Guinea Pig Round Window Membrane Explantation for Ex Vivo Studies.

Efficient and minimally invasive drug delivery to the inner ear is a significant challenge. The round window membrane (RWM), being one of the few entry points to the inner ear, has become a vital focus of investigation. However, due to the complexities of isolating the RWM, our understanding of its pharmacokinetics remains limited. The RWM comprises three distinct layers: the outer epithelium, the middle connective tissue layer, and the inner epithelial layer, each potentially possessing unique delivery properties. Current models for investigating transport across the RWM utilize in vivo animal models or ex vivo RWM models which rely on cell cultures or membrane fragments. Guinea pigs serve as a validated preclinical model for the investigation of drug pharmacokinetics within the inner ear and are an important animal model for the translational development of delivery vehicles to the cochlea. In this study, we describe an approach for explantation of a guinea pig RWM with surrounding cochlear bone for benchtop drug delivery experiments. This method allows for preservation of native RWM architecture and may provide a more realistic representation of barriers to transport than current benchtop models.

A Benchtop Round Window Model for Studying Magnetic Nanoparticle Transport to the Inner Ear.

INTRODUCTION: The round window membrane (RWM) presents a significant barrier to the local application of therapeutics to the inner ear. We demonstrate a benchtop preclinical RWM model and evaluate superparamagnetic iron oxide nanoparticles (SPIONs) as vehicles for magnetically assisted drug delivery. METHODS: Guinea pig RWM explants were inset into a 3D-printed dual chamber benchtop device. Custom-synthesized 7-nm iron core nanoparticles were modified with different polyethylene glycol chains to yield two sizes of SPIONs (NP-PEG600 and NP-PEG3000) and applied to the benchtop model with and without a magnetic field. Histologic analysis of the RWM was performed using transmission electron microscopy (TEM) and confocal microscopy. RESULTS: Over a 4-h period, 19.5 ± 1.9% of NP-PEG3000 and 14.6 ± 1.9% of NP-PEG600 were transported across the guinea pig RWM. The overall transport increased by 1.45× to 28.4 ± 5.8% and 21.0 ± 2.0%, respectively, when a magnetic field was applied. Paraformaldehyde fixation of the RWM decreased transport significantly (NP-PEG3000: 7.6 ± 1.5%; NP-PEG600: 7.0 ± 1.6%). Confocal and electron microscopy analysis demonstrated nanoparticle localization throughout all cellular layers and layer-specific transport characteristics within RWM. CONCLUSION: The guinea pig RWM explant benchtop model allows for targeted and practical investigations of transmembrane transport in the development of nanoparticle drug delivery vehicles. The presence of a magnetic field increases SPION delivery by 45%-50% in a nanoparticle size- and cellular layer-dependent manner. LEVEL OF EVIDENCE: NA Laryngoscope, 134:3355-3362, 2024.

Synaptic transfer from outer hair cells to type II afferent fibers in the rat cochlea.

Type II cochlear afferents receive glutamatergic synaptic excitation from outer hair cells (OHCs) in the rat cochlea. However, it remains uncertain whether this connection is capable of providing auditory information to the brain. The functional efficacy of this connection depends in part on the number of presynaptic OHCs, their probability of transmitter release, and the effective electrical distance for spatial summation in the type II fiber. The present work addresses these questions using whole-cell recordings from the spiral process of type II afferents that run below OHCs in the apical turn of young (5-9 d postnatal) rat cochlea. A "high potassium puffer" was used to elicit calcium action potentials from individual OHCs and thereby show that the average probability of transmitter release was 0.26 (range 0.02-0.73). Electron microscopy showed relatively few vesicles tethered to ribbons in equivalent OHCs. A "receptive field" map for individual type II fibers was constructed by successively puffing onto OHCs along the cochlear spiral, up to 180 µm from the recording pipette. These revealed a conservative estimate of 7 presynaptic OHCs per type II fiber (range 1-11). EPSCs evoked from presynaptic OHCs separated by >100 µm did not differ in amplitude or waveform, implying that the type II fiber's length constant exceeded the length of the synaptic input zone. Together these data suggest that type II fibers could communicate centrally by maximal activation of their entire pool of presynaptic OHCs.

Traumatic axonopathy in spinal tracts after impact acceleration head injury: Ultrastructural observations and evidence of SARM1-dependent axonal degeneration.

