Dietary Supplementation With Omega-3Fatty Acids Showed A Neuroprotective Potential On Repeated Mild TBI In Rats

Background and objectiveTraumatic brain injury (TBI) is the leading cause of mortality and disability inpersons under45years of age in developed countries. It has become a serious clinicalproblem that afflicts approximately1.7million people each year in the United State.And in China, the incidence of TBI and the amount of disable or died patient after TBIincreased during years. More patients currently have a long-term requirement forassistance with daily living activities as a result of TBI. The Glasgow Coma Scale(GCS) GCS reflects the risk of dying from TBI. The TBI severity classification rangesfrom mild (GCS13-15), moderate (GCS9-12), to severe (GCS </=8). Mild traumaticbrain injury (mTBI) or concussions are the most prevalent form of TBI and constitutesgreater than80%of clinical TBI cases. Unlike moderate or severe TBI, mTBI is not aclinically heterogeneous condition. The diagnosis and treatment of mTBI is hinderedby the lack of or subtlety of clinical symptoms. It is estimated that1%mTBI patientsdied when compared to moderate (up to15%) and severe (up to40%) cases. Patientsdeveloped headaches, irritability, sleep disturbances, memory difficulties and cognitivedifficulties as post-concussion symptoms. Significant brain damage sustained fromrepeated mTBIs has become an increasingly common public health problem, especiallyin areas of sports and combat.Currently, little is known about the pathophysiology of mTBI. It has been wellcharacterized in vivo and in vitro models that the initial mechanical insult from TBI cantrigger a cascade of neuroinflammatory and metabolic changes. Metabolic dysfunction,in the absence of ischemia, is present in mTBI. In experimental mild TBI models, asingle concussive brain injury can lead to significant biochemical changes. Thesebiochemical changes can be reversible and the brain does fully recover. There is agrowing body of evidence from both clinical and laboratory settings that suggestrepetitive mTBIs can culminate into permanent brain damage. The concussed brainmay undergo a transient heightened period of susceptibility to a secondary insult duringwhich subsequent concussions may cause irreversible brain damage.In this study, we evaluated the behavioral-cognitive outcome in adult rats following single or repetitive mild fluid percussion injuries. In addition, we examined thelong-term hippocampal cell survival outcome as well as the brain inflammatoryresponse to multiple mild insults. Morever, we investigated the neuroprotectivepotential of dietary supplementation with omega-3fatty acids on repeated mild TBI.We hypothesized that spatial and temporal profiles of multiple mTBIs influencecumulative brain damage and cognitive deficits. Dietary supplementation with omega-3fatty acids prior to injury may have potential neuroprotective benefits on preventing ortreatment for repeated mild brain injuries and improved spatial learning and memoryperformance after TBI. Our paradigm of repeated mTBIs may be a useful model forinvestigating the effects of repeated concussions in athletes or warfighters.Part Ⅰ: To build a repeated mild TBI model and comparision ofmortality and body weight changing between groupsAbstractPurpose: To set up different injury model and reveal the difference in mortality andwight changing afert TBI among groups.Materials and methods:1) Six groups of rats were subjected to a varied number ofrepeated lateral fluid percussion mTBIs either unilaterally or bilaterally as well as atdifferent inter-injury intervals: Group1(2×unilaterlly,6hrs apart); Group2(2×unilaterlly,24hrs apart); Group3(1×bilaterlly,1min apart); Group4(2×bilaterlly,6hrs apart); Group5(2×bilaterlly,24hrs apart); Group6(sham, craniotomy withoutmTBI).2) Rats were initially anesthetized with4%isoflurane in a2:1nitrousoxide/oxygen mixture, intubated, and mechanically normoventilated with a rodentvolume ventilator (Harvard Apparatus model683, Holliston, MA, USA) withisoflurane reduced to2%. Rats were mounted in a stereotaxic frame, a scalp incisionmade along the midline, and a4.8-mm-diameter craniectomy was performed with atrephine on the right parietal bone. For a bilateral mTBI, an additional identicalcraniectomy was made on the left parietal bone. After then, animals were subjected to avaried number of repeated lateral fluid percussion mTBIs either unilaterally orbilaterally as well as at different