The Neuroprotective Effect Of Apolipoprotein E And Apolipoprotein J Following Traumatic Brain Injury

PART 1 Apolipoprotein E improves axonal regeneration in a model oftraumatic brain injury in vitro via activating Dab1 pathway ObjectiveTraumatic brain injury(TBI) is the single large cause of mortality and morbidity, with large direct and indirect costs to society worldwide. Axonal injury has emerged as one of the most common important pathological features of TBI, functional recover following TBI base upon axonal regeneration. Hence strategies to promote axonal regeneration provide promising opportunities for the treatment of TBI. Apolipoprotein E(Apo E)promotes outcome after TBI, suggesting that Apo E plays an important role in neuroprotection. However, the mechanisms of Apo E on TBI recovery remain unknown. The present study attempts to determine whether Apo E affects axonal regeneration in the in vitro model of TBI.Methods1. Primary cortical neurons from APOE gene knockout(APOE KO)mice and APOE gene wild type(APOE WT) mice were cultured.Mechanical scratch injury model was established on neurons to simulate TBI in vitro.1) Neurons were divided into APOE KO group and APOE WT group.Regenerated neurite were marked by immunofluorescence and the total length were measured at 24 h post injury.2) The level of phosphorylated Disabled-1(p Dab1) and growth associated protein 43(GAP43) were determined by western blot at 24 h post injury.2. Lentiviral vectors encoding Dab1-si RNA were made to silence the Dab1 of neurons. Neurons were divided randomly into scramble group,Dab1-si RNA-1, Dab1-si RNA-2 and Dab1-si RNA-3 group. The expression level of Dab1 were detected by western blot 72 hours after lentivirus transduction in order to screen out the Dab1-si RNA with highest silencing efficiency.3. Neurons were divided into four groups as follow:(1) Negative control group: Neurons were transfected with negative virus vector before injury, then treated with bovine serum albumin(BSA) without injury;(2)Positive control group: Neurons were transfected with negative lentivirus vector before injury and treated with BSA after injury;(3) Apo E treatment group: Neurons were transfected with negative lentivirus vector before injury and treated with Apo E after injury;(4) Apo E + LV/Dab1 si RNA group: Neurons were transfected with LV/Dab1 si RNA before injury andtreated with Apo E after injury. The concentration of Apo E in every Apo E treatment group was maintained at 10μg/ml so as to the concentration of BSA.1) The total length of regenerated axon, the number and area of growth cones were detected by immunofluorescence at 24 hours post injury.2) The level of p Dab1, GAP43 and activated CDC42 were detected by western blot at 24 hours post injury.Results1. Axonal regeneration of Neurons lack of Apo E protein(APOE KO group) were significantly lower than control(APOE WT group); the level of p Dab1 in APOE KO group was significantly reduced compared than control; level of GAP43 was significantly lower than control.2. The expression level of Dab1 protein in neurons was significantly inhibited by LV/Dab1 si RNA-1, LV/Dab1 si RNA-2 and LV/Dab1 si RNA-3compared with control. The expression level of Dab1 protein was significantly lower in LV/Dab1 si RNA-2 group compared to either LV/Dab1 si RNA-1 or LV/Dab1 si RNA-3.3. Apo E treatment significantly increased axonal outgrowth and growth cone formation after injury compared with positive control group.4. During the process of axonal regeneration, the level of p Dab1 of neurons underwent scratch injury(both Apo E treatment group and positivecontrol group) were significantly increased compared with negative control group; meanwhile the level of activated Cdc42 and GAP43 were also significantly increased during axonal regeneration. Furthermore, the level of p Dab1, activated Cdc42 and GAP-43 were significantly increased by Apo E treatment compared with positive control group.5. After treated with Apo E protein, the axonal outgrowth and growth cone formation of neurons transfected with LV/Dab1 si RNA(Apo E+LV/Dab1 si RNA) were significantly reduced than Dab1 expressing neurons.During axonal regeneration, the activated Cdc42 and GAP43 of Apo E+LV/Dab1 si RNA group were significantly lower than Apo E treatment group.ConclusionAfter TBI, axonal regeneration of injured neurons are inhibited due to lacking of Apo E protein, while Apo E protein treatment can improve axonal regeneration. Apo E treatment may promote axonal regeneration via increasing Dab1 phosphorylation, which