Hippocampal Slice Cultures Research Articles

The MT1 receptor as the target of ramelteon neuroprotection in ischemic stroke.

Stroke is the leading cause of death and disability worldwide. Novel and effective therapies for ischemic stroke are urgently needed. Here, we report that melatonin receptor 1A (MT1) agonist ramelteon is a neuroprotective drug candidate as demonstrated by comprehensive experimental models of ischemic stroke, including a middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia in vivo, organotypic hippocampal slice cultures ex vivo, and cultured neurons in vitro; the neuroprotective effects of ramelteon are diminished in MT1-knockout (KO) mice and MT1-KO cultured neurons. For the first time, we report that the MT1 receptor is significantly depleted in the brain of MCAO mice, and ramelteon treatment significantly recovers the brain MT1 losses in MCAO mice, which is further explained by the Connectivity Map L1000 bioinformatic analysis that shows gene-expression signatures of MCAO mice are negatively connected to melatonin receptor agonist like Ramelteon. We demonstrate that ramelteon improves the cerebral blood flow signals in ischemic stroke that is potentially mediated, at least, partly by mechanisms of activating endothelial nitric oxide synthase. Our results also show that the neuroprotection of ramelteon counteracts reactive oxygen species-induced oxidative stress and activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Ramelteon inhibits the mitochondrial and autophagic death pathways in MCAO mice and cultured neurons, consistent with gene set enrichment analysis from a bioinformatics perspective angle. Our data suggest that Ramelteon is a potential neuroprotective drug candidate, and MT1 is the neuroprotective target for ischemic stroke, which provides new insights into stroke therapy. MT1-KO mice and cultured neurons may provide animal and cellular models of accelerated ischemic damage and neuronal cell death.

The effects of prolyl hydroxylase inhibition during and post, hypoxia, oxygen glucose deprivation and oxidative stress, in isolated rat hippocampal slices

The contributions of hypoxia and oxidative stress to the pathophysiology of acute ischemic stroke are well established and can lead to disruptions in synaptic signaling. Hypoxia and oxidative stress lead to the neurotoxic overproduction of reactive oxygen species (ROS) and the stabilization of hypoxia inducible factors (HIF). Compounds such as prolyl-4-hydroxylase domain enzyme inhibitors (PHDIs) have been shown to have a preconditioning and neuroprotective effect against ischemic insults such as hypoxia, anoxia, oxygen glucose deprivation (OGD) or H2O2. Therefore, this study explored the effects of two PHDIs, JNJ-42041935 (10 µM) and roxadustat (100 µM) on cell viability using organotypic hippocampal slice cultures. We also assessed the effects of these compounds on synaptic transmission during and post hypoxia, OGD and H2O2 application in isolated rat hippocampal slices using field recording electrophysiological techniques and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit trafficking using immunohistochemistry. Our organotypic data demonstrated a protective role for both inhibitors, where slices had significantly less cell death post anoxia and OGD compared to controls. We also report a distinct modulatory role for both JNJ-42041935 and roxadustat on fEPSP slope post hypoxia and OGD but not H2O2. In addition, we report that application of roxadustat impaired long-term potentiation, but only when applied post-hypoxia. This inhibitory effect was not reversed with co-application of the cyclin-dependent kinase 5 (CDK-5) inhibitor, roscovitine (10 µM), suggesting a CDK-5 independent synaptic AMPAR trafficking mechanism. Both hypoxia and OGD induced a reduction in synaptic AMPA GluA2 subunits, the OGD effect being reversed by prior treatment with both JNJ-42041935 and roxadustat. These results suggest an important role for PHDs in synaptic signaling and plasticity during episodes of ischemic stress.

NTS-105 decreased cell death and preserved long-term potentiation in an in vitro model of moderate traumatic brain injury

Traumatic brain injury (TBI) is a major cause of hospitalization and death. To mitigate these human costs, the search for effective drugs to treat TBI continues. In the current study, we evaluated the efficacy of the novel neurosteroid, NTS-105, to reduce post-traumatic pathobiology in an in vitro model of moderate TBI that utilizes an organotypic hippocampal slice culture. NTS-105 inhibited activation of the androgen receptor and the mineralocorticoid receptor, partially activated the progesterone B receptor and was not active at the glucocorticoid receptor. Treatment with NTS-105 starting one hour after injury decreased post-traumatic cell death in a dose-dependent manner, with 10 nM NTS-105 being most effective. Post-traumatic administration of 10 nM NTS-105 also prevented deficits in long-term potentiation (LTP) without adversely affecting neuronal activity in naïve cultures. We propose that the high potency pleiotropic action of NTS-105 beneficial effects at multiple receptors (e.g. androgen, mineralocorticoid and progesterone) provides significant mechanistic advantages over native neurosteroids such as progesterone, which lacked clinical success for the treatment of TBI. Our results suggest that this pleiotropic pharmacology may be a promising strategy for the effective treatment of TBI, and future studies should test its efficacy in pre-clinical animal models of TBI.

