Cultured Mouse Brain Research Articles

Temporal dynamics of neocortical development in organotypic mouse brain cultures: a comprehensive analysis.

Murine organotypic brain slice cultures have been widely used in neuroscientific research and are offering the opportunity to study neuronal function under normal and disease conditions. Despite the broad application, the mechanisms governing the maturation of immature cortical circuits in vitro are not well understood. In this study, we present a detailed investigation into the development of the neocortex in vitro. Using a holistic approach, we studied organotypic whole hemisphere brain slice cultures from postnatal mice and tracked the development of the somatosensory area over a 5-wk period. Our analysis revealed the maturation of passive and active intrinsic properties of pyramidal cells together with their morphology, closely resembling in vivo development. Detailed multielectrode array (MEA) electrophysiological assessments and RNA expression profiling demonstrated stable network properties by 2 wk in culture, followed by the transition of spontaneous activity toward more complex patterns including high-frequency oscillations. However, culturing weeks 4 and 5 exhibited increased variability and initial signs of neuronal loss, highlighting the importance of considering developmental stages in experimental design. This comprehensive characterization is vital for understanding the temporal dynamics of the neocortical development in vitro, with implications for neuroscientific research methodologies, particularly in the investigation of diseases such as epilepsy and other neurodevelopmental disorders.NEW & NOTEWORTHY The development of the mouse neocortex in vitro mimics the in vivo development. Mouse brain cultures can serve as a model system for cortical development for the first 2 wk in vitro and as a model system for the adult cortex from 2 to 4 wk in vitro. Mouse organotypic brain slice cultures develop high-frequency network oscillations at γ frequency after 2 wk in vitro. Mouse brain cultures exhibit increased heterogeneity and variability after 4 wk in culture.

EcoHIV Infection of Primary Murine Brain Cell Cultures to Model HIV Replication and Neuropathogenesis.

EcoHIV is a chimeric HIV that replicates in mice in CD4+ T cells, macrophages, and microglia (but not in neurons), causing lasting neurocognitive impairment resembling neurocognitive disease in people living with HIV. The present study was designed to develop EcoHIV-susceptible primary mouse brain cultures to investigate the indirect effects of HIV infection on neuronal integrity. We used two EcoHIV clones encoding EGFP and mouse bone marrow-derived macrophages (BMM), mixed mouse brain cells, or enriched mouse glial cells from two wild-type mouse strains to test EcoHIV replication efficiency, the identity of productively infected cells, and neuronal apoptosis and integrity. EcoHIV replicated efficiently in BMM. In mixed brain cell cultures, EcoHIV targeted microglia but did not cause neuronal apoptosis. Instead, the productive infection of the microglia activated them and impaired synaptophysin expression, dendritic density, and axonal structure in the neurons. EcoHIV replication in the microglia and neuronal structural changes during infection were prevented by culture with an antiretroviral. In murine brain cell cultures, EcoHIV replication in the microglia is largely responsible for the aspects of neuronal dysfunction relevant to cognitive disease in infected mice and people living with HIV. These cultures provide a tool for further study of HIV neuropathogenesis and its control.

Localization of the Stem Cells Exosomes in the Brain and in the Cultured Mouse Brain Cells

Localization of the Stem Cells Exosomes in the Brain and in the Cultured Mouse Brain Cells

Involvement of the peroxisome proliferator-activated receptor gamma (Pparγ) and matrix metalloproteinases-2 and -9 (Mmp-2 and -9) in the mechanism of action of di(2-ethylhexyl)phthalate (DEHP) in cultured mouse brain astrocytes and neurons

Di(2-ethylhexyl)phthalate (DEHP) is one of the most widely used phthalates in industry. It has been shown that, after entering the body, DEHP has the ability to cross the blood-placenta and blood-brain barriers. One of the proposed mechanisms of action of DEHP is the activation of peroxisome proliferator-activated receptors (PPARs). Many different functions of PPARγ in cells have been demonstrated, one of which is the modulation of the activation of matrix metalloproteinases (MMPs). The aim of this study was to investigate the role of Pparγ, Mmp-2, and Mmp-9 in the mechanism of action of DEHP. The experiments were performed on in vitro primary murine neurons and astrocytes. The results showed that DEHP has a pro-apototic effect on neurons, causing an increase in caspase-3 activity and in the number of apoptotic bodies. However, in astrocytes, the increase in caspase-3 activity was not related to the apoptosis process, as no increase in the formation of apoptotic bodies was observed. Moreover, DEHP increased the proliferation of astrocytes, which was confirmed by the increase in the amount and expression of the Ki-67 protein. In astrocytes, DEHP decreased the expression of the Pparγ and Mmp-9 proteins but increased the expression of the Mmp-2 protein. In DEHP neurons, it increased the expression of the Pparγ protein but decreased the expression of the Mmp-2 and Mmp-9 proteins.

