Curcumin-releasing mechanically adaptive intracortical implants improve the proximal neuronal density and blood-brain barrier stability.

The cellular and molecular mechanisms by which neuroinflammatory pathways respond to and propagate the reactive tissue response to intracortical microelectrodes remain active areas of research. We previously demonstrated that both the mechanical mismatch between rigid implants and the much softer brain tissue, as well as oxidative stress, contribute to the neurodegenerative reactive tissue response to intracortical implants. In this study, we utilize physiologically responsive, mechanically adaptive polymer implants based on poly(vinyl alcohol) (PVA), with the capability to also locally administer the antioxidant curcumin. The goal of this study is to investigate if the combination of two independently effective mechanisms - softening of the implant and antioxidant release - leads to synergistic effects in vivo. Over the first 4weeks of the implantation, curcumin-releasing, mechanically adaptive implants were associated with higher neuron survival and a more stable blood-brain barrier at the implant-tissue interface than the neat PVA controls. 12weeks post-implantation, the benefits of the curcumin release were lost, and both sets of compliant materials (with and without curcumin) had no statistically significant differences in neuronal density distribution profiles. Overall, however, the curcumin-releasing softening polymer implants cause minimal implant-mediated neuroinflammation, and embody the new concept of localized drug delivery from mechanically adaptive intracortical implants.

Environmentally-controlled microtensile testing of mechanically-adaptive polymer nanocomposites for ex vivo characterization.

Implantable microdevices are gaining significant attention for several biomedical applications. Such devices have been made from a range of materials, each offering its own advantages and shortcomings. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control. To this end, a custom microtensile tester was designed to accommodate microscale samples with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation. The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

The effect of resveratrol on neurodegeneration and blood brain barrier stability surrounding intracortical microelectrodes.

The current study seeks to elucidate a biological mechanism which may mediate neuroinflammation, and decreases in both blood-brain barrier stability and neuron viability at the intracortical microelectrode-tissue interface. Here, we have focused on the role of pro-inflammatory reactive oxygen species. Specifically, adult rats implanted within intracortical microelectrodes were systemically administered the anti-oxidant, resveratrol, both the day before and the day of surgery. Animals were sacrificed at two or four weeks post-implantation for histological analysis of the neuroinflammatory and neurodegenerative responses to the microelectrode. At two weeks post-implantation, we found animals treated with resveratrol demonstrated suppression of reactive oxygen species accumulation and blood-brain barrier instability, accompanied with increased density of neurons at the intracortical microelectrode-tissue interface. Four weeks post-implantation, animals treated with resveratrol exhibited indistinguishable levels of markers for reactive oxygen species and neuronal nuclei density in comparison to untreated control animals. However, of the neurons that remained, resveratrol treated animals were seen to display reductions in the density of degenerative neurons compared to control animals at both two and four weeks post-implantation. Initial mechanistic evaluation suggested the roles of both anti-oxidative enzymes and toll-like receptor 4 expression in facilitating microglia activation and the propagation of neurodegenerative inflammatory pathways. Collectively, our data suggests that short-term attenuation of reactive oxygen species accumulation and blood-brain barrier instability can result in prolonged improvements in neuronal viability around implanted intracortical microelectrodes, while also identifying potential therapeutic targets to reduce chronic intracortical microelectrode-mediated neurodegeneration.

Stab injury and device implantation within the brain results in inversely multiphasic neuroinflammatory and neurodegenerative responses.

An estimated 25 million people in the US alone rely on implanted medical devices, ∼2.5 million implanted within the nervous system. Even though many devices perform adequately for years, the host response to medical devices often severely limits tissue integration and long-term performance. This host response is believed to be particularly limiting in the case of intracortical microelectrodes, where it has been shown that glial cell encapsulation and localized neuronal cell loss accompany intracortical microelectrode implantation. Since neuronal ensembles must be within ∼50 µm of the electrode to obtain neuronal spikes and local field potentials, developing a better understanding of the molecular and cellular environment at the device-tissue interface has been the subject of significant research. Unfortunately, immunohistochemical studies of scar maturation in correlation to device function have been inconclusive. Therefore, here we present a detailed quantitative study of the cellular events and the stability of the blood-brain barrier (BBB) following intracortical microelectrode implantation and cortical stab injury in a chronic survival model. We found two distinctly inverse multiphasic profiles for neuronal survival in device-implanted tissue compared to stab-injured animals. For chronically implanted animals, we observed a biphasic paradigm between blood-derived/trauma-induced and CNS-derived inflammatory markers driving neurodegeneration at the interface. In contrast, stab injured animals demonstrated a CNS-mediated neurodegenerative environment. Collectively these data provide valuable insight to the possibility of multiple roles of chronic neuroinflammatory events on BBB disruption and localized neurodegeneration, while also suggesting the importance to consider multiphasic neuroinflammatory kinetics in the design of therapeutic strategies for stabilizing neural interfaces.

Reduction of autofluorescence at the microelectrode-cortical tissue interface improves antibody detection.

Immunohistochemistry (IHC) remains among the most utilized methods for detection of inflammatory events occurring at the microelectrode-cortical tissue interface. It has further become a standard protocol to quantify the intensity of this resulting fluorescent signal, normalized to "background", as a measurement of the extent of inflammatory events. Unfortunately, several sources of autofluorescence could result in variations in this user-defined "background". Notably, we found that the presence of hemosiderin-laden macrophages (HLMs) at the interface resulted in a variable source of background in both green and red fluorescent channels. The HLM-derived autofluorescence prevented the reproducible detection of presumably low-level antigens at the interface. Here we show that treatment of the native cortical tissue for no less than 10 min, with a minimum of 0.5mM copper sulfate, resulted in at least a 70% reduction in native HLM autofluorescence in both green and red fluorescent channels. In the case of highly expressed antigens, such as glial fibrillar acidic protein (GFAP), treatment of immuno-labeled tissue with copper sulfate reduced tissue background, compared to standard IHC methodology, but did not result in significant differences in the quantification of normalized signal intensity. However, treatment with copper sulfate substantially enhanced the detection efficiency of weakly expressed antigens at the device-tissue interface. This study demonstrates that the inclusion of copper sulfate incubation during IHC tissue preparation significantly reduced HLM-derived autofluorescence, and allowed for more accurate detection and quantification of faintly expressed inflammatory markers at the device-tissue interface.