The Laiyngoscope zyxwvutsrqponm Lippincott Williams zyxwvutsrqpon & Wilkins, Inc., Philadelphia zyxwvutsrqp 0 1999 The American Laryngological, Rhinological and Otological Society, Inc. Glutamate-like Immunoreactivity During Hair Cell Recovery After Gentamicin Exposure in the Chinchilla Vestibular Sensory Periphery Kenley W. Chin, MD; Ivan Lopez, PhD; Seung-Chul Lee, MD, PhD; Vicente Honrubia, MD, DMSc Objective: Determine the expression of glutamate by immunohistochemistry in normal and recovering vestibular hair cells in the chinchilla crista amp- zyxwv ullaris after gentamicin ototoxicity. Study Design: In five groups of three animals each, ototoxicity was pro- duced by placing gentamicin zyxwvu (50 pg)-impregnated Gelfoam pellets within the perilymphatic space of the superior semicircular canal. Animals were sacrificed at 1,2,4,8, and 16 weeks after treatment. A group of normal (n=3) animals was also processed. Methods: For the detection of glutamate the inner ears of these animals were dissected, and the horizontal cristae ampullaris embedded in plastic. Two-micron-thick tissue sections were obtained and incubated with monoclonal antibodies against glutamate. The immu- noreaction was detected using the avidinbiotiny- lated-complex technique and diaminobenzidine was the chromogen. Results: Normal sensory epithelia demonstrated type I and type I1 hair cells with mod- erate glutamate-like immunoreactivity. Supporting cells demonstrated no glutamate-likeimmunoreactiv- ity. Afferent nerve fibers and calyxes surrounding type I hair cells demonstrated strong glutamate-like immunoreactivity. At 1 and 2 weeks after treatment the few type I1 hair cells surviving ototoxic treatment (15O/'a-18%) contained moderate glutamate-like immu- noreactivity, supporting cells showed no immunore- activity, and nerve terminals and fibers displayed strong immunoreactivity. At zyxwvut 4 and 8 weeks after treat- ment, recovered hair cells (80%) had greater - ~~~- ... Presented at the Meeting of the Western Section of the American Laryngological, Rhinological and Otological Society, Inc., Denver Colorado, January 9, 1999, and at the Southern California Resident Research Sym- posium, San Diego, California, April 26, 1997. From the Division of Head and Neck Surgery, Department of Sur- gery, University of California at Los Angeles, School of Medicine, Los Angeles, California. This work was supported by NIWNIDCD grants DC00008, DC02952. and the Oberkotter Foundation. Editor's Note: This Manuscript was accepted for publication March 31, 1999. Send Correspondence to Vicente Honrubia, MD, DMSc, Director of Research, Victor Qnodhill Ear Center, 1000 Veteran Avenue, Los Angeles, CA 90095, U.S.A. glutamate-like immunoreactivity when compared with normal hair cells, supporting cells displayed no glutamate-like immunoreactivity, and afferent fibers contained strong glutamate-like immunoreactivity. At 16 weeks, glutamate-like immunoreactivity in hair cells returned to normal level. Conclusion: Glutamate may be used as an indicator of hair cell differentia- tion and as an index of the molecular recovery of hair cells after ototoxicity. Key Words: Crista ampullaris, gentamicin, hair cell recovery, glutamate-like, immunoreactivity. Laiyngoscope, 109:1037-1044,1999 INTRODUCTION Glutamate is the afferent vestibular hair cell neuro- transmitter based on physiological, pharmacological, and molecular biological studies in different vertebrate spe- cies. Glutamate-like immunoreactivity has been localized to various components of the hair cell-vestibular afferent nerve synapse including hair cells (in frogs,l mice,2 rat~,~-4 and cats2 ), afferent nerve fibers (in frogs,' mice,2 rats,"4 and cats2 1, and Scarpa's ganglion (in frogs,5mice,2 rats,2,3-5 and cats2S6 ). Hair cells are the sensory receptors of the vestibular organs. Aminoglycoside antibiotic exposure is one of sev- eral known causes of vestibular hair cell damage. Previous studies have found that autogenous production of vestib- ular hair cells during postembryonic life does not appear to occur in the mammalian sensory neuroepithelium;7 however, recent anatomic studies suggest that mamma- lian hair cell recovery does occur in the vestibular end organs following injury.8-13 Our laboratory has developed a mammalian model to study the process of hair cell recovery. In this model, a majority of type I1 hair cells and a minority of type I hair cells recover following gentamicin vestibulotoxic exposure.10-l2 During hair cell recovery, newly formed hair cells expe- rience a process of maturation, differentiation, and reinner- vation.10-12 To fulfill their physiological role, new hair cells must develop the capacity to produce neurotransmitters. This study uses immunohistochemistry to identify the puta- Laryngoscope 109: July 1999 Chin et al.: Glutamate lmmunoreactivity During Hair Cell Recovery 1037