Small molecule delivery across a perforated artificial membrane by thermoreversible hydrogel poloxamer 407.

Microperforations in the round window membrane have been suggested for enhancing the rate and reliability of drug delivery into the cochlea. Intratympanic injection, the most common delivery method, involves injecting therapy into the middle ear to establish a reservoir from which drug diffuses across the round window membrane into the cochlea. This process is highly variable because (i) the reservoir, if liquid, can lose contact with the membrane and (ii) diffusion across the membrane is intrinsically variable even with a stable reservoir. To address these respective sources of variability, we compared the thermoreversible hydrogel poloxamer 407 (P407) to saline as a drug carrier and studied the effect of membrane microperforations on drug diffusion rate. We used Rhodamine B as a drug proxy to measure permeance across an artificial membrane in a horizontal diffusion cell. We found that permeance of Rhodamine B from a saline reservoir was an order of magnitude higher than that from a P407 reservoir across unperforated membranes. Moreover, permeance increased with total perforation cross-sectional area regardless of number of perforations (p < 0.05 for all saline-based experiments), but the same association was not found with P407. Rather, for a P407 reservoir, only a large perforation increased permeance (p < 0.001), while multiple small perforations did not (p = 0.749). These results confirm that for drug dissolved in saline, multiple small perforations can effectively enhance diffusion. However, for drug dissolved in P407, larger perforations are necessary.

Utility of electronystagmography in the prediction of post-operative outcome following cochlear implantation.

OBJECTIVE: To examine the relationship between pre-operative electronystagmography and videonystagmography test results and post-operative outcomes in dizziness, auditory sensitivity and speech recognition. METHODS: A retrospective chart review was performed. Auditory sensitivity and speech perception ability were tested pre- and post-operatively in 37 adult cochlear implant recipients. Auditory sensitivity was evaluated using either pure tones (for testing with earphones) or frequency-modulated warble tones (for sound-field testing). Speech perception ability was evaluated using Northwestern University Auditory Test Number 6. RESULTS: No correlation was found between pre-operative electronystagmography test results and post-operative subjective dizziness. However, pre-operative electronystagmography testing and post-operative hearing sensitivity as measured by warble tone average (dB HL) correlated significantly at six months or later after cochlear implant activation (r  ≥  -0.34, n = 34, p < 0.05). CONCLUSION: This study, which has a level of evidence 4, demonstrates that pre-operative electronystagmography testing has a potential use in predicting post-operative outcomes in hearing sensitivity following cochlear implantation. However, larger studies are needed to confirm this novel finding.

Scanning electrochemical microscopy as a novel proximity sensor for atraumatic cochlear implant insertion.

A growing number of minimally invasive surgical and diagnostic procedures require the insertion of an optical, mechanical, or electronic device in narrow spaces inside a human body. In such procedures, precise motion control is essential to avoid damage to the patient's tissues and/or the device itself. A typical example is the insertion of a cochlear implant which should ideally be done with minimum physical contact between the moving device and the cochlear canal walls or the basilar membrane. Because optical monitoring is not possible, alternative techniques for sub millimeter-scale distance control can be very useful for such procedures. The first requirement for distance control is distance sensing. We developed a novel approach to distance sensing based on the principles of scanning electrochemical microscopy (SECM). The SECM signal, i.e., the diffusion current to a microelectrode, is very sensitive to the distance between the probe surface and any electrically insulating object present in its proximity. With several amperometric microprobes fabricated on the surface of an insertable device, one can monitor the distances between different parts of the moving implant and the surrounding tissues. Unlike typical SECM experiments, in which a disk-shaped tip approaches a relatively smooth sample, complex geometries of the mobile device and its surroundings make distance sensing challenging. Additional issues include the possibility of electrode surface contamination in biological fluids and the requirement for a biologically compatible redox mediator.

The cochlear-carotid interval: anatomic variation and potential clinical implications.

