中国科学院昆明动物研究所机构知识库

虽然缺血性卒中带来高昂的社会负担，但是临床一直缺乏促进康复的药物。因此，寻找既具有神经保护又能促进长期功能恢复的候选药物被寄予高度的希望。评估候选药物对缺血卒中诱发长期功能缺陷恢复的影响急需更为合理有效的实验范式。丹参（Salvia miltiorrhiza）是传统中药，长期以来一直用于治疗心脑血管系统疾病，对痴呆也有一定疗效。丹酚酸A（salvianolic acid A，SAA）是丹参的主要活性成分之一，已被多个实验室证明对脑缺血具有神经保护作用。在这里，我们采用光栓诱导大鼠额叶皮层损伤，研究SAA对光栓缺血诱发的感觉运动和记忆功能缺陷的长期影响。脑成像技术被广泛用于临床脑损伤后的诊断和检测。为了解释缺血性损伤后空间记忆是如何受损和SAA是怎样促进损伤后功能恢复，我们采用小动物18F-deoxyglucose-microPET（FDG-PET）成像技术活体跟踪了大鼠脑葡萄糖代谢在光栓缺血后2个月内的动态变化。光栓诱导双侧额叶皮层缺血后立即给予SAA治疗，定点检测相关行为学指标和PET图谱，直到手术后2个月；结果表明单次SAA治疗促进了光栓缺血诱导空间记忆功能障碍和大脑葡萄糖代谢异常的恢复。光栓缺血造成较轻微的前后肢运动功能缺陷仅在1-5天能检测到；SAA治疗对感觉运动功能缺陷的恢复没有影响。3组动物在粘附移除测试和转棒测试中成绩没有差异；进一步的系列水迷宫学习任务测试结果显示，3组大鼠之间游泳速度没有差异；手术后第4天大鼠寻找可视平台的潜伏时间也没有差异。此外，在开放场测试、高架迷宫测试、强迫游泳测试、跑步机测试、糖水偏爱测试中，与假手术组比，生理组或SAA治疗组都没有显著行为缺陷。以上结果提示，双侧额叶皮层缺血轻微损害了大鼠的感觉运动能力，但这种运动缺陷并没有干扰水迷宫测试。在系列水迷宫反转学习任务中，缺血对照组都表现空间记忆损伤，而且记忆损伤一直持续到第64天；同时SAA治疗组在24天和64天的两次测量中各指标与假手术组相比没有差异。结果提示，双侧额叶皮层缺血诱导了持久的空间记忆障碍，急性期应用SAA治疗促进了认知障碍的恢复。小动物FDG-PET成像结果显示，在缺血损伤后第1天，缺血对照组和SAA治疗组显现出相似的脑葡萄糖代谢异常。主要表现是缺血灶所在区域出现葡萄糖代谢抑制，远隔区域也出现葡萄糖代谢抑制，这种现象提示额叶皮层小范围的缺血损伤引起皮层广泛区域神经机能失联。葡萄糖代谢升高区域主要出现在皮层下区域，包括岛叶皮层、背侧丘脑等区域。这种卒中后大脑葡萄糖代谢既有抑制又有升高的现象意味着局灶性皮层损伤破坏了相互联系的神经网络而导致全局效应。在缺血损伤后60天，缺血对照组大脑葡萄糖代谢降低和升高区域仍然持续，尽管异常区域面积变小。相比之下，在相同显著水平下，SAA组没有检测到任何脑区葡萄糖代谢异常；这些研究结果提供了一个直接证据，表明缺血损伤后大脑功能可自行恢复，而且SAA治疗可促进大脑功能恢复。针对单侧额叶皮层光栓缺血模型，为期15天的玻璃缸测试感觉运动功能缺陷，结果显示急性期应用单次SAA治疗促进了缺血诱发的感觉运动缺陷的恢复；并行开展的为期2个月的Morris水迷宫测试结果显示SAA治疗减轻了缺血引起的持续空间记忆障碍。此外，在转棒测试、Morris水迷宫游泳速度、可视平台测试、开放场测试、高架迷宫测试、强迫游泳测试、跑步机测试中，与假手术组比，生理组或SAA治疗组都没有显著行为缺陷；以上结果暗示，Morris水迷宫测试显示的成绩缺陷不是动物焦虑或感觉运动功能障碍混淆导致的结果。光栓诱导单侧额叶皮层缺血模型的应用及SAA促进卒中后功能恢复的机理需要进一步探索总体而言，目前的范式适用于药治疗对缺血性卒中的长期影响的临床前评估。同时，SAA具有治疗缺血性卒中的潜力。

With respect to the high burden of ischemia stroke and the absence of pharmacological treatment for promoting rehabilitation, promising candidates with specific effects on long-term functional recovery are highly desired. Candidates need reasonable experimental paradigms to evaluate the long-term functional outcome focused on ischemia-induced sensorimotor and memory deficits. Danshen, a traditional Chinese herb, has long been used to treat coronary and cerebral vascular diseases as well as dementia. Salvianolic acid A (SAA), one of the major active ingredients of Danshen, was demonstrated to be effective in protecting against cerebral ischemia injury. Here, employing an experimental stroke model induced by photothrombosis in the frontal cortex of rats, we investigated whether SAA has long-term protective effects on ischemia induced sensorimotor and memory deficits. Brain imaging is widely used to detect brain injury after stroke in clinic. To address how ischemic injury can lead to impaired spatial memory and how SAA can promote functional recovery, we used 18F-deoxyglucose (FDG) microPET imaging in adult rats.After the bilateral frontal cortex photothrombosis, a single SAA treatment was administered immediately and appropriate behavioral indices were monitored over a period of 2 months. The results indicated that a single SAA treatment promotes functional recovery from