Impacts of an urban flood control infrastructure on the limnology and ichthyofauna of a basaltic Cuesta stream (southeast Brazil).

Strategies for flood control associated to extreme precipitation events in urban areas are urgent, in order to prevent not only material damages but also to avoid human losses. The construction of flood contention reservoirs ("piscinões") has become a common engineering intervention in urban and peri-urban areas. However, there is a lack of studies focused on the evaluation of environmental impacts of this type of construction. This study intended to verify the ecological effects of a retention reservoir built directly on the course of the Cascata stream, Botucatu (SP). Three sampling sites were selected, located upstream the reservoir, in the reservoir and downstream. Samplings were carried out in July (winter - dry) and November (late spring - rainy) 2020. In situ measurements were obtained through a multiparameter probe (temperature, pH, electrical conductivity, dissolved oxygen, total dissolved solids, and oxidation-reduction potential) and water samples were collected for laboratory determinations (nitrogen, total phosphorus, thermotolerant coliforms, and chlorophyll-a). For fish sampling, manual trawls, sieves and hand nets were used, with a sampling effort of 10 throws per artefact and site. Despite the small distance between the sampling points (~1,300 m) considerable changes in the limnological conditions and fish community structure were observed. The studied environment is originally a small river surrounded by riparian forest, but this characteristic was abruptly changed in the reservoir stretch, with the direct exposition of a much larger water surface to intense solar radiation and atmosphere exchanges. Consequently, as evidenced by the PCA analysis, there was a considerable (stream-reservoir increase) of temperature, dissolved oxygen and chlorophyll. However, this spatial trend was partially disturbed by an accidental sewage-pipe rupture (posteriorly fixed) adjacent to the first sampling point, due to a previous event of extreme precipitation, which resulted in increased values of nutrients, chlorophyll, conductivity and thermotolerant coliforms. Eleven fish species were collected (two non-native), belonging to seven families and five orders. The upstream reference point (despite not be pristine), was characterized by the predominance of native species, while the reservoir condition favored the development of large populations of the non-native species. Despite the urgency of effective actions to prevent floods in urban areas, construction of contention reservoirs directly on stream courses should be avoided, due to their negative ecological impacts.

Allopurinol attenuates endolymphatic hydrops in the guinea pig cochlea.

Based on the hypothesis that Ca(2+) overload in the scala media may produce endolymphatic hydrops and generate free oxygen radicals (FOR), allopurinol, a xanthine oxidase inhibitor and free radical scavenger, was administered to guinea pigs after the surgical obliteration of the endolymphatic duct. Allopurinol was given intraperitoneally (50 mg/kg/day) for 15 days starting 1 day prior to the surgical blockage procedure. Measurements from histological serial sections of these temporal bones showed that the total volume of the scala media was significantly reduced (p = 0.007) compared with control hydropic ears. There was an indication of reduced incidence of atrophy in sensorineural structures and stria vascularis. These findings suggest that allopurinol may attenuate the development of endolymphatic hydrops and cell damage by preventing the formation of FOR or scavenging FOR. This study may lead to a new aspect of treatment for Menière's disease.

Volumetric and dimensional analysis of the guinea pig inner ear.

The objective of this study was to provide accurate volumetric data on the fluid spaces and soft tissue in the guinea pig inner ear by measuring all histologic serial sections by means of Metamorph Imaging Software at 400x to 1,000x magnification. The total endolymph volume of the inner ear was 4.691 mm3, of which 1.501 mm3 was in the cochlea, 3.090 mm3 in the vestibular labyrinth, and 0.100 mm3 in the endolymphatic duct and sac. The total perilymph volume was 15.938 mm3, of which 8.867 mm3 was in the cochlea and 7.071 mm3 in the vestibular labyrinth. The volume of the organ of Corti per millimeter length increased toward the apex, but the volumes of the stria vascularis, spiral ligament, and spiral limbus decreased. The volume of the macula utriculi was larger than that of the macula sacculi. The measurement of the luminal surface area of the stria vascularis was 3.944 mm2, and that of the vestibular dark cells was 5.772 mm2.

Gap junction systems in the mammalian cochlea.

