The Ultrastructure of Hepatic Stellate Cell-Macrophage Intercellular Crosstalk as a New Morphological Insight into Phenomenon of Fibrogenesis in Pediatric Autoimmune Hepatitis.

The aim of the study was the pioneering retrospective ultrastructural evaluation of respective forms of hepatic stellate cells (HSCs) and analysis of their crosstalk with other adjacent nonparenchymal cells (NPCs), especially Kupffer cells/macrophages (KCs/MPs), in pediatric autoimmune hepatitis (AIH). METHODS: Ultrastructural assessment of the HSC population and NPCs was performed in transmission electron microscopy (TEM) using pretreatment liver biopsies from 25 children (8 boys and 17 girls) aged 4-17 with clinic-pathologically diagnosed untreated AIH. RESULTS: Submicroscopic evaluation allowed easy identification of numerous HSCs in the form of transitory cells, i.e., T-HSCs, accompanied by signs of fibrosis. T-HSCs included cells with features of activation initiation (iHSCs) and activation perpetuation (pHSCs), indicating high HSC activation plasticity. The pHSCs were markedly elongated and mainly showed a distinct loss of lipid cytoplasmic material, expanded and dilated channels of granular endoplasmic reticulum, and linear bundles of microfilaments beneath the cell membrane. They were surrounded by usually mature collagen fibers. Frequently activated KCs/MPs adhered directly to T-HSCs. Between them, tight intercellular junctions were formed by means of point desmosomes. CONCLUSIONS: Our qualitative TEM observations indicate a key role of T-HSCs in liver fibrogenesis in pediatric AIH, with the essential involvement of activated KCs/MPs that directly adhere to them. Tight intercellular junctions, being the ultrastructural exponent of the specific cellular mechanisms of the crosstalk between NPCs, can play a vital role in hepatic collagen fibroplasia. A better understanding of HSC population morphology at the ultrastructural level in AIH seems important not only to improve the disease morphological diagnostics but to also provide new insights into therapeutic interventions for the phenomenon of liver fibrogenesis.

Influence of Topiramate on the Synaptic Endings of the Temporal Lobe Neocortex in an Experimental Model of Hyperthermia-Induced Seizures: An Ultrastructural Study.

The objective of this pioneering study was to assess potentially neuroprotective properties of topiramate (TPM), a broad spectrum and newer-generation antiepileptic used against damage to synaptic endings of the temporal lobe neocortex in experimental hyperthermia-induced seizures (HS). TPM (80 mg/kg b.m.) was administered in young male Wistar rats with an intragastric tube before and immediately after HS. Specimens (1 mm3) collected from the neocortex, fixed via transcardial perfusion with paraformaldehyde and glutaraldehyde solution, were routinely processed for transmission-electron microscopic study, i.e., for descriptive and morphometric analysis. The ultrastructure of neocortical neuropil components affected by hyperthermic stress showed distinct swelling of pre and post-synaptic axodendritic and axospinal endings, including total disintegration. Mitochondria were markedly damaged in synaptic structures. Axoplasm of presynaptic boutons contained a decreased number of synaptic vesicles. Synaptic junctions showed active zone-shortening. Preventive administration of TPM before HS induction demonstrated neuroprotective effects against synaptic damage in approximately 1/4 of these structures. Interestingly, beneficial effects on synapsis morphology were more common in perivascular zones close to well-preserved capillaries. They were demonstrated by smaller swelling of both presynaptic and postsynaptic parts, well-preserved mitochondria, an increased number and regular distribution of synaptic vesicles within axoplasm, and a significantly increased synaptic active zones. However, topiramate used directly after HS was ineffective in the prevention of hyperthermia-evoked synaptic injury. Our findings support the hypothesis that topiramate applied before HS can protect some neocortical synapses via the vascular factor by enhancing blood-brain barrier components and improving the blood supply of gray matter in the temporal lobe, which may be significant in febrile seizure-prevention in children.

