Oxidative stress-induced apoptosis of cochlear sensory cells: otoprotective strategies

Oxidative stress-induced apoptosis of cochlear sensory cells:otoprotective strategies

Tina Huanga, Alan G. Chenga, Howard Stupaka, Wei Liua, Ana Kima,Hinrich Staeckerc, Philippe P. Lefebvred, Brigitte Malgranged, Richard Kopkee,

Gustave Moonend, Thomas R. Van De Watera,b,d,*aDepartment of Otolaryngology, Albert Einstein College of Medicine, Bronx, New York, NY, USAbDepartment of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, NY, USA

cDepartment of Otolaryngology, University of Maryland, Baltimore, MD, USAdDepartment of Human Physiology and Pathophysiology, University of Liege, Liege, Belgium

eDepartment of The Army, Naval Medical Center, San Diego, CA, USA

Received 3 March 1999; received in revised form 3 November 1999; accepted 4 November 1999

Abstract

Apoptosis is an important process, both for normal development of the inner ear and for removal of oxidative-stress damaged

sensory cells from the cochlea. Oxidative-stressors of auditory sensory cells include: loss of trophic factor support, ischemia-reperfusion, and ototoxins. Loss of trophic factor support and cisplatin ototoxicity, both initiate the intracellular production ofreactive oxygen species and free radicals. The interaction of reactive oxygen species and free radicals with membrane

phospholipids of auditory sensory cells creates aldehydic lipid peroxidation products. One of these aldehydes, 4-hydroxynonenal,functions as a mediator of apoptosis for both auditory neurons and hair cells. We present several approaches for the preventionof auditory sensory loss from reactive oxygen species-induced apoptosis: 1) preventing the formation of reactive oxygen species;(2) neutralizing the toxic products of membrane lipid peroxidation; and 3) blocking the damaged sensory cells' apoptotic

pathway. 7 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Apoptosis; Auditory system; Neurons; Hair cells; Reactive oxygen species; Caspases; Calpains; Cisplatin; Trophic factor withdrawal;

Ischemia/hypoxia

1. Introduction

Apoptosis, or programmed cell death (PCD), is anactive form of cell death that often requires the syn-thesis of new proteins as is evidenced by the ability of

protein synthesis inhibitors such as cycloheximide to

block many apoptotic pathways. It occurs during nor-

mal development as well as when cells are exposed to

various insults such as toxins, ischemia/hypoxia, or

radiation. An apoptotic cell is de®ned by both its mor-

phologic and biochemical features. Such features

include: nuclear fragmentation, chromatin conden-

sation, cell shrinkage, surface blebbing with the main-

tenance of membrane integrity, formation of apoptotic

bodies, and DNA proteolysis into a distinct ``ladder''

pattern when run on an agarose gel. The remains of

the cell are eventually phagocytosed by neighboring

cells or macrophages. This is in contrast to necrosis

which is a passive form of cell death involving cellular

Int. J. Devl Neuroscience 18 (2000) 259±270

0736-5748/00/$20.00 7 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved.

PII: S0736-5748(99 )00094 -5

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* Corresponding author. Tel.: +1-718-430-4082; fax: +1-718-430-

4258.

E-mail address: [email protected] (T.R. Van De Water).

Abbreviations: Lac, b galactosidase; BDNF, brain derived growth

factor; GSH, glutathione; GSHe, glutathione diethyl esther; HSV,

Herpes amplicon vector; HNE, 4-hydroxynonenal; Met, methionine;

NGF, nerve growth factor; NT-3, neurotrophin 3; PCD, pro-

grammed cell death; ROS, reactive oxygen species; VCN, ventral

cochlear nucleus.

swelling, organelle damage, plasma membrane break-down, and the spillage of cell contents into the inter-cellular environment. The DNA gel pattern of a celldying by necrosis consists of a smear of DNA frag-ments which is in marked contrast to the orderly lad-dering of DNA fragments seen in apoptotic DNAproteolysis Table 1 [6].

