Periodic Reporting for period 1 - NEUROGUT (Targeted insula stimulations as treatment for autoimmune disorders)
Periodo di rendicontazione: 2023-06-01 al 2024-11-30
Inflammatory Bowel Diseases (IBD), including Crohn’s and Ulcerative Colitis, are chronic conditions with rising prevalence, affecting millions globally and causing significant systemic and emotional burdens. Current treatments, focused on immunosuppression, are limited in efficacy, cause severe side effects, and fail to address the root causes.
Our research (ERC-funded NEIMO project, Cell, 2021) identified the insular cortex (InsCtx) as a brain region that stores “memories of past inflammation.” Activating these neurons can trigger inflammation, while suppressing them alleviates disease symptoms in mouse models. In this grant, we proposed developing a closed-loop brain-device system to monitor and suppress InsCtx activity, offering a novel therapeutic strategy.
Our approach may revolutionize the treatment landscape for chronic inflammatory diseases, offering a novel therapeutic category that transcends current immunosuppressive strategies. Additionally, the project has sparked collaborations across disciplines and attracted public interest, as evidenced by opinion pieces in leading scientific journals and popular science outlets.
Our research (ERC-funded NEIMO project, Cell, 2021) identified the insular cortex (InsCtx) as a brain region that stores “memories of past inflammation.” Activating these neurons can trigger inflammation, while suppressing them alleviates disease symptoms in mouse models. In this grant, we proposed developing a closed-loop brain-device system to monitor and suppress InsCtx activity, offering a novel therapeutic strategy.
Our approach may revolutionize the treatment landscape for chronic inflammatory diseases, offering a novel therapeutic category that transcends current immunosuppressive strategies. Additionally, the project has sparked collaborations across disciplines and attracted public interest, as evidenced by opinion pieces in leading scientific journals and popular science outlets.
Mapping insular cortex immune associated neuronal networks:
We employed advanced viral tracing and activity-dependent labeling methods to map the immune-processing neural network associated with the posterior insular cortex (pIC).
Using a dual viral injection strategy, we injected two types of AAV based tracers into the pIC of FosTRAP2 mice:
• A retrograde tracer to identify neural terminals projecting to the pIC.
• An anterograde tracer to label monosynaptic downstream projections from the pIC.
Following a recovery period, inflammation was induced using Zymosan-induced peritonitis, a well-characterized model that previously demonstrated immune memory in the pIC. During peak inflammation, 4-hydroxytamoxifen (4-OHT) was administered to label neurons activated during the immune response. Next, we performed tissue clearing and analysis using the iDisco protocol, allowing for high-resolution mapping of neural circuits. Fluorescent signals were used to identify both input and output pathways of the pIC. Whole-brain imaging quantified labeled neurons based on fluorescence and spherical morphology, providing an unbiased view of the immune-associated neural network.
We were able to create an activity-dependent map of the pIC across the entire brain, largely in accordance with existing literature on pIC connectivity. Moreover, our further investigation into these neural circuits uncovered recruitment or suppression of specific pathways during peripheral inflammation. This additional layer of insight could set the stage for new neural targets, including the olfactory system and oxytocin-expressing neurons as new and unexpected candidates for intervention and potential promising candidates for neuromodulation in the context of immune-related disorders.
Targeted electrical stimulation of the insular cortex in humans:
Patients undergoing invasive recordings for seizure localization (stereo-EEG) were invited to participate in an experiment testing the hypothesis that targeted electrical stimulation of specific brain regions can modulate immune responses and brain connectivity. Ethical considerations and informed consent processes were strictly adhered to, ensuring voluntary participation and minimizing additional risks.
Focusing the InsCtx, Direct Current Stimulation (DCS) was applied to predetermined areas of the insular cortex of 13 epilepsy patients via stereo-EEG electrodes. Stimulation parameters were carefully calibrated to ensure safety while achieving effective modulation of neural activity. Blood samples were collected before and after stimulation to assess changes in key immune markers, such as cytokines, immune cell profiles, and inflammatory mediators. Functional brain network changes induced by DCS were quantified using stereo-EEG recordings and advanced connectivity analysis techniques. Stimulation of non-target brain regions or sham stimulation were included as control conditions.
Analysis of preliminary data collected revealed a change in power correlation values across brain regions, time, and frequency bands subsequent to intracranial insula stimulation. Furthermore, these changes were unique to individual patients and stimulation, revealing bidirectional changes in overall regional brain activity as a result of insula stimulation. We are still analyzing the additional patients to gain statistical power. Nevertheless, as we estimated, we may not find significant correlations under these conditions since the patients have no immune challenge. Nevertheless, the fallback position of this study is estimating safety of these procedures for further therapeutic development.
We employed advanced viral tracing and activity-dependent labeling methods to map the immune-processing neural network associated with the posterior insular cortex (pIC).
Using a dual viral injection strategy, we injected two types of AAV based tracers into the pIC of FosTRAP2 mice:
• A retrograde tracer to identify neural terminals projecting to the pIC.
• An anterograde tracer to label monosynaptic downstream projections from the pIC.