Ultrastructural Profile Combined with Immunohistochemistry of a Hepatic Progenitor Cell Line in Pediatric Autoimmune Hepatitis: New Insights into the Morphological Pattern of the Disease.

Considering that the heterogenic population of a hepatic progenitor cell line (HPCL) can play a vital role in autoimmune hepatitis (AIH), we decided to conduct pioneering retrospective evaluation of these cells in pediatric AIH by means of transmission electron microscopy (TEM). The aim of the study was to assess the ultrastructure of the HPCL in children with untreated AIH. Ultrastructural analysis of the HPCL population, preceded by immunohistochemical staining for cytokeratin 7 (CK7), was performed using pretreatment liver biopsies from 23 children with clinicopathologically diagnosed AIH. Immunohistochemical assessment for CK7 allowed detection of proliferating immature epithelial cells differentiating towards periportal and intralobular intermediate hepatocytes without marked formation of ductular reactions in AIH children. Using TEM, we distinguished three morphological types of HPCs: I-the most undifferentiated progenitor cells; III-intermediate hepatocyte-like cells; II-intermediate bile duct cells. Most frequent were the cells differentiating towards hepatocytes, most rare-those differentiating towards cholangiocytes. The results indicate that an HPCL may be an important source of hepatocyte regeneration. Ultrastructural analyses of the HPCL population, combined with immunohistochemistry for CK7, might be a useful tool to evaluate liver cell regeneration, including fibrogenesis, and may help better understand the morphological pattern of the disease, in pediatric AIH. Frequent appearance of an HPCL in the vicinity of fibrotic foci, often accompanied by hyperactive Kupffer cells and transitional hepatic stellate cells, may indicate their significant involvement in liver fibrogenesis.

Effects of topiramate on the ultrastructure of synaptic endings in the hippocampal CA1 and CA3 sectors in the rat experimental model of febrile seizures: the first electron microscopy report.

The present study aimed at exploring a potentially neuroprotective effect of topiramate (TPM), one of the most commonly used newer-generation, broad-spectrum, antiepileptic drugs against ultrastructural damage of hippocampal synaptic endings in the experimental model of febrile seizures (FS). The study used male young Wistar rats aged 22-30 days, divided into three experimental groups and the control group. Brain maturity in such animals corresponds to that of 1- or 2-year-old children. Hyperthermic stress was evoked by placing animals in a 45°C water bath for four consecutive days. TPM at a dose of 80 mg/kg b.m. was administered with an intragastric tube before and immediately after FS. Specimens (1 mm3) collected from the hippocampal CA1 and CA3 sectors, fixed via transcardial perfusion with a solution of paraformaldehyde and glutaraldehyde, were routinely processed for transmission-electron microscopic analysis. Advanced ultrastructural changes induced by hyperthermic stress were manifested by distinct swelling of hippocampal pre- and post-synaptic axodendritic and axospinal endings, including their vacuolization and disintegration. The axoplasm of the presynaptic boutons contained a markedly decreased number of synaptic vesicles and their abnormal accumulation in the active synaptic region. The synaptic junctions showed a dilated synaptic cleft and a decreased synaptic active zone. TPM used directly after FS was ineffective in the prevention of hyperthermia-induced injury of synaptic endings in hippocampal CA1 and CA3 sectors. However, "prophylactic" administration of TPM, prior to FS induction, demonstrated a neuroprotective effect against synaptic damage in approximately 25% of the synaptic endings in the hippocampal sectors, more frequently located in perivascular zones. It was manifested by smaller oedema of both presynaptic and postsynaptic parts, containing well-preserved mitochondria, increased number and regular distribution of synaptic vesicles within the axoplasm, and increased synaptic active zone. Our current and previous findings suggest that TPM administered "prophylactically", before FS, could exert a favourable effect on some synapses, indirectly, via the vascular factor, i.e. protecting blood-brain barrier components and through better blood supply of the hippocampal CA1 and CA3 sectors, which may have practical implications.

