Acute Rift Valley fever virus infection induces inflammatory cytokines and cell death in ex vivo rat brain slice culture.

Rift Valley fever virus (RVFV) is an emerging arboviral disease with pandemic potential. While infection is often self-limiting, a subset of individuals may develop late-onset encephalitis, accounting for up to 20 % of severe cases. Importantly, individuals displaying neurologic disease have up to a 53 % case fatality rate, yet the neuropathogenesis of RVFV infection remains understudied. In this study, we evaluated whether ex vivo postnatal rat brain slice cultures (BSCs) could be used to evaluate RVFV infection in the central nervous system. BSCs mounted an inflammatory response after slicing, which resolved over time, and they were viable in culture for at least 12 days. Infection of rat BSCs with pathogenic RVFV strain ZH501 induced tissue damage and apoptosis over 48 h. Viral replication in BSCs reached up to 1×107 p.f.u. equivalents/ml, depending on inoculation dose. Confocal immunofluorescent microscopy of cleared slices confirmed direct infection of neurons as well as activation of microglia and astrocytes. Further, RVFV-infected rat BSCs produced antiviral cytokines and chemokines, including MCP-1 and GRO/KC. This study demonstrates that rat BSCs support replication of RVFV for ex vivo studies of neuropathogenesis. This allows for continued and complementary investigation into RVFV infection in an ex vivo postnatal brain slice culture format.

New uses for an old technique: live imaging on the slice organ culture to study reproductive processes†.

Reproductive processes are dynamic and involve extensive morphological remodeling and cell-cell interactions. Live imaging of organs enhances our understanding of how biological processes occur in real time. Slice culture is a type of organ culture where thick slices are collected from an organ and cultured for several days. Slice culture is a useful and easy-to-implement technique for live imaging of reproductive events at cellular resolution. Here we describe a pipeline of live imaging on slice culture to visualize the process of urethra closure in mouse embryonic penis as a proof of principle. In combination with genetic reporter mice, nuclear stains, and exposure experiments, we demonstrate the feasibility of slice culture on a reproductive organ. We also provide a step-by-step protocol and troubleshooting guide to facilitate the adoption of slice culture with live imaging in other reproductive organs. Lastly, we discuss potential utilities and experiments that could be implemented with slice culture in reproductive sciences.

Disease stage-dependent changes in brain levels and neuroprotective effects of neuroactive steroids in Parkinson's disease.

Neuroactive steroids are known neuroprotective agents and neurotransmitter regulators. We previously found that expression of the enzymes synthesizing 5α-dihydroprogesterone (5α-DHP), allopregnanolone (ALLO), and dehydroepiandrosterone sulfate (DHEAS) were reduced in the substantia nigra (SN) of Parkinson's Disease (PD) brain. Here, concentrations of a comprehensive panel of steroids were measured in human post-mortem brains of PD patients and controls. Gas chromatography-mass spectrometry (GC/MS) was used to measure steroid levels in SN (involved in early symptoms) and prefrontal cortex (PFC) (involved later in the disease) of five control (CTR) and nine PD donors, divided into two groups: PD4 (PD-Braak stages 1-4) and PD6 (PD-Braak stages 5-6). In SN, ALLO was increased in PD4 compared to CTR and 5α-DHP and ALLO levels were diminished in PD6 compared to PD4. The ALLO metabolite 3α5α20α-hexahydroprogesterone (3α5α20α-HHP) was higher in PD4 compared to CTR. In PFC, 3α5α20α-HHP was higher in PD4 compared to both CTR and PD6. The effects of 5α-DHP, ALLO and DHEAS were tested on human post-mortem brain slices of patients and controls in culture. RNA expression of genes involved in neuroprotection, neuroinflammation and neurotransmission was analysed after 5 days of incubation with each steroid. In PD6 slices, both 5α-DHP and ALLO induced an increase of the glutamate reuptake effector GLAST1, while 5α-DHP also increased gene expression of the neuroprotective TGFB. In CTR slices, ALLO caused reduced expression of IGF1 and GLS, while DHEAS reduced the expression of p75 and the anti-apoptotic molecule APAF1. Together these data suggest that a potentially protective upregulation of ALLO occurs at early stages of PD, followed by a downregulation of progesterone metabolites at later stages that may exacerbate the pathological changes, especially in SN. Neuroprotective effects of neurosteroids are thus dependent on the neuropathological stage of the disease.

