OBJECTIVETo investigate acrylamide (ACR)-induced subacute neurotoxic effects on the central nervous system (CNS) at the synapse level in rats.

METHODSThirty-six Sprague Dawley (SD) rats were randomized into three groups, (1) a 30 mg/kg ACR-treated group, (2) a 50 mg/kg ACR-treated group, and (3) a normal saline (NS)-treated control group. Body weight and neurological changes were recorded each day. At the end of the test, cerebral cortex and cerebellum tissues were harvested and viewed using light and electron microscopy. Additionally, the expression of Synapsin I and P-Synapsin I in the cerebral cortex and cerebellum were investigated.

RESULTSThe 50 mg/kg ACR-treated rats showed a significant reduction in body weight compared with untreated individuals (P < 0.05). Rats exposed to ACR showed a significant increase in gait scores compared with the NS control group (P < 0.05). Histological examination indicated neuronal structural damage in the 50 mg/kg ACR treatment group. The active zone distance (AZD) and the nearest neighbor distance (NND) of synaptic vesicles in the cerebral cortex and cerebellum were increased in both the 30 mg/kg and 50 mg/kg ACR treatment groups. The ratio of the distribution of synaptic vesicles in the readily releasable pool (RRP) was decreased. Furthermore, the expression levels of Synapsin I and P-Synapsin I in the cerebral cortex and cerebellum were decreased in both the 30 mg/kg and 50 mg/kg ACR treatment groups.

CONCLUSIONSubacute ACR exposure contributes to neuropathy in the rat CNS. Functional damage of synaptic proteins and vesicles may be a mechanism of ACR neurotoxicity.

Acrylamide ; toxicity ; Animals ; Cerebellum ; cytology ; drug effects ; Cerebral Cortex ; cytology ; drug effects ; Drug Administration Schedule ; Gait ; Gene Expression Regulation ; drug effects ; Male ; Neurons ; drug effects ; Neurotoxicity Syndromes ; pathology ; Rats ; Rats, Sprague-Dawley ; Synapses ; drug effects ; Synapsins ; genetics ; metabolism ; Synaptic Vesicles ; drug effects ; physiology ; Weight Loss ; drug effects

Axonal transport of mitochondria is critical for neuronal survival and function. Automatically quantifying and analyzing mitochondrial movement in a large quantity remain challenging. Here, we report an efficient method for imaging and quantifying axonal mitochondrial transport using microfluidic-chamber-cultured neurons together with a newly developed analysis package named "MitoQuant". This tool-kit consists of an automated program for tracking mitochondrial movement inside live neuronal axons and a transient-velocity analysis program for analyzing dynamic movement patterns of mitochondria. Using this method, we examined axonal mitochondrial movement both in cultured mammalian neurons and in motor neuron axons of Drosophila in vivo. In 3 different paradigms (temperature changes, drug treatment and genetic manipulation) that affect mitochondria, we have shown that this new method is highly efficient and sensitive for detecting changes in mitochondrial movement. The method significantly enhanced our ability to quantitatively analyze axonal mitochondrial movement and allowed us to detect dynamic changes in axonal mitochondrial transport that were not detected by traditional kymographic analyses.
Animals ; Axonal Transport ; physiology ; Cerebral Cortex ; cytology ; metabolism ; Drosophila melanogaster ; cytology ; metabolism ; Embryo, Mammalian ; Gene Expression ; Lab-On-A-Chip Devices ; Microscopy, Confocal ; Mitochondria ; metabolism ; ultrastructure ; Motor Neurons ; metabolism ; ultrastructure ; Movement ; Mutation ; Primary Cell Culture ; RNA-Binding Protein FUS ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Software

OBJECTIVETo explore the role of P53 phosphorylation in neuron apoptosis of rats by chronic aluminum exposure.

METHODSA total of male 40 SD rats were divided randomly into 4 groups (n = 10/dose), the exposed groups were fed with normal diet with different concentration of AlCl3 · 6H2O for 6 months respectively. The dosage of low, middle and high groups were 10.73, 107.33, 1073.33 mg/kg in sequence. The control group received normal diet. The neuron apoptosis was measured by method of Tunel. The expressions of P53 and pP53-ser15 protein in the cortex were detected by Western-blot.

