MicroRNA-219 alleviates glutamate-induced neurotoxicity in cultured hippocampal neurons by targeting calmodulin-dependent protein kinase Ⅱ gamma

Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal neurons to model mi R-219 overexpression.A protective effect of mi R-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons,and an underlying mechanism involving calmodulin-dependent protein kinase II γ（Ca MKIIγ） was demonstrated.mi R-219 and Ca MKIIγ m RNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.After neurons were transfected with mi R-219 mimic,effects on cell viability and apoptosis were measured by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide（MTT） assay and flow cytometry.In addition,a luciferase reporter gene system was used to confirm Ca MKIIγ as a target gene of mi R-219.Western blot assay and rescue experiments were also utilized to detect Ca MKIIγ expression and further verify that mi R-219 in hippocampal neurons exerted its effect through regulation of Ca MKIIγ.MTT assay and q RT-PCR results revealed obvious decreases in cell viability and mi R-219 expression after glutamate stimulation,while Ca MKIIγ m RNA expression was increased.MTT,flow cytometry,and caspase-3 activity assays showed that mi R-219 overexpression could elevate glutamate-induced cell viability,and reduce cell apoptosis and caspase-3 activity.Moreover,luciferase Ca MKIIγ-reporter activity was remarkably decreased by co-transfection with mi R-219 mimic,and the results of a rescue experiment showed that Ca MKIIγ overexpression could reverse the biological effects of mi R-219.Collectively,these findings verify that mi R-219 expression was decreased in glutamate-induced neurons,Ca MKIIγ was a target gene of mi R-219,and mi R-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling Ca MKIIγ expression.

Comparison of the severity of injury of hippocampal neuron in rats induced by simulated push-pull maneuver at various degrees

BACKGROUND： Push-pull effect is often caused during maneuver, and the changes of unconsciousness induced can affect or damage cerebral neurons at various degrees. OBJECTIVE： To observe the effect of simulated push-pull maneuver at various degrees on injury of hippocampal neurons in rats and analyze its phase effect. DESIGN： Randomized control study.SETTING ： Physiological Department of Jilin Medical College.MATERIALS： A total of 40 healthy male Wistar rats, of clean grade, weighting 205-300 g, aged 3-4 months, were randomly divided into control group （n=4） and three push-pull experimental groups, including ＋2 Gz group （intensity： -2 Gz to ＋2 Gz, n=12）, ＋6 Gz group （-6 Gz to ＋6 Gz, n=12） and ＋8 Gz group （-8 Gz to ＋8 Gz, n=12）.METHODS： The experiment was completed in the Physiological Department of Jilin Military Medical College from March 2002 to May 2003. ① Rats in the experimental groups were put at the specially rolling arm of animal centrifugal machine. Then, they were pushed and pulled with ±2 Gz, ±6 Gz and ±8 Gz, respectively. The jolt was 1 Gz/s. However, rats in control group were not treated with any ways. ② Stroke index and neurological evaluation were performed on rats in the experimental groups at 0.5, 6 and 24 hours after push-pull. Stroke index was 25 points in total. The higher the scores were, the severer the cerebral injury was. Neurological evaluation was 10 points in total. The higher the scores were, the severer the nerve injury was. ③ Hippocampal tissue in brain of rats were selected to cut into sections at each time points, and form and distribution of neurons were observed in hippocampal areas with HE staining. Degrees of neuronal injury in hippocampal CA1 area were assayed after push-pull at various degrees with electron microscope. ④ Measurement data were compared with t test.MAIN OUTCOME MEASURES：① Stroke index and neurological evaluation; ② form and distribution of neurons in hippocampal areas;③ degrees of neuronal injury in hippocampal CA1 area.RESULTS： A total of 40 rats were involved in the final analysis. ① Stroke index and neurological evaluation of rats in experimental groups： At 30 minutes and 6 hours after push-pull exposure, stroke index and neurological evaluation were higher in ±6Gz group and ±8 Gz group than those in control group （P 〈 0.01）, especially at 6 hours after push-pull exposure, those in ±8 Gz group were the highest at each time points [（11.00±2.16）, （5.75±1.70） points]. At 24 hours after exposure, those were decreased as compared with those within the former two time points, but the values were still higher than those in control group （P 〈 0.05-0.01）. ② Results of HE staining： At 6 and 24 hours after exposure, partially neuronal degeneration was observed in pyramidal layer in ±6 Gz group and ±8 Gz group, including crenation of neurons, tdangle or polygon, and karyopycnosis, especially the injury in ±8 Gz group was the most obvious at 6 hours after exposure. ③ Results of ultrastructure with electron microscope： Partially neuronal degeneration at various degrees was observed in hippocampal CA1 area in ±2 Gz group at 6 hours after exposure and in ±6 Gz group and ±8 Gz group at 6 and 24 hours after exposure. At 6 hours after exposure, nucleus of hippocampal neurons in ±8 Gz group was irregular and umbilication. Caryotin was aggregated, nuclear matrix was swelled and disorder, and vacuolation was also observed. Rough endoplasmic reticulum was expanded, mitochondrium was swelled, and crista was disappeared.CONCLUSION： ① Push-pull cannot damage hippocampal neurons of rats in ±2 Gz group. ② Exposure can cause injury of hippocampal neurons of rats in ±6Gz group and ±8 Gz group, especially the injury is the severest at 6 hours after exposure in ±8 Gz group and relieves gradually 24 hours later.

