Liver sinusoidal endothelial cells in morphogenesis of pediatric autoimmune hepatitis. Ultrastructural characteristics - a novel report.

The pathogenesis of autoimmune hepatitis (AIH) is poorly understood. Up to now, little is known of the involvement of liver sinusoidal endothelial cells (LSECs), accounting for approximately 40% of nonparenchymal hepatic cells, in AIH morphogenesis in pediatric patients. The study objective was ultrastructural analysis of LSECs from pretreatment biopsies of 19 children, aged 4-17 years (14 girls), with clinically and histologically diagnosed AIH. Our study is the first to describe alterations in LSECs, from swelling to necrosis, demonstrating their important role in the morphogenesis and progression of pediatric AIH. Frequently damage to LSECs coexisted with significantly activated Kupffer cells, fibrogenesis and fibrosis, but not cirrhosis, accompanied by the appearance of transitional hepatic stellate cells. Interestingly, even though in half of the AIH children the sinusoidal vessels were found to undergo transformation of discontinuous into continuous endothelium showing features of defenestration, the true basement membrane did not form underneath. The fact that the basement membrane is not formed, even when LSECs are markedly damaged, may seem to indicate some regenerative capacities of these cells and lesion reversibility.

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.

Ultrastructural study of hippocampal cortex neurons in an experimental model of valproate encephalopathy.

Valproate (VPA) is a widely used antiepileptic drug. A serious neurological-outcome defined as valproate encephalopathy (VE) may rarely occur during VPA therapy. Structural abnormalities within neurons are postulated as one of the reasons for VE. The aim of this study was to assess the ultrastructure of neurons in the hippocampal cortex during the course of chronic application of VPA to rats. VPA was chronically administered to rats, intragastrically, once daily at a dose of 200 mg/kg b.w. for 1, 3, 6, 9 and 12 months. The samples of hippocampal cortex, after routine laboratory preparation, were examined by electron microscopy. The drug induced pronounced ultrastructural changes in the population of pyramidal neurons within the hippocampal cortex after 9 and 12 months of VPA administration. The most expressed abnormalities were observed within the mitochondria and manifested by fragmentation of crests and almost complete disappearance of intramitochondrial granules. Mitochondria of numerous neurons resembled large vacuolar structures. Widening, shortening and irregular distribution of rough endoplasmic reticulum was also found. A characteristic feature of damaged neurocytes in the last two phases of the experiment was the disintegration of nuclear chromatin and the presence of numerous lipofuscin deposits within hyaloplasm. These cells assumed the look of "dark neurons" and presented the ultrastructural features of apoptosis and necrosis. Our results indicate that long-term VPA administration to rats leads to aponecrosis of hippocampal neurons.

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 neuroprotective effect of topiramate on the ultrastructure of pyramidal neurons of the hippocampal CA1 and CA3 sectors in an experimental model of febrile seizures in rats.

The objective of the current ultrastructural study was to explore the potentiality of the neuroprotective effect of TPM against damage of pyramidal neurons in the hippocampal CA1 and CA3 sectors in an experimental model of febrile seizures (FS) in rats. The FS group exhibited variously pronounced submicroscopic lesions of the neuronal perikarya, including total cell disintegration. Advanced changes induced by hyperthermic stress were manifested by marked degenerative abnormalities, such as substantial swelling of the mitochondria, dilation, degranulation and disintegration of the granular endoplasmic reticulum, and vacuolar changes in the Golgi complex. The most substantially damaged pyramidal neurons showed features of aponecrosis (so-called "dark neurons"), resulting in a marked neuronal loss in the explored areas of the hippocampal cortex. The neurodegenerative changes were accompanied by distinct damage to the blood-brain barrier components. The administration of topiramate at a dose of 80/kg b.m. prior to the induction of hyperthermic stress (as prevention against febrile seizures) caused a substantial neuroprotective action - the drug efficiently lightened the neuronal damage, basically reduced cell aponecrosis and enhanced cell viability. However, TPM applied directly after FS induction did not exert any distinct neuroprotective effect on the perikarya of pyramidal neurons in the hippocampal cortex.

Chronic non-cholestatic liver disease is not associated with an increased fracture rate in children.