Substantially improved gene transfer to interneurons with second-generation glutamate receptor-targeted DART-AAV vectors

BackgroundAdeno-associated viral vectors (AAVs) are a widely used gene transfer platform in neuroscience. Although naturally AAV serotypes can have preferences for certain tissues, selectivity for particular cell types in the CNS does not exist. Towards interneuron targeting, capsid engineering of AAV2 including display of the designed ankyrin repeat protein (DARPin) 2K19 specific for the glutamate receptor subunit 4 (GluA4) at the N-terminus of the VP2 capsid protein has been established. The resulting AAV-VP2N is highly specific for interneurons, but exhibits rather moderate transduction efficiencies. MethodsTwo alternative insertion sites for 2K19 in the GH2/GH3 loop of capsid proteins VP1 (AAV-VP1L) or VP2 (AAV-VP2L) were exploited to yield second generation GluA4-AAVs. Having packaged reporter genes under ubiquitous promoters, the vectors were characterized for biochemical properties as well as gene delivery into cell lines and rat hippocampal slice cultures. Electrophysiological recordings monitored the functional properties of transduced cells. ResultsCompared to AAV-VP2N, the second-generation vectors, especially AAV-VP1L, achieved about 2-fold higher genomic titers as well as a substantially improved GluA4 binding. Improvements in gene transfer activities were 18-fold on GluA4-overexpressing A549 cells and five-fold on rat hippocampal organotypic slice cultures reaching approximately 60 % of all parvalbumin positive interneurons upon a single administration. The spiking behaviour of transduced cells was unaltered and characteristic for a heterogeneous group of interneurons. ConclusionThe substantially improved gene transfer activity of the second generation GluA4-targeted AAV combined with low toxicity makes this vector an attractive tool for interneuron-directed gene transfer with unrestricted promotor and transgene choice.

P10.01.A A PATIENT-DERIVEDEX VIVO ORGANOID MODEL ALLOWS EVALUATION OF TUMOR TREATING FIELDS (TTFIELDS) EFFECTS ON GLIOBLASTOMA, ANAPLASTIC EPENDYMOMA AND OLIGODENDROGLIOMA SAMPLES

Abstract BACKGROUND The use of TTFields, which are alternating electric fields of low intensity (1-3 V/cm) and intermediate frequency (100-500 kHz), has shown its clinical efficacy in the treatment of newly diagnosed glioblastoma (GBM) patients at 200 kHz. Nevertheless, more knowledge is required for the prediction of individual tumors’ responsiveness to treatment. Recently, we developed patient-derived three-dimensional GBM tissue culture models to screen patient tumors for TTFields treatment susceptibility. Here, we extended these models to brain tumors beyond GBM, namely anaplastic ependymoma (AE) and oligodendroglioma. We cultured tumor-organoids as microtumors on murine organotypic hippocampal slice cultures (OHSCs) and subjected them to TTFields at 200 kHz. MATERIAL AND METHODS We prepared OHSCs by slicing the brains of mice 5-8 days postpartum to a thickness of 350 µm using a vibratome and culturing them for 14 days. We obtained organoids from freshly resected tumor tissue from three GBM, one AE, and one oligodendroglioma patient, each. The organoids were cultured for 14 days as well and then stained with carboxyfluorescein succinimidyl ester. Next, we placed the organoids onto the OHSCs and administered TTFields at 200 kHz and 1.5 V/cm for 72 h using the inovitro™ laboratory research system. We quantified microtumor growth by fluorescence imaging. RESULTS The three tumor entities, in particular organoids from GBM, AE and oligodendroglioma, grew as microtumors after being placed onto OHSCs. TTFields treatment at 200 kHz led to significantly decreased microtumor size of the three analysed GBM samples as well as the AE sample. However, the oligodendroglioma microtumor size was not significantly reduced. CONCLUSION Based on clinical experience, we expected that TTFields lead to a reduction in microtumor size. However, to our knowledge, we were the first to apply TTFields to patient-derived AE and oligodendroglioma samples. The lack of TTFields-effects on the latter might be due to different inter-patient sensitivity or suboptimal TTFields frequency. Nevertheless, our work is proof of principle that an organoid-based patient-derived 3D tissue culture model could be used to evaluate effects of TTFields on different brain tumor entities.

