Exploring the involvement of Na pump inhibitor marinobufagenin in the interactions between age-associated vascular and neurodegenerative processes in cognitive impairment and Alzheimer's disease

实验1. Dahl-S大鼠血管性痴呆模型的建立 中心动脉僵硬度（CAS）与动脉血压（BP）升高相关，并可能与脑动脉壁硬化相关，伴有脑灌注不足、神经元密度丢失和认知能力下降。达尔盐敏感（Dahl-S）大鼠表现出与年龄相关的高血压和记忆丧失，即使在正常的盐摄入量。我们试图探讨是否中央动脉脉搏波速度（PWV），CAS的标志物，与高血压Dahl-S大鼠海马脑血流量（CBF）和神经元密度。 我们测量了收缩压（尾袖体积描记法），主动脉PWV（超声心动图）和CBF和N-乙酰天门冬氨酸（NAA）（磁共振成像）在12个6个月大的雄性Dahl-S大鼠。我们已经证明，较大的PWV与海马中较低的CBF和较低的NAA浓度显著相关，支持CAS在脑血管功能障碍和认知能力随年龄增长而下降中的作用。 这些发现表明，在年龄相关性心血管功能障碍的Dahl-S模型中，脑灌注不足和神经元密度和功能丧失中CAS增加。 实验2. ACE抑制剂对Dahl-S大鼠血管性痴呆模型的影响 随着年龄的增长，正常盐摄入量的Dahl盐敏感大鼠（Dahl-S）的主动脉硬化和BP过早增加，并伴有空间认知障碍。由于一种新的促高血压和促纤维化类固醇，marinobufagenin（MBG），涉及在Dahl-S的血压升高，我们假设，促纤维化因子，包括MBG和血管紧张素II，刺激MBG的水平的变化，将导致动脉壁硬化，这将导致脑动脉和神经元的结构变化，随后认知能力下降。我们认为，针对动脉壁内年龄依赖性促纤维化过程的药物干预，以降低中央动脉僵硬度，将改善脑微血管疾病，认知障碍和痴呆。 对Dahl-S（n=20）雄性大鼠喂食正常盐饮食12个月;其中10只大鼠通过饮水连续给予ACE抑制剂赖诺普利（15 mg/kg/天）6个月，10只Dahl-S大鼠保持常规饮水作为对照（2只未处理的对照动物在实验结束前因高血压死亡）。在6个月（基线）、9个月和12个月时进行收缩压（SBP）、PWV、尿MBG、脑MRI扫描、旷场和注意力测试（图1），而在12个月时进行Morris水迷宫（MWM）测试和脑组织化学。 我们证明了（i）更大的PWV（CAS的标志物）与海马灌注减少和神经元质量（NAA）减少相关。高海马灌注与低焦虑水平相关。这一发现表明，中央动脉硬度可能是预防和治疗痴呆症的潜在治疗靶点。(ii)接下来，我们证明了赖诺普利治疗与PWV和SBP降低相关，并且在治疗大鼠中MBG水平在研究期间保持稳定，而对照动物表现出MBG增加以及SBP和PWV升高。(iii)在高血压Dahl-S大鼠中，赖诺普利治疗改善了空间海马记忆，这与在Morris水迷宫中寻找隐藏平台的行进距离减少以及与未治疗对照相比具有更好的路径效率相关。(iv)注意力测试的初步结果表明，用赖诺普利治疗6个月的Dahl-S大鼠在听觉和视觉分心的挑战期间表现出注意力增加（p=0.04）。进一步的分析将被用来评估注意力测试中的冲动性和强迫性。(v)此外，我们发现，赖诺普利治疗的动物显示海马CBF和NAA（脑MRI数据）的稳定性，而未治疗的动物显示这些值随年龄增长而下降的趋势。MWM中给药动物的表现增加表明了给药的空间海马记忆益处，这支持了海马CBF和NAA的趋势。 这些发现与绿色光片显微镜下获得的初步脑血管密度组织化学数据一致。用赖诺普利处理的Dahl-S大鼠与未处理的对照相比表现出血管密度的增加，这可以解释为在抗高血压和抗纤维化处理后导致向大脑的更高的血液供应。

Experiment 1. Dahl-S rat model of age-associated vascular dementia Central arterial stiffness (CAS) is associated with elevated arterial blood pressure (BP) and is likely associated with stiffening of cerebral artery walls, with attendant cerebral hypoperfusion, neuronal density loss and cognitive decline. Dahl salt-sensitive (Dahl-S) rats exhibit age-associated hypertension and memory loss, even on a normal salt intake. We sought to explore whether central arterial pulse wave velocity (PWV), a marker of CAS, is associated with hippocampal cerebral blood flow (CBF) and neuronal density in hypertensive Dahl-S rats. We measured systolic BP (by tail-cuff plethysmography), aortic PWV (by echocardiography) and CBF and N-acetyl aspartate (NAA) (by magnetic resonance imaging) in twelve 6 months old male Dahl-S rats. We have demonstrated that greater PWV was significantly associated with lower CBF and lower NAA concentration in the hippocampus, supporting a role of CAS in cerebrovascular dysfunction and decline in cognitive performance with aging. These findings implicate increased CAS in cerebral hypoperfusion and loss of neuronal density and function in the Dahl-S model of age-associated cardiovascular dysfunction. Experiment 2. Effect of an ACE inhibitor in the Dahl-S rat model of age-associated vascular dementia A premature increase in aortic stiffening and BP, accompanied by spatial cognitive impairment, occur in Dahl salt-sensitive rats (Dahl-S) on a normal salt intake with advancing