Neuroprotection And Mechanism In Experimental Traumatic Brain Injury

Traumatic brain injury (TBI), with its poor prognosis and long-term consequences, is the leading cause of death among children and young adults. According to the Centers for Disease Control and Prevention, the total annual cost for TBI-related injuries is estimated at 76.5 billion in the United States alone (http://www.cdc.gov/traumaticbraininjury/statistics.html#3). While a cure for this devastating condition still remains elusive, much effort has been invested in translational research to understand and mitigate the clinical symptoms derived from TBI.Part 1. ω-3 PUFAs supplementation improves neurologic recovery and attenuates white matter injury after experimental traumatic brain injuryDietary supplementation with ω-3 PUFAs is a safe, economical mean of preventive medicine that has shown protection against several neurologic disorders.Recent discoveries have demonstrated that ω-3 PUFAs confers neuroprotection against TBI. Some conceivable mechanisms are concerned with normalizing BDNF levels and reducing oxidative damage, maintaining brain cell membrane homeostasis#, decreasing the number of APP-positive axons.We speculated that there must be some other mechanisms, such as anti-inflammatory effect, restoration of white matter damage. The present study aimed to investigate the long term neuroprotective effects of prophylactic supplementation of ω-3 PUFAs against TBI through protection against inflammation-mediated white matter injury using a controlled cortical impact (CCI) mouse model.Traumatic brain injury was induced in C57/BL6 mice by a controlled cortical impact mouse model.ω-3 PUFAs was administered in the diet for 2 months before CCI. Animals were sacrificed on day 1,3,7,28,35 post-TBI. Short and long term motor deficits were determined using hang wire, cylinder and foot fault tests. Cognitive performance was detected in the Morris water maze tests 22-27 days post-TBI. Cresyl violet (CV) staining was used to assess the size of cortical lesion and calculate survival neurons at CA3. Microglial activation was measured using a microglial marker(Ibal), microglial pro-inflammatory response was detected by Real-time PCR and western blot of various pro-inflammatory cytokines. White matter injury was evaluated by immunohistochemistry staining of myelin basic protein (MBP) and Neurofilament H Non-Phosphorylated (SMI32) Monoclonal Antibody, western blot of MBP. Electron microscopic studies were taken to document myelinated or unmyelinated fiber damage in the corpus callosum post-TBI. The conductivity of the myelinated nerve fibers in the corpus callosum surrounding brain regions were evaluated Compound action potential s(CAPs).Microglia and oligodendrocytes were cocultured in 24-well transwell systems by adding microglia in the inserts on top of oligodendrocyte cultures in the lower chamber (1×105/well). A 10:1 ratio of oligodendrocytes to microglia was plated in accordance with the ratio of these two types of glia in the cerebral cortex in vivo.Mice fed with ω3 PUFAs-enriched diet for 2 months exhibited attenuated short and long-term behavioral deficits due to CCI. Although co-3 PUF As did not decrease cortical lesion volume, these fatty acids did protect against hippocampal neuronal loss after CCI and reduced pro-inflammatory response. Interestingly, ω-3 PUF As prevented the loss of myelin basic protein (MBP), preserved the integrity of the myelin sheath, and maintained the nerve fiber conductivity in the CCI model.ω-3 PUF As also directly protected oligodendrocyte cultures from excitotoxicity and blunted the microglial activation-induced death of oligodendrocytes in microglia/oligodendrocyte cocultures.In sum, ω-3 PUF As elicit multifaceted protection against behavioral dysfunction, hippocampal neuronal loss, inflammation, and loss of myelination and impulse conductivity. The present report is the first demonstration that ω3 PUFAs protect against white matter injury in vivo and in vitro. The protective impact of ω-3 PUFAs supports the clinical use of this dietary supplement as a prophylaxis against traumatic brain injury and other nervous system disorders.Part 2. Bumetanide Attenuates White Matter Injury and Protects blood-brain-barrier in a Mouse Model of Traumatic Brain InjuryNa-K-Cl co-transporter isoform 1 (NKCC1) is expressed in neurons (cell body, dendrite, myelinated axon), astrocytes. oligodendrocytes and blood vessel endothelial cells throughout the brain and plays an important role in regulating neuronal volume and ion homeostasis. Administration of the NKCC1 inhibitor bumetanide has been reported to significantly attenuate the contusion volume, brain edema and neuronal damage after TBI. However, recent research appears to shown that bumetanide may protect damage of blood-brain-barrier (BBB) and white matter damage resulting from ischemic injury. In the present study, we investigated the effect of bumetanide on neurobehavioral outcomes, white matter damage and BBB in a mouse model of controlled cortical impact (CCI).TBI was induced in adult male C57BL/6J mice using a CCI model, and the animals were then randomly assigned to sham, vehicle or bumetanide groups. Bumetanide (25 mg/kg) was given intraperitoneally immediately after impact and administered spaced 6 hours apart. For outcome assessments, sensorimotor deficits (hang wire, cylinder and foot fault tests) were determined at 1-35 days after TBI.BBB integrity (Evans blue extravasation and electron microscope) was determined at 48 hour post-TBI, and brain edema(wet and dry weight) and expression of MMPs and occluding (immunostaining and Western blots) were examined at 72 hour post-TBI. The integrity of myelin sheath and axon was assessed by expression of myelin basic protein (Western blots) and accumulation of amyloid precursor protein (APP,DAB staining).The NKCC1 transgenic mouse (SV129/Black Swiss) was established by Flagella previously. The genotypeof each mouse was determined by a polymerase chain reaction (PCR) of DNA from tail biopsies.Our results showed that administration of bumetanide attenuated short and long term (up to 35 days post-TBI) sensorimotor deficits. Nonetheless, bumetanide failed to significantly reduce the size of cortical lesion at 35 day post-TBI. In contrast to the ineffectiveness in sparing cortical tissue loss, bumetanide preserved the integrity of myelin sheath and axon by preventing the loss of MBP and accumulation of APP, thus maintaining the conductivity of the myelinated nerve fibers. TBI induced short-term brain edema resulting from BBB injury within the lesion and the surrounding brain regions, however, bumetanide markedly reduced brain water content and evans blue (EB) extravasation through protection against BBB injury after TBI. On the other hand, short-term and long-term neurological outcomes are improved in NKCC1+/-transgenic black swiss mice compared with NKCC1+/+ transgenic black swiss mice after Traumatic brain injury. IgG extravasation of NKCC1+/-Black Swiss mice was attenuated after TBI.NKCC1+/-Black Swiss mice show significantly increase of NF200 and CNPase immunoreactivity after TBI.In concert, our results suggest that bumetanide and NKCC1+/-improves long term neurological outcomes of TBI mice through protection against injury of BBB and white matter.These observations promote clinical investigations on bumetanide as a potential therapeutic agent for TBI.