Effect of recombinant Lactococcus lactis producing myelin peptides on neuroimmunological changes in rats with experimental allergic encephalomyelitis.

Multiple sclerosis (MS) is a human autoimmune neurodegenerative disease with an unknown etiology. Despite various therapies, there is no effective cure for MS. Since the mechanism of the disease is based on autoreactive T-cell responses directed against myelin antigens, oral tolerance is a promising approach for the MS treatment. Here, the experiments were performed to assess the impact of oral administration of recombinant Lactococcus lactis producing encephalogenic fragments of three myelin proteins: myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein, on neuroimmunological changes in rats with experimental allergic encephalomyelitis (EAE) - an animal model of MS. Lactococcus lactis whole-cell lysates were administered intragastrically at two doses (103 and 106 colony forming units) in a twenty-fold feeding regimen to Lewis rats with EAE. Spinal cord slices were subjected to histopathological analysis and morphometric evaluation, and serum levels of cytokines (IL-1b, IL-10, TNF-α and IFN-γ) were measured. Results showed that administration of the L. lactis preparations at the tested doses to rats with EAE, diminished the histopathological changes observed in EAE rats and reduced the levels of serum IL-1b, IL-10 and TNF-α, previously increased by evoking EAE. This suggests that oral delivery of L. lactis producing myelin peptide fragments could be an alternative strategy to induce oral tolerance for the treatment of MS.

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.

NMDA receptor antagonism does not inhibit induction of ischemic tolerance in gerbil brain in vivo.

Effects of high and moderate affinity uncompetitive NMDA receptor antagonists (+)MK-801 and memantine on ischemic tolerance were compared in relation to telemetrically controlled brain temperature. The tolerance to an injurious 3 min test of global forebrain ischemia in Mongolian gerbils was induced 48 h earlier by 2 min preconditioning ischemia. Normothermic preconditioning was virtually harmless, and greatly reduced neurodegeneration evoked by test ischemia. In hyperthermic animals it was injurious and failed to induce tolerance. Memantine (5 mg/kg) and (+)MK-801 (3 mg/kg) injected i.p. 1 h before preconditioning did not inhibit ischemic tolerance in the normothermic gerbils, while in hyperthermic animals treated with (+)MK-801 ischemic tolerance was partially restored. Subchronic 3 day infusion of memantine (30 mg/kg/day) significantly decreased neurodegeneration, and preconditioning in the normothermic gerbils further reduced neuronal damage. Hyperthermia exacerbated preconditioning ischemia and in this way reduced expression of tolerance, while (+)MK-801 partially reversed this effect. Our results do not confirm previous reports on the role of NMDA receptors in the induction of ischemic tolerance in gerbils.

CDP-choline, but not cytidine, protects hippocampal CA1 neurones in the gerbil following transient forebrain ischaemia.

The effects of CDP-choline (citicoline), cytidine monophosphate or cytidine on the number of CA1 hippocampal neurones surviving five-minute forebrain ischaemia have been evaluated in gerbils. The substances tested were given in daily doses equivalent on a molar basis to 500 mg/kg CDP-choline, starting immediately after ischaemia. On day five the brains were perfused, postfixed, cut into 10 microm slices and stained with cresyl violet, and the number of neurones in the CA1 sectors was counted manually under a light microscope at magnification x 400. The results indicate a significant degree of protection provided by citicoline, but no protection by cytidine monophosphate or cytidine. The choline moiety of CDP-choline appears to be essential for the neuroprotective properties of the drug.

Blood-brain barrier disturbances and morphological changes in rat brain after photochemically induced focal ischaemia.

The disturbances in cerebral circulation lead to focal brain ischaemia and brain infarcts. The pathogenesis of brain ischaemia has been a subject of numerous experimental studies employing different models and focusing on the anatomy of collateral circulation. The aim of this study was the evaluation of our own model of focatcerebral damage caused by a photodynamic reaction, and determination of its utility as a model of brain necrosis and blood-brain barrier damage in the rat. Wistar rats were used for the experiments. The animals were anaesthetised with 3% chloral hydrate (325 mg/kg) and injected intravenously with 40 mg/kg of 3% solution of rose bengal. After removal of the periostium the brain was irradiated through the skull for 30 min with a 250 W halogen, air-cooled light source. The material for morphologic studies was sampled 24 h, 4 days and 7 days after irradiation. The brains were fixed by perfusion, embedded in paraffin, and stained with haematoxilin-eosin (HE), acid vanadium fuchsin, cresyl violet, and GFAP. The results document the usefulness of this method for studying focal brain ischaemia in rats. The observed morphological changes and disturbances in blood-brain barrier provided information about the dynamics of the formation of gliosis, the formation of necrotic foci, and the quality and extent of brain damage in the surrounding tissue.

