It has long been known that certain species possess the capacity to regenerate bony appendages following amputation through a process called epimorphic regeneration. For instance, zebrafish possess the ability to regenerate their tail fin bones. Early regeneration involves the formation of a blastema, similar to the blastema that mediates limb regeneration in salamanders. After the blastema forms, the bone regeneration process resembles the bone developmental process in humans. Thus, a better understanding of epimorphic regeneration holds promise to enhance our understanding of regenerative biology, allow for medical advances in bone tissue engineering, as well as increase understanding of the skeletal developmental processes. A challenge in understanding the regeneration process is the inability to immobilize fish to generate time lapsed images of various stages of regeneration. Typical methods for anesthesia in zebrafish only enable 10-20 minutes of sedation. Several studies (including those by our lab) have developed specialized methods to do long-term imaging with the use of Tricaine Methanesulfonate and Benzocaine. However, both anesthetics are sodium channel inhibitors, which inhibit the regeneration process itself. In order to circumvent this problem, we designed a chamber for lower anesthetic plane imaging with a restraint system, as well as a glass-bottom window to facilitate imaging. The chamber is coupled to peristaltic pumps so water and anesthesia can flow in and out. In pilot studies we have found the zebrafish quickly acclimate to the chamber, and have remained in the chamber for two hours. We have been able to image sp7, NADH and birefringence simultaneously for the first 2 hours of regeneration and have found stability in these signals over time. Our studies indicate that this long-term imaging chamber may allow us to see details of dynamic processes that unfold during bone regeneration, which currently can only be seen through snapshots of short-term imaging.

Impella devices provide temporary mechanical circulatory support (MCS) by means of a catheter consisting of a left ventricular (LV) inlet, a microaxial pump within the catheter, and an outflow port in the proximal aorta. The recommended position of the inlet is 4-4.5cm below the aortic valve annulus. Following initial placement, the device is prone to migration, potentially leading to ineffective support, hemolysis, ventricular arrhythmia, or mitral regurgitation. Positioning is typically monitored by echocardiography. However, the Impella device has a bend between the inflow and outflow ports, and traditional 2D-imaging may not be reliable in assessing the location of the device, as single plane 2D windows may not include both the inflow port and the aortic annulus, leading to foreshortening of the cannula and under-measurement of the distance. We hypothesized that 3D echocardiography provides more accurate characterization of the Impella cannula location. We analyzed 25 echocardiograms of patients with Impella devices placed in LV which had analyzable 2D and 3D images. Measurements of the distance from the aortic annulus to the inflow port were made according to recommendations from established measurement guidelines. Three sets of measurements were made at end systole and at end diastole. Full volume 3D datasets acquired from the parasternal window were acquired an analyzed using Qlab software to overcome possible foreshortening of the Impella cannula. In addition, the 3D dataset was used to measure the angle deviation of the cannula from the long axis of the left ventricular outflow tract. There was no recognizable benefit of 3D techniques in measuring aortic annulus to Impella inflow port distance. A larger sample size may be necessary to detect a significant difference. 3D imaging may have a benefit in observing other relationships, such as the mitral apparatus, and bears ongoing investigation.

Stroke is the leading cause of long-term disability in the USA. Ischemic preconditioning (IPC) is a neuroprotective phenomenon wherein a brief ischemic exposure induces robust neuroprotection against subsequent prolonged ischemia. The Weinstein laboratory has previously demonstrated: (i) a robust type 1 interferon response in cortical microglia following IPC, (ii) type I interferon signaling in microglia is required for IPC-mediated protection and (iii) IPC induces a robust increase in the number of microglia in preconditioned cortex. An initial component of my project was to validate this microglial response by first staining for Iba1 (a microglial marker) alone and then double staining for Iba1 and proliferation marker BrdU. We used immunofluorescent microscopy (IFM) following by quantitative stereology (QS). Our hypothesis was that type I interferon signaling is necessary for IPC-induced microglial proliferation. We carried out IPC on WT and type 1 interferon receptor deficient (IFNAR-/-) mice and quantified cortical microglial number and proliferation as above. Preliminary results were: (i) in naïve WT, 0.643 ± 0.038 (mean ± S.D), Iba1+ cells per position, (ii) in preconditioned WT, 1.113 ± 0.1385, (iii) in naïve IFNAR1-/-, 0.751 ± 0.058 and in preconditioned IFNAR1-/-, 0.903 ± 0.125. Two way ANOVA revealed a significant difference between naïve and IPC-induced cortical microglia numbers [F(1,14)=9.62, p=0.0078], but no significant effect of genotype [F(1,14)=0.258, p=0.619]. IPC induced increases in the number of cortical Iba1+/BrdU+ proliferating microglia in both IFNAR1-/- (0.451 ± 0.107) and WT (0.205 ± 0.069) mice. These results suggest a complex picture in which deficiency in type 1 IFN signaling may not influence IPC-induced microglial proliferation but does attenuate the overall number of cortical microglia. This raises the possibility that type 1 IFN signaling may be required for optimal microglial survival following IPC. More studies will be required to confirm the above findings and explore possible mechanisms.