Traumatic axonal injury (TAI) and the associated axonopathy are common consequences of traumatic brain injury (TBI) and contribute to significant neurological morbidity. It has been previously suggested that TAI activates a highly conserved program of axonal self-destruction known as Wallerian degeneration (WD). In the present study, we utilize our well-established impact acceleration model of TBI (IA-TBI) to characterize the pathology of injured myelinated axons in the white matter tracks traversing the ventral, lateral, and dorsal spinal columns in the mouse and assess the effect of Sterile Alpha and TIR Motif Containing 1 (Sarm1) gene knockout on acute and subacute axonal degeneration and myelin pathology. In silver-stained preparations, we found that IA-TBI results in white matter pathology as well as terminal field degeneration across the rostrocaudal axis of the spinal cord. At the ultrastructural level, we found that traumatic axonopathy is associated with diverse types of axonal and myelin pathology, ranging from focal axoskeletal perturbations and focal disruption of the myelin sheath to axonal fragmentation. Several morphological features such as neurofilament compaction, accumulation of organelles and inclusions, axoskeletal flocculation, myelin degeneration and formation of ovoids are similar to profiles encountered in classical examples of WD. Other profiles such as excess myelin figures and inner tongue evaginations are more typical of chronic neuropathies. Stereological analysis of pathological axonal and myelin profiles in the ventral, lateral, and dorsal columns of the lower cervical cord (C6) segments from wild type and Sarm1 KO mice at 3 and 7 days post IA-TBI (n = 32) revealed an up to 90% reduction in the density of pathological profiles in Sarm1 KO mice after IA-TBI. Protection was evident across all white matter tracts assessed, but showed some variability. Finally, Sarm1 deletion ameliorated the activation of microglia associated with TAI. Our findings demonstrate the presence of severe traumatic axonopathy in multiple ascending and descending long tracts after IA-TBI with features consistent with some chronic axonopathies and models of WD and the across-tract protective effect of Sarm1 deletion.

Traumatic Axonal Injury in the Optic Nerve: The Selective Role of SARM1 in the Evolution of Distal Axonopathy.

Traumatic axonal injury (TAI), thought to be caused by rotational acceleration of the head, is a prevalent neuropathology in traumatic brain injury (TBI). TAI in the optic nerve is a common finding in multiple blunt-force TBI models and hence a great model to study mechanisms and treatments for TAI, especially in view of the compartmentalized anatomy of the visual system. We have previously shown that the somata and the proximal, but not distal, axons of retinal ganglion cells (RGC) respond to DLK/LZK blockade after impact acceleration of the head (IA-TBI). Here, we explored the role of the sterile alpha and TIR-motif containing 1 (SARM1), the key driver of Wallerian degeneration (WD), in the progressive breakdown of distal and proximal segments of the optic nerve following IA-TBI with high-resolution morphological and classical neuropathological approaches. Wild type and Sarm1 knockout (KO) mice received IA-TBI or sham injury and were allowed to survive for 3, 7, 14, and 21 days. Ultrastructural and microscopic analyses revealed that TAI in the optic nerve is characterized by variable involvement of individual axons, ranging from apparent early disconnection of a subpopulation of axons to a range of ongoing axonal and myelin perturbations. Traumatic axonal injury resulted in the degeneration of a population of axons distal and proximal to the injury, along with retrograde death of a subpopulation of RGCs. Quantitative analyses on proximal and distal axons and RGC somata revealed that different neuronal domains exhibit differential vulnerability, with distal axon segments showing more severe degeneration compared with proximal segments and RGC somata. Importantly, we found that Sarm1 KO had a profound effect in the distal optic nerve by suppressing axonal degeneration by up to 50% in the first 2 weeks after IA-TBI, with a continued but lower effect at 3 weeks, while also suppressing microglial activation. Sarm1 KO had no evident effect on the initial traumatic disconnection and did not ameliorate the proximal optic axonopathy or the subsequent attrition of RGCs, indicating that the fate of different axonal segments in the course of TAI may depend on distinct molecular programs within axons.

Insights into Presbycusis From the First Temporal Bone Laboratory Within the United States.

