Nuclear entry of hyperbranched polylysine nanoparticles into cochlear cells.

BACKGROUND: Gene therapy is a potentially effective therapeutic modality for treating sensorineural hearing loss. Nonviral gene delivery vectors are expected to become extremely safe and convenient, and nanoparticles are the most promising types of vectors. However, infrequent nuclear localization in the cochlear cells limits their application for gene therapy. This study aimed to investigate the potential nuclear entry of hyperbranched polylysine nanoparticles (HPNPs) for gene delivery to cochlear targets. METHODS: Rat primary cochlear cells and cochlear explants generated from newborn rats were treated with different concentrations of HPNPs. For the in vivo study, HPNPs were administered to the rats' round window membranes. Subcellular distribution of HPNPs in different cell populations was observed with confocal microscope 24 hours after administration. RESULTS: Nuclear entry was observed in various cochlear cell types in vitro and in vivo. In the primary cochlear cell culture, concentration-dependent internalization was observed. In the cochlear organotypic culture, abundant HPNPs were found in the modiolus, including the spiral ganglion, organ of Corti, and lateral wall tissues. In the in vivo study, a gradient distribution of HPNPs through different layers of the round window membrane was observed. HPNPs were also distributed in the cells of the middle ear tissue. Additionally, efficient internalization of HPNPs was observed in the organ of Corti and spiral ganglion cells. In primary cochlear cells, HPNPs induced higher transfection efficiency than did Lipofectamine(™). CONCLUSION: These results suggest that HPNPs are potentially an ideal carrier for gene delivery into the cochlea.

Immunodefense of the round window.

A systematic analysis using serial sectioning of the round window membrane (RWM) in the cynomolgus monkey was performed. Light and transmission electron microscopy (LM and TEM) revealed that the RWM rim may be endowed with gland-like structures with glyco-protein material secernated into the window niche. This was detected in one third of the specimens. The secreted material displayed waste material and scavenger cells. There was also a rich network of capillaries, lymph channels, and sinusoidal veins containing leukocytes. Their abluminal surfaces displayed mature plasma cells and monocytes. These findings suggest that in certain primates the middle ear may have developed specific immunoprotective means for disposal of foreign and noxious substances before they reach the inner ear.

[Effect of poloxamer 407 on the cochlear morphology and hearing function after perfusion in round window: experiment with guinea pigs].

OBJECTIVE: To observe the effect of sustained-release vehicle-Poloxamer 407 on the function and morphology of the cochlear after perfusion in round window. METHODS: The right ears of 16 guinea pigs as experimental group were perfusion with 20% Poloxamer 407 solusion 100 microl in round window, the left ears as control group with normal saline. Another 4 animals without treatment were in negative control group. Auditory function was investigated before and immediate, 7, 14, 28, 49 days after perfusion, and the cochlear basilar membranes were stained by silver nitrate after ABR examinations. The stained basilar membranes were examined under light microscopy. RESULTS: The ABR threshold to filtered clicks were elevated after perfusion, and were recovered to normal at 49 days after perfusion. The morphology of cochlear hair cells were not significantly damaged after perfusion. CONCLUSION: Poloxamer 407 after topical perfusion in round window can cause temporary changes on auditory function of inner ear, but no inreversible damage on function and morphology of cochlear. Therefore, Polomamer 407 can be used as sustained-release vehicle in middle and inner ear diseases.

[Influence of stapedectomy on the hearing of guinea pigs].

OBJECTIVE: To compare the influences of stapedectomy and small fenestra stapedotomy on the hearing of guinea pigs. METHODS: Twenty-four (48 ears) guinea pigs were randomized equally into two groups, and the left ears were subjected to stapedectomy and total stapes replacement with a prosthesis, or sham operation (12 ears) to expose the footplate of the stapes and the round window. Each guinea pig was tested by ABR perioperatively. Four guinea pigs were chosen randomly from each group and decapitated for morphological examination by light microscopy and scanning electron microscopy after ABR test. RESULTS: In the sham operation group, the post-operative latencies of each wave, the intervals and the hearing threshold exhibited no significant changes other than prolonged latency of wave I. In stapedectomy group, the hearing threshold increased to 23.75-/+3.77 dBSPL 1 h after operation with significantly prolonged post-operative latencies of all the waves and intervals but for III-IV interval, which was shortened. The latencies of each wave (especially waves I and III) in the stapedectomy group were increased by a greater magnitude than those in the sham operation group, but the intervals were comparable between the two groups. No significant difference was noted in the parameters of ABR either 1 h or 1 day after the operation between the two groups, in which the architecture of cochleas remained intact with similar number of spiral ganglion cells. The stereocilia of the outer hearing cells (OHC) were normal in the sham operation group while in stapedectomy group, slight stereocilia disorder occurred but became normal 1 day after operation. No obvious changes were found in the stereocilia of the inner hearing cell (IHC) in either groups. CONCLUSION: Stapedectomy can induce mild hearing loss without seriously damaging the function of the cochlea in guinea pigs.

A frequency-position function for the human cochlear spiral ganglion.

