Waltzing Guinea Pig Research Articles

Vestibular morphology in the German Waltzing guinea pig.

The German waltzing guinea pig is a special strain of animal with a recessively inherited inner ear defect, resulting in deafness and a severe vestibular dysfunction. The hearing loss in the cochlea of the German strain is a result of a collapse of the Reissner membrane and the absence of scala media. The vestibular organ has not yet been described. German waltzing guinea pigs (homozygote and heterozygote) of different ages ranging from embryologic age 25 days to adulthood were investigated. The living animals were tested with four different vestibular tests, and the fetuses were controlled according to breeding. The morphology of the vestibular parts (ampulla, saccule, and utricle) was observed by using the light and transmission electron microscopy. Collapse of the membranous labyrinth was found already at embryologic age 50 days and progressed over time. Vestibular dysfunction was noted already from birth. Vestibular atelectasis has been shown to have the same morphology as the reported vestibular dysfunction in the German waltzing guinea pig. Owing to this similarity, this animal can be a good model for vestibular research.

Reduced noise susceptibility in littermate offspring from heterozygous animals of the German waltzing guinea pig.

The German waltzing guinea pig is a spontaneously mutated strain with severe auditory and vestibular impairment caused by a so far unknown genetic mutation. The animals are born deaf and show a circling behavior. The heterozygote animals of this guinea pig strain have functionally normal hearing and balance. However, these animals have, in earlier studies, shown an increased resistance to noise compared with normal wild-type guinea pigs. In the present study, we explored the functional hearing with auditory brainstem response thresholds before and at different time points after noise exposure. Symptom-free littermates from heterozygote couples of the German waltzing guinea pigs were exclusively used for the study, which, after the hearing test, were sent back for breeding to confirm their genotype (i.e. heterozygote or normal). The aim of this paper was to ascertain that the previously shown reduced susceptibility to noise trauma in the heterozygote animals of the German waltzing guinea pig was also evident when littermates were used as control animals. The findings are important for further analysis of the heterozygote animals of this strain and for future investigations of the underlying mechanisms behind the diverse susceptibility to exposures of loud sound.

Effect of auditory stress agents on heterozygous German waltzing guinea pigs

The German waltzing guinea pig is a strain of animals expressing deafness and severe balance disorders at birth. The mutation arose spontaneously in a breeding facility in Germany and as the affected animals show a characteristic waltzing behavior, the strain is named the German waltzing guinea pig. The strain is presently bred only at Karolinska Institutet.The hereditary inner ear impairment has a recessive mode of inheritance and the strain thus produces not only affected homozygotes but also symptom-free heterozygotes and fully normal offspring. The outcome depends solely on the genotype of the parents. The heterozygotes, which have obtained the “waltzing” gene from one parent only, have normal hearing and no balance dysfunction. The heterozygous animals appear normal but will, in turn, carry the genetic defect to the next generation. The present thesis is focused on these animals.Noise and ototoxic drugs are well known stress factors that interfere negatively with the hearing organ in both humans and animals, causing hearing impairment. However, the inter-individual variability in susceptibility to auditory stress factors is surprisingly large, most likely due to different genetic predisposition. In this study, heterozygous animals of the German waltzing guinea pig, animals carrying a genetic defect known to cause severe hearing impairment, were used to study how an unexplored gene for deafness interacts with auditory stress agents, i.e. noise exposure and the ototoxic drugs gentamicin and cisplatin.Animals were exposed to both narrowband as well as broadband noise at different ages and hearing thresholds were measured using ABRs. Heterozygotes of the German waltzing guinea pig showed less threshold shifts compared to control strains. Older animals were less affected by the noise trauma than younger animals.To test the hypothesis that the efferent system contributes to protection of the inner ear against noise trauma, measurements using a new method of post onset adaptation of DPOAEs and maximum adaptation magnitude were conducted. The post onset adaptation of DPOAEs detected a strain difference at the higher frequency region while in the maximum adaptation magnitude method showed no difference between the strains.The heterozygous German waltzing guinea pigs displayed a distinctly increased resistance to noise exposures, manifested as reduced threshold shifts and faster recovery following acoustic overstimulation. However, when exposed to ototoxic drugs, the heterozygous carriers suffered from a more pronounced hearing loss.It is concluded that endogenous resistance to noise in the heterozygotes does not offer any protection against ototoxic drugs. The detailed mechanisms remain to be explored.