Helicobacter pylori infection selectively attenuates endothelial function in male mice via exosomes-mediated ROS production.

Substantial sex differences exist in atherosclerosis. Excessive reactive oxygen species (ROS) formation could lead to endothelial dysfunction which is critical to atherosclerosis development and progression. Helicobacter pylori (H. pylori) infection has been shown to attenuate endothelial function via exosomes-mediated ROS formation. We have demonstrated that H. pylori infection selectively increases atherosclerosis risk in males with unknown mechanism(s). The present study was to test the hypothesis that H. pylori infection impaired endothelial function selectively in male mice through exosome-mediated ROS formation. Age-matched male and female C57BL/6 mice were infected with CagA+ H. pylori to investigate sex differences in H. pylori infection-induced endothelial dysfunction. H. pylori infection attenuated acetylcholine (ACh)-induced endothelium-dependent aortic relaxation without changing nitroglycerine-induced endothelium-independent relaxation in male but not female mice, associated with increased ROS formation in aorta compared with controls, which could be reversed by N-acetylcysteine treatment. Treatment of cultured mouse brain microvascular endothelial cells with exosomes from H. pylori infected male, not female, mice significantly increased intracellular ROS production and impaired endothelial function with decreased migration, tube formation, and proliferation, which could be prevented with N-acetylcysteine treatment. H. pylori infection selectively impairs endothelial function in male mice due to exosome-mediated ROS formation.

Brain aging is faithfully modelled in organotypic brain slices and accelerated by prions

Mammalian models are essential for brain aging research. However, the long lifespan and poor amenability to genetic and pharmacological perturbations have hindered the use of mammals for dissecting aging-regulatory molecular networks and discovering new anti-aging interventions. To circumvent these limitations, we developed an ex vivo model system that faithfully mimics the aging process of the mammalian brain using cultured mouse brain slices. Genome-wide gene expression analyses showed that cultured brain slices spontaneously upregulated senescence-associated genes over time and reproduced many of the transcriptional characteristics of aged brains. Treatment with rapamycin, a classical anti-aging compound, largely abolished the time-dependent transcriptional changes in naturally aged brain slice cultures. Using this model system, we discovered that prions drastically accelerated the development of age-related molecular signatures and the pace of brain aging. We confirmed this finding in mouse models and human victims of Creutzfeldt-Jakob disease. These data establish an innovative, eminently tractable mammalian model of brain aging, and uncover a surprising acceleration of brain aging in prion diseases.

Phillyrin Prevents Neuroinflammation-Induced Blood-Brain Barrier Damage Following Traumatic Brain Injury via Altering Microglial Polarization.

Background: Phillyrin (Phi) is the main polyphenolic compound found in Forsythia suspensa. Recent studies have revealed that Phi has potent antioxidative and anti-inflammatory effects. However, whether Phi could relieve blood–brain barrier (BBB) damage following traumatic brain injury (TBI) remains unknown. Materials and Methods: Lipopolysaccharide (LPS) was used to activate primary microglia, which were then treated with different doses of Phi or the peroxisome proliferator–activated receptor-gamma (PPARγ) antagonist (GW9662). CCK-8 assay was used for evaluating cell viability, and the cytokines (including IL-1β, IL-6, TNFα, IL-4, IL-10, and TGFβ), microglial phenotypic markers (iNOS, COX2, and CD86 for “M1” polarization; Arg1, Ym1, and CD206 for “M2” polarization), PPARγ, and NF-κB were determined by RT-PCR, Western blot, or cellular immunofluorescence. Primary cultured mouse brain microvascular endothelial cells (BMECs) were stimulated by the condition medium (CM) from microglia. The cell viability, angiogenesis, and tight junction of BMECs were determined via CCK-8 assay, tube formation assay, and Western blot (for detecting MMP3, MMP9, ZO1, claudin-5, and occludin). Furthermore, the mouse TBI model was constructed and treated with Phi and/or GW9662. The BBB integrity was evaluated by H&E staining, Evans blue staining, and tissue immunofluorescence. Results: Phi markedly restrained the pro-inflammatory (“M1” state) cytokines and promoted anti-inflammatory (“M2” polarization) cytokines in LPS-mediated microglia. Phi mitigated “M1” polarization and promoted “M2” polarization of microglia via enhancing PPARγ and inhibiting the NF-κB pathway. The PPARγ antagonist GW9662 significantly repressed Phi-mediated anti-inflammatory effects. Meanwhile, Phi enhanced the viability, tube formation ability, and cell junction of BMECs. In the TBI mouse model, Phi promoted “M2” polarization, whereas it repressed the “M1” polarization of microglia. In addition, Phi reduced TBI-mediated BBB damage. However, the protective effects of Phi were reversed mainly by GW9662 treatment. Conclusion: Phi prevents BBB damage via inhibiting the neuroinflammation of microglia through the PPARγ/NF-κB pathway, which provides a potential therapeutic drug against TBI.

