In vivo gut-brain optogenetic endomicroscopy for digestive disorders management

Project Information

NeurO-GI

Grant agreement ID: 853378

DOI

10.3030/853378

EC signature date 1 December 2020

Start date 1 November 2021

End date 31 October 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 526 438,00

EU contribution

€ 1 526 438,00

Coordinated by

WYSS CENTER FOR BIO AND NEURO ENGINEERING
Switzerland

Periodic Reporting for period 2 - NeurO-GI (In vivo gut-brain optogenetic endomicroscopy for digestive disorders management)

Reporting period: 2023-05-01 to 2024-10-31

Summary of the context and overall objectives of the project

Our digestive system, otherwise known as the gastrointestinal (GI) tract, is made up of a series of organs whose mucosal barriers are exposed to a wide variety of external stimuli including environmental factors. It comprises an intricate network of more than half a billion neurons known as the enteric nervous system (ENS). The ENS controls gut motility, nutrient absorption, immune regulation, and defense. Neurogastroenterology is a new subspecialty of gastroenterology that includes the status of the nervous system of the gut and the gut-brain axis during diagnosis and treatment of patients. It is currently a key approach to investigate, diagnose and treat functional digestive disorders (FGID), as well as inflammatory bowel disease (IBD). Together, these conditions are the most common digestive system disorders that cause temporary or chronic discomfort and if not diagnosed and treated early, can lead to life-threatening complications and thus are a significant public health issue.

Moreover, proper functioning of the brain is also connected to the health of the gut via the gut-brain axis (GBA). The gut-brain axis is a complex, multi-systems network of physiological interactions that enables our brain to integrate stimuli and coordinate responses to and from the gastrointestinal tract. The global impact of the gut-brain axis on health is exemplified by the relatively recent identification of its implication in diseases of the nervous system such as Parkinson’s disease, Alzheimer’s disease, Autism spectrum disorders and amyotrophic lateral sclerosis. In the case of Parkinson’s disease, the direct link between the gut and the brain has been made more evident by the discovery that GI symptoms precede those in the central nervous system and that GI symptom severity is predictive of cognitive decline.

Despite the importance of the status of the gut in efficient diagnosis and treatment of digestive and neurological disorders, it is still not well understood. One of the limiting factors is the lack of tools enabling in-vivo and minimally invasive investigations of the enteric nervous system’s involvement in diseases. Novel treatment methods have mainly targeted the gut microbiota, whose dysbiosis directly affects the GBA. However, ENS activity, whose role is crucial in coordinating gut homeostasis, remains mostly unexplored in these contexts. To provide insights into the functional dynamics of the gut and advance our understanding of its involvement and possible therapeutic potential in the GBA, we are developing an endoscope relying on optical and electrical signals to stimulate and monitor status of the gut. Since this entails the development of a new technology and methodology for the investigation of physiological phenomena that are not well understood, the project’s impact will affect both the device and biological knowledge landscapes surrounding the ENS and gut-brain axis.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The main objective of this action is to address the unmet needs for the investigation of the functional status of the gut to improve management of gastrointestinal (GI) and neurological conditions. To achieve this, we are developing a new method to map morphology and functionality of the gut in-vivo over large segments of the lower GI tract of mice.

To define design inputs for the new technology involving optical and electrical recording, as well as optogenetic stimulation, we first developed a methodology for ex-vivo imaging of large segments of the gut with the advanced microscopy platform at the Wyss Center. This approach gave unprecedent qualitative and quantitative information about the network of neurons and others cells involved in physiological function of the gut in mice (healthy and with Parkinson disease) and in human samples at the network level in 3D volumes. Measurements of neuron distribution in the distal colon were used to refine the design of the endoscope’s design for high-resolution intraluminal opto-electrical recording. By applying this method to tissue samples from healthy and disease animals, we could observe morphological remodeling of the network.

Building on the vast experience in electrical recording of brain at the Wyss Center, in August 2023 we achieved the first preliminary in-vivo recording of colon activity. We found spontaneous activity consisting of regular 10~20s trains of neurogenic spikes, repeating every 30~40s. We reliably modulated this activity with pharmacological stimulation of neuronal cholinergic conductance. This led to the development of the NeurO-GI ephys endoscope to map activity over 3 cm of the gut in-vivo. In parallel, we developed a custom endoscopic OCT system, compatible with the ephys endoscope, with specific scanning protocols to support visualization of enteric neuronal ganglia morphology in-vivo. We successfully collected high-quality OCT data in 5 animals and most recently acquired the first simultaneous OCT and ephys recording in-vivo. We also used the same opto-electrical endoscope for intraluminal optogenetic activation. To test the concept, we first achieved the complicated task of customizing an optogenetic mouse line to our experimental needs, choosing the ReaChR-mCitrine transgenic model expressing a red-shifted opsin sensitive to stimulation and inducing the expression of a fluorescent GCaMP reporter of neuronal activity. To target expression in the ENS we injected custom viral vectors produced by a subcontractor. We successfully delivered opsin-activating light to the gut in living animals and effectuated stimulation. We confirmed homogeneous expression of the opsin in the tissue with our ex-vivo imaging methodology. Overall, this novel neurotechnology has the potential to address the needs of neurogastroenterology research and was guided by our knowledge of neurobiology of the gut obtained with ex-vivo imaging.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

As we started this scientific development, we realized that there is limited knowledge about the morphology of the ENS at the organ level. To fill the gap for network-level analysis of ENS morphology ex-vivo, we developed a methodology for network-scale sample preparation, imaging and data analysis from mouse gastrointestinal tissues and full-thickness human colon surgical resections. The neuroscience-compatible protocol for processing and imaging the gut will hopefully expand the imaging toolbox of laboratories to improve understanding of how gut-brain dynamics are affected by disease with the hopes of identifying novel diagnostic markers.

Since the beginning of the action, we presented our approach and results to key opinion leaders from fields including neurology, gastroenterology, biomedical research and business development at venture capitals. The proposed mindset of neurobiology-driven device development to achieve a minimally invasive approach to understand, diagnose and possibly treat disorders in the gut and the brain has been extremely well received with a lot of interest in scientific collaboration and suggestions for translation to the clinical use. Apart from providing a completely novel research device for small animal investigations, we believe that this approach will allow for expansion to large animal models and ultimately humans.

According to a key expert in the field, a major weakness in neurogastroenterology has been the absence of any technology to record in high resolution the activity from the gastrointestinal (GI) tract of live rodents. The ability of our proposed technology to measure the dynamic changes in electrical activity from the gut with morphological confirmation in live mice intraluminally offers biomedical science a unique opportunity to study transgenic animals and hence manipulate specific genes of interest to determine function in-vivo. Commonly, optical and electrical read/write approaches are plagued by crosstalk artifacts and competing engineering requirements. However, by bringing extended experience of the PI in endoscopic optical catheters with expertise in electrophysiology and neurotechnology of the Wyss Center, we were able to create a novel multimodal device showing that the high risk / high gain idea at the basis of this action is feasible.
The first groundbreaking results obtained with our prototypes of electrical and optical endoscopes showed great potential for the development of a robust tool to advance understanding of the gut-brain axis. To demonstrate this tool’s potential impact, we will apply it in longitudinal studies in small mammals with various sources of GI dysfunction and stimulate at the gut and the brain sites. With the ultimate goal of transferring our findings into the clinic, we will be focused on defining modalities and biomarkers that can be used in the early diagnosis of gut-brain axis disorders in humans.

NeurO-GI ex-vivo characterization and in-vivo endoscopic methodology