Maria Elzbieta Sobaniec-Lotowska

Ultrastructure of Purkinje cell perikarya and their dendritic processes in the rat cerebellar cortex in experimental encephalopathy induced by chronic application of valproate

Long‐term intragastric administration of the antiepileptic drug sodium valproate (Vuprol ‘Polfa’) to rats for 1, 3, 6, 9 and 12 months, once daily at the effective dose of 200 mg/kg body weight showed morphological evidence of encephalopathy, manifested by numerous nonspecific changes within Purkinje cell perikarya and their dendritic processes. The first ultrastructural abnormalities appeared after 3 months. They became more severe in animals with longer survival and were most pronounced after 12 months. The changes were maintained both 1 and 3 months after drug withdrawal. Mitochondria of Purkinje cell perikarya were most severely affected. Damage to mitochondria was accompanied by disintegration and fragmentation of granular endoplasmic reticulum, dilation of channels and cisterns of Golgi apparatus, enlargement of smooth endoplasmic reticulum elements including submembranous cisterns, and accumulation of profuse lipofuscin deposits. Frequently, Purkinje cells appeared as ‘dark’ ischemic neurones, with focally damaged cellular membrane and features of disintegration. Swollen Bergmanns astrocytes were seen among damaged Purkinje cells or at the site of their loss. The general pattern of submicroscopic alterations of Purkinje cell perikarya suggested severe disorders in several intercellular biochemical extents, including inhibition of oxidative phosphorylation and abnormal protein synthesis, both of which could lead to lethal damage. Ultrastructural abnormalities within dendrites were characterized by damage to elements of smooth endoplasmic reticulum, which was considerably enlarged, with formation of large vacuolar structures situated deep in the dendroplasm. Mitochondrial lesions and alterations in cytoskeletal elements – disintegration of microtubules or even their complete loss –were also observed. The general pattern of abnormalities within the organelles and cytoskeletal elements of dendritic processes in Purkinje cells in the VPA chronic experimental model imply that there are disturbances in detoxication processes. Furthermore these changes were irreversible, as they were maintained after drug withdrawal.

Ultrastructure of hepatocyte mitochondria in nonalcoholic steatohepatitis in pediatric patients: Usefulness of electron microscopy in the diagnosis of the disease

Ultrastructure of hepatocyte mitochondria in nonalcoholic steatohepatitis in pediatric patients: usefulness of electron microscopy in the diagnosis of the disease

Ultrastructure of astrocytes in the cortex of the hippocampal gyrus and in the neocortex of the temporal lobe in experimental valproate encephalopathy and after valproate withdrawal

Summary.  The aim of the study was to analyse the astrocyte ultrastructure within the hippocampal gyre cortex and neocortex of the temporal lobe in valproate encephalopathy induced by chronic administration of an anti‐epileptic drug – sodium valproate (VPA) to rats for 1, 3, 6, 9 and 12 months, once daily intragastrically, in a dose of 200 mg/kg b.w. and after its withdrawal for 1 and 3 months. Prolonged application of VPA caused damage to protoplasmic astrocytes of the cortex regions examined, mainly in the pyramidal layer, which intensified in the later stages of the experiment, especially after 9 and 12 months. Ultrastructural alterations in astroglia during this experiment did not differ significantly between the hippocampal cortex and neocortex. The most pronounced astroglial abnormalities, concerning about 2/3 of protoplasmic astrocytes after 9 and 12 months, were characterized by considerable swelling of cells, with the presence of empty vacuolar structures in the cytoplasm, a substantial decrease in the number of gliofilaments or even their complete loss, which indicated fibrillopoietic failure of the cell, and the appearance of astrocytes showing phagocytic activity. The astrocytic changes coexisted with distinct damage to neurones and structural elements of the blood–brain barrier. One month after termination of chronic exposure to the drug, the abnormalities did not subside, whereas after 3 months features of distinct normalization could be observed in a considerable number, more than a half, of astrocytes. In valproate encephalopathy, apart from any direct effect of VPA and/or its metabolites on astrocytes, the main cause of the protoplasmic astroglial damage in the cortex of the CNS structures examined could be associated with changes in microcirculation in the cortex (vasogenic factor), leading to its ischaemia.

