Preyer's Reflex Research Articles

Curcumin attenuates gentamicin and sodium salicylate ototoxic effects by modulating the nuclear factor-kappaB and apoptotic pathways in rats

This study aimed to investigate the effectiveness of curcumin (CCM) against gentamicin (GEN) and sodium salicylates (NaS)-induced ototoxic effects in rats. For 15 consecutive days, seven rat groups were given 1 mL/rat physiological saline orally, 1 mL/rat olive oil orally, 50 mg/kg bwt CCM orally, 120 mg/kg bwt GEN intraperitoneally, 300 mg/kg bwt NaS intraperitoneally, CCM+GEN, or CCM+NaS. The distortion product otoacoustic emission measurements were conducted. The rats’ hearing function and balance have been behaviorally assessed using auditory startle response, Preyer reflex, and beam balance scale tests. The serum lipid peroxidation and oxidative stress biomarkers have been measured. Immunohistochemical investigations of the apoptotic marker caspase-3 and the inflammatory indicator nuclear factor kappa (NF-κB) in cochlear tissues were conducted. GEN and NaS exposure resulted in deficit hearing and impaired ability to retain balance. GEN and NaS exposure significantly decreased the reduced glutathione level and catalase activity but increased malondialdehyde content. GEN and NaS exposure evoked pathological alterations in cochlear and vestibular tissues and increased caspase-3 and NF-κB immunoexpression. CCM significantly counteracted the GEN and NaS injurious effects. These outcomes concluded that CCM could be a naturally efficient therapeutic agent against GEN and NaS-associated ototoxic side effects.Graphical abstract

Gap-induced inhibition of the post-auricular muscle response in humans and guinea pigs

A common method for measuring changes in temporal processing sensitivity in both humans and animals makes use of GaP-induced Inhibition of the Acoustic Startle (GPIAS). It is also the basis of a common method for detecting tinnitus in rodents. However, the link to tinnitus has not been properly established because GPIAS has not yet been used to objectively demonstrate tinnitus in humans. In guinea pigs, the Preyer (ear flick) myogenic reflex is an established method for measuring the acoustic startle for the GPIAS test, while in humans, it is the eye-blink reflex. Yet, humans have a vestigial remnant of the Preyer reflex, which can be detected by measuring skin surface potentials associated with the Post-Auricular Muscle Response (PAMR). A similar electrical potential can be measured in guinea pigs and we aimed to show that the PAMR could be used to demonstrate GPIAS in both species.In guinea pigs, we compare the GPIAS measured using the pinna movement of the Preyer reflex and the electrical potential of the PAMR to demonstrate that the two are at least equivalent. In humans, we establish for the first time that the PAMR provides a reliable way of measuring GPIAS that is a pure acoustic alternative to the multimodal eye-blink reflex. Further exploratory tests showed that while eye gaze position influenced the size of the PAMR response, it did not change the degree of GPIAS.Our findings confirm that the PAMR is a sensitive method for measuring GPIAS and suggest that it may allow direct comparison of temporal processing between humans and animals and may provide a basis for an objective test of tinnitus.

Cisplatin effects on guinea pigs: cochlear histology and genotoxicity

AbstractTo understand how the DNA answers to external agents such as cisplatin may be relevant to the diagnosis and treatment of hearing disorders caused by the administration of such drug. ObjectivesTo investigate the cisplatin influence on the cochlea and DNA of guinea pigs. Material and MethodsExperimental study carried out with 12 guinea pigs (Cavia porcellus). The inclusion criterion was the presence of Preyer's reflex and distortion-product otoacoustic emissions. Guinea pigs were divided into two groups: Control Group (CG) - made up of six guinea pigs, to which we administrated saline solution during six consecutive days, intraperitoneally; and a Study Group (SG) - made up of six guinea pigs, to which we administrated cisplatin during six consecutive doses of 3mg/kg/day intraperitoneally. Twenty-four hours after the last administration of cisplatin the guinea pigs were slaughtered, blood samples were collected and the cochleae were removed. ResultsThe administration of cisplatin did not cause identifiable changes to the DNA. Histological analysis showed changes in the organ of Corti and spiral ganglion. ConclusionCisplatin causes changes in cochlear histology, such as the loss of the normal microcytoarchitecture of the organ of Corti, and reduction of neurons of the spiral ganglion with cell alterations, however, DNA damage was not detected.

