Acoustic overstimulation produces many anatomical, biochemical and physiological changes in the inner ear. However, the changes in gene expression that underlie these biological changes are poorly understood. Our approach to investigating this problem is to use gene microarrays to measure the changes in gene expression in the chinchilla inner ear following a 3 h or 6 h noise exposure (95 dB SPL, 707-1414 Hz). This noise exposure causes a temporary threshold shift (~40 dB) and a temporary reduction in distortion product otoacoustic emissions (DPOAE), but no permanent hearing loss or hair cell loss. Here, we present data showing (1) the suitability of mouse and human complementary DNA (cDNA) clones for detecting chinchilla cochlear gene transcripts, and (2) the change in cochlear gene transcripts in noise exposed chinchillas. Chinchilla cochlear transcript probes exhibited strong and discrete signals on both mouse and human cDNA filter arrays. Since the strongest hybridization occurred with mouse clones, mouse cDNA microarrays were used to study noise-induced changes in gene expression. Chinchilla cDNA probes were differentially labelled with Cy3 (control) or Cy5 (noise exposed) by random primed synthesis, hybridized to 8750 mouse cDNAs arrayed on microscope slides and analysed by laser fluorescent microscopy. Several classes of genes exhibited time-dependent up regulation of transcription, including those involved in protein synthesis, metabolism, cytoskeletal proteins, and calcium binding proteins. The results are discussed in relationship to previous studies showing noise-induced changes in structural proteins, calcium binding proteins, metabolic enzymes and membrane bound vesicles.
Keywords: gene expression, sound conditioning, neurofilaments, heat shock protein, calmodulin, cytoskeletal proteins
|How to cite this article:|
Taggart R T, McFadden SL, Ding DL, Henderson D, Jin X, Sun W, Salvi R. Gene expression changes in chinchilla cochlea from noise-induced temporary threshold shift. Noise Health 2001;3:1-18
|How to cite this URL:|
Taggart R T, McFadden SL, Ding DL, Henderson D, Jin X, Sun W, Salvi R. Gene expression changes in chinchilla cochlea from noise-induced temporary threshold shift. Noise Health [serial online] 2001 [cited 2021 Jan 18];3:1-18. Available from: https://www.noiseandhealth.org/text.asp?2001/3/11/1/31768
| Introduction|| |
The equal energy hypothesis, a fundamental theory in noise research, assumes that the amount of hearing loss and hair cell damage increases in proportion to the intensity and duration of the noise exposure (Henderson et al., 1991). This hypothesis, based largely on engineering concepts of an integrator, ignores the fact that biological tissues have the ability to repair cellular damage caused by environmental stress and to use intracellular mechanisms to cope with future traumatic insults. For example, the amount of hearing loss and cochlear damage that results from a traumatic noise exposure can be reduced significantly by prior exposure to a mildly stressful continuous or intermittent sound (Boettcher et al., 1992; Campo et al., 1991; Canlon et al., 1988; Henderson et al., 1992). The protection afforded by prior acoustic stimulation is generally referred to as "sound conditioning" or "toughening." The molecular changes that occur within the inner ear during sound exposures that give rise to "sound conditioning" or "toughening" as well as acoustic overstimulation in general are poorly understood. Previous studies have identified several biochemical and molecular changes in the inner ear associated with acoustic overstimulation and sound conditioning. For example, Jacono et al. (1998) found increases in several enzymes associated with the antioxidant defence system in the organ of Corti and lateral wall of the cochlea after acoustic overstimulation. The greatest changes in enzyme activity were observed in animals exposed to a conditioning noise followed by a high-level traumatic noise. Other studies have found increased amounts of heat shock proteins (Altschuler et al., 1996; Lim et al., 1996) as well as decreased calbindin in hair cells after acoustic overstimulation (Canlon, 1996). In situ hybridization and RT-PCR have been used to evaluate changes in mRNA levels for three alpha-isoforms and two beta-isoforms of Na, KATPase, an enzyme that plays an important role in ion transport and the generation of the endocochlear potential (Ryan et al., 1996). Only the Na, K-ATPase beta-2 isoform showed a significant decrease 1-2 h after a noise exposure. While these studies have provided valuable insights into the mechanisms involved in noise induced hearing loss, and the auditory "conditioning" phenomenon, the changes in gene expression associated with exposure to high level noise have not yet been investigated.
