In the retina, loss of neurons results in blindness, because the mammalian retina cannot regenerate. Although mammals cannot regenerate neurons, species such as fish and amphibians can make fully functional neurons after injury and restore their vision. Muller Glia (MG) cells in fish and amphibians are the source of this regeneration. These cells respond to injury by dedifferentiating into progenitor cells that then become neurons, replacing the dead neurons and in turn restoring sight. At the Reh Lab, we have found a way to stimulate functional regeneration in mammals through these MG cells by expressing a gene called Ascl1. This technique has limitations however, as it only causes 25% of MG to undergo neurogenesis. One of the variables potentially limiting regeneration is inflammation, as inflammation has been known to be detrimental to neurogenesis. Monocytes invade the retina after injury and potentially cause inflammation that limits retinal regeneration. To determine monocyte impact on retinal regeneration we employed a transgenic technique to ablate monocytes. I then performed our retinal regeneration paradigm and determined whether regeneration is enhanced in the absence of monocyte invasion. Using immunohistochemistry and confocal microscopy I found more regenerated neurons in retinas that lacked monocytes. These data further confirm that inflammation limits the regeneration capacity of the retina, and provides future topics to improve neural repair through modifying the immune response.

Blindness caused by retinal degeneration is untreatable, and is a condition currently suffered by millions. The field of retinal reprogramming aims to establish a way to treat this type of vision loss, by finding a way for the eye to essentially produce new retinal neurons after they have been lost to degeneration. Often, this is done through directing cell fate by expressing or repressing various transcription factors such as Ascl1, a potent pro-neural transcription factor involved in retinal reprogramming. Ascl1 expression in mouse Muller glia, a support cell in the retina, can stimulate the regeneration of certain subtypes of retinal neurons, but the variety of retinal cells produced through this strategy is still limited. The class of transcription factors known as ‘Krüppel-like factors,’ or KLFs, regulates important cell processes, such as cell proliferation and development, with several KLFs functioning in the development of neurons. Due to the inhibitory function of KLFs during neuron development, we propose that the loss of inhibitory KLF genes in Muller glia may allow for activation of neurogenesis. Therefore, coupling the knockout of KLF genes with Ascl1 expression in Muller glia may be a key to enhancing reprogramming capabilities of Ascl1. For this project, I knocked out KLF genes in young mouse Muller glia in culture using the CRISPR/Cas9 system, and induced Ascl1 expression to stimulate reprogramming of the Muller glia. We used scRNA sequencing to determine if the knockout reprogrammed cells were molecularly similar to various retinal cell types that are typically lost with degenerative blindness. Preliminary sequencing results revealed that knockout of one candidate, KLF15, appears to promote neurogenesis. Findings from this experiment will allow us to increase our understanding of the role of KLF genes in retinal cell development as we work towards a future treatment for degenerative vision loss.

Retinal diseases such as macular degeneration and glaucoma lead to various forms of blindness as neurons in the retina die. Unlike amphibians or fish, the neurons of the mammalian retina cannot regenerate on their own and any damage is permanent. Previous research has shown that we can recover some of the lost regenerative capacity in mammalian retinas by mimicking the regeneration process in other species. This is possible by overexpressing transcription factor Ascl1 in Müller glia (MG), which are the main support cells in the retina. However, expressing Ascl1 alone can only lead to the neurogenesis of one type of neuron in the retina. This limits the therapeutic applicability of this approach because in diseases like macular degeneration or glaucoma, specific neuronal cell types are lost such as photoreceptors and ganglion cells respectively. Therefore, to regenerate these neurons we need to identify the right cocktail of transcription factors for MG reprogramming. Recent single-cell analysis from our lab has identified candidate developmental factors that could push reprogrammed cells to photoreceptors or retinal ganglion cells. This project aims to investigate the role of these factors in influencing cell fate after MG cells have been pushed to a progenitor state with Ascl1. Using lentiviral vectors, I will induce the overexpression of these candidate genes in primary cultures of young adult mouse MG that have been engineered to express Ascl1. In order to identify the nature of resulting neurons from these cultures I will perform immunocytochemistry paired with confocal microscopy, and to better understand changes in functionality of these cells I aim to perform calcium imaging. Since the electrophysiological responses of glia and neurons are distinct, cultures with reprogrammed neurons would record differently. Overall, this analysis evaluates the influence of new transcription factors on mammalian retinal regeneration.

In fish and amphibians there is a specialized zone of retinal stem-cells at the edge of the retina, called the ciliary margin zone (CMZ) which replenishes the retina with new cells if it is damaged in adult animals. However, the presence of these stem-cells has not been observed specifically in the CMZ of the developing human. Here, I investigate the developing human retina to understand if it contains stem-cells that could be harnessed for repair. I first utilized CMZ stem-cell markers found in fish and amphibians to assess in the developing human retina, including BLBP, C-myc, cyclin D3, Six3, SMAD1/5, and Zic1. Following the stainings, I observed expression of C-myc and BLBP. To maintain their long-term proliferation, stem-cells will replicate slowly. Therefore I analyzed cell cycle kinetics in the CMZ. Primary cultures of fetal human retina, called retinospheres, were made by dissecting the fetal retina into small pieces containing a portion of the CMZ and growing them in culture with retinal differentiation media. EdU, a dye that is integrated into DNA only in replicating cells, was then added to the media for different incubation intervals with EdU being 30min, 1hr, 2hrs, 4hrs, 6hrs, 8.5hrs and 25hrs, then retinospheres were fixed in PFA. Retinospheres were IHC stained with antibodies and dyes: Pax6 (a stem-cell marker), EdU (marker of cell division), Ki67 (marker of replicating cells) and DAPI. The total Ki67 positive cells in the CMZ and of EdU and Ki67 positive cells were counted so that the S-phase of mitosis could be measured to discover how fast the cells in the CMZ were dividing. I observed that cells in the CMZ were replicating slower than those further away from the CMZ, consistent with the possibility that there is a population of stem-cells in the CMZ of the developing human retina.

