Cellular damage of low-dose combined exposure to mercury, lead and cadmium on hippocampal neurons in rats / 中华预防医学杂志

Objective@#To observe the cellular damage of low-dose combined exposure to Hg, Pb and Cd on hippocampal neurons in rat.@*Methods@#SH-SY5Y cells were randomly divided into 8 groups by 2×2×2 factorial design: control group, Pb exposure group, Hg exposure group, Pb+Hg exposure group, Pb+Cd exposure group, Hg+Cd exposure group and Pb+Cd+Hg exposure group. And the cell viabilities were measured. On this basis, an animal model was established. Twenty eight-week-old SD pregnant rats were randomly divided into four groups by random number table, and five in each group: the control group(distilled water), 1-fold metal mixture exposure group (1×MM, poisoning solution containing mercury chloride 0.15 mg/L, lead acetate trihydrate 25 mg/L, cadmium chloride 7.5 mg/L), 5-fold metal mixture exposure group (5×MM, poisoning solution containing mercury chloride 0.75 mg/L, lead acetate trihydrate 125.00 mg/L, cadmium chloride 37.50 mg/L), 10-fold metal mixture exposure group (10×MM, poisoning solution containing mercury chloride 1.50 mg/L, lead acetate trihydrate 250.00 mg/L, cadmium chloride 75.00 mg/L). Pregnant rats drank water until delivery. Twenty male pups were selected and exposed to these metals through breast milk until weaned. The heavy metals dose of poisoning water was adjusted, and then the weaned rats were exposed to heavy metals via drinking poisoning water until adulthood (postnatal day 83). The blood samples and brain hippocampus samples were collected to observe the ultrastructural changes of hippocampus, and to determine the levels of Hg, Pb and Cd in blood. In addition, apoptosis rate and fluorescence intensity of reactive oxygen species and intracellular free calcium concentration ([Ca2+]i) in hippocampal neurons were measured.@*Results@#Cellular factorial design analysis showed that Hg+Pb+Cd (at no observed adverse effect level, 1.0, 0.5 and 0.1 μmol/L, respectively)had a interaction on cell viability after 48 or 72 hours of combined exposure (P<0.05). The results of ultrastructure showed that mitochondria decreased, ridges and matrixes gradually dissolved in rat hippocampal neurons of 5×MM group; nuclear chromatin aggregated, more ridges and matrixes dissolved and the mitochondria also decreased in rat hippocampal neurons of 10×MM group. The concentration of Hg, Pb and Cd in the blood of 1×MM group, 5×MM group and 10×MM group were higher than those in the control group, and the differences were statistically significant (P<0.001). There was no significant difference in apoptosis rate between the 1×MM group and the control group. The apoptosis rate of 5×MM group and 10×MM group was higher than that in the control group, and the differences were statistically significant (P<0.001). There was no statistically significant difference in the fluorescence intensity of reactive oxygen species in hippocampal neurons of the 1×MM group and the control group. The fluorescence intensity of reactive oxygen species in the 5×MM group and the 10×MM group was higher than that in the control group, the difference was statistically significant (P<0.05). There was no significant difference in the fluorescence intensity of [Ca2+]i between the 1×MM group and the control group. The fluorescence intensity values of [Ca2+]i in the 5×MM group and the 10×MM group were higher than the control group, the differences were statistically significant (P<0.001).@*Conclusion@#Low-level combined exposure to Hg, Pb, and Cd caused synergistic neurotoxic damage, and the process may be related to the changes of neuronal apoptosis, reactive oxide species, and [Ca2+]i levels.

