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The LaryngoscopeVC 2009 The American Laryngological,Rhinological and Otological Society, Inc.

Anti-Intercellular Adhesion Molecule-1Antibody’s Effect on Noise Damage

Michael D. Seidman, MD, FACS; Wenxue Tang, MD; Najeeb Shirwany, MD; Uma Bai, PhD;

Cory J. Rubin, MD; Joseph P. Henig, MS; Wayne S. Quirk, PhD

Objectives/Hypothesis: The purpose of thisstudy was to investigate possible preventive effects ofanti-intercellular adhesion molecule-1 antibody (anti-ICAM-1 Ab) on noise-induced cochlear damage asassessed by changes in auditory thresholds and coch-lear blood flow.

Study Design: A controlled animal study. Pre-treated rats with anti-ICAM-1 Ab or saline control, fol-lowed with exposure to 72 continuous hours of broadband noise (107 dB SPL), and 24 hours after noise ex-posure treated again with anti-ICAM-1 Ab or saline.

Methods: Eighteen healthy male Fischer rats(200–250 g) were used. Sixteen were randomlyselected to study noise-induced temporary thresholdshifts. The remaining two rats were used to studycochlear blood flow (CBF), using laser Doppler flow-metry and blood pressure measurements.

Results: Rats treated with anti-ICAM-1 Ab(1.875 mg/kg, intravenously) showed attenuated tem-porary threshold shifts (TTS) compared to controls.Both groups showed a partial threshold recovery 72hours following noise exposure, normal for this noiseexposure paradigm. Comparisons of baseline andpost-treatment measurements of CBF and mean arte-rial blood pressure revealed no significant changes.Anti-ICAM-1 Ab animals displayed significantly lower

mean auditory threshold shifts at all five test fre-quencies (P < .05) when compared to control.

Conclusions: Blocking the cascade of reactiveoxygen species (ROS) generation by using anti-ICAM-Ab protects against noise-induced hearing loss.

Key Words: ICAM-1, noise-induced hearing loss,temporary threshold shifts, rat.

Laryngoscope, 000:000–000, 2009

INTRODUCTIONIntense noise exposure initiates a cascade of events,

which ultimately results in cell death and cochlear dam-age. A significant body of evidence has revealed thatnoise exposure produces profound cochlear microcircula-tory changes, including hypoperfusion and ischemia.Studies using intravital microscopy, Laser Doppler flow-metry, and microcast techniques have demonstratedreduced cochlear blood flow, decreased red blood cell ve-locity, capillary constriction, and increased vascularpermeability during noise exposure.1–3 The oxidativestress that results from hypoxia and ischemia producesa variety of damaging reactive oxygen species (ROS),which includes hydrogen peroxide, the superoxide anion,and the hydroxyl radical.4,5 The accumulation of ROS,cytokines, and chemokines that are associated with hy-poxia and ischemia promotes the expression ofintercellular adhesion molecule-1 (ICAM-1) in endothe-lial cells, which subsequently leads to neutrophil-endothelial cell adhesion.6 This process results inincreased circulating tissue levels of various cytokines,leukotrienes, thromboxanes, platelet activating factor,complement components, elastases, and other enzymes,as well as additional formation of ROS. Typically, theeffects of these molecules and their activation are delete-rious to the cells and tissues involved. Some of thesesubstances up regulate leukocyte adhesion receptors,increase vascular permeability, damage tissues directly,and impair endothelial function, which leads to edema,vascular insufficiency, and ultimately necrosis.7

Previous studies have demonstrated that anti-oxidants and scavengers of ROS attenuate ischemia/reperfusion-induced and noise-induced cochlear dam-age.4,8 Furthermore, anti-ICAM-1 Ab has been shown to

From the Department of Otolaryngology (W.T.), Emory UniversitySchool of Medicine, Atlanta, Georgia, U.S.A., Department ofOtolaryngology (M.D.S., U.B., C.J.R.), Henry Ford Health System, Detroit,Michigan, U.S.A., Department of Physiology (N.S.), Oklahoma UniversityHealth Sciences Center, Oklahoma City, Oklahoma, U.S.A., BurnhamInstitute for Medical Research (J.P.H.), La Jolla, California, U.S.A., andDepartment of Psychology (W.S.Q.), Central Washington University,Ellensburg, Washington, U.S.A.