Pediatric non-alcoholic steatohepatitis: The first report on the ultrastructure of hepatocyte mitochondria

AIM To investigate the ultrastructure of abnormal hepatocyte mitochondria, including their cellular and hepatic zonal distribution, in bioptates in pediatric non-alcoholic steatohepatitis (NASH). METHODS Ultrastructural investigations were conducted on biopsy liver specimens obtained from 10 children (6 boys and 4 girls) aged 2-14 years with previously clinicopathologically diagnosed NASH. The disease was diagnosed if liver biopsy revealed steatosis, inflammation, ballooned hepatocytes, Mallory hyaline, or focal necrosis, varying degrees of fibrosis in the absence of clinical, serological, or histological findings of infectious liver diseases, autoimmune hepatitis, metabolic liver diseases, or celiac disease. For ultrastructural analysis, fresh small liver blocks (1 mm(3) volume) were fixed in a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer. The specimens were postfixed in osmium tetroxide, subsequently dehydrated through a graded series of ethanols and propylene oxide, and embedded in Epon 812. The material was sectioned on a Reichert ultramicrotome to obtain semithin sections, which were stained with methylene blue in sodium borate. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined using an Opton EM 900 transmission electron microscope. RESULTS Ultrastructural analysis of bioptates obtained from children with non-alcoholic steatohepatitis revealed characteristic repetitive mitochondrial abnormalities within hepatocytes; mainly mitochondrial polymorphisms such as megamitochondria, loss of mitochondrial cristae, and the presence of linear crystalline inclusions within the mitochondrial matrix of an increased electron density. The crystalline inclusions were particularly evident within megamitochondria (MMC), which seemed to be distributed randomly both within the hepatic parenchymal cell and the zones of hepatic lobule, without special variations in abundance. The inclusions appeared as bundles viewed longitudinally, or as an evenly spaced matrix in cross section, and frequently caused mitochondrial deformation. The average diameter of these linear structures was 10 nm and the average space between them 20 nm. Sometimes enlarged intramitochondrial granules were seen in their vicinity. Foamy cytoplasm of hepatocytes was found, resulting from the proliferation of smooth endoplasmic reticulum and glycogen accumulation. The perivascular space of Disse was frequently dilated, and contained transitional hepatic stellate cells, as well as mature and/or newly forming collagen fiber bundles. CONCLUSION Marked ultrastructural abnormalities observed in hepatocyte mitochondria, especially their polymorphism in the form of MMC and loss of mitochondrial cristae, accompanied by foamy cytoplasm, clearly indicate a major role of these organelles in the morphogenesis of pediatric NASH. Our findings seem to prove the high effectiveness of electron microscopy in the diagnosis of the disease.

Aberrant Distributions and Relationships Among E-cadherin, β-catenin, and Connexin 26 and 43 in Endometrioid Adenocarcinomas

