Effect Of Calmodulin Inhibitor On Cognitive Dysfunction In Mouse Model Of Bilateral Carotid Artery Stenosis

Background:Vascular dementia(VD)is a chronic progressive disease with cognitive impairment,induced by a variety of cerebrovascular risk factors.Among these causes of dementia,VD is the second leading cause inferior to Alzheimer's disease(AD).The increased incidence of VD caused economic pressure and burden to the public health care system.In recent years,it is found that cognitive dysfunction is closely related to carotid stenosis,which can lead to chronic cerebral hypoperfusion.Studies have shown that chronic cerebral ischemia associated with the pathogenesis of vascular dementia and other cerebrovascular diseases.The white matter damage and cognitive dysfunction due to chronic cerebral hypoperfusion is very common in the elderly,which serious impact on their quality of life.Therefore,to find a new and viable treatment is particularly important.Currently,VD treatments mainly symptomatic treatment and control of various vascular risk factors or disease,but the treatment cannot retard the progression of the disease and cures it.Although studies have shown that some drugs can improve cognitive function in patients with VD,the improvement of daily living and overall function is not obvious yet,and the lethal dosage for VD patients has not been determined.Diversity in pathphysiology of VD,poor controllability of patient population and inconsistent baseline make it difficult to establish uniform criteria for clinical inclusion or exclusion.The existing animal model for VD widely varies,and each represents some terms of VD pathological features.In recent years,with the application of molecular biology techniques and transgenic animal models,we can explore new animal models which combine new technologies and new theories to study the pathogenesis of VD at the molecular and genetical levels,which is particularly essential to discover new drugs.Chronic lack of cerebral blood flow supply cause deficit in brain glucose and oxygen,it can cause hippocampal neurons membrane phospholipid metabolism,excessive production of free radicals,massive release of excitatory amino acids,intracellular calcium overload,resulting in the dysfunction of cholinergic neurons in the hippocampus and decreased choline acetyltransferase activity,accompanied by astrocyte hypertrophy and hyperplasia,microglia activation,finally leads to vascular dementia.Nevertheless,the underlying molecular mechanisms of VD remain elusive.Before a new strategy can be developed to counter the adverse effects of VD,it is necessary to define the potential factors responsible for VD-induced cognitive deficit.Calmodulin(CaM)can activate a variety of enzymes or channel involved in many biological functions,such as muscle contraction,synaptic transmission,neurotransmitter synthesis and release,hormone secretion and gene expression.CaM has a variety of functions in the nervous system,usually involved in the regulation of metabolism of neurons,axonal transport,synaptic function,and the synthesis and release of neurotransmitters.Ca2+/CaM-dependent multiple pathways involved in the pathological process of brain ischemia.Numerous studies show that neuronal Ca2+overload can cause excessive activation of calmodulin signaling pathway,leading to neuronal death.Intracellular Ca2+ overload also causes abnormal activation of calcium-dependent protein kinases,such as phospholipase A2 and calpain.Among Ca2+/CaM-dependent enzymes,nitric oxide synthase(NOS)and calcineurin exert their deleterious actions by excessive degradation of cellular enzymes to cause abnormal cell structure and function in the brain ischemia and vascular dementia.Ca2+-dependent phospholipase A2 generates arachidonic acid,leading to arachidonic acid generation of prostaglandins,leukotrienes and free radicals,and other active substances destroy biofilms,causing widespread inflammation.The inflammatory cells secrete the proinflammatory cytokines interleukin(IL)-1?,tumoral necrosis factor(TNF)-a and Fas ligand,which induce blood-brain barrier damage via downstream deleterious molecules.Therefor,targeting the key pathway may represent a novel treatment strategy for neurovascular protection in VD.Calmodulin inhibitors(CaMI)in the presence of Ca2+,binds CaM with high affinity and inhibit the activation of CaM enzymes.In fact,the relevant studies indicating that CaMI play an important role in the occurrence and development of