Brain Slice Cultures Research Articles

Tau Internalization is Regulated by 6-O Sulfation on Heparan Sulfate Proteoglycans (HSPGs)

The misfolding and accumulation of tau protein into intracellular aggregates known as neurofibrillary tangles is a pathological hallmark of neurodegenerative diseases such as Alzheimer’s disease. However, while tau propagation is a known marker for disease progression, exactly how tau propagates from one cell to another and what mechanisms govern this spread are still unclear. Here, we report that cellular internalization of tau is regulated by quaternary structure and have developed a cellular assay to screen for genetic modulators of tau uptake. Using CRISPRi technology we have tested 3200 genes for their ability to regulate tau entry and identified enzymes in the heparan sulfate proteoglycan biosynthetic pathway as key regulators. We show that 6-O-sulfation is critical for tau-heparan sulfate interactions and that this modification regulates uptake in human central nervous system cell lines, iPS-derived neurons, and mouse brain slice culture. Together, these results suggest novel strategies to halt tau transmission.

Xenon Protects against Blast-Induced Traumatic Brain Injury in an In Vitro Model.

The aim of this study was to evaluate the neuroprotective efficacy of the inert gas xenon as a treatment for patients with blast-induced traumatic brain injury in an in vitro laboratory model. We developed a novel blast traumatic brain injury model using C57BL/6N mouse organotypic hippocampal brain-slice cultures exposed to a single shockwave, with the resulting injury quantified using propidium iodide fluorescence. A shock tube blast generator was used to simulate open field explosive blast shockwaves, modeled by the Friedlander waveform. Exposure to blast shockwave resulted in significant (p < 0.01) injury that increased with peak-overpressure and impulse of the shockwave, and which exhibited a secondary injury development up to 72 h after trauma. Blast-induced propidium iodide fluorescence overlapped with cleaved caspase-3 immunofluorescence, indicating that shock-wave–induced cell death involves apoptosis. Xenon (50% atm) applied 1 h after blast exposure reduced injury 24 h (p < 0.01), 48 h (p < 0.05), and 72 h (p < 0.001) later, compared with untreated control injury. Xenon-treated injured slices were not significantly different from uninjured sham slices at 24 h and 72 h. We demonstrate for the first time that xenon treatment after blast traumatic brain injury reduces initial injury and prevents subsequent injury development in vitro. Our findings support the idea that xenon may be a potential first-line treatment for those with blast-induced traumatic brain injury.

Interaction of Normal and Antineuronal IgGs with Neurons in Rat Brain Slice Cultures: Comparison of IgG Uptake, Clearance, Intraneuronal Binding to Target Antigens, and Production of Neuronal Death (P2.403)

Interaction of Normal and Antineuronal IgGs with Neurons in Rat Brain Slice Cultures: Comparison of IgG Uptake, Clearance, Intraneuronal Binding to Target Antigens, and Production of Neuronal Death (P2.403)

A novel ex vivo Huntington's disease model for studying GABAergic neurons and cell grafts by laser microdissection.

Organotypic brain slice cultures have been recently used to study neurodegenerative disorders such as Parkinson’s disease and Huntington’s disease (HD). They preserve brain three-dimensional architecture, synaptic connectivity and brain cells microenvironment. Here, we developed an innovative model of Huntington’s disease from coronal rat brain slices, that include all the areas involved in the pathology. HD-like neurodegeneration was obtained in only one week, in a single step, during organotypic slice preparation, without the use of neurotoxins. HD-like histopathology was analysed and after one week, a reduction of 40% of medium spiny neurons was observed. To analyse new therapeutic approaches in this innovative HD model, we developed a novel protocol of laser microdissection to isolate and analyse by RT-qPCR, grafted cells as well as surrounding tissue of fresh organotypic slices. We determined that laser microdissection could be performed on a 400μm organotypic slice after alcohol dehydration protocol, allowing the analysis of mRNA expression in the rat tissue as well as in grafted cells. In conclusion, we developed a new approach for modeling Huntington's disease ex vivo, and provided a useful innovative method for screening new potential therapies for neurodegenerative diseases especially when associated with laser microdissection.