Programmed cell death can be a useful processduring morphogenesis and has been shown to be anessential process for the normal development of thevertebrate inner ear [11,13,38]. If this process of pro-grammed cell death is perturbed by transfecting thedeveloping inner ear with an anti-apoptotic molecule(e.g. a retroviral vector expressing human bcl-2) thendysmorphogenesis of the membranous labyrinth willoccur (e.g. malformed or absent semicircular ducts)[11]. There also appears to be a temporal sequence ofprogrammed cell death within the mammalian mem-branous labyrinth with programmed cell death a�ect-ing ®rst the vestibule and then the auditory sensoryepithelium with a correlation between periods of activecell proliferation and periods of apoptosis [38]. Thepattern of programmed cell death with areas of devel-oping neuroepithelial structures also appears to be cor-related with expression patterns of the low a�nityneurotrophin receptor, p75NGFR, which is a memberof the tumor necrosis factor family of cell death recep-tors. This expression pattern strongly implicates thesphingomyelin pathway as a modulator of cell deathwithin the neuroepithelium in the early stage of oticdevelopment and morphogenesis [13]. A signi®cantprogrammed cell death of ventral cochlear nucleus(VCN) neurons has also been demonstrated which co-incides with the onset of hearing immediately followinga period of rapid increase in the number of VCN neur-ons, and may be a mechanism for balancing auditoryinput of the VIIIth nerve projection and its receptor®eld within the VCN [52]. So it is evident from these

examples, i.e. programmed cell death in otic develop-ment [11,13,38], and maturation [52] that this processis indeed a naturally occurring phenomena that is arequirement for normal formation of the vertebrateinner ear and its central targets. However, once themembranous labyrinth and its central nervous systemnuclei (e.g. VCN) have completed their process ofmaturation, then the loss of auditory sensory cells dueto oxidative stress-induced apoptosis (e.g. amikacinototoxicity) can result in a permanent de®cit since theresponse of the mammalian cochlea to the apoptoticloss of sensory hair cells does not result in a successfulrenewal of the lost sensory hair cells [53], in contrastto the successful renewal of these cells that occurs fol-lowing similar ototoxic damage to the avian cochlea(basilar papilla). It is, therefore, of particular import-ance in the mammalian inner ear to preserve the exist-ing cochlear sensory cells (i.e. both hair cells andneurons) from loss due to oxidative stress-inducedapoptosis.

We review several initiators of the apoptotic path-way as it pertains to the auditory hair cells of theorgan of Corti and the auditory neurons of the spiralganglion. Three examples of oxidative stress-inducedapoptosis of auditory sensory cells are discussed: (1)neurotrophin withdrawal-induced apoptosis of thespiral ganglion neurons, (2) cisplatin exposure-inducedapoptosis of hair cells and spiral ganglion neurons,and (3) hypoxia/ischemia-induced apoptosis of haircells and spiral ganglion neurons. Otoprotective strat-egies against these di�erent types of oxidative stress-induced apoptosis are presented as well as possiblefuture investigations.

2. The role of oxidative stress damage in apoptosis

In the inner ear, damage caused by oxidative stress

Table 1

Working criteria for distinguishing apoptosis from necrosis [6]

Criterion Apoptosis Necrosis

Descriptive

Morphology Cell shrinkage with membrane preservation Formation of

apoptotic bodies

Cell swelling with early membrane

failure

DNA fragmentation Early internucleosomal cleavage Late non-speci®c breakdown

[Ca2+] Typically remains low until late in the cell death process Early increases

Interventional

Raise [Ca2+] Protective No e�ect or death promoting

Lower [Ca2+] Death promoting Protective

Block protein synthesis (e.g. with

cycloheximide)

Often protective No e�ect

Block ICE family members (e.g. with ZVAD-

FMK)

Often protective No e�ect

Alter bcl-2, bax, bad gene expression bcl-2 protective; bax and bad death promoting No e�ect

Administer neurotrophins Often protective (if receptors present) Pretreatment is death promoting

