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Time course of efferent fiber and spiral ganglion cell degeneration following complete hair cell loss in the chinchilla

Time course of efferent fiber and spiral ganglion cell degeneration following complete hair cell loss in the chinchilla
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  Research report  Time course of efferent fiber and spiral ganglion cell degenerationfollowing complete hair cell loss in the chinchilla Sandra L. McFadden*, Dalian Ding, Haiyan Jiang, Richard J. Salvi Center for Hearing and Deafness, University at Buffalo, 215 Parker Hall, Buffalo, NY 14214, USA Accepted 20 October 2003 Abstract Ethacrynic acid (EA) is known to interact with aminoglycoside antibiotics such as gentamicin (GM). In the chinchilla, co-administrationof GM and EA can produce hair cell lesions ranging from a small loss of outer hair cells (OHCs) in the base of the cochlea to completedestruction of all hair cells, depending on dosing parameters. Although hair cell loss has been characterized, little is known about the fate of efferent fibers or spiral ganglion neurons (SGNs) in this model. To study the time course of efferent fiber and SGN loss, chinchillas wereinjected with GM (125 mg/kg IM) followed immediately by EA (40 mg/kg IV). Estimates of efferent fiber loss and density changes weremade after 3 days or 1, 2, 3, or 4 weeks of survival. Estimates of SGN loss and density changes were made after 15 days or 1, 2, 4, or 6months of survival. Cochlear function was rapidly abolished and all cochlear hair cells were missing within 24 h after treatment. Inner hair cells (IHCs) in the middle turn of the cochlea died earlier than cells in the apex or base, and OHCs in Rows 1 and 2 died earlier than OHCs inRow 3. Degeneration of efferent nerve fibers began 3–7 days post-injection, versus 15–30 days for SGNs, and the loss of efferent fibers wasessentially complete within 1 month, versus 2–4 months for SGNs. The rapid time course of efferent fiber and SGN loss in the chinchillamay make it a practical model for studying mechanisms of neural loss and survival in the mammalian inner ear. D  2003 Elsevier B.V. All rights reserved. Theme:  Development and regeneration Topic:  Neuronal death/sensory systems  Keywords:  Aminoglycoside antibiotic; Loop diuretic; Ototoxicity; Inner ear pathology; Ganglion neuron; Nerve fiber; Cochlea 1. Introduction Research on topics related to the protection, rescue andrepair of neurons and their processes is aided by the use of animal models in which neural degeneration occurs in a predictable manner over a well-defined time course. Onemethod for producing an animal model of spiral ganglionneuron (SGN) and nerve fiber degeneration in the cochleais to destroy cochlear hair cells by administering a largedose of an aminoglycoside antibiotic (AAB) and ethacrynicacid (EA) [14–16,21,24–26]. SGNs degenerate followingIHC destruction, with a time course that depends on factorssuch as species and magnitude of IHC loss. The AAB/EAdeafening procedures that have been used with cats,monkeys and guinea pigs typically result in variable andincomplete hair cell loss, thereby confounding studies of SGN degeneration and treatments aimed at preservingthem. Furthermore, SGN degeneration is a slow processthat can take more than a year to complete in cats andguinea pigs [15,21,25]. The slow time course and variable magnitude of SGN degeneration can be a major drawback for studies of neural death and survival mechanisms. Werecently described a GM/EA deafening procedure that reliably destroys all cochlear hair cells in chinchillas [17].In these animals, we also observed rapid degeneration of SGNs and nerve fibers in the cochlea, suggesting that thechinchilla may be a practical model for studies of neural protection, rescue and repair. The current study describesthe time course of SGN and nerve fiber degeneration in thechinchilla cochlea following a single injection of GM (125mg/kg IP) and EA (40 mg/kg IV) that rapidly destroyedhair cells and abolished cochlear function. We were par-ticularly interested in comparing the time course of efferent  0006-8993/$ - see front matter   D  2003 Elsevier B.V. All rights reserved.doi:10.1016/j.brainres.2003.10.031* Corresponding author. Tel.: +1-716-829-2001x13; fax: +1-716-829-2980.  