Dietary restriction reduces age-related degeneration of stria vascularis in the inner ear of the rat

We report here beneficial effects of life-long dietary restriction on the progression of age-associated cochlear degeneration in female Sprague–Dawley rats. Thirty-month old rats on a 70% dietary restriction were compared to ad libitum fed age-matched rats, and three-month old adult rats. As expected, aged dietary restricted rats displayed about 20% higher survival rate than age-matched rats fed ad libitum. This difference was reflected also in the auditory system. In the dietary restricted group, 73% of the subjects had preserved auditory reflexes (Preyer), and only modest degeneration of the stria vascularis of the inner ear was observed. In contrast, aged ad libitum fed animals, of which only 15% had detectable Preyer reflexes, showed a marked thinning, cellular degeneration and loss of cell processes in the stria vascularis. The extent of loss of sensory hair cells (~24%) was similar in both the aged groups, and neither group showed a significant reduction in the number of spiral ganglion neurons across adult life-span. The observations thus demonstrate that dietary restriction delays age-related degradation of the auditory system. The results provide further insights into the mechanisms of strial presbycusis.

The effect of prostaglandin E1 on brainstem blood flow disturbance in an animal model of vertebrobasilar insufficiency

Using an animal model of vertebrobasilar insufficiency (VBI), in which brainstem circulatory disturbance was induced in rats, we examined how prostaglandin E1 (PG-E1) affects brainstem blood flow (BBF) to clarify whether it is effective against VBI. Fifteen healthy male Wister rats that displayed positive responses to Preyer's reflex were used. Their BBF was continuously measured on the left side of the midline of the brainstem using laser Doppler flowmetry. A rat model of VBI, a pathological condition that presents with decreased BBF, was prepared by applying a vertebral artery clamp ipsilateral to the BBF measuring site and inducing hypotension of 60-70mmHg by blood withdrawal. Saline as a control (n=5), 5ng/kg/min (n=5) or 10ng/kg/min (n=5) PG-E1 at a dose was continuously administered to the rats using a motor-driven syringe pump. The effects of the drugs on the rats' BBF were evaluated. BBF, which was decreased by the unilateral vertebral artery clamping combined with blood withdrawal-induced hypotension, recovered in a dose-dependent manner after the administration of 5 or 10ng/kg/min PG-E1, and complete recovery to the baseline level was achieved by 60min treatment; however, no such effect was observed for intravenous saline. These results suggest that PG-E1 acts on local vessels and improves blood flow insufficiency in the brainstem in our animal model of VBI. PG-E1 might be useful as a cerebral vasodilator for VBI.

A novel behavioural approach to detecting tinnitus in the guinea pig

Tinnitus, the perception of sound in the absence of an external stimulus, is a particularly challenging condition to demonstrate in animals. In any animal model, objective confirmation of tinnitus is essential before we can study the neural changes that produce it. A gap detection method, based on prepulse inhibition of the whole-body startle reflex, is often used as a behavioural test for tinnitus in rodents. However, in the guinea pig the whole-body startle reflex is subject to rapid habituation and hence is not an ideal behavioural measure. By contrast, in this species the Preyer or pinna reflex is a very reliable indicator of the startle response and is much less subject to habituation. We have developed a novel adaptation of the gap detection paradigm, which uses the Preyer reflex to measure the startle response, rather than whole-body movement. Using this method, we have demonstrated changes in gap detection, in guinea pigs where tinnitus had been induced by the administration of a high dose of salicylate. Our data indicate that the Preyer reflex gap detection method is a reliable test for tinnitus in guinea pigs.

