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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 immune repair mechanisms of body. OBJECTIVE: To evaluate the effect of pre-stroke DNA immunization against NGIs on the rehabilitation for sensorimotor function of rat models of focal cerebral ischemia. DESIGN: A completely randomized 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 body mass from 180 to 250 g were provided by the 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 the 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. ① The involved rats were randomized into 3 groups: model group (group A), pcDNA3.1(+) group (group B) and pcDNA-NGIs group (group C), with 20 rats in each group. Left focal cerebral ischemia was permanently induced through middle cerebral artery occlusion with the assistance of an operating microscope. Successful middle cerebral artery occlusion 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 before middle cerebral artery occlusion for 6 weeks in group C. As control, pcDNA3.1 (+) was also administrated in the same way in group B and nothing was administrated in group A. 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 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 (BDA). 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; ② The improvement of the sensorimotor deficit. RESULTS: All the involved 60 rats entered the final analysis. ① The number of newly generated axons of corticorubral projection of rats: Only ipsilateral axons of corticorubral projiction were noted with little evidence of fibers crossing to the contralateral red nucleus in rats of groups A and B. More BDA-positive fibers crossing the midline and terminating in the contralateral red nucleus in appropriate target areas mirroring the non-differentiated red nucleus were found in rats of group C. Quantitative analysis showed that BDA-labelled axons in the denervated side of rats in group C were more than those in group B (P < 0.05). ② The improvement of the sensorimotor deficit: At two weeks after middle cerebral artery occlusion, significant improvement in sensorimotor deficit was found in rats of group C. There was significant difference of improvement in sensorimotor deficit of rats between group C and group B or group A at eight and 10 weeks after middle cerebral artery occlusion (P < 0.05). CONCLUSION: Pre-stroke DNA immunization against NGIs led to increased sensorimotor recovery following focal cerebral ischemia and compensatory newly growth of axons from corticorubral projection.
BACKGROUND: Inhibitory signals, ie 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 immune repair mechanisms of body. OBJECTIVE: To evaluate the effect of pre-stroke DNA immunization against NGIs on the rehabilitation for sensorimotor function of rat models of focal cerebral ischemia. SETTING: Brain Injury Research Laboratory, Department of Neurosurgery, National Neuroscience Institute, Singapore. MATERIALS: Sixty adult male Sprague-Dawley rats ranging from 45 to 120 days and in body mass from 180 to 250 g were provided by the 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 the 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. ① The involved rats were randomized into 3 groups: model group (group A), pcDNA3.1 (+) group (group B) and pcDNA-NGIs group (group C) with 20 rats in each group. Left focal cerebral ischemia was permanently induced through middle cerebral artery occlusion with the assistance of an operating microscope. Successful middle cerebral artery occlusion 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 before middle cerebral artery occlusion for 6 weeks in group C. As control, pcDNA3 .1(+) was also administrated in the same way in group B and nothing was administered in group A. 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 0-18, ie 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 (BDA). 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; ② The improvement of the sensorimotor deficit. RESULTS: All the involved 60 rats entered the final analysis. ① The number of newly generated axons of corticorubral projections of rats: Only ipsilateral axons of corticorubral projiction were with little evidence of fibers crossing to the contralateral red nucleus in rats of groups A and B. More BDA-positive fibers crossing the midline and terminating in the contralateral red nucleus in appropriate target areas mirroring the non- differentiated red nucleus were found in rats of group C. Quantitative analysis showed that BDA-labeled axons in the denervated side of rats in group C were more than those in group B (P <0.05). ② The improvement of the sensorimotor deficit: At two weeks after middle cerebral artery occlusion, significant improvement in sensorimotor deficit was found in rats of group C. There was significant difference of i mprovement inSensorimotor deficit of rats between group C and group B or group A at eight and 10 weeks after middle cerebral artery occlusion (P <0.05). CONCLUSION: Pre-stroke DNA immunization against NGIs led to increased sensorimotor recovery following focal cerebral ischemia and prompt newly growth of axons from corticorubral projection.