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Does erythropoietin augment noise induced hearing loss?
Hearing Research
Hearing Research 223 (2007) 129–137
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Research paper
Does erythropoietin augment noise induced hearing loss? Birgitte Lidegaard Frederiksen a, Per Caye´-Thomasen a,*, Søren Peter Lund b, Niels Wagner a, Korhan Asal a, Niels Vidiendal Olsen c, Jens Thomsen a a
Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Gentofte University Hospital of Copenhagen, Niels Andersens Vej 65, 2900 Hellerup, Denmark b The National Institute of Occupational Health (AMI), Copenhagen, Denmark c The Department of Neuroanaesthesia, Copenhagen University Hospital, Denmark Received 21 June 2005; received in revised form 31 October 2006; accepted 2 November 2006 Available online 8 December 2006
Abstract Noise-induced hearing loss may result from excessive release of glutamate, nitrogen oxide and reactive oxygen species. The effects of these factors on the inner ear may potentially be prevented or reduced by erythropoietin (EPO), as indicated by previously demonstrated neuro-protective effects of EPO upon damage to the central nervous system and the retina. This paper reports three separate trials, conducted to investigate the hypothesis that noise-induced hearing loss is prevented or reduced by erythropoietin. The trials employed three different modes of drug application, different administration time windows and different rodent species. In trial 1, guinea pigs were exposed to 110 dB SPL, 4–20 kHz wide band noise (WBN) for 8 h. EPO was administered to the round window membrane 24 h after noise exposure, either sustained by pump for a week or by single dose middle ear instillation. In trial 2, rats were exposed to 105 dB SPL, 4–20 kHz WBN for 8 h. EPO was administered by single dose middle ear instillation 1 or 14 h after noise exposure. In trial 3, rats were exposed to 105 dB SPL, 4–20 kHz WBN for 8 or 3 · 8 h. EPO was injected intraperitoneally 1 h before noise exposure. Oto-acoustic emissions and auditory brainstem responses (at 16 kHz) were recorded before and after noise exposure in all trials. The noise exposure induced a hearing loss in all animals. In trial 1, no recovery and no improvement of hearing occurred in any treatment group. In trial 2 and 3, a partial hearing recovery was seen. However, the hearing loss of the EPO treated animals was significantly worse than controls in trial 2. In trial 3, the hearing of the EPO treated animals exposed for 3 · 8 h was significantly worse than controls. Thus, surprisingly, the results from 2 of the 3 present trials indicate that erythropoietin may in fact augment noise-induced hearing loss. This is contradictory to the beneficial effect of EPO reported by the vast majority of studies on stressed neural tissues. EPO administration may alter the blood flow dynamics of the cochlear vascular bed during or after noise exposure, by a potential induction of vasoconstriction. This may be the cause of the surprising findings. 2006 Elsevier B.V. All rights reserved. Keywords: EPO; NIHL; Inner ear; Auditory function; Guinea pigs; Rats; Blood flow
1. Introduction Intense noise exposure may cause temporary or permanent functional auditory impairment. The cellular and Abbreviations: NIHL, noise-induced hearing loss; EPO, erythropoietin; WBN, wide band noise; NO, nitric oxygen; NOS, nitric oxide synthase; ROS, reactive oxygen species; DPOAE, distortion product otoacoustic emissions; ABR, auditory brainstem response; SPL, sound pressure level; CDP, cubic distortion product * Corresponding author. Tel.: +45 3977 7293; fax: +45 3977 7634. E-mail address: [email protected] (P. Caye´-Thomasen). 0378-5955/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2006.11.002
molecular mechanisms underlying noise-induced hearing loss (NIHL) are not fully disclosed, but available evidence indicates that glutamate receptors, nitric oxygen (NO) and reactive oxygen species (ROS) are involved. Glutamate is considered to be the neurotransmitter at the inner hair cell afferent synapse. It has been suggested that NIHL is due in part to excessive release of excitatory amino acids, such as glutamate, leading to swelling of the postsynaptic nerve ending (Puel, 1995). Nitric oxide synthase (NOS) is expressed in the cochlea (Duan et al., 2002) and NO is believed to mediate the
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effects of excitatory amino acids in the central nervous system. As an example of a role of NO, it has been shown that competitive inhibition of NO production by neuronal NOS attenuates hearing loss caused by kainic acid (a glutamate analog) (Johnson et al., 1998). ROS production is involved in noise trauma and antioxidant treatment has been shown to prevent hair cell damage and hearing loss (reviewed by Henderson et al., 2006). An intervention targeting the referred actions of glutamate, NO and ROS may thus be an appropriate approach to prevent or reduce NIHL. Apart from playing an essential role in erythropoiesis, recent research has shown that erythropoietin (EPO) has neurotrophic and neuroprotective functions in the central nervous system (Sanaka et al., 1998; Marti et al., 2000; Sasaki et al., 2001; Sire´n and Ehrenreich, 2001; Cerami et al., 2002; Juul, 2002; Buemi et al., 2003; Sasaki, 2003) and in the retina (Grimm et al., 2002; Junk et al., 2002). These functions are the result of several effects of EPO receptor binding: decrease of glutamate toxicity (Morishita et al., 1997; Kawakami et al., 2001), generation of neuronal anti-apoptotic factors (Juul et al., 1998; Sire´n and Ehrenreich, 2001; Renzi et al., 2002), reduction of inflammation (Brines et al., 2000), decrease of nitric oxide mediated injury (Digicaylioglu and Lipton, 2001), direct anti-oxidation (Chattopadhyay et al., 2000) and possibly vascular modulation and angiogenesis (Marti et al., 2000; Springborg et al., 2002). Further, EPO regulates migration and phenotypic differentiation of neural stem cells to neuronal progenitors (Shingo et al., 2001). Thus, there are at least three pathways of action, by which EPO theoretically should attenuate or prevent NIHL, i.e. decrease of glutamate toxicity, decrease of nitric oxide mediated injury, and direct anti-oxidation. Anti-apoptosis may be an additional effect of potential significance. The receptor for EPO is expressed widely within the cochlea of the normal guinea pig, by phalangeal cells, supporting cells, spiral ligament fibrocytes and some spiral ganglion cell profiles (Caye´-Thomasen et al., 2005). This background knowledge prompted the present trials, conducted to investigate the hypothesis that noise-induced hearing loss is prevented or reduced by erythropoietin administration. The trials employed different modes of drug application, different drug concentrations, different administration time windows and different rodent species. 2. Materials and methods Three separate trials were conducted, all controlled. 2.1. Trial 1 2.1.1. Animals Twenty-six pigmented male Dunkin–Hartley guinea pigs (Taconic MB, Ry, Denmark) with intact Preyer’s reflex were used. They were kept in standard laboratory conditions and fed water and food (pellets) ad libitum.
