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Growth factor therapy to the damaged inner ear: clinical propects
International Journal of Pediatric Otorhinolaryngology 49 Suppl.1 (1999) S19 – S25 www.elsevier.com/locate/ijporl
Growth factor therapy to the damaged inner ear: clinical propects Brigitte Malgrange a, Jean-Michel Rigo a, Thomas R. Van de Water c,d, Hinrich Staecker d,e, Gustave Moonen a, Philippe P. Lefebvre a,b,d,* a
Department of Human Physiology and Pathophysiology, Place Delcour 17, Uni6ersity of Lie`ge, 4020 Lie`ge, Belgium b Department of Otorhinolaryngology, Uni6ersity of Lie`ge, Lie`ge, Belgium c Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA d Department of Otolaryngology, Albert Einstein College of Medicine, Bronx, NY, USA e Massachussetts Eye and Ear Infirmary, Har6ard Medical School, Boston, MA, USA
Abstract Most hearing loss results from lesions of the sensory cells and/or of the neurons of the auditory part of the inner ear. There is currently no treatment able to stop the progression of a hearing loss or to restore a lost auditory function. In this paper, we review the progress which has been made with respect to the regeneration and the protection of the hair cells and of the auditory neurons in the cochlea. In particular, we emphasize the control by growth factors of the protection/repair mechanisms of the neurosensory structures within the inner ear, in the prospect of the possible clinical use of these molecules. Finally, we discuss the different approaches which can be used to deliver these therapeutic agents to the inner ear. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cell culture; Cochlea; Hearing impairment; Inner ear; Ototoxicity
1. Introduction The frequency of hearing impairment diagnosed by audiometry increases with age and affects nearly half of the population by age 80 years. At least 30% of persons 65 years of age or older have a hearing loss of 50 dB at 4000 Hz, sufficient to interfere substantially with the understanding of speech. Most deafness is of sensorineural origin and is characterized by a loss of hair cells and of * Corresponding author.. E-mail address: [email protected] (P.P. Lefebvre)
spiral ganglion neurons. At the present time, hearing aids are the only satisfactory means available for the hearing impairment. Because of the lack of treatment of neurosensory deafness capable of restoring auditory function or altering the course of a progressive hearing loss, we have developed experimental strategies to approach the treatment of neurosensory deafness: (1) otoprotection, which is designed to prevent a further degradation of auditory function; and (2) regeneration, which is defined as the replacement of lost hair cells in the deafened ear and their reconnection by primary auditory neurons to the central auditory pathway.
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2. Growth factors Very much as acetylcholine is for neurotransmitters, nerve growth factor (NGF) is the prototypic neurotrophic factor. Initially, these molecules were considered as target-derived molecules that were the molecular substratum responsible for the matching between the size of a given neuronal pool and the volume of its innervation territory. Therefore a classical neurotrophic factor is produced within a given tissue at an appropriate time during embryonic development and is able to support the viability of the innervating neurons by interacting with specific receptors (e.g. NGF/TRKA). The spectrum of biological effects of such factors on responding neurons goes well beyond a nerve survival promoting effect. Other effects can include stimulation of neuritic outgrowth, chemotropism (i.e. being able to steer the orientation of neuronal projections), effect on cell adhesion molecule expression, release of proteases/protease inhibitors, commitment to a specific neurotransmitter phenotype. Slowly the neurotrophic factors were considered not only as factors important during the development of a given neuronal population but also to be essential for the maintenance of adult neurons, to protect specific populations of neurons against various insults and if damage occurs to stimulate their regeneration and repair. ‘Factorology’ has thus entered the field of therapeutics. The list of factors endowed with neurotrophic properties has been slowly growing. Some of these factors are primarily derived from neuroscience research and have been characterized either by protein purification monitored using some sort of bioassay for their trophic actions (e.g. NGF, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and glial-derived neurotrophic factor (GDNF)), or directly by molecular biological techniques using polymerase chain reaction (PCR) primers based upon the conserved sequences of previously known factors of a given family (e.g. neurotrophins NT-3, NT-4/ 5, NT-6). Other factors purified using classical protein chemistry methods and bioassay monitoring turned out after sequencing to be identical
with known factors that had already been characterized based on their biological effects on nonneuronal cells (e.g. fibroblast growth factor-1 (FGF1) fibroblasts). Finally, other factors ‘became neurotrophic’ because an effect on nerve cells was discovered after their purification, sequencing and cloning for their effects in non-nervous tissues (e.g. epidermal growth factor (EGF), tumor growth factor-a (TGFa) and other members of the neuroleukin family).
