摘要
研究背景
神经病理性疼痛是神经系统原发性损伤或功能异常所导致的痛觉异常或痛觉过敏。创伤、缺血性损伤、感染或炎症、肿瘤、药物和压迫等原因均可以引起神经病理性疼痛。非甾体类抗炎药和阿片类镇痛药以及三环类抗抑郁药不仅治疗效果不理想，而且副作用很大。因此寻找一种安全有效、作用持久的镇痛方法已成为神经病理性疼痛研究的重要方向。神经病理性疼痛的受体机制研究表明，神经元和神经胶质细胞膜上的NMDA受体(N-methyl-D-aspartate receptor，NMDAR)表达上调或过度激活可引起中枢敏感化，在神经病理性痛的产生如自发性痛，触诱发痛及痛觉过敏中起重要作用。NMDA受体的2B亚基(NR2B)在体内呈选择性分布，主要位于脊髓背角、前脑等与痛觉传递有关的区域，提示NR2B可能是一个潜在的镇痛治疗靶点。据此，我们将NR2B基因插入腺病毒，成功构建了NR2B重组腺病毒镇痛疫苗(recombinantadenovirus serotype 5 mediated NR2B gene transfer，rAd5/NR2B)，并在神经病理性痛模型大鼠体内已经被证实能够表达NR2B抗原以诱导机体产生NR2B特异性抗体，并与神经元或胶质细胞膜上的NR2B结合，下调NR2B的表达或阻断NR2B的激活，在神经病理性疼痛模型中发挥良好的镇痛作用。
然而，NMDA不仅参与了疼痛病理过程，而且广泛参与机体的多种生理活动，如学习记忆、突触可塑性等，并在其中起着重要的作用。因此利用NMDA受体拮抗剂治疗疼痛或多或少都会引起其他生理状态的改变。研究表明，氯胺酮在产生镇痛作用的同时对大鼠的空间学习与记忆能力产生了明显的损害，神经元也发生空泡样改变。同样，应用NMDA受体拮抗剂MK801后大鼠的空间学习和记忆功能下降，神经发育受到影响。基于此，我们拟研究rAd5/NR2B镇痛疫苗在治疗大鼠神经病理性疼痛同时，对部分生理功能的影响，以评价其安全性。
本研究通过四个方面的观察，评价rAd5/NR2B镇痛疫苗应用于大鼠疼痛治疗的安全性。
1．rAd5/NR2B镇痛疫苗对谷氨酸诱导的离体海马神经元凋亡的影响：利用rAd5/NR2B镇痛疫苗免疫大鼠，提取免疫血清，并在体外培养大鼠原代海马神经元，加入免疫血清，观察神经元胞内钙及其凋亡的变化；
2．rAd5/NR2B镇痛疫苗对大鼠体内神经元凋亡的影响：大鼠经rAd5/NR2B镇痛疫苗免疫后，通过免疫组织化学染色和Western blot方法分别检测大鼠脊髓和海马组织中活化Caspase-3蛋白的表达以评价神经元的凋亡情况；
3．rAd5/NR2B镇痛疫苗对大鼠认知功能的影响：利用rAd5/NR2B镇痛疫苗免疫大鼠，Morris水迷宫实验观察大鼠空间学习与记忆功能的改变，并检测大鼠海马组织中NR2B蛋白的表达水平；
4．rAd5/NR2B镇痛疫苗影响大鼠认知功能的电生理学和分子学机制：使用电生理学检测方法观察rAd5/NR2B镇痛疫苗免疫大鼠海马组织长时程增强(Long-termpotentiation，LTP)的诱发和维持，并检测大鼠海马组织中磷酸化tau蛋白和NR2B蛋白表达水平的变化。
研究方法与结果
1．rAd5/NR2B镇痛疫苗对谷氨酸诱导的离体海马神经元凋亡的影响
方法：选取雌性SD大鼠(n=6)，通过灌胃的方法给予rAd5/NR2B镇痛疫苗(1×10~8PFU)免疫大鼠，2周后以相同的剂量加强免疫一次。初次免疫后4周，断尾法取静脉血，制备免疫血清，并通过ELISA检测血清中NR2B特异性抗体的滴度，同时取正常大鼠静脉血制备正常大鼠血清。取24h内新生SD大鼠，分离海马组织，体外培养原代神经元。培养1周后利用Fluo-4/AM标记原代神经元胞内钙，在激光扫描共聚焦显微镜下观察免疫血清对原代神经元胞内钙浓度的影响。神经元培养1周后分为四组，对照组(Naive组)、谷氨酸组(Glu组)，谷氨酸加免疫血清组(Glu+rAd5/NR2B组)、谷氨酸加正常血清组(Glu+Normal组)。四组神经元培养基中分别加入PBS、谷氨酸、免疫血清加谷氨酸、正常血清加谷氨酸，培养3d，Annexin V-PI双标后通过流式细胞仪检测各组中神经元的凋亡，并利用免疫组织化学染色的方法观察活化Caspase-3的表达水平。
结果：①大鼠经rAd5/NR2B镇痛疫苗免疫4周后，能在血清中发现较高滴度的NR2B特异性抗体(12．7±2．8μg/ml)。Fluo-4/AM标记的原代神经元经激光扫描共聚焦显微镜观察发现，经谷氨酸刺激后神经元胞内钙浓度明显升高，rAd5/NR2B镇痛疫苗免疫血清能逆转谷氨酸的这种升高作用；而且，经免疫血清处理后的神经元在谷氨酸的作用下，胞内钙浓度变化不大；但是，正常大鼠血清却对神经元胞内钙浓度没有影响。
