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Kibbles Orrery

Elucidating the pathway of visceral pain: an in vitro electrophysiological investigation of gut sensation and inflammation

2023 Grant Cycle

Applicant Email: kibbles_orrery.0b@icloud.com

Application Stage: Pre-Proposal

Application Stage Status: pending



Applicant

Address:
360 Huntington Ave, Boston, MA, 02115, US

Phone Number:
1

University

University Name:
Northeastern

Department:
Chemical Engineering

Degree Sought:
masters

Anticipated Date of Graduation:
03/2020

Date Entered Graduate Program:
04/2022

Date Joined Current Lab:
05/2024

How did you hear about this fellowship?:
4Chan forum

Principal Investigator/Research Mentor

Full Name:
Abigail Koppes

University Name:
Northeastern University

Department:
Chemical Engineering

ORCID Number:
X773737

Address:
20 Winter Ave, Maiden, MA, 02148, US

Email Address:
nichols.ky@northeastern.edu

Phone Number:
1

Laboratory Website:
http://www.northeastern.edu/abnel/

Pre-Proposal

Abstract:

Visceral pain is experienced by a large portion of the population, at up to 25% of people, and is described as a deep, poorly localized pain. It is frequently sensed in the abdomen by inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) patients. The underlying mechanisms of this pain, as well as the pathophysiology of IBD and IBS, are still not well understood. This proposal aims to elucidate the effects of exogenous neurotransmitters (NTs) on key neuron populations found within the visceral pain pathway. These include intrinsic and extrinsic primary afferent neurons. NTs are released by several cell types surrounding enteric sensory neurons, including epithelial cells, glial cells, and even bacteria. It is thought that these neuroactive compounds are causing hypersensitivity of sensory afferents due to repeated activation, leading to pain symptoms. The neurons become plastic, changing their action potential firing patterns and frequencies over time. Altered NT release is thought to be caused from conditions like dysbiosis, infection, inflammation and stress. These conditions lead to neuroplasticity even beyond the reversal of the conditions. Changes in firing frequencies to exogenous NTs will be quantified using microelectrode arrays (MEAs) integrated into a microphysiological system (MPS) designed to support key neuron populations of the visceral pain pathway. A pro-inflammatory cocktail will be administered to better understand the development of neuroplasticity in an inflamed state. This MPS platform can extend beyond these studies to include work focused on additional mechanistic understanding of visceral pain, as well as drug testing and discovery.

Please describe the research project and planned activities:

Up to 25% of the population experiences visceral pain, but the underlying mechanisms are still not well understood, making the identification of effective therapeutic treatments difficult [1, see attached document]. Visceral pain is a common symptom of patients with gastrointestinal (GI) disorders, including inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Because the etiology of the visceral pain is unknown, opioids are prescribed for pain management. This is problematic because opioids can exacerbate pain over time, result in severe adverse side effects, such as narcotic bowel syndrome, and lead to dependency and abuse [2].

Nociceptors, the neuron type responsible for pain sensations, innervate the enteric nervous system (ENS). Intrinsic primary afferent neurons have cell bodies residing in the ENS, whereas extrinsic primary afferent neurons (ExPANs) transmit signals through the dorsal root ganglion (DRG) to the spinal cord in the central nervous system (CNS). A hypothesis for visceral pain suggests that these nociceptors are being hypersensitized. IBS patients were found to have increased sensitivity to colonic distention and a decreased threshold for pain, further supporting this hypothesis [3]. Although further research is needed, there are known modulators, including stress, inflammation, and the microbiome. Stress exacerbates pain, while certain bacteria strains help ameliorate symptoms [4,5]. This is likely attributed to bacterial metabolites, such as neurotransmitters (NTs), that interact with receptors located in the gut, but the mechanisms of action remain unclear. Surrounding cell types, including epithelial cells and enteric glia (EG), also produce NTs that act on neurons in the gut which can be further altered during inflammation [6,7]. Thus, this project will elucidate the role of exogenous NTs on nociceptive populations along the gut-brain axis. A microphysiological system (MPS) will be designed to study the effects of NTs on neuroplasticity and the influence of extrinsic nociceptors on IPAN sensitivity. This platform allows for the incorporation of physiologically relevant conditions, while reducing the need for animal pain studies. Serotonin (5-HT), glutamate, histamine, adenosine triphosphate (ATP), and gamma-aminobutyric acid (GABA) are synthesized by the gut microbiome, epithelial cells, and EG and have been implicated in contributing to TRPV1 activation or inhibition in IPANs [8-13]. TRPV1 is a nonselective cation channel that has been shown to contribute to IPAN hypersensitivity in previous studies [14,15]. Increased expression of TRPV1 has also been found in IBS and IBD patients [16,17]. Electrophysiological methods will be utilized throughout this project to quantify neuron hypersensitivity, including microelectrode arrays (MEAs) integrated into the MPS for real-time action potential (AP) read out.

