The Influence Of He-Ne Laser Irrigation On Cultured Neurons

ObjectiveAlong with the progress that social advanced of age ,and enhancing of cerebralpalsy. The research of the outbreak mechanism of the cerebral palsy; old-age disease and the health care problem of the old people become more and more urgent demand of the medical science nowadays. In recent years, many scholars carry on the various research, working hard to look for the methods what can be promote the neurons growth and development,prolonging ageing. The neurons is the nervous system structure and basic unit of the functions. The study of neurons growth development and decrepitudes may be provide the mechanisms for it, also providing the theories for the cerebal palsy and old ageing disease as well as valid treatments of the old-age disease of the central nervous system according to. Especially the culture of serum free medium provided vast of foreground for the research that neuronse decrepition and death in recent years. Recently He-Ne laser has been applied in clinic because of its unique biological stimulation extensively currently. But it for the central neurons whether have function to promote the growth and development,prolong ageing .Does its function mechanism is only its biological stimulation effects or blame on the change of an environment factor with tiny surroundings of neurons? There are without hard conclusion nowadays. So this experiment adopts culture of serum free medium for new-born rats Hippocampal and part of frontal lobe neurons, The influence of He-Ne laser irrigation on the growth and development of hippocampal neurons and part of frontal lobe neurons wasobserved. The function and its function mechanisms were further studied for He-Ne laser to the neurons, trying to find out the decrepitude of central nervous system and a new and valid therapeutic method for it. Method12 within postnatal 24h wistar rats weight 6.3 ±0.5 grams were taked randomly. After normal regulations disinfect decapitated the rat head to take the brain quickly, separated the Hippocampal and parts of frontal cerebral cortex from cerebral hemisphere completely ,all succeeding steps must be performed in a laminar flow hood .After dgesting and dispersing diluted it making it become 0.5-1.0X 106 density liquid with plating medium (high sugar DMEM+10 % fetal bovine serum), plating it in 4 pieces plastic Petri dishes of 24 bore which per bore has spreaded over the glass coverslips with poly-L-lysine all. Place in incubator HF90 and maintained at 37°C in an atmosphere containing 5% CO2. After feeding cultures 24h changed the culture medium completely with serum free medium (B27-Supllment). We replace one-third of the medium once every two days.we carry on neurons authenticate with (NSE) immunohistochemical staining of ABC method after plating 3d.Selected fine tow pieces plastic Petri dishes were randomly divided into four groups: I : normal control group only feeding culture with serum free medium without irradiation. Common red light matched control group of II :The irradiation were started at second day of Feeding culture. Wave length:632.8 nm,output power:3 mw,facular diameter 3 nuns, each time: 3 min, distence: 4-5 cm, and perpendicular irradiation, guaranteeing that the irradiation scope is slightly small in each area of bore. IILThe cerebrolysin control group: feeding medium were joined 200 u g/ml cerebrolysin, only feeding culture with serum free medium without treatment. IV The experiment group: The irradiation were started at second day of Feeding culture. Wave length:632.8 nm; output power:3 mw; facular diameter: 3 mms; each time: 3min; distence: 4-5 cm, and perpendicular irradiation, guaranteeing that the irradiation scope is slightly small in each area of bore. When Common red light matched control group and normal control group were irradiated , Another groups were taked out and same placing equally but don't irradiated equally. Observes the circumstance of cultured neurons regularly withtime-lapse video microcopy and sequntial photography everyday after experimented, taked the each group cultured neurons and recorded its survival number neurite outgrowth length ^ soma area and body maximum diameter, and comepared each other in 7^ 14> 2K 28d. it were cultured 14d later, The glass coverslips were taked out from plastic Petri dishes, sectioned and stained by Nissl staining, BDNF and MAP2 immunohistochemical staining. The neurons number of positive neurons counted under microscope and the grey scale values were measured,the data were statistically analysed with SPSS 10.0. Result1 Effect of He-Ne laser irrigation on neurite survival number % soma area <. body maximum diameter and outgrowth length: The neurons have already adhesion after planting 12 hours. In the 7 day it is thus clear that the experiment group and the cerebrolysin control group grow certainly good. The outgrowth rise longer, and making the dense neuronal network of outgrowth because of the extensiv fasciculation . compared each other there was no significant difference between groupIII and group IVas well as between group I and group II .There are neurons number less in I and II groups, and the neuronal network is very sparse of axons. there are obvious difference(/?<0.01) comepared with III and IV; Especially normal control group have already started deteriorate and die but experiment group and cerebrolysin control group to grow certainly good and making dense neuronal network of outgrowth at 2Id. ( number of neurons I 63.5+2.6;II67.4±3.2;III 103.2+2.5;IV 105.1 +3.5 cell piece/ visual fields ). The neurons of I and II have been already all died, but another two groups have parts of survivals still.2 Nissl staining: The neuron cytoplasm was full of dark-blue colored nissl bodies in group III and IV, positive neurons were distributed widely in there.A great number of nissl bodies were lost in I and II groups, the grey scale value appeared a ascending tendency .positive neurons were less in there.And had obvious difference betweenlll IVand III (/K0.05).3 BDNF immunohistochemical staining: BDNF positive neurons were less ingroup I II (I group.- 88.86±2.83 ,11 group: 9.23±2.56), there was a considerable increase in groupIII IV(III: 14.25 + 82.21, IV: 15.85±2.79), There are a marked increase in groupIII IV,and obvious difference between III IV and III ,when compared with group III IVand III (jX0.05).4 MAP2 immunohistochemical staining: MAP2 positive neurons were expression widely in group III and VI,its dendrites are stained hardly.it can be seen clearly that dendrites was compared with I II, There are a marked positive neuronal i increase in groupIII IV,and obvious difference between III IVand I II ,when compared with group III IVand III (p<0.05);5 SOD and MDA: SOD were less in group I II .but MDA were increasing in HI IV.There are obvious difference betweenlll IVandIH(/K0.05). Conclusions:(1 )TheHe-Ne Laser irrigation can ncreasing survival number> length ^somaarea > body maximum diameter and increasing survival number.(2) The He-Ne Laser irrigation may enhance the volum Nissl body obviously . could promote the growth and developement.(3) He-Ne laser acupoint irradiation may increase the expression of BDNF and MAP2 on cultured neurons, which maybe the mechanism of neuronal growths development and prolong ageing ;(4) Activity of the antixoidant enzyme could be inhanced by He-Ne laser irradiation, which maybe the mechanism of neuronal prolong ageing ;(5 ) The experiment as a result expresses that the He-Ne Laser irrigation might play important role in the growth ■. development prolong ageing of neurons.