Peripherally derived T regulatory and γδ T cells have opposing roles in the pathogenesis of intractable pediatric epilepsy.

The pathophysiology of drug-resistant pediatric epilepsy is unknown. Flow cytometric analysis of inflammatory leukocytes in resected brain tissues from 29 pediatric patients with genetic (focal cortical dysplasia) or acquired (encephalomalacia) epilepsy demonstrated significant brain infiltration of blood-borne inflammatory myeloid cells and memory CD4+ and CD8+ T cells. Significantly, proinflammatory (IL-17- and GM-CSF-producing) γδ T cells were concentrated in epileptogenic lesions, and their numbers positively correlated with disease severity. Conversely, numbers of regulatory T (T reg) cells inversely correlated with disease severity. Correspondingly, using the kainic acid model of status epilepticus, we show ameliorated seizure activity in both γδ T cell- and IL-17RA-deficient mice and in recipients of T reg cells, whereas T reg cell depletion heightened seizure severity. Moreover, both IL-17 and GM-CSF induced neuronal hyperexcitability in brain slice cultures. These studies support a major pathological role for peripherally derived innate and adaptive proinflammatory immune responses in the pathogenesis of intractable epilepsy and suggest testing of immunomodulatory therapies.

Visualization of Rostral Migratory Stream in the Developing Rat Brain by In Vivo Electroporation.

Interneurons in the olfactory bulb (OB) are generated from neuronal precursor cells migrating from anterior subventricular zone (SVZa) not only in the developing embryo but also throughout the postnatal life of mammals. In the present study, we established an in vivo electroporation assay to label SVZa cells of rat both at embryonic and postnatal ages, and traced SVZa progenitors and followed their migration pathway and differentiation. We found that labeled cells displayed high motility. Interestingly, the postnatal cells migrated faster than the embryonic cells after applying this assay at different ages of brain development. Furthermore, based on brain slice culture and time-lapse imaging, we analyzed the detail migratory properties of these labeled precursor neurons. Finally, tissue transplantation experiments revealed that cells already migrated in subependymal zone of OB were transplanted back into rostral migratory stream (RMS), and these cells could still migrate out tangentially along RMS to OB. Taken together, these findings provide an in vivo labeling assay to follow and trace migrating cells in the RMS, their maturation and integration into OB neuron network, and unrecognized phenomena that postnatal SVZa progenitor cells with higher motility than embryonic cells, and their migration was affected by extrinsic environments.

Targeting the cMET pathway augments radiation response without adverse effect on hearing in NF2 schwannoma models

Neurofibromatosis type II (NF2) is a disease that needs new solutions. Vestibular schwannoma (VS) growth causes progressive hearing loss, and the standard treatment, including surgery and radiotherapy, can further damage the nerve. There is an urgent need to identify an adjunct therapy that, by enhancing the efficacy of radiation, can help lower the radiation dose and preserve hearing. The mechanisms underlying deafness in NF2 are still unclear. One of the major limitations in studying tumor-induced hearing loss is the lack of mouse models that allow hearing testing. Here, we developed a cerebellopontine angle (CPA) schwannoma model that faithfully recapitulates the tumor-induced hearing loss. Using this model, we discovered that cMET blockade by crizotinib (CRZ) enhanced schwannoma radiosensitivity by enhancing DNA damage, and CRZ treatment combined with low-dose radiation was as effective as high-dose radiation. CRZ treatment had no adverse effect on hearing; however, it did not affect tumor-induced hearing loss, presumably because cMET blockade did not change tumor hepatocyte growth factor (HGF) levels. This cMET gene knockdown study independently confirmed the role of the cMET pathway in mediating the effect of CRZ. Furthermore, we evaluated the translational potential of cMET blockade in human schwannomas. We found that human NF2-associated and sporadic VSs showed significantly elevated HGF expression and cMET activation compared with normal nerves, which correlated with tumor growth and cyst formation. Using organoid brain slice culture, cMET blockade inhibited the growth of patient-derived schwannomas. Our findings provide the rationale and necessary data for the clinical translation of combined cMET blockade with radiation therapy in patients with NF2.

