Abstract: Bilateral Meniere's disease (BMD) is a severe disease that usually results in bilateral severe or profound sensorineural hearing loss and chronic disequilibrium with loss of vestibular function. We examined single nucleotide polymorphisms (SNPs) in the PTPN22 and CTLA4 genes in Caucasian patients with BMD to assess the possible association between these polymorphism and the predisposition and clinical expression of this disease.
Abstract: Dynamic visual acuity (DVA) during the head-thrust test (htDVA) is reliable in normal subjects, having a high specificity for the horizontal canal, so it may be useful to monitor vestibular rehabilitation in patients with vestibular neuritis (VN).
Abstract: To evaluate the usefulness of vestibular evoked myogenic potentials (VEMPs) in subjects with vestibular neuritis (VN) and to determine the impact of the disease in health-related quality of life (HRQoL).
Abstract: Conclusion: The longer alleles (CA)(17-20) of the promoter region of PARP-1 gene may confer some protection against bilateral Meniere's disease (BMD). Objective: To analyze microsatellite (CA)(n) polymorphisms in the promoter region of PARP-1 gene and seek out risk and protective variants for BMD. Subjects and methods: Eighty patients from two ethnically defined groups with definite BMD, according to the diagnostic scale of the American Academy of Otolaryngology Head and Neck Surgery, were compared with a group of 371 normal controls from the same origin in a prospective multicenter study. We developed a specific amplification protocol to determine the PARP1-promotor CA microsatellite polymorphisms. Results: We found that the longer alleles (CA)(17-20) had a very low frequency in BMD (2/160, 1.3%, OR = 7.33 (1.77-30.37, 95% CI), corrected p=0.012), suggesting that it may confer some protection against BMD.
Abstract: Diagnosis and treatment of benign paroxysmal positional vertigo (BPPV) is a mixture of empiricism of particle repositioning with the rationally-based knowledge obtained from clinical observations, histopathology, and neurophysiological experiments. The recently published clinical practice guideline on BPPV makes recommendations on the management of BPPV. One of the statements discourages the use of radiographic or vestibular testing, unless the diagnosis was uncertain or there were additional signs or symptoms unrelated to BPPV. The role of video-oculography in diagnosis and treatment of BPPV is argued, since vestibular testing has provided key relevant information to understand positional nystagmus in patients with BPPV.
Abstract: Benign paroxysmal positional vertigo is the most common vestibular disorder and it has a significant impact on health-related quality of life. The disease is probably caused by the accumulation of lithiasis material from the otolithic membrane of the utricle. Patients experience multiple short vertigo crises lasting seconds when they go to bed or turn over. There are several clinical variants affecting posterior, horizontal or anterior canals and in some cases vestibular lithiasis can occur in two canals simultaneously. Diagnosis is by video-oculographic recording of positional nystagmus during positional tests to identify the canal affected. There are specific treatment manoeuvres for each clinical variant, which a high degree of short-term effectiveness.
Abstract: Time constant and maximum slow phase velocity (SPV) of head-shaking nystagmus (HSN) demonstrated a differential canal response to head shaking in 24% of patients with posterior canal benign paroxysmal positional vertigo (BPPV). We suggest that vestibular lithiasis has a limited contribution to the mechanism that generates HSN.
Abstract: To obtain reference values for the vestibulo-ocular reflex response to the head-shaking nystagmus and the bithermal caloric test in the spanish population.
Abstract: To evaluate the eye movement response to the head auto-rotation test (HART) in the vertical plane in patients with benign paroxysmal positional vertigo.
Abstract: The aim of this study was to describe the clinical features and video-oculographic findings in patients with anterior semicircular canal benign paroxysmal positional vertigo (BPPV).
