Pulmonary delivery of d-methionine is associated with an increase in ALCAR and glutathione in cochlear fluids.

In animals, hearing loss resulting from cochlear mechanosensory cell damage can be mitigated by antioxidants such as d-methionine (d-met) and acetyl-l-carnitine (ALCAR). The systemic routes of administration of these compounds, that must of necessity transit trough the cochlear fluids, may affect the antioxidant levels in the cochlea and the resulting oto-protective effect. In this study, we analyzed the pharmacokinetics of [(14)C]d-met in the cochlea and four other tissues after intratracheal (IT), intranasal (IN), and oral by gavage (OG) administration and compared it to intravenous administration (IV). We then analyzed the effect of these four routes on the antioxidant content of the cochlear fluids after d-met or ALCAR administration, by liquid chromatography/mass spectrometry. Our results showed that the concentration of methionine and ALCAR in cochlear fluids significantly increased after their respective systemic administration. Interestingly, d-met administration also contributed to an increase of ALCAR. Our results also showed that the delivery routes differently affected the bioavailability of administered [(14)C]d-met as well as the concentrations of methionine, ALCAR and the ratio of oxidized to reduced glutathione. Overall, pulmonary delivery via IT administration achieved high concentrations of methionine, ALCAR, and oxidative-related metabolites in cochlear fluids, in some cases surpassing IV administration, while IN route appeared to be the least efficacious. To our knowledge, this is the first report of the direct measurements of antioxidant levels in cochlear fluids after their systemic administration. This report also demonstrates the validity of the pulmonary administration of antioxidants and highlights the different contributions of d-met and ALCAR allowing to further investigate their impact on oxidative stress in the cochlear microenvironment.

[Clinical and anatomical features of the middle ear in the premature infants at different gestational time].

The objective of the present study was to analyse anatomical features of the middle ear in the premature infants of different gestational age. Materials from 100 still-born and live-born babies (200 temporal bones) were available for the investigation. The study has revealed a number of distinctive clinical and morphological peculiarities in the structure of tympanic membranes in both the prematurely born infants depending on the gestational age and in the full-term babies. The fluid from the tympanic cavity was found to contain human beta-chorionic gonadotropin.

Magnetic resonance imaging features of large endolymphatic sac compartments: audiological and clinical correlates.

OBJECTIVES: (1) To study the prevalence and characteristics of large endolymphatic sac internal compartments on thin-section T2- and T2*-weighted magnetic resonance imaging, and to relate these to other large endolymphatic sac magnetic resonance imaging features, and (2) to correlate the compartment imaging features, endolymphatic sac size and labyrinthine anomalies with the patients' clinical and audiological data. METHOD: Magnetic resonance imaging studies for 38 patients with large endolymphatic sac anomalies were retrospectively reviewed in a tertiary referral centre. Endolymphatic sac compartment presence, morphology and imaging signal were assessed. Endolymphatic sac size and labyrinthine anomalies were also recorded. Endolymphatic sac compartments and other imaging features were correlated with clinical and audiological data. RESULTS: Compartments were present in 57 per cent of the imaged endolymphatic sacs, but their presence alone did not correlate with other imaging features or clinical data. The endolymphatic sac : internal auditory meatus signal ratio was associated with a history of sudden or fluctuating hearing loss. Hearing loss correlated with opercular and extraosseous endolymphatic sac size measurements. A larger midpoint intraosseous endolymphatic sac size was associated with clear fluid loss at cochlear implantation. CONCLUSION: The magnetic resonance imaging characteristics of large endolymphatic sac compartments have been defined. The endolymphatic sac size and distal compartment signal should be recorded, as these provide prognostic information and assist the planning of appropriate interventions.

Aquaporins and Meniere's disease.

PURPOSE OF REVIEW: Review of the role of aquaporins in inner ear homeostasis and potential role in the pathogenesis of Meniere's disease. RECENT FINDINGS: Recent findings include the immunolocalization of aquaporins in the inner ear of mouse, rat, and human to cell types that are likely to undergo high ionic perturbances (e.g. potassium flux) and to putative areas of endolymph resorption or cycling. SUMMARY: The expression of aquaporins and related proteins in the human cochlea and vestibular periphery resembles the distribution found in animal models, suggesting a critical role of aquaporins in inner ear water homeostasis and their potential role in the pathogenesis of Meniere's disease.

