Preparation of the intact rodent organ of Corti for RNAscope and immunolabeling, confocal microscopy, and quantitative analysis.

This protocol describes the preparation of the mouse organ of Corti for RNAscope, immunolabeling, confocal microscopy, and quantitative image analysis to examine transcript and protein localization, sensory hair cells, and synapses. This protocol can be applied to mice and other rodents (juvenile and adult) and can be adapted for other techniques, including electrophysiology and RNA sequencing. This protocol features minimal tissue processing to preserve viability for downstream assays, while isolating the organ of Corti is the most challenging step. For additional details on the use and execution of this protocol, please refer to McLean et al. (2009); Schuth et al. (2014); Lingle et al. (2019); Pyott et al. (2020).

Mechanical forces drive ordered patterning of hair cells in the mammalian inner ear.

Periodic organization of cells is required for the function of many organs and tissues. The development of such periodic patterns is typically associated with mechanisms based on intercellular signaling such as lateral inhibition and Turing patterning. Here we show that the transition from disordered to ordered checkerboard-like pattern of hair cells and supporting cells in the mammalian hearing organ, the organ of Corti, is likely based on mechanical forces rather than signaling events. Using time-lapse imaging of mouse cochlear explants, we show that hair cells rearrange gradually into a checkerboard-like pattern through a tissue-wide shear motion that coordinates intercalation and delamination events. Using mechanical models of the tissue, we show that global shear and local repulsion forces on hair cells are sufficient to drive the transition from disordered to ordered cellular pattern. Our findings suggest that mechanical forces drive ordered hair cell patterning in a process strikingly analogous to the process of shear-induced crystallization in polymer and granular physics.

Noise-induced nitrotyrosine increase and outer hair cell death in guinea pig cochlea.

BACKGROUND: Modern research has provided new insights into the biological mechanisms of noise-induced hearing loss, and a number of studies showed the appearance of increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) during and after noise exposure. This study was designed to investigate the noise exposure induced nitrotyrosine change and the mechanism of outer hair cells death in guinea pig cochlea. METHOD: Thirty guinea pigs were used in this study. The experimental animals were either exposed for 4 hours per day to broadband noise at 122 dB SPL (A-weighted) for 2 consecutive days or perfused cochleae with 5 mg/ml of the SIN1 solutions, an exogenous NO and superoxide donor, for 30 minutes. Then the cochleae of the animals were dissected. Propidium iodide (PI), a DNA intercalating fluorescent probe, was used to trace morphological changes in OHC nuclei. The distribution of nitrotyrosine (NT) in the organ of Corti and the cochlear lateral wall tissue from the guinea pigs were examined using fluorescence immunohistochemistry method. Whole mounts of organ of Corti were prepared. Morphological and fluorescent changes were examined under a confocal microscope. RESULTS: Either after noise exposure or after SIN1 perfusion, outer hair cells (OHCs) death with characteristics of both apoptotic and necrotic degradation appeared. Nitrotyrosine immunolabeling could be observed in the OHCs from the control animals. After noise exposure, NT immunostaining became much greater than the control animals in OHCs. The apoptotic OHC has significant increase of nitrotyrosine in and around the nucleus following noise exposure. In the normal later wall of cochleae, relatively weak nitrotyrosine immunolabeling could be observed. After noise exposure, nitrotyrosine immunoactivity became stronger in stria vascularis. CONCLUSION: Noise exposure induced increase of nitrotyrosine production is associated with OHCs death suggesting reactive nitrogen species participation in the cochlear pathophysiology of noise-induced hearing loss.

Identification of cisplatin-binding proteins using agarose conjugates of platinum compounds.

Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.

Regulated vesicular trafficking of specific PCDH15 and VLGR1 variants in auditory hair cells.

