创伤性脑损伤神经源性机制及其干预实验研究
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摘要
研究背景
创伤性脑损伤（TBI）是致死致残率很高的一种常见病。除原发性脑损伤外，继发性脑损伤对TBI的预后也起着重要影响。目前有关TBI的发生发展机理至今尚未完全明确，国内外有多种学说，如：血脑屏障学说，钙通道学说，自由基学说，脑微循环学说，能量代谢学说等。但上述学说没有一种能完全解释清TBI发病机理，这是因为创伤性脑水肿的发生机理是十分复杂的。上述的各种机制并非孤立存在、单独起作用，而是相互影响、多种机制共同起作用的结果。近年来，神经源性炎症在TBI中的作用机制逐渐引起人们的重视，越来越多的研究发现，神经肽Y（NPY）、降钙素相关基因肽（CGRP）、P物质（SP）、水通道蛋白-4（AQP4）和核转录因子-κB（NF-κB）等在TBI后神经源性炎症中扮演重要角色。本课题提出“创伤性脑损伤的神经源性机制”假说，正是基于神经源性炎症。假说主要内容：TBI后，脑组织内许多神经化学和细胞介质发生改变，并且组织内发生类似神经源性炎症反应或者/和疼痛作为应激性刺激通过复杂的神经源性机制诱发的脑组织内神经递质分泌异常，导致或者加剧脑组织内生物学活性物质产生与代谢失衡，引起脑细胞的损伤，从而加剧损伤区脑组织的损伤，并且引起损伤区周围脑组织的损伤反应，引发创伤性脑水肿，造成继发性颅脑损害，即①神经源性炎症或损伤刺激直接或经传导作用于脑细胞→胞体分泌神经肽增加→神经突触间神经递质释放增多→离子通道活动增加→动作电位活动增加，并由突触部位扩散到胞体→胞体钠、钙通道活动增加→导致水钠储留，钙超载→细胞性脑水肿。②神经源性炎症产生的生物学活性物质（如SP）作用于脑血管及水通道-4（AQP4）→AQP4活动增强（在朝向血管面及软脑膜面的胶质细胞膜区有选择性的高表达），脑血管通透性增加，胶质细胞水肿→血脑屏障（BBB）通透性增加，血管内容渗出增多，间隙性水肿→血管源性脑水肿。①②共同导致创伤性脑水肿，引起颅内高压，此时如不采取措施（脱水、激素、手术）阻止其发展，那将会有更多的脑细胞死亡，炎症加剧，水肿加重，形成恶性循环，直至脑疝死亡。如果上述假设成立，通过干预或阻断上述环节均可能减轻组织损伤，这为我们寻找开发新药治疗创伤性脑损伤提供了新的思路。
研究目的：
1、探讨创伤性脑损伤的神经源性机制，即颅脑创伤导致创伤性脑水肿引起脑组织损伤性改变的具体机制。（第一部分实验要解决的问题）
2、探讨使用各种受体拮抗剂，能否减轻和抑制上述损伤性改变。（第二部分实验要解决的问题）
研究方法：
1.第一部分实验，采用Wistar雄性大鼠40只，体重280±10g，随机分为4组：对照组（C组），轻度创伤组（M1组），中度创伤(M2组),重度创伤组（S组），以上4组每组10只。用Feeney按自由落体致伤原理制作TBI大鼠模型，伤后记录丘脑腹后内侧核（VPM）痛敏神经元（PSN）放电频率，1h后断头取血，开颅取脑。通过肉眼大体观测各组大鼠脑皮层损伤处及其周边脑组织损伤程度；光镜苏木精—伊红染色法(HE)从组织细胞水平观测大鼠脑组织损伤程度;透射电镜超微结构水平观测大鼠脑皮层损伤处及其周边脑组织损伤程度;免疫组化检测大鼠脑皮层损伤处及其周边脑细胞中SP、 NPY、CGRP、NF-κB、AQP4阳性表达情况; Western blot检测大脑皮质损伤区AQP4蛋白表达情况; RT-PCR检测SP、NPY、CGRP、NF-κB、NSE和AQP-4基因表达水平; ELISA法测定血清中神经元特异性烯醇化酶（NSE）、缓激肽（BK）、前列腺素E2（PGE2）、组织胺（HA）、基质金属蛋白酶（MMP-9）、高敏C反应蛋白（hs-CRP）含量;激光共聚焦荧光离子成像实验测定脑细胞内Ca++浓度，检测钙超载情况;流式细胞仪检测大鼠脑皮层细胞亚二倍体比率，了解脑细胞凋亡情况;多通道电生理记录仪记录分析VPM核痛敏神经元放电频率。对上述各项指标进行统计学分析，做相关性研究。从组织形态学、蛋白学、基因学、血清学、神经电生理学、超微结构多角度研究创伤性脑损伤的神经源性机制。
2．第二部分第一章干预实验--NK1受体拮抗剂L-703,606对大鼠创伤性脑损伤保护作用机制的实验研究，采用45只Wistar雄性大鼠，体重280±10g，随机分为3组：对照组（C组），L-703,606干预组（L组），TBI创伤未干预组(T组),以上3组每组15只。TBI造模成功后立即尾静脉给予NK1受体拮抗剂L-703,606(250nmol/kg Sigma公司),然后于造模后1h断头取血，开颅取脑，HE染色观察各组鼠脑损伤情况；免疫组化染色检测SP、CGRP、NF-κB表达情况；RT-PCR检测SP、CGRP、NF-κB、NSE、AQP4mRNA基因表达；ELISA法测定血清中NSE、hs-CRP；统计学分析上述指标，研究P物质受体-NK1受体拮抗剂L-703,606干预效果。
3.第二部分第二章干预实验--Rho激酶抑制剂对创伤性脑损伤后脑细胞内亚二倍体比率的影响及其意义，采用健康成年雄性Wistar大鼠45只，体重280±10g,随机分为3组，即假手术组（Sham组），创伤组（TBI组）和Rho激酶抑制剂干预组（FSD组），每组15只。造模后采用流式细胞仪（FCM)检测鼠脑创伤区皮层细胞亚二倍体比率，了解使用Rho激酶抑制剂（Fasudil）后使TBI细胞凋亡改善情况。
研究结果
1．第一部分实验结果显示，随打击程度加重，鼠脑水肿越明显、脑细胞、线粒体肿胀越明显，并可见神经突触间释放神经递质及炎症细胞浸润； SP、NPY、CGRP、NF-κB、AQP4、NSE基因表达与蛋白阳性表达成正相关性，且损伤越重，基因表达越强，其对应的阳性蛋白含量表达也越多;致痛物质BK、PGE2、HA与致痛致炎神经递质-SP基因蛋白表达水平成正相关；SP与VPM核痛敏神经元放电频率成正相关性，致痛物质-P物质表达含量越高，VPM核痛敏神经元放电频率越高；SP与神经肽-NPY、CGRP之间成正相关性，P物质表达及释放越多，神经肽NPY和CGRP表达越多；SP与炎症因子-核转录因子KB(NF-κB)及炎症反应指标-高敏感性C反应蛋白（hs-CRP）之间成正相关性，P物质表达及释放越多，炎症反应指标hs-CRP和NF-κB表达越高，炎症反应越严重，NF-κB与hs-CRP之间也呈正相关性，NF-κB表达越多，血清hs-CRP含量越高，炎症反应越重；SP与基质蛋白水解酶9(MMP-9)、水通道蛋白AQP4表达之间成正相关性，P物质表达及释放越多，MMP-9含量增多，降解和破坏血管内皮基膜的能力越强，引起血管通透性增加，炎性物质渗出增多，导致血管源性脑水肿,P物质表达越多可引起AQP4表达也增多，引起细胞膜通透性增高，导致细胞性脑水肿; SP与损伤皮层脑细胞钙离子含量及细胞凋亡指标-亚二倍体（Hd）比率之间成正相关性，P物质表达及释放越多，损伤皮层脑细胞钙离子含量越高（钙超载现象越严重），脑细胞亚二倍体（Hd）比率也越高（细胞凋亡越严重）；SP与神经元特异性烯醇化酶（NSE）之间成正相关，P物质表达越多，炎症反应越严重，神经细胞坏死越多，NSE表达及含量也越高; NPY、CGRP与NF-κB之间成正相关性，TBI后NPY、CGRP表达越高，NF-κB表达也越高，炎症反应也越重；NF-κB与NSE、AQP4、钙离子浓度、亚二倍体（Hd）比率之间成正相关性，TBI后NF-κB表达越高，炎症反应越重，NSE(反映神经细胞损伤)、AQP4（反映细胞性脑水肿）、钙离子浓度（反映钙超载）和亚二倍体（反映脑细胞凋亡）比率也越高。（P均﹤0.05）
2.第二部分L-703,606对大鼠创伤性脑损伤保护作用机制的实验研究，结果显示L-703,606干预组（L组）明显较TBI未干预组（T组）损伤减轻，仅有轻度充血，少量炎性细胞浸润; L组和T组大鼠脑皮层损伤区脑组织SP、CGRP和NF-κB阳性表达较对照组阳性表达有明显增强（P﹤0.05），L组较T组表达明显降低；L组和T组大鼠脑皮层损伤区脑组织SP、CGRP、NF-κB、AQP-4和NSE基因表达水平较对照组阳性表达有明显增强（P﹤0.05），且L组较T组表达明显降低（P﹤0.05）；L组和T组大鼠血清中hs-CRP和NSE含量较对照组明显增高（P﹤0.05），且L组较T组hs-CRP和NSE含量明显降低。
