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....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.展开更多
基金the National Natural Science Foundation of China, No. 30600702
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.