The Effects of Fibrinogen's Interactions with Its Neuronal Receptors, Intercellular Adhesion Molecule-1 and Cellular Prion Protein.

Neuroinflammatory diseases, such as Alzheimer’s disease (AD) and traumatic brain injury (TBI), are associated with the extravascular deposition of the fibrinogen (Fg) derivative fibrin and are accompanied with memory impairment. We found that during the hyperfibrinogenemia that typically occurs during AD and TBI, extravasated Fg was associated with amyloid beta and astrocytic cellular prion protein (PrPC). These effects coincided with short-term memory (STM) reduction and neurodegeneration. However, the mechanisms of a direct Fg–neuron interaction and its functional role in neurodegeneration are still unclear. Cultured mouse brain neurons were treated with Fg in the presence or absence of function-blockers of its receptors, PrPC or intercellular adhesion molecule-1 (ICAM-1). Associations of Fg with neuronal PrPC and ICAM-1 were characterized. The expression of proinflammatory marker interleukin 6 (IL-6) and the generation of reactive oxygen species (ROS), mitochondrial superoxide, and nitrite in neurons were assessed. Fg-induced neuronal death was also evaluated. A strong association of Fg with neuronal PrPC and ICAM-1, accompanied with overexpression of IL-6 and enhanced generation of ROS, mitochondrial superoxide, and nitrite as well as the resulting neuronal death, was found. These effects were reduced by blocking the function of neuronal PrPC and ICAM-1, suggesting that the direct interaction of Fg with its neuronal receptors can induce overexpression of IL-6 and increase the generation of ROS, nitrite, and mitochondrial superoxide, ultimately leading to neuronal death. These effects can be a mechanism of neurodegeneration and the resultant memory reduction seen during TBI and AD.

Selective striatal cell loss is ameliorated by regulated autophagy of the cortex

AimsThe harmful cellular environment leads to brain damage, and each brain subregion exhibits a differential vulnerability to its effects. This study investigated the causes of selectively striatal cell loss in systemic 3-nitropropionic acid (3-NP) infused mice. Main methodsThis study was performed in the neuronal cell line, primary neuron, cultured mouse brain, and mice brain tissues. The 3-NP solution was delivered using an osmotic mini-pump system for 7 days. ROS in brain tissue were detected and evaluated with the signals of CM-H2DCFDA for total cellular ROS and MitoSOX Red for mitochondrial ROS. Cellular ROS and the functional status of mitochondria were assessed with a detection kit and analyzed using flow cytometry. To quantify oxidative damaged DNA, apurinic/apyrimidinic (AP) site numbers in DNA were measured. The protein expression level was assessed using Western blotting, and immunohistochemistry was performed. Cleaved caspase-3 activities were measured by using an enzyme-linked immunosorbent assay (ELISA) kit. Key findingsBy 3-NP, mitochondrial dysfunction was higher in the striatum than in the cortex, and mitochondria-derived ROS levels were higher in the striatum than in the cortex. However, autophagy that may restore the energy depletion resulting from mitochondrial dysfunction occurred comparably less in the striatum than in the cortex. Inhibition of ASK1 by NQDI1 regulates MAPK signaling, apoptosis, and autophagy. Regulated autophagy of the cortex improved non-cell autonomously striatal damaged condition. SignificanceThis study illustrated that the different vulnerabilities of the brain subregions, striatum or cortex, against 3-NP are rooted in different mitochondria-derived ROS amounts and autophagic capacity.