3.第二部分Rho激酶抑制剂对创伤性脑损伤后脑细胞内亚二倍体比率的影响及其意义实验研究结果显示，Sham组、TBI组和FSD组的创伤区皮层细胞亚二倍体比率（%）分别为1.58±0.35，15.90±3.91和5.35±2.10，三组间差异有统计学意义（P<0.01），且FSD组能较TBI组明显降低脑细胞内亚二倍体比率，因而可减轻TBI后脑细胞凋亡，具有脑保护作用。
结论
1．神经源性炎症、神经源性机制在创伤性脑损伤中发挥重要作用；
2. NK1受体拮抗剂L-703,606对大鼠创伤性脑损伤具有保护作用；
3. Rho激酶抑制剂（Fasudil）能降低脑细胞内亚二倍体比率，对TBI后脑组织具有保护作用；
4.其他受体拮抗剂，如CGRP拮抗剂CGRP8-37、NPY拮抗剂BIBP3226,芬太尼痛觉干预,PDTC干预NF-κB,小剂量多巴胺抑制APQ4表达，骨髓间充质干细胞（BMSC）联合血管内皮生长因子（VEGF）或其他神经营养因子移植治疗TBI，均能起到抑制神经源性炎症，保护脑细胞作用（详见本人已发表之相关论文）。期望通过深入研究创伤性脑损伤神经源性机制，找到更多、更高效的治疗TBI的药物和方法，开发新药，为广大TBI患者带来福音。
BACKGROUND
Traumatic brain injury (TBI) is a common disease which has a high level of lethal anddisability. In addition to the primary brain injury, secondary brain injury also plays animportant influence to the prognosis of TBI. the Development Mechanism of TBI hasnot yet entirely clear. At home and abroad, there are a variety of theories, such as: thedoctrine of blood-brain barrier, calcium channel theory, radical theory, brainmicrocirculation doctrine, energy metabolism theory. But there is no doctrine canfully explain the pathogenesis of TBI, because the occurrence of traumatic brainedema is a very complex mechanism. The various mechanisms described above doesnot exist in isolation and work alone, but influence each other and a variety ofmechanisms to work together. In recent years, the mechanism of neurogenicinflammation in TBI gradually attracted people's attention, a growing number ofstudies found that neuropeptide Y (NPY), calcitonin gene related peptide (CGRP),substance P (SP), aquaporin-4(AQP4) and nuclear transcription factor-kappa B(NF-κB) in neurogenic inflammation plays an important role after TBI. This subjectput forward the hypothesis of " The neurogenic mechanisms of traumatic braininjury”, is just based on neurogenic inflammation. The main content of thehypothesis is follows: When TBI happen, many neurochemical in the brain tissue andcell media is changed within an organization similar to neurogenic inflammatoryresponse, or/and pain as a stress stimulus-induced brain tissue through a complexmechanism of neurogenic abnormal secretion of neurotransmitters, cause orexacerbate the biological active substances in the brain tissue and metabolicimbalance, causing brain cell damage, thus exacerbating the damage to brain tissuedamage, and caused the damage zone surrounding brain tissue response to injurycaused by traumatic cerebral edema, resulting in secondary brain damage. Theneurogenic inflammation or injury stimulate, directly or through conduction in thebrain cells, causing the secretion of neuropeptides from the cell body increase.Neurotransmitter in synaptic is released increase, and activity of ion channelsincreased, then activity of action potential increased. It is proliferated by the synapsesspread to the cell body. The sodium, calcium channel activity of cell bodyincreased, and resulting in sodium and water retention, calcium overload, cellularbrain edema.
The substances (such as SP) which have biological activityproduced by neurogenicinflammation act on the cerebrovascular and aquaporin-4(AQP4), The activity ofAQP4is enhanced (selective toward the vascular surface and the pial surface of glialcell membrane area high expression). Increased vascular permeability in the brain,glial cell edema, blood brain barrier (BBB) permeability increase, vascular exudationincreased interstitial edema, causing vasogenic cerebral edema. Cellular brain edemaand vasogenic cerebral edema common cause the traumatic brain edema,which causedby intracranial hypertension. This time as no measures are taken (for example,dehydration, hormones, surgery) to prevent its development, it will have more braindead cells, inflammation and edema exacerbated. And a vicious circle until theherniation death. If this assumption is true, tissue damage may be reduced, interferewith or block the above link provides a new approach for us to find and develop newdrugs and treatment of traumatic brain injury.
