Protein transduction therapy into cochleae via the round window niche in guinea pigs

Transcription

1 Cittion: Moleculr Therpy Methods & Clinicl Development (6), 655; doi:.8/mtm Article vi the round window niche in guine pigs Hiroki Tked, Tkomi Kuriok, Tku Kitsuk, Kzuhito Tomizw, Tkeshi Mtsunou, Frzn Hkim, Kunio Mizutri, Toru Miw, Tko Ymd, Momoko Ise, Akihiro Shiotni, Eiji Yumoto nd Ryosei Minod Cell-penetrting peptides (CPPs) re short sequences of mino cids tht fcilitte the penetrtion of conjugted crgoes cross mmmlin cell memrnes, nd s such, they my provide sfe nd effective method for drug delivery to the inner er. Simple polyrginine peptides hve een shown to induce significntly higher cell penetrtion rtes mong CPPs. Herein, we show tht peptide consisting of nine rginines ( 9R ) effectively delivered enhnced green fluorescent protein (EGFP) into guine pig cochlee vi the round window niche without cusing ny deteriortion in uditory function. A second ppliction, 4 hours fter the first, prolonged the presence of EGFP. To ssess the fesiility of protein trnsduction using 9R-CPPs vi the round window, we used X-linked inhiitor of poptosis protein (XIAP) onded to 9R peptide (XIAP-9R). XIAP-9R tretment prior to coustic trum significntly reduced puttive hering loss nd the numer of poptotic hir cells loss in the cochlee. Thus, the topicl ppliction of molecules fused to 9R-CPPs my e simple nd promising strtegy for treting inner er diseses. Moleculr Therpy Methods & Clinicl Development (6), 655; doi:.8/mtm.6.55; pulished online 7 August 6 INTRODUCTION Tretment modlities for humn sensorineurl hering loss hve not yet een fully estlished. A mjor ostcle for this is lck of n effective nd sfe delivery method for therpeutic drugs or molecules into cochlee, ecuse mmmlin inner ers hve unique nd complex ntomicl structures: perilymphtic spce; nd, n endolymphtic spce where oth uditory nd vestiulr sensory cells exist. The cochle of the inner er, which contins uditory sensory cells, is one of the most criticl structures in the uditory pthwy. The cochle consists of three fluid filled comprtments: the scl tympni; the scl vestiule; nd the scl medi (Figure ). The surfce of the hering sensory orgn is covered y endolymph tht fills the scl medi. The inner er is isolted from the systemic circultion y the lood-lyrinth rrier, nd from the middle er y the round window memrne (RWM). These complex structures mke drug delivery into the inner er difficult. Given this ckground, there re two routes to chieve inner er drug delivery tht re used cliniclly in humns: the systemic route nd the intrtympnic route. Systemic dministrtion hs een used cliniclly for glucocorticoid tretments of cute sensorineurl hering loss nd Meniere s disese. Intrtympnic dministrtion though ppers to e superior to systemic dministrtion. Systemic dministrtion tends to require higher-dosge regimens ecuse of the lood lyrinth rrier nd the extent of the systemic circultion. Besides such high-dose regimens cn cuse undesirle side effects. 5 Contrrily, intrtympnic drug dministrtion my e le to void the dverse systemic effects thus generting therpeutic effects in the inner er vi the RWM which consists of three lyers: n outer epithelium; middle core of connective tissue; nd n inner epithelium. 6 The RWM is the memrnous septum etween the middle er nd the perilymphtic spce in the cochlee in humns, monkeys, felines, nd rodents. Becuse of the limited permeility nture of the RWM the development of tretment delivery strtegies my e chllenging. Permeility through the RWM cn e ffected y fctors such s moleculr size, configurtion, concentrtion, liposoluility, electricl chrge level, nd memrne thickness. 7,8 Regrding size, in vivo experiments hve reveled tht -μm microspheres cn trverse chinchill RWMs, wheres -μm spheres cnnot. 9 Lundmn et l. demonstrted tht the pssge of endotoxins with moleculr weight >, through norml RWM ws limited. Thus, lower moleculr weight molecules pss through the RWM, while higher moleculr weight molecules do not redily pss through the RWM. Indeed, intrtympnic drug therpy utilizing lower moleculr weight molecules hs een performed successfully cliniclly, s is shown in the two following exmples: minoglycoside ntiiotics (e.g., gentmicin: its moleculr weight is 478) for the tretment of Meniere s disese; nd glucocorticoids (e.g., dexmethsone: its moleculr weight is 9) for the tretment of sensorineurl hering loss. 7 Although the impenetrle nture of the RWM for higher moleculr weight molecules my e defense system for the inner The first three uthors contriuted eqully to this work. Deprtment of Otolryngology-Hed nd Neck Surgery, Kummoto University, Kummoto, Jpn; Deprtment of Otolryngology, Ntionl Defense Medicl College, Tokorozw, Jpn; Deprtment of Moleculr Physiology, Kummoto University, Kummoto, Jpn. Correspondence: R Minod (minod@gpo.kummoto-u.c.jp) Received 4 Mrch 6; ccepted 4 July 6

2 SV S-EGFP-9R hours S-EGFP-9R 48 hours Scl Vestiuli (perilymphtic spce) Nontreted cochle ( hour) TM IHC Scl Medi (endolymphtic spce) OC OHCs SLg S-EGFP-9R 4 hours S-EGFP-9R 7 hours SL SGC Scl Tympni (perilymphtic spce) c d e Apicl turn S-EGFP hours D-EGFP-9R 48 hours Middle turn Bsl turn S-EGFP 4 hours D-EGFP-9R 7 hours Figure Sectionl imges fter enhnced green fluorescent protein (EGFP) or EGFP-9R trnsduction. () Cross-sectionl digrm of norml dult cochle. The dult mmmlin cochle consists of three comprtments: the scl vestiuli; scl tympni; nd, scl medi. The scl medi contins the orgn of Corti (OC). The OC contins inner hir cells (IHCs) nd outer hir cells (OHCs). The IHCs nd outer hir cells (OHCs) ct s mechno-electricl trnsducers nd ply crucil role in hering. The electricl signl is trnsmitted vi the spirl gnglion cells (SGC) to the uditory pthwy of the rin. The stri vsculris (SV), locted in the lterl wll of the scl medi, is responsile for the secretion of K + into the endolymph nd the production of the endocochler potentil. () The imges show sections of the middle turns of the cochlee. In the s-egfp-9r group, EGFP ws detectle strongly in the SV, OC nd SGC t hours. Also EGFP ws detectle strongly in the SV, OC, nd SGC, nd slightly in the spirl limus (SL) nd the spirl ligment (SLg) 4 hours. EGFP levels were modertely detectle t the SV nd SG t 48 hours nd only slightly detectle t the SV t 7 hours. (c) At 4 hours in the s-egfp-9r Group. EGFP ws detectle t ll turns in the cochle. (d) At the middle turn of the cochlee in the s-egfp group, EGFP ws slightly detectle in the SGC t hours nd in the SV t 4 hours. (e) At the middle turn of the cochlee in the d-egfp-9r group, EGFP ws significntly detectle in the SV nd SGC t 48 hours. The scle rs indicte 5 μm. er, the lck of penetrtion y higher moleculr weight molecules could simultneously constitute rrier to good therpeutic outcomes when intrtympnic drug therpy is pplied vi the RWM for inner er dysfunction. The humn immunodeficiency virus type trns-ctivtor of trnscription (TAT) protein cn enter cells upon ddition to culture medium., The trnsduction of TAT, which contins high proportion of rginine nd lysine residues, is responsile for the ility of the virus to e le to penetrte the plsm memrne. Short peptide sequences, such s the protein trnsduction domin of TAT, re referred to s cell-penetrting peptides (CPPs). CPPs re short ctionic peptides with the ility to trverse the cell memrnes of severl types of mmmlin cells. 