摘要
背景
颅脑创伤在一些发达国家已经成为45岁以下人群致死或致残的首要病因。在美国，每年约有170万人遭受不同程度的颅脑外伤。随着社会经济及交通的发展，中国颅脑外伤的发生率以及因颅脑外伤致死及致残率也呈上升趋势。越来越多的脑外伤患者需要接受长期的治疗及生活护理，给家庭及社会带来沉重的经济负担。依据格拉斯哥颅脑创伤评分（GCS），可将颅脑创伤分为3类：轻型颅脑外伤（格拉斯哥评分13-15分）、中度颅脑损伤（格拉斯哥评分9-12分）以及重度颅脑损伤（格拉斯哥评分≤8分）。轻型颅脑外伤或脑震荡是临床最为常见的颅脑创伤类型，约占临床脑外伤病例的80%。相比于中度颅脑损伤约15%和重度颅脑损伤约40%致死率，约1%的患者死于轻度颅脑损伤。与其他类型颅脑创伤临床表现不同，轻型颅脑外伤主要以头疼、情绪变化、睡眠障碍、记忆力及认知功能障碍为临床表现，临床统称为颅脑创伤后综合征。近年来，关于反复性轻型颅脑创伤已越来越引起研究人员的关注，特别是对于运动员和军人等一些反复轻型脑外伤发生的高比例人群。
各种体外或体内实验显示颅脑损伤初始机制均为外伤导致的级联神经炎性反应以及代谢功能异常。在之前的实验性轻型颅脑外伤模型研究中，单次轻型颅脑创伤后实验对象体内可检测到明显的生化改变，但这些变化均为可逆改变，脑组织功能也可得到完全恢复。目前越来越多的临床及实验室证据表明，反复的轻型颅脑创伤可导致永久性的不可逆脑损伤。
本次实验目的通过建立单次或反复性轻型成年大鼠颅脑外伤模型，记录不同组别颅脑创伤后致死率及体重变化，并通过行为学实验评估不同类型颅脑创伤对实验对象空间获取及记忆存储等认知功能方面的影响。同时，利用不同组织染色方法比较各类型颅脑创伤后急性神经元变性凋亡及长期神经细胞存活情况。同时我们根据前述实验结果给予实验大鼠进食一段时间（伤前4周至伤后2周）富含ω3-多不饱和脂肪酸的饲料，研究其对于反复性轻型颅脑创伤的神经保护潜能。最终建立一种稳定可靠的与战创伤及运动性颅脑创伤相关的实验性脑损伤模型并验证ω3-脂肪酸酸在反复性轻型颅脑创伤中的预防及治疗作用。
第一部分建立反复轻型创伤性脑外伤模型并比较不同组别脑创伤后致死率及体重变化
一、目的：建立反复轻型性创伤性脑外伤大鼠模型，并记录不同组别死亡率及创伤后体重变化。
二、材料和方法：1）成年雄性SD大鼠47只，体重范围（300-350克）。根据颅脑创伤类型将实验对象随机分为6组：1组（单侧双次，创伤时间间隔6小时，数量=8）；2组（单侧双次，创伤时间间隔24小时，数量=8）；3组(双侧单次，创伤时间间隔1分钟，数量=5)；4组（双侧双次，创伤时间间隔6小时，数量=10）；5组（双侧双次，创伤时间间隔24小时，数量=8）；6组（对照组，单纯行开颅手术，无颅脑创伤，数量=8）。在动物房内管理饲养一周直至手术。2）实验大鼠行气管插管，呼吸机辅助呼吸，持续性吸入2%异氟烷维持麻醉，术中检测大鼠呼吸、心率及体温变化。根据不同创伤类型，实验对象行单侧或双侧开颅术。实验性颅脑创伤模型由侧方液压打击模型诱导形成，颅脑创伤打击强度根据外接传感示波器测量，本次实验打击强度约为1.25倍大气压力，打击次数及创伤时间间隔根据不同组别创伤类型予以区分。3）记录并比较不同组别实验大鼠颅脑损伤打击强度、脑损伤前后体温及脑温变化以及复苏时间等指标。同时监测各组大鼠脑损伤后恢复情况及体重变化，比较不同组别颅脑外伤后实验大鼠死亡率。
三、结果：1）不同组别实验大鼠颅脑创伤前体重无明显差异，单次颅脑创伤打击强度及颅脑创伤前后体温及脑温变化无显著差异。与单纯行开颅术无脑损伤组术后复苏时间相比，所有类型颅脑损伤组术后复苏时间均显著延长（p<0.05）。双侧轻型颅脑外伤组较单侧轻型颅脑外伤组伤后复苏时间较长，但无明显差异（p=0.578>0.05），同时我们将本次实验数据与之前中度实验性颅脑创伤后复苏时间相比，所有组别伤后复苏时间明显缩短（p<0.05）。2）相比于实验性中度颅脑外伤约20%死亡率，所有不同类型轻型颅脑损伤组中实验大鼠无一例死亡，脑外伤后死亡率为0。各组别实验对象颅脑外伤后恢复良好，无明显肢体运动及平衡功能障碍。3）脑外伤后不同组别体重变化：所有轻型颅脑外伤组伤后体重均呈下降趋势，比较不同组别脑外伤后体重下降程度及恢复时间，统计分析结果显示：1）双侧多次轻型颅脑损伤组较单侧多次轻型颅脑损伤组，脑外伤后体重下降程度更严重，恢复至伤前体重水平所需时间明显延长（5天vs11天）；2）双侧多次轻型颅脑损伤组较双侧单次轻型颅脑损伤组，脑外伤后体重下降程度明显，恢复至伤前体重水平所需时间明显延长（5天vs11天）；3）单侧多次轻型颅脑损伤组与双侧单次轻型颅脑损伤组相比，脑外伤后体重下降程度及恢复至伤前体重水平所需时间无显著差别（5天vs5天）。
四、结论：
1.所有类型轻型颅脑损伤组别术后复苏时间较无损伤组均显著延长(p<0.05)。双侧轻型颅脑外伤组较单侧轻型颅脑外伤组伤后复苏时间较长，无统计学意义。与中度颅脑创伤相比，各类型轻型颅脑外伤伤后复苏时间显著缩短(p<0.05),有统计学意义。由此推测反复性轻型颅脑创伤其脑组织损伤程度较单次轻型颅脑损伤较轻，脑组织功能恢复时间较短。
2.相比于实验性中度颅脑外伤（致伤强度约为2.15倍大气压力）约20%的死亡率，所有不同类型轻型颅脑损伤组中实验大鼠无一例死亡，脑外伤后死亡率为0。各组别实验对象颅脑外伤后恢复良好，无明显肢体运动及平衡功能障。由此推测打击强度是导致实验对象脑外伤后死亡的主要因素，反复性轻型颅脑创伤强度不同于单次中度颅脑创伤。
3.双侧多次轻型颅脑损伤组较其他类型轻型脑损伤组可产生明显的体重下降，其恢复至脑损伤前体重水平所需时间明显延长。可认为多次反复性轻型颅脑损伤脑组织损伤程度较重，恢复时间相对较长。
第二部分评估不同类型反复轻型创伤性脑损伤后对其认知功能的影响
一、目的：通过颅脑创伤后连续5天行为学测试，观察并评估不同类型反复轻型创伤性脑损伤后对其认知功能的影响。
二、材料和方法：将不同类型轻型颅脑外伤组自脑创伤后12-16天连续5天行Morris水迷宫行为学测试。整个行为学测试分为定位航行测试(place navigation)及空间探索测试(spatial probe)。1）定位航行测试：连续5天，每天将大鼠面向池壁分别从4个入水点放入水中若干次,记录其寻找到隐藏在水面下平台的时间及游动速度和距离；2）空间探索测试：是在定位航行试验最后一天测试后去除平台,然后选定一个入水点将大鼠放入水池中,记录其在一定时间内的游泳轨迹,考察大鼠对原平台的记忆。
三、结果：
1）所有不同类型轻型颅脑损伤组别中实验对象的游动速度及视力测试均无明显差异。
2）与无损伤对照组相比较，单侧双次轻型颅脑损伤组中大鼠在测试1-2天存在明显认知功能障碍，双侧单次轻型颅脑损伤组中大鼠在测试1-3天存在明显的认知功能障碍，而双侧多次轻型颅脑损伤组中大鼠则在测试1-5天内存在认知功能障碍。3）与无损伤对照组最终测试结果相比，双侧多次轻型颅脑损伤组中实验大鼠存在明显认知功能障碍(第四组，p=0.003/第五组，p<0.001)，而单侧多次及双侧单次轻型颅脑损伤组中实验大鼠与无损伤组比较无显著差别。
3）空间探索测试显示：多侧反复轻型颅脑创伤组其各区域内游动轨迹相对较平均，而其他各组则表现出目标区域相对集中性。
四、结论
1.单侧多次及双侧单次轻型颅脑损伤组对脑创伤后认知功能无显著影响，可推测其损伤程度较轻，脑组织未造成永久性损伤，脑组织功能未受明显影响。
2.反复性轻型颅脑外伤可造成明显的认知功能障碍，由此推测对于双侧重复性轻型脑创伤，6小时或24小时的时间间隔对于脑组织的功能恢复不够充分，应此连续的轻型脑创伤均可加重颅脑损伤程度。虽然与单次中度创伤性脑损伤相比，反复轻型颅脑创伤损伤程度更轻微，但重复的创伤累积可产生更为严重的长期性认知功能障碍。
第三部分比较不同类型反复性轻型颅脑损伤后脑组织观察区域急性神经元变性及长期神经细胞生存状况
