This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNSRE.2019.2897256, IEEE Transactions on Neural Systems and Rehabilitation Engineering TNSRE-2018-00397 1 Long-Term Functionality of Transversal Intraneural Electrodes Is Improved By Dexamethasone Treatment N. de la Oliva1, J. del Valle1,2, I. Delgado-Martínez1, M. Mueller3, Student Member IEEE, T. Stieglitz3,4,5, Senior Member IEEE, Xavier Navarro1*  Abstract—Neuroprostheses aimed to restore lost functions I. INTRODUCTION after a limb amputation are based on the interaction with the nervous system by means of neural interfaces. Among the different designs, intraneural electrodes implanted in peripheral T he interface of neuroprosthetic systems intended to substitute an amputated limb is based in the stimulation of sensory axons to evoke sensory feedback and in the recording nerves represent a good strategy to stimulate nerve fibers to send sensory feedback and to record nerve signals to control the of motor nerve signals to control the prosthetic device. prosthetic limb. However, intraneural electrodes, as any device Peripheral nerve electrodes are a key component of implanted in the body, induce a foreign body reaction (FBR) that neuroprostheses, as they are the active part that will interact results in the tissue encapsulation of the device. The FBR causes a progressive decline of the electrode functionality over time due to with the nervous tissue [1]. Despite the different designs, most the physical separation between the electrode active sites and the nerve electrodes consist of a polymer substrate where metal axons to interface. Modulation of the inflammatory response has active sites are embedded to deliver current to stimulate nerve arisen as a good strategy to reduce the FBR and maintain fibers or to record nerve signals. Therefore, the relative electrode functionality. In this study transversal intraneural position and distance to the nerve fibers will determine the multi-channel electrodes (TIMEs) were implanted in the rat intensity needed to stimulate the axons and the quality of the sciatic nerve and tested for 3 months to evaluate stimulation and recording capabilities under chronic administration of recorded signals. Among the different designs developed, dexamethasone. Dexamethasone treatment significantly reduced intraneural electrodes, such as the transversal intrafascicular the threshold for evoking muscle responses during the follow-up multichannel electrode (TIME), have shown good capabilities compared to saline-treated animals, without affecting the for selective stimulation and recording in comparison to selectivity of stimulation. However, dexamethasone treatment did extraneural electrodes [2]. not improve the signal-to-noise ratio of the recorded neural However, studies in human subjects and animal signals. Dexamethasone treatment allowed to maintain more working active sites along time than saline treatment. Thus, experiments have shown a progressive decline in the systemic administration of dexamethasone appears as a useful intraneural electrode functionality over time [3]–[5]. This has treatment in chronically implanted animals with neural been attributed, at least in part, to the foreign body reaction electrodes as it increases the number of functioning contacts of (FBR) triggered after the implantation of the device [6], [7]. the implanted TIME and reduces the intensity needed to The FBR against an intraneural electrode is characterized by stimulate the nerve. an initial inflammatory phase, in which polymorphonuclear cells and monocytes/macrophages infiltrate the tissue from 1 Index Terms— dexamethasone, foreign body reaction, intraneural electrode, neuroprosthesis, stimulation. day after the implant. This first phase induces a later anti- inflammatory and tissue-remodeling phase from 8 weeks post- implant, mainly orchestrated by fibroblasts [8]–[11]. This FBR results in tissue encapsulation of the electrodes, leading to a physical separation of the active sites from the nerve fibers 1 Institute of Neurosciences, Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, and Centro de [9], [11] and an increase in the impedance [10], [12]. Since the Investigación Biomédica en Red en Enfermedades Neurodegenerativas relative position and distance to the nerve fibers is critical to (CIBERNED), Bellaterra, Spain. determine the intensity needed to stimulate the axons and the 2 Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and quality of the recorded signals, the capsule tissue around the BIST, Campus UAB, Bellaterra, Spain. 