cerebrovascular disease,epilepsy,brain injury,Alzheimer's disease and other neurological diseases,but the mechanism is not yet clear.According to calmodulin pharmaceutical prospects and possible mechanism of action,here we set out to address the potential role of CaMI on the pathological process of VD and its mechanisms.DY98,3-{2-[4N-(2-methyl-3-chlorophenyl)-lN-piperazinyl]ethyl}-5,6-dimethoxy-yl-1H-indazole oxalate,one of the calmodulin inhibitors,is a low solubility indazole compounds in water which is derivative from DY9760e,is not yet commercially available for the treatment of acute cerebral ischemia.It has been reported that DY9760e protect the blood-brain barrier from damage caused by microsphere embolism,relieve cerebral edema,which is a potent cytoprotective agents.The present study designed to explore whether DY98 has pharmacological effect for the treatment of VD cognitive disorders.Polysialic acid(PSA),a unique carbohydrate polymer located on the cell surface,is a long linear homopolymer of alpha-2,8-linked-N-acetyl neuraminic acid with an enormous hydrated volume and is synthesized by two complementary sialyltransferases,ST8SiaII and ST8Sia?.The major carrier of PSA is neural cell adhesion molecule(NCAM).PSA as a hydrophilic,non-toxic and biodegradable polymer endogenously has been more widely used in drug carriers.Polymeric micelles have the ability to penetrate the blood-brain barrier.Therefore,nano drug combined with the polymer micelle delivery system is expected to improve the efficacy of VD dementia drugs.Here we evaluate whether DY98 combined with the new delivery system PSA prevents impairment of cognitive deficit in VD mice,and whether the treatment prevents impairment of hippocampal phospho-CaMKII in model mice.We also address the potential mechenisms of DY98 against hippocampal neuronal dysfunction in same context.The calmodulin inhibitor DY98 combined with the new delivery system PSA might block Ca2+/calmodulin-dependent protein kinase cascade,might be a new safe effective drugs and formulations,is expected to achieve new breakthroughs.Result.Optimization of vascualr dementia mice model1)Compared to the control group,bilateral carotid artery occlusion plus sodium nitroprusside model of mice,showed no significant difference in local cerebral blood flow before and after surgery within 2 hours.No significant changes of spatial memory function of model mice observed on postoperative 14-18 day and 28-32 day.2)Bilateral carotid artery occlusion 20 minutes three times model of mice group was suffered spatial memory impairment after 7 days.The mice mortality rate is very high.Bilateral carotid artery occlusion 20 minutes three times model of mice group was suffered spatial memory impairment after 28 days.The mice mortality rate remains high.Bilateral carotid artery occlusion 20 minutes two times model of mice group was no significant change after 28 days.Bilateral carotid artery occlusion model of vascular dementia to mice were not suitable for use in chronic disease model.3)Coil method with bilateral carotid artery stenosis(unilateral carotid artery diameter 0.18mm)was performed in mice;spatial memory impairment was obvious on 30 day after surgery,but with very high mortality.Coil method(unilateral carotid artery diameter 0.20mm)was performed in mice;its survival rate was 72.7%.After 30 days surgery sequentially,spatial learning and memory impairment were observed.The result of forced swimming test indicated that model mice have depression-like behavior.The result of open-field test,elevated plus maze,light-dark experiments suggest that anxiety-like behaviors were not obvious.Therefore,the coil method in mice with bilateral carotid artery stenosis(unilateral carotid artery diameter 0.20mm)was optimal VD model in the present experimental context.Synthesis and characterization of poly-sialic acid-DY98 micelles.1)Synthesis of PSA-ODA polymers having amphiphilic characteristics,when the PSA-ODA is higher than the concentration in the aqueous solution of 120?g/mL,can form polymeric micelles spatial structure.The blank micelles in the aqueous phase of the particle diameter of 145.7±2.08nm,and having a narrow particle size distribution.The transmission electron microscope observation of PSA-ODA blank micelle which diameter is about 50 nm.Zeta potential of PSA carboxyl group is-33.4±2.56mV.After ODA modification,zeta potential of micelles PSA-ODA fell to-21.2±1.01mV.Higher micelle surface zeta potential increased