一、目的：比较不同组别脑外伤后神经细胞存活情况，研究不同类型轻型颅脑损伤对脑组织的损伤程度。
二、材料和方法：
更据不同类型组织学染色要求，分别随机抽取不同组别实验大鼠于伤后24小时内或行为学测试最后一天（脑创伤后16天）通过4%多聚甲醛灌流（每只大鼠约150毫升）并固定脑组织，经不同浓度蔗糖溶液浸泡保护后，将脑组织至于干冰冷冻，于切片机上将冰冻脑组织以每层45微米的厚度连续冠状切片，选取10张脑组织贴片。室温晾干组织切片后，分别行Fluoro Jade-B组织荧光染色及甲酚紫（CV）染色评估不同组别神经细胞受损情况，同时与先前实验性中度创伤性脑损伤大体脑组织形态及神经细胞存活情况相比较，并进行统计学分析。
三、结果：
1）脑外伤后16天脑组织大体形态学比较：与中度颅脑创伤后受伤区域脑组织常可见连续性疤痕或组织缺损不同，仅双侧多次或单侧双次轻型颅脑损伤组脑组织损伤局部可见轻微皮层下瘢痕或含铁血黄素沉积，其余单次轻型脑创伤组脑组织打击致伤区域未见明显疤痕或脑组织皮层缺损。光镜下甲酚紫（CV）染色结果同时显示，所有反复性轻型颅脑损伤组大鼠脑组织损伤相关区域未见缺血梗死或出血灶及外伤后瘢痕组织形成。
2）所有不同类型轻型脑创伤组其创伤区域的顶叶皮层及丘脑均可见明显的FJ-B阳性荧光染色(p<0.05)，随着打击次数的增加，脑损伤后急性神经元变性数量随之增加，利用立体测量细胞计数软件分析系统计算比较不同脑损伤组别外伤后急性期变性死亡的神经细胞数量结果显示单次反复性轻型颅脑损伤较单侧单次轻型颅脑损伤相比，表现出增加神经细胞变性数量的趋势。
3），通过甲紫酚染色观察比较不同类型反复性轻型颅脑损伤后同侧脑组织海马CA3区长期神经细胞存活数量结果提示所有不同类性颅脑损伤后均可导致不同程度的长期存活神经细胞数量下降，且各不同组别脑外伤后神经细胞存活数量有显著差异(p<0.01)。单次轻型颅脑损伤后神经细胞存活数量与无损伤组无显著差别(p>0.05)。反复性即同侧2次轻型颅脑损伤，无论创伤时间间隔6或24小时，均可产生最显著的神经细胞数量下降(p<0.01)。
四、结论
1）伤后24小时FJ-B荧光染色结果提示各种类型轻型颅脑损伤均可造成受伤区域海马及皮层不同程度急性神经细胞变性死亡，反复性轻型颅脑由于打击次数增加，可导致急性脑损伤程度加重及表现出增加变性神经细胞数量的趋势。
2）伤后脑组织大体形态学表明，脑组织机械损伤程度主要与瞬间颅脑打击强度有关，轻型颅脑损伤由于脑组织机械性打击程度较轻，相比于中度颅脑损伤，脑组织大体形态病理学无特殊改变。
3）甲酚紫（CV）染色结果显示多次反复轻型颅脑损伤可恶化继发性脑损伤程度，表现为显著增加脑组织损伤相关区域神经细胞丢失数量，造成脑组织不可逆的损伤。
第四部分ω3-鱼脂酸对反复性轻型创伤性脑损伤保护作用的实验研究
一、目的：确立ω3-鱼脂酸对反复性轻型颅脑损伤的神经保护作用并探讨其对脑外伤后大鼠认知功能的影响。
二、材料和方法：
将32只实验大鼠（体重约150克，出生时长：6周）随机根据饲养食物不同分为两组，每组各16只，其中一种食物中富含ω3-鱼脂酸，而另一组食物以普通实验饲料为对照。所有大鼠与脑损伤前4周开始进食不同食物，并持续进食至伤后2周，直至行为学实验完成。实验期间记录每天不同组别实验动物的体重变化。颅脑创伤后10-14天连续5天行Morris水迷宫行为学测试，分别记录每只实验大鼠游向平台时间及游行速度。评估空间认知及记忆功能。行为学测试最后一天（脑创伤后16天）通过4%多聚甲醛灌流（每只大鼠约150毫升）并固定脑组织，经不同浓度蔗糖溶液浸泡保护后，将脑组织至于干冰冷冻，与切片机上冠状切面将冰冻脑组织以每层45微米的厚度切片、贴片。室温晾干组织切片后，行甲酚紫（CV）染色评估不同组别神经细胞受损情况，并进行统计学分析。
三、结果：
1）两组中颅脑外伤打击强度、伤后意识及反射恢复时间、损伤前后体温及脑温变化无显著性差别。颅脑外伤前进食不同食物4周时间，各组别大鼠体重增长比率无差别，但伤后体重记录显示，进食富含ω3-鱼脂酸饲料组大鼠可明显减轻体重减少程度（311±25g vs321±22g），恢复至脑外伤前基础体重时间较对照饮食治疗组缩短。
2）两组不同饮食组于脑损伤后10-14天行水迷宫行为学评估，其中各组中实验对象的游动速度及视力测试均无明显差异。进食富含ω3-鱼脂酸饲料的实验大鼠脑外伤后较进食普通饲料的实验大鼠在认知功能方面有明显改善。比较行为学测试最后一天各组别游向平台时间，ω3-鱼脂酸饮食组平均用时显著缩短(p=0.030，p<0.05)。空间探索测试结果显示，二组实验大鼠均表现出目标区域相对集中性，进食富含ω3-鱼脂酸组大鼠与目标区域游动时间相对较长（22.8±3.6秒vs19.7±2.9秒）。
3）组织学检查结果：各组别大鼠与行为学测试最后一天（脑外伤后16天）处死，提取脑组织标本行甲酚紫（CV）染色，用以评估不同组别神经细胞长期生存状况。脑组织大体形态学比较（4×）：进食普通实验饲料组大鼠较进食富含ω3-鱼脂酸组大鼠相比，其脑组织海马颗粒层厚度及细胞密度减少。光镜下甲酚紫（CV）染色结果同时显示（100×），不同饮食组双侧海马CA2/3区域平均神经细胞数量比较结果上无显著性差异（左侧：p=0.149p＞0.05；右侧：p=0.253p＞0.05），然而，进食富含ω3-鱼脂酸组显示出可减少神经细胞死亡的趋势。
四、结论
1）各组大鼠伤后无死亡率。脑外伤前至伤后一段时间进食ω3-脂肪酸，可满足机体颅脑外伤后的高代谢及能量需求状态，明显减轻反复性轻型颅脑创伤后体重下降，促进神经功能恢复，改善伤后恢复。
2）反复性轻型颅脑损伤组可产生明显的认知功能障碍，进食富含ω3-鱼脂酸饲料可明显改善脑外伤后空间认知及记忆功能，并显示出更明显的目标区域的偏好性。
3）甲酚紫（CV）染色结果显示虽然二组不同饮食组在平均神经细胞数目皮较无显著性差异，但与进食普通实验饲料组大鼠相比，进食富含ω3-鱼脂酸饲料的大鼠显示出增加脑外伤后长期神经细胞存活数量的神经保护潜能。
Background and objective
Traumatic 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 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 groups
Abstract
Purpose: 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 was
recorded 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 weight
changing 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 groups
Abstract
Purpose: 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 Survival
Abstract
Purpose: 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 significant
neuronal 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 loss
3. 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 rats
Abstract
Purpose: 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.
引文