Editor’s Note: This Manuscript was accepted for publicationNovember 11, 2008.

This research was performed at Henry Ford Health System,Department of Otolaryngology, under the supervision of Dr. Michael D.Seidman.

This research was presented as a poster presentation at the Asso-ciation for Research in Otolaryngology Mid-Winter Meeting, St. Peters-burg, Florida, U.S.A. on February 4, 2001. Funding was supported inpart by the following NIH grants: NIDCD-DC00101-05 (MDS),1R21AT001067-01-03, NCCAM, July 2003–2005. Pharmacia & UpjohnCompany donated the anti-ICAM-1 antibody.

Send correspondence to Wenxue Tang, MD, Henry Ford HealthSystem, Department of Otolaryngology – Head & Neck Surgery, Divisionof Otology and Neurotology, 6777 West Maple Road, West Bloomfield, MI48323. E-mail: [email protected]

DOI: 10.1002/lary.20109

J_ID: LARY Customer A_ID: 08-0176.R1 Date: 2-February-09 Stage: Page: 1

ID: kumarpr I Black Lining: [ON] I Time: 19:21 I Path: //xinchnasjn/01Journals/Wiley/3b2/LARY/Vol00000/080117/APPFile/C2LARY080117

Laryngoscope 000: Month 2009 Seidman et al.: Anti-ICAM-1 Ab and Noise Damage

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attenuate damage in brain, heart, kidney, and other tissueswhere ICAM-1 plays a critical role in ischemia-induceddamage.9 Prior studies have shown that ICAM-1 also playsan important role not only in middle ear diseases, includingotitis media10 and cholesteatoma,11 but also in inner earinflammation12 and carcinoma of the head and neck.13 Thepurpose of our study was to assess the possible protectiveeffect of anti-ICAM-1 Ab on noise-induced cochlear damageby evaluating noise-induced TTS.

MATERIAL AND METHODS

SubjectsA total of 18 healthy male Fischer rats (200–250 g) were

used in this study. Sixteen animals were used to study noise-induced TTS as determined by auditory brainstem response(ABR) measurements. These animals were randomly assignedto either the anti-ICAM-1 Ab treatment group (n ¼ 8) or thecontrol group (n ¼ 8). The remaining two rats were used tostudy Cochlear blood flow (CBF) using laser Doppler flowmetryand blood pressure measurements. The use of experimental ani-mals for this study was approved by the Care for ExperimentalAnimals Committee at Henry Ford Health System. Additionally,all procedures were conducted in strict compliance with theNational Institutes of Health guidelines for experimental ani-mal subjects.

ABRBaseline ABR measurements were obtained from 16

healthy male Fischer rats. Animals were anesthetized with amixture of ketamine and xylazine (100 mg/kg and 15 mg/kg,respectively), given subcutaneously. Auditory stimuli were gen-erated using a D/A converter (Model DA3-2; Tucker-DavisTechnologies (TDT), Gainesville, FL) with a sampling frequencyof 100 kHz. The output of the D/A converter was connected to aprogrammable attenuator (Model PA4; TDT), a weightedsummer (Model SM3; TDT), a headphone buffer (Model HB6;TDT), and an earphone (Model DT-48; Beyer Dynamic, Heil-bronn, Germany), which was placed in close approximation tothe tympanic membrane of the animal. The stimuli consisted oftone bursts with a rise/fall time of 1 ms, a duration of 15 ms,and a period of 100 ms. A series of stimuli were produced at 3,6, 9, 12, and 18 kHz test frequencies with intensities rangingfrom 5–100 dB SPL in 5 dB increments. The system was cali-brated at the tympanic membrane using a probe microphone(Model ER-7C; Etymotic Research, Elk Grove Village, IL),which was connected to an A/D converter (Model AD2, TDT)and a computer. Automated calibrating routines were used foronline calibration?

ABRs were collected using subcutaneous electrodes (ModelE2; Grass Instruments, Quincy, MA) and placed at the vertex andunder both pinnae of each animal. This output was channeledinto a biologic amplifier (Model P5 Series; Grass Instruments)with a gain of �100,000. The response was filtered between 0.3and 3.0 kHz, and then the output was sent to an A/D converter(Model AD2; TDT). Custom-designed software allowed theseresponses to be displayed with a sampling rate of 50 kHz in realtime on a computer monitor. For each recording, a 20-ms neuralresponse was averaged 1,024 times. For each of the five test fre-quencies, auditory thresholds were determined by identifying thesmallest intensity (in dB SPL) at which ABR wave forms becameevident.