Greenwood's frequency-position function for the organ of Corti (OC) is commonly used to estimate represented frequencies for cochlear implant (CI) electrodes, both in temporal bone studies and in imaging studies of living CI recipients. However, many contemporary CIs position stimulating electrodes near the modiolus, directly targeting the spiral ganglion (SG) cells within Rosenthal's canal. At the extreme base and apex, the SG does not extend as far as the OC, and the radial nerve fibers take a tangential course into the modiolus resulting in a potential offset between the frequency maps of the OC and SG. In this investigation, human cadaveric cochleae (n = 7) were studied in surface preparations after osmium staining. The OC and SG lengths were measured and radial fiber trajectories traced to identify frequency-matched points on each structure. These data allowed derivation of a mathematical function correlating represented frequency along the OC to position along the SG. A cubic function fit the data with a very high intersubject correlation. Better knowledge of the human SG 'neural frequency map' may help to refine electrode design, and to more accurately map CI channel filter bands to the appropriate cochlear place along the SG, which may be advantageous for more sophisticated CI outcomes, such as music appreciation. These data also could be valuable for electroacoustic stimulation, by defining the insertion distance of a CI electrode required to reach specific frequencies (based upon preoperative imaging) in an individual subject, thus helping to avoid trauma to cochlear regions with residual hearing.

Cochlear implantation via the round window membrane minimizes trauma to cochlear structures: a histologically controlled insertion study.

OBJECTIVE: To evaluate cochlear implant trauma to intracochlear structures when inserting the electrode via the round window membrane. MATERIAL AND METHODS: Eight fresh human temporal bones were evaluated histologically after insertion using two types of cochlear implant array. Bones underwent a special fixation and embedding procedure that allowed sectioning of undecalcified bone with the electrode in situ. Insertions depths were evaluated radiologically and histologically. RESULTS: All arrays were found in the scala tympani of the cochlea. Basal trauma could be avoided in all but one specimen. The mean depth of insertion was 382.5 degrees. Apically, only one implanted bone showed cochlear trauma exceeding lifting of the basilar membrane. CONCLUSION: Insertions through the round window membrane were shown to be atraumatic, even in basal cochlear regions. This route of insertion might be very effective for combined electric and acoustic stimulation of the auditory system.

Round window membrane in young and aged C57BL/6 mice.

Although there have been many studies on the round window membrane (RWM), little information has been reported about changes in the membrane associated with aging. We have undertaken morphological studies of RWMs using young (7-8 weeks old) and aged (27-29 months old) C57BL/6 mice. The RWM was thinner in mice from the aged group compared with that of the young group. The cell density in the epithelial and inner layers was also reduced in the aged group. In the middle layer of the RWM in aged mice, transmission electron microscopy revealed many degenerated short and thick elastic fibers. Confocal laser microscopy using fluorescein isothiocyanate(FITC)-wheat germ agglutinin (WGA) staining was used to identify WGA-positive fibers in the middle layer of the RWM, which changed in a similar manner to the fibers in aging skin.

[Comparison of microfissures between the oval and round window niche areas in human temporal bone].

There are numerous reports on microfissures in the round window niche area, but not many reports have appeared on microfissures in the oval window niche area. In addition, few studies have compared microfissures between the oval and round window niche areas in the same subjects. Hence, the present study investigated the prevalence of microfissures in both areas in human temporal bones. The study was conducted on the 637 ears of 333 cases. Specimens were prepared according to the conventional methods: after fixation, decalcification and dehydration, each specimen was embedded in Celloidin, and the temporal bones were sectioned horizontally at 25-micron intervals. Every 10th section was stained with hematoxylin & eosin, and each section was analyzed under a light microscope. The prevalence of microfissures was analyzed in relation to the sex, left/right differences and age of the subjects. Microfissures were seen in the oval window niche area in 66.2%, and in the round window niche area in 92.0%. When a microfissure was seen in the oval window niche area, it was also detected in the round window niche area in every case. There were no sex or left/right differences in the prevalence of microfissures, and microfissures occurred bilaterally in most cases. The youngest subject with a microfissure in the oval window niche area was a 14-year-old, and the prevalence increased with age up to approximately 60 years. On the other hand, microfissures in the round window niche area were seen in a 3-year-old, and were seen in most cases over the age of ten. These findings suggest that microfissures are more likely to occur in the round window niche area as compared to the oval window niche area. However, the prevalence of microfissures in the oval window niche area was not low either, and it is important to pay attention to both the oval and round niche areas when microfissures are involved in the formation of perilymph fistulae. Given that the prevalence of microfissures in the oval window niche area increased up to about 60 years of age, not only growth, but also mastication, may be involved in the occurrence of microfissures.

Round window niche melanocytes and webby tissue. Possible collateral route of vascular and perilymph circulation.

Two hundred thirteen human temporal bones were studied for characteristics of the round window niche. In 163 specimens melanocytes were found within the attachments of the round window membrane and in the niche, most prominently at the level of the cochlear aqueduct opening. Sixty-two specimens demonstrated discrete webby foci within the lining of the niche, 16 of which completely or partially covered the tympanic surface of the round window membrane, unassociated with fibrous tags or adhesions, and in some cases contained congested vascular channels and melanocytes. The webby foci were sometimes associate; with melanocytes, while melanocytes were present without demonstrable webby foci. These findings may represent a collateral route of vascular or perilymph-related drainage from the inner ear.