The Johns Hopkins Otologic Research Laboratory was founded in 1924 as the first human temporal bone laboratory within the United States. To better understand the contributions of the Johns Hopkins Otologic Research Laboratory to our understanding of presbycusis, we consulted with a medical librarian and archivist to search the Alan Mason Chesney Medical Archives, PubMed, JSTOR, and Johns Hopkins Bulletin for published and unpublished works from the lab. Between 1924 and 1938, Samuel J. Crowe, the Chairman of Otolaryngology, and anatomist Stacy R. Guild amassed a collection of ∼1,800 temporal bones. This collection allowed for an unprecedented period of discovery related to otologic disease. They combined hearing thresholds measured by the recently invented audiometer with new techniques for temporal bone decalcification, sectioning, and staining, and a method for the graphic reconstruction of the cochlea. Crowe and Guild used this unique opportunity to correlate otopathology with hearing and to make the first detailed descriptions of the otopathology of presbycusis. In 1931 and 1934, they observed spiral ganglion neuron and outer hair cell loss in the basal turn of the cochlea in individuals with high-frequency hearing loss. These were the first studies to reveal that stria vascularis degeneration and middle ear pathology were not the most common causes for high-frequency hearing loss. Aside from revealing the primary driving factors of presbycusis, this work provided insight into the tonotopic organization of the cochlea. After initially being recruited to help raise money for the laboratory, medical illustrator Max Brödel used the vertical histologic cross-sections of the cochlea to produce illustrations of the ear. The decision to produce histologic sections in the plane of the superior semicircular canal likely influenced Brödel's illustrations that share a similar orientation and would later become widely circulated. Significant contributions from the Otologic Research Laboratory were also made by Mary Hardy, D.Sc., a woman who has previously received little recognition for her work. The sectioning of temporal bones was stopped in 1938 due to World War II, but much of Crowe's and Guild's work continued into the 1940s until a rift between the two resulted in the temporary closure of the laboratory in 1949. Nearly 100 years after its founding, discoveries from the Johns Hopkins Otologic Research Laboratory remain relevant and emphasize the importance of continued human temporal bone research to improve our understanding and treatment of otologic disease.

Acoustic Trauma Increases Ribbon Number and Size in Outer Hair Cells of the Mouse Cochlea.

Outer hair cells (OHCs) in the mouse cochlea are contacted by up to three type II afferent boutons. On average, only half of these are postsynaptic to presynaptic ribbons. Mice of both sexes were subjected to acoustic trauma that produced a threshold shift of 44.2 ± 9.1 dB 7 days after exposure. Ribbon synapses of OHCs were quantified in post-trauma and littermate controls using immunolabeling of CtBP2. Visualization with virtual reality was used to determine 3-D cytoplasmic localization of CtBP2 puncta to the synaptic pole of OHCs. Acoustic trauma was associated with a statistically significant increase in the number of synaptic ribbons per OHC. Serial section TEM was carried out on similarly treated mice. This also showed a significant increase in the number of ribbons in post-trauma OHCs, as well as a significant increase in ribbon volume compared to ribbons in control OHCs. An increase in OHC ribbon synapses after acoustic trauma is a novel observation that has implications for OHC:type II afferent signaling. A mathematical model showed that the observed increase in OHC ribbons considered alone could produce a significant increase in action potentials among type II afferent neurons during strong acoustic stimulation.

Targeted disruption of dual leucine zipper kinase and leucine zipper kinase promotes neuronal survival in a model of diffuse traumatic brain injury.

BACKGROUND: Traumatic 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. METHODS: Here 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. RESULTS: Our 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. CONCLUSION: The 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.

Effect of platelet rich plasma and fibrin sealant on facial nerve regeneration in a rat model.

