| Objective1.Establish an in vitro neurovascular unit model by co-culture of brain microvascular endothelial cells(BMECs),neurons,and astrocytes.2.Study the protection of ?-caryophyllene(BCP)on in vitro neurovascular unit against oxygen-glucose deprivation and re-oxygenation(OGD/R)-induced injuryMethods1.(1)BMECs were prepared from 2-week-old SD rats,purified by passage,and were identified by immunofluorescence staining of special protein factor ?.Astrocytes were obtained from brain cortices of 1-day-old rats,purified by shaking,and were identified by immunofluorescence staining of special glial fibrillary acidic protein(GFAP).Neurons were prepared from rat embryos at 17-19 days gestation,and culture by DMEM containing 10% FBS.After 4 hours,neurons were maintained in neurobasal medium supplemented with B27,purified by Ara-C and were identified by immunofluorescence staining of special protein MAP-2.(2)In vitro NVU model was established using a transwell.Astrocytes were plated on the exterior of poly-L-lysine-coated inserts.After 5 hours,the inserts were placed into matching wells in which neurons had already been cultured for 5 days.After 24 hours,BMECs were plated on the inner side of the insert membrane coated with Matrigel matrix.The NVU model was maintained for 72 hours prior to analysis.NVU permeability was evaluated by performing a 4-hour leakage experiment,TEER values and Evans Blue(EB).Morphological features of in vitro NVU model was observed by inverted microscopy and scanning electron microscopy.Tight junctions(TJs)and desmosomes between the BMECs were observed by transmission electron microscopy and immunofluorescence staining.2.To induce ischemic insult in vitro,OGD/R treatment in the context of the NVU model was performed as previously described with modifications.NVUs were randomly separated into three groups(n=3):(i)the control group,(ii)the OGD/R group,which was exposed to OGD/R;and(iii)the BCP+OGD/R group,in which BCP(10 ?mol/L)was applied for 24 hours prior to OGD/R and maintained throughout OGD/R.NVU permeability was evaluated by TEER values and EB.MMP-9 activity was assessed by gelatin zymography assay.TJs protein,growth-associated protein-43(GAP-43)and apoptosis protein were analyzed by western blot.Neuronal apoptosis was detected by flow cytometry.NFG-?,SOD?MDA?LDH?NO?TNF-??IL-1??IL-6 were analyzed by ELISA analysis.Results1.(1)Successfully obtain three primary cells.BMECs exhibited a typical spiral growth state,and were detected by specific anti-Factor VIII antibodies,and the purity was >96%.Astrocytes were identified with anti-GFAP antibodies with a purity of >97%.Neuron purity was >90% as measured with anti-MAP-2 antibodies.(2)Compared with monolayer or double cells co-culture,the triple-cell culture had a sound growth state and can facilitated growth each other.4-hour leakage experiment was negative.TEER values were significantly higher than monolayer and double cells co-culture,and triple-cell co-culture exhibited lower EB permeability and intercellular TJs and desmosomes were also formed.2.BCP pretreatment prior to the initiation of OGD/R significantly(i)decreased BBB permeability and neuronal apoptosis,(ii)mitigated oxidative stress damage and the release of inflammatory cytokines,(iii)down-regulated Bax expression,MMP-9 activity and expression,and(iv)up-regulated claudin-5,occludin,ZO-1,GAP-43,NGF-? and Bcl-2 expression.Conclusion1.Triple-cell co-culture model exhibit more sound state,and can better imitation the complex physiological environment in vivo.2.BCP pretreatment exerted multiple protective effects on NVU in the context of OGD/R-induced injury... |
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