Recent findings that a high proportion of non-syndromic hereditary sensorineural hearing loss is due to mutations in the gene for connexin 26 indicate the crucial role that the gene product plays for normal functioning of the cochlea. Excluding sensory cells, most cells in the cochlea are connected via gap junctions and these gap junctions appear to play critical roles in cochlear ion homeostasis. Connexin 26 occurs in gap junctions connecting all cell classes in the cochlea. There are two independent systems of cells, which are defined by interconnecting gap junctions. The first system, the epithelial cell gap junction system, is mainly composed of all organ of Corti supporting cells, and also includes interdental cells in the spiral limbus and root cells within the spiral ligament. The second system, the connective tissue cell gap junction system, consists of strial intermediate cells, strial basal cells, fibrocytes in the spiral ligament, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, mesenchymal dark cells in the supralimbal zone, and fibrocytes in the spiral limbus. One function of these gap junctional systems is the recirculation of K(+) ions from hair cells to the strial marginal cells. Interruption of this recirculation, which may be caused by the mutation in connexin 26 gene, would deprive the stria vascularis of K(+) and result in hearing loss.

Electron microscopic temporal bone histopathology in experimental pneumococcal meningitis.

Bacterial meningitis is one of the most common causes of acquired profound sensorineural deafness in children. Measurement of hearing and examination of the cochlea is limited in patients suffering from acute meningitis. A rabbit model of pneumococcal meningitis was developed to identify the temporal bone histopathologic changes that occur in meningogenic labyrinthitis caused by Streptococcus pneumoniae. Light microscopy was previously performed on temporal bones from acutely meningitic rabbits with profound hearing loss as determined electrophysiologically. Extensive inflammation of the cochlea with endolymphatic hydrops was observed. The organ of Corti, however, showed preserved architecture in the majority of these animals. In order to further investigate these findings, a protocol was used to create meningitic rabbits with hearing loss ranging from early high-frequency loss to profound deafness. The temporal bones from 7 rabbits were examined by transmission electron microscopy. In cases of mild hearing loss, partial degeneration of the inner row of outer hair cells, as well as edema of efferent cochlear nerve endings and marginal cells of the stria vascularis, was seen. With increasing degrees of hearing loss, the remainder of the organ of Corti and intermediate cells of the stria showed ultrastructural abnormalities. Spiral ganglion cells and basal cells of the stria vascularis remained intact in all subjects. This study provides unique information regarding the histology and pathophysiology of meningogenic deafness. The clinical implications of these findings are discussed, with an emphasis on potentially reversible changes and therapeutic intervention.

Vestibular responses of normal and hydropic ears of the guinea pig to middle ear pressure application.

The pressure in the middle ears of normal and hydropic guinea pig ears was increased, and nystagmographic recordings were compared. Two-month unilateral hydropic guinea pigs and normal control guinea pigs underwent pressure treatments in which pressure was introduced into the middle ear. Significantly lower pressure was needed to elicit nystagmus in hydropic ears (mean 1.00 psi or 70.3 cm H2O) compared with normal control ears (mean 1.27 psi or 89.3 cm H2O). All of the normal control guinea pigs showed fast phase nystagmus toward the pressure-applied side, while hydropic guinea pigs showed nystagmus toward the normal ear. The duration of nystagmus was slightly longer in hydropic animals than in normal control animals. The slow phase velocity was slightly higher in the hydropic guinea pigs. Histologic examination revealed that the vestibular sensory cells remained normal and that changes in the organ of Corti were similar between the hydropic ears with and without pressure treatment at equal survival times.

Morphological changes induced by administration of a Na+,K+-ATPase inhibitor in normal and hydropic inner ears of the guinea pig.

The objective of this study was to determine the effects of ouabain, a Na+,K+-ATPase inhibitor, in inner ears. Administering ouabain locally through the round window and vestibule, resulted in degenerative changes in cochlear and vestibular sensory cells and limbal fibrocytes, but the stria vascularis and spiral ligament were less affected. The position of Reissner's membrane was rarely changed. Vacuolar spaces in the sensory epithelia of cristae, maccula utriculi and macula sacculi increased in number. Nystagmus was a common occurrence with or without demonstrating degeneration of vestibular sensory cells. By administering ouabain systemically, the course of developing endolymphatic hydrops could not be altered in the ears with endolymphatic duct blockage. Edema of nerve endings of inner hair cells and vestibular sensory cells was frequently observed with administration of a high concentration of ouabain in both normal and hydropic ears, but edema was reversible. Degeneration of some vestibular sensory cells were observed in hydropic ears with a long survival time. The mechanism of selective sensitivity or non-sensitivity of inner ear tissues to ouabain is discussed.

Middle ear overpressure treatment of endolymphatic hydrops in guinea pigs.

