Traumatic spinal cord injuries (SCI) are devastating and lead to permanent and irreversible functional impairments. While the primary injury causes immediate damage, the progression of secondary injury is characterized by neuroinflammation. Microglia, the resident immune cells of the central nervous system, can further contribute to tissue damage. Although extensive research has been done focusing on microglial activation at the lesion site, their presence in regions distal to the injury at the chronic stage (4-6 weeks after injury) remains understudied. Here, we aimed to investigate the correlation between microglial activation in the lumbar region caudal to the injury site and cervical region rostral to the injury site with different grades of injury severity. We hypothesize that as injury severity increases, microglial activation in the surrounding region will increase as well. Contusion injuries were created at T8/9 in Long Evans rats using three impact forces: 200 kilodynes, 200 kilodynes and 2-second dwell time, and 250 kilodynes and 2-second dwell time. After 8 weeks post injury, our histological analyses revealed a significant increase in microglial activation in the caudal region, with activation levels reaching 136.03%, 205.08%, and 254.81% of sham levels in the 200, 200/2s, and 250/2s groups, respectively (n = 5/group). The most severe injury had the highest microglial inflammation. We anticipate a similar trend in the cervical region rostral to the injury. Combined, our data indicate a significant microglial response in regions distal to the injury, which correlates with impaired functional recovery. We believe that modulating microglial responses in the regions distal to the site of SCI could provide a novel approach to reducing chronic secondary injury and enhancing recovery following traumatic SCI.

Cervical spinal cord injuries (SCIs) often result in impaired locomotion and forelimb mobility. Assessed with a forelimb locomotor scale (FLS), previous research demonstrated improved forelimb locomotion with task-specific physical therapy (PT) training following a unilateral 150 kdyne C6 SCI in a rat model. Improvement in locomotion was calculated using a recovery ratio, which measures the difference in FLS scores normalized to the animal’s baseline score. There was a significant improvement in the recovery ratio (3dpi-14wpi) for animals that underwent task-specific PT training (p ≤ 0.0014). The underlying molecular mechanisms behind this improvement are still unclear. A potential explanation for these functional changes is the task-specific PT-induced increase in brain-derived neurotrophic factor (BDNF) targeted at the injury site. Task-specific PT stimulates neurons, microglia, and astrocytes to release BDNF, promoting neuron survival, growth, and synaptic plasticity. These processes are crucial for mediating the effects of cervical SCI trauma. This study investigates whether BDNF expression in the spinal cord varies in rats that underwent task-specific PT training following cervical SCI. PT-trained rats are hypothesized to have increased BDNF within the spinal cord, especially proximal to the injury site, which would help mitigate SCI trauma and enhance forelimb locomotion and mobility. To this end, we performed immunohistochemistry staining and quantification of BDNF in the spinal cord and compared it across three groups: PT + SCI, SCI-only, and Sham. The expression levels of BDNF in neuronal cells, astrocytes, and microglia were then correlated with their FLS scores to determine if there was a positive correlation between BDNF expression and forelimb locomotion. This would indicate that BDNF supports synaptic plasticity and recovery from SCI-induced trauma.

Hypertrophic Cardiomyopathy is the most common form of hereditary heart disease affecting ~1:500 individuals, characterized by progressive thickening of the left ventricular wall. The first mutation linked to this disease was the heterozygous R403Q mutation in human beta-myosin heavy chain (β-MHC). Conflicting reports of contractile kinetics between human myectomy samples vs transgenic mouse and rabbit models motivated us to study the molecular mechanisms of altered contraction in a CRISPR/Cas9 gene edited human inducible pluripotent stem cell line. Following differentiation to cardiomyocytes (hiPSC-CMs) and maturation in culture, we isolated sub-cellular contractile organelles called myofibrils. Myofibril contractile kinetics from this line had slowed force development and cross-bridge detachment, with reduced maximal force compared to the WT line. hiPSC-CMs were cast into fibrin matrices to form three-dimensional, engineered heart tissue (EHT) for measures of twitch force and contractile kinetics. At 1Hz stimulation, heterozygous mutation EHT’s exhibited a hypercontractile phenotype compared to WT EHTs, with slowed relaxation kinetics. Since the penetrance of our heterozygous R403Q hiPSC-CMs is unknown, we are now studying a homozygous iPSC-CM line where 100% of the β-MHC is mutated. This will allow us to assess the direct contribution of the mutation to the disease contractile phenotype. We will repeat the myofibril and EHT measures of contractile properties and perform stopped flow kinetics analysis on isolated myosin to determine ATP turnover and ATP hydrolysis product release rates. This will provide molecular mechanistic insight of the contractile abnormalities, allowing development of therapeutic interventions that specifically target the mechanisms that alter contractile function. 