During carcinogenesis, loss of intracellular cohesion is observed among cancer cells with altered expression of such adhesion molecules as E-cadherin and β-catenin, and aberrant expression and cellular location of intercellular gap junction proteins-connexins. The aim of this study was to evaluate immunohistochemically the expression and relationship between E-cadherin and β-catenin, and the connexins Cx26 and Cx43 in 86 endometrioid adenocarcinomas. The aberrant cytoplasmic translocation of the studied proteins was a predominant finding, whereas only a minority of cases showed normal, nuclear β-catenin labeling or membranous distribution of the remaining molecules. E-cadherin was positively and significantly associated with β-catenin (P=0.001, r=0.366), as was Cx26 with Cx43 (P<0.001, r=0.719), E-cadherin with Cx26 (P<0.001, r=0.413), and E-cadherin and Cx43 (P<0.001, r=0.434) in all cancers. A subgroup of endometrioid adenocarcinomas (FIGO IB+II) exclusively showed a positive significant association between the expression of β-catenin and Cx26 (P=0.038, r=0.339). In addition, there were significantly more β-catenin-positive carcinomas among superficially spreading cancers (FIGO IA) than among deeper invading neoplasms (FIGO IB+II) (P=0.056). The altered location of the studied proteins indicates impairment of their physiological functions. In particular, normal membranous distribution of E-cadherin and connexins is lost and replaced by abnormal cytoplasmic accumulation in most cancers, and thus intercellular ties are expected to be weakened and loosened as a consequence. In contrast, the lack of relationship between β-catenin and connexins, E-cadherin seems to be closely associated with the expression of Cx26 and Cx43 in endometrioid adenocarcinomas.

The relationships between hypoxia-dependent markers: HIF-1alpha, EPO and EPOR in colorectal cancer

Hypoxia triggers production of several cytoprotective proteins. Hypoxia-inducible factor 1alpha (HIF-1α) is a powerful stimulator of transcription of many genes, including erythropoietin (EPO) in hypoxia-affected cells. Recent data have also implicated signaling by EPO receptor (EPOR) as a new factor influencing tumor progression. The aim of the study was to detect by immunohistochemistry the presence of HIF-1α, EPO and EPOR in colorectal cancer (CRC) in reference to clinicopathological variables. We found the presence of the studied proteins in specimens of all 125 CRC patients which is suggestive of the occurrence of hypoxia in colorectal cancer tissues. The expression of HIF-1α correlated significantly with the presence of EPO and EPOR in all samples (P < 0.001, r = 0.549 and P < 0.001, r = 0.536, respectively). Significant correlations (from P < 0.024 to P < 0.001) were found in the analyses of CRC subgroups such as histopathological type tumor, tumor grade, tumor stage and patients with lymph nodes metastases. The same high significant correlations (P < 0.001) were observed in group of sex, age and tumor location. However, the values of the correlation coefficients (r) which usually ranged from 0.5 to 0.6 suggest the existence of independent or concurrent mechanism stimulating generation of these proteins in colorectal cancer.

The role of Kupffer cells in the morphogenesis of nonalcoholic steatohepatitis – ultrastructural findings. The first report in pediatric patients

Abstract Objective. Until now studies concerning the involvement of hepatic nonparenchymal cells (NPCs), particularly Kupffer cells/macrophages (KCs/MPs), in the pathogenesis of human nonalcoholic steatohepatitis (NASH) have been limited to adult patients; there are no similar reports referring to children. This study aimed to explore, based on ultrastructural analysis, the role of KCs/MPs in the morphogenesis of nonalcoholic steatohepatitis (NASH) in children. Material and methods. Ultrastructural investigations of KCs were conducted on liver bioptates obtained from 10 children, aged 2–14 years, with clinicopathologically diagnosed NASH. Bioptatic material was fixed in solution of paraformaldehyde and glutaraldehyde in cacodylate buffer, routinely processed for transmission-electron microscopic analysis and examined using an Opton EM microscope. Results. The current ultrastructural study revealed within the hepatic sinusoids the presence of numerous enlarged KCs with increased phagocytic activity, which reduced or blocked vascular lumen. Interestingly, the activated KCs not only contained primary and secondary lysosomes, altered mitochondria, and well-developed Golgi apparatus, but also absorbed fragments of erythrocytes. Such macrophages were frequently seen very close to the transformed hepatic stellate cells (T-HSCs) and progenitor/oval cells. Intensive fibrosis was observed in the vicinity of activated KCs/MPs. Bundles of collagen fibers were seen directly adhering to these cells and to other NPCs, especially T-HSCs. Conclusions. KCs are involved in the morphogenesis and development of pediatric NASH. Engulfment of erythrocytes by hepatic macrophages may lead to the accumulation of iron derived from hemoglobin in liver and play a role in triggering the generation of oxidative stress in the disease course.