Since each cell synthesizes thousands of different proteins, it is difficult to predict which proteins and messenger RNAs (mRNAs) will show increased or decreased expression following a noise exposure. Furthermore, the pattern and time course of gene expression is likely to change with exposure conditions (e.g., sound level, spectrum, exposure duration and post-exposure time) since the expression of immediate genes will influence the expression of other genes. How can auditory neuroscientists begin to understand such a complex series of biological events that take place in the inner ear following acoustic overstimulation that leads to sound conditioning or toughening? One approach to this complex problem is to use gene microarray technology to screen for changes in mRNA expression levels in thousands of genes following an acoustic exposure. Here we present preliminary data on the changes in mRNA expression levels in the chinchilla inner ear following a short duration noise (3 or 6 h, 95 dB SPL, 707-1414 Hz) that can "condition" the ear if presented over many days. The noise parameters used in this study produce anatomical and physiological changes that have been well characterized in previous studies. Both the 3-h and 6-h exposure cause a temporary threshold shift (TTS) and a temporary reduction in DPOAE amplitude. However, if the exposure is repeated over a period of 10-15 days, the daily TTS actually decreases rather than remaining constant or increasing, i.e., the ear becomes "conditioned" to the noise exposure. Importantly, a "conditioned" ear develops less PTS from subsequent exposure to high-level noise.
To put the current gene microarray studies into perspective and to develop the rationale for the current studies, the physiological and anatomical effects of the exposure will first be described. This will be followed by an overview of gene microarray technology and a description of the temporal changes in several gene families that showed increased expression following acoustic overstimulation. All of the results presented below were carried out with chinchillas except where explicitly stated.
Interrupted Noise Exposure and
CAP Threshold and Amplitude. The impetus for the present study arose from previous studies from our lab showing that the amount of TTS resulting from 3-h or 6-h of noise exposure decreased significantly if the noise was presented over a period of 10-15 days. The progressive reduction of TTS with repeated exposure has been termed "toughening" or "conditioning." For example, Boettcher et al. (1992) exposed chinchillas to an octave band noise (707-1414 Hz) at 95 dB SPL for 3 h followed by 9 h of quiet. This interrupted noise exposure continued for 15 days. Cochlear function and hearing loss were estimated before and during the course of the exposure by measuring the compound action potential (CAP) from electrodes chronically implanted on the round window of the cochlea. After the first day of exposure, the CAP amplitude was greatly depressed at low sound levels, but began to increase towards the control amplitude between 70 and 85 dB SPL. Surprisingly, after the 15th day of exposure, the CAP amplitude was only slightly reduced relative to pre-exposure values. Thus, the cochlea became much more resistant to the traumatic effects of the noise. By 30 days after the end of the exposure, the CAP amplitude had recovered to pre-exposure levels.
Changes in CAP threshold paralleled changes in CAP amplitude. The maximum threshold shift during the first days of the exposure was 30-45 dB between 0.5 and 8.0 kHz. By day 15 of the exposure, the threshold shift at the two lowest frequencies, 0.5 and 1.0 kHz, was less than 8 dB, an improvement of more than 30 dB. CAP threshold at 2.0 and 4.0 kHz declined by approximately 25 dB during this same period, whereas little change was seen at higher frequencies. Thus, the low frequencies became 25-30 dB more resistant to the noise exposure over the course of the exposure.
