·76· Journal of Otology 2008 Vol. 3 No. 2

Original Article Spontaneous Proliferation in Organotypic Cultures of Mouse Cochleae

1 2,CA 2 1 DING Da-lian , WANG Jian , YU Zhi-ping , JIANG Hai-yan , 1 1 WANG Ping , Richard Salvi 1 Center for and Deafness, State University of New York at Buffalo, NY, USA 2 School of Human Communication Disorders, Dalhousie University, Halifax, NS, Canada

Abstract Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferative regenera⁃ tion. However, substantial proliferation occurs in organotypic cultures of cochleae from postnatal mice. In the present study, we studied the time course of proliferative growth in cultures of mouse explants obtained from up to 12 （ ） postnatal days. The mitotic nature of this growth was confirmed by bromodeoxyuridine BrdU staining and expres⁃ （ ） sion of proliferation cell nuclear antigen PCNA evaluated with real-time quantitative polymerase chain reaction （ ） RT-PCR . Similar growth time course was found in the cochlear explants of different postnatal ages. The new growth reached its maximum at around 2 days in culture followed by a slow-down, and virtually stopped after 5 days of culture. The possible mechanisms and the significance of this proliferation are discussed. Key words （ ） organotypic culture, cochlea, proliferation, bromodeoxyuridine BrdU , proliferation cell nuclear anti⁃ （ ） （ ） gen PCNA , real-time quantitative polymerase chain reaction RT-PCR Introduction

During embryonic development, cells of various desti⁃ regeneration can largely restore inner functions［ after 11, 12, nations increase in numbers through proliferation in the HCs are］ destroyed by intense noise or ototoxic drugs 9, 13, 14, 15 , producing progenitors to supporting cells . Limited regeneration through proliferation has （ ） （ ） SCs ,［ hair］ cells HCs and spiral ganglion neurons been reported［ ］ in the vestibular apparatus of mammalian （ ）1 16-18 SGNs . However, proliferation has virtually stopped［ ］ inner ear , but not in mammalian cochleae. 2-4 immediately after birth in mammalian cochleae . Various efforts are being made to study mechanisms Therefore, hearing impairment is permanent if HCs and/ that promote proliferation in the inner ear in lower level or SGNs in mammalian cochleae are destroyed by haz⁃ animals, in an attempt to understand mechanisms that ardous factors. In contrast,［ ］ HC regeneration［ ］ occurs restrain cell proliferation in mammalian cochleae. Mean⁃ 5 6 throughout lifetime in fish and［ amphibians］ , as well while, various methods are being evaluated to induce 7, 8 as in vestibular receptors in birds . Although sponta⁃ postnatal proliferation［ ］ and HC regeneration in mammali⁃ 19-21 neous proliferation is not seen after birth, SCs in avian ［an cochleae］ . These include the use of mitogen 22-24 cochleae go back into cellular circles［ ］ in response to le⁃ ［ ］, manipulation of cyclin-dependent kinases［ ］ 9, 10 25, 26 27-30 sions that destroy cochlear HCs . Such proliferative , as well as transplantation of neural stem cells . A great portion of such studies are carried out using in Corresponding author: WANG Jian, 5599 Fenwick St. SCHD vitro approaches in［ which various］ types of inner ear ex⁃ 27, 31, 29, 32, 17, 33, 34 G-100 Dalhousie University Halifax, Nova Scotia, Canada B3H plants are cultured . 1R2. Tel: (902)4945149; Fax: (902)4945151 Induction of proliferation is the first step that may Email: [email protected] lead towards successful HC regeneration in the cochlea. Journal of Otology 2008 Vol. 3 No. 2 · 77 ·