The Genetic-Induced Hearing Loss Can Block the Effect of Noise Trauma in Waltzing Guinea pig

The waltzing guinea pig may be a good model to investigate if genetic factor can change the sensitivity in noise-induced hearing loss. A total of 34 waltzig guinea pigs were studied and we found that there is no any significant increased sensitivity to noise trauma if the age-induced hearing loss was considered in waltzing guinea pig.

Spatiotemporal loss of K+ transport proteins in the developing cochlear lateral wall of guinea pigs with hereditary deafness

Genetic deafness is one of the most common human genetic birth defects. To understand the molecular mechanisms underlying human hereditary deafness, deaf animal strains have proved to be invaluable models. The German waltzing guinea pig is a new strain of animals with unidentified gene mutation(s), displaying recessively inherited cochleovestibular impairment. Histological investigations of the homozygous animals (gw/gw) revealed a collapse of the endolymphatic compartment and malformation of stria vascularis. RT-PCR showed a significant reduction in expression of the strial intermediate cell-specific gene Dct and the tight-junction gene Cldn11 in the embryonic day (E)40 and adult gw/gw cochlear lateral wall. Immunohistochemical analysis of the gw/gw cochlea showed loss of the tight junction protein CLDN11 in strial basal cells from E40, loss of the potassium channel subunit KCNJ10 in strial intermediate cells from E50, and loss of the Na-K-Cl cotransporter SLC12A2 in strial marginal cells from E50. In addition, a temporary loss of the gap junction protein GJB2 (connexin 26) between fibrocytes in the spiral ligament of the E50 gw/gw cochlea was observed. The barrier composed of tight junctions between strial basal cells was disrupted in the gw/gw cochlea as indicated by a biotin tracer permeability assay. In conclusion, spatiotemporal loss of K+ transport proteins in the cochlear lateral wall is caused by malformation of the stria vascularis in the developing German waltzing guinea pig inner ear. This new animal strain may serve as a good model for studying human genetic deafness due to disruption of inner ear ion homeostasis.

Type I hair cell degeneration in the utricular macula of the waltzing guinea pig

Waltzing guinea pigs are an inbred guinea pig strain with a congenital and progressive balance and hearing disorder. A unique rod-shaped structure is found in the type I vestibular hair cells, that traverses the cell in an axial direction, extending towards the basement membrane. The present study estimates the total number of utricular hair cells and supporting cells in waltzing guinea pigs and age-matched control animals using the optical fractionator method. Animals were divided into four age groups (1, 7, 49 and 343 day-old). The number of type I hair cells decreased by 20% in the 343 day-old waltzing guinea pigs compared to age-matched controls and younger animals. Two-photon confocal laser scanning microscopy using antibodies against fimbrin and βIII-tubulin showed that the rods were exclusive to type I hair cells. There was no significant change in the length of the filament rods with age. Taken together, our data show that despite rod formation in the type I hair cells and deformation of hair bundles being present at birth, the type I hair cell population is not affected quantitatively until a year after birth.

Augmented Ototoxic Effect of Cisplatin in Heterozygotes of the German Waltzing Guinea Pig

It has previously been demonstrated that the carriers of the German waltzing guinea pig are less susceptible to noise trauma. To explore whether this represents a general resistance to inner ear trauma, carriers of the German waltzing guinea pig were exposed to the ototoxic agent cisplatin. Two doses of cisplatin were injected intravenously into anesthetized carriers and weight-matched control animals. Prior to and 96 h after the injections hearing thresholds were established by recording the auditory brainstem responses at 3.5, 7, 14, and 28 kHz. The cochleae were harvested to estimate hair cell loss and to analyze total platinum content. The carriers of the German waltzing guinea pig strain suffered from a more pronounced cisplatin-induced hearing loss compared to the control animals. The results suggest that mechanisms responsible for the protection against acoustic stress do not provide any protection against cisplatin in carriers of the German waltzing guinea pig.

Malformation of stria vascularis in the developing inner ear of the German waltzing guinea pig

Auditory function and cochlear morphology have previously been described in the postnatal German waltzing guinea pig, a strain with recessive deafness. In the present study, cochlear histopathology was further investigated in the inner ear of the developing German waltzing guinea pig (gw/gw). The lumen of the cochlear duct diminished progressively from embryonic day (E) 35 to E45 and was absent at E50 because of the complete collapse of Reissner's membrane onto the hearing organ. The embryonic stria vascularis, consisting of a simple epithelium, failed to transform into the complex trilaminar tissue seen in normal animals and displayed signs of degeneration. Subsequent degeneration of the sensory epithelium was observed from E50 and onwards. Defective and insufficient numbers of melanocytes were observed in the developing gw/gw stria vascularis. A gene involved in cochlear melanocyte development, Pax3, was markedly reduced in lateral wall tissue of the cochlea of both E40 and adult gw/gw individuals, whereas its expression was normal in the skin and diaphragm muscle of adult gw/gw animals. The Pax3 gene may thus be involved in the pathological process but is unlikely to be the primary mutated gene in the German waltzing guinea pig. TUNEL assay showed no signs of apoptotic cell death in the developing stria vascularis of this type of guinea pig. Thus, malformation of the stria vascularis appears to be the primary defect in the inner ear of the German waltzing guinea pig. Defective and insufficient numbers of melanocytes might migrate to the developing stria vascularis but fail to provide the proper support for the subsequent development of marginal and basal cells, thereby leading to stria vascularis malformation and dysfunction in the inner ear of the German waltzing guinea pig.

