Protective Effect And Mechanism Of Acetylated α-tubulin In Motor Neural Tracts Injury After Intracerebral Hemorrhage

Background and purposes Intracerebral hemorrhage(ICH)is the most severe subtype of stroke,caused by the accumulation of blood in the brain parenchyma after the rupture of blood vessels.The incidence of ICH in the population is 12-15 per 100,000 person per year,accounting for 10 to 15% of all the stroke types.The acute mortality rate of ICH is 30% to 40%,more than 90% of the patients have different degrees of neurological dysfunction.With the improvement of medical technology,the mortality rate of patients with ICH is on the decline,but the disability rate is still high,and there is no effective strategy for treating neurological dysfunction after ICH until now.Therefore,high disability rate is still the choke point of ICH treatment.How to improve the neurological function(such as motor,sensory,cognition,etc.)and reduce the disability rate of the ICH patients has become the focus of ICH research.Hypertensive ICH occurs predominantly in the basal ganglia area.This area contains many white matter fiber tracts which project from cortex to spinal cord,the injury of white matter fibers lead to severe motor dysfunction.Many clinical studies employed diffusion tensor imaging(DTI)and diffusion-weighted imaging(DWI)to observe the damage of white matter fiber tracts-corticospinal tract(CST)after ICH,and found that the degree of CST injury after ICH is closely related to the degree of motor function impairment.The results suggest that reducing CST injury at the acute stage after ICH is conducive to motor function recovery.Besides,it is difficult for the central nervous system to regenerate.Therefore,it is easier and more important to protect nerve fibers at the acute stage of ICH than to promote later nerve regeneration.The remaining nerve fibers are also the structural basis of neurological rehabilitation.We reviewed the previous research directions of the ICH,and found that most of the researches focus on the mechanisms and translational studies of blood-brain barrier protection,gray matter neuron protection,inflammation inhibition,iron chelation,and hematoma absorption after ICH.Significant results have been achieved onexperimental ICH animal models.Interestingly,there were more than 1000 kinds of drugs that had been proved to have protective effects in experimental stroke animal models,and nearly 200 kinds of those drugs had been used for clinical trials,but all of them have no significant therapeutic effects during clinical trials.There are many reasons for this results: first,there are differences between rodents and humans;second,the majority of studies ignore the protection of motor neural tracts;third,with the deepening of research,we find that there are many mechanisms of neural damage after ICH,one single intervention can only achieve moderate results,more significant effect may be achieved if several interventions are combined.The location specificity and multiple injury mechanisms of ICH suggest that we should explore new research directions,intervention targets and appropriate model animals to improve neurological dysfunction after ICH.Therefore,we think that the basic research of ICH should pay more attention to the following aspects: 1)emphasizing the protection of the white matter fiber tracts in the acute stage of ICH to preserve the structural basis for functional recovery;2)using large animals to carry out experiments which have similar anatomical structure to the human nervous system,such as non-human primates,pigs,etc.;3)using a variety of combined treatment.However,the research on the role and mechanism of motor neural tracts injury after ICH is still blank.In chapters 2 and 3 of this paper,we explored the pathological changes of the two key motor neural tracts: CST in pyramidal system that controls fine movements,and the nigrostriatal pathway(NSP)in extrapyramidal system that coordinates fine movements.Recent studies have found that microtubule depolymerization and mitochondrial dysfunction are involved in the development of spinal cord injury,Alzheimer’s disease(AD),Parkinson’s disease(PD)and other diseases.Maintaining the stability of microtubules can promote axon regeneration after spinal cord injury and improve the symptoms of AD and PD in rodents.Research has also found that protecting mitochondrial function after spinal cord injury can promote nerve regeneration and improve motor dysfunction in mice.Acetylated α-tubulin is a marker for stable microtubules,and promoting the expression of acetylated α-tubulin