T. Huang et al. / Int. J. Devl Neuroscience 18 (2000) 259±270260

can induce apoptosis in the cochlear hair cells as wellas in the neurons of the spiral ganglion. Insults suchas neurotrophin withdrawal, ototoxin exposure, andhypoxia/ischemia lead to oxidative stress-induceddamage which then leads to apoptosis. Reactive oxy-gen species (ROS) and free radicals are formed asbyproducts of several metabolic pathways and thesemolecules can themselves cause cell damage by react-ing with cellular proteins. The reaction of ROS withthe plasma membrane leads to the formation of phos-pholipid membrane peroxidation products such as 4-hydroxynonenal (HNE), an aldehydic lipid peroxi-dation product, which is highly reactive and can leadto sensory cell damage and death [26]. Cells protectthemselves against oxidative stress via an antioxidantdefense system which utilizes free radical scavengersand other enzymes that maintain the appropriateredox state of cellular proteins. These free radical sca-vengers and antioxidants enzymes include superoxidedismutase, catalase, glutathione peroxidase, and thethiol tripeptide glutathione (GSH). Glutathione acts toreduce proteins which have been oxidized by ROS andis normally responsible for the detoxi®cation of toxicproducts of this process such as HNE. Oxidative stressto PC12 cells and mature primary hippocampal cellcultures caused a buildup of HNE intracellularly andHNE applied directly to these cells caused mitochon-drial damage and rapid apoptotic cell death of theexposed cells [26]. Other aldehydic products of mem-brane peroxidation (e.g. maliondialdehyde) did notcause the cells to exhibit signs of apoptosis [26]. HNEapplied to auditory hair cells and spiral ganglion neur-ons also caused cell death via apoptosis (unpublisheddata). Antioxidants (Ebselen) protected the cells fromoxidative stress-induced death [25], but not fromHNE-induced death indicating that HNE is down-stream from the formation of ROS. A cell permeantform of glutathione (GSHe) was able to rescue theseand other cells exposed to oxidative stress. ROS-induced damage has also been implicated in the apop-totic death of cochlear sensory cells exposed to neuro-trophin withdrawal, cisplatin administration, hypoxia/ischemia, and sound trauma. Cell permeant GSHe wasalso protective in several of these models [25].

3. The role of caspase in apoptosis

Caspases are cysteine proteases which cleave onlyafter aspartic acid residues. They are normally foundin an inactive pro-form and require cleavage by eitheranother member of the caspase family or granzyme Bfor activation. Caspases were initially linked to apop-tosis after the discovery in C. elegans of two pro-apop-totic genes critical to its developmental programmedcell death, i.e. ced-3 and ced-4, and a suppressor of

ced-3/ced-4 dependent PCD, i.e. ced-9 [14]. Bcl-2 is themammalian homologue of ced-9 [50]. Support for therole of caspases in apoptosis include the ®ndings thatcaspase activity increases in in vitro models of apopto-sis [20], overexpression of caspases induces apoptosisin mammalian cells, and caspase inhibitors can protectcells from apoptotic death [14,43].

Thirteen members of the caspase family have beenidenti®ed thus far and perhaps the reason there are somany is because of the cleavage site speci®city of thecaspases. Caspase-8 can be activated by cell deathreceptors (e.g. Fas) while caspase-9 is activated bycytotoxins and requires interaction with mitochondrialcytochrome c. Both of these caspases are thought toactivate caspase-3 which has been shown to be activein several di�erent models of apoptosis. Caspase-3 actsas a cell death e�ector molecule that fully commits thecell to the apoptotic pathway [1,51]. The fact that cas-pases are so prevalent and crucial in apoptosis [18] hasmade it an appealing target for inhibition. Not onlycan caspase inhibitors prevent apoptosis due to severaldi�erent insults, but caspase inhibition has been shownto protect neurons from direct oxidative stress-inducedapoptosis [10,20,54]. However, a recent study has ques-tioned the e�ectiveness of caspase inhibition as a neu-roprotective strategy since caspases may bedownstream of the release of mitochondrial cyto-chrome c into the cytosol [48].

4. The role of calpains in apoptosis

Another protease group that has been implicated inapoptosis are the calpains. Calpain are calcium acti-vated proteases which under normal conditions arepresent in a pro-form, and are believed to be activatedvia autolysis although some evidence shows that auto-lysis may not be necessary [45]. Calpains exist in twoforms, i.e. calpain I, activated by low calcium concen-trations (mM), and calpain II, activated by higher con-centrations (mM) of calcium in vitro [34]. While thecalcium concentrations needed in vitro for activationare higher than those found physiologically, it hasbeen found that lower calcium concentrations may beneeded to activate calpain when DNA or membranephospholipids are present [4,45]. Substrates of calpainsinclude cytoskeletal proteins (e.g. fodrin and microtu-bule associated proteins), membrane bound proteins(e.g. Ca2+-ATPase), and other cytosolic and nuclearproteins (e.g. protein kinase C, p53).

Calpains have been found to be active in severalapoptotic models including stroke, myocardial ische-mia, cytotoxin-induced apoptosis, and irradiation. Cal-pain inhibitors were also found to be protective inthese models [2,5]. The role of caspases in apoptosis iswell-established and ubiquitous in almost all cell types,

T. Huang et al. / Int. J. Devl Neuroscience 18 (2000) 259±270 261

Fig. 1. E�ect of BDNF deprivation on ROS (DCF dye) and GSH (CMF dye) production in spiral ganglion neurons. Sequential confocal obser-

vations of identi®ed neurons in dissociated cell cultures of P3 rat spiral ganglia with the 0 time point of sampling beginning after 48 h in vitro.