3. Mammalian hair cell regeneration/repair and growth factors Auditory hair cells are the sensory receptor cells that transduce a sound stimulus into a bioelectric signal. Auditory hair cells can be readily damaged and lost after exposure to noxious agents, including noise and ototoxic drugs such as aminoglycosides. In cold blooded animals, where sensory hair cells are continuously produced throughout life, the damaged sensory epithlium is repaired by a spontaneous hair regeneration process [1]. Until recently, regeneration of auditory hair cells was considered to be restricted to lower vertebrates since the auditory hair cells that populate avian and mammalian inner ears are produced only during embryogenesis. Therefore, sensorineural hearing loss due to damage or loss of hair cells was considered to be irreversible. The capacity of spontaneous postembryonic repair of neurosensory epithelium was demonstrated following acoustic overstimulation and ototoxic drug exposure in the neonatal chick basilar papilla [2,3]. The ability of ongoing production of new hair cells was observed in the vestibular receptors of adult epithelia of birds [4]. The factors which control these generative and regenerative processes are not well known. Recently, it has been showed that insulin growth factor 1 (IGF1) and insulin induce proliferation of progenitor cells in the vestibular portion of the bird inner ear [5]. In the auditory portion of the avian inner ear, basic fibroblast growth factor (bFGF or FGF1) is thought to play a role in the regenerative processes after a lesion. This discovery of a capacity to produce new hair cells in birds led to the
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question of how to extend this repair process to the mammalian labyrinth (Fig. 1). In mammals, the production of hair cell stops at birth in the organ of Corti as well as in the vestibule [6]. In the macula of the utricle, the formation of new hair cells has been demonstrated following a lesion by an aminoglycoside [7,8]. This renewal of vestibular hair cells was also demonstrated in vivo in the adult guinea pig vestibule and is stimulated by the perilymphatic perfusion of a combination of retinoic acid, TGFa and IGF1 [9]. Recently, the stimulation of the proliferation of epithelial cells from utricles in culture has been observed using various growth factors (bFGF, FGF4, FGF6, FGF7, IGF1, IGF2, TGFb and EGF [10]), suggesting the involvement of growth factors in the regenerative processes of inner ear sensory epithelium. In the cochlea, the production of new hair cells and the repair of the sensory epithelium seems to be limited to the embryonic and perinatal periods. Retinoic acid was showed to induce the formation of supernumerary hair cells in organ of Corti explants from mouse em-
Fig. 1. Mechanisms of hair cell renewal in the sensory epithelia of the inner ear. When hair cells are lesioned, they can either undergo an apoptotic cell death or be injured and survive the insult. In the first case, the supporting cell may proliferate and a daughter cell differentiate into a new hair cell (A, regeneration mechanism). Alternatively, if no proliferation occurs, a supporting cell may differentiate into a hair cell (B, transdifferentiation mechanism). In the second case, where hair cells survive and are just injured, they may regenerate a new apex with its stereocilia bundle and cuticular plate (C, repair mechanism).
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bryos between E13 and E16 [11]. However, at E18, organ of Corti sensory hair cells killed by exposure to a laser beam could still be replaced with new hair cells [12]. This replacement of hair cells is obtained through a differentiation of preexisting cells in the sensory epithelium to form new replacement hair cells. If this experiment is performed on cochlear explants from newborn mice there is no replacement of lost hair cells. Using an other experimental paradigm, Romand et al. recently showed that EGF or TGFb1 induce the formation of supernumemary hair cells at the level of the organ of Corti in the absence of any lesion [13]. Sobkowicz et al. demonstrated a continuous capability of self-repair of damaged hair cells in the mammalian organ of Corti. Using a micropipette, the auditory sensory epithelium was lesioned. A recovery of the hair cells was observed after delay between 4 h and 7 days with a ‘regeneration’ of the cuticular plate and the production of a new stereociliar bundle [14]. Using explants of Corti’s organ from 3-day-old rat pups, we demonstrated a renewal of hair cells after aminoglycoside treatment of the explant which allowed us to kill almost all of the hair cells [15,16]. This regeneration/repair was observed in the presence of the TGFa and retinoic acid. The regenerated/repaired hair cells had an immature aspect characterized by the presence of long microvillar at the apex. The regeneration/repair of the hair cells in cultured explants from neonatal rats has been controversial. Using another experimental protocol, these results were not reproduced using a different culture technique [17]. Recently, experiments confirming the replacement of hair cells after a lesion of the organ of Corti with an aminoglycoside has been reported [18]. Analysis using a scanning electron microscope confirmed the immature characteristics of the ‘regenerated’ hair cells. The mechanism responsible for the renewal of hair cells in the lesioned postnatal organ of Corti explants is still unknown. The occurrence of immature hair cells (i.e. atypical cells) has also been shown to transiently occur at the lesion margin in the apical region of the cochlear of young rats treated with amykacin between postnatal days 9 and 16 [19]. These results suggest that some regenerative ca-
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pacity exists but is very limited in the absence of any specific intervention. At present there has not been any demonstration of a regenerative capacity of the hair cells in Corti’s organ of any adult mammal.
