effects of growth factors on the hair cells after ototoxic treatment of the neonatal mammalian...

Ž .Brain Research 825 1999 46–58

Research report

Effects of growth factors on the hair cells after ototoxic treatment of theneonatal mammalian cochlea in vitro

R. Romand ), S. ChardinLaboratoire de Neurobiologie, UniÕersite Blaise Pascal-Clermont II, 63177 Aubiere Cedex, France´ `

Accepted 2 February 1999

Abstract

The aim of this study was to test the possible regenerative potential of several molecules and growth factors such as retinoic acidŽ . Ž . Ž . Ž .RA , insulin, epidermal growth factor EGF and transforming growth factors alpha TGFa and beta TGFb on the neonatal cochlea invitro after neomycin intoxication. Our studies show that cochlear sensory epithelium behaves differently while maintained in variousculture conditions, although we did not observe regeneration whatever the molecules or growth factors tested. The ototoxic action ofneomycin in vitro produced a specific death of hair cells, except in the apical region. Organ of Corti of rats 3 days after birth alwayspresented two regions that responded differently to the antibiotic: a widespread scar region extending from the basal cochlea up to thebeginning of the apical turn, where most hair cells had disappeared, and a second region called the resistance region localized in the apex,and which was more or less developed depending on culture conditions. The length of the resistance region was modulated by moleculesor growth factors added to the feeding solution suggesting that some of them could produce a protective action on hair cells againstneomycin. Slight protection effects may be found with RA and insulin, however, the most definite protection results from the combinationof insulin with TGFa as shown by the large increase in the length of the resistance region compared to organ of Corti treated withantibiotic alone. The tested molecules and growth factors did not promote cochlear hair cell regeneration in vitro after neomycintreatment, however some of them may offer a protective action against ototoxicity. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Organotypic culture; Neomycin intoxication; Protective action; Retinoic acid; Insulin; Epidermal growth factor; Transforming growth factors

1. Introduction

Thanks to cellular and molecular biology associatedwith detailed knowledge of mechanisms related to thedevelopment of the cochlea, it could be possible in thefuture to devise original therapeutic tools in order toprevent the devastating outcome of deafness. The regenera-tion of stato-acoustic sensory epithelium after experimentalcreation of lesions in birds has already given very interest-ing clues. For example, regenerated avian hair cells de-rived from supporting cells in the vestibular sensory or-

w xgans 78,82–84 , as well as in the auditory organw x20,23,25,67,68 gave valuable information. The same ob-servation seems to apply for the mammalian vestibular

w xorgan 48 .Hair cell regeneration in the avian cochlea involves

damage of the existing hair cells that will trigger prolifera-tion of surviving supporting cells and possibly initiate

) Corresponding author. Fax: q33-473-40-78-02; E-mail:[email protected]

w xdifferentiation into new hair cells 3,24,65 . In the mam-malian stato-acoustic epithelia, a low level of regeneration

w xis possible for the vestibular receptors in vitro 44,81,90 ,w xas well as in vivo 34,48,62 . However, no regeneration of

wauditory sensory epithelium was observed in vivo 28,39,x56,57 , except a very restricted regeneration observed in

w xneonatal mouse cochlea in vitro 42 .Unlike previous observations, massive hair cell regener-

ation was described in postnatal rat cochlear explantsŽ . w xtreated with retinoic acid RA after ototoxic injury 46 .

Unfortunately, replication of these experiments failed tow xproduce any sign of hair cell regeneration 15 . More

recently, it has been suggested that transforming growthŽ .factor alpha TGFa was able to initiate regeneration or

replacement of auditory hair cells in neonatal rat cochleaw xin vitro 74,92 . Several indications from indirect observa-

tions of young animals also give rise to hope that neodif-ferentiation from existing cells or regeneration could bepossible in the future. It has been shown by severalexperiments in neonatal rodent cochleas in vitro that theauditory sensory epithelium could produce an important

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01211-1

( )R. Romand, S. ChardinrBrain Research 825 1999 46–58 47

number of supernumerary hair cells. These new cells arew xdevelopmentally regulated 1,41 , and possibly under the

w xcontrol of some growth factors 16 . Another more recentobservation made both in vivo and in vitro in young ratcochleas, showed a spontaneous neodifferentiation of haircell-like cells in the scar epithelium after ototoxic damage.Unfortunately, these cells failed to develop into hair cells

w xafter a few weeks 42 , even with TGFa in the culturew xmedium 62 .