Machine learning model for anti-cancer drug combinations: Analysis, prediction, and validation.

Drug combination therapy is a highly effective approach for enhancing the therapeutic efficacy of anti-cancer drugs and overcoming drug resistance. However, the innumerable possible drug combinations make it impractical to screen all synergistic drug pairs. Moreover, biological insights into synergistic drug pairs are still lacking. To address this challenge, we systematically analyzed drug combination datasets curated from multiple databases to identify drug pairs more likely to show synergy. We classified drug pairs based on their MoA and discovered that 110 MoA pairs were significantly enriched in synergy in at least one type of cancer. To improve the accuracy of predicting synergistic effects of drug pairs, we developed a suite of machine learning models that achieve better predictive performance. Unlike most previous methods that were rarely validated by wet-lab experiments, our models were validated using two-dimensional cell lines and three-dimensional tumor slice culture (3D-TSC) models, implying their practical utility. Our prediction and validation results indicated that the combination of the RTK inhibitors Lapatinib and Pazopanib exhibited a strong therapeutic effect in breast cancer by blocking the downstream PI3K/AKT/mTOR signaling pathway. Furthermore, we incorporated molecular features to identify potential biomarkers for synergistic drug pairs, and almost all potential biomarkers found connections between drug targets and corresponding molecular features using protein-protein interaction network. Overall, this study provides valuable insights to complement and guide rational efforts to develop drug combination treatments.

Investigating the Mitoprotective Effects of S1P Receptor Modulators Ex Vivo Using a Novel Semi-Automated Live Imaging Set-Up.

In multiple sclerosis (MS), mitochondrial alterations appear to contribute to disease progression. The sphingosine-1-phosphate receptor modulator siponimod is approved for treating secondary progressive MS. Its preceding compound fingolimod was shown to prevent oxidative stress-induced alterations in mitochondrial morphology. Here, we assessed the effects of siponimod, compared to fingolimod, on neuronal mitochondria in oxidatively stressed hippocampal slices. We have also advanced the model of chronic organotypic hippocampal slices for live imaging, enabling semi-automated monitoring of mitochondrial alterations. The slices were prepared from B6.Cg-Tg(Thy1-CFP/COX8A)S2Lich/J mice that display fluorescent neuronal mitochondria. They were treated with hydrogen peroxide (oxidative stress paradigm) ± 1 nM siponimod or fingolimod for 24 h. Afterwards, mitochondrial dynamics were investigated. Under oxidative stress, the fraction of motile mitochondria decreased and mitochondria were shorter, smaller, and covered smaller distances. Siponimod partly prevented oxidatively induced alterations in mitochondrial morphology; for fingolimod, a similar trend was observed. Siponimod reduced the decrease in mitochondrial track displacement, while both compounds significantly increased track speed and preserved motility. The novel established imaging and analysis tools are suitable for assessing the dynamics of neuronal mitochondria ex vivo. Using these approaches, we showed that siponimod at 1 nM partially prevented oxidatively induced mitochondrial alterations in chronic brain slices.

IL-17A in Human Liver: Significant Source of Inflammation and Trigger of Liver Fibrosis Initiation.

IL-17A is considered to guide liver inflammation and fibrosis. From twenty-two human liver samples of different fibrosis stages (F0 to F4), IL-17A, IL-22, and TGFß1 protein expression in liver tissue lysates were analyzed. Ten paired samples of liver tissue (F0-F1 stage) and blood from the same patient were used to analyze intrahepatic and blood T-lymphoid IL-17A+ cells by flow cytometry. The analyses have been performed regardless of pathology, considering the stage of fibrosis. Human liver tissue was used for the primary human liver slice cultures, followed by subsequent cytokine stimulation and fibrotic markers' analysis by ELISA. IL-17A production in human liver tissue was significantly higher in the early fibrotic stage compared with the advanced stage. Th17 T cells and, to a lesser extent, MAIT cells were the main sources of IL-17A in both compartments, the liver and the blood. Moreover, the presence of liver Th17IL-17A+INFγ+ cells was detected in the liver. IL-17A stimulation of human liver slice culture increased the expression of profibrotic and pro-inflammatory markers. IL-17A, secreted by Th17 and MAIT cells in the liver, triggered fibrosis by inducing the expression of IL-6 and profibrotic markers and could be a target for antifibrotic treatment. Further amplitude studies are needed to confirm the current results.