Chronic liver disease in adults is a risk factor of osteoporosis, but little is known about risk of fractures in children with non-cholestatic liver disease. The aim of this study was to investigate associations among the severity of liver fibrosis, bone mass and low-energy fractures in children. History of fractures, anthropometry, and bone mass and size were examined in 39 Caucasian children (25 boys, 14 girls) aged 7.1-18 years (mean 11.9 ± 3.1) with chronic hepatitis B and liver fibrosis evidenced by liver biopsy. Severity of liver fibrosis was based on histological classification according to the method of Batts and Ludwig (mild, 1-2 scores; advanced, 3 scores) and Ishak (1-3 and 4-5 scores, respectively). Bone mineral content (BMC), density (BMD) and body composition were determined in the total body and lumbar spine using dual energy X-ray absorptiometry. Seven subjects (4 girls, 3 boys; 18% of the sample) had low BMD in the total body and lumbar spine region (Z-scores below -2.0). No associations were found among BMC, BMD, bone size and the severity of liver fibrosis. Nine boys (36% of all boys) and one girl reported repeated fractures (forearm, wrist, tibia, ankle, humerus), showing trends similar to the prevalence in general population. Fractures were neither associated with lower BMD/BMC nor with scores of liver fibrosis. Deficits in BMD in children with chronic hepatitis B are not associated with the severity of liver fibrosis. This study suggests that non-cholestatic liver disease does not increase the risk of low-energy fractures during growth. From the practical perspective, however, children with chronic liver disease should be screened for history and clinical risk factors for fractures rather than referred to bone density testing.

Electron microscopic alterations in intermediate hepatocyte-like cells in children with chronic hepatitis B: the first report in pediatric patients.

OBJECTIVE: The study objective was an in-depth ultrastructural analysis of intermediate hepatocyte-like cells (IHCs), constituting a subpopulation of liver progenitor/oval cells, in children with chronic hepatitis B viral (HBV) infection. METHODS: Ultrastructural investigations were conducted on liver biopsy material, fixed in a solution of 2.5% glutaraldehyde, 2% paraformaldehyde, and 0.1 mol/l cacodylate buffer, obtained from 40 children, aged 3-16 years, with chronic hepatitis B. RESULTS: Transmission-electron microscopic analysis of liver progenitor/oval cells showed, apart from a morphologically unchanged population of oval cells, the presence of IHCs displaying variously pronounced ultrastructural changes, including degeneration. Interesting was that damaged IHCs were mainly observed in patients with a coexisting advanced liver fibrosis, where they frequently adhered to bundles of collagen fibers. Submicroscopic abnormalities in these cells referred mainly to mitochondria and granular endoplasmic reticulum. The most pronounced mitochondrial alterations observed in degenerating IHCs in the course of chronic HBV infection were characterized by distinct swelling, loss of mitochondrial crests, and the presence of myelin structures within the matrix. In granular endoplasmic reticulum, shortening and segmental degranulation of the reticulum were observed. The above changes were accompanied by the appearance of primitive phagosome-like structures with absorbed biliary pigment. In the vicinity of altered IHCs, transitional hepatic stellate cells could be found. CONCLUSION: Our study seems to suggest that chronic HBV infection, lasting from childhood and coexisting with intensive fibrosis may, with the involvement of other carcinogenic factors, promote degenerating IHCs towards neoplastic transformation in adulthood.

Ultrastructural features of astrocytes in the cortex of the hippocampal gyrus and in the neocortex of the temporal lobe in an experimental model of febrile seizures and with the use of topiramate.

The objective of the current study was ultrastructural assessment of astroglia in specimens of the hippocampal cortex and neocortex of the temporal lobe in our own experimental model of febrile seizures (FS) in rats, as well as the analysis of the influence of a structurally novel broad spectrum anticonvulsant, topiramate (TPM), upon these cells in the CNS regions studied. The current study was inspired by some interesting literature reports on the in vitro investigation into the biological effects of TPM in primary cultures of rat cortical astrocytes and by the lack of data concerning astroglial morphology in vivo in an experimental model with this antiepileptic. In the FS group, the most pronounced changes in the study cell population referred to protoplasmic astroglia and were observed in approximately 3/4 of these cells. The abnormalities were similarly expressed in the two CNS regions studied, in terms of both quantity and quality. They were characterized by considerable swelling and degenerative changes, both in astrocytic perikarya and their processes. Changes were visible in the elements of the granular endoplasmic reticulum and mitochondria, which had a condensed configuration. In the group receiving topiramate directly after the induction of FS, submicroscopic changes in protoplasmic astrocytes were similarly expressed as in the FS group. However, in the group receiving the drug prior to the induction of FS its protective action was observed on the morphology of approximately 1/3 of the population of the protoplasmic astroglial cells. The remaining protoplasmic astrocytes still showed features of considerable or moderately pronounced injury. The beneficial effect of TPM on the ultrastructure of part of the population of the protoplasmic astroglia in the group in which the drug was applied prior to the induction of FS can be explained, among others, by a protective effect of the blood-brain barrier enhanced by the drug administration, as indicated by our earlier findings.