Neuroprotective effect of dexmedetomidine on autophagy in mice administered intracerebroventricular injections of Aβ25-35.

Alzheimer's disease (AD), one of the most prevalent neurodegenerative diseases is associated with pathological autophagy-lysosomal pathway dysfunction. Dexmedetomidine (Dex) has been suggested as an adjuvant to general anesthesia with advantages in reducing the incidence of postoperative cognitive dysfunction in Dex-treated patients with AD and older individuals. Several studies reported that Dex improved memory; however, evidence on the effects of Dex on neuronal autophagy dysfunction in the AD model is lacking. We hypothesized that Dex administration would have neuroprotective effects by improving pathological autophagy dysfunction in mice that received an intracerebroventricular (i.c.v.) injection of amyloid β-protein fragment 25-35 (Aβ25-35) and in an autophagy-deficient cellular model. In the Y-maze test, Dex reversed the decreased activity of Aβ25-35 mice. Additionally, it restored the levels of two memory-related proteins, phosphorylated Ca2+/calmodulin-dependent protein kinase II (p-CaMKII) and postsynaptic density-95 (PSD-95) in Aβ25-35 mice and organotypic hippocampal slice culture (OHSC) with Aβ25-35. Dex administration also resulted in decreased expression of the autophagy-related microtubule-associated proteins light chain 3-II (LC3-II), p62, lysosome-associated membrane protein2 (LAMP2), and cathepsin D in Aβ25-35 mice and OHSC with Aβ25-35. Increased numbers of co-localized puncta of LC3-LAMP2 or LC3-cathepsin D, along with dissociated LC3-p62 immunoreactivity following Dex treatment, were observed. These findings were consistent with the results of western blots and the transformation of double-membrane autophagosomes into single-membraned autolysosomes in ultrastructures. It was evident that Dex treatment alleviated impaired autolysosome formation in Aβ mice. Our study demonstrated the improvement of memory impairment caused by Dex and its neuroprotective mechanism by investigating the role of the autophagy-lysosomal pathway in a murine Aβ25-35 model. These findings suggest that Dex could be used as a potential neuroprotective adjuvant in general anesthesia to prevent cognitive decline.

Astaxanthin enhances autophagy, amyloid beta clearance and exerts anti-inflammatory effects in in vitro models of Alzheimer’s disease-related blood brain barrier dysfunction and inflammation

Defective degradation and clearance of amyloid-β as well as inflammation per se are crucial players in the pathology of Alzheimer’s disease (AD). A defective transport across the blood–brain barrier is causative for amyloid-β (Aβ) accumulation in the brain, provoking amyloid plaque formation. Using primary porcine brain capillary endothelial cells and murine organotypic hippocampal slice cultures as in vitro models of AD, we investigated the effects of the antioxidant astaxanthin (ASX) on Aβ clearance and neuroinflammation. We report that ASX enhanced the clearance of misfolded proteins in primary porcine brain capillary endothelial cells by inducing autophagy and altered the Aβ processing pathway. We observed a reduction in the expression levels of intracellular and secreted amyloid precursor protein/Aβ accompanied by an increase in ABC transporters ABCA1, ABCG1 as well as low density lipoprotein receptor-related protein 1 mRNA levels. Furthermore, ASX treatment increased autophagic flux as evidenced by increased lipidation of LC3B-II as well as reduced protein expression of phosphorylated S6 ribosomal protein and mTOR. In LPS-stimulated brain slices, ASX exerted anti-inflammatory effects by reducing the secretion of inflammatory cytokines while shifting microglia polarization from M1 to M2 phenotype. Our data suggest ASX as potential therapeutic compound ameliorating AD-related blood brain barrier impairment and inflammation.

A Dynamic Balance between Neuronal Death and Clearance in an in Vitro Model of Acute Brain Injury.