A New Organotypic 3D Slice Culture of Mouse Meibomian Glands Reveals Impact of Melanocortins.

The meibomian glands (MGs) within the eyelids produce a lipid-rich secretion that forms the superficial layer of the tear film. Meibomian gland dysfunction (MGD) results in excessive evaporation of the tear film, which is the leading cause of dry eye disease (DED). To develop a research model similar to the physiological situation of MGs, we established a new 3D organotypic slice culture (OSC) of mouse MGs (mMGs) and investigated the effects of melanocortins on exocrine secretion. Tissue viability, lipid production and morphological changes were analyzed during a 21-day cultivation period. Subsequently, the effects on lipid production and gene expression were examined after stimulation with a melanocortin receptor (MCR) agonist, α-melanocyte-stimulating hormone (α-MSH), and/or an MCR antagonist, JNJ-10229570. The cultivation of mMGs OSCs was possible without impairment for at least seven days. Stimulation with the MCR agonists induced lipid production in a dose-dependent manner, whereas this effect was tapered with the simultaneous incubation of the MCR antagonist. The new 3D OSC model is a promising approach to study the (patho-) physiological properties of MG/MGD while reducing animal studies. Therefore, it may accelerate the search for new treatments for MGD/DED and lead to new insights, such as that melanocortins likely stimulate meibum production.

Perfusion-Based Bioreactor Culture and Isothermal Microcalorimetry for Preclinical Drug Testing with the Carbonic Anhydrase Inhibitor SLC-0111 in Patient-Derived Neuroblastoma.

Neuroblastoma is a rare disease. Rare are also the possibilities to test new therapeutic options for neuroblastoma in clinical trials. Despite the constant need to improve therapy and outcomes for patients with advanced neuroblastoma, clinical trials currently only allow for testing few substances in even fewer patients. This increases the need to improve and advance preclinical models for neuroblastoma to preselect favorable candidates for novel therapeutics. Here we propose the use of a new patient-derived 3D slice-culture perfusion-based 3D model in combination with rapid treatment evaluation using isothermal microcalorimetry exemplified with treatment with the novel carbonic anhydrase IX and XII (CAIX/CAXII) inhibitor SLC-0111. Patient samples showed a CAIX expression of 18% and a CAXII expression of 30%. Corresponding with their respective CAIX expression patterns, the viability of SH-EP cells was significantly reduced upon treatment with SLC-0111, while LAN1 cells were not affected. The inhibitory effect on SH-SY5Y cells was dependent on the induction of CAIX expression under hypoxia. These findings corresponded to thermogenesis of the cells. Patient-derived organotypic slice cultures were treated with SLC-0111, which was highly effective despite heterogeneity of CAIX/CAXII expression. Thermogenesis, in congruence with the findings of the histological observations, was significantly reduced in SLC-0111-treated samples. In order to extend the evaluation time, we established a perfusion-based approach for neuroblastoma tissue in a 3D perfusion-based bioreactor system. Using this system, excellent tissue quality with intact tumor cells and stromal structure in neuroblastoma tumors can be maintained for 7 days. The system was successfully used for consecutive drug response monitoring with isothermal microcalorimetry. The described approach for drug testing, relying on an advanced 3D culture system combined with a rapid and highly sensitive metabolic assessment, can facilitate development of personalized treatment strategies for neuroblastoma.

Identifying cellular signalling molecules in developmental disorders of the brain: Evidence from focal cortical dysplasia and tuberous sclerosis.

AIMS: We understand little of the pathogenesis of developmental cortical lesions, because we understand little of the diversity of the cell types that contribute to the diseases or how those cells interact. We tested the hypothesis that cellular diversity and cell-cell interactions play an important role in these disorders by investigating the signalling molecules in the commonest cortical malformations that lead to childhood epilepsy, focal cortical dysplasia (FCD) and tuberous sclerosis (TS). METHODS: Transcriptional profiling clustered cases into molecularly distinct groups. Using gene expression data, we identified the secretory signalling molecules in FCD/TS and characterised the cell types expressing these molecules. We developed a functional model using organotypic cultures. RESULTS: We identified 113 up-regulated secretory molecules in FCDIIB/TS. The top 12 differentially expressed genes (DEGs) were validated by immunohistochemistry. This highlighted two molecules, Chitinase 3-like protein 1 (CHI3L1) and C-C motif chemokine ligand 2 (CCL2) (MCP1) that were expressed in a unique population of small cells in close proximity to balloon cells (BC). We then characterised these cells and developed a functional model in organotypic slice cultures. We found that the number of CHI3L1 and CCL2 expressing cells decreased following inhibition of mTOR, the main aberrant signalling pathway in TS and FCD. CONCLUSIONS: Our findings highlight previously uncharacterised small cell populations in FCD and TS which express specific signalling molecules. These findings indicate a new level of diversity and cellular interactions in cortical malformations and provide a generalisable approach to understanding cell-cell interactions and cellular heterogeneity in developmental neuropathology.