②Annexin V-PI双标流式细胞仪检测结果表明：与Glu组相比，加入正常血清和免疫血清后神经元凋亡率均增高(P＜0．05)；但正常血清组神经元凋亡率增高更加明显(P＜0．05)。活化Caspase-3表达水平的检测结果显示：与Glu组相比，Glu+rAd5/NR2B组和Glu+Normal组中活化caspase-3阳性神经元数目增加(P＜0．05)；与Glu+Normal组比较，Glu+rAd5/NR2B组中活化caspase-3减少(P＜0．05)。2．rAd5/NR2B镇痛疫苗对大鼠体内神经元凋亡的影响
方法：实验分为6组(n=10)：空白对照组(Naive组)、单纯rAdS/NR2B干预组(rAd5/NR2B组)、假手术组(Sham组)、SNI模型组(SNI组)、MK801处理的SNI模型组(SNI+MK801组)和rAd5/NR2B处理的SNI模型组(SNI+rAd5/NR2B组)。其中，Naive组不做任何处理；rAd5/NR2B组通过灌胃的方法注射rAd5/NR2B镇痛疫苗(1×10~8PFU)，2周后重复注射；SNI组和SNI+MK801组先构建SNI慢性神经病理性疼痛模型，1周后再分别隔天腹腔注射生理盐水和MK801(1mg/kg)；SNI+rAd5/NR2B组先通过灌胃的方法注射rAd5/NR2B镇痛疫苗(1×10~8 PFU)，2周后重复注射，再1周后构建SNI疼痛模型；Sham组只暴露坐骨神经的分支，不结扎。SNI模型构建后检测大鼠的机械缩爪阈值(Mechanical Withdrawal Threshold，MWT)和热痛缩腿时间(Thermal Withdrawal Duration，)，评价rAd5/NR2B的镇痛效果。
取大鼠海马和脊髓组织，通过免疫组织化学染色和Western blot的方法分别检测大鼠脊髓和海马活化Caspase-3蛋白的表达水平。
结果：SNI模型建立前各组大鼠间MWT和TWD比较差异无统计学意义(P＞0．05)。SNI模型建立后2周，与SNI组相比，SNI+rAd5/NR2B组和SNI+MK801组大鼠MWT增高，TWD缩短(P＜0．05)，这种差异可持续至SNI术后42天。SNI模型建立后3周，SNI+MK801组大鼠MWT较SNI+rAd5/NR2B组增高，TWD缩短(P＜0．05)。
脊髓切片经免疫组化染色后显示，与Naive组相比，rAd5/NR2B组、Sham组、SNI组和SNI+rAd5/NR2B组中脊髓和海马组织中活化Caspase-3阳性细胞数目变化差异无统计学意义(P＞0．05)，而SNI+MK801组中活化Caspase-3阳性细胞数目增加(P＜0．05)。Western blot检测结果显示：与SNI组相比，SNI+rAd5/NR2B组海马组织中活化Caspase-3的表达水平无明显变化(P＞0．05)，但SNI+MK801组中Caspase-3的表达水平升高(P＜0．05)。
3．rAd5/NR2B镇痛疫苗对大鼠认知功能的影响
方法：取雌性SD大鼠(体重200g左右)60只，分组与处理同实验第二部分。SNI模型构建后1周所有大鼠一起进行Morris水迷宫检测。第1～5天进行定位导航实验。记录大鼠自入水到找到平台后四肢爬上平台时所需的时间，即潜伏期(若在规定时间内未找到平台，按60s计算)，同时记录寻找平台的游泳速度。第6天入水点改为平台所在象限的毗邻左侧象限，每只大鼠进行2次平台实验，记录大鼠的潜伏期和游泳速度。之后测定大鼠的空间探索能力，撤去水中的平台，记录60s内大鼠穿越平台的次数。水迷宫实验结束后，断头取脑，利用免疫组织化学染色和Western blot的方法检测大鼠海马组织中NR2B的表达水平。
结果：①Morris水迷宫实验中，与Naive组相比，rAd5/NR2B组和Sham组中大鼠的潜伏期和穿越平台次数无明显变化(P＞0．05)，SNI组、SNI+rAd5/NR2B组和SNI+MK801组中大鼠的潜伏期延长，穿越次数减少(P＜0．05)中。与SNI组相比，SNI+rAd5/NR2B组的潜伏期没有增加，穿越次数也没有缩短(P＞0．05)，而利用MK801镇痛后大鼠的潜伏期延长，穿越次数减少(P＜0．05)。6组大鼠的游泳平均速度比较差异无统计学意义(P＞0．05)。
