Nondestructive evaluation of progressive neuronal changes in organotypic rat hippocampal slice cultures using ultrahigh-resolution optical coherence microscopy.
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ABSTRACT:
SUBMITTER: Li F
PROVIDER: S-EPMC4350448 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Neurogenesis in the adult hippocampus has become an intensively investigated research topic, as it is essential for proper hippocampal function and considered to bear therapeutic potential for the replacement of pathologically lost neurons. On the other hand, neurogenesis itself is frequently affected by CNS insults. To identify processes leading to the disturbance of neurogenesis, we made use of organotypic hippocampal slice cultures (OHSC), which, for unknown reasons, lose their neurogenic potential during cultivation. In the present study, we show by BrdU/Prox1 double-immunostaining that the generation of new granule cells drops by 90% during the first week of cultivation. Monitoring neurogenesis dynamically in OHSC from POMC-eGFP mice, in which immature granule cells are endogenously labeled, revealed a gradual decay of the eGFP signal, reaching 10% of initial values within 7 days of cultivation. Accordingly, reverse transcription quantitative polymerase chain reaction analysis showed the downregulation of the neurogenesis-related genes doublecortin and Hes5, a crucial target of the stem cell-maintaining Notch signaling pathway. In parallel, we demonstrate a strong and long-lasting activation of astrocytes and microglial cells, both, morphologically and on the level of gene expression. Enhancement of astroglial activation by treating OHSC with ciliary neurotrophic factor accelerated the loss of neurogenesis, whereas treatment with indomethacin or an antagonist of the purinergic P2Y12 receptor exhibited potent protective effects on the neurogenic outcome. Therefore, we conclude that OHSC rapidly lose their neurogenic capacity due to persistent inflammatory processes taking place after the slice preparation. As inflammation is also considered to affect neurogenesis in many CNS pathologies, OHSC appear as a useful tool to study this interplay and its molecular basis. Furthermore, we propose that modification of glial activation might bear the therapeutic potential of enabling neurogenesis under neuropathological conditions.
| S-EPMC4870256 | biostudies-literature
Project description:Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.
| S-EPMC5338800 | biostudies-literature
Project description:Contrast agents improve clinical and basic research MRI. The manganese ion (Mn2+ ) is an essential, endogenous metal found in cells and it enhances MRI contrast because of its paramagnetic properties. Manganese-enhanced MRI (MEMRI) has been widely used to image healthy and diseased states of the body and the brain in a variety of animal models. There has also been some work in translating the useful properties of MEMRI to humans. Mn2+ accumulates in brain regions with high neural activity and enters cells via voltage-dependent channels that flux calcium (Ca2+ ). In addition, metal transporters for zinc (Zn2+ ) and iron (Fe2+ ) can also transport Mn2+ . There is also transfer through channels specific for Mn2+ . Although Mn2+ accumulates in many tissues including brain, the mechanisms and preferences of its mode of entry into cells are not well characterized. The current study used MRI on living organotypic hippocampal slice cultures to detect which transport mechanisms are preferentially used by Mn2+ to enter cells. The use of slice culture overcomes the presence of the blood brain barrier, which limits inferences made with studies of the intact brain in vivo. A range of Mn2+ concentrations were used and their effects on neural activity were assessed to avoid using interfering doses of Mn2+ . Zn2+ and Fe2+ were the most efficient competitors for Mn2+ uptake into the cultured slices, while the presence of Ca2+ or Ca2+ channel antagonists had a more moderate effect. Reducing slice activity via excitatory receptor antagonists was also effective at lowering Mn2+ uptake. In conclusion, a hierarchy of those agents which influence Mn2+ uptake was established to enhance understanding of how Mn2+ enters cells in a cultured slice preparation.
| S-EPMC7988546 | biostudies-literature
Project description:Intracellular tau inclusions are a pathological hallmark of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and other sporadic neurodegenerative tauopathies. Recent in vitro and in vivo studies have shown that tau aggregates may spread to neighbouring cells and functionally connected brain regions, where they can seed further tau aggregation. This process is referred to as tau propagation. Here we describe an ex vivo system using organotypic hippocampal slice cultures (OHCs) which recapitulates aspects of this phenomenon. OHCs are explants of hippocampal tissue which may be maintained in culture for months. They maintain their synaptic connections and multicellular 3D architecture whilst also permitting direct control of the environment and direct access for various analysis types. We inoculated OHCs prepared from P301S mouse pups with brain homogenate from terminally ill P301S mice and then examined the slices for viability and the production and localization of insoluble phosphorylated tau. We show that following seeding, phosphorylated insoluble tau accumulate in a time and concentration dependent manner within OHCs. Furthermore, we show the ability of the conformation dependent anti-tau antibody, MC1, to compromise tau accrual in OHCs, thus showcasing the potential of this therapeutic approach and the utility of OHCs as an ex vivo model system for assessing such therapeutics.