OBJECTIVE
1.To explore the neurogenic mechanisms of traumatic brain injury. ie. traumatic braininjury lead to the specific mechanisms of traumatic brain edema caused by changes ofbrain tissue damage.(First part of the experimental problem to be solved)
2.To explore the use of a variety of receptor antagonists,whether it can obtain theresults in mitigation and suppression of these traumatic changes.(Second part of theexperimental problem to be solved)
METHODS
1. In the first part of the experiment,40male Wistar rats weighing280±10g, wererandomly divided into4groups: control group (C), mild trauma group (M1group),moderate trauma (M2group), severe trauma group(S group), the above four groupswith10in each group. TBI rat model using Feeney produced according to theprinciple of free-fall injury, after injury record of the thalamic ventral posterior medialnucleus (VPM) of hyperalgesia neurons (PSN) discharge frequency. Then after TBI1h, decapitation blood and brain craniotomy were finished. The lesion of rat braincortex and its surrounding brain tissue damage in general observation by the nakedeye; By the light microscopy hematoxylin-eosin staining (HE) and TEM, brain tissueinjury of rat were observed from the organization at the cellular level and ultrastructure level; the expression of SP, NPY, of CGRP, of NF-κB of AQP4weredetected by immunohistochemical; AQP4protein expression of the cerebral cortexdamage zone was detected by Western blot; gene expression level of SP, NPY, ofCGRP, of NF-κB, NSE, and AQP-4was detected by RT-PCR; ELISA method for thedetermination of serum neuron-specific enolase (NSE), bradykinin peptide (BK),prostaglandin E2(PGE2), histamine (HA), matrix metalloproteinase (MMP-9),high-sensitivity C-reactive protein (hs-CRP) content; Confocal laser fluorescence ionimaging experimental determination of brain cells, the concentration of calciumoverload is detected; hypodiploid ratio, was measured by flow cytometry tounderstand the brain cell apoptosis; multi-channel electrophysiological recorderrecords the VPM nuclear hyperalgesia neuronal firing frequency. On the aboveindicators for statistical analysis, correlation studies. To explore neurogenicmechanisms of TBI from the multi-angle, such as tissue morphology, protein, genetics,serology, nerve electrophysiology and ultrastructure.
2. The second part of the first chapter of the intervention experiment, A experimentalstudy about the protective mechanism of NK1receptor antagonist L-703,606totraumatic brain injury, using45male Wistar rats, weighing280±10g, were randomlydivided into threegroups: control group (group C), L-703,606intervention group(group L),TBI trauma group (group T),15rats in each group. The tail veinimmediately after TBI model was successful to give the NK1receptor antagonistL-703,606(250nmol/kg, Sigma), after TBI1h, decapitated, blood and braincraniotomy. By HE staining rats brain damage were observed in each group; SP, ofCGRP, NF-κB expression were mesured by immunohistochemical staining; SP,CGRP, NF-κB, NSE, AQP4mRNA gene expression were detected by RT-PCR;ELISA method for the determination of serum NSE of hs-CRP; Statistics analysis ofthese indicators, the intervention effect of receptor-NK1receptor antagonist L-703,606.