New approaches with cellular and molecular tools havefound several growth factors or their receptor mRNAs, in

Žsensory epithelia of the ear for review see Refs.w x.19,21,22 . Moreover, specific growth factors and geneexpression have been found in the inner ear after exposureto noise or ototoxic substances, that could be related to

w xhair cell regeneration 36,60,69 . Investigation of hair cellregeneration on drug damaged inner ear using growth

w xfactors suggested that TGFa 44,75,92 could stimulatecell proliferation and regeneration. Recently, it has beenshown that fibroblast and insulin-like growth factors in-duced cell proliferation in rat inner ear epithelial cell

w xcultures 91 .From our previous observations showing spontaneous

neodifferentiation of hair cell-like cells in vivo and in vitrow x62 , it was possible to postulate that the failure of thesecells to differentiate into hair cells was due to lack ofgrowth factors in the micro environment of the cochlea. Inorder to investigate this hypothesis, we tested the potentialuse of insulin, TGFa and other growth factors such as

Ž .epidermal growth factor EGF and transforming growthŽ .factor beta TGFb . For this purpose, we devised several

in vitro experiments using antibiotic treated cochleas inorder to evaluate the effects of the above molecules on anycochlear hair cell regeneration.

None of the molecules tested such as RA, insulin, EGF,TGFa and TGFb induced hair cell regeneration whentested alone or with insulin. Although this was not theprimary goal of this study, it does demonstrate that somemolecules such as RA and insulin alone might have aprotective effect for apical hair cells in the organ of Cortidamaged by ototoxic antibiotics, but the main observationis the clear protective effect induced by a synergetic actionof insulin and TGFa on hair cells.

2. Materials and methods

2.1. Materials

Cochlear organotypic cultures were derived from morethan 100 cochleas from Sprague–Dawley rats at 3 days

Ž .after birth 3DAB . To Dulbecco’s modified Eagle’sŽ .medium nutrient mixture F-12 HAM DMEMrF12 HAM ,

were added D-glucose, L-glutamine, sodium selenium,Ž .apo-transferrin, albumin bovine fraction V , all from

Sigma. Tested molecules such as insulin, RA, fetal bovineŽ .serum FBS , EGF from mouse submaxillary glands were

purchased from Sigma, as well as TGFa , from a syntheticmolecule of the rat model. TGFb from human plateletswas obtained from Calbiochem. Tetramethylrhodamine

Ž .isothiocyanate TRITC -labeled phalloidin was purchasedfrom Sigma. The plastic substratum was obtained fromThermanoxw , Nunc.

2.2. Organotypic cultures

After isolation of the inner ear, the stria vascularis andReissner’s membrane were removed in order to isolate theorgan of Corti. Two culture techniques were used, the first

Ž .of which is called the support method SM . In thismethod, the organ of Corti was cut in two pieces; the firstone included the hook and the first turn, the second oneincluded the second and the apical turn. Explants weremaintained on a plastic substratum without matricialmolecule. The second technique used the free floating

Ž .method FFM : the organ of Corti was not cut in piecesbut was placed in a single well of a 96-well plate filledwith a culture medium. The organ of Corti was freefloating, so without any contact with adhesion molecules.For both methods, cultures were maintained for 10 days ina humidified 5% CO atmosphere at 378C. Feeding solu-2

tions were changed every day.We used two feeding solutions, one called the basal

Ž .feeding solution BFS , made of Dulbecco’s modifiedŽEagle’s medium nutrient mixture F-12 HAM DMEMrF12

.HAM with a final concentration of 15 mM of HEPESŽ . Žbuffer, to which D-glucose 6 mgrml and L-glutamine 4

.mM were added. When FBS was not added to the previ-ous solution, a mixture with a final concentration of 30 nMNA seleniumq5 mgrml apo-transferrinq2 mgrml albu-

Ž .min bovine fraction V was added to the BFS, and thisŽ .mixture was called the standard feeding solution STA .

Control explants were maintained 10 days in vitro in theSTA. This feeding solution without serum was used be-cause feeding solution with serum has many disadvan-tages, primarily that it is a chemically undefined mixture,w x12 . Moreover, commercial sera contain varying amountsof hormones, vitamins and other constituents that vary notonly from one batch of serum to another, but even frompreparation to preparation when the same batch is usedw x13,77 . Finally, it is worth noting that cells in the organ-ism never come into direct contact with serum, and manycomponents which are normally present in the serum never

w xreach most cells of the body 37 .After 2 days of treatment with 10y3 M of neomycin,

other explants were kept 8 days in culture with supple-mented feeding solution in order to test the possible effectsof molecules or growth factors on the regeneration of haircells. Molecules or growth factors were added after theantibiotic treatment because the primary goal of this study

( )R. Romand, S. ChardinrBrain Research 825 1999 46–5848

was to test their possible regenerative, not protective,effects on hair cells. We tested 10% FBS, 10y8 M RAŽ .all-trans , insulin at final concentrations of 1 and 5 mgrml,EGF at 5, 10, and 20 ngrml, TGFa at 10, 25 and 50ngrml, TGFb at 1, 2.5 and 5 ngrml. TGFa and -b werereconstituted with a solution of 4 mM HClr1 mgrmlbovine serum albumin in order to limit growth factor

w xbinding to vessel walls 8 , and were stored in low proteinbinding Eppendorf tubes at y208C.

