Effects And Regulating Mechanism Of Notch Signal On Neuronal Death Of Mice After Traumatic Brain Injury

BackgroundTraumatic brain injury(TBI) represents physical injury to brain tissue by mechanical forces of shearing, tearing, or stretching, resulting in immediate clinical effects such as contusion,hemorrhage and edema. TBI is a common damaging disease in the central nervous system,which results in extremely high rates of disability and death. Studies found that the main mechanisms of TBI include glutamate excitatory toxicity, inflammation, oxidative stress and so on. And these pathological mechanisms lead to neuronal death and neurological deficit. Because neuron cannot be regenerated,patients with severe brain injury are still lack of effective treatments to restore the various functional defects caused by TBI,which brings about a huge economic and spiritual burden to family and society. The mechanism of molecular regulation after TBI is still unclear. Therefore, exploring the key endogenous molecule, which may regulate neuronal death and survival,become an important strategy for TBI treatment.Notch signal,which is a kind of highly conservative signals in the process of evolution,plays an important role in development and differentiation of the nervous system. Accumulated findings showed that activation of Notch signal existed in different types of brain injury model. Inhibition of Notch signal by chemical inhibitors or gene regulation showed neuroprotective effects in ischemic brain injury. However, some studies reported that inhibition of Notch signal exacerbated neuronal injury after ischemic brain injury. These contradictory conclusions make the role of Notch in neuronal injury controversial. Therefore,it is very important to define the biological functions of the Notch signal and related regulation for the use of Notch as a treatment target in TBI. Purpose(1) To explore temporal and spatial trend and function of Notch after TBI;(2) to explore the effects and related mechanisms of Notch on neuronal apoptosis and autophagy after TBI;(3) to explore the effects of autophagy on neuronal apoptosis after TBI. Methods(1) Cell culture: primary cortical neurons culture from mice;(2) Model: brain fluid percussion impact model in vivo and mechanical neuronal scratch model in vitro;(3) Assessment of neuronal injury: LDH assay and CCK8 assay;(4) Expression and location of key molecule, immunofluorescence, immunohistochemistry and westernblot;(5) Assessment of brain injury: dry-wet method and neurological severity score;(6) Neuronal apoptosis: western blot,caspase test kit and TUNEL staining;(7) Regulation of Notch: down- regulation of Notch by inhibitor GSI and up-regulation of Notch by recombinant rat jagged1 protein;(8) Regulation of ERK pathway: ERK inhibitor PD98059;(9) Regulation of autophagy: up-regulation and down-regulation of autophagy by rapamycin and 3-MA respectively,the expression of LC3 protein was used to reflect the degree of autophagy. ResultsChapter one: Compared to control group, mechanical neuronal scratch model significantly increased the LDH release and decreased the cell viability in neurons 1h after injury. Less cells can be seen using microscope 6h after TBI. In the meantime,Hochest staining showed more apoptotic cells, and more autophagosomes with double layer could be seen by using transmission electron microscopy. Immunofluorescent data showed NICD was mainly localized in the cytoplasm and nucleus. In mouse brain fluid percussion impact model, TBI induced obviously brain edema and neurological impairment compared to control group24 h after TBI.TUNEL staining showed more TUNEL positive cells in damaged cerebral cortex. DAB staining revealed that more NICD positive cells compared to control group, NICD was highly expressed in injured cortex region of neurons and mainly located in nucleus.In addition,western blot assay in vitro and in vivo both indicated the expression of NICD、cleaved caspase 3 and LC3Ⅱ were all up-regulated early after TBI,and then gradually fell back after their peak point.Chapter two: TBI model in vitro and in vivo were pretreated with Notch signal inhibitor GSI,and both showed significantly inhibited expression of NICD. GSI decreased LDH leakage and increased cell survival in cultured neurons 6h after mechanical scratch injury. Western blot assay showed GSI decreased the activation of caspase 3 and LC3 caused by scratch. GSI decreased brain water content and improved neurological functional score in mice after TBI. Western blot assay also showed GSI pretreatment significantly decreased the activation of caspase 3 and LC3.TUNEL staining revealed that less TUNEL positive cellswere identified after GSI pretreatment compared to TBI group.Chapter three: The expression of p-ERK reached the peak at 1h after scratch injury in cultured neurons,then gradually returned to normal levels 24 h after injury. Pretreatment of neuron using ERK inhibitor PD98059 significantly reduced p-ERK level. In the meanwhile, the expression of cleaved caspase 3 and LC3 II were also inhibited. PD98059 significantly decreased LDH leakage in neurons after mechanical scratches and increased cell survival. Moreover, inhibition of Notch signal with GSI markedly decreased p-ERK level. While giving Notch agonist jagged1 and PD98059 at the same time, we found jagged1 could significantly increase the expression of NICD, p-ERK,cleaved caspase 3 and LC3 II after injury. However,these effects could be reversed by PD98059.Chapter four: Cultured neurons received autophagy agonist rapamycin and inhibitor 3-MA 1h before injury respectively. We found 3-MA could significantly reduce LC3 II expression, neuron survival rate and increased LDH leakage,TUNEL staining showed more TUNEL positive cells after 3-MA pretreatment,but rapamycin played opposite effects. At last, we used caspase activity kit and found autophagy might affect apoptosis by regulating the activity of caspase 9 in mitochondria. ConclusionOur data demonstrated that inhibition of Notch signal exerts neuroprotective effects after TBI; Notch Signaling might interact with ERK to regulate neuronal apoptosis and autophagy after TBI; autophagy played a neuroprotective role and affected neuronal apoptosis by regulating the activity of caspase 9 caused by TBI. These results clarified the regulatory mechanism of neuronal death following TBI,and provided a new perspective and direction for the treatment of traumatic brain injury.