2.1.2. Noise exposure Twenty-four hours before osmotic pump implantation, the animals were exposed to 110 dB SPL, 4–20 kHz wide band noise (WBN) for 8 h. This level of noise exposure was chosen after a pilot study using 105 dB in five animals. The assessment of hearing on day 1 after 105 dB noise exposure showed that four animals had a considerable hearing-impairment, while one animal had a modest hearing impairment. On day 8, one animal had no hearing impairment, while one had a slight, one a moderate and two a considerable hearing impairment. From this pilot study we concluded, that the level of noise had been too low. Accordingly, the level of noise exposure was increased to 110 dB. During noise exposure, the animals were kept in wire mesh cages in exposure chambers with climatic control. The noise was generated on a computer with a digital signal processing board (Ariel DSP16+), amplified by an audio amplifier (NAD 216 THX), and delivered by dome tweeters (Vifa D26TG-05-06) situated 56 cm above the floor of each cage. To ensure an equal noise exposure, the animals were kept single in the cages. The sound fields were measured with a B & K 4133 microphone, a HP 35679A dynamic signal analyzer, and a B & K 4230 Sound Level Calibrator. The WBN had a uniform frequency distribution within the pass band, and the level differed less than ±1 dB SPL within and between the cages. 2.1.3. Drug delivery An established guinea pig model for sustained local drug administration to the inner ear was employed (Caye´-Thomasen et al., 2004) and is briefly described as follows. Complete anesthesia was achieved by intraperitoneal administration of Narcoxyl Vet. Xylazin 30 mg/kg bodyweight and Ketaminol Vet. 62 mg/kg bodyweight. The skin behind the right ear was shaved and disinfected, followed by subcutaneous injection of a local anaesthetic (Lidocaine 6 mg/kg bodyweight combined with Noradrenaline 0.6 mg/kg bodyweight) and a retroauricular incision. The posterior aspect of the temporal bone was located by blunt dissection and the middle ear cavity opened through a posterior tympanotomy, whereby the round window niche was visualized. An osmotic micropump (Alzet osmotic pump 1007D, volume 100 ll, pumping rate 0.5 ll/h, Scanbur, Koege, Denmark), was filled with either saline (eight animals – serving as control) or EPO (Epoitin alpha) 2000 IU/ml (600 IU/kg bodyweight) (10 animals), and fitted with a catheter. The pump was submerged in saline at body temperature for at least 6 h before surgery (according to the supplier instructions for pump priming), with the tip of the catheter above surface. This procedure allows a functioning pump at the time of implantation. The pump was placed in a sub-cutaneous pocket on the neck of the animal, behind the skin incision. The tip of the catheter was fixed in the niche of the round window, secured by a suture to the periosteum of the lateral-anterior aspect of the tympanotomy opening
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and by the application of a glass ionomer cement around the catheter in the tympanotomy opening. Care was taken to avoid manipulation of the ossicular chain and to ensure that no cement entered the middle ear cavity. The skin incision was closed by resorbable sutures. Sterile operating techniques were used throughout the operative procedures. Excluding the pump/catheter, an additional eight animals were operated by the same procedure, and a single dose of 100 ll EPO 2000 IU/ml (600 IU/kg bodyweight – equivalent to the dose administered by pump) instilled in the round window niche. 2.1.4. Assessment of hearing Quantitative assessments of hearing were made by measurements of distortion product oto-acoustic emissions (DPOAE) and auditory brainstem responses (ABR) at 16 kHz during full anaesthesia. The assessments were made a week before noise exposure, just before operation on the first day after noise exposure (day 1), and finally on the eighth day after noise exposure (day 8). Testing was performed on the right ear by stimulation directly in the ear canal. During the test procedure the animal was placed on a heating plate. The body temperature was held at a constant level between 37.5 and 38.5 C, using a rectal sensor to control the heating of the plate. The measuring system used has been reported in detail recently (Rasmussen et al., 2005) and can be described shortly as follows. 2.1.5. Acoustic probe A probe for measuring DPOAE contains at least two stimulus transducers and one microphone. Two 1/2-in. condenser microphones (B & K type 4191) were chosen for stimulus transduction. The probe assembly consisted of two parts: a probe body containing the transducers and a socket to be placed in the ear canal during testing. The socket was mounted on a three-dimensional micromanipulator for accurate placement of the probe. First, under otoscope control, the socket for the DPOAE probe was fixed in the outer ear canal. Subsequently, the DPOAE probe, housing a Knowles FG 3329/3452 electret microphone and connected to two stimulus transducers (1/2-in. B & K 4191 condenser microphones) using 25 mm long silicon tubes (diameter 1 mm), was mounted to the prefixed socket in the ear canal. A polarizing voltage of 200 V was utilized, and the maximum driving signal amplitude was limited to 80 V peak to peak, in order not to exceed the maximum peak polarization voltage of the microphone cartridges. The microphone was connected to a preamplifier with a fixed gain of 40 dB. 2.1.6. Calibration An acoustic brass coupler simulating the ear canal was mounted with a 1/8-in. precision microphone (B & K type 4138) in place of the tympanic membrane. The coupler
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microphone was calibrated using a B & K type 4230 Sound Level Calibrator fitted with a 1/8-in. adaptor. 2.1.7. Distortion product oto-acoustic emissions (DPOAE) The primary stimulus tones f1 and f2 were generated using a HP 8904 two-channel tone generator with phase control. A custom-made trigger circuit provided the trigger pulses necessary for time averaging of the responses. The distortion products were measured with a HP-35670A dynamic signal analyzer, performing time averaging and subsequently fast fourier transform (FFT) analysis of the pre-amplified microphone signal from the acoustic probe. All test procedures and equipment were controlled by a computer, programmed in the visual programming language HP VEE (version 5.0). DPOAEs were collected by stimulation with f2 primary input tones from 2 kHz to 45 kHz (f2/f1 = 1.22), using primary levels of L1 = 60 dB SPL and L2 = 50 dB SPL. Determination of DPOAE input/output curves were made at f2 = 16.384 kHz (f2/f1 = 1.22 and L1 = L2 + 10 dB). The noise floor for every DP-frequency was calculated by summing the noise power at frequencies adjacent to the DPOAE frequency (2 · f1 f2) and transformed to the corresponding level in dB SPL. 2.1.8. Auditory brainstem responses (ABR) Determination of hearing thresholds was performed by analysis of the auditory brainstem response (ABR) at 16,384 Hz. The pure tone stimuli were generated with a repetition rate of 19.9 per second, by a programmable function generator (Hameg 8130). After digital attenuation, symmetrical tapered 1.4 ms tone-pips were delivered by the B & K condenser microphones at levels ranging from 20 to 100 dB SPL in 5 dB steps. The ABRs were recorded with a silver wire inserted subcutaneously at the back of the head as active electrode, a small roll of silver wire in the mouth as reference electrode and a stainless steel needle in the tail as ground electrode. The response was amplified 50,000 times, filtered through analogue bandpass filter (10 Hz to 4 kHz), and 15 ms were sampled at a rate of 51.2 kHz by a 16 bit data acquisition board. The ABR consisted of at least 256 artefact free recordings that was averaged and stored for analysis. After further digitally filtering (FIR lowpass filter, 2.0 kHz cut of frequency and 5 kHz stopband) of the stored ABRs, the hearing thresholds were determined as the lowest stimulus level where the first wave and the first through could be clearly identified. 2.1.9. Euthanasia and control of pump placement After the last assessment of hearing on day 8, the animals were sacrificed by intracardiac injection of Pentobarbital 50 mg/kg bodyweight, followed by opening of the middle ear by a lateral tympanotomy to ensure correct placement of the micropump and catheter.
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2.2. Trial 2
2.4. Statistical analysis – trial 1–3
2.2.1. Animals and noise exposure Thirty-six male Wistar albino rats (Taconic MB, Ry, Denmark) with intact Preyer’s reflex were used. Laboratory conditions were as in trial 1. The animals were exposed to 105 dB SPL, 4–24 kHz WBN for 8 h. The conditions of noise exposure were otherwise the same as in trial 1.
Comparison of the mean CDP loss and the mean ABR threshold shift between the different groups in all three trials was performed using the Student’s t test for two samples with unequal variance, with p < 0.05 as the level of significance.
2.2.2. Drug delivery Drug administration was performed by transmembraneous middle ear instillation through the tympanic membrane during full anesthesia achieved by intraperitoneal injection of Mebumal (pentobarbital) 65 mg/kg bodyweight. The anterior, inferior quadrant of the right tympanic membrane was perforated by a needle fitted to a syringe, and EPO 5000 IU/ml or saline slowly instilled into the middle ear cavity, taking care not to sever the tympanic membrane and not to manipulate the ossicular chain. A quantity that filled the middle ear cavity was used, approximately 100 ll (1650 IU/kg body weight). Instillations were performed 1 h after noise exposure (EPO in 10 animals and saline in eight animals), or 14 h after noise exposure (EPO in 10 animals and saline in eight animals). 2.2.3. Assessment of hearing DPOAEs were measured immediately before noise exposure, before instillation and 1 week later (day 8). ABRs were measured before noise exposure, and 1 week after instillation (day 8). The assessments were performed as described in trial 1, although the upper limit of the f2 primary input tone was increased from 45 to 75 kHz when collecting DPOAEs.
2.4.1. Correlation between CDP loss and ABR threshold shift Regression analysis of the CDP loss at 16 kHz on the ABR threshold shift at the same frequency showed a close to one-to-one relationship between the two parameters (Fig. 1). This analysis was performed to test (and confirm) the comparability of results between the two modes of measuring hearing function at 16 kHz and the result of the regression strengthens the validity of the results on CDP loss at other frequencies, when considering ABR threshold shift as the standard of reference. 2.4.2. Calculation of the mean CDP Apart from the regression analysis on the results obtained at 16 kHz (Fig. 1), the mean CDP was calculated as the average of the DPOAEs above the noise floor across all frequencies tested, at the primary stimulus levels (L1 = 60 dB SPL and L2 = 50 dB SPL), followed by calculation of the average across animals.
2.3. Trial 3 2.3.1. Animals and noise exposure Forty-six male Wistar albino rats (Taconic MB, Ry, Denmark) with intact Preyer’s reflex were used. Laboratory conditions as in trial 1. Twelve EPO-treated animals and 11 controls were exposed to 105 dB SPL, 4–20 kHz for 8 h, whereas another 12, respectively 11 animals, were exposed to 105 dB SPL, 4–20 kHz for 3 · 8 h (on three subsequent days). Conditions were otherwise as in trial 1. 2.3.2. Drug delivery One millilitre EPO 5000 IU/ml (16,500 IU/kg body weight) or saline was injected intraperitoneally 1 h before initiating noise exposure. 2.3.3. Assessment of hearing DPOAEs and ABRs were measured before noise exposure and 14 days after drug administration. The assessments were performed as described in trial 1, although the upper limit of the f2 primary input tone was increased from 45 to 75 kHz when collecting DPOAEs.
Fig. 1. Regression of the loss of cubic distortion products (loss of CDP) at f2 = 16,384 kHz on the ABR threshold shift (TS) at the same frequency, for the rats in trial 2 and 3. The outlier marked black on the y-axis is left out of the analysis. There is a close relationship between the loss of CDP and the TS. The loss of CDP reaches maximum near 50 dB TS, and the animals with a TS of more than 50 are left out of the analysis. The regression coefficient is close to one (y = 1.03 + 1.08x; df = 60 and R2 = 0.82). Thus, as long as the CDP can be measured above the noise floor, there is a close to one to one relationship between the two parameters.