Garlic-Supplemented Diet Attenuates Gentamicin-Induced Ototoxicity: An Experimental Study

Gentamicin sulfate is a potent aminoglycoside antibiotic associated with serious side effects, including ototoxicity. Garlic, with its intrinsic antioxidant activity, may prove beneficial in prevention of ototoxicity. In this study, we investigated the effect of a 4% garlic-supplemented diet on the ototoxicity induced by gentamicin in rats by using brain stem evoked response audiometry. Eighteen male Wistar rats with an intact Preyer's reflex and an initial weight of 220 to 260 g were randomly assigned to a group with gentamicin injection and garlic supplementation, a group with gentamicin injection without garlic supplementation, or a control group (6 rats each group). Gentamicin was given by intraperitoneal injection at 120 mg/kg body weight once daily for 16 days. The garlic-supplemented diet was prepared by adding pulverized whole garlic cloves to standard chow in a 4% proportion. After 21 days, hearing thresholds were evaluated by use of brain stem evoked response audiometry at 10 kHz. The mean (+/- SD) amplitudes of the auditory thresholds (sensation level) measured by use of brain stem evoked response audiometry for the group with garlic supplementation, the group without garlic, and the control group were 43.3 +/- 8.16, 78.0 +/- 4.47, and 16.7 +/- 5.16 dB sensation level, respectively. The differences were statistically significant (p < 0.05). A garlic-supplemented diet seems to attenuate aminoglycoside-induced hearing loss.

Geranylgeranylacetone suppresses noise-induced expression of proinflammatory cytokines in the cochlea

Heat shock transcription factor 1 (HSF1) is a master regulator of heat shock response, and also inhibits expression of inflammatory cytokines directly or indirectly. Here, we examined effects of HSF1 activation on the expression of proinflammatory cytokines in mouse cochlea after exposure to noise. Male CBA/N mice with normal Preyer's reflex were exposed to intense noise for 3h. Three hours after noise exposure, bilateral cochleae were removed and expression of major inflammatory cytokines was examined. We found that interleukin-6 (IL-6) and interleukin-1β (IL-1β) expression increased significantly after noise exposure, and the expression was suppressed significantly in mice administered with geranylgeranylacetone (GGA), which activates HSF1. Seven days after noise exposure, thresholds for auditory brainstem response were elevated, and GGA administration significantly suppressed this elevation. These results suggest that HSF1-mediated suppression of proinflammatory cytokines in the cochlea by GGA administration could be an important means of inner ear protection.

Using the Auditory Brainstem Response (ABR) to Determine Sensitivity of Hearing in Mutant Mice.

Measurements of auditory evoked potentials can be used to determine reliably an audiometric representation of hearing sensitivity in mice. In a high-throughput phenotyping screen of mice carrying targeted mutations of single genes, the auditory brainstem response (ABR) is used to gain an estimate of hearing threshold for broadband click stimuli and pure tone frequencies ranging from 6 to 30 kHz. Comparison of thresholds obtained in mutant and wild-type mice give a means to determine mild, moderate, and severe hearing impairment. This gives a clear advantage over using a "clickbox" test to assess hearing by observations of the Preyer reflex. The ABR screen has identified several mutant lines with mild and moderate hearing loss, which appear to demonstrate normal Preyer responses. The ABR technique also allows frequency-selective hearing loss to be identified. Curr. Protoc. Mouse Biol. 1:279-287 © 2011 by John Wiley & Sons, Inc.

Therapeutic efficacy of topical application of dexamethasone to the round window niche after acoustic trauma caused by intensive impulse noise in guinea pigs

