The vestibular calyceal junction is dismantled following subchronic streptomycin in rats and sensory epithelium stress in humans.

Hair cell (HC) loss by epithelial extrusion has been described to occur in the rodent vestibular system during chronic 3,3'-iminodipropionitrile (IDPN) ototoxicity. This is preceded by dismantlement of the calyceal junction in the contact between type I HC (HCI) and calyx afferent terminals. Here, we evaluated whether these phenomena have wider significance. First, we studied rats receiving seven different doses of streptomycin, ranging from 100 to 800 mg/kg/day, for 3-8 weeks. Streptomycin caused loss of vestibular function associated with partial loss of HCI and decreased expression of contactin-associated protein (CASPR1), denoting calyceal junction dismantlement, in the calyces encasing the surviving HCI. Additional molecular and ultrastructural data supported the conclusion that HC-calyx detachment precede HCI loss by extrusion. Animals allowed to survive after the treatment showed functional recuperation and rebuilding of the calyceal junction. Second, we evaluated human sensory epithelia obtained during therapeutic labyrinthectomies and trans-labyrinthine tumour excisions. Some samples showed abnormal CASPR1 label strongly suggestive of calyceal junction dismantlement. Therefore, reversible dismantlement of the vestibular calyceal junction may be a common response triggered by chronic stress, including ototoxic stress, before HCI loss. This may partly explain clinical observations of reversion in function loss after aminoglycoside exposure.

Pre- and intra-operative prognostic factors of facial nerve function in cerebellopontine angle surgery.

PURPOSE: The study assesses whether pre- and intraoperative factors linked to electromyography and direct electrical stimulation (DES) of facial nerve can predict facial nerve function in the short- (12 days) and long-term (1 year) after cerebellopontine angle (CPA) tumor resection. METHODS: 157 patients who underwent surgical resection of CPA tumors with facial nerve monitoring. Pre-operative factors (age, tumor size, pure tone average), surgical time and intra-operative parameters regarding facial function, minimum stimulation threshold (MST), compound muscle action potential (CMAP) and the difference between proximal and distal CMAP (DPDC) were evaluated. RESULTS: A correlation between tumor size, MST, CMAP and facial function in both short and long term was found. A higher grade of immediate facial paralysis corresponded to a higher risk of poor outcome after one year. A postoperative House-Brackmann (HB) score of V or VI was correlated with poor outcome in 88.8% and 93.8% of cases. A risk of HB 3 or more, in the long term, was correlated with a tumor size of 20.2 mm. Using an MST of 0.1 mA, for long-term predictions, sensitivity and specificity were 0.62 (95% CI 0.46-0.75) and 0.73 (95% CI 0.61-0.82), respectively. With a CMAP cut-off < 200 µV, for long-term prediction, sensitivity was 0.73 (95% CI 0.53-0.87) and specificity 0.73 (95% CI 0.55-0.85). CONCLUSION: The assessment based on the cut-offs described increases the ability to predict facial function. Improving predictive accuracy enables surgeons to address patients' expectations and to establish an intervention timeline for planning facial reanimation.

Mitochondrial DNA and TLR9 drive muscle inflammation upon Opa1 deficiency.

Opa1 participates in inner mitochondrial membrane fusion and cristae morphogenesis. Here, we show that muscle-specific Opa1 ablation causes reduced muscle fiber size, dysfunctional mitochondria, enhanced Fgf21, and muscle inflammation characterized by NF-κB activation, and enhanced expression of pro-inflammatory genes. Chronic sodium salicylate treatment ameliorated muscle alterations and reduced the muscle expression of Fgf21. Muscle inflammation was an early event during the progression of the disease and occurred before macrophage infiltration, indicating that it is a primary response to Opa1 deficiency. Moreover, Opa1 repression in muscle cells also resulted in NF-κB activation and inflammation in the absence of necrosis and/or apoptosis, thereby revealing that the activation is a cell-autonomous process and independent of cell death. The effects of Opa1 deficiency on the expression NF-κB target genes and inflammation were absent upon mitochondrial DNA depletion. Under Opa1 deficiency, blockage or repression of TLR9 prevented NF-κB activation and inflammation. Taken together, our results reveal that Opa1 deficiency in muscle causes initial mitochondrial alterations that lead to TLR9 activation, and inflammation, which contributes to enhanced Fgf21 expression and to growth impairment.

Diagnostic Accuracy of Intracochlear Test Electrode for Acoustic Nerve Monitoring in Vestibular Schwannoma Surgery.