3. Chapter II of Part Intervention Study-Rho kinase inhibitor on traumatic braininjury brain cells diploid ratio and its significance. The use of healthy adult maleWistar rats45, weight280±10g, were randomly divided for the three groups, namelysham operation group (Sham), trauma group (TBI group) and Rho kinase inhibitors inthe intervention group (FSD group)(n=15). After the model,the ratio of hypodiploidin the rats brain trauma cortical cells was mesured by flow cytometry (FCM) toevaluate the situation after using the Rho kinase inhibitors (Fasudil).
RESULTS
1.The first part of the experimental results show that, with the increase oftrauma degree, the more obvious cerebral edema, brain cells, mitochondrial swelling,the more obvious and visible between the synapses, the release of neurotransmittersand inflammatory cell infiltration; There are positive correlations between the geneexpression and protein expression of SP, NPY, CGRP, NF-κB，AQP4and NSE. Andthe more severe damage of TBI, the more gene expression, corresponding to thepositive expression and content of protein; There are positive correlations betweenBK, PGE2, HA and SP in protein gene expression level, SP and the firing frequencyof VPM nuclear hyperalgesia neuron. the higher the expression level of pain causedby substance-substance P, the higher the VPM nuclear hyperalgesia neuronal firingfrequency; There are positive correlations between SP and NPY, CGRP, substance Pexpression and release more neuropeptides NPY and CGRP expression in the more;There are positive correlations between SP and inflammatory factors, the nucleartranscription factor KB (NF-κB) and the inflammatory response indicators, highsensitivity C reactive protein (hs-CRP), the higher substance P expression, release ofhs-CRP and expression of NF-κB, the more serious the inflammatory response.There is a positive correlation between NF-κB and hs-CRP, the more NF-κBexpression, the higher content of hs-CRP in serum,and the more severe inflammatoryresponse. There are positive correlations between the expression of SP, matrixproteolytic enzymes (MMP-9) and AQP4. The higher level of the MMP-9in theserum, the more the ability of degradation and destruction of blood vessels, whichcausing increased vascular permeability, exudation of inflammatory substancesincreased, leading to vasogenic cerebral edema. The more expression of substance P,the more AQP4expression, which causing increased membrane permeability, leadingto cellular brain edema; There are positive correlations between SP,calcium contentand cell apoptosis of damaged cortex cell. There are positive correlations between theratio of hypodiploid (Hd), SP expression and calcium content of cortical brain cells,the higher the ratio the more serious of apoptosis. There are positive correlationsbetween SP, neuron-specific enolase (NSE), the more substance P expression, themore serious inflammatory response, the more nerve cell necrosis and the higher NSEexpression; There are positive correlations between NPY, CGRP, and NF-κB afterTBI. The higher NPY and CGRP expression, the higher the expression of NF-κB,the more severe inflammatory response; There are positive correlations between NF-κB and NSE, AQP4, the calcium ion concentration, hypodiploid (Hd) ratio afterTBI. The higher NF-κB expression, the more severe inflammatory response, NSE(reflecting the nerve cell damage), AQP4(reflecting the cell brain edema), calciumion concentration (reflecting the calcium overload) and the higher the ratio ofhypodiploid (reflecting brain cell apoptosis).(P <0.05).
2. The study results of experimental study about the protective mechanism of NK1receptor antagonist L-703,606to traumatic brain injury, showed that L-703,606intervention group (group L) injury was attenuated significantly compared with TBIintervention group (T group), only mild congestion, a small amount of inflammatorycell infiltration; SP, CGRP, and NF-κB expression of L group and T group in ratcerebral cortical lesions in the brain tissue was increased compared with the controlgroup. L group was significantly reduced compared with T group; SP, CGRP,NF-kappa B, AQP-4and NSE gene expression levels in the brain tissue of L groupand T group in rat cerebral cortical lesions significantly increased than the controlgroup, positive expression of the L group compared with Tgroup was significantlylower; serum hs-CRP and NSE levels of L group and T group was significantly higherthan the control group, and the hs-CRP and NSE of L group were significantly lowerthan that in the T group,(P <0.05)
3. The results of Rho kinase inhibitor and its implication of experimental study ontraumatic brain injury of brain cells diploid ratio show that hypodiploid ratio ofcortical cells in Sham group, TBI group and trauma area of the FSD group arerespectively.1.58±0.35,15.90±3.91and5.35±2.10, the differences among the threegroups was statistically significant (P <0.01), and the brain cells hypodiploid ratio ofFSD group was significantly lower than that of the TBI group. Thus Fasudil canreduce the TBI brain cells apoptosis, and have the cerebral protective effects.
CONCLUSION
1.Neurogenic inflammation and neurogenic mechanisms play an important role intraumatic brain injury;
2.NK1receptor antagonist L-703,606has a protective effect on the brain injury inrats with traumatic brain injury;
3.Rho kinase inhibitors (Fasudil) can reduce the rate of brain cells hypodiploid, andhas a protective effect of TBI in brain tissue;(4) receptor antagonist, such as CGRP antagonists of CGRP8-37, NPY antagonistBIBP3226, fentanyl (pain intervention),PDTC intervention of NF-κB, a small dose of dopamine inhibition APQ4, the expression of bone marrow mesenchymal stemcells (BMSC) with vascular endothelial growth factor (VEGF) or other neurotrophicfactor transplantation in the treatment of TBI, can act as a disincentive to neurogenicinflammation, protect brain cells (see I have published papers). Expect in-depth studyof neurogenic mechanisms of traumatic brain injury, to find more and more efficientdrugs and methods of treatment of TBI, the development of new drugs for themajority of TBI patients to bring the Gospel.
