Hearing loss after exposure to simulated gunfire.

<p><i>A:</i> Three examples of CM responses obtained before, during, and immediately after noise exposure. <i>B:</i> CM magnitude as a function of sound level before, during, and after noise exposure. Note the magnitude is plotted on log scale. <i>C:</i> Tone pip-evoked ABR thresholds (mean ± SD, n = 19) for both ears before and seven days after noise exposure. No statistical difference (student's t-test) in thresholds at each frequency was found between left (dot lines) and right (solid lines) ears either before or after noise exposure (p>0.05).</p

Representative SEM pictures and ABR thresholds obtained from cochleae treated with the Atoh1 gene one month after noise deafening.

<p><i>A, B:</i> SEM images obtained from basal and middle turns. The reticular lamina was flat with no sign of stereocilia. Bars represent 10 µm. <i>C:</i> ABR thresholds (means ± SD, n = 4) measured from control and Atoh1-treated ears one month after the treatment.</p

ABR thresholds measured from Atoh1-treated and control ears.

<p><i>A:</i> ABR thresholds (means ± SD, n = 8) were measured at different days after the viral vector carrying the <i>Atoh1</i> gene was introduced. Contralateral ears were used as control. Click was used to evoke ABRs. <i>B:</i> ABR thresholds (means ± SD) of the Atoh1-treated ears (n = 8), contralateral ears (n = 8), and EGFP vector-treated ears (n = 6) at one month after cochlear inoculation. Tone pips with frequencies of 4, 8, 16, and 20 kHz were used to evoke ABRs.</p

Stereocilia bundle counts in the normal (without noise exposure), Atoh1-treated and untreated cochleae.

<p><i>A–B</i>: Examples of damaged bundles. <i>C</i>: Examples of normal-looking bundles. Scale bar represent 5 µm for all images in panels from <i>A</i> to <i>C</i>. <i>D, E</i>: Counts of IHC and OHC bundles in three different cochlear locations, each 1 mm in length. Three cochleae were used for each group (normal, Atoh1-treated, and untreated). Status of the bundles was determined by visual inspection of the bundles under scanning electron microscope. Hair bundles with no clear signs of trunction, fusion, or folding were included in the bundle count. Student's t-test was used for statistical analysis. Two asterisks represent statistical significance with a p-value less than 0.01.</p

Representative SEM pictures obtained from different cochlear locations from Atoh1-treated and untreated (control) ears one month after viral inoculation.

<p><i>A–C:</i> SEM images showing different extent and severity of damage from the control cochleae. <i>D–F:</i> SEM pictures of hair bundles from the Atoh1-treated ears. Missing hair bundles in panel <i>E</i> are marked by asterisks. The arrow indicates a Deiters' cell. Scale bars: 20 µm (<i>A</i>) and 10 µm (<i>B</i> to <i>F</i>).</p

Confocal images of the stereocilia and prestin immunoactivity, and the pattern of Atoh1-EGFP expression in the organ of Corti.

<p><i>A:</i> Stereocilia bundles in one cochlear location. The hair bundle was labeled with rhodamine-phalloidin (in red). The “V”-shaped OHC bundles were missing in the outmost (third) rows of OHCs. One bundleless OHC is indicated by an asterisk. One “V”-shaped hair bundle is marked by a yellow arrow. Prestin immunoactivity was labeled in green. Scale bar: 10 µm. <i>B:</i> Z-axis stack image of prestin immunoactivity at another cochlear location. Scale bar: 10 µm. All the images in <i>A</i> and <i>B</i> were obtained from cochleae 10 days after noise exposure. <i>C:</i> Confocal image of Atoh1-expression in a cochlea. <i>D:</i> Composite images of hair cells (labeled with myo7a antibody, in red) and EGFP-positive cells (from the same location as shown in panel <i>C</i>). The images were acquired seven days after Ad.<i>Atoh1-EGFP</i> inoculation in the noise-damaged cochlea (second turn). A magnified image of the area marked by white lines is presented in panel <i>E</i>. Scale bar: 20 µm for <i>C</i> and <i>D</i>. <i>E:</i> High magnification image of Atoh1 and myo7a expression in the organ of Corti. Scale bar: 10 µm. <i>F:</i> Confocal image obtained from optical sectioning from a basal turn location at 3 days after Atoh1 treatment. Hair cells were labeled with myo7a (in red) and the nuclei were labeled with DAPI. Most preparations examined at 3 days after transfection showed weak or no expression of EGFP in the organ of Corti. For those that expressed EGFP, the expression was in the area of Deiters' cells. <i>G, H:</i> Confocal image using optical sectioning from a basal turn location at 7 and 14 days after Atoh1 treatment. Scale bars (<i>F</i>,<i>G, H</i>): 10 µm.</p

Identification of Tricyclic Agonists of Sphingosine-1-phosphate Receptor 1 (S1P₁) Employing Ligand-Based Drug Design

Fingolimod (<b>1</b>) is the first approved oral therapy for the treatment of relapsing remitting multiple sclerosis. While the phosphorylated metabolite of fingolimod was found to be a nonselective S1P receptor agonist, agonism specifically of S1P₁ is responsible for the peripheral blood lymphopenia believed to be key to its efficacy. Identification of modulators that maintain activity on S1P₁ while sparing activity on other S1P receptors could offer equivalent efficacy with reduced liabilities. We disclose in this paper a ligand-based drug design approach that led to the discovery of a series of potent tricyclic agonists of S1P₁ with selectivity over S1P₃ and were efficacious in a pharmacodynamic model of suppression of circulating lymphocytes. Compound <b>10</b> had the desired pharmacokinetic (PK) and pharmacodynamic (PD) profile and demonstrated maximal efficacy when administered orally in a rat adjuvant arthritis model