4. Mammalian neuronal regeneration and maintenance and growth factors There has been ample evidence that both central and peripheral target tissues act to support the development of auditory neurons [20]. In adult animals, damage to Corti’s organ is followed by a secondary degeneration of the auditory neurons of the spiral ganglion, suggesting that hair cells are one of the sources of trophic factors to support the survival of these neurons [21]. In the cochlea of the adult rat, transcripts for NT-3 have been localized to the inner hair cells only [22]. The same study localized mRNA for TRKB and TRKC (the high affinity receptors for BDNF and NT-3, respectively) to the auditory neurons, suggesting that both NT-3 and BDNF play roles in maintenance of these neurons. The study of cultured spiral ganglion neurons has provided additional information regarding the growth factors which support the survival and repair of injured auditory neurons. Dissociated cultures of the spiral ganglion provide a simple method to study injured auditory neurons which have undergone a double axotomy in the dissociation process and a disruption in the normal cellcell interactions with the non-neuronal cells [23]. Therefore the neurons in the dissociated cultures are deprived of trophic support from both central and peripheral target tissue sources as well as their normal cell–cell interactions. The effect of neurotrophic growth factors was assessed on cultures of dissociated mature spiral ganglion neurons. When FGF2 was bound to substrate in the culture dish, it was found to be a strong promoter of neuronal survival. NGF while unable to promote neuron survival when used alone was found when combined with substrate bound bFGF to be a potent initiator of neurite outgrowth [24]. Dissociated cell cultures of the adult rat spiral ganglion treated with either exogenous BDNF or NT-3
demonstrated that these two neurotrophins were equally effective in promoting the survival of the neurons [25]. Glia-derived neurotrophic factor GDNF was also found to promote the survival of adult auditory neurons in culture [26]. Modiolus spiral ganglion explants from adult rats that closely mirror cell–cell interactions and in situ tissue relationships within this ganglion provide an excellent model for testing neurotrophic factors. In this modiolus explant system, NT-3 is the strongest individual promoter of survival while CNTF with NT-3 was the most effective combination of a cytokine with a neurotrophin for promoting survival of auditory neurons [27]. The in vitro experiments allowed us to determine which of the growth factors are good candidates to be used as therapeutic agents for growth factor therapy in the inner ear. From our data [25,27], neurotrophins were selected (in particular BDNF and NT-3) because of their strong survival promoting effects. When hair cells are destroyed by exposure to ototoxins or noise trauma, a source of growth factor support is lost and auditory neurons undergo apoptotic cell death induced by a lack of trophic support as described in clinical and animal models. In our in vivo study, guinea pigs were exposed to the ototoxic combination of an aminoglycoside antibiotic with a loop diuretic and then received 8 weeks of intracochlear perfusion of either NT3, BDNF or NT3 +BDNF to determine whether site specific application of these neurotophins could prevent the loss of auditory neurons that is known to follow a loss of auditory hair cells [28]. Infusion of either NT-3 or NT-3 + BDNF into the scala tympani resulted in a \ 90% survival of auditory neurons while BDNF infusion yielded a 78% survival rate, compared with a 14–24% neuronal survival rate in untreated ototoxin exposed cochleae (Fig. 2). These results show that loss of auditory neurons that occurs subsequent to a loss of auditory hair cells can be prevented by in vivo neurotrophic therapy with either BDNF or NT-3. In a similar experimental paradigm, GDNF was also found to support neuronal survival in the spiral ganglia after an ototoxic destruction of the organ of Corti in adult guinea pig.
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Fig. 2. Growth factor treatment prevents the degeneration of auditory neurons in the spiral ganglion deprived from their peripheral target territory (i.e. the hair cells of the organ of Corti). (A) Control condition. (B) When adult guinea pig are treated with an aminoglycoside, hair cells disappear in the organ of Corti and a secondary neuronal cell death is observed within the following weeks in the spiral ganglion. (C) If a neurotrophic factor (i.e. BDNF of NT-3) is perfused in the inner ear using a miniosmotic pump, 90% of the auditory neurons are maintained alive.
5. Clinical prospect: is there a future for an application of growth factor therapy for the treatment of inner ear disorders? With the identification of the actions of growth factors such as BDNF, NT-3, CNTF, TGFa, retinoic acid and IGF1, there is the possibility of pharmacological intervention for the treatment of neurosensory disorders of the inner ear. Experimentation using animal models of neurosensory disorders of the inner ear will help to establish which of these growth factors either individually or in combination will be effective in vivo in: (1) preventing hair cell loss; (2) neuronal degeneration; (3) promoting hair cell renewal and repair; (4) neuronal repair; and (5) reestablishment of peripheral innervation via evoking neoneuritogenesis. The clinical goal of identifying the mechanisms that will evoke hair cell regeneration-repair are ambitious with an ultimate goal being the
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restoration of hearing in deaf patients and balance in patients with vestibular disorders through the replacement of lost hair cells and the reestablishment of their neuronal connections to the appropriate pathway within the central nervous system. However, many questions must be addressed prior to applying these concepts to the pharmacological treatment of patients in the clinic. An initial road block to progress into the clinical arena has been a failure to demonstrate that the process of hair cell regeneration occurs in the mature cochlea of a mammal. Neuronal injury is a probable cause of most sudden hearing loss which demonstrates spontaneous improvement over time. This type of spontaneously recovering sudden hearing loss represents about 60% of cases seen in the clinic and can be predicted by the presence of otoacoustic emission [29,30]. Recovery is most probably due to repair and reestablishment of synaptic connections to the hair cells [31,32]. Growth factor therapy should improve the level of recovery through the stimulation of the repair of damaged neuronal processes below the auditory hair cells. Growth factor therapy will also be applicable to the treatment of other inner ear disorders. Indeed, some in vitro studies reported by Zheng et al. using dissociated cell cultures of 5-day-old rat spiral ganglia demonstrate neuroprotective effects of two neurotrophins from cisplatin neurotoxicity. Both BDNF and NT-4/5 provided good neuroprotection of auditory neurons from cisplatin induced damage[33]. Furthermore, GDNF was found to prevent cisplatin damage of adult auditory neurons in culture. This factor has also ben suggested to have some otoprotective activity on hair cells induced toxicity against cisplatin and against neuronal damage in adult guinea pigs [26]. This study demonstrates the therapeutic potential of neurotrophins and GDNF for not only preventing trauma-induced hair cell degeneration but also for the protection of auditory neurons. Therefore, neurotrophins could be used as therapeutic agent to prevent the ototoxic damage of cisplatin on both the neurons of the spiral ganglion and the auditory hair cells. Cochlear implants provide a therapeutic option for patients with profound deafness. This device is implanted into the scala tympani and is able to
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directly stimulate spiral ganglion neurons located within the bony modiolus. One of the major determinants of post-implant hearing perception is the total number of viable auditory neurons available for electric stimulation. Conceivably a mechanism for the direct delivery of neurotrophins into the scala tympani could be added to the cochlear implant for either periodic or chronic infusion of these neurotrophic molecules to prevent ongoing neuronal degeneration secondary to the original insult or subsequent to the trauma of the implant procedure[34].
other growth factors to the inner ear are already being studied. Some success has been obtained with the infusion of BDNF and NT-3 into the scala tympani of deafened guinea pigs using osmotic minipumps. Implantable polymers, implantable genetically engineered cells and introduction of viral gene vectors which express genes for BDNF or NT-3 are also potential methods by which neurotrophins could be delivered to the inner ear on a chronic basis.
