| Background and Objectives: Sensorineural deafness is one of the most common clinical dieases that leads to speech communication barrier. Patients’ quality of life is often seriously affected. Although treatment strategies of sensorineural deafness has a lot of, but the treatment effect is unsatisfactory. To further explore a new direction of future sensorineural deafness treatment, molecular mechnism of development and maintenance of cochlear hearing should understand. Development and maintenance of hearing depends on neurotransmitters released from cochlear hair cells ribbon synapses, which can complete mechanical一electrical transformation. Voice information which was generated primary auditory afferent nerve synapses is done by IHC and ribbon synapses, IHC ribbon synaptic accurately release the neurotransmitter which was demanded normal effective neurotransmitter circulation mechanism, which was named synaptic vesicle cycle. Synaptic vesicle recycling is the basis of the auditory nerve neurotransmitter that was sustainedly released by synaptic vesicle and was accurately recyled by IHC, further the rapid endocytosis is the precondition of recycling. This study used dye FM1-43 to observe endocytosis of cochlear hair cells in vitro. To explore similarities and differences of endocytosis of the inner and outer hair cells in mature mice, FM1-43 were uptake by cochlear IHC and OHC which was monitored by the phase-contrast microscopic and electron microscopic examinations. To explore relationship of cochlea hearing development and endocytosis. Our could find new therapeutic targets that could prevent hair cells apoptosis has become a new trend for the research of therapy against hearing loss.Methods: First, we employed dye FM1-43 and the phase-contrast microscopic and electron microscopic examinations to explore endocytosis in cochlear hair cells of 3days rats postnatally in vitro. Second, through styrene type dye FM1-43 uptake by cochlear hair cells and dynamically observed by electron microscope, our results could provide that endocytosis in IHC may participate the development and maintenance hearing in postnatal 3days mice. Finally, isolated by microscopy to keep good activity and structural integrity of postnatal 1 months IHC and OHC, and dynamically recorded the fluorescent signal of cochlear inner and outer hair cells that was uptake the dye FM1-43, we could compare fluorescent signal change characteristics of inner and outer hair cells in mature mice, endocytosis of IHC and OHC may be involved in cochlea hearing development through interaction with process sound pathways.Results: 1. In vitro culture of C57 BL / 6 j mice in the basement membrane(BM), through dye FM1-43 tracked endocytosis of hair cells, fm1-43 fluorescent signal of inner hair cells was appeared quickly. As change by the time, the fm1-43 fluorescence signal intensity was revealed by the whole hair cells, which was especially enhanced in the basal part of IHC. Within 5 seconds time points, fm1-43 fluorescence intensity peak of hair cells was 521, 535 in 20 seconds, 618 in 80 seconds, 637 in 160 seconds,, 652 in 320 seconds, although fluorescence signal intensity of the basolateral area of the IHC was increased slowly over time, but the increase is not obvious. By comparing fm1-43 fluorescent values of hair cells in vitro, the results showed that fm1-43 fluorescent values were statistically difference at different time points(x±s, p<0.05).2.Fm1-43 fluorescent signal was appeared quickly the top of the inner hair cells in vitro; As change by the time, fm1-43 fluorescence signal intensity was significantly enhanced basal part of IHC and synaptic release active region; In 120 seconds time points,fm1-43 fluorescence intensity peak was 445, 513 in the 480 s, 588 in 1200 s. Well as time goes on, fluorescence peak values of synaptic release active structure region was not enhanced in inner hair cells,and this time point is close to saturation. In this experiment, we measured that the mean fluorescence of the inner hair cells were 165.16±33.16(60 s), 177.63±34.02(120 s), 258.17±32.33(480 s), 385.25± 64.06(1200 s). Fluorescence values of IHC were statistically difference at different time points(x±s, p<0.05).3. Fm1-43 fluorescent signal was firstly expressed at or near the lateral cellular wall and above the nucleus of outer hair cell. As change by the time, fm1-43 fluorescence signal intensity near the lateral cellular wall and above the nucleus of outer hair cell was gradually strengthened. In 480 seconds time points after joining dye fm1-43,the fluorescent signal peak values of OHC was 319, 405 in 1200 seconds time points.Then, fluorescence signal intensity of OHC is no longer a significant increased, however,the strength seems to be weakened. In this experiment, we measured the mean fluorescence of OHC were 132.83 ±15.4(60 s), 150.64± 25.2(120 s), 192.89 ± 35.42(480 s), 246.59 ±51.11(1200 s). Comparing fm1-43 fluorescence value of outer hair cells, the results were statistically difference at different time points(x±s, p<0.05).4. In different observation point fluorescent signal peak values of IHC and OHC in mice, fm1-43 uptake of inner hair cells were significantly higher than that of outer hair cells(P < 0.05).The results were statistically difference at different time points(x±s, p<0.05).Conclusion: Dynamic endocytosis at or near the basolateral area of the IHC suggests that endocytosis in IHCs may taking part in ribbon synapse plasticity. Endocytosis activities near the lateral wall and in the supranuclear area in OHC indicate that endocytosis in OHC involves in tip-links or iron channels. Higher levels of FM1-43 uptake by IHCs may indicate IHC’s vital roles in hearing development and maintenance. |
PDF Full Text Request