An advanced glioma cell invasion assay based on organotypic brain slice cultures

BackgroundThe poor prognosis for glioblastoma patients is caused by the diffuse infiltrative growth pattern of the tumor. Therefore, the molecular and cellular processes underlying cell migration continue to be a major focus of glioblastoma research. Emerging evidence supports the concept that the tumor microenvironment has a profound influence on the functional properties of tumor cells. Accordingly, substantial effort must be devoted to move from traditional two-dimensional migration assays to three-dimensional systems that more faithfully recapitulate the complex in vivo tumor microenvironment.MethodsIn order to mimic the tumor microenvironment of adult gliomas, we used adult organotypic brain slices as an invasion matrix for implanted, fluorescently labeled tumor spheroids. Cell invasion was imaged by confocal or epi-fluorescence microscopy and quantified by determining the average cumulative sprout length per spheroid. The tumor microenvironment was manipulated by treatment of the slice with small molecule inhibitors or using different genetically engineered mouse models as donors.ResultsBoth epi-fluorescence and confocal microscopy were applied to precisely quantify cell invasion in this ex vivo approach. Usage of a red-emitting membrane dye in addition to tissue clearing drastically improved epi-fluorescence imaging. Preparation of brain slices from of a genetically engineered mouse with a loss of a specific cell surface protein resulted in significantly impaired tumor cell invasion. Furthermore, jasplakinolide treatment of either tumor cells or brain slice significantly reduced tumor cell invasion.ConclusionWe present an optimized invasion assay that closely reflects in vivo invasion by the implantation of glioma cells into organotypic adult brain slice cultures with a preserved cytoarchitecture. The diversity of applications including manipulation of the tumor cells as well as the microenvironment, permits the investigation of rate limiting factors of cell migration in a reliable context. This model will be a valuable tool for the discovery of the molecular mechanisms underlying glioma cell invasion and, ultimately, the development of novel therapeutic strategies.

Function of B-Cell CLL/Lymphoma 11B in Glial Progenitor Proliferation and Oligodendrocyte Maturation.

B-cell CLL/lymphoma 11B (Bcl11b) – a C2H2 zinc finger transcriptional factor – is known to regulate neuronal differentiation and function in the development of the central nervous system (CNS). Although its expression is reduced during oligodendrocyte (OLG) differentiation, its biological role in OLGs remains unknown. In this study, we found that the downregulation of Bcl11b gene expression in glial progenitor cells (GPCs) by lentivirus-mediated gene knockdown (KD) causes a reduction in cell proliferation with inhibited expression of stemness-related genes, while increasing the expression of cell cyclin regulator p21. In contrast, OLG specific transcription factors (Olig1) and OLG cell markers, including myelin proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), were upregulated in Bcl11b-KD GPCs. Chromatin immunoprecipitation (ChIP) analysis indicated that Bcl11b bound to the promoters of Olig1 and PLP, suggesting that Bcl11b could act as a repressor for Olig1 and PLP, similar to its action on p21. An increase in the number of GC+- or PLP+- OLGs derived from Bcl11b-KD GPCs or OLG precursor cells was also observed. Moreover, myelin basic protein (MBP) expression in OLGs derived from Bcl11b-KD GPCs was enhanced in hippocampal neuron co-cultures and in cerebellar brain-slice cultures. The in vivo study using a lysolecithin-induced demyelinating animal model also indicated that larger amounts of MBP+-OLGs and PLP+-OLGs derived from implanted Bcl11b-KD GPCs were present at the lesioned site of the white matter than in the scramble group. Taken together, our results provide insight into the functional role of Bcl11b in the negative regulation of GPC differentiation through the repression of OLG differentiation-associated genes.

