Experimental Study Of Nano-paclitaxel Co-assembly System To Alleviate Neuronal Death In Ischemic Stroke By Stabilizing Microtubule Cytoskeleton

ObjectiveNeurons are highly sensitive to ischemia,and after suffering ischemic injury,they undergo progressive microtubule cytoskeleton degradation and lead to neuronal death.However,neuronal death also occurs in the hippocampal CA1 region as a non-blood supply area after middle cerebral artery ischemia,and it is not clear whether microtubule cytoskeleton degradation is related to it.Therefore,this study aimed to investigate the dynamic changes of neuronal microtubule cytoskeleton in hippocampal CA1 area after ischemic stroke and its relationship with neuronal death.At the same time,a nano-paclitaxel co-assembly system was constructed to reduce the neurotoxicity of paclitaxel and to investigate whether it could reduce neuronal death and exert neuroprotective effects in the hippocampal CA1 region of ischemic stroke by inhibiting the degradation of microtubule cytoskeleton,using the drug property of paclitaxel to stabilize the microtubule cytoskeleton.Methods1.Construction and validation of the mouse left middle cerebral artery embolism(MCAO)model:mice were anesthetized with 3%isoflurane and fixed in the supine position to make a midline cervical incision.The external carotid artery,internal carotid artery and common carotid artery were isolated separately and the ligature line was reserved.After ligation of the external carotid artery,both ends of the common carotid artery were simultaneously clamped with vascular clips,and a small incision was made at the proximal end of the common carotid artery with microscissors to insert a wire plug with a black marker point to the bifurcation of the internal and external carotid arteries.After the insertion,the ligature wire pre-placed in the common and internal carotid arteries was tied tightly to prevent the wire plug from slipping out and bleeding,and the neck wound was routinely sutured.After 45 min of ischemia,the mice were anesthetized again,the internal carotid artery was isolated and exposed,the fixation wire was loosened,and the ligature wire was tied tightly after removing the wire plugs.2.HT22 cell oxygen glucose deprivation reperfusion(OGD/R)model establishment and evaluation:Cells in good growth condition and in logarithmic growth phase were selected for plate laying.After 24 h,the cells were replaced with DMEM low-glycemic medium and treated in a hypoxic chamber(99%N2 and CO2 mixture,1%O2)for 4 h.Then the cells were replaced with DMEM high-glycemic medium and incubated in a cell culture incubator for 24 h to establish the HT22 oxygen-glycemic deprivation-reperfusion cell model.3.To study the in vivo distribution of OA-GA-PTX NPs using live imaging:Co6-OA-GAPTX NPs were injected intravenously into MCAO mice through the tail vein.Fluorescence imaging was performed using a multimodal in vivo imaging system.4.The toxicology of OA-GA-PTX NPs on the systemic organs of MCAO mice was detected and analyzed by HE staining.5.To evaluate the volume of brain infarction in mice by TTC staining.6.To detect neuronal death in CA1 region of mouse hippocampus by Nissl staining,F-JC staining and NeuN staining.7.To detect changes in microtubule cytoskeleton by labeling microtubule-associated proteins acetyl-tubulin,β-tubulin Ⅲ and MAP2 using immunofluorescence staining experimental method.8.To detect the effect of OA-GA-PTX NPs on inflammatory response in MCAO mice by immunofluorescence staining and Western Blot.9.To detect the expression of neuronal microtubule-associated proteins acetyl-tubulin,βtubulin III and MAP2 by Western Blot.Results1.The volume of brain infarcts in the MCAO model group mice gradually increased with the prolongation of the extraction time.In addition,the number of neuronal death in the hippocampal CA1 region gradually increased in the MCAO model group compared with the sham-operated group,and was accompanied by the degradation of microtubule-associated proteins β-tubulin,β-tubulin Ⅲ and MAP2.2.Tail vein injection of OA-GA NPs,PTX injection and OA-GA-PTX NPs,respectively,significantly improved neurological dysfunction and reduced neuronal death with OA-GAPTX NPs treatment compared with the MCAO model group.3.HT22 mouse neuronal cells showed reduced survival and microtubule skeleton-related protein expression levels after OGD/R modeling.4.OA-GA-PTX NPs were uniformly distributed in various organs and brain tissues,and a large enrichment of NPs in brain tissues was observed within the following 1 h.The NPs were metabolized by the liver and kidneys around 6 h after entering the body;rapid uptake of NPs by HT22 cells was observed when co-cultured with coumarin 6(Co6)-labeled OA-GA-PTX NPs and HT22 cell lines,and the stay in the cells for more than 5h.5.The optimal dosing concentration of OA-GA-PTX NPs was determined to be 2 mg/kg by HE staining,TTC staining,animal behavior and Doppler blood flow monitoring.6.Treatment with OA-GA-PTX NPs significantly reduced the brain infarct volume and neuronal death in the CA1 region of the hippocampus in MCAO mice.7.Treatment with OA-GA-PTX NPs delayed the degradation of microtubule backbone proteins and apoptosis compared to the model group.8.OA-GA-PTX NPs suppress inflammatory responses while attenuating ischemic strokeinduced blood-brain barrier disruption.ConclusionThe degradation of microtubule-associated proteins may be one of the factors of neuronal death in the hippocampal CA1 region in ischemic stroke;OA-GA-PTX NPs,constructed for the drug properties of paclitaxel to stabilize microtubule structure,can effectively inhibit the degradation of microtubule-associated proteins in the hippocampal CA1 region in ischemic stroke and attenuate neuronal death,reduce infarct volume and improve neurobehavior without significant drug toxicity,and co The OA and GA components of the assembled system may also have a protective effect against ischemic stroke by both inhibiting the inflammatory response and maintaining the integrity of the BBB.