can maintain the stability of microtubules;inhibiting the opening of mitochondrial permeability transition pore(m PTP)can alleviate the swelling of mitochondria and improve the function of mitochondria.We speculate that promoting the expression of acetylated α-tubulin to maintain the stability of microtubules and mitochondrial protection will be conductive to protect the motor neural tracts after ICH.In chapters 2 and 3 of this paper,we explored the protective effects of maintaining microtubule stability and mitochondrial protection in the CST and NSP motor neural tracts.Moreover,it is necessary to establish a stable and reproducible model of ICH in large animals,and to explore the dosage,cycle and timing of preclinical drug intervention.What’s more,after the establishment of the ICH model in large animals,we should establish the corresponding methods to evaluate the severity of ICH,especially MRI analysis of brain edema,iron content,white matter fibers imaging.In the fourth chapter,we focused on the establishment of minipig ICH model,and explores MRI-T2 imaging,quantitative susceptibility mapping(QSM),diffusion tensor imaging(DTI)to analyze brain tissue structure,brain iron content and white matter fiber bundle noninvasively,accurately and quantitatively.Finally,we investigated the protective effects of minocycline(Mino),a promising drug for the treatment of ICH,in minipig ICH model,in order to provide a more appropriate ICH model for clinical research.Part Ⅰ The role and mechanism of microtubule stability and mitochondrial protection in corticospinal tract injury after intracerebral hemorrhageObjective Intracerebral hemorrhage(ICH)occurs predominantly in the basal ganglia area of the thalamus where white matter fiber tracts are concentrated.Cortical spinal tract(CST)is the axon part of white matter fiber tracts,and the most important structure to control fine function.However,the pathological changes of CST axons injury after ICH are still unclear.This part mainly studies the pathological changes of CST axon after ICH.Furthermore,in order to provide theoretical basis and possible treatment strategy for the protection of motor neural tracts of CST after ICH,we explored the role and mechanism of microtubules and mitochondria in CST axon damage after ICH.The expression of acetylated α-tubulin was enhanced to maintain the stability of microtubules and protection of mitochondrial function by transgenic mice,drugs and other technologies,and it was investigated whether the increase of acetylated α-tubulin expression and protection of mitochondrial function can alleviate CST axon damage and motor dysfunction after ICH.This study was to provide a theoretical basis and possible treatment strategy for the protection of motor neural tracts of CST after ICH.Methods The ICH model was established by injecting 25 μLof autogenous blood into the right striatum using Thy1-YFP mice.The morphology of CST injury around hematoma were observed by immunofluorescence and electron microscopy at 24 hours after ICH.Then,MEC17-/-mice were used to reduce the expression of acetylated α-tubulin,and Tubastatin a(Tub A),a specific inhibitor of HDAC6,was used to increase the expression of acetylated α-tubulin to increase the stability of microtubule.Cyclosporin A(Cs A)was used to blocke the formation of m PTP,alleviated the swelling of mitochondria.MEC17-/-mice,Tub A and Cs A were applied to ICH mice alone or in combination.Immunofluorescence staining,electron microscopy,neural circuit tracing and other methods were used to detect the morphology of CST motor neural tracts.Beam walking and irregular ladder walking were used to detect fine motor dysfunction.In addition,the primary cortical neurons were extracted and cultured to observe the movement of mitochondria and the positional relationship between acetylated α-tubulin and mitochondria.Results 1.ICH led to the “retraction bulbs(RBs)” pathological changes of the CST axons.The electron microscopy results showed that there were irregular arrangements of microtubules and swollen mitochondria in RBs,forming a non-nutritive growth cone.Anterograde neural circuit tracing result indicated the damage of CST,and behavioral study found that CST related fine motor dysfunction.2.Within 24 hours after ICH,the damage of white matter fibers only occurred in the axons,and there was no significant damage to the myelin sheath.On the 7th day after ICH,the myelin sheath disintegrated most seriously,and on the 14 th day after ICH,new myelin sheath appeared.3.41% of mitochondria in primary cortical neurons were co-labeled with the "enhanced