RESULTSTunel staining showed that the low, middle and high group rats had increased apoptosis rate than control group (P < 0.01). Western-blot test demonstrated that the expression of P53 protein in the cortex of high group rats were significantly higher than the control and low groups (P < 0.05). The expression of pP53-ser15 protein in the cortex of middle and high group rats were also higher than the control and low groups (P < 0.05).

CONCLUSIONChronic aluminum exposure can lead to over expression of P53 and pP53-ser15 protein in cerebral cortex, which maybe one of the most important mechanisms of neuron apoptosis induced by AlCl3.

Aluminum ; toxicity ; Aluminum Compounds ; toxicity ; Animals ; Apoptosis ; Cerebral Cortex ; metabolism ; Chlorides ; toxicity ; Male ; Neurons ; cytology ; drug effects ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo evaluate the efficiency of a modified culture method for rat cerebral cortical astrocytes in vitro.

METHODSThe astrocytes derived from the cerebral cortex of 3-day-old Sprague-Dawley rats were first purified as described previously, then the cells were replanted at a low density. The culture flask was changed after 1 hour and substratum was replaced after 24 hours. Cells were syncretized to a monolayer, followed by cell passage. After three passages the cells were cultured in DMEM medium containing 10% fetal serum for a long period. The derivation of the cells was identified by immunofluorescent staining with anti-GFAP polyclonal antibodies.

RESULTSA variety of morphologically distinct astrocytes with many long processes and small cell bodies were obtained. Finally an astrocytic network occurred through cellular process connections. The immunofluorescent staining demonstrated the percentage of GFAP-positive cells was above 98%.

CONCLUSIONSThe modified culture method for astrocytes from rat cerebral tissue is reliable, with a high purity. The cultured astrocytes have a similar morphological development to those in vivo.

Animals ; Astrocytes ; physiology ; Cell Culture Techniques ; Cerebral Cortex ; cytology ; Female ; Glial Fibrillary Acidic Protein ; analysis ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the protective effects of insulin-like growth factor-1 (IGF-1) on the nerve cells of neonatal rats under oxidative stress.

METHODSPrimary cortical neurons, oligodendrocytes, and astrocytes from newborn rats were cultured. An oxidative stress model was established with different concentrations of H2O2 (0-60 μmol/L); the degree of damage of nerve cells was evaluated by lactate dehydrogenase assay, and the viability of nerve cells was tested by MTT assay. An oxidative stress model was established with different concentration of H2O2 (0-80 μmol/L). Expression of Akt/p-Akt (Ser473) in neurons was measured by Western blot before and after IGF-1 (25 ng/mL) administration.

RESULTSCompared with those not treated with H2O2, the cortical neurons, oligodendrocytes, and astrocytes treated with different concentrations of H2O2 for 24 hours showed increased damage and decreased cell viability; compared with oligodendrocytes and astrocytes, neurons showed significantly more changes (P<0.01). Compared with those not treated with H2O2, the cortical neurons treated with different concentrations of H2O2 for 5 minutes showed a significant decrease in p-Akt (Ser473) level (P<0.01), which was dependent on the concentration of H2O2. For the neurons treated with low-concentration H2O2, the addition of IGF-1 could reverse the inhibition of Akt phosphorylation, eliminating the difference in p-Akt level compared with the neurons not treated with H2O2, (P>0.05); however, it had no significant effect on the inhibition of Akt phosphorylation by high-concentration H2O2, and the treated neurons still had a lower p-Akt level than untreated neurons (P<0.01 for all). For the cortical neurons that had been treated with different concentration of H2O2 for 1 hour, the addition of IGF-1 (25 ng/mL) could eliminate thedifference in p-Akt level between the treated neurons and untreated neurons (P>0.05).

CONCLUSIONSCortical neurons are more sensitive to oxidative stress induced by H2O2 than other nerve cells. IGF-1 has protective effects on cortical nerve cells under oxidative stress.

