【作者】 刘俊丽；

【导师】 王正洪； 李敏；

【作者基本信息】 第四军医大学 ， 社会医学与卫生事业管理， 2009， 博士

【摘要】 随着突发性灾难事件的增多,创伤后应激障碍(post-traumatic stress disorder,PTSD)成为全球关注的热点。PTSD的本质是一系列与创伤事件相关的记忆(条件性恐惧),创伤性记忆一旦形成,很难自然消失,严重影响日常生活。暴露疗法的原理是创伤记忆的消退,但其对PTSD的疗效难以维持,表现为已消退的恐惧记忆又很快再现。提示消退保持(extinction retention)障碍可能是创伤后应激障碍发生和治疗困难的关键。既往实验表明内侧前额叶皮层(medial prefrontal cortex,mPFC)边缘下区(infralimbic area,IL)与PTSD发生、发展和治疗结局密切相关,然而,其在条件性恐惧消退保持中的作用和机制尚不清楚。我们在以往学者研究的基础上,综合行为药理学、形态学以及Western Blot方法探索条件性恐惧记忆消退的最佳“时间窗”,探讨IL区在条件性恐惧记忆消退保持中的作用及其机制,为提高并巩固PTSD治疗效果提供实验依据。为达成这一目的,设计了以下3个实验。实验一不同消退方式后条件性恐惧大鼠消退保持的动态观察目的:探讨条件性恐惧大鼠经历不同消退训练后,消退记忆保持水平的动态变化。方法:条件刺激(声音)与非条件刺激(不可逃避的足底电击)配对呈现建立条件性恐惧;随后重复呈现单独的条件刺激用于消退训练。48只SD雄性大鼠随机分为3组:对照组(自然消退,Cont组),大鼠建立声音提示的条件性恐惧而不进行消退训练;立即消退组(Imme EXT),建立条件性恐惧后15min进行消退训练;延迟消退组(EXT组),建立条件性恐惧后24h进行消退训练。以上三组大鼠一半于建模后第1、3、7、20天测试僵立行为(大鼠连续10min内僵立时间百分比);另一半分别于消退后第1、3、7、20天通过高架十字迷宫测试焦虑行为。结果:①Cont,Imme EXT和EXT组僵立行为基础值的差异无统计学意义(P>0.05)。EXT组的消退效果好于Imme EXT和Cont组(P<0.001),其远期消退效果持续稳定;EXT组大鼠消退后第1、3天的僵立行为显著下降(P<0.01),第7天已接近正常水平。Imme EXT组和Cont组间消退效果的差异无统计学意义(P >0.05)。各组大鼠在消退后7-20天僵立行为的变化无统计学意义(P>0.05)。②Cont,Imme EXT和EXT组的开放臂进入次数百分比(OA time%)和开放臂停留时间百分比(OA time%)基础值差异无统计学意义(P >0.05);在消退后不同时间点,OA entries%和OA time%两个指标的差异均无统计学意义(P>0.05)。总体来说,各组动物随实验天数延长其焦虑水平逐渐降低,但各组之间在同一时间点上的焦虑水平的改变无统计学意义(P >0.05)。结论:不同的消退训练方式对条件性恐惧大鼠的远期消退效果不同,延迟消退训练方式效果最好。同时,由于不同处理组大鼠的焦虑水平类似,排除了立即消退组僵立评分值较高是焦虑水平升高所致。实验二条件性恐惧消退保持过程中IL区磷酸化CREB的动态变化目的:探讨条件性恐惧大鼠经历不同消退训练后,消退记忆保持过程中,IL区pCREB的变化。方法:实验一观察到条件性恐惧延迟消退训练效果好于立即消退训练,且消退后1-3天的消退效果最为明显。本实验采用声音-足底电击配对(条件恐惧)或非配对(非条件恐惧)刺激后,使用延迟消退方式训练大鼠,比较非条件恐惧和条件恐惧延迟消退后大鼠僵立行为变化和IL区pCREB的表达不同,同时观察条件恐惧延迟消退组在消退后1、3、7天IL区信号分子pCREB的动态变化。将SD雄性成年大鼠随机分为5组:空白组(Naive组,无任何处理),假训练A组(Sham A,声音和足底电刺激不配对呈现),假训练B组(Sham B,仅有足底电刺激),条件性恐惧组(Cont组,无消退训练),延迟消退组(EXT组)。除Cont组外,其余各组均采用与实验一相同的延迟消退训练方式,在消退后24h取材; EXT组在延迟消退后不同时间点(消退后1、3、7天)取材;各组均采用免疫荧光染色及Western Blot定位和半定量检测目标蛋白pCREB的表达水平(3-4只大鼠/时间点)。免疫荧光单一标记各组IL区pCREB以说明非条件恐惧对IL区细胞pCREB表达的影响。免疫荧光三重标记分别指:pCREB显示细胞核(绿),Neu-N显示神经元胞体和胞核(红),hoechst染色细胞核(蓝)。pCREB阳性细胞同时为Neu-N阳性、hoechst阳性的进行记数。以此说明延迟消退后IL区神经元的pCREB表达水平。结果:①EXT组消退后1天僵立时间百分比(48.72±10.27)显著低于Sham A(79.65±6.88)、Sham B(77.53±6.03)和Cont组(61.12±10.22)(P<0.001),而明显高于Naive组(55.77±10.40)(P<0.01)。EXT组消退后1天(823.70±67.31)IL区pCREB阳性细胞表达显著高于Sham A(442.20±53.19),Sham B(453.20±49.47)和Cont组(564.20±55.37) (P<0.001),而低于Naive组(994.00±56.62)pCREB表达(P<0.01)。Sham A组和Sham B组的僵立时间百分比和IL区pCREB阳性细胞表达与Cont组相比差异无统计学意义(P>0.05)。②免疫荧光三重标记IL区pCREB阳性神经元表达数:在延迟消退后1天(45±5.70)、3天(62±8.39)、7天(66.2±7.59)大鼠IL区pCREB阳性表达数呈逐渐增加趋势,其中在消退后第1、3天增加迅速(P<0.001),消退后3、7天的变化无统计学意义(P>0.05),消退后第7天基本回复到正常水平。条件性恐惧组IL区pCREB阳性表达数(28±2.73)显著低于Naive组(73.2±5.31)和延迟消退组在各时间点的表达数(P<0.001)。③Western Blot结果:将β-actin表达强度作为内参,各组大鼠IL区pCREB目标蛋白表达变化如下:延迟消退组在消退后1天(0.86±0.06)、3天(1.11±0.06)、7天(1.22±0.04)的pCREB蛋白表达量持续增加(P<0.01),消退后1、3天增加尤为突出。条件性恐惧组大鼠pCREB蛋白表达显著减少到(0.54±0.03)(P<0.001)。消退后7天的目标蛋白表达与Naive组(1.39±0.05)的差异无统计学意义(P>0.05)。结论:声音提示的条件性恐惧伴随IL区内pCREB表达水平降低,而僵立行为的改善伴随IL区pCREB表达水平的逐渐增强。延迟消退后第1-3天IL区内pCREB的表达水平显著升高,且这些表达变化主要集中在IL区神经元内。提示pCREB可能参与条件恐惧的消退。实验三NMDA受体在大鼠条件性恐惧消退保持中的作用机制研究目的:观察非竞争性NMDA受体拮抗剂MK-801对条件性恐惧大鼠消退保持及IL区pCREB表达的影响,探讨NMDA受体影响消退保持的可能机制。方法:采用延迟消退训练方法,将雄性成年SD大鼠随机分为4组。训练前腹腔注射MK-801(Pre-MK-801)组:延迟消退训练前40min腹腔注射MK-801(0.3 mg/kg);训练后腹腔注射MK-801(Post-MK-801)组:延迟消退训练后4h腹腔注射MK-801(0.3 mg/kg);延迟消退组(EXT):条件性恐惧建立24h后进行消退训练,给予等量生理盐水;对照组(自然消退,Cont):只建立条件性恐惧,不进行消退训练,给予等量生理盐水。一半大鼠于消退当日(EXT-0 d)、消退后1、3、7(EXT-1、3、7d)测定僵立时间,进行条件性恐惧消退保持研究;另一半大鼠在上述时间点进行高架十字迷宫实验,以评价实验处理后大鼠在消退期间焦虑水平的变化。同样处理的大鼠在相同的时间点被处死,用于免疫荧光、Western Blot研究(3-4只大鼠/时间点)。