Usher syndrome is a genetically heterogeneous disorder characterized by hearing and balance dysfunction and progressive retinitis pigmentosa. Mouse models carrying mutations for the nine Usher-associated genes have splayed stereocilia, and some show delayed maturation of ribbon synapses suggesting these proteins may play different roles in terminal differentiation of auditory hair cells. The presence of the Usher proteins at the basal and apical aspects of the neurosensory epithelia suggests the existence of regulated trafficking through specific transport proteins and routes. Immature mouse cochleae and UB/OC-1 cells were used in this work to address whether specific variants of PCDH15 and VLGR1 are being selectively transported to opposite poles of the hair cells. Confocal colocalization studies between apical and basal vesicular markers and the different PCDH15 and VLGR1 variants along with sucrose density gradients and the use of vesicle trafficking inhibitors show the existence of Usher protein complexes in at least two vesicular subpools. The apically trafficked pool colocalized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associated with membrane microdomains and SNAP25. Moreover, coimmunoprecipitation experiments between SNAP25 and VLGR1 show a physical interaction of these two proteins in organ of Corti and brain. Collectively, these findings establish the existence of a differential vesicular trafficking mechanism for specific Usher protein variants in mouse cochlear hair cells, with the apical variants playing a potential role in endosomal recycling and stereocilia development/maintenance, and the basolateral variants involved in vesicle docking and/or fusion through SNAP25-mediated interactions.

Vibratome sectioning for enhanced preservation of the cytoarchitecture of the mammalian organ of Corti.

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in (1,2)).Visualization of this cellular mosaic often requires that the organ of Corti is cross-sectioned. In particular, the nonsensory pillar and Deiters' cells, whose nuclei are located basally with respect to the hair cells, cannot be visualized without cross-sectioning the organ of Corti. However, the delicate cytoarchitecture of the mammalian organ of Corti, including the fine cytoplasmic processes of the pillar and Deiters' cells, is difficult to preserve by routine histological procedures such as paraffin and cryo-sectioning, which are compatible with standard immunohistochemical staining techniques. Here I describe a simple and robust procedure consisting of vibratome sectioning of the cochlea, immunohistochemical staining of these vibratome sections in whole mount, followed by confocal microscopy. This procedure has been used widely for immunhistochemical analysis of multiple organs, including the mouse limb bud, zebrafish gut, liver, pancreas, and heart (see (3-6) for selected examples). In addition, this procedure was sucessful for both imaging and quantitificaton of pillar cell number in mutant and control organs of Corti in both embryos and adult mice (7). This method, however, is currently not widely used to examine the mammalian organ of Corti. The potential for this procedure to both provide enhanced preservation of the fine cytoarchitecture of the adult organ of Corti and allow for quantification of various cell types is described.

Localization of fatty acid binding proteins (FABPs) in the cochlea of mice.

Various fatty acids (FAs) are involved in many different functions in the organism as a source of energy, as essential ingredients of membranous lipids as well as intracellular signaling molecules. Intracellular fatty acid binding proteins (FABPs) comprise a family of soluble lipid binding proteins with low molecular masses and which can make long chain FAs soluble to allow intracellular translocation in the aqueous cytosol. To clarify the possible involvement of FAs and FABPs in hearing function, the present study investigated the localization of FABPs in the cochlea of adult mice using immunohistochemical procedures. Among various FABP species, H (heart-type)-FABP was localized in inner and outer pillar cells and outer phalangeal cells, while B (brain-type)-FABP was localized in border cells and cells of Hensen, and fibrocytes in the spiral limbus and spiral prominence. In the spiral ganglion, moderate to low H-FABP immunoreactivity was observed in almost all neurons, while B-FABP immunoreactivity was found in satellite cells. The discrete localization of the two FABPs in different non-receptor cells in the Organ of Corti suggests that the FABP species and/or their ligands, FAs, play important roles in the regulation of the hearing function.

Regional expression of the ADAMs in developing chicken cochlea.

The expression patterns of five members of the ADAM (a disintegrin and metalloprotease) family including ADAM9, ADAM10, ADAM17, ADAM22, and ADAM23 were analyzed in different anatomical structures of the developing chicken cochlea by in situ hybridization and immunohistochemistry. Results show that ADAM9, ADAM10, and ADAM17 are widely expressed in the sensory epithelium of the basilar papilla, by homogene cells, spindle-shaped cells, and acoustic ganglion cells, and in the tegmentum vasculosum, each with a different pattern. ADAM22 expression is restricted to spindle-shaped cells and acoustic ganglion cells, while ADAM23 is prominently expressed by hair cells and acoustic ganglion cells. Furthermore, ADAM10 protein is coexpressed with several members of the classic cadherins, including cadherin-7, N-cadherin, and R-cadherin in distinct anatomical regions of the cochlea except for acoustic ganglion cells. The expression of the ADAMs in the developing cochlea suggests a contribution of the ADAMs to the development of distinct cochlear structures.

