10 research outputs found
Rhesus Cochlear and Vestibular Functions Are Preserved After Inner Ear Injection of Saline Volume Sufficient for Gene Therapy Delivery
Sensorineural losses of hearing and vestibular sensation due to hair cell dysfunction are among the most common disabilities. Recent preclinical research demonstrates that treatment of the inner ear with a variety of compounds, including gene therapy agents, may elicit regeneration and/or repair of hair cells in animals exposed to ototoxic medications or other insults to the inner ear. Delivery of gene therapy may also offer a means for treatment of hereditary hearing loss. However, injection of a fluid volume sufficient to deliver an adequate dose of a pharmacologic agent could, in theory, cause inner ear trauma that compromises functional outcome. The primary goal of the present study was to assess that risk in rhesus monkeys, which closely approximates humans with regard to middle and inner ear anatomy. Secondary goals were to identify the best delivery route into the primate ear from among two common surgical approaches (i.e., via an oval window stapedotomy and via the round window) and to determine the relative volumes of rhesus, rodent, and human labyrinths for extrapolation of results to other species. We measured hearing and vestibular functions before and 2, 4, and 8 weeks after unilateral injection of phosphate-buffered saline vehicle (PBSV) into the perilymphatic space of normal rhesus monkeys at volumes sufficient to deliver an atoh1 gene therapy vector. To isolate effects of injection, PBSV without vector was used. Assays included behavioral observation, auditory brainstem responses, distortion product otoacoustic emissions, and scleral coil measurement of vestibulo-ocular reflexes during whole-body rotation in darkness. Three groups (N = 3 each) were studied. Group A received a 10 μL transmastoid/trans-stapes injection via a laser stapedotomy. Group B received a 10 μL transmastoid/trans-round window injection. Group C received a 30 μL transmastoid/trans-round window injection. We also measured inner ear fluid space volume via 3D reconstruction of computed tomography (CT) images of adult C57BL6 mouse, rat, rhesus macaque, and human temporal bones (N = 3 each). Injection was well tolerated by all animals, with eight of nine exhibiting no signs of disequilibrium and one animal exhibiting transient disequilibrium that resolved spontaneously by 24 h after surgery. Physiologic results at the final, 8-week post-injection measurement showed that injection was well tolerated. Compared to its pretreatment values, no treated ear’s ABR threshold had worsened by more than 5 dB at any stimulus frequency; distortion product otoacoustic emissions remained detectable above the noise floor for every treated ear (mean, SD and maximum deviation from baseline: −1.3, 9.0, and −18 dB, respectively); and no animal exhibited a reduction of more than 3 % in vestibulo-ocular reflex gain during high-acceleration, whole-body, passive yaw rotations in darkness toward the treated side. All control ears and all operated ears with definite histologic evidence of injection through the intended site showed similar findings, with intact hair cells in all five inner ear sensory epithelia and intact auditory/vestibular neurons. The relative volumes of mouse, rat, rhesus, and human inner ears as measured by CT were (mean ± SD) 2.5 ± 0.1, 5.5 ± 0.4, 59.4 ± 4.7 and 191.1 ± 4.7 μL. These results indicate that injection of PBSV at volumes sufficient for gene therapy delivery can be accomplished without destruction of inner ear structures required for hearing and vestibular sensation
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Targeted disruption of dual leucine zipper kinase and leucine zipper kinase promotes neuronal survival in a model of diffuse traumatic brain injury.
BackgroundTraumatic brain injury (TBI) is a major cause of CNS neurodegeneration and has no disease-altering therapies. It is commonly associated with a specific type of biomechanical disruption of the axon called traumatic axonal injury (TAI), which often leads to axonal and sometimes perikaryal degeneration of CNS neurons. We have previously used genome-scale, arrayed RNA interference-based screens in primary mouse retinal ganglion cells (RGCs) to identify a pair of related kinases, dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) that are key mediators of cell death in response to simple axotomy. Moreover, we showed that DLK and LZK are the major upstream triggers for JUN N-terminal kinase (JNK) signaling following total axonal transection. However, the degree to which DLK/LZK are involved in TAI/TBI is unknown.MethodsHere we used the impact acceleration (IA) model of diffuse TBI, which produces TAI in the visual system, and complementary genetic and pharmacologic approaches to disrupt DLK and LZK, and explored whether DLK and LZK play a role in RGC perikaryal and axonal degeneration in response to TAI.ResultsOur findings show that the IA model activates DLK/JNK/JUN signaling but, in contrast to axotomy, many RGCs are able to recover from the injury and terminate the activation of the pathway. Moreover, while DLK disruption is sufficient to suppress JUN phosphorylation, combined DLK and LZK inhibition is required to prevent RGC cell death. Finally, we show that the FDA-approved protein kinase inhibitor, sunitinib, which has activity against DLK and LZK, is able to produce similar increases in RGC survival.ConclusionThe mitogen-activated kinase kinase kinases (MAP3Ks), DLK and LZK, participate in cell death signaling of CNS neurons in response to TBI. Moreover, sustained pharmacologic inhibition of DLK is neuroprotective, an effect creating an opportunity to potentially translate these findings to patients with TBI