Protective Effect And Mechanisms Of Action Of TBN In Rat Model Of Ischemic Stroke

Objectives To investigated the therapeutic effects of TBN in permanent,transient and global ischemic stroke animal models according to guidelines of CFDA,FDA and STAIR and to study the underlying mechanisms of action in permanent ischemic stroke providing useful information for clinical trials.Meanwhile,a preclinical pharmacodynamic research system will be established for research of other stroke drugs.Methods1.The permanent ischemic stroke model was established using the Laser Doppler flowmetry.The dose response study,therapeutic time windows study,pharmacodynamics study and different routes of drug administration were conducted in the rat permanent ischemic model based on the main outcome measures of Neurological severity score changes and infarct area.Physiological indicators such as body weight,body temperature,venous blood gas analysis were also conducted.2.The rat transient ischemic model was established by inserting the monofilament into middle cerebral artery.TBN was administered by intravenous injection.The effects of TBN on animal behavioral functions of Rotarod,Longa and Neurological severity score and infarct size were measured.Cerebral blood flow,venous blood gas analysis,venous glucose,heart rate,blood pressure,body weight and body temperature parameters were monitored during operation.3.The global ischemic stroke model was established by ligating bilateral common carotid artery.TBN at 30 mg/kg was administered by intravenous injection for one week,twice a day.The Y-maze,novel object recognition and gait analysis as the main indicators and combined with other physiological parameters including cerebral blood flow,blood gas analysis,body weight,body temperature etc.were used to study the long-term pharmacodynamic effects.4.To provide a theoretical basis to illustrate the efficacy,HE staining,immunohistochemistry,and Western blot technology were used to explore the possible protective mechanisms of TBN on ischemic stroke damage.Results1.The body weight of the animals in each group was decreased significantly in permanent ischemic stroke model.However,there is no statistics difference among groups,indicating that TBN has no effect on body weight.The Neurological severity score was used for behavioral evaluation after stroke onset at 3 h and 24 h.The data showed that the rat behavior,feeling,balance and reflection were improved and cerebral infarction size was significantly reduced at 10 mg/kg,30 mg/kg and 90 mg/kg TBN.The protection rate of three doses is 16.64 %,14.50 %and 22.82 %,respectively.The lowest effective dose of TBN was 10 mg/kg and the therapeutic time window was at least 6 h.The therapeutic efficacy of TBN after continuous intravenous injection was better than that given by an intravenous bolus injection.2.In the ischemic-reperfusion rat stroke model,behavior evaluation was assessed by Rotarod,Longa and Neurological severity score.Rotarod result showed that,at both 10 mg/kg and 90 mg/kg,TBN remarkably increased retention time on the rotarod by the ninth day of the experiment and that the efficacy at 90 mg/kg was similar to the positive control Edaravone,No significant improvement was seen in the TMP group.Longa assay proved that 30 mg/kg and 90 mg/kg of TBN can effectively reduce behavioral disturbance of ischemic rat.Neurological severity score demonstrated TBN at all doses could improve the rat behavior,feeling,balance and reflection compared with the model group on the ninth day.Cerebral infarct staining showed 10 mg/kg,30 mg/kg and 90 mg/kg of TBN significantly reduced the area of cerebral infarction with the protective rate of 21.7 %,31.3 % and 36.3 %,respectively.The protective effect of TBN at 10 mg/kg was equal to TMP at 85 mg/kg(equal molarity to TBN)and 6 mg/kg of Edaravone(13 times the dose used in to clinic).3.In global cerebral ischemic stroke model,the learning and memory ability and motor function were assessed by Y maze,novel object recognition test and gait analysis after 1 week of TBN treatment.Results from Y maze and novel object recognition test showed that rat learning and memory ability were both improved.While gait analysis showed no significant difference between the model and sham group,the same as between TBN group and model group.Pathological results showed that TBN significantly increased the number of neurons and protected the nerve fiber damage in rat with global cerebral ischemic stroke.4.In the 24 h permanent ischemic stroke model,the expression of antioxidation indexs 4-HNE,8-OHd G were markedly reduced and the number of neuron cells was significantly increased after treatment with 90 mg/kg of TBN.TBN inhibited neurocyte apoptosis through up-regulation of Bcl-2 expression and down-regulation of the expression of protein Bax,Cyt C and followed by repression of caspase-3 and cleaved caspase-3 expression by up-regulation of p-Akt.TBN also reduced the injury of neuron axons and synapses through increasing the expression of PSD95,CNPase,MEF2 C protein.TBN also protected the microvessel through promoting expression of VEGF.and MEF2 C.TBN also scavenged free radicals generated by antimycin A and inhibited mitochondrial swelling induced by Ca Cl2 in normal isolated rat cortical neuron mitochondria.Conclusions1.TBN can significantly improve neurobehavioral deficits and reduce the infarct area or protection neuronal cell damage in permanent ischemic stroke model,ischemia-reperfusion stroke model and global ischemic stroke model.The efficacy is superior to Edaravone and TMP.The minimum effective dose was 10 mg/kg and the therapeutic time window is at least 6 h in the permanent ischemic stroke model2.The protective effects of TBN on neuron injury may be attributed to its anti-apoptosis function which is also attributed to scavenger free radicals in mitochondria and to inhibit mitochondrial calcium overload.In addition,TBN protects the neuron axons and synapses injury through promotion the expression of PSDS95?CNPase?MEF2C.TBN also protects the microvessel in the parenchyma of stroke penumbra through the up �regulation of VEGF and MEF2 C protein expression.