DPOAEs. The improvement in CAP threshold and amplitude over the 15-day exposure could be due to changes at the auditory nerve itself or earlier stages in the signalling pathway such as the outer hair cells (OHCs), inner hair cells (IHCs) or stria vascularis. Since the OHCs play a critical role in establishing the low-intensity range of hearing (Ryan et al., 1975) and are extremely sensitive to the effects of acoustic trauma (Liberman et al., 1986; Salvi et al., 1982), one might expect that the OHCs would play a critical role in the "toughening" phenomenon. An extremely sensitive, frequency-specific, and convenient method of assessing the functional status of the OHCs is to measure the amplitude of the DPOAEs (Brown et al., 1989; Eddins et al., 1999; Hofstetter et al., 1997). To determine if the OHCs were developing a resistance to the interrupted noise exposure, DPOAE input/output functions were measured before and at regular intervals over the course of a 15-day "toughening" exposure (3 hr on, 9 hr off, 95 dB SPL, 707-1414 Hz) (Subramaniam et al., 1994). The pre-exposure DPOAE input/output function at 1-kHz increased from -10 dB SPL to approximately 30 dB SPL as the level of the primary tones increased from 30 to 60 dB SPL. On day 2 of the interrupted noise exposure, the input/output function was displaced approximately 30 dB to the right of the pre-exposure function (i.e., to higher intensities), reflecting an increase in threshold, and the maximum DPOAE amplitude was reduced by approximately 20 dB. Significantly, by day 10 of the exposure, the maximum amplitude was essentially normal and amplitudes at low intensities were only slightly reduced.
Hair Cell Stereocilia. The preceding results suggest that the OHCs had become more resistant to the noise over the course of the exposure. What morphological features of OHCs change with the recovery of the DPOAEs? Since the OHC stereocilia bundle is often damaged by acoustic overstimulation, we reasoned that the stereocilia bundles might repair themselves over the course of the noise exposure thereby contributing to the recovery of DPOAE amplitude (Saunders et al., 1986). To test this hypothesis, scanning electron micrographs of the stereocilia on OHCs and IHCs were obtained from normal chinchillas and carefully compared to those from chinchillas exposed to the interrupted noise for 1 day, 2 days or 15 days (Subramaniam et al., 1994). Experimenters, blind to the experimental conditions, rated the stereocilia on a scale of 1 to 5 with 1 being completely normal and 5 being severely damaged. The histogram in [Figure - 1] shows the condition of IHC and OHC stereocilia in the 500 Hz region of the cochlea, a frequency where there was a significant improvement in CAP threshold and DPOAE amplitude (Boettcher et al., 1992; Subramaniam et al., 1994). Paradoxically, the anatomical condition of the stereocilia deteriorated over the 15-day exposure while the functional measures such as the CAP and DPOAE recovered. This suggests that cellular mechanisms other than stereocilia repair are responsible for the toughening effect.
Protection and PTS. During the interrupted exposure, the inner ear becomes significantly more resistant and develops significantly less TTS. This raises several important questions. First, is a "toughened" ear more resistant to highlevel traumatic exposures that cause PTS? Second, how long does the "toughening" phenomenon persist? To address these questions, an experimental group received a conditioning noise (707-1414 Hz, 95 dB SPL, 6 hr on, 18 hr off, 10 day) 30 days prior to a traumatic noise (106 dB SPL, 707-1414 Hz) (Henderson et al., 1996). The experimental group was compared to a control group that only received the traumatic noise exposure. The experimental group that received the conditioning noise developed 5-20 dB less PTS than the control group after the traumatic noise exposure. In subsequent experiments, protection was found even when the time between the conditioning exposure and the high-level exposure was increased from 30 days to 60 days, although the amount of protection was diminished somewhat with the longer time interval (McFadden et al., 1997). This suggests that the conditioning noise can produce significant, long-term changes in the ear's susceptibility to acoustic trauma.
Sound toughening and/or protective effects similar to those described above have been observed in a number of different laboratories, with different species (mice, guinea pig, cat and chinchilla) and a variety of sound exposure paradigms (Canlon, 1997; Canlon et al., 1988; Miller et al., 1963; Willott et al., 1999). These results illustrate a general principle of auditory function, namely, the ability of the cochlea to modify its adaptive mechanisms to protect against stressful acoustic stimuli. To better understand the cellular and molecular changes involved in "conditioning" and noise-induced hearing loss, we conducted a series of experiments to identify the changes in gene expression occurring in the inner ear after a 3-h or 6-h noise exposure.