3 Laboratory for Biomedical Microtechnology, Department of electrode clearly hampers its functionality over time. Microsystems Engineering IMTEK, Albert-Ludwig-University Freiburg, Different strategies have been pursued to improve the long- Freiburg, Germany term functional outcome of implanted neural electrodes, 4 BrainLinks-BrainTools Cluster of Excellence, Albert-Ludwig-University Freiburg, Freiburg, Germany including the use of different polymers as a substrate of the 5 Bernstein Center Freiburg, Albert-Ludwig-University Freiburg, Freiburg, electrode [13]–[16], diverse surface coatings [17]–[19] and Germany. pharmacological modulators of the FBR [20], [21]. One of the *X.N. Faculty of Medicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain (correspondence e-mail: Xavier.navarro@uab.cat). most used drugs to modulate the FBR is dexamethasone, a 1534-4320 (c) 2018 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNSRE.2019.2897256, IEEE Transactions on Neural Systems and Rehabilitation Engineering TNSRE-2018-00397 2 glucocorticoid known for its anti-inflammatory and immune- muscles to reduce tethering forces on the electrode. The wires depressant effects [22]. Dexamethasone administration has were routed inside a thin silicon tube subcutaneously and shown good results in reducing the inflammatory reaction to soldered to a Circular Omnetics connector (Nano series) that implanted electrodes in the central nervous system [23], [24] was fixed with a custom-made plastic base on the back of the as well as improving brain recordings [25]. Some studies have animal to facilitate the connection during the applied these strategies to peripheral nerve electrodes, as for electrophysiological tests and covered with a metal protection example, using dexamethasone-releasing cuff [26] or cap (Fig. 1B). The muscles and the skin were closed with dexamethasone-loaded multichannel [27] electrodes. sutures and the wounds disinfected with povidone-iodine. However, these studies did not take into account the time- After the surgical procedure, animals were left to recover in window in which such modulators may be effective or if warm pads. During the follow-up, they were housed at 22±2ºC modulation of the FBR has, indeed, positive effects on the under a 12:12 h light cycle with food and water access ad functional performance of the electrodes for stimulation and libitum. recording. Systemic administration of drugs for reducing the FBR may allow for a better control of dosage and timing without modifications in the design of current nerve electrodes [28]. Thus, this study aims to evaluate if dexamethasone treatment improves the long-term functional outcome of TIME electrodes implanted in the rat sciatic nerve. II. MATERIAL AND METHODS A. Surgical Procedures and Drug Administration Figure 1. Surgical implant of a TIME in the rat sciatic nerve. (A) Photograph All animal experiments conducted in this study were of the TIME inserted across tibial and peroneal fascicles of the sciatic nerve. performed according to protocols approved by the Ethical The tip of the TIME was sutured to the close muscle to avoid motion. Active site 1 (square) is almost out but still within the nerve. Scale bar = 1cm. (B) Committee of the Universitat Autònoma de Barcelona in View of the back of the animal with the subcutaneous wires and the plastic accordance with the European Communities Council Directive base made to house the Omnetics connector. A metal cup was used to prevent 2010/63/EU. damage to the connector. The electrode used was a transversal intrafascicular Half of the rats were administered daily with multichannel electrode (TIME version 3H; developed by the dexamethasone (n=6, 200 µg//kg subcutaneous; Merck) from Department of Microsystems Engineering-IMTEK, University two days before the surgery to ensure systemic levels and of Freiburg, Germany) [29], [30]. It contains seven active sites during all the follow-up. The other half received injections of made of iridium oxide at each arm of the device, with a the same volume of saline vehicle. After 12 weeks post- diameter of 80 µm in one arm and a diameter ranging from 20 implant, animals were deeply anesthetized with an overdose of to 60 µm in the other. In this study, only the active sites of 80 pentobarbital, transcardially perfused with 4% µm at one arm were evaluated. The TIME electrodes were paraformaldehyde in phosphate buffer and the sciatic nerve implanted in the sciatic nerve of female Sprague-Dawley rats including the implanted device harvested for histological (n=12, 220±20g). Animals were anesthetized with ketamine analysis. and xylazine (90/10 mg/kg, i.p.) and the sciatic nerve was B. Histological Studies surgically exposed at the midthigh and freed from adherences to surrounding tissues. Following the same procedure as in The nerve segment including the intraneural electrode was [29], the TIME was transversally inserted across the sciatic embedded in paraffin, and transverse sections (10 µm thick) nerve with the help of a straight needle attached to a 10-0 loop were cut, mounted on silane-coated slides and dried overnight. thread. All the process was monitored under a dissection To identify the location of TIME electrode inside the nerve microscope to ensure the correct position of the active site and to evaluate the presence of foreign