Acknowledgements 6. Application of growth factors for the treatment of cochlear injury Because of the blood labyrinthine barrier, growth factors may have difficulty reaching inner ear tissues. However, since the neuronal axons and synapses are in communication with the perilymphatic fluid of the scala tympani space this route affords a means of growth factor delivery. The inner ear is somewhat unique in being isolated from the rest of the body compartments by a bony protective capsule except through the endolymphatic duct and sac and the cochlear aqueduct. Therefore the inner ear can potentially be treated in its own micro-environment. Methods of delivery of neurotrophic molecules into the perilymphatic fluid of the labyrinth could include introducing growth factor directly into the middle ear cavity with diffusion or transport occurring across the round window membrane or via direct infusion of the factor through openings into the labyrinth itself. Binding growth factors to a carrier molecule, which is transported across the endothelial cells of capillaries is another potential mode of growth factor delivery. While some deafening results from acute hair cell and neuronal damage many other causes of sensorineural hearing loss are associated with a gradual loss of hair cells and/or neurons. In the latter case, or in instances where hair cells are permanently missing, the chronic infusion of growth factors may be required to support the health and viability of the neuronal population of the spiral ganglion. Approaches to chronic delivery of neurotrophins and
This work was supported by the Fonds National de la Recherche Scientifique (FNRS), the Concerted Action of the French Community of Belgium, by the Fondation Me´dicale Reine Elisabeth (FMRE) and the Shulsky Foundation. We thank P. Ernst, M. Wouters and A. Broze for their technical support and expertise.
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B. Malgrange et al. / Int. J. Pediatr. Otorhinolaryngol. 49 (1999) S19 – S25 [9] R. Kopke, P. Garcia, R. Gabaizadeh, J. Feghali, W. Liu, P.P. Lefebvre, T.R. Van De Water, In vivo treatment with TGFa/IGF-1/retinoic acid mixture increases hair cell regeneration/repair in guinea pig utricule, Assoc. Res. Otolaryngol. 789 (1996) 198–198. [10] J.L. Zheng, C. Helbig, W.Q. Gao, Induction of cell proliferation by fibroblast and insulin-like growth factors in pure rat inner ear epithelial cell cultures, J. Neurosci. 17 (1997) 216 – 226. [11] M.W. Kelley, X.M. Xu, M.A. Wagner, M.E. Warchol, J.T. Corwin, The developing organ of Corti contains retinoic acid and forms supernumerary hair cells in response to exogenous retinoic acid in culture, Development 119 (1993) 1041 – 1053. [12] M.W. Kelley, D.R. Talreja, J.T. Corwin, Replacement of hair cells after laser microbeam irradiation in cultured organs of Corti from embryonic and neonatal mice, J. Neurosci. 4 (1995) 3013–3026. [13] S. Chardin, R. Romand, Factors modulating supernumerary hair cell production in the postnatal rat cochlea in vitro, Int. J. Dev. Neurosci. 4/5 (1997) 497–508. [14] H.M. Sobkowicz, B.K. August, S.M. Slapnick, Epithelial repair following mechanical injury of the developing organ of Corti in culture: an electron microscopic and autoradiographic study, Exp. Neurol. 115 (1992) 44–49. [15] H. Staecker, P.P. Lefebvre, B. Malgrange, G. Moonen, T.R. Van De Water, Regeneration and mammalian auditory hair cells. Response, Science 267 (1995) 709–711. [16] P.P. Lefebvre, B. Malgrange, H. Staecker, G. Moonen, T.R. Van De Water, Retinoic acid stimulates regeneration of mammalian auditory hair cells after ototoxic damage in vitro, Science 260 (1993) 692–694. [17] S. Chardin, R. Romand, Regeneration and mammalian auditory hair cells, Science 267 (1995) 707–709. [18] A. Zine, F. De Ribaupierre, Replacement of mammalian auditory hair cells, Neuroreport 9 (1998) 263–268. [19] M. Lenoir, P. Vago, Does the organ of Corti attempt to differentiate new hair cells after antibiotic intoxication in rat pups?, Int. J. Dev. Neurosci. 4/5 (1997) 487–495. [20] P.P. Lefebvre, T. Weber, J.-M. Rigo, H. Staecker, G. Moonen, T.R. Van De Water, Peripheral and central target-derived trophic factor(s) effects on auditory neurons, Hear. Res. 58 (1992) 185–192. [21] E. Bichler, H. Spoendlin, H. Rauchegger, Degeneration of cochlear neurons after amikacin intoxication in the rat, Arch. Otorhinolaryngol. 237 (1983) 201–208. [22] J. Ylikoski, U. Pirvola, M. Moshnyakov, J. Palgi, U. Aruma¨e, M. Saarma, Expression patterns of neurotrophin