Photodynamic studies reveal rapid formation and appreciable turnover of tau inclusions.

Accumulation of the tau protein in fibrillar intracellular aggregates is a defining feature of multiple neurodegenerative diseases collectively referred to as tauopathies. Despite intensive study of tau, there is limited information on the formation and clearance dynamics of tau inclusions. Using rAAV vectors to mediate expression of Dendra2-tagged human wild-type, P301L and pro-aggregant P301L/S320F tau proteins, with and without the addition of exogenous tau fibrillar seeds, we evaluated tau inclusion dynamics in organotypic brain slice culture (BSC) models using long-term optical pulse labeling methodology. Our studies reveal that tau inclusions typically form in 12-96 h in tauopathy BSC models. Unexpectedly, we demonstrate appreciable turnover of tau within inclusions with an average half-life of ~ 1 week when inclusions are newly formed. When BSCs with inclusions are aged in culture for extended periods, tau inclusions continue to turnover, but their half-lives increase to ~ 2 weeks and ~ 3 weeks after 1 and 2 months in culture, respectively. Individual tau inclusions can be long-lived structures that can persist for months in these BSC models and for even longer in the human brain. However, our data indicate that tau inclusions, are not 'tombstones', but dynamic structures with appreciable turnover. Understanding the cellular processes mediating this inclusion turnover may lead to new therapeutic strategies that could reverse pathological tau inclusion formation.

Chronic Treatment of Ascorbic Acid Leads to Age-Dependent Neuroprotection against Oxidative Injury in Hippocampal Slice Cultures.

Increased oxidative damage in the brain, which increases with age, is the cause of abnormal brain function and various diseases. Ascorbic acid (AA) is known as an endogenous antioxidant that provides neuronal protection against oxidative damage. However, with aging, its extracellular concentrations and uptake decrease in the brain. Few studies have dealt with age-related functional changes in the brain to sustained ascorbate supplementation. This study aimed to investigate the susceptibility of hippocampal neurons to oxidative injury following acute and chronic AA administration. Oxidative stress was induced by kainic acid (KA, 5 µM) for 18 h in hippocampal slice cultures. After KA exposure, less neuronal cell death was observed in the 3 w cultured slice compared to the 9 w cultured slice. In the chronic AA treatment (6 w), the 9 w-daily group showed reduced neuronal cell death and increased superoxide dismutase (SOD) and Nrf2 expressions compared to the 9 w. In addition, the 9 w group showed delayed latencies and reduced signal activity compared to the 3 w, while the 9 w-daily group showed shorter latencies and increased signal activity than the 9 w. These results suggest that the maintenance of the antioxidant system by chronic AA treatment during aging could preserve redox capacity to protect hippocampal neurons from age-related oxidative stress.

Hallmarks of NLRP3 inflammasome activation are observed in organotypic hippocampal slice culture.

Microglial inflammation driven by the NACHT, LRR and PYD domain-containing protein 3 (NLRP3) inflammasome contributes to brain disease and is a therapeutic target. Most mechanistic studies on NLRP3 activation use two-dimensional pure microglial cell culture systems. Here we studied the activation of the NLRP3 inflammasome in organotypic hippocampal slices, which allowed us to investigate microglial NLRP3 activation in a three-dimensional, complex tissue architecture. Toll-like receptor 2 and 4 activation primed microglial inflammasome responses in hippocampal slices by increasing NLRP3 and interleukin-1ß expression. Nigericin-induced NLRP3 inflammasome activation was dynamically visualized in microglia through ASC speck formation. Downstream caspase-1 activation, gasdermin D cleavage, pyroptotic cell death and interleukin-1ß release were also detected, and these findings were consistent when using different NLRP3 stimuli such as ATP and imiquimod. NLRP3 inflammasome pathway inhibitors were effective in organotypic hippocampal slices. Hence, we have highlighted organotypic hippocampal slice culture as a valuable ex vivo tool to allow the future study of NLRP3 inflammasomes in a representative tissue section, aiding the discovery of further mechanistic insights and drug development.