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.

Changes in the extracellular profiles of neuroactive amino acids in the rat striatum at the asymptomatic stage of hepatic failure.

Rats were treated with a hepatotoxin thioacetamide (TAA) and examined 21 days later, when they showed moderate fatty metamorphosis of the liver and morphological changes in brain indicative of excitotoxic neuronal damage, but no evident biochemical or neurophysiological symptoms of hepatic encephalopathy (HE). High-performance liquid chromatography (HPLC) analysis of extracellular amino acids in striatal microdialysates of TAA-treated rats revealed a significant increase in the excitatory amino acids glutamate (Glu) and aspartate (Asp) and their amino acid metabolites glutamine (Gln) and alanine (Ala). Microdialysis in the presence of 50 mM K+ triggered in TAA-treated rats an accumulation of Asp and Glu, and diminished the accumulation of Gln. These effects were virtually absent in control rats. None of the treatments affected the accumulation of the nontransmitter amino acid leucine (Leu). The above changes mirror those previously described in symptomatic HE and are likely to contribute to excitotoxic damage. The basal microdialysate content of taurine (Tau), an amino acid with antioxidant and volume regulatory properties, was 60% lower in TAA-treated rats than in control rats despite its increased blood-to-brain transport. The decrease in extracellular Tau may thus reflect Tau redistribution to adjacent central nervous system (CNS) cells manifesting a cell-protective response. Stimulation with 50 mM K+ increased extracellular Tau in control rats by 182% and in TAA-treated rats by 322%. Stimulation with 100 microM N-methyl-D-aspartate (NMDA) increased extracellular Tau in control rats by 27 % and in TAA-treated rats by as much as 250%. The increase of K+- or NMDA-dependent Tau release may reflect improved cell volume regulation and neuroprotection and contribute to attenuation of neurologic symptoms in rats with liver failure.

Retinal gliopathy accompanying thioacetamide-induced liver insufficiency: light and electron microscopic observations.

A recent examination of retinae of patients who had died with symptoms of liver insufficiency (LI) including hepatic encephalopathy (HE) revealed morphological changes in retinal Müller glia similar to the astrocytic changes normally accompanying HE, and the term "hepatic retinopathy" (HR) was coined to define these changes. In the present study, the immunomorphology and ultrastructure of Müller cells were examined in rats in which LI with accompanying HE was induced with a hepatotoxin, thioacetamide (TAA). Light microscopically, retinae of rats with LI were characterized by swelling of the Muller cell cytoplasm. Immunostaining for glia-specific marker proteins in Müller cells from LI rats revealed a strongly enhanced expression of glial fibrillary acidic protein, and a considerable increase in glutamine synthetase immunoreactivity, as compared to control animals. Ultrastructurally, the Müller cells of LI rats showed swelling and vacuolization of cell processes. In particular, the endfeet contained many swollen mitochondria. By contrast, LI produced no morphologically demonstrable changes in retinal neurons and photoreceptor cells. Thus, the retinal changes induced by TAA in the rats strongly resembled those described in human HR, rendering the present rat model suitable for more detailed investigations of the pathomechanism(s) of HR.

Changes in localization of chosen lectins in gerbil's brain submitted to 3 and 4 minute-long CNS ischemia.