OBJECTIVE: To investigate the effects of platelet rich plasma (PRP) and fibrin sealant (FS) on facial nerve regeneration. STUDY DESIGN: Prospective, randomized, and controlled animal study. METHODS: Experiments involved the transection and repair of facial nerve of 49 male adult rats. Seven groups were created dependant on the method of repair: suture; PRP (with/without suture); platelet poor plasma (PPP) (with/without suture); and FS (with/without suture) groups. Each method of repair was applied immediately after the nerve transection. The outcomes measured were: 1) observation of gross recovery of vibrissae movements within 8-week period after nerve transection and repair using a 5-point scale and comparing the left (test) side with the right (control) side; 2) comparisons of facial nerve motor action potentials (MAP) recorded before and 8 weeks after nerve transection and repair, including both the transected and control (untreated) nerves; 3) histologic evaluation of axons counts and the area of the axons. RESULTS: Vibrissae movement observation: the inclusion of suturing resulted in overall improved outcomes. This was found for comparisons of the suture group with PRP group; PRP with/without suture groups; and PPP with/without suture groups (P < .05). The PRP without suture group had a significantly greater degree of recovery than the PPP without suture group (P < .05), but it did not have better performance than suture group (P > .05). The movement recovery of the suture group was significantly better than the FS group (P = .014). The recovery of function of the PRP groups was better than that of the FS groups, although this did not reach statistical significance (P = .09). Electrophysiologic testing: there was a significantly better performance of the suture group when compared with the PRP and PPP without suture groups in nerve conduction velocity (P < .05). The PRP with suture group had the best results when compared with the suture as well as the PPP with suture groups in duration and latency-2 of MAP (P < .05). For the FS groups, no results were found demonstrating a biological effect. The PRP with suture group demonstrated the best performance in the latency-2 and the area under the curve of MAP when compared with the suture and FS with suture groups (P < .05). Histomorphometric analysis: PRP with suture demonstrated the greatest increase in axon counts when compared with suture, FS with suture, and PPP with suture groups (P < .05). There was no statistically significant difference seen in axon diameter. CONCLUSION: The best results for the return of function in our rat facial nerve axotomy models occurred when the nerve ends were sutured together. At the same time, the data demonstrated a measurable neurotrophic effect when PRP was present, with the most favorable results seen with PRP added to suture. There was an improved functional outcome with the use of PRP in comparison with FS or no bioactive agents (PPP). FS showed no benefit over conventional suturing in facial nerve regeneration. Our study provides the potential of a new clinical application for PRP in peripheral nerve regeneration.

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.

Efficient quantification of afferent cochlear ultrastructure using design-based stereology.

The afferent synapse between the auditory nerve fiber and the inner hair cell (IHC) represents a critical junction for hearing. Elucidation of the structure at this site will help establish the substrate for normal sound encoding as well as pathologic processes associated with hearing dysfunction. Previous applications of unbiased (design-based) stereological principles have expanded our knowledge of neuro-morphological changes evident with the light microscope. Applying these principles at the level of the synapse is a promising morphometric approach for the efficient sampling of large reference spaces with electron microscopy. This study tests the accuracy of using ultra-thin sections at a fixed interval, known as disector pairs, to quantify afferent innervation density. We analyzed the total numbers of afferent terminals, synaptic thickenings, and synaptic bodies associated with each IHC in the C57BL/6J mouse cochlea, and confirmed the accuracy of the stereological approach in comparison to three-dimensional reconstructions of serial alternate sections. The higher sampling efficiency of the disector pair method rapidly increases precision while also reducing the largest source of variability, inter-animal differences. We conclude that ultrastructural quantification of afferent innervation can be accomplished in the cochlea using efficient design-based stereology.

Synaptic alterations at inner hair cells precede spiral ganglion cell loss in aging C57BL/6J mice.

Hearing deficits have often been associated with loss of or damage to receptor hair cells and/or degeneration of spiral ganglion cells. There are, however, some physiological abnormalities that are not reliably attributed to loss of these cells. The afferent synapse between radial fibers of spiral ganglion neurons and inner hair cells (IHCs) emerges as another site that could be involved in transmission abnormalities. We tested the hypothesis that the structure of these afferent terminals would differ between young animals and older animals with significant hearing loss. Afferent endings and their synapses were examined by transmission electron microscopy at approximately 45% distance from the basal end of the cochlea in 2-3 month-old and 8-12 month-old C57BL/6J mice. The number of terminals in older animals was reduced by half compared to younger animals. In contrast, there was no difference in the density of SGCs between the age groups. Older animals featured enlarged terminals and mitochondria and enlarged postsynaptic densities and presynaptic bodies. These morphological changes may be a combination of pathologic, adaptive and compensatory responses to sensory dysfunction. Improved knowledge of these processes is necessary to understand the role of afferent connectivity in dysfunction of the aging cochlea.

High efficiency gene delivery into laryngeal muscle with bidirectional electroporation.