(a) E�ect of BDNF deprivation on ROS production (as measured by changes in the intensity of intracellular levels of the ROS sensitive dye

2 ',7 '-dichloro¯uoresceine acetate, DCF; Molecular Probes) in auditory neurons ÿBDNF (n = 32), after 20 min post-BDNF withdrawal the

neurons showed higher levels of ROS than the neurons in the BDNF supplemented group +BDNF at all of the time points examined (n = 31).

(b) E�ect of BDNF deprivation on GSH levels (as measured by changes in the intensity of intracellular levels of thiol compound sensitive dye 5-

chloromethyl¯uorescein, CMF; Molecular Probes) in auditory neurons. Sequential confocal observations of identi®ed neurons in the dissociated

cell cultures of P3 rat spiral ganglia with the 0 time point of sampling beginning after 48 h in vitro. CMF stained (indicator for GSH) auditory

neurons deprived of BDNF, ÿBDNF (n = 37), after 20 min post-BDNF withdrawal showed higher GSH levels at all time points, compared to

the neurons in the BDNF supplemented cultures, +BDNF (n = 30) [15].


while the role of calpains in apoptosis may be morecell-speci®c [36,37]. One of the di�culties in establish-ing the role of calpains in apoptosis is the lack of cal-pain speci®c cell-permeant inhibitors. Calpastatin, theendogenous inhibitor, is extremely speci®c, but lackscell permeability. Newer synthetic inhibitors are morepermeant, but lack speci®city, especially when used athigh concentrations.

5. Neurotrophin withdrawal-induced apoptosis

Neurotrophins are necessary for neuronal survivalboth in the peripheral and the central nervous systems.Neurotrophins are thought to regulate neuronal survi-val and target innervation during development viadi�erential expression of neurotrophins by targetorgans and cells. NGF withdrawal from dissociatedrat superior cervical ganglia neurons results in the nearcomplete apoptotic death of these neurons. In the cen-tral nervous system, Kew et al. showed that septalcholinergic neurons undergo apoptosis when subjectedto NGF withdrawal [24]. Dugan et al. found thatNGF withdrawal produced an increased production ofROS in both superior cervical ganglia neuronal cul-tures as well as in a central nervous system-derivedneuronal cell line (GT1-1 trk) which was quickly inhib-ited by the reapplication of NGF [9]. ROS productionwas also found to increase after NGF deprivation inPC12 cells [20,43]. Of interest was the observation thattreatment of the NGF deprived PC12 cells with anantioxidant and a free radical scavenger was able toblock the NGF withdrawal-induced apoptosis. Wefound that neurotrophin withdrawal (i.e.ÿBDNF) indissociated spiral ganglion cell cultures resulted inhigher levels of intercellular ROS in the neurons thanin the neurons of the control (+BDNF) cultures andthat there was a later rise in GSH production thatoccurred within the neurons in response to an el-evation in the levels of ROS (Fig. 1) [15]. GSH acts toneutralize the deleterious e�ects of ROS within an oxi-datively stressed cell. Caspase inhibitors are also ableto block neurotrophin withdrawal-induced apoptosisin neurons. Several studies have shown protection withcaspase inhibitors in a variety of cell lines [19,43,54].In addition, not only are caspase inhibitors able toblock apoptosis, they are also able to block the for-mation of ROS [43]. This suggests that the activationof caspase is upstream to the production of ROS withthe caveat that they may be downstream of irreversiblechanges in mitochondrial viability [48]. Both neurotro-phin type 3 (NT-3) and BDNF are produced by haircells of the developing cochlea, and exert their actionsin paracrine fashion [17]. Acute withdrawal of neuro-trophins in this system leads to apoptosis [47].