5 Vrious mcromolecules hve een ttched to these peptides nd susequently internlized. The crgo molecules tht penetrte the cell memrnes mintin their iologicl ctivities. 5 CPPs re generlly used for protein trnsduction nd RNA-sed gene silencing to interrupt or suppress gene expression. 6 Among the vrious CPPs, rgininerich peptides hve een the most widely studied.,4,7 Simple polyrginine peptides, with n optiml length of 9 residues, induce significntly higher cell penetrtion rtes thn TAT proteins do. 9 Considering this superior nture of polyrginine peptides, ttchment of polyrginine peptides to higher moleculr weight therpeutic molecules should prove to e useful for fscilitting the penetrtion of therpeutic molecules to therpeutic trgets in the inner er. Apoptosis is progrmmed cell deth tht involves the controlled dismntling of intrcellulr components while voiding inflmmtion nd dmge to surrounding cells. This process is implicted in severl inner er disorders, including drug-induced, noise-induced, ge-relted, nd genetic hering loss conditions. 4 Cspses, fmily of cysteine proteses, re essentil effector molecules for inititing poptosis. 5 8 X-linked inhiitor of poptosis protein (XIAP; its moleculr weight is 84,), the most potent memer of the inhiitors of poptosis (IAP) fmily, intercts physiclly with cspse-9 t its BIR domin nd with cspse- nd cspse-7 t their BIR domins, therey interfering with the poptosis signling pthwys. 9 XIAP lso enhnces the survivl signl directly y forming complex with TAB/TAK vi its BIR domin. XIAP hs protective effect ginst severl types of inner er dmge. Tuchi et l. reported on the protective effect of XIAP ginst gentmicininduced hir cell dmge in n in vitro study utilizing rt cochlee, while Wng et l.,4 reported tht XIAP overexpressed in mice exerted protective effect ginst noise-induced nd ge-relted hering loss in cochler hir cells nd spirl gnglion cells (SGCs). Thus, XIAP ppers to hve protective effects ginst drug- nd noise-induced hering loss. Only one report though hs exmined the tretment efficcy of XIAP in vivo: Cooper et l. 5 demonstrted

3 the successful protective tretment effect of XIAP ginst cispltinmedited ototoxicity using n deno-ssocited virl vector tht ws injected into the scl tympni of the cochle in rts. When considering the possile clinicl pplictions of deno-ssocited virl vector-sed XIAP tretment for humns, the direct injection of virl vectors into the scl tympni, which is prt of the perilymphtic spce, my cuse inner er dmge; moreover, the direct injection of therpeutic molecules into either the perilymphtic or the endolymphtic spces of the cochlee hve not een performed cliniclly ecuse of this dmge risk. Thus, the need exists for simpler nd sfer strtegy to dminister XIAP into the cochlee. CPP-sed XIAP tretment my e such tretment modlity. Additionlly, this CPP-sed XIAP strtegy my e n importnt tretment modlity for dministering other therpeutic molecules for treting vrious inner er diseses. Herein, we first exmined the sfety nd efficcy of protein trnsduction using CPP consisting of nine rginines (9R-CPP) vi the round window (RW) niche. Second, to prove the fesiility of 9R-CPP-sed tretment vi the RW, we performed 9R-CPP-sed XIAP tretment ginst noise-induced hering loss in guine pigs in vivo. RESULTS Protein trnsduction in vitro HEK9 cells were cultured in medium contining EGFP-9R or EGFP for 4 hours. In the cells, which were cultured with EGFP-9R, EGFP-9R protein ws trnsduced into ll of the cytoplsm nd into some of the nuclei. In the cells, which were cultured with enhnced green fluorescent protein (EGFP) without 9R, EGFP ws not trnsduced into ny cells. Western lotting nlyses reveled tht the reltive GFP intensity in the cells which were treted with EGFP-9R ws higher thn tht in EGFP-treted cells (Supplementry Figure S,). The EGFP protein expression levels t 4 hours fter EGFP-9R tretment were equivlent to EGFP protein expression levels t 4 hours fter plsmid trnsfection (Supplementry Figure S,). A chronologicl study fter EGFP-9R tretments reveled tht EGFP protein expression levels fter EGFP-9R tretments were highest t hours nd they still mintin some level of EGFP expression even t 7 hours fter the EGFP-9R tretments (Supplementry Figure Sc,d). Additionlly, the higher concentrtions ( µmol/l) of EGFP-9R induced longer protein expression periods when compred with lower concentrtions ( µmol/l) (Supplementry Figure Se). The EGFP protein expression level within the cells fter EGFP-9R tretments ws pproximtely similr in level to the EGFP protein level of % of the originl (%) EGFP-9R solution (Supplementry Figure S). It ppers tht % of the totl mount of EGFP moved into the cells vi EGFP-9R tretment. WST ssy reveled tht the viility of oth HEK9 cells nd MEFs were unffected even t high dosges (Supplementry Figure Sc,d). Protein trnsduction into the guine pig cochlee in vivo We performed single-protein trnsduction study using the EGFP (s-egfp Group) nd the EGFP-9R (s-egfp-9r Group) (Figure d) nd doule-protein trnsduction study using the doule EGFP-9R (d-egfp-9r Group) (Figure e). For quntifiction of EGFP protein trnsfection, we utilized two strtegies: western lot nlysis nd modified leling index (mli) nlysis. Western lot nlyses were performed utilizing whole cochlee. The GFP trnsduction levels t 4 hours in the s-egfp Group did not show ny significnt chnges compred with those of the norml non-treted cochlee (referred to s hours in Figure,). The GFP trnsduction levels in the s-egfp-9r Group t 4 hours ws significntly higher compred to those t hours nd lso to those t 4 hours in the s-egfp Group, while the GFP trnsduction levels in the s-egfp-9r Group t 48 hours showed no significnt differences compred to those t hours nd those t 4 hours in the s-egfp Group. The GFP trnsduction levels t 48 hours in the d-egfp-9r Group ws significntly higher compred with those t hours nd those t 4 hours in the s-egfp Group, while GFP trnsduction levels t 48 hours in the s-egfp-9r Group indicted no significnt chnges compred with those t hours nd those t 4 hours in the s-egfp Group (Figure ). When we compred GFP trnsduction levels of the s-egfp-9r nd the d-egfp-9r groups, t the sme elpsed time points, fter their finl tretments; reltive intensity t 4 hours in the s-egfp-9r Group versus tht t 48 hours in the d-egfp-9r Group, no significnt differences were detected. These results strongly suggest tht single EGFP-9R ppliction t the RW niche induces significnt protein trnsduction in the cochlee, nd tht the protein trnsduction is mintined for t lest 4 hours fter the plcement. A doule EGFP-9R ppliction is effective t mintining protein trnsduction levels for t lest 48 hours. To ssess the locliztion of the trnsduced proteins in the cochlee, we performed mli ssessments. The mlis of the treted cochlee in the