can induce activation of Cdc42 and promotes GAP43 expression.PART 2 Intraventricular Apolipoprotein Apo J infusion actsprotectively in Traumatic Brain Injury ObjectiveTraumatic brain injury(TBI) is the leading cause of mortality and morbidity in youth, which brings damage to brains by oxidative stress,neuroinflammation, blood brain barrier breakdown, and edema and so on.But to date effective therapies are still lacking. Previous studies revealed a marked response of Apolipoprotein J(Apo J) expression to brain injury.Apo J plays a key role in neuro-immunomodulation and neuro-repair. The aim of this study was to determine the potential roles of Apo J in functional recovery following TBI.Methods1. Controlled Cortex Impact(CCI) model in adult C57BL/6J mice were introduced to mimic TBI in vivo. Apo J expression was first detected by western blot at the time point of before injury and 6 hours, 1 day, 3days, 5 days,7 days, 10 days, 14 days, 28 days post injury.2. Mice were randomly divided into three groups as follow: Sham group underwent sham injury and received 100 μl of 5 μg/m L BSA intraventricularly 30 minutes prior to injury; Vehicle group underwent CCI and 100 μl of 5 μg/m L BSA intraventricularly 30 minutes prior to injury;Apo J group underwent CCI and 100 μl of 5 μg/m L human recombinant Apo J intraventricularly 30 minutes prior to injury. Neurological SevereScore and Morris water maze test were implied to evaluate the functional recovery after injury for each group.3. Mice were divided into three groups as before and protein samples from lesion site were collected. At 24 hours post injury, western blot were applied to detect oxidative stress markers 3-NT and 4-HNE, as well as complement activation products C3 b and C5b-9.4. Mice were divided into three groups as before and RNA samples from lesion site were collected. At 24 hours post injury, m RNA level of cytokines like IL-1β, IL-6, and TNF-α were detected by PCR.5. Mice were divided into three groups as before.Immunohistochemistry(IHC) was applied to detect the glia activation at 24 hours post injury; also neuron maintenance of cortex and hippocampus were measured by IHC at 7 days post injury.6. Mice were divided into three groups as before. Evans blue dye were injected intravenously to detect blood brain barrier permeability and brain water content were measured at 24 hours post injury.Results1. Apo J expression was up-regulated since 6 hours post-injury and sustained for 5 days, and returned to base level since 7 days post injury.2. Better behavior improvements measured NSS were observed in Apo J-treated mice compared with the vehicle group. There was no significant difference between Apo J treated group and vehicle group inMorris water maze performance.3. Both of oxidative stress marker(3-NT and 4-HNE) and complement activation production(C3b and C5b-9) were increased by CCI compared with sham group. Apo J-treated mice showed significantly reduced oxidative stress(3-NT, 4-HNE) compared with control group; Apo J-treated mice also showed significantly reduced C5b-9 compared with control group, while there is no significant difference on C3 b between Apo J group and control group.4. m RNA expression of inflammatory cytokine(IL-1β, IL-6 and TNF-α) were significantly increased by CCI injury compared with sham group. Apo J treatment was shown to significantly suppress the expression of IL-1β and TNF-α compared with control group, no difference of IL-6expression between Apo J group and control were found.5. Glia activation were significantly increased by CCI injury compared with sham group. Apo J treatment was shown to significantly suppress the activation of astrocytes and microglia compared with control group. Neuronal maintenance were decreased by CCI compared with sham group. Apo J treatment was shown to significantly increase the neuron maintenance compared with control group.6. Blood-Brain Barrier(BBB) disruption(Evans blue extravasation)and cerebral edema(water content) were induced by CCI compared with sham group. Apo J treatment was shown to significantly reduce BBBdisruption and edema after injury compared with control group.ConclusionApo J treatment can promote the functional outcome of TBI. These findings support a neuroprotective role of Apo J via multifunctional pathways, including suppression of oxidative stress and complement activation, inhibition of neuroinflammation, reducing BBB breakdown and brain edema, improving neuron survival. Therefore Apo J provides a novel and encouraging treatment strategy for TBI.