[1] Bryant R. Post-traumatic stress disorder vs trauma tic brain injury. Dialogues ClinNeurosci.2011;13(3):251-62.
[2] American Congress of Rehabilitation Medicine. Definition of mild traumatic braininjury. J Head Trauma Rehab.1993;8:86–87.
[3] Carroll LJ., Cassidy JD., Holm L., Kraus J., Coronado VG. Methodological issues andresearch recommendations for mild traumatic brain injury: The WHO CollaboratingCentre Task Force on Mild Traumatic Brain Injury. J Rehab Med.2004;36:113–125.
[4] Ruff RM., Iverson GL., Barth JT., Bush SS., Broshek DK. Recommendations fordiagnosing a mild traumatic brain injury: a National Academy of Neuropsycholo gyeducation paper. Arch Clin Neuropsychol.2009;24:3–10.
[5] Iverson GL., Lange RT. Examination of “postconcussion-like” symptoms in a healthsample. Appl Neuropsychol.2003;10:137–144.
[6] Dhaval Shukla and B Indira Devi. Mild traumatic brain injuries in adults. J NeurosciRural Pract.2010Jul-Dec;1(2):82–88.
[7] Elan J. MS; Inglese; Matilde MD, PhD; Bammer, Roland PhD. Mild Traumatic BrainInjury: Is Diffusion Imaging Ready for Primetime in Forensic Medicine? Topics inMagnetic Resonance Imaging.2010Dec21(6):379–386
[8] Binder LM, RohlingML, Larrabee GJ. A review of mild head trauma. Part I:Meta-analytic review of neuropsychological studies. J Clin Exp Neuropsychol.1997;19:421–431.
[9] Langlois JA, Rutland-Brown W, Wald MM. The epidemiology and impact of traumaticbrain injury: a brief overview. J Head Trauma Rehabil.2006;21:375–378.
[10] Lew HL, Pogoda TK, Baker E, Stolzmann KL, Meterko M, Cifu DX, Amara J, HendricksAM. Prevalence of dual sensory impairment and its association with traumatic brain injuryand blast exposure in OEF/OIF veterans. J Head Trauma Rehabil.2011Mar7;26(2)201;
[11] Hoge CW, McGurk D, Thomas JL, Cox AL, Engel CC, et al. ild raumatic brain injury inU.S. soldiers returning from Iraq. N Engl J Med.2008;31:453–463
[12] Cernak, I. Animal models of headtrauma. NeuroRx.2005;2,410-422.
[13] Kabadi,SV., Hilton, G.D., Stoica, B.A., Zapple, D.N., Faden, A.I. Fluid-percussion-inducedtraumatic brain injury model in rats. Nat Protoc.2010;5,1552-1563.
[14] Vollmer DG and Dacey RG Jr. The management of mild and moderate head injuries.Neurosurg Clin N Am.1991;2(2):437-55.
[15] Berrigan L, Marshall S, McCullagh S, Velikonja D, Bayley M. Quality of clinical practiceguidelines for persons who have sustained mild traumatic brain injury. Brain Inj.2011;25(7-8):742-51.
[16]Nakajima Y. Diagnosis of higher brain dysfunction after traumatic brain injury. NoShinkei Geka.2011;39(8):731-42.
[17] Charrueau C, Belabed L, Besson V, Chaumeil JC, Cynober L, Moinard C. Metabolicresponse and nutritional support in traumatic brain injury: evidence for resistance torenutrition. J Neurotrauma.2009;26(11):1911-20.
[18] Cook AM, Peppard A, Magnuson B. Nutrition considerations in traumatic brain injury.Nutr Clin Pract.2008;23(6):608-20.
[1] Hamm, R.J. Neurobehavioral assessment of outcome following traumatic brain injury inrats: an evaluation of selected measures. J Neurotrauma.2001;18:1207-1216
[2] Smith, D.H., Okiyama, K., Thomas, M.J., Claussen, B., and McIntosh, T.K. Evaluation ofmemory dysfunction following experimental brain injury using the Morris water maze. JNeurotrauma.1991;8:259-269
[3] Fleminger S. Long-term psychiatric disorders after traumatic brain injury. Eur JAnaesthesiol Suppl.2008;42:123-130.