point" of acetylated α-tubulin expression.MEC17 knockout resulted in deletion of acetylated α-tubulin expression,and caused a significant decrease in the number of moving mitochondria along the neural fibers.4.ICH model of MEC17-/-mice showed more severe CST axon damage and motor dysfunction.5.Using Tub A to increase the expression of acetylated α-tubulin and CSA to alleviate the swelling of mitochondria could reduce the injury of CST axon caused by ICH,such as reducing the formation of RBs and alleviating the motor dysfunction.The combination of these interventions could achieve more significant improvement in ICH mice.Conclusion In this part,we explored the pathological damage and mechanism of the pyramidal system-CST motor neural tracts caused by ICH.We found that microtubule depolymerization and mitochondrial damage at the early stage of ICH are the important causes of CST damage.Acetylated α-tubulin and mitochondria complement each other and increase the expression of acetylated α-tubulin and protection of mitochondrial function at the same time significantly alleviate CST injury after ICH.It is considered that combined treatment is the key to the treatment of dyskinesia at the early stage of ICH,which provides a new research strategy and intervention target for the treatment of dyskinesia after ICH.Part Ⅱ The protective effect and mechanism of microtubule stability in the nigrostriatal pathway injury after intracerebral hemorrhageObjective Nigrostriatal pathway(NSP)is one of the main structures of the extrapyramidal system,which is composed of dopaminergic neurons and fibers originating from nigra and projecting to the striatum.Its main function is to regulate muscle tension and coordinate various fine and complex movements.The striatum contains caudate nucleus,lenticular nucleus and many white matter fiber tracts running through the striatum,which is the predilection site of intracerebral hemorrhage(ICH).To date,there is no systematic study and intervention on NSP neural pathway damage after striatal hemorrhage.Therefore,this part focuses on the injury NSP neural pathway after ICH,and explores the protective effect and mechanism of acetylated α-tubulin mediated microtubule stabilization in the injury of NSP after ICH,so as to explore new research ideas for the recovery of fine functional dysfunction after ICH.Methods In this part,the ICH model was established by injecting 25 μL of autogenous blood into the right striatum of mice.The pathological changes of dopaminergic neuron fibers around hematoma in striatum and cell bodies in the substantia nigra were detected by immunofluorescence staining,electron microscopy,western blot and neural circuit tracing on the 1st,3rd and 7th days after ICH.The expression of vesicular monoamine transporter-2 (VMAT2),kinesin and dynein,dopamine neurotransmitter were detected to monitor the function of dopaminergic neurons.In addition,we used gross behaviors(open field,neurological score,rotarod)and fine behaviors(beam walking and irregular ladder walking task)to detect the motor dysfunction of mice after ICH,as well as the correlation between the protein expression level of acetylated α-tubulin and the morphological and functional abnormalities of dopaminergic neurons.What’s more,we gave the microtubule stabilizer Epothilone B(Epo B)to treat the ICH model of mice in vivo and oxyhaemoglobin-mimicked in vitro ICH model and observed Epo B’s protective effect on dopaminergic neurons and fibers in NSP,and its repairing effect on fine behavior.In addition,using the above experimental method,we injected nocodazole into striatum to observe the damage effect of microtubule depolymerization on NSP and the reversal effect of Epo B.Finally,we used lentivirus and adenovirus vector to intervene αTAT1/MEC17(acetyltransferase of α-tubulin)sh RNA on dopaminergic neurons in the substantia nigra in vitro and in vivo to reduce the expression of acetylated α-tubulin,and observed the effect of the decrease of acetylated α-tubulin on the cell body,fiber and related behaviors of substantia nigra neurons.Results 1.The density of dopaminergic fibers around the hematoma and the number of dopaminergic neurons in the substantia nigra of the ipsilateral side were significantly reduced after striatum hemorrhage.Dopaminergic neurons showed "vacuolation" pathological changes,accompanied by decreased expression of VMAT2,kinesin and dynein,decreased content of dopamine neurotransmitter,and decreased expression of acetylated α-tubulin,a stable microtubule marker.2.Anterograde and retrograde tracing showed that striatal hemorrhage caused