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and their receptor mRNAs in the rat inner ear, Hear. Res. 65 (1993) 69 – 78. B. Malgrange, P.P. Lefebvre, T.R. Van De Water, H. Staecker, G. Moonen, Effects of neurotrophins on early auditory neurons in cell culture, Neuroreport 7 (1996) 913 – 917. P.P. Lefebvre, T.R. Van De Water, H. Staecker, T. Weber, V. Galinovic-Schwartz, G. Moonen, R.J. Ruben, Nerve growth factor (NGF) stimulates neurite regeneration but not survival of adult auditory neurons in vitro, Acta Otolaryngol. 112 (1992) 288 – 293. P.P. Lefebvre, B. Malgrange, H. Staecker, M. Mogadassi, T.R. Van De Water, G. Moonen, Neurotrophins affect survival and neuritogenesis by adult injured auditory neurons in vitro, Neuroreport 5 (1994) 865 – 868. E. Magal, R. Kuang, G. Hever, F. Collins, J.C. Louis, GDNF protects both cochlear hair cells and auditory neurons against ototoxins, Abstr. Soc. Neurosci. 22 (1996) 1621 – 1621. H. Staecker, W. Liu, C. Hartnick, P.P. Lefebvre, B. Malgrange, G. Moonen, T.R. Van De Water, NT-3 combined with CNTF promotes survival of neurons in modiolus spiral ganglion explants, Neuroreport 6 (1995) 1533 – 1537. H. Staecker, R. Kopke, B. Malgrange, P.P. Lefebvre, T.R. Van De Water, NT-3 and/or BDNF therapy prevents loss of auditory neurons following loss of hair cells, Neuroreport 7 (1996) 889 – 894. R.J. Stokroos, F.W.J. Albers, The etiology of idiopathic sudden sensorineural hearing loss, Acta Otolaryngol. Belg. 50 (1) (1996) 69 – 76. R.J. Stokroos, F.W.J. Albers, Therapy of idiopathic sudden sensorineural hearing loss, Acta Otolaryngol. Belg. 50 (1) (1996) 77 – 84. R. Pujol, J.L. Puel, C. Gervais d’Aldin, M. Eybalin, Pathophysiology of the glutamatergic synapses in the cochlea, Acta Otolaryngol. 113/3 (1993) 330 – 334. R. Pujol, C. Gervais d’Aldin, R. Leducq, J.L. Puel, Auditory-nerve regeneration and neo-synaptogenesis below inner hair cells after excitotoxic damage, Abstracts of XVIIth Meeting ARO, St Petersburg 1994; 116, c464. J.L. Zheng, R.R. Stewart, W.Q. Gao, Neurotrophin-4/5 enhances survival of cultured spiral ganglion neurons and protects them from cisplatin neurotoxicity, J. Neurosci. 7 (1995) 5079 – 5087. M.A. Marsh, H.A. Jenkins, N.J. Coker, Histopathology of the temporal bone following multichannel cochlear implantation, Arch Otolaryngol. Head Neck Surg. 118/11 (1992) 1257 – 1265.
Growth factor therapy to the damaged inner ear: clinical propects Brigitte Malgrange a, Jean-Michel Rigo a, Thomas R. Van de Water c,d, Hinrich Staecker d,e, Gustave Moonen a, Philippe P. Lefebvre a,b,d,* a
Department of Human Physiology and Pathophysiology, Place Delcour 17, Uni6ersity of Lie`ge, 4020 Lie`ge, Belgium b Department of Otorhinolaryngology, Uni6ersity of Lie`ge, Lie`ge, Belgium c Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA d Department of Otolaryngology, Albert Einstein College of Medicine, Bronx, NY, USA e Massachussetts Eye and Ear Infirmary, Har6ard Medical School, Boston, MA, USA
Abstract Most hearing loss results from lesions of the sensory cells and/or of the neurons of the auditory part of the inner ear. There is currently no treatment able to stop the progression of a hearing loss or to restore a lost auditory function. In this paper, we review the progress which has been made with respect to the regeneration and the protection of the hair cells and of the auditory neurons in the cochlea. In particular, we emphasize the control by growth factors of the protection/repair mechanisms of the neurosensory structures within the inner ear, in the prospect of the possible clinical use of these molecules. Finally, we discuss the different approaches which can be used to deliver these therapeutic agents to the inner ear. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cell culture; Cochlea; Hearing impairment; Inner ear; Ototoxicity
1. Introduction The frequency of hearing impairment diagnosed by audiometry increases with age and affects nearly half of the population by age 80 years. At least 30% of persons 65 years of age or older have a hearing loss of 50 dB at 4000 Hz, sufficient to interfere substantially with the understanding of speech. Most deafness is of sensorineural origin and is characterized by a loss of hair cells and of * Corresponding author.. E-mail address: [email protected] (P.P. Lefebvre)
spiral ganglion neurons. At the present time, hearing aids are the only satisfactory means available for the hearing impairment. Because of the lack of treatment of neurosensory deafness capable of restoring auditory function or altering the course of a progressive hearing loss, we have developed experimental strategies to approach the treatment of neurosensory deafness: (1) otoprotection, which is designed to prevent a further degradation of auditory function; and (2) regeneration, which is defined as the replacement of lost hair cells in the deafened ear and their reconnection by primary auditory neurons to the central auditory pathway.