[4] Bigler ED. Anterior and middle cranial fossa in traumatic brain injury: relevantneuroanatomy andneuropathology in the study of neuropsychological outcome.Neuropsychology2007;21:515-531.
[5]Bigler E. Neuropsychology and clinical neuroscience of persistent post-concussivesyndrome. J Int Neuropsychol Soc2008;14:1-22.
[6] Strangman GE, O’Neil-Pirozzi TM, Goldstein R. Prediction of memory rehabilitationoutcomes in traumatic brain injury by using functional magnetic resonance imaging. ArchPhys Med Rehabil.2008;89:974-981.
[7] Ghajar J, Richard BI. The predictive brain state: timing deficiency in traumatic braininjury? Neurorehabil Neural Repair.2008;22:217-227.
[8] Saunders JC, McDonald S, Richardson R. Loss of emotional experience after traumaticbrain injury findings with the startle probe procedure. Neuropsychology.2006;20:224-231.
[9] Kur a E, Sivák S, Ku era P. Impaired cognitive functions in mild traumatic brain injurypatients with normal and pathologic magnetic resonance imaging. Neuroradiology.2006;48:661-669.
[10] Christodoulou C, DeL uca J,Ricker JH, et al. Functional magnetic resonance imaging ofworking memory impairment after traumatic brain injury. J Neurol Neurosurg Psychiatry.2001;71:161-168.
[11] Mathias J, Wheaton P. Changes in attention and information-processing speed followingsevere traumatic brain injury. Neuropsychology.2007;21:212-223.
[12] Iverson GL. Misdiagnosis of the persistent postconcussion syndrome in patients withdepression. Arch Clin Neuropsychol.2006;21:303-310.
[13] Bogdanova Y and Verfaellie M. Cognitive sequelae of blast-induced traumatic brain injury:recovery and rehabilitation.Neuropsychol Rev.2012;22(1):4-20.
[14] Vasterling JJ, Verfaellie M, Sullivan KD. Mild traumatic brain injury and posttraumaticstress disorder in returning veterans: perspectives fromcognitive neuroscience. ClinPsychol Rev.2009;29(8):674-84. Epub2009Aug21.
[15] Thériault M, De Beaumont L, Gosselin N, Filipinni M, Lassonde M. Electrophysiologicalabnormalities in well functioning multiple concussed athletes. Brain Inj.2009;23(11):899-906.
[16] Brandeis, R., Y. Brandys, and S.Yehuda. The use of the Morris Water Maze in the study ofmemory and learning. Int J Neurosci.1989;48(1-2):29-69.
[17] Carrol Lj,Cassidy JD, Holm L, Kraus J,Coronado VG; Methodological issues and researchrecommendations for mild traumatic brain injury: the WHO Collaborating Centre TaskForce on Mild Traumatic Brain Injury. J Rehabil Med.2004;(43Suppl):113-25.
[18] Jaffee MS and Meyer KS. A brief overview of traumatic brain injury (TBI) andpost-traumatic stress disorder (PTSD) within the Department of Defense. ClinNeuropsychol.2009;23(8):1291-8.
[19] Morris, R. G. M., P. Garrud, J. N. Rawlins, and J. O'Keefe. Place navigation impaired inrats with hippocampal lesions. Nature.1982;297(5868):681-683.
[20] Sutherland, Whishaw, and B. Kolb.A behavioural analysis of spatial localizationfollowing electrolytic, kainate-or colchicine-induced damage to the hippocampalformation in the rat. Behav Brain Res.1983;7(2):133-153.
[1] C. Werner, K. Engelhard. Pathophysiology of traumatic brain injury Br. J. Anaesth.2007;99(1):4-9.
[2] Barclay Morrison III, Benjamin S. Elkin, Yarmush. In Vitro Models of Traumatic Brain InjuryAnnu. Rev. Biomed. Eng.2011.13:91–126.
[3] Globus, M.Y, Alonso, O, Dietrich, W.D, Busto, R., and Ginsberg, M.D. Glutamate release andfree radical production following brain injury: effects of posttraumatic hypothermia. JNeurochem.1995;65:1704-1711.
[4] Park E, Bell JD, Baker AJ. Traumatic brain injury: Can the consequences be stopped?. CanadianMedical Association Journal.2008;178(9):1163–70.
[5] Zhao, X., Ahram, A., Berman, R.F., Muizelaar, J.P., and Lyeth, B.G. Early loss of astrocytes afterexperimental traumatic brain injury. Glia.2003;44:140-152.
[6] Zhong, C., Zhao, X., Sarva, J., Kozikowski, A., Neale, J.H., and Lyeth, B.G. NAAG peptidaseinhibitor reduces acute neuronal degeneration and astrocyte damage following lateral fluidpercussion TBI in rats. J Neurotrauma..2005;22:266-276.
[7] Hallam, T.M., Floyd, C.L., Folkerts, M.M., Lee, L.L., Gong, Q.Z., Lyeth, B.G., Muizelaar, J.P.,mand Berman, R.F. Comparison of behavioral deficits and acute neuronal degeneration in rat lateralfluid percussion and weight-drop brain injury models. J Neurotrauma.2004;21:521-539.
[8] Schmued, L.C., Albertson, C., and Slikker, W., Jr. Fluoro-Jade: a novel fluorochrome for thesensitive and reliable histochemic al localization of neuronal degeneration. Brain Res.1997;751:37-46.
[9] Schmued, L.C., and Hopkins, K.J. Fluoro-Jade B: a high affinity fluorescent marker for thelocalization of neuronal degeneration. Brain Res.2000;874:123-130.
[10] Anderson, K.J., Miller, K.M., Fugaccia, I., Scheff, S.W. Regional distribution of Fluoro-Jade Bstaining in the hippocampus following traumatic brain injury. Experimental Neurology.2005;193(1):125–130.
[11] Anderson, K.J., Fugaccia, I., Scheff, S.W."Fluoro-Jade B stains quiescent and reactive astrocytesin the rodent spinal cord". J of Neurotrauma.2003;20(11):1223–1231.
[12] Hellmich HL, Capra B, Eidson K, Garcia J, Kennedy D, Uchida T, Parsley M, Cowart J, DeWittDS, Prough DS. Dose-dependent neuronal injury after traumatic brain injury. BrainRes.2005;1044:144–154.
[13] Xiong Y, Lee CP, Peterson PL. Mitochondrial dysfunction following traumatic brain injury. InMiller LP and Hayes RL, eds. Co-edited by Newcomb JK.Head Trauma: Basic, Preclinical, andClinical Directions. New York: John Wiley and Sons, Inc..2000; pp.257–80.
[14] Ghajar J. Traumatic brain injury. Lancet.2000;356(9233):923–29.
[15] Morley EJ, Zehtabchi S. Mannitol for traumatic brain injury: Searching for the evidence. Annals ofEm ergency Medicine.2008;52(3):298–300.
[16] Sauaia A, Moore FA, Moore EE et al. Epidemiology of trauma deaths: A reassessment. TheJournal of Trauma.1995;38(2):185–93.
[17] Narayan RK, Michel ME, Ansell B et al. Clinical trials in head injury. Journal of Neurotrauma.2002;19(5):503–57
[18] Pierce, J.E., Smith, D.H., Trojanowski, J.Q., and McIntosh, T.K. Enduring cognitive,neurobehavioral and histopathological changes persist for up to one year following severeexperimental brain injury in rats. Neuroscience.1998;87:359-369.