Gene Expression Changes
Cochlear mRNA. To address the question of noise-induced gene expression, procedures were developed for harvesting the mRNA from the cochleae of normal chinchillas and chinchillas that had been exposed for 3 h or 6 h to our standard conditioning noise (95 dB SPL, 7071414 Hz). Whole cochleae were homogenized in Trizol, extracting the RNA, and purifying the poly-A+RNA with either magnetic beads containing oligo-dT 25 or latex beads containing dC 10 T 30 oligonucleotides. Approximately 2-10 mg of poly-A+RNA was obtained from the cochleae of 4-8 control or noise-exposed chinchillas and used for subsequent analyses. The poly-A+RNA from normal and noiseexposed chinchillas was used to synthesize complementary DNA (cDNA) for subsequent analyses. In addition, mRNA was isolated from the chinchilla brain and used to synthesize cDNA to test for hybridization to mouse and human genes.
Hybridization to Mouse & Human Genes.
Because most genes have been sequenced for either mouse or humans, but not for chinchilla, it was first necessary to determine the degree to which the chinchilla cDNA probes would hybridize to mouse and human gene sequences. This preliminary study was accomplished by using chinchilla brain poly-A+RNA and assessing the degree of hybridization to gene filter arrays. Complementary DNA (cDNA) probes, radioactively labelled with 32 P-a-dCTP (40 mCi, 3000 Ci/mmole), were prepared from poly-A+ RNA by synthesis with oligo-dT and random primer oligonucleotides. Radioactively labelled 32 P-labeled cDNA was synthesized, purified by spin column chromatography, hybridized with filter arrays containing mouse or human cDNAs (Genome Systems), washed with increasing stringency and prepared for autoradiography using x-ray film. Both mouse and human cDNA filter arrays showed strong cross-species labelling [Figure - 2]; however, the intensity was generally stronger for mouse than human, presumably due to closer sequence similarity between mice and chinchillas. Based on these results, we decided to use mouse microarrays to compare the level of gene expression in noise-exposed chinchilla cochleae relative to unexposed cochleae.
Gene Microarray Technology. The gene microarrays used in this study (GEM, Genome Systems, Incyte) consist of 8750 separate gene sequences plated onto a microscopic grid. Fluorescent cDNA probes were generated using cochlear mRNA from unexposed and exposed animals. The cDNA probes competitively hybridized against each gene on the microarray. The poly-A mRNA from 4-8 unexposed chinchilla cochleae was incubated with random oligonucleotides, 5'-labeled with Cy3, to generate Cy3-labeled cDNA probes for control ears (Incyte) whereas the poly-A mRNA from 48 noise-exposed cochleae (3 hr or 6 hr exposures) was incubated with random oligonucleotides, 5'-labeled with Cy5, to generate Cy5 labelled cDNA probes for the noise-exposed ears. Following the procedures of Genome Systems, Cy3 (unexposed) and Cy5 (exposed) cDNA probes were plated on microarrays where they competitively hybridized to the gene transcripts platted on the microarray grid. The amount of Cy3 (unexposed) and Cy5 (exposed) labelled cDNA probe that hybridized to each gene on the microarray was quantified by measuring the fluorescence intensity of Cy3 and Cy5 using a laser and fluorescence detector tuned to the wavelength of fluorochrome.
The Cy3 and Cy5 fluorescence intensity of each transcript located at a specific location on the microarray grid was measured and quantified using internal calibration standards on the microarray. Results were analysed by Genome Systems using GEM Tools (version 2.3, Incyte) software. The ratio of fluorescence intensity of Cy3 to Cy5 was used to quantify the change (fold increase or decrease) in expression of each transcript after the 3-h or 6-h noise exposure.