Epilepsy identification based on EEG signal using RQA method.

PURPOSE: Epilepsy is one of the most common neurological diseases and its cause is not unequivocal. Thus, additional methods and searches that may help to diagnose the disease are used in the clinical practice. In this study, we tested the possibility of using the Recurrence Quantification Analysis (RQA) method to identify epilepsy and present the analysis of EEG signals of healthy patients and epileptic patients by the RQA method. MATERIALS/METHODS: The recordings of signals belong to 13 patients, which were divided into 2 groups: Group A (5 epileptic patients) and Group B (8 healthy patients). In this study Fp1, Fp2, T3 and T4 electrodes were considered in the analysis using the RQA method. RESULTS: It is difficult to explore the dynamics of signals by linear methods. In this study, another way of analyzing the dynamics of signals by the RQA method is presented. The RQA method revealed differences in the dynamics between the epileptic and normal signals, which seemed important in an organoleptic way. It was found that the dynamics of epileptic signals is more periodic than normal signals. To confirm the correctness of the statements issued for the RQA data the Principal Component Analysis mapping was applied. This method showed more clearly the differences in the dynamics of both signals. CONCLUSIONS: The RQA method can be used to identify nonlinear biomedical signals such as EEG signals.

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.

Ultrastructural Characteristics of Rat Hepatic Oval Cells and Their Intercellular Contacts in the Model of Biliary Fibrosis: New Insights into Experimental Liver Fibrogenesis.

PURPOSE: Recently, it has been emphasized that hepatic progenitor/oval cells (HPCs) are significantly involved in liver fibrogenesis. We evaluated the multipotential population of HPCs by transmission electron microscope (TEM), including relations with adherent hepatic nonparenchymal cells (NPCs) in rats with biliary fibrosis induced by bile duct ligation (BDL). METHODS: The study used 6-week-old Wistar Crl: WI(Han) rats after BDL for 1, 6, and 8 weeks. RESULTS: Current ultrastructural analysis showed considerable proliferation of HPCs in experimental intensive biliary fibrosis. HPCs formed proliferating bile ductules and were scattered in periportal connective tissue. We distinguished 4 main types of HPCs: 0, I, II (bile duct-like cells; most common), and III (hepatocyte-like cells). We observed, very seldom presented in literature, cellular interactions between HPCs and adjacent NPCs, especially commonly found transitional hepatic stellate cells (T-HSCs) and Kupffer cells/macrophages. We showed the phenomenon of penetration of the basement membrane of proliferating bile ductules by cytoplasmic processes sent by T-HSCs and the formation of direct cell-cell contact with ductular epithelial cells related to HPCs. CONCLUSIONS: HPC proliferation induced by BDL evidently promotes portal fibrogenesis. Better understanding of the complex cellular interactions between HPCs and adjacent NPCs, especially T-HSCs, may help develop antifibrotic therapies in the future.

Unfavorable effect of levetiracetam on cultured hippocampal neurons after hyperthermic injury.

BACKGROUND: The aim of this study was to examine the viability of neurons and the putative neuroprotective effects of second-generation antiepileptic drug, levetiracetam (LEV), on cultured hippocampal neurons injured by hyperthermia. METHODS: Primary cultures of rat's hippocampal neurons at 7day in vitro (DIV) were incubated in the presence or absence of LEV in varied concentrations under hyperthermic conditions. Cultures were heated in a temperature of 40°C for 24h or in a temperature of 41°C for 6h. Flow cytometry with Annexin V/PI staining as well as fluorescent microscopy assay were used for counting and establishing neurons as viable, necrotic or apoptotic. Additionally, the release of lactate dehydrogenase (LDH) to the culture medium, as a marker of cell death, was evaluated. Assessment was performed after 9DIV and 10 DIV. RESULTS: Incubation of hippocampal cultures in hyperthermic conditions resulted in statistically significant increase in the number of injured neurons when compared with non-heated control cultures. Intensity of neuronal destruction was dependent on temperature-value. When incubation temperature 40°C was used, over 80% of the population of neurons remained viable after 10 DIV. Under higher temperature 41°C, only less than 60% of neurons were viable after 10 DIV. Both apoptotic and necrotic pathways of neuronal death induced by hyperthermia were confirmed by Annexin V/PI staining. CONCLUSIONS: LEV showed no neuroprotective effects in the current model of hyperthermia in vitro. Moreover, drug, especially when used in higher concentrations, exerted unfavorable intensification of aponecrosis of cultured hippocampal neurons.