In in vitro models of acute brain injury, neuronal death may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. Neurons undergoing programmed cell death are in this queue, and are the most visible and frequently quantified measure of neuronal death after injury. However, the size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for 2 weeks. Altering phagocytosis rates (e.g., by changing the number of microglia) dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for stains that label dying neurons. Canonically neuroprotective interventions, such as seizure blockade, and neurotoxic maneuvers, such as perinatal ethanol exposure, were mediated by effects on microglial activity and the membrane permeability of neurons undergoing programmed cell death. These canonically neuroprotective and neurotoxic interventions had either no or opposing effects on healthy surviving neurons identified by the ongoing expression of transgenic fluorescent proteins.SIGNIFICANCE STATEMENT In in vitro models of acute brain injury, microglial phagocytosis is overwhelmed by the number of dying cells. Under these conditions, the assumptions on which assays for neuroprotective and neurotoxic effects are based are no longer valid. Thus, longitudinal assays of healthy cells, such as serial assessment of the fluorescence emission of transgenically expressed proteins, provide more accurate estimates of cell death than do single-time point anatomic or biochemical assays of the number of dying neurons. More accurate estimates of death rates in vitro will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.

Thrombin-Induced Microglia Activation Modulated through Aryl Hydrocarbon Receptors.

Thrombin is a multifunctional serine protein which is closely related to neurodegenerative disorders. The Aryl hydrocarbon receptor (AhR) is well expressed in microglia cells involving inflammatory disorders of the brain. However, it remains unclear as to how modulation of AhR expression by thrombin is related to the development of neurodegeneration disorders. In this study, we investigated the role of AhR in the development of thrombin-induced neurodegenerative processes, especially those concerning microglia. The primary culture of either wild type or AhR deleted microglia, as well as BV-2 cell lines, was used for an in vitro study. Hippocampal slice culture and animals with either wild type or with AhR deleted were used for the ex vivo and in vivo studies. Simulations of ligand protein docking showed a strong integration between the thrombin and AhR. In thrombin-triggered microglia cells, deleting AhR escalated both the NO release and iNOS expression. Such effects were abolished by the administration of the AhR agonist. In thrombin-activated microglia cells, downregulating AhR increased the following: vascular permeability, pro-inflammatory genetic expression, MMP-9 activity, and the ratio of M1/M2 phenotype. In the in vivo study, thrombin induced the activation of microglia and their volume, thereby contributing to the deterioration of neurobehavior. Deleting AhR furthermore aggravated the response in terms of impaired neurobehavior, increasing brain edema, aggregating microglia, and increasing neuronal death. In conclusion, thrombin caused the activation of microglia through increased vessel permeability, expression of inflammatory response, and phenotype of M1 microglia, as well the MMP activity. Deleting AhR augmented the above detrimental effects. These findings indicate that the modulation of AhR is essential for the regulation of thrombin-induced brain damages and that the AhR agonist may harbor the potentially therapeutic effect in thrombin-induced neurodegenerative disorder.

Pharmacological blockade of cannabinoid receptor 2 signaling does not affect LPS/IFN-γ-induced microglial activation

Cannabinoid receptor 2 (CB2) signaling modulates microglial responses to inflammatory stimuli. Our previous studies demonstrated that genetic deletion of CB2 inhibits microglial activation during inflammatory stimulation of toll-like receptors (TLRs) or in neurodegenerative conditions. However, we cannot exclude developmental effects of the constitutive CB2 knockout (CB2−/−), which could mediate compensatory outcomes in CB2−/− mice. In the present study, we therefore tested whether acute pharmacological inhibition of CB2 receptor has a similar effect on microglial activation as in CB2−/− in response to inflammatory stimulation. Our findings suggest that the CB2-specific antagonist SR144528 has little or no effect on LPS/IFN-γ-induced activation in primary microglia or organotypic hippocampal slice cultures at nanomolar concentrations. We show that SR144528 did not alter LPS/IFN-γ-mediated microglial cytokine secretion, Iba1 and CD68 staining intensity or morphology at 1 and 10 nM. Although SR144528 suppressed LPS/IFN-γ-induced microglial activation at 1 µM, this anti-inflammatory effect was not dependent on CB2 receptors and exceeded the Ki on CB2 receptors by more than a thousand-fold. Thus, SR144528 does not mimic the anti-inflammatory effects observed in the CB2−/− microglia after LPS/IFN-γ stimulation. Therefore, we propose that the deletion of CB2 probably triggered an adaptive mechanism, making microglia less responsive to inflammatory stimulation.