inter-injury intervals. Mild experimental TBI (~1.25ATM) was produced using a fluid percussion device (VCU Biomedical Engineering, Richmond, VA) with the lateral orientationI.3) Mortality and body weight wasrecorded daily for2weeks post-injury.Results:1) There were no significant differences between groups in mean TBImagnitude, initial body weight (at the time of surgery), and temperatures values duringsurgery and post TBI.2) There was no mortality associated with any type of mild TBIwhen compared with our previous single moderate TBI with about~20%. Morever, ratssubjected to any kind of mild TBI showed decrease in the time to regain the rightingreflex compared to our previous moderate TBI model.3) Rats following multiplebilateral mild TBIs took a significant longer to recover base line body weight comparedas any other groups. There was no significant difference in the rate of body weightchanging after TBI between single bilateral group and multiple unilateral group.Conclusion:1. All kinds of mTBI groups showed an increase in the time to regain the righting reflexcompared to sham group. There was no significant difference in righting time betweenbilateral group and unilateral group, although animals following bilateral mTBIs need alonger righting time compared with those with unilateral mTBI. Our data s alsorevealed all types of mTBI groups showed a significant decrease in the time to regainthe righting reflex compared to our previous moderate TBI group (p<0.05).2. There was no mortality associated with any type of mild TBI. Repeated mild TBIscould produce more body weight loss when compared with other types of mTBI. Bodyweight loss after all types of mild TBI was less than for a single moderate TBI. Part II: Spatial learning using the Morris Water Maze betweendifferent groupsAbstractPurpose: To investigate the effect of any kind of mild TBI on cognitive development.Materials and methods:1) Acquisition of spatial learning and memory retention afterany kind of mild brain injury was tested by the Morris Water maze on days12-16afterTBI.2) Rats were placed in the pool facing the wall and required to find and mount the escape platform. Each trial allowed a maximum of120seconds to find the escapeplatform. Rats failing to find the platform were placed on it. Rats remained on theplatform for30seconds before being removed from the maze. Then rats were kept inwarmed cages and received a4-minute inter-trial interval. A total of four trials per daywere performed across five consecutive days. The starting location for each trial wasrandomly selected.3) Following the final MWM trail, each animal was run a probe testwithout an escape platform for60seconds to analysis capability for spatial learning.4)To assess visual ability, the platform which located in different quadrant was darkenedand raised1cm above the water surface so the animals can visually find the platform.Results:1) Animals in all groups had a normal visual processing and no significantdifference in swimming speed.2) Repeated mild TBIs distinctly affected theacquisition MWM performance. Compared to sham-injured animals, a single mild TBIresulted in very little or no measurable cognitive deficit. With multiple concussions,spatial learning performance was significantly poorer on training days1-2whencompared to their respective sham control group. However, onlty multiple bilaternalmTBI animals showed asignificantly poorer cognitive deficit on all5days of testing.3)On day16post TBI, we removed the platform and the results of probe trails revealedthat there had no significant difference between groups in the time spent in the platformquadrant, although repeated mild TBIs group showed less in quadrant preference thanother TBI groups.Conclusion:1. We demonstrated that cognitive performance declined with multiple concussions atmore than one location of injury and cognitive deficits are greater after bilateral mildtraumatic brain injuries.2. More importantly, the inter-injury interval is crucial in determing the long-termcognitive outcome. Six or24hrs is not adequate for the bilaterally concussed brain tofully recover from the initial concussion. As a result, each successive concussion maycontribute to the overall severity of the injured brain. Part III: Evaluation of Acute Neuronal Degeneration andLong-term Cell SurvivalAbstractPurpose: Research the effect of differenct type of mild TBI on acute as well aslong-term cell death using histological Measures.Materials and methods:1) For