创伤性脑损伤（TBI）是致死致残率很高的一种常见病。除原发性脑损伤外，继发性脑损伤对TBI的预后也起着重要影响。目前有关TBI的发生发展机理至今尚未完全明确，国内外有多种学说，如：血脑屏障学说，钙通道学说，自由基学说，脑微循环学说，能量代谢学说等。但上述学说没有一种能完全解释清TBI发病机理，这是因为创伤性脑水肿的发生机理是十分复杂的。上述的各种机制并非孤立存在、单独起作用，而是相互影响、多种机制共同起作用的结果。近年来，神经源性炎症在TBI中的作用机制逐渐引起人们的重视，越来越多的研究发现，神经肽Y（NPY）、降钙素相关基因肽（CGRP）、P物质（SP）、水通道蛋白-4（AQP4）和核转录因子-κB（NF-κB）等在TBI后神经源性炎症中扮演重要角色。本课题提出“创伤性脑损伤的神经源性机制”假说，正是基于神经源性炎症。假说主要内容：TBI后，脑组织内许多神经化学和细胞介质发生改变，并且组织内发生类似神经源性炎症反应或者/和疼痛作为应激性刺激通过复杂的神经源性机制诱发的脑组织内神经递质分泌异常，导致或者加剧脑组织内生物学活性物质产生与代谢失衡，引起脑细胞的损伤，从而加剧损伤区脑组织的损伤，并且引起损伤区周围脑组织的损伤反应，引发创伤性脑水肿，造成继发性颅脑损害，即①神经源性炎症或损伤刺激直接或经传导作用于脑细胞→胞体分泌神经肽增加→神经突触间神经递质释放增多→离子通道活动增加→动作电位活动增加，并由突触部位扩散到胞体→胞体钠、钙通道活动增加→导致水钠储留，钙超载→细胞性脑水肿。②神经源性炎症产生的生物学活性物质（如SP）作用于脑血管及水通道-4（AQP4）→AQP4活动增强（在朝向血管面及软脑膜面的胶质细胞膜区有选择性的高表达），脑血管通透性增加，胶质细胞水肿→血脑屏障（BBB）通透性增加，血管内容渗出增多，间隙性水肿→血管源性脑水肿。①②共同导致创伤性脑水肿，引起颅内高压，此时如不采取措施（脱水、激素、手术）阻止其发展，那将会有更多的脑细胞死亡，炎症加剧，水肿加重，形成恶性循环，直至脑疝死亡。如果上述假设成立，通过干预或阻断上述环节均可能减轻组织损伤，这为我们寻找开发新药治疗创伤性脑损伤提供了新的思路。
研究目的：
1、探讨创伤性脑损伤的神经源性机制，即颅脑创伤导致创伤性脑水肿引起脑组织损伤性改变的具体机制。（第一部分实验要解决的问题）
2、探讨使用各种受体拮抗剂，能否减轻和抑制上述损伤性改变。（第二部分实验要解决的问题）
研究方法：
1.第一部分实验，采用Wistar雄性大鼠40只，体重280±10g，随机分为4组：对照组（C组），轻度创伤组（M1组），中度创伤(M2组),重度创伤组（S组），以上4组每组10只。用Feeney按自由落体致伤原理制作TBI大鼠模型，伤后记录丘脑腹后内侧核（VPM）痛敏神经元（PSN）放电频率，1h后断头取血，开颅取脑。通过肉眼大体观测各组大鼠脑皮层损伤处及其周边脑组织损伤程度；光镜苏木精—伊红染色法(HE)从组织细胞水平观测大鼠脑组织损伤程度;透射电镜超微结构水平观测大鼠脑皮层损伤处及其周边脑组织损伤程度;免疫组化检测大鼠脑皮层损伤处及其周边脑细胞中SP、 NPY、CGRP、NF-κB、AQP4阳性表达情况; Western blot检测大脑皮质损伤区AQP4蛋白表达情况; RT-PCR检测SP、NPY、CGRP、NF-κB、NSE和AQP-4基因表达水平; ELISA法测定血清中神经元特异性烯醇化酶（NSE）、缓激肽（BK）、前列腺素E2（PGE2）、组织胺（HA）、基质金属蛋白酶（MMP-9）、高敏C反应蛋白（hs-CRP）含量;激光共聚焦荧光离子成像实验测定脑细胞内Ca++浓度，检测钙超载情况;流式细胞仪检测大鼠脑皮层细胞亚二倍体比率，了解脑细胞凋亡情况;多通道电生理记录仪记录分析VPM核痛敏神经元放电频率。对上述各项指标进行统计学分析，做相关性研究。从组织形态学、蛋白学、基因学、血清学、神经电生理学、超微结构多角度研究创伤性脑损伤的神经源性机制。
2．第二部分第一章干预实验--NK1受体拮抗剂L-703,606对大鼠创伤性脑损伤保护作用机制的实验研究，采用45只Wistar雄性大鼠，体重280±10g，随机分为3组：对照组（C组），L-703,606干预组（L组），TBI创伤未干预组(T组),以上3组每组15只。TBI造模成功后立即尾静脉给予NK1受体拮抗剂L-703,606(250nmol/kg Sigma公司),然后于造模后1h断头取血，开颅取脑，HE染色观察各组鼠脑损伤情况；免疫组化染色检测SP、CGRP、NF-κB表达情况；RT-PCR检测SP、CGRP、NF-κB、NSE、AQP4mRNA基因表达；ELISA法测定血清中NSE、hs-CRP；统计学分析上述指标，研究P物质受体-NK1受体拮抗剂L-703,606干预效果。
3.第二部分第二章干预实验--Rho激酶抑制剂对创伤性脑损伤后脑细胞内亚二倍体比率的影响及其意义，采用健康成年雄性Wistar大鼠45只，体重280±10g,随机分为3组，即假手术组（Sham组），创伤组（TBI组）和Rho激酶抑制剂干预组（FSD组），每组15只。造模后采用流式细胞仪（FCM)检测鼠脑创伤区皮层细胞亚二倍体比率，了解使用Rho激酶抑制剂（Fasudil）后使TBI细胞凋亡改善情况。
研究结果
1．第一部分实验结果显示，随打击程度加重，鼠脑水肿越明显、脑细胞、线粒体肿胀越明显，并可见神经突触间释放神经递质及炎症细胞浸润； SP、NPY、CGRP、NF-κB、AQP4、NSE基因表达与蛋白阳性表达成正相关性，且损伤越重，基因表达越强，其对应的阳性蛋白含量表达也越多;致痛物质BK、PGE2、HA与致痛致炎神经递质-SP基因蛋白表达水平成正相关；SP与VPM核痛敏神经元放电频率成正相关性，致痛物质-P物质表达含量越高，VPM核痛敏神经元放电频率越高；SP与神经肽-NPY、CGRP之间成正相关性，P物质表达及释放越多，神经肽NPY和CGRP表达越多；SP与炎症因子-核转录因子KB(NF-κB)及炎症反应指标-高敏感性C反应蛋白（hs-CRP）之间成正相关性，P物质表达及释放越多，炎症反应指标hs-CRP和NF-κB表达越高，炎症反应越严重，NF-κB与hs-CRP之间也呈正相关性，NF-κB表达越多，血清hs-CRP含量越高，炎症反应越重；SP与基质蛋白水解酶9(MMP-9)、水通道蛋白AQP4表达之间成正相关性，P物质表达及释放越多，MMP-9含量增多，降解和破坏血管内皮基膜的能力越强，引起血管通透性增加，炎性物质渗出增多，导致血管源性脑水肿,P物质表达越多可引起AQP4表达也增多，引起细胞膜通透性增高，导致细胞性脑水肿; SP与损伤皮层脑细胞钙离子含量及细胞凋亡指标-亚二倍体（Hd）比率之间成正相关性，P物质表达及释放越多，损伤皮层脑细胞钙离子含量越高（钙超载现象越严重），脑细胞亚二倍体（Hd）比率也越高（细胞凋亡越严重）；SP与神经元特异性烯醇化酶（NSE）之间成正相关，P物质表达越多，炎症反应越严重，神经细胞坏死越多，NSE表达及含量也越高; NPY、CGRP与NF-κB之间成正相关性，TBI后NPY、CGRP表达越高，NF-κB表达也越高，炎症反应也越重；NF-κB与NSE、AQP4、钙离子浓度、亚二倍体（Hd）比率之间成正相关性，TBI后NF-κB表达越高，炎症反应越重，NSE(反映神经细胞损伤)、AQP4（反映细胞性脑水肿）、钙离子浓度（反映钙超载）和亚二倍体（反映脑细胞凋亡）比率也越高。（P均﹤0.05）
2.第二部分L-703,606对大鼠创伤性脑损伤保护作用机制的实验研究，结果显示L-703,606干预组（L组）明显较TBI未干预组（T组）损伤减轻，仅有轻度充血，少量炎性细胞浸润; L组和T组大鼠脑皮层损伤区脑组织SP、CGRP和NF-κB阳性表达较对照组阳性表达有明显增强（P﹤0.05），L组较T组表达明显降低；L组和T组大鼠脑皮层损伤区脑组织SP、CGRP、NF-κB、AQP-4和NSE基因表达水平较对照组阳性表达有明显增强（P﹤0.05），且L组较T组表达明显降低（P﹤0.05）；L组和T组大鼠血清中hs-CRP和NSE含量较对照组明显增高（P﹤0.05），且L组较T组hs-CRP和NSE含量明显降低。
3.第二部分Rho激酶抑制剂对创伤性脑损伤后脑细胞内亚二倍体比率的影响及其意义实验研究结果显示，Sham组、TBI组和FSD组的创伤区皮层细胞亚二倍体比率（%）分别为1.58±0.35，15.90±3.91和5.35±2.10，三组间差异有统计学意义（P<0.01），且FSD组能较TBI组明显降低脑细胞内亚二倍体比率，因而可减轻TBI后脑细胞凋亡，具有脑保护作用。