BACKGROUND AND PURPOSE: A temporal bone CT study in a patient with episodic mid-tone sensorineural hearing loss and tinnitus demonstrated absence of bone between the petrous internal carotid artery and the basal turn of the cochlea. The potential implications with respect to increasingly popular cochlear implant surgery compelled us to retrospectively analyze a series of temporal bone CT scans to establish typical measurements for this region, which we termed the "cochlear-carotid interval" (CCI). METHODS: After IRB exemption, 2 observers independently measured the bony interval between the cochlea and the petrous internal carotid artery canal on coronal images from 30 consecutive temporal bone CT studies. The 1-mm thick coronal images were either acquired directly or were reconstructed from an axial dataset acquired at 0.75 or 0.6 mm section thickness. All measurements were performed by using electronic calipers on a Sienet MagicView VE 42 Siemens PACS station. Mixed model analysis of variance was used to evaluate differences between readers and sides with respect to the mean CCI but adjusted for age and accommodating the correlation among observations generated for the same subject. RESULTS: The patient in our case report had a right CCI of 0.2 mm and left CCI of 0.0 mm. In the other 30 patients, the right CCI ranged from 0.2 to 3.8 mm (mean, 1.2 +/- 0.8 mm; median, 0.9) and the left CCI from 0.2 to 5.0 mm (mean, 1.1 +/- 0.9 mm; median, 0.8). The CCI did not exhibit a significant association with subject age (P = .1336), and there were no significant differences between readers (P = .824) or sides (P = .350) in terms of mean CCI. CONCLUSION: The CCI varies widely between patients and may be as small as zero. Analysis of anatomic relationships suggests a potential relationship between small CCI and mid-tone sensorineural hearing loss, as in our reported patient. Preoperative knowledge of thin or absent bone between the cochlea and petrous carotid canal may help prevent inadvertent penetration of the carotid canal during cochlear implant surgery.

SDHB, SDHC, and SDHD mutation screen in sporadic and familial head and neck paragangliomas.

Mutations within three genes, SDHB, SDHC, and SDHD, encoding distinct subunits of a hetero-oligomeric protein known as the mitochondrial complex II, a component of the mitochondrial electron transport chain and the Krebs cycle have been implicated in the pathogenesis of hereditary paraganglioma (PGL). This study describes a mutation screen of SDHB, SDHC, and SDHD in blood and tumor samples of 14 sporadic and three familial cases of head and neck PGL (HNP). Germline mutations in SDHB and SDHD were identified in two of the three affected individuals with familial HNP. The SDHB mutation was a novel 3 base pair, in-frame deletion of AGC at nucleotide 583-585 encoding serine (delS195). The SDHD mutation was a C to T transition within codon 81 causing substitution of proline with leucine (P81L). In contrast to familial cases, no germline or somatic mutations were identified in the 14 sporadic cases of HNP. The presence of mutations within SDHB and SDHD in two of the three samples of familial PGLs and absence of mutations in sporadic cases is consistent with the significant contribution of these genes to familial but not sporadic PGL. The etiology of sporadic PGL remains to be elucidated.

Refinement of the DFNA4 locus to a 1.44 Mb region in 19q13.33.

Many forms of autosomal dominant non-syndromic hearing impairment are known. While the underlying gene defects and causative mutations have been discovered for some forms, the gene responsible for DFNA4 has remained elusive to date. Examination of a German four-generation kindred led to the identification of a 1.44 Mb map segment in contig NT_011109 as being the most likely DFNA4 candidate region in 19q13.33. The recombination breakpoints in this family and the intervals of two previously reported DFNA4 families allowed us to delineate a minimum consensus region between the markers D19S879 and D19S246. In our family, a maximum two-point LOD score of 4.5 was obtained at theta = 0 for the marker D19S867. Within the refined DFNA4 interval the public databases list more than 50 genes, from which several appear to be promising DFNA4 candidates due to similarities with animal models and with other causative genes involved in hearing disability.

Mutation analysis of Connexin 31 (GJB3) in sporadic non-syndromic hearing impairment.