免疫荧光三重标记分别指:pCREB显示细胞核(绿),Neu-N显示神经元胞体和胞核(红),hoechst染色细胞核(蓝)。pCREB阳性细胞同时为Neu-N阳性、hoechst阳性的进行记数。以此说明延迟消退后IL区pCREB神经元表达的水平。结果:①各组大鼠僵立行为基础值的差异无统计学意义(P>0.05)。Post-MK-801组大鼠在EXT-1、3、7天的僵立行为高于EXT组的相同时间点(P<0.01),但明显低于Pre-MK-801组和对照组(P<0.05)。Pre-MK-801组在EXT-1、3、7天的僵立行为与对照组相同时间点比较无统计学意义(P >0.05)。Cont组在EXT-7d的僵立行为低于EXT-0d(P<0.05)。在消退后各时间点上Pre-MK-801组和Post-MK-801组的僵立行为改变无统计学意义(P>0.05)。②各组大鼠进入开放臂次数百分数(OA entries%)和开放臂停留时间百分数(OA time% )基础值的差异无统计学意义(P>0.05)。在消退后不同时间点,OA entries%和OA time%两个指标的变化无统计学意义(P>0.05)。各组大鼠焦虑水平随实验天数延长有所改善,但各组之间在同一时间点上的焦虑水平的改变无统计学意义(P>0.05)。③免疫荧光三重标记IL区pCREB的神经元数表达: Post-MK-801组(42.80±4.32)IL区pCREB阳性神经元数低于EXT组(48.80±2.39)(P<0.05),但明显高于Pre-MK-801组(34.80±3.11)和对照组(35.60±4.16)(P<0.01)。Pre-MK-801组IL区阳性细胞数与对照组比较无统计学意义(P>0.05)。④Western Blot结果显示:将β-actin表达强度作为内参,不同给药方式大鼠IL区pCREB目标蛋白表达变化如下:Post-MK-801组(0.71±0.06)IL区pCREB阳性带明显低于EXT组(0.87±0.08)(P<0.01),但明显高于Pre-MK-801组(0.49±0.05)和对照组(0.52±0.03)(P<0.01)。Pre-MK-801组IL区pCREB阳性带与对照组比较其差异无统计学意义性(P>0.05)。结论:在消退训练前40min、消退训练后4h腹腔注射MK-801不影响大鼠的焦虑行为,而影响其条件恐惧消退的巩固和保持。消退训练前40min腹腔注射MK-801的消退效果明显差于消退训练后4h给药,这些行为学改变伴随IL区pCREB神经元表达水平的相应改变。大鼠IL区的pCREB可能参与了条件性恐惧记忆的建立和消退过程,MK-801可能通过拮抗NMDA受体从而减弱大鼠条件性恐惧记忆的消退保持过程。这些改变主要局限于该区的神经元,提示MK-801通过拮抗NMDA受体活性,进而减弱条件性恐惧记忆消退保持过程。更多还原

【Abstract】 With the accidental disaster on a rise, the post traumatic stress disorder (PTSD) has become a worldwide focus. Actually, PTSD is a series of disorders caused by traumatic-related memories (conditioned fear). The disorder is difficult to vanish naturally once it comes into being, and seriously affects people’s daily life. The exposure therapeutics is based on erasing the conditioned fear and can not maintain the effect in treating PTSD, because the extinct fear recurs in a short time, suggesting that extinction retention disorder may be the key factor to cause PTSD and the difficulty in its consequent therapy. Previous experiments have shown that infralimbic (IL) in medical prefrontal cortex (mPFC) is closely interrelated to the morbility, development and therapy of PTSD, but its mechanism of retention of conditioned fear extinction is not clear.Based on the previous studies, in the current study, the best‘time window’of conditioned fear memory extinction was investigated using behavioral pharmacology, morphological and Western Blot methods to illuminate the IL area mechanism of the retention of conditioned fear memory extinction. experimental proof to improve and consolidate PTSD therapy effect, and researching. To achieve this purpose, the following three experiments were performed. The first experiment Dynamic Observation on the extinction retention after operations with different extinction protocolsObjective: To investigate the dynamic change of memory retention of conditioned fear extinction after different extinction protocols.Methods: The rats’conditioned fear model was established with conditioned stimulus (classical auditory fear conditioning) pairing with unconditioned stimulus (an aversive foot shock). Later the repeated single conditioned stimulus was used for extinction training. 