Expression of MAP2, GFAP, and calcium in CA3 area of a modified organotypic hippocampal slice culture treated with kainic acid

Background: Neurodegeneration due to neurotoxicity is one of the phenomena in temporal lobe epilepsy. Experimentally, hippocampal excitotoxicity process can occur due to kainic acid exposure, especially in the CA3 area. Neuronal death, astrocyte reactivity and increased calcium also occur in hippocampal excitotoxicity, but few studies have investigated immediate effect after kainic acid exposure. The organotypic hippocampal slice culture (OHSC) is a useful model for studying the neurodegeneration process, but there are still many protocol differences. In this study, minor modifications were made in the OHSC protocol. Methods: OHSC was obtained from two healthy wild type Wistar rats aged P10. Healthy culture slices were obtained and lasted up to 10 days in vitro (DIV 10). Bath application of kainic acid for 48 hours in DIV 10 followed by observation of its initial effects on neurons, astrocytes, and calcium via the expression of MAP2, GFAP, and intracellular calcium, subsequently. Results: After 48 h of kainic acid administration, there was a significant increase in intracellular calcium (p = 0.006 < α), accompanied by a significant decrease in MAP2 (p = 0.003 < α ) and GFAP (p = 0.010 < α) expression. Conclusion: These findings suggest early neuronal and astrocyte damage at the initial onset of hippocampal injury. This implies that astrocyte damage occurs early before an increase in GFAP that characterizes reactive astrogliosis found in other studies. Damage to neurons and astrocytes may be associated with increased intracellular calcium. It is necessary to develop further research regarding expression of calcium, MAP2, and GFAP at a spatial time after exposure to kainic acid and strategies to reduce damage caused by excitotoxicity.

Organotypic Hippocampal Slice Cultures from Adult Tauopathy Mice and Theragnostic Evaluation of Nanomaterial Phospho-TAU Antibody-Conjugates.

Organotypic slice culture models surpass conventional in vitro methods in many aspects. They retain all tissue-resident cell types and tissue hierarchy. For studying multifactorial neurodegenerative diseases such as tauopathies, it is crucial to maintain cellular crosstalk in an accessible model system. Organotypic slice cultures from postnatal tissue are an established research tool, but adult tissue-originating systems are missing, yet necessary, as young tissue-originating systems cannot fully model adult or senescent brains. To establish an adult-originating slice culture system for tauopathy studies, we made hippocampal slice cultures from transgenic 5-month-old hTau.P301S mice. In addition to the comprehensive characterization, we set out to test a novel antibody for hyperphosphorylated TAU (pTAU, B6), with and without a nanomaterial conjugate. Adult hippocampal slices retained intact hippocampal layers, astrocytes, and functional microglia during culturing. The P301S-slice neurons expressed pTAU throughout the granular cell layer and secreted pTAU to the culture medium, whereas the wildtype slices did not. Additionally, cytotoxicity and inflammation-related determinants were increased in the P301S slices. Using fluorescence microscopy, we showed target engagement of the B6 antibody to pTAU-expressing neurons and a subtle but consistent decrease in intracellular pTAU with the B6 treatment. Collectively, this tauopathy slice culture model enables measuring the extracellular and intracellular effects of different mechanistic or therapeutic manipulations on TAU pathology in adult tissue without the hindrance of the blood-brain barrier.

Umbelliferone Ameliorates Memory Impairment and Enhances Hippocampal Synaptic Plasticity in Scopolamine-Induced Rat Model.

Alzheimer's disease (AD) is a neurodegenerative disorder, characterized by memory loss and cognitive decline. Among the suggested pathogenic mechanisms of AD, the cholinergic hypothesis proposes that AD symptoms are a result of reduced synthesis of acetylcholine (ACh). A non-selective antagonist of the muscarinic ACh receptor, scopolamine (SCOP) induced cognitive impairment in rodents. Umbelliferone (UMB) is a Apiaceae-family-derived 7-hydeoxycoumarin known for its antioxidant, anti-tumor, anticancer, anti-inflammatory, antibacterial, antimicrobial, and antidiabetic properties. However, the effects of UMB on the electrophysiological and ultrastructure morphological aspects of learning and memory are still not well-established. Thus, we investigated the effect of UMB treatment on cognitive behaviors and used organotypic hippocampal slice cultures for long-term potentiation (LTP) and the hippocampal synaptic ultrastructure. A hippocampal tissue analysis revealed that UMB attenuated a SCOP-induced blockade of field excitatory post-synaptic potential (fEPSP) activity and ameliorated the impairment of LTP by the NMDA and AMPA receptor antagonists. UMB also enhanced the hippocampal synaptic vesicle density on the synaptic ultrastructure. Furthermore, behavioral tests on male SD rats (7-8 weeks old) using the Y-maze test, passive avoidance test (PA), and Morris water maze test (MWM) showed that UMB recovered learning and memory deficits by SCOP. These cognitive improvements were in association with the enhanced expression of BDNF, TrkB, and the pCREB/CREB ratio and the suppression of acetylcholinesterase activity. The current findings indicate that UMB may be an effective neuroprotective reagent applicable for improving learning and memory against AD.