significant damage to the NSP neural pathway.Open field and neurological score methods could only detect the motor dysfunction of ICH mice within 3 days,but the fine fine motor(beam walking and irregular ladder walk task)still had dysfunction at 14 th day after ICH.3.The intervention of Epo B could increase the expression of acetylated α-tubulin,improve the damage of NSP structure and function caused by ICH,and improve the fine motor function after ICH.4.The injection of nocodazole into striatum can cause the injury of NSP and motor dysfunction after ICH.The results suggested that the single factor of microtubule depolymerization has damage effect on NSP.Maintaining microtubule stability is an important target to protect the injury of NSP after ICH.5.αTAT1/MEC17 sh RNA intervention could reduce the expression of acetylated α-tubulin in dopaminergic neurons and aggravate the damage of the structure and function of NSP caused by ICH and oxyhaemoglobin.Epo B could also protect dopaminergic neurons from damage caused by αTAT1 sh RNA and oxyhaemoglobin intervention.Conclusion Striatal hemorrhage can lead to severe damage to the NSP neural pathway,and the decrease of acetylated α-tubulin expression after ICH is the important reason for the damage to the NSP neural pathway.The intervention of the microtubule stabilizer Epo B can increase the expression of acetylated α-tubulin,significantly reduce the damage to the NSP neural pathway caused by ICH and improve the motor dysfunction of ICH mice.In this part,we first explored the damage to the extrapyramidal system-NSP caused by ICH and its possible mechanism,which provided a new way to improve the motor dysfunction after ICH.Part Ⅲ Establishment of intracerebral hemorrhage model in minipig and exploration of MRI detection method for large animals Objective The intracerebral hemorrhage(ICH)model was mainly established in rodents.However,the white matter fibers were not rich enough,and there was no obvious internal capsule anatomical structure in rodents.Therefore,in this experiment,we attempted to establish a stable,repeatable and easy to operate minipig model of ICH.At the same time,we observed and analyzed brain edema,ventricular volume change and iron deposition after hemorrhage at multiple time points.We also used MRI-T2 imaging,quantitative susceptibility mapping(QSM),diffusion tensor imaging(DTI)and other sequences to analyze brain tissue structure,iron content and white matter fiber tract.Furthermore,in order to evaluate the stability and reliability of the minipig ICH model,we observed the therapeutic effect of minocycline on this ICH model,to provide a more clinical ICH model for clinical transformation studies.Methods We selected 10-12 Kg male Bama pig,fixed the pig to the large animal stereotaxic instrument after isoflurane anesthesia,drilled a hole about 1mm in diameter on the surface of the skull above the inner capsule(bregma coordinates:11 mm anterior and 11 mm lateral from the midline),and slowly injected 2m L of autogenous arterial blood into the right inner capsule area of the pig with the micro injection pump,5m L syringe and 33 g needle,with the depth of 18 mm.MRI-T2,QSM,DTI,brain water content,iron content around hematoma and immunohistochemical staining were performed on minipig at 1st,7th and 28 th days after ICH.In addition,we tested the protective effect of minocycline in minipig model of ICH.Results 1.The intracerebral hematoma,ventricular distention,brain edema,white matter fiber injury and motor dysfunction were caused by the injection of autogenous blood into the inner capsule of minipig,which well simulated the clinical characteristics of intracerebral hemorrhage.This minipig ICH model was a repetitive and reliable model.2.There was a good correlation between the quantitative magnetic susceptibility of QSM and the iron content around hematoma.QSM could make a noninvasive and accurate quantitative analysis of iron content around hematoma after ICH.DTI could be used for quantitative analysis of white matter fiber tracts.3.Minocycline could perform the function of iron chelation,reduce the iron content around the hematoma,and alleviate brain edema,lateral ventricles expansion,white matter fiber tracts damage and neural dysfunction.Conclusion The ICH model of Bama minipig is repeatable and reliable,and QSM is a noninvasive and accurate method to evaluate the iron content of hematoma in acute and chronic phase after ICH.Minocycline can reduce brain edema,ventricular expansion,iron overload,white matter fiber damage,glial cell activation and nerve dysfunction in minipig ICH model.