2.3. Tissue processing

Cultures were fixed for 1 h in 4% paraformaldehyde inŽ .phosphate buffered saline PBS , pH 7.2. After washing,

explants were incubated, without Triton, in a bufferedsolution containing 3 mgrml TRITC-labeled phalloidin for

45 min at room temperature. Explants were mounted onglass slides after being spread out in order to observe theentire length of the organ of Corti. Observations weremade with a Nikon light microscope with a 541 mmwavelength.

2.4. QuantitatiÕe analysis

The length of the organ of Corti was measured from theapex and the size of the resistance region was expressed asa mean length. Quantitative analysis of inner hair cellsŽ . Ž .IHCs and outer hair cells OHCs was performed on theentire organ of Corti divided in segments of 200 mm. Atleast six cochleas were used for each test, from whichseveral tens of segments were studied. In each segment the

Ž .Fig. 1. A Schematic distribution of hair cells along the organ of Corti after neomycin treatment and 1 mgrml of insulin in the culture medium. The mostimportant region is localized in the basal region extending toward the apex and is called the scar region, where most hair cells are absent and are replacedby a scar epithelium. The apical region of the cochlea is made of resistant hair cells and is called the resistance region. This last region is made up of twoareas: the basal end area appearing just above the scar region with exclusively OHCs. The second, called the extreme apical end area, includes OHCs and

Ž .IHCs, and is the most apical area of the resistance region. B–D Micrographs of surface preparations of an organ of Corti treated by neomycin andŽ .maintained in 1 mgrml of insulin and labeled by phalloidin. B The extreme apical end area of the resistance region from the apical turn of the cochlea

shows three to four rows of OHCs and one row of IHCs. Notice the length of the stereocilia and the disorganization of the stereocilia bundle that is typicalŽ .of apical hair cells after histological processing. This area is only visible when explants are treated with growth factors. C The basal end area from the

Ž .resistance region is localized below the extreme apical end area and is only made of OHCs. D The scar region corresponds to the former sensoryŽ .epithelium where most hair cells were destroyed by neomycin and are replaced by cells showing an apical polygonal shape brackets . Bars10 mm.


two types of hair cells were counted under light micro-scopic observations at =600.

Statistical analysis was assessed first by one way Stu-Ž .dent’s t-test followed by analysis of variance ANOVA .

Statistical differences are inferred with p value -0.01.

3. Results

3.1. The reactiÕity of the organ of Corti to neomycinototoxicity

Organotypic cochleas were treated for 2 days with 10y3

M of neomycin, and subsequently maintained in STA withthe tested molecules. Neomycin-treated organ of Cortipresented two main regions at all times: one was termedthe scar region, where few hair cells remained and whichsometimes showed stereocilia as described in hearing im-

w xpaired ears 10,29 . The former sensory epithelium wasreplaced by a scar epithelium made of supporting cell

w xexpansions 32 . This region was mainly restricted to thebasal organ of Corti, extending in some cases depending

Ž .on the culture conditions, toward the apical turn Fig. 1 .The second region was termed the resistance region, wherehair cells were still visible and showed a more or lessregular spatial organization that resembled normal sensory

epithelium with several rows of OHCs. This region wasdivided into a basal end area where only OHCs werepresent and an extreme apical end area where both types ofhair cells were visible. This last area was only visiblewhen growth factors were added to the culture mediumŽ .Fig. 1 . The resistance region could extend to the secondturn depending on the culture conditions. A transitionalregion was seen between these two regions, made of oneor two rows of hair cells ranging in length from 100 to 200mm. Because of its small size, the transitional region wasnot included either in the scar or the resistance regions forstatistical analysis. From the point of view of morphologi-cal criteria, some of these cells presented the same patho-logical features as those observed in the scar region.

Results were compared to control experiments. The firstŽ .main control was the standard control STA control corre-

sponding to cochlear explants maintained 10 days in vitroin STA, the second was the neomycin control made withexplants treated 2 days with neomycin and maintained for8 days in culture. Histology of the STA control revealednormal organization of the organ of Corti with one row ofIHCs and three rows of OHCs, although sometimes extra

w xrows of OHCs could be observed 1 .In the organ of Corti maintained with the FFM in STA

and treated with neomycin for 2 days, the resistance regionwas always localized in the apical part of the cochlea,

Fig. 2. Comparison of the length of the scar and the resistance regions from organ of Corti maintained in STA with two models of cultures, the FFM andthe SM methods, or supplemented with different molecules or growth factors after antibiotic treatment. The bar in black corresponds to the scar region withvery few hair cells left, the white bar is the resistance region including the apical end and the extreme apical end areas. The two methods of cultures tested:

Ž . Ž .the FFM 48 h—Neo and the SM support did not produce any statistical difference in the length of the resistance region. All other tests were performedwith the FFM after neomycin treatment. One can observe variations of the resistance region in relation with the molecules tested. The length of the

Ž .resistance region of the explants maintained with TGFa 50 ngrml with insulin is statistically different from all other tests. Bar: "1 S.D.