Conformational stabilities of guinea pig OCP1 and OCP2.

OCP1 and OCP2, the most abundant proteins in the cochlea, are evidently subunits of an SCF E3 ubiquitin ligase. Although transcribed from a distinct gene, OCP2 is identical to Skp1. OCP1 is equivalent to the F-box protein known as Fbs1, Fbx2, or NFB42 - previously shown to bind N-glycosylated proteins and believed to function in the retrieval and recycling of misfolded proteins. The high concentrations of OCP1 and OCP2 in the cochlea suggest that the OCP1-OCP2 heterodimer may serve an additional function independent of its role in a canonical SCF complex. At 25 degrees C, urea-induced denaturation of OCP1 is slow, but reversible. The data suggest that the protein possesses one or more disordered regions, a conclusion supported by analysis of the far-UV circular dichroism spectrum and the appearance of the (1)H, (15)N-HSQC spectrum. Thermal denaturation of OCP1 is irreversible, evidently due to formation of high molecular weight aggregates. Analysis with a kinetic model yields an estimate for the activation energy for unfolding of 49 kcal/mol. Urea denaturation data for OCP2 returns DeltaG(o) and m values of 6.2 kcal/mol and 1.5 kcal mol(-)(1) M(-1), respectively. In contrast to OCP1, thermal denaturation of OCP2 is reversible. In phosphate-buffered saline, at pH 7.40, the protein exhibits a DeltaH(vH)/DeltaH(cal) ratio of 1.69, suggesting that denaturation proceeds largely from the native dimer directly to the unfolded state. OCP1 and OCP2 associate tightly at room temperature. However, DSC data for the complex suggest that they denature independently, consistent with the highly exothermic enthalpy of complex formation reported previously.

Expression of peripherin in the pig spiral ganglion--aspects of nerve injury and regeneration.

CONCLUSION: Peripherin protein may be important not only for developing neurons but also for the maintenance and regeneration of axonal processes in the mature cochlea. More knowledge about its expression and function could improve our understanding with reference to axonal regrowth and nerve restoration in the damaged cochlea. OBJECTIVE: To investigate the existence of peripherin protein in adult pig spiral ganglion and cultured spiral ganglion neurons of the guinea pig. MATERIALS AND METHODS: Immunohistochemistry with anti-peripherin antibodies was performed on sections of adult pig spiral ganglion and guinea pig spiral ganglion cell (SGC) culture. RESULTS: In pig auditory neurons, both type I and type II SGCs showed expression of the protein peripherin. These cells were not preferentially located near the intraganglionic spiral bundle (IGSB). The IGSB consisted of thin calibre fibres showing intense peripherin and thicker fibres that were TUJ-1 positive. Only a few fibres within the IGSB co-expressed both peripherin and TUJ-1. Cultured guinea pig neurons displayed a rich expression of peripherin, including the nuclei. This protein was expressed in regions such as the perikaryon and axons but there was also a segmental expression of peripherin in some regions. Peripherin was more expressed in areas of axon branching and in the centre of the active growth cones and lammelipodia.

WDR1 presence in the songbird basilar papilla.