OBJECTIVE: The impact of polarity change on the efficiency of in vivo electroporative (EP) gene transfection was assessed in rat laryngeal muscle. STUDY DESIGN AND SETTING: High (HV) and low field voltage (LV) were combined with polarity changes to determine transfection in 5 different conditions: 1) without EP (EP[-]), 2) HV+LV (HL), 3) HV+LV followed by HV+LV with no change in polarity (HLHL unidirectional), 4) HV+LV followed by HV+LV with opposite polarity (HLHL bidirectional), 5) HV+LV followed by LV with opposite polarity (HLL bidirectional). RESULTS: HLL bidirectional sequence showed the best result with less interindividual variability and extended expression period. With the exception of repeated high voltage sequences, EP parameters were not likely to induce cell injury or inflammation. CONCLUSION: HLL bidirectional electroporative gene delivery produces high transfection rates with limited tissue trauma. SIGNIFICANCE: Bidirectional EP provides a safe and highly efficient method for therapeutic gene delivery into skeletal muscle.

Intratympanic Iodine Contrast Injection Diffuses Across the Round Window Membrane Allowing for Perilymphatic CT Volume Acquisition Imaging.

HYPOTHESIS: Whether the round window membrane (RWM) is permeable to iodine-based contrast agents (IBCA) is unknown; therefore, our goal was to determine if IBCAs could diffuse through the RWM using CT volume acquisition imaging. INTRODUCTION: Imaging of hydrops in the living human ear has attracted recent interest. Intratympanic (IT) injection has shown gadolinium's ability to diffuse through the RWM, enhancing the perilymphatic space. METHODS: Four unfixed human cadaver temporal bones underwent intratympanic IBCA injection using three sequentially studied methods. The first method was direct IT injection. The second method used direct RWM visualization via tympanomeatal flap for IBCA-soaked absorbable gelatin pledget placement. In the third method, the middle ear was filled with contrast after flap elevation. Volume acquisition CT images were obtained immediately postexposure, and at 1-, 6-, and 24-hour intervals. Postprocessing was accomplished using color ramping and subtraction imaging. RESULTS: After the third method, positive RWM and perilymphatic enhancement were observed with endolymph sparing. Gray scale and color ramp multiplanar reconstructions displayed increased signal within the cochlea compared with precontrast imaging. The cochlea was measured for attenuation differences compared with pure water, revealing a preinjection average of -1,103 HU and a postinjection average of 338 HU. Subtraction imaging shows enhancement remaining within the cochlear space, Eustachian tube, middle ear epithelial lining, and mastoid. CONCLUSION: Iohexol iodine contrast is able to diffuse across the RWM. Volume acquisition CT imaging was able to detect perilymphatic enhancement at 0.5-mm slice thickness. The clinical application of IBCA IT injection seems promising but requires further safety studies.

Repetitive mild traumatic brain injury with impact acceleration in the mouse: Multifocal axonopathy, neuroinflammation, and neurodegeneration in the visual system.

Repetitive mild traumatic brain injury (mTBI) is implicated in chronic neurological illness. The development of animal models of repetitive mTBI in mice is essential for exploring mechanisms of these chronic diseases, including genetic vulnerability by using transgenic backgrounds. In this study, the rat model of impact acceleration (IA) was redesigned for the mouse cranium and used in two clinically relevant repetitive mTBI paradigms. We first determined, by using increments of weight dropped from 1m that the 40g weight was most representative of mTBI and was not associated with fractures, brain contusions, anoxic-ischemic injury, mortality, or significant neurological impairments. Quantitative evaluation of traumatic axonal injury (TAI) in the optic nerve/tract, cerebellum and corpus callosum confirmed that weight increase produced a graded injury. We next evaluated two novel repetitive mTBI paradigms (1 time per day or 3 times per day at days 0, 1, 3, and 7) and compared the resulting TAI, neuronal cell death, and neuroinflammation to single hit mTBI at sub-acute (7days) and chronic time points (10weeks) post-injury. Both single and repetitive mTBI caused TAI in the optic nerve/tract, cerebellum, corticospinal tract, lateral lemniscus and corpus callosum. Reactive microglia with phagocytic phenotypes were present at injury sites. Severity of axonal injury corresponded to impact load and frequency in the optic nerve/tract and cerebellum. Both single and repeat injury protocols were associated with retinal ganglion cell loss and optic nerve degeneration; these outcomes correlated with impact load and number/frequency. No phosphorylated tau immunoreactivity was detected in the brains of animals subjected to repetitive mTBI. Our findings establish a new model of repetitive mTBI model featured by TAI in discrete CNS tracts, especially the visual system and cerebellum. Injury in retina and optic nerve provides a sensitive measure of severity of mTBI, thus enabling further studies on mechanisms and experimental therapeutics. Our model can also be useful in exploring mechanisms of chronic neurological disease caused by repetitive mTBI in wild-type and transgenic mice.

