The Effect And Mechanism Of RADA16-I And CDNF In The Neurogenesis And Neuroprotection Of Cerebral Ischemic Injury

BackgroundStroke is one of the most common cardiovascular and cerebrovascular diseases,with a high incidence and many sequelae.It is mainly divided into hemorrhagic stroke and ischemic stroke.The current treatment methods still have some limitations.For example,traditional Chinese herbal medicines have toxic side effects,and tissue plasminogen activators are only used in the acute phase.Traditional wisdom holds that nerve cell death can cause irreversible brain damage.However,in recent years,it has been found that brain injury can stimulate neurogenesis and repair of the central nervous system in adult mammals over a period of several months,which makes it particularly important to find effective drug treatment in the chronic phase of stroke If a viable method is selected to stimulate the neurogenesis of endogenous neural stem cells,which can migrate to the ischemic penumbra and differentiate,and generate new neurons instead of apoptotic cells,the effect of nerve repair can be achievedScaffold materials,neurotrophic factors,and seed cells,as the three elements of neural tissue engineering,play an important role in the field of regenerative medicine Hydrogel was first proposed by Zhang et al.as early as 1993.Because of the good histocompatibility,it can provide cells with a three dimensional growth environment and become an excellent scaffold material.In 1999,the research team discovered that RADA16-I can trigger its self-assembly under appropriate conditions to form a three-dimensional network structure,which is conducive to cell adhesion and growth Cerebral dopamine neurotrophic factor was discovered by Lindholm et al.in 2007.It contains 187 amino acids and has two unique domains.CDNF,as a new type of neurotrophic factor,can provide nutrition to nerve cells and play an important role in the growth of neurons.A large number of literatures have reported that CDNF is effective for the treatment of Parkinson's disease and has a good neural repair effect.Neural stem cells,as a kind of seed cells,have become important cells for studying the central nervous system because of their characteristics of migration,differentiation,immunogenicity,and wide sourceAfter cerebral ischemia-reperfusion injury,methods need to be found to stimulate neurogenesis and improve neuroprotection.It has been reported in the literature that RAD A16-1 hydrogel is effective for the regeneration of recurrent laryngeal nerves.CDNF can reduce damage to midbrain dopaminergic neurons in the PD model.In addition,CDNF can achieve neuroprotection by reducing endoplasmic reticulum stress of neurons in cerebral ischemic injury.These suggest that RADA 16-1 and CDNF have the effect of promoting neurogenesis and neuroprotection,but in the cerebral ischemia-reperfusion injury model,whether they have effects on neurogenesis and neuroprotection has not been reported.In the lateral ventricle,the microinjection scaffold material RADA 16-I and the neurotrophic factor CDNF were used to explore the effects of both on the characteristics of neural stem cellsObjective:1?Effects of RADA 16-1 and CDNF on neurogenesis in cerebral ischemia-reperfusion injury.2?Effects of RADA 16-I and CDNF on the proliferation and differentiation of neural stem cells in vitro.3?Neuroprotective effects of RADA 16-1 and CDNF on cerebral ischemia-reperfusion injury4?The molecular mechanism of CDNF for neurogenesis and neuroproteciton in cerebral ischemia-reperfusion injury.Methods:1.MCAO modelA modified middle cerebral artery occlusion(MCAO)model was prepared using a modified suture method.After 2 hours of ischemia,the suture was removed to achieve cerebral ischemia-reperfusion2.MicroinjectionRats were anesthetized by intraperitoneal injection of chloral hydrate.After no righting reflex,the rat was fixed on a stereotaxic apparatus.By adjusting the ear stick and the height of rat head,the difference of Z value between the anterior condyle point and the herringbone point does not exceed 0.05.Then adjust the positioning pin to the bregma,and return the