The gerbils brains after 3- and 4-minute-long ischemia caused by bilateral common carotid artery occlusion and 14 days survival were investigated using lectin techniques. Chosen lectins, represented by synthetic plant glycoproteids, which are specifically bound to particular sugar residues (receptors) located on the cell surfaces were examined. Lectins recognizing the following sugar residues have been used in our experiment: 1. N-acetyl-D-galactosyl (using Helix pomatia agg., HPA) 2. alpha-D-mannosyl and alpha-D-glucosyl (using Concanavalin A, Con A). 3. beta-D-galactosyl (using Ricinus communis agg., RCA-120). 4. beta-D-galactosyl and neuraminic acid (using Arachis hypogaea-Peanut agg., PNA). 5. N-acetyl-galactosaminyl and N-acetyl-neuraminic acid (using Wheat germ agg., WGA). Changes in the glycoconjugates localization were found in neurons, glial cells, vessels, white matter fibers or neuropil. They were expressed by the weaking of the reaction with HPA in the hippocampus and white matter comparing to control. Con A receptors were considerably less susceptible to ischemia, though appearance of positively stained glial cells not found in the control group has been observed. Reaction with RCA-120 localized selectively in the network of capillary vessels decreased considerably. Similarly, receptors marked using PNA revealed reduction of staining reaction in white matter as well as in hippocampal interneurons. Using WGA we have also observed that staining reaction was reduced in capillary as well as in neuropil. That lectin indicated additionally strong accumulation of its sugar residues in glial cells appearing as a result of ischemia in particular sectors of hippocampus. The same cells were also Con A positive. The presented results indicate functionally and histochemically perceptible changes taking place in particular CNS elements as a result of short ischemia, expressed by the disturbances in the localization and accumulation of specific sugar residues examined with the use of lectin technique.

Lead-induced abnormalities in blood-brain barrier permeability in experimental chronic toxicity.

The aim of this paper was to determine whether prolonged drinking of lead acetate-containing water by adult rats, which imitates environmental exposure to lead (Pb), affects some morphological and biochemical properties of rat brain microvessels. We noted a significant increase of lead level in capillaries and synaptosomes obtained from brains of rats under chronic toxicity conditions. Intravenously injected horseradish peroxidase (HRP) was used to evaluate the functional state of the blood-brain barrier (BBB). The results indicate that, systematically administered at low doses, lead induces BBB dysfunction. The changes, revealed in light microscopy and confirmed by electron microscopic studies, are typical for "leaky" microvessels, reported for variety of neuropathological conditions associated with BBB damage. Enhanced pinocytotic activity of the endothelial cells and the opening of interendothelial tight junctions, together with enormous phagocytizing action of the pericytes, are the most characteristic ultrastructural features noted. The presence of specific type of perivascular cells containing droplets of lipids in the cytoplasm, together with changes in phospholipid profile in brain capillaries, suggest that altered lipid composition of membranes may, at least in part, be responsible for changes in observed membrane permeability.

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.

Protection against post-ischaemic neuronal loss in gerbil hippocampal CA1 by glycineB and AMPA antagonists. Short communication.

Novel antagonists of the glycineB site of the NMDA receptor (MRZ 2/570, MRZ 2/576), and an AMPA receptor antagonist, NBQX were tested in 3-min global ischaemia in gerbils. Untreated animals showed after 14 days a loss of almost 90% of pyramidal neurones in the CA1 region, which was prevented by NBQX, and reduced to 50% by both glycineB antagonists. NBQX produced a delayed, long lasting (up to 24 hr) hypothermia while hypothermia with both glycineB antagonists was transient.

Differential effects of N-methyl-D-aspartate on Ca2+ homeostasis in developing and adult rat striatum: in vivo microdialysis approach.

This in vivo study concerns developmental differences in the sensitivity of striatal neurons to N-methyl-D-aspartate (NMDA). Changes in calcium homeostasis in adult vs immature rats at postnatal days 8-10, evoked by NMDA, were evaluated by measurements of 45Ca efflux and of Ca2+ taurine and phosphoethanolamine concentrations in striatal microdialysates. The efflux of [14C]sucrose was employed in order to measure changes in extracellular space volume. In adult rats the addition of 5 mM NMDA for 20 min to the perfusion medium resulted in a 30-40% increase in 45Ca efflux, and in a 15% increase in [14C]sucrose efflux. Ten minutes after NMDA perfusion, 45Ca and [14C] sucrose efflux returned to the baseline. No significant changes in Ca2+ or amino acid concentrations were observed in the dialysate of the adult rat striatum. NMDA perfusion in the striatum of immature rats initially induced a transient (5 min) increase in the efflux of 45Ca (by 13%) and [14C]sucrose (by 9%). This was followed by a prolonged (lasting 45-50 min) 45% decrease in 45Ca efflux, an 80% reduction of Ca2+ concentration, and increases in taurine and phosphoethanolamine concentrations in the dialysate, whereas [14C]sucrose efflux recovered within 10 min. These data illustrate differences in the NMDA response between developing and adult rat striatum. Only in developing rats did NMDA induce a large and prolonged influx of extracellular calcium to neurons that may explain the enhanced NMDA neurotoxicity in immature rats.

Concanavalin A binding by the hippocampal neurons following brief cerebral ischemia in gerbils.