To assess the therapeutic efficacy of dexamethasone administered topically to the round window niche, following acoustic trauma induced by intensive impulse noise, in guinea pigs. Adult, male, albino guinea pigs with a normal Preyer's reflex were exposed to 80 impulse noises (peak value 167 dB, duration 0.5 ms, interval 2 s). Dexamethasone (40 mg/ml) or saline was then topically applied to the round window niche. Each animal's auditory brainstem response was measured before and one day after exposure, and three weeks after topical treatment. Cochlear morphology was examined to assess hair cell loss and spiral ganglion cell damage. To assess oxidative activity, cochlear malondialdehyde and superoxide dismutase concentrations were determined three weeks post-treatment. Following topical application, the pharmacokinetic characteristics of dexamethasone in cochlear perilymph were analysed using high-performance liquid chromatography. Animals receiving dexamethasone showed reduced noise-induced outer hair cell loss (three weeks post-treatment), and significant attenuation of noise-induced auditory brainstem response threshold shifts (one day post-exposure and three weeks post-treatment), compared with controls. There was no difference in spiral ganglion morphology. Animals receiving dexamethasone also showed a significantly lower malondialdehyde concentration and a higher superoxide dismutase concentration, post-exposure. Following topical application, the perilymph dexamethasone level peaked at 5330.522 µg/ml (15 minutes post-treatment), and was 299.797 µg/ml 360 minutes later. Topical application of dexamethasone to the round window niche has protective effects against intensive impulse noise induced trauma in the guinea pig cochlea. This drug can diffuse into the inner ear through the round window membrane and persist in the perilymph for a relatively long period. The mechanism of protection may involve an anti-oxidant effect.

The quantification of kanamycin ototoxicity in the rat using conditioned tone discrimination.

Twelve male Lister hooded rats were conditioned to discriminate an 8 kHz tone (56.5 +/- 0.5 dB re 0.0002 dyne cm-2) and were subsequently injected subcutaneously with kanamycin (400 mg kg-1 day-1) for 28 days; during this time and for five weeks after dosage was stopped, the nature and extent of the resultant hearing deficits were studied. The animals' Preyer reflex thresholds were determined at intervals during the study. Only one rat was unaffected by the kanamycin dosage. The onset of hearing impairment (reduced discrimination performance), which was gradual in some rats and sudden in others, generally occurred during the fourth week of kanamycin dosage although the earliest onset was towards the end of the second week. In most animals the hearing impairment progressed after kanamycin was stopped and in one rat there was a latency between the end of drug dosage and onset of hearing impairment. Hearing impairment was irreversible in five rats. One rat, whose impairment was slight, recovered normal hearing. Some rats showed a reduced discrimination performance at a time when their Preyer reflex threshold showed no elevation suggesting that kanamycin, at least initially, caused a threshold elevation rather than reduced sensitivity to intense sounds.

Exome sequencing identifies a missense mutation in Isl1associated with low penetrance otitis media in dearisch mice

BackgroundInflammation of the middle ear (otitis media) is very common and can lead to serious complications if not resolved. Genetic studies suggest an inherited component, but few of the genes that contribute to this condition are known. Mouse mutants have contributed significantly to the identification of genes predisposing to otitis mediaResultsThe dearisch mouse mutant is an ENU-induced mutant detected by its impaired Preyer reflex (ear flick in response to sound). Auditory brainstem responses revealed raised thresholds from as early as three weeks old. Pedigree analysis suggested a dominant but partially penetrant mode of inheritance. The middle ear of dearisch mutants shows a thickened mucosa and cellular effusion suggesting chronic otitis media with effusion with superimposed acute infection. The inner ear, including the sensory hair cells, appears normal. Due to the low penetrance of the phenotype, normal backcross mapping of the mutation was not possible. Exome sequencing was therefore employed to identify a non-conservative tyrosine to cysteine (Y71C) missense mutation in the Islet1 gene, Isl1Drsh. Isl1 is expressed in the normal middle ear mucosa. The findings suggest the Isl1Drshmutation is likely to predispose carriers to otitis media.ConclusionsDearisch, Isl1Drsh, represents the first point mutation in the mouse Isl1 gene and suggests a previously unrecognized role for this gene. It is also the first recorded exome sequencing of the C3HeB/FeJ background relevant to many ENU-induced mutants. Most importantly, the power of exome resequencing to identify ENU-induced mutations without a mapped gene locus is illustrated.

