CRISPR Editing Technology in Biological and Biomedical Investigation.

The CRISPR or clustered regularly interspaced short palindromic repeats system is currently the most advanced approach to genome editing and is notable for providing an unprecedented degree of specificity, effectiveness, and versatility in genetic manipulation. CRISPR evolved as a prokaryotic immune system to provide an acquired immunity and resistance to foreign genetic elements such as bacteriophages. It has recently been developed into a tool for the specific targeting of nucleotide sequences within complex eukaryotic genomes for the purpose of genetic manipulation. The power of CRISPR lies in its simplicity and ease of use, its flexibility to be targeted to any given nucleotide sequence by the choice of an easily synthesized guide RNA, and its ready ability to continue to undergo technical improvements. Applications for CRISPR are numerous including creation of novel transgenic cell animals for research, high-throughput screening of gene function, potential clinical gene therapy, and nongene-editing approaches such as modulating gene activity and fluorescent tagging. In this prospect article, we will describe the salient features of the CRISPR system with an emphasis on important drawbacks and considerations with respect to eliminating off-target events and obtaining efficient CRISPR delivery. We will discuss recent technical developments to the system and we will illustrate some of the most recent applications with an emphasis on approaches to eliminate human viruses including HIV-1, JCV and HSV-1 and prospects for the future. J. Cell. Biochem. 118: 3586-3594, 2017. © 2017 Wiley Periodicals, Inc.

Prevalence of radiographic semicircular canal dehiscence in very young children: an evaluation using high-resolution computed tomography of the temporal bones.

BACKGROUND: Previous studies suggest that semicircular canal dehiscences (SCDs) have a developmental origin. OBJECTIVE: We hypothesized that if SCDs originate during development, incidence of radiographic SCDs in young children will be higher than in adults. MATERIALS AND METHODS: Thirty-four temporal bone HRCTs of children younger than 2 years and 40 temporal bone HRCTs of patients older than 18 years were reformatted and re-evaluated for presence of SCD or canal thinning. Results were compared with indications for HRCT and clinical information. RESULTS: SCDs were detected in 27.3% of children younger than 2 years of age (superior, 13.8%; posterior, 20%) and in 3% of adults (P < 0.004). Of children with one radiographic dehiscence, 55.6% had multiple and 44% had bilateral SCDs on HRCT. No lateral canal SCDs were present. Thinning of bone overlying the semicircular canals was found in 44% of children younger than 2 years and 2.5% of adults (P < 0.0001). CONCLUSION: SCDs are more common on HRCTs of very young children. This supports the hypothesis that SCDs originate from discontinuation of bone deposition/maturation. However, SCDs on imaging do not necessarily correlate with canal dehiscence syndrome and should therefore be interpreted carefully.

Actinomycosis of the temporal bone and brain: case report and review of the literature.

OBJECTIVES: Actinomycosis is a rare disease with a typically indolent course in the head and neck. During the modern era, only 12 cases within the ear and temporal bone and 75 intracranial cases have been reported. We present a case of actinomycosis of the petrous apex that led to meningitis and encephalitis. METHODS: The patient was a 12-year-old girl who presented with mental status changes. After 48 hours of treatment with empiric antibiotics for meningitis without improvement, imaging revealed an enhancing mass in the right petrous apex, destruction of the cochlea, meningeal enhancement, and left temporoparietal encephalitis. RESULTS: The initial therapy included broad-spectrum antibiotic, antifungal, and antiviral agents, as well as myringotomy and tympanostomy tube placement. When the patient's clinical status worsened, she underwent subtotal petrosectomy with drainage of the petrous apex. The final pathologic findings were consistent with actinomycosis. CONCLUSIONS: Actinomycosis is a rare infection in the temporal bone and central nervous system that can have a high mortality risk if not treated appropriately. Often, these bacteria do not grow well in culture, and diagnosis must be made on the basis of histopathologic features. Good clinical outcomes can be obtained with surgical debridement followed by long-term antibiotic treatment.

Osseointegrated Auditory Devices: Bone-Anchored Hearing Aid and PONTO.