body giant cells inside the nerve fascicles (Fig. 1A). Since the TIME used was (FBGCS) and the encapsulating tissue, sections were originally designed for human nerves, in most cases, only 2-3 deparaffinized and stained, first overnight with standard luxol of seven contacts were positioned within the rat sciatic nerve fast blue (LFB) for myelin visualization, and then with at the same time due to dimension mismatch. Then, the hematoxylin Harris solution (Fluka, Sigma) for 7 min, washed electrode tip was fixed to the closest muscle with a 10-0 suture with water and stained with Eosin Y (Merck Millipore) for 3 to avoid the electrode sliding from the nerve and reduce min. Sections were dehydrated with series of graded ethanol motion. The fixation flaps in the arm of the TIME3H were not rinses and mounted with DPX (Sigma). For encapsulation used to fix it to the epineurium, as done in human nerve measurement, the area and the surface occupied by the implants [3], to avoid nerve damage. This fixation was chosen encapsulating tissue around the electrode were measured, and in order to assess functional stability over time, i.e. to maintain a mean thickness of the capsule was obtained. The number of the electrode inside the nerve, but without further damaging it. foreign body giant cells (FBGCs) was counted in each section The ceramic interconnector, on which the TIME tracks are (4 sections/animal, n=6/group). connected to wires, was fixed subcutaneously over the gluteus 1534-4320 (c) 2018 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNSRE.2019.2897256, IEEE Transactions on Neural Systems and Rehabilitation Engineering TNSRE-2018-00397 3 C. Nerve Stimulation Protocol the noise (no stimulus applied) was obtained after applying the To assess the stimulation performance of the implanted short-time Fourier transform with a window of 1 ms, and an TIMEs, biphasic rectangular current pulses with a width of overlap of 87.5%. Only the best recording active site in each 100 µs and an intensity up to 1 mA were delivered (Stimulator TIME was used to calculate the signal-to-noise ratio (SNR), as DS4, Digitimer) through each one of the active sites against a the ratio between the mean of the total power when the stimuli small needle electrode placed near the nerve. The compound are applied and the mean of the total power when there are no muscle action potentials (CMAP) were recorded from stimuli applied. gastrocnemius medialis (GM), tibialis anterior (TA) and Animal body temperature was maintained constant using a plantar interossei (PL) muscles using small needle electrodes thermostatic heating pad during the electrophysiological tests. placed in each muscle [2]. The CMAPs were amplified Both stimulation and recording protocols were performed (P511AC amplifiers, Grass), band-pass filtered (3 Hz to 3 acutely (0 days, 30 minutes after the implantation) and at 7, kHz) and digitized with a Powerlab recording system 14, 30, 60 and 90 days after implant or until failure of the (PowerLab16SP, ADInstruments) at 20 kHz. The amplitude of electrode. each CMAP (M wave) was measured peak to peak and E. Statistical Analysis normalized to the maximum CMAP amplitude obtained in each experiment by stimulation of the sciatic nerve with a Results are expressed as the average ± SEM. Normality of stainless-steel needle electrode. For each active site, the distribution was tested by the Shapiro-Wilk test. Differences threshold current of stimulation that elicited a 5 and 95% of between groups or times in normally distributed data were the maximum CMAP was determined. The active site with the analyzed by two-way ANOVA followed by Sidak`s post hoc lowest threshold value in each electrode (best AS) was used tests. For non-normal variables the analysis was performed for data analysis and comparison between groups with the Kruskal–Wallis test followed by Dunn’s test. (1AS/animal, n=6 at day 0, 7 and 14 and n=5 at day 30 for Statistical significance was considered at p < 0.05. The Dex group and n=6 at day 0 and n=4 at day 7, 14 and 30 for GraphPad Prism software was used for all statistical analyses. Saline group). To measure the electrode displacement over time, the best active site from each TIME at each time point III. RESULTS was determined, the difference with respect to the previous Animals implanted with TIME electrodes and administered position calculated and the result multiplied by the distance with dexamethasone or saline were followed for 3 months to between active sites (400μm). Finally, the selectivity index evaluate electrode functionality. Unfortunately, the wiring (SI) was calculated to quantify the specific activation of a system got damaged before the final date in some cases. single muscle among the set of three muscles (GM, TA, PL) Therefore, the comparison between the two groups with when stimulating from each active site, as previously respect to the electrophysiological results was made up to 1 described [2], [31] and the maximum SI (SImax) from each month of implantation only (subchronic time-point, [34]), TIME for each muscle was used. whereas histology was performed in all animals at 3 months (chronic time-point). D. Nerve Signals Recording Protocol To assess the recording capabilities of TIME electrodes A. Nerve Stimulation Results over Time over time, two different protocols were performed as The current needed to achieve 5% of the maximal CMAP previously described [32]. First, the compound nerve action (5% threshold) for each active site in each TIME implanted potentials (CNAPs) were recorded from the TIME following showed variations over time depending on the active site (Fig. electrical stimulation of the distal tibial nerve. Rectangular 2A). For contacts located inside the nerve immediately after pulses of 100 µs and up to 5 mA (DS4, Digitimer) were implantation, the 5% threshold averaged 70 ± 10 µA in the delivered using two small needles inserted on the medial side saline-treated group and 60 ± 9 µA in the dexamethasone- of the paw. Recordings were made from each active site of the treated group (n=6 per group), whereas half of the active sites TIME against the counter electrode in the ribbon part of the were outside the nerve and had 5% threshold above 200 µA. TIME (outside the nerve), with a small needle electrode Interestingly, the active site with the lowest threshold changed subcutaneously inserted as ground. The CNAP amplitude during the follow-up in each case, indicating a relative motion recorded from each site was measured peak to peak. The from the original position (at day 0) of the implanted maximum amplitude recorded from each TIME was analyzed electrodes (Fig. 2A-B) and suggesting a dynamic integration for comparison between groups (N per groups as detailed in of the electrode within the nerve rather than a fixed implant, the nerve stimulation part). Second, neural activity evoked by that could damage the tissue with its rigidity. Thus, we scratching with a blunt plastic probe the plantar surface of the quantified the displacement of the electrodes with respect to paw (10 repetitions) was recorded from each active site [33]. its original position for the two groups. Saline-treated animals Signals were amplified x5000, band-pass filtered (between presented longer displacement of the electrode than the 300 Hz and 10 kHz) and fed to a power-line noise eliminator dexamethasone-treated group (1400 ± 200 μm vs 720 ± 366 (Hum Bug, Quest Scientific), then digitized at 20 kHz and μm) during the first week of follow-up, equating to the shift of recorded with Chart software (PowerLab System, 2 or 3 active sites Differences, however, did not reach ADInstruments). The total power of the recorded signals and statistical significance (p>0.05, Kruskal-Wallis test). In fact, 1534-4320 (c) 2018 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNSRE.2019.2897256, IEEE Transactions on Neural Systems and Rehabilitation Engineering TNSRE-2018-00397 4 the position of the electrodes in animals treated with significantly lower values for GM and TA muscles in dexamethasone was more stable over time (400 ± 179 μm and comparison with the saline-treated group (p<0.05) (Fig. 3), 160 ± 98 μm after 14 and 30 days, respectively) than the indicating that the treatment limited the initial increase in electrodes in the saline-treated group (300 ± 191 μm and 500 tissue resistance. The treated group still maintained lower ± 252 μm after 14 and 30 days, respectively) with respect to values after 1 month of implant (Fig.3). the previous position (Fig. 2B). Figure 3. Values of stimulation current needed to elicit (A) 5%, (B) 50% and (C) 95% of the maximum CMAP amplitude of gastrocnemius (GM), tibialis anterior (TA) and plantar interossei (PL) muscles in saline and dexamethasone treated groups over time. Two-way ANOVA, *p<0.05 vs saline-treated group, #p<0.05 vs time. Additionally, the number of functioning active sites able to evoke CMAPs in the muscles recorded decreased over time for both groups. However, there were more functioning sites during all the follow-up in the dexamethasone-treated than in the control group, although the difference was not significant (p>0.05, Kruskal-Wallis test, Fig. 4). Figure 2. Changes in stimulation threshold and active sites position within the sciatic nerve over time. (A) Schematic view of a given TIME implanted in the fascicles of the sciatic nerve and its shift over time, as derived from values found in the stimulation protocol. Changes in the active site (AS) position correspond to changes in stimulation threshold. (B) Quantification of the displacement of implanted TIMEs in saline- and dexamethasone-treated groups over time. Each column corresponds to the mean change with respect to the previous tested time. Because of the relative displacement, we used the best active site (i.e., the one with the lowest threshold value which indicates the best position inside the