Although the inner ear has long been reported to be susceptible to middle ear disease, little is known of the inflammatory mechanisms that might cause permanent sensorineural hearing loss. Recent studies have shown inner ear tissues are capable of expressing inflammatory cytokines during otitis media. However, little quantitative information is available concerning cytokine gene expression in the inner ear and the protein products that result. Therefore, this study was conducted of mouse middle and inner ear during acute otitis media to measure the relationship between inflammatory cytokine genes and their protein products with quantitative RT-PCR and ELISA, respectively. Balb/c mice were inoculated transtympanically with heat-killed Haemophilus influenzae and middle and inner ear tissues collected for either quantitative RT-PCR microarrays or ELISA multiplex arrays. mRNA for several cytokine genes was significantly increased in both the middle and inner ear at 6 hours. In the inner ear, these included MIP-2 (448 fold), IL-6 (126 fold), IL-1β (7.8 fold), IL-10 (10.7 fold), TNFα (1.8 fold), and IL-1α (1.5 fold). The 24 hour samples showed a similar pattern of gene expression, although generally at lower levels. In parallel, the ELISA showed the related cytokines were present in the inner ear at concentrations higher by 2 to 122 fold higher at 18 hours, declining slightly from there at 24 hours. Immunohistochemistry with antibodies to a number of these cytokines demonstrated they occurred in greater amounts in the inner ear tissues. These findings demonstrate considerable inflammatory gene expression and gene products in the inner ear following acute otitis media. These higher cytokine levels suggest one potential mechanism for the permanent hearing loss seen in some cases of acute and chronic otitis media.

Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.

Hypothesis The inner ear endothelium is capable of responding to therapeutic steroids by altering local expression of cytokine and ion homeostasis genes that impact inflammation and fluid regulation. Background Glucocorticoids are often given trans-tympanically for hearing disorders because of their anti-inflammatory effects, but their direct impact on inner ear ion homeostasis and cytokine gene expression has not been studied. Methods The middle ears of Balb/c mice were transtympanically injected with 5 µl of either phosphate-buffered saline (PBS), prednisolone or dexamethasone. Untreated mice were used as controls. Mice were euthanized at 6, 24 and 72 hours, the cochleas harvested and total RNA isolated from the inner ear tissues. Expression of eight cytokine genes and 24 ion homeostasis genes were analyzed with qRT-PCR. Results PBS caused upregulation of inflammatory cytokine genes that peaked at 6 hours. Surprisingly, prednisolone and dexamethasone also caused upregulation of most cytokine genes. Interestingly, ion homeostasis genes were predominantly upregulated with dexamethasone and prednisolone, with prednisolone having the larger effect. Conclusion In the murine model, intratympanic steroids caused an initial upregulation of inflammatory cytokine genes in the inner ear, as well as predominantly upregulation of ion homeostasis genes. These findings suggest glucorticoids do not suppress inner ear inflammation, but rather cause an initial inflammatory response in the inner ear. Thus, inflammatory gene suppression is not a likely mechanism for their hearing restorative effects. On the other hand, these steroids have a significant mineralocorticoid function, as demonstrated by increased function of ion homeostasis genes, implicating their ionic and fluid regulatory properties as a mechanism for their therapeutic effects.

Mutations in the MYO7A gene lead to Usher syndrome type 1B (USH1B), a disease characterized by congenital deafness, vision loss, and balance impairment. To create a nonhuman primate (NHP) USH1B model, CRISPR/Cas9 was used to disrupt MYO7A in rhesus macaque zygotes. The targeting efficiency of Cas9 mRNA and hybridized crRNA-tracrRNA (hyb-gRNA) was compared to Cas9 nuclease (Nuc) protein and synthetic single guide (sg)RNAs. Nuc/sgRNA injection led to higher editing efficiencies relative to mRNA/hyb-gRNAs. Mutations were assessed by preimplantation genetic testing (PGT) and those with the desired mutations were transferred into surrogates. A pregnancy was established from an embryo where 92.1% of the PGT sequencing reads possessed a single G insertion that leads to a premature stop codon. Analysis of single peripheral blood leukocytes from the infant revealed that half the cells possessed the homozygous single base insertion and the remaining cells had the wild-type MYO7A sequence. The infant showed sensitive auditory thresholds beginning at 3 months. Although further optimization is needed, our studies demonstrate that it is feasible to use CRISPR technologies for creating NHP models of human diseases.

An overview of recent topics under investigation in our laboratory is presented. Topics include the cytokine and ion homeostasis gene response to steroids in the middle ear in a murine model of otitis media and gene chip analysis of inner ear genes affected by trans-tympanic and systemic steroid administration to the mouse.