s-egfp-9r Group were significntly higher thn those in the s-egfp Group, in the SVs, OCs (inner hir cells (IHCs), outer hir cells (OHCs), nd supporting cells; Supplementry Figure S4,), nd SGCs t nd 4 hours (Figure nd Supplementry Figure S4), while there were no significnt differences etween the mlis in the s-egfp Group nd in the norml nontreted cochlee (referred to s hours in Figure ). There were no significnt differences etween the mli vlues of the treted-side cochlee t 7 hours in the s-egfp-9r Group nd the mli t hours (Figure ). These findings suggest tht the mlis of the treted cochlee in the s-egfp-9r Group returned to the seline level y 7 hours. Additionlly, when we compred the mli t ech time point in the s-egfp-9r Group, the vlues t 48 hours were significntly lower thn those in the SVs t 4 hours, nd the mlis t 7 hours were significntly lower thn those in the SGCs t hours nd those in the SVs, OCs, nd SGCs t 4 hours (Figure ). We ssessed the mli t ech turn of the cochle fter 4 hours in the s-egfp-9r Group. The mlis of the SVs nd SGCs t the sl turns in the s-egfp-9r Group were significntly higher thn those t the picl turns, nd the mlis of the SVs t the middle turns were significntly higher thn those t the picl turns (Figure ). Thus, in the s-egfp-9r Group, the mlis t the sl turns, where the inner surfce of the RWM directly fces the scl tympni nd which contins the perilymph, ws the highest mong the three turns. Regrding the doule-protein trnsduction study, the mlis of the treted cochlee in the SVs t 48 hours in the d-egfp-9r Group were significntly higher thn those in the SVs t 48 hours in the s-egfp-9r Group or those t hours (Figure ). The mlis of the treted cochlee in the SGCs t 7 hours in the d-egfp-9r Group were lso significntly higher thn those t 7 hours in the SGCs of the s-egfp-9r Group (Figure ). Next, we compred the mlis in the s-egfp-9r Group nd in the d-egfp-9r Group t the sme elpsed time points fter their finl tretments. Consequently, the mlis t 48 nd 7 hours in the d-egfp-9r Group were not significntly different from those t 4 nd 48 hours in the s-egfp-9r Group, respectively. These findings suggest tht doule EGFP-9R ppliction is effective t reproducing protein trnsduction levels of the first ppliction.

4 4 GFP β-ctin hour 4 hours 4 hours 48 hours 48 hours 4 hours 4 hours 48 hours 48 hours EGFP S-EGFP-9R D-EGFP-9R EGFP S-EGFP-9R D-EGFP-9R Treted side Nontreted side * * Reltive intenisty ** ** hour 4 hours 4 hours 48 hours 48 hours 4 hours 4 hours 48 hours 48 hours S-EGFP S-EGFP-9R D-EGFP-9R S-EGFP S-EGFP-9R D-EGFP-9R Treted side Nontreted side Figure Quntifiction of protein trnsduction levels vi western lot. () GFP nd β-ctin protein expression ws detected utilizing western lotting in the s-egfp-9r nd d-egfp-9r groups. () Quntifiction of GFP protein trnsduction levels ws performed utilizing the reltive intensity. The reltive intensity in s-egfp-9r Group t 4 hours ws significntly higher thn tht t hours nd t 4 hours in the s- enhnced green fluorescent protein (EGFP) groups. The EGFP trnsduction level t 48 hours in the d-egfp-9r Group ws significntly higher compred with tht t hours nd tht t 4 hours in the s-egfp Group, while EGFP trnsduction level t 48 hours in the s-egfp-9r Group indictes no significnt chnge compred with tht t hours nd tht t 4 hours in the s-egfp group. 8 mli 6 4 hour hours 4 hours hours 4 hours 48 hours 7 hours 48 hours 7 hours SV OC SGC mli Bsl turn Middle turn Apicl turn SV OC SGC S-EGFP S-EGFP-9R D-EGFP-9R Figure Quntifiction of protein trnsduction levels vi mli. () The mlis in the s-egfp, s-egfp-9r nd d-egfp-9r groups re shown. The mlis of the treted-side cochlee in the s-egfp-9r Group peked t 4 hours, decresed over time, nd ecme pproximtely equl to tht in hours y 7 hours. () The mlis t ech turn t 4 hours in the s-egfp-9r Group re shown. The mli in the SV nd SGC t the lower turns ws generlly higher, versus t the higher turns. Ech n = 5. *P <.5; **P <..

5 Thickness of the RWM At 4 hours in the s-egfp-9r Group, the thicknesses of the RWMs of the treted-side cochlee were significntly greter thn those of the nontreted side cochlee (Figure 4). The increse in RWM thickness my hve ffected the slight decrese in protein trnsduction fter the second trnsduction. Impct of protein trnsduction on inner er function nd morphology To ssess the impct of protein trnsduction on inner er function, we ssessed uditory thresholds, the numer of HCs, nd the numer of SGCs t 8 dys in the s-egfp nd s-egfp-9r Groups. ABR testing reveled tht there ws no significnt chnge in the uditory thresholds t 4,, or khz etween the s-egfp nd s-egfp-9r Groups (Supplementry Figure S5). Both groups hd norml surfce morphology in the OCs (Supplementry Figure S6). There were no sttisticlly significnt differences in the hir cells nd SGCs numers etween the groups (Supplementry Figure S6 nd S7). Protein trnsduction of XIAP or XIAP-9R in vivo Western lot nlyses were performed periodiclly fter protein ppliction (Figure 5). The quntifiction indicted tht the trnsduction level in the s-xiap-9r Group incresed, with pek t hours fter ppliction (Figure 5). The trnsduction level of XIAP in the s-xiap Group did not show ny significnt chnges. Protective effect of XIAP-9R tretment ginst noise-induced hering loss To exmine the fesiility of 9R-CPP-sed tretment vi the RW niche, we performed XIAP-9R tretment to protect ginst noiseinduced hering loss. The uditory thresholds in the s-xiap-9r Group t 4 dys fter noise exposure were significntly etter thn those in the Group t khz, wheres no significnt differences were found etween the s-xiap nd sline groups (Figure 6). Effects of XIAP-9R tretment on hir cell loss To clrify the morphologicl effects of the s-xiap-9r Group, we counted the numer of OHCs t 4 dys fter noise exposure. A surfce morphologicl study reveled tht the OHCs loss rte t the se of the cochle, which corresponds to khz in the s-xiap- 9R Group, decresed significntly, compred to tht in the sline Group, while there ws no significnt difference in the OHCs loss rte etween the s-xiap nd sline Groups (Figure 7). 5 ** ST TC ST TC µm Treted side Nontreted side Treted side Nontreted side Figure 4 RWM thickness. () Typicl sectionl imges of the round window memrne (RWM) t 4 hours in the s-egfp-9r group. The RWM thickness in treted-side cochlee ppers to e thicker thn tht in nontreted cochlee. Scle rs indicte μm. ST: scl tympni; TC: tympnic cvity. () The thickness of the RWM in the s-egfp-9r group. The thickness of the RWM in treted-side cochlee ws significntly greter thn tht in nontreted cochlee in the s-egfp-9r group. Ech n = 5. **P <.. Time (hours) XIAP β-ctin Reltive intensity * * 6 4 Hours 48 Figure 5 Chronologicnl chnges of XIAP protein expression levels. () Representtive western lot results showing the trnsduction levels of X-linked inhiitor of poptosis protein (XIAP) in the whole cochlee. () Quntifiction of the trnsduction levels of XIAP in the cochlee. The trnsduction levels for XIAP in s-xiap-9r-treted cochlee were significntly higher thn those in XIAP-treted cochlee t nd 4 hours. Ech n = 4. *P <.5.