Auditory function and cochlear morphology in the German waltzing guinea pig

The German waltzing guinea pig is a new strain of animals with a recessively inherited inner ear defect resulting in deafness and severe vestibular dysfunction. Measurements of auditory brainstem responses (ABRs) demonstrated that the homozygotes ( gw/ gw) are deaf while the heterozygotes ( gw/+) have normal hearing. In the gw/gw cochlea, a collapse of Reissner’s membrane leads to the absence of scala media. Melanin pigment accumulation was clearly observed in the gw/ gw stria vascularis, and both the height and width of stria vascularis were significantly reduced. Ultrastructural observations further detailed the disorganization of stria vascularis in the gw/gw animals: marginal cells lacked basolateral infoldings; intermediate cells (melanocytes) were scarce and degenerated; and basal cells were difficult to identify. The level of degeneration of the organ of Corti varied between individual gw/ gw animals. The density of spiral ganglion neurons was significantly decreased in old (1–2 years of age) gw/ gw animals. In contrast, no pathological changes were observed in the cochleae of gw/+ animals. Our data suggest that the degeneration originates in the stria vascularis (most likely in the melanocytes), and that this is the primary cause for inner ear defects in the German waltzing guinea pig. Here, we describe the auditory function and cochlear morphology in this spontaneously mutated guinea pig strain.

A Guinea Pig Strain with Recessive Heredity of Deafness, Producing Normal-Hearing Heterozygotes with Resistance to Noise Trauma

A new strain of waltzing guinea pigs arose spontaneously in a guinea pig breeding facility in Germany in 1996. In addition to obvious vestibular dysfunction, the waltzing animals appear deaf already at birth. Histological analysis revealed that the waltzers lack an open scala media due to the collapse of Reissner’s membrane onto the surface of the hearing organ. Subsequent breeding has shown that this strain has a recessive mode of inheritance. The homozygotes are deaf and display a waltzing behaviour throughout their lives while the heterozygotes show no significant signs of inner ear injury despite being carriers of this specific mutated gene of hearing impairment. However, the heterozygous animals offer the opportunity to study how hereditary factors interact with auditory stress. In the present study, the susceptibility of the carriers to noise was investigated. Auditory brainstem responses were obtained prior to and after noise exposure (4 kHz, 110 dB, 6 h). The carriers were significantly less affected by the noise as compared to control animals. This difference was still significant at 4 weeks following noise exposure. It is suggested that the heterozygous animals have an endogenous resistance to auditory stress.

遺伝性内耳障害モデルにおける前庭形態異常と遺伝子導入の試み―ｓｈａｋｅｒ‐２マウス，ｗａｌｔｚｉｎｇ ｇｕｉｎｅａ Ｐｉｇの検討から―

We reviewed the hair cell pathology of genetic hearing loss and circling behavior and discussed possibility of gene therapy in human genetic inner ear disease through genetic inner ear disease animal models. First, we focused on the cytocauds containing pathological actin filaments in the vestibular type 1 hair cell of waltzing guinea pigs (WGP) and in auditory and vestibular hair cells of shaker-2 mice. The cytocaud, which seems like to be an actin filaments ladder, penetrates the basement membrane and runs along nerve fiber. One possible explanation is that hair cells detach from the basement membrane during development but do not in WGP and shaker-2 mice. However, the molecular signals that are required for detachment are unknown.Furthermore, we tested the possibility of gene transudation in shaker-2 mice, the model of DFNB3, i.e. human deafness, in order to treat the hair cell pathology including cytocaud. Vestibular sensory epithelia of explant organ culture obtained from mouse pups at P1, P3, and P5 were transfected by adenovirus vector containing the bacterial lacZ in vitro and processed to detect the reporter gene expression. Vestibular hair cells can be transduced at all stages in shaker-2 mice. Our data demonstrate that differentiating hair cells in mouse explants can be virally transduced, suggesting that wild-type genes can be inserted into these cells to correct the phenotype. We need further investigations of gene therapy using animal in vivo data to discuss the possibility of clinical application for the genetic inner ear diseases.