（ ） But results obtained from culture studies need to be 10 × basal medium Eagle BME and 2% sodium interpreted with caution. Spontaneous proliferation of carbonate in the ratio of 9:1:1. A drop of gel was placed certain cell types occurs in organotypic culture of on a 35-mm culture dish and allowed to gel for cochlear explants,［ － ］ possibly in response to injury during approximately 15 min. After placement of the cochlear 35 37 （ ） dissection . In this study, we re-visited this issue by preparation with and without collagen gel , 1 ml of extending the observation of culture of mouse cochlea culture medium was added and the explant incubated at explants up to 12 postnatal days(P12) under different 37 ℃ in 5% CO2 for 6 hours. Then, 1 ml of cultural culture conditions. In addition, the nature of mitosis was medium was added to completely cover the tissue, studied using specific molecular markers. followed by incubation until the planed time of Methods termination. The culture medium was replaced every day. Subjects To rule out proliferation stimulated by ingredients in A total of 210 C57BL/10J mice were used. At lease SFM or the supporting materials such as type I rat-tail 10 cochlear preparations were obtained from P0,P1,P2, collagen gel, control experiment was performed using P5 and P12 mice, respectively. Basilar membranes were DMEM/F-12 medium plus 10% serum and no gel. F-actin staining dissected to include cells from spiral ganglion to the Cultured preparations were fixed in 4% parafo- Hensen/Claudius cell boundary. Care was taken to （ ） exclude the and stria vascularis. The rmaldehyde in 0.1 M phosphate buffer pH 7.4 for 4 hours, rinsed in 0.1 M phosphate-buffered saline preparations were cultured for various durations from 12 （ ） PBS , incubated in 0.25% Triton X-100 for 5 min, hours to 12 days. The growth was examined daily using （ ） and immersed in TRITC-labeled Sigma P1951 or light microscope. The cultured specimens were stained （ ） with FITC-conjugated phalloidin or FITC-labeled phalloidin Sigma P-5282, 1:200 in PBS for 30 min. Specimens were mounted in Fluoromount rhodamine-conjugated phalloidin for cellular skeleton （ ） structures. Proliferation was identified with EMS Inc. , examined and photographed under a Zeiss （ ） bromodeoxyuridine BrdU staining and detection of fluorescence microscope. （ ） BrdU labeling proliferation cell nuclear antigen PCNA by quantitative real-time polymerase chain reaction To confirm the involvement of proliferation in this （ ） （ ） RT-PCR . The growth of cultured explants was also growth, BrdU Sigma B5002 was added in culture viewed in cross section with the help of toluidine blue medium to identify new synthesis of DNA associated staining. with mitosis. In order to estimate the time course of Organotypic culture proliferation in culture, chronological BrdU labeling was （ performed in a series of cultures from P5 mice Figure Most cochlear preparations were flattened and ） （ 3 terminated after different durations of culture 12 to immerged in collagen gel, which provided mechanical ） support to the to prevent collapse in 48 hours . BrdU was applied during the last 12 hours of culture. The gel was transparent in the culture medium, culturing, so only newly proliferated cells were labeled. providing an ideal environment for culturing organ of The distribution of cells with BrdU labeled nuclei would indicate the area of new growth. Corti. To rule out unexpected effect of collagen gel on （ After adding BrdU with a final concentration of proliferation, the gel was not used in a few cases, where ） the preparations were simply placed on the bottom of 2.5% , the cochlear tissue was fixed in 3.5% dishes. For the same purpose, serum-free medium paraformaldehyde for 4 hours at 4℃ and then treated （ ） （ ） SFM, Sigma I1884, 1% was occasionally replaced with 90% methanol containing 0.3%H2O2 for 20 min with DMEM/F-12 serum medium. and 2 N hydrochloric acid for 30 min to inactivate The collagen gel was freshly prepared immediately endogenous peroxidase. The specimens were then before the experiment. Type I rat-tail collagen blocked with a solution containing 2% normal horse （ ） Collaborative Research of 3.76 mg/ml was mixed with serum, 2% BSA and 5% nonfat dry milk to eliminate ·78· Journal of Otology 2008 Vol. 3 No. 2