Several strategies have been used in the mammalian

cochlea to prevent neurotrophin withdrawal-inducedapoptosis. Our laboratory has demonstrated that directinfusion of suprapharmalogical levels of either NT-3or BDNF into the scala tympani could rescue thespiral ganglion neurons after loss of trophic support(i.e. auditory hair cells), but the osmotic pump has alimited lifespan. The use of a Herpes simplex viral vec-tor (HSV) with a bdnf gene and a gene for the markerprotein b galactosidase (i.e. HSVbdn¯ac ) was thenused to deliver BDNF to the neurons of the spiralganglion resulting in prolonged protection of the audi-tory neurons after destruction of the cochlear hair cells(Fig. 2) [47]. Transduction of these ototoxin-damagedmouse cochleae with a marker protein gene therapyvector (i.e. HSVlac ) but lacking a gene that encodesfor a therapeutic protein (e.g. bdnf ) did not supportthe survival of the spiral ganglion neurons. An ad-ditional proof of the gene therapy vector delivery ofthe therapeutic protein (i.e. BDNF) to the neurons ofthe spiral ganglion is immunostaining of these neuronswith an anti-BDNF antibody demonstrating the pre-sence of BDNF within the somas of the HSVbdn¯actransduced neurons. Transduced neurons were ident-i®ed by the presence of the marker protein b galactosi-dase. Caspase inhibitors as well as calpain inhibitorsprotect dissociated spiral ganglion neuronal cell cul-tures when applied concurrently with the neurotrophinwithdrawal (Cheng et al., in press). Current work isaimed at determining the activity levels, types, andsequence of activation of caspases and calpains in-itiated by a loss of neurotrophic support (i.e. auditoryhair cells).

6. Cisplatin-induced apoptosis

Cisplatin is a widely used chemotherapeutic agent,but its use is limited by dose-related side e�ects suchas ototoxicity which can lead to irreversible sensori-neural hearing loss.

Cisplatin-induced hearing loss is most frequent inthe higher frequencies which is consistent with the baseto apex pattern of cochlear hair cell damage. The mostsevere damage is present in the third row of outer haircell stereocilia bundles at the basal turn of the organof Corti. The stereocilia tip links become damaged,and the stereocilia then become disarrayed and/orfused, and decrease in number [7]. At even more higherdoses, the remaining rows of outer hair cells and theinner hair cells become damaged, shrink and disap-pear. At even higher doses of cisplatin, the stria vascu-laris becomes damaged and the entire organ of Cortiand Reissner's membrane collapses [29]. Additionally,changes in auditory brainstem-evoked responses fol-lowing cisplatin exposure show signi®cant increases inhearing thresholds [29].


Fig. 2. Toluidine blue-stained cochlea sections from control, neomycin-injected, and neomycin- injected and HSVbdn¯ac transduced (Herpes vec-

tor expressing bdnf and b galactosidase genes) mouse inner ears. (A) Control, untreated cochlea. Note that Rosenthal's canal (arrows) is ®lled

with toludine blue stained auditory neurons. The organ of Corti (arrowhead) appears normal. (B) Cochlea exposed to intracochlear injection of

neomycin showed destruction of Corti's organ (arrowhead) with loss of hair cells, and damage to the stria vascularis (S). In this cochlear speci-

men there was a signi®cant decrease in the density of auditory neurons in Rosenthal's canal (arrows) with the surviving neurons appearing

unhealthy, i.e. poorly stained with toludine blue. (C) Cochlea of a mouse that was injected with neomycin followed by injection of HSFbdn¯ac

gene therapy vector. Four weeks after injection, the spiral ganglion neurons in Rosenthal's canal (arrows) appear similar in both density and

toludine blue staining characteristics to those in Rosenthal's canal of the control mice (A). Note that the organ of Corti (arrowhead) and stria

vascularis (S) are both extensively damaged with loss of hair cells from Corti's organ [47].


Oxidative stress has also been shown to be involvedin cisplatin-induced apoptosis [15,25,41,42]. Cisplatinexposure in vivo caused an increase in lipid peroxi-dation byproducts and decreased levels of antioxidantenzyme activities in the cochlea which were normalizedby antioxidants and free radical scavengers [41,42].Ford et al. found that there may be a dissociation

between acute cisplatin ototoxicity as evidenced byendocochlear potentials and compound action poten-tials and lipid peroxidation which occurs after theacute functional damage [12]. Our laboratory foundthat cisplatin exposure of cochlear sensory cells invitro led to increased amounts of ROS and decreasedlevels of antioxidant enzyme activities [15,16,25]. Ad-