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2.5. Ethics The experiments were approved and supervised by The Committee for Animal Experiments under the Danish Ministry of Justice (approval 220801-083). 3. Results 3.1. Trial 1 DPOAE measurements are visualized in Fig. 2, displaying the mean DPOAE amplitudes plotted as a function of f2 frequency (stimulus levels of L1 = 60 dB and L2 = 50 dB). The recordings before noise exposure have a somewhat ragged appearance with peaks and throughs, which owes to the fact that the calibration of the sound stimuli was performed in an acoustic coupler and not directly in the external ear canal. This approach allows measurements of hearing over the entire frequency range.
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In general, the figures show an increasing level of the cubic distortion product (CDP) as the frequency of f2 increases to peak at 30 kHz, with a relatively steep decline above this frequency. The signal-to-noise ratio of the CDP was greatest in the f2 = 15–25 kHz range, and reached a maximum of almost 40 dB (input/output curve, Fig. 2b). The ABR measured at 16 kHz displayed hearing thresholds around 30 dB SPL. The mean threshold shift at 16 kHz following noise exposure amounted to 45–60 dB (Table 1), explaining the rather limited oto-acoustic emissions at this frequency, and overall there were limited emissions at all frequencies. At the last assessment of hearing a week following the insertion of the osmotic pump, only sparse oto-acoustic emissions could be measured. For one third of the animals, the hearing threshold at 16 kHz exceeded the dynamic range of the testing equipment. Treatment had no significant impact on hearing outcome (Table 1).
Fig. 2. (a) Cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23, L1 = 60 and L2 = 50 dB SPL) across frequencies in groups of Guinea pigs before (Init Contr and Init EPO), and 8 days after ending noise exposure (Contr and EPO). NF is the noise floor. All the Guinea pigs were exposed to 110 dB SPL, 4– 20 kHz wide band noise for 8 h. The animals were administered saline (control) or erythropoietin (EPO) intratympanically by instillation or sustained (by pump), starting 24 h after noise exposure. (b) Input/ouput-curves of the cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23) at f2 = 16,384 Hz for the same groups as in a. The figure displays the outcome of increasing intensities of sound stimulation. The error bars represent the 95% confidence intervals.
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Table 1 Hearing in noise exposed guinea pigs treated with intratympanic EPO or saline Guinea pigs treatment (intratympanic)
Pre-noise measurements day 1 (dB)
Post-noise measurements day 8 (dB)
Loss (dB)
CDP
ABR
CDP
ABR
CDP
ABR
Sustained EPO (n = 10) Sustained H2O (n = 8) Instilled EPO (n = 8)
15.6 ± 4.6 14.3 ± 4.9 14.2 ± 6.5
32.5 ± 3.4 30.7 ± 1.4 30.6 ± 2.3
85.0 ± 11.2 89.3 ± 8.0 79.4 ± 3.6
22.5 ± 4.9 21.2 ± 5.1 19.8 ± 5.8
52.5 ± 10.6 58.6 ± 8.9 48.8 ± 12.5
6.9 ± 0.8 6.9 ± 0.4 5.6 ± 1.9
Trial 1. Results (means) of pre- and post-noise measurements of the cubic distortion product (CDP) and auditory brainstem response (ABR) in guinea pigs treated intratympanically with erythropoietin (EPO) or saline (H2O). The mean loss of hearing is shown to the right. There was no significant effect of EPO treatment. The ±values are the 95% confidence intervals.
3.2. Trial 2 The curve of the CDP before noise exposure shows an increasing level as the frequency of f2 increases to peak
at 30 kHz, with a relatively steep roll off above this frequency and no detectable CDP above 85 kHz (Fig. 3). Just before drug administration 1 or 14 h after noise exposure there were only limited oto-acoustic emissions at all
Fig. 3. Cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23, L1 = 60 and L2 = 50 dB SPL) across frequencies in groups of rats before (Init Contr and Init EPO), and 8 days after ending noise exposure (Contr and EPO). NF is the noise floor. All the rats were exposed to 105 dB SPL, 4–20 kHz wide band noise for 8 h. (a) Rats treated by middle ear instillation of either saline (control) or erythropoietin (EPO) 1 h after ending noise exposure. (b) Rats treated by middle ear instillation 14 h after ending noise exposure. (c) The controls and EPO treated rats from figure a and b grouped together. (d–f) Input/ouputcurves of the cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23) at f2 = 16,384 Hz for the same groups as in a, b and c, respectively. The figures display the outcome of increasing intensities of sound stimulation. The CDP increases with intensity to peak around 55 dB SPL, above which the curve rolls somewhat off. The error bars represent the 95% confidence intervals.
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frequencies in all groups, as a result of the noise exposure. At the last assessments 8 days following drug administration, the hearing had partly recovered in the control groups, whereas a sparse recovery in the EPO treated animals was seen primarily at low frequencies. Compared to the controls, the mean loss of CDP was significantly higher in the early EPO treatment group, (p = 0.025, Students ttest, degrees of freedom: 16) and there was a tendency to
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a higher loss in the late treatment group (p = 0.096, Students t-test, degrees of freedom: 16). When all the EPO treated animals were cumulated, the decrease in the CDP was highly significant (p = 0.006, Students t-test, degrees of freedom: 32) (Fig. 3, Table 2). The ABRs showed a tendency to a greater mean permanent threshold shift by EPO treatment overall (p = 0.1, Students t-test, degrees of freedom: 32) (Table 2).
Table 2 Hearing in noise exposed rats treated with intratympanic EPO or saline Rats treatment (intratympanic instillation)
Pre-noise measurements day 1 (dB)
Post-noise measurements day 8 (dB)
Loss (dB)
CDP
ABR
CDP
ABR
CDP
ABR
EPO 1 h post-noise (n = 10) H2O 1 h post-noise (n = 8) EPO 14 h post-noise (n = 10) H2O 14 h post-noise (n = 8)
27.5 ± 1.5 27.5 ± 2.1 26.4 ± 2.4 27.5 ± 8.5
27.5 ± 1.9 25.0 ± 3.2 28.3 ± 1.8 29.2 ± 1.7
0.1 ± 4.3 12.6 ± 9.4 0.4 ± 2.8 7.3 ± 5.0
68.8 ± 13.7 57.0 ± 19.4 64.4 ± 14.5 54.2 ± 16.2
27.4* ± 4.8 14.9* ± 9.1 26.0§ ± 4.2 20.3§ ± 7.0
41.3 ± 14.0 32.0 ± 21.6 36.1 ± 14.0 25.0 ± 16.9
All EPO (n = 20) All H2O (n = 16)
26.9 ± 1.4 27.5 ± 1.0
27.9 ± 1.2 27.3 ± 2.8
0.2 ± 2.4 9.7 ± 5.6
66.5 ± 9.5 55.5 ± 16.1
26.7# ± 3.1 17.8# ± 5.6
38.5 ± 9.4 28.2 ± 17.5
Trial 2. Results (means) of pre- and post-noise measurements of the cubic distortion product (CDP) and auditory brainstem response (ABR) in rats treated by intraperitoneal injection of erythropoietin (EPO) or saline (H2O). The mean loss of hearing is shown to the right. EPO treatment augmented in the hearing loss in the early treatment group (p = 0.025. Students t-test, degress of freedom: 16)(*) and overall (p = 0.006, Students t-test, degrees of freedom: 32)(#). § symbolizes a p-value of 0.09 (Students t-test, degrees of freedom: 16) and symbolizes a p-value of 0.1 (Students t-test, degrees of freedom: 32). The ±values are the 95% confidence intervals.
Fig. 4. Cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23, L1 = 60 and L2 = 50 dB SPL) across frequencies in groups of rats before (Init Contr and Init EPO), and 14 days after ending noise exposure (Contr and EPO). NF is the noise floor. One hour before initiating noise exposure, the rats were administered intraperitoneal erythropoietin (EPO) or an equivalent volume of saline (controls). (a) Rats exposed to 105 dB SPL, 4–20 kHz wide band noise for 8 h. (b) Rats exposed to 105 dB SPL, 4–20 kHz wide band noise for 3 consecutive days, 8 h/day. (c, d) Input/ouput-curves of the cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23) at f2 = 16,384 Hz for the same groups as in a and b, respectively. The figures display the outcome of increasing intensities of sound stimulation. The CDP increases with intensity to peak around 55 dB SPL, above which the curve rolls somewhat off. The error bars represent the 95% confidence intervals.