[Expression and its significance of aquaporins in normal guinea pig inner ears].

OBJECTIVE: To investigate the expression and its significance of aquaporins (AQPs) in normal guinea pig inner ears. METHOD: Ten healthy guinea pigs were used. Immunohistochemistry were employed to detect AQP0, 1, 2, 3, 5, 7, 8 in normal guinea pig inner ears,using rabbit anti-rat polyclonal antibodies. RESULT: AQP0, 1, 2, 3, 5, 7, 8 were all expressed in guinea pig inner ears. AQP0 was only located in stria vascularis and spiral ganglion. The distribution of AQP1 consisted of cells lining the bony labyrinth, fibrocytes lining the endolymphatic duct and sac, cells under the basilar membrane, fibrocytes of the spiral ligament and the spiral limbus,Corti's organ, inner and outer spiral sulcus, stria vascularis, saccular and utricular wall, and spiral ganglion. AQP2 located in stria vascularis, Corti's organ, spiral ganglion and endolymphatic sac. AQP3,7,8 distributed in a similar manner which was surrounding the membranous labyrinth, including Corti's organ, inner and outer spiral sulcus, stria vascularis, fibrocytes of the spiral ligament and the spiral limbus, saccular and utricular wall, endolymphatic sac and spiral ganglion. AQP5 located at Corti's organ, inner and outer spiral sulcus, spiral ganglion, fibrocytes in spiral ligament. CONCLUSION: Diverse AQPs are distributed in the normal guinea pig inner ears,which may co-work to maintain the homeostasis of the inner ear.

Aquaporin-mediated fluid regulation in the inner ear.

1. The sensory functions of the inner ear (hearing and balance) critically depend on the precise regulation of two fluid compartments of highly desparate ion composition, i.e., the endolymph and the perilymph. 2. The parameters volume, ion composition, and pH need to be held at homeostasis irrespective of the hydration status of the total organism. 3. Specific cellular water channels, aquaporins, have been shown to be essential for the fluid regulation of several organs, e.g., kidney, lung, and brain. 4. Because of functional similarities of water regulation in the kidney and inner ear this review initially summarizes some aquaporin functions in the kidney and then focuses on 6 out of 11 mammalian aquaporins that are present in the inner ear (AQP1-6). 5. Their potential role in the inner ear fluid control will be discussed on the basis of the respective expression patterns and individual pore properties. 6. Further, a working model is presented of how the endolymphatic sac may contribute to inner ear fluid regulation.

Expression of G protein alpha subunits in the lateral wall of the rat cochlea.

Expression of five G protein alpha subunits was investigated in the rat cochlea by reverse transcription-polymerase chain reaction (RT-PCR) in order to understand their role in the cochlear signal transduction mechanisms. Immunohistochemical techniques were employed to study their distribution in the lateral wall of the cochlea. Total RNA was extracted with guanidine thiocyanate from cochleas and brains of 14-21-day-old rats. The extract was treated with DNase to degrade genomic DNA. After RT, the resulting cDNA was amplified by PCR using primers specific for the nucleotide sequences representing alpha subunits of heterotrimeric G proteins. The results indicated that mRNA for all five alpha subunits was expressed in the brain and cochlear samples. For immunohistochemical localization, temporal bones of 6-week-old rats were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for embedding in paraffin wax. The dewaxed, midmodiolar sections of the cochlea were incubated with subunit-specific polyclonal antibodies. The pattern of immunoreactivity varied for the five G protein alpha subunits studied in the stria vascularis and spiral ligament. The significance of these findings and the role of G protein alpha subunits in cochlear fluid homeostasis are discussed.

An in vivo tracer study of noise-induced damage to the reticular lamina.

