| ObjectiveThe heroin dependent mice model was established by the conditioned place preference(CPP)method,the effective ultrasonic energy parameters derived in our previous study were applied,the nucleus accumbens was damaged by combined microbubble cavitation,the CPP score,the open field test,the elevated plus maze test were used to evaluate the behavioral changes,and the changes of pathological structure,synaptic ultrastructure,as well as neurotransmitter content were analyzed as controls,The mechanism by which a certain energy of ultrasound improves the ethology of heroin dependent mice was preliminarily elaborated to provide new ideas and methods for drug abstinence and treatment.Methods1 Establishment of a heroin dependent model: 52 healthy mice were randomly divided into a control group(Control group),a heroin dependent group(Heroin group),a heroin dependent+ultrasound stimulation group(US group),and a heroin dependent+ultrasound stimulation combined with microbubbles group(US+MBs group),with 13 mice in each group.A heroin dependent mouse model was established by dose increasing injection combined with CPP.2 Ultrasound combined with microbubble cavitation stimulation: After successful modeling,ultrasound was used to stimulate the nucleus accumbens region of the mouse.The probe frequency was 2 MHz,the duty ratio was 5%,and the pulse amplitude was 25%.It lasted for 15 minutes per day for a total of 2 days.The US group was given simple ultrasound stimulation,while the US+MBs group was given ultrasound stimulation and then injected sulfur hexafluoride microbubbles(200μL),Herion group and Control group were subjected to false stimulation(no energy output,only irradiation without power).3 Behavioral evaluation: After treatment,retest CPP to assess the intensity of mice’s addictive memory through CPP scores,measure the time the mice stay in the central area through the open field experiment,evaluate the degree of anxiety through the number of times they cross the central area,evaluate the autonomous activity ability of the mice through the total distance of movement in the open field,and evaluate the degree of anxiety and fear through the elevated test to detect the time the mice stay in the open arm and the number of times they enter the open arm.4 Pathology,synaptic structure,and neurotransmitter detection: Evaluate the pathological changes of the nucleus accumbens using hematoxylin and eosin(HE)staining,observe the changes in synaptic ultrastructure of the nucleus accumbens using transmission electron microscopy,and detect changes in the content of neurotransmitters(dopamine,5-hydroxytryptamine,glutamate)in the nucleus accumbens using high-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS).Results1 Successfully established a heroin dependence model: Before administration,there was no statistically significant difference in CPP scores among the groups(P>0.05).After a continuous week of CPP training,compared with the Control group,the CPP scores of the Herion group,the US group,and the US+MBs group significantly increased(P<0.05),and the activity trajectory of the drug box was significantly increased.There was no statistically significant difference in CPP scores among the Herion group,the US group,and the US+MBs group.2 Behavioral evaluation: The results of the retest of CPP showed that compared with the Herion group,the CPP value in the US group was lower(P>0.05),while the CPP value in the US+MBs group was significantly lower(P<0.05);The results of the open field experiment showed that compared with the Control group,the total movement distance,the number of times of entering the central region,and the dwell time of the mice in the Herion group were significantly reduced(P<0.05);Compared with the Herion group,there was an increase after US treatment(P>0.05),and a significant increase after US+MBs treatment(P<0.05);The results of the elevated cross maze experiment showed that compared with the Control group,the percentage of mice entering the open arm and the percentage of mice staying in the open arm in the Herion group were significantly reduced(P<0.05),compared with the Herion group,the treatment with US was prolonged(P>0.05),and the treatment with US+MBs was significantly prolonged(P<0.05).3 HE staining: In the Control group,the morphology and structure of neurons in the nucleus accumbens region were normal,and no red blood cell exudation was observed;There was no red blood cell extravasation and no damage to the nucleus accumbens in both the Herion group and the US group;In the US+MBs group,red blood cells exuded and gathered in the nucleus accumbens area,and vacuolar degeneration of the nerve fiber network was observed.4 Transmission electron microscopy: In the Control group,the synaptic cleft structure in the brain region of the nucleus accumbens was clear,and the mitochondrial cristae were obvious;In the Herion group,the structure of synaptic cleft was normal,with slight swelling of mitochondria and broken cristae;In US group,the synaptic cleft was locally unclear,mitochondria swelled,and cristae ruptured;In the US+MBs group,the synaptic gap was blurred,mitochondria were significantly swollen,cristae were broken,and the number of synapses was reduced compared to that in the Herion group(P<0.05).5 Detection of neurotransmitter content by UPLC-MS/MS: Compared with the Control group,the contents of DA,5-HT,and Glu in the nucleus accumbens of mice in the Herion group increased(P>0.05);Compared with the Herion group,the levels of DA,5-HT,and Glu were decreased after US treatment(P>0.05),and significantly decreased after US+MBs treatment(P<0.05).Conclusion1 Stimulation of the nucleus accumbens in mice by ultrasound combined with microbubble cavitation reduced the degree of drug preference,improved addictive memory,alleviated anxiety like symptoms and improved locomotor activity in heroin dependent mice.2 Ultrasound combined with microbubble cavitation impaired the nucleus accumbens in heroin dependent mice,leading to erythrocyte extravasation,vacuolar degeneration of the neural fiber network,ultrastructural changes in synapses in the nucleus accumbens region,decreased synapse number,and significantly decreased levels of dopamine,5-hydroxytryptamine,and glutamate,which may be the reason why ultrasound combined with microbubble cavitation improved addictive memory and anxiety behavior. |
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