Microwave exposure impairs synaptic plasticity in the rat hippocampus and PC12 cells through over-activation of the NMDA receptor signaling pathway / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>The aim of this study is to investigate whether microwave exposure would affect the N-methyl-D-aspartate receptor (NMDAR) signaling pathway to establish whether this plays a role in synaptic plasticity impairment.</p><p><b>METHODS</b>48 male Wistar rats were exposed to 30 mW/cm2 microwave for 10 min every other day for three times. Hippocampal structure was observed through H&E staining and transmission electron microscope. PC12 cells were exposed to 30 mW/cm2 microwave for 5 min and the synapse morphology was visualized with scanning electron microscope and atomic force microscope. The release of amino acid neurotransmitters and calcium influx were detected. The expressions of several key NMDAR signaling molecules were evaluated.</p><p><b>RESULTS</b>Microwave exposure caused injury in rat hippocampal structure and PC12 cells, especially the structure and quantity of synapses. The ratio of glutamic acid and gamma-aminobutyric acid neurotransmitters was increased and the intracellular calcium level was elevated in PC12 cells. A significant change in NMDAR subunits (NR1, NR2A, and NR2B) and related signaling molecules (Ca2+/calmodulin-dependent kinase II gamma and phosphorylated cAMP-response element binding protein) were examined.</p><p><b>CONCLUSION</b>30 mW/cm2 microwave exposure resulted in alterations of synaptic structure, amino acid neurotransmitter release and calcium influx. NMDAR signaling molecules were closely associated with impaired synaptic plasticity.</p>

Antinociceptive effects of gabapentin & its mechanism of action in experimental animal studies.

Background & objectives: Several studies have shown the possible analgesic effects of gabapentin, widely used as an antiepileptic. Thus, clinical studies have been carried out especially for neuropathic syndroms. This study was undertaken to investigate experimentally whether gabapentin has analgesic effects in mice and rats. Methods: The mice were divided into 10 groups (n=7) with various treatments to assess central and peripheral antinociceptive activity of gabapentin. Hot plate, tail clip and tail flick tests were applied for the investigation of central antinociceptive activity and the writhing test was applied for the investigation of peripheral antinociceptive activity. In addition, we also evaluated the levels of PGE2 and nNOS on perfused hippocampus slices of rats. Results: Gabapentin showed a peripheral antinociceptive effect at all doses and a central antinociceptive effect at 30mg/kg dose. While the L-NAME and cyproheptadine changed the central and peripheral effects of gabapentin, naloxone did not change these effects. In vitro studies showed that gabapentin significantly increased nNOS level. PGE2 and nNOS were found to have an important role in the antinociceptive effects of gabapentin at all doses and its combinations with L-NAME, cyproheptadine, indomethacine, and naloxone. As expected, PGE2 levels decreased in all groups, while nNOS levels increased, which is believed to be an adaptation mechanism. Interpretation & conclusions: Our findings indicate that arachidonate, nitrergic and serotonergic systems play an important role in the antinociceptive activity of gabapentin except for the opioidergic system. Additionally, this effect occured centrally and peripherally. These effects were also mediated by nNOS and PGE2.

Effects of Embedding Medicine in Acupoints and Electro-acupuncture on Expression of HSP70 in Hippocampus of Rat with Focal Cerebral Ischemia Reperfusion / 中国中医药信息杂志

Objective To observe the effects of embedding implantation of collagen and Tetramethylpyrazine in acupoints and electro-acupuncture on the expression of HSP70 in hippocampus of rat with focal cerebral ischemia reperfusion. Methods SD rats were randomly divided into normal group, sham operation group, model group, electro-acupuncture group, embedding medicine in acupoints group. Model of local ischemia was made by middle cerebral artery occlusion, preparation of implantation collagen and electro acupuncture and Tetramethylpyrazine was embedded in the acupoints of Dazhui and Neiguan two-sided. The expression of HSP-70 protein in hippocampus of different time point (24, 72, 120 h) was assayed by immunohistochemistry. Results There were only few positive cells of HSP-70 in normal group and sham operation group. The expression of HSP-70 in each therapy group were all heightened, and achieved the peak 24 h later and then declined in brain tissue after focal cerebral ischemiareperfusion, the differences were relatively prominent compared with the same phase model group (P

Proteomic analysis of hippocampus in the rat / 中国脑血管病杂志

Objective To analyze the protein expression in the rat hippocampus by the proteomic approach.Methods Proteins from hippocampal tissue homogenates of the rat were separated by two-dimensional gel electrophoresis(2-DE),and stained with colloidal Coomassie blue to produce a high-resolution map of the rat hippocampus proteome.Selected proteins from this map were digested with trypsin,and the resulting tryptic peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).The mass spectrometric data were used to identify the proteins through searches of the NCBI protein sequence database.Results 37 prominent proteins with various functional characteristics were identified.The identified brain protein classes covered metabolism enzymes,cytoskeleton proteins,heat shock proteins,antioxidant proteins,signalling proteins,proteasome-related proteins,neuron-specific proteins and glial-associated proteins.Furthermore,3 hypothetical proteins,unknown proteins so far only proposed from their nucleic acid structure,were identified.Conclusion This study provides the first unbiased characterization of proteins of the rat hippocampus and will be used for future studies of differential protein expression in rat models of neurological disorders.