Hepatotoxicity caused by montelukast in a paediatric patient

Montelukast is a selective and competitive cysteinyl leukotriene receptor antagonist (CystLTRA) which is increasingly used for the treatment of allergic asthma. Recently, hepatotoxicity has been reported with this drug in adult patients, but only one letter to the editor has reported a case of probable montelukast-induced hepatotoxicity in a child. We present a case of a 3.5-year-old boy, receiving treatment with montelukast, who developed hepatocellular injury. The exclusion of other causes of increased activity of aminotransferases (viral, metabolic, autoimmune), improvement after dechallenge, the morphological findings and previous reports of comparable cases support the diagnosis of montelukast-induced liver injury in this boy. Physicians should strictly analyse indications for this drug and be aware of potential drug-induced liver disease caused by this agent. Therefore, the periodical assessment of aminotransferases should be recommended during treatment with this leukotriene modifier.

Hypoxia related growth factors and p53 in preoperative sera from patients with colorectal cancer - evaluation of the prognostic significance of these agents

Abstract Background: Insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) belong to a group of hypoxia related proteins. IGF-I induces expression of VEGF and decomposes wild type p53 in cancer cell lines. The goal of our study was to evaluate serum IGF-I, VEGF and p53 with respect to overall and disease free survival of patients with colorectal cancer (CRC) patients compared with healthy volunteers. Methods: Preoperative blood samples from 125 patients with CRC and 16 healthy volunteers were examined using ELISA for serum IGF-I, p53 and VEGF concentrations. Results: Concentrations of p53 and VEGF were significantly higher in CRC patients than in controls (p<0.0006 and p<0.0001, respectively). IGF-I was not statistically different between both groups. Serum IGF-I showed negative correlation with p53 in CRC patients (p<0.04, r=−0.193). IGF-I and VEGF showed negative correlation in poorly differentiated cancers (G3) (p<0.03, r=−0.339). Patients with VEGF concentrations that were above average for the cancer population survived for a shorter period of time (p=0.065 in evaluation of overall survival and 0.071 in estimation of disease-free survival during a 3-year follow-up) compared with patients with serum VEGF lower than the highest values seen in controls. Conclusions: Comparisons between serum IGF-I and p53 appear to confirm the metabolism of p53 by IGF-I. Serum VEGF showed prognostic significance in our study. Serum concentrations of IGF-I and VEGF did not show positive correlation, as expected due to IGF-I induction of VEGF in malignant colon cell lines. Clin Chem Lab Med 2009;47:1439–45.

A transmission electron microscopic study of microglia/macrophages in the hippocampal cortex and neocortex following chronic exposure to valproate

In chronic administration of sodium valproate to rats, significant disorders of structural integrity of the hippocampal gyrus and the neocortex of the temporal lobe, observed in the last two stages of the experiment (after 9 and 12 months), coexisted with increased number of microglial cells and, especially after 12 months, with intense phagocytic activity within these cells. At the ultrastructural level, phagocyte microglial cells were hypertrophied with several broadened processes. Their cytoplasm contained rich lysosomal apparatus, numerous lipofuscin‐like structures, lipid droplets and multilaminated bodies. The nuclei of these cells were characteristic oval or round and sometimes triangle in shape with dense and highly clumped heterochromatin, distinctly accumulated under nuclear envelope, and sparse euchromatin. Microglia/macrophages were frequently present in a close vicinity of changed neuronal somata and also close to the altered elements of the neuropil pyramidal layer of the cortex. Microglial response may, together with abnormalities in neurones, astroglia and blood–brain barrier, play a significant role in the development of experimental valproate encephalopathy.