ABSTRACT
Background Oscillatory potentials (OPs) of scotopic electroretinogram (ERG) plays an important role in the evaluation of visual function in multiple retinal diseases.However,the origin of OPs is uncompletely clear.It is essential to analyze the time domain and frequency domain components for the further study of OPs.Objective The present study was to investigate the change characteristics of frequency spectrum of scotopic OPs with age and stimulating intensity.Methods RCS-rdy+-p+rats with the ages of 21,25,32,35,37,46,60,90 days were selected iu this study and 3 rats for each.Scotopic flash ERG were recorded from all the rats with RETI-scan system.Gold-foil ring cornea recording electrode was used as the recording electrode and the steel needle electrode was used as the reference and earth electrode during the record.The intensity of stimulating light was set at-20,-10,-5,0 and 5 dB respectively.Data were output into the computer and processed by the software Matlab7.0.Results The principle frequency corresponding to maximum amplitude component was 80-120 Hz in the various ages of rats under the different stimulating conditions above.With the increase of the intensity of stimulating light,high frequency component (200-250 Hz) began to appear and the amplitudes showed a gradually raise upon the intensity of light.The major component was subdivided into two peaks at 0 dB stimulation.Further,the age affected the major frequency peak with the maximum value at 60-day-old rats and the minimum value at 25-day-old rats.Also,the pass-band width of main amplitude appeared to be maximal at 60-day-old rats and minimal at 25-day-old rats.Conclusions OPs in Rcsrdy+-p+ rats are influenced by stimulating intensity and agc.Stimulating intensity affects the amplitude and age lead to the change of distribution of primary frequency of OPs.It is possible to know the influences of the degeneration of rods and be helpful to diagnosis this kind of disease.
ABSTRACT
Background Channelrhodopsin-2 (ChR2)is a cation channel isolated from the eyespot of Chlamydomonas algae and has been used to control neuron activity.The light stimulation is a more precise fashion whether space or time than that of electrical,magnetic and ultrasound stimulation. Objective This study was to construct a replication deficient recombinant adenovirus cxpression vector of ChR2 and to determine its function.Methods Human embryo kidney 293 (HEK293) cell line was cultured and passaged in DF12 medium containing 10％ fetal bovine serum(FBS).The ChR2 gene was cloned at the downstream of cytomegalovirus(CMV)promoter of the adenoviral shuttle plasmid pSB291 in sense direction,and the resultant recombinant plasmid pSB291-hChR2- GFP was transfected into HEK293 cell together with plasmid pBHG lox ( deltaE1,3 ) containing adenoviral genome,then small amounts replication deficient recombinant adenovirus expression vector of ChR2 (Ad-ChR2) was obtained.Through amplification gradient centrifugation and dialysis,pure Ad-ChR2 was obtained.Visual cortex cells derived from 4 1-day-old clean Long Evans rats were primary cultured with serum-free culture media and infected by AdChR2.When expressing green fluorescencc,those cells received the stimulated of blue light with 460 nm.Patch clamp technique was applied to record an action potential. Results After purification and concentration,the titer of AdhCHR2 reached 7.9×1010 PFU/ml.Twenty-four hours after transfect of Ad-ChR2,HEK293 cell membrane showed the green fluorescence for the recombinant plasmid with green fluorescence protein under the inversed fluorescence microscope.The HEK293 cells change their shape from flat to round 13 days after transfected.The primary cultured visual cortex cells exhibited the green fluorescence 3-5 days after infected by Ad-ChR2.The action potentials evoked by blue light stimulation were recorded with patch clamp on those cells expressing green fluorescence. Conclusions Ad-ChR2 expressing vector is constructed successfully in this study.It is verified that Ad-ChR2 expressing vector can infect visual cortex cells with visual function.This result is very important for visual plasticity study.
ABSTRACT
Background RCS-rdy--P+ rat occur retinitis pigmentosa (PR) with the aging and development.To find OUt the retinal functional change using electrophysiological technique is useful for the further study of RCS-rdy--P+ rat. Ohjectlve The goal of this experiment was to investigate the dark-adapted electroretinogram (ERG) of RCS rats with aging. Methods The series of seotopic ERG were recorded in postnatal 21-,32-,37-,45-,60-day-old RCS-rdy--P+ rats respectively,and the age-matched RCS-rdy+-P+ rats were simultaneously recorded as control group.The ERG record was performed by a series of flash with RETI-scan system,gold-foil ring form cornea recording electrode and home-made stainless steel needle electrode.The use of animals complied with the Regulations of the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results Increase dark adaptation duration led to an ascended amplitude in ERG b-wave within 12 hours under the same light intensity,frequency and body temperature.Compared with the RCS-rdy+-P+ rats,the amplitudes of scotopic ERG a-wave and b-wave in RCS-rdy--P+ rats with postnatal 21 days age were apparently declined,showing significant difference between them(P<0.01).The implicit time of both RCS-rdy+-P+ rats and RCS-rdy--P+ rats were delayed, especially for the a-wave. The amplitudes of a-wave and b-wave were declined drastically with the growth of RCS-rdy--P+ rats and progression of their retinal degeneration, and the ERG responses were undetectable at the postnatal 60 days. Both the b-wave and a-wave amplitudes were lowered at P21-day RCS-rdy+-P+ rats and after that,significant increase amplitudes were noted till the P32-day rats. Thereafter, the b-wave amplitude and a-wave amplitude were stable from P32 d to P45 d,and much more ascending of b-wave and a-wave amplitudes was at P60 d RCS-rdy+-P+ rats. Conclusion RCS-rdy--P+ rats occur the retrogression of retinal function along with the ages growth. The scotopic ERG changes of RCS-rdy--P+ rats with aging is evidently different and is in accordance to the characteristics of the RP.