Human in vitro systems for examining synaptic function and plasticity in the brain.

The human brain shows remarkable complexity in its cellular makeup and function, which are distinct from nonhuman species, signifying the need for human-based research platforms for the study of human cellular neurophysiology and neuropathology. However, the use of adult human brain tissue for research purposes is hampered by technical, methodological, and accessibility challenges. One of the major problems is the limited number of in vitro systems that, in contrast, are readily available from rodent brain tissue. With recent advances in the optimization of protocols for adult human brain preparations, there is a significant opportunity for neuroscientists to validate their findings in human-based systems. This review addresses the methodological aspects, advantages, and disadvantages of human neuron in vitro systems, focusing on the unique properties of human neurons and synapses in neocortical microcircuits. These in vitro models provide the incomparable advantage of being a direct representation of the neurons that have formed part of the human brain until the point of recording, which cannot be replicated by animal models nor human stem-cell systems. Important distinct cellular mechanisms are observed in human neurons that may underlie the higher order cognitive abilities of the human brain. The use of human brain tissue in neuroscience research also raises important ethical, diversity, and control tissue limitations that need to be considered. Undoubtedly however, these human neuron systems provide critical information to increase the potential of translation of treatments from the laboratory to the clinic in a way animal models are failing to provide.

Transient appearance of Ca2+ -permeable AMPA receptors is crucial for the production of repetitive LTP-induced synaptic enhancement (RISE) in cultured hippocampal slices.

We have previously shown that repetitive induction of long-term potentiation (LTP) by glutamate (100 µM, 3 min, three times at 24-hr intervals) provoked long-lasting synaptic enhancement accompanied by synaptogenesis in rat hippocampal slice cultures, a phenomenon termed RISE (repetitive LTP-induced synaptic enhancement). Here, we examined the role of Ca2+ -permeable (CP) AMPA receptors (AMPARs) in the establishment of RISE. We first found a component sensitive to the Joro-spider toxin (JSTX), a blocker of CP-AMPARs, in a field EPSP recorded from CA3-CA1 synapses at 2-3 days after stimulation, but this component was not found for 9-10 days. We also observed that rectification of AMPAR-mediated current appeared only 2-3 days after stimulation, using a whole-cell patch clamp recording from CA1 pyramidal neurons. These findings indicate that CP-AMPAR is transiently expressed in the developing phase of RISE. The blockade of CP-AMPARs by JSTX for 24 hr at this developing phase inhibited RISE establishment, accompanied by the loss of small synapses at the ultrastructural level. These results suggest that transiently induced CP-AMPARs play a critical role in synaptogenesis in the developing phase of long-lasting hippocampal synaptic plasticity, RISE.

GM-CSF induces noninflammatory proliferation of microglia and disturbs electrical neuronal network rhythms in situ.

BACKGROUND: The granulocyte-macrophage colony-stimulating factor (GM-CSF) (or CSF-2) is involved in myeloid cell growth and differentiation, and, possibly, a major mediator of inflammation in body tissues. The role of GM-CSF in the activation of microglia (CNS resident macrophages) and the consequent impacts on neuronal survival, excitability, and synaptic transmission are widely unknown, however. Here, we focused on electrical neuronal network rhythms in the gamma frequency band (30-70 Hz). Gamma oscillations are fundamental to higher brain functions, such as perception, attention, and memory, and they are exquisitely sensitive to metabolic and oxidative stress. METHODS: We explored the effects of chronic GM-CSF exposure (72 h) on microglia in male rat organotypic hippocampal slice cultures (in situ), i.e., postnatal cortex tissue lacking leukocyte invasion (adaptive immunity). We applied extracellular electrophysiological recordings of local field potential, immunohistochemistry, design-based stereology, biochemical analysis, and pharmacological ablation of microglia. RESULTS: GM-CSF triggered substantial proliferation of microglia (microgliosis). By contrast, the release of proinflammatory cytokines (IL-6, TNF-α) and nitric oxide, the hippocampal cytoarchitecture as well as the morphology of parvalbumin-positive inhibitory interneurons were unaffected. Notably, GM-CSF induced concentration-dependent, long-lasting disturbances of gamma oscillations, such as slowing (beta frequency band) and neural burst firing (hyperexcitability), which were not mimicked by the T lymphocyte cytokine IL-17. These disturbances were attenuated by depletion of the microglial cell population with liposome-encapsulated clodronate. In contrast to priming with the cytokine IFN-γ (type II interferon), GM-CSF did not cause inflammatory neurodegeneration when paired with the TLR4 ligand LPS. CONCLUSIONS: GM-CSF has a unique role in the activation of microglia, including the potential to induce neuronal network dysfunction. These immunomodulatory properties might contribute to cognitive impairment and/or epileptic seizure development in disease featuring elevated GM-CSF levels, blood-brain barrier leakage, and/or T cell infiltration.