Joubert syndrome (JS) is a neurodevelopmental disorder characterized by developmental delay and a specific hindbrain malformation resulting in the “molar tooth sign” (MTS) as seen on MRI. Individuals with JS have variable intellectual disabilities, polydactyly, retinal dystrophy and cystic kidney disease. JS is classified as a ciliopathy—disorders with overlapping phenotypes affecting 1/1000 live births combined. Currently, 36 genes when mutated cause JS, and all their encoded products localize in and around the primary cilium—a cellular organelle important for signaling and human development. We recently identified the newest JS-associated gene ARMC9 and its function, like the molecular mechanism(s) underlying JS, is unknown. Our preliminary data suggest that ARMC9 is required for cilium stability by promoting post-translational modifications (PTM) of ciliary microtubules. In stably ciliated, fixed (non-living) cells, ARMC9 localizes to the ciliary base, but to understand ARMC9’s role in cilium stability, we must determine ARMC9 localization during ciliogenesis (ciliary growth). We hypothesize that ARMC9 redistributes to the ciliary tip to promote tubulin PTMs during ciliogenesis. To test this hypothesis, I am using GFP and BFP (green and blue fluorescent protein) tags to visualize ARMC9 and primary cilia respectively. I created 5HT₆-BFP, a DNA vector encoding a BFP-tagged serotonin receptor fragment called 5HT₆ which naturally localizes to cilia. Currently, I am optimizing the transfection of 5HT₆-BFP and ARMC9-GFP into control cells for live imaging. As suggested by our data, I expect to see ARMC9 at the ciliary tip during growth, then redistribution to the base once the organelle is built. Determining where ARMC9 localizes during ciliogenesis will give us additional insight into its mechanism of action and role in JS. Ultimately, this work will allow us to identify therapeutic targets to mitigate the progressive features of JS and improve the quality of life of affected individuals.

As one of the five leading causes of blindness, uveitis is the reoccurring inflammation of the uvea, demonstrating pathological changes in the eye. In recent years, optical coherence tomography (OCT) has been recognized as the leading, clinically accepted imaging modality for diagnosing major human optical diseases. Despite this, research on the clinical usage of OCT, primarily spectral domain OCT (SD-OCT) and swept source OCT (SS-OCT), for uveitis diagnosis remains sparse due to the lack of a quantitative-based set of parameters to assist with OCT image analysis. As a result, there is a need to develop an index of parameters that quantifies the microvasculature and structural changes associated with uveitis. To address this need, a novel five parameter quantitative-based metric consisting of distance of retinal detachment, retinal thickness, vessel area density, vessel diameter, and vessel perimeter was evaluated. Through layer segmentation of SD-OCT and SS-OCT scans, application of optical microangiography, and quantitative analysis of structural and microvasculature changes for healthy and uveitis cases, the clinical potential of SD-OCT and SS-OCT for diagnosing uveitis was evaluated. This project introduced a metric for evaluating changes associated with uveitis in a qualitative and quantitative manner to further understand the abnormalities that accompany the disease. Assessing the clinical efficacy of SD-OCT and SS-OCT for uveitis detection can provide insights on the most effective method for diagnosing this disease.

Giardia, a protozoan parasite which resides in human intestine, can cause Giardiasis, a common parasitic disease associated with severe diarrhea. It possesses a simple life cycle including the motile trophozoite stage and infectious cyst stage. Once a human consumes unsanitary drinking water or food with Giardia cysts, the low pH in stomach drives excystation to release the trophozoites. When they perceive stimuli from the high bile and alkalinized pH environment, they will initiate encystation by turning on the Cyst Wall Protein gene to form cysts. Our purpose is to understand the spatiotemporal encystation process and the encystation rate of Giardia in a physiological environment (murine intestine) via observing the ratio metric change of encysting and non-encystation cells in the Dual-color Bioluminescence Imaging (BLI) System. BLI is essential to monitor noninvasive biological processes in living organisms via detecting photon emissions that requires the enzyme luciferase to catalyze a substrate, usually luciferin, with oxygen, ATP, and magnesium. To test if dual-color BLI can report the ratio metric change of encysting and non-encysting cells in vitro, we have generated the in vitro encystation assay. The promoter of Glutamate Dehydrogenase, a constantly expressed gene at all stages of Giardia, is combined with a luciferase gene to represent non-encysting cells when transcribed in normal media. The promoter of cyst specific gene, Cyst Wall Protein 1, is combined with another luciferase gene to represent encysting cells when expressed only in encystation media. By successfully observing the ratio metric change of luciferase expressions in vitro, we can then proceed to future mouse experiments. Once the technique has been confirmed feasible in the mouse model, it can be applied to monitor the life cycle of all cyst forming parasites and understand when and where they are at infectious stage, thereby improving the accuracy for alternative drug screening.