The Neuroprotection And Mechanism Of TAT-Neuroglobin In A Rabbit Model Of Aneurysmal Subarachnoid Hemorrhage By The Ear Artery-Intracranial Shunt

Background and object: Aneurysmal subarachnoid hemorrhage(a SAH)is a life-threatening hemorrhagic cerebrovascular disease.Despite continous advances in treament of cerebral vasospasm,the mortality and morbidity after a SAH have not changed in delayed ischemic neurological deficits.For the past years,the concept of early brain injury(EBI),was more and more accepted by researchers.EBI was induced by the sharply increased intracranial press(ICP)and low cerebral perfusion press(CPP)with the cerebral global ischemia fllowing rupture of aneurysm and may be the key factor due to bad outcome.They have not been well studied to date,partly for lack of a good animal a SAH model.Accumulated evidence showed neuroglobin(Ngb),a monomeric globin located in normal neuron,is up-regulated by ischemic/hypoxic and trauma conditions,meanwhile is an endogenous neuroprotective molecule.There is seldom study between EBI of a SAH and Ngb.The aim of this study is to set up a new rabbit model of a SAH which is more corresponded with the pathophysiologic damage of EBI including the sharply increased intracranial press and low cerebral perfusion press;to evaluate the expression changes of neuroglobin(Ngb)after a SAH with EBI;to generate the TAT-Ngb or Ngb fusion proteins in Escherichia coli BL21(DE3)with p ET-TAT-human Ngb or p ET-human Ngb expression vectors.Then study evaluate whether TAT-neuroglobin fusion protein could cross the blood brain barrier and protect the brain from cerebral ischemia,and how to work.Methods 1.Right frontal craniotomy was operated to the newzland rabbits with operating microscope,the ICP probe was located at the anterior cranial fossa,and the PE-50 tube was located at the supra sella cistern,the ear central artery was punctured and blood was shunt into the supra sella cistern.Then monitor the ICP,blood pressure,CPP and heart rate before and after a SAH.The rabbit were examined for neurological deficits at 6h/24 h then sacrificed 24 h latter.Brain coronal sections which tissue was taken at 24 h after a SAH were respectively assessed by HE staining,Cresyl violet staining,and TUNEL staining.2.The expressions of Ngb m RNA and protein in brain tissue of rabbits after a SAH with EBI at different time group(contral,3h,6h,12 h,24h,48 h,72h)were detected by immuohistochemistry staining,Real-Time PCR and Western blotting.3.The p ET-TAT-human Ngb or p ET-human Ngb expression vectors was amplified by Escherichia coli DH5? and induced into BL21(DE3),and the TAT-Ngb and Ngb fusion proteins were generated.The fusion proteins were purified by a Ni-NTA resin column,desalted by a PD-10 desalting column and verified by Western blotting.4.The rabbits were injected intravenously with TAT-Ngb or Ngb and sacrificed 4 h latter.Immunofluorescence staining was applied to the cerebral frozen section to observe the transduction of TAT-Ngb and Ngb.5.The a SAH rabbits with EBI were divided into 3 groups,and injected from ear marginal vein with TAT-Ngb,Ngb,and saline respectively,just after operation,At 6h and 72 h after a SAH,the rabbits were examined for neurological deficits,then sacrificed and brain coronal sections were respectively assessed by HE staining,Cresyl violet staining,and TUNEL staining.The expression of bcl-2,bax,caspase-3 and caspase-9 in brain tissue at different group were detected by Western blotting.Results 1.A new rabbit model of a SAH was set up successfully through the bleeding driven by the ear central artery-intracranial shunt,which had sharply increased ICP,decreased CPP,lowed neurological deficits and pathalogic brain injury.2.Boths of Ngb m RNA and protein expressed in normal brain tissue.Compared to the control group,at 3h group,the expressions of boths increased a little but no statistically significant difference;at 6h group,the expressions of m RNA increased more and reached statistically significant difference(p<0.01),while the protein had no statistically significant difference;at 12 h group,the expressions of boths increased and reached statistically significant difference(p<0.01);they reached the peak at 24 h group.Then the expressions decreased slightly at 48 h group and more in 72 h group,but was still higher than the contral(p<0.01).Immunohistochemical staining of Brain tissue sections displayed corresponding change of Ngb which located in the neuronal cytoplasm.3.The fusion proteins were produced and purified,then identified by Western Blotting analysis with 6x His-tag and Ngb antibodies.4.The TAT-Ngb fusion protein was efficiently delivered into the rabbit brain tissue and located at neuronal cytoplasm by intravenous injection as demonstrated by double-labeling immunofluorescence staining with NSE monoclonal antibody and 6×His-tag monoclonal antibody,but Ngb was not achieve detectable at brain tissue.5.Compared to the Ngb-or saline-group,the TAT-Ngb group at 72 h after a SAH with EBI had better neurologic outcomes,more Nissl bodies and neron survial,less apoptosis.Western blotting analysis showed the TAT-Ngb group had up-regulated protein expression of bcl-2,down-regurated protein expression of Bax,caspase-3 and caspase-9 in brain tissue,and had statistically significant difference Compared to the Ngb-or saline-group(p<0.01).Conclusions 1.A new rabbit model of a SAH corresponding with the a SAH with EBI in patients was set up.This model was simply operable,controllable,and had the advantage of high success rate,low death rate,wide experimental animal sources,and low price of animal.2.EBI of a SAH up-regulate the expression boths m RNA and protein of Ngb which are changed in a time-dependent manner.3.The TAT-Ngb fusion protein was produced,purified and efficiently delivered into the rabbit brain tissue and located at neuronal cytoplasm,while not Ngb.4.Neurological deficits and pathalogic brain injury of a SAH rabbits with EBI can be alleviated by TAT-Ngb treatment.Mechanism of neuroprotection related to reduce mitochondrial pathways of apoptosis.