Effects of infrasound on hippocampus ultrastructure of rats / 中国康复理论与实践

@#ObjectiveTo investigate effects of 8Hz 90dB and 8Hz 130dB infrasound on hippocampi ultrastructure of SD rats.Methods60 male SD rats were randomly divided into the control group and experimental group. Rats of experimental group were exposed to 8Hz 90dB or 8Hz 130dB infrasound 2 hours every day for 1,7,14, 21,28,35 and 42 days. As soon as the exposure finished,the right hippocampus of rat was taken and examined by electron microscopy.ResultsHippocampus ultrastructure of rats was changed after only one day exposure to infrasound and the longer exposure to infrasound, the worse ultrastructure changed. But during the latter period,damages of hippocampus ultrastructure became lighter. Different rules were observed between 90dB and 130dB groups.ConclusionInfrasound of 8Hz 90dB and 8Hz 130dB can make hippocampus ultrastructure of rats damaged mainly as denaturalizations. These changes are nonliner with time, and the hippocampus of rats has adaptability to infrasound.

Effect of High Temperature on Electrical Seizure-induced Neuronal Damage in Explant Culture of Rat Hippocampus / 대한소아신경학회지

PURPOSE: Epileptic patients have a increasing tendency to develop seizure attack in high temperature. This finding suggests that high temperature may have an effect on neuronal hyperexcitability and injury of epileptic brain. Therefore, the influence of high temperature on normal and epileptic brain was studied in organotypic explant cultures of rat. METHODS: Fourteen days-in-vitro cultures from 8 day-old rat pups were perfused with standard aCSF bubbled with 95%/5% O2/CO2 in a microchamber. Stimulus train(0.3 sec, 60 Hz) was applied to Schaffer collaterals in CA3 and extracellular field potential was recorded in the CA1 pyramidal layer. At 36degrees C initially, AD was evoked. In high temperature(HT) group, the cultures were subjected to 39degrees C for a period of 8 min before the second stimulus train was applied. They were then restored to 36degrees C for 10 min. In normal temperature group, temperature was maintained at 36degrees C for the second stimulus train. The cultures were returned to the incubator and observed serially for neuronal damage. Intensity of propidium iodide fluorescence indicative of neuronal injury was quantitated by digital image analysis. The cultures on the same insert that were not stimulated served as the unstimulated groups. RESULTS: There was not a statistically significant difference in neuronal damage between the unstimulated high-temperature(HT) and normal-temperature(NT) group. In CA1 sector, % damage(mean+/-SEM) was 0.42+/-0.20 vs 0.27+/-0.05 at 24 hrs(HT vs NT group, n=16 each, P>0.05, Student t-test); 1.81+/-0.79 vs 1.43+/-0.27 at 48 hrs(P>0.05); 3.50+/-1.32 vs 3.35+/-0.56 at 72 hrs(P>0.05). In CA3 sector, % damage was 0.34+/-0.10 vs 0.20+/-0.03 at 24 hrs(P>0.05); 0.99+/-0.20 vs 0.83+/-0.23 at 48 hrs(P>0.05); 2.00+/-0.38% vs 2.26+/-0.35% at 72 hrs(P>0.05). Neuronal damage on AD induced cultures during febrile setting(n=16) was significantly higher than in nonfebrile setting(n=16). In CA1 sector, % damage was 6.63+/-2.56 vs 0.92+/-0.45 at 24 hrs(febrile setting vs nonfebrile setting, P= 0.036); 26.37+/-7.44 vs 4.99+/-2.23 at 48 hrs(P=0.010); 38.59+/-9.63 vs 6.48+/-2.30 at 72 hrs (P=0.003). In CA3 sector, % damage was 1.23+/-0.48 vs 3.91+/-2.37 at 24 hrs(P=0.277); 13.09+/-5.75 vs 5.93+/-3.27 at 48 hrs(P=0.288); 27.86+/-8.68 vs 7.54+/-3.74 at 72 hrs(P=0.04). CONCLUSION: At high temperature, seizures in epileptic brain may be more injurious than seizures in normal temperature.