Animals ; Animals, Newborn ; Cerebral Cortex ; cytology ; drug effects ; Hydrogen Peroxide ; pharmacology ; Insulin-Like Growth Factor I ; pharmacology ; Neurons ; drug effects ; metabolism ; Oxidative Stress ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism

To investigate the role and possible molecular mechanism of astrocytes in inflammation and amyloid β-protein (Aβ) formation, in this research, by using LPS to stimulate cultured rat astrocytes in vitro with or without anti-Toll-like receptor 4 (TLR4) antibody pretreatment, we first detected the TLR4, TNF-α, IL-1β, β-amyloid precursor protein (β-APP) and β-site APP clearing enzyme 1 (BACE1) mRNA with real-time PCR, and TLR4, NF-κB/P65 protein in cultured astrocytes by Western blot, and then further probed the translocation of NF-κB/P65 using immunofluorescence and the contents of TNF-α, IL-1β and Aβ in culture supernatant through ELISA. We found that all of these indexes increased at different degrees after LPS-stimulation. However, if pretreatment with anti- TLR4 antibody, such stimulating effects of LPS on the nuclear translocation of NF-κB/P65 and TNF-α, IL-1β, Aβ contents in astrocytic culture supernatant were reduced significantly or disappeared in comparison with the group with only LPS-administration. Our results suggest that TLR4 in astrocytes might play an important role in the inflammation and Aβ formation through the TLR4/NF-κB signaling pathway, thus providing new knowledge and understanding of the inflammatory hypothesis of AD pathogenesis.
Amyloid Precursor Protein Secretases ; metabolism ; Amyloid beta-Protein Precursor ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; metabolism ; Astrocytes ; metabolism ; Cells, Cultured ; Cerebral Cortex ; cytology ; Inflammation ; metabolism ; Interleukin-1beta ; metabolism ; RNA, Messenger ; Rats ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Toll-Like Receptor 4 ; metabolism ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

In the present study, we examined the effect of oxygen glucose deprivation (OGD) post-conditioning (PostC) on neural cell apoptosis in OGD-PostC model and the protective effect on primary cortical neurons against OGD injury in vitro. Four-h OGD was induced by OGD by using a specialized and humidified chamber. To initiate OGD, culture medium was replaced with de-oxygenated and glucose-free extracellular solution-Locke's medium. After OGD treatment for 4 h, cells were then allowed to recover for 6 h or 20 h. Then lactate dehydrogenase (LDH) release assay, Western blotting and flow cytometry were used to detect cell death, protein levels and apoptotic cells, respectively. For the PostC treatment, three cycles of 15-min OGD, followed by 15 min normal cultivation, were applied immediately after injurious 4-h OGD. Cells were then allowed to recover for 6 h or 20 h, and cell death was assessed by LDH release assay. Apoptotic cells were flow cytometrically evaluated after 4-h OGD, followed by re-oxygenation for 20 h (O4/R20). In addition, Western blotting was used to examine the expression of heat-shock protein 70 (HSP70), Bcl-2 and Bax. The ratio of Bcl-2 expression was (0.44±0.08)% and (0.76±0.10)%, and that of Bax expression was (0.51±0.05)% and (0.39±0.04)%, and that of HSP70 was (0.42±0.031)% and (0.72±0.045)% respectively in OGD group and PostC group. After O4/R6, the rate of neuron death in PostC group and OGD groups was (28.96±3.03)% and (37.02±4.47)%, respectively. Therefore, the PostC treatment could up-regulate the expression of HSP70 and Bcl-2, but down-regulate Bax expression. As compared with OGD group, OGD-induced neuron death and apoptosis were significantly decreased in PostC group (P<0.05). These findings suggest that PostC inhibited OGD-induced neuron death. This neuro-protective effect is likely achieved by anti-apoptotic mechanisms and is associated with over-expression of HSP70.
Animals ; Apoptosis ; drug effects ; Blotting, Western ; Cell Hypoxia ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; blood supply ; cytology ; embryology ; Flow Cytometry ; Glucose ; pharmacology ; HSP70 Heat-Shock Proteins ; metabolism ; Ischemic Postconditioning ; methods ; Neurons ; cytology ; drug effects ; metabolism ; Oxygen ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the antioxidative effects of two cysteinyl leukotriene receptors antagonists (CysLT1R and CysLT2R) montelukast and HAMI 3379 on ischemic injury of rat cortical neurons in vitro.

METHODSCultured rat cortical neurons were pretreated with CysLT1R antagonist montelukast and CysLT2R antagonist HAMI 3379, and then exposed to oxygen-glucose deprivation/recovery (OGD/R）or H2O2. Reactive oxygen species (ROS) mitochondrial membrane potential (MMP) depolarization, neuronal viability and lactate dehydrogenase (LDH) release were determined. Meanwhile, RNA interference was used to inhibit the expression of CysLT1R and CysLT2R，and the effects were observed.