[19] Smith, D.H., Chen, X.H., Pierce, J.E., Wolf, J.A., Trojanowski, J.Q., Graham, D.I., andMcIntosh, T.K. Progressive atrophy and neuron death for one year following brain traumain the rat. J Neurotrauma.1997;14:715-727.
[20]Kim, B.T., Rao, V.L., Sailor, K.A., Bowen, K.K., and Dempsey, R.J. Protective effects ofglial cell line-derived neruotrophic factor onhippocampal neurons after traumatic braininjury in rats. J Neurosurg.2001;95:674-679
[21] Smith, D.H., Soares, H.D., Pierce, J.S., Perlman, K.G., Saatman, K.E., Meaney, D.F., Dixon, C.E.,and McIntosh, T.K. A model of parasagittal controlled cortical impact in the mouse: cognitiveand histopathologic effects. J Neurotrauma.1995;12:169-178.
[22]Kotapka, M.J., Graham, D.I., Adams, J.H., and Gennarelli, T.A. Hippocampal pathology infatal human head injury without high intracranial pressure. J Neurotrauma.1994;11:317-324.
[1] Yehuda S, Rabinovitz S, Crasso RL, Mostofsky DI. The role of polyunsaturated fattyacids in restoring the ageing neuronal membrane. Neurobiol Ageing.2002;23:843–53.
[2] Holman RT. The slow discovery of the importance of omega3essential fatty acids inhuman health. J. Nutr.1998；128(2Suppl):427S–433S.
[3]Von Schacky C. The role of omega-3fatty acids in cardiovascular disease. Curr.Atheroscler.2003;5(2):139–45.
[4] Wang, C.; Harris, W. S.; Chung, M.; Lichtenstein, A. H.; Balk, E. M.; Kupelnick, B.;Jordan, H. S.; Lau, J. N-3Fatty acids from fish or fish-oil supplements, but notalpha-linolenic acid, benefit cardiovascular disease outcomes in primary-andsecondary-prevention studies: a systematic review. The American journal of clinicalnutrition.2006;84(1):5–17.
[5] G. Fontani, F. Corradeschi, A. Felici, F. Alfatti, S. Migliorini and L. Lodi. Cognitive andphysiological effects of Omega-3polyunsaturated fatty acid supplementation in healthysubjects. European Journal of Clinical Investigation.2005;35,691–699.
[6] PETRA S. H and Isaacs. Nutrition for the Brain. PEDIATRIC RESEARCH.2008;63(3):299-230
[7] Le′pine JP, Briley M. The increasing burden of depression. Neuropsychiatr Dis Treat.2011;7:3–7.
[8] Harsharan Singh Bhatia, Rahul Agrawal, Sandeep Sharma, Yi-Xin Huo, Zhe Ying, andFernando Gomez-Pinilla. PLoS One.2011;6(12): e28451.
[9] Lin, Pao-Yen; Kuan-Pin Su. A Meta-Analytic Review of Double-Blind, Placebo-Controlled Trialsof Antidepressant Efficacy of Omega-3Fatty Acids.2007; J Clin Psychiatry68(7):1056–1061.
[10] Wu A, Ying Z, Gomez-Pinilla F: Dietary omega-3fatty acids normalize BDNF levels,reduce oxidative damage, and counteract learning disability after traumatic brain injury inrats. J Neurotrauma.2004;21:1457–1467.
[11] James D. Mills, M.D., Julian E. Bailes, M.D., Cara L. Sedney, M.D., Heather Hutchins,M.S., R.D., and Barry Sears, Ph.D. Omega-3fatty acid supplementation and reduction oftraumatic axonal injury in a rodent head injury model. J Neurosurg.2011;114:77–84.
[12] Perel P, Yanagawa T, Bunn F, Roberts I, Wentz R, Pierro A. Nutritional support forhead-injured patients. Cochrane Database Syst Rev.2006;3:CD001530.
[13] Roger H, Linda M. G, Quanhong N, and Jamshid G, Effect of early nutrition on deaths dueto severe traumatic brain injur y. J Neurosurg.2008;109:50–56.
[14] Young B, Ott L, Twyman D, Norton J, Rapp R, Tibbs P. The effect of nutritional supporton outcome from severe head injury. J Neurosurg.1987;67:668–676.
[15] Hovda DA, Lee SM, Smith ML, Von Stuck S, Bergsneider M, Kelly D. The neurochemicaland metabolic cascade following brain injury: moving from animal models to man. JNeurotrauma.1995;12:903–906.
[16] Deutschman CS, Konstantinides FN, Raup S, Thienprasit P, Cerra FB: Physiological andmetabolic response to isolatedclosed-head injury. Part1: Basal metabolic state:correlations of metabolic and physiological parameters with fasting and stressed controls.J Neurosurg.1986;64:89–98.
[17]Morris, R. G. M. Spatial Localization Does Not Require the Presence of Local Cues.Learning and Motivation.1981;12(2),239-260.
[18] Hamm, R.J. Neurobehavioral assessment of outcome following traumatic brain injury inrats: an evaluation of selected measures. J Neurotrauma.2001;18:1207-1216.
[19]Smith, D.H., Okiyama, K., Thomas, M.J., Claussen, B., and McIntosh, T.K. Evaluation ofmemory dysfunction following experimental brain injury using the Morriswater maze. JNeurotrauma.1991;8:259-269.
[20] Eser O, SongurA, Yaman M, Cosar M, Fidan H, Sahin O, Mollaoglu H, Buyukbas S.Theprotective effect of avocado soybean unsaponifilables on brain ischemia/reperfusion injuryin rat prefrontal cortex.Br J Neurosurg.2011;25(6):701-6.
[21] Mehmet Yaman, Olcay Eser, Murat Cosar, Orhan Bas, Onder Sahin, Hakan Mollaoglu,Huseyin Fidan, Ahmet Songur. Oral Administration of Avocado Soybean Unsaponifiables(ASU) Reduces Ischemic Damage in the Rat Hippocampus. Archives of Medical Research.2007;7:489-494.
[22] Yimit D, Hoxur P, Amat N, Uchikawa K, Yamaguchi N. Effects of soybean peptide onimmune function, brain function and neurochemistry in healthy volunteers.Nutrition.2012;28(2):154-9.
[23] Kim, B.T., Rao, V.L., Sailor, K.A., Bowen, K.K., and Dempsey, R.J. Protective effects ofglial cell line-derived neruotrophic factor onhippocampal neurons after traumatic braininjury in rats. J Neurosurg.2001;95:674-679
[24] Kotapka, M.J., Graham, D.I., Adams, J.H., and Gennarelli, T.A. Hippocampal pathology infatal human head injury without high intracranial pressure. J Neurotrauma.1994;11:317-324.
[1] Rutland-Brown W, Langlois JA, Thomas KE, Xi YL. Incidence of traumatic brain injuryin the United States,2003. J Head Trauma Rehabil.2006;21:544–8.
[2] Jennett B. Epidemiology of head injury. J Neurol Neurosurg Psychiatry.1996;60:362–9.
