| In China and the Western world，stroke is the first or the second cause of death anddisability. Moreover, the survivors suffered the disability such as hemiplegia and aphasiawhich seriously affect the quality of life after stroke in China. At present, theadministration tissue type plasminogen activator is thought as an effective treatmentagainst cerebral ischemic stroke. But only10%of patients are able to receive thisbeneficial therapy due to its narrow therapeutic window (<4.5h). For now, the approbatorytreatment of disability caused by stroke is rehabilitation exercise. Thus, the most importantis seemed to solve the permanent dysneuria fundamentally.The Nogo-A protein which brings a hope for the treatment of dysneuria has animportant role in inhibiting axonal remodeling after central nervous system (CNS) injuries.Inhibition of NgR1which is Nogo-A receptor could promote axonal regeneration. Ourprevious study showed that TAT-M9which had a special structure domain of Nogo-Acould inhibit oxidative damage and cell apoptosis in vivo and in vitro. At the same time, TAT-NEP1-40(antibody of NgR1) could enhance synaptic plasticity and acceleratefunctional recovery. Recently reported, the content of neuronal Nogo-A was transientreduced in adult rats at7d, ratchets up later in ischemic damage areas and high expressedat28days after stroke. According to the spatial characteristics of Nogo-A expression, weused TAT-M9at acute stage to anti-apoptotic, and used TAT-NEP1-40when the expressionof Nogo-A increase to promote neuritis outgrowth. Lucubrate the long termneuroprotection of sequential treatment with TAT-M9and TAT-NEP1-40, and provide anew treatment for ischemic stroke.PirB is also a receptor of myelin proteins. The previous study found that there wasmore axon regeneration after knockout PirB than knockout NgR1. It seems that PirB has amore important role in inhibition of myelin proteins, which remind us that PirB may beanother treatment target of rehabilitation after stroke. To inhibit the function of PirB, weused the extracellular domain of PirB to synthesize PEP protein. But PEP which has alarge molecular weight cannot trought the BBB. Thus, we used protein transductiontechnology fusing with PEP and acquired TAT-PEP fusion protein that could across BBB.We used methods of immunohistochemistry, western blot and behavioristics toinvestigate the long term neuroprotection of sequential treatment with TAT-M9andTAT-NEP1-40and injection of TAT-PEP individually in mice of bilateral common carotidartery occlusion (BCCAO) model and oxygen and HT22cell in glucose deprivation (OGD)model, intend to find a new targets and medicine and a new way for the treatment ofischemic cerebral insult.Experiment1. Protective effect of TAT-M9againstischemic cerebral insult in acute stage in miceObjective: To observe the neuoprotection of TAT-M9after7d of BCCAO in miceMethods: C57BL/6mice were randomly divided to three groups after BCCAO：Sham group (sham operation group, n=6), Control group (normal saline, n=6), TAT-M9group(10mg/kg, n=6). The drug was intraperitoneally injected immediately after reperfusion atthe first time and continuing injected for7d. The mice were executed at6h after injectionand investigated whether the drug acrossed the BBB by immunofluorescence. Motorfunction after7d of BCCAO mice was evaluated by TMS. The viable neurons andTUNEL-positive cells were assessed by HE staining and TUNEL staining after7dtreatment, while the expression of Bcl-2and Bax was measured by western blot inhippocampus.Result: We found that TAT-M9could through BBB and express at the ischemia insultarea. TAT-M9enhanced TMS scroes after7d of BCCAO mice (P<0.01). The viableneurons were increased after TAT-M9treatment (P<0.01). TUNEL-positive cells and theratio of Bax/Bcl-2were decreased (P<0.01).Conclusion: Our results indicated that TAT-M9could through BBB and express inneurons. TAT-M9treatment not only had an anti-apoptotic effect and promoted neuronsurvival, but also reduced neuronal loss and improved early motor function recovery.Experiment2. Neuroprotective effect of sequential treatment withTAT-M9and TAT-NEP1-40against cerebral ischemia in miceObjective: To investigate the long term neuroprotective effect of sequential treatmentwith TAT-M9and TAT-NEP1-40after cerebral ischemia.Methods: C57BL/6mice were randomly divided to four groups after BCCAO：Congroup (0.5ml normal saline every day for28d, n=30), TAT-M9group(10mg/kg TAT-M9every day for first7days, n=30), TAT-NEP1-40group (6mg/kg TAT-NEP1-40for last21days, n=30), TAT-M9+TAT-NEP1-40group(10mg/kg TAT-M9every day for the first7days and6mg/kg TAT-NEP1-40for last21days, n=30). The short memory of BCCAOmice was measured by step-down passive avoidance test, the elevated plus maze and fear conditioning. Open-field test was used to evaluate the motor function of BCCAO mice.Morris water maze test and T-maze were taken to test the long term memory after28d.The