0165-5876/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 5 8 7 6 ( 9 9 ) 0 0 1 2 6 - 3
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B. Malgrange et al. / Int. J. Pediatr. Otorhinolaryngol. 49 (1999) S19 – S25
2. Growth factors Very much as acetylcholine is for neurotransmitters, nerve growth factor (NGF) is the prototypic neurotrophic factor. Initially, these molecules were considered as target-derived molecules that were the molecular substratum responsible for the matching between the size of a given neuronal pool and the volume of its innervation territory. Therefore a classical neurotrophic factor is produced within a given tissue at an appropriate time during embryonic development and is able to support the viability of the innervating neurons by interacting with specific receptors (e.g. NGF/TRKA). The spectrum of biological effects of such factors on responding neurons goes well beyond a nerve survival promoting effect. Other effects can include stimulation of neuritic outgrowth, chemotropism (i.e. being able to steer the orientation of neuronal projections), effect on cell adhesion molecule expression, release of proteases/protease inhibitors, commitment to a specific neurotransmitter phenotype. Slowly the neurotrophic factors were considered not only as factors important during the development of a given neuronal population but also to be essential for the maintenance of adult neurons, to protect specific populations of neurons against various insults and if damage occurs to stimulate their regeneration and repair. ‘Factorology’ has thus entered the field of therapeutics. The list of factors endowed with neurotrophic properties has been slowly growing. Some of these factors are primarily derived from neuroscience research and have been characterized either by protein purification monitored using some sort of bioassay for their trophic actions (e.g. NGF, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and glial-derived neurotrophic factor (GDNF)), or directly by molecular biological techniques using polymerase chain reaction (PCR) primers based upon the conserved sequences of previously known factors of a given family (e.g. neurotrophins NT-3, NT-4/ 5, NT-6). Other factors purified using classical protein chemistry methods and bioassay monitoring turned out after sequencing to be identical
with known factors that had already been characterized based on their biological effects on nonneuronal cells (e.g. fibroblast growth factor-1 (FGF1) fibroblasts). Finally, other factors ‘became neurotrophic’ because an effect on nerve cells was discovered after their purification, sequencing and cloning for their effects in non-nervous tissues (e.g. epidermal growth factor (EGF), tumor growth factor-a (TGFa) and other members of the neuroleukin family).
3. Mammalian hair cell regeneration/repair and growth factors Auditory hair cells are the sensory receptor cells that transduce a sound stimulus into a bioelectric signal. Auditory hair cells can be readily damaged and lost after exposure to noxious agents, including noise and ototoxic drugs such as aminoglycosides. In cold blooded animals, where sensory hair cells are continuously produced throughout life, the damaged sensory epithlium is repaired by a spontaneous hair regeneration process [1]. Until recently, regeneration of auditory hair cells was considered to be restricted to lower vertebrates since the auditory hair cells that populate avian and mammalian inner ears are produced only during embryogenesis. Therefore, sensorineural hearing loss due to damage or loss of hair cells was considered to be irreversible. The capacity of spontaneous postembryonic repair of neurosensory epithelium was demonstrated following acoustic overstimulation and ototoxic drug exposure in the neonatal chick basilar papilla [2,3]. The ability of ongoing production of new hair cells was observed in the vestibular receptors of adult epithelia of birds [4]. The factors which control these generative and regenerative processes are not well known. Recently, it has been showed that insulin growth factor 1 (IGF1) and insulin induce proliferation of progenitor cells in the vestibular portion of the bird inner ear [5]. In the auditory portion of the avian inner ear, basic fibroblast growth factor (bFGF or FGF1) is thought to play a role in the regenerative processes after a lesion. This discovery of a capacity to produce new hair cells in birds led to the
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question of how to extend this repair process to the mammalian labyrinth (Fig. 1). In mammals, the production of hair cell stops at birth in the organ of Corti as well as in the vestibule [6]. In the macula of the utricle, the formation of new hair cells has been demonstrated following a lesion by an aminoglycoside [7,8]. This renewal of vestibular hair cells was also demonstrated in vivo in the adult guinea pig vestibule and is stimulated by the perilymphatic perfusion of a combination of retinoic acid, TGFa and IGF1 [9]. Recently, the stimulation of the proliferation of epithelial cells from utricles in culture has been observed using various growth factors (bFGF, FGF4, FGF6, FGF7, IGF1, IGF2, TGFb and EGF [10]), suggesting the involvement of growth factors in the regenerative processes of inner ear sensory epithelium. In the cochlea, the production of new hair cells and the repair of the sensory epithelium seems to be limited to the embryonic and perinatal periods. Retinoic acid was showed to induce the formation of supernumerary hair cells in organ of Corti explants from mouse em-
Fig. 1. Mechanisms of hair cell renewal in the sensory epithelia of the inner ear. When hair cells are lesioned, they can either undergo an apoptotic cell death or be injured and survive the insult. In the first case, the supporting cell may proliferate and a daughter cell differentiate into a new hair cell (A, regeneration mechanism). Alternatively, if no proliferation occurs, a supporting cell may differentiate into a hair cell (B, transdifferentiation mechanism). In the second case, where hair cells survive and are just injured, they may regenerate a new apex with its stereocilia bundle and cuticular plate (C, repair mechanism).