Guinea pigs placed outside or inside a pressure chamber and exposed to 49.2 cm H2O overpressure continuously for 24 h immediately after blockage of the endolymphatic duct showed no significant difference in the magnitude of endolymphatic hydrops when compared to controls, although there was a slight indication of a volume decrease in the outside-treatment group and an increase in the inside-treatment group. A pressure increase of 49.2 cm H2O in the external auditory canal for 1 h twice a day for 2 weeks outside the chamber significantly inhibited the development of hydrops. The latter result supports the merit of pressure application through the external auditory canal as a treatment for Meniere's disease.

Inhibition of experimentally induced endolymphatic hydrops by middle ear ventilation.

The tympanic membrane was perforated (n = 19) or a tube was inserted into the middle ear through the wall of the tympanic bulla (n = 6) immediately after blockage of the endolymphatic duct in guinea pigs. Total cochlear endolymph volume and volumes in each cochlear turn were determined from serial sections of the temporal bones and volume changes were analyzed statistically by comparison with hydropic controls without treatment (n = 12). Results showed that both middle ear ventilation procedures significantly reduced the subsequent development of endolymphatic hydrops. This inhibition of hydrops was presumed to be due to pressure release into the middle ear and/or improved oxygenation of the middle and inner ears. Findings suggest the possible merit of a tympanostomy as a treatment for Meniere's disease in carefully selected patients refractory to medical management.

Vein of the vestibular aqueduct in the gerbil.

The vein of the vestibular aqueduct in the gerbil runs along the lateral side of the endolymphatic duct, deviates from this course to enter the middle ear cavity, and connects with the lateral sinus. In 20 animals, the vein of the vestibular aqueduct was obliterated by drilling from the middle ear side. Histopathology of these specimens after 2 months' survival revealed consistent sensory cell atrophy in the posterior canal cristae, frequent loss of sensory cells in a small superior portion of the macula sacculi and in the basal end of the cochlea, and fibrosis and osteogenesis in the three semicircular canals. The endolymphatic sacs contained colloidal substances in some specimens which were otherwise normal. Endolymphatic hydrops was absent except in some specimens which showed additional surgical damage to the endolymphatic sac and canals. The blocked vein re-opened occasionally and connected with the vessel formed in new bone from which it attached to the lateral sinus. The sensory cell degeneration and canal fibrosis reflects the pattern of blood drainage by the vein of the vestibular aqueduct. Vascular disorder in the vestibular labyrinth initiates vestibular symptoms; however, it will not produce endolymphatic hydrops unless function of the endolymphatic sac is impaired.

Comparison of endolymph cross-sectional area measured histologically with that measured in vivo with an ionic volume marker.

In order to establish how endolymph volume is regulated, it is essential to be able to measure endolymph volume or cross-sectional area in vivo. We have developed methods to accomplish this by injecting the volume marker ion hexafluoroarsenate (AsF6) into endolymph by iontophoresis. For an injection at a constant rate, the endolymph concentration is inversely dependent on the cross-sectional area of the scala into which injection occurred. Marker concentrations were monitored by inserting ion-selective microelectrodes into endolymph near the injection site. In a previous study we quantified the degree of hydrops in animals following ablation of the endolymphatic sac. In the present study we validated the technique by comparing the endolymphatic cross-sectional area measured in vivo with AsF6 with that measured by established histologic procedures. The correlation between the two measures was good, with a coefficient of .903, although the area measured histologically was a little lower than that measured in vivo.

Gap junctions in the rat cochlea: immunohistochemical and ultrastructural analysis.

Gap junctions in the rat cochlea were investigated using immunostaining for connexin26 and transmission electron microscopy. Electron microscopy of normal and pre-embedded immunostained material showed that there were gap junctions between and among all cells that light microscopy showed to have immunostained appositions. Light microscopy showed immunostaining between and among cell types that electron microscopy showed to be joined by gap junctions. Immunostaining for connexin26 was therefore taken as providing a reasonable approximation of the locations of gap junctions throughout the cochlea and was used to provide an overview of the extent of those locations. Cells interconnected via gap junctions fell into one of two groups. The first group consists of nonsensory epithelial cells and includes interdental cells of the spiral limbus, inner sulcus cells, organ of Corti supporting cells, outer sulcus cells, and cells within the root processes of the spiral ligament. The second group consists of connective tissue cells and includes various fibrocyte types of the spiral limbus and spiral ligament, basal and intermediate cells of the stria vascularis, and mesenchymal cells which line the scala vestibuli. The present work represents a first attempt towards a description of how serial gap junctions among cochlear cells reflect a level of organization of the tissue. The organization described here, together with a great deal of information from previous investigators, suggest that serially arranged gap junctions of both epithelial and connective tissue cells serve as the structural basis for recycling endolymphatic potassium ions that pass through the sensory cells during the transduction process.

