Intranasal deferoxamine to treat stroke in young and older, male and female rats

描述（由申请人提供）： 拟议研究的目的是确定卒中后通过鼻内（IN）途径给予去铁胺（DFO）是否对年轻和老年雄性和雌性大鼠的长期功能缺陷提供神经保护，并评估卒中发作后多久IN DFO治疗可以延迟，同时仍保持疗效。这项研究非常重要，因为中风最常发生在老年人群中，女性比男性更频繁发生，并且在中风发作后开始治疗之前总是有一些延迟。拟议研究的假设是，IN DFO将为年轻和老年大鼠（两种性别）卒中后的长期功能缺陷提供神经保护，功能恢复将更快，并且治疗开始可延迟至卒中发作后4小时。第1阶段将使用年轻（3月龄）雄性和雌性大鼠;第2阶段将使用年长（14月龄）雄性和雌性大鼠。所有雌性大鼠将被切除卵巢，以消除雌激素的神经保护作用和发情周期期间发生的激素变化。动物将习惯于被处理，并在手术前接受行为测试培训。中风将通过管腔内缝合大脑中动脉闭塞（MCAO）模型诱导。在MCAO前和再灌注时测量血压、心率、直肠温度、血液pH值、PO 2、PCO 2、乳酸盐和葡萄糖。大鼠将经历120分钟的MCAO，并在中风发作后60分钟进行急性神经学评估，包括姿势反射和前肢放置测试，以确认神经功能缺损。为了绕过血脑屏障，减少全身暴露，并迅速靶向中枢神经系统，DFO将在中风和再灌注后的不同时间鼻内给药。将大鼠再次麻醉，并将DFO甲磺酸盐（10%水溶液）或水对照以10滴6- 5 l的形式给药，总体积为605 l，交替鼻孔，滴之间间隔2分钟。605 l被认为是一个剂量，含有6 mg DFO（9.35 mol）。每个给药方案将包括水对照大鼠。在MCAO后24小时和此后每周，将对大鼠进行神经学评估，沿着慢性功能和精细运动控制评估，包括本体感受放置、独立前肢伸展和抓握能力以及触觉粘合剂去除试验。将使用巴恩斯迷宫评估认知功能。在MCAO后28天对大鼠实施安乐死，取出脑，切片，染色，并计算梗死体积。神经保护将通过存活率、神经和功能评估、体重变化、生理测量和梗死体积来确定。将评价28天生存期内DFO治疗组和对照组之间行为-功能评分改善的时间进程。在第1阶段，年轻的雄性和雌性大鼠将在再灌注时立即开始接受6次IN DFO剂量，再灌注是MCAO发作后2小时，随后以2小时间隔接受2次剂量，并在第二天以3小时间隔再接受3次剂量。给药方案的开始将延迟至3小时，然后在2小时MCAO发作后4小时，以确定治疗窗是否可以延长。在第2阶段，将使用与第1阶段相同的方案对老年雄性和雌性大鼠给药。 公共卫生相关性： 中风是美国第三大死亡原因，也是成年人严重、长期残疾的主要原因之一。每年有795，000人，大多数是60岁及以上的人，经历中风。此外，55，000多名女性比男性经历中风，更多的女性比男性死于中风。2010年中风的估计费用为737亿美元。中风是一个特别相关的VA卫生保健问题，因为患者人群年龄较大， 女性退伍军人的数量正在增加，越来越多的士兵从伊拉克和阿富汗返回时患有创伤性脑损伤，这增加了中风的风险。目前可用于治疗中风的药物具有非常显著的风险和局限性。目标 该研究的目的是在男性和女性以及年轻人和老年人中评估一种安全、非侵入性、有前途的治疗中风的药物的疗效，该药物可以鼻内给药。如果成功，过渡到临床试验可能是迅速的，导致数百万美元的节省VA医疗保健系统。

DESCRIPTION (provided by applicant): The goal of the proposed study is to determine whether deferoxamine (DFO), administered via the intranasal (IN) route following stroke, provides neuroprotection against long-lasting functional deficits in young and older male and female rats and to evaluate how long after the onset of stroke, IN DFO therapy can be delayed while still maintaining efficacy. This study is extremely important because stroke most often occurs in the older population, occurs more frequently in women than in men, and there is always some delay after onset of stroke before treatment can be initiated. The hypothesis of the proposed study is that IN DFO will provide neuroprotection against long-lasting functional deficits following stroke in young and older rats o both sexes, that functional recovery will be sooner, and that the start of treatment can be delayed for up to 4 hours after the onset of stroke. Young (three month-old) male and female rats will be used in phase 1; older (14 month-old) male and female rats will be used in phase 2. All female rats will be ovariectomized to eliminate the neuroprotective effect of estrogen and the hormonal changes that occur during the estrous cycle. Animals will be accustomed to being handled and will have been trained in the behavioral tests prior to surgery. Stroke will be induced by the intraluminal suture middle cerebral artery occlusion (MCAO) model. Blood pressure, heart rate, rectal temperature, blood pH, PO2, PCO2, lactate, and glucose will be measured before MCAO and at reperfusion. Rats will undergo 120 minute MCAO and be given an acute neurological