[Observation of the efficacy of autologous mucosal transplantation to prevent esophageal stricture after near-circumferential endoscopic submucosal dissection for early esophageal cancer].

Objective: To investigate the efficacy of autologous mucosal transplantation to prevent esophageal stricture after near-circumferential endoscopic submucosal dissection (ESD) for early esophageal cancer. Methods: The case data of 33 patients, who underwent near-circumferential ESD for early esophageal cancer and were followed up regularly in the First Affiliated Hospital of Zhengzhou University from April 2017 to July 2022, were analyzed retrospectively, including 14 males and 19 females, aged (66.4±7.4) (47-77) years. According to the different treatment methods, they were divided into 4 groups: group A (6 cases) were treated with autologous mucosa transplantation and fully covered metal stent implantation, combined with oral, intravenous and local injection of hormone; Group B (8 cases) were treated with autologous mucosa transplantation and fully covered metal stent implantation; Group C (11 cases) were treated with fully covered metal stent implantation combined with oral or intravenous hormone; Group D (8 cases) were treated with fully covered metal stent implantation. After the operation, the growth of the transplanted mucosa, esophageal stricture and surgical complications were observed by endoscopy, so as to understand the efficacy of automucosa transplantation in preventing esophageal stricture after near-circumferential ESD for early esophageal cancer. Results: The gastroscopic operation was successful in 33 patients. The times of expansion in groups B, C and D were more than that in group A, and the times of expansion [M(Q1,Q3)] in group A were 0(0, 1.8) times, while the times of expansion in group B, C and D were 5.5(4.3, 6.8), 4.0(4.0, 7.0) and 5.5(3.5, 10.8) times, respectively, with statistical significance (all P<0.05). There was no significant difference in times of expansion among groups B, C and D (all P>0.05). The stent placement time [M(Q1,Q3)] in group B [7.5(6.3, 8.8) days] was shorter than that in group A [64.5(41.5, 75.5) days] (P=0.006). There was no significant difference in stent placement time between group C [38.0(28.0, 50.0) days] and group D [31.5(27.3, 66.3) days] and group A (both P>0.05). The stent placement time in group C was longer than that in group B (P<0.05).There was no significant difference in stent placement time between group B, C and D (all P>0.05). There was no significant difference in the incidence of complications among the groups (all P>0.05). Conclusions: Autologous mucosal transplantation is safe and effective in preventing stenosis after near-circumferential ESD for early esophageal cancer. The effect of autologous mucosal transplantation combined with fully covered metal stent placement, systemic and local steroid application in preventing esophageal stricture after near-circumferential ESD for early esophageal cancer is better than that of single application.

Intracellular calcium and outer hair cell electromotility.

The influence of increased intracellular calcium level on outer hair cell (OHC) electromotility was examined by means of transcellular electrical stimulation in a partitioning microchamber. Electromotile activity was measured before and after application of the calcium ionophore ionomycin, which promotes the inflow of extracellular calcium, as well as its release from intracellular calcium stores. The ionomycin solvent, dimethyl sulphoxide (DMSO), by itself elicited a significant decrease in the magnitude of OHC electromotility. The DMSO effect was counteracted by 10 microM ionomycin and was reversed by 50 microM ionomycin. The increase in electromotility is partially mediated by a calmodulin-dependent mechanism, since W7, a calmodulin antagonist, attenuated the 50 microM ionomycin-induced motility increase. Our results suggest that the electromotility magnitude increase in isolated OHCs due to ionomycin is a calcium/calmodulin-dependent phenomenon.

Development of acetylcholine receptors in cultured outer hair cells.

