Non-human primate (NHP) research has become an essential step in the translation of medical technologies from animal models to clinical trials. This is especially so in neural research, as there is a large discrepancy between rodent and human brains in both anatomy and size. For some techniques such as optogenetics, which requires viral transduction of neurons, traditional diffusion-based viral injection approaches are effective in rodent brains but are impractical for large NHP ones. Convection-enhanced delivery (CED), a large-scale injection approach, currently lacks a practical quantitative bench-side injection modeling method to guide neurosurgical preparation. We aim to develop a gel model of the NHP brain and replicate surgical injections of it in order to reduce the risks of directly injecting into a NHP without sufficient preparation. We are testing the validity of our model by monitoring the spread of the injection through the gel and comparing the data with those from MRI scans of the injections in NHP. Since CED can behave differently depending on the location of injection in the brain, we are testing bench-side injections at different depths to validate the versatility of our model. We are seeing that the injections in the gel model mirror that of the injections in NHP brains as expected. Our next steps are to test the effectiveness of smaller injection cannula sizes with our bench-side model to assess if injection results remain consistent. This would indicate that tissue damage could be minimized in surgeries while still achieving desired injection parameters.

Stroke, when the blood supply to the brain is reduced or disrupted, is a leading cause of disability among adults. Brain plasticity, also known as neural plasticity, can aid in the way that the brain recovers from a traumatic injury such as a stroke by creating newer, stronger synapses (connections) between neurons and thus increasing cell functionality. This literature review explores how brain plasticity informs new bioengineering solutions to stroke rehabilitation and asks, “What type of novel stroke treatments have been developed using the concept of brain plasticity?” Preliminary findings indicate that a breakthrough in this field is using optogenetics to trigger and control the neural connections that the brain can make through promoting motor function after ischemic stroke. Other innovations in this field include repetitive transcranial magnetic stimulation, transcranial direct current stimulation, and epidural cortical stimulation, which have all been shown to make permanent changes in neural synaptic transmission. These methods partially restore brain function while being less invasive and more effective in comparison to older interventions. This literature review indicates that new bioengineering treatments informed by brain plasticity are promising and could promote better rehabilitation outcomes for those suffering from stroke and potentially other traumatic brain injuries.

Drug delivery-enhancing platforms are vital to overcoming the blood-brain barrier that prevents sufficient accumulation of drugs in the brain during treatment. Although nanoparticles can improve the treatment of neurological diseases, such as Alzheimer’s disease, the formulation process must be optimized because an inadequate amount of drug can currently be loaded in nanoparticles. The goal of my project is to 1) optimize nanoparticle formulation parameters to maximize therapeutic enzyme activity, and 2) characterize the extent of nanoparticle degradation due to sonication. Within part 1), formulation methods were composed of poly(lactic-co-glycolic) acid copolymerized with poly(ethylene glycol), cholic acid (CHA) or polyvinyl alcohol surfactant, and the enzyme catalase. I formulated each of the nanoparticles at varied sonication times during the emulsion step to measure differences in therapeutic activity using UV-Vis spectroscopy. I found that the 30s sonicated CHA double emulsion and nanoprecipitation methods yielded the greatest enzymatic activity, 2.10% and 3.72% activity, respectively. In the presence of degradative pronase, CHA double emulsion nanoparticles exhibited better retention of enzymatic activity than nanoprecipitation, 75.66% and 9.22% retention, respectively. Within part 2), I monitored heat flow to identify the melting temperature in a sample of PEG exposed to varying sonications and assessed PEG degradation using differential scanning calorimetry. 5kDa PEG exhibited a melting temperature of 60.792C, while 5kDa PEG exposed to 2x150s of sonication exhibited a melting temperature of 59.450C, correlating to 4.8kDa. The 30s sonication CHA double emulsion formulation yielded the highest enzymatic activity with protection from degradation from external proteases. Thus, my results point to a promising polymeric nanoparticle design that may help in the development of more powerful and effective treatment options for neurological diseases.

Optogenetic stimulation is a technique that modulates the activity of genetically modified neurons with light of a particular wavelength. Optogenetic modulation has high temporal resolution and cell-type specificity that enables precise stimulation of cortical neurons and allows us to conduct artifact-free recording during stimulation. Using a large-scale optogenetic interface, we stimulated and recorded across the primary somatosensory (S1) and motor (M1) cortices of non-human primates (NHP). We conducted our investigation on NHPs because their cortical organization is particularly similar to that of humans. The goal of this study is to determine the effect of various spatial and temporal patterns of optogenetic stimulation on the neural response and network dynamics across the two cortical regions. Delivering stimulus pulses via two lasers placed on top of the cortical surface, we found that stimulation of one cortical region evoked neural responses across both S1 and M1, which we then classified into primary and secondary responses based on their delays. While our previous work has established that optogenetic stimulation strengthened functional connectivity between S1 and M1, we wanted to further investigate the distribution of primary and secondary neural responses after repeated stimulation. We examined two measures of neural responses, the temporal delay between the trough of evoked response and onset of light stimulation, and the distribution of power across cortical networks up to 50ms after the stimulation. Our preliminary results indicate that optogenetic stimulation changed the delay of the primary and secondary response. We also observed that different temporal patterns of paired laser pulses evoked distinct neural activity. Identifying different neural responses after complex spatiotemporal patterns of stimulation would help us predict network changes post cortical modulation and contribute significantly to the development of stimulation-based clinical therapies and rehabilitation strategies for neural disorders.