Fibrinogen Interaction with Astrocyte ICAM-1 and PrPC Results in the Generation of ROS and Neuronal Death

Many neuroinflammatory diseases, like traumatic brain injury (TBI), are associated with an elevated level of fibrinogen and short-term memory (STM) impairment. We found that during TBI, extravasated fibrinogen deposited in vasculo-astrocyte interfaces, which was associated with neurodegeneration and STM reduction. The mechanisms of this fibrinogen-astrocyte interaction and its functional role in neurodegeneration are still unclear. Cultured mouse brain astrocytes were treated with fibrinogen in the presence or absence of function-blocking antibody or peptide against its astrocyte receptors intercellular adhesion molecule-1 (ICAM-1) or cellular prion protein (PrPC), respectively. Fibrinogen interactions with astrocytic ICAM-1 and PrPC were characterized. The expression of pro-inflammatory markers, generations of reactive oxygen species (ROS) and nitric oxide (NO) in astrocytes, and neuronal death caused by astrocyte-conditioned medium were assessed. Data showed a strong association between fibrinogen and astrocytic ICAM-1 or PrPC, overexpression of pro-inflammatory cytokines and overproduction of ROS and NO, resulting in neuronal apoptosis and death. These effects were reduced by blocking the function of astrocytic ICAM-1 and PrPC, suggesting that fibrinogen association with its astrocytic receptors induce the release of pro-inflammatory cytokines, resulting in oxidative stress, and ultimately neuronal death. This can be a mechanism of neurodegeneration and the resultant STM reduction seen during TBI.

Ultrastructural Imaging of Activity-Dependent Synaptic Membrane-Trafficking Events in Cultured Brain Slices

SummaryElectron microscopy can resolve synapse ultrastructure with nanometer precision, but the capture of time-resolved, activity-dependent synaptic membrane-trafficking events has remained challenging, particularly in functionally distinct synapses in a tissue context. We present a method that combines optogenetic stimulation-coupled cryofixation (“flash-and-freeze”) and electron microscopy to visualize membrane trafficking events and synapse-state-specific changes in presynaptic vesicle organization with high spatiotemporal resolution in synapses of cultured mouse brain tissue. With our experimental workflow, electrophysiological and “flash-and-freeze” electron microscopy experiments can be performed under identical conditions in artificial cerebrospinal fluid alone, without the addition of external cryoprotectants, which are otherwise needed to allow adequate tissue preservation upon freezing. Using this approach, we reveal depletion of docked vesicles and resolve compensatory membrane recycling events at individual presynaptic active zones at hippocampal mossy fiber synapses upon sustained stimulation.

Development and validation of an LC-MS/MS method for the quantitative analysis of milciclib in human and mouse plasma, mouse tissue homogenates and tissue culture medium

Milciclib is a promising cyclin-dependent kinase inhibitor currently in phase II clinical trials to treat several types of cancer. The first bioanalytical method for the quantitative analysis of milciclib in several biomatrices using liquid chromatography-tandem mass spectrometry is described here. This method was fully validated in human plasma according to FDA and EMA guidelines, and partially validated in mouse plasma, homogenates of mouse brain, kidney, liver, small intestine, spleen, and tissue culture medium. Palbociclib, an analog compound, was used as internal standard. A simple and fast sample pre-treatment by protein precipitation with acetonitrile was used, leading to efficient extraction of the analyte with recoveries between 95–100%. Chromatographic separation was achieved with a C18 analytical column and a gradient elution using 10 mM ammonium bicarbonate in water and 10 mM ammonium bicarbonate in water-methanol (1:9, v/v). This assay was selective, accurate, precise and linear in the concentration range of 1−1000 ng/mL. Moreover, samples above the upper limit of quantification can be integrally diluted up to 10-fold prior to analysis. The use of human plasma as a surrogate matrix to quantify milciclib in tissue culture medium and mouse matrices resulted in acceptable accuracy and precision, however tissue culture medium samples required a dilution with human plasma prior the pre-treatment. All performance parameters of the method complied with the acceptance criteria recommended by the guidelines, except for the carry-over, which was slightly above (22.9% of the lower limit of quantification) the recommended percentage (20%). Therefore, additional measures were taken to assure data integrity. Stability of milciclib in all matrices was evaluated, and in some matrices the analyte was unstable under the tested conditions. It is therefore recommended to keep these samples as briefly as possible at room temperature during the pre-treatment, and to store them at –70 °C to avoid analyte degradation. This method was successfully applied to support preclinical pharmacokinetic studies of milciclib.

