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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.Objective.To uncover physiological changes potentially affecting device function and longevity, spatial transcriptomics (ST) 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.Approach.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 ST processing. The tissue was immunolabeled for neurons and astrocytes, which provided both a spatial reference for ST and a quantitative measure of glial fibrillary acidic protein and neuronal nuclei immunolabeling surrounding each implant.Main results. Results from rat motor cortex within 300µm of the implanted electrodes at 24 h, 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.Significance. Our analysis has shed new light onto the potential mechanisms involved in the tissue response to implanted electrodes while generating hypotheses regarding potential biomarkers related to recorded signal quality. A new approach has been developed to understand the tissue response to electrodes implanted in the brain using genes identified through transcriptomics, and to screen those results for potential relationships with functional outcomes.

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RESUMEN

Implantation of electrodes in the brain can be used to record from or stimulate neural tissues to treat neurological disease and injury. However, the tissue response to implanted devices can limit their functional longevity. Recent RNA-seq datasets identify hundreds of genes associated with gliosis, neuronal function, myelination, and cellular metabolism that are spatiotemporally expressed in neural tissues following the insertion of microelectrodes. To validate mRNA as a predictor of protein expression, this study evaluates a sub-set of RNA-seq identified proteins (RSIP) at 24-hours, 1-week, and 6-weeks post-implantation using quantitative immunofluorescence methods. This study found that expression of RSIPs associated with glial activation (Glial fibrillary acidic protein (GFAP), Polypyrimidine tract binding protein-1 (Ptbp1)), neuronal structure (Neurofilament heavy chain (Nefh), Proteolipid protein-1 (Plp1), Myelin Basic Protein (MBP)), and iron metabolism (Transferrin (TF), Ferritin heavy chain-1 (Fth1)) reinforce transcriptional data. This study also provides additional context to the cellular distribution of RSIPs using a MATLAB-based approach to quantify immunofluorescence intensity within specific cell types. Ptbp1, TF, and Fth1 were found to be spatiotemporally distributed within neurons, astrocytes, microglia, and oligodendrocytes at the device interface relative to distal and contralateral tissues. The altered distribution of RSIPs relative to distal tissue is largely localized within 100µm of the device injury, which approaches the functional recording range of implanted electrodes. This study provides evidence that RNA-sequencing can be used to predict protein-level changes in cortical tissues and that RSIPs can be further investigated to identify new biomarkers of the tissue response that influence signal quality. STATEMENT OF SIGNIFICANCE: Microelectrode arrays implanted into the brain are useful tools that can be used to study neuroscience and to treat pathological conditions in a clinical setting. The tissue response to these devices, however, can severely limit their functional longevity. Transcriptomics has deepened the understandings of the tissue response by revealing numerous genes which are differentially expressed following device insertion. This manuscript provides validation for the use of transcriptomics to characterize the tissue response by evaluating a subset of known differentially expressed genes at the protein level around implanted electrodes over time. In additional to validating mRNA-to-protein relationships at the device interface, this study has identified emerging trends in the spatiotemporal distribution of proteins involved with glial activation, neuronal remodeling, and essential iron binding proteins around implanted silicon devices. This study additionally provides a new MATLAB based methodology to quantify protein distribution within discrete cell types at the device interface which may be used as biomarkers for further study or therapeutic intervention in the future.

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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.