long-lasting impairment of both spatial memory and glucose metabolism. Compared with Sham group, the tapered/ledged beam-walking test was disrupted on days 1-5 in the forelimb and day 1 in the hindlimb, while adhesive-tape-removal and rotarod tests were both intact, after ischemic injury. This was consistent with the finding in the spatial learning task of the Morris water maze (MWM), during which the velocity and the latency were all not different among three groups. The visual platform task of MWM on day 4 was also not different among the groups. These results suggested that neither motor activity nor learning ability was impaired after ischemic cortical injury. Saline but not SAA group exhibited impairment of spatial memory on both of the measurements on day 24 and 64 as compared with Sham group, strongly suggesting that a single treatment of SAA promoted later cognitive functional recovery. MicroPET imagings showed that the areas with ischemic injury displayed similar decreases of glucose metabolism in both Saline and SAA groups relative to Sham group on day 1. However, it is surprising that other brain regions mainly in the subcortical areas showed increases of glucose metabolism with a similar pattern in both Saline and SAA groups compared with Sham group, implicating that the focal cortical injury resulted in the global effects possibly via the disruption of the top-down control onto the interconnected neural networks. At 60 days post ischemic injury, both the decreased and increased glucose metabolism were still persistent, although the size of the decreased became smaller but those of the increased were largely remained, in Saline group as compared with Sham group. In marked contrast, no differences of glucose metabolism in any brain regions between SAA and Sham groups were detected. These findings provided a direct evidence that a partial self-functional recovery in the penumbra might occur after ischemic injury in Saline group, but that was largely promoted by a single treatment of SAA. In the unilateral frontal cortex photothrombosis model, a single SAA treatment improved the cortical ischemia induced sensorimotor deficits during 15-days cylinder test period, and alleviated ischemia induced sustained spatial memory impairments during the 2-months dependent MWM tests. In addition, either ischemia injury or SAA treatment did not show any changes compared with sham group in other behavioral tests including rotarod tests, swimming speed in MWM tests, open field tests, elevated plus maze tests, tread mill tests and forced swimming tests. The results reveal that the cognitive deficits are not the results of animal’s anxiety or confounding motor impairments. Overall, the present paradigm appears suitable for the preclinical evaluation of the long-term effects of pharmacological treatments on ischemia stroke. Meanwhile, SAA might have therapeutic potential for the treatment of memory deficits associated with ischemia stroke.