stability of the polymer micelles.2)DY98 has sustained release characteristics in vitro release from the drug-loaded micelles.Effect of calmodulin inhibitor and its polysialic acid micelles against cognitive deficit and its potential mechanisms1)Morris maze analysis Compared with sham group mice,escape latency of DY98(1mg/Kg)high-dose treatment group,DY98(0.5mg/Kg)of low-dose treatment group,PSA(7mg/Kg)treated group,PSA+DY98(7+0.25)mg/Kg treatment group were reduced in space exploration of Morris water maze;escape latency of DY98(1mg/Kg)high-dose treatment group,PSA(7mg/Kg)treatment group were reduced in navigation test of Morris water maze;escape latency of DY98(lmg/Kg)high dose treatment group,PSA(7mg/Kg)treated group,PSA+DY98(7+0.25)mg/Kg treated group were reduced in working memory test of Morris water maze.2)Effect of drugs on phosphorylation of CaMKII in hippocampus Compared with the sham group,bilateral carotid artery stenosis mice shown that impaired spatial learning and memory,significantly reduced CaMKII phosphorylation of pyramidal neurons in CAl,CA3,DG regions.DY98(1mg/Kg)high-dose treatment group,PSA(7mg/Kg)-treated group,PSA+DY98(7+0.25)mg/Kg-treated group show a significant restoration of phospho-CaMK II in pyramidal neurons of hippocampal CAl,CA3,DG regions than VD mice.3)Effect of drugs on protein tyrosine nitration and caspase-1/IL-1? signal in hippocampusNLRP3 and STAT3 protein levels were up-regulated in hippocampus of model control group mice.The disturbance of nNOS,nitrotyrosine,caspase-1 and IL-1?processing level were observed in the hippocampus region of VD mice.Compared with model control mice,DY98(lmg/Kg)high-dose treatment group,PSA(7mg/Kg)treated group,PSA+DY98(7+0.25)mg/Kg treatment groups decreased the NLRP3 protein levels;PSA(7mg/Kg)treated group,PSA+DY98(7+0.25)mg/Kg treatment group decreased the STAT3 protein levels in VD mice.To visualize involvement of nitrosative stress in pyramidal neurons,brain slices containing the hippocampal region were stained with anti-nitrotyrosine antibody.We found that the number of nitrotyrosine-positive pyramidal neurons in the hippocampus was markedly increased 45 days in VD group,whereas DY98(lmg/Kg)or PSA+DY98(7+0.25)mg/Kg treatment effectively prevented the increasing,which accompanied by decreasing of IL-1? processing after drug treatment.Conclusion:1)The coil method(unilateral carotid artery diameter 0.20mm)is the suitable model to mimic the VD.Morris water maze was used as evaluation criteria for spatial learning and memory impairment for vascular dementia in mice model.Among bilateral carotid artery occlusion plus sodium nitroprusside,bilateral carotid artery occlusion with different time,the coil method(single carotid artery side diameter 0.18,0.20mm)caused by chronic hypoperfusion of cerebral blood flow,coil method(unilateral carotid artery diameter 0.20mm)is the optimal model.2)Treatment of VD mice with DY98 and DY98-loaded PSA micelles prevents impairment of hippocampus-dependent spatial learning and memory function in model mice.3)This is the first study using poly sialic pharmacodynamic studies calmodulin inhibitors as drug carrier's clathrate compounds.The study found that 0.25mg/kg by calmodulin inhibitor DY98-loaded PSA micelles have efficacy in compared to high-dose calmodulin inhibitor(1mg/kg)group.Poly-sialic acid as a drug carrier has potential applications in improving aspects of drug effects.DY98 and DY98-loaded PSA micelles increase pyramidal neurons phospho-CaMKII(Thr286)levels,might related its inhibitory effect on VD-induced nitrosative stress injury and NLRP3/caspase-1/IL-1? inflammation.In summary,the present study established the coil method with bilateral carotid artery stenosis(unilateral carotid artery diameter 0.20mm)mice model,which a suitable VD mice modelto mimic pathological process of VD.Calmodulin inhibitor DY98 and DY98-loaded PSA micelles were synthesized and their pharmacological effect was evaluated by using Morris water maze and biochemical analysis.Treatment of VD mice with DY98 and DY98-loaded PSA micelles prevents the impairment of hippocampus-dependent cognition impairment in model mice through inhibiting nitrosative stress and its downstream caspase-1/IL-1? pathway.Protection of hippocampal pyramidal neurons against hypoperfusion injury represents a potential strategy to prevent or delay cognitive impairments in VD disorders.Calmodulin inhibitor is expected to become clinical drug candidate for VD treatment.