②免疫组织化学染色和Western blot检测发现：与Naive组相比，rAd5/NR2B组、Sham组和SNI+rAd5/NR2B组中大鼠海马组织中NR2B蛋白的表达水平无明显变化(P＞0．05)，而SNI组中NR2B表达水平增高(P＜0．05)，SNI+MK801组中表达水平下降(P＜0．05)。与SNI组相比，SNI+rAd5/NR2B组中NR2B表达水平下降(P＜0．05)，而SNI+MK801组下降更明显(P＜0．05)。
4．rAd5/NR2B镇痛疫苗影响大鼠认知功能的电生理学和分子学机制
方法：取雌性SD大鼠(体重200g左右)36只，分为6组(n=6)：Naive组、rAd5/NR2B组、Sham组、SNI组、SNI+MK801组和SNI+rAd5/NR2B组。每组的处理同实验第二部分。SNI模型构建后2周，断头取脑，制备海马脑片，记录大鼠海马CA1区兴奋性突触后电位(Field Excitatory Postsynaptic Potential，fEPSP)的变化。利用诱发最大幅度fEPSP 30～40％的刺激电压作为单刺激，并稳定刺激20min以上，记录fEPSP曲线，作为fEPSP的基础值。给予强直刺激(High-frequency stimulation，HFS)后记录fEPSP幅度和斜率的变化，如增加20％并持续30min以上视为LTP诱发成功，同时比较各组大鼠的LTP幅度，依此评价rAd5/NR2B镇痛疫苗对大鼠LTP的影响。电生理实验结束后，提取海马组织中总蛋白，利用Western-blot的方法检测磷酸化Tau蛋白和NR2B蛋白的表达水平。
结果：①在电生理实验中，与Naive组相比，rAd5/NR2B组、Sham组和SNI组中大鼠海马CA1区fEPSP和LTP幅度均无明显变化(P＞0．05)，但SNI+rAd5/NR2B组中海马脑片经HFS处理后虽成功诱发出LTP，但LTP幅度下降(P＜0．05)，SNI+MK801组中，海马CA1区LTP幅度明显下降，不仅低于SNI+rAd5/NR2B组(P＜0．05)，甚至低于20％，视之为LTP诱发不成功。
②海马磷酸化Tau蛋白Western blot检测发现：6组中大鼠海马组织中磷酸化tau蛋白的表达水平差异无统计学意义(P＞0．05)。
③海马NR2B蛋白Westernblot检测发现：与Naive组相比，rAd5/NR2B组、Sham组、SNI+rAd5/NR2B组和SNI+MK801组中大鼠海马组织中NR2B蛋白的表达水平无明显变化(P＞0．05)，而SNI组中NR2B表达水平增高(P＜0．05)，SNI+MK801组中表达水平下降(P＜0．05)。与SNI组相比，SNI+rAd5/NR2B组中NR2B表达水平下降(P＜0．05)，而SNI+MK801组下降更明显(P＜0．05)。
5、统计学处理
采用SPSS13．0统计软件进行处理。计量资料以均数±标准差((?)±S)表示，组内比较采用重复变量数据方差分析，组间比较采用单因素方差分析，P＜0．05为差异有统计学意义。
研究总结
一主要研究结果
1．大鼠经rAd5/NR2B镇痛疫苗免疫4周后，产生较高滴度的NR2B特异性抗体，后者能够抑制或逆转谷氨酸造成的钙超载。rAd5/NR2B镇痛疫苗免疫血清与正常血清相比，能够减少谷氨酸诱导的原代神经元凋亡。
2．在体实验中，rAd5/NR2B镇痛疫苗不会诱发在体脊髓和海马神经元的凋亡，但MK801却增加了大鼠脊髓和海马神经元的凋亡。
3．通过Morris水迷宫实验发现，经rAdS/NR2B镇痛疫苗免疫后大鼠的潜伏期、穿越平台次数没有显著变化，MK801则明显延长了大鼠的潜伏期，穿越平台的次数减少。rAd5/NR2B镇痛疫苗免疫后的SNI大鼠NR2B表达水平下降。
4．正常大鼠经rAd5/NR2B镇痛疫苗免疫后海马CA1区LTP幅度无明显变化。但疼痛模型大鼠经免疫后，fEPSP斜率以及LTP幅度降低，MK801则显著降低fEPSP斜率，LTP诱发不成功。rAd5/NR2B镇痛疫苗对tau蛋白的磷酸化水平无明显影响，而注射MK801后Tau蛋白的磷酸化水平增高。rAd5/NR2B镇痛疫苗免疫后的SNI大鼠NR2B表达水平下降。
二研究结论
1．大鼠经rAd5/NR2B镇痛疫苗免疫后产生含较高滴度NR2B特异性抗体的免疫血清，该免疫血清能抑制或逆转谷氨酸造成的钙超载，并减少谷氨酸诱导的原代神经元凋亡，对神经元具有一定的保护作用。
2．rAd5/NR2B镇痛疫苗不会增加体内海马和脊髓神经元的凋亡。
3．rAd5/NR2B镇痛疫苗对大鼠的空间学习与记忆功能没有明显的损害。
4．rAd5/NR2B镇痛疫苗一定程度上降低了大鼠LTP的诱导和维持，但对tau蛋白的磷酸化水平无明显影响。