FMRP phosphorylation and interactions with Cdh1 regulate association with dendritic RNA granules and MEF2-triggered synapse elimination

Fragile X Messenger Ribonucleoprotein (FMRP) is necessary for experience-dependent, developmental synapse elimination and the loss of this process may underlie the excess dendritic spines and hyperconnectivity of cortical neurons in Fragile X Syndrome, a common inherited form of intellectual disability and autism. Little is known of the signaling pathways that regulate synapse elimination and if or how FMRP is regulated during this process. We have characterized a model of synapse elimination in CA1 neurons of organotypic hippocampal slice cultures that is induced by expression of the active transcription factor Myocyte Enhancer Factor 2 (MEF2) and relies on postsynaptic FMRP. MEF2-induced synapse elimination is deficient in Fmr1 KO CA1 neurons, and is rescued by acute (24 h), postsynaptic and cell autonomous reexpression of FMRP in CA1 neurons. FMRP is an RNA binding protein that suppresses mRNA translation. Derepression is induced by posttranslational mechanisms downstream of metabotropic glutamate receptor signaling. Dephosphorylation of FMRP at S499 triggers ubiquitination and degradation of FMRP which then relieves translation suppression and promotes synthesis of proteins encoded by target mRNAs. Whether this mechanism functions in synapse elimination is not known. Here we demonstrate that phosphorylation and dephosphorylation of FMRP at S499 are both necessary for synapse elimination as well as interaction of FMRP with its E3 ligase for FMRP, APC/Cdh1. Using a bimolecular ubiquitin-mediated fluorescence complementation (UbFC) assay, we demonstrate that MEF2 promotes ubiquitination of FMRP in CA1 neurons that relies on activity and interaction with APC/Cdh1. Our results suggest a model where MEF2 regulates posttranslational modifications of FMRP via APC/Cdh1 to regulate translation of proteins necessary for synapse elimination.

Abstract 4573: Evaluation of tumor treating fields (TTFields) effects at 200 kHz on a glioblastoma, an anaplastic ependymoma and an oligodendroglioma sample in a patient-derived ex vivo organoid model

Abstract TTFields are alternating electric fields of low intensity (1-3 V/cm) and intermediate frequency (100-500 kHz), which are effective and approved for the treatment of glioblastoma (GBM) using 200 kHz frequency. However, there is a lack of ex vivo models to evaluate effects on patients’ tumor biology or to screen patients for treatment efficacy. Therefore, we adapted patient-derived three-dimensional GBM tissue culture models to be compatible with TTFields application and recently published the feasibility of such an approach (Nickl, et al., 2022, doi: 10.3390/cancers14215177). Here, we applied one of those models, i.e. tumor-organoids cultured as microtumors on murine organotypic hippocampal slice cultures (OHSCs), to additional brain tumor entities, namely a sample of an anaplastic ependymoma (AE) patient and an oligodendroglioma patient. Organoids were generated from fresh intra-operatively obtained tumor tissue and cultured for 2 weeks. OHSCs were prepared by slicing the brains of mice 5-8 days postpartum to sections with a thickness of 350 µm using a vibratome, and culturing them for 2 weeks as well. Subsequently, organoids were placed onto the OHSCs. The inovitro™ laboratory research system was used for TTFields administration at 200 kHz and 1.5 V/cm for 72 h. Microtumor growth was evaluated on fluorescence images. Viable organoids formed from the GBM, AE and oligodendroglioma sample and grew to microtumors when placed onto OHSCs. Application of TTFields at 200 kHz led to a significant decrease of microtumor size of the GBM and AE (both p<0.0001), but not the oligodendroglioma sample. This proof-of-principle investigation proved that the application of patient-derived organoids cultured on OHSCs is feasible to investigate the effects of TTFields on different kinds of brain tumors. To our knowledge, this is the first evaluation of TTFields efficacy on patient derived AE and oligodendroglioma tissue cultures. While TTFields at 200 kHz led to a decrease in the microtumor size of the AE sample, the non-responsiveness of the oligodendroglioma sample may be due to different inter-patient sensitivity to TTFields or a suboptimal TTFields frequency. Citation Format: Vera Nickl, Ellina Schulz, Ellaine Salvador, Laureen Trautmann, Leopold Diener, Almuth F. Kessler, Camelia M. Monoranu, Ralf-Ingo Ernestus, Mario Löhr, Carsten Hagemann. Evaluation of tumor treating fields (TTFields) effects at 200 kHz on a glioblastoma, an anaplastic ependymoma and an oligodendroglioma sample in a patient-derived ex vivo organoid model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4573.

