Physiology and Morphological Correlates of Excitatory Transmission are Preserved in Glutamine Transporter SN1-Depleted Mouse Frontal Cortex.

Glutamine is an astroglia-derived precursor of the neurotransmitter glutamate, and its astroglia-to-neuron transfer is controlled by distinct glutamine transporters on the astrocytic and neuronal sites. In this study, we focused on the role of astrocytic glutamine efflux-mediating system N transporter SN1 in the maintenance of glutamatergic neurotransmission by analyzing the electrophysiological parameters ex vivo in the brain slices from control mice and mice in which vivo-morpholino technique was used to diminish SN1 protein. The glutamatergic transmission was characterized by electrophysiological recordings, ultrastructure of neuron terminals, and determination of proteins related to glutamate synaptic transmission: synaptophysin, synaptotagmin, and vit1A. The space-restricted ∼51,5% reduction of SN1 protein did not affect the expression of the neuronal glutamine transporter SAT2. SN1 depletion resulted in a reduction of field potentials (FPs), unaltered frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs/mEPSCs), and presented a tendency towards a decrease of long-term potentiation (LTP). Ultrastructurally, preserved number of synaptic vesicles, primarily localized centrally of the cell body, correlates with unchanged levels of synaptic proteins. Collectively, the study indicates that glutamatergic transmission proceeds relatively independently of the SN1 - mediated glutamine transfer to the synapse.

Mammalian cell defence mechanisms against the cytotoxicity of NaYF4:(Er,Yb,Gd) nanoparticles.

Water-soluble upconversion nanoparticles (UCNPs), based on polyvinylpyrrolidone (PVP)-coated NaYF4:Er3+,Yb3+,Gd3+, with various concentrations of Gd3+ ions and relatively high upconversion efficiencies, were synthesized. The internalization and cytotoxicity of the thus obtained UCNPs were evaluated in three cell lines (HeLa, HEK293 and astrocytes). No cytotoxicity was observed even at concentrations of UCNPs up to 50 µg ml-1. The fate of the UCNPs within the cells was studied by examining their upconversion emission spectra with confocal microscopy and confirming these observations with transmission electron microscopy. It was found that the cellular uptake of the UCNPs occurred primarily by clathrin-mediated endocytosis, whereas they were secreted from the cells via lysosomal exocytosis. The results of this study, focused on the mechanisms of the cellular uptake, localization and secretion of UCNPs, demonstrate, for the first time, the co-localization of UCNPs within discrete cell organelles.

Pulmonary surfactant: ultrastructural features and putative mechanisms of aging.

Pulmonary surfactant is essential for maintaining lung function. In the present study we attempted to gain insight into the mechanisms underlying changes in surfactant in old age. We examined the ultrastructure of surfactant-producing lamellar bodies of the alveolar epithelial cells and of extracellular tubular myelin unfolding from the lamellar bodies in the lungs of two contrasting age-groups of rats: young, 2-3 months old and senescent, 26 months old. The study also focused on the plausible role of surfactant protein insufficiency in the process of surfactant aging. To this end, puromycin, a protein synthesis inhibitor, was used in vivo in young rats (12 mg/100 g body weight, i.p.) and its effects on surfactant ultrastructure were compared with the surfactant status in senescent rats. Lungs were rapidly dissected after being perfused with a mixture of aldehyde fixative and the tissue was subjected to the routine transmission electron microscopic procedures. Electronograms of the senescent lungs show that the alveolar epithelial lining layer and the lamellar bodies of type II cells, producing surfactant, displayed profound degenerative alterations. No regularly shaped myelin-tubular mesh, so characteristic of young lungs, could be recognized in the old ones. The aqueous, protein-containing hypophase of the alveolar epithelial lining, consisting of myelin tubules, no longer formed a solid layer integrated with the plasma membrane of type II cells. The effects of puromycin-induced inhibition of protein synthesis on the alveolar lining layers in the young lungs were reminiscent of the picture seen in the untreated aged lungs. The similarity of surfactant changes after puromycin to those present in senescent lungs is suggestive of the possible role of decaying surfactant proteins in the natural process of surfactant aging. We conclude that protein deficiency possibly developing in old age may underlie surfactant degradation which may impact lung function in old age.