| S-EPMC8119691 | biostudies-literature
Project description:We hypothesize that peptide-containing solutions pulled through tissue should reveal the presence and activity of peptidases in the tissue. Using the natural zeta-potential in the organotypic hippocampal slice culture (OHSC), physiological fluids can be pulled through the tissue with an electric field. The hydrolysis of the peptides present in the fluid drawn through the tissue can be determined using capillary HPLC with electrochemical detection of the biuret complexes of the peptides following a postcolumn reaction. We have characterized this new sampling method by measuring the flow rate, examining the use of internal standards, and examining cell death caused by sampling. The sampling flow rate ranges from 60 to 150 nL/min with a 150 microm (ID) sampling capillary with an electric field (at the tip of the capillary) from 30 to 60 V/cm. Cell death can be negligible with controlled sampling conditions. Using this sampling approach, we have electroosmotically pulled Leu-enkephalin through OHSCs to identify ectopeptidase activity in the CA3 region. These studies show that a bestatin-sensitive aminopeptidase may be critical for the hydrolysis of exogenous Leu-enkephalin, a neuropeptide present in the CA3 region of OHSCs.
| S-EPMC2920223 | biostudies-literature
Project description:We have developed a novel sampling technique that allows both introduction and removal of fluid from the extracellular space of living tissue. This method is based on the fluidics of push-pull perfusion but flow is driven by electroosmosis. We have applied this method to organotypic hippocampal cultures. A source capillary is inserted into the tissue and a collection capillary is in contact with the tissue surface through a thin layer of fluid. A voltage is applied across the proximal ends of source and collection capillary. In the applied field, fluid will move from source, into the tissue, and then be collected. In this process, damage to cells may occur. To understand better what sampling conditions influence damage most, we tested various sampling geometries and applied voltages, quantifying damage 16-24 h later using propidium iodide as a cell death marker. We found that damage correlates with both voltage drop and power dissipated in the tissue, but that voltage drop is a better indicator of damage when comparing models in which capillary arrangement and length are different.
| S-EPMC3656745 | biostudies-literature
Project description:BackgroundPyrethroids, such as bifenthrin (BF), are among the most widely used class of insecticides that pose serious risks to human and wildlife health. Pyrethroids are proposed to affect astrocytic functions and to cause neuron injury in the central nervous system (CNS). Microglia are key cells involved in innate immune responses in the CNS, and microglia activation has been linked to inflammation and neurotoxicity. However, little information is known about the effects of BF-induced toxicity in primary microglial cells as well as in organotypic hippocampal slice cultures (OHSCs).MethodsOxidative stress and inflammatory responses induced by BF were evaluated in primary microglial cells and OHSCs incubated with different concentrations of BF (1-20 μM) for 4 and 24 h. mRNA and protein synthesis of cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), nuclear erythroid-2 like factor-2 (Nrf-2), and microsomal prostaglandin synthase-1 (mPGES-1) was also studied by qPCR and Western blot. Cell viability was analyzed by MTT-tetrazolio (MTT) and lactate dehydrogenase (LDH) assays. Neurotoxicity in OHSCs was analyzed by propidium iodide (PI) staining and confocal microscopy.ResultsExposure of microglial cells to BF for 24 h resulted in a dose-dependent reduction in the number of viable cells. At sub-cytotoxic concentrations, BF increased reactive oxygen species (ROS), TNF-alpha synthesis, and prostaglandin E2 (PGE2) production, at both 4- and 24-h time points, respectively. Furthermore, BF incubation decreased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and increased lipid peroxidation, protein oxidation, and H2O2 formation. In addition, BF significantly induced protein synthesis and mRNA expression of oxidative and inflammatory mediators after 4 and 24 h, including Nrf-2, COX-2, mPGES-1, and nuclear factor kappaB (NF-kappaB). A 24-h exposure of OHSCs to BF also increased neuronal death compared to untreated controls. Furthermore, depletion of microglia from OHSCs potently enhanced neuronal death induced by BF.ConclusionsOverall, BF exhibited cytotoxic effects in primary microglial cells, accompanied by the induction of various inflammatory and oxidative stress markers including the Nrf-2/COX-2/mPGES-1/NF-kappaB pathways. Moreover, the study provided evidence that BF induced neuronal death in OHSCs and suggests that microglia exert a protective function against BF toxicity.