WD40 repeat 1 protein (WDR1) was first reported in the acoustically injured chicken inner ear, and bioinformatics revealed that WDR1 has numerous WD40 repeats, important for protein-protein interactions. It has significant homology to actin interacting protein 1 (Aip1) in several lower species such as yeast, roundworm, fruitfly and frog. Several studies have shown that Aip1 binds cofilin/actin depolymerizing factor, and that these interactions are pivotal for actin disassembly via actin filament severing and actin monomer capping. However, the role of WDR1 in auditory function has yet to be determined. WDR1 is typically restricted to hair cells of the normal avian basilar papilla, but is redistributed towards supporting cells after acoustic overstimulation, suggesting that WDR1 may be involved in inner ear response to noise stress. One aim of the present study was to resolve the question as to whether stress factors, other than intense sound, could induce changes in WDR1 presence in the affected avian inner ear. Several techniques were used to assess WDR1 presence in the inner ears of songbird strains, including Belgian Waterslager (BW) canary, an avian strain with degenerative hearing loss thought to have a genetic basis. Reverse transcription, followed by polymerase chain reactions with WDR1-specific primers, confirmed WDR1 presence in the basilar papillae of adult BW, non-BW canaries, and zebra finches. Confocal microscopy examinations, following immunocytochemistry with anti-WDR1 antibody, localized WDR1 to the hair cell cytoplasm along the avian sensory epithelium. In addition, little, if any, staining by anti-WDR1 antibody was observed among supporting cells in the chicken or songbird ear. The present observations confirm and extend the early findings of WDR1 localization in hair cells, but not in supporting cells, in the normal avian basilar papilla. However, unlike supporting cells in the acoustically damaged chicken basilar papilla, the inner ear of the BW canary showed little, if any, WDR1 up-regulation in supporting cells. This may be due to the fact that the BW canary already has established hearing loss and/or to the possibility that the mechanism(s) involved in BW hearing loss may not be related to WDR1.

Energetics of OCP1-OCP2 complex formation.

OCP1 and OCP2, the most abundant proteins in the cochlea, are putative subunits of an SCF E3 ubiquitin ligase. Previous work has demonstrated that they form a heterodimeric complex. The thermodynamic details of that interaction are herein examined by isothermal titration calorimetry. At 25 degrees C, addition of OCP1 to OCP2 yields an apparent association constant of 4.0 x 10(7) M(-1). Enthalpically-driven (DeltaH=-35.9 kcal/mol) and entropically unfavorable (-TDeltaS=25.5 kcal/mol), the reaction is evidently unaccompanied by protonation/deprotonation events. DeltaH is strongly dependent on temperature, with DeltaC(p)=-1.31 kcal mol(-1) K(-1). Addition of OCP2 to OCP1 produces a slightly less favorable DeltaH, presumably due to the requirement for dissociation of the OCP2 homodimer prior to OCP1 binding. The thermodynamic signature for OCP1/OCP2 complex formation is inconsistent with a rigid-body association and suggests that the reaction is accompanied by a substantial degree of folding.

MRNA expression of members of the IGF system in the organ of Corti, the modiolus and the stria vascularis of newborn rats.

We analyzed the mRNA expression of the insulin-like growth factor (IGF) family genes and of selected downstream pathway genes using the Affymetrix microarray system and confirmatory RT-PCR in the freshly prepared organ of Corti (OC), modiolus (MOD) and stria vascularis (SV) from neonatal rats (3-5 days old) and after 24h in culture. Among the seven members of the IGF family analyzed in this paper, IGF1, IGF2 and IGF-binding protein (IGFBP2) had the highest basal expression in all regions. Preparatory stress and culture increased the expression of IGF2, IGFBP2, IGFBP3, IGFBP5, glucose transporterl (GLUT1), signal transducer, and activator of transcription3 (STAT3), phosphoinositide-3-kinase regulatory subunit (Pik3r1), Jun oncogene (c-jun) and decreased that of mitogen-activated protein kinases MAPK3 and MAPK14 in all regions. Region-specific changes were observed in OC (GLUT1), MOD (IGFBP3 and c-jun) and SV (IGF2 and IGFBP2).

Intratympanic injection of dexamethasone: time course of inner ear distribution and conversion to its active form.