Fibrinogen-cellular prion protein complex formation on astrocytes.

Traumatic brain injury (TBI) is one of the most common neurological disorders causing memory reduction, particularly short-term memory (STM). We showed that, during TBI-induced inflammation, increased blood content of fibrinogen (Fg) enhanced vascular protein transcytosis and deposition of extravasated Fg in vasculo-astrocyte interfaces. In addition, we found that deposition of cellular prion protein (PrPC) was also increased in the vasculo-astrocyte endfeet interface. However, association of Fg and PrPC was not confirmed. Presently, we aimed to define whether Fg can associate with PrPC on astrocytes and cause their activation. Cultured mouse brain astrocytes were treated with medium alone (control), Fg (2 mg/mL or 4 mg/mL), 4 mg/mL of Fg in the presence of a function-blocking anti-PrPC peptide or anti-mouse IgG, function-blocking anti-PrPC peptide, or anti-mouse IgG alone. After treatment, either cell lysates were collected and analyzed via Western blot or coimmunoprecipitation was performed, or astrocytes were fixed and their activation was assessed with immunohistochemistry. Results showed that Fg dose-dependently activated astrocytes, increased expressions of PrPC and tyrosine (tropomyosin) receptor kinase B (TrkB), and PrP gene. Blocking the function of PrPC reduced these effects. Coimmunoprecipitation demonstrated Fg and PrPC association. Since it is known that prion protein has a greater effect on memory reduction than amyloid beta, and that activation of TrkB is involved in neurodegeneration, our findings confirming the possible formation of Fg-PrPC and Fg-induced overexpression of TrkB on astrocytes suggest a possible triggering mechanism for STM reduction that was seen previously during mild-to-moderate TBI.NEW & NOTEWORTHY For the first time we showed that fibrinogen (Fg) can associate with cellular prion protein (PrPC) on the surface of cultured mouse brain astrocytes. At high levels, Fg causes upregulation of astrocyte PrPC and astrocyte activation accompanied with overexpression of tyrosine receptor kinase B (TrkB), which results in nitric oxide (NO) production and generation of reactive oxygen species (ROS). Fg/PrPC interaction can be a triggering mechanism for TrkB-NO-ROS axis activation and the resultant astrocyte-mediated neurodegeneration.

UV irradiation of Type I collagen gels changed the morphology of the interconnected brain capillary endothelial cells on them.

We cultured mouse brain capillary endothelial cell line bEnd.3 on the UV-irradiated Type I collagen gel. Morphology of bEnd.3 cells on the Type I collagen gel was drastically changed if the gel was crosslinked by UV irradiation. The interconnecting network of bEnd.3 cells which have cord-like morphology on the soft collagen gels was converted to the monolayer of the flat cells, tightly-bound each other covering the gel surface, in a confluent state. The collagen gels were mechanically stiffened by UV irradiation for 15min with UV light at 254nm showing approximately two times higher value of Young's modulus E (1.51±0.58kPa) than the control gel (3.17±1.17kPa). AFM images of the collagen fibrils were not severely changed after irradiation. Collagen subunit proteins were crosslinked and degraded simultaneously under UV irradiation proved by results of SDS-PAGE and separation by centrifugation. Expression of Integrin gene was measured by quantitative real-time PCR. Expression of the integrin α2 gene, tight junction protein 1 gene, and claudin 5 gene were down-regulated in cells on the UV irradiated collagen gel in comparison with the unirradiated one while expression of the integrin β1 gene and Integrin α1 gene did not significantly change. Thick actin filaments were more clearly observed in the cells on the UV-irradiated collagen gel than the unirradiated one by fluorescent microscopy. We conclude that UV irradiation made the collagen gel stiffened and changed the physiological state of bEnd.3 cells including their adhesion, extension, and proliferation.