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Table 3 Hearing in noise exposed rats treated with intraperitoneal EPO or saline Rats treatment (intraperitoneal, day 1)
Pre-noise measurements day 1 (dB)
Post-noise measurements day 14 (dB)
Loss (dB)
CDP
ABR
CDP
ABR
CDP
ABR
8 h noise + EPO (n = 12) 8 h noise + H2O (n = 11) 3 · 8 h noise + EPO (n = 12) 3 · 8 h noise + H2O (n = 11)
26.3 ± 1.2 26.2 ± 1.0 24.8 ± 4.1 27.0 ± 0.7
30.4 ± 1.9 31.4 ± 1.4 31.7 ± 1.5 31.4 ± 2.4
4.7 ± 4.9 5.0 ± 5.2 2.1 ± 2.8 6.0 ± 4.3
65.4 ± 10.3 66.4 ± 9.4 71.7 ± 4.9 62.3 ± 8.8
21.7 ± 5.3 21.2 ± 5.8 22.7 ± 2.7 21.0 ± 4.1
35.0 ± 10.4 35.0 ± 9.0 40.0* ± 5.1 30.9* ± 9.0
Trial 3. Results (means) of pre- and post-noise measurements of the cubic distortion product (CDP) and auditory brainstem response (ABR) in rats treated by intraperitoneal injection of erythropoietin (EPO) or saline (H2O H2O). The mean loss of hearing is shown to the right. EPO treatment in combination with noise for 3 · 8 h produced a significant increase of the ABR threshold shift (*) (p = 0.048, Students t-test, degrees of freedom: 21). The ± values are the 95% confidence interval.
3.3. Trial 3 The initial measurements of hearing were as described in trial 2 (Fig. 4, Table 3). Fourteen days after drug administration and noise exposure the hearing had recovered partially. There was no statistical effect of EPO treatment prior to noise exposure for 8 h. However, in the groups exposed to noise for 3 · 8 h, the ABR of the EPO group displayed a significantly higher threshold shift than the control group (p = 0.048, Students t-test, degrees of freedom: 21) (Table 3). 4. Discussion This paper tested the hypothesis that noise-induced hearing loss can be prevented or reduced by erythropoietin. The three included trials employed three different modes of drug application (systemic, single dose or sustained intratympanic), different drug concentrations, different administration time windows (from 1 h pre to 24 h post-noise exposure) and two different rodent species (guinea pig and rat). Surprisingly, the results from two of the three trials indicate that erythropoietin may in fact augment noiseinduced hearing loss. This is contradictory to the protective effect of EPO reported by the vast majority of studies on stressed neural tissues, e.g. cerebral or retinal neurons exposed to ischemia (Sanaka et al., 1998; Marti et al., 2000; Sasaki et al., 2001; Sire´n and Ehrenreich, 2001; Cerami et al., 2002; Juul, 2002; Grimm et al., 2002; Junk et al., 2002; Buemi et al., 2003; Sasaki, 2003). Theoretically, EPO should attenuate or prevent NIHL through at least three pathways of action, i.e. decrease of glutamate toxicity, decrease of nitric oxide mediated injury, and direct anti-oxidation. In addition, EPO-induced anti-apoptosis is an effect with a potential to mitigate hearing loss following noise exposure. The reason for the unexpected detrimental effect is likely to be of vascular origin. A number of alterations occur in the cochlear vascular bed during excessive noise exposure and these are probable pathophysiologic contributors to a resultant temporary or permanent hearing loss (Quirk and Seidman, 1995; Latoni et al., 1996; Miller et al., 2003). The alterations include capillary vasoconstriction and reduced cochlear blood flow, which induce localised periods of stasis, alterations in vascular permeability, and
local ischemia (Quirk and Seidman, 1995; Latoni et al., 1996; Miller et al., 2003). These events are at least in part caused by noise induced release of the vasoconstrictor 8isoprostaglandin F2-alfa (Miller et al., 2003), which is a by-product of the formation of reactive oxygen species during oxidative stress. The vasoconstrictor tromboxane is another potential regulator of cochlear microcirculation (Umemure et al., 1993). EPO modulates cerebral blood flow autoregulation (Springborg et al., 2002), but has also a vasoconstrictive effect, which reduces the blood flow (Morakkabati et al., 1996; Wu et al., 1999; Scherer, 2001). This is mediated directly through increased calcium-ion influx to the vascular cell cytosol (Morakkabati et al., 1996; Marrero et al., 1998) and activation of vascular endothelin receptors (Ishikawa et al., 1999; Scherer, 2001), and indirectly by an increased release of vasoconstrictive prostanoids, such as prostaglandin F2-alfa and tromboxane (Bode-Boger et al., 1996; Wu et al., 1999). Thus, the vasoconstrictive effect of EPO is mediated, at least in part, through the same pathways activated and causing vasoconstriction during noise exposure, and this may lead to a detrimental additive or synergistic reduction of cochlear blood flow and a resultant increase of the hearing loss. This hypothesis is supported by the fact that the EPO induced augmentation of hearing loss was seen in the rat experiments, but not in the guinea pig experiment (which showed no effect of EPO). The rat cochlea has been documented to be more sensitive to a decrease in blood flow during noise exposure (Miller et al., 2003). In one of the rat trials (trial 2), the EPO was administered after noise exposure. Thus, a hypothesised post-exposure vasoconstriction may be detrimental to the partial recovery of hearing. The conclusion of the present trials is that EPO may augment noise induced hearing loss and that this surprising and detrimental effect by theory is due to induction of an additive or synergistic decrease of cochlear blood flow. This finding may be important, as it suggests that individuals or patients receiving EPO treatment may be more susceptible to a hearing loss associated with exposure to noise, although the results from these animal studies only cautiously and with proviso can be transferred as conceivably valid for human conditions.
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Acknowledgements Thanks are due to laboratory assistant Gitte Bondega˚rd Kristiansen, who performed the hearing tests. Financial support was provided by The Oda Pedersen Foundation. References Bode-Boger, S.M., Boger, R.H., Kuhn, M., Radermacher, J., Frolich, J.C., 1996. Recombinant human erythropoietin enhances vasoconstrictive tone via endothelin-1 and constrictor protanoids. Kidney Int. 50, 1255–1261. Brines, M.L., Ghezzi, P., Keenan, S., Agnello, D., de Lanerolle, N.C., Cerami, C., Itri, M.L., Cerami, A., 2000. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc. Natl. Acad. Sci., USA 97, 10526–10531. Buemi, M., Cavallaro, E., Floccari, F., Sturiale, A., Aloisi, C., Trimarchi, M., Corica, F., Frisina, N., 2003. The pleiotropic effects of erythropoietin in the central nervous system. J. Neuropathol. Exp. Neurol. 62, 228–236. Caye´-Thomasen, P., Wagner, N., Laure´ll, G., Bagger-Sjo¨ba¨ck, D., Thomsen, J., 2004. An animal model for continous drug administration to the inner ear. Audiological Med. 2, 174–178. Caye´-Thomasen, P., Wagner, N., Frederiksen, B.L., Asal, K., Thomsen, J., 2005. Erythropoietin and erythropoietin receptor expression in the guinea pig inner ear. Hear. Res. 203, 21–27. Cerami, A., Brines, M., Ghezzi, P., Cerami, C., Itri, L.M., 2002. Neuroprotective properties of epoitin alfa. Nephrol. Dial. Transplant. (Suppl 1), 8–12. Chattopadhyay, A., Choudhury, T.D., Bandyopadhyay, D., Datta, A.G., 2000. Protective effect of erythropoietin on the oxidative damage of erythrocyte membrane by hydroxyl radical. Biochem. Pharmacol. 59, 419–425. Digicaylioglu, M., Lipton, S.A., 2001. Erythropoietin-mediated neuroprotection involves cross-talk between JAK2 and NF-kappaB signaling cascades. Nature 412, 641–647. Duan, M.L., Ulfendahl, M., Laurell, G., Counter, A.S., Pyykko¨, I., Borg, E., Rosenhall, U., 2002. Protection and treatment of sensorineural hearing disorders caused by exogenous factors: experimental findings and potential clinical application. Review. Hear. Res. 169, 169–178. Grimm, C., Wenzel, A., Groszer, M., Mayser, H., Seeliger, M., Samardzija, M., Bauer, C., Gassman, M., Reme, C.E., 2002. HIF-1 induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration. Nat. Med. 8, 718–724. Henderson, D., Bielefeld, E.C., Harris, K.C., Hu, B.H., 2006. The role of oxidative stress in noise-induced hearing loss. Ear Hear. 27, 1–19. Ishikawa, A., Suzuki, K., Fujita, K., 1999. A preventive effect of a selective endothelin-A receptor antagonist, S-0139, on the erythropoietininduced reduction of the renal cortical blood flow. Urol. Res. 27, 312–314. Johnson, K.L., Carrasco, V., Prazma, J., Zdanski, C.J., Durland, W.F., Pillsbury, H.C., 1998. Role of nitric oxide in kainic acid-induced elevation of cochlear compound action potential thresholds. Acta Otolaryngol. 118 (5), 660–665. Junk, A.K., Mammis, A., Savitz, S.I., Singh, M., Roth, S., Malhotra, S., Rosenbaum, P.S., Cerami, A., Brines, M., Rosenbaum, D.M., 2002. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proc. Natl. Acad. Sci. USA 99, 10659– 10664. Juul, S., 2002. Erythropoietin in the central nervous system, and its use to prevent hypoxic-ischemic brain damage. Acta Pædiatr. (Suppl 438), 36–42.
