Neuroprotective Effects Of MSC-EV Against Early Brain Injury And Cognitive Function Impairments After Subarachnoid Hemorrhage

BackgroundSubarachnoid hemorrhage(S AH)is a life-threatening neurological disease with high mortality and disability rates.However,those patients who survive acute bleeding and rebleeding often suffer from cognitive deficits with decreased life quality for years after SAH.Increasing evidence indicates that early brain injury(EBI)may account for the poor outcomes observed in SAH patients.Recently,an increasing amount of attention has been paid to mesenchymal stem cell(MSC)-derived EVs,as messengers that function in intercellular communication and promising therapeutic vesicles used for regeneration medicine,which are believed to be able to replace MSC cell therapy.In addition,theoretically,using secreted EVs rather than cells themselves could overcome some safety problems with MSC therapy.EVs contain proteins,lipids,mRNAs and noncoding RNAs that can modulate the pathophysiological state of receiver cells.MSC EV-based therapy has shown promise in experimental models of liver injury,renal injury,acute myocardial infarction,wound healing and so on.MSC cell therapy has been used in the restorative process after stroke and tra?matic brain injury(TBI)since the early 2000s.Additionally,much evidence has suggested that neuroprotective effects are mediated by EVs secreted by MSCs.For example,Michael Chopp et al.demonstrated that MSC EVs could be taken up by neural cells.These miR-133b-enriched EVs could help restore neurological function.However,only a few studies concerning the role of MSC therapy in SAH have been reported,and these have demonstrated that the neuroprotective effects partly resulted from the alleviation of microglia-mediated neuroinflammation.However,whether MSC EVs play a part in SAH therapy remains unclear.MiRNA is one of the important cargoes encapsulated in EVs.These miRNAs serve as messengers for intercellular communications,biomarkers for clinical diagnosis and prognosis,and therapeutic agents for diseases.Bache et al.reported that miRN A profile changed after SAH,while they didn't explore the mechanisms of miRNA upregulation.The aim of this study was to investigate the therapeutic potential of MSC EVs in SAH,especially for cognitive function recovery,and to explore the underlying mechanisms.We demonstrated that MSC EVs can alleviate EBI and improve cognitive function after SAH and that the mechanism involved mainly depends on the transfer of MSC-derived EV-derived miR-21-5p to neurons to activate AKT signaling to inhibit apoptosis.Part ?:Neuroprotective effects of MSC-EV against early brain injury after Subarachnoid hemorrhage1.Objectives:(1)To isolate and characterize MSC-EV.(2)To investigate the effect of MSC-EV on early brain injury after subarachnoid hemorrhage.(3)To clarify the role of MSC-EV on alleviating cognitive impairments after subarachnoid hemorrhage.(4)To identify the target cell type of MSC-EV in SAH-insulted rats.(5)To reveal the mechanism of MSC-EV inhibiting neuronal apoptosis.2.Methods:(1)Set up experimental SAH model using endovascular perforationAdult male SD rats weighing 300-320 g were anesthetized through an intraperitoneal injection of chloral hydrate(340 mg/kg body weight).After the left carotid artery and associated branches were dissected,a 3-0 monofilament nylon suture was inserted into the stretched external carotid artery,then guided into the internal carotid artery wall and was withdrawn following the perforation.Rats in the sham group underwent the same surgical procedure without perforation.(2)Isolation and characterization of MSC-EVBone marrow-derived MSCs were cultured with a-modified MEM medi?um containing 10%fetal bovine ser?m(FBS)and penicillin-streptomycin in 175 cm2 tissue culture flasks.For EVs isolation,we replaced the conventional culture medi?m with 20 ml medi?m containing 10%EV-depleted FBS when the cells reached 60%-80%confluence.Following an additional 48 hours of culturing,the media were then collected for centrifugation.The EVs were stored at-80?.The concentration of EVs was determined by using Enhanced BCA Protein Assay Kit.To determine the size distribution of the EVs,nanoparticle tracking analysis was performed using the qNano system on samples diluted with PBS.And antibodies of EV markers and negative controls were used for Western blot analysis(3)GroupingAdult male rats were randomly assigned into three groups:sham,SAH and SAH+MSC-EV.100ug MSC-EV was slowly injected into the caudal vein of each SAH+MSC-EV rat,while equal vol?me of PBS was administered to every rat in SAH group.