Isolated Brain Tissue Research Articles

Bi-directional regulation of AIMP2 and its splice variant on PARP-1-dependent neuronal cell death; Therapeutic implication for Parkinson's disease

BackgroundParthanatos represents a critical molecular aspect of Parkinson's disease, wherein AIMP2 aberrantly activates PARP-1 through direct physical interaction. Although AIMP2 ought to be a therapeutic target for the disease, regrettably, it is deemed undruggable due to its non-enzymatic nature and predominant localization within the tRNA synthetase multi-complex. Instead, AIMP2 possesses an antagonistic splice variant, designated DX2, which counteracts AIMP2-induced apoptosis in the p53 or inflammatory pathway. Consequently, we examined whether DX2 competes with AIMP2 for PARP-1 activation and is therapeutically effective in Parkinson’s disease.MethodsThe binding affinity of AIMP2 and DX2 to PARP-1 was contrasted through immunoprecipitation. The efficacy of DX2 in neuronal cell death was assessed under 6-OHDA and H2O2 in vitro conditions. Additionally, endosomal and exosomal activity of synaptic vesicles was gauged in AIMP2 or DX2 overexpressed hippocampal primary neurons utilizing optical live imaging with VAMP-vGlut1 probes. To ascertain the role of DX2 in vivo, rotenone-induced behavioral alterations were compared between wild-type and DX2 transgenic animals. A DX2-encoding self-complementary adeno-associated virus (scAAV) was intracranially injected into 6-OHDA induced in vivo animal models, and their mobility was examined. Subsequently, the isolated brain tissues were analyzed.ResultsDX2 translocates into the nucleus upon ROS stress more rapidly than AIMP2. The binding affinity of DX2 to PARP-1 appeared to be more robust compared to that of AIMP2, resulting in the inhibition of PARP-1 induced neuronal cell death. DX2 transgenic animals exhibited neuroprotective behavior in rotenone-induced neuronal damage conditions. Following a single intracranial injection of AAV-DX2, both behavior and mobility were consistently ameliorated in neurodegenerative animal models induced by 6-OHDA.ConclusionAIMP2 and DX2 are proposed to engage in bidirectional regulation of parthanatos. They physically interact with PARP-1. Notably, DX2's cell survival properties manifest exclusively in the context of abnormal AIMP2 accumulation, devoid of any tumorigenic effects. This suggests that DX2 could represent a distinctive therapeutic target for addressing Parkinson's disease in patients.

Prednisolone Targets Claudins in Mouse Brain Blood Vessels.

Endothelial cells in brain capillaries are crucial for the function of the blood-brain barrier (BBB), and members of the tight junction protein family of claudins are regarded to be primarily responsible for barrier properties. Thus, the analysis of bioactive substances that can affect the BBB's permeability is of great importance and may be useful for the development of new therapeutic strategies for brain pathologies. In our study, we tested the hypothesis that the application of the glucocorticoid prednisolone affects the murine blood-brain barrier in vivo. Isolated brain tissue of control and prednisolone-injected mice was examined by employing immunoblotting and confocal laser scanning immunofluorescence microscopy, and the physiological and behavioral effects were analyzed. The control tissue samples revealed the expression of barrier-forming tight junction proteins claudin-1, -3, and -5 and of the paracellular cation and water-channel-forming protein claudin-2. Prednisolone administration for 7 days at doses of 70 mg/kg caused physiological and behavioral effects and downregulated claudin-1 and -3 and the channel-forming claudin-2 without altering their localization in cerebral blood vessels. Changes in the expression of these claudins might have effects on the ionic and acid-base balance in brain tissue, suggesting the relevance of our findings for therapeutic options in disorders such as cerebral edema and psychiatric failure.

Effects of hesperetin on dimethylsulphoxide induced cognitive dysfunction mediated through oxidative stress marker and neuroinflammation in hippocampus