Mutations in GJB3, the gene encoding the gap junction protein Connexin 31 (CX31), have been pathogenically linked to erythrokeratodermia and non-syndromic autosomal dominant (DFNA2) or recessive hereditary hearing impairment (HHI). To determine the contribution of CX31 to sporadic deafness, we assessed 63 individuals with non-syndromic hearing impairment for CX31 mutations. Single coding exon of CX31 was amplified from genomic DNA and then sequenced. Single nucleotide sequence alteration was present in 15 out of 63 patients (24%), all of the positives being heterozygous for the four different single base pair changes that were detected: C94T, C201T, C357T and C798T. Of these, only C94T transition, identified in two patients, results in amino acid change, R32W, while the other three changes are single nucleotide polymorphisms (SNPs). The R32W substitution in CX31 has been previously documented and is speculated to manifest variable penetrance, similar to the polymorphic allele encoding CX26M34T. Over one-third of all samples were also screened with denaturing high-performance liquid chromatography (dHPLC). Seven out of 25 individuals screened were determined to be positive for CX31 sequence variation. Sequence analysis of the 25 individuals screened identified nucleotide alterations in all of the 7 'positives' and in none of the 16 'negatives' yielding a specificity and sensitivity of 100%. Thus, dHPLC represents a highly efficient CX31 screening technique. This study suggests that while sequence alterations are common, pathogenic mutations of CX31 are infrequent in sporadic non-syndromic hearing impairment.

The use of Preyer's reflex in evaluation of hearing in mice.

Preyer's reflex, the elicitation of startle response to auditory stimuli, has been widely used for the evaluation of hearing in rodents and other animals. Surprisingly, however, the sensitivity and specificity of Preyer's reflex in the assessment of hearing has not been adequately studied. The aim of this study was to investigate the utility of Preyer's reflex in the evaluation of auditory function in mice. Forty-six adult albino mice on an FVB background with variable hearing loss were used for this study. Two different methods for eliciting a Preyer's reflex were tested: a handclap and a sharp metallic sound. The reflex was considered positive when a rapid movement of the whole body of the animal was clearly noticed. Thereafter, the mice underwent auditory brain stem response (ABR) testing with broadband clicks. The presence or absence of Preyer's reflex was compared with the corresponding ABR thresholds. Five of the 46 animals studied (11%) showed a negative Preyer's reflex, while the remaining 41 animals demonstrated a positive Preyer's reflex. There was no difference between the abilities of the two different stimuli to elicit a Preyer's reflex. The click-evoked ABR thresholds in the test animals varied between 8 and 136 (mean 50) dB sound pressure level (SPL). Preyer's reflex was positive in all animals with an ABR threshold of < or = 76 dB SPL, but was absent in animals with an ABR threshold of > or = 81 dB SPL. Preyer's reflex is effective for identifying profound sensorineural hearing loss in experimental mice, but is insensitive for detecting less severe auditory dysfunction. For definitive hearing assessment, and for defining the hearing thresholds. objective electroacoustical methods such as ABR should be used.

Assessment of denaturing high-performance liquid chromatography (DHPLC) in screening for mutations in connexin 26 (GJB2).

Mutations in the gene GJB2 encoding connexin 26 (Cx26), a gap junction protein, have been shown to be responsible for a majority of recessive nonsyndromic hereditary hearing impairment in children. Over 60 different mutations in Cx26 have been reported. To obviate the need for direct sequencing of each specimen, a variety of screening techniques have been used to detect mutations in Cx26. However, each of these methods has significant shortcomings including expense, time consumption, and limited sensitivity. Denaturing high-performance liquid chromatography (DHPLC) has been recently introduced as a rapid and highly sensitive method of detecting sequence alterations. We have assessed the efficacy of DHPLC as a screening assay for detecting mutation in Cx26 coding region in 154 patients with hereditary hearing impairment. The GJB2 coding exon was amplified in one or two fragments, analyzed by DHPLC, and sequenced. Sequence analysis identified sequence variations in 34 patients concordant with abnormal DHPLC results. Three novel Cx26 mutations were identified: a single base pair substitution 511G>A, a 4 bp insertion 504insAACG, and a 3 bp deletion 358delAGG in three unrelated patients. In 120 patients with normal Cx26 sequence, DHPLC was normal. These results yield sensitivity and specificity of 100% for DHPLC-based detection of Cx26 mutations, and demonstrate that DHPLC is a highly sensitive and specific method of screening for sequence variations in Cx26 that is time and labor efficient. Further, our experience suggests that DHPLC screening alone followed by DNA sequencing only when DHPLC is abnormal may be adequate for identification of all sequence alterations in Cx26.

Conservative management of infections in cochlear implant recipients.