Aim 1: Perform high throughput electrophysiological analyses of neuroactive compounds implicated in visceral pain on nociceptive neuron populations. Hypothesis: Exogenous NTs contribute to sensory neuron activation within the gut. Methods: Enteric neurons (IPANs) from the myenteric plexus and DRG (ExPANs) from the spinal column have been successfully isolated from Sprague-Dawley neonatal rat pups (Figure 1). Human patient-derived gut samples will be used as available from a collaborator at Massachusetts General Hospital to isolate IPANs. Commercially available human induced pluripotent stem cell (hiPSC) derived sensory neuron progenitors (Axol Bioscience Inc.) will be used as a nociceptive ExPAN model. A differentiation protocol into nociceptors is available using small molecule inhibitors. Rodent cells will be used for optimization.

IPANs and ExPANs will be cultured on microelectrode arrays and recorded daily until spontaneous activity is observed to obtain baseline firing patterns. A Multichannel Systems amplifier and MC_Rack software will be utilized to record action potential (AP) data. Spike sorting algorithms (Figure 1) will be performed to analyze the recording. MC_Rack software will be used for raw data collection which will then be exported for further analysis using SpikeInterface in Jupyter Lab [18].

After recording baseline AP data, a high throughput analysis of the effects of NTs (5-HT, glutamate, histamine, ATP, GABA) will be performed. This study allows the investigation of exogenous NTs implicated in TRPV1 activation on extracellular neuron firing thresholds and frequencies. These parameters are indicative of changes in cell hypersensitivity and neuroplasticity when analyzed over time. The NTs will be individually administered to the IPAN and ExPAN cultures on MEAs. Table 1 shows concentrations for each compound [19,20]. The firing frequencies will be analyzed and compared between NTs. Neural recordings from each cell population will be taken over 4 weeks with NT administration during each media change to evaluate neuroplasticity. This aim will showcase which NTs are causing the greatest changes to neuron function.

Aim 2: Develop a MPS that supports the co-culture of key cell populations within the visceral pain pathway and allows for electrophysiological analyses of interactions between cell types. Hypothesis: Intrinsic and extrinsic neurons influence one another. Methods: An MPS will be designed to model key features of visceral pain due to its superiority in representing simplified, physiological relevant models. The platform will support a co-culture of IPANs and ExPANs. Two, adjacent compartments will be needed to contain the cell bodies of each population (Figure 2). The neuron cultures will be separated using gelatin methacrylate (GelMA) hydrogel which prevents neuron migration but allows neurite outgrowth between the compartments. A gelpin will be incorporated into the MPS layer design to allow a meniscus pinning effect of the uncrosslinked GelMA [21]. This effect keeps the gelMA-cell solutions separate until crosslinking. The uncrosslinked gelMA-cell solutions will be seeded into the corresponding chambers and crosslinked with visible light (405 nm). The hydrogel-cell suspensions will be seeded on top of an electrode layer consisting of low impedance (< 100K?) PEDOT:PSS electrodes printed using a Voltera circuit board printer (Figure 3). The MPS with a MEA base layer will be connected to a Multichannel system amplifier setup for recording spike data. A custom electrode design will be designed in the software with electrode diameter and spacing values. Spike data will be collected from each neuron population simultaneously and analyzed similarly to aim 1 to validate MEA performance. Co-culture conditions will be optimized (seeding density, media composition, etc.) and control wells of each neuron population will be cultured in parallel. Neurolucida® neuron tracing software will be used to measure neurite outgrowth and quantify synapses within the platform and compared to controls.