Ž .reaching a mean length of 530 mm, range: 300–800 mm ,Ž .Fig. 2 . There was no IHC in this resistance region andmost of these hair cells also had disappeared in the scarregion. For an entire cochlea, only two or three IHCs wereto be found. The density of OHCs was decreased by 20.2%in the resistance region and by up to 98.6% in the scarregion compared to the STA control without neomycin

Ž .treatment Fig. 3 . With a different culture method, thelength of the resistance region was not statistically modi-fied, although with the SM the mean length of this region

Ž .increased slightly to 660 mm, range: 250–1600 mm . Thecell density between the two culture methods was notstatistically different.

Depending on the molecules added to the culture media,the length of the resistance region can be modulated. Forinstance, by adding 10% FBS to the BFS, the length of theresistance region was almost tripled, reaching a mean

Ž .length of 1460 mm, Fig. 2 . The density of OHCs in theresistant region decreased by 20.3% compared to the STA

Fig. 3. Mean density of hair cells obtained from 3-day-old cochlearpartitions maintained for 10 days in vitro in the STA control, withoutneomycin treatment. The density is given for a 200 mm length reportedall over the organ of Corti from several cochlear partitions taken together.

Ž .From the neomycin control cochleas Neomycin control , two regions areobserved: the scar region with very few hair cells left, and the resistanceregion. With the neomycin control, the resistance area is only made ofOHCs, while very few hair cells are visible in the scar region. Tests with10% FBS were made with the BFS and antibiotic treatment for 2 days, nostatistical difference in hair cell density can be seen on the cochlearpartition compared to the neomycin control for both regions. Bar: "1S.D.

control. However, there was no statistical difference com-pared with the neomycin control. In the scar region, lessthan 0.5% of hair cells were left.

3.2. Tests with RA

Adding 10y8 M of RA to neomycin treated explantsincreased the length of the resistance region to a mean

Ž . Ž .length of 1350 mm, range: 1200–1600 mm , Fig. 2 .IHCs were present in the extreme apical part of theresistance region with a mean length of 370 mm, but witha decreased density of 55% compared to the standardcontrol. In the basal end of the resistance region, there wasno statistical difference of cell density compared to the

Ž .STA control and the neomycin control Figs. 3 and 4 . Themean number of hair cells was 4.4"3.9r200 mm in thescar region while a 94.6% decrease was observed with theSTA control. No hair cell regeneration was observed.Some of the few cells seen in this region show pathologi-cal features.

3.3. Tests with insulin

Neomycin treated explants supplemented with 1 mgrmlof insulin showed a mean length of the resistance region of

Ž . Ž .1060 mm, range: 600–1400 mm , Fig. 2 , while 40% ofIHCs were still present over a mean length of 250 mm,Ž .range: 200–400 mm . Only a mean of 4.0"4.7 haircellsr200 mm was present in the scar region representinga decrease of 95.6% compared to the STA control. Thedensity of OHCs in the resistance region did not differstatistically from the neomycin control, which was alsotrue for the 5 mgrml of insulin. Using a concentration of 5mgrml insulin increased the length of the resistance region

Ž .by between 1400 and 2000 mm, mean: 1680 mm , whilethe extreme apical area reached a length up to 600 mmŽ .mean 325 mm , although only 17.8% of IHC were left

Ž .compared to the STA control Figs. 3 and 4 . The numberof hair cells present in the scar region was similar to thesituation with 1 mgrml of insulin with a mean of 4.6"3.5hair cellsr200 mm.

3.4. Effects of growth factors on neomycin treated cochleas

Several growth factors alone or in combination withprevious molecules were tested in neomycin treated ex-plants in order to study their possible effects on hair cellregeneration.

( )3.5. Epidermal growth factor EGF

Three doses of EGF were tested, 5, 10, and 20 ngrml.The length of the resistance region whatever the dosesused was almost double compared to the neomycin controlŽ .data not shown . No statistical difference in hair cell


Fig. 4. Tests of RA and two doses of insulin. With RA, the OHC density is not statistically different, while only 45% of IHCs are still present in the apicalregion compared to the STA control. The most striking feature is the remaining IHCs in the apical region compared to the neomycin control. Tests withtwo doses of insulin, 1 and 5 mgrml gave approximately the same results which are not statistically different from the neomycin control, except someIHCs that are present in the resistance region, especially with 1 mgrml of insulin, and a few hair cells in the scar region. Bar: "1 S.D.

density was observed in the resistance nor in the scarŽ .regions compared to the neomycin control Fig. 5 , except

for the scar region with a 20 ngrml treatment, where inaverage less than one hair cellr200 mm was present.