The scatter plots in [Figure - 3] show intensity of Cy5 labelling for each transcript in the noise-exposed cochleae (ordinate) versus the intensity of Cy3 labelling of the same transcript in the control cochlea (abscissa) for the 3-h noise exposure (top panel) and the 6-h noise exposure (bottom panel). Competitive hybridization of cochlear transcript probes from control and animals exposed for 3 h and 6 h exhibited similar distributions. Signal intensities (transcript levels) in the noiseexposed cochleae and the control cochleae were distributed over a range of 30 to 20,000 with most intensities falling between 50-5000. The series of diagonal lines, labelled from +10 to -10 in the upper right of each panel, show the relative level of expression of Cy5 to Cy3. The diagonal line labelled 1 indicates that the gene was expressed at equal levels in unexposed and control ears. A value of -2 indicates that gene expression was twice as great in the noise-exposed cochleae as in the unexposed ears; a value of +2 indicates that gene expression was twice as high in the unexposed control ears as in the noise-exposed ears.
Greater than 97% of the transcripts in the exposed ears and unexposed ears were clustered along the diagonal and exhibited expression ratios between -1.5 and 1.5. Thus, most gene transcripts in the exposed cochleae showed expression changes of 50% or less. However, a small proportion of transcripts showed roughly a two-fold (-2) increase in expression. These genes showed a time-dependent increase in expression between the 3-h and 6-h exposure. This suggests that the changes are actually due to the noise exposure rather than to chance alone. The number of transcripts that exhibited a 2-fold or greater increase in gene expression increased from 35 genes after the 3-h exposure to 146 genes after the 6-h exposure. This indicates that the "toughening" exposure selectively activates a subset of genes and that the number of activated genes increases with exposure time. Since the data set is extremely large, data are presented from a subset of gene families and related expressed sequence tags (EST) based on their cellular function and/or cellular distribution. In general, data are shown if the transcription level showed a 2-fold or greater increase after either the 3-h or 6-h exposure.
Gene Families Showing Increased Expression
Ribosomal Genes & Protein Synthesis. Ten ribosomal gene transcripts associated with protein synthesis showed a 1.2 and 1.7 fold increase in expression after the 3-h exposure; these increased to between 2.0 and 2.7 fold after the 6-h exposure. [Figure - 4] shows the fold increase in expression (post-exposure level relative to control level) for six transcripts: (1) EST, similar to 40S ribosomal protein S4, X isoform, (2) ribosomal protein L35a, (3) ribonuclear protein A2/B1, (4) 60S ribosomal protein L3; (5) ribonuclear protein 70 kD polypeptide A, (6) peptidyl-prolyl cis-trans isomerase A. All six transcripts showed a systematic increase in expression with increase in exposure duration from 3 to 6 h. These results demonstrate an upregulation of the protein synthesis machinery for the increased production of certain cellular proteins in response to the noise exposure. Identifying the sites of increased protein synthesis in the cochlea, in particular the regulation of these genes, will require further anatomical studies utilizing in situ hybridization techniques with probes directed against these specific gene sequences.
Metabolic Genes. The 3-h and 6-h exposures would undoubtedly increase the level of metabolic activity in many cell types within the cochlea. As shown in [Figure - 5], several enzymes in the glycolytic pathway and the tricarboxylic acid cycle (mitochondria) exhibited 1.0 to 1.7 fold increases after the 3-h exposure and 2.0 to 2.3 fold increases after the 6-h exposure. The gene transcripts that showed significant, time dependent increases included fructosebiphosphate aldose A, glucose-6-phosphaste isomerase, EST similar to succinate dehydrogenase, glutamine fructose-6-phosphate transaminase and mitochondrial malate dehydrogenase. Previous studies using radiolabelled markers of deoxyglucose uptake and activities of hexokinase and glucose-6phosphatase identified significant increases in these metabolic markers in the auditory nerve, organ of Corti and stria vascularis following moderate to high levels of acoustic stimulation (Canlon et al., 1983). Other studies have shown that the degree of histochemical labelling of succinate dehydrogenase in the hair cells was inversely correlated with the degree of impulse noise-induced TTS (Zhai et al., 1998). Future in situ hybridization studies with probes directed against the specific metabolic gene sequences identified here would allow us to identify the anatomical sites of increased metabolic activity.