Following a recovery period, inflammation was induced using Zymosan-induced peritonitis, a well-characterized model that previously demonstrated immune memory in the pIC. During peak inflammation, 4-hydroxytamoxifen (4-OHT) was administered to label neurons activated during the immune response. Next, we performed tissue clearing and analysis using the iDisco protocol, allowing for high-resolution mapping of neural circuits. Fluorescent signals were used to identify both input and output pathways of the pIC. Whole-brain imaging quantified labeled neurons based on fluorescence and spherical morphology, providing an unbiased view of the immune-associated neural network.
We were able to create an activity-dependent map of the pIC across the entire brain, largely in accordance with existing literature on pIC connectivity. Moreover, our further investigation into these neural circuits uncovered recruitment or suppression of specific pathways during peripheral inflammation. This additional layer of insight could set the stage for new neural targets, including the olfactory system and oxytocin-expressing neurons as new and unexpected candidates for intervention and potential promising candidates for neuromodulation in the context of immune-related disorders.
Targeted electrical stimulation of the insular cortex in humans:
Patients undergoing invasive recordings for seizure localization (stereo-EEG) were invited to participate in an experiment testing the hypothesis that targeted electrical stimulation of specific brain regions can modulate immune responses and brain connectivity. Ethical considerations and informed consent processes were strictly adhered to, ensuring voluntary participation and minimizing additional risks.
Focusing the InsCtx, Direct Current Stimulation (DCS) was applied to predetermined areas of the insular cortex of 13 epilepsy patients via stereo-EEG electrodes. Stimulation parameters were carefully calibrated to ensure safety while achieving effective modulation of neural activity. Blood samples were collected before and after stimulation to assess changes in key immune markers, such as cytokines, immune cell profiles, and inflammatory mediators. Functional brain network changes induced by DCS were quantified using stereo-EEG recordings and advanced connectivity analysis techniques. Stimulation of non-target brain regions or sham stimulation were included as control conditions.
Analysis of preliminary data collected revealed a change in power correlation values across brain regions, time, and frequency bands subsequent to intracranial insula stimulation. Furthermore, these changes were unique to individual patients and stimulation, revealing bidirectional changes in overall regional brain activity as a result of insula stimulation. We are still analyzing the additional patients to gain statistical power. Nevertheless, as we estimated, we may not find significant correlations under these conditions since the patients have no immune challenge. Nevertheless, the fallback position of this study is estimating safety of these procedures for further therapeutic development.
In this project, we achieved two experimental aims:
1. We mapped the posterior insular cortex (pIC) immune-associated neuronal network using dual viral tracing and advanced tissue clearing techniques.
2. We performed the first clinical intervention in 13 epilepsy patients, targeted insular cortex stimulation was performed. Blood samples are still under analysis, and results will be conclusive after completing the cohort.
These results are critical for the development of the therapeutic potential of the project in several ways:
1. Identification of additional targets for stimulation
2. Safety of the stimulations in humans
3. A potential benefit of brain manipulations in humans
In order to further develop this novel therapeutic direction, we will to:
1. Complete cohort evaluation and design a large scale study
2. Determine, based on the immune profile the specific human condition we want to do our proof of concept. We are now considering Chrone’s patients with psoriasis or severe allergies for this aim. We are considering these conditions because they present a major unmet need and strong psychosomatic correlations.
3. Analyze different potential devices that will allow real-time, closed-loop stimulations in patients. There are multiple options and we are discussing with industry leaders what will be the most effective collaboration.
4. Work with regulators to set safety standards and ethical guidelines for clinical use of neuromodulation.
5. Secure funding for device development and commercialization
6. Protect intellectual property for novel techniques and devices.
Overview of Results:
This project established groundbreaking insights into the potential for brain-targeted therapies in humans. The findings pave the way for a new category of precision treatments for inflammatory diseases, addressing unmet clinical needs and creating a platform for future innovation.
1. We mapped the posterior insular cortex (pIC) immune-associated neuronal network using dual viral tracing and advanced tissue clearing techniques.
2. We performed the first clinical intervention in 13 epilepsy patients, targeted insular cortex stimulation was performed. Blood samples are still under analysis, and results will be conclusive after completing the cohort.
These results are critical for the development of the therapeutic potential of the project in several ways:
1. Identification of additional targets for stimulation
2. Safety of the stimulations in humans
3. A potential benefit of brain manipulations in humans
In order to further develop this novel therapeutic direction, we will to:
1. Complete cohort evaluation and design a large scale study
2. Determine, based on the immune profile the specific human condition we want to do our proof of concept. We are now considering Chrone’s patients with psoriasis or severe allergies for this aim. We are considering these conditions because they present a major unmet need and strong psychosomatic correlations.
3. Analyze different potential devices that will allow real-time, closed-loop stimulations in patients. There are multiple options and we are discussing with industry leaders what will be the most effective collaboration.
4. Work with regulators to set safety standards and ethical guidelines for clinical use of neuromodulation.
5. Secure funding for device development and commercialization
6. Protect intellectual property for novel techniques and devices.
Overview of Results:
This project established groundbreaking insights into the potential for brain-targeted therapies in humans. The findings pave the way for a new category of precision treatments for inflammatory diseases, addressing unmet clinical needs and creating a platform for future innovation.