The same experiment was carried out with the threedoses of EGF, adding 10% FBS in the BFS. As in theformer experiment with FBS alone, no IHC was present inthe resistance region. In this region, we found an increaseof 10.2% and 22.6% of the OHC density with 5 and 10ngrml of EGF, respectively compared to the neomycin

Ž .control supplemented with 10% FBS Figs. 3 and 5 . Therewas no difference with the 20 ngrml test. In the scarregion, the density of hair cells left was very low, andthere was no statistical difference in hair cell density for10 and 20 ngrml of EGF compared to neomycin and

Ž .neomycin plus 10% FBS controls Figs. 3 and 5 .Replacing the FBS in the culture medium by 1 mgrml

of insulin with the same experimental conditions as beforeproduced a resistance region of 1050 mm mean lengthŽ .range: 800–1200 mm , with a restricted presence of IHCsŽ .Fig. 2 . Insulin did not produce a statistical difference inthe hair cell density in the resistance region whatever the

concentration of EGF compared to the neomycin controlŽ .with 1 mgrml insulin Figs. 4 and 5 . By adding 5 mgrml

of insulin, we obtained a significant decrease of up to28.3% in OHC density in the resistance region, comparedto the neomycin control supplemented with 5 mgrml of

Ž .insulin alone data not shown . The density of hair cells inthe scar region did not present any statistical difference.

( )3.6. Transforming growth factor alpha TGFa

Organotypic cochlear cultures treated as usual withneomycin for 2 days were tested with three doses ofTGFa: 10, 25 and 50 ngrml supplemented with 10y8 Mof RA or 1 mgrml of insulin. TGFa tested alone at 10ngrml presented a resistance region similar to neomycin

Ž .control data not shown , with no hair cell regeneration inthe scar area.

3.7. Tests with RA

There was no statistical difference in the length of theresistance region compared to the neomycin control with


Fig. 5. Tests with EGF in combination with FBS and insulin. There is no effect of EGF on the hair cell density in the scar region suggesting that thismolecule does not promote regeneration whatever the combination with FBS or insulin. For the resistance region, tests with EGF alone did not produce astatistically significant modification of OHC density compared to the neomycin control, although a large variability was observed between cochleas as seenby the large standard deviation. Bar: "1 S.D. By adding 10% FBS to the feeding solution, the OHC density reached a value close to the STA control, then

Ž .higher than with FBS alone see Fig. 3 . With insulin and 10 ngrml of EGF, IHCs are quite well-preserved in the apical region, but there is no statisticalŽ .difference with the neomycin control plus 1 mgrml of insulin see Fig. 4 , almost no hair cells are present in the scar region. Bar: "1 S.D.

Ž .RA data not shown . In the extreme apical end of theresistance region, no difference in IHC density was ob-served, whereas an important drop was observed for theOHCs which reached 33.2% with 50 ngrml of TGFa

compared with the neomycin control supplemented withŽ .RA only Figs. 4 and 6 . In the resistance region, the

density of OHCs showed a statistical difference betweencontrol explants with RA alone and with 50 ngrml ofTGFa . Very few hair cells were present in the scar region.

3.8. Tests with insulin

The most obvious observation with 1 mgrml insulinand TGFa was a considerable increase in the length of theresistance region of the neomycin treated explant whichshowed a mean length of 2450 mm with 10 ngrml ofTGFa , 2520 mm with 25 ngrml and 2800 mm with 50

Ž .ngrml, that extended toward the second turn Fig. 2 . So itwould appear that there is a dose relationship between theincrease in the resistance region and the amount of TGFa .Moreover, the extreme apical end of the resistance region

showed 52.5% IHC remaining when 25 ngrml of TGFa

was added to the culture medium and up to 86.6% with 50Žngrml of TGFa compared to the STA control Figs. 3 and

.6 . In this area, there was no statistical modification in theOHC density compared to the neomycin control supple-mented with 1 mgrml insulin. No cell density modifica-tion was observed in the basal end of the resistance regionwhatever the dose of TGFa used, whereas there seemed tobe a drop in hair cells in the scar region 50 ngrml TGFa ,

Ž .but this was not statistically significant Figs. 4 and 6 .

( )3.9. Transforming growth factor beta TGFb

Ž .Three doses of TGFb 1.0, 2.5 and 5.0 ngrml weretested on cochlear explants after these had been exposed toneomycin and maintained in the STA supplemented with 1mgrml of insulin.

The length of the resistance region did not differ statis-tically with different doses and remained stable around a

Ž .mean length of 1000 mm Fig. 2 . The IHC density of theresistance region was stable whatever the doses of TGFb


Fig. 6. Tests with TGFa and TGFb. The outcome of a mixture of 50 ngrml of TGFa with RA added to the STA shows a statistical significant decrease inŽ .OHC density of the resistance region compared with the neomycin control supplemented with RA alone see Fig. 4 . No hair cells are present in the scar

region. Tests performed with two doses of TGFa and insulin do not show any statistical difference in OHC density compared with the neomycin controlŽ .supplemented with insulin see Fig. 4 . The main observation is a large percentage of IHCs left in the resistance region which is close to the STA control.