6 6 Threshold (db SPL) Bseline 4 khz 8 khz 6 khz khz Figure 6 ABR testing results t 4 dys fter noise exposure. A significnt difference ws found etween the s-xiap-9r nd sline groups t khz. *P <.5. * Effects of XIAP-9R tretment on noise-induced poptosis A TdT-medited dutp nick end leling (TUNEL) ssy ws used to ssess the mechnism of cochler HC loss nd the protective effect of XIAP-9R ginst coustic trum. Previous studies hve shown tht TUNEL-positive nuclei were oservle s erly s hour fter noise exposure nd tht they were oserved continuously up to 7 hours fter noise exposure. 6 Thus, we performed TUNEL leling for HCs nd supporting cells of the OC in the sl turns of the cochlee in ech group t 48 hours fter noise exposure (Figure 7). TUNEL-positive nuclei were clerly pprent in the region of the OHCs in the sline Group. The TUNEL-positive nuclei showed the chrcteristic chnges of poptosis, including shrunken ppernce nd the formtion of micronuclei. There were fewer TUNEL-positive nuclei in the s-xiap nd s-xiap-9r Groups (Figure 7). Susequently, quntittive nlysis reveled tht the numer of TUNEL-positive OHC nuclei decresed significntly in the s-xiap-9r Group, ut not in the s-xiap Group, compred with tht in the sline Group (Figure 7c). These results suggest tht XIAP-9R tretment ginst noise-induced cochler dmge hd protective effect vi the inhiition of poptosis. TUNEL DAPI TUNEL / DAPI OHCs loss (%) * 4 khz 8 khz 6 khz khz c d e Cspse- / F-ctin TUNEL positive OHCs (%) 4 * IHCs 5 IHCs % of cspse- positive OHCs 5 5 * IHCs Figure 7 XIAP experiment results. () Quntifiction of outer hir cells (OHC) loss t ech frequency region t 4 dys fter tretment is shown. OHC loss t the -khz region in the group ws significntly lower thn tht in the sline group. Ech n = 5. *P <.5. () Surfce preprtions from the sl turns of cochlee t dys fter noise exposure. TUNEL-positive nuclei re indicted y white rrowheds. IHCs: inner hir cells.,, nd : first, second, nd third rows of OHCs. The scle r indictes μm. (c) Quntifiction of TUNEL-positive cells in the -khz region. The numer of TUNEL-positive cells in the s-xiap-9r group ws significntly lower thn tht in the sline group. Ech n = 5. *P <.5. (d) Surfce preprtions of the -khz region of cochle t dy fter noise exposure. Cleved cspse--positive cells re indicted y white rrowheds.,, nd : first, second, nd third rows of OHCs. The scle r indictes μm. (e) Quntifiction of cleved cspse--positive OHCs from the sl regions. The numer of cleved cspse--positive OHCs in the s-xiap-9r group ws significntly lower thn tht in the sline group. Ech n = 5. *P <.5.

7 To further exmine the mechnism of the ntipoptotic effects of XIAP in noise exposure in the cochlee, we exmined cleved cspse- in the cochlee t 4 hours fter noise exposure. It hs een reported tht cleved cspse- is detectle in the cochlee from immeditely fter noise exposure up to dys fter noise exposure, 7,8 nd it is therefore prime trget for the ntipoptotic effects of XIAP. Cleved cspse--positive OHCs were found more frequently in the sl prts of the OCs thn in the middle nd picl turns; this is consistent with the finding tht poptotic OHCs were found most frequently t the sl turns fter noise exposure (dt not shown). Cleved cspse--positive cells were found less frequently in the s-xiap-9r Group thn in the s-xiap Group or in the sline Groups. The numer of cleved cspse--positive OHCs in the s-xiap-9r Group ws significntly lower thn in the sline group (Figure 7d,e). These results suggest tht n poptotic mechnism, vi cspse-, ws suppressed y the XIAP-9R tretment. DISCUSSION Western lot nlyses reveled tht single EGFP-9R ppliction t the RW niche induced significnt protein trnsduction in the cochlee. The protein trnsduction in the whole cochlee ws mintined for t lest 4 hours fter the ppliction when compred with the nontreted cochlee, wheres, single EGFP ppliction did not induce significnt protein trnsduction. In the XIAP-9R study, the western lot nlysis dt similrly reveled tht XIAP trnsduction ws mintined for t lest 4 hours fter the tretment. Doule EGFP-9R pplictions were effective t mintining protein trnsduction levels for t lest 48 hours fter the first pplictions. To ssess the locliztion of the trnsduced proteins in the cochlee fter 9R-CPP-medited protein trnsfer vi the RWM, we utilized mli ssessment, which is photoshop-sed imge nlysis method, 9 which hs een utilized for ssessing protein expression levels in in vivo nd in in vitro experiments Consequently, we found tht protein trnsduction signls were significntly detectle in ll ssessed loctions (the SV, SGC, nd OC regions) until 4 hours fter the EGFP-9R ppliction, when compred with nontreted cochlee. While t 48 hours fter the EGFP-9R pplictions, significnt protein trnsduction levels were detectle in the SV nd SGC regions in the cochlee. In the doule ppliction study, significnt protein trnsduction signls were detectle in the SVs t 48 hours fter the initil pplictions, when compred with the nontreted cochlee. Thus, sequentil chnges of the protein trnsduction levels found in our mli nlysis in the EGFP-9R experiment were similr to those found in the western lot nlysis; in oth nlyses, we could detect significnt protein trnsduction signls in the cochlee for t lest 4 hours fter the single EGFP-9R tretments, nd for t lest 48 hours fter the doule EGFP-9R tretments, ut ccording to the results, there were some minor differences in the sequentil chnges of the protein trnsduction levels etween the western lot nlysis dt nd the mli nlysis dt, (for exmple, t 48 hours fter the single EGFP-9R ppliction, significnt protein trnsduction signls were not detectle in western lot nlysis, ut significnt signls were detectle in two of the three loctions in the cochlee with mli nlysis), we think tht these differences reflect differences in the trget smples: whole cochlee in the western lot nlyses nd selected loctions in the cochle in the mli nlyses. We therefore mintin tht protein trnsduction vi the RWM utilizing 9R-CPP is proven, vlid, nd relile protein trnsduction strtegy into the cochlee. Our in vitro studies reveled tht the EGFP protein expression levels t 4 hours fter EGFP-9R tretment were equivlent to the EGFP protein expression levels t 4 hours fter plsmid trnsfection vi ctionic lipid-medited trnsfection. Recently, Zuris et l. 49 hve reported successful protein trnsfection into the cochler tissues vi ctionic lipid medited trnsfection. Although they injected trget proteins nd ctionic lipid into the scl medi, the direct injection into the cochle hs potentil risk to cuse inner er dmge. Additionlly, Zuris et l. 49 hve found cytotoxic effects in oth in vitro nd in vivo studies when utilizing the higher concentrtion of ctionic lipid. Contrrily, 9R-CPP-sed protein trnsfection did not show ny cytotoxic effects in either HEK9 cells or MEFs even under t higher concentrtions of 