Degeneration of vestibular sensory cells caused by ablation of the vestibular aqueduct in the gerbil ear.

The vestibular aqueduct of the gerbil has a unique anatomic feature that makes it possible to selectively obliterate the endolymphatic sac with or without interfering with its venous drainage. In animals in which only the endolymphatic sac was ablated, endolymphatic hydrops was slight in the cochlea and was absent in the vestibular labyrinth. The cochlear and vestibular sensory cells were normal. In animals in which both the endolymphatic duct and the vein were obliterated, hydrops was slight, with the exception of a few cochleas that showed moderate hydrops. The sensory cells of the posterior canal cristae had degenerated in all specimens, while varied pathologic changes in cochlear and vestibular sensory cells were present in some specimens. These results suggest that hydrops is primarily due to blockage of the endolymphatic duct and sac and that degeneration of sensory cells occurs when blood flow in the vestibular aqueduct is impeded. Pathologic changes in the endolymphatic sac, including the vascular plexus at the endolymphatic sac, may play an important role in the production of endolymphatic hydrops and vestibular symptoms in Meniere's disease.

Gap junction systems in the rat vestibular labyrinth: immunohistochemical and ultrastructural analysis.

The distribution of gap junctions within the vestibular labyrinth was investigated using immunohistochemistry and transmission electron microscopy. Connexin26-like immunoreactivity was observed among supporting cells in each vestibular sensory epithelium. Reaction product was also present in the transitional epithelium of each vestibular endorgan and in the planum semilunatum of crista ampullaris. No connexin26-like immunoreactivity was observed among thin wall epithelial cells or among vestibular dark cells. In addition, fibrocytes within vestibular connective tissue were positively immunostained. Reaction product was also detected in the melanocyte area just beneath dark cells. Ultrastructural observations indicated that a gap junction network of vestibular supporting cells extends to the transitional epithelium and planum semilunatum and forms an isolated epithelial cell gap junction system in each vestibular endorgan. In contrast, no gap junctions were found among wall epithelial cells or among dark cells. Fibrocytes and melanocytes were coupled by gap junctions and belong to the connective tissue cell gap junction system, which is continuous throughout the vestibular system and the cochlea. The possible functional significance of these gap junction systems is discussed.

Changes in immunostaining of cochleas with experimentally induced endolymphatic hydrops.

Cochleas with experimentally induced endolymphatic hydrops were immunostained for Na+,K(+)-ATPase, intracellular Ca(++)-ATPase, carbonic anhydrase, aldehyde dehydrogenase, calcium-binding proteins, vimentin, and the gap junction protein, connexin 26. No changes in immunostaining of hydropic ears were observed 1 week after blockage of the endolymphatic duct. Two weeks to 1 month after the operation, immunostaining of type I fibrocytes in the spiral ligament, which are positive for all but Na+,K(+)-ATPase, was slightly decreased on the operated side. These changes became more pronounced 3 months after the operation. However, staining for Na+,K(+)-ATPase of the stria vascularis and of type II fibrocytes of the spiral ligament was not reduced until 6 months postoperative. The reduction of enzymes and other cell constituents that may be involved in ion balance of cochlear fluids indicates that cells in the spiral ligament play an important role in cochlear homeostasis and that they merit further study in animal and human otopathology.

Ultrastructural changes of the vestibular sensory organs after streptomycin application on the lateral canal.

Early changes in the vestibular sense organs resulting from the application of a streptomycin sulfate soaked Gelfoam pledget on the fenestra of the lateral semicircular canal were studied by transmission and scanning electron microscopy. Three days after the application, lesions were present in the central part of the lateral crista. The type I sensory cells were more affected than the type II cells. These sensory cells showed mitochondrial swelling, cytoplasm protrusion at the cell apex, inclusion of multiple vacuoles, fusion or loss of stereocilia, and pyknotic nuclei. Seven days after the drug application, the sensory cell damage extended to all three cristae and macula utriculi. The lesions were very extensive after ten days and the sensory cells had almost equally disappeared in all three cristae; the lesion in the macula utriculi was smaller and the macula sacculi was unaffected. At fourteen days, the lesions appeared less severe. Thus, a single application of a small amount of streptomycin on the lateral canal fenestra affected all vestibular sense organs, except the saccule, in a short time. The strong affinity of aminoglycosides for the cristae suggests possible entrapment of the drug at the ampullae. This local drug application technique to the canal will be useful in studying vestibular function in animals, and it is applicable to controlling severe vestibular symptoms in human patients.

