The Mechanisms Underlying The Effects Of Combining Use Captopril And Losartan On Tympanosclerosis As Well As The Establishment Of A Novel Model For The Culture Of Spring Ganglion Neurons

Part I Study on the mechanisms underlying the effects of combining use Captopril and Losartan on Tympanosclerosis induced by Streptococcus PneumoniaeObjectiveTympanosclerosis (TS) is a common disease that affects the middle ear and tympanic membrane (TM) and is one of the important reason of conductive hearing loss. The development of TS can affect normal communications, then reduce the patients’ quality of living, studying and working in different degrees, and even influence the psychological health of patients. However, up to now, there is no effective drug to prevent or treat TS. Recently, studies find that various tissues and organs have the same features in development of sclerosis:beginning with damaging of parenchymal cells, and then inducing extracellular matrix deposits and tissue remodeling eventually. Captopril or losartan, such a typical drug, which belongs to the category of ACEI or ARB, respectively, is frequently employed in clinic or animal experiments to treat with the sclerosis in various organs, such as arteriosclerosis and nephrosclerosis but have never been tried to prevent TS. In our study, we set up the animal model of TS using the inoculation of type-3 Spneumoniae pneumonia microorganisms in the right ears of animals, thereby laying a solid foundation for gaining insight into such mechanisms for TS, with special attention given to whether captopril and losartan is effective in the prevention of TS development in animal models of TS.Methods1. Male albino guinea pigs and male rats were utilized in this study and divided into three groups respectively. Some were inoculated type-3 Streptococcus pneumoniae microorganisms into the right ears, then were further divided into two subgroups on the basis of drug intervention or not, with twenty subjects in each group respectively. The left ears of animals and the control group were untreated. All experimental animals were kept at standard laboratory conditions with normal feeding for 6 weeks.2. The middle ears were observed by otomicroscope to examine the development of otitis media and TS every week.3. At the 6 week, auditory brainstem response (ABR) was used to assess hearing loss in response to various stimuli. For ABR, mice were stimulated with clicks and 4kHz,8kHz,16kHz,32kHz pure tones.4. After undergoing the ABR, these animals were placed in a stereotaxic frame and removed the temporal bones. The middle ear mucosa was microscopically separated. Hematoxylin-eosin (HE) staining was performed for morphological observation, and von Kossa staining was performed to evaluate the calcified deposits.5. Immunofluorescence and western-blot were used to evaluate the variation of the protein expression of TGF-β1 in the middle ear mucosa in three groups.6. RT-PCR was used to detect the expression of TGF-β1 in RNA level in three groups.Results1. The results of otomicroscope suggested that combining use of captopril and losartan reduced the incidence of TS. No effusion of the middle ear was otomicroscopically observed in untreated animals and the left ears of animals in all experimental groups. At the sixth week,100% of the animals both in guinea pigs and rats appeared calcification in TMs, whereas, with the administration of captopril and losartan, only 53% of guinea pigs and 63% in rats had calcification in TMs.2. ABR threshold with click was significantly increased in the TS group (guinea pigs, 62.37±1.06 dB and rats,57±1.83 dB), but was unaffected in control group (guinea pigs,10.2±1.17 dB and rats,7.75±0.85 dB). With the captopril and losartan treatment, ABR threshold significantly decreased (guinea pigs,36.39±1.44 dB and rats,35.5±1.92 dB) compared with that in TS group without treatment.3. HE staining showed that obvious otitis media manifestations were observed in the middle ear mucosa of animals in TS group at week 6, and great improvements were evidenced in in captopril and losartan treated group.4. von Kossa staining showed that marked calcium crystal accumulation was located in the middle ear mucosa of animals in TS groups, while no sclerosis was found in control animals and slight calcium deposition in captopril and losartan treated groups.5. Immunofluorescence and western-blot showed that the protein expression of TGF-β1 in the middle ear mucosa was apparently upregulated in TS groups at week 6 compared with that in the control groups and significantly decreased in the captopril