Twenty two Mongolian gerbils after 5 min bilateral carotid artery occlusion and 6, 12, 24, 48, 72, 94 hours and 5 days survival were investigated for the neuronal changes in dorsal hippocampus. Paraffin sections were stained with cresyl-violet and marked by their binding of Concanavalin A (Con A) labelled with peroxidase. The degeneration and neuronal loss was observed only in CA1 sectors in almost all experimental groups, whereas the decreased binding of Con A by the neurons of CA1 sector corresponded to the intensity of histologic changes but appeared also only in this sector even without any histological changes. These observation can point at the subthreshold damage of CA1 neurons as result of either diminished supply or increased metabolism of d-glucose, or diminished number of Con A receptors or changes of their specificity after ischemic period.

N-methyl-D-aspartate-induced 45Ca2+ release from pre-labelled adult rat hippocampus in vivo.

We report the results of microdialysis experiments investigating the NMDA-induced release of intracellular Ca2+ in different brain regions. Microdialysis probes were implanted stereotaxically into the striatum, thalamus and hippocampus dentate gyrus (DG) of adult rats. Dialysates were analysed for alterations in the concentration of ionized Ca2+ in an initially calcium-free medium and for changes in 45Ca efflux from the pre-labelled endogenous Ca2+ pools. The application of 5 mM of NMDA to the dialysis medium for 20 min in the striatum, resulted in increases in Ca2+ and 45Ca concentrations by 25% and 35% respectively. After NMDA perfusion in the hippocampus DG and in the thalamus, decreases in the Ca2+ concentration to 65.6% and 38.6% of the basal level respectively, were accompanied by increases in 45Ca efflux, exceeding 1,500% of the basal level in the hippocampus. Cell swelling, and the corresponding reduction of the extracellular space volume was insufficient to explain the huge increase in 45Ca efflux. Thus, our experiments demonstrated that in vivo in the rat hippocampus DG, NMDA induces the release of 45Ca to the extracellular space from unidentified intracellular calcium stores.

Immuno-electron microscopic demonstration of GABA and glutamate synapses in Mongolian gerbils hippocampus after ischemia.

The subcellular distribution of glutamate and GABA in synapses of Mongolian gerbils hippocampus was examined using post-embedding immunogold staining method for electron microscopy. Immunolabelling was performed with 10 nm gold-antibody complexes for glutamate and GABA. The gold particle densities gave reliable information about the relative concentrations of these amino acid neurotransmitters. Our results indicate that ischemia leads to the temporal decrease of GABA-like immunoreactivity in symmetric synapses and a slight enhancement of the level of glutamate-like reactivity in asymmetric ones. The striking finding was a redistribution of glutamate-like immunoreactivity from neurons to glia. This suggests the capacity of glia to metabolize the excess of glutamate after ischemia. The disturbances at the level of neurotransmitters and their possible role in hippocampal neuronal injury were stressed.

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.

Changes of activity and ultrastructural localization of alkaline phosphatase in cerebral cortical microvessels of rat after single intraperitoneal administration of mercuric chloride.

Inorganic mercury salts administered systemically at low mg/ml doses produce neurotoxic effects without penetrating the cerebral microvascular endothelial cells which form the blood-brain barrier (BBB). This phenomenon promoted investigations testing a hypothesis relating inorganic mercury-induced brain dysfunction to its interference with the BBB transport. In the present study, we tested the effect of a single i.p. administration of mercuric chloride (MC) (6 mg/kg body weight) on the activity and ultrastructural localization of cerebral alkaline phosphatase (AP), a cerebromicrovascular marker enzyme primarily located on luminal plasmalemma of endothelial cells. At 1h after MC administration, light microscopy revealed a virtual absence of AP in cerebral cortical layers II and III, and its dramatic reduction in the remaining layers. Electron microscopy confirmed the disappearance of the AP reaction product from luminal endothelial cell membranes, and luminal phasmalemma revealed pinocytic vesicles and invaginations likely to manifest changes in BBB transport. At 18h post-treatment, a moderate enzyme activity appeared on abluminal endothelial plasmalemma and on basement membrane, but remained absent from luminal plasmalemma. A similar picture persisted through day 5 post-treatment. The inhibition and subsequent translocation of AP activity from luminal to abluminal site and the accompanying ultrastructural changes are typical of the formation of "leaky" microvessels, previously reported for a variety of neuropathological conditions associated with BBB damage.