Study of the protective effects of nootropic agents against neuronal damage induced by amyloid-beta (fragment 25-35) in cultured hippocampal neurons.

BACKGROUND: Alzheimer's disease (AD) is a common neurodegenerative disorder, in which progressive neuron loss, mainly in the hippocampus, is observed. The critical events in the pathogenesis of AD are associated with accumulation of ß-amyloid (Aß) peptides in the brain. Deposits of Aß initiate a neurotoxic "cascade" leading to apoptotic death of neurons. Aim of this study was to assess a putative neuroprotective effects of two nootropic drugs: piracetam (PIR) and levetiracetam (LEV) on Aß-injured hippocampal neurons in culture. METHODS: Primary cultures of rat's hippocampal neurons at 7 day in vitro were exposed to Aß(25-35) in the presence or absence of nootropics in varied concentrations. Flow cytometry with Annexin V/PI staining was used for counting and establishing neurons as viable, necrotic or apoptotic. Additionally, release of lactate dehydrogenase (LDH) to the culture medium, as a marker of cell death, was evaluated. RESULTS: Aß(25-35) caused concentration-dependent death of about one third number of hippocampal neurons, mainly through an apoptotic pathway. In drugs-containing cultures, number of neurons injured with 20 µM Aß(25-35) was about one-third lesser for PIR and almost two-fold lesser for LEV. When 40 µM Aß(25-35) was used, only LEV exerted beneficial neuroprotective action, while PIR was ineffective. CONCLUSIONS: Our results suggest the protective potential of both studied nootropics against Aß-induced death of cultured hippocampal neurons with more powerful neuroprotective effects of LEV.

Study of the protective effect of calcium channel blockers against neuronal damage induced by glutamate in cultured hippocampal neurons.

BACKGROUND: The aim of this study was to examine the putative protective effect of calcium channel blockers on hippocampal neurons in the experimental model of excitotoxic damage. METHODS: Seven-day old primary dissociated cultures of rat hippocampal neural cells containing one of the following calcium channel blockers: cinnarizine, flunarizine or nimodipine were exposed to glutamate-induced injury. Quantitative assessments of neuronal injury were accomplished by measuring lactate dehydrogenase (LDH) activity in the media 24 h after exposure to glutamate and by counting and establishing the apoptotic and necrotic cells in flow cytometry with Annexin V-FITC/PI staining. RESULTS: In our experiment, glutamate induced a 339% elevation of apoptotic cells and a 289% increase of necrotic cells in hippocampal neurons as compared to control cultures without drugs. In cultures containing flunarizine, glutamate-induced cell apoptosis was suppressed by 62% while necrosis showed no significant alternation. Cinnarizine exerted no anti-apoptotic effects on glutamate-injured cultured hippocampal neurons, while nimodipine intensified the apoptotic pathway of cell death and promoted an increase in the number of apoptotic neurons by 26%. When cinnarizine or nimodipine were used, the percentage of necrotic cells was significantly lower when compared with glutamate-injured cultures and it amounted to 44% and 24% for cinnarizine and nimodipine, respectively. CONCLUSIONS: The obtained results suggest the beneficial anti-apoptotic potential of flunarizine and the anti-necrotic potential of cinnarizine against glutamate-induced death of cultured hippocampal neurons. Nimodipine can protect neurons against necrosis, but has an intensified adverse pro-apoptotic effect on cultured neurons in the experimental model of excitotoxic injury.

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.