Nanoparticle Delivery of CD147 Antagonistic Peptide-9 Protects against Acute Ischemic Brain Injury and tPA-Induced Intracerebral Hemorrhage in Mice.

CD147 has emerged as a potential therapeutic target in many human diseases. We have demonstrated that inhibition of CD147 using its function-blocking antibody ameliorates acute ischemic brain injury and promotes long-term functional recovery in mice. Recently, peptide-nanoparticle conjugates have emerged as powerful tools for biomedical applications. The present study aimed to investigate the therapeutic potential of CD147 antagonist peptide-9 (AP9) in acute ischemic stroke in mice using nanomaterial as the drug delivery vehicles. AP9-conjugated nanoparticles (APN), with an average size of about 40 nm, were fabricated by maleimide linkage and characterized using dynamic light scattering and transmission electron microscopy. We found that APN specifically bound to CD147 in cultured mouse brain endothelial cells (bEnd.3) and to ischemia-induced CD147 in mouse cerebral microvessels. Using a mouse model of transient middle cerebral artery occlusion (tMCAO), we demonstrated, for the first time, that systemic delivery of APN (2.5 mg/kg, I.V.) initiated at 1 h after tMCAO significantly reduced brain infarct size, improved functional outcome, and attenuated delayed (5 h after tMCAO) tPA-induced intracerebral hemorrhage in acute ischemic stroke. These protective effects were associated with profound inhibition of MMP-9 and MMP-3 in both ischemic brain and plasma. In conclusion, the CD147 antagonist peptide-9 represents a potentially promising therapeutic candidate for the treatment of ischemic stroke.

Inhibition of glycogen synthase kinase 3β improves cognitive function in aged mice by upregulating claudin presences in cerebral endothelial cells.

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, is widely distributed in mammalian brains. Since GSK-3β plays a vital role in the development of neurodegenerative disorders, the present study was designed to investigate the role of GSK-3β in the blood-brain barrier (BBB) permeability in aged mice. Morris water maze test was used to examine mouse cognitive function. BBB permeability was examined by the leakage of fluorescence signals of low-molecular weight dextran. GSK-3β inhibitor, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), was administrated in aged mice and in cultured mouse brain microvascular endothelial cells (bEnd.3). Compared with young mice, aged mice had increased leftover signals of dextran in the hippocampus and a lower score in the maze test, suggesting that aged mice have abnormal leakage of BBB and cognitive dysfunction. The protein expression of Toll-like receptor 4 (TLR4) was increased, whereas the protein expressions of junction proteins (claudin1 and claudin5) were reduced in endothelial cells of BBB in aged mice. Phosphorylated level of serine 9, an inhibitory residue in GSK-3β protein, was decreased. TDZD-8 treatment downregulated TLR4 protein expression, upregulated claudin1 and claudin5 protein expressions, and significantly improved cognitive function in aged mice. In bEnd.3 cells, TDZD-8 treatment reduced TLR4 expression and increased claudin5 expression in cells stimulated with lipopolysaccharides. In conclusion, the inhibition of GSK-3β activity downregulates aging-induced TLR4 expression and restores the BBB integrity, resulting in the improvement of cognitive function in aged mice.

Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion.

Prion or PrPSc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrPSc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89–230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrPSc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion.

Dendritic Polyglycerols Are Modulators of Microglia-Astrocyte Crosstalk

Aim: To determine the ability of sulfated dendritic polyglycerols (dPGS) to modulate neuroglia activation challenged with lipopolysaccharide (LPS). Materials & methods: Microglia/astrocyte activation in vivo was determined in transgenic animals expressing TLR2-/GFAP-luciferase reporter. Mechanisms implicated in microglia-astrocyte crosstalk were studied in primary mouse brain cultures. Results & discussion: dPGS significantly reduced microglia activation in vivo, and decreased astrocytic LCN2 production. Activated microglia are necessary for astrocyte stimulation and increase in LCN2 abundance. LCN2 production in astrocytes involves signaling via toll-like receptor 4, activation of NF-κB, IL6 and enhancement of reactive oxygen species. Conclusion: dPGS are powerful modulators of microglia-astrocyte crosstalk and LCN2 abundance; dPGS are promising anti-inflammatory dendritic nanostructures.

