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a Smooth and hierarchical Pt-Ir electrode surfaces (sPt-Ir and hPt-Ir). PU polyurethane. b Fixation base of the electrode¹⁷. c X-ray photo of the implanted electrode. d Experimental design. WE working electrode, RE reference electrode, CE counter electrode, CV cyclic voltammetry, EIS electrochemical impedance spectrum, HE hematoxylin–eosin staining, IHC immunohistochemistry, SEM scanning electron microscopy.

Electrochemical performance was tested using an electrochemical workstation (CHI 660E, CHI, China). In vitro tests were performed in phosphate-buffered saline (0.01 M, Solarbio, China). In vivo tests were conducted during surgery and before sacrifice with a physiological saline bath on the skull formed by medical skimmed cotton. A three-electrode system was used for both in vitro and in vivo testing (Fig. 1d): an sPt-Ir or hPt-Ir electrode as the working electrode, a titanium sheet (20 mm × 30 mm × 0.3 mm) as the counter electrode, and an Ag/AgCl electrode filled with saturated KCl (Model 218, Leici, China) as the reference electrode. During the in vivo test, a ball of medical skimmed cotton soaked with physiological saline was first placed on the skull to form a bath, and then the counter electrode and reference electrode were immersed into it. After testing, they were removed.

After electrochemical testing at 1, 8, and 16 weeks, the animals were euthanized with an overdose of sodium pentobarbital and systemically perfused with phosphate buffer and 4% formaldehyde solution. Then, the electrodes were gently explanted, and the brain tissues were carefully removed. The brain tissues and electrodes were fixed separately in 4% formaldehyde for 24 h. The brain tissue was embedded in paraffin wax. Perpendicular to the electrode axis, consecutive sections were made at a thickness of 5 μm. After dewaxing with xylene, some sections were stained with hematoxylin–eosin (HE). The other sections were incubated with antibodies: rabbit anti-neuronal nuclei (NeuN, AB177487, Abcam, UK, 1:1000), mouse anti-glial fibrillary acidic protein (GFAP, MAB360, Millipore, USA, 1:800) and rabbit anti-ionized calcium binding adaptor molecule 1 (Iba-1, AB178846, Abcam, UK, 1:2000) at 4 °C overnight to label neurons, astrocytes, and microglia, respectively. Then, appropriate dilutions of horseradish peroxidase-conjugated secondary antibodies were added, followed by 3,3′-diaminobenzidine (DAB, PV-9001 and PV-9005, ZSGB-Bio, China).

Photographs were taken at ×20 magnification using an automated digital slide scanner (Axio Scan.Z1, Zeiss, Germany). The measurements were conducted on one NeuN-stained slide for each animal. The normalized neuron number was calculated as a function of distance from the electrode-tissue interface using the image-processing program ImageJ (National Institute of Health)^20,21,22. An outline of the electrode-tissue interface was defined automatically. NeuN-positive cells were counted at 100 μm increment bins away from the interface up to 1000 μm using a marker-controlled watershed algorithm in ImageJ. The results were normalized to the background cellular count (1000 μm away from the interface). The average values and standard deviations were calculated.

The explanted electrodes were dehydrated using graded ethanol and tert-butanol and then lyophilized at −20 °C. The electrodes were observed using a scanning electron microscope (SEM) operating at 20 kV (Quanta 200, FEI, Netherlands). Each electrode was photographed at magnifications of ×100, ×1k, and ×10k to examine the surface structure and biological adhesion.

Electrochemical performance and biocompatibility are critical for neural electrodes. To assess clinical feasibility, hierarchical Pt-Ir electrodes structured by a femtosecond laser were implanted in rat brains to study their in vivo acute and chronic performance compared with existing smooth Pt-Ir electrodes²³. Although both electrodes experienced a decrease in charge storage capacity after implantation, the hierarchical Pt-Ir electrode showed superior long-term electrochemical performance and comparable biocompatibility to the smooth electrode, demonstrating its potential for application as a clinical neural electrode.