ObjectiveHesperetin is a natural flavonoid with antioxidant potential and can be used to treat disorders of the brain. Therefore, present study is designed to investigate effect of hesperetin on dimethylsulphoxide induced cognitive dysfunction mediated through oxidative stress marker and neuroinflammation in hippocampus MethodologyThe experiment was conducted using five treatment groups, each group consisting six rats. Group 1 (control) received 1 ml/kg normal saline interperitoneally for 14 days. Group 2 received 1 ml/kg DMSO per oral for14 days. Group 3 received donepezil 3 mg/kg per day for 14 days and DMSO (1 ml/kg p.o.) for 14th day. Group 4 and 5 received a dose of 25 mg /kg of hesperetin (H1) and 50 mg/kg hesperetin (H2) per oral, per day for 14 days, respectively. Hesperetin was dissolved in DMSO to get 25 mg/ml solution. On 14th day, the memory of rats was assessed by Cook's pole climbing apparatus and Morris water maze test. On 15th day, six rats of each group were sacrificed and the isolated brain tissues were subjected for the estimation of biochemical parameters, beta actin blot analysis and histopathological examination. ResultsThe DMSO treated rats showed significantly enhanced escape latency time (P < 0.0001) acetylcholinesterase (AchE) activity (P < 0.0001), lipid peroxidation (P < 0.0001), TNF-α level and decreased reduced glutathione levels (p < 0.001), attenuated beta actin blot density, degradation and inflammation in the hippocampus region as compared to the control group. These parameters were significantly recovered in the rats pretreated with donepezil and hesperetin. ConclusionThe findings of this study demonstrated that DMSO induced cognitive dysfunction in rats and hesperetin has potential to improve the cognitive performance against DMSO via reduced oxidative stress and neuroinflammation in hippocampus.

381 The role of leucine-rich PPR motif-containing protein (LRPPRC) in myelin lipid metabolism

OBJECTIVES/GOALS: Leigh Syndrome, French Canadian-Type (LSFC) is a neurometabolic disorder caused by mutation of mitochondria-related gene, LRPPRC. White matter lesions and demyelination in central nervous system are common in LSFC. LRPPRC is enriched in myelinating glial cells, yet its role is not known. Our goal is to elucidate its mechanistic role in myelination. METHODS/STUDY POPULATION: We crossed C57BL/6N mice bearing a LRPPRC-loxP allele with mice bearing a Plp-CreERT2 allele. Mice with the Plp-CreERT2 allele expresses a tamoxifen-inducible Cre under the control of the Plp promoter, which drives expression in oligodendrocytes. Using these strains, we can target the deletion of LRPPRC, via tamoxifen injection, in both newly formed myelin and mature myelin. Plp-CreERT2; LRPPRCL/L (LRPPRC-KO) or control littermate mice will be injected for LRPPRC deletion at developmental and maturation stages of myelin. Immunofluorescence and electron microscopy of isolated brain tissues will be used for myelin integrity analysis. Cognitive functions of the mice will be measured via behavioral tests. Lastly, we will submit tissues for lipidomic analyses to observe any lipid metabolite variation. RESULTS/ANTICIPATED RESULTS: Behavioral and motor defects would be expected in LRPPRC-KO mice performing in cognitive function tasks across myelin maturation stages. Electron microscopy-based structure analysis of optic nerve, corpus callosum, and spinal cord should reveal thin or loss of myelin on the axons of LRPPRC-KO compared to control. Immunofluorescence staining of major myelin structural proteins, including myelin proteolipid protein (PLP), myelin basic protein (MBP), and myelin-associated glycoprotein (MAG) would be expected have lower levels in LRPPRC deficient tissues. Since myelin is a lipid-rich species, we would also expect lipid concentrations to be affected. LRPPRC-KO lipidomic analyses of myelin-related lipids should depict lower levels in comparison to control, which would imply dysfunctional lipid metabolism. DISCUSSION/SIGNIFICANCE: There are limited studies in ameliorating neural deficits caused by LS and LSFC. Successful completion of this project would help elucidate the functions of LRPPRC in myelination and lipid metabolism and potentially provide insights for developing novel therapeutic strategies for alleviating the demyelination and neural deficits in LSFC.

Neuroprotective effects of Lasmiditan and Sumatriptan in an experimental model of post-stroke seizure in mice: Higher effects with concurrent opioid receptors or KATP channels inhibitors