Efferents, originating in the superior olivary complex, preferentially synapse with cochlear outer hair cells (OHCs), with acetylcholine (ACh) as their primary neurotransmitter. The OHC ACh receptors (AChRs), which have unusual pharmacology, have been cloned and identified as a new subunit (alpha9) of the nicotinic AChR family. The expression of alpha9 AChRs is first detected before birth and peaks between 6 and 10 days after birth (DAB) in developing mice and rats, while functional maturation of the receptor, as determined by measuring the ACh-induced currents, takes place between 6 and 12 DAB. In this study we attempted to examine the development of AChRs in OHCs grown in explanted cultures, deprived of efferent innervation. ACh-induced currents were used as an assay. Reverse transcription-PCR analysis was also performed to detect the expression of alpha9 subunit from cultured OHCs. PCR study indicates that mRNA of the alpha9 subunit was expressed in primary cochlear cultures, similar to that seen in the cochleae of developing animals. Measurement of whole-cell currents showed that ACh-induced outward current was first detected around 5 days in a fraction of cultured OHCs. The number of responsive cells increased between 5 and 12 days in culture. The size of ACh-induced currents also increased during this period. These results suggest that the development of AChRs in cultured OHCs is not affected by removal of efferent innervation.

Intracellular anions as the voltage sensor of prestin, the outer hair cell motor protein.

Outer hair cells (OHCs) of the mammalian cochlea actively change their cell length in response to changes in membrane potential. This electromotility, thought to be the basis of cochlear amplification, is mediated by a voltage-sensitive motor molecule recently identified as the membrane protein prestin. Here, we show that voltage sensitivity is conferred to prestin by the intracellular anions chloride and bicarbonate. Removal of these anions abolished fast voltage-dependent motility, as well as the characteristic nonlinear charge movement ("gating currents") driving the underlying structural rearrangements of the protein. The results support a model in which anions act as extrinsic voltage sensors, which bind to the prestin molecule and thus trigger the conformational changes required for motility of OHCs.

Isolation of cochlear inner hair cells.

In order to identify hair cell specific genes, it is essential to obtain isolated hair cells in quantity. While whole-cell recordings have been made from isolated inner hair cells (IHCs) from guinea pigs, detailed methods for obtaining a fairly large amount of isolated inner hair cells have not been published. Here we describe a protocol that can yield a fairly large amount of isolated gerbil IHCs. This technique can provide sufficient numbers of solitary IHCs for either electrophysiological studies of the cell's membrane properties or identifying genes related to IHC functions using techniques of molecular biology.

Prestin is the motor protein of cochlear outer hair cells.

The outer and inner hair cells of the mammalian cochlea perform different functions. In response to changes in membrane potential, the cylindrical outer hair cell rapidly alters its length and stiffness. These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by inner hair cells. Here we have identified an abundant complementary DNA from a gene, designated Prestin, which is specifically expressed in outer hair cells. Regions of the encoded protein show moderate sequence similarity to pendrin and related sulphate/anion transport proteins. Voltage-induced shape changes can be elicited in cultured human kidney cells that express prestin. The mechanical response of outer hair cells to voltage change is accompanied by a 'gating current', which is manifested as nonlinear capacitance. We also demonstrate this nonlinear capacitance in transfected kidney cells. We conclude that prestin is the motor protein of the cochlear outer hair cell.

Development of the gerbil inner ear observed in the hemicochlea.

A frequency-dependent change in hearing sensitivity occurs during maturation in the basal gerbil cochlea. This change takes place during the first week after the onset of hearing. It has been argued that the mass of a given cochlear segment decreases during development and thus increases the best frequency. Changes in mass during cochlear maturation have been estimated previously by measuring the changes in cochlear dimensions. Fixed, dehydrated, embedded, or sputter-coated tissues were used in such work. However, dehydration of the tissue, a part of most histological techniques, results in severe distortion of some aspects of cochlear morphology. The present experiments, using a novel preparation, the hemicochlea, show that hydrated structures, such as the tectorial membrane and the basilar membrane hyaline matrix, are up to 100% larger than estimated previous studies. Therefore, the hemicochlea was used to study the development of cochlear morphology in the gerbil between the day of birth and postnatal day 19. We used no protocols that would have resulted in severe distortion of cochlear elements. Consequently, a detailed study of cochlear morphology yields several measures that differ from previously published data. Our experiments confirm growth patterns of the cochlea that include a period of remarkably rapid change between postnatal day 6 and 8. The accelerated growth starts in the middle of the cochlea and progresses toward the base and the apex. In particular, the increase in height of Deiters' cells dominated the change, "pushing" the tectorial membrane toward scala vestibuli. This resulted in a shape change of the tectorial membrane and the organ of Corti. The tectorial membrane was properly extended above the outer hair cells by postnatal day 12. This time coincides with the onset of hearing. The basilar membrane hyaline matrix increased in thickness, whereas the multilayered tympanic cover layer cells decreased to a single band of cells by postnatal day 19. Before and after the period of rapid growth, the observed gross morphological changes are rather small. It is unlikely that dimensional changes of cochlear structures between postnatal days 12 and 19 contribute significantly in the remapping of the frequency-place code in the base of the cochlea. Instead, structural changes affecting the stiffness of the cochlear partition might be responsible for the shift in best frequency.