Comparative analysis of adeno-associated virus serotypes for gene transfer in organotypic heart slices.

BACKGROUND: Vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo and have gained increasing interest as shuttle systems to deliver therapeutic genes to the heart. However, there is little information on their tissue penetration and cytotoxicity, as well as the optimal AAV serotype for transferring genes to diseased hearts. Therefore, we aimed to establish an organotypic heart slice culture system for mouse left ventricular (LV) myocardium and use this platform to analyze gene transfer efficiency, cell tropism, and toxicity of different AAV serotypes. METHODS: LV tissue slices, 300 µm thick, were prepared from 15- to 17-day-old transgenic alpha-myosin heavy-chain-mCherry mice using a vibrating microtome. Tissue slice viability in air-liquid culture was evaluated by calcein-acetoxymethyl ester staining, mCherry fluorescence intensity, and the tetrazolium assay. Four recombinant AAV serotypes (1, 2, 6, 8) expressing green fluorescent protein (GFP) under the CAG promoter were added to the slice surface. Gene transfer efficiency was quantified as the number of GFP-positive cells per slice. AAV cell tropism was examined by comparing the number of GFP-positive cardiomyocytes (CMs) and fibroblasts within heart slices. RESULTS: Slices retained viability in in vitro culture for at least 5 days. After adding AAV particles, AAV6-infected slices showed the highest number of GFP-expressing cells, almost exclusively CMs. Slice incubation with AAV1, 2, and 8 resulted in fewer GFP-positive cells, with AAV2 having the lowest gene transfer efficiency. None of the AAV serotypes tested caused significant cytotoxicity when compared to non-infected control slices. CONCLUSIONS: We have established a readily available mouse organotypic heart slice culture model and provided evidence that AAV6 may be a promising gene therapy vector for heart failure and other cardiac diseases.

Neuronal activity modulates alpha-synuclein aggregation and spreading in organotypic brain slice cultures and in vivo.

Alpha-synuclein (αSyn) preformed fibrils (PFF) induce endogenous αSyn aggregation leading to reduced synaptic transmission. Neuronal activity modulates release of αSyn; however, whether neuronal activity regulates the spreading of αSyn pathology remains elusive. Here, we established a hippocampal slice culture system from wild-type (WT) mice and found that both Ca2+ influx and the uptake of αSyn PFF were higher in the CA3 than in the CA1 sub-region. Pharmacologically enhancing neuronal activity substantially increased αSyn pathology in αSyn PFF-treated hippocampal or midbrain slice cultures and accelerated dopaminergic neuron degeneration. Consistently, neuronal hyperactivity promoted PFF trafficking along axons/dendrites within microfluidic chambers. Unexpectedly, enhancing neuronal activity in LRRK2 G2019S mutant slice cultures further increased αSyn pathology, especially with more Lewy body (LB) forming than in WT slice cultures. Finally, following injection of αSyn PFF and chemogenetic modulators into the dorsal striatum of WT mice, both motor behavior and αSyn pathology were exacerbated likely by enhancing neuronal activity, since they were ameliorated by reducing neuronal activity. Thus, a greater understanding of the impact of neuronal activity on αSyn aggregation and spreading, as well as dopaminergic neuronal vulnerability, may provide new therapeutic strategies for patients with LB disease (LBD).

Organotypic slice culture based on in ovo electroporation for chicken embryonic central nervous system.

Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)-positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 µm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30-mm culture dish and 1 ml of slice culture media was added. We show that during serum-free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured-tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum-free medium cultured-tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.