nerve) for each muscle in each electrode for comparison between dexamethasone and saline-treated groups. All animals showed a clear increase in the threshold current needed for muscle activation, at 5% and 95% of maximal CMAP, during the first week of follow-up, with later stabilization at 14 and 30 days after implant (p<0.05). However, rats receiving dexamethasone had 1534-4320 (c) 2018 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNSRE.2019.2897256, IEEE Transactions on Neural Systems and Rehabilitation Engineering TNSRE-2018-00397 5 differences between both groups (Fig. 5C). Figure 5. Maximal selectivity index (SImax) for gastrocnemius (GM), tibialis anterior (TA) and plantar interossei (PL) muscles over time in saline and dexamethasone treated groups. Two-way ANOVA, #p<0.05 vs time. Figure 4. Number of working active sites (AS) in the TIME that were able to B. Nerve signal recording results evoke a (A) 5%, (B) 50% and (C) 95% of the maximum CMAP amplitude of gastrocnemius (GM), tibialis anterior (TA) and plantar interosseus (PL) The maximum amplitude of the CNAP, elicited by electrical muscles in saline and dexamethasone treated groups at different time points.. stimulation of the distal tibial nerve and recorded from active sites in the TIME, decreased over time in both groups (Fig. The selectivity of stimulation for the three different muscles 6A). Dexamethasone treatment allowed recording CNAPs of tested innervated by the sciatic nerve ranged between 0.56 and slightly higher amplitudes, yet not significantly than in control 0.9. The SImax was maintained during the follow-up for GM rats during the first two weeks of follow-up (Fig. 6A). (Fig. 5A) and TA (Fig. 5B) muscles, whereas it decreased Electroneurographic recordings were performed during from 0 to 30 days after the implant for the PL muscle, without mechanical stimulation of the hind paw skin. The SNR 1534-4320 (c) 2018 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNSRE.2019.2897256, IEEE Transactions on Neural Systems and Rehabilitation Engineering TNSRE-2018-00397 6 markedly decreased from 0 to 7 days after the implant and remained unchanged during the follow-up (Fig. 6 B, C), without differences between both groups at any time point. However, dexamethasone treatment resulted in higher number of active sites from which afferent nerve signals could be recorded during follow-up, yet not significantly (p>0.05, Kruskal-Wallis test, Fig.6D). Figure 7. Histological transverse nerve sections stained with LFB-HE showing TIMEs implanted in the sciatic nerve of rats after 12 weeks with (A) saline or (B) dexamethasone treatment. Dotted lines delimit nerve fascicles: TN = tibial nerve, PN = peroneal nerve. Arrow in A points to active sites in the implanted electrode. Scale bars in (A-B) 150µm. (C) Quantification of the thickness of the capsule formed around the electrode in saline and dexamethasone treated groups. (D) Number of FBGCs around the surface of the implanted electrodes in each studied group. T-test vs saline-treated group. IV. DISCUSSION Neural electrodes are a key component of interfaces for the communication between the nervous system and bionic prostheses. Despite the considerable advances achieved in Figure 6. Recording of nerve signals with TIME. (A) Representative CNAP biomaterial and electrode designs [1], [35], improved stability recordings after 0 and 7 days of implantation (dpi). (B) Maximum CNAP is desired to maintain the functionality of implanted electrodes amplitude recorded from active sites in TIMEs implanted in the sciatic nerve over long periods of time. One of the factors that contribute to of saline and dexamethasone treated rats. (C) Representative neural activity recordings after mechanical stimuli in the paw at 0 and 7 days of implantation. the failure or decline of function of neural implants is the FBR (D) Signal-to-noise (SNR) ratio over time in recordings made with TIMEs [9]. The encapsulation of the implanted devices enlarges the implanted in saline or dexamethasone treated rats. (E) Number of active sites distance between the axons membrane and the active sites and (AS) from which recording of nerve signals was obtained with a SNR above also increases the tissue resistance [36], resulting in an 1.01 over time. Two-way ANOVA, #p<0.05 vs time. increase in the current needed to stimulate the axons and also a C. Nerve histology decrease of the SNR during recordings. This study combines The histological evaluation of TIMEs implanted after 3 two of the most promising strategies to improve the long-term months showed that the TIMEs were within the sciatic nerve, functionality of neural implants in peripheral nerves: traversing peroneal and tibial fascicles (Fig. 7A, B). Although transversal and flexible intraneural electrodes (i.e., TIME) and axons were still close to the active sites, the electrodes were anti-inflammatory treatment (i.e., dexamethasone) by systemic surrounded by a thick