9R-CPP. Additionlly, in in vivo studies, 9R-CPP-sed protein trnsfection did not show ny cytotoxic effects t either the highest concentrtion levels of EGFP-9R or XIAP-9R, which were ville to us. Thus, when considering clinicl pplictions of protein trnsduction into the cochlee, 9R-CPP-sed protein trnsduction ppers to e superior to ctionic lipid-medited trnsfection ecuse of the lck of cytotoxicity with 9R-CPP-sed trnsfections. There re two known routes for the diffusion of sustnces from the tympnic cvity, the norml ir spce surrounding the ony cochlee, to the perilymphtic spce in the cochlee of rodents. One is vi the RWM nd the other is vi the ony wll of the cochle. It hs een reported tht gentmicin nd steroid hormones plced on the RW niche of guine pigs re distriuted into the perilymphtic spce of the cochle. The concentrtion t the sl turn of the cochle ws,-fold higher thn t the picl turn. 5 This suggests tht sustnces plced on the RW niche pss through the RWM nd diffuse into the perilymphtic spce from the sl turn to the picl turn. In guine pigs, the lterl ony wll of the cochler pex is thinner thn t the sl turn. 5 Additionlly, lcuno-cnliculr system hs een descried in the cochler wlls of oth humns nd mice. 5 These ntomicl structures in guine pigs pper to induce higher concentrtion of sustnce t the picl turn when the tympnic cvity is filled with the sustnce. 5 The existence of the two routes (vi the RWM nd vi the ony wll of the cochle) my hve contriuted to the distriution of the sustnces used in the present study. The EGFP intensities t the sl turns in the cochlee were significntly higher thn t the picl turns fter plcement of n EGFP-9R-soked geltin sponge. Considering our distriution pttern of EGFP intensity, we concluded tht EGFP-9R proly diffuses minly vi the RWM nd ecomes distriuted in the perilymphtic spce in the cochle, rther thn vi the cochler ony wll. Although the generl mechnism y which CPPs enter cells hs yet to e determined, it hs een suggested tht they use direct penetrtion nd endocytosis for cellulr internliztion. 54,55 Additionlly, CPPs pper to use prcellulr pthwys to pss through the mucos nd rin endothelil cells. 54,56 In the present study, EGFP-9R nd XIAP-9R, which were plced t the RW niche, proly reched the perilymphtic spce using these cellulr nd prcellulr pthwys. Generlly, it is known tht sustnces which exist in the perilymphtic spce in the scl tympni rech the modious, the Rosenthl cnls nd the OC y pssing successively through the cnliculi perforntes. 57 It is lso known tht there is communiction etween the scl tympni nd the scl vestiuli vi the spirl ligment nd the stri vsculris In our experiments, EGFP-9R nd XIAP-9R, which were plced t the RW niche, my hve extended to ech prt in the cochle vi these routes. To ssess the fesiility of 9R-CPP-sed tretment vi the RW niche, we performed protection study using XIAP-9R ginst noise-induced hering loss in guine pigs in vivo. XIAP-9R pplied 7

8 8 topiclly to the RWM successfully prevented uditory deteriortion cused y noise-induced cochler dmge t higher frequencies. The presumed mechnism underlying this protective effect is tht 9R-CPP coupled to XIAP fcilitted XIAP permeility cross the RWM nd diffusion into the cochler tissues, including OHCs; susequently, 9R-XIAP chieved protective effect ginst noise-induced hering loss y suppressing the cspse- pthwy. Considering this, 9R-XIAP tretment vi the RW niche could e simple nd promising tretment modlity for decresing cochler dmge in sensorineurl hering loss cused y n poptotic mechnism. The finding tht XIAP-9R showed significnt protective effects t higher frequencies ut limited protective effect t lower frequencies my limit the clinicl ppliction of XIAP-9R tretment. This finding my hve een cused y differences in the XIAP distriution pttern dependent upon ech specific cochler turn; in the s-egfp-9r Group, the EGFP intensity t lower turns showed significntly higher EGFP trnsduction. The concentrtion of XIAP-9R ws. mg/ml, while the concentrtion of EGFP-9R ws 4 mg/ml. Appliction of such lower concentrtion of XIAP-9R t the RW niche should hve lowered the locl concentrtion of XIAP in the cochle, especilly t the higher turns. Tünnemnn et l. 55 hs lso reveled tht the trnsduction ility of rginines cn e ffected not only y their concentrtion, ut lso y the numer of consecutive residues. Considering these points, optimiztion of the concentrtion levels nd the numer of consecutive rginine residues my increse the efficcy of trnsduction, nd this in turn should increse the therpeutic efficcy of crgos coupled with the CPP. Furthermore, in the noise-induced hering loss model used in the present study, the protective effect ws lower t lower frequencies thn t higher frequencies. Our model produced uditory deteriortion primrily t higher frequencies nd less so t lower frequencies. This difference in the uditory deteriortion pttern my explin our finding of reduced protective effect t lower frequencies in XIAP-9R-treted guine pigs. To further explore this issue, we need to optimize the conditions for CPPs nd to evlute the effects of CPP tretment on other inner er dmge models, including models tht produce uditory deteriortion t lower frequencies. A single 9R-CPP-sed protein trnsduction vi the RW niche exerts more efficient protein trnsduction compred with protein trnsduction without 9R-CPP for t lest 4 hours fter tretment. When we look t time points of the peks of protein expression levels in our studies, there were some differences: 4 hours in in vivo mli study nd hours in WB results of in vivo study nd in XIAP study. We think tht this difference proly cones from difference of ssessing methods (mli versus WB) nd loction of the smples (second turns of the cochlee versus whole cochlee). In either cse, to 4 hours of expression periods fter single 9R-CPP-sed protein trnsduction my limit the usefulness of this method. There re two possile solutions for this issue. Our in vitro study reveled tht higher concentrtion level of 9R-CPP induced longer protein expression periods. In our in vivo study, we utilized the highest concentrtion of 9R-CPPs which we hd ville in our institution, though, if we might hve used higher concentrtion of 9R-CPPs, proly we could hve chieved longer expression periods. Additionlly, repeted pplictions my lso help to extend expression periods. In our study, following n dditionl EGFP-9R ppliction t 4 hours fter the first, EGFP protein trnsduction levels t 48 hours fter the first ppliction were significntly higher compred to those in the controls. Additionlly, regrding the EGFP protein trnsduction levels t the sme elpsed time fter one versus two pplictions, no significnt differences were oserved etween the s-egfp nd s-egfp-9r groups. These findings indicte tht multiple consecutive pplictions through the RW niche re effective in terms of extending the expression