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Juul, S.E., Anderson, D.K., Li, Y., Christensen, R.D., 1998. Erythropoietin and erythropoietin receptor in the developing human central nervous system. Pediatr. Res. 43, 40. Kawakami, M., Sekiguchi, M., Sato, K., Kozaki, S., Takahashi, M., 2001. Erythropoietin receptor-mediated inhibition of exocytotic glutamate release confers neuroprotection during chemical ischemia. J. Biol. Chem. 276, 39469–39475. Latoni, J., Shivapuja, B., Seidman, M.D., Quirk, W.S., 1996. Pentoxifylline maintains cochlear microcirculation and attenuates temporary threshold shifts following acoustic overstimulation. Acta Otolaryngol. 116, 388–394. Marrero, M.B., Venema, R.C., Ma, H., Ling, B.N., Eaton, D.C., 1998. Erythropoietin receptor-operated Ca2+ channels: activation by phospholipase C-gamma 1. Kidney Int. 53, 1259–1268. Marti, H.H., Bernaudin, M., Petit, E., Bauer, C., 2000. Neuroprotection and angiogenesis: dual role of erythropoietin in brain ischemia. News Physiol. Sci. 15, 225–229. Miller, J.M., Brown, J.N., Schacht, J., 2003. 8-Iso-prostaglandin F-2-alfa, a product of noise exposure, reduces inner ear blood flow. Audiol. Neurotol. 8, 207–221. Morakkabati, N., Gollnick, F., Meyer, R., Fandrey, J., Jelkmann, W., 1996. Erythropoietin induces Ca2+ mobilization and contraction in rat mesangial and aortic smooth muscle cultures. Exp. Hematol. 24, 392– 397. Morishita, E., Masuda, S., Nagao, M., Yasuda, Y., Sasaki, R., 1997. Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamateinduced neuronal death. Neuroscience 76, 105–116. Puel, J.L., 1995. Chemical synaptic transmission in the cochlea. Prog. Neurobiol. 47 (6), 449–476. Quirk, W.S., Seidman, D.S., 1995. Cochlear vascular changes on response to loud noise. Am. J. Otol. 16, 322–325. Rasmussen, A.N., Osterhammel, P.A., Lund, S.P., Kristiansen, G.B., Andersen, S., 2005. A system for measuring distortion product otoacoustic emissions at ultra-sonic frequencies in rodents. Int. J. Audiol. 44, 237–243. Renzi, M.J., Farrell, F.X., Bittner, A., Galindo, J.E., Morton, M.T., Trinh, H., Jolliffe, L.K., 2002. Erythropoietin induces changes in gene expression in pc-12 cells. Mol. Brain Res. 104, 86–95. Sanaka, M., Wen, T.-C., Matsuda, S., Masuda, S., Morishita, E., Nagao, M., Sasaki, R., 1998. In vivo evidence that erythropietin protects neurons from ischemic damage. Proc. Natl. Acad. Sci., USA 95, 4635– 4640. Sasaki, R., 2003. Pleiotropic functions of erythropoietin. Int. Med. 42, 142–149. Sasaki, R., Masuda, S., Nagao, M., 2001. Pleiotropic functions and tissuespecific expression of erythropoietin. News Physiol. Sci. 16, 110–113. Scherer, E.Q., Wonneberger, K., Wangemann, P., 2001. Differential desensitization of Ca2+ mobilization and vasocontriction by ET(A) receptors in the gerbil spiral modiolar artery. J. Membr. Biol. 182, 183–191. Shingo, T., Sorokan, T., Shimazaki, T., Weiss, S., 2001. Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J. Neurosci. 21, 9733–9743. Sire´n, A.-L., Ehrenreich, H., 2001. Erythropoietin – a novel concept for neuroprotection. Eur. Arch. Psychiatr. Clin. Neurosci. 251, 179–184. Springborg, J.B., Ma, X., Rochat, P., Knudsen, G.M., Amtorp, O., Paulson, O.B., Juhler, M., Olsen, N.V., 2002. A single subcutaneous bolus of erythropoietin normalizes cerebral blood flow autoregulation after subarachnoid haemorrhage in rats. Br. J. Pharmacol. 135, 823–829. Umemure, K., Asai, Y., Uematsu, T., Nakashima, M., 1993. Role of thromboxane A2 in a microcirculation disorder of the rat inner ear. Eur. Arch. Otorhinolaryngol. 250, 342–344. Wu, X.C., Richards, N.T., Johns, E.J., 1999. The influence of erythropoietin on the vascular responses of rat resistance arteries. Exp. Physiol. 84, 917–927.
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Research paper
Does erythropoietin augment noise induced hearing loss? Birgitte Lidegaard Frederiksen a, Per Caye´-Thomasen a,*, Søren Peter Lund b, Niels Wagner a, Korhan Asal a, Niels Vidiendal Olsen c, Jens Thomsen a a
Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Gentofte University Hospital of Copenhagen, Niels Andersens Vej 65, 2900 Hellerup, Denmark b The National Institute of Occupational Health (AMI), Copenhagen, Denmark c The Department of Neuroanaesthesia, Copenhagen University Hospital, Denmark Received 21 June 2005; received in revised form 31 October 2006; accepted 2 November 2006 Available online 8 December 2006
Abstract Noise-induced hearing loss may result from excessive release of glutamate, nitrogen oxide and reactive oxygen species. The effects of these factors on the inner ear may potentially be prevented or reduced by erythropoietin (EPO), as indicated by previously demonstrated neuro-protective effects of EPO upon damage to the central nervous system and the retina. This paper reports three separate trials, conducted to investigate the hypothesis that noise-induced hearing loss is prevented or reduced by erythropoietin. The trials employed three different modes of drug application, different administration time windows and different rodent species. In trial 1, guinea pigs were exposed to 110 dB SPL, 4–20 kHz wide band noise (WBN) for 8 h. EPO was administered to the round window membrane 24 h after noise exposure, either sustained by pump for a week or by single dose middle ear instillation. In trial 2, rats were exposed to 105 dB SPL, 4–20 kHz WBN for 8 h. EPO was administered by single dose middle ear instillation 1 or 14 h after noise exposure. In trial 3, rats were exposed to 105 dB SPL, 4–20 kHz WBN for 8 or 3 · 8 h. EPO was injected intraperitoneally 1 h before noise exposure. Oto-acoustic emissions and auditory brainstem responses (at 16 kHz) were recorded before and after noise exposure in all trials. The noise exposure induced a hearing loss in all animals. In trial 1, no recovery and no improvement of hearing occurred in any treatment group. In trial 2 and 3, a partial hearing recovery was seen. However, the hearing loss of the EPO treated animals was significantly worse than controls in trial 2. In trial 3, the hearing of the EPO treated animals exposed for 3 · 8 h was significantly worse than controls. Thus, surprisingly, the results from 2 of the 3 present trials indicate that erythropoietin may in fact augment noise-induced hearing loss. This is contradictory to the beneficial effect of EPO reported by the vast majority of studies on stressed neural tissues. EPO administration may alter the blood flow dynamics of the cochlear vascular bed during or after noise exposure, by a potential induction of vasoconstriction. This may be the cause of the surprising findings. 2006 Elsevier B.V. All rights reserved. Keywords: EPO; NIHL; Inner ear; Auditory function; Guinea pigs; Rats; Blood flow
1. Introduction Intense noise exposure may cause temporary or permanent functional auditory impairment. The cellular and Abbreviations: NIHL, noise-induced hearing loss; EPO, erythropoietin; WBN, wide band noise; NO, nitric oxygen; NOS, nitric oxide synthase; ROS, reactive oxygen species; DPOAE, distortion product otoacoustic emissions; ABR, auditory brainstem response; SPL, sound pressure level; CDP, cubic distortion product * Corresponding author. Tel.: +45 3977 7293; fax: +45 3977 7634. E-mail address: [email protected] (P. Caye´-Thomasen). 0378-5955/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2006.11.002
molecular mechanisms underlying noise-induced hearing loss (NIHL) are not fully disclosed, but available evidence indicates that glutamate receptors, nitric oxygen (NO) and reactive oxygen species (ROS) are involved. Glutamate is considered to be the neurotransmitter at the inner hair cell afferent synapse. It has been suggested that NIHL is due in part to excessive release of excitatory amino acids, such as glutamate, leading to swelling of the postsynaptic nerve ending (Puel, 1995). Nitric oxide synthase (NOS) is expressed in the cochlea (Duan et al., 2002) and NO is believed to mediate the
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effects of excitatory amino acids in the central nervous system. As an example of a role of NO, it has been shown that competitive inhibition of NO production by neuronal NOS attenuates hearing loss caused by kainic acid (a glutamate analog) (Johnson et al., 1998). ROS production is involved in noise trauma and antioxidant treatment has been shown to prevent hair cell damage and hearing loss (reviewed by Henderson et al., 2006). An intervention targeting the referred actions of glutamate, NO and ROS may thus be an appropriate approach to prevent or reduce NIHL. Apart from playing an essential role in erythropoiesis, recent research has shown that erythropoietin (EPO) has neurotrophic and neuroprotective functions in the central nervous system (Sanaka et al., 1998; Marti et al., 2000; Sasaki et al., 2001; Sire´n and Ehrenreich, 2001; Cerami et al., 2002; Juul, 2002; Buemi et al., 2003; Sasaki, 2003) and in the retina (Grimm et al., 2002; Junk et al., 2002). These functions are the result of several effects of EPO receptor binding: decrease of glutamate toxicity (Morishita et al., 1997; Kawakami et al., 2001), generation of neuronal anti-apoptotic factors (Juul et al., 1998; Sire´n and Ehrenreich, 2001; Renzi et al., 2002), reduction of inflammation (Brines et al., 2000), decrease of nitric oxide mediated injury (Digicaylioglu and Lipton, 2001), direct anti-oxidation (Chattopadhyay et al., 2000) and possibly vascular modulation and angiogenesis (Marti et al., 2000; Springborg et al., 2002). Further, EPO regulates migration and phenotypic differentiation of neural stem cells to neuronal progenitors (Shingo et al., 2001). Thus, there are at least three pathways of action, by which EPO theoretically should attenuate or prevent NIHL, i.e. decrease of glutamate toxicity, decrease of nitric oxide mediated injury, and direct anti-oxidation. Anti-apoptosis may be an additional effect of potential significance. The receptor for EPO is expressed widely within the cochlea of the normal guinea pig, by phalangeal cells, supporting cells, spiral ligament fibrocytes and some spiral ganglion cell profiles (Caye´-Thomasen et al., 2005). This background knowledge prompted the present trials, conducted to investigate the hypothesis that noise-induced hearing loss is prevented or reduced by erythropoietin administration. The trials employed different modes of drug application, different drug concentrations, different administration time windows and different rodent species. 2. Materials and methods Three separate trials were conducted, all controlled. 2.1. Trial 1 2.1.1. Animals Twenty-six pigmented male Dunkin–Hartley guinea pigs (Taconic MB, Ry, Denmark) with intact Preyer’s reflex were used. They were kept in standard laboratory conditions and fed water and food (pellets) ad libitum.