An in vivo tracer was used to determine if the reticular lamina and/or the cell membranes abutting the endolymphatic space are temporarily disrupted after intense noise exposure (4-kHz OBN, 108-dB SPL, 1.75 h). Using a double-barreled micropipette, the endolymphatic potential (EP) was recorded and artificial endolymph containing 10% carbon particles was injected into the endolymphatic space either 0 days or 28 days post-exposure. The cochleae were fixed 30-45 min post-injection, then dehydrated, embedded in plastic and dissected as flat preparations. Damage in the organ of Corti (OC) was quantified, the location of carbon was determined, and some OC segments were then sectioned radially. EP averaged 72+/-5 mV in five controls. These cochleae had carbon tracer in the endolymphatic space only. Four of five noise-exposed chinchillas examined 3-4 h post-exposure had a low EP (30+/-6 mV). The cochleae from these 0-day animals had several focal lesions in which nearly all outer hair cells had just degenerated. At these lesions, carbon was attached to cell membranes and debris between the reticular lamina and basilar membrane. By transmission electron microscopy, discontinuities were found in the apical membranes of sensory and supporting cells. Carbon particles were found in the cytoplasm of these cells. Four of five animals examined at 28 days had an average EP of 70+/-11 mV. The cochleae from these animals had multiple lesions in the basal turn, all of which were healed by phalangeal scars or squamous epithelial cells. In these cochleae, no carbon was found within the OC. Acute disruption of the reticular lamina and the apical membranes of sensory and supporting cells from noise appears to be a major mechanism to account for degeneration in the cochlea that spreads or continues for days to weeks post-exposure.

Corticosteroid pharmacokinetics in the inner ear fluids: an animal study followed by clinical application.

OBJECTIVE: Autoimmune disease (e.g., Cogan syndrome) and other inflammatory inner ear diseases may ravage the labyrinth if not treated aggressively with antiinflammatory medication. Corticosteroids are the mainstay of treatment, yet, partly because of the existence of the blood-labyrinthine barrier, the ideal drug, dose, and route of administration are currently unknown. STUDY DESIGN: In the present study, we established cochlear fluid pharmacokinetic profiles of hydrocortisone, methylprednisolone, and dexamethasone in the guinea pig following oral, intravenous, and topical (intratympanic) administration. High-performance liquid chromatography was used to determine the drug concentrations, and comparisons were made with simultaneous pharmacokinetic profiles from blood and cerebrospinal fluid. RESULTS: Our findings demonstrated a much higher penetration of all three drugs into the cochlear fluids following topical application as compared with systemic administration, with methylprednisolone showing the best profile. DISCUSSION: The results suggested that intratympanic administration of corticosteroids might be more efficacious while avoiding high blood levels and therefore the deleterious side effects of systemic use. CLINICAL APPLICATION: Thirty-seven patients with various inner ear disorders causing sensorineural hearing loss were subsequently treated using intratympanic corticosteroids, 20 with dexamethasone, and 17 with methlyprednisolone. Patients with immune-mediated hearing losses showed the best results, with notable improvement also seen in several cases of a "sudden deafness." No benefit was seen in patients with cochlear hydrops or those with sudden deterioration of a preexisting hearing loss. Three patients developed a transient otitis media related to the treatments, easily controlled with antibiotics. There were no cases of treatment-induced hearing loss and no permanent tympanic membrane perforations. CONCLUSIONS: Overall injection of intratympanic corticosteroids for the treatment of hearing loss in inner ear disorders appears to be both safe and highly effective for certain disorders. The concept of this technique is supported by animal experimental data. The findings from the present study warrant further clinical application and experimental investigation.

Salt-load electrocochleography.

