Epithelial migration on the tympanic membrane. An experimental study

The existence of an epidermal migration pattern was studied in mice using a whole-mount autoradiographical technique. The mitotic activity appeared to be confined to an area close to the annulus tympanicus. A very slow migratory activity of the labelled cells, was observed, situated in the basal cell layer. Perforation of the tympanic membrane showed no effect on the position of the labelled cells. This slow migration described by several authors. The latter migration was suggested to represent a mechanism for cleaning the tympanic membrane.     

用羟基磷灰石纳米粒作内耳基因治疗新载体的体内外研究

目的 研制羟基磷灰石(hydroxyapatite,HAT)纳米粒,评价其作为新的内耳基因转染载体的可能性.方法 用化学共沉淀-水热合成法制备HAT载体.经DNA电泳做不同pH值和HAT含量的DNA结合实验,了解HAT纳米粒对DNA的结合和保护作用;用四甲基偶氮唑盐法观察HAT纳米粒对宫颈癌上皮Hela细胞的毒性;用Hela细胞和小鼠耳蜗原代螺旋神经节细胞做神经营养素3(neurotrophin 3,NT3)体外转染实验(绿色荧光蛋白基因荧光显微镜法)及NT3的蛋白印迹分析,观察HAT介导的NT3基因对活细胞的体外转染能力和基因表达情况;经实验性兴奋性耳毒性损伤豚鼠的耳蜗外淋巴腔灌注HAT纳米载体-含增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)质粒载体-NT3基因复合物(HAT-pEGFPC2-NT3),用免疫组化(二氨基联苯胺法)观察NT3在耳蜗的表达,以了解HAT纳米载体在活体动物耳蜗介导基因转染的能力.结果 含量250.0 μg/ml,pH值3～7是这种长约40～50 nm的短棒状HAT纳米粒能完全结合重组质粒pEGFPC2-NT3的最低含量混悬液和最佳pH环境,并可有效保护基因不被核酸酶(DNase Ⅰ)破坏.四甲基偶氮唑盐法观察HAT纳米粒从62.5 μg/ml至500.0 μg/ml 4种混悬液对Hela细胞存活率无明显影响,细胞形态无变化.EGFP荧光显微镜观察显示,HAT纳米载体对Hela细胞的基因转染率为15.7%±2.6%(-x±s);将HAT-pEGFPC2-NT3转染培养的小鼠耳蜗神经元,也可见明显的EGFP表达.NT3免疫组化显示,向实验性兴奋性耳蜗损害豚鼠的耳蜗外淋巴腔灌注HAT-pEGFPC2-NT3,可见活体耳蜗神经元内有明显的NT3蛋白表达.结论 HAT纳米粒不仅可介导NT3基因的体外转染,而且可介导其耳蜗活体转染;尽管转染率有待提高,但由于没有病毒载体转染造成的生物威胁,仍不失为一种很有研究价值的非病毒型内耳基因治疗载体.     

Tracheal reconstruction: In vitro and in vivo animal pilot study

In order to further address the problem of tracheal stenosis, an animal model was created to simulate both local and regional tracheal injury. An epithelial equivalent created from a fibroblast-collagen matrix was used to attempt to either inhibit or lessen the degree of tracheal stenosis that was evident in animal models. Preliminary data appears to show that the epithelial equivalent is effective in limiting and perhaps even abolishing the development of tracheal stenosis.     

