultrathin sections at the ultrastructural level has shown that specific reaction product was confined to free and lateral surfaces of thyroid follicular cells. Endothelial cell membranes of thyroid capillary vessels were totally devoid of the reaction product. The reaction was weakly positive in thyroid follicular and papilllary carcinomas but absent from medullary carcinoma.

Clinical and morphological aspects of Buerger's disease.

BACKGROUND: Thirty-five patients with TAO and 35 with ASO have been studied. To determine differences between these two arterial diseases, we used clinical criteria, arteriographic and morphological methods. The peripheral (below the bifurcation of the popliteal artery) angiographic changes were found in TAO with 94% sensitivity, 94% specificity and with 96% of positive predictive value. Other clinical diagnostic criteria for differential diagnosis had a lower value (they had high sensitivity and low specificity or low sensitivity and high specificity). RESULTS: The histologic studies confirmed the clinical diagnoses of Buerger's disease in 92,9% cases. The pathologic findings in TAO and ASO were different. CONCLUSIONS: Our investigations proved that the characteristics of Buerger's disease include the development of changes not only in small and medium size arteries or veins, but also in microcirculation.

The experimental investigations of the toxic influence of tobacco smoke affecting progeny during pregnancy.

1. Tobacco smoke contains around 4000 substances, most of which are described as toxic, and they may have an influence on the development of progeny. 2. The present studies concentrate on the measurement and calculation of indices describing the new-born's survival, rearing of pups, weight of foetuses, young animals, placenta and females in relation to different doses of tobacco smoke (carbon monoxide levels). The morphological studies of placenta, foetal and newborn lungs were done as a supplement. Biochemical placenta study was also done. 3. The results of the experiment proved that some indices for animals in groups which were passively exposed to the highest concentrations of tobacco smoke were lower, others fluctuated (4 day, 12 day and total survival) and some did not reveal any changes (rearing). Direct correlation between maternal passive exposure to tobacco smoke and the presence of acute respiratory distress syndrome symptoms in new-borns was observed. A decrease of body weight of pregnant females passively exposed to tobacco smoke was also observed. An increase of placenta-foetal factor was found. A decrease of rat weight was observed after passive exposure to tobacco smoke. 4. We concluded that there is correlation between passive exposition to tobacco smoke during pregnancy and delayed lung maturation in the offspring. Exposure of the pregnant rats to cigarette smoke increases the activity of isocitric and glucose-6-phosphate dehydrogenases in placenta.

The influence of simultaneous whole body exposure to cigarette smoke and low-protein diet feeding on fertility, reproduction, and progeny of rats.

The aim of the present study was to evaluate the toxicological influence of different concentrations of tobacco smoke and low-protein diet on fertility, reproduction, and survival of offspring of Wistar female rats. We studied three generations, two litters in each. There were 192 animals in parent generation. Animals were exposed to three different concentrations of tobacco smoke that were reflected by the carbon monoxide (CO) concentration (500, 1000, and 1500 mg CO/m3 air) during 11 weeks (six before and two weeks during mating, and three weeks of pregnancy). Additionally, animals were divided into two subgroups that were fed normal or low-protein diet. In the present study, we concentrated on the indices describing the reproduction ability, fertility, and rearing of pups of mothers exposed to tobacco smoke. The parameters of newborns survival were estimated also. We concluded that neither tobacco smoke exposure nor the low-protein diet changed the duration of pregnancy. The tobacco smoke in all doses and low-protein diet had a negative influence on fertility, reproduction, and survival of newborns from exposed mothers. This influence was mainly attributed to the tobacco smoke exposure. The levels of carboxyhaemoglobin were increasing with increasing tobacco smoke concentration.

Development of the fetal and newborn lung--morphometric studies (rat model).

The aim of this study was to investigate the development of the fetal lung by morphometric means. Lung specimens from 16-20 day-old fetuses, and 1, 3, 5, 14 and 21 day-old newborns were used. Tissue specimens were evaluated by light microscopy and transmission electron microscopy. The material was divided into four groups--related to four main stages of lung development (pseudoglandular, tubular, saccular and alveolar). For each developmental stage, 30 photographs of semi-thin sections were saved on computer hard disk for future morphometric measurements. OPTIMAS 4.02 software was used for evaluation of the relative area occupied by vascular, epithelial, stromal and fluid/gaseous compartments. GraphPad InStat software was used for statistical analysis. All measurements and calculations were done per observation field. The area occupied by vessels was greatest at the pseudoglandular stage (11.7%), and smallest at alveolar stage (5.7%). The stroma also occupied the largest area at the pseudoglandular stage (58.4%) and the smallest at alveolar stage (17.0%). The area occupied by epithelial cells was most extensive at the tubular stage (52.5%) and smallest at the alveolar stage (6.1%). The fluid/gas compartment was largest at the alveolar stage (70.5%) and smallest at the pseudoglandular stage (0.58%). The present results indicate that the most dynamic lung development occurs during thin air-blood barrier formation (19th day of intrauterine life to 5th day after delivery--saccular and the beginning of alveolar stage).