No regeneration of hair cells is present in the scar region, although few hair cells are present. TGFb added to the culture medium with 1 mgrml of insulindid not produce any effect on hair cell regeneration in the scar region. An increase of OHC density is observed in the extreme apical end area of the

Ž .resistance region compared to the neomycin control with insulin see Fig. 4 . Bar: "1 S.D.

used. However, contradictory results were obtained for theOHC density of the extreme apical end. An increase of35.5% of hair cells was observed at 1 ngrml of TGFb,while a decrease in hair cell density respectively of 25.5and 34.9% was seen at 2.5 and 5 ngrml, compared to theneomycin control with insulin. There was no modificationof hair cell density in the basal end with 1 and 2.5 ngrmlof TGFb, nor was any modification in hair cell densityobserved either in the scar region compared to the neomycin

Ž .control with insulin Figs. 4 and 6 .

4. Discussion

Our in vitro results correspond well to previous in vitroobservations where hair cells from the base were moresensitive to neomycin ototoxicity than hair cells from the

w xapical region 33 , and the same observation holds for thew xchick in vivo 38 . This leads to the distinction of two

regions in the organ of Corti after neomycin treatment: a

scar region where most of the sensory cells disappear, andthe resistance region where many hair cells remain present.It has been shown in young rats that both in vivo and invitro cochleas showed a spontaneous neodifferentiation ofhair cell-like cells in the scar epithelium after ototoxicdamage. Unfortunately, these cells failed to develop into

w xhair cells after a few weeks 47,62 . It was thought that thefailure of these cells to reach full development was due toa lack of growth factors. Adding these molecules to thefeeding solutions could initiate regeneration in a controlledenvironment, such as in our cochlear organotypic cultures,and boost regeneration of hair cells. Several growth factorsare known to be present during development of the inner

Ž w x.ear for review see Refs. 19,21,22 , and some of themcould be potential candidates.

It has been shown by qualitative observations aftermechanical injury of the apical pole of hair cells, that

w xpersistent cell bodies could lead to hair cell repair 72 . Sowe may ask the following question: does observation ofthe apical surface of hair cells by phalloidin staining give a


good estimation of hair cell loss? However, observationsof cross-sections of the organ of Corti from similar in vitroexperiments after antibiotic treatment have shown degener-

w xation of hair cells 50,59 . Moreover, other observations inyoung rats have demonstrated that hair cells undergo apop-

w xtosis after antibiotic intoxication 79 . From our observa-Žtions even 2 weeks after the antibiotic treatment personal

.observation , we observed no hair cell repair, as these cellsshould present a typical immature stereocilia bundle. Thisis not to say that no such repair phenomenon occurs inyoung cochlea in vitro, but we believe its incidence mustbe very limited.

4.1. Regeneration and RA

One of the first molecules tested was RA as initiated byw xa study of Kelley et al. 41 on the overproduction of hair

cells in neonatal mouse cochlea in vitro. Although it wasclaimed that RA induced regeneration of hair cells in vitro

w xafter neomycin treatment in rats 46 , this claim is notw xconfirmed by the present study and a previous one 15 ,

when RA failed to produce new hair cells in the scarŽ .region Fig. 3 even in combination with TGFa . The only

effect that could be related to RA in the neomycin treatedcochlea in vitro was maybe a protective effect on apicalhair cells as observed by an increase of the length of theresistance region.

RA is the most active metabolite of retinol and isindeed present in the embryo with pleotropic actions on

w xvarious tissues 14 depending of nuclear receptor combi-w xnations 18 . A well-documented effect of RA is its mor-

phogenetic action during embryonic developmentw x18,26,52 . For instance, it has been shown that applicationof exogenous RA to zebra fish during the initial stages ofphotoreceptor differentiation results in a precocious devel-opment of rod photoreceptors and an inhibition of conephotoreceptor maturation, but does not initiate cell divisionw x40 . RA receptor gene transcripts are indeed present in the

w xcochlea during early and late embryogenesis 49,63 . Thus,it is possible to imagine that RA is highly implicated in themorphogenesis of the inner ear and maybe in hair cell

w xdifferentiation 41,58,63 , but this is less obvious for haircell regeneration. However, it has been suggested that thepresence of cellular retinoid binding proteins in rodentneonatal cochleas could promote hair cell regenerationw x41,88 . Adult cochlear phenotype observation of inacti-vated genes of cellular retinoid binding proteins by homol-ogous recombination showed functional and morphological

Ž .normal hair cells unpublished observations . This observa-tion suggests that the cellular retinoid binding proteinsmay not be very important during cochlear development,and thus also during regeneration. However, RA mightpresent a protective effect on apical hair cells againstneomycin ototoxicity through nuclear retinoid receptors asshown by the increase of the length of the resistance

region when RA is added in the culture medium. However,more experiments are needed to explore this hypothesis.