capsule of fibrotic tissue, as shown by administration. the hematoxylin-eosin staining in samples taken after 3 TIME electrodes have been previously shown to provide months of implantation. The capsule thickness was slightly good stimulation capabilities in animal models [2], [32] and lower in dexamethasone-treated rats (54 ± 18 μm) than in the humans [3], [4]. Although these studies reported adequate saline rats (74 ± 13 μm) (Fig. 7C). Finally, foreign body giant values of impedance and injected charge for useful stimulation cells (FBGCs) were present in close contact with the in human patients, an increase in the stimulation threshold was polyimide substrate, but without significant differences in observed during the first month of implant. The present study number between groups (Fig. 7D). is in agreement since the stimulation current needed to activate the nerve increased during the first weeks of implant, followed by stabilization of the threshold values. Moreover, the selectivity indices found in this work are similar to those shown in previous studies with intraneural electrodes [2], [10], [37], confirming the stable selectivity in subchronic implants. However, few works have focused on the recording 1534-4320 (c) 2018 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNSRE.2019.2897256, IEEE Transactions on Neural Systems and Rehabilitation Engineering TNSRE-2018-00397 7 capabilities of this type of electrode and only in acute reasons for the loss of functional contacts over time of conditions [32]. Our results on subchronically implanted implantation, as previously reported [10]. Indeed, we TIME electrodes show a decrease in the amplitude of the minimized the fixation of the TIME to the rat nerve, compared recorded signals, either compound or single action potentials to the surgical procedure used in human implants [3] or the from one week post-implant, although it was stabilized in the implant of SELINE electrodes in rats [10] that secure a stable following weeks. position, in order to assess the effect of intraneural motion. Several studies have investigated the effect of anti- A limitation of the present study is the lack of evaluation of inflammatory drugs on the FBR to neural implants, with the electrode itself with e.g. impedance analysis. It is true that positive effects on electrode functionality and on the amount neural interfaces can loss its functional capabilities not only of FBR [23]–[26]. However, this is the first work in which it due to biological causes but material degradation or active has been evaluated with intraneural electrodes in the sites corrosion. Further experiments should consider the peripheral nerve. Dexamethasone administration substantially analysis of these parameters. Nevertheless, the reduction of decreased the stimulation intensity needed to activate the FBR with dexamethasone treatment yielded better results with nerve during the first month of implant. We have previously the electrodes over time. described the time course of the FBR to nerve implants, and On the other hand, dexamethasone treatment did not found that the first phase of the FBR peaks at one month [9], improve the capability for recording neural signals. The and that dexamethasone treatment decreases the amount of quality of the recorded signal relies on the amplitude of that infiltrating macrophages within the nerve and also the signal and the placement of the active site with respect to the thickness of the capsule formed around intraneural devices activated axons. Since the amplitude of nerve signals is on the [28]. In this study, we observed that dexamethasone-treated scale of microvolts, the distance between the axons and the animals also had a tendency, yet not significant, to present active site and the capsule tissue characteristics become thinner tissue capsule. These changes can be related to the critical to have a good SNR. While the FBR adds about 30 µm decrease in macrophage infiltration in the nerve tissue, as of separation between the active sites and the axons in demonstrated by lower presence of FBGCs after 3 months of longitudinal implants [9], in transversal implants the FBR adds implant. Dexamethasone treatment also resulted in better about 60 µm of tissue capsule, which would explain the rapid functionality of the implanted TIMEs. In particular, there was decay in electrode recording capability. In addition, the less displacement of the electrode within the nerve during the fibrotic capsule formed around the electrode will presumably first weeks of implant, confirmed by the maintenance of the offer a higher resistivity, that might be comparable to that of active sites stimulation profile in the follow-up testing. This the perineurium [39]. Even though, probably due to the fact may be in disagreement with the reduction of the tissue decrease in capsule thickness produced by dexamethasone capsule, since fewer adherences would be expected to be treatment (75 µm