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bryos between E13 and E16 [11]. However, at E18, organ of Corti sensory hair cells killed by exposure to a laser beam could still be replaced with new hair cells [12]. This replacement of hair cells is obtained through a differentiation of preexisting cells in the sensory epithelium to form new replacement hair cells. If this experiment is performed on cochlear explants from newborn mice there is no replacement of lost hair cells. Using an other experimental paradigm, Romand et al. recently showed that EGF or TGFb1 induce the formation of supernumemary hair cells at the level of the organ of Corti in the absence of any lesion [13]. Sobkowicz et al. demonstrated a continuous capability of self-repair of damaged hair cells in the mammalian organ of Corti. Using a micropipette, the auditory sensory epithelium was lesioned. A recovery of the hair cells was observed after delay between 4 h and 7 days with a ‘regeneration’ of the cuticular plate and the production of a new stereociliar bundle [14]. Using explants of Corti’s organ from 3-day-old rat pups, we demonstrated a renewal of hair cells after aminoglycoside treatment of the explant which allowed us to kill almost all of the hair cells [15,16]. This regeneration/repair was observed in the presence of the TGFa and retinoic acid. The regenerated/repaired hair cells had an immature aspect characterized by the presence of long microvillar at the apex. The regeneration/repair of the hair cells in cultured explants from neonatal rats has been controversial. Using another experimental protocol, these results were not reproduced using a different culture technique [17]. Recently, experiments confirming the replacement of hair cells after a lesion of the organ of Corti with an aminoglycoside has been reported [18]. Analysis using a scanning electron microscope confirmed the immature characteristics of the ‘regenerated’ hair cells. The mechanism responsible for the renewal of hair cells in the lesioned postnatal organ of Corti explants is still unknown. The occurrence of immature hair cells (i.e. atypical cells) has also been shown to transiently occur at the lesion margin in the apical region of the cochlear of young rats treated with amykacin between postnatal days 9 and 16 [19]. These results suggest that some regenerative ca-
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pacity exists but is very limited in the absence of any specific intervention. At present there has not been any demonstration of a regenerative capacity of the hair cells in Corti’s organ of any adult mammal.
4. Mammalian neuronal regeneration and maintenance and growth factors There has been ample evidence that both central and peripheral target tissues act to support the development of auditory neurons [20]. In adult animals, damage to Corti’s organ is followed by a secondary degeneration of the auditory neurons of the spiral ganglion, suggesting that hair cells are one of the sources of trophic factors to support the survival of these neurons [21]. In the cochlea of the adult rat, transcripts for NT-3 have been localized to the inner hair cells only [22]. The same study localized mRNA for TRKB and TRKC (the high affinity receptors for BDNF and NT-3, respectively) to the auditory neurons, suggesting that both NT-3 and BDNF play roles in maintenance of these neurons. The study of cultured spiral ganglion neurons has provided additional information regarding the growth factors which support the survival and repair of injured auditory neurons. Dissociated cultures of the spiral ganglion provide a simple method to study injured auditory neurons which have undergone a double axotomy in the dissociation process and a disruption in the normal cellcell interactions with the non-neuronal cells [23]. Therefore the neurons in the dissociated cultures are deprived of trophic support from both central and peripheral target tissue sources as well as their normal cell–cell interactions. The effect of neurotrophic growth factors was assessed on cultures of dissociated mature spiral ganglion neurons. When FGF2 was bound to substrate in the culture dish, it was found to be a strong promoter of neuronal survival. NGF while unable to promote neuron survival when used alone was found when combined with substrate bound bFGF to be a potent initiator of neurite outgrowth [24]. Dissociated cell cultures of the adult rat spiral ganglion treated with either exogenous BDNF or NT-3
demonstrated that these two neurotrophins were equally effective in promoting the survival of the neurons [25]. Glia-derived neurotrophic factor GDNF was also found to promote the survival of adult auditory neurons in culture [26]. Modiolus spiral ganglion explants from adult rats that closely mirror cell–cell interactions and in situ tissue relationships within this ganglion provide an excellent model for testing neurotrophic factors. In this modiolus explant system, NT-3 is the strongest individual promoter of survival while CNTF with NT-3 was the most effective combination of a cytokine with a neurotrophin for promoting survival of auditory neurons [27]. The in vitro experiments allowed us to determine which of the growth factors are good candidates to be used as therapeutic agents for growth factor therapy in the inner ear. From our data [25,27], neurotrophins were selected (in particular BDNF and NT-3) because of their strong survival promoting effects. When hair cells are destroyed by exposure to ototoxins or noise trauma, a source of growth factor support is lost and auditory neurons undergo apoptotic cell death induced by a lack of trophic support as described in clinical and animal models. In our in vivo study, guinea pigs were exposed to the ototoxic combination of an aminoglycoside antibiotic with a loop diuretic and then received 8 weeks of intracochlear perfusion of either NT3, BDNF or NT3 +BDNF to determine whether site specific application of these neurotophins could prevent the loss of auditory neurons that is known to follow a loss of auditory hair cells [28]. Infusion of either NT-3 or NT-3 + BDNF into the scala tympani resulted in a \ 90% survival of auditory neurons while BDNF infusion yielded a 78% survival rate, compared with a 14–24% neuronal survival rate in untreated ototoxin exposed cochleae (Fig. 2). These results show that loss of auditory neurons that occurs subsequent to a loss of auditory hair cells can be prevented by in vivo neurotrophic therapy with either BDNF or NT-3. In a similar experimental paradigm, GDNF was also found to support neuronal survival in the spiral ganglia after an ototoxic destruction of the organ of Corti in adult guinea pig.