OBJECTIVE: The goal of this study was to evaluate a conservative management strategy of postoperative infection after cochlear implantation. METHODS: A retrospective review of the medical records of 108 cochlear implant patients operated on at the University of California, San Francisco between 1991 and 2000 and 133 cochlear implant patients from the University of Iowa between 1997 and 2000 showed 4 patients with evidence of postoperative infections. The clinical presentation, intervention, laboratory results, and outcome are analyzed in each case. RESULTS: Minimal surgical intervention with limited incision and drainage with prolonged postoperative antibiotics was effective in treating postoperative cochlear implant infections without the need for device removal. Implant function remained unaffected after surgery. CONCLUSION: Postoperative cochlear implant infections can be effectively controlled with limited surgical and prolonged medical management. Chronic implant infections may be explained by a primary immunodeficiency. With appropriate treatment leading to infection control, a conservative management strategy is advocated before consideration of device explantation.

Facial nerve monitoring in middle ear and mastoid surgery.

HYPOTHESIS: Intraoperative electromyographic facial nerve monitoring, long accepted as the standard of care in surgery for acoustic neuroma and other cerebellopontine angle tumors, may be of aid in middle ear and mastoid surgery. STUDY DESIGN: Retrospective series of 262 cases of middle ear/mastoid surgery in which monitoring was performed by a neurophysiologist. METHODS: Neurophysiological monitoring events were classified as mechanical or electrical. The voltages producing facial nerve stimulation were compiled and compared with observed facial nerve dehiscence. RESULTS: The most common use of monitoring was localization of the facial nerve by electrical stimulation (60%) or identification of mechanically evoked activity (39%). In 57 cases (36%), the first electrical stimulation event evoked a facial nerve response at less than 1 V threshold, indicating little or no bony covering. The minimum stimulation threshold throughout each of these cases was less than 1 V in 88 of the 159 cases (55%) in which stimulation was attempted. In contrast, the facial nerve was visibly dehiscent in only 35 cases (13%). Neurophysiological monitoring confirmed aberrant facial nerve course through the temporal bone in four cases resulting in cancellation of surgical treatment in two cases. Postoperative facial nerve function was preserved in all cases when present preoperatively. CONCLUSIONS: An electrical stimulation threshold of less than 1 V is a more useful criterion of dehiscence than observation under the operating microscope. The absence of monitoring events allows safe dissection. Monitoring can help locate the facial nerve, guide the dissection and drilling, and confirm its integrity, thereby allowing more definitive surgical treatment while preserving neural function.

Cochlear gene delivery through an intact round window membrane in mouse.

Cochlear gene transfer studies in animal models have utilized mainly two delivery methods: direct injection through the round window membrane (RWM) or intracochlear infusion through a cochleostomy. However, the surgical trauma, inflammation, and hearing loss associated with these methods lead us to investigate a less invasive delivery method. Herein, we studied the feasibility of a vector transgene-soaked gelatin sponge, Gelfoam, for transgene delivery into the mouse cochlea through an intact RWM. The Gelfoam absorbed with liposomes and adenovirus, but not with adeno-associated virus (AAV), was successful in mediating transgene expression across an intact RWM in a variety of cochlear tissues. The Gelfoam technique proved to be an easy, atraumatic, and effective, but vector-dependent, method of delivering transgenes through an intact RWM. Compared with the more invasive gene delivery methods, this technique represents a safer and a more clinically viable route of cochlear gene delivery in humans.

Detection of fungi in the nasal mucosa using polymerase chain reaction.

HYPOTHESIS: Fungi have been increasingly recognized as important pathogens in sinusitis. However, detection of fungus with conventional culture techniques is insensitive and unreliable. Polymerase chain reaction (PCR) is an exquisitely sensitive assay that can detect the DNA of 10 or less fungal elements. The aim of this study was to compare the sensitivity of conventional culture techniques using PCR analysis. METHODS: Nasal swabs and DNA samples were collected from the nasal cavities of control subjects and patients with chronic sinusitis. Fungal-specific PCR analysis and standard cultures were performed on every sample. chi2 analysis was used to test for statistical differences between groups. RESULTS: PCR analysis detected fungal DNA in 42% and 40% of control subjects and patients with chronic sinusitis while standard cultures were positive in 7% and 0%, respectively. There was no statistically significant difference in the prevalence of fungi in the normal volunteers and patients with chronic rhinosinusitis. CONCLUSION: PCR is significantly more sensitive than nasal swab cultures in detecting the presence of fungi in nasal mucosa. In addition, our study suggests that the presence of fungi alone is insufficient to implicate it as the pathogen in chronic sinusitis.

