Study On Common Brain Nuclei Of Two-Way Neural Pathways In Rat Feishu And Lower Respiratory Tract Based On The Technique Of Neurotropic Virus Labeling

Objective:Based on the clinical efficacy of acupuncture in treating asthma,the common brain nuclei of Feishu and Lower Respiratory Tract in rat were screened by technique of neurotropic virus labeling.Methods:Experimental animals grouping:SD rats were divided into 5 groups,which were normal group,anterograde labeling group of Feishu,retrograde labeling group of Feishu,anterograde labeling group of Lower Respiratory Tract,and retrograde labeling group of Lower Respiratory Tract group.The normal group was not labeled;HSV-EGFP was used to label the afferent nerve pathway in the Lower Respiratory Tract group and the Feishu group;PRV531-CAG-GFP was used to label the efferent nerve pathway in the Lower Respiratory Tract group and the Feishu group.Anterograde labeling method of HSV-EGFP in afferent nerve pathway of Feishu:Feishu locates on the side of the third thoracic vertebrae 0.5cm,slowly inject 2?l of HSV-EGFP stock solution(virus amount 4×10~7PFU)to the Feishu.Retrograde labeling method of PRV531-CAG-GFP in afferent nerve pathway of Feishu:The method was the same as the marking method of afferent nerve pathway of Feishu,the virus was PRV531-CAG-GFP(virus amount 4×10~7 PFU).Anterograde labeling method of HSV-EGFP in afferent nerve pathway of Lower Respiratory Tract:Micro syringe needle is inserted into the middle and lower 1/3intersection of the trachea,injecting 6?l dilution of HSV-EGFP(virus amount 2.4×10~7PFU)to the Lower Respiratory Tract of rats.The micro syringe was immediately removed from the rat airway.Retrograde labeling method of PRV531-CAG-GFP in afferent nerve pathway of Lower Respiratory Tract:The method was the same as the marking method of afferent nerve pathway of Feishu,the virus was PRV531-CAG-GFP(virus amount 2.4×10~7PFU).The criteria for successful estimate of neurotropic virus marker:The symptoms of HSV-EGFP labeled group is dull of eye expression,nasal bleeding,stumbling gait,and slow activity.The symptoms of PRV531-CAG-GFP labeled group is nasal bleeding,limb bleeding,and excited spirit.Localization analysis of neurotropic virus labeled brain nucleus:Perfusion time node was determined according to the onset state of rats.The whole body was fixed by 4%paraformaldehyde through the heart.The brain tissue was cut into a coronal section by a frozen slicer,cell nuclei were labeled by DAPI.Then using automatic tissues fluorescence scan technology to observe the distribution of two dyes in visceral organs and the nuclei was localized by a stereotaxic map of the rat brain.Results:1.The distribution of fluorescence signals in the organs of rats labeled with neurotropic virus at Feishu:(1)Feishu of rat was injected with HSV-EGFP into the onset state.Organs of rat compared with the normal organs.There were no fluorescence signal distribution in the Feishu of the virus marker and the visceral tissue heart,liver,spleen,kidney,trachea,esophagus,intestine,stomach and lung.(2)Feishu of rat was injected with PRV531-CAG-GFP into the onset state.Organs of rat compared with the normal organs.There were no fluorescence signal distribution in the Feishu of the virus marker and the visceral tissue heart,liver,spleen,kidney,trachea,esophagus,intestine,stomach and lung.2.The distribution of fluorescence signals in the organs of rats labeled with neurotropic virus at Lower Respiratory Tract:(1)HSV-EGFP was injected into the Lower Respiratory Tract of rats,and compared with normal tissue sections,there was no fluorescence signal in trachea and lung.There was no fluorescence signal distribution in heart,liver,spleen,kidney,esophagus,stomach,intestine and Feishu.(2)PRV531-CAG-GFP was injected into the Lower Respiratory Tract of rats,and compared with normal tissue sections,there was no fluorescence signal distribution in trachea and lung There was no fluorescence signal distribution in heart,liver,spleen,kidney,esophagus,stomach,intestine and Feishu.3.The distribution of fluorescent signals in brain of rats labeled with neurotropic virus(Lower Respiratory Tract or Feishu):The distribution of fluorescent signals was observed in cerebral cortex,telencephalon,midbrain and pons,The distribution density of fluorescence signal can be divided into small distribution,dispersed distribution and concentrated distribution from low to high.4.Distribution results of nuclei in brain of Lower Respiratory Tract and Feishu labeled with neurotropic virus:(1)The results of HSV-EGFP labeling brain nucleus by the afferent pathway from the Feishu showed that the midbrain was distributed in hypothalamus,periaqueductal gray,dorsal tegmental chiasmi,Edinger-Westphal nucleus,interstitial nucleus and thalamus;The pons were distributed in the nucleus oculomotor,red nucleus,pontine reticular nuclei,posterior vertebral nucleus,reticularis nucleus,facial nucleus,paracellular laterals nucleus,medial longitudinal fasciculus,dorsal motor nucleus of vagus,inferior olivary nucleus,solitary tract,spinal trigeminal tract.