Cytoskeletal Genes. Many different cytoskeletal proteins contribute to the highly organized structure of the inner ear (Flock et al., 1981; Gillespie et al., 1993; Slepecky et al., 1990; Slepecky et al., 1992a). However, noise can result in many types of cytoskeletal protein changes, including disruption of the three dimensional organization of cytoskeletal proteins (Avinash et al., 1993) and increased expression of F-actin in OHCs and their supporting cells (Hu et al., 1997). Notably, the changes in F-actin immunolabelling observed by Hu and Henderson were caused by a 6-h exposure to conditioning noise similar to the one used in this study. The structural integrity of the inner ear clearly depends on the synthesis of cytoskeletal proteins to replenish those degraded over time and by environmental stressors such as noise exposure.
In response to the 3-h and 6-h exposures used here, we observed significant time-dependent increases in the level of gene transcripts for several cytoskeletal proteins [Figure - 6]. Greater than two-fold increases were seen for: (1) neurofilament, light polypeptide, (2) tubulin beta-5 chain, (3) actin-related protein 1 alphaisoform, (4) muscle myosin heavy chain and (5) smooth muscle form of alpha-actinin. Transcripts increased 1.5 to 1.8 fold after 3-h of exposure and rose to 2.1 to 2.4 fold after 6 h of exposure. The neurofilament light polypeptide transcript, which showed one of the largest increases after the 3-h exposure, encodes the 68 kD neurofilament protein which is abundantly expressed in type I and type II spiral ganglion neurons (Hafidi et al., 1989). The mRNA for neurofilament light polypeptide was localized within the cochlea tissue by in situ hybridization (ISH) using a cDNA probe (Life Technologies) 5'-labeled with FITC. An in situ hybridization detection kit for FITC-labelled probes (Sigma ISH-F1) was used to localize the mRNA for neurofilaments light polypeptide in paraffin embedded sections. Paraffin sections were obtained from normal chinchilla cochlea that had been fixed (10% buffered formalin), decalcified and sectioned (4 mm). Microscopic examination revealed significant in situ labelling confined mainly to the soma of spiral ganglion neurons. These results confirm previous neurofilament immunolabelling studies and, together with our microarray data, suggest that there is significant turnover of this structural protein in the auditory nerve in response to acoustic overstimulation.
Beta tubulin shows a complex pattern of developmental expression in the hair cells and supporting cells of the organ of Corti (Hallworth et al., 2000). This suggests that noise exposure increases the synthesis of tubulin in the organ of Corti during the early stages of acoustic overstimulation. Several different myosin isoforms have been identified in the cochlea by immunolabelling and they appear to play a critical role in its development and function (Crozet et al., 1997; Gillespie et al., 1993; Self et al., 1999). Our results suggest that noise exposure causes a significant increase in the protein encoded by muscle myosin heavy chain. Alpha actinin immunolabelling has been identified in the greater and lesser epithelial ridges of the developing cochlea (Anniko et al., 1989) and in the hair cells and supporting cells of the mature cochlea (Slepecky et al., 1992b). Our results suggest that noise exposure causes a significant increase in the synthesis of the protein encoded by the gene for the smooth muscle form of alpha actinin. In situ probes against the specific neurofilament transcripts that showed a significant increase in expression during the exposure can be used to identify the cochlear regions where these proteins are synthesized.
Calcium Related Genes. A number of calciumrelated transcripts showed a two-fold or greater increase in expression after 3 or 6 h of exposure [Figure - 7]. The most robust, early response occurred for the mRNA coding for the calciumbinding protein S100A1. Interestingly, the expression of this transcript decreased to 1.8 after the 6-h exposure suggesting an early signalling response of this gene. Previous immunolabelling studies have identified alpha and beta isoforms of the S100 protein in the developing avian ear, mainly in neurons and secretory cells (Fermin et al., 1995). Moreover, vestibular nuclei show increased expression of this protein within an hour following destruction of the vestibular epithelium suggesting that these proteins may play a role in neural plasticity (Rickmann et al., 1995). Calcium ions play a critical role in a variety of cellular functions and calcium-binding proteins regulate the intracellular calcium ion concentration.
