Kasra Zarei

Automated Axon Counting in Rodent Optic Nerve Sections with AxonJ

We have developed a publicly available tool, AxonJ, which quantifies the axons in optic nerve sections of rodents stained with paraphenylenediamine (PPD). In this study, we compare AxonJ’s performance to human experts on 100x and 40x images of optic nerve sections obtained from multiple strains of mice, including mice with defects relevant to glaucoma. AxonJ produced reliable axon counts with high sensitivity of 0.959 and high precision of 0.907, high repeatability of 0.95 when compared to a gold-standard of manual assessments and high correlation of 0.882 to the glaucoma damage staging of a previously published dataset. AxonJ allows analyses that are quantitative, consistent, fully-automated, parameter-free, and rapid on whole optic nerve sections at 40x. As a freely available ImageJ plugin that requires no highly specialized equipment to utilize, AxonJ represents a powerful new community resource augmenting studies of the optic nerve using mice.

A method for detailed movement pattern analysis of tadpole startle response

Prolonged space flight, specifically microgravity, presents a problem for space exploration. Animal models with altered connections of the vestibular ear, and thus altered gravity sensation, would allow the examination of the effects of microgravity and how various countermeasures can establish normal function. We describe an experimental apparatus to monitor the effects of ear manipulations to generate asymmetric gravity input on the tadpole escape response. To perform the movement pattern analysis, an imaging apparatus was developed that uses a high-speed camera to obtain time-resolved, high-resolution images of tadpole movements. Movements were recorded in a temperature-controlled test chamber following mechanical stimulation with a solenoid actuator, to elicit a C-start response. Temperature within the test cell was controlled with a recirculating water bath. Xenopus laevis embryos were obtained using a standard fertilization technique. Tadpole response to a controlled perturbation was recorded in unprecedented detail and the approach was validated by describing the distinct differences in response between normal and one-eared tadpoles. The experimental apparatus and methods form an important element of a rigorous investigation into the response of the tadpole vestibular system to mechanical and biochemical manipulations, and can ultimately contribute to improved understanding of the effects of altered gravity perception on humans.

Sonic hedgehog antagonists reduce size and alter patterning of the frog inner ear

Sonic hedgehog (Shh) signaling plays a major role in vertebrate development, from regulation of proliferation to the patterning of various organs. In amniotes, Shh affects dorsoventral patterning in the inner ear but affects anteroposterior patterning in teleost ears. It remains unknown how altered function of Shh relates to morphogenetic changes that coincide with the evolution of limbs and novel auditory organs in the ear. In this study, we used the tetrapod, Xenopus laevis, to test how increasing concentrations of the Shh signal pathway antagonist, Vismodegib, affects ear development. Vismodegib treatment dose dependently alters the development of the ear, hypaxial muscle, and indirectly the Mauthner cell through its interaction with the inner ear afferents. Together, these phenotypes have an effect on escape response. The altered Mauthner cell likely contributes to the increased time to respond to a stimulus. In addition, the increased hypaxial muscle in the trunk likely contributes to the subtle change in animal C‐start flexion angle. In the ear, Vismodegib treatment results in decreasing segregation between the gravistatic sensory epithelia as the concentration of Vismodegib increases. Furthermore, at higher doses, there is a loss of the horizontal canal but no enantiomorphic transformation, as in bony fish lacking Shh. Like in amniotes, Shh signaling in frogs affects dorsoventral patterning in the ear, suggesting that auditory sensory evolution in sarcopterygians/tetrapods evolved with a shift of Shh function in axis specification.

An image processing framework for automated analysis of swimming behavior in tadpoles with vestibular alterations

Micogravity, as experienced during prolonged space flight, presents a problem for space exploration. Animal models, specifically tadpoles, with altered connections of the vestibular ear allow the examination of the effects of microgravity and can be quantitatively monitored through tadpole swimming behavior. We describe an image analysis framework for performing automated quantification of tadpole swimming behavior. Speckle reducing anisotropic diffusion is used to smooth tadpole image signals by diffusing noise while retaining edges. A narrow band level set approach is used for sharp tracking of the tadpole body. The use of level set method for interface tracking provides an inherent advantage of using level set based image segmentation algorithm (active contouring). Active contour segmentation is followed by two-dimensional skeletonization, which allows the automated quantification of tadpole deflection angles, and subsequently tadpole escape (or C-start) response times. Evaluation of the image analysis methodology was performed by comparing the automated quantifications of deflection angles to manual assessments (obtained using a standard grading scheme), and produced a high correlation (r2 = 0.99) indicating high reliability and accuracy of the proposed method. The methods presented form an important element of objective quantification of the escape response of the tadpole vestibular system to mechanical and biochemical manipulations, and can ultimately contribute to a better understanding of the effects of altered gravity perception on humans.

Corrigendum: Automated Axon Counting in Rodent Optic Nerve Sections with AxonJ

Scientific Reports 6: Article number: 26559; published online: 26 May 2016; updated: 19 October 2016 In the Supplementary Information file originally published with this Article, Supplemental Figure 1 was omitted. In addition, “Supplemental Software” was incorrectly given as “Software Legend” These errors have now been corrected in the Supplementary Information that now accompanies the Article.