BackgroundEarly post-stroke seizure frequently occurs in stroke survivors within the first few days and is associated with poor functional outcomes. Therefore, efficient treatments of such complications with less adverse effects are pivotal. In this study, we investigated the possible beneficial effects of lasmiditan and sumatriptan against post-stroke seizures in mice and explored underlying mechanisms in their effects. MethodsStroke was induced by double ligation of the right common carotid artery in mice. Immediately after the ligation, lasmiditan (0.1 mg/kg, intraperitoneally [i.p.]) or sumatriptan (0.03 mg/kg, i.p.) were administered. Twenty-four hours after the stroke induction, seizure susceptibility was evaluated using the pentylenetetrazole (PTZ)-induced clonic seizure model. In separate experiments, naltrexone (a non-specific opioid receptor antagonist) and glibenclamide (a KATP channel blocker) were administered 15 min before lasmiditan or sumatriptan injection. To evaluate the underlying signaling pathways, ELISA analysis of inflammatory cytokines (TNF-α and IL-1β) and western blot analysis of anti- and pro-apoptotic markers (Bcl-2 and Bax) were performed on mice isolated brain tissues. ResultsLasmiditan (0.1 mg/kg, i.p.) and sumatriptan (0.03 mg/kg, i.p.) remarkably decreased seizure susceptibility in stroke animals by reducing inflammatory cytokines and neuronal apoptosis. Concurrent administration of naltrexone (10 mg/kg, i.p.) or glibenclamide (0.3 mg/kg, i.p.) with lasmiditan or sumatriptan resulted in a higher neuroprotection against clonic seizures and efficiently reduced the inflammatory and apoptotic markers. ConclusionLasmiditan and sumatriptan significantly increased post-stroke seizure thresholds in mice by suppressing inflammatory cytokines and neuronal apoptosis. Lasmiditan and sumatriptan seem to exert higher effects on seizure threshold with concurrent administration of the opioid receptors or KATP channels modulators.

3,4‐methylenedioxymethamphetamine (MDMA) stimulates activation of TAAR1 and subsequent neurotransmitter transporter internalization in serotonin neurons

The use of 3,4‐methylenedioxymethamphetamine (MDMA) has recently gained interest as a potential therapeutic tool for the treatment of post‐traumatic stress disorder (PTSD). However, the mechanism of action of this drug has not yet been fully elucidated. Here we demonstrate that MDMA acts by targeting the trace amine associated receptor 1 (TAAR1), an intracellular G‐protein coupled receptor (GPCR) expressed throughout the brain and in peripheral tissues. In previous studies, we have shown that a closely‐related compound, amphetamine (AMPH), enters neurons through the dopamine and norepinephrine transporters (DAT and NET) and activates TAAR1. This activates the small GTPase RhoA that in turn increases internalization of the DAT and NET. We have also found that a neuronal glutamate transporter, EAAT3, is also internalized. These actions result in an increase in extracellular DA, NE and glutamate concentrations. In this study, we show that MDMA enters serotonin neurons through the 5‐HT transporter (SERT) and activates a similar signaling cascade.We isolated brain tissue encompassing the Raphe nucleus from E15 mice and prepared primary cultures of serotonin (5‐HT) neurons. 5‐HT neurons were identified by immunocytochemistry for 5‐HT. Functional SERT activity was confirmed by citalopram sensitive 3H‐5‐HT uptake. Pre‐treatment of these cultures for thirty minutes with 1 mM MDMA resulted in a decrease of SERT transport activity. This loss of function was blocked by an inhibitor of RhoA, indicating that MDMA regulates SERT through a Rho‐dependent signaling pathway.Using total internal reflection fluorescence (TIRF) microscopy in HEK293 cells we confirmed that the loss in SERT activity produced by MDMA was due to internalization of the SERT. The application of 1 mM MDMA led to a decrease in GFP‐tagged SERT at the cell membrane. Similarly, GFP‐tagged EAAT3 was also internalized in response to MDMA. Parallel studies in HEK293 cells in which the TAAR1 gene was deleted by CRISPR‐Cas9 showed no effects of MDMA treatment on either SERT or EAAT3 trafficking and confirmed that TAAR1 is an obligate target for the actions of MDMA,To examine the effects of MDMA on endogenous SERT and EAAT3 in situ, we prepared acute brain slices from wildtype and TAAR1 knockout mice. Slices containing the Raphe nucleus were treated with either vehicle control or MDMA (1 mM) and subsequently treated with a cell‐impermeable biotin reagent that would label all surface proteins. By western blot analysis, we observed internalization of SERT and EAAT3 in response to the MDMA treatment. However, neither transporter exhibited any change in cell‐surface expression in response to MDMA in the TAAR1 knockout tissue.Our data indicate that in serotonergic neurons MDMA potentiates the action of both serotonin and glutamate by activating TAAR1 signaling and triggering SERT and EAAT3 internalization. These observations point to new mechanisms to target for the development of drugs to treat PTSD.