Somatosensory neurons innervate the skin, where their peripheral axons detect signals like touch and pain. The neurons relay stimuli to the brain via peripheral axons in the skin and spinal cord axons in the spinal cord. Given their superficial location, somatosensory axons are susceptible to damage. Axon damage can cause tingling, increased pain, or sensory inhibition, and reinnervation in mammals is often slow or incomplete. I use injury models in zebrafish to study the mechanisms of successful axon regeneration in an adult vertebrate with optically accessible skin. I aim to reveal conserved regeneration patterns of somatosensory neurons. Furthermore, I seek to understand the extent of reinnervation success and observe the prevalence of hyperinnervation post-injury. Using in vivo confocal microscopy and adult zebrafish skin models, I created a methodology to capture somatosensory reinnervation over a three-week span following a scale pluck injury. Zebrafish scales separate epidermal and dermal layers of skin, and scale removal induces regeneration of epidermal skin and surrounding dermal tissue. I use transgenic zebrafish with fluorescent labels for dorsal root ganglion DRG neurons and osteoblast cells Tg(p2rx3a:mCherry);Tg(sp7:EGFP). DRG neurons are the primary somatosensory neuron in adult zebrafish, and osteoblasts allow me to view the scale alongside axon reinnervation. For image acquisition, I designed a 3d-printed chamber for zebrafish mounting and intubation within our confocal microscope. For analysis, I developed Image J macros which use threshold analysis to quantify changes in axon density of specific regions of regenerating axons. Dermal axons tend to regenerate first while superficial axons in the epidermis regenerate secondarily in conjunction with the novel scale. To examine skin layer differences, I separate epidermal and dermal layers to compare the reinnervation trends between superficial and dermal axons. With this data, I can gain insight in the regeneration potential of somatosensory neurons.

Alzheimer's Disease (AD) is a neurodegenerative disorder with classical behavioral and pathophysiological presentations such as memory impairment, cerebral vascular alterations, and most notably, the accumulation of amyloid-beta plaques and tau fibers in humans. While these symptoms are hallmark signs of AD, the severity of these symptoms differs for individual patients. As such, clinicians face challenges in misdiagnosing or giving a late diagnosis using a few of these AD markers. My research project aims to characterize AD through 3 concurrent modalities with the goal of early diagnosis and better quality of care for AD patients: [1] motor activity using a motion capture chamber, [2] spatial memory and learning using Barnes Maze Trials, and [3] real-time cerebral hemodynamics with ultrafast contrast-enhanced ultrasound (CEUS) in a rat model of AD. This project utilized control wild-type (WT; n=3) and transgenic (AD; n=3) rats. We hypothesize that the WT and AD rats will express similar movement and cognitive metrics measured until they are 12 months old, whereas the AD rats show a gradual motor decline and cognitive impairment. The 12-month metric is used as a benchmark set by prior research using this rat model. Intravital CEUS imaging will be conducted at 16-19 months old. We expect CEUS imaging will reveal that cerebral blood flow in the parietal and hippocampal regions of the brain is reduced, and microvascular response is impaired in AD rats. The expected outcome of all 3 experiments is that there is a strong correlation between motor and cognitive decline with impaired hemodynamics in AD rats. Results from our study will allow for systematically chronologizing the progression of AD in greater detail than before which allows for greater diagnostic ability.  

According to the Centers for Disease Control and Prevention, the U.S. has more than 2.8 million antibiotic-resistant infections each year. The rise of multidrug resistance in bacteria poses an urgent clinical threat contributing to these various infections. UPAB1 is a specific strain of a notoriously drug-resistant bacteria Acinetobacter baumannii associated with catheter-associated urinary tract infections (CAUTI). UPAB1 infects the urinary tract through the introduction of a foreign object, such as a catheter. In response, the immune system coats the catheter with fibrinogen, a glycoprotein complex that assists in wound healing. UPAB1 uses its bacterial adhesin proteins, such as Abp2D, to bind to fibrinogen, deplete essential nutrients, and infect the urinary tract. By designing Abp2D inhibitors as de novo miniproteins, we hypothesize that A. baumannii will be prevented from establishing a bacterial infection and allow us to offer a potential alternative in combating antibiotic resistance in CAUTIs. Targeting UPAB1 Abp2D, we first developed designs of Abp2D inhibitors utilizing computational software like RoseTTAFold Diffusion (RFdiffusion) for miniprotein backbone design, ProteinMPNN for sequence design, and AlphaFold2 (AF2) for structure prediction of the sequences to validate and filter. Afterwards, in the laboratory, we expressed and purified the miniprotein designs. We are currently testing these designs as Abp2D inhibitors via E. coli cultures to determine their success in binding to UPAB1 Abp2D.