Modeling human pregnancy-associated developmental neurotoxicity in vitro using pluripotent stem cell-derived neurons and astrocytes

Developmental neurotoxicity is any toxic effect on the developing nervous system interfering with normal nervous system structure or function before or after birth and can be associated with neurodevelopmental disorders such as autism, attention deficit disorder, mental retardation and cerebral palsy. Currently, developmental neurotoxicity testing relies heavily on neurobehavioral and neuropathological studies in rodent models; however, an existing backlog of chemicals lack appropriate neurotoxicity information due to the low throughput nature and subsequent high cost of in vivo animal testing. Alternative models have been developed to replace rodent models (including non-mammalian models, brain slice culture, and primary and transformed cell models) though limitations to increasing throughput and reproducibility remain. In this review, we focus on the use of human pluripotent stem cells as a cell source for in vitro chemical screening. Pluripotent stem cell-based assays are derived from a virtually unlimited and uniform cell source making studies highly reproducible and relatively low cost compared to rodent models. Moreover, directed differentiation to distinct neural lineages, including neurons and astrocytes, provides isogenic, precise and tunable cell mixtures to mimic in vivo brain composition for high throughput screening. Thus, human pluripotent stem cell-derived neural models are poised to vastly increase throughput and decrease costs for developmental neurotoxicity screening.

RGS4 deficit in prefrontal cortex contributes to the behaviors related to schizophrenia via system xc--mediated glutamatergic dysfunction in mice.

Rationale: Although molecular investigations of regulator of G-protein signaling 4 (RGS4) alterations in schizophrenia patients yielded partially inconsistent findings, the previous studies suggested that RGS4 is both a positional and functional candidate gene for schizophrenia and is significantly decreased in the prefrontal cortex. However, the exact role of RGS4 in the pathophysiology of schizophrenia is unclear. Moreover, a whole genome transcription profile study showed the possibility of RGS4-regulated expression of SLC7A11(xCT), a component of cysteine/glutamate transporter or system xc-. We hypothesized that system xc- is a therapeutic target of RGS4 deficit-mediated schizophrenia.Methods: Pharmacological and genetic manipulation of RGS4 in organotypic brain slice cultures were used as an ex vivo model to investigate its role in system xc- and glutamatergic function. Lentiviral-based mouse models with RGS4 deficit in the prefrontal cortex and treatment with system xc- activator, N-acetyl cysteine (NAC), were utilized to observe their impacts on glutamatergic function and schizophrenic behaviors.Results: Genetic and pharmacological inhibition of RGS4 resulted in a significant decrease in SLC7A11 (xCT) expression and hypofunction of system xc- and reduced glutamatergic function in organotypic brain slice cultures. However, NAC restored the dysregulation of RGS4-mediated functional deficits of glutamate. Moreover, knockdown of RGS4 specifically in the prefrontal cortex caused mice to exhibit behaviors related to schizophrenia such as increased stereotypy, impaired prepulse inhibition, deficits in social interactions, working memory, and nesting behavior, while enhancing sensitivity to the locomotor stimulatory effect of MK-801. These mice displayed glutamatergic dysfunction in the prefrontal cortex, which may have contributed to the behavioral deficits. RGS4 knockdown mice that received NAC treatment had improved glutamatergic dysfunction and schizophrenia behaviors.Conclusion: Our results suggest that RGS4 deficit induces dysregulation and dysfunction of system xc-, which further results in functional deficits of the glutamatergic system and subsequently to schizophrenia-related behavioral phenotypes. Activation of system xc- offers a promising strategy to treat RGS4 deficit-mediated schizophrenia.

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-β (Aβ) peptide.

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-β (Aβ) peptide.

