摘要
目的：本课题组先期研究已证实提高循环血内皮祖细胞(endothelial progenitor cells, EPCs)水平能改善创伤性颅脑损伤的预后。本实验进一步研究弥漫性轴索损伤(diffuse axonal injury, DAI)的EPCs的变化趋势。结合临床患者和DAI大鼠模型,我们观察了循环血EPCs在DAI的变化规律并尝试通过孕酮(progesterone, PROG)调节DAI大鼠的循环血EPCs水平,以观察孕酮促进DAI大鼠损伤区脑组织的血管生成和神经功能恢复的作用。
方法：1)选取DAI患者15例和出血性脑外伤(traumatic intracerebral hemorrhage, TIH)患者26例。利用流式细胞仪测定患者伤后1d、4d、7d、14d、21d的循环血EPCs数量,以格拉斯哥预后评分(GOS)为患者伤后6个月的预后评价标准。2)建立Marmarou DAI大鼠模型,选用Wistar雄性大鼠,随机分为假手术组、单纯DAI组、DAI安慰剂组和DAI孕酮治疗组。其中孕酮治疗组于伤后1h立即给予腹腔注射孕酮16mg/kg,随后于伤后6h,24h给予皮下注射相同剂量的孕酮,随后每日一次连续给予5d；安慰剂组在相同的给药时间点给予等量安慰剂(二甲基亚砜,DMSO);假手术组与单纯DAI组均不予任何治疗。应用流式细胞仪测定大鼠伤后3h,6h,24h,3d,7d,14d循环血中的EPCs数量。取损伤脑组织应用免疫荧光进行CD34染色,以观察创伤区脑组织血管新生情况。伤后1d、7d、14d和21d利用改良神经损伤评分法(Modified Neurological Severity Scores, mNSS)连续对四组大鼠进行评分,评定其行为学功能变化。伤后14d开始连续5d以Morris水迷宫(MWM)试验检测各组大鼠定位航行试验的逃避潜伏期和空间搜索试验的目标象限逗留时间百分比及跨越原平台位置次数。第20天记录大鼠脑部穿通纤维到海马齿状回通路的长时程增强(long-term potentiation, LTP),结果以兴奋性突触后电位(excitatory post-synaptic potential, EPSP)斜率与EPSP基线值比较的百分数表示。
结果：1)DAI患者与前期TBI和同期TIH患者一样,其循环血EPCs水平呈现先升高后降低的变化趋势,但是增加的幅度不如TIH患者明显,且于7d、14d和21d与TIH患者的差异有统计学意义(P<0.05)。Logistic回归分析、线性相关分析以及受试者工作特征曲线(ROC)均发现,EPCs数量与DAI预后相关性不强。2)分别用HE染色和p-淀粉样前体蛋白(β-APP)免疫组织化学染色检查结合伤后24h的MRI,证实成功建立了DAI动物模型。检测单纯DAI组和安慰剂组大鼠的EPCs变化与DAI患者相似,呈现相对平坦的趋势,而同一个时间点的孕酮治疗组的大鼠的EPCs水平明显升高,并于治疗7d后到达高峰(P<0.05),后逐渐下降,但仍然维持在一个较高水平一直持续到伤后14d。孕酮治疗组伤后7d创伤区周围及伤侧海马的CD34+细胞与安慰剂组相比较均不同程度升高,有统计学意义(P<0.05)。mNSS评分结果显示,与安慰剂组比较,孕酮治疗组大鼠伤后7d的行为学功能得到明显改善(P<0.05)。水迷宫检测发现,孕酮治疗组大鼠伤后14d的逃避潜伏期明显少于安慰剂组和单纯DAI组,而目标象限逗留时间百分比和跨越原平台位置次数明显比安慰剂组和单纯DAI组升高(P<0.05)。长时程增强实验显示孕酮治疗组大鼠伤后20d的LTP明显高于安慰剂组和单纯DAI组(P<0.05)。
结论：DAI患者的循环血EPCs水平在伤后没有明显升高,呈现相对平坦的趋势。我们分析正是由于DAI患者EPCs总体水平持续性偏低,可能是导致临床上DAI患者预后不良的一个重要因素,但具体机制还需要进一步研究。我们在成功建立的DAI大鼠模型上发现了同样的EPCs变化趋势。在给予DAI大鼠孕酮治疗后,结果显示PROG能明显增强DAI大鼠骨髓EPCs的动员。在其后的行为学和神经电生理的结果显示PROG能促进脑创伤区周围及海马区周围的血管新生,改善DAI大鼠行为学功能和学习记忆功能。这一结果验证了我们最初的假设,即通过上调EPCs数量能够改善DAI预后。同时,药物性干预上调EPCs可能成为促进DAI后血管修复与神经再生的新策略。
Objective:
The previous research by our group has confirmed that circulating endothelial progenitor cells (EPCs) can improve the prognosis of traumatic brain injury (TBI). This study aims to identify correlations between circulating EPCs levels and outcomes in diffuse axonal injury (DAI) patients, then to explore the effects of progesterone on EPCs levels, lesion revascularization and neurological function recovery in rats with DAI.