(4)Mortality and Neurological assessmentMortality was recorded everyday and at 48h after SAH,modified Garcia scoring system was used to assess neurological function of every group of rats.(5)Pathological Histology determinationAll rats were deeply anaesthetized and perfused with 4%paraformaldehyde for sectioning.HE and Nissl staining were performed respectively to determine pathological changes.Meanwhile,TUNEL staining and cleaved-caspase-3/NeuN double staining were performed to detect neuronal apoptosis.(6)Detection of apoptosis-related proteins and pathwayAfter sacrificing animals,prefrontal lobe and hippocampus were respectively collected to extract total protein.Then the expression levels of apoptosis-related proteins were measured by Western blot.(7)Morris water mazeThe Morris Water Maze(MWM)test was performed to evaluate cognitive functional impairment in a relatively long term after SAH.Two weeks after perforation,a four-day hidden platform test and a one-day probe test were performed successively.(8)EV tracing assayEVs dyed with PKH67 were administered to SAH-suffered rats.The brain tissue slides were obtained from frozen sectioning and stained with neuronal markers for observation under microscopy.3.Results:(1)MSC-EV isolated from ultracentrifuge meets the qualifications of EVqNano analysis showed that the diameters of isolated EVs ranged from 50 to 200 nm.Moreover,Western blot showed that EV markers were highly-expressed in isolated EVs and the cellular marker calnexin was rarely expressed.(2)MSC-EV alleviates early brain injury after SAHBrain injury was also determined by HE staining and Nissl staining.Many more neurons exhibited a contracted appearance in the SAH group,while MSC-EV alleviated this type of damage.The brain water content also decreased after MSC-EV administration,indicating that brain edema resulted from SAH was alleviated by MSC-EV(3)MSC-EV alleviates neurological impairments after SAH and improve cognitive function recovering.Compared with the SAH+PBS group,the SAH+EV group achieved higher neurological scores at 48 hours after SAH insult,implying improved neurological performance.In Morris water maze test,MSC-EV shortened the time spent seeking the hidden island and extended the time staying in the island zone.(4)MSC-EV is taken up by damaged neurons after SAH,inhibiting neuronal apoptosis.EV tracing assay using PKH67-labelled MSC-EV revealed that fluorescent signal mainly located in neurons and also distributed in liver,kidney and spleen.Moreover,TUNEL staining and NeuN/Caspase3 staining showed that the n?mber of total apoptosis cells and apoptotic neurons were reduced in SAH+EV group compared with SAH group.Western blot also showed that both in prefrontal lobe and hippocampus the expression of Bcl-2 increased as a result of the effects of MSC-EV.while the expression of Bax and cleaved-caspase-3 decreased compared with that in the SAH-PBS group.4.Conclusions:(1)MSC-EV isolated from ultracentrifuge meets the qualifications of EV(2)MSC-EV alleviates early brain injury after SAH(3)MSC-EV alleviates neurological impairments after SAH and improve cognitive function recovering.(4)MSC-EV is taken up by damaged neurons after SAH,inhibiting neuronal apoptosis.Part ?:miR-21 mediated neuroprotective effects of MSC-EV after SAH1.Objectives:(1)To explore the mechanism of neuroprotective effects of MSC-EV after SAH.(2)Combining sequencing data and online database,to identify the key cargo in MSC-EV.(3)To reveal the alteration of miR-21 expression level in SAH rat.(4)To clarify the effects of EV-derived miR-21 and the downstream signaling pathway in SAH.2.Methods:(1)EV-derived RNA sequencing MSC conditioned medi?m was collected for ultracentrifuge to isolate MSC-EV.Then total RNA was extracted from MSC-EV and constructed into a library for sequencing.After sequencing,we screened and annotated all reads obtained.(2)Data analysis of miRNA sequencing from SAH patients' CSF The data of miRNA sequencing from SAH patients' CSF was downloaded from exRNA atlas database and marked up.Then miRNA expression was recorded to identify potential SAH-related miRNA.