2.1.2. Noise exposure Twenty-four hours before osmotic pump implantation, the animals were exposed to 110 dB SPL, 4–20 kHz wide band noise (WBN) for 8 h. This level of noise exposure was chosen after a pilot study using 105 dB in five animals. The assessment of hearing on day 1 after 105 dB noise exposure showed that four animals had a considerable hearing-impairment, while one animal had a modest hearing impairment. On day 8, one animal had no hearing impairment, while one had a slight, one a moderate and two a considerable hearing impairment. From this pilot study we concluded, that the level of noise had been too low. Accordingly, the level of noise exposure was increased to 110 dB. During noise exposure, the animals were kept in wire mesh cages in exposure chambers with climatic control. The noise was generated on a computer with a digital signal processing board (Ariel DSP16+), amplified by an audio amplifier (NAD 216 THX), and delivered by dome tweeters (Vifa D26TG-05-06) situated 56 cm above the floor of each cage. To ensure an equal noise exposure, the animals were kept single in the cages. The sound fields were measured with a B & K 4133 microphone, a HP 35679A dynamic signal analyzer, and a B & K 4230 Sound Level Calibrator. The WBN had a uniform frequency distribution within the pass band, and the level differed less than ±1 dB SPL within and between the cages. 2.1.3. Drug delivery An established guinea pig model for sustained local drug administration to the inner ear was employed (Caye´-Thomasen et al., 2004) and is briefly described as follows. Complete anesthesia was achieved by intraperitoneal administration of Narcoxyl Vet. Xylazin 30 mg/kg bodyweight and Ketaminol Vet. 62 mg/kg bodyweight. The skin behind the right ear was shaved and disinfected, followed by subcutaneous injection of a local anaesthetic (Lidocaine 6 mg/kg bodyweight combined with Noradrenaline 0.6 mg/kg bodyweight) and a retroauricular incision. The posterior aspect of the temporal bone was located by blunt dissection and the middle ear cavity opened through a posterior tympanotomy, whereby the round window niche was visualized. An osmotic micropump (Alzet osmotic pump 1007D, volume 100 ll, pumping rate 0.5 ll/h, Scanbur, Koege, Denmark), was filled with either saline (eight animals – serving as control) or EPO (Epoitin alpha) 2000 IU/ml (600 IU/kg bodyweight) (10 animals), and fitted with a catheter. The pump was submerged in saline at body temperature for at least 6 h before surgery (according to the supplier instructions for pump priming), with the tip of the catheter above surface. This procedure allows a functioning pump at the time of implantation. The pump was placed in a sub-cutaneous pocket on the neck of the animal, behind the skin incision. The tip of the catheter was fixed in the niche of the round window, secured by a suture to the periosteum of the lateral-anterior aspect of the tympanotomy opening
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and by the application of a glass ionomer cement around the catheter in the tympanotomy opening. Care was taken to avoid manipulation of the ossicular chain and to ensure that no cement entered the middle ear cavity. The skin incision was closed by resorbable sutures. Sterile operating techniques were used throughout the operative procedures. Excluding the pump/catheter, an additional eight animals were operated by the same procedure, and a single dose of 100 ll EPO 2000 IU/ml (600 IU/kg bodyweight – equivalent to the dose administered by pump) instilled in the round window niche. 2.1.4. Assessment of hearing Quantitative assessments of hearing were made by measurements of distortion product oto-acoustic emissions (DPOAE) and auditory brainstem responses (ABR) at 16 kHz during full anaesthesia. The assessments were made a week before noise exposure, just before operation on the first day after noise exposure (day 1), and finally on the eighth day after noise exposure (day 8). Testing was performed on the right ear by stimulation directly in the ear canal. During the test procedure the animal was placed on a heating plate. The body temperature was held at a constant level between 37.5 and 38.5 C, using a rectal sensor to control the heating of the plate. The measuring system used has been reported in detail recently (Rasmussen et al., 2005) and can be described shortly as follows. 2.1.5. Acoustic probe A probe for measuring DPOAE contains at least two stimulus transducers and one microphone. Two 1/2-in. condenser microphones (B & K type 4191) were chosen for stimulus transduction. The probe assembly consisted of two parts: a probe body containing the transducers and a socket to be placed in the ear canal during testing. The socket was mounted on a three-dimensional micromanipulator for accurate placement of the probe. First, under otoscope control, the socket for the DPOAE probe was fixed in the outer ear canal. Subsequently, the DPOAE probe, housing a Knowles FG 3329/3452 electret microphone and connected to two stimulus transducers (1/2-in. B & K 4191 condenser microphones) using 25 mm long silicon tubes (diameter 1 mm), was mounted to the prefixed socket in the ear canal. A polarizing voltage of 200 V was utilized, and the maximum driving signal amplitude was limited to 80 V peak to peak, in order not to exceed the maximum peak polarization voltage of the microphone cartridges. The microphone was connected to a preamplifier with a fixed gain of 40 dB. 2.1.6. Calibration An acoustic brass coupler simulating the ear canal was mounted with a 1/8-in. precision microphone (B & K type 4138) in place of the tympanic membrane. The coupler
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microphone was calibrated using a B & K type 4230 Sound Level Calibrator fitted with a 1/8-in. adaptor. 2.1.7. Distortion product oto-acoustic emissions (DPOAE) The primary stimulus tones f1 and f2 were generated using a HP 8904 two-channel tone generator with phase control. A custom-made trigger circuit provided the trigger pulses necessary for time averaging of the responses. The distortion products were measured with a HP-35670A dynamic signal analyzer, performing time averaging and subsequently fast fourier transform (FFT) analysis of the pre-amplified microphone signal from the acoustic probe. All test procedures and equipment were controlled by a computer, programmed in the visual programming language HP VEE (version 5.0). DPOAEs were collected by stimulation with f2 primary input tones from 2 kHz to 45 kHz (f2/f1 = 1.22), using primary levels of L1 = 60 dB SPL and L2 = 50 dB SPL. Determination of DPOAE input/output curves were made at f2 = 16.384 kHz (f2/f1 = 1.22 and L1 = L2 + 10 dB). The noise floor for every DP-frequency was calculated by summing the noise power at frequencies adjacent to the DPOAE frequency (2 · f1 f2) and transformed to the corresponding level in dB SPL. 2.1.8. Auditory brainstem responses (ABR) Determination of hearing thresholds was performed by analysis of the auditory brainstem response (ABR) at 16,384 Hz. The pure tone stimuli were generated with a repetition rate of 19.9 per second, by a programmable function generator (Hameg 8130). After digital attenuation, symmetrical tapered 1.4 ms tone-pips were delivered by the B & K condenser microphones at levels ranging from 20 to 100 dB SPL in 5 dB steps. The ABRs were recorded with a silver wire inserted subcutaneously at the back of the head as active electrode, a small roll of silver wire in the mouth as reference electrode and a stainless steel needle in the tail as ground electrode. The response was amplified 50,000 times, filtered through analogue bandpass filter (10 Hz to 4 kHz), and 15 ms were sampled at a rate of 51.2 kHz by a 16 bit data acquisition board. The ABR consisted of at least 256 artefact free recordings that was averaged and stored for analysis. After further digitally filtering (FIR lowpass filter, 2.0 kHz cut of frequency and 5 kHz stopband) of the stored ABRs, the hearing thresholds were determined as the lowest stimulus level where the first wave and the first through could be clearly identified. 2.1.9. Euthanasia and control of pump placement After the last assessment of hearing on day 8, the animals were sacrificed by intracardiac injection of Pentobarbital 50 mg/kg bodyweight, followed by opening of the middle ear by a lateral tympanotomy to ensure correct placement of the micropump and catheter.