结论
1．神经源性炎症、神经源性机制在创伤性脑损伤中发挥重要作用；
2. NK1受体拮抗剂L-703,606对大鼠创伤性脑损伤具有保护作用；
3. Rho激酶抑制剂（Fasudil）能降低脑细胞内亚二倍体比率，对TBI后脑组织具有保护作用；
4.其他受体拮抗剂，如CGRP拮抗剂CGRP8-37、NPY拮抗剂BIBP3226,芬太尼痛觉干预,PDTC干预NF-κB,小剂量多巴胺抑制APQ4表达，骨髓间充质干细胞（BMSC）联合血管内皮生长因子（VEGF）或其他神经营养因子移植治疗TBI，均能起到抑制神经源性炎症，保护脑细胞作用（详见本人已发表之相关论文）。期望通过深入研究创伤性脑损伤神经源性机制，找到更多、更高效的治疗TBI的药物和方法，开发新药，为广大TBI患者带来福音。
BACKGROUND
Traumatic brain injury (TBI) is a common disease which has a high level of lethal anddisability. In addition to the primary brain injury, secondary brain injury also plays animportant influence to the prognosis of TBI. the Development Mechanism of TBI hasnot yet entirely clear. At home and abroad, there are a variety of theories, such as: thedoctrine of blood-brain barrier, calcium channel theory, radical theory, brainmicrocirculation doctrine, energy metabolism theory. But there is no doctrine canfully explain the pathogenesis of TBI, because the occurrence of traumatic brainedema is a very complex mechanism. The various mechanisms described above doesnot exist in isolation and work alone, but influence each other and a variety ofmechanisms to work together. In recent years, the mechanism of neurogenicinflammation in TBI gradually attracted people's attention, a growing number ofstudies found that neuropeptide Y (NPY), calcitonin gene related peptide (CGRP),substance P (SP), aquaporin-4(AQP4) and nuclear transcription factor-kappa B(NF-κB) in neurogenic inflammation plays an important role after TBI. This subjectput forward the hypothesis of " The neurogenic mechanisms of traumatic braininjury”, is just based on neurogenic inflammation. The main content of thehypothesis is follows: When TBI happen, many neurochemical in the brain tissue andcell media is changed within an organization similar to neurogenic inflammatoryresponse, or/and pain as a stress stimulus-induced brain tissue through a complexmechanism of neurogenic abnormal secretion of neurotransmitters, cause orexacerbate the biological active substances in the brain tissue and metabolicimbalance, causing brain cell damage, thus exacerbating the damage to brain tissuedamage, and caused the damage zone surrounding brain tissue response to injurycaused by traumatic cerebral edema, resulting in secondary brain damage. Theneurogenic inflammation or injury stimulate, directly or through conduction in thebrain cells, causing the secretion of neuropeptides from the cell body increase.Neurotransmitter in synaptic is released increase, and activity of ion channelsincreased, then activity of action potential increased. It is proliferated by the synapsesspread to the cell body. The sodium, calcium channel activity of cell bodyincreased, and resulting in sodium and water retention, calcium overload, cellularbrain edema.
The substances (such as SP) which have biological activityproduced by neurogenicinflammation act on the cerebrovascular and aquaporin-4(AQP4), The activity ofAQP4is enhanced (selective toward the vascular surface and the pial surface of glialcell membrane area high expression). Increased vascular permeability in the brain,glial cell edema, blood brain barrier (BBB) permeability increase, vascular exudationincreased interstitial edema, causing vasogenic cerebral edema. Cellular brain edemaand vasogenic cerebral edema common cause the traumatic brain edema,which causedby intracranial hypertension. This time as no measures are taken (for example,dehydration, hormones, surgery) to prevent its development, it will have more braindead cells, inflammation and edema exacerbated. And a vicious circle until theherniation death. If this assumption is true, tissue damage may be reduced, interferewith or block the above link provides a new approach for us to find and develop newdrugs and treatment of traumatic brain injury.
