Neuroprotective and regenerative effects of small molecules

（一） 二腺苷四磷酸（AP 4A）。本研究的目的是确定AP 4A是否对甲基苯丙胺（MA）介导的多巴胺能神经元毒性在体外和体内的保护。从大鼠胚胎（E15）腹侧中脑组织制备原代神经元培养物。用2 mM MA处理细胞48小时。应用MA增加LDH水平，减少TH免疫反应性，并增加TUNEL。AP 4A预处理可降低上述变化。还在体内检查了AP 4A的保护作用。向成年Sprague道利大鼠脑室内注射AP 4A或溶剂，然后注射4次MA（5 mg/kg）。MA给药增加了纹状体和皮质中caspase-3的免疫反应性。AP 4A预处理可显著降低caspase-3（+）细胞密度。采用微透析技术，对自由活动大鼠背侧纹状体多巴胺（DA）的释放进行了监测。AP 4A没有急性改变MA诱发的DA释放，表明AP 4A诱导的保护作用不是通过DA溢出的变化直接介导的。总之，这些数据表明，AP 4A在体外和体内均对MA介导的神经元损伤具有保护作用。其作用机制可能与抑制MA诱导的细胞凋亡有关. （二） 虾青素（ATX）。 我们发现ATX对缺血性脑损伤具有保护作用。 在成年大鼠大脑中动脉闭塞（MCAo）60分钟前10-20分钟，脑室内给予ATX或溶剂。在MCAo后2天，接受ATX的大鼠的运动活动增加。ATX可显著减少缺血性脑中的脑梗死和TUNEL标记。ATX拮抗缺血介导的乌头酸酶活性丧失（乌头酸酶活性是产生活性氧的间接标志物）和减少的谷氨酸释放、脂质过氧化、Chrome C易位以及缺血皮质中的TUNEL标记。ATX未改变生理参数，如血气、血液pH值、血压、体温、脑温和脑血流量。总的来说，我们的数据表明，虾青素通过抑制氧化应激、抑制谷氨酸释放和抗凋亡，对体内自由基毒性和缺血相关损伤具有保护作用。 （三） 匹非亭-a（PFT-a）：（A）我们发现PFTa对多巴胺能神经元中MA介导的损伤具有保护作用。高剂量MA降低原代多巴胺能神经元培养中TH免疫反应性和纤维密度。与PFTa联合治疗显著减弱了这些退行性变化。我们的数据表明，用p53抑制剂或抑制p53表达的治疗限制了暴露于多巴胺能神经毒素诱导的神经元功能障碍和细胞死亡。(B)PFT-a在体内和体外均增强神经祖细胞（NPC）的存活。首先在组织培养的SVZ神经球中检查NPC的存活和增殖的增强。PFT-a剂量依赖性地增加新神经球形成的数量和大小。用PFT-a延迟治疗（即损伤后6至9天）增强了成年大鼠脑室下区内源性神经祖细胞（NPC）的存活。PFTa促进内源性NPC的增殖、存活、迁移和分化，并促进中风动物运动行为的功能恢复。PFT-抑制中风动物SVZ内p53依赖性促凋亡基因的表达，称为p53上调的凋亡调节因子（p53-upregulated modulator of apoptosis）。我们的数据表明，用p53抑制剂PFT-a延迟治疗能够改变内源性神经发生并改善脑损伤后的功能恢复。(c)全身给予PFT-1可提高体内多巴胺能神经元移植物的存活率。PFT治疗抑制细胞死亡，增加TH（+）细胞的存活，增强VM移植物的TH神经突生长，并增强帕金森病大鼠的行为恢复。总之，我们的数据表明，激活p53是一个重要的介质损伤后或神经修复过程中的细胞死亡。 p53的瞬时抑制可增加脑损伤后神经元或NPC的存活。 （四） 视黄酸（RA）：9 cis RA（9 cRA）预处理增加自发活动，减轻神经功能缺损，减少脑梗死和TUNEL标记在中风大鼠。9 cRA可增加脑组织骨形态发生蛋白7（BMP 7）的表达。BMP拮抗剂noggin可拮抗9 cRA的保护作用。我们的数据表明，RA可以在培养的细胞和体内诱导保护性反应，可能通过BMP 7机制。 （五） （-）纳洛酮：神经变性可通过炎症过程的激活而发生。从过去一年进行的实验中，我们观察到在大鼠大脑中动脉闭塞后一天开始每天给予（+）纳洛酮持续1周时，卒中后行为恢复（身体不对称和神经评分）得到改善。 我们目前正在跟进这些研究，比较（-）纳洛酮以及检查缺血后上调的炎症标志物的变化。

(1) Diadenosine tetraphosphate (AP4A). The purpose of this study was to determine whether AP4A is protective against methamphetamine (MA) -mediated toxicity in dopaminergic neurons in vitro and in vivo. Primary neuronal cultures were prepared from rat embryonic (E15) ventral mesencephalic tissue. Cells were treated with 2 mM MA for 48 hours. Application of MA increased LDH levels, decreased TH immunoreactivity, and increased TUNEL. All these changes were reduced by pretreatment with AP4A. The protective effect of AP4A was also examined in vivo. Adult Sprague Dawley rats were injected with AP4A or vehicle intracerebroventricularly followed by 4 doses of MA (5mg/kg). Administration of MA increased caspase-3 immunoreactivity in striatum and cortex. Pretreatment with AP4A significantly reduced the density of caspase-3 (+) cells. Using microdialysis, dopamine (DA) release was monitored in dorsal striatum in freely moving rats. AP4A did not acutely alter MA-evoked DA release, suggesting that AP4A -induced protection is not directly mediated through a change in DA overflow. Taken together, these data show that AP4A has protective effects against MA-mediated neuronal injury both in vitro and in vivo. The mechanism of action may involve suppression of MA -induced apoptosis. (2) Astaxanthin (ATX). We found that ATX has protective effects against ischemic brain injury. ATX or vehicle was administered intracerebroventricularly 10-20 minutes prior to a 60-min middle cerebral artery occlusion (MCAo) in adult rats. At 2 days after MCAo, rats that received ATX had an increase in locomotor activity. ATX significantly reduced cerebral infarction and TUNEL labeling in ischemic brain. ATX antagonized ischemia -mediated loss of aconitase activity, an