Influence of implantation on electrochemical performance

Compared with the in vitro results (Fig. 3b), the intraoperative CSCs for both the sPt-Ir and hPt-Ir electrodes decreased at a similar rate. This decrease is reported to be associated with the reduced ionic conductivity and oxygen concentration of the biological environment²⁴. Over the course of the experiment, the CSCs of the sPt-Ir electrodes remained stable, but the values of the hPt-Ir electrodes further decreased by ~50% from 0 to 8 weeks (Fig. 3b), similar to that of the electrodeposited Pt-Ir coating on cochlear electrodes²⁵. Given that the hierarchical structures showed no signs of shedding, the deterioration should be related to the glial scarring and biological adhesion on the electrode surface, as seen in the histology and SEM characterizations (Figs. 4 and 5)^26,27,28. In the surrounding blood and extracellular matrix, serum albumin and procollagen are the most common proteins, respectively, both with diameters of a few nanometers¹⁶. These proteins might enter the submicron-sized pores of the hPt-Ir electrode surface, leading to an increase in the pore resistance and limiting the inward diffusion of ions^16,29,30. With complete encapsulation, the electrochemical performance of hPt-Ir tended to be stable after 8 weeks³¹.

The differences in EIS among various implantation times were smaller than those between in vitro and intraoperative measurements (Fig. 3c). This was consistent with previous studies^16,32. Not only in vitro but also in vivo, the hPt-Ir electrodes had a similar impedance above the cutoff frequency compared to sPt-Ir electrodes. The impedance in this range was nearly frequency-independent and mainly determined by the geometric surface area and the surrounding electrolytes¹⁹. On the other hand, below the cutoff frequency, the hPt-Ir electrodes had a lower impedance. The impedance in this range was frequency-dependent and related to the real surface area¹⁹. In addition, the postoperative f_cut-off of the hPt-Ir electrode was slightly higher than the intraoperative f_cut-off reflected by the rightward shift of the phase curve. This result suggested an increased real part of the impedance, which might result from increased biological adhesion³¹. Nevertheless, after 16 weeks of implantation in the brain, the hPt-Ir electrode still had greater strengths in impedance amplitude and cutoff frequency.

Intriguingly, the hPt-Ir electrode had a rather stable EIS over 16 weeks, but the CSC value decreased significantly over the first 8 weeks. Similar results have also been found in conductive hydrogel electrodes in rats^22,33. It is assumed to be associated with varying diffusion processes under CV and EIS tests. Given the complex effects of electrode materials, surface structures, and implantation environments on electrochemical processes, it would be worthwhile to conduct more research in the future.

Biocompatibility of the structured electrodes

Biocompatibility is another key aspect of neural implants, which is characterized by increased surrounding neuron density and limited glial encapsulation and inflammation^34,35. Smooth Pt-Ir electrodes have been widely used in long-term implants and have shown good biocompatibility¹⁹. Nevertheless, according to ISO 10993-22: 2017^36,37, medical devices containing nanostructured surfaces should prevent nanoparticle release and should be assessed for their potential effects on cells and tissues due to direct interactions.

To investigate the effects during acute and long-term implantation, we examined the progression from 1 to 16 weeks^21,34,35. In histological analysis, there were similar neuronal densities and thicknesses of neuron-depleted areas around both electrodes, indicating no distinctly negative impact of hPt-Ir (Fig. 4). Furthermore, previous studies have shown that activated microglia and an inflammatory response would be observed if implants were causing the shedding and spreading of micro/nanoparticles^38,39. In this study, no particulate material from micro/nanostructures or abnormal inflammation was observed in the tissues, indicating that the hierarchically structured Pt-Ir electrodes had good mechanical stability and biocompatibility.