E-mail address: (S.L. McFadden) Research 997 (2004) 40–51  fiber degeneration and afferent fiber/SGN degenerationcaused by the GM/EA combination, given speculationabout a trophic role of lateral olivocochlear (LOC) efferent fibers in the cochlea [18]. 2. Materials and methods 2.1. Subjects Subjects were 41 healthy adult chinchillas obtained froma commercial breeder. Four chinchillas were prepared for electrophysiological recording by implanting chronic re-cording electrodes at the round window niche (see below).Thirty-six chinchillas, including the four animals with roundwindow electrodes, were anesthetized with ketamine (55mg/kg IM) and acepromazine (0.5 mg/kg IM), then injectedwith GM (gentamicin sulfate, Sigma Chemicals; 125 mg/kgIM), followed immediately by EA (Sodium Edecrin R ,Merck & Co., Inc., West Point, PA; 40 mg/kg into the right  jugular vein). For counts of efferent nerve fibers, animalswere sacrificed after survival times of 3 days or 1, 2, 3, or 4weeks (  N  =15, three animals at each time point). For countsof SGNs and examination of nerve fibers in the habenula perforata of the osseous spiral lamina (OSL), animals weresacrificed after survival times of 15 days or 1, 2, 4, or 6months (  N  =15, three animals at each time point). Sixchinchillas were sacrificed at 6, 12 or 24 h post-injectionfor verification of rapid hair cell loss [17]. Five untreatedchinchillas served as normal controls for SGN counts. 2.2. Electrophysiology Compound action potentials (CAP), cochlear microphon-ics (CM) and summating potentials (SP) were monitored infour chinchillas to observe the time course of decline of cochlear function following GM/EA injection. The chin-chillas were prepared for electrophysiological recording byimplanting a Teflon-coated silver wire ball electrode at theround window niche of the right ear under surgical anes-thesia (ketamine, 55 mg/kg IM; acepromazine, 0.5 mg/kgIM). Animals were allowed to recover for at least 1 week  prior to GM/EA injection. Stimuli were digitally generatedtones at 1, 2, 4 and 8 kHz, presented at a rate of 19 per sfrom a loudspeaker located 25 cm directly in front of theanimal in a double-walled sound attenuating booth. For CAP, tones were 7 ms duration, 1 ms rise/fall; for CM andSP, tones were 17 ms duration, 1 ms rise/fall. Electricalactivity recorded from the round window electrode wasamplified (20,000   for CAP and CM, 5000   for SP),filtered (100–3000 Hz for CAP; 10–10,000 for CM; 1–300Hz for SP), digitized by an A/D converter on a signal processing board in a computer, and averaged for 100 presentations at each stimulus level. Stimulus level wasincremented in 10 dB steps, from 0 to 70 dB SPL for CAP,from 50 to 90 dB SPL for CM, and from 40 to 90 dB SPLfor SP. Responses were recorded prior to GM/EA injection,and at 0.5, 1.0, 1.5, 2.0, 24, and 72 h following injection. 2.3. Morphological assessment  Animals were sacrificed with CO 2  and decapitated, andthe bullae were quickly removed. As described in detail byAzeredo et al. [1], two distinct populations of olivocochlear efferent fibers, referred to as LOC and medial olivocochlear (MOC) efferents, innervate the chinchilla cochlea. The vast majority of olivocochlear efferent fibers are small, unmy-elinated fibers that arise from neurons located within thelateral superior olivary nucleus; these LOC efferent fibers project almost exclusively to the ipsilateral cochlea, wherethey form synapses with the afferent fibers beneath the IHCs.A smaller number of olivocochlear efferent fibers are large,myelinated fibers that arise from neurons located in themedial region of the superior olive, particularly the dorso-medialperi-olivarynucleus;theseMOCfibersprojecttobothcochleas (in a 1:4 ipsilateral to contralateral ratio in chinchil-la), lose their myelin at the habenula perforata, and cross thetunnel of Corti to form synapses directly with the OHCs.For determination of LOC and MOC efferent fiber loss,cochleas were prepared using acetylcholine esterase (AChE)histochemistry as described previously [2,30]. In AChE stained cochleas, MOC fibers can be seen crossing thetunnel of Corti en route to the OHCs, whereas LOC fiberstravel in the inner spiral bundle (ISB) in the IHC region (seeFig. 