A standardized polyherbal preparation POL-6 diminishes alcohol withdrawal anxiety by regulating Gabra1, Gabra2, Gabra3, Gabra4, Gabra5 gene expression of GABAA receptor signaling pathway in rats

BackgroundAlcohol abuse is a major problem worldwide and it affects people’s health and economy. There is a relapse in alcohol intake due to alcohol withdrawal. Alcohol withdrawal anxiety-like behavior is a symptom that appears 6–24 h after the last alcohol ingestion.MethodsThe present study was designed to explore the protective effect of a standardized polyherbal preparation POL-6 in ethanol withdrawal anxiety in Wistar rats. POL-6 was prepared by mixing the dried extracts of six plants Bacopa monnieri, Hypericum perforatum, Centella asiatica, Withania somnifera, Camellia sinesis, and Ocimum sanctum in the proportion 2:1:2:2:1:2 respectively. POL-6 was subjected to phytochemical profiling through LC-MS, HPLC, and HPTLC. The effect of POL-6 on alcohol withdrawal anxiety was tested using a two-bottle choice drinking paradigm model giving animals’ free choice between alcohol and water for 15 days. Alcohol was withdrawn on the 16th day and POL-6 (20, 50, and 100 mg/kg, oral), diazepam (2 mg/kg) treatment was given on the withdrawal days. Behavioral parameters were tested using EPM and LDT. On the 18th day blood was collected from the retro-orbital sinus of the rats and alcohol markers ALT, AST, ALP, and GGT were studied. At end of the study, animals were sacrificed and the brain was isolated for exploring the influences of POL-6 on the mRNA expression of GABAA receptor subunits in the amygdala and hippocampus.ResultsPhytochemical profiling showed that POL-6 contains major phytoconstituents like withaferin A, quercetin, catechin, rutin, caeffic acid, and β-sitosterol. In-vivo studies showed that POL-6 possesses an antianxiety effect in alcohol withdrawal. Gene expression studies on the isolated brain tissues showed that POL-6 normalizes the GABAergic transmission in the amygdala and hippocampus of the rats.ConclusionThe study concludes that POL-6 may have therapeutic potential for treating ethanol-type dependence.

Fibrin As a Target for Glioblastoma Detection and Treatment

Glioblastoma is a highly aggressive brain tumor characterized by diffuse growth and resistance to therapy. Angiogenesis in glioblastoma is poorly organized and therefore, tends to be associated with tumor cell necrosis, hemorrhage and thrombosis. This leads to the formation of a fibrin-rich extracellular matrix, which could provide important adhesive cues for glioblastoma growth and proliferation. To establish that blood clotting takes place in the extracellular matrix of malignant brain tumors, we assessed fibrin formation in tumor samples from patients with astrocytoma and glioblastoma using immunohistochemistry. Compared to normal brain tissue, which is essentially fibrin-free, this analysis revealed a marked upregulation of clot formation in the interstitial spaces of patients with high-grade tumors (i.e. astrocytoma 3 and GBM). The low-grade astrocytoma 2, however, expressed 3-3.5 fold less fibrin than was found in tissues from patients with astrocytoma 3 and GBM indicating that the degree of clot formation positively correlates with tumor grade. Paralleling these data, we found that primary GBM cells, that were freshly isolated from patients after tumor surgery, infiltrated and proliferated strongly after embedding in a three dimensional (3D) matrix of clotted plasma ex vivo. Primary tumor cells from patients with astrocytoma 2 and 3, on the other hand, infiltrated clot but were unable to proliferate in 3D. GBM proliferation in 3D depended on fibrin, which mediated upregulation of the stem cell marker nestin, whereas culturing glioblastoma cells in a 3D matrix of matrigel™ failed to promote nestin expression as well as glioblastoma proliferation. Therefore, these data suggest that the presence of clotted plasma in the tumor extracellular matrix represents a niche for glioblastoma stem cells and, as such, contributes to GBM progression. To determine the interaction of GBM cells with fibrin on a molecular basis, we transfected GBM cells with siRNA against integrin β3, which completely abolished invadopodia formation and, at the same time, caused a sustained growth inhibition. GBM cell proliferation in 3D fibrin also depended on the formation of a fibronectin matrix as knockdown of fibronectin led to complete growth arrest. These findings appear to be clinically relevant since freshly isolated tumor cells from patients with glioblastoma colonized 3D fibrin most efficiently when they express fibronectin in combination with integrin β3. This suggests that fibrin stimulates adhesive interactions between integrin β3 and fibronectin and that these interactions in turn support glioblastoma stemness. To assess fibrin formation in glioblastoma in vivo, mice with orthotopic U87MG xenografts were injected intravenously with the fluorescein-coupled dekapeptide CGLKIQKNEC, which is a derivative of the clot-binding peptide CLT1. Using a fluorescence endoscope in situ, we detected strong green fluorescence over the parietal lobe of the right cerebral hemisphere, where tumor growth had been established by MRI beforehand. Subsequently, we confirmed tumor binding of the peptide in isolated brain tissue by fluorescence microscopy ex vivo, which demonstrated specific green fluorescence in the tumor xenograft while adjacent normal brain tissue as well as tissues from distant organs only exhibited background fluorescence. Together, our data demonstrate a specific upregulation of fibrin in high-grade astrocytoma, which promotes infiltration and proliferation of glioblastoma stem cells via integrin β3 and fibronectin. Moreover, we present a strategy to identify fibrin in the tumor extracellular matrix as a possible means to identify astrocytoma progression in vivo. Disclosures Eichler: Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Pilch:CSL Behring: Other: Grants (investigator initiated), Speakers Bureau; ASPIRE Award/Pfizer: Other: Grants (investigator initiated); Bayer: Consultancy, Speakers Bureau; Roche: Consultancy.