Osseointegrated auditory devices (OADs) are hearing devices that use an external receiver/processor that stimulates bone conduction of sound via a titanium prosthesis that is drilled into the bone of the cranium. Since their introduction in 1977, OADs have undergone substantial evolution, including changes in manufacturing of the implant, improvements in the external sound processor, and simplification of implantation techniques. Expansion of criteria for patient candidacy for implantation has occurred corresponding with changes in the implants and processors.

Percutaneous Threshold of Facial Nerve Stimulation Predicts Facial Canal Dehiscence.

Iatrogenic facial nerve (FN) injury is one of the most feared complications of otologic surgery. Dehiscence of the bony covering of the FN within the temporal bone increases FN vulnerability to accidental injury. High-resolution computed tomography (HRCT) of the temporal bone is used preoperatively to assess middle ear and mastoid anatomy; however, it is unreliable for detecting facial canal dehiscence. In this study, our aim was to determine if preoperative percutaneous FN stimulation could predict middle ear facial canal dehiscence. Between January 2015 and February 2017, we performed preoperative HRCT and percutaneous FN stimulation on adult patients who underwent otologic surgery at our institution. Stimulation was performed with a monopolar probe placed on the skin over the stylomastoid foramen. Electrical stimuli ranged from 0 to 40 milliamperes (mA). Recordings were made from ipsilateral facial muscles. Dependent variables included threshold to compound muscle action potential (CMAP), threshold to maximum amplitude of CMAP, and maximum amplitude of CMAP for each muscle. A retrospective chart review was performed. Seventy patients met inclusion criteria. Of the 24 with an intraoperatively confirmed dehiscence, 10 were identified preoperatively by the attending surgeon on HRCT. Averages of the lowest recorded threshold to CMAP (5.1mA v. 9.1mA), and an average of the threshold to CMAP (8.9 mA. 11.8 mA) of dehiscent versus non-dehiscent nerves were significantly different (p < .05). In conclusion, percutaneous FN stimulation is a simple and cost-effective tool that can give the surgeon important preoperative information about FN anatomy.

Endoscope-assisted repair of CSF otorrhea and temporal lobe encephaloceles via keyhole craniotomy.

OBJECTIVE Temporal lobe encephaloceles and cerebrospinal fluid otorrhea from temporal bone defects that involve the tegmen tympani and mastoideum are generally repaired using middle fossa craniotomy, mastoidectomy, or combined approaches. Standard middle fossa craniotomy exposes patients to dural retraction, which can lead to postoperative neurological complications. Endoscopic and minimally invasive techniques have been used in other surgeries to minimize brain retraction, and so these methods were applied to repair the lateral skull base. The goal of this study was to determine if the use of endoscopic visualization through a middle fossa keyhole craniotomy could effectively repair tegmen defects. METHODS The authors conducted a retrospective review of 6 cases of endoscope-assisted middle fossa repairs of tegmen dehiscences at a tertiary care medical center within an 18-month period. RESULTS All cases were successfully treated using a keyhole craniotomy with endoscopic visualization and minimal retraction. Surgical times did not increase. There were no major postoperative complications, recurrences of encephaloceles, or cerebrospinal fluid otorrhea in these patients. CONCLUSIONS Endoscopic visualization allows for smaller incisions and craniotomies and less risk of brain retraction injury without compromising repair integrity during temporal encephalocele and tegmen repairs.

Management of acoustic neuromas in patients 65 years or older.

OBJECTIVE: To analyze an optimal management protocol for patients 65 years or older at the time of acoustic neuroma diagnosis. STUDY DESIGN: Retrospective case review. SETTING: Tertiary care hospital. PATIENTS: Two hundred sixteen patients with acoustic neuroma 65 years or older at time of diagnosis. INTERVENTION: Patients with smaller tumors (<2.5 cm) were followed with serial magnetic resonance imaging. If significant growth occurred, they were treated with surgery. Surgery was performed at initial diagnosis on patients with larger tumors or in selected patients for hearing preservation. Stereotactic radiotherapy was performed for poor surgical candidates and for patient choice. OUTCOME MEASURES: Measurement of acoustic neuroma growth and tabulation of complications. RESULTS: One hundred fourteen patients were initially managed by observation, 80 with surgery, and 3 with radiation therapy, with an average follow-up of 35.4 months. For patients in the observation group, average tumor growth was 1.2 mm/yr. Thirty-two patients required crossover to surgery or radiotherapy due to tumor growth (average growth, 4.1 versus 0.3 mm/yr for those remaining in the observation group). One of the patients in the observation group had a complication (0.9%). CONCLUSION: Management of acoustic neuromas in elderly patients can be based on size and "biological age" criteria. Surgical treatment can safely be reserved for the few patients who have significant tumor growth.