Methods:
1) EPCs levels in15patients with DAI and26cases of traumatic intracerebral hemorrhage (TIH) brain injury were counted by FACS (Fluorescence Activating Cell Sorter) at Id,4d,7d,14d, and21d post-injury, and the patients prognosis were evaluated by Glasgow Outcome Scale (GOS) at6months after discharge.
2) Male Wistar rats were randomly divided into4groups:sham operation group (SHAM), DAI alone group (DAI), DAI placebo group (placebo) and DAI progesterone treatment group (PROG). DAI rat models were established as Marmarou's descripition. Rats in PROG group recieved16mg/kg progesterone intraperitoneally within1h post-insult, then subcutaneously after6h and24h and a once-daily dose for up to5d. Same amount of dimethyl sulfoxide (DMSO) were administered via same route and at same time points to placebo group. The circulating EPCs levels were enumerated by FACS after3h,6h,24h,3d,7d, and14d. Lesion angiogenesis were identified by immunofluorescence stained for CD34. At1d,7d,14d and21d after injury, changes of motor functions were evaluated by the Modified Neurological Severity Score (mNSS). From the14th day after injury, the Morris water maze (MWM) experiment was performed to assess the spatial learning and memory functions of the four groups. At day20after the-operation, the rats were subjected to long-term potentiation (LTP) recording in hippocampus to measure the percentage of slope and baseline of excitatory post-synaptic potential (EPSP).
Results:
1) Circulating EPCs levels in DAI patients showed an initial up-regulation and subsequent reduction. However, these dynamic changes were significantly more pronounced in TIH patients than DAI paitents (P<0.05) at7d,14d and21d post-injury. Regression analysis, linear correlation analysis and receiver operating characteristic curve (ROC) failed to establish significant correlation between the EPCs level and outcomes, while the placebo group and the DAI patients had comparable changes in EPCs levels.
2) The EPCs level in progesterone-treated rats increased significantly compared to placebo rats (P<0.05), peaked after7days of treatmentand then gradually decreased, but remained higher than the placebo group through14d post-injury. Rats treated with progesterone for7d after injury had significantly increased numbers of CD34+cells in lesion and ipslateral hippocampus (P<0.05). The mNSS indicated significantly improved behavioral function in the progesterone-treated rats at7d post-injury, compared with the placebo group and DAI alone group (P<0.05). Progesterone-treated rats showed significantly less escape latencies, higher target quadrant time staying percentage and the number of times the original platform position was crossed after14d (P<0.05), and higher LTP at20d post-injury, than the placebo group and DAI alone group (P<0.05).
Conclusion:
The circulating EPCs levels in the DAI patients did not experience signifincantly fluctuations after injury or correlated with paitents outcome. And same changes were found in DAI rat model. Progesterone administration significantly enhanced mobilization of EPCs, promoted angiogenesis in lesion and hippocampus and thus improved behavioral function and learning and memory abilities. Therapies targeting to increase EPCs levels may provide a new efficient strategy for promoting vascular repair and nerve regeneration in DAI patients.
引文
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