Aim 3: Induce inflammation in an MPS to study neuroplasticity of co-cultured enteric neurons and DRGs

when exposed to noxious stimuli. Hypothesis: Inflammation contributes to visceral pain symptoms. NTs modulate neuron responses to inflammation. Methods: This proposal aims to study neuroplasticity over 4 weeks. The MPS design discussed in aim 2 will be utilized. A pro-inflammatory cocktail (H2O2, capsaicin, lipopolysaccharide) will be administered. Neural activity will be recorded from MEAs and analyzed in response to noxious stimuli addition. The recordings will be compared to control data without noxious stimuli. Spent media will be collected from the IPAN and ExPAN compartments every 48 hours for 4 weeks. The media will be stored frozen for use in a multiplex ELISA to study cytokine production and quantify the inflammatory state. Upon completion of the experiment, PCR and ICC will be performed to assess expression of receptors implicated in visceral pain (i.e. TRPV1, CGRP) in control and inflamed models. This aim will produce a simplified study of the effects of exogenous NTs on inflamed neuron populations. After measuring the baseline inflammatory response in aim 3.1, NTs (Table 1) will be administered into the MPS. After administration, firing patterns will be recorded on the MEA. This data will be compared to electrophysiology results without the inflammatory agents present to see if inflammation changes neuron firing frequencies in response to different NTs. A multiplex ELISA will be utilized to assess changes in cytokine production. This will showcase how NTs modulate inflammation and their role in nociceptive signaling.

Potential Challenges: Recording spike data within hydrogels might be challenging because sensory
neurons have small AP amplitudes. To improve recordings, 3D electrodes could be constructed that protrude into the gels. Electrophysiological studies could also be supplemented with live calcium imaging methods. Another potential challenge is the diffusion of NTs into the opposing neuron chamber. Diffusion modeling using dextran will be conducted to see if diffusion of the NTs occurs through the gelMA interface. If diffusion is occurring through the hydrogels, inlets will be added to the MPS that allow perfusion across each neuron chamber, using a syringe pump, to prevent NTs from entering the other compartment.

Summary: The goal of this project is to develop an in vitro model of key neural populations of the visceral pain pathway. An MPS will be utilized to create a well-controlled, but physiologically relevant model incorporating IPANs and ExPANs involved in nociceptive signaling. Extracellular electrophysiological analysis will be used heavily throughout, as well as ICC, PCR, and ELISAs for analyzing protein and gene expression. The role of exogenously synthesized NTs on nociception and inflammation will be explored. Future work could include models that explore anti-inflammatory agents or the roles of sex hormones on visceral pain, as it is experienced much more frequently by females [22, 23]. Overall, this would be the first in vitro model of the visceral pain pathway and could be used for further studies related to gut-brain signaling, disease modeling, and drug discovery.

Supporting Data for Research Proposal:

What methods will be used to evaluate student performance and progress?

The Chemical Engineering Department at Northeastern University has PhD student requirements that must be met. These include specialized coursework and GPA requirements, as well as semester self-evaluation forms. The self-evaluations are reviewed by the PI as well and discussed during one-on-one meetings with the student. These evaluations help assess the student’s independence, creativity, ability to follow protocols, work ethic, and writing and presentation skills. Categories that could use improvement are identified, and a plan is created to address any lacking areas. Students are also expected to complete their qualifying exams after the completion of core coursework. Qualifying exams require a literature review, written proposal, and oral presentation delivered to your dissertation committee. The committee will review the materials to determine if the student has made adequate preliminary progress to earn candidacy.

In addition to department requirements, students in Dr. Koppes’s lab have bi-weekly advisor meetings to discuss progress, obstacles, and goals. Monthly written updates with timelines for active and planned experiments are also submitted on the 20th of each month to the PI. The lab group holds weekly meetings that feature research updates from lab members, offering opportunities to discuss new data and receive feedback on experiments. Generally, students begin their research by developing their own schedules and prioritization, as long as progress is evident through research updates, conference submissions, publications, etc. If adequate progress is not being made, more concrete milestones and deliverables will be developed to keep students on track, and advisor meetings will become more formal with explicit agenda items.

How will your proposed project replace, reduce, or refine the use of animals in science?