（ -△△CT ） nonspecific binding. Subsequently, the specimens were method 2 method . incubated overnight in mouse anti-BrdU monoclonal The animal-use protocol of this experiment was （ antibody 1:50 dilution, Becton Dickinson, Mt. View, approved by the animal use committee of State ） CA , 2% normal horse serum and 1% Triton X-100 in University of New York at Buffalo. PBS. After washing in BSA solution, specimens were Results （ treated with biotinylated horse anti-mouse IgG Vector ） Labs., Burlingame, CA for 1 hour, followed by a Spontaneous proliferation in organotypic culture of complex of avidin-biotinylated horseradish peroxidase （ ） cochlear preparations from postnatal mice was indicated HRP; Vector Labs, Elite ABC Kit for 45 min. Then, by: (1) morphological observation of cell growth in the specimens were immersed for 4 min in citrate buffer （ ） ’ culture dishes, (2) BrdU staining demonstrating inc- pH 5.1 containing 0.2% 3, 3 -diaminobenzidine and orporation of thymidin analogues into newly synthesized （ 0.01%H2O2. DNA during mitosis allowing determination of cell Toluidine blue staining of cross section ） （ cycle characteristics , and (3) PCNA gene expression a Specimens were fixed with 3.5% paraformaldehyde method widely employed［ ］ for the assessment of pro- 38 for 4 hours, dehydrated with gradient alcohol, and liferative activities) . embedded in Epon 812 resin. Serial sections were cut in Rapid tissue growth was evident around 6 hours after a plane vertical to the longitude axis of basilar culture, as demonstrated first by fluorescent dyes membrane at a thickness of 3μm. Sections were stained against F-actin, a widely distributed protein seen in the with 0.1% toluidine blue. form of micro-filament, which is an important skeleton Real-time quantitative PCR structure of cochlear cells. Staining specific to F-actin TM Platinum Quantitative RT-PCR Thermo Script （ ） demonstrated HC and the outline of HCs and One-Step System Cat No:11731015 and a program of supporting cells. The outer border of the explant was R （ ） amplification on the Opticon 2 MJR company were typically established laterally to the third row OHC used. The primers were designed using LUXTM including a narrow belt of outer sulcus region when Designer from Invitrogen company based on published （ harvesting cochlear tissues. Figure 1A shows a freshly mRNA sequences forward sequence: 5 ’ ’ dissected specimen from a P1 mouse stained with -gaatttgcacgtatatgccg-3 , reverse sequence: 5 ’ ’） （ TRITC-phalloidin. Figure 1B shows a preparation from -ctaagatgcttcctcatctt-3 . β-actin primers set served ） a P1 mouse after 72 hours of culture. An outward growth as an internal control was provided by Invitrogen （ ） from the edge of dissection is clearly visible when company Cat No: 101M01, 101M02 . Total RNA was compared to the freshly dissected preparation in Figure extracted from 6 cultured cochlear samples using the 1A. Figure 1C focuses on the upper layer of the newly RNeasy Mini kit following manufacturer's instructions （ ） generated cells of the new growth in Figure 1B. All Qiagen, Germany . Each 25 μL PCR reaction solution （ ） epithelial cells in this region show a hexagonal shape, contained 5 μL 100 ng total RNA after treatment similar to that of Claudius cells normally occupying the （ ） with DNAse I, 20 pmol of each primer, 12.5 μL 2 × upper layer facing scala media in the outer sulcus TM ThermoScript Reaction Mix, 0.5 μL ROX Reference region. TM Dye, and 0.5 μL ThermoScript Plus/Platium Taq Mix. The cochlear preparations from mice of different （ ） The program consisted of 1 cycle of denaturation at （ ） postnatal ages P0-P12 showed similar ability of 95 ℃ 5 min followed by 35 cycles of 30 seconds growth in culture. Figure 2 shows a series of cochlear denaturation at 95 ℃ , 30 seconds anneal at 55 ℃ , cultures from P5 mice stained with FITC-labeled and 1 minute extension at 72℃ . The results were phalloidin after different durations of culture in SFM. analyzed using Opticon software 1.0 and normalized to Growth was visible in all cultures, starting from the edge levels of expression relative to β-actin. In each case, of the dissection. There was no expansion in the organ of data were shown relative to the levels of non-cultured Corti and other areas of the preparations. The growth （ ） （ ） cochlear tissue with comparative CT Threshold Cycle accelerated during the 12-24 hours Figure 2B and 2C Journal of Otology 2008 Vol. 3 No. 2 · 79 ·

Fig. 1 Growth of outer sulcus in cultured cochlear explants from a P1 mouse. The specimens are labeled with TRITC-phalloidin. A: （ ） newly dissected preparation with a narrow belt of outer sulcus cells Scale bar: 100 μm . B: a preparation fixed and stained after 72 （ ） hours of culturing with dramatic growth in outer sulcus region Scale bar: 100 μm . C: magnified from image B showing focal plane of （ ） photomicrograph at upper layer of outer sulcus, Scale bar: 30 μm .

Fig. 2 Cochlear preparations from P5 mice labeled with FITC-labeled phalloidin after culturing in SFM for 6, 12, 24 and 48 hours re⁃ （ ）（ ） spectively A, B, C and D Scale bar: 100 μm . Arrows indicate the frontier of new growth, visible after 6 hours of culture. The growth is dramatic after 24 hours of culture, followed by a slow-down. OC: organ of Corti