X,Y,and Z values to zero as the reference zero point.Move the positioning pin to the designated position and mark,and drill with a skull drill.Move the positioning pin to the designated position and mark,and drill with a skull drill.Sterile distilled water,10 ?g RADA16-I,6 ?g CDNF,and 10 ?g RADA 16-1+6 ?g CDNF were injected into the lateral ventricle of rat brain3.BrdU labeling and immunofluorescence histochemical stainingEstablishment model of cerebral ischemia-reperfusion in rats.After 3 days,the rats were administered with 5-bromo-2-deoxyuracil nucleoside(BrdU)at a concentration of 10 mg/mL at a dose of 50 mg/kg for 7 consecutive days.On the 11th or 25th day after the administration,the rats were perfused for brain extraction The brains were fixed in 4%PFA solution for 24 hours,and then dehydrated with 10%,20%,and 30%sucrose solution.Finally,the frozen section was cut into 40 ?m slices,and the brain slices at the specified positions were taken for immunofluorescence staining experiments.Antibody source properties and concentrations used were as followd:BrdU(Sheep,1:500),nestin(Ms,1:500),DCX(Rb,1:500),NeuN(Ms,1:250),GFAP(Rb,1:500),Caspase-3(Rb,1:500).4.TTC staining and calculation of infarct volume percentageRats were decapitated.The brain was cut into 2 mm brain slices with a razor blade and placed in a 2%TTC(PBS as solvent)solution prepared in advance Incubate in a 37? oven for 30 min.Scan the front and back with a scanner and save the picture,and analyze the ischemic infarct volume with Photoshop.Ischemic infarct volume percentage=(white infarct area � thickness)/(full-thickness area � thickness)�100%.5.Protein extraction and SDS-PAGESelect the tissues of rat SVZ brain area and ischemic penumbra area,grind and lyse the tissue to extract protein.The expression of related proteins in the pathway was detected by immunoblotting6.Neurological deficit scoreAccording to the five-point method of Longa and Bederon,rats were scored for neurological deficits 24 h after cerebral ischemia and reperfusion.Normal performance,no neurological damage,recorded as 0 points;tail suspension observation,contralateral forelimbs of the contralateral operation can not be fully extended,recorded as 1 point;circle to the contralateral side of the operation,can not walk straight,recorded as 2 points;dump to the contralateral side of the operation,with walking disturbance,recorded as 3 points;loss of consciousness,unable to walk autonomously,recorded as 4 points.Rats with 1 to 2 points were selected for subsequent experiments.7.Primary neural stem cells cultureThe forebrain tissues of embryo on day 13.5 from pregnancy SD rats were taken and digested into single cells.Cells were plated in a 6 cm dish with appropriate amount of neural stem cell culture medium in a 37?,5%CO2 incubator.The medium was changed every 3 days and cells were passaged every 5 days.8.The proliferation and differentiation of neural stem cellsAbout 3,000 cells were seeded in a 24-well plate and divided into four grroups randomly.On the fifth day of the experiment,neurospheres were photographed to count the numbers and measure the diameters of each groupNeurospheres cultured in vitro for 5 days were inoculated into 24-well plates,dividing into four groups and adding with differentiation medium to induce differentiation.Immunofluorescent staining was performed on the 7th day of the experiment.The antibody source properties and concentration used were as followd:GFAP(Rb,1:300),Tuj1(Ms,1:200)Results:1.Effects of RADA16-I and CDNF on neurogenesis in cerebral ischemia-reperfusion injury1.1 Injecting RADA16-I and CDNF recombinant proteins into the lateral ventricle could promote the cell proliferation in SVZThe rat brain ischemia-reperfusion model was established and randomly divided into four groups.After 2 h of ischemia/70 h of reperfusion,sterile distilled water,10?g RADA 16-1,6 ?g CDNF,and 10 ?g RADA 16-1+6 ?g CDNF were injected into the lateral ventricle by a stereotaxic apparatus.On the eleventh day after the administration,the rats were perfused to fix the brain.Selected frozen slices with SVZ brain regions were co-stained with BrdU and