Effects of Schisandrin B on Rod Photoreceptors in the pde6c Larval Retina

Current Rapid prototyping tools although reduce development time, they still have several restrictions such as print volume and print times. Through this thesis, two prototyping kits are proposed, a rod connector based, user controlled computational support system that re-purposes surface models and transforms them into structurally scaffolded constructs with movable joints; and a planar connector based system that implements movable joints to create functional prototypes. The rod based system enables users to interactively personalize scaffolded structures by re-purposing existing surface mesh models; analyze scaffolded constructs in-situ for better structurality, add movable joints to increase functionality, and attach personalized appearances to the scaffolding for increased customization.vThe planar connector system uses movable and electronic embedded connectors to functionalize planar surfaces to create multi-fidelity prototypes capable of interacting with the environment

3D In Vitro Neuron on a Chip for Probing Calcium Mechanostimulation

The evolution of tissue on a chip systems holds promise for mimicking the response of biological functionality of physiological systems. One important direction for tissue on a chip approaches are neuron‐based systems that could mimic neurological responses and lessen the need for in vivo experimentation. For neural research, more attention has been devoted recently to understanding mechanics due to issues in areas such as traumatic brain injury (TBI) and pain, among others. To begin to address these areas, a 3D Nerve Integrated Tissue on a Chip (NITC) approach combined with a Mechanical Excitation Testbed (MET) System is developed to impose external mechanical stimulation toward more realistic physiological environments. PC12 cells differentiated with nerve growth factor, which were cultured in a controlled 3D scaffolds, are used. The cells are labeled in a 3D NITC system with Fluo‐4‐AM to examine their calcium response under mechanical stimulation synchronized with image capture. Understanding the neural responses to mechanical stimulation beyond 2D systems is very important for neurological studies and future personalized strategies. This work will have implications in a diversity of areas including tissue‐on‐a‐chip systems, biomaterials, and neuromechanics.Developing more physiologically relevant 3D neuron integrated systems with external mechanics with synchronized image capture enables understanding of cellular responses that are important for advancing future neural studies and personalized strategies. This work examines calcium response with respect to voice coil actuator controlled force profiles.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163437/3/adbi202000080-sup-0001-SuppMat.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163437/2/adbi202000080_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163437/1/adbi202000080.pd

Utilizing Zebrafish Visual Behaviors in Drug Screening for Retinal Degeneration

Zebrafish are a popular vertebrate model in drug discovery. They produce a large number of small and rapidly-developing embryos. These embryos display rich visual-behaviors that can be used to screen drugs for treating retinal degeneration (RD). RD comprises blinding diseases such as Retinitis Pigmentosa, which affects 1 in 4000 people. This disease has no definitive cure, emphasizing an urgency to identify new drugs. In this review, we will discuss advantages, challenges, and research developments in using zebrafish behaviors to screen drugs in vivo. We will specifically discuss a visual-motor response that can potentially expedite discovery of new RD drugs

Normalization of large-scale behavioural data collected from zebrafish

Many contemporary neuroscience experiments utilize high-throughput approaches to simultaneously collect behavioural data from many animals. The resulting data are often complex in structure and are subjected to systematic biases, which require new approaches for analysis and normalization. This study addressed the normalization need by establishing an approach based on linear-regression modeling. The model was established using a dataset of visual motor response (VMR) obtained from several strains of wild-type (WT) zebrafish collected at multiple stages of development. The VMR is a locomotor response triggered by drastic light change, and is commonly measured repeatedly from multiple larvae arrayed in 96-well plates. This assay is subjected to several systematic variations. For example, the light emitted by the machine varies slightly from well to well. In addition to the light-intensity variation, biological replication also created batch-batch variation. These systematic variations may result in differences in the VMR and must be normalized. Our normalization approach explicitly modeled the effect of these systematic variations on VMR. It also normalized the activity profiles of different conditions to a common baseline. Our approach is versatile, as it can incorporate different normalization needs as separate factors. The versatility was demonstrated by an integrated normalization of three factors: light-intensity variation, batch-batch variation and baseline. After normalization, new biological insights were revealed from the data. For example, we found larvae of TL strain at 6 days post-fertilization (dpf) responded to light onset much stronger than the 9-dpf larvae, whereas previous analysis without normalization shows that their responses were relatively comparable. By removing systematic variations, our model-based normalization can facilitate downstream statistical comparisons and aid detecting true biological differences in high-throughput studies of neurobehaviour