Systematic Review of Ossicular Chain Anatomy: Strategic Planning for Development of Novel Middle Ear Prostheses.

Objective To systematically review the anatomy of the ossicular chain. Data Sources Google Scholar, PubMed, and otologic textbooks. Review Methods A systematic literature search was performed on January 26, 2015. Search terms used to discover articles consisted of combinations of 2 keywords. One keyword from both groups was used: [ ossicular, ossicle, malleus, incus, stapes] and [ morphology, morphometric, anatomy, variation, physiology], yielding more than 50,000 hits. Articles were then screened by title and abstract if they did not contain information relevant to human ossicular chain anatomy. In addition to this search, references of selected articles were studied as well as suggested relevant articles from publication databases. Standard otologic textbooks were screened using the search criteria. Results Thirty-three sources were selected for use in this review. From these studies, data on the composition, physiology, morphology, and morphometrics were acquired. In addition, any correlations or lack of correlations between features of the ossicular chain and other features of the ossicular chain or patient were noted, with bilateral symmetry between ossicles being the only important correlation reported. Conclusion There was significant variation in all dimensions of each ossicle between individuals, given that degree of variation, custom fitting, or custom manufacturing of prostheses for each patient could optimize prosthesis fit. From published data, an accurate 3-dimensional model of the malleus, incus, and stapes can be created, which can then be further modified for each patient's individual anatomy.

Preliminary Model for the Design of a Custom Middle Ear Prosthesis.

HYPOTHESIS: Custom prostheses could be used to recreate the ossicular chain and improve hearing. BACKGROUND: Ossicular discontinuity or fixation occurs in 55% of cases of conductive hearing loss, with most cases involving the incus. Reconstruction has been achieved by a variety of methods; however, there has been little improvement in hearing outcomes in decades. METHODS: Precise measurements of anatomic dimensions, weight, and center of gravity were taken from 19 cadaveric incudes. These measurements were combined with measurements from the medical literature and micro-computed tomography (micro-CT) of cadaveric temporal bones to generate a rasterizable incus model. As a proof of concept, incudal replacements including possible anatomic variations were then three-dimensionally (3-D) printed and inserted into a cadaveric temporal bone. RESULTS: Our measurements of cadaveric incudes corresponded well with those from the medical literature. These measurements were combined with anatomical information from micro-CT allowing identification of critical features of the incus, which remained constant. Other model features were modified to increase stability and facilitate synthesis, including broadening and thickening of the lenticular process and the incudomalleolar articulation. 3-D printed incudal replacements based on this model readily fit into a cadaveric temporal bone and successfully bridged the gap between malleus and incus. CONCLUSION: We have generated a model for custom 3-D synthesis of incudal prostheses. While current 3-D printing in biocompatible materials at the size required is limited, the technology is rapidly advancing, and 3-D printing of incudal replacements with polylactic acid (PLA) is of the correct size and shape.

Cochlear implant explantation as a sequela of severe chronic otitis media: case report and review of the literature.

INTRODUCTION: In the 1980s, intracranial and inner ear infections were feared complications in patients with recurrent or chronic otitis media (COM) who had undergone cochlear implantation. Current studies show a low incidence of such complications. We present a case of a patient who developed severe COM requiring cochlear explantation. CASE: Our patient had a previous cleft palate repair and as a three-year-old was implanted with a Nucleus-24 implant. She developed chronic otorrhea in the implanted ear, which was managed by her pediatrician until her cochlear implant stopped functioning. Radiographic imaging revealed erosion of the cochlea and extrusion of the distal electrode medially in the petrous apex. SETTING: Tertiary care university hospital. INTERVENTION/RESULTS: The patient underwent cochlear explantation, subtotal petrosectomy, obliteration of ear, and intravenous antibiotic therapy. One month later she was implanted in the contralateral ear. CONCLUSION: COM poses potentially severe complications in patients receiving cochlear implants. Patients receiving cochlear implants who are at high risk for COM require follow-up for an extended period of time.