Two models of outer hair cell stiffness and motility.

Cochlear outer hair cells change their axial dimension and theiraxial stiffness when their membrane potential is altered. These changes appear to be highly correlated. Because of this, we endeavored to produce models that would yield both phenomena via a single mechanism. Two models are proposed. In one, it is assumed that elementary motor molecules can be in either of two conformational states, these having different physical lengths and stiffnesses. The state of the molecule is taken to be a stochastic function of membrane potential and is expressed by a Boltzmann relationship. In the other model, a similar dependence is assumed to occur between membrane potential and stiffness, but no dimensional change isassigned to the molecule. Length changes can be had by preloading the cell. We show that either general model can produce realistic length and stiffness changes with an appropriate selection of parameters. One particular realization of the first model is proposed as an example. In this--the boomerang model--the molecule is assumed to be L-shaped, with two different angles between the two legs representing the conformational states. Finally, the behavior of the model is compared with available data when the voltage stimulus comprises a brief sinusoid upon a DC pedestal.

Properties of voltage-dependent somatic stiffness of cochlear outer hair cells.

We have shown recently that isolated cochlear outer hair cells change their axial stiffness when their membrane potential is altered under voltage-clamp. Here we extend those observations, using a more stable mechanical platform, the microchamber, to hold the cells and to deliver voltage commands. Cell stiffness is determined by opto-electronically measuring the amplitude of motion of a flexible fiber as it is loaded by the cell. Cell stiffness is decreased by depolarization and increased by hyperpolarization. The stiffness changes have been measured with sinusoidal electrical command signals up to 1750 Hz and fiber motion up to 2000 Hz. It is shown that electrically evoked stiffness changes and length changes (electromotility) have very similar characteristics and may arise in a common process.

Cyclic GMP and outer hair cell electromotility.

The aim of this study is to examine the effect of phosphorylation pathways on the electrically evoked fast motile response of isolated outer hair cells (OHCs). Transcellular electrical stimulation was applied in the microchamber to guinea pig OHCs and motility was measured before and after drug application. Forskolin (adenylate cyclase activator), phorbol 12-myristate 13-acetate (PMA, protein kinase C activator) and dibutyryl 3',5'-cyclic guanosine monophosphate (cGMP agonist) were studied. As controls, L15 medium and dimethyl-sulfoxide (DMSO) were used. In each group, 12 cells were measured. Forskolin and PMA were dissolved in 0.1% DMSO to render them membrane permeable. DMSO by itself caused a statistically significant electromotility magnitude decrease. Forskolin and PMA could not reverse the motility decrease due to DMSO, the effects seen in their presence were the same as observed with DMSO alone. Thus, neither 3',5'-cyclic AMP-dependent protein kinase nor calcium/phospholipid-dependent protein kinase appear to have modulatory effects on electromotility. Dibutyryl cGMP (DBcGMP), in concentrations of 200 microM, elicited a significant electromotility magnitude increase. The DBcGMP effect could be inhibited by co-application of 200 microM DBcGMP and 100 microM 8-Rp-pCPT-cGMPS (8-4-chlorophenylthio-guanosine 3',5'-cyclic monophosphothioate, Rp isomer, a cGMP antagonist). Our results suggest that OHC electromotility is modulated by a cGMP-dependent pathway.

Somatic stiffness of cochlear outer hair cells is voltage-dependent.

The mammalian cochlea depends on an amplification process for its sensitivity and frequency-resolving capability. Outer hair cells are responsible for providing this amplification. It is usually assumed that the membrane-potential-driven somatic shape changes of these cells are the basis of the amplifying process. It is of interest to see whether mechanical reactance changes of the cells might accompany their changes in cell shape. We now show that the cylindrical outer hair cells change their axial stiffness as their membrane potential is altered. Cell stiffness was determined by optoelectronically measuring the amplitude of motion of a flexible vibrating fiber as it was loaded by the isolated cell. Voltage commands to the cell were delivered in a tight-seal whole-cell configuration. Cell stiffness was decreased by depolarization and increased by hyperpolarization.