Effect of Burow's Solution on the Guinea Pig Inner Ear

We examined the changes in the inner ear hair cells following intratympanic injection of Burow's solution. Thirty-one albino guinea pigs with a normal Preyer's reflex were used. Burow's solution was applied and allowed to remain on the round window membrane for 30 minutes (30-minute group), 1 hour (1-hour group), or 2 hours (2-hour group). Seven days later, the left temporal bone was removed. Auditory brain stem responses were recorded at 4, 8, and 20 kHz before application of Burow's solution and again immediately before decapitation. The cochlea and utricle were dissected, stained with rhodamine-phalloidin, and examined under a fluorescence microscope. The postoperative auditory brain stem response thresholds at 20 kHz in the 1-hour group and those at 8 and 20 kHz in the 2-hour group were increased significantly compared to the baseline thresholds. Surface preparations of the organ of Corti revealed no hair cell loss in the 30-minute group, loss of outer hair cells in the lower half of the basal turn in half of the animals in the 1-hour group, and loss of outer hair cells in the basal turn in almost all animals in the 2-hour group. In the 2-hour group, the microthin sections of the round window membrane showed degeneration of the outer epithelium. The retention of Burow's solution on the round window membrane for 2 hours induces degeneration of the outer epithelium and damage to the cochlear outer hair cells.

마우스에서 고려 홍삼의 구강내 섭취를 통한 소음성 난청의 예방효과

It is well known that the saponin of Korean red ginseng (KRG) has an anti-oxidant effect and could suppress the accumulation of lipid peroxidation. The aim of the present study was to observe the inhibitory effect of KRG on mice with noise-induced hearing loss, and to determine its optimal dose. BALB/c mice with a normal hearing level and normal Preyer's reflexes were used in the study. The mice in the permanent-threshold-shift (PTS) group were exposed to noise (120-㏈ SPL, white noise band) in a noise booth for 3 h a day, for three consecutive days. The mice in the experimental group were given heat-processed red-ginseng extract (50 ㎎/㎏, 100 ㎎/㎏, and 200 ㎎/㎏), and those in the control group were given normal saline alone during their noise exposure. The mice in the temporary-threshold-shift (TTS) group were exposed to noise (120 ㏈SPL, white noise band) in a noise booth for 3 h. The mice in the experimental group were given heat-processed red-ginseng extract (50 ㎎/㎏, 100 ㎎/㎏, and 200 ㎎/㎏), and those in the control group were given normal saline alone before their noise exposure. The hearing levels of the mice were measured through auditory brainstem response (ABR) immediately and 1, 3, 5, 7, and 14 days after their noise exposure. Cochleae were removed from the mice 14 days after their noise exposure. Immunochemical and immunofluorescent staining were performed to observe the expression of 8-oxoG in cochlea. In the PTS group, the hearing function of the mice in all the groups was not recovered after their noise exposure. In the TTS group, however, the hearing function of the mice in all the groups was recovered within 14 days. Reduced hearing impairment and early recovery were observed in the mice that were given 200 ㎎/㎏ KRG, and early recovery was observed in the mice that were given 100 mg/kg KRG. The immunopositive staining of 8-oxoG was detected in the stria vascularis in the control group but was diminished in the mice that were given 200 ㎎/㎏ KRG. The ingestion of more than 100 ㎎/㎏ KRG demonstrated a protection and recovery effect on the noiseinduced-TTS group. Since KRG has been reported to be a safe compound even up to hundreds of mg/kg, a higher concentration of it may effectively protect and recover TTS.

