| Objectives:Dry eye is a commom ocular surface disease in clinic and abnormal nerve regulation is an important cause of dry eye.The purpose of this study is to investigate the distribution of tear-secreting reflex center in the brain of rabbits and the effect of acupuncture on the expression of central cholinergic neurons in tears of dry eye model rabbits,and to determine whether acupuncture can regulate the cholinergic receptor-mediated signal transduction pathway in the lacrimal gland of the dry eye model rabbits,thereby promoting the role and mechanism of tear secretion.Methods:(1)Healthy adult New Zealand rabbits were randomly divided into 3 groups.The sterilized dry cotton swabs were used to take the appropriate amount of peppermint camphor mixture to wipe the lower eyelids of the rabbits as the model group,and the saline group and the completely blank group were set as the control.After 2 hours,the animals in each group were sacrificed.Immunofluorescence histochemical staining combined with computer image analysis system were used to observe the expression of c-fos protein in the frontal,hippocamptus,hypothalamus,pons and medulla oblongata neurons of each group.(2)Trace amount of pesudorabies virus(PRV)was injected into the lacrimal gland of the rabbit and the distribution of fluorescently labeled neurons in the brain were observed after surviving for 30 hours,38 hours,46 hours separately through the way of immunofluorescence histochemistry.(3)New Zealand rabbits were randomly divided into normal group,model group,western medicine group and acupuncture group,and each group had four rabbits.Normal group did not accept any treatment.According to 2.0mg each time and four times each day until the end of the experiment,subcutaneous injection of scopolamine hydrobromide was made in model group.Western medicine group and acupuncture group were conducted 1%pilocarpine eye drops and acupuncture treatment respectively started with the success of dye eye model,and treatment for seven days in a row.The tear secretion of experimental rabbits from different groups were tested by Schirmer Test in different time.The changes of ChAT content in brain neurons of rabbits were observed by immunofluorescence histochemical staining method.(4)Twenty-four New Zealand rabbits were randomly divided into 4 groups:normal group,model group,western medicine group,and acupuncture group,with 6 rabbits in each group.The treatment methods of each group were the same as above.Tear secretion examination was performed on the 1th,4th,7th,and 14th after acupuncture treatment.Half of the experimental rabbits were sacrificed on the 7th and 14th day after acupuncture.Real-time PCR was used to detect the expression of M3AChR,PLC?,PKC,Ip3,PLD1,Ras,Raf,MEK,MAPK and PyK2 mRN A in lacrimal gland.Results:(1)A large number of c-fos immunoreactive cells were observed in the model group.A small amount of c-fos immunoreactive cells were observed in the saline group,and the mean number of c-fos immunoreactive cells in the brain regions of the two groups were significantly difTerent(p<0.05).In the completely blank group,a small amount of c-fos immunopositive cells were observed,and compared with the model group,the mean number of c-fos immunoreactive cells in each brain region were significantly different(p<0.05).Compared with the saline group,the mean number of c-fos immunoreactive cells in frontal lobe,hippocampus and pons were significantly different(p<0.05),but there was no significant difference in the mean number of hypothalamus and medulla oblongata(p>0.05).(2)Positive and marked neurons were found only in superior salivatory nucleus of pons(SSN)after injected for 30 hours.While after 38 hours,the secondary neurons infected PRV across synapses were observed in lateral nucleus of superior olive(LSO)and medial nucleus of superior olive(MSO)of superior olivary complex.After 46 hours,positive and marked cells were found in nucleus of trapezoid body(Tz)and hypothalamic paraventricular nucleus(PVN)and their distributions were dense in LSO and MSO.(3)The results from Schirmer I test showed that the tears of rabbits of acupuncture group and western medicine group increased significantly on the 28th day,compared with the 21th day.Acupuncture group and model group had significant differences on the 28th day in the experiment and so did the western medicine group(p<0.05).According to the expression and distribution of ChAT immune positive neurons,ChAT immune positive reaction products were located in dorsal tegmental nucleus and lateral parabrachial nucleus in the pons.Compared with the model group,the count of ChAT immune positive neurons of the acupuncture group was increased and had significant differences in dorsal tegmental nucleus and lateral parabrachial nucleus(p<0.05),and so did the western medicine group.(4)The results of PCR showed that on the 14th day of acupuncture,the mRNA transcription level of M3AChR in the lacrimal gland of the model group was significantly lower than that of the blank group,the difference was significant(p<0.05).Compared with the model group,the mRNA transcription levels of M3AChR in the lacrimal gland of the acupuncture group and western medicine were signi:ficantly increased in the group(p<0.05).Compared with the blank group,the mRNA transcription levels of PLD1,MAPK and Ras in the lacrimal gland of the model group were significantly increased,and the difference was significant(p<0.05).Compared with the model group,the mRNA transcription levels of PLD1,MAPK and Ras in the acupuncture group and western medicine group were significantly decreased,and the difference was significant(p<0.05).Conclusions:(1)The acupuncture information is uploaded to the dry eye model rabbit's tear secretion reflex center,and then the cholinergic neurons in it will be excited to release ACh from the nerve endings supporting the lacrimal gland to promote tear secretion.(2)Acupuncture can cause changes in key gene activities in the acetylcholine M receptor and its signal transduction pathway in the lacrimal gland of dry eye model rabbits,and promote tear secretion. |
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