age. Because a novel pro-hypertensive and pro-fibrotic steroid, marinobufagenin (MBG), is implicated in BP increases in Dahl-S, we hypothesized that the changes in the levels of the pro-fibrotic factors, including MBG and angiotensin II, which stimulates MBG, will cause the arterial wall stiffening, which will lead to the structural changes in the cerebral arteries and neurons following by cognitive decline. We posited that pharmacologic interventions targeting age-dependent pro-fibrotic processes within the arterial wall to reduce central arterial stiffness, will ameliorate brain microvascular disease, cognitive impairment and dementia. Dahl-S (n=20) male rats were fed a normal salt diet for 12 months; 10 of them were continuously treated with an ACE inhibitor Lisinopril (15 mg/kg/day) administered through their water for 6 months, and 10 Dahl-S rats remained on regular water as a control (2 non-treated control animals died before the end of the experiment due to hypertension). Systolic BP (SBP), PWV, urine MBG, brain MRI scans, open field and attention tests (Fig. 1) were performed at 6 (baseline), 9 and 12 months, while Morris water maze (MWM) test and brain histochemistry were performed at 12-mo. We demonstrated that (i) greater PWV, a marker for CAS, was associated with a decreased hippocampal perfusion and a decreased neuronal mass (NAA). Higher hippocampal perfusion was associates with the lower anxiety level. This finding suggests that central arterial stiffness may be a potential therapeutic target for the prevention and treatment of dementia. (ii) Next, we demonstrated that Lisinopril treatment was associated with a decrease in PWV and SBP, and that MBG level was stable for the duration of the study in the treated rats whereas control animals exhibited increase in MBG and higher SBP and PWV. (iii) In hypertensive Dahl-S rats, Lisinopril treatment improved spatial hippocampal memory, which was associated with a decrease of distance traveled to find a hidden platform in Morris water maze and with a better path efficiency vs. non-treated controls. (iv) Preliminary results in attention test suggest that Dahl-S rats treated with Lisinopril for 6 months demonstrated increased attention during a challenge with auditory and visual distractions (p=0.04). Further analysis will be done to assess impulsivity and compulsivity in this attention test. (v) In addition, we found, that Lisinopril-treated animals demonstrates stabilization of hippocampal CBF and NAA (brain MRI data) while untreated animals exhibited trend to decline of these values with aging. Increased performance for treated animals in MWM points to spatial hippocampal memory benefits of treatment that support trends seen for hippocampal CBF and NAA. These findings are consistent with the preliminary brain vessels density histochemical data, obtained by a green light sheet microscopy. Dahl-S rats treated with Lisinopril, exhibited an increase in blood vessel density vs. non-treated controls, which can be translated that a higher blood supply to the brain is resulted after anti-hypertensive and anti-fibrotic treatment.