nestin,BrdU and DCX.The results showed that the three groups of RADA 16-1,CDNF,RADA 16-I+CDNF could significantly increase the number of neonatal neural stem cells and neonatal neural progenitor cells compared to the CON group.1.2 Injecting RADA16-I and CDNF recombinant proteins into the lateral ventricle could promote cell migration to the ischemic penumbra areaThe rat brain ischemia-reperfusion model was established and randomly divided into four groups.After 2 h of ischemia/70 h of reperfusion,sterile distilled water,10?g RADA 16-I,6 ?g CDNF,and 10 ?g RADA 16-I+6 ?g CDNF were injected into the lateral ventricle by a stereotaxic apparatus.On the eleventh day after the administration,the rats were perfused to fix the brain.Selected frozen slices with the ischemic penumbra were co-stained with BrdU and DCX.The results showed that the three groups of RADA16-I,CDNF,RADA16-I+CDNF could significantly promote the cell migration to the ischemic penumbra area compared to the CON group1.3 Injecting RADA16-I into the lateral ventricle could promote the differentiation of neural stem cells into neuronsThe rat brain ischemia-reperfusion model was established and randomly divided into four groups.After 2 h of ischemia/70 h of reperfusion,sterile distilled water,10?g RADA16-I,6 ?g CDNF,and 10 ?g RADA 16-I+6 ?g CDNF were injected into the lateral ventricle by a stereotaxic apparatus.On the eleventh day after the administration,the rats were perfused to fix the brain.Selected frozen slices with the ischemic penumbra were co-stained with BrdU and NeuN.It was found that the injection of RADA16-I significantly increased the number of neurons compared to the CON group.1.4 Injecting RADA16-I and CDNF recombinant proteins into the lateral ventricle could promote the differentiation of neural stem cell into neuronsThe rat brain ischemia-reperfusion model was established and randomly divided into four groups.After 2 h of ischemia/70 h of reperfusion,sterile distilled water,10?g RADA16-I,6 ?g CDNF,and 10 ?g RADA16-I+6 ?g CDNF were injected into the lateral ventricle by a stereotaxic apparatus.On the 25th day after the administration,the rats were perfused to fix the brain.Selected frozen slices with the ischemic penumbra were co-stained with BrdU and GFAP,BrdU and NeuN.The results showed that in the ischemic penumbra area,the three groups of RADA16-I,CDNF,RADA 16-I+CDNF had no significant effect on the number of astrocytes but all three groups could significantly increase the number of neurons compared to the CON group.2.Effects of RADA16-I and CDNF on the proliferation and differentiation of neural stem cells in vitro2.1 RADA16-I and CDNF could promote the proliferation of neural stem cells cultured in vitroThe forebrain tissues of embryo on day 13.5 from pregnancy SD rats were taken and digested into single cells.Each well of a 24-well plate is seeded with about 3000 cells.Neural stem cell culture medium was added for culture,and the neurospheres in each well were photographed under the inverted microscope on the fifth day.Count the numbers and measure the diameters of neurospheres between 50 and 200 nm.The results showed that the numbers and diameters of neurospheres in the three groups of RADA 16-I,CDNF,RADA 16-I+CDNF were significantly increased compared to the CON group2.2 RADA16-I and CDNF could promote neural stem cell differentiation in vitroThe forebrain tissues of embryo on day 13.5 from pregnancy SD rats were taken and digested into single cells.Cultured with neural stem cell culture medium for 5 days,and the cells were seeded in 24-well plates(24-well plates were added with fly sheet and hydrogel in advance).Neural stem cell differentiation medium were added to induce differentiation.Immunofluorescence staining was performed on the 7th day.The results showed that the proportion of astrocytes and neurons in the three groups of RADA 16-I,CDNF,RADA 16-I+CDNF were significantly increased compared to the CON group.3.Neuroprotective effects of RADA16-I and CDNF on cerebral ischemia-reperfusion injury3.1 CDNF could reduce neurological score after cerebral ischemia-reperfusionThe rat brain ischemia-reperfusion