Prenatal Exposure to Benzophenone-3 Impairs Autophagy, Disrupts RXRs/PPAR\u03b3 Signaling, and Alters Epigenetic and Post-Translational Statuses in Brain Neurons

The UV absorber benzophenone-3 (BP-3) is the most extensively used chemical substance in various personal care products. Despite that BP-3 exposure is widespread, knowledge about the impact of BP-3 on the brain development is negligible. The present study aimed to explore the mechanisms of prenatal exposure to BP-3 in neuronal cells, with particular emphasis on autophagy and nuclear receptors signaling as well as the epigenetic and post-translational modifications occurring in response to BP-3. To observe the impact of prenatal exposure to BP-3, we administered BP-3 to pregnant mice, and next, we isolated brain tissue from pretreated embryos for primary cell neocortical culture. Our study revealed that prenatal exposure to BP-3 (used in environmentally relevant doses) impairs autophagy in terms of BECLIN-1, MAP1LC3B, autophagosomes, and autophagy-related factors; disrupts the levels of retinoid X receptors (RXRs) and peroxisome proliferator-activated receptor gamma (PPARγ); alters epigenetic status (i.e., attenuates HDAC and sirtuin activities); inhibits post-translational modifications in terms of global sumoylation; and dysregulates expression of neurogenesis- and neurotransmitter-related genes as well as miRNAs involved in pathologies of the nervous system. Our study also showed that BP-3 has good permeability through the BBB. We strongly suggest that BP-3-evoked effects may substantiate a fetal basis of the adult onset of neurological diseases, particularly schizophrenia and Alzheimer’s disease.

Abstract P205: Age-dependent Alteration in Expression of Brain-derived Neurotrophic Factor and its Receptors in Central Nervous System of Spontaneously Hypertensive Rats.

Background and Aim: Essential hypertension is a cause unknown disease leading to heart disease, stroke and renal failure. Spontaneously hypertensive rats (SHR) are animal model of human essential hypertension and develop hypertension at 7-15-week-old. Several studies reported expression pattern of brain-derived neurotrophic factor (BDNF) changes in central nervous system (CNS) in SHR, suggesting that BDNF and its receptors might be related to increased blood pressure. However, age-dependent change in BDNF and receptors (TrkB and p75NTR) remains to be elucidated. This study aimed to clarify it. Method: Isolated brain tissues from SHR and control Wistar Kyoto Rats (WKY) at 4-, 7- and 15-week-old were dissected into five regions (cerebral cortex: CC; cerebellum: CB; brainstem: BS; hypothalamus: HT; hippocampus: HC). Protein and mRNA expression of BDNF, TrkB isoforms (FL, T1 and T2) and p75NTR as well as an inflammatory marker NOX-2 was examined by Western blotting and real-time PCR. Results: The following data showed significant difference in SHR compared with WKY (n = 4): pro-BDNF protein was lower by 49.9% (4-week, p &lt; 0.05) and 38.2% (7-week, p &lt; 0.05) in HT, while mature BDNF protein was lower by 21.9% in HC (15-week, p &lt; 0.05). TrkB FL mRNA was higher by 32.5% in CB (7-week, p &lt; 0.05) and 43.1% in BS (7-week, p &lt; 0.05), while TrkB FL protein was higher by 51.4% in BS (15-week, p &lt; 0.05). TrkB T1 mRNA was higher by 22.8% in BS (7-week, p &lt; 0.05) and 17.2% in HT (15-week, p &lt; 0.05), while TrkB T1 protein was higher by 56.1% in BS (4-week, p &lt; 0.01). p75NTR protein was higher by 46.9% in CB (7-week, p &lt; 0.05). NOX-2 mRNA was higher by 29.1% (4-week, p &lt; 0.01), 104.2% (7-week, p &lt; 0.01) and 75.8% (15-week, p &lt; 0.05) in CC, 27.0% in CB (15-week, p &lt; 0.05), 34.6% in BS (4-week, p &lt; 0.05), 326.1% in HT (15-week, p &lt; 0.01) and 29.8% in HC (7-week, p &lt; 0.05). Conclusion: We for the first time revealed age-dependent changes in expression of BDNF and its receptors in CNS of SHR. Specifically, our results suggest that alteration of BDNF and TrkB expression is already occurred at juvenile before a stable blood pressure increase. The upregulation of NOX-2 was also identified. Further detailed study might identify whether and how BDNF-receptor-oxidative stress axis is related to pathogenesis of essential hypertension.