Diversity of immunophenotypes of endothelial cells participating in new vessel formation following surgical rat brain injury.

Surgical brain injury causes neovascularization in the disrupted brain parenchyma, which occurs with the participation of endothelial-like cells. Differentiation of angioblasts from embryonic mesothelial cells has been proposed on the ground of biochemical and antigenic similarities between mesothelial and endothelial cells. Therefore, a transient localization of cytokeratin, the main mesothelial intermediate filament protein, to some embryonic vessels and endothelial progenitors, prompted us to use it to identify the source of cells participating in vessel formation after surgical brain injury. To determine the immunophenotypes of immature endothelial cells involved in new vessel formation following surgical rat brain injury, we used immunohistochemical and electron microscopic immunocytochemical techniques. Subcellular localization of protein markers: Flk-1, cytokeratin, and vimentin was examined in the cells investigated. Our results confirmed the existence of a diversity of immunophenotypes of immature endothelial cells in case of surgical-related brain injury.

Ultrastructural features of the neurovascular unit in Alzheimer's neurodegeneration.

Recent studies suggest that capillaries, neurons, and astrocytes form a functional unit that serves to maintain cerebral homeostasis. Physiological interactions between all these components of the neurovascular unit control cerebral microcirculation, while abnormal regulatory mechanisms lead to cerebral dysfunction and disease states, such as Alzheimer's disease (AD). Using electron microscopy, we studied a fragment of the frontotemporal cortex obtained intraoperatively from a patient with established AD. The objective of our study was to assess the ultrastructure of the components of the neurovascular unit. Such ultrastructural studies allow analyzing the structural process of new blood vessels formation and also the appearance of neurons and astrocytes contributing to the neurovascular unit. We suggest that dysfunction of particular components of the neurovascular unit underlies AD and ultimately leads to neurodegeneration.

Glial scar instability after brain injury.

Glial scar is formed following surgical damage to the cerebral cortex. In the present study we examined the ultrastructural status of the cerebral cortex 14 to 180 days following surgical damage to cerebral parenchyma. The results showed a contribution of astrocytes, but also mesodermal cells, to the process of scar formation. Furthermore, our study showed that the process initiated by trauma did not terminate with the formation of a glial scar. Late phases of repair following tissue damage were associated with lytic processes and a disassembly of the cerebral parenchyma. These findings indicate a changing and unstable nature of the glial scar and its components.

Mechanisms of vascular dysfunction after subarachnoid hemorrhage.

The main consequence of subarachnoid hemorrhage, for those who survive bleeding, is delayed, persistent vasospasm of intracranial conduit arteries which occurs between the third and seventh day after the insult and results in symptomatic brain ischemia in about 40% of cases. This vasospasm is considered to be a major cause of disability of post-SAH patients. Despite extensive experimental and clinical research, mechanisms of vasospasm are not fully understood. Dysfunction of the endothelium resulting in enhanced production of vasoconstrictors, phenotypic changes of the receptors in endothelium and smooth muscle cells, increased sensitivity of vascular smooth muscle cells to vasoconstrictors, release of spasmogens from lysed blood clot and inflammatory response of the vascular wall have been demonstrated and discussed as pathological mechanisms participating in the development of spasm. In recent years more attention is paid to the functional and structural changes in microcirculation and a concept of microvascular spasm is evolving. Our experimental studies in rat model of SAH strongly suggest that microcirculatory dysfunction and delayed vasospasm are related to the severity of acute, transient ischemia caused by critical decrease of perfusion pressure and active vasoconstriction immediately after the bleeding.

Ultrastructural alterations of endothelium covering advanced atherosclerotic plaque in human carotid artery visualised by scanning electron microscope.