4.2. Insulin

Insulin is involved in the regulation of insulin stockingand plays a key role in growth and cell differentiation.This cellular effect takes place through one membranereceptor which is involved in amino acid transport, lipidmetabolism, DNA synthesis, various protein synthesismechanisms related to metabolism, and cellular growth in

w x Ž .various tissues 17 . Insulin-like growth factor IGF tran-scripts have been found in the cochlear sensory epithelium

w xduring inner ear ontogenesis 6 , and it stimulates prolifera-tion of the vestibular epithelial cells during developmentw x53,91 .

Insulin added to the culture medium increased the lengthof the resistance region up to around three times comparedto the neomycin control. This may suggest a protectiveaction on the treated cochlea that is dose dependent,because with a higher dose, the resistance region increasesin size. This protective effect of insulin could be mediatedthrough the phosphoinositide pathway that is used by bothmolecules. The toxicity of aminoglycosides may be relatedto their capacity to inhibit some enzymes of this pathwayw x7,43,80 . The involvement of insulin in the resistance ofhair cells from the apical region of the cochlea could berelated to its capacity to limit the accumulation of phos-phoinositides due to aminoglycosides.

Recently, it has been proposed that the ototoxic effectsof gentamicin are related to the ability of anironrgentamicin complex to catalyze free radical reactionw x55 . Similar phenomena have been postulated for neuronaldamage in ischemia, alongside sustained elevation of cal-cium levels, where iron may play a central role in thegeneration of the radicals by initiating lipid peroxidation

w xreactions in membranes 31 . The findings that administra-tion of hydroxyl radical scavengers or iron chelatorsrbind-ing proteins is effective in reducing post-ischemic tissue

w xdysfunction 27,71 , and that they attenuate gentamicinw xototoxicity 73 tend to support this hypothesis. It has been

shown that several growth factors, including IGF, protectw xneurons against iron-induced degeneration 89 . It may be

possible that insulin and related molecules produce thesame protective effect on the inner ear through the samemechanisms.

4.3. EGF and TGFa

EGF and TGFa are two molecules from the family ofepidermal growth factors. They both interact with the same

w xhigh affinity receptor 11,30 . These growth factors areknown to be involved in proliferation, migration and dif-

w xferentiation of epithelial and mesenchymal cells 5 . TGFa

w xwas detected in the mouse otic vesicle 85 and stimulatesthe proliferation of the mouse vestibular sensory epithe-


w x w xlium during development 91 , as well as in the adult 87 .EGF alone or with insulin did not induce hair cell regener-ation nor protection against neomycin, although it pre-sented an increase of cell density in the resistance region

Ž .in combination with FBS Fig. 5 . TGFa alone did notshow any greater effect on hair cell regeneration in thebasal cochlear region and protection for the apical ones.

With RA, TGFa did not increase the length of theresistance region nor hair cell regeneration in the scar area,which confirms the previous observation that RA did not

w xinitiate nor potentiate hair cell regeneration 15 . Despitemany trials with different doses of TGFa with RA andinsulin, our observations do not agree with those previ-

w xously published 75,92 . These authors reported an impor-tant mitosis response in the scar cochlear sensory epithe-lium after ototoxic treatment when TGFa and RA wereadded together to the culture medium. These results could

w xbe related to reported data by Lambert 44 in the adultmouse vestibular epithelium treated by TGFa afterneomycin intoxication, where some instances of mitosisrelated to hair cells were observed, although much lessnumerous and temporally restricted. However, in this lastexperiment, FBS was added to the culture medium whichcould have produced complex and unknown interactions.Hair cell regeneration in the mammalian vestibular systemis not surprising since it has been demonstrated by several

w xreports 34,66,81 , and it is possible that specific growthw xfactors could trigger hair cell regeneration 44,87,91 . In

the cochlea, no hair cell regeneration has been observed tow xdate in the scar region after ototoxic damage 15,61 , and

this is confirmed by the present study.An explanation can be put forward concerning these

conflicting results. No precise information was given abouthair cell distribution on the organ of Corti after neomycin

w xtreatment in these two reports 46,74 . We have shown thatthe combination of TGFa with insulin results in the lengthof the resistance region being promoted to an unusual sizeŽ .Fig. 2 . Hair cells from this very well-developed resis-tance region can extend to the second turn and could havebeen mistaken for regenerating hair cells. This is particu-larly striking with the experiment of Zine and de Rib-

w xaupierre 92 , where younger rat pups were used for theirŽ .experiments 1–3 days postnatal . In fact, we have ob-

served that the length of the resistance area depends on theage of the animal, and rats younger than 3 days after birthtend to produce a much larger resistance area after antibi-

Ž .otic treatment unpublished data . Two observations sup-port this hypothesis. The first comes from a picture pub-

w xlished by Staecker et al. 74 as being regenerated hair cellsbut that look like apical hair cells; the second comes from

w xthe same authors 75 , where they described that a highdose of TGFa with insulin has a protective effect on thecochlear hair cells after neomycin treatment. This lastobservation could correspond to what we have seen with acombination of a 10-fold inferior dose of TGFa , whichinitiates a very large resistance region. Their second article

w x75 describing the protective effect of TGFa is in contra-diction with the first one related to the regenerative effectw x74 , although the most recent conclusion seems to be morerealistic and corresponds well to our observation.