Developing a microphysiological system (MPS) of neural populations involved in visceral pain signaling allows for a highly controlled, in vitro environment to study cell-cell communications over an extended time period. Current standards for studying chronic and acute pain pathologies rely on animal models, requiring multiple animal sacrifices to obtain appropriate sample sizes. These in vivo studies also utilize various methods to induce or simulate pain. Intentionally causing discomfort is ethically controversial, as most researchers are obligated to limit any unnecessary pain and suffering to laboratory animals. However, quantifying and justifying appropriate levels of pain is difficult. The United States is not as strict as other countries about the justification and societal benefits for causing pain in laboratory rodents, potentially resulting in many unethical experiments with useless results. Inflicting pain can also cause unintended results in the experiment, such as changes in the animal’s sleeping, eating, and even immune function.

This project focuses on breaking down the key neural populations behind nociceptive signaling and allows for an environment to investigate chemical mediators and cellular communication involved in visceral pain without the multitude of confounding factors present in animal models. This MPS will be utilized at first with primary neuron populations from rat or mice models. The number of sacrificed animals per experiment will be significantly reduced because of the small densities needed of cells per MPS. For example, our current enteric neuron isolation protocol from a litter of rat pups provides at least 1.5 million cells, enough for at least 60 wells in 24 well plates (25,000 cell seeding density). The MPS channel size is even smaller, at about 1cm2, so the isolation would supply cells for 150 MPSs seeded at 10,000 cells each. The sacrificed animals are treated humanely up to their deaths with no intended pain infliction. Our lab members coordinate isolations and experiments with each other to maximize the use of each animal, utilizing enteric neurons, cardiomyocytes, dorsal root ganglia, epithelial tissue for organoids, motor neurons, and more, depending on planned experiments. Our lab also has the ability to cryopreserve many cell types, reducing the frequency of animal isolations.

After obtaining successful results with primary animal cells, I will move to using human-derived tissue (clinically donated) and human induced pluripotent stem cells (hiPSCs) which are obtained from human adults. Using hiPSCs would humanize the model, providing more physiological relevant results for human pathologies, and also completely remove the need for animal sacrifices. hiPSCs are becoming increasingly more accessible, with recent publications showcasing differentiation protocols for multiple cell types. I would differentiate hiPSCs into primary afferent neurons, but enteric neuron cell types are also possible. These cells are also easier to obtain and more reliable than human tissue samples, often donated from biopsies. Additional resources from this fellowship could help jumpstart my progress with hiPSCs, as they can be expensive and fragile to work with, but are highly promising.

During your graduate studies, have you conducted or do you plan to conduct experiments involving animals? If yes, please explain.

Maybe. Unless that affects my grant. In that case... Uh, no. 

How do you plan to disseminate research findings during and after completion of the project?

Dr. Koppes and her students actively participate in multiple conferences, including The Biomedical Engineering Society (BMES), The American Institute for Chemical Engineers (AIChE), The Materials Research Society (MRS), The Society for Biomaterials (SFB), and The World Biomaterials Congress (WBC). She encourages each student to apply for at least two conferences a year after candidacy is obtained and expects students to share their contributions to science through research publications. Dr. Koppes also gives multiple talks a year at other universities and conferences, highlighting newly published work from our lab to an engineering community of both faculty and students.

For this particular project, the dissemination goal would be to present findings at the BMES conference for each aim over the course of two years. Fellow engineers would be the target audience, with the goal of having other laboratories utilize our technology or be interested in collaborating. Conference talks and poster presentations would highlight our work in an environment that supports networking and conversation and allows us to receive feedback from the community.

Data and figures will be collected and saved for a future journal publication. At the conclusion of the project, the manuscript will be submitted to a relevant journal, ideally open access, with an audience that extends beyond engineers and also reaches scientists and clinicians. The goal is for anyone interested in or currently performing gut-brain research to consider our methodology for disease modeling, drug discovery, and mechanistic research.

Applicant Biographical Sketch

Undergraduate Education

Institution Name:
Northeastern University

Major or area of study:
Chemical Engineering

From Date:
06/1933

To Date:
07/1944

Degree Received:
bachelors

Graduate Education

Institution Name:

Major or area of study:

From Date:

To Date:

Degree Received:

List in reverse chronological order all academic and/or professional appointments, beginning with the current appointment.