Following baseline ABR testing, treatment group animals(n ¼ 8) were intravenously infused with 1.25 mg/kg of anti-ICAM-1 Ab (Gift of Discovery Research, Upjohn Co., Kalama-

zoo, MI) over a 2-minute interval, while control group animals(n ¼ 8) were infused in the same manner with an equivalentvolume of the vehicle control (saline). Animals were then imme-diately placed in an acoustically-insulated noise booth(Industrial Acoustics, New York, NY) and exposed to 107 dBSPL broadband noise for 72 continuous hours. Twenty-fourhours after the onset of the noise exposure, the treatment groupanimals received an additional half-dose of anti-ICAM-1 Ab(0.625 mg/kg), while the control group animals received anequivalent volume of saline. Caging was designed to be acousti-cally transparent, and calibration measurements throughoutthe cage revealed a variance of 0.4–0.8 dB from the front of thecage to the back. To determine TTS, ABR measurements wererecorded 10 minutes after the completion of the noise exposure.Final ABR measurements were recorded 72 hours after thenoise exposure.

CBFCBF experiments were conducted on two healthy male Fi-

scher rats. The animals were anesthetized as previouslydescribed. A small opening was made in the right bulla and a24-gauge laser Doppler needle probe was positioned over the ba-sal turn of the cochlea. A small amount of petroleum jelly wasapplied to the tip of the laser probe to enhance optical couplingof the laser signal to the otic capsule. The hole in the bulla wasthen covered with bone wax. A laser Doppler flow meter (BLF21D; Transonic Systems Inc., Ithaca, NY) was used to recordCBF measurements (represented as tissue perfusion units orTPU). Mean arterial blood pressure (BP) was monitored by ablood pressure transducer connected to a catheter that wasinserted into the left femoral artery. Baseline CBF and BPmeasurements were recorded at 1-minute intervals for 33minutes. anti-ICAM-1 Ab (1.25 mg/kg) was then intravenouslyadministered. CBF and BP measurements were continued foran additional 75 minutes.

RESULTS

ABRABR measurements were recorded at five test fre-

quencies (3, 6, 9, 12, and 18 kHz) as previouslydescribed.14 Baseline ABR measurements obtained priorto anti-ICAM-1 Ab treatment and noise exposure indi-cated that there were no significant differences in meanauditory thresholds between the treatment and controlgroups (Fig. F11). Ten minutes following exposure to72 hours of continuous 107 dB SPL broadband noise,anti-ICAM-Ab (1.25 mg/kg) treatment group animals dis-played significantly lower mean auditory thresholdshifts at all five test frequencies (3 kHz (P ¼ .02), 6 kHz(P ¼ .047), 9 kHz (P ¼ .042), 12 kHz (P ¼ .0095) and18 kHz (P ¼ .0098)) when compared to control (Fig. F22).Seventy-two hours following noise exposure, a reductionin mean auditory threshold shifts was observed in boththe treatment and control groups (Fig. F33), indicating apartial recovery of auditory sensitivity. Intergroup com-parison showed little or no significant differences inauditory threshold shifts between treatment and control.This type of recovery is characteristic of the noise expo-sure paradigm, which primarily produces TTS.




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CBFCBF and BP measurements were obtained as pre-

viously described. FigureF4 4 depicts CBF shifts (repre-sented as a percentage change from mean baseline CBF)and FigureF5 5 depicts a BP recording from a representa-tive animal taken over a period of 108 minutes. Nosignificant changes in CBF or mean BP were observedbetween baseline and post-treatment measurements.

DISCUSSIONThe premise of this study was that intense noise ex-

posure causes reduced CBF and ischemia, leading to theproduction of ROS. The accumulation of ROS then pro-motes the expression of ICAM-1 to initiate a cascade ofevents, which ultimately leads to cochlear damage. ABRresults indicated that noise-induced TTS could be signifi-cantly attenuated by administering anti-ICAM-1 Abintravenously (Fig. 2). This protective effect suggests amechanism of inflammatory prevention, whereby anti-ICAM-1 Ab prevents ICAM-1 from eliciting a deleteriouseffect that would otherwise lead to cochlear damage.