Ultrastructural changes in the lung alveoli after cardiac surgical operations with the use of cardiopulmonary bypass (CPB).

Cardiac surgical procedures with the use of cardiopulmonary bypass (CPB) are commonly complicated by pulmonary dysfunction. The mechanisms of such injury are not well understood. The aim of the present study is to analyze morphologically (mainly ultrastructurally) alveolar injury, which occurred during cardiac surgical operations involving CPB, equipped with a hollow fiber oxygenator. Our study included 20 patients, aged 45-72, who underwent coronary artery bypass grafting. Lung biopsies were taken from the left upper lobe 20 minutes after stopping CPB. Pre-CPB biopsies served as controls. Tissue specimens used for electron microscopy were processed according to standard procedures. Light microscopy revealed only a few alterations in the terminal part of the respiratory tract. Frank edema was seen in some of the alveoli. Extravasated erythrocytes as well as some neutrophils were present in the alveoli and several alveolar capillaries were congested. Ultrastructural observations confirmed the above mentioned changes. Moreover, in many alveoli, extensive injury to air-blood barrier was observed. Type I pneumocytes and endothelial cells appeared swollen or necrotically changed. The cytoplasm of type II pneumocytes was swollen. In many alveoli, pulmonary surfactant could not distribute over the alveolar surface because of edema. Structures of pulmonary surfactant were also seen in alveolar capillaries. The results of this investigation suggest that CPB is associated with some injury to lung tissue. However, this injury seems to be temporary since all examined patients had an uneventful post-operative course.

Retardation of lung development after maternal cigarette smoking and low protein diet in animal model.

This study has been aimed at showing the influence of tobacco smoke, together with low protein diet on the lungs of newborn rats. The studied animals were Wistar rats. They were exposed to the tobacco smoke for 11 weeks (6 weeks before mating, 12 days during mating, and 21 days of pregnancy). Morphological analysis and interpretation of 48 one-day old rats was based on observation under the light and electron microscope. The study showed atelectasis of the lungs: the lung tissue was insufficiently extended, the alveoli were geometrically irregular and the alveolar septa were thick. Many extravasated erythrocytes and edema (latent as well as overt) were also present. The II type pneumocytes and their surfactant were poorly developed. Tobacco smoke caused the serious pathological effects and together with poor protein diet all changes worsened. For comparison, the lungs of control group were studied (lack of exposure to tobacco smoke); they were well aerated and their structure well-developed.

Studies on tissue expression of HBV in children with chronic hepatitis type B using Immunomax technique.

The study aimed at employing the Immunomax technique to detect the markers of HBV replication (HBcAg and HBV-DNA) in liver biopsy material, obtained from children with chronic hepatitis type B. In line with the currently modified classification of chronic hepatitis and with the increasing potential of antiviral therapy it seemed purposeful to supplement routine staining techniques with studies at the molecular level. Our studies demonstrated the effective detection of both the core antigen and HBV-DNA in liver tissue in children using immunocytochemical techniques and in situ hybridization, amplified with the Immunomax technique. HBcAg was detected in 26 out of 27 liver biopsies from patients with chronic hepatitis type B and with replication of the virus. HBV-DNA was detected in all study children with HBV infection and in 2 out of 5 cases of chronic hepatitis of a distinct etiology. No significant relationships could be found between the detection of tissue HBV markers on the one hand and the intensity of inflammatory lesions or severity of fibrosis on the other.

Is vacuolization of podocytes and glomerular endothelial cells of prognostic value with respect to FSGS?