Phenotypic and functional complexity of brain-infiltrating T cells in Rasmussen encephalitis

Objective:To characterize the brain-infiltrating immune cell repertoire in Rasmussen encephalitis (RE) with special focus on the subsets, clonality, and their cytokine profile.Methods:The immune cell infiltrate of freshly isolated brain tissue from RE was phenotypically and functionally characterized using immunohistology, flow cytometry, and T-cell receptor (TCR) deep sequencing. Identification of clonally expanded T-cell clones (TCCs) was achieved by combining flow cytometry sorting of CD4+ and CD8+ T cells and high-throughput TCR Vβ-chain sequencing. The most abundant brain-infiltrating TCCs were isolated and functionally characterized.Results:We found that CD4+, CD8+, and also γδ T cells infiltrate the brain tissue in RE. Further analysis surprisingly revealed that not only brain-infiltrating CD8+ but also CD4+ T cells are clonally expanded in RE. All 3 subsets exhibited a Tc1/Th1 phenotype characterized by the production of interferon (IFN)-γ and TNF. Broad cytokine profiling at the clonal level showed strong production of IFN-γ and TNF and also secretion of interleukin (IL)-5, IL-13, and granzyme B, both in CD4+ and CD8+ T cells.Conclusions:CD8+ T cells were until now considered the central players in the immunopathogenesis of RE. Our study adds to previous findings and highlights that CD4+ TCCs and γδ T cells that secrete IFN-γ and TNF are also involved. These findings underline the complexity of T-cell immunity in RE and suggest a specific role for CD4+ T cells in orchestrating the CD8+ T-cell effector immune response.

Protective effect of grifolin against brain injury in an acute cerebral ischemia rat model

Purpose: To evaluate the protective effects of grifolin against brain injury in an acute cerebral ischemia rat model.Methods: Rats were assigned to five groups: control, negative control, and grifolin (50, 100, and 200 mg/kg, p.o.) treated groups, which received the drug for 2 weeks. All the animals were sacrificed at the end of the protocol, and tissue homogenates were prepared from isolated brain tissue. Glutathione peroxidase (GPX), superoxide dismutase (SOD), malondialdehyde (MDA), and nitric oxide (NO), as oxidative stress indicators, were determined for the tissue homogenates of the ischemic rats. Inflammatory mediators (cytokines and nuclear factor kappa B p65, NF κB), DNA damage, and ATP and caspase 3 levels in the tissue homogenates were also assessed.Results: Treatment with grifolin increased SOD and GPX significantly and decreased MDA and NO levels in tissue homogenates of the cerebral ischemic rats compared with those in the negative control group (p &lt; 0.05). Treatment with grifolin also attenuated the altered levels of inflammatory mediators (cytokines and NF-κB), caspase 3, and ATP levels in the tissue homogenate of cerebral ischemic rats (p &lt; 0.05). The results of comet assay on the tissue homogenate suggest that treatment with grifolin reduced or prevented damage.Conclusions: The results show that treatment with grifolin protects against neuronal damage in acute cerebral ischemic rats via its anti-inflammatory and anti-oxidant properties.Keywords: Neuroprotection, Cerebral ischemia, Brain injury, DNA, Grifolin, Anti oxidant

Optogenetic Visualization of Presynaptic Tonic Inhibition of Cerebellar Parallel Fibers.