Abstract: A prospective cohort study was designed to evaluate the long-term outcome and health-related quality of life (HRQoL) in patients with posterior canal benign paroxysmal positional vertigo (PC-BPPV) treated by the particle repositioning maneuver (PRM) in the outpatient clinic of a general community hospital. Fifty individuals with PC-BPPV were included, and 45 (90%) completed the study. The diagnosis was based on the history of short episodes of vertigo and a positional nystagmus during the Dix-Hallpike test (DHT). All patients were treated by a single PRM, and relapses were evaluated by DHT at 30, 180 and 360 days post-treatment; a new PRM was performed if the DHT was positive. The main outcome measures were: percentage of patients with a negative DHT after treatment, scores obtained on the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36) and the Dizziness Handicap Inventory Short Form (DHI-S) before and 30, 180 and 360 days post-treatment. The DHT was found negative in 80% (40/50) of individuals at 30 days. Ten, seven and five patients presented a positive DHT at 30, 180 and 360 days, respectively. Persistent BPPV was observed in 5% (2/50) of patients at 360 days, despite repeated PRM. Relapses (DH+ after successful PRM) were observed in 7.5% (3/50) at 180 days and 360 days. Both questionnaires showed a reliability Cronbach's alpha >0.7. The average standardized score for each SF-36 scale was compared with the reference population normative data, showing differences with norms for all scales except for vitality. After PRM, patients improved their scores with both instruments, indicating a restoration of HRQoL at 30 days. Physical dimension scores of the SF-36 improved from day 30 to 360. DHI-S scores were statistically better after PRM (P < 0.001). Our results show that the effectiveness of PRM is 88% after 1 year of follow-up. Patients with BPPV experienced a decrease in HRQoL, which was restored after PRM. Although relapses were observed in 7.5% of individuals, they did not affect HRQoL.
Abstract: Video-oculography demonstrates a higher occurrence of atypical positional nystagmus in patients with benign paroxysmal positional vertigo (BPPV). This includes anterior and horizontal canal variants and multiple positional nystagmus, suggesting combined lesions affecting several canals.
Abstract: Missense mutations in the coagulation factor C homology (COCH) gene (14q12-q13) cause the autosomal dominant sensorineural hearing loss and vestibular disorder DFNA9 (OMIM 603196), and a high prevalence of symptoms of Meniere disease (MD) has been described in families with a mutation in the COCH gene. In this study, we search for mutations in the COCH gene in peripheral blood from patients with definite MD. DNA was extracted from peripheral blood cells of 30 individuals with MD and 30 controls. Exons 4 and 5 of the COCH gene were amplified by PCR reaction, using primer pairs flanking both exons. Sequences were analysed by a DNA sequencing system and compared with the published COCH cDNA sequence. No differences were found in the nucleotide sequences of exons 4 and 5 in the COCH gene in patients with definite sporadic MD when they were compared with the control group. Patients with definite MD have a low prevalence of mutations in exons 4 and 5 of the COCH gene.
Abstract: Benign paroxysmal positional vertigo (BPPV) is a common cause of dizziness within the geriatric population causing disability. The diagnosis is established by the position-induced rotatory nystagmus, related to the involved ear, demonstrated by the Dix-Hallpike test (DHT). Although the Epley's modified manoeuvre, or particle respositioning manoeuvre (PRM), is an effective treatment for this disorder, its impact on health outcome in patients over 60 years old is unknown.
Abstract: To determine the impact of the particle repositioning maneuver on posterior canal benign paroxysmal positional vertigo-related quality of life using the Medical Outcomes Study 36-Item Short Form Health Survey and the Dizziness Handicap Inventory Short Form.
Abstract: The purpose of this study was to identify pre- or intraoperative markers for post-tonsillectomy haemorrhage (PTH) that may help to define in-/outpatient tonsillectomy.
Abstract: To count the scientific contributions that were carried out at the Otorhinolarngology Departments in Spain from 1998 to 2002 years and to measure their scientific relevance using different bibliometrics tools.
Abstract: To identify pre or intraoperative risk factors that could indicate a higher risk for post-tonsillectomy hemorrhage (PTH) in children undergoing electrocautery dissection. SET-UP: Primary referral hospital.