acute neuronal degeneration: Several animals fromeach group are being used to study the acute pathology of different kind of mTBIs.Neuronal degeneration was detected using the histofluorescent stain, Fluoro Jade-B(FJ-B) at24hrs after the initial mTBI. Brain tissues were collected at24hrs post-injuryand stained for neuronal degeneration.2) For long-term cell survival: Brain tissueswere collected on day16post-mTBI after the completion of the MWM test and stainedwith Cresyl violet. CV-staining can manifest the number of pyramidal neurons in theCA2-3region of the hippocampus associated with TBI and is used for long-termneruonal survival analysis. For bilateral brain injury, long-term survival neuronsstained with Cresyl Violet were quantified in the CA2-3regions of the hippocampus forboth sides using stereological techniques.Results:1) Significant numbers of FJ-B positive cells were observed in the injuredparietal cortex and thalamus in all injury conditions (P<0.05compared to sham-TBI).For multiple bilaternal mTBIs, FJ-B staining showed a noticeable and more extent cellinjury than we observed with other mild brain injury.2)Gross pathologic examinationshowed a consistent area of scarring on the bilaternal parietal cortex of a multiple mildTBIs brain. Light microscopy using Cresyl-violet revealed extensive areas of infarctlocalized primarily near the impact site. However, cortical infarct is not visible in othertype's mTBIs brains.3) There was no significant difference in the mean numbers ofneuronal cell for each side among any group, however, group of multiple mild TBIsshowed a trend to decrease the numbers of surviving neurons as compared to groupswith any different type mild TBI.Conclusion:1. The findings demonstrated that multiple mild brain injuries provided significantincrease in the numbers of degenerating neurons in the dorsal bilateral CA2-3at24hours post-injury. 2. Analysis of cumulative neuronal cell loss16days after mild TBI revealed significantneuronal loss in the bilateral CA2-3regions compared to sham-injury group. Unlike theacute neuronal degeneration analysis, repeated mild TBIs did not significantly increasethe cumulative neuronal cell loss3. Damage of brain using gross pathologic examination showed multiple could producemore severity damage than any other kinds of mild brain injury. Part Ⅳ: Neuroprotective potential of dietary supplementation withomega-3fatty acids on repeated mild TBI in ratsAbstractPurpose: To investigate the therapeutic benefits of omega-3fatty acids on improvingcognitive development and reducing cell death associated with multiple mild TBIswhen oral administrated in the form of fish oil.Research the effect of differenct type ofmild TBI on acute as well as long-term cell death using histological Measures.Materials and methods: Two group rats received either standard rat chow containingwith soybean or special diet high in omega-3fatty acids for4weeks prior toexperimental repeated mild TBI and for2weeks after injury. Body weight wasrecorded pre-TBI and also recorded daily for2weeks post-injury. Acquisition of spatiallearning and memory retention was assessed using the Morris Water Maze (MWM) ondays10–14after TBI. Brain tissues were collected and stained with Cresyl-violet (CV)for long-term cell survival analysis.Results: There was no difference in weight gaining of pre-TBI for each group.However, rats fed on the diet with fish oil resulted in significant less weight losscompared to those received diet with soybean following multiple mTBIs. The animalsfed on soybean showed a poorer cognitive deficit on all5days and had a significantdifference in spatial learning performance on last day of testing when compared withthose fed on the diet with fish oil. Histology analysis on survival neurons in theCA2/CA3hippocampus on14days after TBI revealed no significant difference werefound between two group, although there was a trend for an increase in neuronal numbers in animals fed on the diet with fish oil.Conclusion: Our present data highlighted the fact that multiple mild TBIs would causecumulative brain damage resulting in significant cognitive deficits. Dietarysupplementation with omega-3fatty acids showed neuroprotective effect and improvedspatial learning and memory performance after TBI. Although the potentialneuroprotection was not apparent in terms of preventing long-term neuronal cell death,the omega-3fatty acids may produce a trend toward reduce neuronal loss associatedwith TBI.