Cryogel scaffolds for regionally constrained delivery of lysophosphatidylcholine to central nervous system slice cultures: A model of focal demyelination for multiple sclerosis research

The pathology of multiple sclerosis (MS) is typified by focal demyelinated areas of the brain and spinal cord, which results in axonal degeneration and atrophy. Although the field has made much progress in developing immunomodulatory therapies to reduce the occurrence of these focal lesions, there is a conspicuous lack of licensed effective therapies to reduce axonal degeneration or promote repair. Remyelination, carried out by oligodendrocytes, does occur in MS, and is protective against axonal degeneration. Unfortunately, remyelination is not very efficient, and ultimately fails and so there is a research focus to generate new therapeutics to enhance remyelination leading to neuroprotection.To develop these therapies, we need preclinical models that well reflect remyelination in MS. We have previously characterized an ex vivo model that uses lysophosphatidylcholine (LPC) to cause acute and global demyelination of tissue slices, followed by spontaneous remyelination, which has been widely used as a surrogate for in vivo rodent models of demyelination. However, this ex vivo model lacks the focal demyelinated lesions seen in MS, surrounded by normal tissue from which the repairing oligodendrocytes are derived. Therefore, to improve the model, we have developed and characterized small macroporous cryogel scaffolds for controlled/regional delivery of LPC with diameters of either 0.5, 1 or 2 mm. Placement of LPC loaded scaffolds adjacent to ex vivo cultured mouse brain and spinal cord slices induced focal areas of demyelination in proximity to the scaffold. To the best of our knowledge, this is the first such report of spatial mimicry of the in vivo condition in ex vivo tissue culture. This will allow not only the investigation into focal lesions, but also provides a better platform technology with which to test remyelination-promoting therapeutics. Statement of SignificanceThis manuscript is the first report of using macroporous hydrogels (cryogels) as a research tool for lysophosphatidylcholine (LPC) delivery, in order to create an ex vivo model of focal demyelination in the brain and spinal cord, which is of great relevance to multiple sclerosis research.Here, we transform an existing ex vivo model of demyelination by delivering LPC to focal regions of brain and spinal cord slice cultures. We have developed an easy-to-handle cylindrical and macroporous PEG-based sponge-like scaffold material (cryogel) that can deliver LPC only to a small area of the slice. Such cryogels are ideal as a delivery system in this culture model as they exhibit a soft but robust nature, with high mechanical deformability in their dry and swollen state, with no need to stay permanently hydrated. In addition, the synthesis of these cryogels is simple and easy to reproduce via photochemical cryopolymerisation using a PEG-diacrylate monomer and a photoinitiator, which are both commercially available.This more accurate model of demyelination will not only allow researchers to gain a better understanding of the CNS remyelination process in diseases such as MS, but also provides a platform technology, which could be utilized to screen and test pro-remyelination compounds which may help to find new therapeutics for progressive MS.

Involvement of Toll-like receptor 2 in the cerebral immune response and behavioral changes caused by latent Toxoplasma infection in mice.

Subacute and chronic infections with the intracellular protozoan parasite Toxoplasma gondii are associated with an increased risk of psychiatric diseases like schizophrenia. However, little is known about the mechanisms involved in T. gondii-induced neuronal disorders. Recently, we reported that Toll-like receptor 2 (TLR2) was required to initiate the innate immune response in cultured mouse brain cells. However, how TLR2 contributes to latent infection with T. gondii remains unclear. Therefore, we examined the role of TLR2 in brain pathology and behavior using wild-type (TLR2+/+) and TLR2-deficient (TLR2-/-) mice. The behavioral analyses showed that TLR2 deficiency increased the anxiety state of the uninfected and infected animals alike, and TLR2 deficiency showed no relationship with the infection. In the contextual and cued fear-conditioning tests, T. gondii infection decreased the mouse freezing reaction while TLR2 deficiency increased it, but there was no interaction between the two factors. Our histopathological analysis showed that the TLR2+/+ and TLR2-/- mice had similar brain lesions at 30 days post infection (dpi) with T. gondii. Higher numbers of parasites were detected in the brains of the TLR2-/- mice than in those from the TLR2+/+ mice at 30 dpi, but not at 7 and 14 dpi. No significant differences were observed in the proinflammatory gene expression levels in the TLR2+/+ and TLR2-/- mice. Therefore, it appears that TLR2 signaling in the brain might contribute to the control of parasite growth, but not to brain pathology or the impaired fear memory response induced by infection with T. gondii.