OBJECTIVE: To introduce a new protocol for diagnostic electrocochleography using a pretest oral salt load to improve test sensitivity in patients with suspected inner ear fluid imbalance. STUDY DESIGN: A retrospective review of patients who reported vertigo that, by medical history, was suggestive of an inner ear fluid imbalance was preformed. The patients received a complete audiovestibular evaluation that included a baseline electrocochleogram. Despite the incapacitating nature of their vertigo, there were no symptoms or electrophysiologic abnormalities that would isolate an etiologic ear. After the baseline studies, the patients received 4 g of sodium chloride daily for 3 days before repeat electrocochleography. A control group of 13 healthy volunteers with normal baseline electrocochleography and pure tone audiometry was tested under similar conditions. SETTING: This study was conducted at an ambulatory care clinic associated with a tertiary referral medical center. INTERVENTION: Electrocochleography was performed using alternating polarity clicks presented at a rate of 9.7/sec at 95 dB nHL by an extratympanic TIPtrode electrode and recorded with a Nicolet Spirit (Nicolet Instrument Corp., Madison, WI, U.S.A.). Responses were averaged for 1000 sweeps using a 10-msec time base with bandpass filtering from 5 to 1500 Hz. A summating potential/action potential (SP/AP) ratio of 0.37 was considered the upper limit of normal. MAIN OUTCOME MEASURES: Enhancement in the SP/AP ratio from a normal baseline value to > 0.37 after oral salt loading was indicative of a positive test. RESULTS: None of the ears from control subjects had a positive salt load electrocochleogram, and one or both ears in 38% of the patients in the study group with normal baseline SP/AP ratios and symptoms of inner ear fluid imbalance converted to abnormal. The mean SP/AP ratio of the control group for the conditions before and after salt-load was not statistically different (p = 0.48), although the difference in the mean SP/AP ratio in the study group after salt loading was statistically significant (p = 1.329 x 10(-5)). CONCLUSIONS: A group of patients who reported vertigo with no localizing abnormalities had a statistically significant increase in the mean SP/AP ratio after ingestion of a large quantity of sodium chloride. A modest percentage had elevation of the SP/AP ratio above the upper limit of normal for our audiovestibular lab. The localization of a "salt-senstitive" ear could assist the clinician in the management of these difficult problems with long-term medical therapy or surgical treatment when alternative measures fail.

A new immunohistochemical method for the detection of gentamicin in inner ear fluid compartments.

A new method was developed for frozen section detection of antigens that natively occur in the cochlear peri- and endolymph. A combination of immuno-histochemistry and immunoblot assay enabled topological and quantitative detection of small and hydrophilic molecules (such as the aminoglycoside antibiotics) in frozen sections of the inner ear compartments (scala tympani, scala vestibuli and cochlear duct). A selective localization is possible in the peri- and endolymphatic region of each coil of the cochlea. During sectioning of the cochlea, a small piece of a nitrocellulose membrane is placed to the surface of the intersection and briefly warmed. The sections are cut, simultaneously attached to a nitrocellulose membrane on which the aminoglycoside antibiotics remain adsorbed without any fixation procedure. Using this method, immunoincubation to detect gentamicin was performed in a way usually done in western blot analysis. Results with two different enzyme reactions with the enzyme conjugated to a second antibody (i.e., dye as substrate and the chemiluminescence detection system) are presented and compared. This histoimmunoblot assay provides a general non-radioactive and sensitive immunohistochemical tool for the localization of compounds occurring in extracellular body fluid compartments. For inner ear research this method now enables the investigation of the penetration and distribution of therapeutics in peri- and endolymphatic sites and can even be applied to separately quantifying concentrations of a substance in different coils of the same cochlear section.

Accuracy of an automatic impedance apparatus in a population with secretory otitis media: principles in the evaluation of tympanometrical findings.

INTRODUCTION: The primary demand for a screening procedure for secretory otitis media (SOM) is to identify an objective test with sufficient sensitivity and specificity to meet the needs of the screening program. This study was designed to estimate the value of a portable automatic impedance apparatus to meet these needs. SUBJECTS AND METHODS: Two hundred twenty children with unilateral or bilateral SOM were evaluated in a double-blind, cross-sectional study. All have fulfilled two of the following three criteria for surgical intervention: (1) SOM by physical examination, (2) greater than 20 dB hearing impairment, and/or (3) adenoid symptoms. All ears were evaluated using tympanometry, following which myringotomy was performed. RESULTS: Otomicroscopy showed fluid behind the tympanic membrane in 393 ears. All underwent myringotomy. Thirteen percent (51 ears) were empty. Of the otomicroscopic findings consistent with SOM, 82% had B curves, 6% had C2 curves, 8% had C1 curves, and 4% had A curves. Both C2 and B curves were considered abnormal. Sensitivity of the apparatus is 94.4% and of the specificity is 52.9%. There was no statistical correlation between tympanometrical findings and fluid viscosity. RESULTS: A test intended for screening has to be simple, easy, and quick to perform. Furthermore, the test should have high sensitivity. The automatic impedance apparatus is able to meet these demands; however, the individual results have to be confirmed by another method, eg, otomicroscopy, pure-tone audiometry, and/or manual tympanometry.

Ionic activities of the inner ear fluid and ionic permeabilities of the cochlear duct in endolymphatic hydrops of the guinea pig.