[3] Kraus JF, Nourjah P. The epidemiology of mild, uncomplicated brain injury. J Trauma.1988;28:1637–43.
[4] Halstead ME, Walter KD. Sport-related concussion in children and adolescents. Pediatrics.2010;126:597–615.
[5] McKee AC, Cantu RC, Nowinski CJ, Hedley-Whyte ET, Gavett BE, Budson AE, San tiniVE, Lee HS, Kubilus CA, Stern RA. Chronic traumatic encephalopathy in athletes:progressive tauopathy after repetitive head injury. J Neuropathol Exp Neurol.2009;68:709–35.
[6] Pellman EJ, Viano DC, Tucker AM, Casson IR, Waeckerle JF. Concussion in professionalfootball: reconstruction of game impacts and injuries. Neurosurgery.2003b;53:799–812.
[7] Fedorova I, Salem N, Jr.. Omega-3fatty acids and rodent behavior. Prostaglandins LeukotEssent Fatty Acids.2006;75:271–289.
[8] Crawford MA, Hassam AG, Williams G. Essential fatty acids and fetal brain growth.Lancet.1976;1:452–453.
[9]Barker DJ, Eriksson JG, Forse′n T, Osmond C. Fetal origins of adult disease: strength ofeffects and biological basis. Int J Epidemiol.2002;31:1235–1239.
[10] Page KC, Malik RE, Ripple JA, Anday EK. Maternal and postweaning diet interactionalters hypothalamic gene expression and modulates response to a high-fat diet in maleoffspring. Am Physiol Regul Integr Comp Physiol.2009;297: R1049–1057.
[11] World Health Organization. Neurotrauma.2008. Available at http://www.who.int/violenc e_injury_prevention/road_traffic/activities/neurotrauma/en/.
[12] McIntosh, T.K. et al. Traumatic brain injury in the rat: characterization of a lateralfluid-percussion model. Neuroscience.1989;28,233-244.
[13] Teasdale G, Jennett B.1974. Assessment of coma and impaired consciousness. a practicalscale. Lancet.1974;2:81–4.
[14] Alexander MP. Mild traumatic brain injury: pathophysiology, natural history, and clinicalmanagement. Neurology.1995;45:1253–60.
[15] American Congress of Rehabilitation Medicine. Definition of mild traumatic brain injury.J Head Trauma Rehabil.1993;8:86–7.
[16] Ruff RM, Jurica P. In search of a unified definition for mild traumati brain injury. Brain Inj.1999;13:943–52.
[17] Arciniegas DB, Silver JM. Regarding the search for a unified definition of mild traumaticbr ain injury.Brain Inj.2001;15:649–52.
[18] Borczuk P. Predictors of intracranial injury in patients with mild head trauma. Ann EmergMed.1995;25:731–6.
[19] Haydel MJ, Preston CA, Mills TJ, et al. Indications for computed tomography in patientswi th minor head injury.N Engl J Med.2000;343:100–5.
[20] Miller EC, Holmes JF, Derlet RW. Utilizing clinical factors to reduce head CT scanordering for minor head trauma patients. J Emerg Med.1997;15:453–7.
[21] Stein SC, Ross SE. Mild head injury: a plea for routine early CT scanning. J Trauma.1992;33:11–13.
[22] Schynoll W, Overton D, Krome R, et al. A prospective study to identify high-yield criteriaassociated with acute intracranial computed tomography findings in head-injured patients.Am J Emerg Med.1993;11:321–6.
[23] J.J. Bazarian, J.McClung, M.H. Shah, Y.T.Cheng, W. Flesher and J. Kraus. Mild traumaticbrain injury in the United States,1998–2000, Brain Injury.2005;85–91.
[24] Bo gdanova Y and VerfaellieM. Cognitive sequelae of blast-induced traumatic brain injury:recovery and rehabilitation.Neuropsychol Rev.2012;22(1):4-20.
[25] Okie S. Traumatic brain injury in the war zone. N Engl J Med.2005;352:2043–7.
[26] Tanielian T, Jaycox LH, editors. Invisible Wounds of War: Psychological and CognitiveInjuries, Their Consequences, and Services to Assist Recover. Santa Monica, CA:2008;RAND Corporation.
[27] Guskiewicz KM, Weaver NL, Padua DA, Garrett Jr WE. Epidemiology of concussion incollegiate and high school football players. Am J Sports Med.2000;28:643–50.
[28] Halstead ME, Walter KD. Sport-related concussion in children and adolescents. Pediatrics.2010;126:597–615.
[29] Guskiewicz KM, Marshall SW, Bailes J, McCrea M, Cantu RC, Randolph C, JordanBD.Association between recurrent concussion and late-life cognitive impairment in retiredprofessional football players. Neurosurgery.2005;57:719–26.
[30] Pellman EJ, Viano DC, Casson IR, TuckerAM, Waeckerle JF, Powell JW, Feuer H.Concussion in professional football: repeat injuries. Part4. Neurosurgery.2004;55:860–73R.
[31] Cernak I. Animal models of head trauma. NeuroRx.2005;2:410–22.
[32] Finnie J. Animal models of traumatic brain injury: a review. Aust Vet J.2001;79:628–33.
[33] Lighthall JW, Dixon CE, Anderson TE. Experimental models of brain injury. J Neurotra-uma.1989;6:83–97.
[34] Viano DC, Casson IR, Pellman EJ. Concussion in professional football: biomechanic ofthe struck player. Part14. Neurosurgery.2007;61:313–27.
[35] Meaney DF, Smith DH. Biomechanics of concussion. Clin Sports Med.2011;30:19–31.
[36] Marmarou A, Foda MA, van den Brink W, Campbell J,Kita H, Demetriadou K. A newmodel of diffuse brain injury in rats. Part I. Pathophysiology and biomechanics.JNeurosury.1994;80:291–300.
[37] Chen AJ, D’Esposito M. Traumatic brain injury: from bench to bedside to society. Neuron.2010;66:11–4.
[38] Flierl MA, Stahel PF, Beauchamp KM, Morgan SJ, Smith WR, Shohami E. Mouse closedhead injury model induced by a weight-drop device. Nat Protoc.2009;4:1328–37.
[39] Kilbourne M, Kuehn R, Tosun C, Caridi J, Keledjian K, Bochicchio G, Scalea T,Gerzanich V, Simard JM. Novel model of frontal impact closed head injury in the rat. JNeurotrauma2009;26:2233–43.
[40] Thompson HJ, Lifshitz J, Marklund N, Grady MS, Graham DI, Hovda DA, McIntosh TK.Lateral fluid percussion brain injury: a15-year review and evaluation. J Neurotrauma2005;22:42–75.
[41] Kabadi, SV., Hilton, G.D., Stoica, B.A., Zapple, D.N., Faden, A.I. Fluid-percussion-inducedtraumatic brain injury model in rats. Nat Protoc.2010;5,1552-1563.
[42] Michael J. Kanea, Mariana Angoa-Péreza, Denise I. Briggs David C. Vianoc, ChristianW. Kreipke. Donald M. A mouse model of human repetitive mild traumatic brain injury.Journal of Neuroscience Methods.2012;(203):41-49.