An in vivo tracer study of noise-induced damage to the reticular lamina

An in vivo tracer was used to determine if the reticular lamina and/or the cell membranes abutting the endolymphatic space are temporarily disrupted after intense noise exposure (4-kHz OBN, 108-dB SPL, 1.75 h). Using a double-barreled micropipette, the endolymphatic potential (EP) was recorded and artificial endolymph containing 10% carbon particles was injected into the endolymphatic space either 0 days or 28 days post-exposure. The cochleae were fixed 30-45 min post-injection, then dehydrated, embedded in plastic and dissected as flat preparations. Damage in the organ of Corti (OC) was quantified, the location of carbon was determined, and some OC segments were then sectioned radially. EP averaged 72+/-5 mV in five controls. These cochleae had carbon tracer in the endolymphatic space only. Four of five noise-exposed chinchillas examined 3-4 h post-exposure had a low EP (30+/-6 mV). The cochleae from these 0-day animals had several focal lesions in which nearly all outer hair cells had just degenerated. At these lesions, carbon was attached to cell membranes and debris between the reticular lamina and basilar membrane. By transmission electron microscopy, discontinuities were found in the apical membranes of sensory and supporting cells. Carbon particles were found in the cytoplasm of these cells. Four of five animals examined at 28 days had an average EP of 70+/-11 mV. The cochleae from these animals had multiple lesions in the basal turn, all of which were healed by phalangeal scars or squamous epithelial cells. In these cochleae, no carbon was found within the OC. Acute disruption of the reticular lamina and the apical membranes of sensory and supporting cells from noise appears to be a major mechanism to account for degeneration in the cochlea that spreads or continues for days to weeks post-exposure.     

Alcohol and ultrastructural changes in the developing inner ear. An in vitro study

Inner ear explants from the CBA/CBA mouse were used in an organ culture system. The explants were cultured from the 16th gestational day until one day post partum. They were exposed to 1.5% or 3% ethanol in organ culture medium in order to determine any possible toxic effects upon the differentiating sensory structures of the sensory epithelium of the inner ear, that could be correlated to fetal alcohol syndrome. The higher concentration of ethanol caused a general and possibly unspecific destruction of the sensory epithelium, while the lower concentration caused characteristic changes including intracellular edema or vacuolization, especially confined to hair cells. Pathologic changes seemed dose-related but not time-related.     

The effect of fibrin glue on the healing of hydroxyapatite ceramics. An animal experiment study

Calcium phosphate ceramics have been proposed for obliteration of the mastoid cavity in middle ear surgery. To our knowledge no experimental data are available concerning the use of homologous fibrin glue associated with hydroxyapatite (HA) in animals. Obliteration of the epitympanic space and closure of defects in the femur of rabbits by HA granules with and without homologous fibrin glue were performed on 17 animals, and the animals followed up for 2 weeks to 6 months. X-ray and histological examinations of the tissue integration of the implants were performed by semi-quantitative measurements. Fibrin glue accelerates the development of soft tissue and slows down the osteointegration of the ceramic implants.     

An in vitro growth study on cholesteatoma and normal skin

The ability of cholesteatoma to grow rapidly within the ear is well recognized. This study aimed to quantify the in vitro growth of cholesteatoma derived cells. Following removal of cholesteatoma explant cultures were established. Cellular outgrowths were subcultured and a colony forming assay performed. Cells were repeatedly passaged every 14 days until senescence was observed. In comparison with cells derived from normal scalp skin, cholesteatoma derived cells demonstrated a lower colony forming efficiency in both primary and secondary subcultures, achieved fewer passages and cell generations in serial culture, and achieved a lower total population expansion. No evidence was found to suggest an intrinsic loss of growth control. It is proposed that the majority of cholesteatoma is composed of cells with a limited capacity for growth. Explant studies suggested that these may be the progeny of more highly proliferative cells situated at the neck of the cholesteatoma sac.     

甲壳胺无纺布的降解性及生物相容性观察

目的观察甲壳胺无纺布在体外、体内降解性能和规律及其生物相容性。方法将甲壳胺无纺布称重后分组置于1%溶菌酶和0.1%溶菌酶溶液中,37℃恒温震荡水浴,按时间顺序取出,80℃烘干4h后称重,观察其体外降解速度,将其植入兔皮下,分别于2、4、8、12周取材进行大体观察和HE染色光镜检查。结果体外在1%溶菌酶溶液中1周平均降解5.52%,2周降解9.36%。体内植入大体观察及组织形态学检查显示其具有良好的可降解性和生物相容性。结论甲壳胺无纺布具有良好的可降解性和生物相容性,可作为理想的组织工程细胞支架。     