GABAA receptor subunit modulation reversed electrophysiological network alterations after blast exposure in rat organotypic hippocampal slice cultures

Throughout training and deployment, some military service members are frequently exposed to shock waves due to blasts, and some complain of myriad neurological symptoms. In rat organotypic hippocampal slice cultures (OHSCs), blast-induced traumatic brain injury (bTBI) causes deficits in some electrophysiological measures, like long term potentiation, a neuronal correlate for learning and memory. In this study, we further characterized the alterations in the hippocampal network of OHSCs following a single moderate blast exposure. Connectivity and clustering coefficients were reduced across the hippocampal network following bTBI, despite the lack of changes in the firing rate, spike amplitude, spike duration, or inter-spike interval. However, interrogation with the GABAA receptor antagonist, bicuculline, revealed additional significant differences between injured and control slices in measures of spike amplitude, spike duration, connectivity, and clustering. bTBI also significantly reduced expression of the α1 and α5 GABAA receptor subunits. Treatment with the FDA-approved histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) restored the α1 subunit and attenuated deficits in network measures, like connectivity and clustering coefficients. These findings suggest that GABAA receptors may be implicated in neuronal network changes in OHSCs following bTBI, and their recovery may be a viable therapeutic intervention to mitigate injury-induced neurological symptoms.

Inhibition of cyclooxygenase and EP3 receptor improved long term potentiation in a rat organotypic hippocampal model of repeated blast traumatic brain injury.

Blast-induced traumatic brain injury (bTBI) is a health concern in military service members who are exposed to multiple blasts throughout their training and deployment. Our group has previously reported decreased long term potentiation (LTP) following repeated bTBI in a rat organotypic hippocampal slice culture (OHSC) model. In this study, we investigated changes in inflammatory markers like cyclooxygenase (COX) and tested the efficacy of COX or prostaglandin EP3 receptor (EP3R) inhibitors in attenuating LTP deficits. Expression of COX-2 was increased 48h following repeated injury, whereas COX-1 expression was unchanged. EP3R expression was upregulated, and cyclic adenosine monophosphate (cAMP) concentration was decreased after repeated blast exposure. Post-traumatic LTP deficits improved after treatment with a COX-1 specific inhibitor, SC-560, a COX-2 specific inhibitor, rofecoxib, a pan-COX inhibitor, ibuprofen, or an EP3R inhibitor, L-798,106. Delayed treatment with ibuprofen and L-798,106 also prevented LTP deficits. These findings suggest that bTBI induced neuroinflammation may be responsible for some functional deficits that we have observed in injured OHSCs. Additionally, COX and EP3R inhibition may be viable therapeutic strategies to reduce neurophysiological deficits after repeated bTBI.

Expression of MAP2, GFAP, and calcium in CA3 area of a modified organotypic hippocampal slice culture treated with kainic acid

Background: Neurodegeneration due to neurotoxicity is one of the phenomena in temporal lobe epilepsy. Experimentally, hippocampal excitotoxicity process can occur due to kainic acid exposure, especially in the CA3 area. Neuronal death, astrocyte reactivity and increased calcium also occur in hippocampal excitotoxicity, but few studies have investigated immediate effect after kainic acid exposure. The organotypic hippocampal slice culture (OHSC) is a useful model for studying the neurodegeneration process, but there are still many protocol differences. In this study, minor modifications were made in the OHSC protocol. Methods: OHSC was obtained from two healthy wild type Wistar rats aged P10. Healthy culture slices were obtained and lasted up to 10 days in vitro (DIV 10). Bath application of kainic acid for 48 hours in DIV 10 followed by observation of its initial effects on neurons, astrocytes, and calcium via the expression of MAP2, GFAP, and intracellular calcium, subsequently. Results: After 48 h of kainic acid administration, there was a significant increase in intracellular calcium (p = 0.006 < α), accompanied by a significant decrease in MAP2 (p = 0.003 < α ) and GFAP (p = 0.010 < α) expression. Conclusion: These findings suggest early neuronal and astrocyte damage at the initial onset of hippocampal injury. This implies that astrocyte damage occurs early before an increase in GFAP that characterizes reactive astrogliosis found in other studies. Damage to neurons and astrocytes may be associated with increased intracellular calcium. It is necessary to develop further research regarding expression of calcium, MAP2, and GFAP at a spatial time after exposure to kainic acid and strategies to reduce damage caused by excitotoxicity.