本研究中，我们发现rAd5/NR2B镇痛疫苗能够对谷氨酸诱导的体外神经元凋亡产生一定的保护作用，同时对在体神经元的凋亡、大鼠的认知功能以及tau蛋白的磷酸化没有明显影响；电生理和分子学机制研究显示，rAd5/NR2B镇痛疫苗削弱了大鼠LTP的诱导和维持，而且降低了NR2B的表达水平。这表明经rAd5/NR2B镇痛疫苗免疫所产生的NR2B特异性抗体，能够拮抗NR2B并下调NR2B蛋白表达水平，以发挥镇痛作用，并影响到神经元的突触可塑性，但其幅度有限，不足以诱发在体神经元的凋亡，也不足以影响到大鼠的认知功能。这些结果证明rAd5/NR2B镇痛疫苗应用大鼠镇痛是安全的，相比其他NMDA受体拮抗剂有明显的优势。三展望
rAd5/NR2B镇痛疫苗应用于神经病理性痛大鼠有良好的镇痛效果，是一种新颖、有效的镇痛方法。同时，rAd5/NR2B对大鼠的生理功能没有明显影响，安全性较高，具有极大的临床推广价值。但是，我们的观察仅仅限于动物实验水平，且观察的时间有限，因此仍需要更客观更全面的观察和评价。
Background
Neuropathic pain is defined as pain initiated or caused by a primary lesion ordysfunction in the nervous system.The reasons responsible for neuropathic pain include trauma,ischaemic injury,infection/inflammation,cancer,drugs and compression etc.Patients withneuropathic pain do not respond to non-steroidal anti-inflammatory drugs and haveresistance or insensitivity to opiates.The current pharmacological mainstays of clinicalmanagement are tricyclic anti-depressants and certain anticonvulsants,but these onlyachieve clinical significant pain relief in less than 50% of patients and are associated withsuboptimal side effect profiles.Therefore,an important issue of pain research is to find anovel method of analgesia that is effective and safe.Peripheral nerve injury may result in apersistent pain due to abnormal activity in peripheral afferents and a sustained input to thespinal cord that leads to alterations in the activity and excitability of spinal cord neurons.These spinal changes involve central sensitization of dorsal horn neurons.N-methyl-D-aspartate (NMDA) receptor subunit 2B(NR2B) plays an important role in thefacilitation and maintenance of central sensitization.NMDA receptor antagonists such asketamine,dextromethorphan and MK-801 have been reported to produce symptomaticrelief in a number of neuropathies including postherpetic neuralgia,central pain caused byspinal cord injury and phantom limb pain.So,we constructed recombinant adenovirusserotype 