Laryngeal muscle surface receptors identified using random phage library.

OBJECTIVES: The ultimate goal of this study is to improve the efficiency of gene transfer in mammalian muscle by developing targeted adenoviral vectors. Altering the tropism of viral vectors to recognize tissue specific antigens is one method to achieve this goal. This approach requires identification of cell-surface receptors and the insertion of target peptide sequences into the adenoviral fiber protein. In this study, phage biopanning was performed on cultured rat skeletal and laryngeal muscle to identify cell-surface receptors. STUDY DESIGN: In vitro cell culture and in vivo animal model. METHODS: M-13 Phage biopanning was used for muscle cell-surface receptor analysis on cultured rat skeletal and laryngeal muscle. Nonbinding and binding phage to cultured skeletal and laryngeal muscle were screened for muscle specific surface peptides. In vivo studies were then performed using muscle specific phage. RESULTS: Skeletal muscle specific binding by the YASTNPM phage was observed by in vivo immunostaining. Phage titering demonstrated a 10(9)-fold increase in skeletal muscle binding compared with nontarget tissue. A peptide sequence (NPSQVKH) specific for laryngeal muscle yielded a 10(7)-fold increase in laryngeal muscle phage titer compared with nontarget tissue. CONCLUSIONS: These results identify muscle cell-surface receptors that may be used as potential targets for genetic modification of adenovirus tropism. Moreover, phage specificity for skeletal and laryngeal muscle indicates specific muscle groups may be targeted.

Histopathologic Changes of the Inner ear in Rhesus Monkeys After Intratympanic Gentamicin Injection and Vestibular Prosthesis Electrode Array Implantation.

Bilateral vestibular deficiency (BVD) due to gentamicin ototoxicity can significantly impact quality of life and result in large socioeconomic burdens. Restoring sensation of head rotation using an implantable multichannel vestibular prosthesis (MVP) is a promising treatment approach that has been tested in animals and humans. However, uncertainty remains regarding the histopathologic effects of gentamicin ototoxicity alone or in combination with electrode implantation. Understanding these histological changes is important because selective MVP-driven stimulation of semicircular canals (SCCs) depends on persistence of primary afferent innervation in each SCC crista despite both the primary cause of BVD (e.g., ototoxic injury) and surgical trauma associated with MVP implantation. Retraction of primary afferents out of the cristae and back toward Scarpa's ganglion would render spatially selective stimulation difficult to achieve and could limit utility of an MVP that relies on electrodes implanted in the lumen of each ampulla. We investigated histopathologic changes of the inner ear associated with intratympanic gentamicin (ITG) injection and/or MVP electrode array implantation in 11 temporal bones from six rhesus macaque monkeys. Hematoxylin and eosin-stained 10-µm temporal bone sections were examined under light microscopy for four treatment groups: normal (three ears), ITG-only (two ears), MVP-only (two ears), and ITG + MVP (four ears). We estimated vestibular hair cell (HC) surface densities for each sensory neuroepithelium and compared findings across end organs and treatment groups. In ITG-only, MVP-only, and ITG + MVP ears, we observed decreased but persistent ampullary nerve fibers of SCC cristae despite ITG treatment and/or MVP electrode implantation. ITG-only and ITG + MVP ears exhibited neuroepithelial thinning and loss of type I HCs in the cristae but little effect on the maculae. MVP-only and ITG + MVP ears exhibited no signs of trauma to the cochlea or otolith end organs except in a single case of saccular injury due to over-insertion of the posterior SCC electrode. While implanted electrodes reached to within 50-760 µm of the target cristae and were usually ensheathed in a thin fibrotic capsule, dense fibrotic reaction and osteoneogenesis were each observed in only one of six electrode tracts examined. Consistent with physiologic studies that have demonstrated directionally appropriate vestibulo-ocular reflex responses to MVP electrical stimulation years after implantation in these animals, histologic findings in the present study indicate that although intralabyrinthine MVP implantation causes some inner ear trauma, it can be accomplished without destroying the distal afferent fibers an MVP is designed to excite.

Two types of afferent terminals innervate cochlear inner hair cells in C57BL/6J mice.

