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.
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.