Statistical Analysis of Zebrafish Locomotor Response

Zebrafish larvae display rich locomotor behaviour upon external stimulation. The movement can be simultaneously tracked from many larvae arranged in multi-well plates. The resulting time-series locomotor data have been used to reveal new insights into neurobiology and pharmacology. However, the data are of large scale, and the corresponding locomotor behavior is affected by multiple factors. These issues pose a statistical challenge for comparing larval activities. To address this gap, this study has analyzed a visually-driven locomotor behaviour named the visual motor response (VMR) by the Hotelling's T-squared test. This test is congruent with comparing locomotor profiles from a time period. Different wild-type (WT) strains were compared using the test, which shows that they responded differently to light change at different developmental stages. The performance of this test was evaluated by a power analysis, which shows that the test was sensitive for detecting differences between experimental groups with sample numbers that were commonly used in various studies. In addition, this study investigated the effects of various factors that might affect the VMR by multivariate analysis of variance (MANOVA). The results indicate that the larval activity was generally affected by stage, light stimulus, their interaction, and location in the plate. Nonetheless, different factors affected larval activity differently over time, as indicated by a dynamical analysis of the activity at each second. Intriguingly, this analysis also shows that biological and technical repeats had negligible effect on larval activity. This finding is consistent with that from the Hotelling's T-squared test, and suggests that experimental repeats can be combined to enhance statistical power. Together, these investigations have established a statistical framework for analyzing VMR data, a framework that should be generally applicable to other locomotor data with similar structure

Effects of Schisandrin B on Rod Photoreceptors in the pde6c Larval Retina

Degeneration of photoreceptors (PRs) in the retina is a common cellular consequence in inherited retinal degenerative diseases. In human patients and animal models, the loss of PRs expedites loss of vision. Some of the causal mutations for PR degeneration, result in the death of only one PR type leaving the other functionally intact; whereas some mutations result in death of both PR types. As such there is no definitive chemical therapy to preserve the function of PRs or to prevent them from dying in such degenerative conditions. Our lab previously showed that an active compound extracted from the five-flavored fruit (Fructus Schisandrae) called Schisandrin B (SchB) improves light sensation in the pde6cw59 zebrafish mutant model, as assessed by Visual Motor Response (VMR) assay. We also showed that the SchB treatment reduces the size of the morphologically abnormal rods usually observed in pde6cw59 larval retina, whereas no significant difference was found on cones. The pde6cw59 mutants harbor a mutation in a cone specific phosphodiesterase enzyme subunit gene which causes the cones to degenerate. Additionally, due to unknown reasons, the rods also degenerate non-autonomously thereby causing the pde6c w59 to lose vision starting 4 days post fertilisation (dpf). Thus, the pde6cw59 mutants provide a unique model for studying PRs degeneration. Based on these results, I hypothesized that SchB improves light sensation in dystrophic rods thereby enhancing VMR in pde6cw59 larvae. Moreover, I hypothesized that SchB reduces size of dystrophic rods thereby reducing apoptosis in larval pde6cw59. Thus, the overarching goal of my thesis was to build a mechanistic framework behind action of SchB treatment in pde6cw59 mutants. In order to examine the cellular changes caused by SchB treatment in rods of pde6c w59, I first sought to establish that rods are apoptotic in larval pde6cw59 and then test if SchB could reduce this apoptosis of rods in larval pde6cw59. Consistent with previous reports, I observed increased overall TUNEL staining in pde6cw59 larvae compared with WT between 4 dpf and 5 dpf. However, I found the higher apoptotic signals were observed in the Outer Nuclear Layer (ONL) and Inner Nuclear Layer (INL), indicating that photoreceptors and other second order neurons in INL (but not ganglion cells) were affected in these mutants. Using a transgenic reporter that expresses GFP in rods bred into the pde6cw59 mutants, I observed co-localisation of apoptotic signals with rods. This showed us that rods are indeed apoptotic in the pde6cw59 larvae. Having established that rods are apoptotic in pde6cw59 larval retinas, I sought to determine if treatment with SchB could reduce or prevent this. To test this, I subjected the pde6cw59 mutants to previously established concentrations of SchB from 3 dpf to 6 dpf and assayed apoptosis on 6 dpf. I observed that the SchB treatment seemed to cause a reduction in number of apoptotic rods in 6 dpf pde6c w59 larval retinas compared to untreated counterparts. In order to examine effects of SchB on rods directly, using visual behavior, I first sought to define conditions that stimulate only rods to drive VMR and then to use these conditions to directly test the effect of SchB in pde6c w59 larvae. By using light stimuli with different irradiance together with nof/gnat2w21 mutants (that lack functional cones) I defined the irradiance that could stimulate only the rods to drive the VMR. I reasoned that since nof/gnat2w21 mutants lack cone function, only rods could be stimulated to drive the VMR. As expected, I observed the light-on VMR of WT and the nof/gnat2 w21 mutant to be identical using a very low light stimulus. This systematic approach allowed me to define the conditions required to drive scotopic light-on VMR. Following this, the light intensity to drive the scotopic VMR was used to re-evaluate the VMR of untreated and SchB treated pde6c w59 mutants. I observed that SchB treatment caused an enhanced light-on scotopic VMR in SchB treated pde6cw59 but not in the untreated mutants. Taking together my results and observations, I show that SchB does exert a preserving effect on rods in the pde6c w59 mutants. Furthermore, I propose that SchB treatment reduces apoptotic rods in these mutants. To summarize, I propose that SchB treatment could induce enhanced phagocytosis of apoptotic debris in the pde6cw59 mutants which in turn causes an improvement in their vision as assayed by the VMR. Thus, my thesis study provides a foundation for a mechanistic framework of action of SchB in pde6c w59 larvae

