ABSTRACT
Objective To establish a method for the simultaneous determination of Forsythoside A, Phillyrin, (R,S)-Epigoitrin, Chlorgenic Acid and Isochlorogenic Acid A by HPLC, and test 16 batches of samples from 14 manufacturers. Methods The test was performed on Kinetex EVO C18 column (150 mm × 4.6 mm, 5 μm) with the column temperature at 35 ℃ . The gradient elution was adopted with the mobile phase of acetonitrile and 0.3% phosphoric acid aqucous at a flow rate of 1.0 ml/min. The detection wavelength of (R,S)-Epigoitrin and Phillyrin were set as 236 nm, the detection wavelength of Forsythoside A, Cholorogenic Acid and Isochlorogenic Acid A were set as 327 nm. Results The good linear relationships were displayed within the linear range of 0.050 45-2.018 00 μg for Forsythoside A (r=0.999 9), 0.018 21-0.728 40 μg for Phillyrin (r=0.999 9), 0.010 16-0.406 40 μg for (R,S)-Epigoitrin (r=0.999 9), 0.006 60-0.263 90 μg for Cholorogenic Acid (r=0.999 9) and 0.0040 44~0.161 76 μg for Isochlorogenic Acid A ( r=0.999 5). The RSDs of reproducibility and stability tests were lower than 2%; recoveries were 97.01%, 98.28%, 99.35% and 96.21%, RSD were 3.19%, 1.19%, 0.81%, 2.88% and 2.96%. The content ranges of Forsythoside A, Phillyrin, (R,S)-Epigoitrin, Chlorgenic Acid and Isochlorogenic Acid A from 16 batches of samples from 14 manufacturers were 0.057 43-1.508 71 mg/g, 0.017 72-0.350 15 mg/g, 0.005 68-0.177 13 mg/g, 0.007 53-0.226 33 mg/g and 0.00308-0.11908 mg/g. Conclusions The established method is simple and accurate, and has a good repeatability. It can be used for the quality analysis of Forsythoside A, Phillyrin, (R,S)-Epigoitrin, Chlorgenic Acid and Isochlorogenic Acid A. The content of the tested chemical components from 16 batches of samples from 14 manufacturers have significant differences which indicate that a reinforcement of the quality control is needed.
ABSTRACT
OBJECTIVE@#To observe whether bFGF could cross the blood-labyrinth barrier (BLB) after intra-abdominal injection and to establish an experimental basis for its clinical applications.@*METHOD@#Thirty guinea pigs were divided into three groups. Animals in group 1 were administered o I-bFGF, while animals in group 2 and 3 were administered 125 and saline, respectively, via intra-abdominal injection. The both cochlea, blood, liver, brain, thyroid gland and kidney were collected and weighted. A radioimmunoassay analyzer was employed to measure counts per minute (CPM) of each sample, and autoradiography was performed on both cochlea.@*RESULT@#The CPM value of organ samples in the 125I group was higher than that in other groups, and radioactive grain was observed in cochlear samples of this group. In the 125I-bFGF group, blood demonstrated the highest CPM value, while cochlea and brain demonstrated the lowest CPM value, with no radioactive grain observed in cochlear samples.@*CONCLUSION@#bFGF has some difficulties in getting across BLB, so the way of bFGF application in clinics need further study.
Subject(s)
Animals , Autoradiography , Cochlea , Cell Biology , Metabolism , Fibroblast Growth Factor 2 , Guinea Pigs , Injections, Intraperitoneal , Iodine RadioisotopesABSTRACT
Objective To evaluate the safety of Ad-Mathl administration to inner ear and provide the base data for vestibular dysfunction gene therapy.Methods Ten mature Wistar rats were divided into normal control group(srats) and adenovirus(E1,E3-Deleted and carried mathl and enhanced green fluorescent protein report gene,Ad-Mathl-EGFP)scala vestibuli transfer group(5 rats).Right ears of the Ad-Mathl-EGFP transfering group rats were deliveried 5ul Ad-Mathl-EGFP(physieal tite 2.1 10~(11)v.P./ml)into cochleas through the way of drilling scala vestibuli of cochlear basal turn.As a control,the normal group received nothing to inner ear.In order to estimate functional condition of vestibule and cochlea,the click-evoked potentials on the surface of the cervical dura mater(CDM-CEP),auditory brain stem response(ABR)and swimming time were recorded in all rats at 7 days after treatment,and then histologic and morphologic observation were carried out after animals were sacrificed.Results All animals' morphologic observation showed that inner ear hair cells were normal after transfer.Seven days after transfer,the swimming time was 4.0±0.71 s in normal control group and 5.0±0.71 s in scala vestibuli delivery group.The threshold of CDM-CEP and ABR were 85±3.54 dB SPL and 37±4.47 dB SPL in normal control group,and 89±6.52 dB SPL and 40±3.54 dB SPL in Ad-Mathl-EGFP scala vestibuli delivery group,respectively.There was no significant difference existed between control group and Ad-Mathl-EGFP scala vestibuli delivery group.Conclusion The Ad(E1,E3-Deleted)is safe for vestibular and cochlea hair cells and can be used as an ideal vector of gene transfer.