5-mediated NR2B(rAd5/NR2B) by inserting NR2B gene into the adenovirus serotype 5.rAd5/NR2B vaccine targeting NR2B epitope could induce the fluidimmunological response in vivo,the NR2B-specific antibodies were produed,whichpenetrated the blood-brain barrier at the time of pain,reacted with NR2B protein todownregulate the activity of NR2B and fight against neuropathic pain.
Over the last several years,an increasing number of reports have demonstrated theimportance of the NR2B subunit in determining the pharmacological and functionalproperties of the NMDA receptor.Consequently,NR2B has been implicated in modulatingfunctions such as learning,memory processing,and synaptic plasticity,as well as beinginvolved in a number of human disorders which include pain perception,hypoxia/ischemia,parkinson's disease,Alzheimer's disease,seizure disorder,diabetes,schizophrenia et al.So,the agents such as ketamine and MK801 induce unacceptable side effects at analgesicdoses including hallucinations,dysphoria and disturbances of cognitive and motor function,which exclude their widespread use.On the base of these researches,we detected whetherrAd5/NR2B targetting at NR2B produce any side effects accompanying the analgesic effect,to consider the safety of rAd5/NR2B.
So our objective is to evaluate the safety of rAd5/NR2B in rat base on following 4parts.
1.The influence of rAd5/NR2B on the primary neurons apoptosis induced by glutamicacid in vitro:Immuned serum was obtained from the rats immuned with rAd5/NR2B byintragastric gavage,then the primary neurons were separated and cultured from neonatalrats' hippocampus.After administrated with immuned serum,the intracellular calciumconcentration and apoptosis of primary neurons were detected.
2.The influence of rAd5/NR2B on the neurons apoptosis in vivo:after immuned withrAd5/NR2B by intragastric gavage,the rats were stained by immunohistochemistry toexplore the apoptosis of neurons.