Significant data exists showing that ROS promotethe expression of ICAM-1 in endothelial cells and subse-quently neutrophil-endothelial cell adhesion.6,15 Forexample, hydrogen peroxide-induced polymorphonuclearneutrophil (PMN) adhesion is dependent on the rapidinduction of the ICAM-1 mRNA signal and the surfaceexpression of ICAM-1 on the endothelial cell.15 Inaddition, hydrogen peroxide-induced expression ofhyperadhesivity may amplify PMN attachment to theendothelium.15 Furthermore, one study demonstrated

that: 1) ischemia in vivo followed by reperfusion in iso-lated perfuse kidneys resulted in neutrophil retention; 2)retained neutrophils adversely affect renal function; and

Fig. 1. Baseline auditory brainstem response thresholds. Mean au-ditory threshold levels of treatment group rats (dashed line) andcontrol group rats (solid line) at five test frequencies (3, 6, 9, 12,and 18 kHz). Measurements were obtained prior to treatment withanti-intercellular adhesion molecule-1 antibody (anti-ICAM-1 Ab)and noise exposure. SPL ¼ sound pressure level.

Fig. 2. Auditory threshold shifts (post-noise: 10 min.). Mean audi-tory threshold shifts of anti-intercellular adhesion molecule-1 anti-body (anti-ICAM-1 Ab) (1.25 mg/kg) treatment group rats (dashedline) exposed to 72 hours of continuous 107 dB sound pressurelevel (SPL) broad band noise and control (solid line). Measure-ments were obtained 10 minutes following noise exposure at fivetest frequencies (3, 6, 9, 12, and 18 kHz).

Fig. 3. Auditory threshold shifts (post-noise: 72 hours). Mean audi-tory threshold shifts of anti-intercellular adhesion molecule-1 anti-body (anti-ICAM-1 Ab) (1.25 mg/kg) treatment group rats (dashedline) exposed to 72 hours of continuous 107 dB sound pressurelevel (SPL) broad band noise and control (solid line). Measure-ments were obtained 72 hours following noise exposure at fivetest frequencies (3, 6, 9, 12, and 18 kHz).




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3) neutrophil retention was dependent on ICAM-1 andoxygen metabolites.6 These results indicate that theeffect of hydrogen peroxide (i.e., ROS) in instigating neu-trophil adhesion is mediated by ICAM-1. Utilizing thisknowledge, studies were conducted to show that ROSscavengers and blockers can attenuate ischemia/reperfu-sion-induced and noise-induced cochlear damage,4,8

presumably by preventing ROS from inducing theexpression of ICAM-1. Our results suggest that anti-ICAM-1 Ab has a similar effect in preventing noise-induced cochlear damage. This effect is mediated byblocking ICAM-1 and thereby attenuating the inflamma-tory cascade. Inflammatory damage time course of thecochlea is consistent with the time courses in othertissues.16

Although the effect of anti-ICAM-1 Ab attenuatesnoise-induced cochlear damage, it was not completelyprotective. This may be due to inappropriate concentra-tion or timing of drug administration or possibly becauseother adhesion molecule pathways play a role in noise-induced hearing loss. ICAM-1 is a single polypeptidechain (MW ¼ 90 kD) that is glucosulated.17 It has fiveimmunoglobulin homology domains and is closely relatedto neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG). The primary sites ofICAM-1 expression are the microvasculature, peritubu-lar capillaries, and the vascular endothelium of largevessels. In addition, ICAM-1 is expressed by leukocytes.ICAM-2, on the other hand, is a glycoprotein of approxi-mately 60 kD with only two immunoglobulin-likedomains. It is constitutively expressed on endothelial

cells, but unlike ICAM-1, it is not up regulated by cyto-kines. ICAM-2 is a ligand for CD11a/CD18 but not forthe other B2-integrins. Another member of the immuno-globulin superfamily important for leukocyte-endothelialcell interactions is vascular cell adhesion molecule-1(VCAM-1), which interacts with the a4b1-integrin(VLA4) on mononuclear leukocytes. Neutrophils do notexpress VLA4. Other members of the immunoglobulinsuperfamily such as CD31 and mucosal addressing celladhesion molecule-1 (MAdCAM-1) may also contribute toleukocyte-endothelial cell interactions.