Development of acetylcholine-induced responses in neonatal gerbil outer hair cells.

Cochlear outer hair cells (OHCs) are dominantly innervated by efferents, with acetylcholine (ACh) being their principal neurotransmitter. ACh activation of the cholinergic receptors on isolated OHCs induces calcium influx through the ionotropic receptors, followed by a large outward K+ current through nearby Ca2+-activated K+ channels. The outward K+ current hyperpolarizes the cell, resulting in the fast inhibitory effects of efferent action. Although the ACh receptors (AChRs) in adult OHCs have been identified and the ACh-induced current responses have been characterized, it is unclear when the ACh-induced current responses occur during development. In this study we attempt to address this question by determining the time of onset of the ACh-induced currents in neonatal gerbil OHCs, using whole cell patch-clamp techniques. Developing gerbils ranging in age from 4 to 12 days were used in these experiments, because efferent synaptogenesis and functional maturation of OHCs occur after birth. Results show that the first detectable ACh-induced current occurred at 6 days after birth (DAB) in 12% of the basal turn cells with a small outward current. The fraction of responsive cells and the size of outward currents increased as development progressed. By 11 DAB, the fraction of responsive cells and the current size were comparable with those of adult OHCs. The results indicate that the maturation of the ACh-induced response begins around 6 DAB. It appears that the development of ACh-induced responses occur during the same time period when OHCs develop motility but before the onset of auditory function, which is around 12 DAB when cochlear microphonic potentials can first be evoked with acoustic stimulation in gerbils.

A novel, simple organotypic culture method to study the organ of Corti from the neonatal gerbil.

An original, simple organotypic culture method was developed to grow the organ of Corti from the neonatal gerbil on the bottom of a Petri dish. In comparison with the commonly used Maximov slide assembly method, this method is easier, less time-consuming, and more economic. Our results in this study using fluorescent live/dead viability assay and fluorescein-conjugated antineurofilament antibodies show that the cultured organ of Corti and spiral ganglion cells not only survived for at least 14 days but also maintained their basic organization and normal development in vitro. Therefore, our method can serve as a reliable and easier alternative to the traditional techniques for studying the development as well as other physiological properties of the cultured organ of Corti.

Expression of potassium channels in gerbil outer hair cells during development does not require neural induction.

Mammalian outer hair cells (OHCs) contain Ca and K channels in their synaptic pole. We questioned if the ontogeny of potassium currents of OHCs depends on the neural induction of early afferent contact. By recording whole-cell currents of OHCs grown in organotypic cultures deprived of afferent innervation, we show that a Ca-activated K channel is expressed in these cells, suggesting that the ontogeny of the K channel is an intrinsic process.

Relationship between the development of outer hair cell electromotility and efferent innervation: a study in cultured organ of corti of neonatal gerbils.

Outer hair cell (OHC) electromotility, which powers the cochlear amplifier, develops at a later stage of hearing ontogeny. There has been speculation whether efferents play a necessary role in directing or achieving OHC maturation in mammals. In this study, we examine whether the development of OHC motility depends on the establishment of efferent innervation of the cells' synaptic pole by measuring electromotility of OHCs grown in cultures, deprived of efferent innervation. Tissue cultures of the organ of Corti were prepared from the cochleas of newborn gerbils. Solitary OHCs were obtained from 4- to 15-d-old cultures by enzymatic digestion and mechanical trituration. Length changes evoked by transcellular electrical stimulation were detected and measured with a photodiode sensor. Results show that OHCs develop electromotility between 6 and 13 d in culture without the presence of efferent innervation. The timetable for the onset of OHC electromotility is comparable with that in vivo. This demonstrates that the ontogeny of OHC electromotility is an intrinsic process that does not require the influence of efferent innervation.

Acetylcholine, outer hair cell electromotility, and the cochlear amplifier.