Disseminated histoplasmosis presenting as a unilateral cranial nerve VIII mass: a case report.

OBJECTIVE: To report a unique presentation of disseminated histoplasmosis. STUDY DESIGN: Case report. SETTING: University hospital, tertiary referral center. PATIENT: Our patient presented with vertigo, tinnitus, and unilateral hearing loss, and was initially found to have a 5-mm enhancing left internal auditory canal mass, as revealed by a magnetic resonance imaging (MRI) scan. Subsequently, the patient developed multiple focal neurologic deficits. INTERVENTIONS: Magnetic resonance imaging and treatment with intravenously administered amphotericin B, with subsequent oral administration of itraconazole. MAIN OUTCOME MEASURES: Clinical presentation and imaging findings of Histoplasmosis involving the cranial nerve VIII. RESULTS: A subsequent MRI scan revealed enlargement of the initial lesion and multiple parenchymal lesions. Further workup revealed a pulmonary lesion; the diagnosis of disseminated histoplasmosis was made on the basis of bronchoalveolar lavage culture. CONCLUSION: Infectious processes, including disseminated histoplasmosis, should be considered in the differential of internal auditory canal masses, especially in the setting of rapid progression of symptoms.

Hemostasis in Otologic and Neurotologic Surgery.

Hemostasis is a critical component of otologic and neurotologic surgery. In these surgeries the surgical field is small; thus, even a small amount of bleeding can obstruct the view of critical and extremely small structures. Additionally, relatively large vascular structures traverse the area; if they are encroached on by trauma or disease, bleeding must be controlled within a very small space in a meticulous fashion that does not encroach on structures of the middle ear and mastoid. The authors discuss several hemostatic agents in the middle ear, mastoid, and lateral skull base, highlighting their origins, mechanisms, advantages, and complications.

Inhibition of HSV-1 Replication by Gene Editing Strategy.

HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases.

Strategies to preserve or regenerate spiral ganglion neurons.

PURPOSE OF REVIEW: Degeneration of spiral ganglion neurons following hair cell loss carries critical implications for efforts to rehabilitate severe cases of hearing loss with cochlear implants or hair cell regeneration. This review considers recently identified neurotrophic factors and therapeutic strategies which promote spiral ganglion neuron survival and neurite growth. Replacement of these factors may help preserve or regenerate the auditory nerve in patients with extensive hair cell loss. RECENT FINDINGS: Spiral ganglion neurons depend on neurotrophic factors supplied by hair cells and other targets for their development and continued survival. Loss of this trophic support leads to spiral ganglion neuron death via apoptosis. Hair cells support spiral ganglion neuron survival by producing several peptide neurotrophic factors such as neurotrophin-3 and glial derived neurotrophic factor. In addition, neurotransmitter release from the hair cells drives membrane electrical activity in spiral ganglion neurons which also supports their survival. In animal models, replacement of peptide neurotrophic factors or electrical stimulation with an implanted electrode attenuates spiral ganglion neuron degeneration following deafferentation. Cell death inhibitors can also preserve spiral ganglion neuron populations. Preliminary studies show that transfer of stem cells or neurons from other ganglia are two potential strategies to replace lost spiral ganglion neurons. Inducing the regrowth of spiral ganglion neuron peripheral processes to approximate or contact cochlear implant electrodes may help optimize signaling from a diminished population of neurons. SUMMARY: Recent studies of spiral ganglion neuron development and survival have identified several trophic and neuritogenic factors which protect these specialized cells from degeneration following hair cell loss. While still preliminary, such strategies show promise for future clinical applications.

Viral causes of hearing loss: a review for hearing health professionals.