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Fig. 2. Growth factor treatment prevents the degeneration of auditory neurons in the spiral ganglion deprived from their peripheral target territory (i.e. the hair cells of the organ of Corti). (A) Control condition. (B) When adult guinea pig are treated with an aminoglycoside, hair cells disappear in the organ of Corti and a secondary neuronal cell death is observed within the following weeks in the spiral ganglion. (C) If a neurotrophic factor (i.e. BDNF of NT-3) is perfused in the inner ear using a miniosmotic pump, 90% of the auditory neurons are maintained alive.
5. Clinical prospect: is there a future for an application of growth factor therapy for the treatment of inner ear disorders? With the identification of the actions of growth factors such as BDNF, NT-3, CNTF, TGFa, retinoic acid and IGF1, there is the possibility of pharmacological intervention for the treatment of neurosensory disorders of the inner ear. Experimentation using animal models of neurosensory disorders of the inner ear will help to establish which of these growth factors either individually or in combination will be effective in vivo in: (1) preventing hair cell loss; (2) neuronal degeneration; (3) promoting hair cell renewal and repair; (4) neuronal repair; and (5) reestablishment of peripheral innervation via evoking neoneuritogenesis. The clinical goal of identifying the mechanisms that will evoke hair cell regeneration-repair are ambitious with an ultimate goal being the
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restoration of hearing in deaf patients and balance in patients with vestibular disorders through the replacement of lost hair cells and the reestablishment of their neuronal connections to the appropriate pathway within the central nervous system. However, many questions must be addressed prior to applying these concepts to the pharmacological treatment of patients in the clinic. An initial road block to progress into the clinical arena has been a failure to demonstrate that the process of hair cell regeneration occurs in the mature cochlea of a mammal. Neuronal injury is a probable cause of most sudden hearing loss which demonstrates spontaneous improvement over time. This type of spontaneously recovering sudden hearing loss represents about 60% of cases seen in the clinic and can be predicted by the presence of otoacoustic emission [29,30]. Recovery is most probably due to repair and reestablishment of synaptic connections to the hair cells [31,32]. Growth factor therapy should improve the level of recovery through the stimulation of the repair of damaged neuronal processes below the auditory hair cells. Growth factor therapy will also be applicable to the treatment of other inner ear disorders. Indeed, some in vitro studies reported by Zheng et al. using dissociated cell cultures of 5-day-old rat spiral ganglia demonstrate neuroprotective effects of two neurotrophins from cisplatin neurotoxicity. Both BDNF and NT-4/5 provided good neuroprotection of auditory neurons from cisplatin induced damage[33]. Furthermore, GDNF was found to prevent cisplatin damage of adult auditory neurons in culture. This factor has also ben suggested to have some otoprotective activity on hair cells induced toxicity against cisplatin and against neuronal damage in adult guinea pigs [26]. This study demonstrates the therapeutic potential of neurotrophins and GDNF for not only preventing trauma-induced hair cell degeneration but also for the protection of auditory neurons. Therefore, neurotrophins could be used as therapeutic agent to prevent the ototoxic damage of cisplatin on both the neurons of the spiral ganglion and the auditory hair cells. Cochlear implants provide a therapeutic option for patients with profound deafness. This device is implanted into the scala tympani and is able to
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directly stimulate spiral ganglion neurons located within the bony modiolus. One of the major determinants of post-implant hearing perception is the total number of viable auditory neurons available for electric stimulation. Conceivably a mechanism for the direct delivery of neurotrophins into the scala tympani could be added to the cochlear implant for either periodic or chronic infusion of these neurotrophic molecules to prevent ongoing neuronal degeneration secondary to the original insult or subsequent to the trauma of the implant procedure[34].
other growth factors to the inner ear are already being studied. Some success has been obtained with the infusion of BDNF and NT-3 into the scala tympani of deafened guinea pigs using osmotic minipumps. Implantable polymers, implantable genetically engineered cells and introduction of viral gene vectors which express genes for BDNF or NT-3 are also potential methods by which neurotrophins could be delivered to the inner ear on a chronic basis.
Acknowledgements 6. Application of growth factors for the treatment of cochlear injury Because of the blood labyrinthine barrier, growth factors may have difficulty reaching inner ear tissues. However, since the neuronal axons and synapses are in communication with the perilymphatic fluid of the scala tympani space this route affords a means of growth factor delivery. The inner ear is somewhat unique in being isolated from the rest of the body compartments by a bony protective capsule except through the endolymphatic duct and sac and the cochlear aqueduct. Therefore the inner ear can potentially be treated in its own micro-environment. Methods of delivery of neurotrophic molecules into the perilymphatic fluid of the labyrinth could include introducing growth factor directly into the middle ear cavity with diffusion or transport occurring across the round window membrane or via direct infusion of the factor through openings into the labyrinth itself. Binding growth factors to a carrier molecule, which is transported across the endothelial cells of capillaries is another potential mode of growth factor delivery. While some deafening results from acute hair cell and neuronal damage many other causes of sensorineural hearing loss are associated with a gradual loss of hair cells and/or neurons. In the latter case, or in instances where hair cells are permanently missing, the chronic infusion of growth factors may be required to support the health and viability of the neuronal population of the spiral ganglion. Approaches to chronic delivery of neurotrophins and
This work was supported by the Fonds National de la Recherche Scientifique (FNRS), the Concerted Action of the French Community of Belgium, by the Fondation Me´dicale Reine Elisabeth (FMRE) and the Shulsky Foundation. We thank P. Ernst, M. Wouters and A. Broze for their technical support and expertise.
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