Nitric Oxide Microbubble Combined With Focused Ultrasound To Explore Effect Of Nitric Oxide On Nerve Injury In Parkinson’s Disease

Objective:The purpose of this study is Focused Ultrasound（FUS）was used to open the blood-brain barrier（BBB）to deliver exogenous NO to the brain of experimental mice,and compared with 6-OHDA-induced Parkinson’s model rat,and evaluated the regulatory role of NO in Parkinson’s disease neurological injury by various methods.Methods:1.Firstly,by introducing a macromolecular inert gas—Sulfur hexafluoride（SF₆）mixed with NO gas,using a microscope to observe the shape of the microbubbles,dynamic light scattering to detect the particle size of the microbubbles,and a particle counter to detect the initial concentration of the microbubbles,etc.NO microbubbles were characterized;and the NO content detection kit was used to ultrasonically blast NO microbubbles in vitro,and the NO content curve produced by NO microbubbles with blasting time was monitored to observe its stability.2.First,by introducing a macromolecular inert gas—sulfur hexafluoride（SF₆）mixed with NO gas,observe the shape of microbubbles with a microscope,detect the particle size of microbubbles by dynamic light scattering,and detect the initial concentration of microbubbles by a particle counter,multi-angle detection NO microbubbles were characterized.And the NO content detection kit was used to ultrasonically blast NO microbubbles in vitro,and the NO content curve produced by NO microbubbles with blasting time was monitored to observe its stability.3.Grouping experiments by orthogonal analysis design,irradiating the unilateral brain of rats with FUS with different ultrasound parameters,and then perfusing the heart,evaluating the openness of FUS from the general image,HE staining,EB stimulation experiment,Tunel staining and biochemical indicators Safety and efficacy of the BBB,and determination of optimal ultrasound parameters for an open BBB.4.Treat SH-SY5Y cells with different concentrations of NO microbubbles in vitro,use CCK-8 to detect cytotoxicity,and Annexin V-IF488/PI method to detect cell apoptosis,to explore the influence of different NO concentrations on nerve cells.In vivo experiments,the experimental rat were randomly divided into control group,NO@MBs group,6-OHDA model group,through apomorphine-induced rotation test,tumbler test and water maze test.Evaluate the effect of NO@MBs on the behavior of rats compared with PD model rat,and evaluate the role of NO in nerve injury by detecting the distribution of tyrosine hydroxylase neurons,mitochondrial membrane potential levels,and oxidative stress levels by immunohistochemistry At the same time,the safety of the experimental protocol was evaluated by HE staining of major organs,serum liver and kidney function and myocardial enzyme spectrum.Results:1.By mixing SF₆and NO gas at a volume ratio of 9:1,it is possible to prepare NO microbubbles with a particle size range of 1-2μm,an initial concentration of about 6.68×10⁸/ml,and a stable release of NO.2.When the ultrasonic parameters are set to pulse frequency 1.0MHz,amplitude800m Vpp,and irradiation time 90s.FUS can safely and effectively open the rat BBB.3.In vitro experiments found that SH-SY5Y cells decreased cell viability and even apoptosis with the increase of NO microbubble concentration in a dose-dependent manner.In vivo experiments found that FUS combined with NO@MBs can simulate the same behavioral changes in PD model mice,which can reduce the ability of spatial learning and memory and coordination and balance of limbs in rats.The apomorphine-induced rotation experiment was used as a standard to judge whether the PD model was formed.Although the NO@MBs group also showed the behavior of end-to-end connection and rotation to the healthy side,it did not meet the standard of PD modeling.Considering the possibility of PD nerve damage,in addition to the involvement of NO signaling pathways,and other pathways that cause damage.FUS combined with NO@MBs can also significantly reduce the expression of TH and the number of neurons in the substantia nigra of rat brain,and reduce the level of mitochondrial membrane potential.It can also reduce the activity of SOD and CAT enzymes,significantly reduce the content of GSH,and increase the content of MDA,which verifies that NO can play a role in neuronal damage by forming oxidative stress.Conclusion:FUS combined with NO@MBs can safely and effectively open the BBB,and can cause experimental mice to develop the same behavioral changes as PD rats.High concentrations of NO@MBs have obvious toxic effects on nerve cells..It plays a role in neuronal damage in terms of oxidative stress,TH expression,and mitochondrial membrane potential.