5A). Cochleas were first perfused with 4% paraformal- Fig. 1. Surface preparation from the middle turn of a normal cochlea,‘‘stained’’ with silver nitrate for hair cell counting. Note the presence of three rows of OHCs (OHC1, OHC2, OHC3) and one row of IHC, separated by pillar cells (PC). The arrow shows where one OHC is missing from thethird row. OHC were replaced by a phalangeal scar (arrow) formed bysupporting cells. Scale bar=20  A m. S.L. McFadden et al. / Brain Research 997 (2004) 40–51  41  dehyde in 0.1 M phosphate buffer (pH 7.2) for 30 min to 1h, then dissected in 70% ethanol to remove the organ of Corti as a flat surface preparation. The organ of Corti was pre-incubated in 0.1 M acetate buffer (pH 6.0) for 30 min,then incubated in AChE staining solution for 1 h. Specimenswere washed three times with acetate buffer, then treatedsequentially (1 min in each solution, at room temperature)with 1 ml of 1% ammonium sulfide in distilled water; fivewashes of 0.1 M sodium nitrate; 1 ml of 0.1% silver nitratein distilled water; five washes of 0.1 M sodium nitrate andthree washes of 0.1 M acetate buffer. Specimens weremounted in glycerin on glass slides and examined under alight microscope.AChE-positive tunnel crossing nerve fibers (MOC effer-ents) were counted in successive 0.24 mm segments from theapex of the cochlea to the base. Counts were made at a point close to the outer spiral bundle toward the medial edge of thetunnel, where individual MOC fibers are more easily distin-guished before they converge to form the tight bundle that isvisible in the middle of the tunnel. Counts from each ear were entered into a software program that computed fiber density (fibers per mm) in 10% segments from apex to base(thereby compensating for differences in overall cochlear length among animals). Averages for each group (normal, 3days, 1, 2, 3 and 4 weeks) were computed separately for apical and basal halves of the cochlea for comparisons of MOC fiber density as a function of group and cochlear region. MOC efferent fiber loss was estimated by comparingdata from GM/EA-treated chinchillas to our laboratorynorms based on counts from five normal chinchillas. Efferent fibers were counted in both cochleas of each animal, then anaverage loss was computed for each animal. Statistical testsare, therefore, based on the number of animals in each grouprather than the number of ears examined. Fig. 3. Mean cochleogram (  N  =3 ears) showing the percentage of inner hair cells (IHC) and OHCs in Rows 1, 2 and 3 (OHC1, OHC2, OHC3) missing12 h after GM/EA treatment. Note that IHC loss (solid line) was greater inthe middle region of the cochlea than in the base or the apex. Based on thefrequency–place map of Greenwood [9], the place where IHC loss peaked(approximately 55% distance from the apex) transduces energy around 2kHz. Note also that OHC loss in Row 3 (heavy dashed line) lags behindOHC loss in Rows 1 or 2 (light dashed and dotted lines, respectively).Standard deviations of each 10% segment are as follows: IHC, 2.51, 10.59,17.43, 7.17, 15.11, 7.05, 0, 0, 5.67, 0; OHC1, 9.06, 0.60, 0, 0, 0, 0, 0, 0, 0,0; OHC2, 2.68, 1.21, 0, 0, 0, 0, 0, 0, 0, 0; OHC3, 5.07, 16.37, 0.89, 4.26,7.12, 5.87, 0, 0, 0, 0.Fig. 2. Mean input/output functions for the CAP (A), CM (B), and SP (C)for 4 kHz stimuli, measured before and at various times after GM/EAinjection. Note the rapid decrease in response amplitude within 30 min post-injection, and the transient partial recovery of amplitudes at 1.5 h.Responses at other tested frequencies (1, 2 and 8 kHz) were also abolishedwithin 24 h after injection. Bars show S.E.M. S.L. McFadden et al. / Brain Research 997 (2004) 40–51 42  It is not possible to count individual AChE-labeled fibersin the ISB, and both cochlear afferents and medial efferent tunnel crossing fibers (TFC) can be interspersed among ISBfibers [12]; therefore, LOC efferent loss could only beroughly estimated from our material, based on changes inthe width of the ISB over time. The ISB was examined in10- A m long samples (one from the apical turn and one fromthe basal turn of the cochlea), and measurements were takenat the narrowest and widest points along this segment.Average