A surgical approach appropriate for targeted cochlear gene therapy in the mouse.

Therapeutic manipulations of the mammalian cochlea, including cochlear gene transfer, have been predominantly studied using the guinea pig as the experimental model. With the significant developments in mouse genomics and the availability of mutant strains of mice with well-characterized hearing loss, the mouse justifiably will be the preferred animal model for therapeutic manipulations. However, the potential advantages of the mouse model have not been fully realized due to the surgical difficulty of accessing its small cochlea. This study describes a ventral approach, instead of the routinely used postauricular approach in other rodents, for accessing the mouse middle and inner ear, and its application in cochlear gene transfer. This ventral approach enabled rapid and direct delivery of liposome-transgene complex to the mouse inner ear while avoiding blood loss, facial nerve morbidity, and mortality. Transgene expression at 3 days was detected in Reissner's membrane, spiral limbus, spiral ligament, and spiral ganglion cells, in a pattern similar to that previously described in the guinea pig. The successful access and delivery of material to the mouse cochlea and the replication of gene expression seen in the guinea pig demonstrated in this study should promote the use of the mouse in future studies investigating targeted cochlear therapy.

Dominant modifier DFNM1 suppresses recessive deafness DFNB26.

More than 50% of severe childhood deafness is genetically determined, approximately 70% of which occurs without other abnormalities and is thus termed nonsyndromic. So far, 30 nonsyndromic recessive deafness loci have been mapped and the defective genes at 6 loci, DFNB1, DFNB2, DFNB3, DFNB4, DFNB9 and DNFB21, have been identified, encoding connexin-26 (ref. 3), myosin VIIA (ref. 4), myosin XV (ref. 5), pendrin, otoferlin and alpha-tectorin, respectively. Here we map a new recessive nonsyndromic deafness locus, DFNB26, to a 1.5-cM interval of chromosome 4q31 in a consanguineous Pakistani family. A maximum lod score of 8.10 at theta=0 was obtained with D4S1610 when only the 8 affected individuals in this family were included in the calculation. There are seven unaffected family members who are also homozygous for the DFNB26-linked haplotype and thus are non-penetrant. A dominant modifier, DFNM1, that suppresses deafness in the 7 nonpenetrant individuals was mapped to a 5.6-cM region on chromosome 1q24 with a lod score of 4.31 at theta=0 for D1S2815.

Cochlear microinjection and its effects upon auditory function in the guinea pig.

Microinjection through the round window membrane has been found to represent a method for vector delivery in intracochlear gene transfer in animal models but breaches the round window membrane, making it necessary to evaluate animals for possible postinjection hearing loss. In the present study healthy guinea pigs were evaluated for their baseline click auditory brainstem response (ABR) thresholds. Each animal was then injected with saline via the round window membrane. After 1 week auditory function was evaluated by click ABR. Animals with increased ABR thresholds were retested at 4 weeks. Animals with 1-week postoperative ABRs similar to baseline were not retested. Results showed that postoperative ABR thresholds in five animals (71%) remained unchanged from baseline, while two animals had increases of 20-25 dB in ABRs after 1 week but recovered baseline ABRs after 4 weeks. The mean baseline ABR threshold was 25.7 dB and was 27.9 dB after 1 week after injection. The difference between preoperative and 1-week postoperative averages was not significant (P = 0.707). In this preliminary study saline microinjection through the round window membrane did not cause permanent hearing loss in the guinea pigs tested, and any damage caused by microinjection appeared to be reversible.

Role of angiography in the evaluation of patients with pulsatile tinnitus.