ABSTRACT
Objective To assess the feasibility of adenoviral vectors mediate cochlear gene transfer by postau-ricular microinjection through the round window membrane in mouse. Methods Twelve 5-week old C57BL/6J mice were selected for the study: 8 were implanted with Ad-EGFP by postauricular microinjection through the round window membrane, and 4 with artificial perilymphatic fluid. On postoperative days 5 and 14, the animals were sac-rificed and the surface preparation of cochleae was observed. Results Two animals died after operation. Bright green fluorescence in the cochleae was observed in Ad- EGFP groups. Gene expression on day 14 after operation was higher than that on day 5. However, the control group was free of fluorescence. Oonclusion The postauricular route of the cochlear gene transfer in mice is simple to operate with little side-effect. The technique of transgenic delivery into the inner ear through RWM by mieroinjection is feasible and effective.
ABSTRACT
OBJECTIVE To investigate the effect of Hath1 on epithelial ridge (GER) cells from postnatal rat cochlear. METHODS An experimental method was developed which allowed the isolation and culture of GER cells from P1 rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation. The dissociated GER cells were cultured in DMEM+10% FBS. The GER explants infected with ad-Hath1 for 3 hours were cultured for 10 days and observed under scanning electron microscope. RESULTS The GER cell cultures tended to attach to the substratum and grow in patches which assume a polygonal morphology similar to that of epithelial cells in medium containing 10% serum. Stereociliary bundle-like structures were observed in the boundary of GER cell patches forced to express Hath1. CONCLUSION That GER cells which are likely hair cell progenitors could be cultured in vitro and generate stereociliary bundle-like structure when forced to express Hath1 suggests that the misexpression of Hath1 probably can induce the predifferentiation of pure hair cell progenitors into hair cells in vitro.
ABSTRACT
This study was aimed at to investigating the protective effect of a combined treatment with glial cell line derived neurotrophic factor (GDNF) and neurotrophin 3 (NT 3) on noise induced outer hair cell (OHC) damage. Guinea pigs were subjected to receiving infusion of an artificial perilymph containing GDNF (100ng/ml) and NT 3 (2 5?g/ml) into one cochlea via a mini osmotic pump. Three days later, the animals were exposed to a 4kHz narrow band noise at 115 dB SPL for 4h. The control animals received the same treatment except GDNF and NT 3. Thresholds of auditory brainstem responses (ABRs), elicited by clicks, were measured before and 3 days after the surgery of the pump implantation, and 10 days following noise exposure. Then, the subjects were sacrificed and the cochleas were stained with Hoechst 33342. The specimens were examined under a fluorescence microscope for quantitative assessment of the OHC nuclear morphology. The results showed that compared with the control animals, the drug treated ones had significant less swollen OHC nuclei ( P
ABSTRACT
Objective To investigate the expression of Smad5 gene in the cochlea and to identify its location. Methods Reverse transcription polymerase chain reaction (RT-PCR) was employed to detect the expression of Smad5, the in situ hybridization and the immunohistochemical technique were used to localize the Smad5 messenger RNA and the Smad5 protein in mouse cochlea. Results Smad5 gene expressed in cochlea at a high level. Smad5 expression was concentrated in supporting cell, hair cell, spiral ganglion, epithelium of stria vascularis and basilar membrane. Conclusion The Smad5 proteins exist in the mouse cochlea and it may be involved in the cochlear formation and the differentiation of hair cell. Smad5 might be an essential factor for the development of normal cochlea.
ABSTRACT
Objective To understand the structural characteristics and significance of the outer tunnel of corti's organ.Methods The structural characteristics of the outer tunnel were observed and analyzed with celloidin section and scanning electron microscope(SEM).Results The inner wall of outer tunnel consisted of the third row of outer hair cells(OHC) and the stalk of Deiter's cells in the basal and the first turn.Hensen's cells made up the outer wall of the outer tunnel.The stalks of Deiter's cells gradually moved to outer side to constitute the outer and top walls of outer tunnel from the basal turn to the top turn.The inner wall of outer tunnel consisted of the third OHC.The stalks of Deiter's cells moved to Hensen's cells to make the outer wall of the outer tunnel.The space of outer tunnel was gradually decreased.The outer tunnel was full of Deiter's cells in the third and fourth turn.The stalks of Deiter's cells tightly contacted with the Hensen's cells.Conclusion The structural characteristics of the outer tunnel play an important role in maintaining the stability of Corti's organ.
ABSTRACT
Objective To observe the space-time patterns of damaged outer hair cells(OHCs) in rat cochlea at the early stage after exposure to impulse noise. Methods Wistar rats were exposed to 100 emissions of impulse noise (3 seconds interval between each emission) at 154 dB SPL. Four times (10 min, 30 min, 3h and 6h) after the noise exposure, the animals were sacrificed and the organs of Corti were processed for detection of OHC death modes. The apoptotic and necrotic OHCs were distinguished by propidium iodide (PI), a fluorescent probe specifically labeling the nuclear DNA. The specimens were examined under a fluorescence microscope for assessment of OHC damage. Results Nuclear chromatin began to shrink as the chromatin condensed around the nuclear periphery. The peripheral chromatin ring condensed into discrete mass. Chromatin masses appeared to bleb off from the nuclear surface, forming apoptotic bodies at 10 min after the noise exposure. There were a few swollen nuclei appeared 30 min after the noise exposure. Loss of OHC nuclei could be seen 3 h after the noise exposure. The cochlear lesion expanded to contain a large number of missing OHCs and seriously shrunken nuclei at 6 h after the noise exposure. Conclusions The results of the study indicate that death of OHCs takes place extremely rapid after the impulse noise exposure. The apoptosis of OHCs precedes necrosis. OHC apoptosis is a quick process. Most of dead outer hair cells were eliminated 6 h after the noise exposure.