Drug therapy in otomycosis: An in vitro study

Otomycosis represents a small percentage of clinical external otitis. This well documented entity is often a stubborn clinical problem and, in contrast to bacterial external otitis, there is no otic preparation with specific antifungal activity. In response to this lack of otic preparations, we have surveyed in vitro a variety of available preparations to determine the general spectrum of activity against appropriate bacterial and fungal species. An agar-disc diffusion system was used testing the drugs against (1.) bacteria common in external otitis, and (2.) a variety of yeast and filamentous fungi. Aqueous Merthiolate and Cresylate demonstrated good non-specific antimicrobial activity, while nystatin and clotrimazole demonstrated specific antifungal activity. Otic preparations can now be used which have demonstrated in vitro effectiveness and give an alternate means of therapy to the now empirically selected otic preparation used for otomycosis.     

Effects of Hangeshashinto on the nasal physiological function: An in vitro study.

ObjectiveHangeshashinto is a Japanese Kampo medicine applied for the treatment of oral mucositis and gastroenteritis. Hangeshashinto exhibits broad-spectrum antibacterial activity and suppresses prostaglandin (PG)E2 production in the mucosa and has the ability to improve the inflammatory condition. In addition to these effects, because cAMP, a composition of Hangeshashinto, facilitates ciliary beat, Hangeshashinto could also improve the physiological function of the nasal mucosa, consist of ciliated epithelium, but details were unknown.MethodsThis study was aimed to investigate the effects of Hangeshashinto on the nasal mucosa. Healthy nasal mucosal sections were collected from the nasal septum of ten Japanese white rabbits, placed in a collagen dish for tissue culture, and rinsed with two different concentrations of Hangeshashinto solution (1.0%, n = 10 and 2.5%, n = 10) and cAMP solution (50µM, n=10 and 100 µM, n=10) or saline (control, n = 10). Ciliary beat frequency (CBF) as a physiological function of the nasal mucosa was recorded at 1, 3 and 7 days after rinsing, and histological evaluation of epithelial damage was performed at 7 days after rinsing.ResultsCBF in the 1.0% but not in the 2.5% Hangeshashinto group, increased at 3 and 7 days compared with that in the control group (p < 0.05). This trend was also observed in the CBF in the 100 µM cAMP group, significant difference was not observed between the CBF of the 1.0% Hangeshashinto group and the 100 µM cAMP group at 1, 3 and 7 days after rinsing (p > 0.05). Histological score only in the 2.5% Hangeshashinto group was lower than that in the control group (p < 0.05), while a significant decline was not observed in the other groups compared to that in the control group (p > 0.05).ConclusionOur results suggest that 1.0% Hangeshashinto solution facilitates the physiological function of the nasal mucosa by promoting ciliary functions without histological damage of cilia epithelium. When applied with the appropriate concentration, Hangeshashinto could have ability to improve the physiological functions of the nasal mucosal epithelium.     

ATP induces respiratory ciliostimulation in rat and guinea pig in vitro and in vivo.

Adenosine triphosphate (ATP) has been shown to revitalize the disturbed nasal mucociliary function in man. We investigated the effects of ATP on the ciliary beat frequency (CBF) in animals by immersing tracheal explants from rats in various concentrations of ATP, and by infusing ATP intravenously to guinea pigs. CBF was measured with a photodetector technique from the surface of the explants or from the incised trachea. ATP (from 0.01 to 1 mg/ml) in vitro increased CBF in rat tracheal explants up to 10.5% (p less than 0.05). In vivo ATP (1 mg/kg) increased the CBF by 29% (p less than 0.01) in the guinea pig trachea. As the CBF was increased by ATP, both in vitro and in vivo, it can be suggested that the improvement in mucociliary transport by exogenous ATP as shown in previous studies is caused by the ciliostimulatory effect of ATP.     