Ultrastructure of the blood-brain barrier of the gyrus hippocampal cortex in an experimental model of febrile seizures and with the use of a new generation antiepileptic drug--topiramate.

The ultrastructure of the blood-brain barrier (BBB) of the gyrus hippocampal cortex in an experimental model of febrile seizures in rats and the effect of a new generation antiepileptic drug, topiramate, on the morphological status of this barrier were investigated. Advanced changes indicating a substantial increase in BBB permeability were observed in the animals with induced febrile seizures (FS), with approximately 2/3 of capillaries and perivascular astroglial processes being affected. Almost total occlusion of the capillary lumen was frequently seen, caused by damaged endothelial lining and by external pressure from markedly swollen perivascular astrocytic processes. Mitochondrial changes predominated among the abnormalities found in endoplasmic organelles of endothelial cells. Lesions in the BBB coexisted with damage to pyramidal neurons, mainly with features of aponecrosis ("dark neurons"). The study on topiramate seems to demonstrate its protective action on the BBB components of the ammonal cortex in the group receiving the drug as prevention, i.e. against febrile seizures. It was found to prevent marked BBB damage in over half of the capillaries. However, the application of topiramate directly after FS induction had no distinct beneficial effect on the structural BBB components.

Mast cells in neoangiogenesis.

Mast cells (MCs) always accompany connective tissue and are located in the proximity of lymphatic and blood vessels and nerve fibers. They are round or oval mononuclear cells with a diameter of 4-20 microm containing in their cytoplasm specific exocrine granules (storing neutral proteases) enclosed by a single membrane, whose presence is regarded as an index of the MC's static state. In view of their wide distribution in the organism, they play various roles in, for example, type I hypersensitivity reactions, chronic inflammatory processes, tissue reconstruction and wound healing, and pathological pulmonary fibrosis. They also play a role in angiogenesis, both in normal conditions during tissue regeneration and in pathological neoplastic states. The microcirculation provides building and nutritional substances to cancer cells and enables cancer spread via the blood. On the other hand, a tumor with good vascularization is more prone to penetration by cytostatics, which is why angiogenesis is a very important process in the course of neoplastic disease. Many authors indicate a close association between mast cells and angiogenesis. Some substances contained in the cytoplasm of these cells are potent stimulators of angiogenesis (tryptase, heparin), while others may inhibit it (protamine, platelet factor 4), and this conditions cancer growth and the development of the metastatic process. It is not known, however, what interactions occur between stimulants and inhibitors and what the proportional involvement of particular mediators in the formation of new vessels is.

Ultrastructural study of cerebellar dentate nucleus astrocytes in chronic experimental model with valproate.

The current study focuses on the morphogenesis of changes in the cerebellum dentate nucleus in the course of experimental valproate encephalopathy. Valproate - a broad spectrum antiepileptic and antipsychotic drug - chronically used in rats, intragastrically, once daily at a dose of 200 mg/kg b. w. for 1, 3, 6, 9 and 12 months, induced pronounced ultrastructural changes in the population of glial cells and nerve cells of the dentate nucleus of the cerebellum in the last two phases of the experiment. Astrocytic and neuronal lesions coexisted with a considerable damage to the elements of the blood-brain barrier of the cerebellar structure examined. The changes affected mainly the population of protoplasmic astrocytes lying loosely in a neuropile as well as astrocytes adhering to damaged large multipolar neurons. Focal proliferation of astrocytes was observed. Abnormal astrocytes showed marked swelling expressed by significantly decreased electron density of the cytoplasm that contained almost empty vacuolar structures and by a considerably reduced number of intracellular organelles. It was accompanied by dilation of endoplasmic reticular channels, loss of fibrillopoietic capacity of the cell and features of autophagocytosis. It should be assumed that the essential cause of protoplasmic astroglial damage of the cerebellar dentate nucleus could be associated, apart from the direct effect of valproate and/or its metabolites on these cells, with changes in structural elements of the blood-brain barrier of this CNS region.