（ ） period and became dramatic at 48 hours Figure 2D . This was followed by a gradual slow-down. Growth virtually stopped after 5 days. There was no BrdU labeled nuclei in the region of or⁃ gan of Corti, neither in the area closely lateral to the third row of OHCs, indicating that cells in these regions were those harvested from the cochleae, not newly gener⁃ Fig. ３ ated through proliferation in the culture. Figure 3A to C BrdU staining after different times of organotypic cul⁃ （ show an outward shifting of cells with BrdU labeled nu⁃ turing in cochlear preparations from P5 mice Scale bar: 100 ） clei as culture time increased. Cells with strong BrdU μm . The BrdU is added to the culture medium 12 hour before staining occupy large area in the outer sulcus region in culture termination. From A to C, the culture is terminated at （ the samples terminated after 24-48 hours of culture Fig⁃ 12, 24 and 48 hours respectively. Proliferation is evidented by ） ure 3B and C . BrdU-labeled nuclei. The BrdU labeled cells move laterally in the specimens cultured for longer duration. OC: organ of Corti. Cell growth was also evident in control cultures using OS: outer sulcus. DMEM/F-12 medium plus 10% serum and no gel. Fig⁃ ure 4 shows a dramatic expansion of outer sulcus region in a preparation from a P0 mouse that is cultured for 5 growth stopped gradually after 5 days. However, epitheli⁃ days under this condition. Similar to other cultures, al cells did not appear to grow well in the DMEM/F-12 ·80· Journal of Otology 2008 Vol. 3 No. 2

Fig. 4 Expansion of the outer sulcus cells seen in cultures using DMEM/F-12 serum medium without collagen gel, non-stained image. The preparation is from a P0 mouse co⁃ （ ） chlea and cultured for 5 days Scale bar: 100 μm .

Fig. 6 Cross section of cochlear preparation from P2 mice stained with toluidine blue after different durations of organo⁃ typic culturing in SFM with the support of rat-tail collagen （ ） Scale bar: 100 μm . A: a sample that cultured for 24 hours. and B: a sample cultured for 72 hours. Note the expansion of outer sulcus from A to B. Also note the flattening of cells in both layers, as well the shortening of Henson's cells near HCs. OS: outer sulcus, IS: inner sulcus, OC: organ of Corti, ORT8OTM: .

Fig. 5 A preparation from a P0 mouse cochlea cultured in DMEM/F-12 culture medium containing 10% serum for 24 （ ） hours Scale bar: 30 μm . Few epithelial cells are present along the edge of . Numerous fibroblast cells were growing towards the outside. serum culture medium. Also, most proliferating cells in culture medium containing serum were fibroblast cells （ ） Figure 5 . Fig. 7 Proliferation in outer sulcus region was associated Enhanced PCNA-mRNA expression in organotypic with changes in cell shapes. After 24 hours of culturing, culture. The averaged value and standard deviation are calcu⁃ lated from 6 cochlear preparations at each time point. cells in the upper layer of the outer sulcus (facing scala （ ） media), maintained their pillar shape Figure 6A . After layer and grew beyond the area covered by the upper lay⁃ （ ） longer culture periods, the newly proliferated cells be⁃ （ ） er as culture time increased Figure 3B . After 5 days of came flattened Figure 6B, 3 days of culture . The tall culturing, the proliferation in upper layer had mostly Henson's cells lateral to OHCs also became （ ） ended, while cells in the lower layer continued to grow. shortened after long period of culture Figure 6B . When PCNA-mRNA level was evaluated chronologi⁃ In the organotypic cultures, the two layers of outer sul⁃ cally in a series of cultured explants from P2 mice, as an cus region appeared to grow at different speeds: the fi⁃ additional measure to verify proliferation in the new broblast-like cells grew faster than those in the upper growth, an increase along with culture time was noticed, Journal of Otology 2008 Vol. 3 No. 2 · 81 ·