In vivo and in vitro study of co-expression of LMP1 and Cripto-1 in nasopharyngeal carcinoma

IntroductionNasopharyngeal carcinoma, an epithelial-derived malignant tumor which because of its anatomical location and atypical early symptoms, when diagnosed invasion and metastasis often have occurred. This requires a better understanding of the development mechanism, identifying diagnostic markers, and developing new treatment strategies.ObjectiveTo study the relationship of LMP1 and Cripto-1 in nasopharyngeal carcinoma.MethodsThe expression of LMP1 and Cripto-1 in specimens obtained from nasopharyngeal carcinoma patients (n = 42) and nasopharyngitis patients (n = 22) were examined. The expression of LMP1 and Cripto-1 in LMP1-negative and LMP1-positive (CNE1-LMP1) cells were also examined.ResultsThe expression of LMP1 and Cripto-1 was significantly higher in nasopharyngeal carcinoma than in nasopharyngitis (p < 0.05). Their expression in nasopharyngeal carcinoma with metastasis were significantly higher than that without metastasis (p < 0.05), which was correlated with TNM staging (p < 0.05). High Cripto-1 expression and high proliferation rate were seen in CNE1-LMP1 cells.ConclusionsThe expression of LMP1 and Cripto-1 in nasopharyngeal carcinoma is positively related. Their co-expression might contribute to the proliferation and metastasis of nasopharyngeal carcinoma.     

Tissue binding kinetics of tobramycin. An experimental study in the mouse inner ear in vitro

The tissue-binding capacity of tritiated tobramycin (TM) was analysed in an organ culture system in the embryonic inner ear of the mouse. A rapid and probably irreversible binding of TM occurs within 10 min, showing about 100 micrograms TM per gram protein in the vestibular half and about 75 micrograms TM per gram protein in the cochlear half of the labyrinth. A steady state is reached within 60 min. The TM uptake was then 390 micrograms TM per gram protein in the vestibular part and 270 micrograms in the cochlear part of the inner ear. A minor fraction of TM, of the same magnitude as initially bound, becomes irreversibly bound, whereas the slow accumulation which reaches the steady state level seems reversible.     

Tracheal anastomosis with the diode laser and fibrin tissue adhesive: An in vitro and in vivo investigation

Absorbable sutures have been advocated for tracheal anastomosis to reduce fibrosis and foreign body reaction leading to recurrent stenosis. Fibrin tissue adhesive (FTA) and diode laser welding with indocyanine green-dyed fibrinogen were evaluated in tracheal anastomosis to reduce the number of sutures and to improve healing. In vitro studies demonstrated strong anastomoses with a combination of laser welding and FTA with minimal tissue damage. In a controlled in vivo study, circumferential resections of canine tracheas were repaired with laser welding and FTA augmented with a few stay sutures. These anastomoses had less fibrosis and tissue damage than anastomoses in control animals repaired with sutures alone. This study supports investigation of laser welding and FTA in human beings for tracheal anastomosis and other procedures in which suturing may be difficult.     

Head and neck carcinoma models. In vivo reproduction in athymic mice and in vitro culture

683 tumour fragments from 63 head and neck carcinoma patients were cultured in vitro. Two laryngeal carcinomas and two salivary gland carcinomas were established into permanent cell lines. Malignancy of these cultured cells was proved by cloning, by chromosomal analysis and by transplantation into athymic (nu/nu) mice. Experiments demonstrating preservation of histological, biochemical and antigenic properties in the tumour models counter the objection that tumour-specific characteristics may be lost.     

Round window membrane permeability. An in vitro model

The round window membrane is regarded to be the main route for passage of potentially ototoxic substances from the middle ear cavity into the inner ear. This may be of clinical importance in acute otitis media and chronic otitis media, where sensorineural hearing impairment sometimes develops. The accuracy and reliability of an in vitro round window membrane permeability model was studied. The round window membrane of the mongolian gerbil was resected, together with its bony niche. The preparation was mounted between two glass chambers representing the middle ear and the inner ear. Passage through the round window membrane did not occur within 3 h for low density lipoprotein with a molecular weight of 2,300 kD. Only minute amounts of highly density lipoprotein, of molecular weight 115-350 kD, passed the round window membrane. The passage rate of horseradish peroxidase, which has previously been shown to pass the round window membrane in vivo, was estimated. The design of the present model is considered to make feasible controlled permeability studies on the round window membrane. Passage rates for different substances through the round window membrane can be calculated under controlled conditions by using this type of in vitro model.