RESULTSROS production in neurons was significantly increased after 1 h OGD, which reached the peak at 30 min and lasted for 1.5 h after recovery. Montelukast and HAMI 3379 at 0.01-1μmol/L moderately decreased OGD/R-induced ROS production (P<0.05). Montelukast mildly attenuated OGD/R-induced MMP depolarization (P<0.05)，but HAMI 3379 had no effect. H2O2 reduced neuronal viability and increased LDH release, namely inducing neuronal injury. Montelukast and HAMI 3379 at 0.1-1μmol/L moderately attenuated H2O2-induced neuronal injury (P<0.05). However, both CysLT1R siRNA and CysLT2R shRNA did not significantly affect the responses mentioned above.

CONCLUSIONIn ischemic neuronal injury, montelukast and HAMI 3379 exert a moderate antioxidative effect, and this effect may be receptor-independent.

Acetates ; pharmacology ; Animals ; Antioxidants ; pharmacology ; Cell Hypoxia ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; cytology ; Cyclohexanecarboxylic Acids ; pharmacology ; Leukotriene Antagonists ; pharmacology ; Neurons ; drug effects ; metabolism ; Phthalic Acids ; pharmacology ; Quinolines ; pharmacology ; Rats ; Reactive Oxygen Species ; metabolism

The present paper is aimedto investigate the effect of basic fibroblast growth factor (bFGF) on proliferation, migration and differentiation of endogenous neural stem cell in rat cerebral cortex with global brain ischemia-reperfusion. A global brain ischemia-reperfusion model was established. Immunohistochemistry was used to observe the pathological changes and the expression of BrdU and Nestin in cerebral cortex. RT-PCR was used to measure the NSE mRNA in brain tissue. The results of measurements indicated that in sham operation group, there was no positive cell in cerebral cortex, and the content of NSE mRNA did not change. In the operation group, the expression of BrdU and Nestin increased significantly at the end of the 3rd day, and peaked on the 7th day. NSE mRNA expression did not significantly increase. In bFGF group, compared with sham operation group and model group, the number of BrdU-positive and Nestin-positive cells increased significantly at each time point (P<0. 05), and peaked at the end of the 11th day, and the content of NSE mRNA increased significantly (P<0. 05). This research demonstrated that the proliferation of endogenous neural stem cells in situ could be induced by global cerebral ischemia and reperfu- sion, and could be promoted and extended by bFGF. In additiion, bFGF might promote endogenous neural stem cells differentiated into neurons.
Animals ; Brain Ischemia ; pathology ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Cerebral Cortex ; cytology ; metabolism ; pathology ; Fibroblast Growth Factor 2 ; pharmacology ; Nestin ; metabolism ; Neural Stem Cells ; drug effects ; Rats ; Reperfusion Injury

To investigate the electrophysiology mechanisms of new anxiolytic and antidepressant drug: 4-butyl-alpha-agarofuran (AF-5), patch clamp-recording was used to test the effects of AF-5 on voltage-dependent sodium currents, voltage-dependent potassium currents, L-type voltage-dependent calcium currents and GABA dependent Cl(-) currents in primary cultured rat cortical neurons. Effects of AF-5 on Kv2.1 currents, expressed stably in HEK293 cells, were also tested. Our results showed that, delayed rectifier potassium currents (I(K(DR, L-type voltage-dependent calcium currents (I(LC-ca)) in primary cultured rat cortical neurons and Kv2.1 currents in HEK293 cells were significantly inhibited by AF-5, with IC50 as 6.17, 4.4 and 5.29 micromol x L(-1) respectively. However, voltage-dependent sodium currents (I(Na)), GABA dependent Cl(-) currents and transient outward potassium currents (I(K(A)) in primary cultured rat cortical neurons were not significantly blocked by AF-5. Our results concluded that, blocked I(K(DR)) and I(L-Ca) currents may be one of the mechanisms of anxiolytic and antidepression actions of AF-5.
Animals ; Antidepressive Agents ; pharmacology ; Calcium Channels, L-Type ; drug effects ; Cells, Cultured ; Cerebral Cortex ; cytology ; Chloride Channels ; drug effects ; Delayed Rectifier Potassium Channels ; drug effects ; HEK293 Cells ; Humans ; Neurons ; cytology ; Patch-Clamp Techniques ; Potassium Channels, Voltage-Gated ; drug effects ; Rats ; Rats, Wistar ; Sesquiterpenes ; pharmacology ; Shab Potassium Channels ; drug effects ; Voltage-Gated Sodium Channels ; drug effects