48 male SD rats were randomly divided into three groups: control group (natural extinction, Cont) in which rats were subjected to auditory conditioned fear but no fear extinction conditioning; immediate extinction group (Imme EXT) in which fear extinction started 15min after establishment of conditioned fear; delayed extinction group (EXT) in which fear extinction started 24h after establishment of conditioned fear. The freezing behavior was determined on half of rats in the three groups on 1st, 3rd, 7th, and 20th day after fear extinction. And the anxiety behavior was determined by elevated plus maze (EMP) method on the other rats before experiment and on 1st, 3rd, 7th, and 20th day after fear extinction.Results (1) There were no significant differences in baseline freezing levels among three groups (Cont, Imme EXT, EXT) (P>0.05). Fear extinction caused a significant stable and long-lasting extinction effect in EXT group compared to that in immediate extinction group and control group (P<0.001). Freezing behavior was decreased significantly from 1st to 3rd day after fear extinction in EXT group(P<0.01). Between Imme EXT group and Cont group was no significant difference(P>0.05).There was no significant difference in freezing behavior among the three groups from 7th to 20th day after fear extinction.(2) There was no statistically significant difference in baseline levels of The percentage of open arm entries (OA entries % )and The percentage of the time spent in the open arm entries( OA time % )among three groups (P>0.05). There was no significant difference between OA entries % and OA time% at different time period after fear extinction (P>0.05). Generally, the anxiety behavior level was decreased with the experiment time lasting, but there was no significant difference at the same time point among 3 groups (P>0.05). Conclusion The different extinction protocols cause different long-lasting extinction effects with the best effect in the delayed extinction group. Besides, with the similar anxiety behavior level among 3 groups, it is reasonable to rule out the possibility that the increase in anxiety level causes high level of freezing behavior in Immi EXT group.The second experiment Dynamic Change of pCREB in IL during retention of conditioned fear memory extinctionObjective: To investigate the change of extinction retention and expression of pCREB in rat’s infralimbic area (IL) in different retention period after different extinction protocols.Methods: Experiment 1 showed that delayed extinction of conditioned fear had the significant effect compared with immediate extinction and the effect was obvious during 1st-7th day after fear extinction. The experiment 2 was to find out the dynamic change of pCREB in IL on 1st, 3rd, 7th day after delayed fear extinction for those rats subjected to auditory-foot shock pairing stimulus and unpaired stimulus. Male adult SD rats were randomly divided into 5 groups: natural comparison group (Na?ve, without any disposal), shame A group (Sham A, unpaired auditory and foot shock stimulus), shame B group (Sham B, only by foot shock stimulus), conditioned fear group (natural extinction, Cont)and delayed extinction group (EXT). Except in the conditioned fear group, the delayed extinction method in experiment 1 was employed in the other groups. Twenty-four hours after extinction, samples were collected in each group. Samples were collected in EXT group at different time point after delayed fear extinction (1st, 3rd and 7th day). Immunoflourence staining and Western Blot methods were conducted to locate neurons and determine the expression of pCREB in IL (3-4 rats/time point). The effect of conditioned fear on the expression of pCREB in IL was much more significant than that of unconditioned fear