3.The effect of rAd5/NR2B on the congnitive function of rats:the congnitive functionof rats immuned with rAd5/NR2B by intragastric gavage was detected through Morriswater maze testing,and the expression of NR2B was detected by immunohistochemistryand Western blot.
4.The electrophysiological and molecular mechanism of rAd5/NR2B influencing on the cognitive function:the hippocampus sections were separated from rAd5/NR2Bimmuned rats,and long term potentiation(LTP) was observed by recording fEPSP inhippocampus sections,then the expression of phosphorylated tau protein was detected byWestern blot.
Methods and Results
1.The influence of rAd5/NR2B on the primary neurons apoptosisinduced by glutamic acid in vitro
Methods Female SD rats (n=6) were choosed and immuned with rAd5/NR2B byintragastric gavage.Two weeks after the first gavage,we administrated this vaccine again.Two weeks later,NR2B-specific IgG titers in sera obtained from immuned rats weredetermined by ELISA,the normal serum was prepared and determined at the same time.Primary cultured neurons were separated from the hippocampus of neonatal rats in 24h,andcultured for 1 week,then neurons were stained with Fluo-4/AM and observed with confocallaser scaning to determine the effects of immuned serum,normal serum and glutamic acidon the intracellular calcium concentration of neurons.Following this,primary neurons wererandomly divided into 4 groups:Naive group,glutamic acid group(Glu group),immunedserum and glutamic acid group(Glu+rAd5/NR2B serum group),normal serum and glutamicacid group(Glu+Normal serum group),which were respectively administrated with saline,glutamic acid,immuned serum and glutamic acid,normal serum and glutamic acid.Aftercultured for more 3 days,the apoptosis percentage of primary neurons stained by AnnexinV-PI were explored with flow cytometer,and the neurons were stained byimrnunohistochemistry to detect the expression level of Caspase-3,then the apoptosis ofneurons was determined.
Results①Following the primary immunization and the booster immunization,NR2Bspecific antibodies in serum was 12.7+2.8μg/ml.In contrast,NR2B specific antibodieswere not detected in normal serum from the norma rats.The intracellular calciumconcentration of neurons was increased after the stimulation of glutamic acid,thisincreasing effect of glutamic acid was reversed by rAd5/NR2B immuned serum,furthermore,rAd5/NR2B immuned serum decreased the concentration of intracellular calcium,and the concentration didn't increase obviously after injecting glutamic acid.But,normal serum hadn't this effect.
②The results of flow cytometer showed:Compared with Glu group,the apoptosispercentage of primary neurons was increased in Glu+rAd5/NR2B group and Glu+Normalgroup(P＜0.05);Compared with Glu+Normal group,the apoptosis percentage wasdecreased in Glu+rAd5/NR2B group(P＜0.05).In the research about effect of rAd5/NR2Bimmuned serum on the Caspase-3 experssion,compared with Naive group,the expressionlevels were upregulated in the other 3 groups (P＜0.05).Compared with Glu group,expression levels of Caspase-3 were upregulated in Glu+rAd5/NR2B group andGlu+Normal group (P＜0.05).Compared with Glu+Normal group,expression level ofcaspase-3 in Immuned serum group was downregulated in Glu+rAd5/NR2B group(P＜0.05).
2.The influence of rAd5/NR2B on the neurons apoptosis in vivo
Methods 60 SD rats weighing about 200g were choosed and randomly divided into 6groups(n=10):Naive group,rAd5/NR2B group,Sham group,SNI model group(SNI group),SNI model treated with MK801 group(SNI+MK801 group),and SNI model treated withrAd5/NR2B group(SNI+rAd5/NR2B group).The rats were received saline in Naive groupand rAd5/NR2B in rAd5/NR2B group by intragastric gavage,the administration wererepeated 2 weeks later.After SNI model surgery,the rats were respectively injected withsaline and MK801 in SNI group and SNI+MK801 group.Rats of SNI+rAd5/NR2B groupwere administrated with rAd5/NR2B by intragastric gavage at first,then received SNImodel surgery.The sciatic nerves were separated and exposured,not ligated in Sham group.The mechanical withdrawal threshold(MWT) and thermal withdrawal duration(TWD) ofrats were detected after surgery.Two weeks after SNI model surgery,the spinal cords andhippocampuses of rats were obtained,then the expression level of caspase-3 and NR2Bproteins were detected by immunohistochemistry and Western blot to evaluate the apoptosisof neurons in vivo.