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2.2. Trial 2
2.4. Statistical analysis – trial 1–3
2.2.1. Animals and noise exposure Thirty-six male Wistar albino rats (Taconic MB, Ry, Denmark) with intact Preyer’s reflex were used. Laboratory conditions were as in trial 1. The animals were exposed to 105 dB SPL, 4–24 kHz WBN for 8 h. The conditions of noise exposure were otherwise the same as in trial 1.
Comparison of the mean CDP loss and the mean ABR threshold shift between the different groups in all three trials was performed using the Student’s t test for two samples with unequal variance, with p < 0.05 as the level of significance.
2.2.2. Drug delivery Drug administration was performed by transmembraneous middle ear instillation through the tympanic membrane during full anesthesia achieved by intraperitoneal injection of Mebumal (pentobarbital) 65 mg/kg bodyweight. The anterior, inferior quadrant of the right tympanic membrane was perforated by a needle fitted to a syringe, and EPO 5000 IU/ml or saline slowly instilled into the middle ear cavity, taking care not to sever the tympanic membrane and not to manipulate the ossicular chain. A quantity that filled the middle ear cavity was used, approximately 100 ll (1650 IU/kg body weight). Instillations were performed 1 h after noise exposure (EPO in 10 animals and saline in eight animals), or 14 h after noise exposure (EPO in 10 animals and saline in eight animals). 2.2.3. Assessment of hearing DPOAEs were measured immediately before noise exposure, before instillation and 1 week later (day 8). ABRs were measured before noise exposure, and 1 week after instillation (day 8). The assessments were performed as described in trial 1, although the upper limit of the f2 primary input tone was increased from 45 to 75 kHz when collecting DPOAEs.
2.4.1. Correlation between CDP loss and ABR threshold shift Regression analysis of the CDP loss at 16 kHz on the ABR threshold shift at the same frequency showed a close to one-to-one relationship between the two parameters (Fig. 1). This analysis was performed to test (and confirm) the comparability of results between the two modes of measuring hearing function at 16 kHz and the result of the regression strengthens the validity of the results on CDP loss at other frequencies, when considering ABR threshold shift as the standard of reference. 2.4.2. Calculation of the mean CDP Apart from the regression analysis on the results obtained at 16 kHz (Fig. 1), the mean CDP was calculated as the average of the DPOAEs above the noise floor across all frequencies tested, at the primary stimulus levels (L1 = 60 dB SPL and L2 = 50 dB SPL), followed by calculation of the average across animals.
2.3. Trial 3 2.3.1. Animals and noise exposure Forty-six male Wistar albino rats (Taconic MB, Ry, Denmark) with intact Preyer’s reflex were used. Laboratory conditions as in trial 1. Twelve EPO-treated animals and 11 controls were exposed to 105 dB SPL, 4–20 kHz for 8 h, whereas another 12, respectively 11 animals, were exposed to 105 dB SPL, 4–20 kHz for 3 · 8 h (on three subsequent days). Conditions were otherwise as in trial 1. 2.3.2. Drug delivery One millilitre EPO 5000 IU/ml (16,500 IU/kg body weight) or saline was injected intraperitoneally 1 h before initiating noise exposure. 2.3.3. Assessment of hearing DPOAEs and ABRs were measured before noise exposure and 14 days after drug administration. The assessments were performed as described in trial 1, although the upper limit of the f2 primary input tone was increased from 45 to 75 kHz when collecting DPOAEs.
Fig. 1. Regression of the loss of cubic distortion products (loss of CDP) at f2 = 16,384 kHz on the ABR threshold shift (TS) at the same frequency, for the rats in trial 2 and 3. The outlier marked black on the y-axis is left out of the analysis. There is a close relationship between the loss of CDP and the TS. The loss of CDP reaches maximum near 50 dB TS, and the animals with a TS of more than 50 are left out of the analysis. The regression coefficient is close to one (y = 1.03 + 1.08x; df = 60 and R2 = 0.82). Thus, as long as the CDP can be measured above the noise floor, there is a close to one to one relationship between the two parameters.
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2.5. Ethics The experiments were approved and supervised by The Committee for Animal Experiments under the Danish Ministry of Justice (approval 220801-083). 3. Results 3.1. Trial 1 DPOAE measurements are visualized in Fig. 2, displaying the mean DPOAE amplitudes plotted as a function of f2 frequency (stimulus levels of L1 = 60 dB and L2 = 50 dB). The recordings before noise exposure have a somewhat ragged appearance with peaks and throughs, which owes to the fact that the calibration of the sound stimuli was performed in an acoustic coupler and not directly in the external ear canal. This approach allows measurements of hearing over the entire frequency range.
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In general, the figures show an increasing level of the cubic distortion product (CDP) as the frequency of f2 increases to peak at 30 kHz, with a relatively steep decline above this frequency. The signal-to-noise ratio of the CDP was greatest in the f2 = 15–25 kHz range, and reached a maximum of almost 40 dB (input/output curve, Fig. 2b). The ABR measured at 16 kHz displayed hearing thresholds around 30 dB SPL. The mean threshold shift at 16 kHz following noise exposure amounted to 45–60 dB (Table 1), explaining the rather limited oto-acoustic emissions at this frequency, and overall there were limited emissions at all frequencies. At the last assessment of hearing a week following the insertion of the osmotic pump, only sparse oto-acoustic emissions could be measured. For one third of the animals, the hearing threshold at 16 kHz exceeded the dynamic range of the testing equipment. Treatment had no significant impact on hearing outcome (Table 1).
Fig. 2. (a) Cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23, L1 = 60 and L2 = 50 dB SPL) across frequencies in groups of Guinea pigs before (Init Contr and Init EPO), and 8 days after ending noise exposure (Contr and EPO). NF is the noise floor. All the Guinea pigs were exposed to 110 dB SPL, 4– 20 kHz wide band noise for 8 h. The animals were administered saline (control) or erythropoietin (EPO) intratympanically by instillation or sustained (by pump), starting 24 h after noise exposure. (b) Input/ouput-curves of the cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23) at f2 = 16,384 Hz for the same groups as in a. The figure displays the outcome of increasing intensities of sound stimulation. The error bars represent the 95% confidence intervals.
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Table 1 Hearing in noise exposed guinea pigs treated with intratympanic EPO or saline Guinea pigs treatment (intratympanic)
Pre-noise measurements day 1 (dB)
Post-noise measurements day 8 (dB)
Loss (dB)
CDP
ABR
CDP
ABR
CDP
ABR
Sustained EPO (n = 10) Sustained H2O (n = 8) Instilled EPO (n = 8)
15.6 ± 4.6 14.3 ± 4.9 14.2 ± 6.5
32.5 ± 3.4 30.7 ± 1.4 30.6 ± 2.3
85.0 ± 11.2 89.3 ± 8.0 79.4 ± 3.6
22.5 ± 4.9 21.2 ± 5.1 19.8 ± 5.8
52.5 ± 10.6 58.6 ± 8.9 48.8 ± 12.5
6.9 ± 0.8 6.9 ± 0.4 5.6 ± 1.9
Trial 1. Results (means) of pre- and post-noise measurements of the cubic distortion product (CDP) and auditory brainstem response (ABR) in guinea pigs treated intratympanically with erythropoietin (EPO) or saline (H2O). The mean loss of hearing is shown to the right. There was no significant effect of EPO treatment. The ±values are the 95% confidence intervals.
3.2. Trial 2 The curve of the CDP before noise exposure shows an increasing level as the frequency of f2 increases to peak
at 30 kHz, with a relatively steep roll off above this frequency and no detectable CDP above 85 kHz (Fig. 3). Just before drug administration 1 or 14 h after noise exposure there were only limited oto-acoustic emissions at all
Fig. 3. Cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23, L1 = 60 and L2 = 50 dB SPL) across frequencies in groups of rats before (Init Contr and Init EPO), and 8 days after ending noise exposure (Contr and EPO). NF is the noise floor. All the rats were exposed to 105 dB SPL, 4–20 kHz wide band noise for 8 h. (a) Rats treated by middle ear instillation of either saline (control) or erythropoietin (EPO) 1 h after ending noise exposure. (b) Rats treated by middle ear instillation 14 h after ending noise exposure. (c) The controls and EPO treated rats from figure a and b grouped together. (d–f) Input/ouputcurves of the cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23) at f2 = 16,384 Hz for the same groups as in a, b and c, respectively. The figures display the outcome of increasing intensities of sound stimulation. The CDP increases with intensity to peak around 55 dB SPL, above which the curve rolls somewhat off. The error bars represent the 95% confidence intervals.
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frequencies in all groups, as a result of the noise exposure. At the last assessments 8 days following drug administration, the hearing had partly recovered in the control groups, whereas a sparse recovery in the EPO treated animals was seen primarily at low frequencies. Compared to the controls, the mean loss of CDP was significantly higher in the early EPO treatment group, (p = 0.025, Students ttest, degrees of freedom: 16) and there was a tendency to
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a higher loss in the late treatment group (p = 0.096, Students t-test, degrees of freedom: 16). When all the EPO treated animals were cumulated, the decrease in the CDP was highly significant (p = 0.006, Students t-test, degrees of freedom: 32) (Fig. 3, Table 2). The ABRs showed a tendency to a greater mean permanent threshold shift by EPO treatment overall (p = 0.1, Students t-test, degrees of freedom: 32) (Table 2).
