Protective Effects And Mechanisms Of Poloxamer188on Cerebral Ischemia/reperfusion Injury

Aim: To explore whether P188has a protective effect against cerebralischemia/reperfusion injury and its underlying mechanisms.Methods:Part1: The effects of P188on the cerebral ischemia/reperfusion injuryFirstly we established the mouse middle cerebral artery occlusion (MCAO) modelby intraluminal occlusion using monofilament. P188was intravenously injected justbefore reperfuion, mice were sacrificed to determine the infarct volume using TTCstaining, brain water content and neurological symptoms24h after ischemia/reperfusion.To learn the long-term protective effects, P188were administered by IP injection once aday for three weeks, the survival rate was determined, motor function was evaluatedwith wire hanging and pole test and the loss of brain volume was calculated using cresylviolet staining. In addition, we also apply the oxygen and glucose deprivation cellmodel to evaluate the effects of P188on the cell viability and LDH leakage.Part2: The underlying mechanisms of P188against the cerebralischemia/reperfusion injury.In this part we firstly applied the Propidium Iodide (PI) to evaluate the membraneintegrity24h after ischemia/reperfusion. Furthermore, in vitro0.03%Triton X-100, anonionic detergent, was used to directly disrupt neuronal membranes. Two differentmembrane-impermeant fluorescence dyes (PI and SYTOX Green) were sequentiallyadded to cell medium to observe the changes in membrane integrity after P188treatment. Secondly, we applied Evans Blue Dye Extravasation to assess thepermeability of blood-brain barrier (BBB), and analyzed the activity and expressionlevels of MMP-9using zymogram and western blot. Thirdly, to explore whether P188can penetrate the cell membrane into the cytosol, P188was labeled with Ir complexeswhich can emit the fluencerence and observe its distribution in HT-22cell and in mousebrain suffered from ischemia-reperfusion injury. Furthermore, mitochondrial and lysosome were separated from ischemic hemisphere of MCAO mice to analyze theleakage of cytochrome c and Cathepsin L.Part3: The effects of combined P188and Ginkgolide B on theischemia/reperfusion injuryCombined P188and Ginkgolide B were compared with same dosage of P188orGinkgolide B by the infarct volume, brain water content and neurological symptoms.Results:Part1: The effects of P188on the cerebral ischemia/reperfusion injuryThe results from in vivo MCAO model showed that P188significantly reduced theinfarct volume, ameliorated the brain edema and neurological symptoms24h afterischemia/reperfusion. In the long-term outcome study, P188markedly alleviated brainatrophy and motor impairments and increased survival rate in3weeks of post strokeperiod. Additionally, P188protected cultured hippucampal HT22cells againstoxygen–glucose deprivation and reoxygenation (OGD/R) injuryPart2: The underlying mechanisms of P188against the cerebralischemia/reperfusion injury.Firstly, P188treatment significantly reduced the PI-positive cells followingischemia/reperfusion injury and repaired the HT22cell membrane rupture induced byTriton X-100. Secondly, P188inhibited ischemia/reperfusion-induced activation ofmatrixmetalloproteinase (MMP)-9and leakage of Evans blue, which suggests that P188can preserve the integrity of blood-brain barrier. Thirdly, we found P188labeled with Ircomplexes can penetrate the cell membrane, thus continued to assess the effects of P188on organelles including mitochondrial and lysosomes and also discovered that P188caninhibit the leakage of Cyt c and Cathepsin L.Part3: The effects of combined P188and Ginkgolide B on theischemia/reperfusion injuryCombined P188and Ginkgolide B had greater protective effects on theischemia/reperfusion than that of P188or Ginkgolide B alone.Conclusions:P188can protect against cerebral ischemia/reperfusion injury, and the protectionmechanisms involve:1) directly sealing the cell membranes;2) preserve the integrity ofblood-brain barrier and3) penetrate the membrane and render the protection on mitochondrial and lysosomes. In addition, the combination of P188and Ginkgolide Bcan produce greater protective effects on ischemia/reperfusion injury.