ISB widths were computed for each group basedon 5–10 samples per group.For counts of SGNs in Rosenthal’s canal and examina-tion of nerve fibers in the OSL, the cochleas were perfusedthrough the round window with approximately 1 ml of 4% paraformaldehyde in 0.1 M phosphate buffered saline (pH7.4), then immersed in fixative for approximately 24 h. Thefixed cochleas were decalcified by immersion in Decal(Baxter Scientific Products) for 2 days, dehydrated, andembedded in Epon 812 resin. Serial sections were cut in a plane parallel to the modiolus at a thickness of 3  A m(Reichert Super Nova microtome), mounted on slides andstained with toluidine blue [2]. For estimates of SGNnumbers, every neuron with a nucleus was counted in everyfifth section through the modiolus [26]. To sample the entirearea of Rosenthal’s canal, approximately 40–50 sections per modiolus were examined. Counts were obtained from fivenormal animals, and from 15 animals treated with GM andEA. To estimate the density of SGNs, modiolar sectionsfrom the apical and basal turns of the cochlea were exam-ined. The number of SGNs in a 0.2-mm diameter visual Fig. 4. Structure of the organ of Corti at various times after the loss of all cochlear hair cells (all sections from the lower middle turn). (A) Cross section throughthe normal organ of Corti shows OHCs from rows 1, 2 and 3 (OHC1, OHC2, OHC3), supporting cells (Dieter’s cells (DC) beneath the OHCs, Hensen’s cells(HC), and outer pillar cells (OPC) bordering the tunnel of Corti (asterisk)), and an inner hair cell (IHC). The tectorial membrane (TM) extends over the top of thehair cells. B–F. Cross sections at various times after GM/EA injection: (B) 2 days; (C) 15 days; (D) 1 month; (E) 2 months and (F) 4 months. Scale bar=10  A m. S.L. McFadden et al. / Brain Research 997 (2004) 40–51  43  field was counted for five samples from the apical turn, andfive samples from the basal turn of each cochlea. Groupaverages for the number of SGNs per square millimeter, based on two to five animals per group, were computedseparately for the apical and basal turns. In addition, everyfifth section containing habenula perforatae was examinedto determine the condition of nerve fibers in the OSL in themiddle turn. Because most nerve fibers were absent even at 2 weeks post-injection, counts of habenular nerve fibers of GM/EA-treated animals were not made. The nerve fiber counts from normal control animals that are presented in this paper were obtained from six normal chinchillas as part of aseparate study [4]. Nerve fibers passing through 10 basal, 10middle turn, and 10 apical openings in the OSL of eachcochlea were counted and averaged for the group.The opposite cochlea of each animal was used to confirmhair cell loss, using succinate dehydrogenase (SDH) histo-chemistry as described previously [2]. The round and ovalwindows were opened and 0.2 M sodium succinate in 0.1 M phosphate buffer (pH 7.4) was slowly perfused through theround window. The cochlea was immersed in sodiumsuccinate solution for 1 h at 37  j C, then in 10% formalde-hyde for 24 h. The organ of Corti was dissected out,mounted in glycerin as a flat surface preparation, andexamined under a light microscope at 400   magnification. No hair cells were present in any cochlea examined.A separate group of six animals was used to examine thetime course of hair cell loss following GM/EA treatment.Cochleas from these animals were removed at 6, 12, or 24h after GM/EA treatment and prepared as described previ-ously [2]. Briefly, cochleas were perfused through the roundwindow with 0.5% silver nitrate in phosphate buffered saline(pH 7.4), then fixed in 10% formalin for 2 h. The organ of Corti was dissected out and mounted on a glass slide as a Fig. 5. Surface preparations from the lower middle turn of the organ of Corti showing AChE-positive (efferent) fibers in the normal cochlea (A) and the progressive loss of efferent fibers after GM/EA injection (B–F). In A, thinner white arrow points to TCF that are MOC efferents crossing the tunnel of Corti;large white arrow points to the ISB which contains LOC efferent fibers. Scale bar=10  A m. S.L. McFadden et al. / Brain Research 997 (2004) 40–51 44
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