Poststroke DNA immunization against neurite growth inhibitors is beneficial to the recovery from local cerebral ischemia in rats

摘要

BACKGROUND: Inhibitory signals, i.e. neurite growth inhibitors (NGIs), presenting on central nervous system (CNS) myelin have been shown to play a crucial role in inhibiting lesioned axonal sprouting and leading to less functional recovery. Vaccines targeting NGIs may provide multifactorial protection against brain insults by overcoming the inhibitory effects of these NGIs and boosting the body's immune repair mechanisms. OBJECTIVE: To evaluate the effect of poststroke DNA immunization against NGIs on the rehabilitation for sensorimotor function of rat models of local cerebral ischemia. DESIGN: Completely randomized grouping design, and controlled experiment. SETTING: Brain Injury Research Laboratory, Department of Neurosurgery, National Neuroscience Institute, Singapore. MATERIALS: Sixty adult male Sprague-Dawley rats ranging in age from 45 to 120 days and in weight from 180 to 250 grams were provided by Animal Center of Department of Anatomy, Faculty of Medicine, National University of Singapore. pcDNA3.1(+)-neurite growth inhibitors (pcDNA-NGIs) a gift was provided by Dr. Xiao from Department of Clinical Research, Singapore General Hospital, Singapore. METHODS: The experiment was carried out at Brain Injury Research Laboratory, Department of Neurosurgery, National Neuroscience Institute, Singapore from August 2003 to April 2005. ①The involved rats were randomized into 3 groups: pcDNA-NGIs group (group A), pcDNA3.1 (+) group (group B) and model group (group C), with 20 rats in each group. Left focal cerebral ischemia (FCI) was permanently induced through middle cerebral artery occlusion (MCAO) with the assistance of an operating microscope. Successful MCAO was determined by a 20% decrease to baseline in the ipsilateral cerebral blood flow. 100 μg of pcDNA-NGIs eluted in phosphate-buffered saline (PBS) was intramuscularly injected into the tibial muscle once a week after MCAO for 6 weeks in group A. As control, pcDNA3.1 (+) was also administrated in the same way in group B and nothing was administrated in group C. ② The modified neurological severity score (mNSS), a composite of motor, sensory, reflex and balance tests, was used to test the sensorimotor deficit. The mNSS was graded on a scale of 0–18, i.e. normal score was 0, maximal deficit score was 18, and 1 point was warded for the inability to perform the tasks or the lack of a tested reflex. ③ The newly generated axons of corticorubral projection were traced by stereotaxic guided injection of 100 g/L biotinylated dextran amine. Rats were sacrificed two weeks after tracing, and cryostat coronal sections of midbrains (30 μm) were reacted to BDA according to the manufacturer's instruction by the free-floating method. Images were captured on a DM RXA2 LEICA Microscope with a Spot Digital Camera system (Germany), and the numbers of labeled axons on the denervated side in four standard coronal sections including the red nucleus were manually quantified. MAIN OUTCOME MEASURES: ① The number of newly generated axons of corticorubral projection. ②The improvement in sensorimotor deficit. RESULTS: All the involved 60 rats entered the stage of final analysis. ① The number of newly generated axons of corticorubral projection of rats: Only ipsilateral axons of CRP were noted with little evidence of fibers crossing to the contralateral red nucleus in rats of groups B and C. More BDA-positive fibers crossing the midline and terminating in the contralateral red nucleus in appropriate target areas mirroring thenon-differentiated red nucleus were found in rats of group A. Quantitative analysis showed that BDA-labeled axons in the denervated side of rats in group A were more than those in group B (P < 0.05). ② Improvement in sensorimotor deficit of rats: At 2 weeks after immunization, significant improvement in sensorimotor deficit was found in rats of group A. There were significant differences of improvement in sensorimotor deficit of rats between group A and group B or group C at 12 and 14 weeks after immunization (P < 0.05). CONCLUSION: ① Poststroke DNA immunization against NGIs leads to increased sensorimotor recovery following FCI and compensatory newly growth of axons from corticorubral projection.