HYPOTHESIS: Intratympanically injected dexamethasone 21-phosphate is converted to its active form dexamethasone in the inner ear and follows the distribution of the glucocorticoid receptor. BACKGROUND: Although dexamethasone is routinely delivered intratympanically for hearing loss, we know little of its inner ear pharmacokinetics. Dexamethasone 21-phosphate is the pharmaceutical compound available for injection, but it must be converted to its biologically active form (dexamethasone) to bind to the glucocorticoid receptor. Therefore, the current study was conducted to determine the time course of dexamethasone 21-phosphate movement from the middle ear into the inner ear, its conversion to dexamethasone, and the distribution of both forms relative to the glucocorticoid receptor. METHODS: BALB/c mice were injected intratympanically with the prodrug dexamethasone 21-phosphate and inner ears collected at postinjection times ranging from 5 minutes to 7 days. Ears were immunohistochemically stained for dexamethasone 21-phosphate, dexamethasone, and the glucocorticoid receptor. RESULTS: Both forms of dexamethasone were seen in the inner ear within 15 minutes, reaching their highest staining intensity at 1 hour. Neither drug was seen after 24 hours. The strongest staining occurred in the spiral ligament, organ of Corti, spiral ganglion, and vestibular sensory epithelia. Distribution of the drug paralleled locations of the glucocorticoid receptor except in the stria vascularis marginal cells, which stained heavily for the receptor but not the drug. CONCLUSION: Dexamethasone rapidly travels from the middle ear into the inner ear and converts to its active form. The drug distribution follows that of the glucocorticoid receptor. However, it probably has little impact on ear tissues after 24 hours.

Microscale analysis of proteins in inner ear tissues and fluids with emphasis on endolymphatic sac, otoconia, and organ of Corti.

Here we describe preparatory techniques adapted for the study of proteins of inner ear tissues and fluids that have allowed us to apply state-of-the-art analytical techniques in spite of the minute size and anatomical complexities of this organ. Illustrative examples address unresolved issues of functional and clinical significance. First, we demonstrate how quick-freezing and freeze drying prevents artifacts that arise from sampling endolymphatic sac (ES) content in the liquid state. This set the stage for the generation of the first protein profile of the ES. Identification of crucial proteins will help elucidate mechanisms of endolymph volume regulation and pathogenesis of Meniere's disease. Second, we show how a unique situation allowed identification of otoconial proteins by mass spectrometric analysis without prior separation and we discuss possible roles for these minor otoconins in otoconial development and prevention of degenerative diseases that affect balance. Finally, we demonstrate techniques for the precise dissection of organ of Corti and its substructures, while preserving their near normal chemical state. We extended an earlier study in which we identified a novel calcium-binding protein by IEF, oncomodulin, localized in the outer hair cells and show here the applicability of prefractionation for the screening of calcium-binding proteins of organ of Corti. These studies demonstrate how advanced preparatory and analytical techniques can be applied to studies of the inner ear.

Collagen changes in the cochlea of aged Fischer 344 rats.

Hearing function in the Fischer 344 (F344) albino inbred strain of rats deteriorates with aging faster than in other strains, in spite of the small hair cell loss in old F344 animals [Popelar, J., Groh, D., Pelanova, J., Canlon, B., Syka, J., 2005. Age-related changes in cochlear and brainstem auditory function. Neurobiol. Aging, in press.]. This study was aimed at elucidating the structural changes in the inner ear of this rat strain during aging. Cochlear histopathology was examined in 20-24-month-old F344 rats and compared with that of young F344 rats (4 months) and of old rats of the Long-Evans (LE) strain. Hematoxylin/eosin staining in aged F344 rats showed degenerative changes in the organ of Corti, consisting of a damaged layer of marginal cells, reduced vascularization of the stria vascularis and a distorted tectorial membrane detached from the organ of Corti. Age-related changes in collagen distribution were observed with Masson's trichrome staining in the spiral ligament of old F344 rats. The results of immunohistochemical staining for type II collagen revealed a marked decrease in collagen fibers in the area connecting the spiral ligament and stria vascularis and a decrease in area IV fibrocytes in old F344 but not in LE rats. These findings may contribute to an explanation of the substantial hearing loss found in old F344 rats.

Post-exposure administration of edaravone attenuates noise-induced hearing loss.

We investigated the effects of the antioxidant edaravone against acoustic trauma in guinea pigs. Edaravone (1.722 x 10(-2) M) was infused into the right ear by an osmotic pump, and the left ear was untreated for control. Animals received edaravone 9 h before (-9 h group, n = 7) and 9 h (+9 h group, n = 8), 21 h (+21 h group, n = 7) and 33 h (+33 h group, n = 4) after 3-h exposure to 130-dB noise. Seven days after noise exposure, we examined the shift in auditory brainstem response thresholds and histopathologic characteristics of the sensory epithelia. The smallest shift in auditory brainstem response threshold and smallest proportion of missing outer hair cells were observed in the +9 h group. This result was supported by immunohistochemical analysis of 4-hydroxy-2-nonenal. Our data suggest that edaravone may be clinically effective in the treatment of acoustic trauma, especially if given within 21 h of noise exposure.