(2)The results of PRV531-CAG-GFP labeling brain nucleus by the afferent pathway from the Feishu showed that the telencephalon is distributed in motor cortex,hypothalamus,diagonal band nucleus,septal nucleus,globus pallidus,thalamic nucleus and amygdala.The midbrain was distributed in the internal capsule,A13dopamine cell,visceral nucleus,substantia nigra striatum bundle,dorsal medial nucleus,optic nerve bundle,periaqueductal gray,reticular nucleus,medial papillary nucleus,petrous nucleus,habenular nucleus,red nucleus,oculomotor nucleus,trigeminal crista midbrain bundle,dorsal raphe nucleus,tectal nucleus,tegmental nucleus,raphe nucleus and medial thalamus.The Pons were distributed in the A5noradrenaline cells,superior olivary nucleus,locus coeruleus,superior vestibular nucleus,abducent nucleus,cuneiform nucleus,gracilis nucleus,solitary tract nucleus,dorsal vagus nucleus,trigeminal nerve nuclei,ventral respiratory group and inferior olivary nucleus.(3)Lower Respiratory Tract afferent pathway labeled with neurotropic virus HSV-EGFP showed that the telencephalon is distributed in cortex,thalamus,corpus callosum,caudate nucleus,globus pallidus,hypothalamus,reticular nucleus,internal capsule,amygdala,optic nerve bundle,periaqueductal gray,oculomotor nucleus.The midbrain is distributed in habenular nucleus,Edinger-Westphal nucleus and red nucleus.The Pons is distributed in locus coeruleus,nucleus raphe magnus,tegmental nucleus,trigeminal nerve nuclei,solitary tract,dorsal motor nucleus of vagus and inferior olivary nucleus.(4)Lower Respiratory Tract afferent pathway labeled with neurotropic virus PRV531-CAG-GFP showed that hypothalamic,locus coeruleus,medial longitudinal fasciculus,rostral ventral respiratory group,inferior olivary nucleus,solitary tract,dorsal motor nucleus of vagus.5.Common Nucleus of Neurotropic virus labeled Feishu and Lower Respiratory Tract nerve pathway:(1)Common nucleus of afferent nerve pathway of Feishu and efferent nerve pathway of Lower Respiratory Tract.It's mostly focused on dorsal hypothalamic area,medial longitudinal fasciculus,dorsal motor nucleus of vagus,solitary nucleus ventral part,nucleus of the solitary tract intermediate part,nucleus of the solitary tract medial part.(2)Common nucleus of afferent nerve pathway of Lower Respiratory Tract and efferent nerve pathway of Feishu.It's mostly focused on paraventricular hypothalamic nucleus medial parvicellular part,globus pallidus,internal capsule,pre-Edinger-Westphal nucleus,medial accessory oculomotor nucleus,red nucleus parvicellular part,red nucleus magnocellular part,periaqueductal gray,Edinger-Westphal nucleus,dorsomedial hypothalamic nucleus dorsal part,perifornical part of lateral hypothalamus,A5 noradrenaline cells,locus coeruleus,raphe magnus nucleus,spinal trigeminal nucleus interpolar part,medial longitudinal fasciculus,solitary nucleus,dorsal motor nucleus of vagus.(3)Common nucleus of afferent nerve pathway of Lower Respiratory Tract and afferent nerve pathway of Feishu.It's mostly focused on periaqueductai gray,ventral posteromedial thalamic nucleus,pre-Edinger-Westphal nucleus,medial accessory oculomotor nucleus,Edinger-Westphal nucleus,red nucleus parvicellular part,intermediate reticular nucleus,medial longitudinal fasciculus,dorsal motor nucleus of vagus,spinal trigeminal nucleus interpolar part,parvicellular reticular nucleus,solitary nucleus dorsolateral part,solitary tract,inferior olive medial nucleus.(4)Common nucleus of efferent nerve pathway of Lower Respiratory Tract and efferent nerve pathway of Feishu.It's mostly focused on dorsal hypothalamic area,perifornical part of lateral hypothalamus,medial longitudinal fasciculus,dorsal motor nucleus of vagus,solitary nucleus ventral part,nucleus of the solitary tract intermediate part,nucleus of the solitary tract medial part.6.Common Nucleus of the nerve pathway of Feishu and lower respiratory tract:solitary nucleus,dorsal motor nucleus of vagus and medial longitudinal fasciculus take participate in 4 neural pathways(afferent pathway of Feishu,efferent pathway of Feishu,afferent pathway of Lower Respiratory Tract and efferent pathway of Lower Respiratory Tract);It is significant relay nucleus of Feishu and Lower Respiratory Tract in the nerve pathway.Conclusion:(1)Common nucleus of afferent nerve pathway of Feishu and efferent nerve pathway of Lower Respiratory Tract that are dorsal hypothalamic area,medial longitudinal fasciculus,dorsal motor nucleus of vagus,solitary tract.(2)The visceral-body surface reaction of the lower respiratory tract and the Feishu point has a neuronal basis of the brain nucleus.These brain nuclei include paraventricular hypothalamic nucleus medial parvicellular part,globus pallidus,internal capsule,perifornical part of lateral hypothalamus,dorsomedial hypothalamic nucleus,dorsal part,A5 noradrenaline cells,locus coeruleus,raphe magnus nucleus,pre-Edinger-Westphal nucleus,medial accessory oculomotor nucleus,periaqueductal gray,red nucleus parvicellular part,red nucleus magnocellular part,Edinger-Westphal nucleus,dorsal motor nucleus of vagus,solitary nucleus,medial longitudinal fasciculus and spinal trigeminal nucleus interpolar part.(3)Common Nucleus of the nerve pathway of Feishu and lower respiratory tract that is solitary nucleus,dorsal motor nucleus of vagus and medial longitudinal fasciculus.