| Objective: To explore the feasibility of MRI(Magnetic Resonance Imaging)in the evaluation of the therapeutic effect of recombinant human MANF(Mesencephalic Astrocyte derived Neurotrophic Factor)on ischemia/reperfusion injury in rats dynamically.Methods: Recombinant MANF protein was expressed in E.coli BL21 using 1 m M IPTG induction and purified using a Ni2+-affinity column.The purity was evidented by SDS-PAGE,and the concentration was measured by BCA kit.The focal cerebral ischemia/reperfusion injury models were estabilished successfully and the human recombinant MANF protein was injected intracerebroventricularlly and intravenously at3 h postreperfusion when obvious injury was observed.Besides,edaravone(3 mg/kg)was selected as for the positive control.MRI was conducted at 1 d,2 d,3 d,5 d and 7 d after MANF protein injected.Combined with MRI technology,mortality,body weight,neurological score,TTC Staining and immunohistochemistry were also used to evaluate the therapeutic effect of recombinant human MANF on ischemia/reperfusion injury in Rats.The results of infarct size and signal strength on MRI were analyzed by using MRIcro 2.0 software,and the infarction volume based on TTC staining results are analyzed by using Image J software.All the results were analyzed by using SPASS 16.0software to determine the significant difference.Results: Rat focal ischemic models were established by using middle cerebral artery occlusion(MCAO).The Bederson's score of ischemia rat was 3 points,the rats couldn't unbend the contralateral forelimb and rotate to the opposite side;MRI has been performed to investigate the dynamics of lesion formation at the early period and the signal intensity of right hemisphere has significantly increased at 3 h post-reperfusion,which provides the basis for the time point of MANF administration.The human recombinant MANF protein was injected intracerebroventricularlly and intravenously at3 h pos-treperfusion.Besides,edaravone is a free radical scavenger and has neuroprotective effects on acute cerebral infarction,which was selected as the positive control,PBS as the negative control.MRI was conducted at 1 d,2 d,3 d,5 d and 7 d after MANF protein injected.The MRI images showed that the volume of cerebral infarction in the lateral ventricle of rats(45.31±21.13)was lower than in the tail vein group(97.86±8.64)on the third day(P<0.05);We also found that MANF(10 or 20 ?g)reduced the injury induced by ischemia/reperfusion,and the therapeutic effect of 20 ?g MANF protein treatment was more effective than that of 10 ?g MANF protein treatment.The body weight of rats treated with MANF(10 and 20 ?g)increased to a significant value compared with the PBS group.MANF protein also significantly increased the survival of rats,and played a key role in improving neurological function in ischemia/reperfusion rats.Quantitative analysis showed that infarct volume calculated from T2-weighted image and TTC staining in MANF groups significantly reduced as compared with PBS group on the seventh day after drug treatment.MRI manifestation was confirmed by histologic analyses in evaluating the therapeutic effect of MANF on focal cerebral ischemia.Additionly,the number of Neu N-positive neurons in the MANF groups was larger than that in the PBS group.Conclusions: The present study uniquely reported a dynamic MRI study that monitors and evaluates the effect of MANF protein on ischemia/reperfusion injury in rats with MCAO.Our data demonstrated that intracerebroventricular injection of MANF protein at 3h after reperfusion could reduce the infarct volume,increase the survival rate after ischemia/reperfusion injury,and speed up the locomotor functional recovery.MRI manifestation was confirmed by histologic analyses and TTC staining,which suggest that MRI was an effective method in evaluating the therapeutic effect of MANF on focal cerebral ischemia,... |
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