Focal segmental glomerulosclerosis (FSGS) poses a major problem both from the clinical and pathomorphological viewpoint. The diagnosis of FSGS in its early stage is vital mainly because of rapidly developing therapeutic modalities. In the literature various changes are discussed which may be of prognostic value (may predict the development of FSGS). One the these changes in vacuolization, mainly of podocytes and less frequently of endothelial cells. The purpose of the present study was to analyse biopsy specimens to find out to what extent vacuolization of podocytes and endothelial cells is associated with FSGS. We compared vacuolization in minimal change disease (MCD), mesangial glomerulonephritis (GNMES) and FSGS. A similar analysis was made also with respect to those cases of MCD and GNMES, in which electron microscopy suggested an early stage of FSGS. In each group electron micrographs obtained from 15 children were analysed. Electron micrographs (12-15 on average) were obtained most frequently from 3 glomeruli. Each case electron micrographs contained 90-100 podocytes. Based upon the same electron micrographs we counted capillary lumina and defined the percentage of those which contained vacuolized endothelia (we counted the capillary lumina, and not the cells, because it is most frequently impossible to identify the border of vacuolized endothelial cells). The number of capillary cross-sections was 60 on average. The results of the analysis were compared with the clinical data. This comparison did not confirm the hypothesis of other investigators that vacuolization is of a prognostic value. Additionally we evaluated the character of vacuoles. Within podocytes the vacuoles were varying in shape. Surrounded by a single membranous layer most frequently they contained material corresponding to proteins or proteoglycans, rarely to lipids. Sometimes the vacuoles were autophagosomal, occasionally they consisted of the dilated rough endoplasmic reticulum. Vacuole-like changes within the capillary lumina were related to the swelling of endothelial cytoplasm or mesangial processes. The reasons for a discrepancy between our results and those reported by other investigators necessitate further studies.

Morphological changes in femoropopliteal venous grafts.

Morphological examination of specimens obtained from 82 venous grafts implanted into the femoropopliteal section were done. Intimal oedema, necrosis of single smooth muscle cells and partial exfoliation of the endothelium with as occurrence of parietal thrombi may lead to early graft impatency. The late cause of stenosed inner diameter below 4 mm in the venous graft was in 14,6% the hyperplasia of the smooth muscle cells and collagen and elastin fibres in the intima, whereas in 4,9% the changes were defined as graft atherosclerosis.

Morphological aspects of the aortofemoral graft thrombosis.

Sixty seven patients underwent aorto-femoral double velour grafting for aortoiliac occlusive disease and 38 patients were treated for recurrent lower limb ischemia following aortofemoral grafting. Apart from various clinical examinations the patients underwent angiography as well as the morphological investigation of specimens obtained from abdominal aortae, common femoral arteries and from grafts implanted into the aorto-femoral section. The examinations showed that diffuse intimal thickening may, in the course of atherosclerosis, convert itself into uncomplicated fibrous and further into complicated plaques impairing mainly graft outflow. The drawbacks of grafts are: 1) persistens of fibrin deposits on the luminal surface of the prosthesis which may contribute to thrombosis development, 2) appearance of foci or bands of hyalinized connective tissue causing the stiffening of the graft, 3) development of lesions resembling atheromatous changes at the anastomotic areas and, 4) anastomotic hyperplasia which may contribute to stenosis at the femoral implantation site.

Morphology of brain stem lesion and bera findings after 60Co irradiation.

Ionizing rays, utilized in radiological diagnostics and oncological therapy affect the central nervous system and may injure auditory pathways and cause hearing disturbances which vary in intensity. On the basis of a stereotactic atlas of the brain of a guinea pig, the trapezoid and geniculate bodies were identified in the skull X-ray pictures. The irradiated region was found to have 10 x 6 x 5 mm in dimensions and to be situated at the depth of 11 mm away from the animals occiput. After introductory recording of the potentials obtained from the brain stems (BERA), the stems of 60 guinea pigs were irradiated in the groups with the doses of 5, 10, 20 Gy. The hearing potentials from the brain stems were recorded on the 1st, 4th, 10th, 21st and 84th day after irradiation. The findings of measurements of hearing potentials were compared with the morphologic picture of the brain stems in the examined animals.

Application of electron microscopy and immunocytochemistry in lung cancer diagnosis.

Using bronchofiberoscopy the biopsies of '50 small cell carcinomas of the lung (SCLC) were diagnosed. The material was studied by immunocytochemical methods for cytokeratins and in routine transmission electron microscopy. The presence of bundles of cytokeratins was specific for squamous epithelium, in all histological subtypes of small cell carcinomas. The number and distribution of cytokeratins were described. In whole material we found the neuroendocrine cells in which polymorphic neuroendocrine granules were present.