Table 2 Hearing in noise exposed rats treated with intratympanic EPO or saline Rats treatment (intratympanic instillation)
Pre-noise measurements day 1 (dB)
Post-noise measurements day 8 (dB)
Loss (dB)
CDP
ABR
CDP
ABR
CDP
ABR
EPO 1 h post-noise (n = 10) H2O 1 h post-noise (n = 8) EPO 14 h post-noise (n = 10) H2O 14 h post-noise (n = 8)
27.5 ± 1.5 27.5 ± 2.1 26.4 ± 2.4 27.5 ± 8.5
27.5 ± 1.9 25.0 ± 3.2 28.3 ± 1.8 29.2 ± 1.7
0.1 ± 4.3 12.6 ± 9.4 0.4 ± 2.8 7.3 ± 5.0
68.8 ± 13.7 57.0 ± 19.4 64.4 ± 14.5 54.2 ± 16.2
27.4* ± 4.8 14.9* ± 9.1 26.0§ ± 4.2 20.3§ ± 7.0
41.3 ± 14.0 32.0 ± 21.6 36.1 ± 14.0 25.0 ± 16.9
All EPO (n = 20) All H2O (n = 16)
26.9 ± 1.4 27.5 ± 1.0
27.9 ± 1.2 27.3 ± 2.8
0.2 ± 2.4 9.7 ± 5.6
66.5 ± 9.5 55.5 ± 16.1
26.7# ± 3.1 17.8# ± 5.6
38.5 ± 9.4 28.2 ± 17.5
Trial 2. Results (means) of pre- and post-noise measurements of the cubic distortion product (CDP) and auditory brainstem response (ABR) in rats treated by intraperitoneal injection of erythropoietin (EPO) or saline (H2O). The mean loss of hearing is shown to the right. EPO treatment augmented in the hearing loss in the early treatment group (p = 0.025. Students t-test, degress of freedom: 16)(*) and overall (p = 0.006, Students t-test, degrees of freedom: 32)(#). § symbolizes a p-value of 0.09 (Students t-test, degrees of freedom: 16) and symbolizes a p-value of 0.1 (Students t-test, degrees of freedom: 32). The ±values are the 95% confidence intervals.
Fig. 4. Cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23, L1 = 60 and L2 = 50 dB SPL) across frequencies in groups of rats before (Init Contr and Init EPO), and 14 days after ending noise exposure (Contr and EPO). NF is the noise floor. One hour before initiating noise exposure, the rats were administered intraperitoneal erythropoietin (EPO) or an equivalent volume of saline (controls). (a) Rats exposed to 105 dB SPL, 4–20 kHz wide band noise for 8 h. (b) Rats exposed to 105 dB SPL, 4–20 kHz wide band noise for 3 consecutive days, 8 h/day. (c, d) Input/ouput-curves of the cubic distortion products (2f1 f2, CDP; f2/f1 = 1.23) at f2 = 16,384 Hz for the same groups as in a and b, respectively. The figures display the outcome of increasing intensities of sound stimulation. The CDP increases with intensity to peak around 55 dB SPL, above which the curve rolls somewhat off. The error bars represent the 95% confidence intervals.
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Table 3 Hearing in noise exposed rats treated with intraperitoneal EPO or saline Rats treatment (intraperitoneal, day 1)
Pre-noise measurements day 1 (dB)
Post-noise measurements day 14 (dB)
Loss (dB)
CDP
ABR
CDP
ABR
CDP
ABR
8 h noise + EPO (n = 12) 8 h noise + H2O (n = 11) 3 · 8 h noise + EPO (n = 12) 3 · 8 h noise + H2O (n = 11)
26.3 ± 1.2 26.2 ± 1.0 24.8 ± 4.1 27.0 ± 0.7
30.4 ± 1.9 31.4 ± 1.4 31.7 ± 1.5 31.4 ± 2.4
4.7 ± 4.9 5.0 ± 5.2 2.1 ± 2.8 6.0 ± 4.3
65.4 ± 10.3 66.4 ± 9.4 71.7 ± 4.9 62.3 ± 8.8
21.7 ± 5.3 21.2 ± 5.8 22.7 ± 2.7 21.0 ± 4.1
35.0 ± 10.4 35.0 ± 9.0 40.0* ± 5.1 30.9* ± 9.0
Trial 3. Results (means) of pre- and post-noise measurements of the cubic distortion product (CDP) and auditory brainstem response (ABR) in rats treated by intraperitoneal injection of erythropoietin (EPO) or saline (H2O H2O). The mean loss of hearing is shown to the right. EPO treatment in combination with noise for 3 · 8 h produced a significant increase of the ABR threshold shift (*) (p = 0.048, Students t-test, degrees of freedom: 21). The ± values are the 95% confidence interval.
3.3. Trial 3 The initial measurements of hearing were as described in trial 2 (Fig. 4, Table 3). Fourteen days after drug administration and noise exposure the hearing had recovered partially. There was no statistical effect of EPO treatment prior to noise exposure for 8 h. However, in the groups exposed to noise for 3 · 8 h, the ABR of the EPO group displayed a significantly higher threshold shift than the control group (p = 0.048, Students t-test, degrees of freedom: 21) (Table 3). 4. Discussion This paper tested the hypothesis that noise-induced hearing loss can be prevented or reduced by erythropoietin. The three included trials employed three different modes of drug application (systemic, single dose or sustained intratympanic), different drug concentrations, different administration time windows (from 1 h pre to 24 h post-noise exposure) and two different rodent species (guinea pig and rat). Surprisingly, the results from two of the three trials indicate that erythropoietin may in fact augment noiseinduced hearing loss. This is contradictory to the protective effect of EPO reported by the vast majority of studies on stressed neural tissues, e.g. cerebral or retinal neurons exposed to ischemia (Sanaka et al., 1998; Marti et al., 2000; Sasaki et al., 2001; Sire´n and Ehrenreich, 2001; Cerami et al., 2002; Juul, 2002; Grimm et al., 2002; Junk et al., 2002; Buemi et al., 2003; Sasaki, 2003). Theoretically, EPO should attenuate or prevent NIHL through at least three pathways of action, i.e. decrease of glutamate toxicity, decrease of nitric oxide mediated injury, and direct anti-oxidation. In addition, EPO-induced anti-apoptosis is an effect with a potential to mitigate hearing loss following noise exposure. The reason for the unexpected detrimental effect is likely to be of vascular origin. A number of alterations occur in the cochlear vascular bed during excessive noise exposure and these are probable pathophysiologic contributors to a resultant temporary or permanent hearing loss (Quirk and Seidman, 1995; Latoni et al., 1996; Miller et al., 2003). The alterations include capillary vasoconstriction and reduced cochlear blood flow, which induce localised periods of stasis, alterations in vascular permeability, and
local ischemia (Quirk and Seidman, 1995; Latoni et al., 1996; Miller et al., 2003). These events are at least in part caused by noise induced release of the vasoconstrictor 8isoprostaglandin F2-alfa (Miller et al., 2003), which is a by-product of the formation of reactive oxygen species during oxidative stress. The vasoconstrictor tromboxane is another potential regulator of cochlear microcirculation (Umemure et al., 1993). EPO modulates cerebral blood flow autoregulation (Springborg et al., 2002), but has also a vasoconstrictive effect, which reduces the blood flow (Morakkabati et al., 1996; Wu et al., 1999; Scherer, 2001). This is mediated directly through increased calcium-ion influx to the vascular cell cytosol (Morakkabati et al., 1996; Marrero et al., 1998) and activation of vascular endothelin receptors (Ishikawa et al., 1999; Scherer, 2001), and indirectly by an increased release of vasoconstrictive prostanoids, such as prostaglandin F2-alfa and tromboxane (Bode-Boger et al., 1996; Wu et al., 1999). Thus, the vasoconstrictive effect of EPO is mediated, at least in part, through the same pathways activated and causing vasoconstriction during noise exposure, and this may lead to a detrimental additive or synergistic reduction of cochlear blood flow and a resultant increase of the hearing loss. This hypothesis is supported by the fact that the EPO induced augmentation of hearing loss was seen in the rat experiments, but not in the guinea pig experiment (which showed no effect of EPO). The rat cochlea has been documented to be more sensitive to a decrease in blood flow during noise exposure (Miller et al., 2003). In one of the rat trials (trial 2), the EPO was administered after noise exposure. Thus, a hypothesised post-exposure vasoconstriction may be detrimental to the partial recovery of hearing. The conclusion of the present trials is that EPO may augment noise induced hearing loss and that this surprising and detrimental effect by theory is due to induction of an additive or synergistic decrease of cochlear blood flow. This finding may be important, as it suggests that individuals or patients receiving EPO treatment may be more susceptible to a hearing loss associated with exposure to noise, although the results from these animal studies only cautiously and with proviso can be transferred as conceivably valid for human conditions.
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