OBJECTIVE
1.To explore the neurogenic mechanisms of traumatic brain injury. ie. traumatic braininjury lead to the specific mechanisms of traumatic brain edema caused by changes ofbrain tissue damage.(First part of the experimental problem to be solved)
2.To explore the use of a variety of receptor antagonists,whether it can obtain theresults in mitigation and suppression of these traumatic changes.(Second part of theexperimental problem to be solved)
METHODS
1. In the first part of the experiment,40male Wistar rats weighing280±10g, wererandomly divided into4groups: control group (C), mild trauma group (M1group),moderate trauma (M2group), severe trauma group(S group), the above four groupswith10in each group. TBI rat model using Feeney produced according to theprinciple of free-fall injury, after injury record of the thalamic ventral posterior medialnucleus (VPM) of hyperalgesia neurons (PSN) discharge frequency. Then after TBI1h, decapitation blood and brain craniotomy were finished. The lesion of rat braincortex and its surrounding brain tissue damage in general observation by the nakedeye; By the light microscopy hematoxylin-eosin staining (HE) and TEM, brain tissueinjury of rat were observed from the organization at the cellular level and ultrastructure level; the expression of SP, NPY, of CGRP, of NF-κB of AQP4weredetected by immunohistochemical; AQP4protein expression of the cerebral cortexdamage zone was detected by Western blot; gene expression level of SP, NPY, ofCGRP, of NF-κB, NSE, and AQP-4was detected by RT-PCR; ELISA method for thedetermination of serum neuron-specific enolase (NSE), bradykinin peptide (BK),prostaglandin E2(PGE2), histamine (HA), matrix metalloproteinase (MMP-9),high-sensitivity C-reactive protein (hs-CRP) content; Confocal laser fluorescence ionimaging experimental determination of brain cells, the concentration of calciumoverload is detected; hypodiploid ratio, was measured by flow cytometry tounderstand the brain cell apoptosis; multi-channel electrophysiological recorderrecords the VPM nuclear hyperalgesia neuronal firing frequency. On the aboveindicators for statistical analysis, correlation studies. To explore neurogenicmechanisms of TBI from the multi-angle, such as tissue morphology, protein, genetics,serology, nerve electrophysiology and ultrastructure.
2. The second part of the first chapter of the intervention experiment, A experimentalstudy about the protective mechanism of NK1receptor antagonist L-703,606totraumatic brain injury, using45male Wistar rats, weighing280±10g, were randomlydivided into threegroups: control group (group C), L-703,606intervention group(group L),TBI trauma group (group T),15rats in each group. The tail veinimmediately after TBI model was successful to give the NK1receptor antagonistL-703,606(250nmol/kg, Sigma), after TBI1h, decapitated, blood and braincraniotomy. By HE staining rats brain damage were observed in each group; SP, ofCGRP, NF-κB expression were mesured by immunohistochemical staining; SP,CGRP, NF-κB, NSE, AQP4mRNA gene expression were detected by RT-PCR;ELISA method for the determination of serum NSE of hs-CRP; Statistics analysis ofthese indicators, the intervention effect of receptor-NK1receptor antagonist L-703,606.