by using single Immunoflourence signal of pCREB in IL. In the triple immunoflourence signal, pCREB indicated cell nucleus (green), Neu-N perikaryons and nucleus (red) and Hoechst nucleus (blue). Cells with pCREB-positive, Neu-N-positive and Hoechst-positive staining were counted and recorded to show the expression of pCREB in IL neurons.Results (1) The percentage of freezing time in EXT group 1d (48.72±10.27)after extinction was significantly lower than with those in Sham A (79.65±6.88), Sham B (77.53±6.03) and Cont group (61.12±10.22) respectively, while it was significantly higher than that in Na?ve group (55.77±10.40)( P<0.01). There was no significant difference among Shame A, Shame B and Cont group(P >0.05).The count of pCREB-positive cells in IL in EXT group 1d (823.70±67.31)after extinction was significantly higher than that in Sham A(442.20±53.19), Sham B (453.20±49.47) and Cont group (564.20±55.37) respectively(P<0.001), while it was significantly lower than that in Na?ve group (994.00±56.62)(P<0.01).(2) The expression of pCREB-positive neurons in IL with triple immunofluerence staining: The expression of pCREB-positive neurons in IL was gradually increased on 1d (45±5.70), 3d (62±8.39) and 7d (66.2±7.59) after delayed extinction, of which the expression was increased dramatically from 1d to 3d (P<0.001), while there was no significant difference between the expressions from 3d to 7d (P>0.05). Seven days after the extinction, the expression recovered almost to the same level as that in Na?ve. The expression of pCREB-positive neurons in IL in conditioned fear group (28±2.73) was significantly lower than that of na?ve(73.2±5.31) and delayed extinction group at different time points (P<0.001).(3) Western Blot results showed: Gray scale of pCREB protein toβ-actin showed gradual increase from 1st (0.86±0.06), 3rd (1.11±0.06) to 7th (1.22±0.04) day in delayed extinction group(P<0.01), of which it was increased dramatically from 1d to 3d. The expression of pCREB was significantly decreased in conditioned fear group (0.54±0.03) (P<0.001), and recovered to the expression level in na?ve(1.39±0.05) 7d after extinction (P>0.05).Conclusion: The auditory fear conditioning can cause the reduced expression of pCREB in IL, while the improved freezing behavior can lead to the increased expression. The expression of pCREB in IL was significantly increased 1-3d after the extinction in IL neurons , suggesting that neurons may be implicated in the extinction retention.The third experiment NMDA receptor’s function in retention of conditioned fear extinctionObjective: To observe the effects of MK-801, noncompetitive NMDA receptor antagonist, on the conditioned fear extinction retention and the expression of pCREB in IL for investigating the possible mechanism.Methods: Delayed extinction method was employed on male adult SD rats which were randomly divided into 4 groups. Pre-MK-801 group: Intraperitoneal injection of MK-801(0.3mg/kg) was given to rats 40 min before extinction training; Post-MK-801 group: Intraperitoneal injection of MK-801(0.3mg/kg) was given to rats 4 h after extinction training; EXT group: Extinction training was conducted 24 h after conditioned fear memory was established and intraperitoneal injection of equal amount of saline was given to rats; Cont group: The conditioned fear memory was established without any extinction training, and intraperitoneal injection of equal