OBJECTIVES: Cochlear implants (CIs) are a well-known hearing restoration option for patients with vestibular schwannoma (VS) in cases of neurofibromatosis type-2 and, more recently, for patients with sporadic VS. One of the main limitations when performing CI during VS surgery is the capability to preserve the acoustic nerve (AN) anatomically and functionally. Significant efforts have been directed toward developing an intraoperative testing method for monitoring the AN function to determine if, after tumor removal, it is suitable for conducting stimuli delivered by a CI. However, all these methods have significant limitations, and none of them have documented diagnostic efficacy. To overcome these limitations and to obtain reliable information before CI insertion, a minimally invasive intracochlear test electrode (TE) has been recently developed. This TE has demonstrated to be suitable to test the integrity of the AN before CI in patients without any residual hearing by recording electrically evoked auditory brainstem responses (EABR). The present study constitutes the next phase of this research, which was to determine the usefulness of EABR obtained intraoperatively with the intracochlear TE after the resection of a VS and to calculate its diagnostic accuracy to assess the functionality of the AN for CI. DESIGN: This was a prospective, multicenter study of diagnostic accuracy. It was conducted in three tertiary referral centers between January 2015 and 2018. This study was designed following the Standards for Reporting of Diagnostic Accuracy Studies (STARD) statement guidelines. The STARD statement are guidelines to improve the completeness and transparency of reports of diagnostic accuracy studies. The diagnostic accuracy of the EABR evoked with the intracochlear TE after tumor removal was studied. Accuracy, sensitivity, specificity, positive predictive value, and negative predictive value were calculated. Patients eligible for the study were consecutive adults undergoing surgery for VS with simultaneous CI. The test under evaluation (index test) was the EABR obtained with the intracochlear TE after resection of the tumor. The reference test (gold standard) was the presence of auditory perception with the CI, defined as the presence of sound detection on an audiogram at 500, 1000, 2000, and 4000 Hz of no greater than 50 dB. In all the cases, auditory perception was verified by the presence of a positive EABR evoked with the CI. RESULTS: Twenty-one patients were included during the study period; seven patients were excluded from the diagnostic efficacy analysis due to inconclusive EABR results or absence of the gold standard to compare (they did not finally receive the CI). Thus, the outcome of the gold standard was assessed in 14 cases: 9 cases had positive EABR, all of them obtained auditory perception with the CI, and 5 cases had negative EABR, only one case had auditory perception with the CI, which constitutes the only false negative of this study. Accuracy of the TE was 93% (95% confidence interval, 66 to 100%), sensitivity 90% (95% confidence interval, 71 to 100%), specificity 100% (95% confidence interval, 100 to 100%), positive predictive value 100% (95% confidence interval, 100 to 100%), and negative predictive value 80% (95% confidence interval, 45 to 100%). CONCLUSIONS: EABR elicited with the intracochlear TE had a diagnostic accuracy of 93% for predicting auditory perception with CIs after VS removal. These results suggest that the intracochlear TE can be used intraoperatively after tumor removal to test the integrity of the AN as a useful tool to complement the surgeon's perception for decision-making regarding implantation.

Lactate induces FGF21 expression in adipocytes through a p38-MAPK pathway.

FGF21 (fibroblast growth factor 21), first described as a main fasting-responsive molecule in the liver, has been shown to act as a true metabolic regulator in additional tissues, including muscle and adipose tissues. In the present study, we found that the expression and secretion of FGF21 was very rapidly increased following lactate exposure in adipocytes. Using different pharmacological and knockout mice models, we demonstrated that lactate regulates Fgf21 expression through a NADH/NAD-independent pathway, but requires active p38-MAPK (mitogen activated protein kinase) signalling. We also demonstrated that this effect is not restricted to lactate as additional metabolites including pyruvate and ketone bodies also activated the FGF21 stress response. FGF21 release by adipose cells in response to an excess of intermediate metabolites may represent a physiological mechanism by which the sensing of environmental metabolic conditions results in the release of FGF21 to improve metabolic adaptations.

FGF21 expression and release in muscle cells: involvement of MyoD and regulation by mitochondria-driven signalling.

Although the liver is generally considered the main site of production of FGF21 (fibroblast growth factor-21), high FGF21 levels have been found to be associated with neuromuscular mitochondrial genetic diseases, and there are indications that the muscle may be a relevant site of FGF21 production under conditions of muscular mitochondrial stress. In the present study, we found that expression and release of FGF21 was associated with myogenic differentiation, and we identified MyoD as a major controller of FGF21 gene transcription. Mimicking mitochondrial dysfunction using respiratory chain/oxidative phosphorylation inhibitors resulted in enhanced expression and release of FGF21 by muscle cells. The increased production of reactive oxygen species, subsequent induction of p38 MAPK (mitogen-activated protein kinase) and activation of an ATF2 (activating transcription factor 2)-binding site at the proximal promoter region of the FGF21 gene was found to be a major mechanism linking mitochondrial dysfunction with enhanced FGF21 gene transcription in myogenic cells. The myogenic factor MyoD was required for the induction of FGF21 gene transcription by mitochondrial dysfunction, thus explaining the preferential response of muscle cells to mitochondrial dysfunction-induced FGF21 expression and secretion. FGF21 release by muscle cells in response to mitochondrial alterations may represent a physiological mechanism by which the sensing of internal energetic status by muscles results in the release of FGF21 to favour systemic metabolic adaptations.

FGF21 mediates the lipid metabolism response to amino acid starvation.

Lipogenic gene expression in liver is repressed in mice upon leucine deprivation. The hormone fibroblast growth factor 21 (FGF21), which is critical to the adaptive metabolic response to starvation, is also induced under amino acid deprivation. Upon leucine deprivation, we found that FGF21 is needed to repress expression of lipogenic genes in liver and white adipose tissue, and stimulate phosphorylation of hormone-sensitive lipase in white adipose tissue. The increased expression of Ucp1 in brown adipose tissue under these circumstances is also impaired in FGF21-deficient mice. Our results demonstrate the important role of FGF21 in the regulation of lipid metabolism during amino acid starvation.

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha controls transcription of the Sirt3 gene, an essential component of the thermogenic brown adipocyte phenotype.

Sirt3 (silent mating type information regulation 2, homolog 3), a member of the sirtuin family of protein deacetylases with multiple actions on metabolism and gene expression is expressed in association with brown adipocyte differentiation. Using Sirt3-null brown adipocytes, we determined that Sirt3 is required for an appropriate responsiveness of cells to noradrenergic, cAMP-mediated activation of the expression of brown adipose tissue thermogenic genes. The transcriptional coactivator Pgc-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) induced Sirt3 gene expression in white adipocytes and embryonic fibroblasts as part of its overall induction of a brown adipose tissue-specific pattern of gene expression. In cells lacking Sirt3, Pgc-1α failed to fully induce the expression of brown fat-specific thermogenic genes. Pgc-1α activates Sirt3 gene transcription through coactivation of the orphan nuclear receptor Err (estrogen-related receptor)-α, which bound the proximal Sirt3 gene promoter region. Errα knockdown assays indicated that Errα is required for full induction of Sirt3 gene expression in response to Pgc-1α. The present results indicate that Pgc-1α controls Sirt3 gene expression and this action is an essential component of the overall mechanisms by which Pgc-1α induces the full acquisition of a brown adipocyte differentiated phenotype.

CT Assessment of Otic Capsule Bone Density in Paget's Disease of the Temporal Bone and Its Relationship With Hearing Loss.

OBJECTIVES: To study computed tomography findings in Paget's disease of temporal bone (PDTB) and analyze the relations between otic capsule bone mineral density values measured in Hounsfield Units (HU) and hearing loss (HL). STUDY DESIGN: Observational case-control study. SETTING: Tertiary referral center. PATIENTS: Radiographically confirmed PDTB cases and control group. INTERVENTION: Diagnostic. MAIN OUTCOME MEASURES: Hearing thresholds and computed tomography bone density values. RESULTS: Twenty-three ears in the case group (PDTB) and 27 control ears were included. In the PDTB group, HL was found in 87% of the ears (43% mixed) and an air-conduction threshold of 50.7 dB (SD = 19.8). In the control group, 48% of the ears showed HL (7% mixed) and an air-conduction threshold of 34.5 dB (SD = 20.6) was found; the difference was statistically significant (p < 0.05). Measurements of bone density (HU) in the otic capsule (regions of interest [ROI] 1 and 2) and in the petrous bone (ROI 3) were significantly lower (p < 0.05) in the PDTB group than in controls.The PDTB group presented a significant association between otic capsule bone density in ROI 1 and mean otic capsule density with air and bone-conduction thresholds (p < 0.05). In controls, no association was observed between any density value and audiometric thresholds. CONCLUSION: PDTB patients showed more frequent HL, lower thresholds, and a higher proportion of mixed HL than controls. Bone density (HU) was decreased in all ROIs in PDTB patients in comparison with controls. Bone density in the otic capsule was associated with HL in PDTB patients, but no association was observed between bone density and HL in controls.