A number of viral infections can cause hearing loss. Hearing loss induced by these viruses can be congenital or acquired, unilateral or bilateral. Certain viral infections can directly damage inner ear structures, others can induce inflammatory responses which then cause this damage, and still others can increase susceptibility or bacterial or fungal infection, leading to hearing loss. Typically, virus-induced hearing loss is sensorineural, although conductive and mixed hearing losses can be seen following infection with certain viruses. Occasionally, recovery of hearing after these infections can occur spontaneously. Most importantly, some of these viral infections can be prevented or treated. For many of these viruses, guidelines for their treatment or prevention have recently been revised. In this review, we outline many of the viruses that cause hearing loss, their epidemiology, course, prevention, and treatment.

Extracellular and intracellular melanin in inflammatory middle ear disease.

OBJECTIVES/HYPOTHESIS: Melanin is a pigmented polymer with a known role in dermal solar protection. In vertebrates, melanogenesis has been reported in leukocyte populations, suggesting a potential role in innate immunity. In this study, we report the novel finding of melanin associated with chronic inflammation and speculate on its potential role in the middle ear and mastoid. STUDY DESIGN: Retrospective review of case series. METHODS: Medical records of six patients who demonstrated melanin in the ear were reviewed. RESULTS: Six patients from 1 to 63 years of age were identified with extracellular melanin and melanin-laden histiocytes within the middle ear and/or mastoid air cells at time of surgery. Concurrent intraoperative findings included cholesteatoma (n = 3), chronic suppurative otitis media (n = 2), and coalescent mastoiditis (n = 1). Histologically, extracellular melanin and melanin-laden histiocytes were identified by Fontana-Masson stain; absence of melanocytes was confirmed by the absence of Melan-A staining. One patient had a positive stain for CD163 (a marker for macrophages). CONCLUSION: This case series is the first demonstration of melanin within middle ear mucosa without melanocytes in immediate proximity or metastatic melanocytic lesions. Melanin's presence in the setting of inflammation suggests that there may be a heretofore unreported link between the pigmentary and immune systems in the middle ear. LEVEL OF EVIDENCE: 4.

Identification of endolymphatic hydrops in Ménière's disease utilizing delayed postcontrast 3D FLAIR and fused 3D FLAIR and CISS color maps.

OBJECTIVE: The preferential delayed enhancement of the perilymphatic space enables detection of the non-enhancing endolymphatic hydrops present in patients with Ménière's disease. The aim of this study was to evaluate the diagnostic utility of delayed postcontrast 3D FLAIR images and a color map of fused postcontrast FLAIR and constructive interference steady state (CISS) images in the identification of endolymphatic hydrops in patients with clinically diagnosed Ménière's disease. STUDY DESIGN: Case control, blinded study. SETTING: Tertiary referral center. PATIENTS: Ten patients with Ménière's disease and five volunteer controls. INTERVENTION: Diagnostic. MAIN OUTCOME MEASURE: Two neuroradiologists blinded to the clinical history independently evaluated for the presence of endolymphatic hydrops on the images of both inner ears for test and control subjects. Both the standard gray-scale FLAIR images and the fused color map images were independently reviewed. RESULTS: The gray-scale 3D FLAIR images demonstrated 68.2% sensitivity and 97.4% specificity, and the fused color map images demonstrated 85.0% sensitivity and 88.9% specificity in the identification of endolymphatic hydrops in Ménière's disease. There was significant correlation between the gray-scale 3D FLAIR images and fused color map images with the categorization of involvement (p = 0.002). Inter-evaluator reliability was excellent (kappa = 0.83 for gray-scale images, kappa = 0.81 for fused color map). CONCLUSION: Delayed 3D FLAIR and fused 3D FLAIR-CISS color map images of the inner ears after intravenous contrast administration are potentially useful diagnostic tools in the evaluation of patients with suspected Ménière's disease.

Imaging the facial nerve: a contemporary review.

Imaging plays a critical role in the evaluation of a number of facial nerve disorders. The facial nerve has a complex anatomical course; thus, a thorough understanding of the course of the facial nerve is essential to localize the sites of pathology. Facial nerve dysfunction can occur from a variety of causes, which can often be identified on imaging. Computed tomography and magnetic resonance imaging are helpful for identifying bony facial canal and soft tissue abnormalities, respectively. Ultrasound of the facial nerve has been used to predict functional outcomes in patients with Bell's palsy. More recently, diffusion tensor tractography has appeared as a new modality which allows three-dimensional display of facial nerve fibers.