assessment comprised of postural reflex and forelimb placing tests at 60 minutes after the onset of stroke to confirm a neurological deficit. To bypass the blood-brain barrier, reduce systemic exposure, and rapidly target the central nervous system, DFO will be administered intranasally at various times following stroke and reperfusion. Rats will be re-anesthetized, and DFO mesylate (10% solution in water) or water control will be administered as ten 6-5l drops for a total volume of 605l, alternating nares with 2 minutes between drops. The 605l is considered one dose and contains 6 mg of DFO (9.3 5mol). Water control rats will be included with each dosing regimen. At 24 hours following MCAO and weekly thereafter, rats will be undergo neurological assessment, along with assessments of chronic functional and fine motor control comprised of proprioceptive placing, independent forelimb reaching and grasping abilities, and tactile adhesive-removal tests. Barnes maze will be used to evaluate cognitive function. Rats will be euthanized 28 days after MCAO, brains removed, sectioned, stained, and infarct volume calculated. Neuroprotection will be determined by survival, neurological and functional assessment, body weight changes, physiological measures, and infarct volume. The time course for improvement in behavioral- functional scores between the DFO-treated and the control groups during the 28-day survival will be evaluated. In phase 1, young male and female rats will receive 6 IN DFO doses starting immediately at reperfusion, which is 2 hours after onset of MCAO, followed by 2 doses at 2-hour intervals and an additional 3 doses the following day at 3-hour intervals. The start of the dosing regimen will then be delayed until 3 hours, and then 4 hours after onset of the 2 hour MCAO to determine if the treatment window can be extended. In phase 2, older male and female rats will be dosed utilizing the same regimens as phase 1. PUBLIC HEALTH RELEVANCE: Stroke is the third leading cause of death in the United States and one of the leading causes of severe, long- term disability in adults. Each year, 795,000 people, mostly individuals 60 years and older, experience a stroke. In addition 55,000 more women than men experience stroke, and more women than men die of stroke. The estimated cost of stroke for 2010 is $73.7 billion. Stroke is a particularly relevant VA health care issue because the patient population is older, the number of women veterans is increasing, and an increasing number of soldiers are returning from Iraq and Afghanistan with traumatic brain injury that increases the risk for stroke. The current drug available for treatment of stroke has very significant risks and limitations. The goal of the proposed study is to evaluate, in both males and femaies and in both young and old, the efficacy of a safe, non-invasive, promising therapeutic agent for stroke that can be administered intranasally. If successful, transition to clinical trials could be rapid, resulting in millions ofdollars in savings to the VA healthcare system.