durtion. However, when we compred EGFP protein trnsduction levels t the sme elpsed time etween single EGFP-9R ppliction nd doule EGFP-9R ppliction in detil, the EGFP protein trnsduction level t 48 hours in the d-egfp-9r group ws somewht lower thn tht t 4 hours in the s-egfp-9r group. This slight decrese in EGFP protein trnsduction level my e cused y thickening of the RWM: the thickness of the RWM t 4 hours in the treted cochlee ws significntly greter thn tht in the nontreted cochlee. The thickening of the RWM might e limiting fctor in trnsduction efficcy in more multiple consecutive pplictions. This thickening phenomenon might hve een cused y the geltin sponge, 6 which ws used s crrier of the EGFP-9R. If so, the use of other mterils insted of geltin sponges, or the ppliction of 9R-CPP-conjugted crgos without other mterils, my diminish the thickening of the RWM. Consequently, these strtegies my increse the trnsduction efficcy y repeted pplictions. Mny studies hve shown tht CPPs re efficient crriers of ioctive crgos, including proteins, peptides, nd oligonucleotides. Although the most common ppliction of CPPs is the trnsduction of proteins nd peptides, other pplictions include CPPmedited oligonucleotide delivery. 6,6 The non-specific nture of CPP-medited delivery my e drwck when considering clinicl pplictions. Lkkonen et l. 64 reported tht LyP- peptide, CPP, induced strong ccumultion in primry MDAMB-45 rest cncer xenogrfts fter intrvenous peptide injections. Tn et l. 65 reported tht CPP conjugted with n nti-her-/neu peptide mimetic (AHNP), n ErB extrcellulr domin-inding peptide, preferentilly trgeted ErB-overexpressing in rest cncer cells. The Mtsushit nd Kemp et l. 66 reported tht the ddition of nucler locliztion signl (NLS) resulted in delivery of the peptide exclusively to the nucler comprtment of the cells. Thus, the ttchment of such homing peptides or other trgeting motifs to CPPs my increse retention in specific tissues, cell types, or intrcellulr loctions. We utilized one octve nd noise centered t 4 khz t 6 db sound pressure level (SPL) for hours to generte noise-induced hering loss model. This noise exposure level induced deteriortion of the uditory thresholds, minly t high frequencies. It hs een well known tht there re differences in the frequencies of noise nd the loction of susequent cochler dmge. Hir cell loss t the sl turns seem to e independent of the noise frequency nd used in exposure. 67 The upwrd spred of cochler dmge with respect to the exposure spectrum is typicl of n coustic injury. 68 It hs lso een well known tht the hering impirment level depends on the sound intensity, the types of sound, nd the susceptiility of the nimls. Thus, the reltionship etween the noise frequency nd the loction of the susequent dmge is resonle nd logicl. When considering clinicl pplictions of CPP medited tretments, in humns, it is reltively esy to ccess the RW niche vi the tympnic memrne, nd repeted pplictions vi the tympnic memrne re lso fesile. Although we used geltin sponges s crrier for EGFP-9R in the current study, we suggest tht the repeted ppliction of 9R-CPP-conjugted crgos vi erdrops without using crrier through n incised tympnic memrne could e cliniclly pplicle method. In summry, we demonstrted tht the use of 9R-CPP vi the RW niche is n effective nd sfe method for protein trnsduction

9 into guine pig cochlee. We lso demonstrted some effectiveness of repeted pplictions. Additionlly, we showed tht 9R- CPP-sed tretment is fesile, using XIAP-9R in guine pigs. Thus, we elieve tht 9R-CPP-sed trnsduction vi the RW niche is promising potentil method for the delivery of therpeuticlly relevnt molecules into the cochle in humns. MATERIALS AND METHODS Ethics sttement All niml experiments were pproved y the Committee on the Use nd Cre of Animls t Kummoto University nd the Ntionl Defense Medicl College. They were performed ccording to ccepted veterinry stndrds. Recominnt proteins EGFP nd EGFP fused to 9R (EGFP-9R) were prepred s descried previously.,69 Briefly, the constructed plsmids were trnsfected into BL-DE Escherichi coli cells, nd then protein expression ws induced y. mmol/l isopropyl -thio-β-d-glctopyrnoside. The proteins were purified using TALON resin (Clontech, Mountin View, CA) nd stored t 8 C fter dilysis ginst phosphte-uffered sline (PBS). For the purifiction of recominnt XIAP nd XIAP fused to 9R (XIAP-9R), mouse full-length Xip cdna ws mplified utilizing polymerse chin rection using pproprite linker primers nd sucloned into the EcoRI-XhoI sites of pet(+) (Novgen, Mdison, WI) or the BmHI-EcoRI sites of pet(+), fused with the 9R sequence t the croxyl terminus. Similrly, the proteins were generted nd stored. Experimentl protocol in vivo Regrding the EGFP nd EGFP-9R experiments, we used Hrtley guine pigs, weighing 5 g ech (Kyudo, Sg, Jpn). The EGFP nd EGFP-9R experiments involved three groups: single EGFP ppliction (s-egfp group); single EGFP-9R ppliction (s-egfp-9r group); nd, doule EGFP-9R pplictions (d-egfp-9r group), in which the second ppliction ws performed 4 hours fter the first. EGFP nd EGFP-9R pplictions were chieved in the following mnner. Guine pigs were nesthetized vi the intrperitonel dministrtion of mg/kg of xylzine (Byer; Shwnee Mission, KS) nd 4 mg/kg of ketmine (Diichisnkyo, Tokyo, Jpn) in.9% NCl. The nimls underwent posturiculr incision, nd then the mstoid ulle were opened. Susequently, geltin sponge of 5 mm (Astells Phrm, Tokyo, Jpn) tht ws soked in 5 μl of EGFP (4.4 mg/ml) or EGFP-9R (4. mg/ml) ws plced on the RW niche of the right er, followed y the intrperitonel dministrtion of mg/kg of chlormphenicol (Diichisnkyo, Tokyo, Jpn). The holes in the mstoid ulle were seled immeditely with muscle tissue nd the skin ws closed. In ech group, the cochlee were extrcted fter the tretment t ech time point: the nimls were euthnized with deep nesthesi using n overdose of xylzine nd ketmine. Norml nontreted cochlee were used s controls nd the dt of them were expressed s the dt t hours. At 8 dys fter tretment, uditory functionl nlyses were performed in the s-egfp Group nd in the s-egfp-9r Group. Susequently, the nimls in those groups were euthnized for further morphologicl nlyses. The XIAP nd XIAP-9R experiments involved three groups of 4-week-old Hrtley guine pigs (Jpn SLC, Shizuok, Jpn): single XIAP ppliction (s-xiap group); single XIAP-9R ppliction (s-xiap-9r Group); nd sline ppliction (sline group). Under generl nesthesi with ketmine nd xylzine, geltin sponge of 5 mm soked in XIAP or XIAP-9R t concentrtion of. mg/ml ws plced on the RW niche in the right er of the nimls t hours efore noise exposure. As control, geltin sponge immersed in sline ws used. Susequently, uditory functionl nlyses nd morphologicl nlyses were then performed. Western lotting In the EGFP nd EGFP-9R experiments, five cochler smples were evluted t ech time