Ionic activities (K+, Na+, and Cl-) of the perilymph and endolymph of the basal turn were measured using ion-selective microelectrodes in experimentally induced endolymphatic hydrops of the guinea pig. Three months following the obstruction of the endolymphatic duct and sac, the endocochlear potential (EP) of hydroptic ears was measured at 59.7 +/- 9.6 mV (N = 12) which was significantly lower than the EP of the contralateral control ears (84.4 +/- 2.8 mV, N = 12). A paired t-test (P greater than 0.05) showed no significant differences of ion concentrations of the inner ear fluid between the hydroptic and contralateral ears. Ion permeabilities of the cochlear duct following anoxia were calculated according to the Nernst-Planck equation. Comparing hydroptic and normal ears following anoxia, a statistically significant decrease was observed in the permeability coefficients for K+. Similarly, K+ conductance was significantly lower in the hydroptic ears than in the normal ears. Total conductance of the cochlear duct, defined as the sum of each ion conductance, was 0.560 siemens in the normal ears and 0.217 siemens in the hydroptic ears. On the basis of the Goldman-Hodgkin-Katz equation, preexisting negative EP in the normal state was calculated to be -24.5 mV in normal ears and -21.4 mV in hydroptic ears. Therefore, the positive component of the EP was 108.9 mV in normal ears and 81.1 mV in hydroptic ears. These findings suggest that the pathophysiology of hydrops involves changes in K+ permeability and the inhibition of the electrogenic transport processes.

Pathophysiology of inner ear fluid imbalance.

Maintenance of homeostasis of inner ear fluids and biochemical integrity of inner ear tissue are essential for proper functioning of the auditory and vestibular end organs. Although various regulatory mechanisms exist in a different portion of the labyrinth, the inner ear is known to respond to systemic challenges. The association of Meniere's syndrome with an imbalance of inner ear fluid homeostasis has been hypothesized for the past century. Among many factors, the effects of hormonal imbalance on inner ear fluid composition and inner ear function have however scarcely been studied. The purpose of this study was to explore the relationship between the autonomic nervous system and inner ear function and possible mechanisms of functional disturbances in an experimental condition. An infusion of supraphysiologic amounts of epinephrine, a stress related hormone, resulted in an elevation of osmolality in serum and perilymph. Furthermore, the infusion of epinephrine resulted in elevation of threshold, prolongation of latency, and depression of amplitude in the compound action potential of the auditory nerve. These findings were most marked at high frequencies. We hypothesized that the epinephrine-induced hearing loss was brought about by an increase in perilymphatic osmolality, as well as by the ionic imbalance caused by the osmotic gradient. Since emotional stress has been implicated as a mechanism of inducing a Meniere's attack, evaluation of the relationship between the autonomic system and cochlear function may contribute to the understanding of possible mechanisms of inner ear dysfunction caused by hormonal imbalances.

Concentration of salicylate in serum and perilymph of the chinchilla.

The concentration of salicylate in serum and perilymph was measured in chinchillas after intraperitoneal administration of sodium salicylate. Serum salicylate concentration peaked 2 to 4 hours after a single injection. Clearance was approximately complete after 16 hours. The within-subject variability of serum concentration 4 hours after injection was very low. Subject weight significantly affected serum salicylate concentration, with heavier animals showing higher serum levels. The relationship of perilymph to serum salicylate concentration was approximately linear, with a high correlation between measures.

[Results of trapezoid rotation test in guinea pigs with unilateral endolymphatic hydrops].

Obliteration of the right endolymphatic sac was performed by Kimura's method in 57 guinea pigs with normal hearing and vestibular function. However, 43 animals exhibited postoperative cerebellopontile disturbance and labyrinthitis detected by gait test, ABR test and histological examination. The remaining 14 guinea pigs were evaluated periodically by the trapezoid rotation test. Two weeks after the operation, 11 of these 14 guinea pigs exhibited prolonged right beating nystagmus based on the labyrinthine preponderance of impaired side (Lpi). In addition, endolymphatic hydrops was histological detected in the labyrinth on the operation side. A positive relation was observed between the degree of endolymphatic hydrops and the degree of Lpi. Lpi at 4 weeks after the operation was markedly lower than that at 2 weeks (P = 0.05). Therefore, the degree of Lpi was thought to be influenced by the rate of hydrops development. These results corresponded with the clinical findings that the degree of Lpi increases prior to vertiginous episodes in Meniere's patients.

