Traumatic Brain Injury-induced MANF Expression And Its Neuroprotection In Rats

Traumatic brain injury(TBI)remains a common disease that contributes significantly to the disability and morbidity worldwide.The secondary brain injury is defined as reversible pathological damage after initial traumatic insulting,opening a door for feasible therapeutic intervention to prevent progressive neural damage.The secondary injury cascade after trauma involves many activated pathological reactions,and neuroinflammation is always considered a key mechanism here.Mesencephalic astrocyte-derived neurotrophic factor(MANF),a ~20 k Da secreted protein with highly conserved sequence,belongs to the superfamily of neurotrophic factors(NTFs),and Gene Chip arrays show that it is the most sensitive genes to the endoplasmic reticulum stress(ERS)among the screened 10000 genes.The previous studies indicated that MANF played a protective role in cerebral ischemic model,while it could be massively expressed in microglial cells which may be associated with the modulation of neuroinflammtion.However,up to now,we have not found the report about the role and mechanism of MANF in animal TBI model.In this study,we reveal not only the new pathological process after TBI,but also a new therapeutic strategy for TBI,which may be benefit for the patients with TBI in the future.Part I:Pathological characteristic of TBI and MANF expression in TBI ratsObjective: To establish a rat TBI model and estimate its pathological characteristic and reproducibility.To observe TBI-induced MANF expression in neural cells and its distribution in different brain regions.Methods: Forty eight Sprague Dawley male rats were randomly divided into sham group(n=24)and TBI group(n=24).The model of TBI was induced by Feeney free falling methods(50g×20cm),while sham-operated rats underwent the same procedures except that the weight was hanged without puncture.The rats in both groups were given neurobehavioral test,brain MRI,HE and immunofluorescent staining at 24 hrs post-TBI to verify the damage and reproducibility of the model.Then the brain tissue around the cerebral contusion zone was quickly removed and frozen at-80°C,and the expression of MANF was detected by immunohistochemistry,immunofluorescence,western blot and RT-PCR techniques.Results: At 24 hours after trauma,the modified Garcia score of rats in TBI group was obviously decreased,and diffuse hyperintense signal in lesion of ipsilateral brain was noted.HE staining showed the brain tissue damage after TBI.Besides,we also found excessive activation of microglial cells stained by immunofluorescent.The endogenous MANF was widely expressed in the rat’s brain tissues,including the superficial cortex and deep hippocampus.MANF was also found in different types of cells,such as neurons,microvessel endothelial cells and microglia,which was mainly localized in the cytoplasm.Conclusions: The rat TBI model reflects many characteristics of human brain injury after trauma and shows reproducibility.The endogenous MANF was widely expressed in the rat’s brain tissues and different types of neural cells after TBI,which suggest it may be the intrinsic protective mechanism of brain in the acute phase in response to the damage.Part II:Neuroprotection of MANF and its preliminary mechanism in TBI ratsSection I: Protective effect of MANF on TBI in ratsObjective: To verify whether MANF has a neuroprotective role in rat TBI model.Methods: Ninety-six Sprague Dawley male rats were randomly divided into sham group(n=24),PBS group(n=24),5 μg-MANF group(n=24)and 20 μg-MANF group(n=24).The model of TBI was induced by Feeney free falling methods(50g×20cm),while sham-operated rats underwent the same procedures except that the weight was hanged without puncture.The rats in MANF groups were given different doses of recombinant human MANF protein(5 μg/20 μL or 20 μg/20 μL)intracerebroventricularlly,while an equal volume of 0.01% phosphate-buffered saline solution(20 μL)was injected intracerebroventricularlly in PBS group.Then,neurological score,MRI,immunofluorescent staining,mortality and brain water content were performed to evaluate the therapeutic effect of recombinant human MANF in TBI rats at 24 hrs after trauma.Results: At 24 hours after trauma,we found the decreased modified Garcia score,the increased brain water content,the less surviving neurons,and the high mortality of rats in PBS group,compared with sham group.The above parameters were alleviated in 20μg-MANF group with significant statistical significance(P<0.05)except for mortality.In 5 μg-MANF group,although the above parameters were improved,there is no statistical significance,compared with PBS control group.Conclusions: These results suggest that MANF provides the neuroprotective effect against traumatic brain injury in the acute stage,which was in dose-dependent manner.Section II:MANF inhibits excessive neuroinflammation and blood-brain barrier damage in TBI ratsObjective: To investigate whether the neuroprotective role of MANF is associated with anti-inflammation via NF-?B and inhibiting of the permeability of blood-brain barrier.Methods: Fifty-four Sprague Dawley male rats were randomly divided into sham group(n=18),PBS group(n=18)and 20 μg-MANF group(n=18).The model of TBI was induced by Feeney free falling methods(50g×20cm),while sham-operated rats underwent the same procedures except that the weight was hanged without puncture.The rats in MANF group were given high dose of recombinant human MANF protein(20 μg in 20 μL)intracerebroventricularlly,while an equal volume of 0.01%phosphate-buffered saline solution(20 μL)was injected intracerebroventricularlly in PBS group.Then,the brain tissue around cerebral contusion was quickly removed at 24 hrs after TBI to detect the expressions of CD68,IL-1β,TNF-α,and p65 by using immunofluorescence,RT-PCR,and western blot.In addition,the EB dye was intravenously injected in vivo to test the blood-brain barrier at 24 hrs after TBI.Results: At 24 hours after TBI,we found that a large number of microglial cells became amoeboid-like,and IL-1β,TNF-α and p65 were up-regulated in the brain tissue around cerebral contusion.However,in the MANF group,the number of activated microglial cells was modestly decreased(P<0.05),while the up-regulated expression of IL-1β,TNF-α and p65 in the brain was also attenuated compared with PBS grou(P<0.05).Besides,the brain around cerebral contusion in PBS group had more EB dye leakage compared with sham group(P<0.01),while the leakage was significantly reduced in MANF group compared with PBS group(P<0.05).Conclusions: These results suggest that MANF provides neuroprotective effect against traumatic brain injury is at least partically associated with attenuating BBB disruption and intracranial inflammation and NF-κB signaling pathway.