Peter A. Santi

Morphological alteration of the stria vascularis after administration of the diuretic bumetanide.

Chinchillas were given either a single injection of the diuretic bumetanide (18 mg/kg body weight) or saline-sodium hydroxide and sacrificed at 10 min, 1 hr and 24 hr after the injection. Slight stria edema was present at 10 min, marked edema at 1 hr and no edema 24 h after bumetanide. The edema began in the first cochlear turn at 10 min and spread to the second turn by 1 hr. Along with edema, marginal cell bulging, potential capillary constriction and the formation of marginal cell membranous structures occurred after bumetanide treatment.

Light sheet fluorescence microscopy: A review

Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with subcellular resolution. This method is well suited for imaging deep within transparent tissues or within whole organisms, and because tissues are exposed to only a thin plane of light, specimen photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy. LSFMs produce well-registered serial sections that are suitable for three-dimensional reconstruction of tissue structures. Because of a lack of a commercial LSFM microscope, numerous versions of light sheet microscopes have been constructed by different investigators. This review describes development of the technology, reviews existing devices, provides details of one LSFM device, and shows examples of images and three-dimensional reconstructions of tissues that were produced by LSFM.

Thin-sheet laser imaging microscopy for optical sectioning of thick tissues

We report the development of a modular and optimized thin-sheet laser imaging microscope (TSLIM) for nondestructive optical sectioning of organisms and thick tissues such as the mouse cochlea, zebrafish brain/inner ear, and rat brain at a resolution that is comparable to wide-field fluorescence microscopy. TSLIM optically sections tissue using a thin sheet of light by inducing a plane of fluorescence in transparent or fixed and cleared tissues. Moving the specimen through the thinnest portion of the light sheet and stitching these image columns together results in optimal resolution and focus across the width of a large specimen. Dual light sheets and aberration-corrected objectives provide uniform section illumination and reduce absorption artifacts that are common in light-sheet microscopy. Construction details are provided for duplication of a TSLIM device by other investigators in order to encourage further use and development of this important technology.

A newly identified surface coat on cochlear hair cells

Routine electron microscope methods do not well preserve or stain the surface coat or glycocalyx on cochlear hair cells. In other tissues, enhanced preservation and staining of these glycoconjugates was obtained following fixation with glutaraldehyde containing a cationic dye (e.g., Alcian blue and ruthenium red). When cochleas were fixed with glutaraldehyde containing Alcian blue, the endolymphatic surface of hair cells, but not the supporting cells, displayed an extensive (approximately 90 nm thick) surface coat. Alcian blue positive material was also observed in the tectorial and basilar membranes and in a portion of the spiral ligament. In addition, acellular bands of Alcian blue positive material were observed between the tectorial membrane and the reticular lamina or inner sulcus cells. Although the function of these cochlear glycoconjugates is not yet known, it is proposed that they serve to attach the tectorial membrane to the organ of Corti, and they are involved in stereocilia fusion following sound exposure and ototoxic drug administration.

Conditional deletion of Atoh1 using Pax2-Cre results in viable mice without differentiated cochlear hair cells that have lost most of the organ of Corti.

Atonal homolog1 (Atoh1, formerly Math1) is a crucial bHLH transcription factor for inner ear hair cell differentiation. Its absence in embryos results in complete absence of mature hair cells at birth and its misexpression can generate extra hair cells. Thus Atoh1 may be both necessary and sufficient for hair cell differentiation in the ear. Atoh1 null mice die at birth and have some undifferentiated cells in sensory epithelia carrying Atoh1 markers. The fate of these undifferentiated cells in neonates is unknown due to lethality. We use Tg(Pax2-Cre) to delete floxed Atoh1 in the inner ear. This generates viable conditional knockout (CKO) mice for studying the postnatal development of the inner ear without differentiated hair cells. Using in situ hybridization we find that Tg(Pax2-Cre) recombines the floxed Atoh1 prior to detectable Atoh1 expression. Only the posterior canal crista has Atoh1 expressing hair cells due to incomplete recombination. Most of the organ of Corti cells are lost in CKO mice via late embryonic cell death. Marker genes indicate that the organ of Corti is reduced to two rows of cells wedged between flanking markers of the organ of Corti (Fgf10 and Bmp4). These two rows of cells (instead of five rows of supporting cells) are positive for Prox1 in neonates. By postnatal day 14 (P14), the remaining cells of the organ of Corti are transformed into a flat epithelium with no distinction of any specific cell type. However, some of the remaining organ of Corti cells express Myo7a at late postnatal stages and are innervated by remaining afferent fibers. Initial growth of afferents and efferents in embryos shows no difference between control mice and Tg(Pax2-Cre)::Atoh1 CKO mice. Most afferents and efferents are lost in the CKO mutant before birth, except for the apex and few fibers in the base. Afferents focus their projections on patches that express the prosensory specifying gene, Sox2. This pattern of innervation by sensory neurons is maintained at least until P14, but fibers target the few Myo7a positive cells found in later stages.