vs 55 µm), there were more active sites able related with greater displacement. However, the anti- of recording small nerve signals. inflammatory treatment may decrease not only the cell infiltration in the nerve tissue but also the edema after the V. CONCLUSION implantation. This edema and the subsequent changes due to Overall, our results show a positive effect of dexamethasone cell infiltration in the saline-treated group may result in treatment on the capability of intraneural electrodes to weaker adherences and larger electrode motion. Thus, the stimulate nerve axons during the first month of implant. This reduction of the stimulation intensity may be due to the is the critical time window during which FBR develops and decrease in capsule thickness around TIME implanted in electrodes show the most important shift in threshold charge nerves under dexamethasone treatment. In addition to the and impedance [10], [28]. Other previously proposed reduced distance between the active sites and the axons to strategies try to deliver different drugs locally on the implant stimulate, changes in tissue deposition and cellular infiltration site. However, these strategies have some limitations in the may imply changes in the electrical properties of the case of intraneural electrodes, such as the nerve size, since surrounding tissue. In fact, we found more functioning local delivery systems are difficult to adapt to intraneural thin contacts able to evoke a muscle response in the treated group. electrodes. Our results demonstrate that systemic Besides, less motion of the electrode within the nerve would administration of anti-inflammatory drugs may be useful and imply more steady stimulation and recording in subchronic easy to apply. The dosage used in this work, after translation implants. This is of particular interest since improved to human equivalent dose (HED) [40] is: bidirectional control of upper limb prostheses is based on HED = 0.20 mg/kg * 6/37 = 0.032 mg/kg stimulating specific motor axons and on decoding nerve which is equal to 2.27 mg/day for a 70 kg adult subject. While signals using featured-detection algorithms [3], [32], [38]. this dose lies within the range of inflammatory diseases Thus, stable electrodes within the peripheral nerves offer more treatments [41], possible side-effects should be monitored for steady signals in subchronic implants. It is worth noting that long-term administration. Since dexamethasone is reducing the impact of electrode motion and of tethering forces are the cellular infiltration during the first month [28], coinciding much larger in rats compared to humans due to the electrode- with the improvement in electrode functionality, short-term nerve size relationship, adding challenges for the resistance of administration can be considered for future experiments. electrodes, wires and connectors, which are often among the Moreover, stronger effects could be expected in more stable 1534-4320 (c) 2018 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNSRE.2019.2897256, IEEE Transactions on Neural Systems and Rehabilitation Engineering TNSRE-2018-00397 8 models of electrode implantation, such those observed in brain 4650–4660, 2014. implants [25], or in larger nerves (i.e., human implants), since [9] N. de la Oliva, X. Navarro, and J. del Valle, “Time the size mismatch between rat nerves and the implanted course study of long-term biocompatibility and foreign electrodes is more prominent than in larger species. body reaction to intraneural polyimide-based implants,” J. Biomed. Mater. Res. Part A, vol. 106, no. ACKNOWLEDGMENT 3, pp. 746–757, Mar. 2018. [10] S. Wurth et al., “Long-term usability and bio- This research was supported by the European Union FPT- integration of polyimide-based intra-neural stimulating ICT projects NEBIAS (contract number FP7-611687), electrodes,” Biomaterials, vol. 122, pp. 114–129, Apr. EPIONE (FP7-602547), FEDER and by the European Union’s 2017. Horizon 2020 Graphene Flagship Core Project 2 (grant [11] N. de la Oliva, M. Mueller, T. Stieglitz, X. Navarro, agreement 785219) and FLAG-ERA JTC 2017 project and J. del Valle, “On the use of Parylene C polymer as GRAFIN, by TERCEL (RD12/0019/0011) and CIBERNED substrate for peripheral nerve electrodes,” Sci. Rep., (CB06/05/1105) funds from the Instituto de Salud Carlos III of vol. 8, no. 1, p. 5965, Dec. 2018. Spain, and by Fundación Ramón Areces (CIVP18A3897). The [12] T. S. Davis et al., “Restoring motor control and ICN2 is supported by the Severo Ochoa program from Spanish sensory feedback in people with upper extremity MINECO (Grant SEV-2013-0295), and by the CERCA amputations using arrays of 96 microelectrodes Programme/Generalitat de Catalunya. The authors thank implanted in the median and ulnar nerves,” J. Neural Monica Espejo and Jessica Jaramillo for technical help. Eng., vol. 13, no. 3, p. 036001, 2016. [13] Y. Kim and M. I. 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