The neuron distribution around the electrodes is related to the biocompatibility and functionality of the electrodes. After 16 weeks of implantation, there were fewer neurons within 200~500 µm of both electrodes than there were farther away from the electrodes. A similar neuronal distribution has been found in previous studies^21,22. Neuronal migration is an important issue for microelectrodes due to their limited accessible tissue volume. However, this migration has less of an effect on the macroelectrodes used in this study. These electrode sizes can be used to stimulate neurons and axons⁴⁰ and record local field potentials⁴¹ within a few millimeters. Although the electrodes reported here are functional, improvements in biocompatibility are of great value and deserve further investigation.

Using nanostructured surfaces to control protein and cell adhesion on electrodes has also attracted increasing attention^15,42,43. Several in vitro studies have shown that nanostructures may preferentially provide architectural cues for neuronal adhesion compared to astrocytes^44,45. In addition, structured microelectrodes have been reported to enhance glial resistance⁴⁶ and reduce the expression of some inflammation-related genes after implantation within the brain⁴⁷. These results illuminate the improved integration between neurons and electrodes, which enhances stimulation efficiency and recording quality^34,43,44,48. In this study, hierarchical Pt-Ir electrodes supported more cellular and ECM adhesion, suggesting that they exhibited no apparent biotoxicity (Fig. 5). However, histological results showed similar distributions around hPt-Ir compared to sPt-Ir electrodes (Fig. 4), as reported in some previous works²⁰. This may result from the differences in the implantation regions as well as surface structures¹⁵. Investigating the mechanisms by which micro/nanostructures affect molecular and cellular activities would help to improve the design of Pt-Ir electrode surface structures⁴³. In addition, electrode structure at larger levels may also impact cell morphology and cytoskeletal structure^49,50. A femtosecond laser has been proven to be a feasible tool for preparing various micro- and nanostructures¹⁴. Improving both the in vivo electrochemical and biological properties of electrodes through multiscale structures is an exciting research direction and worth further exploration.

Compared to a rat brain, the electrode in this study is somewhat large. Thus, it might displace brain tissue and induce chronic mechanical stresses, which could affect the function of the neural interface and the long-term health of neural tissue. The maximum occupation volume of electrodes that does not cause damage has not yet been determined⁵¹. It is estimated that multiple implanted microelectrodes occupying 1–2% of the enclosed volume of the brain will not cause significant damage to brain tissue viability⁵². No direct discussion about the occupation effects of the macroelectrode was found. In this study, the occupied volume of the electrode was ~0.6% of the entire rat brain (always less than 1%). None of the experimental rats showed any functional impairment due to brain herniation or brain tissue damage. No increase in chronic inflammatory responses or astrocyte activation was observed. In previous studies that adopted electrodes of the same size, magnetic resonance imaging confirmed that there was no significant midline displacement of the brain^21,53. The cerebrospinal fluid circulation was unobstructed, and there was no change in the size of the ventricles after electrode insertion. This finding suggests that there was no overall effect on brain function in the rats after electrode implantation. In vivo studies for electrodes of similar sizes were also reported using rat models^21,22,54,55. Nevertheless, the brain size and anatomy of rats differ significantly from those of humans. How the electrodes behave in large animal models is worth future exploration.

Implications for clinical applications

The postoperative electrode-tissue interface plays an important role in clinical DBS treatment⁵⁶. Decreasing the impedance amplitude and cutoff frequency by more than an order of magnitude would facilitate improved stimulation efficiency and recording quality¹⁹. Notably, in clinical practice, an hPt-Ir electrode with a lower impedance amplitude may change the effective stimulation parameters under voltage-controlled DBS. Therefore, in this case, current-controlled DBS would be particularly helpful to stabilize the therapeutic efficacy⁵⁷.

Furthermore, clinical DBS studies have reported a decrease in impedance after cyclic electrical stimulation compared to the passive implantation used in this work^58,59. Based on in vitro and in vivo experiments, this effect was reported to probably arise from the desorption of attached proteins and cells, resulting in the cleaning of the electrode⁵⁶. Considering the negative effect of biological adhesion on the electrochemical performance of hPt-Ir electrodes in this study, cyclic stimulation may provide a way to reactivate the implanted electrodes, which deserves further investigation.