Zebrafish Locomotor Response

The VMR assay was implemented based on the design by Emran and colleagues as described below. The assay was conducted inside the ZebraBox system (ViewPoint Life Sciences, Lyon, France). In the system, the 96-well plate with the animals was isolated from environmental light, and stimulated by white light emitted by a light-controlling unit from the bottom of the plate. The animal movement was recorded by an infra-red camera at a rate of 30 frames per second under infra-red light illumination at 850 nm, which the animals could not perceive. Before the actual experiment, the 96-well plate with the animals was placed in the ZebraBox system for 3.5 hours of dark adaption to acclimatize the animals. The data collection was started at 0.5 hours before the first light onset. The actual test consisted of three consecutive trials of light onset (Light-On) and light offset (Light-Off) periods with each period lasted for 30 minutes. The light change (On or Off) was abrupt and was not fading. The Light-On stimulus was set at 100% of the output intensity, which was measured by a LX1010B light meter (Mastech, Taipei, Taiwan). The measurements were taken at nine evenly distributed locations across the surface of the light-controlling unit that would be covered by the 96-well plate. The mean illuminance of these nine locations was 1390.94 Lux and the standard deviation of the measurement was 155.05 Lux. Using this experimental scheme, the VMR was measured from the three WT strains (AB, TL, TLAB). For each strain, two biological repeats were conducted. Each repeat was started with 96 embryos in the 96-well plate from 3 dpf to 9 dpf

A Naturally-Derived Compound Schisandrin B Enhanced Light Sensation in the pde6c Zebrafish Model of Retinal Degeneration.

Retinal degeneration is often progressive. This feature has provided a therapeutic window for intervention that may extend functional vision in patients. Even though this approach is feasible, few promising drug candidates are available. The scarcity of new drugs has motivated research to discover novel compounds through different sources. One such example is Schisandrin B (SchB), an active component isolated from the five-flavor fruit (Fructus Schisandrae) that is postulated in traditional Chinese medicines to exert prophylactic visual benefit. This SchB benefit was investigated in this study in pde6cw59, a zebrafish retinal-degeneration model. In this model, the pde6c gene (phosphodiesterase 6C, cGMP-specific, cone, alpha prime) carried a mutation which caused cone degeneration. This altered the local environment and caused the bystander rods to degenerate too. To test SchB on the pde6cw59 mutants, a treatment concentration was first determined that would not cause morphological defects, and would initiate known physiological response. Then, the mutants were treated with the optimized SchB concentration before the appearance of retinal degeneration at 3 days postfertilization (dpf). The light sensation of animals was evaluated at 6 dpf by the visual motor response (VMR), a visual startle that could be initiated by drastic light onset and offset. The results show that the VMR of pde6cw59 mutants towards light onset was enhanced by the SchB treatment, and that the initial phase of the enhancement was primarily mediated through the mutants' eyes. Further immunostaining analysis indicates that the treatment specifically reduced the size of the abnormally large rods. These observations implicate an interesting hypothesis: that the morphologically-improved rods drive the observed VMR enhancement. Together, these investigations have identified a possible visual benefit of SchB on retinal degeneration, a benefit that can potentially be further developed to extend functional vision in patients

The Hotelling’s T-squared test of VMR between different strains at 6 dpf.

<p>The Hotelling’s T-squared test of VMR between different strains at 6 dpf.</p