and losartan treated groups.6. The mRNA expression of TGF-β1 was apparently increased in the middle ear mucosa of animals in TS group compared with the normal middle ear mucosa, and significantly decreased in captopril and losartan treated group compared with the TS group at the end of week 6.ConclusionIn conclusion, we demonstrated, for the first time, that the combining use of captopril and losartan obviously attenuates the Streptococcus pneumonia-induced TS progress, probably via inhibiting the over-expression of TGF-β1.Part II Study on establishment of a novel model for the Culture of Spring Ganglion Neurons in vitroObjectiveWithin the auditory system, spiral ganglion neurons (SGNs) are the primary afferent neurons that transcribe sound input in the auditory transduction pathway within the inner ear. They deliver signals from hair cells, the peripheral auditory receptors in the organ of Corti, to the brain through the cochlear nerve. Sensorinueral hearing loss (SNHL) as seen in patients with noise-induced hearing loss, presbyacusis or age-associated hearing loss and, in certain cases, auditory neuropathy results from SGN damage, degeneration through hair cells loss and apoptosis characteristic of chemotherapy. Recently more and more studies are centralized on the development and regeneration of bipolar functionality of SGNs, and the most important method, which being utilized now, is the primary SGN cell culture. Traditional primary SGN cell culture method involves opening the bulla of the temporal bone, removing the capsule of the inner ear, dissecting out the spiral ligament and the organ of corti, and then harvesting the modiolus for digested single cells. However, this method inevitably extracts other cells such as glial cells and fibroblast. Many limitations are shown in the conventional two-dimensional (2D) conditions, which can not truly exhibit the complex interactions between SGN cells and the microenvironment. Even though extensive work has been reported using 3D culture for understanding tissue architecture, little has been published on the use of 3D culture as an in vitro model for the SGN cells. Here, we devised a novel protocol that isolated a pure SGN population and compared the neuronal morphology and neuronal length of SGN cells in 3D and 2D culture conditions.Methods1. Bhlhb5cre mice were crossed with ROSA 26-td Tomato reporter mice to trace the lineage of Bhlhb5+cells.2. Both frozen sections and whole mount immunofluorescence were performed to test and verify the specificity and sensitivity of Tomato protein.3. Bhlhb5cre+-tdTomato+mice at P3 were dissected and ganglias were isolated for digestion into cell suspension. After flow sorting, Tomato+and Tomato" cells were collected, respectively.4. The purity of the Tomato+cells isolated by flow sorting was also assessed by quantitative reverse transcriptionpolymerase chain reaction and immunofluorescence.5. The Tomato+cells were cultured in 2D and 3D conditions. The growing states were compared between 3D and 2D culture by the survival rate, the neuronal morphology, neuronal length of SGN cells at 1d、2d、3d、4d、5d、6d、7d.Results1. The results of frozen sections immunofluorescence showed that all Tomato-positive cells were also marked as Tuj-1-positive cells or Neun-positive cells, which showed green-red fluorescence, hence characterized as SGNs.2. Bhlhb5+cells using Tomato reporter gene were isolated from the prepared modiolus cell suspension by flow cytometry. The results of immunofluorescence indicated that almost all Bhlhb5+cells after FCCS were Tomato labeled cells and marked by anti-Tuj-1 antibody. In comparison, Sox-2 expression was only observed in Bhlhb5- cells3. Both Bhlhb5+and Bhlhb5- cells were collected for qRT-PCR and immunofluorescence. The mRNA expression of Tuj-1, the specific marker of nerve cells, was 10×greater in Bhlhb5+cells more than in Bhlhb5- cells. In addition, the mRNA level of Sox 2, expressed in glial cells and supporting cells but not in SGN cells, was almost 300×greater in Bhlhb5- than that in Bhlhb5+ cells.4. The results of immunofluorescence showed that 3D culture improves SGN survivability in vitro.5. The results of immunofluorescence showed that higher proportion of bipolar cells, longer neurite extention and more health grown cone in 3D culture condition.ConclusionIn the present study, we harvest a pure population of SGN cells and keep SGNs activity to the maximum extent by transferred mouse. Cells grown in the 3D culture environment exhibit greater survival, morphology, and dendritic extention compared to those of the 2D culture.