The Effects Of NogoA-PirB Signal Path On The Axonal Regeneration And Recovery Of Disability In Rat With Intracranial Hemorrhage

Objective:Through making the models of rats with intracranial hemorrhage(ICH),this study aimed to observe the expression of PirB and NogoA in the brain of rats within different times,the orientation of PirB expression,anxonal regeneration,and the neurological deficits of rats after acute ICH.We will research that whether the NogoA-PirB signal path is responsibal for the neurological deficits,the mechanism of repairmen of brain injury,and the novel target of therapy after ICH by exploring the effects of Pir B on anxonal regeneration and neurological disability of rats with intracranial hemorrhage,and the relationshipbetween PirB and NogoA.This research will provide powerful basis for clinical therapy.Methods: 75 male SD rats were randomly divided into 2 groups: Sham group(n=25)and ICH group(n=50),each group was randomly divided into five subgroups as 1d,3d,7d,14 d,28d through the differen time after ICH;in the ICH subgroups,rats were randomly divided into western blot group(WB)and immunofluorescence group(IF).Sham group rats were not injected autologous blood;while in the ICH group,the method of modified two times injection was adopted to make the ICH models by injecting 50 ul uncoagulated autologous blood.Three hours after modeling,we would evaluate whether the model was successful by Bederson scoring method and selected the successful models bring into ICH group.The rats of ICH group were graded the neurological dysfunction scoresby standard for evaluation named Garcia at different time point subgroups,following decapitation and collecting the brain.The brain of WB group immediately freezed with liquid nitrogen at the time of gaining the brain and then cryopreserved within-80?,following grinding the tissues and extracting the protein for immunoblotting to measure the expression of PirB and NogoA;while the IF group would be poured PBS and 4% paraformaldehyde into vessels and obtained the brain,following after-fixing,dehydration,frozen section and paraffin section for HE staining and double-immunofluorescence respectively.Result:(1)The neurological deficits scores of ICH group is obviously lower compared with Sham group at each time point;and Garcia's score was lowest at 3d subgroup in ICH group,and the statistical difference was obviously significant(P<0.05).(2)Pathologic change of brain tissue surrounding hematoma: structure of brain tissue was intact in Sham group.There were a large number of red blood cells deposition in the hematoma with obvious edema and inflammatory cell infiltration around the focus at 1d subgroup.At 3d subgroup,the red blood cells reduced and the edema and necrosis of neurons was severe following vast inflammatory cell infiltration.The edema and inflammatory cells around focus were obviously decreased and glial proliferation appeared at 7d subgroup.The hematoma thoroughly faded away substituted with collagenous fiber and cicatrization at 14 d and 28 d subgroups.(3)Quantitative analysis of NogoA and PirB: the expression of NogoA and PirB at each time point of ICH group was higher than corresponding Sham group.Among the different time point subgroup,the peak of expression of Nogo A and PirB both appeared in 7d subgroup,and the expression persistently increased till 28 d,P < 0.05 was considered to be statistically significant?(4)Double-immunofluorescence: PirB expressed in the brain tissue of Sham group and ICH group including each time point subgroup,and mainly located in neurons,axon and astrocytes.The axon of brain tissue in ICH group showed distinct breakage and disorder relative to Sham group,the one whose axon was intact and orderly;the length of axon was obviously shorter compared with Sham group and there was no improvement till 28 d after ICH,there was significant different relative to Sham group(P<0.05).(5)The the expression levels of NogoA and PirB were negatively correlated to neurological dysfunction scores and the length of axon in ICH group.CONCLUSIONS:(1)The PirB and NogoA can express in neuron and astrocyte of brain tissues in adult rats to keep the functional stability of CNS.(2)The expression of NogoA and PirB was obviously increasing after ICH and NogoA-PirB path way would impede functional recovery through inhibition of axon regeneration.