point. Proteins were extrcted with n extrction kit (Thermo Fisher Scientific, Wlthm, MA). The superntnts of the homogentes from ech cochle were sujected to sodium dodecyl sulfte-polycrylmidegel electrophoresis (SDS-PAGE), nd the proteins in the gel were trnsferred to Polyvinylidene Difluoride (PVDF) memrnes (Bio-rd, Hercules, CA). The lots were immunorected with Horse rdish peroxidse-conjugted nti- GFP (Bio-rd) nd with Horse rdish peroxidse-conjugted nti-β-ctin (Bio-rd) ntiodies, nd the protein nds were then visulized using specific chemiluminescence detection system (Bio-rd). The immunolot signls were detected using n LAS4 digitl imging system (Fujifilm, Tokyo, Jpn). The detected nds were nlyzed with Imge J softwre (NIH, Bethesd, MD). For quntifiction of protein trnsduction levels, we utilized the reltive intensity otined y dividing GFP intensity y the β-ctin intensity; susequently, the vlues were normlized to tht t hours. In the XIAP nd XIAP-9R experiments, four cochler smples were evluted t ech time point. The superntnts of the homogentes from ech cochle were sujected to SDS-PAGE. The proteins were trnsferred to n Immoilon-P memrne (Millipore, Billeric, MA). The lots were immunorected with nti-xiap (BD Biosciences, Sn Jose, CA) nd with nti-β-ctin (Sigm, St. Louis, MO) ntiodies, nd the protein nds were then visulized using specific chemiluminescence detection system (Pierce, Rockford, IL). The immunolot signls were detected using n LAS4 digitl imging system (Fujifilm). The detected protein nds were then nlyzed using Imge Qunt TL softwre (GE Helthcre, Little Chlfont, UK). The GFP intensity ws divided y the β-ctin intensity; susequently, the vlues were normlized to control cochler smples tken from nontreted nimls. mli In the s-egfp nd s-egfp-9r Groups, whole cochlee were hrvested nd fixed in 4% PFA (Wko, Osk, Jpn) for hours t 4 C. The inner ers were declcified in disodium ethylenediminetetrcetic cid (EDTA) for 4 dys. The cochlee were emedded in optiml cutting temperture (OCT) medium (Skur Finetek Jpn, Tokyo, Jpn) nd sectioned serilly t thickness of 8 μm utilizing cryostt (GMI, Bellevue, WA). The cryosectioned slices were fixed with 4% PFA nd locked with.% Triton-X (IBI Scientific Kpp Court Peost, IA) in PBS, nd then incuted with Hoechst 58 Dye (Moleculr Proes) for seconds for nucler stining. During ech process, the tissues were wshed three times for 5 minutes ech with PBS. The smples were exmined under BZ-9 fluorescence microscope (Keyence, Osk, Jpn), nd the imges were cptured nd stored on computer. We used the mli to evlute the EGFP protein trnsduction levels in ech cryosectioned slice of the cochlee. The originl leling index method ws reported y Lehr. 9 The mli method used is descried s follows: ech imge ws cptured t resolution of,6,4 pixels t the sme photogrphic exposure condition; tissue-free res outside of the ony cochlee were then selected using the Mgic Wnd tool in Adoe Photoshop (Adoe Systems, Sn Jose, CA); nd, the tolernce level ws then djusted to e le to select the entire outside res of the ony cochlee. Thus, the determined tissue-free res were used s the ckground. Susequently, ech stined re in the orgn of Corti (OC), SGCs, nd stri vsculris (SV) ws determined similrly. These imges were converted to 8-it gryscle imges. The opticl densities of the ckground res nd ech stined re were ssessed using the histogrm tool in Adoe Photoshop; consequently, the men stining intensity nd the numer of pixels in ech re were determined. The finl stining intensity ws clculted s the difference etween the men stining intensity nd the men ckground intensity. Next, the stining rtios were clculted s the rtios of the numer of pixels in ech stined re to tht in the entire imge. These mesuring processes of mli were performed in linded mnner. Noise exposure Guine pigs were nesthetized y n intrperitonel injection of ketmine (5 mg/kg) nd medetomidine (. mg/kg). They were exposed to one octve nd noise centered t 4 khz t 6 db sound pressure level (SPL) for hours in ventilted sound exposure chmer. The sound chmer ws fitted with spekers (Model 8A; JBL, Northridge, CA) driven y noise genertor (DANAC-; Dn Jpn, Tokyo, Jpn) nd power mplifier (D-45; Crown Interntionl, Elkhrt, IN). Sound levels were clirted (Type 64 precision sound level meter; Aco Instruments, Tokyo, Jpn) t multiple loctions within the sound chmer to ensure uniformity of the stimulus. Auditory thresholds Auditory thresholds were mesured using the ABR (System ; Tucker-Dvis Technologies, Alchu, FL). The nimls were nesthetized y the intrperitonel dministrtion of xylzine nd ketmine. Electrodes were plced eneth the pinn of the treted er nd t the vertex just elow the surfce of the skin, nd then the ground electrode ws plced under the contrlterl er. An verge of,4 sweeps ws clculted for 4, 8, nd khz in 9

10 the s-egfp nd s-egfp-9r Groups, nd for 4, 8, 6, nd khz in the s-xiap nd s-xiap-9r Groups. The stimulus levels ner the threshold were vried in 5-dB steps, nd the threshold ws defined s the lowest level t which wves in the ABR could e clerly detected y visul inspection. In the s-egfp nd s-egfp-9r Groups, the hering thresholds were mesured t 8 dys fter protein trnsduction. In the s-xiap nd s-xiap-9r Groups, the ABR ws mesured dy efore noise exposure nd on 4 dys fter noise exposure. Morphologicl nlyses In the s-egfp nd s-egfp-9r groups, the thicknesses of the RWM t 4 hours fter tretment were ssessed. The ilterl cochlee were serilly cryosectioned to thickness of 8 μm in ech group. Nomrski imges of the whole RWM were cptured using BZ-9 microscope (Keyence, Osk, Jpn) nd then they were stored on computer t density of 96 dpi. The center prt of the RWM ws cut out from the cptured RWM imges. Susequently, the lengths nd res of the RWM in the cut-off imges were mesured utilizing ImgeJ softwre (NIH, Bethesd, MD). The vlues otined y dividing the res y the length were utilized for sttisticl nlyses. For hir cell counting, in the s-egfp nd s-egfp-9r Groups, the cochlee were removed from the temporl onest 8 dys fter tretment. After fixtion overnight t 4 C, the ony cpsules nd lterl wlls of the cochlee were removed. The OCs were incuted with Texs Red-X phlloidin (Moleculr Proes) for minutes fter locking with.% Triton-X in PBS for minutes. During ech process, the tissues were wshed three times for 5 minutes ech with PBS. The surfce imges of the OCs were cptured nd stored on computer. The numers of IHCs nd OHCs in μm lengths of the OCs in ech turn were counted, nd the survivl rte of the hir cells (HCs) ws mesured. In the s-xiap, s-xiap-9r, nd sline groups, similrly, OCs were hrvested on 4 dys fter noise exposure. After immersion in the fixtive gent overnight t 4 C, the temporl ones were declcified in. M EDTA (ph 7.4) contining 5% sucrose nd stirred t 4 C for dys. After declcifiction, the cochle ws microdissected. The OCs were incuted with primry ntiodies overnight t 4 C fter locking with.