[43] Reilly, P.L. Brain injury: the pathophysiology of the first hours.'Talk and Die revisited'. JClin Neurosci.2001;8,398-403.
[44] Hovda, D.A. The increase in local cerebral glucose utilization following fluid percussionbrain injury is prevented with kynurenic acid and is associated with an increase in calcium.Acta Neurochir Suppl.(Wien).1990;51,331-333.
[45] Whiting, M.D., Baranova, A.I., and Hamm, R.J. Cognitive Impairment followingTraumatic Brain Injury, Animal Models of Cognitive Impairment.2006.
[46] McIntosh, T.K. et al.Traumatic brain injury in the rat: characterization of a lateralfluid-percussion model. Neuroscience.1989;28,233-244.
[47] Adams, J.H. et al. Diffuse axonal injury in head injury: definition, diagnosis and grading.Histopathology.1989;15,49-59.
[48] Cordobes, F. Post-traumatic diffuse axonal brain injury. Analysis of78patients studiedwith computed tomography. Acta Neurochir.(Wien).1986;81,27-35.
[49] Maxwell, W.L. Povlishock, J. T. and Graham, D.L. Amechanistic analysis of non-disruptive axonal injury: a review. J Neurotrauma.1997;14,419-440.
[50] Lighthall, J.W. and Anderson, T.E. The neurobiology of cenral nervous system trauma.Oxford University Press.1994;3-12.
[51] McIntosh, T.K. et al. Neuropathological sequelae of traumatic brain injury: relationship toneurochemical and biomechanical mechanisms. Lab Invest.1996;74,315-342.
[52] Morganti-Kossmann, M.C., Satgunaseelan, L., Bye, N.,&Kossmann, T. Modulation ofimmune response by head injury. Injury.2007;38,1392-1400.
[53] Capruso, D.X.&Levin, H.S. Cognitive impairment following closed head injury.NeurolClin.1992;10,879-893.
[54]Levin, H.S., Goldstein, F.C., High, W.M., Jr.,&Eisenberg, H.M. Disproportionatelysevere memory deficit in relation to normal intellectual functioning after closed headinjury. J Neurol Neurosurg Psychiatry.1988;51,1294-1301.
[55]Christman CW, Grady MS, Walker SA, et al. Ultrastructural studies of diffuse axonalinjury in humans. J Neurotrauma.1994;11:173–86.
[56] Maxwell WL, Povlishock JT, Graham DL. A mechanistic analysis of nondisruptive axonalinjury: a review. J Neurotrauma.1997;14:419–40.
[57] Povlishock JT. Pathophysiology of neural injury: therapeutic opportunities and challenges.Clin Neurosurg.2000;46:113–26.
[58] Povlishock JT, Jenkins LW. Are the pathobiological changes evoked by traumatic braininjury immediate and irreversible? Brain Pathol.1995;5:415–26.
[59] Povlishock JT, Christman CW. The pathobiology of traumatically induced axonal injury inanimals and humans: a review of current thoughts. J Neurotrauma.1995;12:555–64.
[60]Povlishock JT, Becker DP, Cheng CL. Axonal change in minor head injury. J NeuropatholExp Neurol.1983;42:225–42.
[61] Povlishock JT, Becker DP, Miller JD. The morphopathologic substrates of concussion?Acta Neuropathol (Berl).1979;47:1–11.
[62] Jordan BD, Relkin NR, Ravdin LD. Apolipoprotein E e4associated with chronic traumaticbrain injury in boxing. JAMA.1997;278:136–40.
[63] Nathoo N, Chetty R, van Dellen JR. Genetic vulnerability following traumatic brain injury:the role of apolipoprotein E. Mol Pathol.2003;56:132–6.
[64] Das M, Leonardo CC, Rangooni S, Mohapatra SS, Mohapatra S, Pennypacker KR. Lateralfluid percussion injury of the brain induces CCL20inflammatory chemokine expression inrats. J Neuroinflammation.2011;31;8:148.
[65] Shojo H, Kaneko Y, Mabuchi T, Kibayashi K, Adachi N, BorlonganCV. Genetic andhistologic evidence implicates role of inflammation in traumatic brain injury-inducedapoptosis in the rat cerebral cortex following moderate fluid percussion injury.Neuroscience.201029;171(4):1273-82.
[66] Raghupathi R, Conti AC, Graham DI, Krajewski S, Reed JC, TrojanowskiJQ, McIntosh TK. Mild traumatic brain injury induces apoptotic cell death in the cortexthat is preceded by decreases in cellular Bcl-2immunoreactivity. Neuroscience.2002;110(4):605-16.
[67] Raghupathi R, Graham DI, McIntosh TK. Apoptosis after traumatic brain injury. JNeurotrauma.2000;17(10):927-38.
[68]Shitaka Y, Tran HT, Bennett RE, Sanchez L, Levy MA, Dikranian K, Brody DL.Repetitive closed-skull traumatic brain injury in mice causespersistent multifocal axonalinjury and microglial reactivity. J Neuropathol Exp Neurol.2011;70(7):551-67.
[69] Shimamura M, Garcia JM, Prough DS, Dewitt DS, Uchida T, Shah SA, AvilaMA, Hellmich HL. Analysis of long-term gene expression in neurons of thehippocampal subfields following traumatic braininjury in rats. Neuroscience.2005;131(1):87-97.
[70] Arciniegas DB. The cholinergic hypothesis of cognitive impairment caused by traumaticbrain injury. Curr Psychiatry Rep.2003;5:391–9.
[71] Jones E, Fear NT, Wessely S. Shell shock and mild traumatic brain injury: A historicalreview. The American Journal of Psychiatry.2007;164(11):1641–1645.
[72] Tori, Knutw, Svend MJ. Quantification of post-concussion symptoms3months after minorheadi njury in100consecutive patients. JournalofNeurology.1998;245:609-612.
[73] Iverson GL, Lange RT. Examination of "postconcussion-like" symptoms in a healthysample. Applied Neuropsychology.2003;10(3):137–144.
[74] Jeffrey B and Shannonn M, Gender and Outcome after mild traumatic brain injury. AcadEmerg Med.2007;14(51):142.
[75] McKee AC, Cantu RC, Nowinski CJ, Hedley-Whyte ET, Gavett BE, Budson AE,SantiniVE, Lee HS, Kubilus CA, Stern RA. Chronic traumatic encephalopathy inathletes:progressive tauopathy after repetitive head injury. J Neuropathol Exp Neurol.2009;68:709–35.
[76]Masel BE, DeWitt DS. Traumatic brain injury: a disease process, not an event. JNeurotrauma.2010;27:1529–40.
[77] Omalu BI, Hamilton RL, Kamboh MI, DeKosky ST, Bailes J. Chronic traumaticenceph-alopathy (CTE) in a National Football League Player: case report andemergingmedicolegal practice questions. J Forensic Nurs2010;6:40–6.
[78] Plassman BL, Havlik RJ, Steffens DC,Helms MJ, Newman TN, Drosdick D, PhillipsC,Gau BA,Welsh-Bohmer KA, Burke JR,Guralnik JM, Breitner JC.Documented headinjury in early adulthood and risk of Alzheimer’s disease and other dementias.Neurology.2000;55:1158–66.