Lipopolysaccharide-induced neuroinflammation induces presynaptic disruption through a direct action on brain tissue involving microglia-derived interleukin 1 beta.

BACKGROUND: Systemic inflammation has been linked to synapse loss and cognitive decline in human patients and animal models. A role for microglial release of pro-inflammatory cytokines has been proposed based on in vivo and primary culture studies. However, mechanisms are hard to study in vivo as specific microglial ablation is challenging and the extracellular fluid cannot be sampled without invasive methods. Primary cultures have different limitations as the intricate multicellular architecture in the brain is not fully reproduced. It is essential to confirm proposed brain-specific mechanisms of inflammatory synapse loss directly in brain tissue. Organotypic hippocampal slice cultures (OHSCs) retain much of the in vivo neuronal architecture, synaptic connections and diversity of cell types whilst providing convenient access to manipulate and sample the culture medium and observe cellular reactions. METHODS: OHSCs were generated from P6-P9 C57BL/6 mice. Inflammation was induced via addition of lipopolysaccharide (LPS), and cultures were analysed for changes in synaptic proteins, gene expression and protein secretion. Microglia were selectively depleted using clodronate, and the effect of IL1ß was assessed using a specific neutralising monoclonal antibody. RESULTS: LPS treatment induced loss of the presynaptic protein synaptophysin without altering PSD95 or Aß protein levels. Depletion of microglia prior to LPS application prevented the loss of synaptophysin, whilst microglia depletion after the inflammatory insult was partially effective, although less so than pre-emptive treatment, indicating a time-critical window in which microglia can induce synaptic damage. IL1ß protein and mRNA were increased after LPS addition, with these effects also prevented by microglia depletion. Direct application of IL1ß to OHSCs resulted in synaptophysin loss whilst pre-treatment with IL1ß neutralising antibody prior to LPS addition prevented a significant loss of synaptophysin but may also impact basal synaptic levels. CONCLUSIONS: The loss of synaptophysin in this system confirms LPS can act directly within brain tissue to disrupt synapses, and we show that microglia are the relevant cellular target when all major CNS cell types are present. By overcoming limitations of primary culture and in vivo work, our study strengthens the evidence for a key role of microglia-derived IL1ß in synaptic dysfunction after inflammatory insult.

Cell viability in three ex vivo rat models of spinal cord injury.

Spinal cord injury (SCI) is a devastating disorder that has a poor prognosis of recovery. Animal models of SCI are useful to understand the pathophysiology of SCI and the potential use of therapeutic strategies for human SCI. Ex vivo models of central nervous system (CNS) trauma, particularly mechanical trauma, have become important tools to complement in vivo models of injury in order to reproduce the sequelae of human CNS injury. Ex vivo organotypic slice cultures (OSCs) provide a reliable model platform for the study of cell dynamics and therapeutic intervention following SCI. In addition, these ex vivo models support the 3R concept of animal use in SCI research - replacement, reduction and refinement. Ex vivo models cannot be used to monitor functional recovery, nor do they have the intact blood supply of the in vivo model systems. However, the ex vivo models appear to reproduce many of the post traumatic events including acute and secondary injury mechanisms. Several well-established OSC models have been developed over the past few years for experimental spinal injuries ex vivo in order to understand the biological response to injury. In this study, we investigated cell viability in three ex vivo OSC models of SCI: stab injury, transection injury and contusion injury. Injury was inflicted in postnatal day 4 rat spinal cord slices. Stab injury was performed using a needle on transverse slices of spinal cord. Transection injury was performed on longitudinal slices of spinal cord using a double blade technique. Contusion injury was performed on longitudinal slices of spinal cord using an Infinite Horizon impactor device. At days 3 and 10 post-injury, viability was measured using dual staining for propidium iodide and fluorescein diacetate. In all ex vivo SCI models, the slices showed more live cells than dead cells over 10 days in culture, with higher cell viability in control slices compared with injured slices. Although no change in cell viability was observed between time-points in stab- and contusion-injured OSCs, a reduction in cell viability was observed over time in transection-injured OSCs. Taken together, ex vivo SCI models are a useful and reliable research tool that reduces the cost and time involved in carrying out animal studies. The use of OSC models provides a simple way to study the cellular consequences following SCI, and they can also be used to investigate potential therapeutics regimes for the treatment of SCI.