% Triton-X in PBS supplemented with 5% got serum for hour. During ech process, the tissues were wshed three times for minutes ech with PBS. Primry ntiodies were detected using secondry ntiodies nd viewed under confocl fluorescence microscopy (Nikon C system; Tokyo, Jpn). Hir cells were identified using nti-myosin VII ntiodies (Proteus Biosciences, Sn Diego, CA) s the primry ntiody nd Alex 5 (Invitrogen, Crlsd, CA) s the secondry ntiody. ImgeJ softwre (NIH) ws used to mesure the totl length of the cochler whole mounts, nd cochler frequency mp ws computed to precisely loclize IHCs sed on frequency-specific regions. ImgeJ plugin ( org/reserch/otolryngology/investigtors/lortories/eton-peodylortories/epl-histology-resources/imgej-plugin-for-cochler-frequencympping-in-whole-mounts) tht trnsltes cochler position into specific frequency ccording to the pulished mp of the guine pig. 7 High-power imges of frequency-specific regions ccording to the computed frequency mp were ssemled nd nlyzed. The numers of IHCs nd OHCs in μm were counted in four segments in the cochle. In the sme mnner s descried for the hir cell counts, the wholemounted smples were stined to detect DNA frgmenttion in the nuclei of poptotic cells using the TUNEL technique with n ApopTg Plus Fluorescein In Situ Apoptosis Detection Kit (Millipore) ccording to the mnufcturer s recommended protocol. Briefly, residues of digoxigenin-deoxyrionucleotide triphosphte were dded to the -OH ends of the DNA using terminl deoxynucleotidyl trnsferse. The incorported digoxigenin-nucleotides were then immunostined with FITC-conjugted nti-digoxigenin ntiody from the TUNEL kit. The tissues were counterstined with DAPI. The FITC nd DAPI fluorescent signls were oserved y confocl fluorescence microscopy using the Nikon C system. To quntify TUNEL-positive OHCs, the totl numer of OHCs nd the numer of TUNEL-positive OHCs were counted in ech imge. Counts were otined from five cochlee. For ech condition, the OHC counts were otined from three loctions in the sl turns of ech cochle. After whole-mount preprtion of the cochlee, cspse--positive cells were identified using cleved cspse- ntiodies (Cell Signling Technology, Dnvers, MA) s the primry ntiody nd Alex 488 (Invitrogen) s the secondry ntiody. Susequently, counterstining ws performed using F-ctin (Invitrogen). The numers of cspse--positive OHCs per μm in the sl turns were counted in s-xiap, the s-xiap-9r, nd in the sline groups. Sttisticl nlyses The dt were nlyzed sttisticlly using the Mnn-Whitney U-test. Dt re presented in the text nd figures s mens ± stndrd error. The sttisticl significnce level ws set t P <.5. CONFLICT OF INTEREST The uthors declre no conflict of interest. References. Liu, X, Zheng, G, Wu, Y, Shen, X, Jing, J, Yu, T et l. (). Led exposure results in hering loss nd disruption of the cochler lood-lyrinth rrier nd the protective role of iron supplement. Neurotoxicology 9: Liu, H, Ho, J nd Li, KS (). Current strtegies for drug delivery to the inner er. Act Phrm Sin B : Swn, EE, Mescher, MJ, Sewell, WF, To, SL nd Borenstein, JT (8). Inner er drug delivery for uditory pplictions. Adv Drug Deliv Rev 6: McCll, AA, Swn, EE, Borenstein, JT, Sewell, WF, Kujw, SG nd McKenn, MJ (). Drug delivery for tretment of inner er disese: current stte of knowledge. Er Her : Bowe, SN nd Jco, A (). Round window perfusion dynmics: implictions for intrcochler therpy. Curr Opin Otolryngol Hed Neck Surg 8: Goycoole, MV nd Lundmn, L (997). Round window memrne. Structure function nd permeility: review. Microsc Res Tech 6:. 7. Borkholder, DA, Zhu, X nd Frisin, RD (4). Round window memrne intrcochler drug delivery enhnced y induced dvection. J Control Relese 74: Goycoole, MV, Muchow, D nd Schchern, P (988). Experimentl studies on round window structure: function nd permeility. Lryngoscope 98(6 Pt Suppl 44):. 9. Goycoole, MV, Muchow, DD, Sirvio, LM, Winndy, RM, Cnfx, DM nd Hue, M (99). Extended middle er drug delivery. Act Otolryngol Suppl 49: Lundmn, L, Bgger-Sjöäck, D, Juhn, SK nd Morizono, T (99). Pseudomons eruginos exotoxin A nd Hemophilus influenze type endotoxin. Effect on the inner er nd pssge through the round window memrne of the chinchill. Act Otolryngol Suppl 49: Green, M nd Loewenstein, PM (988). Autonomous functionl domins of chemiclly synthesized humn immunodeficiency virus tt trns-ctivtor protein. Cell 55: Frnkel, AD nd Po, CO (988). Cellulr uptke of the tt protein from humn immunodeficiency virus. Cell 55: Vocero-Akni, A, Lissy, NA nd Dowdy, SF (). Trnsduction of full-length Tt fusion proteins directly into mmmlin cells: nlysis of T cell receptor ctivtion-induced cell deth. Methods Enzymol : Schmidt, N, Mishr, A, Li, GH nd Wong, GC (). Arginine-rich cell-penetrting peptides. FEBS Lett 584: Schwrze, SR, Ho, A, Vocero-Akni, A nd Dowdy, SF (999). In vivo protein trnsduction: delivery of iologiclly ctive protein into the mouse. Science 85: Eguchi, A, Mede, BR, Chng, YC, Fredrickson, CT, Willert, K, Puri, N et l. (9). Efficient sirna delivery into primry cells y peptide trnsduction domin-dsrna inding domin fusion protein. Nt Biotechnol 7: Tkenou, T, Tomizw, K, Mtsushit, M, Li, ST, Moriwki, A, Lu, YF et l. (). Development of p5 protein trnsduction therpy using memrne-permele peptides nd the ppliction to orl cncer cells. Mol Cncer Ther : Futki, S (5). Memrne-permele rginine-rich peptides nd the trnsloction mechnisms. Adv Drug Deliv Rev 57: Futki, S (). Arginine-rich peptides: potentil for intrcellulr delivery of mcromolecules nd the mystery of the trnsloction mechnisms. Int J Phrm 45: 7.. Mtsushit, M, Tomizw, K, Moriwki, A, Li, ST, Terd, H nd Mtsui, H (). A highefficiency protein trnsduction system demonstrting the role of PKA in long-lsting long-term potentition. J Neurosci : Wender, PA, Mitchell, DJ, Pttirmn, K, Pelkey, ET, Steinmn, L nd Rothrd, JB (). The design, synthesis, nd evlution of molecules tht enle or enhnce cellulr uptke: peptoid moleculr trnsporters. Proc Ntl Acd Sci USA 97: 8.. Lindsy, MA (). Peptide-medited cell delivery: ppliction in protein trget vlidtion. Curr Opin Phrmcol : McIlwin, DR, Berger, T nd Mk, TW (). Cspse functions in cell deth nd disese. Cold Spring Hr Perspect Biol 5: Op de Beeck, K, Schcht, J nd Vn Cmp, G (). Apoptosis in cquired nd genetic hering impirment: The progrmmed deth of the hir cell. Her Res 8: Nicholson, DW, Ali, A, Thornerry, NA, Villncourt, JP, Ding, CK, Gllnt, M et l. (995). Identifiction nd inhiition of the ICE/CED- protese necessry for mmmlin poptosis. Nture 76: Slvesen, GS. () Cspses: cell signling y proteolysis. In: Hndook of Cell Signling. nd edn. Brdshw, RA nd Dennis, EA. Oxford, pp Thornerry, NA nd Lzenik, Y (998). Cspses: enemies within. Science 8: Wng, J nd Lenrdo, MJ (997). Molecules involved in cell deth nd peripherl tolernce. Curr Opin Immunol 9: