Oligodendrocyte transcription factor 1 mRNA and protein expression in organotypic rat brain slices

Numerous studies have confirmed that oligodendrocyte transcription factor 1 （Olig-1） is vital for myelin repair. However, the effects of hypoxia and ischemia on Olig-1 expression remain unknown. In this study, Olig-1 mRNA and protein expressions were analyzed by in situ hybridization and immunohistochemistry, to determine the expression profile of Olig-1 in rat brain slices exposed to hypoxia and ischemia. Brains were obtained from 2-day-old Sprague-Dawley rats, and sections were randomly assigned to control and hypoxia/ischemia groups. Hematoxylin-eosin staining revealed karyorrhexis and karyopyknosis in cells from the hypoxia/ischemia group. Under electron microscopy, mitochondria swelling and neuropil edema were observed in the hypoxiaJischemia group. Olig-1 mRNA and protein expressions were increased at 1 day after hypoxia and ischemia treatment. These results suggest that in situ hybridization and immunohistochemistry could be used simultaneously to detect mRNA and protein expression in brain slices.

Expression of hypoxia-inducible factor 1 alpha and oligodendrocyte lineage gene-1 in cultured brain slices after oxygen-glucose deprivation

Oligodendrocyte lineage gene-1 expressed in oligodendrocytes may trigger the repair of neuronal myelin impairment, and play a crucial role in myelin repair. Hypoxia-inducible factor la, a transcription factor, is of great significance in premature infants with hypoxic-ischemic brain damage There is little evidence of direct regulatory effects of hypoxia-inducible factor le on oligodendrocyte lineage gene-l. In this study, brain slices of Sprague-Dawley rats were cultured and subjected to oxygen-glucose deprivation. Then, slices were transfected with hypoxia-inducible factor la or oligodendrocyte lineage gene-1. The expression levels of hypoxia-inducible factor la and oligodendrocyte lineage gene-1 were significantly up-regulated in rat brains prior to transfection, as detected by immunohistochemical staining. Eight hours after transfection of slices with hypoxia-inducible factor la, oligodendrocyte lineage gene-1 expression was upregulated, and reached a peak 24 hours after transfection. Oligodendrocyte lineage gene-1 transfection induced no significant differences in hypoxia-inducible factor la levels in rat brain tissues with oxygen-glucose deprivation. These experimental findings indicate that hypoxia-inducible factor la can regulate oligodendrocyte lineage gene-1 expression in hypoxic brain tissue, thus repairing the neural impairment.

Protective effect of bone marrow-derived mesenchymal stem cells on dopaminergic neurons against 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in rat brain slices

BACKGROUND： To date, the use of bone marrow-derived mesenchymal stem cells （MSCs） for the treatment of Parkinson’s disease have solely focused on in vivo animal models. Because of the number of influencing factors, it has been difficult to determine a consistent outcome. OBJECTIVE： To establish an injury model in brain slices of substantia nigra and striatum using 1-methyl-4-phenylpytidinium ion （MPP＋）, and to investigate the effect of MSCs on dopaminergic neurons following MPP＋ induced damage. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, animal experiment using I mmunohistochemistry was performed at the Laboratory of the Department of Anatomy, Fudan University between January 2004 and December 2006. MATERIALS： Primary MSC cultures were obtained from femurs and tibias of adult Sprague Dawley rats. Organotypic brain slices were isolated from substantia nigra and striatum of 1-day-old Sprague Dawley rat pups. Monoclonal antibodies for tyrosine hydroxylase （TH, 1：5 000） were from Santa Cruz （USA）; goat anti-rabbit IgG antibodies labeled with FITC were from Boster Company （China）. METHODS： Organotypic brain slices were cultured for 5 days in whole culture medium supplemented with 50% DMEM, 25% equine serum, and 25% Tyrode’s balanced salt solution. The medium was supplemented with 5 μg/mL Ara-C, and the culture was continued for an additional 5 days. The undergrowth of brain slices was discarded at day 10. Eugonic brain slices were cultured with basal media for an additional 7 days. The brain slices were divided into three groups： control, MPP＋ exposure, and co-culture. For the MPP＋ group, MPP＋ （30 μmol/L） was added to the media at day 17 and brain slices were cultured for 4 days, followed by control media. For the co-culture group, the MPP＋ injured brain slices were placed over MSCs in the well and were further cultured for 7 days. MAIN OUTCOME MEASURES： After 28 days in culture, neurite outgrowth was examined in the brain slices under phase-contrast microscopy. The percent of area containing dead cells in each brain slice was calculated with the help of propidium iodide fluorescence. Brain slices were stained with antibodies for TH to indicate the presence of dopaminergic neurons. Transmission electron microscopy was applied to determine the effect of MSCs on neuronal ultrastructure. RESULTS： Massive cell death and neurite breakage was observed in the MPP＋ group. In addition, TH expression was significantly reduced, compared to the control group （P 〈 0.01）. After 7 days in culture with MSCs, the co-culture group presented with less cell damage and reduced neurite breakage, and TH expression was increased. However, these changes were not significantly different from the MPP＋ group （P 〈 0.01）. Electron microscopy revealed reduced ultrastructural injury to cells in the brain slices. However, vacuoles were present in cells, with some autophagic vacuoles. CONCLUSION： Bone marrow-derived MSCs can promote survival of dopaminergic neurons following MPP＋-induced neurotoxicity in co-cultures with substantia nigra and striatum brain slices.

Inhibitory effect of morphine on excitatory synaptic transmission via presynaptic mechanism in rat SON neurons in brain slices

Objective: To observe the effects of morphine on the excitatory postsynaptic currents (EPSCs) and miniature EPSCs (mEPSCs) in rat supraoptic nucleus (SON) neurons and to explore its synaptic mechanism. Methods: Using whole-cell voltage-clamp recording technique in the brain slices, the EPSCS and mEPSCs of rat SON neurons were recorded, respectively. Results: Morphine (20μmol/L) decreased the frequency of EPSCs and mEPSCs (by 65% for EPSCS and by 45% for mEPSCs), and reduced the amplitude of EPSCs by 44% in all SON neurons, but the amplitude distribution of mEPSCs was not affected. Conclusion: Morphine inhibits the excitatory transmissions via presynaptic mechanisms in SON neurons from rat brain slices.

Negative regulation of miRNA-9 on oligodendrocyte lineage gene 1 during hypoxic-ischemic brain damage

Oligodendrocyte lineage gene 1 plays a key role in hypoxic-ischemic brain damage and myelin repair, miRNA-9 is involved in the occurrence of many related neurological disorders. Bioin- formatics analysis demonstrated that miRNA-9 complementarily, but incompletely, bound oligodendrocyte lineage gene 1, but whether miRNA-9 regulates oligodendrocyte lineage gene 1 remains poorly understood. Whole brain slices of 3-day-old Sprague-Dawley rats were cultured and divided into four groups： control group; oxygen-glucose deprivation group （treatment with 8% O2 ＋ 92% N2 and sugar-free medium for 60 minutes）; transfection control group （after oxygen and glucose deprivation for 60 minutes, transfected with control plasmid） and miRNA-9 transfection group （after oxygen and glucose deprivation for 60 minutes, transfected with miRNA-9 plasmid）. From the third day of transfection, and with increasing culture days, oligodendrocyte lineage gene 1 expression increased in each group, peaked at 14 days, and then decreased at 21 days. Real-time quantitative PCR results, however, demonstrated that oligoden- drocyte lineage gene 1 expression was lower in the miRNA-9 transfection group than that in the transfection control group at 1, 3, 7, 14, 21 and 28 days after transfection. Results suggested that miRNA-9 possibly negatively regulated oligodendrocyte lineage gene 1 in brain tissues during hypoxic-ischemic brain damage.

Protective effects of costunolide on mouse brain slice injury induced by oxygen glucose deprivation/reoxygenation

OBJECTIVE To investigate the protective effects and mechanisms of costunolide against mousebrain slice injury induced by oxygen-glucose deprivation/reoxygenation(OGD/R).METHODS Mouse brain slice injury was induced by OGD/R in vitro,and the degree ofinjury was evaluated by measuring the release of lactate dehydrogenase(LDH)and 2,3,5-triphenyltetrazolium chloride(TTC)staining.Western blotting was used to analyze the expression of Bax,Bcl-2,Cyt-c,caspase-9,caspase-7 and caspase-3.RESULTS Compared with OGD/R,1,5,and 10μmol·L^-1 costu⁃nolide decreased the LDH levels,increased the TTC staining intensity,inhibited Bax,Cyt-c,caspase-9,caspase-7,caspase-3 expression levels,and enhanced Bcl-2 expression level.CONCLUSION Costunolide has latent neuroprotective activi⁃ties by the regulation of apoptosis via the mitochondrial apoptosis pathway.

Protection of Cactus Polysaccharide against H_2O_2-induced damage in the rat cerebral cortex and hippocampus Differences in time of administration

BACKGROUND： Pharmacological research has shown that cactus polysaccharide （CP） has anti-oxidant, anti-inflammatory, anti-tumor, anti-aging, and immune-stimulating activities. It may also provide protective effects against oxidative stress injuries in the rat brain. OBJECTIVE： To validate the effects of CP on H2O2-induced oxidative stress injuries in the rat cerebral cortex and hippocampal slices 30 minutes prior to injury, as well as 30 minutes and 2.5 hours after injury. DESIGN： A randomized controlled experiment. SETTINGS： Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology; Department of Pharmacology, College of Medical Science, Yangtze University. MATERIALS： A total of 50 male Sprague Dawley （SD） rats, normal grade and weighing 200 300 g, were provided by the Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology. The protocol was performed in accordance with ethical guidelines for the use and care of animals. Cactus polysaccharide, a dried needle crystal, was extracted from Opuntia milpa alta at the Chemistry and Environment Engineering School of Yangtze University. The following chemicals and instruments were used： 2,3,5-triphenyl tetrazolium chloride （Sigma, St Louis, Missouri, USA）; lactate dehydrogenase （LDH）, superoxide dismutase （SOD）, glutathione （GSH）, and total antioxidant competence （T-AOC） assays （Jiancheng-Bioeng Institute, Nanjing）; McIllwain tissue chopper （Mickle Laboratory Engineering, USA）; and ELISA reader and Magellan software （TECAN, Austria）. METHODS： This experiment was performed at the Department of Pharmacology, Medical College of Yangtze University, between March and June 2006. All rats were sacrificed after anesthesia. The cerebral cortex and hippocampus were dissected. Several cerebral cortex and hippocampus slices were selected as controls, while other sections were co-incubated with H2O2 for 30 minutes to induce an oxidative stress injury. The experimental slices were randomly divided into 3 groups： model group, low-dose group, and high-dose group. Slices from the low- and high-dose group were incubated with CP 30 minutes prior to injury, as well as 30 minutes and 2.5 hours after injury. The number of cerebral cortex and hippocampus slices at each time point was greater than 10 and 7, respectively. The control group slices were incubated with artificial cerebrospinal fluid （aCSF） for 3 hours. The model group slices were incubated with aCSF for 30 minutes, followed by 30 minutes of aCSF with H2O2 incubation to establish the oxidative stress injury model. Thereafter, the slices were re-incubated with aCSF for 2 hours. The slices in the low- and high-dose group were co-incubated with 0.33 mg/L CP or 1.67 mg/L CP, respectively. CP was applied either for 30 minutes prior to the H2O2 treatment, co-incubated with H2O2 for 30 minutes, or applied 2 hours after H2O2 treatment. MAIN OUTCOME MEASURES： Brain slice activity was determined by TTC staining. Biochemical markers, such as LDH, SOD, GSH, and T-AOC, were analyzed in clear supernatant liquid to study the pharmacological mechanisms. RESULTS： （1） Effects of CP on H2O2-injured brain slices： TTC absorption level at 490 nm decreased with a 30-minute CP administration before and after injury. The TTC absorption values in the low- and high-dose group were significantly lower than the control group （P 〈 0.05 - 0.01 ）. CP administration prior to insult had greater neuroprotective effects on brain slices than administration during the insult. CP Administration after insult had no obvious protective effects on the brain slices. There was no significant difference in TTC absorption levels between administration after 2.5 hours and the model group （P 〉 0.05）. （g） Effects of CP on substances released from H2O2-induced injury slices were as follows： following incubation with 2 mmol/L H2O2 for 30 minutes, LDH release was significantly increased from the rat cortical and hippocampal brain slices. T-AOC and GSH were decreased, which is in accordance with the TTC assay for evaluating the degree of slice injury. Prior administration of 0.33 mg/L CP and 1.67 mg/L CP decreased LDH release and enhanced T-AOC and GSH release in a dose-dependent manner. After H2O2-induced damage for 30 minutes, the release of SOD increased to some extent. SOD levels were further raised by pre-incubation with 0.33 mg/L CP or 1.67 mg/L CP, compared to the model group （P 〈 0.05）. CONCLUSION： CP can protect rat cerebral cortex and hippocampal slices from H2O2-induced injury, This protective effect is dose-dependent. CP administration before injury is more effective than during or after injury.

Protective Effectof GSH on PD Model Induced by 6-OHDA In Vitro

To study the effects of6 - hydroxydopamine(6 - OHDA) and reduced glutathione(GSH) on the nigral dopaminergic neurons in brain slices in vitro,immolunohistochem ical technique was used to observe the changes of TH - stained neurons,including cell bodies and the dendrites,in the substantia nigra (SN) of midbrain slices of rats after incubation for 1h in the presence of GSH 15 m in before and during the period of incubation with6 - OHDA.The results showed thatcell bodies rem ained intact but dendrites were fragm ented and truncated after treatment with6 - OHDA.The antioxidant GSH alone did not significantly affect the dendrites of SN neurons but prevented6 - O- HDA- induced damage of dendrites.It was concluded that glutathione m ay prevent6 - OHDA- in- duced dopam inergic neurodegeneration and play a protective role in dopaminergic neurons.

Seeing the wood for the trees:towards improved quantification of glial cells in central nervous system tissue

The following mini-review attempts to guide researchers in the quantification of fluorescently-labelled proteins within cultured thick or chromogenically-stained proteins within thin sections of brain tissue.It follows from our examination of the utility of Fiji Image J thresholding and binarization algorithms.Describing how we identified the maximum intensity projection as the best of six tested for two dimensional（2 D）-rendering of three-dimensional（3 D） images derived from a series of z-stacked micrographs,the review summarises our comparison of 16 global and 9 local algorithms for their ability to accurately quantify the expression of astrocytic glial fibrillary acidic protein（GFAP）,microglial ionized calcium binding adapter molecule 1（IBA1） and oligodendrocyte lineage Olig2 within fixed cultured rat hippocampal brain slices.The application of these algorithms to chromogenically-stained GFAP and IBA1 within thin tissue sections,is also described.Fiji’s Bio Voxxel plugin allowed categorisation of algorithms according to their sensitivity,specificity accuracy and relative quality.The Percentile algorithm was deemed best for quantifying levels of GFAP,the Li algorithm was best when quantifying IBA expression,while the Otsu algorithm was optimum for Olig2 staining,albeit with over-quantification of oligodendrocyte number when compared to a stereological approach.Also,GFAP and IBA expression in 3,3′-diaminobenzidine（DAB）/haematoxylin-stained cerebellar tissue was best quantified with Default,Isodata and Moments algorithms.The workflow presented in Figure 1 could help to improve the quality of research outcomes that are based on the quantification of protein with brain tissue.

Nervous System and Gastrointestinal Effects of the Insecticide Esfenvalerate on the Rat:An Ex Vivo Study

Esfenvalerate belongs to the pyrethroid group of insecticides which display significant selective toxicity against insects compared to mammalian species, nevertheless, they may pose health risks, especially in case of accidental exposure. The aim of the present study was to model the effect of acute, relatively high-dose exposure of the esfenvalerate-containing formulation Sumi-Alpha?. Eventual functional alterations in the central nervous system and in the gastrointestinal tract were studied on in vitro tissue preparations at different delays after intragastric administration to rats. Neuronal effects were characterized by field potential recording in cortical and hippocampal brain slices, while gastrointestinal effects were examined by analyzing the motility and excitability of isolated ileum segments. On the brain slices originating from esfenvalerate-treated animals, changes in excitability of both inhibitory and excitatory type could be observed. Voltage thresholds necessary to evoke responses in neocortex slices were elevated, and population spike amplitudes were lower in hippocampal slices. However, epileptiform potentials with pronounced late components were also observed. A decreased long-term potentiation (LTP) could be seen in both brain areas after esfenvalerate treatment. Seizure susceptibility of the slices was not significantly altered, but tended to be somewhat higher in slices originating from treated rats. In ileum segments, both spontaneous and acetyl-choline (ACh)-elicited contractions were modified by treatment. Esfenvalerate raised the amplitude of contractions in the low ACh concentration range. However, the solvent xylene also considerably contributed to the detected changes. We can conclude that a relatively high, single oral dose of Sumi-Alpha? exerted mild and temporary effects on the elementary brain functions and intestine functions of the rat.

Dissecting brain tumor growth and metastasis in vitro and ex vivo

Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system(CNS)tumors and greatly influence mortality and therapy-induced long-term morbidity in survivors.A number of in vitro and ex vivo assay systems have been established to better understand the infiltration and metastatic processes,to search for molecules that specifically block tumor cell infiltration and metastatic dissemination and to pre-clinically evaluate their efficaciousness.These systems allow analytical testing of tumor cell viability and motile and invasive capabilities in simplified and well-controlled environments.However,the urgent need for novel anti-metastatic therapies has provided an incentive for the further development of not only classical in vitro methods but also of novel,physiologically more relevant assay systems including organotypic brain slice culture.In this review,using publicly available peer-reviewed primary research and review articles,we provide an overview of a selection of in vitro and ex vivo techniques widely used to study growth and dissemination of primary metastatic brain tumors.Furthermore,we discuss how our steadily increasing knowledge of tumor biology and the tumor microenvironment could be integrated to improve current research methods for metastatic brain tumors.We believe that such rationally improved methods will ultimately increase our understanding of the biology of brain tumors and facilitate the development of more efficacious anti-metastatic treatments.

EFFECTS OF GLUTAMATE ON SODIUM CHANNEL IN ACUTELY DISSOCIATED HIPPOCAMPAL CA1 PYRAMIDAL NEURONS OF RATS

Objective To observe the effects of glutamate on sodium channel in acutely dissociated hippocampal CA1 pyramidal neurons of rats.Methods Voltage-dependent sodium currents (INa) in acutely dissociated hippocampal CA1 pyramidal neurons of neonate rats were recorded by whole-cell patchclamp of the brain slice technique when a series of doses of glutamate (100-1000μmol/L) were applied.Results Different concentrations of glutamate could inhibit INa,and higher concentration of glutamate affected greater inhibition.In same concentration on INa at different times,longer time glutamate affected greater inhibition.And glutamate could significantly shift activation curve of INa to the right and make inactivation curve of INa negative drift.Conclusion Glutamate can inhibit voltage-dependent sodium channel.Its blockage on INa has voltage-dependent,time-dependent,and dose-dependent characteristics.