Human atherosclerotic plaque morphology at its various stages was extensively documented using light microscopy. However, much less is known of the ultrastructure of the human atherosclerotic plaque, in particular of ultrastructure of endothelial cells in atherosclerosis. Here, we analysed alterations of endothelial cells covering advanced atherosclerotic plaque in carotid artery using scanning electron microscope. Examination was performed on specimens from atherosclerotic lesions of the interior carotid artery, collected from 8 patients who had undergone endarterectomy. We found wide spectrum of pathological alterations of the luminal surface of atherosclerotic plaque. In dominant part of the vessel, endothelial layer was preserved but displayed pronounced irregularities in endothelial architecture including appearance of cuboidal cells. Some endothelial cells were covered by numerous microvilli and/or contained "craters" disrupting continuous surface of the endothelium. Platelets and leukocytes adhering to endothelium were frequently observed. There were also areas of the vessel lumen with endothelial denudation, in which the subendothelial surface containing fibrin proteins and collagen fibrils were visible. Interestingly, signs of proliferation of endothelial cells tending to cover the partially denuded vessel were observed. In summary, in scanning electron microscope, preserved endothelial cells of advanced atherosclerotic plaque displayed pronounced pathology; whether any of these changes represent the ultrastructural correlate of endothelial dysfunction remains to be established.

Focal ischemia in the cerebral cortex has an effect on the neurohypophysis. I. Ultrastructural changes in capillary vessels of the neurohypophysis after focal ischemia of the cerebral cortex.

OBJECTIVES: In our investigations we have reported that photochemical reaction leading to brain ischemia can also be precipitated with visible light from a non-coherent light source. It was revealed that focal cerebral ischemia after photochemical reaction cause the alterations in the capillaries ultrastructure and perivascular spaces of the barrier-competent regions of the brain. The purpose of this study is to first characterize the ultrastructural morphological consequences of photochemically induced ischemia in the cerebral cortex on the capillaries of neurohypophysis as the barrier-free region of the brain. METHOD: We used a model of ischemic brain damage due to obliteration of microvessels following the photochemical reaction. Rats were treated with an intravenous injection of rose bengal and irradiated from a halogen lamp source through an intact cranium to precipitate microvascular damage. Material for electron microscopic studies were sampled from the neurohypophysis 1 and 4 days after irradiation (4 animals in each group) in experimental group and 1 and 4 days after a rose bengal injection in control group. RESULTS: Investigations in transmission electron microscopy revealed platelet aggregation on the endothelium preceded by its early ultrastructural damage. In the capillaries of the neurohypophysis, one and four days after irradiation, numerous microthrombi adhering to the damaged endothelium were present. The capillary vessels contained a continuous, rather than a fenestrated endothelium. The basement membrane was thickened, blurred and locally multiplicated. CONCLUSION: Our results show that experimentally-induced thrombosis of cortical microvessels leads to alterations in the capillaries of neurohypophysis.

Focal ischemia in the cerebral cortex has an effect on the neurohypophysis. II. Angiogenesis in the neurohypophysis is a consequence of the focal ischemia in the cerebral cortex.

OBJECTIVES: Focal ischemia in the cerebral cortex has an effect on neurohypophysis. The morphological changes of microvessels of neurohypophysis were evaluated in a model of the cerebral infarction initiated by a photochemical reaction in the cerebral cortex. After photochemically induced platelet aggregation, we observed the morphological features of angiogenesis. METHOD: The model of photochemically-induced cerebral ischemia was used. Seven days after intravenous injection of rose bengal and irradiation from a halogen lamp source through an intact cranium, the sampled material from neurohypophysis is processed for transmission electron microscopy using standard procedures. RESULTS: We observed morphological features of the new vessel formation: the alterations in the endothelium and extracellular matrix during separation of the endothelial cell from each other in a "mother" vessel, the migration of the endothelial cells in the extracellular matrix, the communication of the lumen of the new and the "mother" vessels. CONCLUSION: We observed development of the angiogenic phenotype in the neurohypophysis after focal ischemia in the cerebral cortex. The endothelium, basement membrane and extracellular matrix undergo morphological alterations which lead to new blood vessel formation.

Beneficial effects of GM1 ganglioside on photochemically-induced microvascular injury in cerebral cortex and hypophysis in rat.

Gangliosides, the glycophospholipids which are abundantly present in the central nervous system, have been shown to stimulate neuronal regeneration and counteract the deleterious effects of ischemia on cerebral neurons. The further elucidate the mechanism of action of gangliosides in cerebral ischemia, we investigated the influence of GM1 ganglioside in the model of photochemically-induced microvascular injury in rat brain. The animals were injected with rose Bengal and illuminated through cranium with halogen lamp. This treatment resulted in the development of microthrombi and alterations in endothelial cells in the microvessels. Administration of 20 mg/kg GM1 ganglioside, 1 h before the photochemical reaction, largely reduced subsequent microvascular damage. In conclusion, the GM1 ganglioside is able to prevent microvascular damage in the central nervous system.