The dominant efferent innervation of the cochlea terminates on outer hair cells (OHCs), with acetylcholine (ACh) being its principal neurotransmitter. OHCs respond with a somatic shape change to alterations in their membrane potential, and this electromotile response is believed to provide mechanical feedback to the basilar membrane. We examine the effects of ACh on electromotile responses in isolated OHCs and attempt to deduce the mechanism of ACh action. Axial electromotile amplitude and cell compliance increase in the presence of the ligand. This response occurs with a significantly greater latency than membrane current and potential changes attributable to ACh and is contemporaneous with Ca2+ release from intracellular stores. It is likely that increased axial compliance largely accounts for the increase in motility. The mechanical responses are probably related to a recently demonstrated slow efferent effect. The implications of the present findings related to commonly assumed efferent behavior in vivo are considered.

Effect of acetylcholine and GABA on the transfer function of electromotility in isolated outer hair cells.

Outer hair cells (OHC) from high- and low-frequency regions were separately isolated from guinea pig cochleas. The cells were inserted with their ciliary pole first into a partitioning microchamber so that only 20-50% of the cell length was excluded. Somatic length changes due to transcellular electrical stimulation were measured at the cuticular plate in the inserted portion of the cells. Transfer curves of electromotility of the OHCs were obtained by both a series of brief (2.5 ms) and longer (30 ms) square pulses with opposite polarity and linearly increasing size from 40 to 280 mV in both negative and positive directions. Alterations in the transient and steady-state electromotility transfer curves were examined by application of acetylcholine (ACh) and gamma-aminobutyric acid (GABA) to the synaptic pole. ACh, in the concentration range of 10-30 microM, evoked a significant magnitude and gain increase of electromotility in both transient and steady-state responses without a measurable shift in the operating point of the displacement-voltage transfer curve. A tonotopic response magnitude difference is found for ACh challenge. Basal turn OHCs responded with greater magnitude increase (+90% increase from control) than apical turn OHCs (+40%). GABA exerted an opposite effect, again in a location-dependent manner. Magnitude response decreased about 30% for long cells and 14% for short ones. Atropin, a muscarinic receptor antagonist, completely blocked the increase in electromotility response due to ACh. However, D-tubocurarine, a nicorinic receptor antagonist, while not blocking the ACh effect, altered the cell's apparent operating point. Bicuculline methiodide, a GABAA-receptor antagonist, completely arrested GABA influences on the electromotility response. These results suggest that both ACh and GABA can change the electromotile activity of OHCs, in a tonotopically biased manner. ACh challenge evokes greater magnitude responses in basal turn OHCs, whereas GABA induces greater motility response decrease in apical turn OHCs. The control of the gain and magnitude of electromotility by the transmitter substances appear to involve at least two mechanisms. One is probably related to conformational changes of the voltage-to-movement converter molecules and a change in their number in an effective operational pool, the other operates via changing the electrical resistance of the basolateral cell membrane.

First appearance and development of electromotility in neonatal gerbil outer hair cells.

With the purpose of pinpointing the time of onset of electromotility, outer hair cells (OHCs) from apical and basal turns of the cochleae of postnatal gerbils, ranging in age from 6 to 19 days, were isolated and drawn into a glass microchamber. Length changes evoked by transcellular electrical stimulation were detected and measured with a photodiode detector. Motile responses first appeared in 3 out of 14 basal turn OHCs at 7 days after birth (DAB). At 8 DAB, 3 out of 13 apical turn cells also responded to the electrical stimulation. By 12 DAB, all the OHCs from both turns showed motile responses. Input-output functions relating applied stimulus and change in cell length revealed that the motile response threshold improved from 7 DAB to 12 DAB and the response amplitude kept increasing from 7 DAB until 13-14 DAB, when mature amplitudes were reached. Measurements of OHC length revealed only minor changes in basal turn hair cell length while apical hair cells continued to elongate until approximately 16 DAB. Since the onset of auditory function in gerbils occurs around 12 DAB and fine tuning develops between 14 and 17 DAB, our results suggest that the onset of OHC motility occurs earlier than that of auditory function and the maturation of the motility amplitude occurred earlier than the development of fine tuning. The maturation of OHC motility and the development of otoacoustic emissions are also compared and discussed.