The effect of increased inner ear pressure on tympanic membrane vibration

It has been suggested that increased inner ear pressure can affect the stiffness of the tympano-ossicular conductive system. However, the published literature contains few studies on changes of middle ear mechanics caused by increased inner ear pressure. In this study, we evaluated the effects of increased inner ear pressure on tympanic membrane vibration in guinea pigs using a laser Doppler vibrometer (LDV). We used 10 male guinea pigs (weighing 250 g each) with normal eardrums and Preyer reflexes were used for the experiment. Inner ear pressure was modified by two approaches--directly via a tube placed in the inner ear (n=5) and indirectly via a tube placed in the subaracnoid space (n=5). Slow uniform pressure (microl/min) was infused until constant pressure was maintained. At this time, an artificial endolymph or perilymph was injected with a constant pressure of 100 and 300 mm H2O. We measured the umbo velocity using a LDV. A positive pressure load decreased the umbo vibration at low frequencies but did not change the umbo vibration at mid and high frequencies. The umbo velocity had a greater reduction for an endolymphatic pressure load than for a perilymphatic pressure load. The largest change in velocity was noted at a frequency of 0.1 kHz. There was no significant change in velocity at higher frequencies, except for 5 kHz. LDV is a good tool to analyze middle ear function and it has the potential indirectly to diagnose an increase in endolymphatic or perilymphatic pressure.

Unilateral intra-perilymphatic infusion of substance P enhances ipsilateral vestibulo-ocular reflex gains in the sinusoidal rotation test

Previous studies have reported localization of substance P (SP) within the inner ear and that SP exists abundantly within vestibular endorgans. While SP's functional role in the inner ear remains unclear, SP can act as a neuromodulator in the CNS and directly influences neuronal excitability. We hypothesized that SP might influence neuronal excitability within the vestibular periphery. The present study used the sinusoidal rotation test to investigate the influence of SP after its local application in the guinea pig unilateral inner ear. A tiny hole was made adjacent to the round window in the right ears of Hartley white guinea pigs that had normal tympanic membranes and Preyer reflexes. An osmotic pump infused SP (10−4M, 10−3M, and 10−2M), neurokinin-1 (NK-1) receptor antagonist (10−3M) alone, or SP (10−3M)+NK-1 receptor antagonist (10−3M) through this hole, with rotation tests performed before, and 12h and 24h after the treatment. Results were used to calculate the vestibulo-ocular reflex (VOR) gains. After administration of 10−3M and 10−2M SP, significant increases in the VOR gains were noted at 12h after treatment, with these gains disappearing by 24h after treatment. This increase was not observed when there was simultaneous NK-1 receptor antagonist administration. There were also no changes in the VOR gains noted after administration of 10−4M SP or the NK-1 receptor antagonist alone. These results indicate the possibility that SP may act on vestibular endorgans as an excitatory factor via the NK-1 receptors.

Ototoxicity in Guinea Pigs: NO Levels in Cochlear Fluids

Problem Ototoxicity continues to be the most common side effect of gentamicin, and its physiopathology is still unclear. Currently the main accepted theory relates the ototoxicity with over production of free radicals, like nitric oxide (NO). Objective: To analyze the NO levels in the cochlear fluids of guinea pigs exposes to gentamicin and its relation with ototoxicity and propose an experimental model to study ototoxicity and drugs that may prevent it. Methods Six guinea pigs were used, divided in two groups: a) Study – submitted to intraperitoneal injections of gentamicin, (135mg/kg/day) for 14days; b) Control - submitted to intraperitoneal injections of distilled water, daily, for 14days; the hearing loss was evaluated with Preyer's reflex. On 14 day the guinea pigs were sacrificed and the cochlear fluids were collected for dosage of the nitric oxide through the chemiluminescence method (NOA-TM 280). Results The Preyer's reflex was absent at the end of the experiment in the Study group, what did not happen in the control group. The average of the nitric oxide levels of the guinea pigs of the study group was 26.2μ and the Control group 11.26μM. Conclusion It was possible to measure nitric oxide levels in guinea pigs cochlear fluids. The results reinforce the role of nitric oxide in ototoxicity induced by gentamicin. Significance Experimental study (Level 2).