Alterations in rat's brain capillaries in a model of focal cerebral necrosis.

Focal brain compression causes cerebral tissue damage. In this study we followed alterations in capillary ultrastructure in the rat cortex and neurohypophysis caused by 40 mm Hg compression for 15 minutes. One day after experiment we observed clogging of capillaries, accumulation of collagen fibrills under the basement membrane and necrosis or apoptosis of endothelial cells. Four days after it the basement membrane was multiplicated, blurred and thickened. In the neurohypophysis the formation of vessels lined with the atypical continuous endothelium was seen. There was also evidence for the migration of pericytes through the blurred basement membrane and the differentiation of pericytes into endothelial cells. Thus, vascular injury in the compressed brain is followed by a highly ordered sequence of processes in the basement membrane and perivascular cells leading to capillary repair.

The experimental squalene encephaloneuropathy in the rat.

Accumulation of squalene in the CNS is observed after administration of tellurium and squalene has been proposed to be a mediator of tellurium encephaloneuropathy. The aim of this study was to investigate the effects of squalene on the central and peripheral nervous systems in rat at the ultrastructural level. Squalene was administered at a dose of 20 g/kg body weight, once daily for 4 days, and the animals were sacrificed 7 days and 30 days after the initiation of the experiment. After 7 days a mild swelling of mitochondria and dilation of the Golgi complex cisterns in few neurons in the cerebral cortex and hippocampus were observed. The swelling of astrocytes and their processes was also seen. Some myelin sheaths in the cerebral white matter were disintegrated. In the peripheral nervous system (the sciatic nerve), a damage of the Schwann cells, a destruction of the myelin sheaths, and lipid-like deposits between myelin lamellae causing a secondary compression of axons were present. Squalene administration caused a stimulation of fibroblast to synthesize collagen and an activation of macrophages in the perineurium. After 30 days, the lipid-like material was present in some neurons as well as in the myelin sheaths in the central nervous system. Endothelial cells were hypertrophic and a few demonstrated features of apoptosis. Endothelial cell hypertrophy caused a narrowing of vessel lumen associated with an aggregation of blood morphological elements. Disturbances in myelination and swelling of astrocytic processes persisted in the central nervous system. In the peripheral nervous system, lipid-like deposits were localized in some fibroblasts and extracellularly between the collagen fibers in the perineurium. In conclusion, our electron microscopic studies indicate that squalene produces characteristic pathological changes both in the central and peripheral nervous systems. However, these alterations differ in some aspects (changes in endothelia, accumulation of lipid-like material) from the known features of tellurium encephaloneuropathy.

Photochemically-induced vascular damage in brain cortex. Transmission and scanning electron microscopy study.

Morphological changes of microvessels of cerebral cortex were evaluated in a model of cerebral infarction initiated by a photochemical reaction. Rats were treated with intravenous injection of rose Bengal and irradiated from a halogen lamp source through an intact cranium to precipitate microvascular damage. Investigations in transmission and scanning electron microscopy revealed platelet aggregation on endothelial cells preceded by its early ultrastructural damage. Other typical microscopic features of brain ischaemic injury were present suggesting that the present method may be used as a model for investigating ischaemic brain damage. Since the photochemical activation of the rose Bengal dye results in formation of reactive oxygen species this model may be particularly useful to elucidate the role of free radical-mediated endothelial damage in the formation of microthrombi and blood-brain-barrier integrity.

Effects of staphylococcal alpha-toxin on the ultrastructure of the rat hypothalamo-neurohypophyseal system.

The influence of staphylococcal alpha-toxin on the ultrastructure of hypothalamo-neurohypophysical system in the brain (nucleus supraopticus, nucleus paraventricularis, neurohypophysis) was studied in the rat. In neurohypophysis, an area lacking blood-brain barrier, alpha-toxin damaged both neuronal endings and capillary vessels. On the other hand in hypothalamus, where blood-brain barrier is present structural alterations were much less pronounced. Reactive gliosis, accordant with cell damage, was observed in the entire neurosecretory system. Putative mechanisms leading to brain damage after systemic administration of alpha-toxin, including direct disruption of cell membrane and induction of nitric oxide synthesis, are discussed.