4.4. The protectiÕe effect of TGFa and insulin

In the present study, we show that TGFa alone did nothave much effect on hair cell regeneration and protectionagainst neomycin, while with insulin at different dosesconspicuous hair cell protection was provided at the apical

Ž .turn, that could extend basal-ward, Fig. 2 . Moreover,insulin alone increased the length of the resistance regionsuggesting that TGFa acts synergically with insulin andenhances the protective action against the long-term dam-aging outcome of aminoglycosides. It may be possible tohypothesize that if insulin and TGFa were present in thedish during the antibiotic treatment, the protective effectwould have been more important. In the mouse utricle,insulin is known to potentiate the effect of TGFa and act

w xin synergy to stimulate cell proliferation 87 . The protec-tive effect described could be mediated throughEGFrTGFa receptor via common pathways of insulin andTGFa through protein kinases such as the mitogen-

w xactivated protein kinase 11,17 .Although emphasis on the role of TGFa concerned

w xotoprotection of hair cells 75 and hair cell replacementw x92 , both culture experiments used media supplementedwith larger doses of insulin. However, we have shown thatTGFa alone did not enhance hair cell resistance afterantibiotic treatment. The observations by Staecker et al.w x75 on hair cell resistance corresponded exactly to ourresults because they used a cocktail of molecules in theirfeeding solution including insulin. It could be possible thatthis resistance andror replacement phenomena is mediated

Ž .by insulin or insulin-like growth factor IGF receptors.Insulin exerts its activity through a surface receptor butmay preset IGF-like activity through the type 1 IGF recep-

w xtor 35 . For example, it has been shown that insulin’s roleas trophic agent in the chick embryo brain cells is medi-

w xated by IGF-I receptor 9 . The insulin and IGF-I receptorsmediate their intracellular effects through a tyrosine kinaseactivity which leads to phosphorylation of endogenoussubstrates. Beside its pleotropic actions, recent findingsindicate that the IGF system has a protective effect on

w xneuronal cell death and other cell types 76 . This mecha-nism of action of IGF-I is still poorly understood, but it istempting to speculate that this growth factor, like the nerve

w xgrowth factor, might prevent apoptosis 4,76 , togetherwith the detrimental effects of free radicals as previouslydiscussed.

Another intracellular pathway that is common to theaction of aminoglycosides in the inner ear and TGFa mayalso be involved, the polyphosphoinositide pathwayw x43,45,70 . The effect of insulin in the resistance of hair


cells after antibiotic treatment could involve activation ofphosphoinositide 3-kinase that would reduce the effects ofsome phosphoinositides produced by antibiotics. Whateverthe exact cellular pathway involved in the hair cell resis-tance is, it seems obvious that the presence of insulin andTGF promote a high level of rescue of hair cells of youngmammalian cochlea in vitro.

4.5. Transforming growth factor beta

TGFb belongs to a super-family made up of tens ofw xmolecules 64 . Four members belong to the TGFb sub-

family that is involved in a wide variety of cellular pro-cesses such as growth and cellular differentiation, morpho-

w xgenesis and cicatrisation 2,54 . Its action is mediatedw xthrough two membrane receptors 86 . TGFb is expressed

w xin the stato-acoustic receptors during ontogenesis 51 .Whatever the doses used, we did not observe any

change in the length of the resistance region nor any haircell regeneration in the scar region. In the resistanceregion, there was no change in the IHC density, while anincrease of 25 to 30% of OHC density could be seen.Thus, it seems that this growth factor did not have anygreat effect on the size of the resistance region, although itdid have a protective effect on OHCs, but without anyregeneration after neomycin treatment. It had been shownpreviously that TGFb could promote overproduction of

w xhair cells in vitro 16 .In conclusion, one can say that none of the molecules

and growth factors tested promoted any cell differentiationfrom the spontaneous attempt of cell neodifferentiationobserved in the organ of Corti in vitro after ototoxic

w xdamage 62 . Moreover, this study shows that no regenera-tion of hair cells in our culture conditions was initiated bythe molecules tested. However, we were able to observethat some molecules provided a relative protection of haircells from the apical part of the cochlea after antibiotictreatment, while the association of TGFa and insulinproduced a significant protective effect related to haircells.

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effects of growth factors on the hair cells after ototoxic treatment of the neonatal mammalian...

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