Early Genetic Diagnosis of Neurofibromatosis Type 2 From Skin Plaque Plexiform Schwannomas in Childhood.

Importance: Neurofibromatosis type 2 (NF2) is a devastating genetic condition characterized by the development of multiple tumors of the nervous system. An early diagnosis of individuals with NF2 would facilitate treatment and reduction of disease impact because most severe effects of the disease do not usually develop before adolescence. Little attention has traditionally been paid to dermatological signs in NF2. However, skin plaques are commonly seen in patients with NF2, normally appearing either at birth or early childhood, providing an opportunity for early NF2 detection and testing. Objective: To determine the clinical utility of skin plaque identification and characterization in children for reaching an early diagnosis of patients with NF2 and to evaluate their molecular pathogenesis and their use in the genetic diagnostics of NF2. Design, Setting, and Participants: Diagnostic test study by the histological and genetic characterization of skin plaques from patients with NF2. Patients were 7 individuals with NF2 or clinical suspicion of NF2 treated at the Spanish Reference Center on Phakomatoses. Main Outcomes and Measures: Histological evaluation of all skin plaques was performed. Fresh skin plaques were cultured to obtain Schwann cells and the NF2 gene was genetically analyzed. For all 7 patients, NF2 clinical history was reviewed. Results: In all 7 patients (4 male and 3 female), all skin plaques analyzed were histologically characterized as plexiform schwannomas. Genetic analysis of primary Schwann cell cultures derived from them allowed the identification of a constitutional and a somatic NF2 mutation. Genetic testing allowed the early diagnosis of NF2 in a child only exhibiting the presence of skin plaques. Most of the patients with NF2 analyzed had an early presentation of skin plaques and a severe NF2 phenotype. Conclusions and Relevance: This work emphasizes the clinical utility of a careful dermatological inspection and the correct identification of skin plaques in children for an early diagnosis of NF2. We show for the first time that Schwann cells derived from skin plaque plexiform schwannomas bear the double inactivation of the NF2 gene and thus constitute an excellent source of tissue for genetic testing, especially in the context of mosaicism.

Fibroblast growth factor 21 protects the heart from oxidative stress.

AIMS: Oxidative stress mediated by reactive oxygen species (ROS) plays a striking role in the pathogenesis of heart failure, and antioxidants have been shown to attenuate cardiac remodelling in experimental models of cardiac damage. We recently showed that fibroblast growth factor 21 (Fgf21) is produced by the heart and exerts protective effects, preventing cardiac hypertrophy development. The aim of the study was to determine the effects of Fgf21 during oxidative stress signalling in the heart. METHODS AND RESULTS: Fgf21 treatment in cardiomyocytes in culture induced the expression of genes encoding proteins involved in antioxidative pathways, including mitochondrial uncoupling proteins (Ucp2 and Ucp3) and superoxide dismutase-2 (Sod2) and reduced ROS production. In keeping with this, expression of antioxidant genes in response to lipopolysaccharide (LPS)-induced stimulation of pro-inflammatory pathways or isoproterenol-induced cardiac hypertrophy in the heart was reduced in Fgf21-null mice. Moreover, we found that Fgf21 is expressed in and released by cardiomyocytes in response to LPS, and its expression is under the control of the Sirt1 (sirtuin-1) pathway. This Fgf21 released by cardiomyocytes acts in an autocrine manner to protect cells against oxidative stress. Finally, failing human hearts showed up-regulation of Fgf21, Ucp3, and Sod2, confirming the association between Fgf21 induction and the control of cardiac oxidative stress pathways. CONCLUSION: Our data indicate that Fgf21 regulates genes involved in antioxidant pathways in an autocrine manner, thus preventing ROS production in cardiac cells. Therefore, Fgf21 acts as an antioxidant factor in the heart, preventing induction of pro-oxidative pathways by inflammatory or hypertrophic conditions.

Segmental neurofibromatosis type 2: discriminating two hit from four hit in a patient presenting multiple schwannomas confined to one limb.