The critical role of ICAM-1 and neutrophil-endothe-lial cell adhesion in ischemic injury is strengthened,however, by several studies. Mutant mice that do notexpress ICAM-1 are protected against ischemic injury.17

Furthermore, antibodies directed against ICAM-1 havebeen shown to protect against ischemic injury.9,17

Another study demonstrated that ICAM-1-deficient andPMN-depleted mice were relatively resistant to cerebralischemia/reperfusion injury.17 Depletion of neutrophilsalone results in protection against ischemia-induced def-icits in renal function, and although administration ofanti-ICAM-1 Ab has been found to be protective, itdoes not confer protection beyond that observed withneutrophil depletion.17 Anti-ICAM-1 Ab added to apulmonary flush and initial reperfusate resulted in adose-dependent enhancement of the reperfused lung’sability to oxygenate blood, possibly as a result ofdecreased leukocyte sequestration.18 Thus, it can be con-cluded that the recruitment of circulating PMN intoischemic tissue initially requires the interaction of mi-crovascular endothelial cells with circulating leukocytes

Fig. 4. Cochlear blood flow (CBF) shifts from a representative ani-mal, where the dotted line depicts the 33-minute baseline CBFshifts and the solid line depicts CBF shifts following intravenousadministration of anti-intercellular adhesion molecule-1 antibody(anti-ICAM-1 Ab) (1.25 mg/kg). The simple spline curve was gener-ated from data points separated by 1-minute intervals.

Fig. 5. Blood pressure (BP) recording from a representative ani-mal, where the dotted line depicts the 33-minute baseline BP andthe solid line depicts BP following intravenous administration ofanti-intercellular adhesion molecule-1 antibody (anti-ICAM-1 Ab(1.25 mg/kg). The simple spline curve was generated from datapoints separated by 1-minute intervals.




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through specific intercellular adhesion molecules, suchas ICAM-1.F1

Studies have also shown that ICAM-1 plays an im-portant role in diseases of the head and neck, includingotitis media,10 cholesteatoma,11 inner and middle earinflammation,12,19,20 nasal polyposis,21,22 and carcinomaof the head and neck.13,23 Systemic administration ofanti-ICAM-1 antibody in experimental immune-mediatedlabyrinthitis showed reduced inflammatory cell infiltra-tion in the scala tympani and the perisaccular tissue ofthe endolymphatic sac.24 Our results depicting the pro-tective effect of anti-ICAM-1 Ab on noise-induced TTSimply that ICAM-1 also plays a role in noise-inducedcochlear damage.

Our results indicate that pretreatment with anti-ICAM-1 Ab following noise exposure followed with abooster dose 24 hours afterwards may reduce noise-induced hearing loss without altering CBF or mean BP.However, its long-term effectiveness is not clear and needsfurther investigation. Many other important issues alsoneed to be addressed. For example, what effect does anti-ICAM-1 Ab have on cochlear histology? Is ICAM-1expressed during noise exposure or after noise exposure orboth? It is also important to determine if anti-ICAM-1 Abcould prevent the degeneration of hair cell, if it were givenafter a traumatic noise exposure. What is the optimaltreatment window following noise exposure? Lastly, theoptimal dose of anti-ICAM-1 Ab needs to be determined.Clearly, additional studies are necessary to determine ifanti-ICAM-1 Ab represents a possible therapeuticapproach to noise-induced hearing loss.

CONCLUSIONIntravenous administration of anti-ICAM-1 Ab sig-

nificantly attenuates noise-induced TTS and has nosignificant effect on CBF and mean BP. These resultssupport our earlier conclusions, which demonstrate theprotective effects of ROS scavengers and blockers in in-

terrupting the cascade of events that leads to noise-induced cochlear damage.

AcknowledgmentsThis research was supported in part by the following

NIH grant: NIDCD-DC00101-05 (MDS); 1R21AT001067-01-03, NCCAM, July 2003–2005. The authors gratefullyacknowledge the invaluable assistance of Dr. David A.Komjathy and Francis Leong in the preparation of themanuscript, and Pharmacia & Upjohn Company for thegift of anti-ICAM-1 antibody.

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Fig. 6. Schematic.




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