indirect marker for the production of reactive oxygen species and reduced glutamate release, lipid peroxidation, translocation of chrome C, as well as TUNEL labeling in the ischemic cortex. ATX did not alter physiological parameters, such as blood gases, blood pH, blood pressure, body temperature, brain temperature and cerebral blood flow. Collectively, our data suggest that astaxanthin has protective effects against free radical toxicity and ischemia-related injury in vivo through the inhibition of oxidative stress, inhibition of glutamate release, and anti-apoptosis. (3) pifithrin-a (PFT-a): (A) We found that PFTa has protective against MA mediated injury in dopaminergic neurons. High dose of MA reduced TH immunoreactivity and fiber density in primary dopaminergic neuronal culture. Co-treatment with PFTa significantly attenuated these degenerative changes. Our data suggest that treatment with a p53 inhibitor or suppression of p53 expression limits neuronal dysfunction and cell death induced by exposure to dopaminergic neurotoxins. (B) PFT-a enhanced the survival of neural progenitor cells (NPCs) both in vivo and in vitro. The enhancement of survival and proliferation of NPCs was first examined in SVZ neurospheres in tissue culture. PFT-a dose-dependently increased the number and size of new neurosphere formation. Delayed treatment (i.e. days 6 to 9 after injury) with PFT-a enhanced survival of endogenous neural progenitor cells (NPCs) in the subventricular zone in adult rats. PFTa enhanced proliferation, survival, migration, and differentiation of endogenous NPCs and enhanced the functional recovery in locomotor behavior in stroke animals. PFT- inhibited the expression of a p53-dependent pro-apoptotic gene, termed PUMA (p53-upregulated modulator of apoptosis), within the SVZ of stroke animals. Our data suggest that delayed treatment with a p53 inhibitor PFT-a is able to modify endogenous neurogenesis and improve the functional recovery after brain injury. (c) Systemic administration of PFT- enhanced the survival of dopaminergic neuronal transplants in vivo. PFT- treatment suppressed cell death, increased survival of TH (+) cells, enhanced TH neurite outgrowth from VM grafts, and augmented behavioral recovery in Parkinsonian rats. In conclusion, our data suggest that activation of p53 is an important mediator for cell death after injury or during neural repair. Transient suppression of p53 may increase the survival of neurons or NPCs after brain injury. (4) Retinoic acid (RA): Pretreatment with 9 cis RA (9cRA) increased locomotor activity, attenuated neurological deficits, and reduced cerebral infarction and TUNEL labeling in stroke rats. 9cRA increased the expression of bone morphogenetic protein 7 (BMP7) in brain. The protective response of 9cRA can be antagonized by BMP antagonist noggin. Our data suggest that RA can induce protective responses in cultured cells and in vivo, possibly through BMP7 mechanism. (5) (-)Naloxone: Neurodegeneration can occur through the activation of inflammatory processes. From experiments conducted this past year, we have observed improved behavioral recovery (body asymmetery and neurological score) following stroke when (+)naloxone was administered daily for 1 week starting one day after middle cerebral artery occlusion in rats. We are currently following up these studies comparing (-)naloxone as well as examining changes in markers of inflammation that are upregulated following ischemia.