In vivo effects of L1 coating on inflammation and neuronal health at the electrode–tissue interface in rat spinal cord and dorsal root ganglion
Introduction
Neural prosthetic devices implanted into the nervous system to bypass and/or restore sensory-motor or cognitive functions have enormous clinical potential. There are a variety of situations in which such devices can be of use, with proposed applications in the fields of gerontology, rehabilitative medicine, psychiatry, neurology and clinical research [1]. More specifically, neural interface systems can be used for communication [2], to restore lost functional movement [3], to reinnervate target locations for bladder control and for the treatment of neurological conditions such as epilepsy [4], [5] and Parkinson’s disease [6], among others. While much effort has been devoted to brain interfaces, both the spinal cord (SC) and dorsal root ganglion (DRG) are also target implantation regions for these promising rehabilitative and therapeutic devices. For example, SC stimulation has been investigated for pain control [7] and restoration of motor functions [8], [9], while the DRG is an attractive site for recording or stimulating primary afferent neurons to provide sensory feedback [10], [11].
Irrespective of the implant location, these neural interfaces must remain stable throughout the lifespan of the user. However, biocompatibility issues have limited the success of chronically implanted devices [12], [13], [14], [15]. The fate of implanted devices is often determined by the effective integration with the surrounding neural tissue, a current and major roadblock in neuroengineering [1], [16], [17], [18]. In brain tissue, immune and inflammatory reactions, including gliosis at the implant site, result in decreased performance of microelectrodes. Gliosis is thought to be mediated by macrophages, activated microglia and reactive astrocytes, resulting in the formation of a glial sheath that can encapsulate and isolate the implanted probe from the surrounding tissue [19]. In addition, significant decreases in neuronal density in the area immediately surrounding the implant site (the “kill zone”) are problematic. For the long-term success of chronically implanted electrodes, maintaining neurons close to the implant site (within 50–100 μm), minimizing astrogliosis and reducing or eliminating microglial activation are necessary. In the peripheral system, manipulation or damage to a neural structure also leads to anatomic, metabolic and physiological alterations [20]. However, the reactions surrounding these peripheral interfaces highlight the potential for nerve regeneration and recovery following initial damage [20], [21], [22]. Although valuable for multiple applications, the SC and DRG are less well-studied than the brain and peripheral nerve. Therefore, the first aim in this study is to fully characterize the tissue responses in the SC and DRG at both acute and chronic time points in an effort to understand better the cellular responses specific to each.
Surface modifications of implanted electrodes are one approach used to promote favorable interactions between the neural implants and neural cells, and a variety of biomaterial designs have been investigated [2], [16]. Previous work in the authors’ laboratory indicates that the immobilization of L1, a neural adhesion molecule, onto the surface of probes implanted in the rat cortex specifically promotes neuronal survival and neurite outgrowth while inhibiting glial cell proliferation [23]. L1, a transmembrane cell surface glycoprotein, mediates cell–cell recognition by interacting with L1 molecules on the surfaces of neighboring cells (“hemophilic interactions”) or with non-L1 molecules on the surfaces of these cells (“heterophilic interactions”) [24], [25]. It is one of the molecular cues that promotes neurite outgrowth [26], [27] thereby contributing to the formation of the complex neuronal connections of the nervous system [25]. It is also involved in neuronal migration and synaptic plasticity, with essential roles in the maintenance of nervous system functions [24], [28]. L1 serves as a survival factor for neurons of the central nervous system (CNS) [29], [30], [31] and is involved during regenerative responses in the adult CNS [32]. In fact, transplantation of cells engineered to express L1 was associated with improved recovery following SC injury [33], [34], [35]. Furthermore, L1 mediates peripheral myelin formation [36]. This evidence indicates that coating electrodes with L1 may improve their biocompatibility when implanted into either the SC or DRG. Therefore, the second goal of the current study was to investigate the ability of L1 to improve the implant–tissue interface. Previous in vitro [19] and in vivo [23] work indicates that L1 is able to reduce inflammatory gliosis while promoting/maintaining neuronal health. It was hypothesized that electrodes coated with L1 and implanted into the SC or DRG would exhibit a reduced inflammatory response and an increase in neuronal density when compared with non-modified (NM) control probes at both acute and chronic time points. Immunohistological evaluation of the tissue response associated with each probe was quantified and compared by implant site (SC vs. DRG), time point (1 week vs. 4 weeks) and coating (L1 vs. NM).
Section snippets
Neural probes and surface modification
Standard tip tungsten microelectrodes (MicroProbes, Gaithersburg, MD) were used for both in vitro experiments and in vivo implants. Each microelectrode was cut to a 3 mm length for chronic insertion into the neural tissue. The shaft diameter of these tips was ∼0.081 mm (with a parylene-C coating of 3 μm) and an exposed tip diameter of 1–2 μm (25:1 taper).
L1 protein was purified as described previously [19], [27] and concentrations determined with the FluoroProfile (Sigma–Aldrich, St. Louis, MO)
In vitro studies
To determine whether the L1 coating could increase neuronal density and decrease gliosis, the cellular attachment of neurons, astrocytes and microglia was quantified and compared for the different surface conditions (Fig. 1). Parylene-C insulated probes were chosen as the implant model, because a number of widely used neural electrode arrays include parylene-C as the insulator. A two-step approach was used to immobilize proteins onto the parylene-C surface. Plasma treatment was first used to
Discussion
One of the remaining challenges in the development of long-term neural interfaces or neuroprosthetics is maintenance of the cellular environment surrounding the implant. In particular, preventing neuronal cell death, promoting neuronal health and minimizing the inflammatory response are critical for success. Both central and peripheral nervous system sites, including the SC and DRG, respectively, are important target implant sites for such devices. This study has compared the cellular response
Conclusions
The current study reported on the cellular responses associated with implanting L1-coated tungsten microelectrodes into the SC and DRG. It demonstrated that immobilization of neuron-specific L1 protein significantly promotes neuronal density and neuronal health at the tissue interface at both acute and chronic time points. These results are consistent with previous work in the present authors’ laboratory and suggest that immobilization of L1 may increase the biocompatibility of neural probes
Disclosures
The authors have no potential conflicts of interest.
Acknowledgements
The authors wish to thank Simon Watkins PhD and the Center for Biologic Imaging of the University of Pittsburgh for providing confocal training and assistance. They also wish to thank Noah Snyder and Cassandra Weaver for proofreading the article. Funding for this work was provided in part by the Department of Defense TATRC grant WB1XWH-07-1-0716 and the National Institute of Health R01NS062019.
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2020, Biosensors and BioelectronicsCitation Excerpt :L1 coated probe significantly decreased microglia activation (Fig. 6 C&D). This observation was consistent with our previous studies (Azemi et al., 2011; Kolarcik et al., 2012). Besides, in response to injury and signals from the activated microglia/macrophage, astrocytes are activated and increase in GFAP expression, changes in appearance and forms a glial sheath in an attempt to wall of the injury and promote healing (Sofroniew, 2014; Wellman and Kozai, 2017), but could also isolate the recording electrodes from the neurons.