(3)Establishing a rat SAH model of time course.Male SD rats were randomly divided into eight subgroups:sham,3,6,12,24,48,72 hours and one week post SAH.Each group of rats was sacrificed at the indicated time post SAH for RNA extraction from the prefrontal cortex and hippocampus.(4)Administration of MSC-EV(-21)or MK2206 to rat SAH model.Male adult SD rats were randomly divided into the following groups:sham,SAH+PBS,SAH+MSC-EV,SAH+MSC-EV(-21)and SAH+MSC-EV+MK2206.miR-21-5p inhibitors was transfected into cultured MSCs and then MSC-EV(-21)was collected from these transfected MSCs' conditioned medi?m.MSC-EV(-21)was given to SAH rats through the caudal vein injection and MK2206 was administered at 100ug per rat dissolved in DMSO through stereotaxic injection into lateral ventricle.(5)In vitro model of SAH.Primary cultured neurons were treated with OxyHb and then used for SAH model in vitro.miR-21 expression in both neuron and neuron-derived EVs were respectively measured using qPCR.Moreover,miR-21 expression was also determined with treatment of MSC-EV or GW4869.SAH-induced neuronal apoptosis was assessed by TUNEL staining and Western blot detection of apoptosis-related proteins.(6)Neurological scoring and assessment of neuronal apoptosis in vivo.Each group of rats was assessed for neurological function using modified Garcia system at 48 h after SAH.Then all rats were deeply anaesthetized and sacrificed for total protein extraction or paraformaldehyde perfusion.In this way,neuronal apoptosis was detected by Western blot,HE staining,TUNEL assay and double immunostaining of NeuN and cleaved caspase-3 in vivo.3.Results:(1)miR-21 was highly enriched in MSC-EV and also increased in SAH patients' CSF.High-throughput sequencing data showed that microRNA accounted for a important part of EV cargoes and more than half of microRNAs was in mature form.Further analysis showed that miR-21,miR-let-7i,let-7c were in abundance.However,according to sequencing data derived from SAH patient cerebrospinal fluid(CSF)from the exRNA Atlas,miR-21-5p expression increased significantly after SAH compared with that found in healthy donors,indicating that miR-21 participated in pathological progress of SAH and MSC-EV may exert its neuroprotective effects through transferring miR-21.(2)The expression of miR-21 was upregulated in prefrontal lobe and hippocampus of SAH rats.qPCR showed that the expression level of miR-21-5p increased and peaked at 3 h post SAH.However,it was decreased at 3 h and increased at 12 h in the hippocampus.The expression and distribution of miR-21-5p was further identified by FISH 48 h after SAH.(3)The expression of miR-21 was upregulated in neuron-derived EVs,leading to enhanced neuronal apoptosis.qPCR showed that OxyHb increased miR-21 expression in neurons and neuronal EVs and that miR-21 upregulation was greater in neuronal EVs than in neuronal cytoplasm.Administration of GW4869 or MSC-EV could increase the expression of miR-21-5p in neuronal cytoplasm,which further inhibited neuronal apoptosis resulted from OxyHb treatment.(4)miR-21 mediated neuroprotective effects of MSC-EV in SAH.Neurological assessment demonstrated that rats in SAH+MSC-EV(-21)group received lower scores than those in SAH+EV group.Furthermore,TUNEL assay showed that more neurons went through apoptosis in SAH+MSC-EV(-21)group than in SAH+EV group.And Western blot results showed that the expression of Bcl-2 and PTEN decreased as a result of the effects of MSC-EV(-21)and that the expression of Bax and cleaved-caspase-3 increased compared with SAH+EV group.(5)miR-21 exerted its neuroprotective effects through PTEN/Akt pathway.HE staining showed that MK2206 inhibited the neuroprotective effects of MSC-EV and cortical neurons became more contracted in shape.TUNEL staining and double staining of NeuN/Caspase 3 showed that MK2206 also increased apoptotic neurons.Moreover,Western blot revealed that AKt phosphorylation and Bcl2 expression were decreased and that the expression of Bax and active caspase3 was increased with the treatment of MK2206 in vivo.4.Conclusions:(1)MiR-21 was highly enriched in MSC-EV and also increased in SAH patients'CSF.(2)The expression of miR-21 was upregulated in prefrontal lobe and hippocampus of SAH rats.(3)The expression of miR-21 was upregulated in neuron-derived EVs,leading to enhanced neuronal apoptosis.(4)miR-21 mediated neuroprotective effects of MSC-EV in SAH.(5)miR-21 exerted its neuroprotective effects through regulating PTEN/Akt pathway.