） reaching 4.4, 8.21 and 9.14 times of the baseline level ure 2 , followed by a gradual slow-down and cessation （ ） after 24, 48 and 72 hours respectively Figure 7 , indi⁃ after 5 days of culture. Because BrdU was added in the cating remarkably enhanced cell proliferation in the cul⁃ medium 12 hours before culture termination, only cells ture. Baseline PCNA-mRNA in this study probably re⁃ dividing during this time would be labeled. BrdU label⁃ flected limited postnatal proliferation of few cells in the ing indicates that nuclei shifted from the organ of Corti ’ Reissner［ ］ s membrane, basilar membrane, and limbus as culture time increased, suggesting higher capability 1 stroma . of proliferation in cells along the growth front line than Discussion other cells, which can last for several generations and is then transferred to daughter cells along the new border. The results of this study support previous reports Measures of PCNA expression seem to suggest in⁃ showing that cells around dissection edge may grow in creasing proliferation along with time up to 72 hours of cultures, the longest PCNA observation time in this organotypic［ － ］ culture of cochlear explants from postnatal 35 37 mice . Our results also show that this growth can oc⁃ study. But this may not be true. Rather, it is likely due cur in explants from mice as old as P12. In addition, the to accumulation of PCNA in extended duration cultures study demonstrates that this new growth is largely due to because of the long half-life of this marker. Anti-PCNA proliferation. The observed variation in rates of cellular staining has been reported to persist［ １ for － a］ long period af⁃ 4 43 proliferation suggests that it is not a methodological arti⁃ ter mitosis due to its long half-life . PCNA testing fact. Different levels of fibroblast cell proliferation asso⁃ therefore tends to over-evaluate the number of dividing ciated with presence or absence of serum in the medium cells and is not an optimal method to evaluate dynamic indicate that serum medium enhances fibroblast cell pro⁃ changes of cellular proliferation. liferation, whereas, serum-free medium inhibits fibro⁃ Extensive BrdU labeling in nuclei of cells in the up⁃ blast cell growth. Therefore different culture medium per layer of the outer sulcus region suggests that much may lead to distinct cell types to grow in the dish. of the expansion in this region is due to proliferation of In general tissue grows as a result of (1) increase of cells, most likely the Claudius cells. At this moment, cell size and/or (2) increase of cell numbers as the result however, it is not clear whether the fast growth of fibro⁃ of cell division. The growth in the upper layer of outer blast cells in the lower layer is from cellular prolifera⁃ sulcus region in this study is probably a result of mitotic tion or processes elongation. The outgrowth of fibro⁃ blasts beyond the upper layer was not accompanied by cell division, because of positive BrdU staining and （ ） changes in PCNA gene expression. BrdU staining has BrdU staining of nuclei Figure 3B in this study, cellu⁃ lar processes elongation is more likely the case, rather been validated as［ an］ effective and principle tool for dem⁃ 39 onstrating mitosis . PCNA gene expression can be an than cellular division. indicator of proliferative activities in all cell types in co⁃ Currently, we do not have a clear idea regarding possi⁃ chlear explants, including fibroblast and epithelial cells. ble causes for the spontaneous proliferation seen in the PCNA gene activation causes a increase of PCNA pro⁃ organotypic culture of the postnatal cochlea explants. tein, which is an auxiliary protein of DNA polymerase δ Two explanations are possible. Firstly, the proliferation may be a response to mechanical lesion during dissec⁃ that is［ mainly］ expressed during the S phase of cellular ［ ］ 40 36, 37 circle . The rate of PCNA synthesis has been shown to tion, as suggested by previous studies . The remark⁃ correlate well with that of cell proliferation (41). PCNA able new growth of cultured explants was initiated at the expression is an obligatory event in the G1-to-S transi⁃ edge of dissection along the outer sulcus region. In most of the explants, the inner border of dissection was cut tion. The changes in PCNA expression in this study （ ） therefore suggest that the expansion seen in the outer along the the bony tissue . In such prepa⁃ sulcus region is largely due to an increase in the number rations, generally there was no growth along the inner of cells from new cell proliferation. border. However, in a few explants in which the dissec⁃ While not quantitatively measured, the growth appears tion was accidentally made along the inner sulcus re⁃ （ （ faster in the early stage of culture up to 24 hours Fig⁃ gion, growth along the inner border was also evident da⁃ ·82· Journal of Otology 2008 Vol. 3 No. 2

） tion: winging our way towards a sound future. Curr Opin Neurobi⁃ ta not shown , suggesting proliferation related with the １ injury from dissection. ol, 2003, 13: 119- 26. 10 Staecker H, Van De Water TR. Factors controlling hair-cell Secondly, the proliferation may be due to loss of con⁃ （ ） regeneration/repair in the inner ear. Curr Opin Neurobiol, 1998, 8: tact or lateral inhibition at the border of the dissection. ４８ 480- 7. During development, contact inhibition among cells ap⁃ 11 Adler HJ, Raphael Y. New hair cells arise from supporting pears［ to］ play a role in limiting over prolifera［ －⁃ 44 45 cell conversion in the acoustically damaged chick inner ear. Neu⁃ tion］ and in the formation of SC-HC mosaic rosci Lett, 1996, 205, 17-20. 49 . Loss of contact with other cells may be the reason for 12 Adler HJ, Komeda M, Raphael Y. Further evidence for sup⁃ porting cell conversion in the damaged avian basilar papilla. 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