Fig. 3. P3 rat organ of Corti explants after 72 h in vitro. FITC-phalloidin-stained stereocilia bundles and cuticular plates of wholemount organo-

typic cultures viewed with a Zeiss Axiophot epi¯uorescent microscope. (a) Control explant demonstrating orderly row of IHCs with organized

stereocilia bundles (white arrow) and three rows of OHCs with V-shaped stereocilia bundles (white arrowhead). (b) An explant exposed to cispla-

tin (10 mg/ml), demonstrating a loss of a majority of its hair cells, a disrupted pattern of cytoarchitecture, and a loss of all stereocilia bundles. A

large white arrow points to the cuticular plate of a remaining hair cell. (c) An explant exposed to cisplatin plus D-Met (10ÿ2 mol/l). Three orderly

rows of OHCs are evident, as are a row of IHCs. The stereocilia bundles of both the IHCs (white arrow) and OHCs (white arrowhead) appear

to have a normal morphology. (d) An explant exposed to cisplatin plus D-Met (10ÿ3 mol/l). Overall cytoarchitecture has remained organized, but

IHCs are missing from some segments of the cochlear duct as indicated by an area bracketed by the black arrows. The white arrowhead points

to an OHC with fused and missing stereocilia. Otoprotection by D-Met at this concentration is reduced compared with the higher concentration

(i.e. 10ÿ2 mol/l) seen in the photomicrograph of (c). (e) In an explant exposed to cisplatin plus L-Nac (10ÿ2 mol/l), three rows of normal appear-

ing OHCs are evident. A row of IHCs shows some areas with missing hair cells as indicated by an area bracketed by the black arrows. The

white arrowhead points to an OHC that has lost its stereocilia bundle. (f) An explant exposed to cisplatin plus L-Nac, 10ÿ2 mol/l plus RPIA

(100 mM/l). Note the intact orderly rows of IHCs and OHCs with normal stereocilia bundles (white arrow and white arrowhead, respectively).

(Bar in (e) = 20 mm for the photomicrographs in (a)±(f) [25].


dition of glutathione diethyl ester (GSHe) lowered theROS to control levels and resulted in signi®cant pro-tection [25]. The addition of BDNF to dissociatedspiral ganglion cell cultures, exposed to cisplatin,resulted in the protection of the auditory neurons fromapoptosis, as well as a reduction in their levels of ROS[15]. Finally, HNE was found to be a mediator of cis-platin-induced apoptosis for both auditory hair cellsand spiral ganglion neurons (unpublished data).

Cisplatin exposure of the kidney resulted in caspaseactivation, while overexpression of bcl-2 had a protec-tive e�ect on the kidney tubule cells, but caspase in-hibitors were not attempted in this model of cisplatinnephrotoxicity [49]. Our laboratory has shown thatcaspase inhibitors o�ers signi®cant protection from cis-platin-induced apoptosis for both the hair cells inorgan of Corti explants and the neurons in the disso-ciated spiral ganglion cell cultures [31]. However, treat-ment with caspase inhibitors did not protect theauditory sensory epithelium from HNE-induced apop-tosis (unpublished data). Another caveat to be con-sidered when using caspase inhibitors as anotoprotective strategy is that these inhibitors may onlyprovide short term protection from apoptotic celldeath and that the caspase inhibitor-protected cellsmay eventually die via another cell death pathway ifthey have sustained irreversible damage to their mito-chondria [48].

Another molecule shown to be protective against cis-platin-induced apoptosis is the thiol-containing aminoacid, methionine (Met), i.e. both the D- and the L-iso-forms (Reser et al. Neurotoxicology, 1999, 20, 731±748;Fig. 3) [3,16,25]. D-Met and L-Met, free radical scaven-gers, may act by several di�erent mechanisms includ-ing direct binding to cisplatin itself or by reversing thebinding of cisplatin to GSH [25]. A combination of D-Met and BDNF provided better protection than eitherof these agents by themselves [16]. However, a caveatwhen considering the use of either L-Met or D-Met asan otoprotective strategy for cisplatin chemotherapy isits apparent ability to directly bind to cisplatin. Thismay limit the e�ectiveness of systemic administrationof this otoprotective aminoacid, however, local appli-cation (e.g. L-Met round window membrane) may bean e�ective otoprotective strategy that does not inter-fere with cisplatin's chemotherapeutic value while pro-viding protection against cisplatin ototoxic damage(unpublished results).

7. Ischemia/hypoxia and noise-induced apoptosis

It has long been thought that neuronal cell death-induced by ischemia was due to necrosis. The neuronsin the core of the ischemic infact undergo hyperpolar-ization and a calcium in¯ux which is thought to be

mediated by the release of excitotoxic molecules. Theischemic neurons exhibited swelling of their somas anddendrites with breakdown of the plasma and internalcellular organelle membranes. However, recent evi-dence also points to a role for apoptosis in ischemic/hypoxic cell death, particularly in the area of thepenumbra, i.e. the area surrounding the core of theinfarct. Apoptosis, most likely, is also responsible fordelayed neuronal death following transient globalischemia as well as neuronal death in transient focalischemia [35,40]. Gottron et al. found that cortical cellcultures exposed to conditions of transient ischemiadisplayed characteristics of both excitotoxicity-inducednecrosis as well as ischemia-induced apoptosis [19].Ischemia in vitro is usually mimicked by combininghypoxia with glucose deprivation of the exposed cells.Mild hypoxic insults result in the apoptosis of exposedneurons, and more closely mimic the conditions withinthe ischemic penumbra [5].

Oxidative stress has been shown to be involved inthe damage to nervous system cellular componentssuch as neurons, microglia, and astrocytes in ischemia/hypoxia-induced apoptosis during reperfusion[32,33,46]. An increase in ROS was found when tissueswere reperfused in vivo or returned to normoxic con-ditions in vitro. Studies on transgenic mice expressingincreased levels of antioxidants (i.e. SOD+) showed anattenuation of DNA degradation, membrane lipid per-oxidation, and neuronal death [8,23]. Kruman et al.found evidence that HNE production occurred duringmembrane lipid peroxidation phase of cell damage in-itiated by oxidative stress (e.g. ROS), and was a me-diator of resultant apoptosis [26]. As a corollary tothis ®nding, Kuntsmann et al. demonstrated that capil-lary endothelial cells in the brain have an increasedability to degrade HNE which they propose may be aneuroprotective strategy in the CNS against ischemiaand hypoxia [5,27].

Caspases and calpains have both been implicated inthe ischemic/hypoxic apoptotic pathway. Caspase in-hibitors have been shown to be e�ective in protectingprimary cultures of rat cortical neurons from hypoxiaas well as in in vivo models of transient global andfocal ischemia [36,49]. Calpain inhibitors have beenshown to be protective in both in vitro as well as invivo models of ischemia/hypoxia [25,21].

In the ear, hypoxia is used to model some of thedeleterious e�ect on the ear produced by noise-inducedtrauma. Lamm and Arnold found that the partialpressure of oxygen as well as cochlear blood ¯owdecreased during broad-band noise exposure and Pujolet al. showed that the acute damage caused in theorgan of Corti by either hypoxia or loud noise ex-posure was similar [28,39]. Noise exposure has beenlinked to oxidative stress [28], in particular the acti-vation of the GSH defense system [30]. GSH was


found to increase in the lateral wall of the cochleaafter loud noise exposure and an inhibitor of GSHsynthesis increased hearing loss while a GSH replen-isher (N6 phenylisopropyl adenosine, RPIA) decreasedhearing loss [55,56]. Our laboratory has shown thatGSH antioxidant enzyme systems are activated duringsound conditioning. Recent work has demonstrated

that apoptosis of hypoxia-damaged hair cells and neur-ons can be protected by treatment with calpain inhibi-tors (Cheng et al., in press). Data obtained supportsthe hypothesis that changes in glutathione reductase(GSSG-red) g-glutamyl cysteine synthetase (gGCS)and catalase play a role in attenuating hearing loss as-sociated with sound conditioning (toughening) fol-lowed by a high level noise (Fig. 4) [22]. Levels ofHNE increase after hypoxia and ischemia, anddecrease in accordance with the level of otoprotectiona�orded by the di�erent calpain inhibitors (unpub-lished data). Seidman and Quirk also found that an in-hibitor of lipid peroxidation was otoprotective againstischemic/reperfusion injury in the inner ear [44].

8. Conclusions and future investigations

Both, the auditory neurons and the inner and outerhair cells of the mammalian cochlea, are at presentconsidered to be non-renewable sensory cells. Thismay change in the future but at present it is of greatimportance to protect these valuable sensory cells fromoxidative stress-induced apoptosis thereby conservinghearing. Apoptosis (programmed cell death) is anactive form of cell death that is important both in nor-mal development as well as after a toxic insult. Indevelopment, apoptosis ensures that the correct num-ber of cells develop in the sensory receptor epithelia(e.g. organ of Corti), and their central targets (e.g.VCN) is an active force in the process of morphogen-esis (e.g. formation of the semicircular ducts). After atoxic insult, apoptosis protects the organism by remov-ing cells which have sustained enough damage tobecome potentially harmful. However, an agent suchas cisplatin induces apoptosis not only in the cancer-ous cells (therapeutic target), but also in other suscep-tible cells throughout the body causing undesirableside e�ects (e.g. loss of auditory hair cells with a resul-tant permanent hearing loss). The ability to protectnon-targeted cells from cisplatin damage-inducedapoptosis would not only decrease these unwanted sidee�ects, but perhaps increase the e�cacy of useful che-

Fig. 4. (A) Glutathione reductase (GSSG-red) activity in organ of

Corti (O. Corti) and Stria vascularis (stria vasc.) tissue fractions. (B)

g-glutamyl cysteine synthetase (gGCS) activity in O. Corti and Stria

vasc. fractions. (C) Catalase activity levels in O. Corti and Stria

vasc. fractions. Speci®c activities are expressed as a percent of that in

unexposed control animals, i.e. exposed to neither conditioning noise

nor high noise paradigms. The Stria vasc. tissue fraction contained

the spiral ligament. The O. Corti tissue fraction excluded the spiral

limbus. P values for comparison of test group to the control: � <0.05, + < 0.01, # < 0.001. The mean values are plotted. The error

bars indicate the standard error [22].


motherapeutic agents such as cisplatin, thereby widen-ing its therapeutic window.

Oxidative stress with the production of ROS andfree radicals and the activation of apoptotic proteases,e.g. caspases and calpains, have been shown to beinvolved in apoptosis. These mechanisms have severaldi�erent points at which inhibition could be targetedto protect cells from apoptosis. To prevent oxidativestress-induced apoptosis, strategies for protectioninclude: (1) preventing the formation of ROS either bybinding the toxin (e.g. L-Met interaction with cisplatin)or replacing the missing neurotrophic factors (e.g.HSVbdn¯ac, a gene therapy vector) in the case of neu-rotrophin withdrawal, (2) preventing the toxic e�ectsof lipid peroxidation byproducts (e.g. GSHe neutraliz-ation of HNE), (3) adding exogenous free radical sca-vengers and antioxidant enzymes and molecules (e.g.overexpressing SOD) to prevent ROS interactions withcellular proteins, and (4) increasing the activity of theendogenous antioxidant system (e.g. treatment with L-Nac). Caspase and calpain activation may be inhibitedby adding exogenous inhibitors of these cell death-e�ector proteases or by using gene therapy to upregu-late anti-apoptotic gene products such as bcl-2 (e.g.HSVbcl2 gene therapy vector). In the inner ear, insultssuch as neurotrophin withdrawal, sound trauma, hy-poxia/ischemia, ototoxic antibiotics, and ototoxic che-motherapeutic agents have all been shown to induceapoptosis both in the auditory hair cells as well as inthe neurons of the spiral ganglion. Previous exper-iments have shown that all the strategies outlinedabove can be used to protect the auditory system fromapoptosis.

Future studies include de®ning the apoptotic path-ways activated by di�ering insults to the auditory sys-tem (e.g. Fas pathway vs. JNK/c-Jun pathway) in anattempt to ®nd other targets for inhibition. We havefound that caspases and calpains may be involved indi�erent apoptotic pathways or that they may be acti-vated at di�erent points along the apoptotic pathway(Cheng et al., Brain Research, 1999, 850, 234±243).Since the cysteine proteases are thought to act as acascade, we hope to ®nd the most upstream caspase inthe cascade (e.g. caspase 8) and target that caspase forinhibition by a speci®c inhibitor. Also, while inhibitorsare often added prior to or concurrent with the insult,most clinical interventions occur after an insult. Thenext step after in vitro experiments is to move to invivo experiments in pursuit of the ®nal goal of design-ing therapeutic interventions for human use. We arehopeful that some of the molecular strategies outlinedabove will eventually be useful for protection againstoxidative stress-induced human hearing loss. Rescue ofdamaged cochlear sensory cells that still possess thepotential for long term survival may also provide theopportunity to use growth factor therapy in combi-

nation with otoprotection to initiate self-repair andsubsequently return the damaged sensory cell to fullfunction.

Acknowledgements

This work was supported by the Hearing ResearchFund of Monte®ore Medical Center to TRV, andAlbert Einstein College of Medicine NeuropathologyTraining grant NS07098 support to Tina Huang, AlanG. Cheng, Howard Stupak, and Ana Kim.

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Oxidative stress-induced apoptosis of cochlear sensory cells: otoprotective strategies

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