Aortic aneurysms form when the aorta, the large artery that carries blood from the heart, weakens and expands. Smooth muscle cells (SMC) in the aortic wall regulate aortic structure and contractility and help preserve normal aortic diameter. Transforming growth factor beta (TGF-β) signaling is integral to SMC function and abnormal TGF-β signaling and is implicated in aneurysm formation. However, whether excessive or deficient SMC TGF-β signaling promotes aneurysms is controversial. To determine if increased SMC TGF-β signaling is sufficient to cause aneurysms, we expressed a Constitutively Active Type I TGF-β Receptor (TβR1-CA) transgene in SMC of male and female mice (both hemizygous). Because the transgene is integrated into the X chromosome, and half of female X chromosomes undergo inactivation, expression of TβR1-CA in males and females may differ. This difference could affect the interpretation of studies that investigate the role of SMC TGF-β signaling in aneurysmal disease. We hypothesize that hemizygous females will have lower levels of TβR1-CA expression than males due to random X-inactivation. To test our hypothesis, we will quantify the mRNA levels of TβR1-CA relative to that of Gapdh (a reference gene for normalization) in aortic SMC of male and female hemizygous transgenic mice using real time (RT)-qPCR. To confirm that we can detect X chromosome inactivation using RT-qPCR, we will also measure mRNA levels of a gene that is known to undergo X-chromosome inactivation (Pgk1) and a gene that is known to escape X-chromosome inactivation (Kdm5c). If our hypothesis is correct, we will find that the amount of aortic TβR1-CA mRNA is ~50% lower in aortas of female versus male transgenic mice. The results of this project will be essential in helping us to interpret results of parallel experiments that investigate whether TβR1-CA expression (and activated SMC TGF-β signaling) causes aneurysms in male and female mice.

In the Chavkin Lab, nalfurafine is a biased kappa agonist of interest and binds to and activates kappa opioid receptors (KOR). Nalfurafine is of interest because through the activation of KOR, it can alleviate pain and has been shown to be non-addictive. KOR signals through G-proteins and can exhibit biased signaling; this biased signaling can activate production of reactive oxygen species (ROS). We hypothesize that ROS may be a necessary signaling mechanism for biased kappa agonists such as nalfurafine, but the dynamic processes of ROS and their impact on cellular physiology remains unknown. To investigate where and how ROS are produced, I utilized a novel HyPerRed imaging technique. HyPerRed is a red fluorophore attached to a specific hydrogen peroxide sensor that is genetically encoded in an adeno-associated virus (AAV). The HyPerRed virus was injected into the ventral tegmental area (VTA) of male mice and horizontal brain slices were taken. The HyPerRed responses to nalfurafine and hydrogen peroxide were imaged under a fluorescent microscope and recorded over time. Significant HyPerRed responses to ROS production were observed in VTA cell bodies but not in nerve terminals, suggesting that different subcellular components may contribute to the robust ROS production observed in the VTA. This study provides a framework for a new technique that can be used to visualize and study reactive oxygen species production in real-time. Understanding the dynamics of ROS production could inspire and improve future research and development of kappa therapeutics to treat chronic pain. The pain-alleviating and non-addictive properties of kappa agonists provide safer alternatives to the traditional prescription opioids that have contributed to the current opioid epidemic.

Non-human primate (NHP) research is a pivotal step in the progression of neuroscientific and neural engineering research from animal models to human trials. In most NHP neuroscience experiments, neurosurgery is required to implant devices such as head posts, recording arrays, and optical windows. Current practices for these surgeries use methods for surgical preparation that carry a degree of unavoidable uncertainty. This comes from an inability to visualize and test the physical compatibility of complex components and anatomy prior to neurosurgery. This project details methods for creating 3D printed models of a subject’s brain and skull, as well as an agarose gel model of the brain. These models can be obtained from magnetic resonance imaging (MRI) using brain extraction software for the brain model, and custom code for the skull. The preparation protocol takes advantage of state-of-the-art 3D printing technology to combine models of the brain and skull with neuroprosthesis. With the addition of a craniotomy using the custom code, the skull and brain models can visualize brain tissue inside the skull, enabling better preparation for surgeries. Using the methods outlined in the protocol, the accuracy of the 3D printed brain, skull, and craniotomy placement were successfully validated through a comparison to the original MRI scan. The gel brain was additionally used to visualize delivery of a mock viral vector through the craniotomy of a skull model. By preoperatively fitting a headpost to the physical model of the skull, we successfully shortened the implantation surgery time by 40% and greatly reduced the risk of operative complications. These methods are designed for surgeries involving neurological stimulation and recording as well as injection in NHPs, but the versatility of the system allows for future expansion of the protocol, extraction techniques, and models to a wider scope of surgeries.