After the 6-h exposure, the transcript coding for calmodulin 3 showed a 2.7 fold increase in expression [Figure - 7]. Antibodies to calcium binding protein, calmodulin, are expressed in hair cells, ganglion cells and in the lateral wall and spiral ligament (Imamura et al., 1996) and calmodulin has been identified in a rat OHC cDNA library (Harter et al., 1999). The mRNA for calmodulin 3 was localized within paraffin sections (4 mm) of the cochlea by ISH using a cDNA probe (Life Technologies) 5'-labeled with FITC and an ISH kit (Sigma ISH-F1) as described above. Examination of cross sections of the cochlea revealed strong ISH labelling for the calmodulin 3 probe in the spiral ganglion and lateral wall of the cochlea, with less intense labelling of the organ of Corti. These results suggest that acoustic overstimulation leads to increased synthesis of calmodulin 3. Calpactin showed a 2-fold increase in gene expression after the 6-h exposure. Calpactin, also known as lipocortin I, is a family member of eight annexins that binds phospholipids in the presence of calcium. Immunolabelling studies have revealed high levels of lipocortin I in the nonsensory cells facing the endolymph (Reiber et al., 1994). Annexin VI also demonstrated a 2.7 fold increase in expression after the 6-h exposure. The mRNA for annexin VI was identified within paraffin section (4 mm) of the cochlea by ISH using a cDNA probe (Life Technologies) 5'-labeled with FITC and an ISH kit (Sigma ISH-F1) as described above. Strong labelling against the cDNA probe for annexin VI was seen in the stria vascularis, spiral ganglion, and organ of Corti. The expression of sarcoplasmic/endoplasmic reticulum Ca2+ - ATPase (SERCA) showed a 2.1 fold increase of expression after the 6-h exposure. The location of different SERCA isoforms within the inner ear is currently unknown, although various SERCA isoforms have been described in other brain regions (Baba-Aissa et al., 1996). SERCA isoforms are presumably present in the inner ear since thapsigargin and cylcopiazonic acid, potent inhibitors of endoplasmic reticulum Ca 2+ ATPase, cause a significant decline in the cochlear microphonic, negative summating potential and CAP, but not the endocochlear potential (Nario et al., 1998). The 6-h exposure also caused a 2.7 fold increase in the ATP binding cassette 2.
Heat Shock Proteins. Heat shock proteins are a family of proteins that are expressed in many different cell types in response to stress (Tytell et al., 1993; Welch, 1992). Previous studies have found significant increases in immunolabelling of heat shock protein 72 kD and heat shock protein 27 kD in OHCs. The microarray employed in our study contained DNA sequences for heat shock protein 74 kD and 84 kD. The 74 kD transcript showed a 1.5 fold increase in expression after the 3-h exposure and a 2-fold increase in expression after the 6-h expression [Figure - 8] consistent with previous immunolabelling results (Altschuler et al., 1996; Lim et al., 1993). However, the transcript for heat shock protein 84 kD failed to show a change in expression at these two time points. The result for the 72 kD transcript is intriguing since prior conditioning with heat protects against subsequent exposure to noise-induced hearing loss (Yoshida et al., 1999). Maximum protection, a 40 dB reduction in threshold shift, occurred when the heat stress-sound exposure interval was 6 h; protection completely disappeared when the time interval between heat stress and noise exposure was 24 h. Quantitative PCR experiments revealed a 100 to 200-fold increase in heat shock mRNAs 30 minutes after heat stress; mRNA levels returned to control values after 6 h (Yoshida et al., 1999).
Antioxidant Enzymes. Previous studies have identified significant increases in three antioxidant enzymes, glutathione reductase, gamma-glutamyl cysteine synthetase, and catalase, in the organ of Corti and stria vascularis after conditioning exposures, traumatic exposures or the combination of the two (Jacono et al., 1998). The increase in these antioxidant molecules was correlated with increased resistance to subsequent traumatic exposures (Jacono et al., 1998). Surprisingly, none of the genes on our array presumably involved in the synthesis of antioxidant enzymes (EST similar to superoxide dismutase, glutathione S transferase, glutathione synthetase A1, glutathione S transferase alpha 3 and EST glutathione reductase) showed a 2-fold increase in expression after the 3-h or 6-h exposure [Figure - 9]. Failure to identify increased expression in these genes could be due to a number of factors.