Transient receptor potential channels in the inner ear: presence of transient receptor potential channel subfamily 1 and 4 in the guinea pig inner ear.

CONCLUSION: The results of this study indicate that transient receptor potential subfamily 1 (TRPV1) may play a functional role in sensory cell physiology and that TRPV4 may be important for fluid homeostasis in the inner ear. OBJECTIVE: To analyze the expression of TRPV1 and -4 in the normal guinea pig inner ear. MATERIAL AND METHODS: Albino guinea pigs were used. The location of TRPV1 and -4 in the inner ear, i.e. cochlea, vestibular end organs and endolymphatic sac, was investigated by means of immunohistochemistry. RESULTS: Immunohistochemistry revealed the presence of TRPV1 in the hair cells and supporting cells of the organ of Corti, in spiral ganglion cells, sensory cells of the vestibular end organs and vestibular ganglion cells. TRPV4 was found in the hair cells and supporting cells of the organ of Corti, in marginal cells of the stria vascularis, spiral ganglion cells, sensory cells, transitional cells, dark cells in the vestibular end organs, vestibular ganglion cells and epithelial cells of the endolymphatic sac.

Localization of neurotensin immunoreactivity in neurons and organ of Corti of rat cochlea.

The distribution of neurotensin-containing cell bodies and fibers has been observed in the central and peripheral nervous system, including sensory ganglia, but no description has been found in the peripheral auditory system. Here, we investigated the presence of neurotensin immunoreactivity in the cochlea of the adult Wistar rat. Strong neurotensin immunoreactivity was detected in the cytoplasm of the inner hair cells (IHC) and Deiters' cells of the organ of Corti. Outer hair cells (OHC) show weak immunoreaction. Neurotensin immunoreactivity was also found in the neurons and fibers of the spiral ganglia. Quantitative microdensitometric image analysis of the neurotensin immunoreactivity showed a strong immunoreaction in the hair cells of organ of Corti and a moderate to strong labeling in the spiral ganglion neurons. A series of double immunolabeling experiments demonstrated a strong neurotensin immunoreactivity in the parvalbumin immunoreactive IHC and also in the calbindin immunoreactive Deiters' cells. Weak neurotensin immunoreactivity was seen in the calbindin positive OHC. Neurofilament and parvalbumin immunoreactive neurons and fibers in the spiral ganglia showed neurotensin immunoreactivity. Calbindin immunoreactivity was not detected in the spiral ganglion neurons, which are labeled by neurotensin immunoreactivity. The presence of neurotensin in the cochlea may be related to its modulation of neurotransmission in the peripheral auditory pathway.

Multivariate search for differentially expressed gene combinations.

BACKGROUND: To identify differentially expressed genes, it is standard practice to test a two-sample hypothesis for each gene with a proper adjustment for multiple testing. Such tests are essentially univariate and disregard the multidimensional structure of microarray data. A more general two-sample hypothesis is formulated in terms of the joint distribution of any sub-vector of expression signals. RESULTS: By building on an earlier proposed multivariate test statistic, we propose a new algorithm for identifying differentially expressed gene combinations. The algorithm includes an improved random search procedure designed to generate candidate gene combinations of a given size. Cross-validation is used to provide replication stability of the search procedure. A permutation two-sample test is used for significance testing. We design a multiple testing procedure to control the family-wise error rate (FWER) when selecting significant combinations of genes that result from a successive selection procedure. A target set of genes is composed of all significant combinations selected via random search. CONCLUSIONS: A new algorithm has been developed to identify differentially expressed gene combinations. The performance of the proposed search-and-testing procedure has been evaluated by computer simulations and analysis of replicated Affymetrix gene array data on age-related changes in gene expression in the inner ear of CBA mice.