| 1 | Phenotypic differentiation of neonatal rat cochlear spiral ganglion neurons following trypsin dissociation and culture显示文摘BACKGROUND: Under laboratory conditions, cochlear spiral ganglion neurons are commonly isolated and cultured by mechanical dissociation. However, these neurons are extremely fragile and survive for only a short time. OBJECTIVE: To establish a trypsin dissociation and culture method for studying neonatal rat cochlear spiral ganglion neurons. DESIGN: A single sample study. SETTING: Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA. MATERIALS: This study was performed at the central laboratory for Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA from February to May 2006. A total of 40 neonatal Sprague Dawley rats of either gender, aged 2-5 days, were provided by the Laboratory Animal Center of the Fourth Military Medical University of Chinese PLA. Trypsin and neuronal-specific nuclear protein (NeuN) monoclonal antibodies were purchased from Sigma Company, USA. Culture medium was synthesized using Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco Company, USA) supplemented with 10% fetal bovine serum (Sigma Company, USA), 100 000 U/L penicillin, and 1 mol/L NaOH. The following protocol was performed in accordance with ethical guidelines for the use and care of animals. METHODS: After anesthesia, rats were sacrificed by neck dislocation. A complete cochlear axis with spiral ganglion tissue was removed. The cochlear axis was rinsed three times in a culture dish with a diameter of 35 mm using Hank’s balanced solution. After washings, the tissue was cut into pieces, digested with 0.25% trypsin for about 20 minutes, and incubated in a 37 ℃ water bath. The tissue was centrifuged, then mixed with serum-containing culture medium. Using a transfer pipette, the cell suspension was transferred to polylysine (0.1%)-treated culture dishes with a diameter of 35 mm. The culture dish was incubated at 37 ℃, with a 5% CO2-air environment. Once the cells adhered to the culture dish wall, DMEM/F12 supplemented with 10% fetal bovine serum was added. MAIN OUTCOME MEASURE: Using an inverted microscope, the adherent cultured cells were observed and neurite growth index was calculated. Immunocytochemistry was performed to identify the spiral ganglion neurons, and NeuN-positive cells were analyzed. Following immunofluorescence, cochlear spiral ganglion neurons were identified through a microscope. RESULTS: Observation of cellular morphology: after digestion, inoculated cells exhibited were spherical, well stacked, and had a transparent appearance. Six hours later, some cells adhered to the culture dish wall, and small neurites were detected in a small number of cells. Twelve hours later, the adherent cells developed into polarized cells. Eighteen hours after inoculation, the adherent cells presented an ellipsoidal appearance, clear cell membranes, homogeneous cytoplasm, good refraction, and a transparent cell body surrounded by a marked halation. Twenty-four hours later, most of the cochlear spiral ganglion neurons exhibited a bipolar neuronal morphology with neurite length ranging from 2-5 times the length of a cell body. Some cochlear spiral ganglion neurons exhibited a tripolar neuronal morphology with neurites that stretched in three directions; neurite length was several times greater than the transverse diameter. Forty-eight to seventy-two hours later, the cells further differentiated and exhibited interwoven neurites, with a length that was 7-8 times greater than the cell body length. Seven days later, cells began to degenerate and underwent apoptosis. Identification of cochlear spiral ganglion neurons: immunocytochemical staining revealed whole cochlear spiral ganglion neurons that were green-colored and exhibited an ellipsoidal cell body with clear neurites. Measurement of neurite growth index: neurite growth index was 0.52±0.13, 0.86±0.21, 1.22±0.33, and 1.05±0.26 for 24, 48, 72, and 120 hours after inoculation, respectively. CONCLUSION: Under cell culture conditions of serum and trypsin dissociation, neonatal rat cochlear spiral ganglion neurons grow well, survive for long periods in vitro, and exhibit normal phenotypic differentiation. | Dingjun Zha Li Qiao Lianjun Lu Xue Gao Tao Xue Wenjuan Mi Shunli Liu Jianhua Qiu | 2008 | Neural Regeneration Research2008，3，3： | 1 |
| 2 | Expression and subcellular localization of cysteine- and glycine-rich protein-2 in rat undifferentiated olfactory stem cells显示文摘BACKGROUND: As a member of the LIM protein family Ⅱ, cysteine- and glycine-rich protein-2 (CRP2) has been demonstrated to play a role in the regulation of growth and differentiation of eukaryotic cells. Our previous study has demonstrated that CRP2 can be detected in the embryonic rat inner ear but not in the adult rat inner ear. However, at present, the expression of LIM protein family Ⅱ members in stem or precursor cells has not been described. OBJECTIVE: To determine the expression and sub-cellular localization of CRP2 in olfactory stem cells. DESIGN, TIME AND SETTING: An experiment with repeated measures was performed in the Laboratory of Otorhinolaryngology, Head and Neck Surgery, Xijing Hospital, the Fourth Military Medical University from February 2008 to April 2008. MATERIALS: Olfactory stem cells, and rabbit-anti-CRP2 polyclonal antibody were prepared and kept in our laboratory. METHODS: Reverse transcription polymerase chain reaction and Western blot analysis were used to detect expression of CRP2 in olfactory stem cells. Immunocytochemistry was also used to localize CRP2 in olfac- tory stem cells. MAIN OUTCOME MEASURES: The expression and sub-cellular localization of CRP2 in rat olfactory stem cells. RESULTS: CRP2 expression was found in olfactory stem cells, and CRP2 was distributed in both the nu- cleus and the cytoplasm. CONCLUSION: Confirmation of the expression and distribution of CRP2 in olfactory stem cells. | Xue Gao Zengyu Cao Jun Chen Shunli Liu Dingjun Zha Feng Wang Yang Chen Li Qiao Lianjun Lu Jianhua Qiu | 2008 | Neural Regeneration Research2008，3，5： | 0 |
| 3 | Translocation of telomerase reverse transcriptase coincided with ATP release in postnatal cochlear supporting cells显示文摘The spontaneous bursts of electrical activity in the developing auditory system are derived from the periodic release of adenosine triphosphate(ATP)by supporting cells in the Kölliker’s organ.However,the mechanisms responsible for initiating spontaneous ATP release have not been determined.Our previous study revealed that telomerase reverse transcriptase(TERT)is expressed in the basilar membrane during the first postnatal week.Its role in cochlear development remains unclear.In this study,we investigated the expression and role of TERT in postnatal cochlea supporting cells.Our results revealed that in postnatal cochlear Kölliker’s organ supporting cells,TERT shifts from the nucleus into the cytoplasm over time.We found that the TERT translocation tendency in postnatal cochlear supporting cells in vitro coincided with that observed in vivo.Further analysis showed that TERT in the cytoplasm was mainly located in mitochondria in the absence of oxidative stress or apoptosis,suggesting that TERT in mitochondria plays roles other than antioxidant or anti-apoptotic functions.We observed increased ATP synthesis,release and activation of purine signaling systems in supporting cells during the first 10 postnatal days.The phenomenon that TERT translocation coincided with changes in ATP synthesis,release and activation of the purine signaling system in postnatal cochlear supporting cells suggested that TERT may be involved in regulating ATP release and activation of the purine signaling system.Our study provides a new research direction for exploring the spontaneous electrical activity of the cochlea during the early postnatal period. | Yukai Zhang Keyong Tian Wei Wei Wenjuan Mi Fei Lu Zhenzhen Liu Qingwen Zhu Xinyu Zhang Panling Geng Jianhua Qiu Yongli Song Dingjun Zha | 2024 | Neural Regeneration Research2024，19，5： | 0 |
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