The Cytocaud: A Hair Cell Pathology in the Waltzing Guinea Pig

The waltzing guinea pig displays severe inner ear dysfunction that involves both an auditory and a vestibular manifestation. The aim of this study was to characterize a pathological tail-like extension of the vestibular hair cells, the cytocaud. Our data suggest that nearly all type I hair cells in the waltzing guinea pig have cytocauds, which appear as membrane-bound tails containing mitochondria and cytoplasm that proceed in a basal direction toward the basement membrane. The extensions either attach to the basement membrane or penetrate it, and further proceed into the extracellular matrix. A core made of a thick and long (30 µm) actin-rich structure supports the slender long process. The actin core has cross-links that are periodically placed along the length of the cytocaud. Our data suggest that the cytocauds in vestibular hair cells of the waltzing guinea pig are highly organized structures associated with a failure to detach from the basement membrane.

Dissociation between the calcium-induced and voltage-driven motility in cochlear outer hair cells from the waltzing guinea pig

The waltzing guinea pig, possessing an hereditary progressive deafness, shows pathology to the actin-bearing structures within the hair cells of the organ of Corti. In particular, the affected structures include the stereocilia, the cuticular plate and, as shown in the present study, swollen and disorganized subsurface cisternae. To test whether this pathology affected outer hair cell motility, cells were isolated from waltzing guinea pigs and their age-matched controls and were subjected to either electrical or chemical stimulation. Visual detection thresholds and the magnitude of the electrically-induced length changes were equivalent for both groups. However, when intracellular calcium was increased with either the calcium ionophore, ionomycin or Ca2+/ATP (under permeabilized conditions with DMSO), length changes were significantly reduced for the outer hair cells from waltzing guinea pigs compared to the controls. The average percent length increase induced by 10 microM ionomycin for the outer hair cells from control animals was 2.3 +/- 1.7 whereas for postnatal day 4 waltzing guinea pigs it was 1.3 +/- 1.7. Postnatal day 7 and 10 waltzing guinea pigs responded with significantly smaller percent length changes. The intracellular concentration of ionic calcium increased similarly for both groups after the application of ionomycin as revealed with the indicator fluo-3. In the permeabilized cells in the presence of Ca2+/ATP, control cells responded with a percent length change of 3.5, whereas, age-matched waltzing outer hair cells responded with barely detectable length changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of gross cochlear potentials to hair cell pathology in the waltzing guinea pig.

Electrophysiological recordings were made from the hearing organs of young waltzing guinea pigs of different ages. Age-related morphological changes in the cochleas of the same animals were studied with scanning and transmission electron microscopy. Results were compared with the results of studying the cochleas of normal guinea pigs of the same age. Waltzing guinea pigs are born with a hearing loss, as can be concluded from the magnitude of the whole-nerve action potential and their lower than normal summating potentials. However, their cochlear microphonics are nearly normal until the time in the degeneration process when hair cells start to disappear. This degeneration process begins at the tops of the hair cells and is evident upon electron microscopical examination of these structures.

Response of cochlear potentials to presumed alterations of ionic conductance: endolymphatic perfusion of barium, valinomycin and nystatin.

Two models ('single-pump' and 'two-pump') of transepithelial potassium movement by the marginal cells of the stria vascularis have been proposed in the literature. Their validity was considered by exposing the endolymphatic (luminal) surface to agents (barium, valinomycin and nystatin) which are known to alter specific cellular membrane conductances in other tissues. This was accomplished by the use either of injections or of a relatively satisfactory technique for perfusion of scala media, which is described. Injection of barium caused the endocochlear potential (EP) to increase in normal animals and had no effect on the EP of deaf, Waltzing guinea pigs. Perfusion of the ionophores caused a decline in the EP in both normal and Waltzing guinea pigs. Only the 'two-pump' model (Na/K-ATPase-mediated cation pump on the basolateral membrane and rheogenic K transporter at the luminal membrane) is consistent with the results. The cellular heterogeneity of the cochlear duct, however, introduces a measure of uncertainty into this interpretation.

Immunohistochemical identification and localization of actin and fimbrin in vestibular hair cells in the normal guinea pig and in a strain of the waltzing guinea pig.

