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

描述(由申请人提供)： 这项拟议研究的目的是确定卒中后经鼻腔(IN)途径给药的去铁胺(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长时间可以推迟IN DFO治疗，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，女性比男性更常见，而且中风发作后总是会有一些延迟，才能开始治疗。这项拟议研究的假设是，IN DFO将为中风后长期存在的功能缺陷提供神经保护，无论男女，功能恢复将更快，治疗可以在中风发作后推迟长达4小时。第一阶段将使用年轻(三个月大)的雄性和雌性大鼠；第二阶段将使用年龄较大(14个月大)的雄性和雌性大鼠。所有雌性大鼠都将被切除卵巢，以消除雌激素的神经保护作用和发情周期中发生的激素变化。动物将习惯于被摆弄，并在手术前接受过行为测试培训。采用线栓法建立大鼠大脑中动脉闭塞(MCAO)模型。在MCAO前和再灌流时测量血压、心率、直肠温度、血pH、PO2、二氧化碳分压、乳酸和血糖。大鼠将接受120分钟的大脑中动脉阻塞，并在中风发作后60分钟接受急性神经学评估，包括姿势反射和前肢放置测试，以确认神经功能缺陷。为了绕过血脑屏障，减少全身暴露，并快速靶向中枢神经系统，DFO将在中风和再灌注后的不同时间进行鼻腔给药。将大鼠再次麻醉，甲磺酸DFO(10%水溶液)或水对照组按6~5L滴注10次，总容量605L，每滴间隔2分钟。605L被认为是一剂，含有6毫克DFO(9.35摩尔)。水控制大鼠将包括在每种剂量方案中。在MCAO后24小时和此后每周，大鼠将接受神经学评估，以及慢性功能和精细运动控制的评估，包括本体感觉放置、独立的前肢伸展和抓取能力以及触觉粘连移除测试。将使用巴恩斯迷宫来评估认知功能。MCAO后第28天处死大鼠，取脑，切片、染色，计算脑梗塞体积。神经保护将通过存活、神经和功能评估、体重变化、生理指标和脑梗塞体积来确定。将评估DFO治疗组和对照组在28天存活期内行为功能评分改善的时间进程。在第一阶段，年轻的雄性和雌性大鼠将在再灌注时立即开始注射6剂IN DFO，即MCAO发作后2小时，然后是2剂，间隔2小时，第二天再注射3剂，间隔3小时。然后，开始给药方案将推迟到3小时，然后在2小时MCAO发生后4小时，以确定是否可以延长治疗窗口。在第二阶段，老年雄性和雌性大鼠将使用与第一阶段相同的方案给药。

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.