3. Chapter II of Part Intervention Study-Rho kinase inhibitor on traumatic braininjury brain cells diploid ratio and its significance. The use of healthy adult maleWistar rats45, weight280±10g, were randomly divided for the three groups, namelysham operation group (Sham), trauma group (TBI group) and Rho kinase inhibitors inthe intervention group (FSD group)(n=15). After the model,the ratio of hypodiploidin the rats brain trauma cortical cells was mesured by flow cytometry (FCM) toevaluate the situation after using the Rho kinase inhibitors (Fasudil).
RESULTS
1.The first part of the experimental results show that, with the increase oftrauma degree, the more obvious cerebral edema, brain cells, mitochondrial swelling,the more obvious and visible between the synapses, the release of neurotransmittersand inflammatory cell infiltration; There are positive correlations between the geneexpression and protein expression of SP, NPY, CGRP, NF-κB，AQP4and NSE. Andthe more severe damage of TBI, the more gene expression, corresponding to thepositive expression and content of protein; There are positive correlations betweenBK, PGE2, HA and SP in protein gene expression level, SP and the firing frequencyof VPM nuclear hyperalgesia neuron. the higher the expression level of pain causedby substance-substance P, the higher the VPM nuclear hyperalgesia neuronal firingfrequency; There are positive correlations between SP and NPY, CGRP, substance Pexpression and release more neuropeptides NPY and CGRP expression in the more;There are positive correlations between SP and inflammatory factors, the nucleartranscription factor KB (NF-κB) and the inflammatory response indicators, highsensitivity C reactive protein (hs-CRP), the higher substance P expression, release ofhs-CRP and expression of NF-κB, the more serious the inflammatory response.There is a positive correlation between NF-κB and hs-CRP, the more NF-κBexpression, the higher content of hs-CRP in serum,and the more severe inflammatoryresponse. There are positive correlations between the expression of SP, matrixproteolytic enzymes (MMP-9) and AQP4. The higher level of the MMP-9in theserum, the more the ability of degradation and destruction of blood vessels, whichcausing increased vascular permeability, exudation of inflammatory substancesincreased, leading to vasogenic cerebral edema. The more expression of substance P,the more AQP4expression, which causing increased membrane permeability, leadingto cellular brain edema; There are positive correlations between SP,calcium contentand cell apoptosis of damaged cortex cell. There are positive correlations between theratio of hypodiploid (Hd), SP expression and calcium content of cortical brain cells,the higher the ratio the more serious of apoptosis. There are positive correlationsbetween SP, neuron-specific enolase (NSE), the more substance P expression, themore serious inflammatory response, the more nerve cell necrosis and the higher NSEexpression; There are positive correlations between NPY, CGRP, and NF-κB afterTBI. The higher NPY and CGRP expression, the higher the expression of NF-κB,the more severe inflammatory response; There are positive correlations between NF-κB and NSE, AQP4, the calcium ion concentration, hypodiploid (Hd) ratio afterTBI. The higher NF-κB expression, the more severe inflammatory response, NSE(reflecting the nerve cell damage), AQP4(reflecting the cell brain edema), calciumion concentration (reflecting the calcium overload) and the higher the ratio ofhypodiploid (reflecting brain cell apoptosis).(P <0.05).
2. The study results of experimental study about the protective mechanism of NK1receptor antagonist L-703,606to traumatic brain injury, showed that L-703,606intervention group (group L) injury was attenuated significantly compared with TBIintervention group (T group), only mild congestion, a small amount of inflammatorycell infiltration; SP, CGRP, and NF-κB expression of L group and T group in ratcerebral cortical lesions in the brain tissue was increased compared with the controlgroup. L group was significantly reduced compared with T group; SP, CGRP,NF-kappa B, AQP-4and NSE gene expression levels in the brain tissue of L groupand T group in rat cerebral cortical lesions significantly increased than the controlgroup, positive expression of the L group compared with Tgroup was significantlylower; serum hs-CRP and NSE levels of L group and T group was significantly higherthan the control group, and the hs-CRP and NSE of L group were significantly lowerthan that in the T group,(P <0.05)
3. The results of Rho kinase inhibitor and its implication of experimental study ontraumatic brain injury of brain cells diploid ratio show that hypodiploid ratio ofcortical cells in Sham group, TBI group and trauma area of the FSD group arerespectively.1.58±0.35,15.90±3.91and5.35±2.10, the differences among the threegroups was statistically significant (P <0.01), and the brain cells hypodiploid ratio ofFSD group was significantly lower than that of the TBI group. Thus Fasudil canreduce the TBI brain cells apoptosis, and have the cerebral protective effects.
CONCLUSION
1.Neurogenic inflammation and neurogenic mechanisms play an important role intraumatic brain injury;
2.NK1receptor antagonist L-703,606has a protective effect on the brain injury inrats with traumatic brain injury;
3.Rho kinase inhibitors (Fasudil) can reduce the rate of brain cells hypodiploid, andhas a protective effect of TBI in brain tissue;(4) receptor antagonist, such as CGRP antagonists of CGRP8-37, NPY antagonistBIBP3226, fentanyl (pain intervention),PDTC intervention of NF-κB, a small dose of dopamine inhibition APQ4, the expression of bone marrow mesenchymal stemcells (BMSC) with vascular endothelial growth factor (VEGF) or other neurotrophicfactor transplantation in the treatment of TBI, can act as a disincentive to neurogenicinflammation, protect brain cells (see I have published papers). Expect in-depth studyof neurogenic mechanisms of traumatic brain injury, to find more and more efficientdrugs and methods of treatment of TBI, the development of new drugs for themajority of TBI patients to bring the Gospel.
引文
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