HMGB1/IL-1β complexes regulate neuroimmune responses in alcoholism

Neuroimmune activation is a key feature of the pathologies of numerous psychiatric disorders including alcoholism, depression, and anxiety. Both HMGB1 and IL-1β have been implicated in brain disorders. Previous studies find HMGB1 andIL-1β form heterocomplexes in vitro with enhanced immune responses, lead to our hypothesis that HMGB1 and IL-1β heterocomplexes formed in vivo to contribute to the pathology of alcoholism. HMGB1/IL-1β heterocomplexes were prepared in vitro and found to potentiate IL-1β receptor proinflammatory gene induction compared to IL-1β alone in hippocampal brain slice culture. These HMGB1/IL-1β complexes were found to be increased in post-mortem human alcoholic hippocampus by co-immunoprecipiation. In mice, acute binge ethanol induced both HMGB1 and IL-1β in the brain and plasma. HMGB1 and IL-1β complexes were found only in mouse brain, with confocal microscopy revealing an ethanol-induced HMGB1 and IL-1β cytoplasmic co-localization. Surprisingly, IL-1β was found primarily in neurons. Studies in hippocampal brain slice culture found ethanol increased HMGB1/IL-1β complexes in the media. These studies suggest a novel neuroimmune mechanism in the pathology of alcoholism. Immunogenic HMGB1/IL-1β complexes represent a novel target for immune modulatory therapy in alcohol use disorders, and should be investigated in other psychiatric diseases that involve a neuroimmune component.

Replication of early and recent Zika virus isolates throughout mouse brain development

Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus-associated injury. Therefore, a critical aspect of ZIKV-induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and midgestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly elongated basal processes of the radial glial progenitor cells and impairment of postmitotic neuronal migration, were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.

Magnesium therapy improves outcome in Streptococcus pneumoniae meningitis by altering pneumolysin pore formation.

Streptococcus pneumoniae is the most common cause of bacterial meningitis in adults and is characterized by high lethality and substantial cognitive disabilities in survivors. Here, we have studied the capacity of an established therapeutic agent, magnesium, to improve survival in pneumococcal meningitis by modulating the neurological effects of the major pneumococcal pathogenic factor, pneumolysin. We used mixed primary glial and acute brain slice cultures, pneumolysin injection in infant rats, a mouse meningitis model and complementary approaches such as Western blot, a black lipid bilayer conductance assay and live imaging of primary glial cells. Treatment with therapeutic concentrations of magnesium chloride (500mg·kg-1 in animals and 2mM in cultures) prevented pneumolysin-induced brain swelling and tissue remodelling both in brain slices and in animal models. In contrast to other divalent ions, which diminish the membrane binding of pneumolysin in non-therapeutic concentrations, magnesium delayed toxin-driven pore formation without affecting its membrane binding or the conductance profile of its pores. Finally, magnesium prolonged the survival and improved clinical condition of mice with pneumococcal meningitis, in the absence of antibiotic treatment. Magnesium is a well-established and safe therapeutic agent that has demonstrated capacity for attenuating pneumolysin-triggered pathogenic effects on the brain. The improved animal survival and clinical condition in the meningitis model identifies magnesium as a promising candidate for adjunctive treatment of pneumococcal meningitis, together with antibiotic therapy.

Organotypic brain explant culture as a drug evaluation system for malignant brain tumors.

Therapeutic options for malignant brain tumors are limited, with new drugs being continuously evaluated. Organotypic brain slice culture has been adopted for neuroscience studies as a system that preserves brain architecture, cellular function, and the vascular network. However, the suitability of brain explants for anticancer drug evaluation has been unclear. We here adopted a mouse model of malignant glioma based on expression of H‐RasV12 in Ink4a/Arf −/− neural stem/progenitor cells to establish tumor‐bearing brain explants from adult mice. We treated the slices with cisplatin, temozolomide, paclitaxel, or tranilast and investigated the minimal assays required to assess drug effects. Serial fluorescence‐based tumor imaging was sufficient for evaluation of cisplatin, a drug with a pronounced cytotoxic action, whereas immunostaining of cleaved caspase 3 (a marker of apoptosis) and of Ki67 (a marker of cell proliferation) was necessary for the assessment of temozolomide action and immunostaining for phosphorylated histone H3 (a marker of mitosis) allowed visualization of paclitaxel‐specific effects. Staining for cleaved caspase 3 was also informative in the assessment of drug toxicity for normal brain tissue. Incubation of explants with fluorescently labeled antibodies to CD31 allowed real‐time imaging of the microvascular network and complemented time‐lapse imaging of tumor cell invasion into surrounding tissue. Our results suggest that a combination of fluorescence imaging and immunohistological staining allows a unified assessment of the effects of various classes of drug on the survival, proliferation, and invasion of glioma cells, and that organotypic brain slice culture is therefore a useful tool for evaluation of antiglioma drugs.