Results Compared with SNI group,the MWT were increased,and the TWD wereshortened in SNI+rAd5/NR2B group.There was no difference on the the apoptosis of neurons in vivo between Naive group and rAd5/NR2B group (P＞0.05).Compared withSham group,apoptosis of neurons didn't increase obviously in SNI group andSNI+rAd5/NR2B group (P＞0.05),but increased in SNI+MK801 group (P＜0.05).Therewas no difference on the expression levels of NR2B in rats' spinal cord and hippocampusbetween Naive group and rAd5/NR2B group(P＞0.05).Compared with SNI group,theexpression of NR2B was decreased in SNI+rAd5/NR2B group and SNI+MK801 group(P＜0.05).
3.The effect of rAd5/NR2B on the congnitive function of rats
Methods 60 SD rats weighing about 200g were choosed,divided and administrated asthe part 2.One week after SNI model surgery,all rats were detected with Morris watermaze testing.Training trials were given on the first 5 consecutive days,and rats underwentthree training trials each day.In eaeh training trial,if the rat found the platform,it wasallowed to remain there for 30s.If the rat was unable to find the platform within 60s,after itwas made to stay there for 30s.The time from diving to finding the hiden platform (escapetime) was recorded (escape time of the rat which was unable to find the platform wasrecorded 60s),swimming path length and the swimming velocity were recorded.On thenext day,each rat was exposed to an additive training trails test after the starting point waschanged to the adjacent left quadrant of the platform.The latency and distance werereeorded in the same way as the previous five training trials.Furthermore,rats performed aspatial probe test trial with the same starting point as in the five training trails.This trialconsisted of removing the platform from the pool and allowing the rat to swim for 60s insearch of it.The time spent in each of the four equal quadrants,and the times of throughingplatform was reeorded.After Morris water maze testing,the hippocampus segments of ratswere harvested,and the expression levels of NR2B were detected usingimmunohistochemistry and Western blot.
Results There was no difference on the escape times between Naive group andrAd5/NR2B group(P＞0.05).Compared with Sham group,the escape time was prolongedin SNI group,SNI+rAd5/NR2B group and SNI+MK801 group(P＜0.05).Compared withSNI group,the escape time didn't change in SNI+rAd5/NR2B group(P＞0.05),however,it was prolonged in SNI+MK801 group(P＜0.05).There was no difference on the swimmngpath length and velocity among these rats of 6 groups.
The expression level of NR2B in rats hippocampus didn't change between Naive groupand rAd5/NR2B group(P＞0.05).Compared with SNI group,the expression of NR2B wasdecreased in SNI+rAd5/NR2B group and SNI+MK801 group.
4.The electrophysiological and molecular mechanism of rAd5/NR2Binfluencing on the cognitive function
Methods 60 SD rats weighing about 200g were choosed,divided and administrated asthe part 2.Two weeks after SNI model surgery,the rats' hippocampuses were seperated andslicinged into 400μm thickness sections.Incubated in ACSF,the hippocampal sections weredetected with electrophysiological method.In all experiments,base line synaptictransmission was monitored for 20 min before high frequency stimulation(HFS).Thestimulus intensity was set to evoke 50% of the maximal amplitude of PS.LTP was inducedusing HFS,which included 300 pluses at 100Hz,on Schaffer-collateral pathway.Theamplitude and slope of fEPSP was recorded from the CA1 region of rat hippocampal slices.If the fEPSP was increased more than 20% after HFS,and maintained more than 30min,itwas considered as a successful LTP induction.The influence of rAd5/NR2B on LTP wasevaluated by comparing the amplitude,slope of fEPSP and the achievement ratio of LTPinduction.Following electrophysiological research,the protein were extracted fromhippocampus,and the expression levels of phosphorylated tau protein and NR2B wereexplored using Western bolt.