Naringin enhances long-term potentiation and recovers learning and memory deficits of amyloid-beta induced Alzheimer’s disease-like behavioral rat model

Alzheimer’s disease (AD), as the most typical type of dementia, is a chronic neurodegenerative disorder characterized by progressive learning and memory impairment. It is known that the main causes of AD are the accumulation of β-amyloid (Aβ) plaques and neurofibrillary tangles (NFT) containing hyperphosphorylated tau protein. Naringin is a flavonoid from citrus fruits, especially in grapefruit, which has anti-inflammatory, antioxidant, anti-apoptotic, and neuroprotective activities. However, the effect of naringin in AD caused by Aβ has not been clearly studied, and there are few studies on the electrophysiological aspect. Thus, we investigated the ex vivo neuroprotective effect of naringin through the long-term potentiation (LTP) on organotypic hippocampal slice cultures. We evaluated the in vivo effects of naringin (100 mg/kg/day) orally treated for 20 days on learning, memory, and cognition which was impaired by bilateral CA1 subregion injection of Aβ. Cognitive behaviors were measured 2 weeks after Aβ injection using behavioral tests and the hippocampal expression of apoptotic and neurotrophic regulators were measured by immunoblotting. In hippocampal tissue slices, naringin dose-dependently increased the field excitatory postsynaptic potential (fEPSP) after theta burst stimulation and attenuated Aβ-induced blockade of fEPSP in the hippocampal CA1 area. In Aβ injected rats, naringin improved object recognition memory in the novel object test, avoidance memory in the passive avoidance test and spatial recognition memory in the Morris water maze test. In the hippocampus, naringin attenuated the Aβ-induced cyclooxygenase-2, Bax activation and Bcl-2, CREB, BDNF and TrkB inhibition. These results suggest that naringin has therapeutic potential to reduce neuronal inflammation and apoptosis induced by Aβ related with the BDNF/TrkB/CREB signaling.

Neuroinflammation regulates the balance between hippocampal neuron death and neurogenesis in an ex vivo model of thiamine deficiency

BackgroundThiamine (vitamin B1) is a cofactor for enzymes of central energy metabolism and its deficiency (TD) impairs oxidative phosphorylation, increases oxidative stress, and activates inflammatory processes that can lead to neurodegeneration. Wernicke–Korsakoff syndrome (WKS) is a consequence of chronic TD, which leads to extensive neuronal death, and is associated with neuropathological disorders, including cognitive deficits and amnesia. The hippocampus is one of the brain areas most affected by WKS. B1 replacement may not be enough to prevent the irreversible cognitive deficit associated with WKS.Materials and methodsAn organotypic hippocampal slice culture (OHC) model was developed to investigate, using immunofluorescence and confocal microscopy and transcriptome analysis, the molecular mechanisms underlying the neurodegeneration associated with TD. The effect of anti-inflammatory pharmacological intervention with resveratrol (RSV) was also assessed in B1-deprived OHCs.ResultsIn OHCs cultured without B1, neuronal density decayed after 5 days and, on the 7th day, the epigenetic markings H3K4me3 and H3K9me3 were altered in mature neurons likely favoring gene transcription. Between the 7th and the 14th day, a pulse of neurogenesis was observed followed by a further massive neuron loss. Transcriptome analysis at day nine disclosed 89 differentially expressed genes in response to B1 deprivation. Genes involved in tryptophan metabolism and lysine degradation KEGG pathways, and those with Gene Ontology (GO) annotations related to the organization of the extracellular matrix, cell adhesion, and positive regulation of synaptic transmission were upregulated. Several genes of the TNF and FoxO signaling pathways and with GO terms related to inflammation were inhibited in response to B1 deprivation. Nsd1, whose product methylates histone H3 lysine 36, was upregulated and the epigenetic marking H3K36me3, associated with negative regulation of neurogenesis, was increased in neurons. Treating B1-deprived OHCs with RSV promoted an earlier neurogenesis pulse.ConclusionNeuroregeneration occurs in B1-deficient hippocampal tissue during a time window. This phenomenon depends on reducing neuroinflammation and, likely, on metabolic changes, allowing acetyl-CoA synthesis from amino acids to ensure energy supply via oxidative phosphorylation. Thus, neuroinflammation is implicated as a major regulator of hippocampal neurogenesis in TD opening a new search space for treating WKS.