Abstract: To analyze the associations of HLA class I and II antigens in patients with Meniere's disease (MD) in southern Spain, 54 patients with definite MD according to the diagnostic scale of the AAO-HNS were compared with 534 normal controls in a prospective multicenter study. We performed a serological typing for A, B, C and DR specificities of the major histocompatibility complex and allele-specific amplification for HLA-DRB1. No differences were found in the distribution of class I antigens or DR antigens in patients with definite MD when they were compared with the control group. Our results suggest that HLA antigens do not determine an increased susceptibility to develop MD.
Abstract: Benign paroxysmal positional vertigo (BPPV) is a recurrent chronic disease and its handicap is usually underestimated. The aim of this study is to determine the impact of the treatment by Epley maneuver on short-term BPPV-related quality of life. Forty-two individuals with BPPV were included: 39 with posterior canal affected, 2 with the lateral canal and one with the anterior canal. Diagnosis was established if a consistent clinical history was found and Dix-Hallpike test (DHT) in cases with canal posterior involvement. Subjects with positive DHT were treated by a single Epley maneuver and were recommended to avoid supine for the next 48 hours. The BPPV relapses were investigated at 7th and 30th day post-treatment. BPPV-associated quality of life was evaluated by the Dizzness Handicap Inventory Short-form (DHI-S) at days 1st and 30th post-treatment. Total and partial scores for emotional, physical and functional subscales were compared by Wilcoxon test for paired samples. Dix-Hallpike test was found positive in el 59% individuals (23/39), and 41% cases did not required any treatment. Among 23 patients treated with Epley maneuver, DHT was found negative in 90% at 30th day follow-up. Mean and standard deviation of the total scores obtained in the DHI-S at the first day were 19.22 +/- 9.66 in the DHT positive-patients and 19.79 +/- 10.14 in the whole group (DHT positive or negative). These scores significantly decreased to 10.84 +/- 10.99 at 30 days post-treatment (p = 0.002 and p = 0.001, respectively). In conclusion, the DHI-S is a specific health questionnaire able to assess BPPV-related health and the effectiveness of treatment.
Abstract: The structured clinical history is the most sensitive test for diagnosing vertigo. Its diagnostic effectiveness on the first visit was analyzed and key signs and symptoms with high predictive value for common causes of vertigo were identified. One hundred outpatients who complained of dizziness or loss of balance were evaluated using a structured clinical interview. Each questionnaire was examined independently by three blinded investigators, who assigned a diagnosis and identified the elements of the history that figured most prominently in the diagnosis. The gold standard was defined as independent selection of the same diagnostic category by all three investigators. A first-visit diagnosis was obtained in 40% of patients (95% confidence interval 30-50%): 38% women and 42% men. Causes included benign positional paroxysmal vertigo (BPPV, 13 patients), headache-associated vertigo (9), Meniere disease (7), cervical vertigo (3), psychiatric dizziness (2), post-traumatic vertigo (2), vertebro-basilar transient ischemic attack (1), vestibular neuritis (1), convulsive seizure (1), and presyncope (1). The best predictors of BPPV were the precipitating mechanism (specificity [SP] 100%), positional nystagmus (sensitivity [SE] 90%, SP 63%), and the Dix-Hallpike test (SE 82%, SP 71%). Elements predictive of headache-associated vertigo were duration of the attack (minutes) and a personal history of headache (both, SP 100%). Other predictors were facial hypoesthesia (SE 92%, SP 47%) and associated neurological disease (SE 82%, SP 58%).
Abstract: Headache and episodic vertigo are common symptoms in adults. Migraine-related dizziness and vertigo is a clinical process that is difficult to assess because specific diagnostic criteria are lacking. Although studies have demonstrated a relationship between migraine and dizziness, the prevalence of headache among patients with episodic vertigo is underestimated. A clinical inventory was used to determine the prevalence of headache among 150 patients with episodic vertigo and the impact that dizziness had on the quality of life using an ordinal scale for disability. Patients were questioned about drug use and self-perceived effectiveness. Twenty-three percent of the patients experienced a severe problem that disabled them for work. The prevalence of headache was 68.6% (71% of women and 64% of men). Forty-five percent of patients with episodic vertigo and 44% with headache did not obtain any benefit from symptomatic treatment. A subset of patients suffered from migraine-related dizziness and vertigo and were able to control their balance disorder with migraine therapy.
Abstract: A structured questionnaire of vertigo (SQV) was used to search patients with Menière's disease (MD) in the first visit. One-hundred consecutive outpatients that complained of dizziness or vertigo were studied. Diagnosis of MD was accepted if three investigators independently recorded it among a list of 29 diagnostic categories after reviewing the SQV. The AAO criteria for definite MD defined by the Committee on Hearing and Equilibrium after 10 months or more of follow-up were the gold standard to contrast questionnaire-based diagnosis. Seven patients were considered as MD with the SQV. The sensitivity (Sn), specificity (Sp) and positive predictive value of the SQV for definite MD were 80%, 97% and 57% respectively, being the false positive rate 3%. The probability for diagnosis of definite MD using the SQV in the first visit was 58% in a set up with a 5% of prevalence. The best predictor selected by all investigators was the presence of hearing loss (Sn 100%, Sp 85%, positive likelihood ratio 6.67). Because of the high specificity, the SQV of vertigo results a useful test to advance the diagnosis of definite MD.
Abstract: Sensorineural hearing loss (SHL) of autoimmune origin is characterized by a rapidly progressive onset. Diagnosis is difficult and the condition responds to corticoids or immunosuppressant agents. Two cases with an onset over weeks and progressive hearing loss are reported. Both patients had SHL associated with another autoimmune disease. Laboratory findings were abnormal (immunoglobulins and lymphocyte populations), suggesting an immune disorder. Treatment with 1 mg/kg/day of deflazacort restored hearing to normal. Both patients are currently asymptomatic. The clinical and laboratory response of patients to deflazacort supported the diagnosis of autoimmune SHL.
Abstract: Latencies of auditory brainstem response are known to be affected by age and sex. We have investigated the effects of age and sex on absolute latencies of waves I, III and V, the interpeaks intervals I-V, I-III and III-V for left and right ears in 67 healthy subjects with normal hearing. We evaluated the reproducibility of the response between ears which showed consistent data. Latency-intensity plots for waves I, III and V were compared among men and women by estimating the regression equations, with no significant difference in the slopes. Regression analysis revealed that latencies of waves I and V were increased with age in men from childhood up to fifties, according to a linear function. However, in women latencies were not dependent on age. The intervals I-V, I-III and III-V were independent on age for both men and women. The latencies of waves III and V and the I-III and I-V intervals were significantly shorter in women than in men in both ears. The findings obtained in the present study show that gender have a stronger effect on latencies of waves III and V, shortening the I-III and I-V intervals in women. Moreover, latencies of waves I and V increase with age in men.
Abstract: An occult, laterocervical papillary thyroid carcinoma tissue was found in a functional neck dissection for larynx cancer. The patient was a 76-year-old man with a history of smoking and alcohol ingestion who presented with a supraglottic carcinoma of the larynx located at the laryngeal surface of the epiglottis, left aryepiglottic fold, band and left ventricle with extension to the left vocal cord. Light microscopy showed a lymph node with a fibrous stroma with lymphoid follicles that presented a total substitution of the parenchyma by a papillary thyroid carcinoma. Although examination of the thyroid gland by seriated sections did not reveal any neoplasm, we argue that the papillary thyroid tissue is metastatic.
Abstract: A case of left horizontal-canal benign paroxysmal positional vertigo (HC-BPPV) in a woman with a 7-year history of episodic vertigo when turning over in bed is reported. The diagnosis was established with the patient lying down with her head raised 30 degrees with respect to the horizontal plane. Treatment with positional maneuvers (270 degrees contralateral head rotation in 3 steps) resolved the attacks. Positional testing failed to elicit symptoms 7 days, 14 days, 1 month, and 2 months after treatment. Four months after treatment, the patient remains asymptomatic. Published cases of HC-BPPV are discussed, as well as the differential diagnosis with posterior semicircular canal BPPV.
Abstract: A case of permanent pulsatile tinnitus of the left ear in a patient with hypertriglyceridemia is reported. The combined radiological study with computed tomography, magnetic resonance imaging, and digital angiography excluded a glomus tumor and revealed an enlarged, high-position jugular bulb with slow blood flow. Causes of pulsatile tinnitus are discussed. We conclude that imaging techniques play a major role in the diagnosis of head and neck vascular abnormalities.
Abstract: Intracellular Ca2+([Ca2+]i) is elevated by depolarization or mechanical stimulation in some hair cell systems. It is not clear whether both these stimuli promote Ca2+ entry in mammalian vestibular hair cells. We monitored [Ca2+]i with the indicator fluo-3 in isolated type I vestibular hair cells of the guinea pig maintained in Hanks' balanced salt solution (HBSS). Mechanical stimulation by bolus application of HBSS led to an immediate rise of [Ca2+]i. The effect depended upon the presence of extracellular Ca2+([Ca2+]o) and no increase occurred in calcium-free HBSS supplemented with calcium-chelators. When the cells were depolarized by bolus application of KCl (final concentration, 100 mM KCl in modified HBSS), the increase in [Ca2+]i was similar to that elicited by HBSS. In the absence of [Ca2+]o, the application of KCI/HBSS led to a slow sustained increase in the fluorescence of the cells suggesting release of calcium from intracellular stores. Finally, treatment of cells with BAPTA prior to mechanical stimulation prevented the rise in [Ca2+]i indicating the need for intact stereociliary tip-links. The results are consistent with the hypothesis that mechanical stimulation elevates [Ca2+]i in isolated vestibular hair cells via calcium influx through mechanotransduction channels.
Abstract: Gentamicin-induced changes in ionic composition in the otolithic membrane of adult OF1 mice were evaluated in the gelatinous layers of the saccule and utricle by quantitative electron probe X-ray microanalysis. The otolithic membranes were plunge-frozen and freeze-dried to prevent the redistribution of elements. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background (P/B) ratio method and different salts dissolved in dextran as standards to calibrate the P/B ratio against the concentration of the elements P, S and K in the microprobe. Gentamicin selectively decreased the concentrations of P (P < 0.001) and S (P < 0.01) in the gelatinous membrane of the saccule, and had no effect in the utricle. The concentration of K also increased in the utricular gelatinous membrane (P < 0.05). The mechanism of ototoxicity in the gelatinous membrane is unknown, but the ability of aminoglycosides to block calcium channels may induce disturbances in the ionic equilibrium of the endolymphatic fluid, and thus affect the biochemical composition of the gelatinous membrane. This technique can be useful to evaluate the distribution of ions in the process of drug-induced ototoxicity.
Abstract: The ability of electron probe X-ray microanalysis (EPMA) to solve biological problems often depends on the use of a quantitative approach. EPMA allows the quantitative determination of chemical elements of biological materials by using reference standards which resemble the specimen in the mode of interaction with the electron beam. Although there is a large experience in the quantification of elements in biological thin specimens, experience with standards for X-ray microanalysis of bulk specimens is limited, especially for calcified structures where the density of the specimen is difficult to estimate. The quality of the results in EPMA depends on obtaining accurate calibration curves which allow the establishment of the relationship between the signal measured and the concentration of the element of interest. The different methods for specimen preparation and the thickness of the specimen will also determine the precise nature of the standardization technique to be adopted. The physics of the electron beam-specimen interactions impose limitations upon the accuracy of calibration, and the choice of an unstable standard can result in large errors in the quantification of elements. We have reviewed the different types of compounds that have been used as standards for biological EPMA of thin and bulk specimens and discuss their potential use for quantitative analysis of mineralized tissues, with special reference to otoconia, the calcified structures of the vestibular system.
Abstract: Chronic gentamicin ototoxicity was evaluated in the otolithic membrane of adult OF1 mice at the otoconial layer of the saccule and utricle by quantitative electron probe X-ray microanalysis of Ca and K. The otolithic membranes were plunge-frozen and freeze-dried. The analysis was carried out with an energy dispersive detector using the peak-to-back-ground ratio method and different inorganic salts of Ca and K as standards to calibrate the microprobe. Ca and K in the otoconia are related via a linear function in both the saccule and the utricle. This association is not maintained after exposure to gentamicin, which suggests that this aminoglycoside antibiotic interferes with the Ca-K equilibrium in the otoconia. A dose of 200 mg/kg gentamicin twice a day for 5 days did not affect Ca in the mineral phase of the otoconia, but did increase K in both saccular (p < 0.05) and utricular (p < 0.01) otoconia. These increases in K may reflect a modification in the composition of the endolymph, resulting from cellular damage at the plasma membrane.
Abstract: Human natural killer cells and polymorphonuclear neutrophils constitutively express the low-affinity IgG Fc receptor (Fc gamma RIII, CD16 molecule). To investigate cell surface morphology, antigenic receptor density, and topographical distribution of Fc gamma RIII on the plasma membrane of natural killer cells and polymorphonuclear neutrophils, conventional scanning electron microscopy (SEM), flow cytometry, and immunoscanning electron microscopy were used. Fc gamma RIII was detected with an indirect immunogold labeling procedure, and receptors were visualized in the backscattered and secondary electron imaging mode of SEM. Natural killer cells showed a cell surface morphology compatible with lymphocytic differentiation characterized by microvilli and microridges. Polymorphonuclear neutrophils showed surface features characterized by ridges with folds and scattered short microvilli. Natural killer cells displayed a lower cell labeling density, whereas polymorphonuclear neutrophils showed a high level of expression of Fc gamma RIII on the plasma membrane by quantitative analysis with SEM in the backscattered electron imaging mode. Flow cytometry analysis confirmed these findings. Analysis of the topographical distribution of Fc gamma RIII antigenic receptor sites by SEM in the backscattered and secondary electron imaging modes showed that Fc gamma RIII on natural killer cells are randomly distributed, whereas Fc gamma RIII are located on ridges and folds of the plasma membrane of polymorphonuclear neutrophils. These observations suggest that natural killer cells and polymorphonuclear neutrophils differ in their levels of expression and topographic distribution of Fc gamma RIII on the plasma membrane. This different spatial distribution of Fc gamma RIII would provide morphological evidence of certain cellular functions mediated by natural killer cells and polymorphonuclear neutrophils.
Abstract: We used scanning electron microscopy to study the morphological surface patterns of cells that cover the attached gingiva and intervestibular papilla of the human oral gingival epithelium. Five patterns are described on the basis of the overall appearance of morphological surface markers: microvilli, parallel, fingerprint, reticular and pitted. Statistical analyses detected significant differences in the frequency of each pattern in both regions of the oral gingival epithelium, and showed the reticular and fingerprint types to predominate. We propose that our description of the different morphological surface types may be of use as a standard for subsequent cytological studies and characterizations of morphological alterations in diseased gingiva.
Abstract: Electron probe X-ray microanalysis was used to study the phosphorus concentration in the otolithic gelatinous membrane of the saccule and the utricle with scanning electron microscopy. The otolithic membranes were plunge-frozen in liquid N2 and freeze-dried. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background ratio method and different concentrations of KH2PO4 salts dissolved in dextran solutions. The otolithic gelatinous membrane consists of a 25-30 microns-thick layer overlying the cilia of the hair cells. Elements detected in the gelatinous membrane are: Na, P, S, Cl, K and Ca. Although Student's t-test did not show significant differences between saccular and utricular concentrations of phosphorus, the distribution of this element in the two organs was different. Regression analysis established that the concentrations of phosphorus in the saccular and utricular gelatinous membrane were dependent. The regression equation was: y = 18.02x2 + 133.9 (r = 0.83, P < 0.05) where y is the concentration of phosphorus in the utricle, and x2 the concentration of phosphorus in the saccule. The findings obtained in the present study could be related to structural differences in organic phosphate residues of the phosphoproteins associated to collagen, or to different polyphosphoinositide turnover rates in the cell membrane.
Abstract: Electron probe X-ray microanalysis was used to determine the concentrations of P, S and K (Cp, CS, CK) in the gelatinous membrane of the mouse utricle. The otolithic membranes were plunge-frozen in liquid N2, freeze-dried and carbon-coated. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background ratio method and different concentrations of KH2PO4 and K2SO4 salts dissolved in dextran solutions to calibrate the microprobe. P, S and K were measured and their concentrations plotted as bar graphs to study the frequency distributions. Regression analysis revealed a dependence between the concentrations of P and K (CK = 1454.10 - 2.83 CP, r = -0.68745, p < 0.05), and P and S (CS = 43.18 + 0.23 CP, r = 0.66949, p < 0.05); however, no correlation was found between CK and CP (r = -0.25424). The findings obtained in the present study show an inverse relationship between P and K ions, and direct relationship between P and S in the gelatinous membrane of the utricle.
Abstract: An absolute quantitative standardization technique has been developed to measure Ca and K weight fractions (WF) in the otolithic membrane of the saccule and utricle by scanning electron microscopy and electron probe X-ray analysis using the peak-to-background (P/B) ratio method. Microcrystalline salt standards were used to calibrate Ca and K K alpha P/B or Y = (P/B).Z2/A (Z = atomic number; A = atomic weight) against WF at 10, 15, 20 and 25 kV accelerating voltage. The effect of voltage on the calibration, plotting the coefficient of correlation (r) as a function of voltage, was not dependent on the voltage in the range 10-25 kV for Ca standards. K standards were also independent when P/B was corrected for Z2/A. Background counts in the otoconia (Bo) were obtained at 5, 25, 50, 100, 200 and 500 s and used to test the electron beam sensitivity of saccular and utricular otoconia. Bo was not dependent on the spectra acquisition time, with the exception of Bo under K alpha K peak in the saccule at 10 kV. Ca and K WF were determined at 10, 15, 20 and 25 kV in the saccule and utricle, showing similar values regardless of the voltage used. This method of calibration offers several advantages, such as stability, homogeneity, known composition of the standards, high reproducibility at different voltages even without Z2/A correction and the similarity between the otoconia and crystal standards. We recommend the application of this method for other elements and biomineral systems.
Abstract: The effect of tissue preparation on calcium and potassium weight percent in the otoconial layer in the utricle and saccule was studied in four groups of OF1 mice with electron probe X-ray microanalysis. Glutaraldehyde and freeze-drying, glutaraldehyde and air-drying, air-drying, and cryo-fixation and freeze-drying were compared. Ca and K changed significantly in the utricle depending on the method used (P < 0.001), and K changed significantly in the saccule (P < 0.001). We chose cryo-fixation with freeze-drying for the quantitative analysis of the otolithic membrane because this method provided the highest values of Ca and K with minimum loss of Ca and K. Microcrystalline salt standards mounted on scanning electron microscopy holders were used for the quantification of Ca and K by the peak-to-local-background (P/B) ratio method. The P/B ratio in standards with reproducible results, when plotted against weight percent, gave a straight line for Ca (r = 0.99, P < 0.001) and K (r = 0.98, P < 0.001). The Ca and K weight percent in otoconia showed similar frequency distributions in the utricle and saccule.
