Characterization of cochlear degeneration in the inner ear of the German waltzing guinea pig : a morphological, cellular, and molecular study

Sammanfattning: The German waltzing guinea pig is a new strain of animals with yet unknown gene mutation(s) displaying recessively inherited cochleovestibular impairment. The homozygous animals (gw/gw) are deaf already at birth and display Wpical waltzing behavior throughout life. The heterozygous animals (gw/+) do not suffer from hearing loss and vestibular symptoms. The present thesis is focused on the homozygous German waltzing guinea pig (gw/gw). Cochlear histological analysis in the postnatal animals revealed a characteristic cochleosaccular defect: collapse of scala media compartment, atrophy of stria vascularis, degeneration of sensory hair cells, and slow loss of spiral ganglion neurons. The stria vascularis was atrophic and appeared a single layer composed of only marginal cells, as further evidenced by morphometric measurement of strial height and width. The abnormal appearance of strial intermediate cells (melanocytes) were sparsely scattered in the spiral ligament, whereas strial basal cells were difficult to identify. The degree of sensory hair cells degeneration varied even among animals of the same age. Morphometric analysis of the spiral ganglion neuron profile density showed a significant reduction in the old (1-2 years of age) animals. An ensuing morphological study in the cochlea of prenatal embryos showed a progressive reduction of the scala media from embryonic day (E) 35 and its complete absence by E50. The degeneration of hair cells was first observed at E50 and onwards. The stria vascularis failed to transform into a multilayered epithelium but consisted of one layer of underdeveloped/degenerate marginal cells. The strial intermediate cells were sparsely distributed in the spiral ligament and showed signs of degeneration Strial basal cells were not easily recognized. RT-PCR analysis of the expression of genes regulating strial melanocyte development showed that Pax3 mRNA was remarkably decreased in the cochlear lateral wall but remained intact in the diaphragm muscle and skin tissue. The loss of the endolymphatic compartment in the gw/gw cochlea suggests a disruption of cochlear fluid and ion homeostasis. The mRNA and protein expression of several key players in cochlear homeostasis were thus investigated in the cochlear lateral. wall by semi-quantitative RT-PCR and immunohistochemistry. RT-PCR analysis showed a significant reduction in expression of the strial intermediate cellspecific gene Dct and the tight junction gene Cldn11 in the gwIgw 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 conclusion, dysfunctional strial cells, in particular intermediate cells, fail to maintain the integrity of the stria vascularis and eradicate the main cochlear K+ recycling pathway in the German waltzing guinea pig inner ear, ultimately resulting in the disruption of cochlear homeostasis and cochlear degeneration.