Tonic inhibition was imaged in cerebellar granule cells of transgenic mice expressing the optogenetic chloride indicator, Clomeleon. Blockade of GABAA receptors substantially reduced chloride concentration in granule cells due to block of tonic inhibition. This indicates that tonic inhibition is a significant contributor to the resting chloride concentration of these cells. Tonic inhibition was observed not only in granule cell bodies, but also in their axons, the parallel fibers (PFs). This presynaptic tonic inhibition could be observed in slices both at room and physiological temperatures, as well as in vivo, and has many of the same properties as tonic inhibition measured in granule cell bodies. GABA application revealed that PFs possess at least two types of GABAA receptor: one high-affinity receptor that is activated by ambient GABA and causes a chloride influx that mediates tonic inhibition, and a second with a low affinity for GABA that causes a chloride efflux that excites PFs. Presynaptic tonic inhibition regulates glutamate release from PFs because GABAA receptor blockade enhanced both the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs at the PF-Purkinje cell synapse. We conclude that tonic inhibition of PFs could play an important role in regulating information flow though cerebellar synaptic circuits. Such cross talk between phasic and tonic signaling could be a general mechanism for fine tuning of synaptic circuits. This paper demonstrates that an unconventional form of signaling, known as tonic inhibition, is found in presynaptic terminals and affects conventional synaptic communication. Our results establish the basic characteristics and mechanisms of presynaptic tonic inhibition and show that it occurs in vivo as well as in isolated brain tissue.

Posterior quadrantic disconnection maintains the activity of isolated temporal-parietal-occipital nerve tissue: neuroprotective measures in the surgical treatment of epilepsy.

Posterior quadrantic disconnection maintains the activity of isolated temporal-parietal-occipital nerve tissue: neuroprotective measures in the surgical treatment of epilepsy.

Interactions of atenolol with alprazolam/escitalopram on anxiety, depression and oxidative stress

Anxiety and depression are highly comorbid disorders possibly sharing a common neurobiological mechanism. The dysfunction of serotoninergic, noradrenergic and dopaminergic neurotransmission, abnormal regulation in the hypothalamic–pituitary–adrenal axis (HPA), disturbance of cellular plasticity including reduced neurogenesis, or chronic inflammation connected with high oxidative damage play a crucial role in the development of anxiety and depression. The present study was aimed to investigate the effects of atenolol alone and in combination with alprazolam/escitalopram on anxiety, depression and oxidative stress. Wistar albino rats were subjected to 21day treatment of drugs then exposed to elevated-plus maze (EPM) and modified forced swim test (MFST), and oxidative stress markers were estimated in isolated brain tissue of all groups. The results indicated that atenolol in combination with alprazolam/escitalopram exhibited antidepressant effects by significantly decreasing the immobility and increasing the swimming behavior in the MFST and anti-anxiety effects by increasing the percentage preference and number of open arm entries as well as time spent in open arm in EPM. Pretreatment with atenolol alone and combination with alprazolam/escitalopram also ameliorated tissue glutathione (GSH) and decreased malondialdehyde (MDA) level significantly which explore antioxidant properties of drugs, and combination augments the therapeutic response of monotherapy in depression. In conclusion behavioral and biological findings indicate that the combination of atenolol with alprazolam/escitalopram has the potential of being highly efficacious in treating anxiety and depressive disorders as well as oxidative stress.

Brain slice on a chip: opportunities and challenges of applying microfluidic technology to intact tissues

Isolated brain tissue, especially brain slices, are valuable experimental tools for studying neuronal function at the network, cellular, synaptic, and single channel levels. Neuroscientists have refined the methods for preserving brain slice viability and function and converged on principles that strongly resemble the approach taken by engineers in developing microfluidic devices. With respect to brain slices, microfluidic technology may 1) overcome the traditional limitations of conventional interface and submerged slice chambers and improve oxygen/nutrient penetration into slices, 2) provide better spatiotemporal control over solution flow/drug delivery to specific slice regions, and 3) permit successful integration with modern optical and electrophysiological techniques. In this review, we highlight the unique advantages of microfluidic devices for in vitro brain slice research, describe recent advances in the integration of microfluidic devices with optical and electrophysiological instrumentation, and discuss clinical applications of microfluidic technology as applied to brain slices and other non-neuronal tissues. We hope that this review will serve as an interdisciplinary guide for both neuroscientists studying brain tissue in vitro and engineers as they further develop microfluidic chamber technology for neuroscience research.