Gentamicin Attenuates Gentamicin-Induced Ototoxicity – Self-Protection

Aminoglycoside antibiotics cause considerable toxicity to the inner ear. A progressive hearing loss at high frequencies resulted from the loss of hair cells in the base of the cochlea and a constant preoccupation with finding a treatment that protects against their toxic effects. A self-protection phenomenon to high ototoxic doses of gentamicin is proposed in this paper. Thirty-eight adult guinea pigs with normal hearing were tested using Preyer's reflex and the distortion product otoacoustic emission (DPOAE) test, and their cochleae were analyzed by scanning electron microscopy. To the four groups investigated, group I (control) and group II (low dose, 10 mg/kg/day for 30 days) showed a normal DPOEA and normal outer hair cells; group III (high dose, 160 mg/kg/day for 10 days) showed the absence of DPOEA and damage to the outer hair cells; and group IV (low dose, 10 mg/kg/day for 30 days followed by a high dose of 160 mg/kg/day for 10 days) showed a normal DPOEA and normal outer hair cells. These results demonstrate that there was a considerable self-protection phenomenon by gentamicin.

Whirler Mutant Hair Cells Have Less Severe Pathology than Shaker 2 or Double Mutants

MYOSIN XV is a motor protein that interacts with the PDZ domain-containing protein WHIRLIN and transports WHIRLIN to the tips of the stereocilia. Shaker 2 (sh2) mice have a mutation in the motor domain of MYOSIN XV and exhibit congenital deafness and circling behavior, probably because of abnormally short stereocilia. Whirler (wi) mice have a similar phenotype caused by a deletion in the third PDZ domain of WHIRLIN. We compared the morphology of Whrn (wi/wi) and Myo15 (sh2/sh2) sensory hair cells and found that Myo15 (sh2/sh2) have more frequent pathology at the base of inner hair cells than Whrn (wi/wi), and shorter outer hair cell stereocilia. Considering the functional and morphologic similarities in the phenotypes caused by mutations in Myo15 and Whrn, and the physical interaction between their encoded proteins, we used a genetic approach to test for functional overlap. Double heterozygotes (Myo15 (sh2/+), Whrn (wi/+)) have normal hearing and no increase in hearing loss compared to normal littermates. Single and double mutants (Myo15 (sh2/sh2), Whrn (wi/wi)) exhibit abnormal persistence of kinocilia and microvilli, and develop abnormal cytoskeletal architecture. Double mutants are also similar to the single mutants in viability, circling behavior, and lack of a Preyer reflex. The morphology of cochlear hair cell stereocilia in double mutants reflects a dominance of the more severe Myo15 (sh2/sh2) phenotype over the Whrn (wi/wi) phenotype. This suggests that MYOSIN XV may interact with other proteins besides WHIRLIN that are important for hair cell maturation.

Ush1c216A knock-in mouse survives Katrina

Usher syndrome is the most common cause of inherited deafness found in combination with blindness. All Usher patients suffer progressive retinitis pigmentosa, with the degree of hearing impairment and the presence or absence of vestibular function differing among subtypes. A cryptic splice site mutation (216G → A) in exon 3 of the USH1C gene on chromosome 11p, which encodes a PDZ-domain protein, harmonin, was found in Acadian Usher type IC patients in south Louisiana. In vitro analysis using constructs containing the mutant 216A and subsequent analysis of patient cell lines revealed a deletion of 35 bases in the transcript. In order to analyze the impact of this frame-shift mutation, we created a knock-in mouse model containing the human 216G → A mutation. A targeting construct was made containing 5′ and 3′ homology arms, each 4 kb in length, and a 650 base pair fragment containing exons 3 and 4 of human USH1C cloned from an Acadian patient homozygous for the 216A mutation. W4/129S6 embryonic stem (ES) cells were electroporated with the targeting construct, and after 10 days of neomycin selection, clones were picked and screened by polymerase chain reaction (PCR) and Southern blot analysis for homologous recombination. Two positive clones for targeted insertion were microinjected into C57BL/6 blastocysts which were then transplanted into pseudo-pregnant females. Chimeras were bred with Cre recombinase-expressing mice for simultaneous deletion of the neomycin gene and germline transmission of the 216A allele. Homozygous Ush1c216A (216AA) mice are hyperactive, display circling and head tossing behavior, and do not have a Preyer reflex at 21–25 days old. RT-PCR analysis of the cochlea and retina from 216AA mice shows the same 35 base deletion characteristic of Usher IC patients.