ability of TAT-NEP1-40that acrossed the BBB after6h injection and the axon growthof neuron in four groups were assessed by immunofluorescence. Western blot was used todetect the expression of axon growth-associated factors (Tau, GAP43and MAP2).Result: The study showed that TAT-NEP1-40could through BBB and express at theischemia insult area. Sequential treatment with TAT-M9and TAT-NEP1-40improved thecontent of Tau, GAP43and MAP2(P<0.01), enhanced the axon growth of neuron inhippocampus. Sequential treatment with TAT-M9and TAT-NEP1-40improved short termspatial learning and memory ability (P<0.01), context and cue memory ability (P<0.05).This therapeutic method could also enhance the long term spatial learning and memoryability (P<0.01), working memory and reference memory ability (P<0.01).Conclusion: Sequential treatment with TAT-M9and TAT-NEP1-40could enhance theaxon reconstruction after cerebral ischemia. Moreover, the short and long term spatiallearning and memory ability was improved after treatment, promoted neurologicalfunction recovery after ischemic injury.Experiment3. Cytoprotective effect of TAT-PEP against HT22cellsexposed to OGDObjective: To comfirmed the axonal regenerate effects of TAT-PEP on HT22cellsexposed to OGDMethods: Using MTT to measure the effective concentration of TAT-PEP with HT22cells. HT22cells were divided to three groups: Sham group, OGD group, OGD+TAT-PEPgroup. At24h after OGD, the axonal regeneration of HT22cells was observed byimmunofluorescence.Result: we found that the quantity of viable cell peaked when the concentration of TAT-PEP was150μg/L, and TAT-PEP could significant enhanced the axonal regenerationof HT22cells after OGD.Conclusion: TAT-PEP enhanced the axonal regeneration of HT22cells in vitro.Experiment4. The long-term treatment effects of TAT-PEP on cerebralischemic injury and neurological functionObjective: To evaluate the long term neuroprotection of TAT-PEP after ischemia injury.Methods: C57BL/6mice were randomly divided to three groups after BCCAO：Shamgroup(sham operation group, n=40), Control group(normal saline, n=40), TAT-PEPgroup(150μg/kg, n=40). The drug was intraperitoneally injected for28d. Observing theability of TAT-PEP across the BBB at6h,24h,48h after injection. The number of viableneurons was counted by Nissl staining and HE staining, TUNEL staining was used tomeasure the apoptosis in injury area and FJC staining was used to evaluate degeneration ofneurons after7d treatment. Western blot was used to detect the expression of axongrowth-associated factors (Tau and GAP43). The axon growth of neuron was assessed byimmunofluorescence. The short memory of BCCAO mice was measured by step-downpassive avoidance test, the elevated plus maze and fear conditioning. Open-field test wasused to evaluate the motor function of BCCAO mice. Morris water maze test and T-mazewere taken to test the long term memory after28d.Result: The result showed that TAT-PEP could through BBB and express at theischemia insult area. The viable neurons were increased after TAT-PEP treatment (P<0.01),TAT-PEP also increased the Nissl-positive cells (P<0.01). At the same time,TUNEL-positive cells FJC-positive cells were decreased (P<0.01) after TAT-PEPtreatment. TAT-PEP could improve the content of Tau (P<0.05) and GAP43(P<0.01),enhance the axon growth of neuron in hippocampus. TAT-PEP could improve short termspatial learning and memory of mice (P<0.05, P<0.01), context and cue memory ability (P<0.05). TAT-PEP could also enhance the long term spatial learning and memory ability(P<0.05, P<0.01), working memory and reference memory ability (P<0.05, P<0.01).Conclusion: Our results indicated that TAT-PEP could through BBB and inhibitedapoptotic and degeneration of neurons, promoted neuron survival and reduced neuronalloss at acute stage. TAT-PEP enhanced the axon reconstruction after cerebral ischemia.The short and long term spatial learning and memory ability was improved after treatment,promoted neurological function recovery after ischemic injury.All these experiment proved that TAT-M9, TAT-NEP1-40and TAT-PEP could throughBBB. TAT-M9had an anti-apoptotic effect and promoted neuron survival, reducedneuronal loss and improved early motor function recovery after ischemia injury.Sequential treatment with TAT-M9and TAT-NEP1-40enhanced the axon reconstructionafter cerebral ischemia, promoted the short and long term spatial learning and memoryability, remarkably strengthen neurological function after ischemic injury. TAT-PEP notonly inhibited apoptotic and degeneration of neurons, promoted neuron survival andreduced neuronal loss at acute stage, but also enhanced the axon reconstruction aftercerebral ischemia, improved the short and long term spatial learning and memory ability atlate stage after ischemic injury. The present study provided a new therapeutic targets andtreatment methods and drugs for patients with dysneuria. |
PDF Full Text Request