Spatial transcriptomics at the brain-electrode interface in rat motor cortex and the relationship to recording quality.

Study of the foreign body reaction to implanted electrodes in the brain is an important area of research for the future development of neuroprostheses and experimental electrophysiology. After electrode implantation in the brain, microglial activation, reactive astrogliosis, and neuronal cell death create an environment immediately surrounding the electrode that is significantly altered from its homeostatic state. To uncover physiological changes potentially affecting device function and longevity, spatial transcriptomics was implemented to identify changes in gene expression driven by electrode implantation and compare this differential gene expression to traditional metrics of glial reactivity, neuronal loss, and electrophysiological recording quality. For these experiments, rats were chronically implanted with functional Michigan-style microelectrode arrays, from which electrophysiological recordings (multi-unit activity, local field potential) were taken over a six-week time course. Brain tissue cryosections surrounding each electrode were then mounted for spatial transcriptomics processing. The tissue was immunolabeled for neurons and astrocytes, which provided both a spatial reference for spatial transcriptomics and a quantitative measure of glial fibrillary acidic protein (GFAP) and neuronal nuclei (NeuN) immunolabeling surrounding each implant. Results from rat motor cortex within 300µm of the implanted electrodes at 24 hours, 1 week, and 6 weeks post-implantation showed up to 553 significantly differentially expressed (DE) genes between implanted and non-implanted tissue sections. Regression on the significant DE genes identified the 6-7 genes that had the strongest relationship to histological and electrophysiological metrics, revealing potential candidate biomarkers of recording quality and the tissue response to implanted electrodes .

Gene Expression Changes in Cultured Reactive Rat Astrocyte Models and Comparison to Device-Associated Effects in the Brain.

Implanted microelectrode arrays hold immense therapeutic potential for many neurodegenerative diseases. However, a foreign body response limits long-term device performance. Recent literature supports the role of astrocytes in the response to damage to the central nervous system (CNS) and suggests that reactive astrocytes exist on a spectrum of phenotypes, from beneficial to neurotoxic. The goal of our study was to gain insight into the subtypes of reactive astrocytes responding to electrodes implanted in the brain. In this study, we tested the transcriptomic profile of two reactive astrocyte culture models (cytokine cocktail or lipopolysaccharide, LPS) utilizing RNA sequencing, which we then compared to differential gene expression surrounding devices inserted into rat motor cortex via spatial transcriptomics. We interpreted changes in the genetic expression of the culture models to that of 24 hour, 1 week and 6 week rat tissue samples at multiple distances radiating from the injury site. We found overlapping expression of up to ∼250 genes between in vitro models and in vivo effects, depending on duration of implantation. Cytokine-induced cells shared more genes in common with chronically implanted tissue (≥1 week) in comparison to LPS-exposed cells. We revealed localized expression of a subset of these intersecting genes (e.g., Serping1, Chi3l1, and Cyp7b1) in regions of device-encapsulating, glial fibrillary acidic protein (GFAP)-expressing astrocytes identified with immunohistochemistry. We applied a factorization approach to assess the strength of the relationship between reactivity markers and the spatial distribution of GFAP-expressing astrocytes in vivo . We also provide lists of hundreds of differentially expressed genes between reactive culture models and untreated controls, and we observed 311 shared genes between the cytokine induced model and the LPS-reaction induced control model. Our results show that comparisons of reactive astrocyte culture models with spatial transcriptomics data can reveal new biomarkers of the foreign body response to implantable neurotechnology. These comparisons also provide a strategy to assess the development of in vitro models of the tissue response to implanted electrodes.

Spatial Transcriptomics as a Novel Approach to Redefine Electrical Stimulation Safety.

Current standards for safe delivery of electrical stimulation to the central nervous system are based on foundational studies which examined post-mortem tissue for histological signs of damage. This set of observations and the subsequently proposed limits to safe stimulation, termed the "Shannon limits," allow for a simple calculation (using charge per phase and charge density) to determine the intensity of electrical stimulation that can be delivered safely to brain tissue. In the three decades since the Shannon limits were reported, advances in molecular biology have allowed for more nuanced and detailed approaches to be used to expand current understanding of the physiological effects of stimulation. Here, we demonstrate the use of spatial transcriptomics (ST) in an exploratory investigation to assess the biological response to electrical stimulation in the brain. Electrical stimulation was delivered to the rat visual cortex with either acute or chronic electrode implantation procedures. To explore the influence of device type and stimulation parameters, we used carbon fiber ultramicroelectrode arrays (7 µm diameter) and microwire electrode arrays (50 µm diameter) delivering charge and charge density levels selected above and below reported tissue damage thresholds (range: 2-20 nC, 0.1-1 mC/cm2). Spatial transcriptomics was performed using Visium Spatial Gene Expression Slides (10x Genomics, Pleasanton, CA, United States), which enabled simultaneous immunohistochemistry and ST to directly compare traditional histological metrics to transcriptional profiles within each tissue sample. Our data give a first look at unique spatial patterns of gene expression that are related to cellular processes including inflammation, cell cycle progression, and neuronal plasticity. At the acute timepoint, an increase in inflammatory and plasticity related genes was observed surrounding a stimulating electrode compared to a craniotomy control. At the chronic timepoint, an increase in inflammatory and cell cycle progression related genes was observed both in the stimulating vs. non-stimulating microwire electrode comparison and in the stimulating microwire vs. carbon fiber comparison. Using the spatial aspect of this method as well as the within-sample link to traditional metrics of tissue damage, we demonstrate how these data may be analyzed and used to generate new hypotheses and inform safety standards for stimulation in cortex.

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model.

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound blood brain barrier opening mediated delivery of MRI-visible albumin nanoclusters to the rat brain for localized drug delivery with temporal control.

There is an ongoing need for noninvasive tools to manipulate brain activity with molecular, spatial and temporal specificity. Here we have investigated the use of MRI-visible, albumin-based nanoclusters for noninvasive, localized and temporally specific drug delivery to the rat brain. We demonstrated that IV injected nanoclusters could be deposited into target brain regions via focused ultrasound facilitated blood brain barrier opening. We showed that nanocluster location could be confirmed in vivo with MRI. Additionally, following confirmation of nanocluster delivery, release of the nanocluster payload into brain tissue can be triggered by a second focused ultrasound treatment performed without circulating microbubbles. Release of glutamate from nanoclusters in vivo caused enhanced c-Fos expression, indicating that the loading capacity of the nanoclusters is sufficient to induce neuronal activation. This novel technique for noninvasive stereotactic drug delivery to the brain with temporal specificity could provide a new way to study brain circuits in vivo preclinically with high relevance for clinical translation.