Afferent synapses on inner hair cells (IHC) transfer auditory information to the central nervous system (CNS). Despite the importance of these synapses for normal hearing, their response to cochlear disease and dysfunction is not well understood. The C57BL/6J mouse is a model for presbycusis and noise-induced hearing loss because of its age-related hearing loss and susceptibility to acoustic over-exposure. In this context, we sought to establish normal synaptic structure in order to better evaluate synaptic changes due to presbycusis and noise exposure. Ultrastructural analysis of IHCs and afferent terminals was performed in a normal hearing 3-month-old C57BL/6J mouse at cochlear sites corresponding to 8, 16 and 32 kHz using semi-serial sections. A stereologic survey of random sections was conducted of IHCs in 11 additional mice. Two morphologically distinct groups of afferent terminals were identified at all 3 frequency locations in 11 out of 12 animals. "Simple" endings demonstrated classic features of bouton terminals, whereas "folded" endings were larger in size and exhibited a novel morphologic feature that consisted of a fully internalized double membrane that partially divided the terminal into two compartments. In many cases, the double membrane was continuous with the outer terminal membrane as if produced by an invagination. We still must determine the generality of these observations with respect to other mouse strains.

Differential expression of myosin heavy chain isoforms between abductor and adductor muscles in the human larynx.

OBJECTIVE: This study examines the differential expression of myosin heavy chain (MyHC) components in human laryngeal muscle groups. STUDY DESIGN: A battery of monospecific monoclonal antibodies in Western blots was used to determine expression of IIX, extraocular-specific (EOM), and IIB MyHCs for the thyroarytenoid (TA), vocalis (VOC), lateral cricoarytenoid (LCA), cricothyroid (CT), and posterior cricoarytenoid (PCA) muscles obtained from fresh cadaver specimens. RESULTS: Fast IIX MyHC was only expressed in the TA, VOC, and LCA muscles. Fast IIA and slow MyHCs were expressed in all laryngeal muscles including the CT and PCA. The CT with mixed phonatory and respiratory function and the PCA with respiratory function did not express IIX MyHC. The 2 MyHC isoforms associated with the highest speeds of contraction in rat laryngeal muscle, namely, the EOM MyHC and IIB MyHC, were not detected in human laryngeal muscles. Novel MyHC bands were not detected in SDS-PAGE gels or Western blots using a broad specificity MyHC antibody. CONCLUSION: The profile of MyHC expression in human laryngeal muscles differs from that observed in human extraocular and masticator muscles, and other mammalian species. Our data demonstrate that IIX MyHC expression is associated primarily with muscles affecting glottic closure and is absent in CT and PCA. SIGNIFICANCE: A higher percentage of IIX MyHC is expected to impart a high speed of shortening to the TA and LCA muscles. The absence of IIX MyHC in muscles with respiratory (PCA) and mixed respiratory/phonatory function (CT) further supports the inference that the physiologic difference between laryngeal muscles is reflected in the molecular composition of contractile protein.

Ultrastructure of cisternal synapses on outer hair cells of the mouse cochlea.

C (cisternal) synapses with a near membrane postsynaptic cistern are found on motor neurons and other central neurons, where their functional role is unknown. Similarly structured cisternal synapses mediate cholinergic inhibition of cochlear hair cells via α9α10-containing ionotropic receptors and associated calcium-activated (SK2) potassium channels, providing the opportunity to examine the ultrastructure of genetically altered cisternal synapses. Serial section electron microscopy was used to examine efferent synapses of outer hair cells (OHCs) in mice with diminished or enhanced cholinergic inhibition. The contact area of efferent terminals, the appositional area of the postsynaptic cistern, the distance of the cistern from the plasma membrane, and the average width of the cisternal lumen were recorded. The synaptic cisterns of wild-type OHCs were closely aligned (14-nm separation) with the hair cell membrane and coextensive with the micrometers-long synaptic terminals. The cisternal lumen averaged 18 nm so that the cisternal volume was approximately 30% larger than that of the cytoplasmic space between the cistern and the plasma membrane. Synaptic ultrastructure of α9L9'T knockin OHCs (acetylcholine receptor gain of function) were like those of wild-type littermates except that cisternal volumes were significantly larger. OHCs of SK2 knockout mice had few small efferent terminals. Synaptic cisterns were present, but smaller than those of wild-type littermates. Taken together, these data suggest that the cistern serves as a sink or buffer to isolate synaptic calcium signals.