OBJECTIVES/HYPOTHESIS: Pulsatile tinnitus in the face of normal findings on otoscopy is a common otological diagnostic dilemma and can be due to serious vascular malformations such as transverse or sigmoid sinus dural arteriovenous fistula (transverse or sigmoid sinus [TS] DAVF). Left untreated, TS DAVF may result in significant morbidity and mortality. TS DAVF can be suspected or diagnosed with computed tomography (CT), magnetic resonance imaging (MRI), and magnetic resonance angiography (MRA), with the gold standard being angiography. Our objective was to assess the utility of these various diagnostic modalities in the diagnosis of dural arteriovenous fistula. STUDY DESIGN: Retrospective clinical review. METHODS: Between 1986 and 1996, 54 patients were evaluated and treated for TS DAVF. Between 1996 and 1999, an additional 33 patients underwent MRI combined with MRA for the evaluation of pulsatile tinnitus. A retrospective review of the medical records for both groups, with special attention to clinical presentation, diagnostic evaluation, therapy, and outcome, was performed. RESULTS: All patients had pulsatile tinnitus with normal findings on otoscopy. CT scan was relatively insensitive in the detection of TS DAVF. MRI and MR/MRA were significantly more sensitive than CT. In the evaluation of patients with subjective pulsatile tinnitus, MRI/MRA defined anatomical abnormalities that may contribute to pulsatile tinnitus in 63% of patients. CONCLUSIONS: In the absence of objective pulsatile tinnitus, MRI/MRA is an appropriate initial diagnostic step. When a patient has an objective bruit, the clinician may choose to proceed directly to angiography to make certain that a TS DAVF is not missed.

Safety of adeno-associated virus as cochlear gene transfer vector: analysis of distant spread beyond injected cochleae.

The adeno-associated virus (AAV), inoculated into the perilymph, has been shown to be an effective vector for mediating intracochlear transgene expression. The unexpected finding of transgene expression in the contralateral cochlea in previous work raised concern about dissemination of the virus from the target tissue. The current study was undertaken to assess the extent of AAV dissemination following its introduction into the inner ear. Adult male guinea pigs were injected with recombinant AAV into their left ears and sacrificed at 2 or 4 weeks. Various organs including the cochleae were harvested to characterize the presence and expression of the viral DNA. Virus DNA was detected via polymerase chain reaction in the infused and contralateral cochlea and in the cerebellum but not in any other organs, including cortex, heart, lung, liver, spleen, and kidney. Although the viral presence was established in the cerebellum, transgene expression in this organ was undetectable with either Western blot or immunohistochemistry. Transgene expression was demonstrated via immunohistochemistry in multinucleated giant cells in the bone marrow spaces adjacent to the infused and contralateral cochleae. Collectively, these results suggest potential routes for AAV dissemination from the infused cochlea via the cochlear aqueduct or by extension through the temporal bone marrow spaces. This study reinforces the need to investigate factors that mitigate viral leakage.

Sensorineural and conductive hearing loss associated with lateral semicircular canal malformation.

OBJECTIVE: Lateral semicircular canal (LSCC) malformation is one of the most common radiological inner ear malformations. Traditionally, inner ear malformations are thought to be associated with sensorineural hearing loss (SNHL). Recent experience with patients with LSCC malformation suggested that LSCC malformation may be associated with both SNHL and conductive hearing loss (CHL). The auditory phenotype associated with LSCC malformation is not well delineated. The objective of this study is to define the nature of the hearing loss associated with LSCC malformation. STUDY DESIGN: Retrospective review METHODS: Retrospective review of clinical records, audiological evaluation, and imaging studies. RESULTS: Two patients with unilateral and 13 patients with bilateral LSCC malformation were identified. LSCC malformation was associated with CHL in 14% (4 ears), SNHL in 71% (20 ears), normal hearing in 11% (3 ears) and CHL due to atresia in one ear. Hearing loss varied from mild to profound but did not correlate with the severity of LSCC malformation. In bilateral malformation, the hearing loss was asymmetric in half of the cases. Malformation of the posterior limb of the LSCC was always associated with a large vestibular aqueduct. An absent or rudimentary LSCC was invariably associated with a cochlear abnormality. CONCLUSIONS: LSCC malformation, like other inner ear malformations such as large vestibular aqueduct and X-linked mixed deafness with perilymph gusher, can be associated with CHL, SNHL, or normal hearing. Children with unexplained conductive hearing loss often undergo exploratory surgery to improve hearing. Given that inner ear malformations may be associated with a pure CHL, it is critical that children undergo computed tomography scan of the temporal bone prior to undergoing exploratory surgery.