Cochlear Fluid Balance A Clinical/Research Overview

Stria edema, and in some cases atrophy, follows osmotic agents, loop-inhibiting diuretics, acoustic trauma, and rupture of Reissners membrane. All have in common an imbalance of fluid and electrolytes in the cochlear duct. The glycerol test causes temporary improvement in hearing in Menières disease. Glycerol causes stria edema and collapse of Reissners membrane in the chinchilla. Stria edema, as well as stria atrophy, are found in Menières disease. Metabolic manipulation of the stria might be the best approach in the search for successful treatment of Menières disease.

Conditional deletion of N-Myc disrupts neurosensory and non-sensory development of the ear.

Ear development requires interactions of transcription factors for proliferation and differentiation. The proto‐oncogene N‐Myc is a member of the Myc family that regulates proliferation. To investigate the function of N‐Myc, we conditionally knocked out N‐Myc in the ear using Tg(Pax2‐Cre) and Foxg1KiCre. N‐Myc CKOs had reduced growth of the ear, abnormal morphology including fused sensory epithelia, disrupted histology, and disorganized neuronal innervation. Using Thin‐Sheet Laser Imaging Microscopy (TSLIM), 3D reconstruction and quantification of the cochlea revealed a greater than 50% size reduction. Immunochemistry and in situ hybridization showed a gravistatic organ‐cochlear fusion and a “circularized” apex with no clear inner and outer hair cells. Furthermore, the abnormally developed cochlea had cross innervation from the vestibular ganglion near the basal tip. These findings are put in the context of the possible functional relationship of N‐Myc with a number of other cell proliferative and fate determining genes during ear development. Developmental Dynamics 240:1373–1390, 2011.

Ultrastructure and immunohistochemical identification of the extracellular matrix of the chinchilla cochlea.

The molecular composition and three-dimensional organization of the extracellular matrix (ECM) was studied by immunofluorescent microscopy, transmission and scanning electron microscopy in three connective tissue structures of the cochlea: the spiral limbus, basilar membrane and spiral ligament. Type II collagen, fibronectin, tenascin, chondroitin sulfate proteoglycans, alphav and beta1 integrins were immunolocalized in the ECM of these connective tissue structures. Electron micrographs showed a continuum of cross-striated collagen fibrils having a similar diameter and axial periodicity that spread from the spiral limbus via the basilar membrane and into the spiral ligament. Some of collagen fibrils were aggregated laterally into bundles. Bundle images, and their digital Fourier transformations, showed a major 67-nm axial D-repeat characteristic for collagen fibrils. Transmission electron microscopy showed numerous proteoglycans associated with the collagen fibrils. The spiral limbus, basilar membrane and spiral ligament demonstrated regional differences in molecular composition and structural organization of their ECM. The glycoproteins fibronectin, tenascin and alphav integrin were immunolocalized mainly in the basilar membrane. Collagen fibrils of the spiral limbus and spiral ligament did not appear to be strongly oriented. However, most of the collagen fibrils in the basilar membrane were arranged into radially directed bundles. Collagen fibrils in the basilar membrane were also surrounded by a homogeneous matrix, which was immunoreactive to fibronectin and tenascin antibodies. A more complete understanding of the composition and structural organization of the ECM in these connective tissue structures in the cochlea provides a foundation upon which micromechanical models of cochlear function can be constructed.

A microcomputer system for the control of behavioral experiments

A self-contained microcomputer control system (MCS) is described for controlling and recording events from behavioral experiments using a low-cost microcomputer, the Rockwell AIM 65, and the BASIC language. Features of the system include a printer, alphanumeric display, 4K RAM, 8K BASIC on read only memory (ROM), a cassette interface, a power supply, and a control interface, all contained in a compact enclosure. The control interface allows for 16 inputs and 16 outputs, and includes a real-time clock and versatile machine language subroutines for controlling and recording real-time events. These features make BASIC a suitable user language for this application. The use of this microcomputer control system for operant research in behavioral pharmacology is described.

The effect of bumetanide on the stria vascularis: A stereological analysis of cell volume density ☆

The purpose of this research was to determine the effect of bumetanide on the volume density (Vv) of the cells, capillaries and intercellular spaces of the stria vascularis (SV). 29 chinchillas were divided into seven groups. There were 3 experimental groups, three control groups and one normal, untreated, group of animals. After either a 20 mg/kg intravenous injection of bumetanide or an injection of a control solution, the animals were killed at 10 min, 1 h and 24 h. One complete radial section of the SV was analyzed in each animal. This section was located at 70% of the length of the basilar membrane as measured from the cochlear apex. Marginal cell volume decreased by 24% and 15% at 10 min and 1 h, respectively, after bumetanide administration. Intermediate cell volume increased by 31% and 27% at 10 min and 1 h, respectively, after bumetanide administration. Intercellular space volume increased by 14% and 21% at 10 min and 1 h, respectively, after bumetanide administration. No significant alteration in the Vv was observed in the strial capillaries or basal cells. A hypothetical model of the ion transporting properties of the SV is presented.