Overexpression of C-terminal fragment of glutamate receptor 6 prevents neuronal injury in kainate-induced seizure via disassembly of Glu R6-PSD95-MLK3 signaling module

Our previous study showed that when glutamate receptor （GluR）6 C terminus-containing peptide conjugated with the human immunodeficiency virus Tat protein （GluR6）-9c is delivered into hippocampal neurons in a brain ischemic model, the activation of mixed lineage kinase 3 （MLK3） and c-Jun NH2-terminal kinase （JNK） is inhibited via GluR6-postsynaptic density protein 95 （PSD95）. In the present study, we investigated whether the recombinant adenovirus （Ad） carrying GluR6c could suppress the assembly of the GluR6-PSD95-MLK3 signaling module and decrease neuronal cell death induced by kainate in hippocampal CA1 subregion. A seizure model in Sprague-Dawley rats was induced by intraperitoneal injections of kainate. The effect of Ad- Glur6-9c on the phosphorylation of INK, MLK3 and mitogen-activated ldnase kinase 7 （MKK7） was observed with western immunoblots and immunohistochemistry. Our findings revealed that overexpression of GluR6c inhibited the interaction of GluR6 with PSD95 and prevented the kainate-induced activation of INK, MLK3 and MKK7. Furthermore, kainate-mediated neuronal cell death was significantly suppressed by GluR6c. Taken together, GluR6 may play a pivotal role in neuronal cell death.

Crocetin Potentiates Neurite Growth in Hippocampal Neurons and Facilitates Functional Recovery in Rats with Spinal Cord Injury

Crocetin is an ingredient of traditional Chinese medicine and has therapeutic potential in various diseases due to its pharmacological properties, such as neuroprotection, anti-oxidative stress, and anti-inflammation. These properties might benefit the treatment of spinal cord injury.In the present study, we tested the effect of crocetin on neurite growth and sensorimotor dysfunction in a rat model of spinal cord injury. We evaluated the viability of cultured hippocampal neurons with tetrazolium dye and lactate dehydrogenase assays, visualized neurites and axons with antibody staining, and monitored motor and sensorimotor functions in rats with spinal cord injury using the Basso,Beattie, and Bresnahan assay and the contact plantar placement test, respectively, and measured cytokine expression using enzyme-linked immuno-absorbent assays.We found that crocetin（1） did not alter the viability of cultured hippocampal neurons;（2） accelerated neurite growth with preference for the longest process in individual hippocampal neurons;（3） reversed the inhibition of neurite growth by chondroitin sulfate proteoglycan and Nogo A;（4） facilitated the recovery of motor and sensorimotor functions after spinal cord injury; and（5） did not inhibit pro-inflammatory responses, but restored the innervation of the descending 5-HT system in injured spinalcord. Crocetin promotes neurite growth and facilitates the recovery of motor and sensorimotor functions after spinal cord injury, likely through repairing neuronal connections.