The Neuroprotection And Molecule Mechanisms Of The Neuroglobin Gene Mediated By Adeno-associated Virus On Spinal Cord Injury In Rats

Objectives1. To isolate, culture and identify primary spinal neurons in vitro.2. To construct the Ngb recombinant adeno-associated virus (AAV-Ngb) and infectprimary spinal neurons in vitro or inject into spinal cord tissue for infecting cellsin vivo.3. To observe the protective effect of AAV-Ngb on the primary spinal neurons afteroxidative stress injury.4. To observe the protective effect of AAV-Ngb on SCI of rats.5. To investigate the effect of AAV-Ngb on JAK2/STAT3signaling pathway afterSCI.Methods1. Primary spinal neurons were cultured by the enzyme of accutase digestive methodcombined with mechanical method. The markers of neurons were detected byNissl staining and immunofluorescent staining.2. The recombinant adeno-associated viral vector carrying Ngb gene was constructedand the adeno-associated viral titer was detected by Real-time PCR. Primaryspinal neurons were infected with Ngbrecombinant adeno-associated virus, thenthe multiplicities of infection (MOI) was determined by expressions offluorescence. The spinal cord tissue was injected with Ngb recombinantadeno-associated virus, then the expressions of EGFP in the spinal cord tissuewere judged bythefluorescence microscope andthe expressions of Ngb weredetected by western blot. 3. The oxidative stress injury model of the primary spinal neurons was establishedby using H2O2. Then they were divided into the sham group, vehicle group,AAV-EGFP group and AAV-Ngb group according to different factors. CCK-8reagent was used to observe the cell activity. Malondialdehyde (MDA) reagentwas used to measure the level of MDA. SOD reagent was used to measure thelevel of SOD. Superoxide anions fluorescent probe (DHE) reagent was used todetect level of reactive oxygen species. The apoptosis of neurons were measuredby staining with hoechst33342and flow cytometer. TMRM reagent was used todetect the mitochondrial membrane potential. The expressions of theBcl-2,Bax,cytochrome c of mitochondria or cytoplasm, cleaved caspases-3weremeasured by Western blot.4. The rat compression injury model was established,then the rats were divided intothe sham group, vehicle group, AAV-EGFP group and AAV-Ngb group accordingto different factors. The hindlimb motor function was evaluated according to theBBB scale. The changes of apoptosis rate were detected by in situ terminaldeoxymudeotityl transferase mediated dUTP nick end labeling (TUNEL). Thesurvival neurons in the spinal cord lesion were measured by immunofluorescentstaining of neuN. Malondialdehyde (MDA) reagent was used to measure the levelof MDA in the spinal cord tissue. SOD reagent was used to measure the level ofSOD in the spinal cord tissue. The expressions of the Bcl-2,Bax,cytochrome c ofmitochondria or cytoplasm, cleaved caspases-3were measured by western blotand immunofluorescent staining.5. The spinal cord tissues were collected to detect the expression level of JAK2,STAT3, p-JAK2, p-STAT3by western blot at pre-injury and1,3,6,12,24,72,156hours post-injury. The time of the peak expression of p-JAK2and p-STAT3was chosen as the observing point of the later experiment. The rats were dividedinto the sham group, vehicle group, AAV-EGFP group, AAV-Ngb group andAAV-Ngb+AG490group according to different factors. The expression levels of JAK2, p-JAK2, STAT3, p-STAT3were detected by western blot at12hourspost-injury.Results1. Primary spinal neurons were successfully isolated and cultured. TheNisslbodiesand the expressions ofβtubulin-Ⅲwere observed in most of these culturedcells, which coincided with the characteristics of neurons.2. The Ngb recombinant adeno-associated viral vector was constructed, and its titerwas9.00x1012v.g/ml. The best MOI that Ngb recombinant adeno-associated virusinfected primary spinal neurons was105, and the expressions of Ngb in the spinalcord tissue were stable and high.3. The model of the oxidative stress injury on the primary spinal neurons wassuccessfully established. Compared to sham group, the level of the cell activity,SOD, the mitochondrial membrane potential, Bcl-2and mitochondrial cytochromec decreased significantly, and the level of the intracellular ROS, MDA, apoptosisindex, Bax, cytoplasmic cytochrome c and cleaved caspase-3increased obviouslyof the vehicle group, AAV-EGFP group and AAV-Ngb group. Compared to vehiclegroup, the level of the cell activity, SOD, the mitochondrial membrane potential,Bcl-2and mitochondrial cytochrome c increased significantly, and the level of theintracellular ROS, MDA, apoptosis index, Bax, cytoplasmic cytochrome c andcleaved caspase-3decreased obviously of the AAV-Ngb group.4. The model of the spinal compression injury was successfully established.Compared to the sham group, the level of SOD, Bcl-2and mitochondrialcytochrome c in the lesion and the motor function decreased significantly of thevehicle group, AAV-EGFP group and AAV-Ngb group, and the level of MDA,apoptosis index,Bax, cytoplasmic cytochrome c and cleaved caspase-3increasedobviously in the lesion. Compared to the vehicle group, the level of SOD, Bcl-2and mitochondrial cytochrome c in the lesion and the motor function increasedsignificantly in the lesion of the AAV-Ngb group, and the level of MDA, apoptosis index,Bax, cytoplasmic cytochrome c and cleaved caspase-3reduced obviously inthe lesion of the AAV-Ngb group.5. The expressions of p-JAK2and p-STAT3achieved to the peaklevel at12h afterSCI. Compared to the sham group, the expression of p-JAK2and p-STAT3increased obviously in the vehicle group, AAV-EGFP group, AAV-Ngb group andAAV-Ngb+AG490group. Compared to the vehicle group, the expression ofp-JAK2and p-STAT3increased significantly in the AAV-Ngb group and theAAV-Ngb+AG490group. AG490can obviously reduced the expression ofp-JAK2and p-STAT3even though it treated with the AAV-Ngb.Conclusions1. Primary spinal neurons of the high yield andpurity were cultured by the accutaseenzymatic digestion combined with the mechanical method.2. The Ngb recombinant adeno-associated virus was successfully structured. TheNgb gene was integrated with primary spinal neurons or the cells of spinal cordand the expressions of Ngb were stable and high.3. Overexpression of Ngb can alleviate the cytotoxic effect inducedby oxidativestress, reduce the intracellular reactive oxygen species, increase the mitochondrialmembrane potential and inhibit the mitochondrial apoptotic pathway to alleviatethe apoptosisof the primary spinal neurons in vitro.4. Overexpression of Ngb can promote the recovery of motor function, alleviate theoxidative stress,increase the survival rate of neurons and inhibit the mitochondrialapoptotic pathway to decrease the apoptotic rate after SCI in vivo.5. Overexpression of Ngb has the protective effect on SCI by promoting to activatethe JAK2/STAT3pathway.