model was established and randomly divided into four groups.After 2 h of ischemia/70 h of reperfusion,sterile distilled water,10?g RADA16-I,6 ?g CDNF,and 10 ?g RADA16-I+6 ?g CDNF were injected into the lateral ventricle by a stereotaxic apparatus.On the 0,1st,2nd,3rd after the administration,the rats were scored for neurological deficits according to the five-point method of Longa and Bederon.The first day after the administration,there was no significant difference between the RADA16-I group and the CON group.The CDNF,RADA 16-I+CDNF groups could significantly reduce the neurological deficit score than the CON group,and there was no significant difference between the two groups.3.2 CDNF could reduce cerebral infarction volume after cerebral ischemia-reperfusionThe rat brain ischemia-reperfusion model was established and randomly divided into four groups.After 2 h of ischemia/70 h of reperfusion,sterile distilled water,10?g RADA16-I,6 ?g CDNF,and 10 ?g RADA16-I+6 ?g CDNF were injected into the lateral ventricle by a stereotaxic apparatus.On the 4th after the administration,rats were decapitated and stained with TTC.The infarct volume was analyzed using Photoshop.The percentage of infarct volume=(white infarct area � thickness)/(full-layer area � thickness)� 100%.The resutls showed that there was no significant difference between the RADA16-I group and the CON group.The CDNF,RADA16-I+CDNF group had a significantly lower percentage of infarct volume than the CON group,and there was no significant difference between the two groups.3.3 CDNF could reduce neuron apoptosis in ischemic penumbra after cerebral ischemia-reperfusionThe rat brain ischemia-reperfusion model was established and randomly divided into four groups.After 2 h of ischemia/70 h of reperfusion,sterile distilled water,10?g RADA16-I,6 ?g CDNF,and 10 ?g RADA16-I+6 ?g CDNF were injected into the lateral ventricle by a stereotaxic apparatus.On the 4th day after the administration,rats were perfused to fix the brain.Selected frozen slices with the ischemic penumbra were stained with Caspase3.The results showed that there was no significant difference between the RADA 16-I group and the CON group;CDNF,RADA 16-I+CDNF could significantly reduce the number of Caspase3 positive cells in the ischemic penumbra compared to the CON group4.The Molecular mechanism of CDNF on neurogenesis and neuroprotection after cerebral ischemia-reperfusion injuryIn order to explore the molecular mechanism of CDNF involved in neurogenesis and neuroprotection after cerebral ischemia-reperfusion injury,establish a rat model of cerebral ischemia-reperfusion.After 2 h of ischemia/70 h of reperfusion,CDNF was injected into the lateral ventricle by a stereotaxic apparatus.The tissues of the ischemic side SVZ and ischemic penumbra were lysed and the protein was extracted.Related proteins in ERK and STAT3 pathway were detected by SDS polypropylene gel electrophoresis.The results showed that CDNF significantly reduced the expression level of p-STAT3 in the SVZ region.In addition,CDNF significantly increased the level of p-ERK in the ischemic penumbra and significantly reduced the expression of p-STAT3.These suggested that CDNF participated in the neurogenesis process in cerebral ischemia-reperfusion injury mainly through the STAT3 signaling pathway,and in neuroprotection through the ERK and STAT3 pathways.Conclusion:1.RADA16-I and CDNF could promote neurogenesis in SVZ brain region in cerebral ischemia-reperfusion injury2.RADA16-I and CDNF recombinant proteins could promote the proliferation and differentiation of neural stem cells in vitro3.CDNF could play a neuroprotective role in cerebral ischemia-reperfusion injury.4.CDNF could regulate neurogenesis and neuroprotection after cerebral ischemia through ERK and STAT3 signaling pathwaysSignificance:1.The role of RADA 16-I and CDNF in neurogenesis in cerebral ischemia-reperfusion injury.2.RADA 16-I and CDNF promote the proliferation and differentiation of neural stem cells in vitro.3.The potential molecular mechanism of CDNF in regulating neurogenesis and neuroprotection in cerebral ischemia-reperfusion injury.