Cerebrospinal fluid absorption in the rabbit. Inner ear pathways.

Fifteen adult rabbits were perfused intrathecally with horseradish peroxidase (HRP) for 20-30 min under conditions that prevented any increase in cerebrospinal fluid (CSF) pressure. Histologic and ultrastructural examination of the cochlea disclosed HRP deposits along the cochlear and vestibular branches of the auditory nerve and beyond their ganglia, in a) epineural and perineural spaces; b) intraneural spaces reaching the membrane of myelinated axons via nodes of Ranvier; and c) extending beyond the epineurium into area lymphatics. HRP was also found in the basilar membrane, along with deposits in the scalae tympani, vestibuli, media and the spiral ligament. The endolymph also received HRP which followed vestibular nerve fibers and penetrated between sustentacular and hair cells of the cristae ampullaris and both maculae. HRP permeated interendothelial spaces lining the modiolus to reach the scala vestibuli lymphatics close to all the above areas were also permeated by HRP, but the inner tunnel was devoid of the marker.

[Experimental endolymphatic hydrops and its related morphological and functional changes in guinea pigs].

The endolymphatic hydrops was induced successfully in 13 guinea pigs by blocking the endolymphatic duct and sac, and the morphological and functional alterations (especially on semicircular canal function) after hydrops were studied by means of ENG, EcochG and serial section techniques. Hydrops in the cochlear duct was evident in the apical and/or near apical turns, especially in earlier stages. Dilation of the saccule was seen in all samples, but was barely noticeable in the utricle and semicircular canal's endolymphatic space. Degeneration in the corti's apparatus, stria vasculi and spiral ganglion occurred late. No evident changes of the sensory components in the saccule, utricle and semicircular canals were seen under the light microscope. The hearing loss in earlier stages of hydrops was evident at lower frequencies: later stages showed effects at all frequencies. The cochlear recruitment and fluctuation phenomena were observed in a few of the operated animals. The value of -SP/CAP in a few animals was significantly after operation. The functional changes of semicircular canals were demonstrated by the semicircular paralysis (CP), directional preponderance (DP), vestibular recruitment (VR) and elevated threshold of nystagmus to angular acceleration stimuli. There was no significant correlation between the degrees of hearing loss and impaired semicircular function. The criteria of ENG elicited by the quantitative angular acceleration stimuli for evaluating the semicircular canal function in guinea pigs is proposed for the first time in our experiment.

Electrochemical aspects of cations in the cochlear hair cell of the chinchilla: a cellular model of the ion movement.

The dc potential and ion composition (K+, Na+, and Ca++) in the hair cells of the cochlea were examined using ion-selective microelectrodes. The K+, Na+, and Ca++ concentrations were 124.0 +/- 29.8 mM, 6.9 +/- 4.1 mM, and 1.7 +/- 1.4 microM in the hair cells, respectively. The electrochemical potential gradients for K+, Na+, and Ca++ across the apical membranes of the hair cells were calculated to be 160.0 +/- 29.8 mV, 87.6 +/- 27.0 mV, and 194.4 +/- 35.2 mV, respectively. Those for K+, Na+, and Ca++ across the basolateral membranes of the hair cells were -12.6 +/- 33.3 mV, 126.8 +/- 28.3 mV, and 170.8 +/- 30.1 mV, respectively. These findings were discussed in light of the transport mechanism necessary to maintain the ionic composition of hair cells.

Round window membrane and perilymph in experimental otitis media with effusion.

To investigate the influence of middle ear effusion (MEE) on perilymph (PL), an experimental otitis media with effusion (OME) was manufactured in chinchillas by injecting the tympanic cavity with immune complexes. The presence of MEE lasted for up to 9 days after the injection of immune complexes. Perilymph was aspirated on the fourth, tenth, and 21st days after the inoculation. The mean concentrations of albumin, immunoglobulin G, histamine, and prostaglandin E2 (PGE2) were significantly greater in PL from ears with induced OME than in that from normal control ears. The 3H-PGE2 placed on the round window membrane of pathologically affected ears passed into PL in significantly greater amounts than in normal control ears. The findings indicate that the immune complexes placed in the middle ear cavity affect the biochemical milieu of PL, and that MEE is a result of immune complexes.