First, longer noise exposures may be needed to induce expression of these gene transcripts. Second, expression of these genes may be delayed relative to the onset of the noise due to delays in the intracellular signalling cascade. Third, in order to obtain sufficient mRNA for our analysis, mRNA was extracted from the whole cochlea. Thus, the mRNA contribution of a particular group of cells is diluted by the mRNA of the entire pool of mRNA. This reduces the overall sensitivity of the gene expression assay.
| Summary|| |
The impetus for the current noise-induced gene expression studies was an outgrowth of previous research showing that the inner ear can be made more resistant to acoustic trauma by preexposure to low-to-moderate level noise that only induced TTS. Previous efforts at elucidating the mechanisms involved in "toughening" identified a number of potential candidates on a one-by-one basis such as metabolic factors, calcium-binding proteins, heat-shock protein, and antioxidant molecules (Altschuler et al., 1996; Canlon, 1996; Canlon et al., 1984; Jacono et al., 1998; Lim et al., 1996; Yoshida et al., 1999). The gene microarray studies employed here allow researchers to search for potential candidate molecules related to noise-induced hearing loss and the "toughening" process by detecting changes in expression level in thousands of gene transcripts at different time points during and after a sound exposure. In our preliminary study, we identified approximately 150 gene transcripts that showed more than a two-fold increase in gene expression following a 6-h exposure that is known to make the ear more resistant to TTS. Thus, one of the major advantages of microarray studies is that it allows researchers to identify many more genes and candidate molecules involved in noiseinduced hearing loss than would be possible with classic hypothesis driven experiments involving a handful of candidate molecules.
Several of the genes showing increased expression were ones that had been implicated in earlier studies using immunolabelling or histochemical techniques, for example, heatshock proteins, calcium-binding proteins and metabolic enzymes. This lends credence to the current scientific approach. In addition, a large number of previously unidentified genes were shown to increase their level of expression during the early stages of acoustic overstimulation. Researchers can use this information to carry out in depth studies on a specific gene or protein. For example, anatomical studies using in situ hybridization and probes against specific mRNAs will allow scientists to identify which cells in the inner ear express specific genes, if there is a significant increase in mRNA synthesis during an exposure, and if new cells begin to express a particular mRNA as a result of acoustic overstimulation.
Limitations. Because we are using mRNA isolated from whole cochlear extracts, the change in gene expression in a particular group of cells could be larger or smaller than that seen in the total mRNA sample from the entire cochlea. We expect that our analysis is biased towards changes in gene expression that occur in many cells or very large changes in gene expression that occur in small populations of cells. Small changes in gene expression or moderate changes that occur in a small subgroup of cells could be missed in our analysis. More precise estimates of the change in gene expression could be achieved by: (1) identifying the cell types expressing a particular mRNA, (2) isolating the individual cell types and (3) measuring the changes in gene expression in a homogenous population of cells or a single cell using more sensitive measuring techniques.
Microarrays may open up many new avenues of research and ways of identifying important intracellular signalling pathways activated by acoustic stimuli. Although the vast amount of data generated by gene microarrays has created a formidable problem in terms of synthesizing and interpreting the results (Brazma et al., 2000; Colantuoni et al., 2000), the rapid advances being made in bioinformatics are likely to alleviate this problem in the future. The specific genes that are activated by acoustic overstimulation are likely to vary with the frequency, level and duration of the exposure, the post-exposure recovery time, subject variables such as age, genetic background and prior noise-exposure history. Unravelling the role of all of these variables will be a major challenge, but one that is likely to yield important new results and understanding.
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Hearing Research Lab, 215 Parker Hall, SUNY University of Buffalo, Buffalo, NY 14214
Source of Support: None, Conflict of Interest: None
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7], [Figure - 8], [Figure - 9]