Developmental Changes in the Activation of Signal Transduction Pathway via JNK in Rat Hippocampus after Kainic Acid-Induced Seizure / 신경정신의학

OBJECTIVE: We observed the developmental pattern of activation of MAPK signal transduction pathways known to be activated by electroconvulsive shock(ECS) in young rat hippocampus after kainic acid(KA)-induced seizure. METHODS: We used the method of immunoblotting for examining the basal protein amount and basal level of phosphorylation of MAPK kinase(SAPK/ERK kinase -1, SEK-1), MAPK(c-Jun N terminal protein kinase, JNK), transcription factor(c-Jun) and immediate early gene proteins(Fos) in rat hippocampus at postnatal day 7, 14, and 21, respectively. We also examined the changes of phosphorylation of those proteins after kainic acid-induced seizure in the same way. RESULTS: The basal protein amounts of SEK-1, JNK, and c-Jun did not show age-dependent changes and basal level of phosphorylation of JNK and c-Jun remains unchanged throughout the early developmental period. The basal level of phosphorylation of SEK-1 was peaked at postnatal 7 days and then decreased with aging. After kainic acid-induced seizure, the change of phosphorylation of JNK was not observed but those of SEK-1 and c-Jun increased after postnatal day 14. The expression of Fos was observed at postnatal day 7 and also increased with aging. CONCLUSION: These results show that the MAPK signal transduction system in rat hippocampus matures in accordance with aging, but the process of maturation differs depending specific proteins. This study suggests the signal transduction cascade(SEK-1 - JNK - c-Jun - Fos) which is well established in cell line studies may not be applied to rat hipposcampus because we could not observe the activation of JNK after KA-induced seizure in young rat hippocampus.

Comparison of Fos Protein Expressions Rat Hippocampus Following Single and Repeated Intraperitoneal Administration of Kainic Acid / 대한간질학회지

BACKGROUND:Acute seizures that increase neuronal activity cause a rapid and transient induction of the immmediate early c-fos in specific brain regions. C-fos gene may mediate long-term changes in cell function, such as growth, differeniation, and development, in response to acute extracellular stimulation. This study is designed to compare the expression of Fos protein in hippocampus after single and repeated injections of kainic acid (KA). METHODS:In KA-single injection model, twelve adult male Sprague-Dawley rats were treated with single intraperitoneal injection of convulsive dose(20-30 mg/kg) of KA, and. in KA-multiple injections model, seven rats received KA by repeated daily intraperitoneal injections for 15 days. Eight control rats received normal saline. Expression of Fos protein was tested in hippocampus by immunohistochemical staining, and was scored by the degree of staining intensity and the ratio of stained cells to tested ones. RESULTS:The scores tended to increase in CA3 and dentate gyrus were significantly higher in KA-single injection model than in control (p<0.05). In comparison with scores in KA-model. CONCLUSION:These results show that repeated seizure produces some blockade of c-fos induction in CA2, CA3 and dentate gyrus. This may be a long-term adaptive response by the nervous system to repated neuronal activation

Effect of White Noise on Expression of NOS Positive Neurons in Hippocampus of Rats during the Processing of Learning and Memory / 中国心理卫生杂志

Objective: To observe the effects of exposure to white noise on the procession of learning and memory, and the expression of NOS (nitric oxide synthase) in hippocampus of rats Method: 20 rats were randomly divided into two groups for observation of spatial learning and memory, one group exposed to 80 dB white noise during Y-maze training, the other group received Y-maze training under normal condition Another 24 rats were randomly divided into 3 groups, they experienced normal Y-maze training, noise Y-maze training and sole noise as control respectively Immunohistochemical assay was used to investigate the expression of NOS positive neurons Result: Noise hampered the ability of rats' spatial learning and memory, furthermore, the number of NOS positive neurons in hippocampus of noise training group was less than that of normal training group Conclusion: The activity of NOS positive neuron in hippocampus was decreased by exposure to noise, suggesting the inhibition of learning dependent long term procession in hippocampus and corresponding difficulty in obtaining and maintaining of memory