BACKGROUND: A clinical overlap exists between mosaic Neurofibromatosis Type 2 and sporadic Schwannomatosis conditions. In these cases a molecular analysis of tumors is recommended for a proper genetic diagnostics. This analysis is challenged by the fact that schwannomas in both conditions bear a somatic double inactivation of the NF2 gene. However, SMARCB1-associated schwannomas follow a four-hit, three-step model, in which both alleles of SMARCB1 and NF2 genes are inactivated in the tumor, with one of the steps being always the loss of a big part of chromosome 22 involving both loci. CASE PRESENTATION: Here we report a 36-year-old woman who only presented multiple subcutaneous schwannomas on her right leg. To help discriminate between both possible diagnoses, an exhaustive molecular genetic and genomic analysis was performed on two schwannomas of the patient, consisting in cDNA and DNA sequencing, MLPA, microsatellite multiplex PCR and SNP-array analyses. The loss of a big part of chromosome 22 (22q12.1q13.33) was identified in both tumors. However, this loss involved the NF2 but not the SMARCB1 locus. SNP-array analysis revealed the presence of the same deletion breakpoint in both schwannomas, indicating that this alteration was actually the first NF2 inactivating hit. In addition, a distinct NF2 point mutation in each tumor was identified, representing independent second hits. In accordance with these results, no deletions or point mutations in the SMARCB1 gene were identified. None of the mutations were present in the blood. Two of the patient's children inherited chromosome 22 deleted in schwannomas of the mother, but in its wild type form. CONCLUSIONS: These results conclusively confirm the segmental mosaic NF2 nature of the clinical phenotype presented.

Cerebral salt wasting syndrome as a postoperative complication after surgical resection of acoustic neuroma.

STUDY DESIGN: Retrospective case review. SETTING: Tertiary care, university hospital. PATIENTS: The authors describe two patients operated on for acoustic neuroma in whom hyponatremia occurred after intervention. INTERVENTIONS: Acoustic neuroma surgery. RESULTS: Wrong diagnosis in case of central nervous system disorder associated with hyponatremia can have fatal consequences. CONCLUSIONS: To the authors' knowledge, this is the first case report associating cerebral salt wasting syndrome with previous surgical resection of acoustic neuroma.

[Use and validity of the 14-3-3 protein test in the diagnosis of prion diseases: a 4-year prospective study]. / Utilización y validez de la prueba de la proteína 14-3-3 en el diagnóstico de enfermedades por priones: estudio prospectivo de 4 años.

BACKGROUND AND OBJECTIVES: The 14-3-3 test shows a high efficiency for the diagnosis of Creutzfeldt-Jakob disease (CJD), as long as an appropriate clinical setting exists. We analyze the evolution of the use and the validity of this test in Spain. PATIENTS AND METHOD: From January 1997 to June 2001, 1,092 samples were tested in our laboratory; 674 samples were selected for the study. Diagnoses were obtained by the referring physicians and the national CJD surveillance system according to standard criteria (results of the test were not included). RESULTS: The number of samples analyzed (% of follow-up) was: 75 (96%) in 1997, 138 (82.3%) in 1998, 197 (73%) in 1999 and 264 (59%) in 2000. The negative predictive value, sensitivity and specificity for a diagnosis of prion disease revealed stable values (95, 86 and 86%, respectively). The positive predictive value (PPV) decreased from 77% in 1997 to 51% in 2000 because of an increase of false-positive (FP) results. Up to 25% of FP might have been excluded through neuroimaging or CSF characteristics. CONCLUSIONS: The observed increase of the demand suggests that the test is used as a screening technique. In this setting, the PPV of the test decreases due to an increase of false-positive results.

In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2.

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, ∼25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T>A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2.

[Neurofibromatosis type 2]. / Neurofibromatosis tipo 2.

Type 2 neurofibromatosis (NF2) is an invalidating, inherited, dominant, autosomal disease. It is commonly confused with type 1 neurofibromatosis, although the two disorders are different. All subjects who inherit a mutated NF2 gene will develop the disease, which is characterised by the growth of schwannomas, generally affecting the vestibular nerve bilaterally, as well as meningiomas and other benign central nervous system tumours, before their third decade of life. It is currently possible to identify the NF2 mutation in most affected families. Up to about 20% of NF2 patients with no family history, apparently sporadic cases, are actually individuals with mosaicism for this mutation. Much of the morbidity from these tumours results from their treatment, which is primarily surgical. Small vestibular schwannomas can often be completely resected with preservation of both hearing and facial function. In case of large tumours it is possible to place a cochlear or brain stem implant during the schwannoma surgery. Age at diagnosis, the presence of intracranial meningiomas, and whether the patient was treated at a specialty centre or not, have been cited as the strongest prognostic factors.