In neonates, hypoxic-ischemic encephalopathy (HIE) increases the risk of impaired neurodevelopmental outcomes and death. In high resource settings, HIE occurs in 1-4 per 1,000 live births, and in low resource settings, 12 per 1,000 live births. Where available, therapeutic hypothermia (TH) is the standard of care for neuroprotection in HIE. However, the protection provided by TH is incomplete, motivating the search for adjunctive therapies. To assist in testing new therapies, researchers have attempted to predict injury severity in animals before ex vivo quantification, but the association between early injury and long-term outcomes has not been studied in the Vannucci model of hypoxic-ischemic brain injury - the most widely used HIE model in rats. This study sought to determine the association of early injury identified by in vivo magnetic resonance imaging (MRI) with weight gain and behavioral testing data. Using the Vannucci model, postnatal day (P) 10 rats underwent ligation of the left carotid artery followed by 3 hours of hypoxia and then 5 hours of normothermia in room air. Animals were then randomized, with half receiving an additional 3 hours of TH while the other half were returned to their cage. On P12, MRIs were performed, and injury quantification was determined using imaging software FSLeyes. Animals were weighed daily for one week following the HIE injury and then every 2-3 days until P43. Behavioral testing was conducted on P42 and P43 and included open field, novel object recognition, and CatWalk. We hypothesize that greater injury on the early MRI will be associated with reduced weight gain after injury and worse performance on behavioral testing.

Hypoxia ischemia (HI) is one of the leading injuries in term neonates and can lead to death or long-term disability. The current standard of care is therapeutic hypothermia (TH). Our lab has developed a model for HI injury in rats which is used to test the efficacy of potential treatments. At postnatal day (P) 10, which is the equivalent of a full-term human neonate, rats undergo unilateral carotid artery ligation followed by hypoxia. The animals are given a 30-minute rest period before rectal temperatures are gauged to determine the post-surgical (time 0) temperatures. Animals are then placed in warm water baths for a period of normothermia (NT). Every 15-30 minutes rectal temperatures are recorded, and water baths are adjusted in order for the rats to maintain the goal temperature of 36.5°C (± 0.5°C). After 6 hours of NT, a randomized subset of animals undergo 3 hours of TH to reach 32°C (± 0.5°C). On P43 the rats are euthanized and perfused transcardially for brain removal and formalin fixation. The brains are assigned an injury severity score between 0 and 4, and % injury by volume is detected by MRI. The correlation between time 0 temperature and injury score will be calculated, as well as the correlation between the coefficient of variation of the temperatures for each animal throughout NT and the injury score. Further statistical analysis will be conducted throughout this experiment as the project is currently ongoing, including analysis by treatment group (NT vs TH). This project aims to determine if time 0 temperatures and variation of temperature are indicative of injury severity. Based on a similar study observing the relationship between the time 0 temperatures of ferrets and HI injury severity score, we expect to see that rats with lower time 0 temperature and greater temperature variation will have more severe brain injuries.

This research project involves analyzing planetesimal accretion through the use of an N-body simulation. A terrestrial planet passes through many stages of growth including: dust grains, pebbles, planetesimals, embryos, to planets. This study focuses on the formation process between planetesimals and embryos. Current simulations demonstrating terrestrial formation use parameters similar to those of our own solar system. This investigation attempts to envision this process at a more “bunched” up scale, such as in the case of the Trappist-1 system. Our inner solar system, a radial distance from the Sun to Mars, is about 25 times larger than the entire Trappist-1 system, meaning that its planets were formed much closer to its star. Through the use of N-body simulations, we can begin to understand the unknown formation of this system as well as others with similar characteristics. These N-body simulations are processed through the University of Washington’s supercomputer Hyak, approximating the motion of the particles that represent the planetesimals and detect if any are in a collision course. Two short period simulations were run using a number of sophisticated collision models that differ in how the particles interact and formation efficiency. The previous collision model used parameters calculated in 2005. The second newer model uses parameters from 2021, which I have programmed into the model's initial condition files. The study’s purpose is to compare the outputs of the collision models through a variety of quantitative and qualitative factors, concentrating on particle growth and runaway growth. More specifically, the data is measured through plots that depict the semi major axis vs. eccentricity, max mass over mean mass as a function of time, and the ratio of collisions that result in a merger. This will later lead to the investigation of which models can accurately replicate terrestrial formations such as the Trappist-1 system.

Computing solutions to partial differential equations using the fast Fourier transform can lead to unwanted oscillatory behavior. Because of the periodic nature of the Fourier transform, waves that leave the interval on one side reappear on the other. However, the fast Fourier transform is a very efficient numerical tool, so it is important to find a way to damp these oscillations so that this transform can still be used. Our goal is to accurately model nonlinear partial differential equations on an infinite domain by considering a finite interval and implementing various damping techniques outside of the interval. We consider the Korteweg-de Vries equation with an initial condition that produces leftward traveling oscillations and a rightward traveling soliton. To damp the wrap-around oscillations, we have used the Strang-splitting method to solve the heat equation with a non-zero diffusion coefficient on the left side of the interval. To stop the soliton from wrapping around, we judiciously multiply the solution values by a decaying exponential on the right side of the interval. We have found that this damping process produces much more accurate solutions at larger times than the undamped solution when modeling solutions on an infinite interval. This method also applies to the nonlinear Schrodinger equation with some additional modifications.