Immunolocalization of Na+, K(+)-ATPase, Ca(++)-ATPase, calcium-binding proteins, and carbonic anhydrase in the guinea pig inner ear.

The distribution of Na+, K(+)-ATPase, Ca(++)-ATPase, carbonic anhydrase, and calcium-binding proteins were investigated immunohistochemically in paraffin sections of guinea pig inner ears. Marginal cells of the stria vascularis, type II fibrocytes of the spiral ligament, and cells in supralimbal and suprastrial regions, were positive for Na+, K(+)-ATPase. Type I fibrocytes of the spiral ligament were positive for Ca(++)-ATPase, carbonic anhydrase, calmodulin and osteopontin. In the vestibular system, dark cells were positive for Na+, K(+)-ATPase. However, these cells and subepithelial fibrocytes were negative for Ca(++)-ATPase, carbonic anhydrase, and the calcium-binding proteins. In the endolymphatic sac, epithelial cells in intermediate and distal portions were positive for Na+, K(+)-ATPase, but the reaction was less than that in the stria. The same endolymphatic sac cells that were positive for Na+, K(+)-ATPase were also positive for Ca(++)-ATPase and calcium-binding proteins, but negative for carbonic anhydrase. The presence of Ca(++)-ATPase and calcium-binding proteins in the type I fibrocytes of the spiral ligament suggests that these cells are involved in mediating Ca++ regulation. Lower levels of Na+, K(+)-ATPase and the co-existence of Ca(++)-ATPase and calcium-binding proteins in the epithelial cells of the endolymphatic sac indicate that these cells have a distinctive role in ion transport that is different from that of the cells of the stria vascularis and vestibular dark cells.

Morphology of the membrana limitans.

The morphology of the membrana limitans (ML) was studied by light and electron microscopy to further understand its functional significance. The ML is a porous shield composed of a few to several layers of tightly packed fibrocytes that separate the perilymphatic space of the cochlea and saccule from that of the utricle and canal system. These fibrocytes are thinner on the side of the vestibule. They show many desmosomes and gap junctions and are interspersed with fibrils. The 3-dimensional reconstruction from celloidin sections shows that the ML, as it is viewed from the anterior side, is rectangular in shape, with various surface curvatures and an invagination toward the internal aperture of the vestibular aqueduct. Horseradish peroxidase, a tracer substance, placed on either side of the ML fails to pass to the opposite side. The ML and trabecular meshwork may be a factor contributing to differential sensitivity of cochlear and vestibular sensory cells in the presence of noxious substances.

Ischemia of the endolymphatic sac.

A decrease in vascular density in the endolymphatic sac was suspected as a factor in the pathogenesis of endolymphatic hydrops in Meniere's disease. The present study was undertaken to explore this possibility by cutting the posterior meningeal artery and the sigmoid sinus above and below the external aperture of the vestibular aqueduct or by incision of the dura adjacent to the sinus in 18 guinea pigs. The lesions in the sac were greater in the segmental ablation of the artery and sinus and were consistently associated with the development of endolymphatic hydrops. Among the lesions shown in the sac epithelia, the intermediate portion was most often and most severely affected with a decrease in rugose formation and a flattening of the tall epithelial cells or replacement of epithelial cells by squamous type cells. A high correlation between the lesions in the intermediate portion and occurrence of hydrops suggests that the intermediate portion plays a greater role in the pathogenesis of endolymphatic hydrops. The sac luminal precipitates known to be increased in human Meniere's cases were decreased or absent in this study, which suggests that the increased amount is unlikely to be the cause of endolymphatic hydrops. The evidence supports the hypothesis that these substances are secreted by the endolymphatic sac. The limited sensory cell lesions seen in the cochleae and saccules are likely to be due to a temporary vascular ischemia and endolymphatic hydrops.

Effect of ototoxic drug administration to the endolymphatic sac.

Aminoglycosides were applied to the endolymphatic sac of guinea pigs, and the inner ears were studied by light microscopy. In group 1 gentamicin sulfate application on the lateral surface of the endolymphatic sac produced sensory cell atrophy limited to the macula sacculi. In group 2 gentamicin injection into the sac by extradural and intradural approaches often showed lesions in the maculae sacculi and to the sac from a lateral opening resulted in a similar pattern of degeneration but with less severity in comparison to the injection method. Mild to severe endolymphatic hydrops was sometimes observed. The study shows diffusion of drugs taking place against the longitudinal flow of endolymph. This procedure may be applicable for the treatment of Meniere's disease.