amount of saline was given to rats. The freezing behavior time and retention study of fear extinction were performed with half quantity of the rats separately before experiment, on the day of extinction (EXT-0d), and 1st, 3rd, 7th day after extinction (EXT-1, 3, 7d). The rats were executed to death for immunohistochemistry and western blot analysis (3-4 rats/time). In the triple immunoflourence signal, pCREB indicated cell nucleus (green), Neu-N perikaryons and nucleus (red) and Hoechst nucleus (blue). Cells with pCREB-positive, Neu-N-positive and Hoechst-positive staining were counted and recorded to show the expression of pCREB in IL neurons.Results (1) There were no significant differences in freezing behavior among four groups (Pre-MK-801, Post-MK-801, EXT, Cont) (P>0.05). Freezing behavior on EXT-7d was significantly lower than that on EXT-0d in Cont group. There were no significant differences in freezing behavior between Pre-MK-801 group and Cont group on the 1st, 3rd and 7th day after fear extinction(P>0.05). Freezing behavior in Post-MK-801 group was significantly higher than that in EXT group, but lower than that in Pre-MK-801 group and Cont group on the 1st, 3rd and 7th day after fear extinction (P<0.05). There were no significant differences in freezing behavior between Pre-MK-801 group and Post-MK-801 group at different time points after fear extinction.(2) There was no statistically significant difference in baseline levels of the percentage of open arm entries (OA entries % )and the percentage of the time spent in the open arm entries( OA time % ) among four groups (P>0.05). There was no significant difference between OA entries % and OA time% at different time period after fear extinction (P>0.05). Generally, the anxiety behavior level was decreased with the experiment time lasting, but there was no significant difference at the same time point among 4 groups (P >0.05).(3) The expression of pCREB-positive neurons in IL with triple immunofluerence staining: The expression of pCREB-positive neurons in IL in Post-MK-801 group (42.80±4.32) was lower than that in EXT group(48.80±2.39) (P<0.05), but significantly higher than that in Pre-MK-801 group (34.80±3.11) and Cont group (35.60±4.16) (P<0.05), There was no significant difference between Pre-MK-801 group and Cont group of the expression of pCREB-positive neurons in IL (P>0.05). (4) Western Blot results showed: Gray scale of pCREB protein toβ-actin showed that the expression of pCREB protein in Post-MK-801 group (0.71±0.06) was significantly lower than that in EXT group(0.87±0.08) (P<0.01), but significantly higher than that in Pre-MK-801 group (0.49±0.05)and Cont group(0.52±0.03) (P<0.01). There was no significant difference between Pre-MK-801 group and Cont group (P>0.05).Conclusion Intraperitoneal injection of MK-801(0.3mg/kg) 40 min before extinction training or 4h after extinction training was given to rats that has no influence on the change of anxiety behavior of rats but affects their extinction retention of conditioned fear. The way of administration causes thedifferent change of the expression of pCREB in IL, which is limited to the neuron. The effect of extinction in Pre-MK-801 is worse than that in Post-MK-801. The expression of pCREB in IL may participate in the extinction of conditional fear memory. MK-801 may antagonize NMDA receptor to restrain the extinction of conditional fear memory.更多还原