Structural mass spectrometry analysis of lipid changes in a Drosophila epilepsy model brain

Phosphatidylethanolamine (PtdEtn) is one of the most abundant phospholipids in many animal cell types. The Drosophila easily shocked (eas(2)) mutant, used as an epilepsy model, is null for the PtdEtn biosynthetic enzyme, ethanolamine kinase. This mutant displays bang sensitive paralysis, and was previously shown to have decreased levels of PtdEtn. We have developed a highly selective and sensitive measurement strategy using ion mobility-mass spectrometry for the relative quantitation of intact phospholipid species directly from isolated brain tissue of eas mutants. Over 1200 distinct lipid signals are observed and within this population 38, including PtdEtn, phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) species are identified to have changed significantly (p < 0.03) between mutant and control tissue. This method has revealed for the first time the structural complexity and biosynthetic interconnectedness of specific PtdEtn and PtdIns lipid species within tissue, and provides great molecular detail compared to traditionally used detection techniques.

Microwave effects on the nervous system.

Studies have evaluated the electroencephalography (EEG) of humans and laboratory animals during and after Radiofrequency (RF) exposures. Effects of RF exposure on the blood-brain barrier (BBB) have been generally accepted for exposures that are thermalizing. Low level exposures that report alterations of the BBB remain controversial. Exposure to high levels of RF energy can damage the structure and function of the nervous system. Much research has focused on the neurochemistry of the brain and the reported effects of RF exposure. Research with isolated brain tissue has provided new results that do not seem to rely on thermal mechanisms. Studies of individuals who are reported to be sensitive to electric and magnetic fields are discussed. In this review of the literature, it is difficult to draw conclusions concerning hazards to human health. The many exposure parameters such as frequency, orientation, modulation, power density, and duration of exposure make direct comparison of many experiments difficult. At high exposure power densities, thermal effects are prevalent and can lead to adverse consequences. At lower levels of exposure biological effects may still occur but thermal mechanisms are not ruled out. It is concluded that the diverse methods and experimental designs as well as lack of replication of many seemingly important studies prevents formation of definite conclusions concerning hazardous nervous system health effects from RF exposure. The only firm conclusion that may be drawn is the potential for hazardous thermal consequences of high power RF exposure.

Tecido cerebral ectópico na órbita: relato de dois casos

Tecido cerebral na órbita, tendo continuidade com o conteúdo intracraniano, é relativamente incomum. Tecido cerebral isolado na órbita, entretanto, é muito mais raro. No presente trabalho são apresentados dois casos de tecido cerebral ectópico na órbita, com cisto ependimal. São analisadas as similaridades e diferenças com outros casos da literatura, bem como as dificuldades no diagnóstico desta malformação. Não conhecemos nenhum caso descrito na literatura nacional.

Nitric Oxide Stimulates ACTH Secretion and the Transcription of the Genes Encoding for NGFI-B, Corticotropin-Releasing Factor, Corticotropin-Releasing Factor Receptor Type 1, and Vasopressin in the Hypothalamus of the Intact Rat

We investigated the effect of the intracerebroventricular injection of the nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on the release of adrenocorticotropin hormone (ACTH) and the neuronal response of hypothalamic neurons responsible for this release. Rats that were administered SIN-1 showed significant elevations in plasma ACTH levels, a response that was virtually abolished by antibodies against corticotropin-releasing factor (CRF) and significantly blunted by vasopressin (VP) antiserum. SIN-1 also upregulated heteronuclear (hn) transcripts for CRF and VP and messenger RNA (mRNA) levels for the immediate early gene NGFI-B and for CRF receptor type 1 (CRF-R(1)) in the parvocellular portion of the paraventricular nucleus (PVN) of the hypothalamus. Blockade of prostaglandin synthesis with ibuprofen did not alter the ACTH or the PVN response to SIN-1. The central nucleus of the amygdala and the supraoptic nucleus, regions that are involved in autonomic adjustments to altered cardiovascular activity, also responded to SIN-1 with elevated NGFI-B mRNA levels. However, the only change in mean arterial blood pressure caused by this NO donor was a transient and modest increase. To our knowledge, this is the first demonstration that in the intact rat NO stimulates the activity of PVN neurons that control the hypothalamic-pituitary-adrenal axis. It must be noted, however, that our results do not allow us to determine whether this effect was direct or mediated through PVN afferents. This study should help resolve the controversy generated by the use of isolated brain tissues to investigate the net effect of NO on hypothalamic peptide production.