Results①Compared with Naive group,the slopes of fEPSP didn't change inrAd5/NR2B group,Sham group and SNI group(P＞0.05),but they were decreased inSNI+rAd5/NR2B group and SNI+MK801 group(P＜0.05).Compared with SNI group,theslopes of fEPSP were decreased in SNI+rAd5/NR2B group and SNI+MK801 group(P＜0.05).Compared with SNI group,they were also decreased in SNI+rAd5/NR2B groupand SNI+MK801 group(P＜0.05).
②There was no difference on the expression levels of phosphorylated tau proteinamong these 6 groups(P＞0.05).Compared with SNI group,the expression level of NR2B was down-regulated in SNI+rAd5/NR2B group(P＜0.05),and the down-regulation wasmore obvious in SNI+MK801 group(P＜0.05.
5.Statistical Analysis
All of the analyses were performed by SPSS 13.0 software package.Measurement datawere presented as means+standard deviation (SD).The differences between multipleindividual means were analyzed by one-way ANOVA.P＜0.05 was considered statisticallysignificant in all tests.
Conclusion
1.rAd5/NR2B vaccine could induce the humoral immunological response andproduction of NR2B-specific antibody in sera.NR2B-specific antibody could react withNR2B subunit and inhibit or reverse the calcium overload induced by glutamic acid,furthermore,rAd5/NR2B immuned serum decreased the apoptosis of primary neuronsinduced by glutamic acid,provided partial protection for primary neurons.
2.rAd5/NR2B vaccine couldn't influence the apoptosis of neurons in vivo,however,MK801 obviously increased the apoptosis of neurons.
3.The administration of rAd5/NR2B didn't prolong the escape time of rats in Morriswater maze testing,and the frequency of throughing platform didn't increase.But theescape time was apparently extended after injecting of MK801,and the times of throughingplatform increased.The expression level of NR2B was down-regulated after immuned withrAd5/NR2B.
4.After the rats were immuned with rAd5/NR2B vaccine,the LTP was inductedsuccessfully,but the amplitude and slope of fEPSP were lowered.This phenomenon wasmore obvious after administration of MK801.rAd5/NR2B vaccine didn't influence on theexpression level of phosphorylated tau protein in hippocampus,but decreased theexpression of NR2B simultaneously.
In this study,the safety of rAd5/NR2B vaccine was explored in rats.We foundrAd5/NR2B vaccine could induce the humoral immunological response and production ofNR2B-specific antibody in serum,which could react with NR2B subunit and inhibit orreverse the calcium overload induced by glutamic acid.rAd5/NR2B immuned serum coulddecrease the apoptosis of primary neurons induced by glutamic acid compared with thenormal serum of rats,and provide partial protection for primary neurons,rAd5/NR2Bvaccine couldn't influence the apoptosis of neurons in vivo,however,MK801 obviouslyincreased the apoptosis of neurons.In Morris water maze testing,the administration ofrAd5/NR2B didn't impair the spatial learning and memory of rats.Furthermore,rAd5/NR2B vaccine was detected to inhibit the induction and maintance of LTP in rats,butdidn't upregulate the phosphorylation level of tau protein in hippocampus.The expressionlevels of NP2B were detected to explore this phenomenon.We found,though theexpression level of NR2B wasn't affected in normal rats immuned with rAd5/NR2B,theexpression were down-regulated after in SNI model rats immuned with rAd5/NR2B.Theseresults suggested:the antagonism and down-regulation of NR2B-specific antibody werepartial effective,it could lead to analgesic effects,but didn't obviously induced theapoptosis in vivo and impair the cognitive function of rats.So we concluded theadministration of of rAd5/NR2B vaccine was partly safe in the time of analgesia,whichproduced less side effects than MK801.
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