| S-EPMC5968622 | biostudies-literature
Project description:Organotypic slice cultures of brain or spinal cord have been a longstanding tool in neuroscience research but their utility for understanding Alzheimer's disease (AD) and other neurodegenerative proteinopathies has only recently begun to be evaluated. Organotypic brain slice cultures (BSCs) represent a physiologically relevant three-dimensional model of the brain. BSCs support all the central nervous system (CNS) cell types and can be produced from brain areas involved in neurodegenerative disease. BSCs can be used to better understand the induction and significance of proteinopathies underlying the development and progression of AD and other neurodegenerative disorders, and in the future may serve as bridging technologies between cell culture and in vivo experiments for the development and evaluation of novel therapeutic targets and strategies. We review the initial development and general use of BSCs in neuroscience research and highlight the advantages of these cultures as an ex vivo model. Subsequently we focus on i) BSC-based modeling of AD and other neurodegenerative proteinopathies ii) use of BSCs to understand mechanisms underlying these diseases and iii) how BSCs can serve as tools to screen for suitable therapeutics prior to in vivo investigations. Finally, we will examine i) open questions regarding the use of such cultures and ii) how emerging technologies such as recombinant adeno-associated viruses (rAAV) may be combined with these models to advance translational research relevant to neurodegenerative disorders.
| S-EPMC6889333 | biostudies-literature
Project description:BACKGROUND AND PURPOSE: Earlier studies had demonstrated that tonic-clonic seizure-like events (SLEs) resembling electrographic correlates of limbic seizures in animals and humans can be induced in organotypic hippocampal slice cultures (OHSCs). We have explored OHSCs for their suitability to serve as in vitro models of limbic seizures for studying seizure mechanisms and screening new antiepileptic compounds. EXPERIMENTAL APPROACH: OHSCs were cultivated according to the interface method. Neuronal activity and extracellular potassium concentration were recorded under submerged conditions. SLEs were induced by lowering magnesium concentration or by applying the potassium channel blocker 4-aminopyridine. The effects of standard antiepileptic drugs (AEDs), carbamazepine, phenytoin, valproic acid, clonazepam, diazepam and phenobarbital sodium on SLEs were analysed. KEY RESULTS: In more than 93% of OHSCs, AEDs did not prevent the induction of SLEs or stop ongoing seizure activity even when toxic concentrations were applied. This pharmacoresistance was independent of the method of seizure provocation, postnatal age at explantation (P2-P10) and cultivation time in vitro (2 months). SLEs were reversibly blocked by glutamate antagonists or the GABA(A)-agonist muscimol. CONCLUSIONS AND IMPLICATIONS: We present a simple to establish in vitro model of tonic-clonic SLEs that is a priori pharmacoresistant and thus has an advantage over animal models of pharmacoresistant seizures in which responders and non-responders can be sorted out only after an experiment. OHSCs could be suitable for exploring mechanisms of pharmacoresistant seizures and be used for the identification of new anticonvulsive compounds eventually effective in drug refractory epilepsy.
| S-EPMC2439516 | biostudies-other
Project description:N-arachidonoyl glycine (NAGly) is an endocannabinoid involved in the regulation of different immune cells. It was shown to activate the GPR18 receptor, which was postulated to switch macrophages from cytotoxic to reparative. To study GPR18 expression and neuroprotection after NAGly treatment we used excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). The effect of NAGly was also tested in isolated microglia and astrocytes as these cells play a crucial role during neuronal injury. In the present study, the GPR18 receptor was found in OHSC at mRNA level and was downregulated after N-Methyl-D-aspartate (NMDA) treatment at a single time point. Furthermore, treatment with NAGly reduced neuronal damage and this effect was abolished by GPR18 and cannabinoid receptor (CB)₂ receptor antagonists. The activation but not motility of primary microglia and astrocytes was influenced when incubated with NAGly. However, NAGly alone reduced the phosphorylation of Akt but no changes in activation of the p44/42 and p38 MAPK and CREB pathways in BV2 cells could be observed. Given NAGly mediated actions we speculate that GPR18 and its ligand NAGly are modulators of glial and neuronal cells during neuronal damage.
| S-EPMC6470786 | biostudies-literature