Using immunohistochemical methods, actin and fimbrin were identified and localized in vestibular epithelia iin the normal guinea pig and in the waltzing guinea pig. In the normal guinea pig, actin was found in the stereocilia and in the cuticular plate. Fimbrin was detected in the stereocilia but surprisingly not in the cuticular plate. As fimbrin was found in the cuticular plates in cochlear hair cells it is suggested that the hair cells in the two organs have different mechanoreceptor properties which can demand different cuticular plate stability. In the waltzing guinea pig, actin was found in the stereocilia, in the rod and n the cuticular plate. Fimbrin was seen in the stereocilia and in the rod, but could not be detected in the cuticular plate. These results emphasize the resemblance between the rod and the stereocilia. It is suggested that the rod is a genetically induced pathological intracellular type of stereocilium which grows in an uncontrolled manner.

A morphological study on vestibular sensory epithelia in a strain of the waltzing guinea pig.

The crista ampullaris, the macula utriculi and the macula sacculi in the waltzing guinea pig were investigated with light microscopy, transmission and scanning electron microscopy. The degenerative changes consist of increasing degrees of apical protrusion of the type I hair cell into the endolymphatic space and of sensory hair fusion. The development of an intracellular actin filament rod in the type I hair cell is coupled to the degenerative changes of the apical part of the cell. Progressive hair cell degeneration with actin rod formation and sensory hair fusion as two important findings in the degenerative pattern is observed in type I hair cells of all sensory areas in the vestibular part of the labyrinth in the waltzing guinea pig. Scanning electron microscopy, shows a concentration of the more advanced stages of degenerative changes to the central part of the crista ampullaris and to the striolar area in the macula utriculi and in the macula sacculi. The fact that there is a concentration of sensory cell damage in the central areas of the vestibular end organs in genetically induced inner ear disease as well as in other inner ear damage supports the concept that the central areas have a different function. This paper further supports earlier suggestions that the type I and type II hair cells are genetically different cell types having different functions.

Freeze-fracturing of vestibular sensory epithelia in a strain of the waltzing guinea pig.

The waltzing guinea pig exhibits genetically induced inner ear disease which develops into deafness and vestibular disorder. The type I hair cell in vestibular end organs shows specific pathology, featuring protrusion of the hair cell apex, sensory hair fusion and an intracellular actin filament rod. With the freeze-fracturing technique, consecutive stages of degeneration of the apical part of the type I hair cell are shown. Different possible mechanisms underlying these changes are discussed. It is probable that the genetically induced prenatally developed hair cell pathology is related to a pathological development of actin filaments and their connections in the apical cytoskeleton.

Embryonic development of the specific vestibular hair cell pathology in a strain of the waltzing guinea pig.

The cytodifferentiation and maturation of vestibular hair cells, and the pathological development of vestibular type I hair cells, were followed in a strain of the waltzing guinea pig. The hair cells are first identified on the 30th gestational day. During the next 7-10 days the number of hair cells increases considerably in parallel with cytodifferentiation: the development of stereocilia, the cuticular plate, the afferent nerve terminals and the formation of synaptic contacts. On the 48th gestational day efferent nerve terminals are identified as are also a large number of fully developed nerve calyces. Differentiation into hair cells type I and type II was clearly evident at this stage. The first pathological finding appeared on the 38th gestational day as fusion of stereocilia. Actin filament rods in type I hair cells are identified with certainty on the 60th gestational day. In parallel with the in vivo development, inner ear explants (30-50th gestational days) were cultured in vitro. In this material actin filament rods were found already in 30th gestational day explants cultured 8 days in vitro. At this stage also hair cell protrusion and sensory hair fusion occurred.

Changes in rapidly transported proteins in the auditory nerve after hair cell loss

In an effort to further characterize the proteins of the auditory nerve, the effects of hair cell loss on rapidly transported proteins of the auditory nerve were studied. The effects were studied in the waltzing guinea pig, a genetically deaf animal which displays an age-dependent loss of sensory cells, and in normal guinea pigs treated with neomycin. In both cases hair cell loss is followed by a slow degeneration of spiral ganglion cells and corresponding auditory nerve fibers. Rapidly transported proteins in the cochlear nucleus were analyzed by two dimensional electrofocusing/electrophoresis 3 h after cochlear injection of [ 35S]methionine. In both the waltzing guinea pig and the neomycin-treated animals, a significant increase in labeling of two series of polypeptides (average molecular weights of 27,000 and 36,000 daltons) was apparent. Quantitation of the 36,000 dalton protein by extracting from dried gels showed a 2-fold increase in the 10-day-old waltzing guinea pig and a 6-fold increase in the 80-day-old waltzing guinea pig. Further analysis shows these proteins to be membrane-associated glycoproteins.