Serum fibrosis markers as predictors of an antifibrotic effect of interferon alfa in children with chronic hepatitis B.

OBJECTIVE: Interferon alfa (IFN-alpha) may retard hepatic fibrogenesis in adults with chronic hepatitis C. We evaluated prospectively four selected serum fibrosis markers before, immediately after and 12 months after IFN treatment of children with chronic hepatitis B (CHB). METHODS: Forty-seven children (mean age 8 years, range 4-16) with CHB underwent IFN-alpha treatment (3 MU t.i.w.) for 5 months. Fibrosis and inflammation were assessed blindly before and 12 months after the end of treatment. Serum laminin-2, collagen IV, MMP-2 and MMP-9/TIMP-1 complex were determined using automated assays. RESULTS: Twelve months after treatment had been discontinued levels of laminin-2, collagen IV and MMP-2 were decreased, and serum MMP-9/TIMP-1 complex was increased. Levels did not differ between sustained responders (42.5%) and non-responders. Similarly, fibrosis did not progress in both groups, whereas histological inflammation improved only in responders. CONCLUSIONS: A 5 month IFN-alpha treatment has no marked effect on histological liver fibrosis in children with CHB, irrespective of virological response. The evolution of serum fibrosis markers suggests they may be more sensitive to detect minor antifibrotic effects than semiquantitative follow-up histology.

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.

Morphometry of synaptophysin immunoreactive ganglion cells in Auerbach plexus in patients with colorectal cancer. Is this a new prognostic factor?

The aim of our study was to estimate morphometric parameters of synaptophysin (Syn-38) immunoreactive ganglion cells in colorectal cancer (within and at various distances from neoplastic infiltration) in postoperative material from 60 patients. We analysed the intensity of Syn-38 expression in Auerbach ganglion neurones, mean number of these cells in the ganglion, and their longitudinal and transverse diameters. The results showed a statistically significant reduction in the number of neurones in intramural ganglia of the large intestine located in neoplastic infiltration and in its close proximity. The size of ganglion cells was directly proportional to the distance from cancer infiltration and inversely proportional to Syn-38 content, which may be explained by degenerative changes and dysfunction of these cells. This correlation was significant in the case of cells with the cytoplasmatic Syn-38 immunoreactivity pattern, but did not refer to the cells with perimembranous pattern, which seemed to be undamaged. Morphometric analysis of synaptophysin immunoreactive ganglion cells in Auerbach plexus in colorectal cancer may be a new useful marker for the description of changes in the intestinal nervous system as well as a prognostic factor for colorectal cancer.

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.

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.

Ultrastructure of synaptic junctions in the cerebellar cortex in experimental valproate encephalopathy and after terminating chronic application of the antiepileptic.

The aim of the study was to analyse in TEM the evolution of changes in structural elements of synaptic junctions of the cerebellar cortex in rats in valproate encephalopathy induced by chronic 12-month administration of sodium valproate - VPA (once daily intragastrically, in a dose of 200 mg/kg b.w.) and after withdrawal of this antiepileptic for 1 and 3 months. After 9 and 12 months of the experiment, synaptic endings of both the symmetrical and asymmetrical synapses in the neuropil of the cerebellar cortex, especially in the molecular layer, showed signs of severe damage (mainly swelling) and even disintegration. They were mostly observed in axodendritic endings and axospinal endings on the dendritic spines of Purkinje cells, being manifested in the presence of large vacuolar structures, electron lucent areas and swollen mitochondria within the cytoplasm. A reduced number of axonal synaptic vesicles (with more type F vesicles preserved) could be seen. One and 3 months after the end of chronic application of VPA, the synaptic junctions did not show morphological exponents of the repair processes. The alterations observed in the synapticjunctions of the cerebellar cortex may suggest disorders in neurotransmission processes, such as exhaustion and damage caused by ischaemia due to damage to the blood-brain barrier induced by VPA and/or its toxic metabolites.