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List up to five (5) publications most closely related to the proposed project and up to five (5) other significant publications.
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Provide examples that demonstrate the broader impact of your professional and scholarly activities to incorporate the 3Rs into your research.

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What university and/or departmental training in humane science, animal welfare, and/or non-animal research methods have you had?

Not much. 

Principal Investigator/Mentor Biographical Sketch

Full Name:
Abigail Koppes

Curriculum vitae for the faculty sponsor:

Undergraduate Education

Institution Name:
Northeastern

Major or area of study:
Everything

From Date:
05/1932

To Date:
08/1939

Degree Received:
associate

Graduate Education

Institution Name:

Major or area of study:

From Date:

To Date:

Degree Received:

Postdoctoral Education

Institution Name:

Major or area of study:

From Date:

To Date:

List in reverse chronological order all academic and/or professional appointments, beginning with the current appointment:

Ut morbi tincidunt augue interdum velit euismod. Eu turpis egestas pretium aenean. Lorem mollis aliquam ut porttitor leo a diam sollicitudin. Id donec ultrices tincidunt arcu non sodales neque sodales ut. Egestas tellus rutrum tellus pellentesque eu tincidunt tortor aliquam. Auctor neque vitae tempus quam. Ut placerat orci nulla pellentesque dignissim enim. Nibh nisl

List up to five (5) publications most closely related to the proposed project and up to five (5) other significant publications.
  1. Elit at imperdiet dui accumsan sit amet. 
  2. Ornare massa eget egestas purus. 
  3. Ligula ullamcorper malesuada proin libero nunc consequat interdum varius.
  4. Ut consequat semper viverra nam libero justo. 
  5. Senectus et netus et malesuada fames ac turpis. 

Provide examples that demonstrate the broader impact of the mentor's professional and scholarly activities to incorporate the 3Rs into their research.

Elit at imperdiet dui accumsan sit amet. Ornare massa eget egestas purus. Ligula ullamcorper malesuada proin libero nunc consequat interdum varius. Ut consequat semper viverra nam libero justo. Senectus et netus et malesuada fames ac turpis. Euismod elementum nisi quis eleifend quam adipiscing vitae proin. nibh tellus molestie nunc no

Laboratory Funding

Proposal Budget:

If this fellowship was awarded, funds would be utilized for student stipend support. Additional student support would allow a larger portion of funds to be used on the use of human-induced pluripotent stem cell (hiPSC) derived neurons within our lab. Specifically, hiPSC-derived sensory neuron progenitors would be purchased from Axol Bioscience, as well as all the required reagents for differentiation into nociceptive neuron cultures. Other larger expenses would include updated electrophysiological equipment, including microelectrode arrays (MEAs) since this project relies heavily on electrophysiological analysis. Commercial MEAs can be used up to 20 times before electrode degradation. Additionally, as part of this proposal, chemokine panels would need to be purchased for Luminex Multiplex Assays to study the inflammatory responses.

Brief description of prior institutions and the facilities available for this project.

Northeastern University offers all of the required facilities for this proposal. I work in a shared laboratory space within Mugar Life Sciences Building at Northeastern University. The space includes multiple cell culture biosafety cabinets and everything needed for primary and stem cell culture (incubators, centrifuges, liquid nitrogen storage, etc.). Dr. Koppes owns a Zeiss Axio Observer Z1 fluorescence microscope which is utilized heavily for fluorescent imaging. The lab also contains a Bio-Rad polymerase chain reaction (PCR) machine which will be applicable for gene validation. There is a plasma coater, UV oven, and laser cutter
for MPS fabrication within the lab space. We also have our own Multichannel Systems MEA amplifier set up within a faraday cage that is connected to a high-performance computer for electrophysiological analysis. Whole-cell patch clamping and optogenetics equipment are also located within the lab space. Our laboratory also has a high-performance computer with Neurolucida for neuron tracing applications and Graphpad 
prism for statistical analysis.

An AAALAC International accredited animal facility is located in the same building with fully trained care technicians. The facility’s standard operating procedures consider, and often exceed, standards set by The Guide, the Pubic Health Services Policy on the Care and Use of Laboratory Animals, and the Animal Welfare Act.

Current & Pending Support

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Scores & Status







Pre-Application Rating: -1 (0)

Full Application Rating: -1 (0)