Overexpression of TIMP-1 and Sensitivity to Topoisomerase Inhibitors in Glioblastoma Cell Lines.

The multifunctional protein - tissue inhibitor of metalloproteinases-1 (TIMP-1) - has been associated with a poor prognosis in several types of cancers including glioblastomas. In addition, TIMP-1 has been associated with decreased response to chemotherapy, and especially the efficacy of the family of topoisomerase (TOP) inhibitors has been related to TIMP-1. As a second line treatment of glioblastomas, the vascular endothelial growth factor (VEGF) antibody bevacizumab is administered in combination with the TOP1 inhibitor irinotecan and glioblastoma cell levels of TIMP-1 could therefore potentially influence the efficacy of such treatment. In the present study, we aimed to investigate whether a high TIMP-1 expression in glioblastoma cell lines would affect the sensitivity to TOP inhibitors, and whether TIMP-1 overexpressing cells would have alterered growth and invasion. We established TIMP-1 overexpressing subclones from two human glioblastoma cell lines. TIMP-1 overexpressing U87MG cells were significantly more resistant than low TIMP-1 expressing clones and parental cells when exposed to SN-38 (TOP1 inhibitor) or epirubicin (TOP2 inhibitor). No significant differences were observed for the TIMP-1 transfected A172 cells. Implantation of both U87MG and A172 spheroids into organotypic brain slice cultures revealed a reduced growth of TIMP-1 overexpressing U87MG spheroids, however, no significant differences in invasion were observed. The present study suggests that TIMP-1 overexpression reduces the effect of TOP inhibitors in glioblastoma. TIMP-1 also appeared to reduce spheroid growth, but did not influence invasion. Whether TIMP-1 plays a role in irinotecan resistance and has a predictive potential in glioblastoma patients remains to be elucidated.

Human Cerebrospinal fluid promotes long-term neuronal viability and network function in human neocortical organotypic brain slice cultures

Pathophysiological investigation of CNS-related diseases, such as epilepsy or neurodegenerative disorders, largely relies on histological studies on human post mortem tissue, tissue obtained by biopsy or resective surgery and on studies using disease models including animal models, heterologous expression systems or cell culture based approaches. However, in general it remains elusive to what extent results obtained in model systems can be directly translated to the human brain, calling for strategies allowing validation or even primary investigation in live human CNS tissue. In the work reported here, we prepared human organotypic slice cultures from access tissue of resective epilepsy surgery. Employing different culture conditions, we systematically compared artificial culturing media versus human cerbrospinal fluid (hCSF) obtained from patients with normal pressure hydrocephalus (NPH). Presented data demonstrates sustained cortical neuronal survival including not only maintenance of typical cellular electrophysiological properties and activity, such as robust action potential generation and synaptic connectivity, but also preservation of tonic and phasic network activity up to several weeks in vitro. As clearly delineated by immunocytochemistry, single cell patch clamp and extracellular recordings, we find that in contrast to artificial culturing media, hCSF significantly enhances neuron viability and maintenance of network activity.

Isolation of Peptides from Calf Brain Tissue and Evaluation of their Tissue-Specific and Stimulatory Activity

A method for separating a mixture of calf brain peptides by extraction with aqueous HOAc followed by fractionation of the extract by cross-flow (tangential) microfiltration and solid-phase extraction was developed. The isolated peptide mixture contained constituents with molecular masses within 0.1 – 3 kDa. Low-pressure ion-exchange chromatography of this mixture over Dowex 50WX8 sulfonated cation exchanger isolated a pure peptide consisting of the five amino acids Arg, Lys, Gly, Ser, and Leu. The biological activity of the obtained pentapeptide was estimated on organotypic brain slice culture explants of Wistar rats. It was established that the peptide possessed high tissue-specific and stimulatory activity for brain explants that was greater than those of the commercial preparations Cortexin and Cerebrolysin.