[79] Ryan LM, Warden DL. Post concussion syndrome. International Review of Psychiatry.2003;15(4):310–316.
[80] L. Carroll, J. Cassidy, P.M. Peloso, J. Borg, H. von Holst and L. Holm. Prognosis for mildtraumatic brain injury: results of theWHO Collaborating Centre Task Force onMildTraumatic Brain Injury, Journal of Rehabilitation Medicine.2004;84–105.
[81] H. Belanger, G. Curtiss, J. Demery, B. Lebowitz and R. Vanderploeg. Factors moderatingneuropsychological outcomes following mild traumatic brain injury: A meta-analysis,Journal of the International Neuropsychological Society.2005;11:215–227.
[82]Hibbard MR, Uysal S, Kepler K. Axis I psychopathology in individuals with traumaticbrain injury. J Head Trauma Rehabil.1998;13:24–39.
[83] O’Donnell ML, Creamer M, Pattison P. Psychiatric morbidity following injury. Am JPsychiatry.2004;161:507–14.
[84] Dikmen SS, Bombardier CH, Machamer JEl. Natural history of depression in traumaticbrain injury. Arch Phys Med Rehabil.2004;85:1457–64.
[85] Holman RT. The slow discovery of the importance of omega3essential fatty acids inhuman health. J. Nutr.1998;128(2Suppl):427S–433S.
[86] Dyerberg J, Bang HO, Hjorne N. Fatty acid composition of the plasma lipids in GreenlandEskimos. Am J Clin Nutr.1975;28(9):958–66.
[87] Yehuda S, Rabinovitz S, Crasso RL, Mostofsky DI. The role of polyunsaturated fatty acidsin restoring the ageing neuronal membrane. Neurobiol Ageing.2002;23:843–53.
[88] Horrobin DF, Jenkins K, Bennett CN, Christie WW.Eicosapentaenoic acid and arachidonicacid: collaboration and not antagonism is the key to biological understanding.Prostaglandins Leukot Essent Fatty Acids.2002;66:83–90.
[89] Piomelli D, Pilon C, Giros B, Sokoloff P, Martres MP, Schwartz YC. Dopamine activationof the arachidonic acio cascade as basis for D1/D2receptor synergism. Nature1991;353:164–7.
[90] Mori, Trevor A, Bao, Danny Q, Burke, Valerie, Puddey, Ian B, Beilin, Lawrence J. D-ocosahexaenoic acid but not eicosapentaenoic acid lowers ambulatory blood pressure andheart rate in humans. Hypertension.1993;34(2):253–260.
[91] G. Fontani, F. Corradeschi, A. Felici, F. Alfatti, S. Migliorini and L. Lodi. Cognitive andphysiological effects of Omega-3polyunsaturated fatty acid supplementation in healthysubjects. European Journal of Clinical Investigation.2005;35,691–699.
[92] Piomelli D. Eicosanoids in synaptic transmission. Crit Rev Neurobiol.1994;8:65–83.
[93] Haag M. Essential fatty acids and the brain. Can J Psychiat.2003;48:195–203.
[94] Martin RE, Bazan NG. Changing fatty acid content of growth cone lipids prior to synaptogenesis. J Neurochem.1992;59:318–25.
[95]Jones CR, Arai T, Rapoport SI. Evidence for the involvement of docosahexanoic acid incholinergic stimulated signal transduction at the synapse. NeurochemRes.1997;22:663–70
[96]Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F. Ca2+-inducedapoptosis through calcineurin dephosphorylation of BAD.Science.1999;284:339–343.
[97] Sarsilmaz M, Songur A, Kus I, Ozyurt B, Gulec M, Sogut S. The regulatory role of dietaryomega-3essential fatty acids on oxidant/antioxidantbalance in the rat hippocampus.Neurosci Res Commun.2003;33:114–123.
[98] Songur A, Sarsilmaz M, Sogut S, Ozyurt B, Ozyurt H, Zararsiz I.Hypothalamic superoxidedismutase, xanthine oxidase, nitric oxide, and malondialdehyde in rats fed with fishomega-3fatty acids. Prog Neuropsychopharmacol Biol Psychiatry.2004;28:693–698.
[99] Cao DH, Xu JF, Xue RH, Zheng WF, Liu ZL. Protective effect of chronic ethyl docosahexaenoate administration on brain injury in ischemic gerbils. Pharmacol Biochem Behav.2004;79:651–659.
[100] Lonergan PE, Martin DS, Horrobin DF, Lynch MA. Neuroprotective actions ofeicosapentaenoic acid on lipopolysaccha ride-induced dysfunction in rat hippocampus. JNeurochem.2004;91:20–29.
[101] Needleman P, Raz A, Minkes MS, Ferrendelli JA, Sprecher H. Triene prostaglandins:prostacyclin and thromboxane biosynthesis and unique biological properties. Proc NatlAcad Sci.1979;76:944–948.
[102] H gyes E, Nyakas C, Kiliaan A, Farkas T, Penke B, Luiten PG. Neuroprotective effect ofdevelopmental docosahexaenic acid supplement against excitotoxic brain damage ininfant rats. Neuroscience.2003;119:999–1012.
[103] Lauritzen I, Blondeau N, Heurteaux C, Widmann C, Romey G, Lazdunski M. Polyunsaturated fatty acids are potent neuroprotectors. EMBO J.2000;19:1784–1793.
[104] Wu A, Ying Z, Gomez-Pinilla F: Omega-3fatty acids supplementation restores mechan-isms that maintain brain homeostasis in traumatic brain injury. J Neurotrauma.2007;24:1587–1595.
[105] Bazan NG: Neuroprotectin D1(NPD1): a DHA-derived mediator that protects brain andretina against cell injury-induced oxidative stress. Brain Pathol.2005;15:159–166.
[106] Blondeau N, Widmann C, Lazdunski M, Heurteaux C: Polyunsaturated fatty acids induceischemic and epileptic tolerance. Neuroscience.2002;109:231–241.
[107] Calderon F, Kim HY: Docosahexaenoic acid promotes neurite growth in hippocampalneurons. J Neurochem.2004;90:979–988.
[108]Amminger GP, Sch fer M, Papageorgiou K. Long-Chain omega-3Fatty Acids for Ind-icated Prevention of Psychotic Disorders: A Randomized, Placebo-Controlled Trial. ArchGen Psychiatry.2010;67(2):146-154.
[109] Green, Pnina; Hermesh, Haggai; Monselisec, Assaf; Maromb, Sofi; Presburgerb, Gadi;Weizman, Abraham. Red cell membrane omega-3fatty acids are decreased innondepressed patients with social anxiety disorder. Eur Neuropsychopharmacol.2006;16(2):107–113.
[110] Bhatia HS, Agrawal R, Sharma S, Huo Y-X, Ying Z. Omega-3Fatty Acid Deficiencyduring Brain Maturation Reduces Neuronal and Behavioral Plasticity in Adulthood. PLoSONE.2011;6(12): e28451.
[110] James D, Juliane B, Caral S, Heather H, Barry S. Omega-3fatty acid supplementationand reduction of traumatic axonal injury in a rodent head injury model. J Neurosurg.2011;114:77–84.